1631172980 aflatoxin

FOOD SCIENCE AND TECHNOLOGY

AFLATOXINS

FOOD SOURCES, OCCURRENCE

AND TOXICOLOGICAL EFFECTS

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AFLATOXINS

FOOD SOURCES, OCCURRENCE

AND TOXICOLOGICAL EFFECTS

ADINA G. FAULKNER

EDITOR

New York

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CONTENTS

Preface vii

Chapter 1 Bio-Prevalence, Determination and Reduction

of Aflatoxin B1 in Cereals 1 Jelka Pleadin, Ksenija Markov, Jadranka Frece,

Ana Vulić and Nina Perši

Chapter 2 Aflatoxin Occurrence 35 Elham Esmaeilishirazifard and Tajalli Keshavarz

Chapter 3 Aflatoxins in Food and Feed: Contamination

Exposure, Toxicology and Control 63 Marta Herrera, Antonio Herrera and Agustín Ariño

Chapter 4 Immunosuppressive Actions of Aflatoxin

and Its Role in Disease Susceptibility 91 Johanna C. Bruneau, Orla Hayden, Christine E. Loscher and Richard O’Kennedy

Chapter 5 Aflatoxins Hazards and Regulations Impacts

on Brazil Nuts Trade 107 Otniel Freita-Silva, Renata Galhardo Borguini and Armando Venâncio

Contents vi

Chapter 6 Polymorphisms of DNA Repair Genes

and Toxicological Effects of Aflatoxin

B1 Exposure 125 Xi-Dai Long, Jin-Guang Yao, Qian Yang,

Cen-Han Huang, Pinhu Liao, Le-Gen Nong,

Yu-Jin Tang, Xiao-Ying Huang, Chao Wang,

Xue-Ming Wu, Bing-Chen Huang, Fu-Zhi Ban,

Li-Xia Zeng, Yun Ma, Bo Zhai, Jian-Jun Zhang,

Feng Xue, Cai-Xia Lu and Qiang Xia

Chapter 7 Incidence of Aspergillus Section Flavi

and Interrelated Mycoflora in Peanut

Agroecosystems in Argentina 157 María Alejandra Passone, Andrea Nesci,

Analía Montemarani and Miriam Etcheverry

Chapter 8 Toxicological Effects, Risk Assessment and

Legislation for Aflatoxins 191 Marina Goumenou, Dimosthenis Axiotis, Marilena Trantallidi, Dionysios Vynias, Ioannis Tsakiris, Athanasios Alegakis, Josef Dumanov and Aristidis Tsatsakis

Chapter 9 Food Sources and Occurrence of Aflatoxins:

The Experience in Greece 233 Ioannis N. Tsakiris, Elisavet Maria Renieri, Maria Vlachou, Eleftheria Theodoropoulou, Marina Goumenou and Aristides M. Tsatsakis

Chapter 10 Aflatoxins As Serious Threats to Economy and Health 259 Lipika Sharma, Bhawana Srivastava, Shelly Rana,

Anand Sagar and N. K. Dubey

Index 287

PREFACE

Progress in understanding the biology of Aspergillus has greatly improved

with the new techniques in genome sequencing and the developed molecular

tools that enable rapid genetic analysis of individual genes. Particularly, the

genetics of aflatoxin synthesis is regarded as a model to gain insight into

fungal secondary metabolism. This compilation discusses topics that include

the prevalence of aflatoxin B1 in cereals; contamination exposure, toxicology

and control of aflatoxins in food and feed; immunosuppressive actions of

aflatoxin; hazards and regulations; toxicological effects, risk assessment and

legislation for aflatoxins; and the threat aflatoxins have on the economy and

health.

Chapter 1 - Moulds of Aspergillus genus are among the most important

causes of food and feed spoilage and can produce mycotoxins as toxic

secondary metabolites when under adverse conditions. Aflatoxins are a group

of mycotoxins that commonly contaminate maize and groundnuts, and are

categorized by the International Agency for Research on Cancer under Class

1A human carcinogens. From the food safety standpoint, one of the most

important mycotoxins is aflatoxin B1 (AFB1). Due to its potent carcinogenic,

teratogenic and mutagenic effects dependent on the level and length of

exposure, the presence of this contaminant in food and feed should be kept as

low as achievable. In order to investigate the occurrence of AFB1, determine

its concentrations and explore the possibility of its reduction using different

methods, samples of maize, wheat, barley and oat were collected from

different cultivation fields during a three-year period. The immunoassay

(ELISA) as a screening method and high performance liquid chromatography

tandem mass spectrometry (LC-MS/MS) as a confirmatory method were used

to determine AFB1 concentrations. Maize contamination seen with AFB1

Adina G. Faulkner viii

concentrations higher than permitted was associated with climate conditions

established in the period of concern, which was extremely warm and dry, and

might had favored mould production and AFB1 formation. Substantial to

almost absolute AFB1 reduction in the maize samples was achieved using

gamma radiation. A strong antifungal effect was also obtained upon the use of

essential oils and lactic acid bacteria as biological AFB1-reduction alternatives.

As the presence of AFB1 in cereals could be dangerous for human and animal

health, in order to prevent its harmful effects and huge economic problems, the

prevention of formation of this contaminant and consistent control over it are

of major interest. Based on these substantiated grounds, possibilities of

implementing new methods of AFB1 determination and reduction within the

frame of safe food production are virtually countless.

Chapter 2 - Toxigenic fungi in crops have been divided historically into

two groups, field and storage fungi. Mycotoxins are produced by toxigenic

fungi at the fields and in the storage. Although many compounds are termed as

―mycotoxin‖, there are only five agriculturally-important fungal toxins:

deoxynivalenol, zearalenone, ochratoxin A, fumonisin and aflatoxin.

Penicillium and Aspergillus species are the most important storage fungi.

However, they can also invade stressed plants in the field. The main

mycotoxins produced by Aspergillus species are aflatoxins, citrinin and

patulin. The word ‗aflatoxin‘ comes from ‗Aspergillus flavus toxin‘, based on

the fact that A. flavus and A. parasiticus are the predominant species

responsible for aflatoxin contamination of crops prior to harvest or during

storage. Aflatoxins B1, B2, G1, and G2 are the four major isolated aflatoxins

from food and feed commodities.

A. flavus and A. parasiticus have distinct affinity for nuts and oilseeds

including peanuts, maize and cotton seed. Cereals are a general substrate for

growth of A. flavus but, unlike nuts, small grain cereal spoilage by A. flavus is

the result of poor handling. Moreover, aflatoxin M1 as a milk contaminant has

potential risk for animal and human health. The character of the aflatoxin

problem varies by region. For instance, aflatoxin accumulation in stored maize

in subtropical Asia has risen rapidly in post-harvest conditions whereas in the

US, the issue is pre-harvest condition of maize. Therefore, the exposure to

aflatoxins differs between countries particularly due to different diets. Food

contamination with Aspergillus is associated with warm and dry climates.

However, in variable environmental conditions, the aflatoxin contamination

may differ from one year to another at the same location.



Preface ix

tools that enable rapid genetic analysis of individual genes. Particularly, the

genetics of aflatoxin synthesis is regarded as a model to gain insight into

fungal secondary metabolism. Well-designed research on production of the

aflatoxin precursor sterigmatocystin with the genetic model A. nidulans, has

contributed greatly to our knowledge of the aflatoxin pathway and the global

regulatory mechanisms. According to the recent studies, fungal pathogenesis is

related to lipid-mediated fungal-host crosstalk, suggesting that secondary

metabolism may be controlled by oxylipins at the transition level. Also, some

oxylipins have been reported to be engaged in the signalling mechanism like

quorum sensing responses in Aspergillus. Quorum sensing molecules and their

genes which are responsible for intra and inter kingdom communications could

be applied in the future aflatoxin bio-control strategies.

Chapter 3 - Aflatoxins (AFs) are secondary metabolites produced by

various fungal species of the genus Aspergillus such as Aspergillus flavus and

Aspergillus parasiticus. The most important compounds are aflatoxins B1, B2,

G1 and G2, as well as two metabolic products secreted in milk, M1 and M2.

The worldwide occurrence of aflatoxins contamination in raw agricultural

products has been well documented; such contamination occurs in a variety of

food and feed, such as cereals, nuts, dried fruits, spices and also in milk as a

consequence of the ingestion of contaminated feed. However, pistachios,

peanuts and corn are the most frequently contaminated food items reported in

the Rapid Alert System for Food and Feed (RASFF) of the European Union.

The occurrence of aflatoxins is mainly affected by environmental factors such

as climatic conditions, geographic location, agricultural practices, and

susceptibility of the products to fungal growth during harvest, storage and

processing. High contamination levels of aflatoxins are mainly associated with

post-harvest growth of Aspergillus moulds in poorly stored commodities.

Aflatoxins can cause adverse effects to the health of animals and humans.

These toxins have been reported to be associated with acute liver damage,

liver cirrhosis, induction of tumors and teratogenic effects. Aflatoxin B1

(AFB1) is usually predominant and the most toxic among aflatoxins because it

is responsible for hepatocarcinoma in animals and strongly associated with the

incidence of liver cancer in humans. AFB1 is a genotoxic and mutagenic

chemical, and it has been classified by the International Agency of Research

on Cancer (IARC) as human carcinogen (group 1). The toxic effects of the

ingestion of aflatoxins in both humans and animals depend on several factors

including intake levels, duration of exposure, metabolism and defense

mechanisms, and individual susceptibility. Aflatoxins affect not only the

health of humans and animals but also the economics of agriculture and food.

Adina G. Faulkner x

Because of the multiple adverse health effects to humans and animals

caused by aflatoxin consumption, many nations worldwide have regulatory

standards on aflatoxin in food and feed. The European Union (EU) regulation

on aflatoxins in foodstuffs is among the strictest in the world (Commission

Regulation (EC) nº 1881/2006 and successive amendments). Maximum

contents of aflatoxins in feeds are also established by Commission Regulation

(EU) nº 574/2011 on undesirable substances in animal feed.

Throughout the world there are many advisory bodies concerned with

food safety, including the World Health Organization (WHO), the Food and

Agriculture Organization of the United Nations (FAO), the Codex

Alimentarius Joint Expert Committee for Food Additives and Contaminants

(JECFA), and many others, which regularly assess the risk from mycotoxins,

advise on controls to reduce consumer exposure and establish different

regulations for these toxins in different countries.

Chapter 4 - Aflatoxins are secondary metabolites produced by fungi of the

Aspergillus species. They occur as contaminants in a variety of food and feed

stuffs that have been infected with the producing fungi. Aflatoxin exposure is

known to cause a number of acute and chronic effects in both humans and

animals, including immunosuppression, liver and other cancers, and failure of

vaccination regimens. The immunomodulatory effects of the aflatoxins have

been shown to affect cell-mediated immunity more than humoral immunity. In

particular, aflatoxin exposure modulates secretion of inflammatory cytokines

and phagocytic function. Decreases in phagocytosis and inflammation

observed following aflatoxin exposure may reduce the effectiveness of the

host immune response to infection, thereby increasing susceptibility to

infection in individuals exposed to these toxins. The aim of this chapter is to

summarise the immunomodulatory effects of aflatoxin exposure in order to

better understand its potential immunosuppressive effects in humans and

animals. The relationship between these immunosuppressive actions and

susceptibility to infection will also be discussed.

Chapter 5 - Brazil nut is an important non-timber forest product produced

in Amazon region. This nut is used as food with high value in the international

market, due to its high nutritional and flavor characteristic and to their

association with environmental conservation and alleviation of poor people

living from Amazonia. Annually, several hundred tons of Brazil nuts are

produced in Brazil. However, they are susceptible to aflatoxins (AF)

contamination. Because of the detection of unacceptable level of AF in Brazil

nuts consignments arriving in European Union ports, in 2003, special

conditions were imposed on Brazil nuts entering the European Union,

Preface xi

decreasing the acceptable levels of AF. In 2010, the European Union revised

AF regulation on nuts; these new limits are more adequate when considering

the complexity of Brazil nut chain and the low risk related to its low

consumption. This chapter points data on the occurrence of AF in Brazil nuts,

as reported by the Rapid Alert System for Food and Feed (RASFF), and

evaluates the efforts made by all sectors involved in the agribusiness of Brazil

nuts, in Brazil, in order to contribute to protection of both domestic and

international consumers from possible health hazard caused by AF.

Chapter 6 - Aflatoxin B1 (AFB1) is an important genic toxin produced by

the moulds Aspergillus parasiticus and Aspergillus flavus. AFB1 is

metabolized by cytochrome P450 enzymes to its reactive form, AFB1-8,9-

epoxide (AFB1-epoxide), which covalently binds to DNA and induces DNA

damage. DNA damage induced by AFB1, if not repaired, may cause such

genic tox toxicological Effects as DNA adducts formation, gene mutations and

hepatocellular carcinoma (HCC). During the repair process of DNA damage

produced by AFB1, DNA repair genes play a central role, because their

function determines DNA repair capacity. In this study, the authors

investigated the association between seven polymorphisms (including rs25487,

rs861539, rs7003908, rs28383151, rs3734091, rs13181, and rs2228001) in

DNA repair genes XPC, XRCC4, XRCC1, XRCC4, XPD, XRCC7, and

XRCC3, and toxicological effects of AFB1 using a hospital-based case-control

study. Toxicological effects of AFB1 were analyzed by means of the levels of

AFB1-DNA adducts, the mutant frequency of TP53 gene, and the risk of

AFB1-related HCC. The authors found that the mutants of XPC, XRCC4,

XRCC1, XRCC4, XPD, XRCC7, and XRCC3 had higher AFB1-DNA adducts

levels, compared with the wilds of these genes (3.276 vs 3.640 μmol/mol DNA

for rs25487, 2.990 vs 3.897 μmol/mol DNA for rs861539, 2.879 vs 3.550

μmol/mol DNA for rs7003908, 3.308 vs 3.721 μmol/mol DNA for

rs28383151, 3.229 vs 3.654 μmol/mol DNA for rs3734091, 2.926 vs 4.062

μmol/mol DNA for rs13181, and 3.083 vs 3.666 μmol/mol DNA for

rs2228001, respectively). Furthermore, increasing risk of TP53 gene mutation

and HCC was also observed in these with the mutants of DNA repair genes.

These results suggested that polymorphisms of DNA repair genes might

modify the toxicological effects of AFB.

Chapter 7 - Studies in typical and new Argentinean peanut areas showed

that toxigenic Aspergillus section Flavi strains are widely distributed in soils

and seeds, with high probability of being transferred to the storage ecosystem.

Mycological analyses of soil showed that Aspergillus section Flavi population

were present in the two areas at similar counts (3.2x102 cfu g

-1). Within this

Adina G. Faulkner xii

section, two fungal species were frequently isolated with isolation percentages

of 73 and 90% for A. flavus and of 27 and 9% for A. parasiticus in soil

samples from traditional and new areas, respectively. The percentages of the

different A. flavus phenotypes from both peanut-growing areas showed that L

strains were recovered in the highest percentage and represented 59 and 88%

of the isolates with variable ability to produce aflatoxins (AFs). Peanut kernels

collected at harvest time from different localities of Córdoba and Formosa

provinces showed A. flavus and A. parasiticus contamination. The 42.8 and

70% were classified as type L and the percentages of aflatoxigenic A. flavus

strains were 68.6 and 80.0% in samples from traditional and recent peanut-

growing areas, respectively. Highly toxigenic A. flavus S strains were isolated

with major frequency from soil and kernel samples coming from traditional

peanut-growing area. Aflatoxin contamination was detected in peanut kernels

from typical peanut growing area. Harvested peanut were stored during 5

months in three storage systems (big bags, wagons of conditioning and drying

and stockpiled warehouse) and mycological population succession was

analyzed. Fungal isolation was greater from pod (95%) than from kernel

tissues. The most common fungi identified included Penicillium, Aspergillus,

Eurotium and Fusarium spp. Within Aspergillus genus, the section Flavi had

the greatest mean counts of 1.4x104, 9.4x10

2, 5.2x10

2 cfu g

-1 for big bags,

wagon and warehouse, respectively. A. flavus and A. parasiticus strains with

variable ability to produce AFs were isolated from peanut kernels stored in the

three systems at all sampling periods in the order of 1.5x102, 2.3x10

2 and 4.5

cfu g-1

, respectively. .A. flavus S and L strains contributed to silo community

toxigenicity during all storage period. Total AF levels ranging from 1.1 to

200.4 ng g-1

were registered in peanuts conditioned at the higher aW values

(0.94–0.84 aW) and stored in big bags. Despite the water stress conditions

registered in the stockpiled warehouse throughout the storage period, AFB1

levels ranging between 2.9 and 69.1 ng g-1

were registered from the third

sampling.

Therefore, the interaction between biological and abiotic factors and

substrate may promote the Aspergillus contamination and the subsequent AF

accumulation in peanut from sowing to storage, highlighting the need to

promote good practices in order to avoid the risk of these metabolites

contamination in peanut food chain.

Chapter 8 - Aflatoxins are toxic metabolites produced by the fungus

Aspergillus. The main representatives are aflatoxins B1, B2, G1, G2. Their

occurrence in food like nuts, cereals and cereal-derived products is a result of

fungal contamination before harvest and during storage. Milk can also be

Preface xiii

contaminated by aflatoxin M1 (main metabolite of B1) as a result of animals‘

exposure to feed contaminated by the aflatoxin B1.

Aflatoxins manifest acute and chronic toxicity. Evidence of acute

aflatoxicosis in humans involving a range of symptoms from vomiting to death

has been reported mainly in Third World Countries. In relation to chronic

toxicity aflatoxins are well known for their genotoxic and carcinogenic

properties while recent studies evident a series of other possible effects like

reprotoxicity, impaired growth in children, intestinal functions, chronic fatigue

syndrome, compromise immunity and interfere with protein metabolism and

multiple micronutrients that are critical to health.

The critical step for aflatoxins‘ risk assessment is the estimation of the real

exposure. For this reason a number of surveys are conducted globally using

tools like biomarkers of exposure and modeling. In addition new parameters

like the climate change are now taken into consideration in order to predict

possible current and future changes of exposure to aflatoxins. As aflatoxins are

compounds of natural origin and their presence in food cannot be totally

eliminated the risk management is based on keeping the total exposure as low

as reasonably achievable taking into account the social-economic impact of

crop and livestock losses. Exposure reduction is achieved mainly by reducing

the number of highly contaminated foods reaching the market by regulatory

control but also applying detoxification strategies. According to the EU

regulatory framework minimization of the exposure to aflatoxins is based on

setting maximum levels of aflatoxins in different foodstuffs (4 – 10 µg/kg total

aflatoxins) and feed (EC/1881/2006, Directive 2002/32/EC). Products

exceeding the maximum levels should not be placed on the EU market.

Methods of sampling and analysis for the official control of aflatoxins, are also

set (EC/401/2006) in order to ensure common sampling criteria to the same

products and that certain performance criteria are fulfilled. The United States

Food and Drug Administration (FDA) has established the action levels for

aflatoxin present in food to the 20 µg/kg (0.5 µg/kg for milk) and up to 300

µg/kg for feed. Finally an action level of 10 µg/kg total aflatoxins is also used

from Japan authorities.

Chapter 9 - This paper presents a review of the occurrence of aflatoxins in

different food commodities in Greece, based both on results represented in

literature as well as results derived from monitoring programs of the Center of

Toxicology Science & Research, Medical School, University of Crete.

Aflatoxins, can pose a severe threat to food safety, since they are characterized

carcinogenic to humans, IARC Group 1. They may be formed or developed in

any stage of the agricultural production (primary production, processing and

Adina G. Faulkner xiv

storage) as a result of transitional weather conditions or of poor storage.

Studies, monitoring programs and surveys, which have been carried out in

Greece, are mainly focused in milk and dairy products. In this context, several

studies have been conducted in animal feeds as well, since there is notable

evidence that they are potential sources of aflatoxins in milk production.

Additionally, both black and green olives have been examined for possible

contamination by aflatoxins, due to the fact that they are damaged during

harvest and processing and thus providing a substrate for aflatoxin

development. Finally, a limited number of studies investigate the presence of

aflatoxins in different processed products like breakfast cereals. The above

foodstuffs have been studied on account of their high nutritional value and the

fact that they are consumed by different population groups. Results indicate

that residue levels of aflatoxins which are presented in fresh as well as

processed agricultural products, do not pose any considerable risk for the

Greek population groups. The most important factors influencing the levels of

aflatoxins in major agricultural products appear to be the growing and

cultivation techniques, as well as the food safety parameters during harvesting,

storage and processing. An additional issue, which seems to raise concern

internationally, is the fact that climate change in combination with

modifications in the cultivation techniques may affect the frequency and

severity of aflatoxin residues in agricultural products.

Chapter 10 - This review deals with the aflatoxins especially with their

food sources, wide occurrence and toxicological effects on animals and

humans. Aflatoxins are highly oxygenated, heterocyclic, difuranocoumarin

compounds and are an important group of mycotoxins produced by the fungi.

There are almost 20 different types of aflatoxins identified till now; among

these AFB1 is considered to be the most toxic. Aflatoxins persist to some

extent in food even after the inactivation of the fungi by food processing

methods, such as ultra-high temperature products, due to their significant

chemical stability. Aflatoxins can affect a wide range of commodities

including cereals, oilseeds, spices, and tree nuts as well as milk, meat, and

dried fruits. Twenty-five percent of the world‘s crops are affected with

mycotoxins. On a worldwide scale, the aflatoxins are found in stored food

commodities and oil seeds. Some of the foods on which aflatoxin producing

fungi grow well include cereals (maize, sorghum, pearl millet, rice, wheat,

corn, oats, barley), oilseeds (peanut, soybean, sunflower, cotton), spices (chile

peppers, black pepper, coriander, turmeric, ginger), and tree nuts (almond,

pistachio, walnut, coconuts), sweet potatoes, potatoes, sesame, cacao beans,

almonds, etc., which on consumption pose health hazards to animals, including

Preface xv

aquaculture species of fish, and humans. Food commodities affected by

aflatoxins are also susceptible to other types of mycotoxins and multiple

mycotoxins can co-exist in the same commodity. Various cereals affected by

aflatoxins are also susceptible to contamination by fumonisins, trichothecenes

(especially deoxynivalenol), zearalenone, ochratoxin A and ergot alkaloids.

More than 5 billion people in developing countries worldwide are at risk

of chronic exposure to naturally occurring aflatoxins through contaminated

foods. Aflatoxin is a potent liver toxin causing hepatocarcinogenesis,

hepatocellular hyperplasia, hepatic necrosis, cirrhosis, biliary hyperplasia, and

acute liver damage in affected animals. Effects of aflatoxins in animals depend

on age, dose and length of exposure, species, breed and nutritional status of the

animal. Health effects occur in fish, companion animals, livestock, poultry and

humans because aflatoxins are potent hepatotoxins, immunosuppressants,

mutagens, carcinogens and teratogens. Aflatoxin– B1 has been shown to cause

significant morphological alterations along with reduced phagocytic potential

in chicken and turkey macrophages. Aflatoxin- B1 exposure to chicken

embryos causes significant suppression in macrophage phagocytic potential in

chicks after hatch. Aflatoxin intercalates into DNA and alkylates the DNA

bases through its epoxide moiety resulting in liver cancer. Other effects

include mutagenic and teratogenic effects. Exposure of biological systems to

harmful levels of aflatoxin results in the formation of epoxide, which reacts

with proteins and DNA leading to DNA-adducts, thus causing liver cancer.

The primary target of aflatoxins is the hepatic system. Acute effects include

hemorrhagic necrosis of the liver and bile duct proliferation while chronic

effects include hepatocellular carcinoma (HCC). HCC is the sixth most

prevalent cancer worldwide with a higher incidence rate within developing

countries. Preliminary evidence suggests that there may be an interaction

between chronic aflatoxin exposure and malnutrition, immunosuppression,

impaired growth, and diseases such as malaria and HIV/AIDS. Outbreaks of

acute aflatoxin poisoning are a recurrent public health problem. The discussion

of this problem and its remedies must be held in the context of the associated

question of food insufficiency and more general economic challenges in

developing countries. Aflatoxin constitutes a serious health concern to the

entire food chain, necessitating a multidisciplinary approach to analysis,

action, and solution.

In: Aflatoxins ISBN: 978-1-63117-298-4

Editor: Adina G. Faulkner © 2014 Nova Science Publishers, Inc.

Chapter 1

BIO-PREVALENCE, DETERMINATION

AND REDUCTION OF AFLATOXIN B1

IN CEREALS

Jelka Pleadin1,

, Ksenija Markov2, Jadranka Frece

2,

Ana Vulić1 and Nina Perši

1

1Croatian Veterinary Institute,

Laboratory for Analytical Chemistry, Zagreb, Croatia 2Faculty of Food Technology and Biotechnology,

Zagreb, Croatia

ABSTRACT

Moulds of Aspergillus genus are among the most important causes of

food and feed spoilage and can produce mycotoxins as toxic secondary

metabolites when under adverse conditions. Aflatoxins are a group of

mycotoxins that commonly contaminate maize and groundnuts, and are

categorized by the International Agency for Research on Cancer under

Class 1A human carcinogens. From the food safety standpoint, one of the

most important mycotoxins is aflatoxin B1 (AFB1). Due to its potent

carcinogenic, teratogenic and mutagenic effects dependent on the level

and length of exposure, the presence of this contaminant in food and feed

should be kept as low as achievable. In order to investigate the

occurrence of AFB1, determine its concentrations and explore the

Corresponding Author: Tel: +38516123626; Fax: +38516123670; E-mail: [email protected].

Jelka Pleadin, Ksenija Markov, Jadranka Frece et al. 2

possibility of its reduction using different methods, samples of maize,

wheat, barley and oat were collected from different cultivation fields

during a three-year period. The immunoassay (ELISA) as a screening

method and high performance liquid chromatography tandem mass

spectrometry (LC-MS/MS) as a confirmatory method were used to

determine AFB1 concentrations. Maize contamination seen with AFB1

concentrations higher than permitted was associated with climate

conditions established in the period of concern, which was extremely

warm and dry, and might had favored mould production and AFB1

formation. Substantial to almost absolute AFB1 reduction in the maize

samples was achieved using gamma radiation. A strong antifungal effect

was also obtained upon the use of essential oils and lactic acid bacteria as

biological AFB1-reduction alternatives. As the presence of AFB1 in

cereals could be dangerous for human and animal health, in order to

prevent its harmful effects and huge economic problems, the prevention

of formation of this contaminant and consistent control over it are of

major interest. Based on these substantiated grounds, possibilities of

implementing new methods of AFB1 determination and reduction within

the frame of safe food production are virtually countless.

1. INTRODUCTION

Cereal grains may become contaminated by moulds that produce

mycotoxins as toxic chemical compounds while in the field and during

storage. This group of compounds represents a significant food safety issue

and poses as a risk to health and wellbeing of humans and animals. Food and

feed contamination with mycotoxins, as toxins of frequent incidence in

agricultural goods, has a negative impact on economies of the affected regions,

especially in the developing countries where harvest and post-harvest

techniques of mould growth prevention are not adequately implemented

(Rustom, 1997).

Cereals such as maize, wheat, barley and oat represent a significant part of

not only human, but also animal diet, and play a role in industrial food & feed

processing. Cereal grains balance the nutrition by virtue of providing a low-fat

diet that has a number of advantages, especially when whole-grain foodstuffs

are consumed. However, grains are a common source of contaminants,

especially mycotoxins, which, under favorable temperature and humidity

conditions, may produce mycotoxins before and/or during harvest, handling,

shipment and storage. The most important mycotoxins are aflatoxins B1, B2,

G1 and G2, fumonisin B1, T-2 toxin, zearalenone, ochratoxin A and

Bio-Prevalence, Determination and Reduction of Aflatoxin B1 … 3

deoxynivalenol. Maize and maize products are known to be prone to

contamination by fungi that produce secondary metabolites such as aflatoxins

(Groopman and Donahue, 1988).

Among food & feed contaminants, aflatoxins are of current concern and

have received a great deal of attention during the last three decades. They were

first heavily researched and truly understood after the death of more than

100,000 young turkeys on poultry farms in England that was found to be

related to the consumption of Brazilian peanut meal (Goldblatt, 1969).

Aflatoxins are known to be produced by two species of Aspergillus genus,

specifically Aspergillus flavus and Aspergillus parasiticus, and represent

highly toxic, mutagenic, teratogenic and carcinogenic compounds that exhibit

an immunosuppressive activity, causing both acute and chronic toxicity in

humans and animals (Eaton and Gallagher, 1994; Massey et al., 1995; EFSA,

2004; Meggs, 2009). Among them, aflatoxin B1 (AFB1) is the most potent

liver carcinogen known in mammals, and is classified by the International

Agency for Research on Cancer (IARC) as Group 1 carcinogen (IARC, 1993).

Factors that promote fungal infection and AFB1 production are inoculum

availability, weather conditions and pest infestation during crop growth,

maturation, harvesting and storage (Lopez-Garcia and Park, 1998). Generally

speaking, crops stored for more than a few days become a potential target for

mould growth and mycotoxin formation (Turner et al., 2009).

In general, mycotoxins, aflatoxins included, are stable compounds not

destroyed during most of the food processing operations, which might lead to

the contamination of cereals and their final products. However, aflatoxin

presence can sometimes be reduced by making improvements in farming

practices, such as providing better storage conditions or using modified seeds,

or by making improvements in manufacturing processes.

Due to the fact that aflatoxins represent the type of mycotoxins most

commonly found in cereals, many studies have attempted to define multiple

aspects of contamination of human food and animal feed chains, and still do

so, so that the topic is a very hot one. Such a contamination is often

unavoidable and still poses as a serious problem associated with important

agricultural goods, which emphasizes the need for suitable processing capable

of inactivating the toxin. Maize, as the most widely grown crop extensively

used for animal feeding and human consumption, represents a particular

problem. Due to its nutritional value, a high percentage of the world maize

production is destined for animal feeding.

The European Food Safety Authority document (EFSA, 2013), prepared

based on analytical data on four aflatoxins (B1, B2, G1, and G2) recovered in


food samples collected between 2007 and 2012, reports that the collection of

data on the occurrence of aflatoxins in relevant foodstuffs should be continued

in order to gather a representative number of samples in different food

categories; in addition, the document draws attention to the need for

harmonizing the reporting formats across the European countries.

This chapter presents the results of AFB1 determination in four types of

commonly used cereals intended for food and feed, collected during a three-

year period from different cultivation fields, as well as the results of an

investigation into the possibilities of contamination reduction and/or

avoidance. For the sake of AFB1 determination, the immunoassay (ELISA) as

a screening method and high performance liquid chromatography tandem mass

spectrometry (LC-MS/MS) as a confirmatory method were used. Gamma

radiation and essential oils & lactic acid bacteria, on the other hand, were used

to investigate the possibilities of AFB1 reduction in contaminated maize

samples.

1.1. Exposure to AFB1 through the Food Chain

The Food and Agriculture Organization (FAO) estimated that 25% of the

world food-intended crops are contaminated with mycotoxins, and that

aflatoxins, as the most toxic among them, are the trickiest to deal with because

of their widespread occurrence in maize, peanuts and its products, cottonseed,

chilies, peppers, pistachio nuts and some other foodstuffs (Scholthof, 2003).

Contaminated feed also represents the main source of AFB1 infestation in farm

animals, which get to be contaminated through parasites living on plants even

prior to harvesting or on stored harvested crops (Huwing et al., 2001; Gareis

and Wolff, 2000). As fodder, cereals and seeds used for dairy cattle feeding

are inevitably in contact with yeasts and filamentous fungi, contamination of

these raw materials frequently occurs already in the field. AFB1 contamination

can also occur during harvesting, transport and storage of cereals and their

products, as well as due to post-harvest mishandling that can lead to rapid feed

spoilage (Alonso et al., 2011).

In animals intended for meat production that had consumed contaminated

feed, the ingestion of AFB1 leads to substantial degradation of meat quality

(Bonomi et al., 1994). Cattle exposure to mycotoxins generally occurs through

the consumption of contaminated feed. Nelson et al.. (1993) described

mycotoxicoses arising on the grounds of exposure to mycotoxin-contaminated

rations. Ruminants, such as cattle and sheep, are generally more resistant to


mycotoxins than most animals, especially pigs, as ruminal microbial

population plays a role in detoxification process. This assumption is based on

the finding that rumen flora is able to convert a number of mycotoxins into

metabolites that are less potent or even biologically inactive at common

exposure levels (Kiessling et al., 1984). The first identified source of

mycotoxins in ruminant diets was the contamination of feed concentrates with

aflatoxins. AFB1 occurs in many typical energy-rich concentrates such as grain

maize, sorghum, pearl millet, rice, soybean products, peanuts, sunflower &

cotton seeds, palm kernels and copra (Vargas et al., 2001; Abbas et al., 2002;

Attala et al., 2003).

Humans are exposed to AFB1 either directly through the consumption of

contaminated food or indirectly through the consumption of animal products

(i.e. milk and eggs) coming from animals that had consumed contaminated

feed (Rustom, 1997; Bennett and Klich, 2009; Markov et al., 2013). Since it

was first observed that dairy animals consuming feeds contaminated with

AFB1 excrete aflatoxin M1 (AFM1) in their milk, studies have established that

variations in carry-over rates are significant both at high- and low-level AFB1

feed contamination (Prandini et al., 2009).

1.1.1. AFB1-Related Effects Seen in Humans and Animals

Although animal species may vary in their susceptibility to aflatoxins,

toxic effects of the latter, known as aflatoxicoses, can generally be divided into

acute and chronic, based on some determinants such as the duration and level

of exposure, entry route, environmental factors, age, health, nutritional status,

and other factors such as stressors affecting the animal (Leeson et al., 1995;

FDA, 2002). In case of humans, exposure to AFB1 occurs mainly through the

consumption of contaminated food such as corn, peanuts, sorghum, copra and

rice, cashew, hazel, peanuts, walnuts, pistachios and almonds (Busby and

Wogan, 1984; Abdel-Gawad and Zohri, 1993; Mahoney and Rodriguez,

1996). AFB1 also exhibits its toxicity through the metabolite AFM1, which

was first determined in human urine while elucidating the etiology of liver

cancer caused by AFB1 (Campbell et al., 1970). It has been reported that about

1.3 to 1.5% of the ingested AFB1 converts into AFM1 that gets to be excreted

in human urine (Zhu et al., 1987).

As the contamination of foodstuffs and feedstock with aflatoxigenic

moulds and their toxins is very common, toxic effects of AFB1 on animal

health are encountered worldwide (FAO, 2004). Many animal species such as

turkeys, ducklings, rainbow trout, guinea pigs, rabbits, rats and dogs show

high susceptibility to aflatoxins. AFB1 can cause liver and other cancers in


humans and livestock; this has been well established in several animal species

including rodents, nonhuman primates and fish, the first symptoms thereby

being a lack of appetite and weight loss (Busby and Wogan, 1984; Eaton and

Gallagher, 1994). Several research reports have agreed that AFB1 is more

toxic for young animals (IARC, 1993, Vainio et al., 1994). It has been

observed in many parts of the world that AFB1 poses a major etiological factor

in the development of hepatocellular carcinoma in individuals infected with

hepatitis B virus (Wild and Hall, 2000). Particularly high incidences of AFB1

contamination have been seen in tropical and subtropical regions, where warm

and humid weather provides for conditions optimal for mould growth.

Chronic ingestion of AFB1 causes various adverse effects such as

increased susceptibility to diseases, loss of reproductive performance and, in

case of dairy cattle, a decrease in quantity and quality of milk production.

Animal exposure to AFB1 leads to a decrease in feed consumption or even to

feed refusal, as well as to the reduction in nutrients‘ absorption, metabolic

impairments, decreases in protein synthesis, and endocrine and immune

system suppression. Acute intoxication is often fatal for both humans and

livestock. In poultry and livestock, severe and sudden anorexia, convulsions,

feed refusal, weight loss, discolored liver, reduced egg production, reduced

energy conversion rate and milk contamination can be encountered. On top of

that, the consumed feed loses its common nutritional value and efficiency,

leading to reduced livestock growth rates (Waliyar et al., 2007).

1.1.2. Conditions under Which AFB1 Gets to Be Produced in Cereals

Accumulation of mycotoxins before and after cereal harvesting largely

reflects actual climate conditions. Fusarium toxins are known to be produced

during cereal harvesting under high moisture conditions (Munkvold and

Desjardins, 1997), whereas pre-harvest aflatoxin contamination of crops,

including peanuts (Dorner, 2008) and maize (Payne, 1998), is associated with

high temperatures, insect-mediated damage and prolonged drought. Chronic

contamination occurs in warm, humid, tropical, and subtropical maize-

growing environments (Widstrom, 1996). The degree of moisture mostly

depends on the water content available at the harvesting point, but also on the

frequency and extensiveness of drying, aerating, and turning of the grain

before and during storage, and the respiration of insects and microorganisms

harbored by stored grain (Bryden, 2012). Since Aspergillus can tolerate lesser

water activity than Fusarium, these contaminations may occur both pre- and

post-harvesting, whereas Fusarium contamination is more specific to the pre-

harvesting period (Abramson, 1998). Stored cereals may become infested with


fungi and insects; such an infestation is also affected by climatic factors such

as temperature and humidity, geographical location, type of storage container,

and handling & transport procedures (Chelkowski, 1991; Jayas et al., 1995).

Climate changes can alter the dynamics of insect populations that facilitate

fungal crop infections (Wu et al., 2011).

Earlier studies have pointed toward significant dependence of AFB1

occurrence on country or region in which the cereals are grown, as well as on

high AFB1 concentrations found in maize, peanuts, tree nuts, rice and

cottonseed (Rustom, 1997; Reddy et al., 2009). It has been pointed out that the

growth of A. flavus and the production of aflatoxins in various biological

materials are influenced by many factors including the type of substrate, its

moisture content, ―culpable‖ fungal species, presence of minerals, relative

humidity of the surroundings, temperature, and physical damage of the kernels

(Viquez et al., 1994). It has been shown that the type of mould and its conidial

concentration, as well as maize moisture content, play critical interactive roles

in the initiation of mould infestation, spoilage and AFB1 production in maize

(Oyebanji and Efiuvwevwere, 1999).

Limitation of AFB1 occurrence in crops before harvest can be achieved

through the reduction of drought and temperatures, weed control, insect

damage reduction, effective harvesting techniques and Aspergillus spore

reduction in soil by virtue of crop turnover. Genetic engineering and the

development of hybrids resistant to Aspergillus spp infection (Widstrom,

1996) may offer new ways of limiting pre-harvest aflatoxin contamination of

certain crops. Post-harvesting control of AFB1-susceptible crops can be

achieved through the control of factors that affect fungal growth, e.g. water

activity, temperature, gas atmospheres, and through the use of insecticides or

food preservatives. The prime concern relative of the storage of grains and

nuts should be to maintain water activity below the limit favoring fungal

growth (which is achievable by virtue of moisture control) (IARC, 2002). The

risk of kernel damaging and consequent AFB1 production can be reduced by

harvesting solely grains having the moisture of around 24% (Prandini et al.,

2009).

1.1.3. The Occurrence of AFB1 in Feed

In many cases, the levels of AFB1 naturally occurring in feeds intended

for dairy cows have been shown to exceed the regulation limits. The contents

of feed intended for milking cows slightly vary dependent on the season and

geographical area; some 10% of feed is commonly intended for these

purposes. Rye, oats, mocha, wheat and sorghum are selected on dairy farms


based on the acreage and selected pasture; the use of commercial pelleted feed

is not uncommon either (Alonso et al., 2011).

Given the fact that in geographical regions having a tropical or sub-

tropical climate the risk of AFB1 contamination has generally been

acknowledged as high, monitoring of feed ingredients for the presence of

AFB1 has been focused on imported feeds such as extracted copra, peanut

cake, sunflower cakes, corn gluten, rice bran, cottonseed, palm kernel and soy

beans, which seem to be the major carriers of AFB1. In some countries,

contamination levels above legal limits were linked to high contamination of

locally grown maize that was used as animal feed (EFSA, 2004).

In different countries AFB1 has been found to be a contaminant of dairy,

cottonseed, barley, soy bean, pellet wheat, peanut shells, corn silage and

sorghum silage (Decastelli et al., 2007; Sassahara et al., 2005). Certain cases

pointed toward an outbreak of acute aflatoxicosis, with high levels of AFB1

observed in maize stored under high humidity conditions (Lewis, 2005). As

for dairy cattle, the problem does not end with animal diseases or production

losses, since AFB1 presence in feed leads to the presence of its metabolic

product AFM1 in milk and dairy products, possibly affecting human health as

well (Boudra et al., 2007; Veldman et al., 1992).

1.2. Current EU Legislation

Since the discovery of aflatoxins in the 1960s, regulations have been

enforced in many countries so as to protect the consumers from harmful

effects of these toxins that may contaminate both foodstuffs and feedstuffs.

Various factors play a role in defining permissible mycotoxin levels. These

include evidence-based data underpinning the risk assessment, such as the

availability of toxicological data, food consumption data, data on the level and

distribution of mycotoxins in goods intended for human and animal

consumption, and data on analytical methodology. Economic factors such as

commercial and trade interests and food safety issues also have an impact

(FAO/WHO, 2008). Compared to other regions of the world, the European

Union (EU) has the most extensive and most detailed regulations governing

AFB1 presence in various types of food and feed. Also, many of the EU

candidate member states have mycotoxin presence-governing regulations

covering the topic as much in depth as the regulations currently in force across

the EU itself.


Methods of sampling and analysis used within the frame of the official

mycotoxin control, AFB1 included, are laid down under the Commission

Regulation No 401/2006, amended by the Commission Regulation (EU) No

178/2010. This ensures that the same sampling criteria are applied for the

same products by the competent authorities throughout the EU and that certain

performance criteria, such as recovery and precision, are fulfilled. Maximum

permitted levels (MPLs) of aflatoxins in food, including those of AFB1 and

total aflatoxins, are laid down under the Commission Regulation (EC) No

1881/2006, amended by the Commission Regulation (EU) No 165/2010.

Legal limits for AFB1 in feedstuffs currently adopted by the EU member

states and set under the Commission Directive 2003/100/EC that amends the

Directive 2002/32/EC, are substantially different from those in other countries

that have enforced AFB1 MPLs for animal feeding stuffs. As AFB1 is a

genotoxic carcinogen and a strong acute toxin that affects various animal

species, it is the only individual mycotoxin whose MPLs are set under the

Directive. Some countries have a number of limits, often dictated by the

destination of the feedstuff. From the human health‘s point of view, the most

stringent criteria apply to feedstuffs intended for dairy cattle because of

AFB1‘s conversion into AFM1 that takes place in milk and dairy products

(MPL= 5 µg/kg across the EU).

1.3. Analytical Methods of AFB1 Determination

For the purpose of AFB1 determination, different screening and

confirmatory analytical methods are used. Most of these analytical methods

have to be performed using the appropriate cleanup procedures, except

perhaps for the immunological assay called the ELISA, with which this step

might be skipped. The development of semi-quantitative ELISA as a screening

method was a major step forward in the development of rapid, repeatable and

sensitive assays suitable for AFB1 determination. Gaur et al. (1981) produced

and characterized AFB2-antiserum, equally specific for AFB2 and AFB1, and

used it within the ELISA for AFB1 quantification.

In the recent years, the ELISA has been described to have advantages over

other methods when it comes to AFB1 determination; these advantages mostly

lie within its rapidity, high specificity, simplicity of use, low cost and the use

of safe reagents (Pestka, 1994; Zheng et al., 2005; Goryacheva et al., 2007,

Ayejuyo et al., 2011). Commercially available ELISA kits suitable for the

detection of mycotoxins are based on the competitive assay format that uses


either a primary antibody specific for the target molecule or an enzyme

conjugate and the required target. The advantage of micro-titer plate-based

immunoassays lies within the fact that these can be used to analyze a large

number of samples simultaneously. The complex formed on the occasion then

interacts with a chromogenic substrate to give a measurable result. The

disadvantage of the ELISA mostly comes as a result of its limited detection

range consequential to the narrow scope of the antibodies‘ sensitivity (Turner

et al., 2009).

Other methods for AFB1 quantification require sophisticated laboratory

equipment, including high performance liquid chromatography (HPLC), gas

chromatography (GC), liquid chromatography/mass spectrometry (LC/MS) or

gas chromatography/mass spectrometry (GC/MS) (Xiang et al., 2006; Krska et

al., 2008; Rahmani et al., 2009; Stephard et al., 2011).

HPLC has a high efficiency, sensitivity and resolution (Herzallah, 2009;

Peiwu et al., 2011). Modern analysis of components heavily relies on HPLC

that employs various adsorbents depending on physical and chemical structure

of different components. The most commonly used HPLC detectors are

fluorescence detectors (FLDs). In order to widen the detection limits, HPLC is

used in combination with mass spectrometry (MS) (Li et al., 2009). MS

represents a method that allows for a highly accurate and specific detection of

AFB1, with limiting factors such as high cost of equipment, complex

laboratory requirements, and limitations in the type of solvents used for

extraction and separation (Turner et al., 2009).

1.4. Methods of AFB1 Reduction

As the presence of moulds and/or mycotoxins in food can be dangerous

for human health and represents a huge economic problem, one has all the

reasons to allow for the implementation of new methods providing for a safe

food production. Methods of control can be classified into two categories: (1)

prevention of mould contamination and growth, and (2) detoxification of

contaminated products (Riley and Norred, 1999; Mishra and Das, 2003). The

prevention of mould growth can be achieved either through pre- or post-

harvesting strategies. The applied AFB1-reduction procedure must effectively

inactivate or remove the toxin, maintaining at the same time both nutritional

and technological properties of the product, while not generating reactive toxic

products (López-García and Park, 1998). These methods can be divided into

chemical, biological and physical (Kabar et al., 2006). Investigation into the


methods of AFB1 inactivation in contaminated food and feed has revealed that

pre-harvest contamination can be reduced by virtue of proper curing, drying,

sorting and storage, all of the aforementioned limiting the growth of

aflatoxigenic fungi. However, the implementation of unique, totipotent method

of aflatoxin reduction, capable of effectively performing in any given

biological material, is virtually impossible.

The efficiency of methods of AFB1 inactivation depends on many

parameters such as the nature of food and feed, their moisture content and

composition, and the level of contamination. Some studies have attempted to

achieve detoxification of, or toxin inactivation in, AFB1-contaminated

feedstuff using gamma irradiation, thermal inactivation, physical separation,

microbial degradation and different chemical treatments (Piva et al., 1995;

Rustom, 1997).

1.4.1. Biological Reduction

Many microorganisms including bacteria, yeasts and acid-producing

moulds can metabolize and inactivate AFB1, Flavobacterium aurantiacum

being the most active among them. AFB1 production is also inhibited by lactic

acid bacteria, Bacillus subtilis and many moulds. As shown in the fermenting

industry settings, aflatoxins do not degrade during fermentation, but have been

proven absent from alcohol fraction after distillation. Aflatoxins are usually

concentrated in spent grains. When contaminated products are used for

fermentation, it is important to determine the end-use of the contaminated by-

products. A specific compound found to be a good decontaminating agent is

usually more biologically- and cost-efficient if added directly. Literature has

revealed that true efficacy of biological methods demonstrating effective

decontaminating properties is usually dependent on specific compounds

produced by selected microorganisms (Waliyar et al., 2007), as well as on the

competition for nutrients required for toxin production, space competition and

the production of anti-aflatoxigenic metabolites coming from coexisting

microorganisms. Studies have suggested that certain fungi, including A.

parasiticus, degrade AFB1, possibly through fungal peroxidases (López-

Garcia and Park 1998).

1.4.2. Physical Reduction

Inactivation of AFB1 using physical methods involves extraction with

solvents, adsorption, and heat- or irradiation-based inactivation. AFB1 levels

can be reduced in stored goods using physical procedures such as color

sorting, density flotation, blanching and roasting.


Despite the debate on safety of irradiated foods, food irradiation is

becoming a technique of a commercial-scale application, employed so as to

render food products sterile (Diehl, 1990). Gamma radiation as a sterilizing

treatment with a high penetrating power passes through materials without

leaving any residues and causes a direct damage to cell DNA through

ionization, inducing mutations and cell killing. There exist a number of reports

on increased, decreased or even unaffected production of mycotoxins after

irradiation of fungi under various conditions. The Joint FAO/IAEA/WHO

Expert Committee pointed out that the irradiation of any food up to the

average total dose of 10 kGy poses no toxicological hazard and no special

nutritional or microbiological problem (WHO, 1991; Mariotti et al., 2011). In

light of the foregoing, in 1999 the European Community authorized this dose

as the maximum total radiation dose allowed to be absorbed by irradiated food

on average.

1.4.3. Chemical Reduction

The use of chemicals to inactivate, bind or remove aflatoxins has been

studied extensively using different chemicals such as propionic acid,

ammonia, copper sulfate, benzoic acid, urea, citric acid and some other

chemicals capable of reacting with aflatoxins (Gowda et al., 2004). These

chemicals convert AFB1 to less toxic and less mutagenic compounds including

acids, bases, oxidizing agents, bisulfites and gases. Where approved, AFB1

levels in goods destined for animal feeds can be reduced by agents such as

adsorbent clays, as well as by ammonization. The main purpose of

ammonization is the elimination of AFB1 from feed intended for dairy cows

(IARC, 2002). As for the chemical methods of AFB1 reduction, they have

generally been labeled as impractical as they call for drastic conditions in

terms of temperature and pressure, as well as unsafe because of toxic residues,

and unfavorable since leading to degradation of nutritional, sensory and

functional properties of the product (Rustom, 1997). To date, chemical

methods have been approved only for the reduction of AFB1 in animal feed.

Techniques other than the use of chemical sorbents and ammonization have

achieved reduction in AFB1 bioavailability that comes as a result of hydrated

sodium calcium aluminosilicate binding (Phillips et al., 1988). Ammonization

is the only chemical inactivation process that has been shown to efficiently

destroy AFB1 in cottonseed and cottonseed meal, peanuts and peanut meal,

and maize (Park et al., 1988; Park and Price, 2001).


2. SURVEY OF AFB1 BIO-PREVALENCE IN CEREALS

2.1. Samples under Study

In order to investigate the bio-prevalence, i.e. the occurrence of AFB1 in

cereals, a total of 792 samples of maize (388), wheat (155), barley (148) and

oat (101) were collected during a three-year period (2010-2012) from different

fields situated in northwestern and eastern part of Croatia. Sampling and

preparation of the test samples were performed in line with ISO 6497:2002

and ISO 6498:1998, respectively.

Determination of moisture content in the sampled materials was

performed as well. Prepared test portions were ground into a fine powder

having a particle size of 1.0 mm using an analytical mill (Cylotec 1093,

Tecator, Sweden) and stored at +4 ºC prior to AFB1 analysis that made use of

ELISA and LC-MS/MS methods.

2.2. Implementation of the ELISA Method

2.2.1. Validation of the ELISA Method

Validation parameters of the ELISA method were determined using

control maize and wheat samples. AFB1 standards employed with the

validation of analytical methods were provided by Sigma-Aldrich Chemie

GmbH (Steinheim, Germany).

The limit of detection (LOD), calculated from the mean value of ten

determinations of blank maize and wheat samples plus three standard

deviations, was 1 µg/kg in both cases. The recovery rate was determined at

four different levels (2, 5, 10 and 50 µg/kg) by virtue of spiking the control

maize and wheat samples with the prepared standard mycotoxin working

solution (100 µg/L) adopted for in-house use (six replicates per concentration

level per day). For the determination of intermediate precision, the same steps

were repeated on two additional occasions by two independent analysts within

a three-month period and under the same analytical conditions.

Validation results (given in Table 1) proved the applied ELISA method to

be efficient and suitable for the determination of AFB1 in cereals under

consideration.


Table 1. Results of the ELISA method validation

Material

Spiked

concentration

(μg/kg)

Mean recovery

(%)

CV

(%)

Intermediate

precision

(%)

CV

(%)

Maize

2 85.4 6.1 88.5 8.4

5 90.7 5.7 93.2 7.3

10 92.2 6.3 93.6 7.1

50 95.5 4.9 95.9 6.7

Wheat

2 86.7 4.6 82.6 6.7

5 88.5 5.8 88.9 7.7

10 93.6 7.4 94.6 8.2

50 96.8 6.8 95.2 8.8

2.2.2. Employment of the ELISA Method

2.2.2.1. Sample Preparation

Samples were prepared using 5 g of the homogenized sample

supplemented with 25 mL of 70%- methanol and shaken vigorously head-

over-head on a shaker for three minutes. The extract was then filtrated

(Whatman, black ribbon); in the further course, 1 mL of the obtained filtrate

was diluted with the appropriate volume of deionized water. When calculating

the final AFB1 concentration in the analyzed sample, the applied dilution

factor was duly taken into account.

2.2.2.2. ELISA Assay

All study samples were first analyzed for AFB1 concentration using the

ELISA method that made use of AFB1 ELISA Ridascreen kits provided by R-

Biopharm (Darmstadt, Germany). ELISA was also used after the

implementation of AFB1 reduction methods. Each kit contains a micro-titer

plate with 96 wells coated with antibodies against AFB1, aqueous solutions of

AFB1 standard (0, 1, 5, 10, 20, and 50 μg/L), peroxidase-conjugated AFB1,

substrate/chromogen (urea peroxide), a stop-reagent (1 N-sulfuric acid), and

the washing buffer (10 mM-phosphate buffer, pH=7.4). All other chemicals

used for the analysis were of an analytical grade. The ELISA assay employed

with the determination of AFB1 in the analyzed cereals, was performed in full

line with the kit manufacturer‘s instructions, and made use of an auto-analyzer

ChemWell 2910 (Awareness Technology, Inc, USA). The obtained AFB1


concentrations were calculated from a six-point calibration curve and

corrected for recovery.

2.3. The Implementation of LC-MS/MS Method

2.3.1. LC-MS/MS Validation

LC-MS/MS validation was carried out according to the Commission

Decision 2002/657/EC using an alternative approach of matrix-comprehensive

in-house factorial design validation. The software used for the factorial design

and calculation was InterVal Plus (quo data, Gesellschaft für

Qualitätsmanagement und Statistik GmbH, Dresden, Germany). Within the

frame of the validation process, decision limit (CCα), detection capability

(CCβ), precision, recovery, repeatability, in-house reproducibility, matrix

effects, specificity and ruggedness of the method were studied. The validation

process started with the factorial design (Table 2).

For the determination of AFB1 in cereals, 8 runs, each at 6 concentration

levels, were done within 8 days using different factor combinations. In total,

48 measurements were performed. Within each run, blank samples were

fortified at six concentration levels: 2.5, 5, 7.5, 15, 30, and 60 μg/kg. In

addition, a blank matrix sample, blank reagent sample and a fortified matrix

sample were included into each run. For maize, CCα and CCβ of 5.86 μg/kg

and 6.70 μg/kg were determined, respectively.

Validation results observed with maize (Table 3), as also the results of

other validation parameters determined with both cereals under study, proved

LC-MS/MS suitable for AFB1 determination.

Table 2. Factors of interest and their levels used for the determination

of AFB1 in cereals

Factor Level

Operator Analyst 1 / Analyst 2

Cereal Maize / Barley

Extraction 2h / 3h

Storage of extracts

(injection solution)

24 hours, +4 °C before injection/

without

RC filter Producer 1- Agilent Technologies/

Producer 2 - Phenomenex


Table 3. Repeatability (sr), in-house reproducibility (sWR) and recovery

established for LC-MS/MS used for the analysis of AFB1 in maize

Spiked AFB1

concentration

(μg/kg)

sr

(μg/kg) RSD (%)

sWR

(μg/kg) RSD (%)

Recovery

(%)

2.5 0.43 17.2 0.43 17.3 101.1

5.0 0.44 8.9 0.44 8.9 100.5

7.5 0.47 6.2 0.47 6.2 100.3

15 0.57 3.8 0.57 3.8 100.1

30 0.86 2.9 0.88 2.9 100.0

60 1.56 2.6 1.63 2.7 99.9

2.3.2. LC-MS/MS Implementation

2.3.2.1. Sample Preparation

To 25 g of the sample, a 100 mL of the extraction solution

(ACN/H2O=80/20) were added. The mixture was shaken for 2 hours on a

horizontal shaker and afterwards filtrated through the Whatman black ribbon

filter paper. One mL of the obtained filtrate was diluted with 3 mL of ultrapure

water, mixed and filtrated through 0.45µm-RC filter. Forty µL of the sample

were injected into the HPLC system.

2.3.2.2. Conditions under Which LC-MS/MS Was Implemented

LC-MS/MS method was used to confirm the presence of AFB1 in the

samples in which this mycotoxin was initially determined at levels higher than

MPLs using the ELISA method (that is to say, in the maize samples only).

The HPLC (LC) system (1260 Infinity, Agilent Technologies, Santa Clara,

USA) consisted of a degasser, a binary pump, an auto-sampler and a column

compartment, and was coupled with a 6410 QQQ-mass spectrometer (MS)

(Agilent Technologies, Santa Clara, USA). HPLC separation was performed

on XBridge BEH C18 columns (150x4.6, particle size 2.5 μm) at the flow rate

of 0.80 mL/min and the temperature of +40 °C. The mobile phase consisted of

the constituent A (0.1%-formic acid dissolved in water) and the constituent B

(acetonitrile). A gradient elution program was employed as follows: 0-3 min:

90%-A, 18 min: 10%- A, 18.1 min: 90%-A, with the post-run time of 4 min

and the injection volume of 40 μL. The conditions under which the mass

spectrometry was performed were as follows: electro-spray ionization, positive

polarity, capillary voltage of 6 kV, source temperature of +350 °C, nebulizer

operating pressure of 45 psi, and the gas flow rate of 9 L/min. The mass


spectrometer was operated in the multiple reaction monitoring mode, the

protonated molecular AFB1 ion at m/z = 313 being the precursor ion. Two

product ions at m/z = 285 and m/z = 241 were monitored. The quantification

was performed during the most intense transition (m/z 313 → 285) by virtue

of extrapolation from six-point calibration curves.

2.4. AFB1 Concentrations Determined in Cereals

Statistical analysis of data on AFB1 concentrations obtained by the two

methods, was performed using the Statistica Software Ver. 10.0 (StatSoft Inc.

1984-2011, USA), with the statistical significance set at 95%-level (p=0.05).

AFB1 presence detected using ELISA was confirmed by virtue of LC-MS/MS,

indicating a high concordance between these two methods when employed to

the effect of AFB1 determination.

The results of AFB1 analyses per each investigated cereal harvested during

2010-2012 period on different fields, together with the determined number

(No) and percentage of positive samples, the average (mean), as well as

minimal and maximal concentrations and the accompanying standard

deviations (SDs) obtained within this investigation, are summarized in

Table 4.

Table 4. Concentrations of AFB1 in cereals harvested during 2010-2012

period on different fields

Cereal

No. of

total

samples

No. of

positive

samplesa

Percentage

of positive

samples

(%)

Mean of

positive

samplesb

(μg/kg)

SD

(μg/kg)

Mine

(μg/kg)

Maxf

(μg/kg)

Maize 388 63 16.2 18.5c 20.3 1.9 97.5

Wheat 155 11 7.1 2.2d 1.0 1.1 3.0

Barley 148 8 5.4 1.5d 0.5 1.2 2.4

Oat 101 2 2.0 1.1 0.1 0.9 1.2 a AFB1 is detected (>LOD).

b Mean AFB1 concentrations determined using ELISA and LC-MS/MS.

c In 32/25 maize samples, AFB1 concentrations were higher than MRLs applicable to

food / feed. d

In 2 wheat/1 barley sample, AFB1 concentrations were slightly higher than MRL

applicable to food. e Minimal AFB1 concentration determined among the positive samples.

f Maximal AFB1 concentration determined among the positive samples.


Among the investigated cereals, maize was proven to be most

contaminated, with AFB1 determined in 16.2% of samples, as compared to

7.1% AFB1-positive wheat, 5.4% AFB1-positive barley, and 2.0% AFB1-

positive oat samples. Taking into account the contamination level of AFB1 in

cereal samples detected in this research, and given the MPL for cereals

intended for foodstuffs, which is 2 μg/kg for all cereals except for maize (to

which the MPL of 5 μg/kg applies), it can be concluded that 32 maize samples

(8.2%), 2 wheat samples (1.3%) and 1 barley sample (0.7%) had AFB1

concentrations over the MPL, whereas all oat samples met the stipulated value.

Comparing the obtained AFB1 level to the MPL of 20 μg/kg, applicable to all

cereals intended for feed, it can be concluded that levels higher than MPL

were determined in 25 maize samples (6.4%), whereas all wheat, barley and

oat samples had satisfied the given criterion.

The maximal AFB1 level detected in the maize samples was 97.5 μg/kg,

which is around 5 times higher than allowed for feed and even 20 times higher

than allowed for food. The lowest number of positive samples and the lowest

average concentration of AFB1 were observed with oat, AFB1 thereby being

determined in only two samples at concentrations approximating to, or being

slightly higher than, the ELISA limit of detection. In general, AFB1 levels

higher than maximally allowed were exclusively found in the maize samples

of 2012 genus, sampled mostly from fields in the eastern part of the country,

i.e. the part known to have the largest grain production and the most developed

farming in Croatia. The results of the analysis of variance (ANOVA) revealed

statistically significant differences (p<0.05) in AFB1 concentrations between

various types of samples under investigation (significantly higher in maize),

but no differences (p>0.05) either in AFB1 concentrations determined across

the same cereal group (barley, wheat or oat), or between the sampling regions,

except for maize under any given scenario.

Given the fact that elevated mycotoxin concentrations are usually

associated with humidity and temperature as the factors most critical for

mould formation and thus also mycotoxin production (Pleadin et al., 2013), the

explanation of the results of this investigation could also be sought among

these factors. In conclusion, such a high cereal contamination, especially that

of maize, could likely be associated with climate conditions established in the

investigated regions in the period of concern, which was extremely warm and

dry (data obtained from the Croatian Meteorological and Hydrological

Institute), which might had favored mould production and AFB1 formation.

Therefore, an inadequate food/feed control could result in the consequent

contamination of food and feed, which is even more worrying should one bear


in mind that the affected region is famous for its production of cereals,

particularly that of maize, and its wide-scale use of the latter.

3. INVESTIGATION INTO THE POSSIBILITIES OF AFB1

REDUCTION IN MAIZE

3.1. Reduction of AFB1 Using Gamma Radiation

The use of gamma (γ) radiation to inactive aflatoxins has already been

investigated on some other materials; the results have shown that fungi that

produce AFB1 can successfully be deactivated in paper, wood and soil using

irradiation doses ranging from 6 to 15 kGy (da Silva et al., 2006). It has also

been observed that doses higher than 10 kGy inhibit seed germination (Chiou

et al., 1990). Aziz et al. (1997) reported that the dose required for the complete

inhibition of fungi in different food and feed range from 4 to 6 kGy. After

gamma irradiation with the dose of 1 and later on of 10 kGy, the toxicity of a

peanut meal contaminated with AFB1 was reduced by 75% and 100%,

respectively (Temcharoen and Thilly, 1982).

The presence of water plays an important role in γ ray-based AFB1

destruction, since the radiolysis of water leads to the formation of highly

reactive free radicals. These radicals can readily attack AFB1 at the terminal

furan ring, yielding the material of lower biological activity (Rustom, 1997).

Van Dyck et al. (1982) established the mutagenic activity of AFB1 in an

aqueous solution (5 μg/mL water) to be reduced by 34%, 44%, 74% and 100%

after the exposure to 2.5, 5, 10, and 20 kGy γ-rays, respectively. The dose of

10 kGy completely inactivated AFB1, and destroyed 95% of AFG1 in

dimethyl-sulphoxide-water (1:9, v/v) solution (Mutluer and Erkoc, 1987).

AFB1 degradation in range from 37% to 100% was observed after the addition

of 1 mL of 5%-hydrogen peroxide to an aqueous AFB1 solution (50 μg/mL)

under 2 kGy γ-irradiation.

As the prevention of pathogenic fungi growth and the production of AFB1

in agricultural goods represents a very important issue, this study included the

investigation into possibilities of reducing AFB1 detected in maize samples

using γ-irradiation at the doses of 5 and 10 kGy (which were applied to 25

maize samples containing AFB1 in concentrations over MPLs set for feed).

The radiation source was the 60Co γ-irradiation chamber situated at Rudjer

Boskovic Institute, Zagreb, Croatia.

Table 5. Concentrations of AFB1 in maize before and after γ-irradiation

Range of

AFB1 level in

maize (μg/kg)

Number of

samplesa

Mean AFB1 level

before irradiation

(μg/kg)

Dose of 5 kGy Dose of 10 kGy

Mean AFB1

concentration (μg/kg)

Reduction

(%)

Mean AFB1

concentration (μg/kg)

Reduction

(%)

20-40 4 28.1 n.d.b 100 n.d.

b 100

40-60 6 53.1 8.71 83.6 1.87 96.5

60-80 6 67.6 15.3 77.4 5.01 92.6

80-100 9 93.0 32.5 65.1 16.4 82.4 a Maize samples in which AFB1 concentrations were higher than MPL set for feed (20 µg/kg).

b After maize samples‘ irradiation, AFB1 was not detected.


The exposure time was calculated based on the natural decay rate (the half-

life) of the source and the location of the sample. The absorbed dose was

measured using a dosimeter. The results obtained in our earlier preliminary

studies showed that the dose of 2, 3 and 5 kGy can effectively stop the

germination of aflatoxicogenic mould spores both in vitro and in situ

(unpublished data). After γ-irradiation with the doses of 5 and 10 kGy, AFB1

level in the contaminated maize samples was determined using the ELISA

method, as described earlier. The mean reduction of AFB1 achieved in the

contaminated maize samples under this investigation using γ-radiation doses

of 5 kGy and 10 kGy, ranged from 65.1% to 100%, and from 82.4% to 100%,

respectively. As can be seen from the obtained results, gamma irradiation

yielded a significant AFB1 reduction with both applied doses, especially with

that of 10 kGy. It was also observed that the level of AFB1 reduction depends

on the level of maize contamination, i.e. the higher the level of maize

contamination, the lower the rate of AFB1 reduction, irrespective of the

radiation dose applied (Table 5).

3.2. The Reduction of AFB1 Using Essential Oils and Lactic Acid

Bacteria

A novel way of reducing the proliferation of microorganisms and/or the

production of their toxins is the use of essential oils. These oils are natural

products extracted from plant materials, which have been proven to inhibit a

wide range of food-spoiling microorganisms and the Aspergillus (Bluma et al.,

2005). Essential oils applied to that effect insofar have shown a significant

impact on AFB1 accumulation, their ultimate effect thereby being dependent

on water activity, AFB1 concentration, and the length of incubation (Bluma

and Etcheverry, 2008). In the study by Bluma et al. (2009), the effects of

essential oils added to maize grains, in terms of their influence on mould

growth rate, lag phase and AFB1 accumulation by Aspergillus section Flavi,

were evaluated under different water activity conditions. The results showed

that essential oils do influence the lag phase length and the mould growth rate,

their efficacy thereby being dependent mainly on their concentration and water

activity of the substrate; a significant impact on AFB1 accumulation was

demonstrated as well.

For the purpose of this investigation, the essential oils extracted from wild

thyme, cinnamon, sage, lavender, and rosemary were used to examine the

potential of controlling the aflatoxigenic fungi A. parasiticus 2999, A. flavus


305, A. niger 388 and their AFB1 production. Essential oils obtained from a

local pharmacy were dissolved in 96 % (by volume) - ethanol (Kemika,

Croatia) to the final concentration of 100 µL/mL. The inhibition of mould

colonies‘ growth was determined on a PDA supplemented with an essential

oil. The results showed that the growth and survival of food/feed-spoiling and

AFB1-producing Aspergillus species can be controlled using essential oils,

particularly that of wild thyme and cinnamon, which were the most effective

in their inhibiting action. In the descending order of efficiency, these were

followed by lavender, sage and rosemary essential oils. Wild thyme essential

oil inhibited mould growth by about 85%, while cinnamon essential oil

completely (100%) inhibited the growth of all tested moulds (Table 6).

Soliman and Badeaa (2002) reported a complete inhibition of A. flavus, A.

parasiticus and A. ochraceus by thyme and cinnamon essential oils added in

concentrations lower than 500 mg/kg. In their research, inhibitory effects of

essential oils or their components on mould growth were proportional to their

concentration in the cultivation medium. It has been suggested that the mode

of antifungal activity of essential oils could include their attack on the fungal

cell wall and the retraction of hyphal cytoplasm, ultimately resulting in the

mycelium‘s death. Montes-Belmont and Carvajal (1998) investigated the

effect of eleven plant essential oils used for the protection of maize against A.

flavus and found that the essential oils of cinnamon (C. zeylanicum),

peppermint (Mentha piperita), basil (Ocimum basilicum), thyme (Thymus

vulgaris), oregano (Origanum vulgare), flavoring herb epazote (Teloxys

ambrosiodes) and clove (Syzygium aromaticum) caused a total inhibition of

fungal development in maize kernels.

In this investigation, the verification of AFB1 production was performed

after 21 days of mould incubation in the YES broth (yeast extract 2%, sucrose

20%, and distilled water up to 1 L) into which essential oils were added in pre-

defined concentrations. The results showed that only cinnamon oil completely

inhibited the production of AFB1 in all tested moulds (Table 6). The addition

of wild thyme essential oil significantly inhibited AFB1 production (about

75%) by A. parasiticus 2999, A. flavus 305 and A. niger 388. Approximately

68% of AFB1 production inhibition was attained by the addition of lavender

essential oil. Rosemary and sage essential oils showed similar results, their

addition inhibiting from 45 to 57% of the toxin production. The obtained

results are in agreement with the data published by Atanda et al. (2007). These

authors showed that essential oils of the aforementioned plant species can

reduce the concentration of the produced AFB1 by about 90%.


Table 6. Inhibitory effects (%) of essential oils on mould growth

and AFB1 production

Moulds/ AFB1 Inhibition (%)

Wild thyme Cinnamon Lavender Sage Rosemary

A. parasiticus 2999

AFB1

87 100 61 47 25

77 100 70 62 48

A. flavus 305

AFB1

89 100 72 53 27

80 100 68 58 43

A. niger 388

AFB1

81 100 68 58 42

74 100 65 51 43

The results presented in this section suggest that wild thyme, cinnamon

and lavender essential oils could be efficiently used against fungi growth and

AFB1 production in food and feed during the storage period.

Several lactic acid bacteria have been found to be able to bind AFB1 in

vitro and in vivo, their efficiency dependent on the bacterial strain. The

inhibition of AFB1 accumulation was not related to the pH-decrease, but rather

to the occurrence of low molecular weight metabolite produced by the lactic

acid bacteria at the beginning of the exponential growth phase (Dalié et al.,

2010). The investigation by El-Nezami et al. (1998) showed that probiotic

strains such as Lb. rhamnosus GG and Lb. rhamnosus LC-705 are very

efficient in removing AFB1, with more than 80% of the toxin trapped in a 20

μg/mL solution (Haskard et al., 1998). It has also been shown that other

organisms such as Saccharomyces cerevisiae have the potential to bind AFB1

(Baptista et al., 2004) and are most efficient in AFB1 quenching (Bueno et al.,

2006). In order to investigate the possibility of AFB1 reduction, several

bacterial strains of lactic acid bacteria (LAB), originally isolated from the

traditional Croatian fermented milk and meat products, were tested for their

ability to bind aflatoxins. Lactobacillus delbrueckii S1, Lactococcus lactis

subsp. lactis SA1, L. plantarum B and L. plantarum A1 were isolated from

milk products, while L. plantarum 1K, Leuconostoc mesenteroides K5, Lactoc.

lactis subsp. lactis 5K1 and L. acidophilus K6 were isolated from meat

products and stored in the Collection of Microorganisms kept by the

Laboratory of General Microbiology and Food Microbiology of the Faculty of

Food Technology and Biotechnology, University of Zagreb (Croatia). Lactic

acid bacteria were cultivated in 5 mL of the de Mann-Rogosa-Sharpe (MRS)

broth at +37 °C for 24 h. Bacterial growth was determined using MRS agar

plates after a 24 hour- incubation at +37 °C by virtue of traditional plate


counting (CFU/mL). Ten mL of the MRS broth were inoculated with 10%-

inoculums of each bacterial strain and artificially contaminated with AFB1 in

the final concentration of 5 μg/mL. The bacteria and AFB1 introduced into the

MRS broth were incubated (at +37oC) for 48 h. After centrifugation (3,500 x g

for 10 min), the sample supernatants were collected at 12-, 24-, and 48-h time

points. The unbound AFB1 was quantified using the ELISA method.

Many studies have suggested that significantly different binding abilities

of the LABs can be attributed to different cell – wall structures. In our study,

L. plantarum A strain (isolated from cow cheese) exhibited a weaker binding

ability (25.1 to 34.3%) than L. plantarum B (isolated from sheep cheese) in

spite of their equal genetic structure, which could be explained by differences

in their biological activities (Peltonen et al., 2001). Among eight LAB strains,

L. delbrueckii S1 and L. plantarum 1K appeared to be the most efficient

binders of AFB1, removing approximately 70% of the latter from the liquid

media after 0 hours of incubation, which implies that the binding process runs

swiftly. The inter-strain differences in AFB1 binding can probably be

explained by different bacterial cell wall and cell casing structure. AFB1 is

bound to LAB surface components; it appears that this binding involves a

number of components (Haskard et al., 2001). In summary, the obtained

results clearly show that probiotic strains L. delbrueckii S1, L. plantarum B, L.

plantarum 1K and Leuco. mesenteroides K5 bind over 50% of AFB1 present in

the MRS broth after a 48-h incubation (Table 7).

Table 7. AFB1 binding by lactic acid bacteria

AFB1 bound ± SDa (%)

Incubation period (h)

LAB 0b 12 24 48

L. delbrueckii S1 67.8±0.5 48.3±0.6 53.2±0.3 59.1±1.3

Lactoc. lactis subsp. lactis

SA1

21.6±0.2 18.1±0.3 27.5±1.1 28.2±0.5

L. plantarum A 25.1±0.2 21.1±0.4 30.1±2.1 34.3±1.3

L. plantarum B 29.7±1.6 45.3±0.5 50.1±0.5 56.6±0.5

L. plantarum 1K 78.3±0.6 51.6±0.6 60.1±0.4 71.3±0.7

Leuco. mesenteroides K5 47.2±0.5 31.3±0.6 43.2±0.5 51.3±0.8

L. acidophilus K6 22.1±0.4 18.4±0.4 29.2±0.6 32.3±1.1

Lactoc. lactis subsp. lactis 5K1 19.8±0.8 16.3±0.2 25.5±0.6 27.2±0.5 a The results are expressed as the average values ± SDs obtained with triple assays.

b0-h sample collected after centrifugation.


CONCLUSION

The highest level of cereal AFB1 contamination was observed with maize

in comparison to wheat, barley and oat (in which the lowest AFB1 levels were

observed). Radiation-based technology could be used as an effective method

of mould growth & development prevention and the reduction of AFB1 in food

and feed. The results pointed towards the possibility of essential oils usage as

an alternative method of AFB1 reduction in agro-industries. Lactic acid

bacteria, characterized as functional cultures and proven to bind mycotoxins,

could also be used for human and animal protection against harmful effects of

mycotoxins. The toxicity of AFB1 and its seemingly unavoidable occurrence

in cereals later used as food and feed components, make the prevention and

detoxification of this mycotoxin the most challenging toxicological problem

that needs further studying and the establishment of an effective control using

screening and confirmatory analytical methods, so as to arrive at accurate

detection and prevention strategies.

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Chapter 2

AFLATOXIN OCCURRENCE

Elham Esmaeilishirazifard and Tajalli Keshavarz Faculty of Science and Technology, University of Westminster, London

ABSTRACT

Toxigenic fungi in crops have been divided historically into two

groups, field and storage fungi. Mycotoxins are produced by toxigenic

fungi at the fields and in the storage. Although many compounds are

termed as ―mycotoxin‖, there are only five agriculturally-important

fungal toxins: deoxynivalenol, zearalenone, ochratoxin A, fumonisin and

aflatoxin. Penicillium and Aspergillus species are the most important

storage fungi. However, they can also invade stressed plants in the field.

The main mycotoxins produced by Aspergillus species are aflatoxins,

citrinin and patulin. The word ‗aflatoxin‘ comes from ‗Aspergillus flavus

toxin‘, based on the fact that A. flavus and A. parasiticus are the

predominant species responsible for aflatoxin contamination of crops

prior to harvest or during storage. Aflatoxins B1, B2, G1, and G2 are the

four major isolated aflatoxins from food and feed commodities.

A. flavus and A. parasiticus have distinct affinity for nuts and

oilseeds including peanuts, maize and cotton seed. Cereals are a general

substrate for growth of A. flavus but, unlike nuts, small grain cereal

spoilage by A. flavus is the result of poor handling. Moreover, aflatoxin

M1 as a milk contaminant has potential risk for animal and human health.

The character of the aflatoxin problem varies by region. For instance,

aflatoxin accumulation in stored maize in subtropical Asia has risen

rapidly in post-harvest conditions whereas in the US, the issue is pre-

harvest condition of maize. Therefore, the exposure to aflatoxins differs

Elham Esmaeilishirazifard and Tajalli Keshavarz 36

between countries particularly due to different diets. Food contamination

with Aspergillus is associated with warm and dry climates. However, in

variable environmental conditions, the aflatoxin contamination may differ

from one year to another at the same location.

Progress in understanding the biology of Aspergillus has greatly

improved with the new techniques in genome sequencing and the

developed molecular tools that enable rapid genetic analysis of individual

genes. Particularly, the genetics of aflatoxin synthesis is regarded as a

model to gain insight into fungal secondary metabolism. Well-designed

research on production of the aflatoxin precursor sterigmatocystin with

the genetic model A. nidulans, has contributed greatly to our knowledge

of the aflatoxin pathway and the global regulatory mechanisms.

According to the recent studies, fungal pathogenesis is related to lipid-

mediated fungal-host crosstalk, suggesting that secondary metabolism

may be controlled by oxylipins at the transition level. Also, some

oxylipins have been reported to be engaged in the signalling mechanism

like quorum sensing responses in Aspergillus. Quorum sensing molecules

and their genes which are responsible for intra and inter kingdom

communications could be applied in the future aflatoxin bio-control

strategies.

TOXIGENIC FUNGI AND THEIR MYCOTOXINS

Toxigenic fungi in crops have been divided historically into two groups.

The first group called ―field fungi‖ invade the crops and produce their

mycotoxins before harvest. The second group, cause post-harvest diseases, and

are known as storage fungi. The preliminary source of these fungi, in both

cases, is the environment field. Invasion by fungi in pre-harvest diseases is

regulated mainly by host-fungus and other biological interactions (e.g.

insects), while fungal post-harvest diseases are governed by factors which act

as nutrients, physical conditions (temperature, moisture) and biotic agents

(insects, competitive interference). Therefore, it has been suggested that

toxigenic fungi could be classified into four types; a) Plant pathogens such as

F. graminearum, b) Mycotoxin producers on stressed plants, such as F.

moniliforme and Aspergillus flavus, c) Colonisers, e.g. A. flavus, that colonise

plants for subsequent mycotoxin infection after harvest, and d) Fungi living in

the soil or decomposing plants e.g. Penicillium verrucosum and Aspergillus

ochraceous. These fungi inoculate the developing kernels of the crops in the

field and proliferate in storage under favourable conditions (Miller, 1995).

Aflatoxin Occurrence 37

Although many compounds are referred to as ―mycotoxin‖, there are only

five agriculturally-important fungal toxins: deoxynivalenol, zearalenone,

ochratoxin A, fumonisin and aflatoxin. Mycotoxins reproduced by Fusarium

include fumonisins, deoxynivalenol and zearalenone. Although Penicillium

and Aspergillus species are storage fungi, they can also invade stressed plants

in the field as well. Penicillium species produce ochratoxins, citrinin and

patulin. Aspergillus species produce aflatoxins, citrinin and patulin. A. flavus

and A. parasiticus are the predominant species responsible for aflatoxin

contamination of crops prior to harvest or during storage. The word ‗aflatoxin‘

comes from ‗Aspergillus flavus toxin’ (IARC, 1993; Gökmen et al., 2005;

Miller, 1995; Sinha & Sinha, 1992; Yu et al., 2004b).

Aflatoxins are considered as the most important mycotoxins because of

their occurrence, toxicological effects and impact on human well-being and

crop trade (Gnonlonfin et al., 2013). A variety of soil inhabiting Aspergillus

strains are aflatoxin producers. These include A. flavus, A. parasiticus, A.

nomius (Wilson et al., 2002), A. pseudotamarii (Ito et al., 2001), and A.

bombycis (Peterson et al., 2001).

Aflatoxins found in food are classified as B1, B2, G1, and G2. 'B' and 'G'

refer to the blue and green fluorescent colours produced by these toxins under

UV light during the thin layer chromatography plate visualization; the

subscript numbers 1 and 2 indicate major and minor compounds, respectively.

When aflatoxins B1 and B2 are ingested by lactating cows, a proportion (about

1.5%) is hydroxylated and excreted in the milk as aflatoxins M1 and M2.

These two compounds have lower toxicity than the parent molecules but this

toxicity would be significant due to the widespread consumption of cows' milk

by infants. Because of their high toxicity, low levels of aflatoxins have been

set and regulated in foods and feeds by many countries. Aflatoxin M1 has been

detected in human breast milk from Victoria, Australia and Thailand as well as

in raw milk from cows and water buffaloes in Iran at high concentrations

(Rahimi et al., 2010; Lanyasunya et al., 2005; Pitt, 2000).

Two Aspergillus species, A. flavus and A. pseudotamarii, produce only B

aflatoxins. They are unable to synthesize G aflatoxins due to deletion (0.8- to

1.5-kb) in the aflatoxin biosynthesis 28-gene cluster (Ehrlich et al., 2004). The

other aflatoxigenic species including A. nomius, A. parasiticus and A.

bombycis produce all four aflatoxins (El-Nezami et al. 1995).

Since the discovery of aflatoxins, A. flavus has become the most widely

reported food-borne microorganism. This reflects its importance in health care

and economy (Pitt, 2000). A. flavus is a ubiquitous and morphologically

complex species including two groups based on its sclerotia size: L strains


(Group I) with sclerotia >400 mm in diameter and S strains (Group II) with

sclerotia <400 mm in diameter (Cotty, 1989). Both A. flavus strains produce

aflatoxins B1 and B2, but A. flavus S strains can also produce aflatoxins G1

and G2. S strains are geographically distributed worldwide but rare in the

United States (Tran-Dinh, et al., 1999). The sexual stage of A. flavus has been

identified as Petromyces where ascospores are found to develop within

sclerotia (Horn, et al., 2009a). A. parasiticus is an important plant pathogen

and produces B and G aflatoxins. Although its sexual stage also belongs to

Petromyces sp., its host specificity is generally limited to ground crops

whereas A. flavus infects a wide range of plant hosts (Horn, et al., 2009b).

Some species belonging to section Ochraceorosei including A. ochraceoroseus

and A. rambellii have been reported to produce aflatoxin (Cary et al., 2005 and

2009). Moreover, several other Aspergilli produce aflatoxin precursors, such

as sterigmatocystin and o-methylsterigmatocystin, which have similar

biological properties to aflatoxin (Brown, et al., 1996).

Aflatoxins are thermo-stable. So they may contaminate the dairy products

and fermented food despite pasteurization and sterilization. It has been

reported recently that aflatoxin M1 (hydroxylated metabolites of aflatoxin B1)

contamination in milk is a potential risk for animal and human health (Prandini

et al., 2009). The occurrence of aflatoxin M1 in raw milk depends on the

climatic conditions. Milk contamination by this mycotoxin was notably

affected during dry periods. In this study, raw milk samples contained

aflatoxin M1 since the food provided to cows was probably contaminated with

the toxin. This contamination occurred particularly during the dry period with

<8.0 mm rainfall and moderate temperatures. Under these climatic conditions,

as the cattle are usually kept in confinement, there is a need for supplementary

feedstuff. It appears that the additional feed was contaminated. However, in

the rainy period, when the animals are usually free to roam on pasture land, the

risk of contamination decreased (Picinin et al., 2013).

A recent report suggests that certain food and food ingredients in African

countries are highly susceptible to contamination by several mycotoxins.

Among these, maize is the main source of fumonisin, deoxynivalenol and

zearalenone while groundnuts are the main source of contamination by

aflatoxin and ochratoxin A. Aflatoxin (primarily aflatoxin B1) exerts toxic

effects on humans, pose the major threat as a potential risk factor for many

human diseases in Cameroon. This threat is likely to be more profound if

aflatoxins co-exist with other mycotoxins (Abia et al., 2013).


DIFFERENT CROPS AND AFLATOXIN CONTAMINATION

A. flavus and A. parasiticus have a distinct affinity for nuts and oilseeds.

Peanuts, maize and cotton seed are the three most important crops affected by

these Aspergillus species. Initial investigation assumed that invasion by

Aspergillus was mainly due to inadequate drying or inappropriate storage.

These factors are important in the occurrence of aflatoxins in the humid tropics

(Pitt, 2000). However, in temperate climate, these crops get infected by A.

flavus before harvest. Invasion of peanuts occurs as a result of drought stress

(Cole et al., 1982). Preharvest invasion in maize rely partly on insect damage

to cobs, but the fungus can also invade the silks of the developing ears

(Lillehoj et al., 1980). Most other nuts are also susceptible to the inoculation

(Pitt & Hocking, 1997). Cereals are a general substrate for growth of A. flavus

but, unlike nuts and oilseeds, small grain cereal spoilage by A. flavus is the

result of poor handling. Aflatoxin levels in small grains are rarely significant

but aflatoxin contamination of spices by A. flavus could be at high levels

(Lillehoj et al., 1980).

AFLATOXINS IN CROPS AND ITS DISTRIBUTION

IN DIFFERENT COUNTRIES

Aflatoxin is mainly a problem in maize because this crop is colonized in

the field depending on environmental conditions. Of the other grains, rice is an

important dietary source of aflatoxin with poor storage in tropical and

subtropical areas (Miller, 1995).

The character of the aflatoxin problem varies by region. In the US, the

issue is pre-harvest contamination of maize as the storage systems are good

(Payne, 1992). In tropical countries, such as Thailand and the Philippines,

storage of maize is an additional, substantial problem (Siriacha et al., 1991;

Quitco, 1991). As notable attention has been paid to the management of

aflatoxin in food/feed by systems which help to detect contamination and

perform better storage, it is generally agreed that a reduction in the levels of A.

flavus inoculum would help to reduce final aflatoxin contamination in storage

(Payne, 1992).

Extensive attention has been paid to the ecology of the aflatoxin-

producing fungi. In the US, Mexico and South America, A. flavus and A.

parasiticus infect corn, although A. parasiticus is relatively uncommon


(Payne, 1992). In corn-belt of the US, corn contamination with A. flavus

occurs in two fundamental ways: a) contamination of the silks with airborne

conidia, and b) colonisation of damaged kernels. In either case, drought-,

nutrient-, or temperature-stressed plants are more susceptible to colonisation

by A. flavus and accumulation of aflatoxin (Diener et al., 1987). In the US, A.

flavus conidia do not overwinter in soil, but sclerotia can survive for several

years in soil. A. flavus sclerotia in the plant debris provide a source of inocula

for the subsequent crop. At the time of silk emergence in the plant, the

survived sclerotia germinate and produce abundant conidia (Wicklow &

Wilson, 1986). This fungus also endures as mycelia in plant debris left in the

field during the winter. A. flavus has been isolated from maize silks in all

stages of plant growth, but principally when pollen is present. The fungus does

not enter the kernel from contaminated silks, but it acts as a source of

inoculum for the further colonisation of the base of the kernels (Payne, 1992;

Wicklow, 1994). A. flavus can then enter the kernels through the attachment

point of the kernel to the rachis. Once there, the fungus spreads throughout the

rachis entering additional kernels (Smart et al., 1990). While colonisation of

maize silk is a common phenomenon, such colonisation plays an important

role in contaminating insects associated with the developing ear not leading to

kernel colonisation in the field (Payne, 1992).

As an experiment, inoculated corn seeds with spores of A. flavus were

planted in a Missouri field. It was observed that compared to the control (non-

contaminated corn seeds), there was a reduction in the germination of seeds.

Furthermore, it was found that the infestation was systemic through the leaf,

stem and roots. However, some differences were found in the spread of

contamination among the tissues, suggesting that the fungus started to spread

through the meristem (Kelly & Wallin, 1987). The occurrence of A. flavus was

investigated in vegetative tissues of cotton plants. The contaminated mature

cotton bolls were collected with their subtending stems and peduncles. The

bolls were inoculated through the carpel wall 30 days after anthesis were

allowed to mature in the field. A high percentage (78 percent) of the naturally

contaminated cotton bolls with A. flavus in the seed had the fungus present in

the stem and peduncle whereas only 31 percent of these contaminated bolls

with no fungus in the seed had A. flavus in the stem. As this difference was

significant (p=0.001), a positive relationship between seed infection and

stem/peduncle infection was suggested. Moreover, it was found that all bolls

inoculated through the carpel wall had fungus in the seed but only 11 percent

of the stem sections were infected, indicating that the fungus did not grow

downward from the boll into the supporting stem. That result was supported


following further experiments on inoculated cotton seedling of cotyledonary

leaf scar. Fungal isolation from flower buds, developing bolls and stem in the

upper parts of the plant but not from roots or stem below the cotyledonary

node indicated that the fungus did not readily move downward through the

plant (Klich et al., 1986).

The ecology of A. flavus in subtropical regions of Asia is different from

that described above regarding crop contamination with Aspergillus in the US.

In Asia, A. parasiticus does not appear to occur in maize (Pitt et al., 1993;

Siriacha et al., 1991). As a result, there is an absence of aflatoxin G from

human samples collected in Asia (IARC, 1993). In the Philippines, maize

inoculated with A. flavus resulted in an infestation of 10% of the ears (Ilag,

1975). Such a high rate of infestation from conidial inoculation has not been

reported in the US in similar studies (Payne, 1992; Wicklow, 1989).

Aflatoxin accumulation in stored maize in subtropical Asia has risen

rapidly in post-harvest conditions (Kawashima et al., 1993; Quitco, 1991). In

contrast to the US corn-belt, A. flavus conidia are common in soil samples

from maize-growing regions of Thailand throughout the year, i.e. in both the

rainy and dry seasons. This important difference seems to lead to variation in

colonisation of different parts of the plants: in Thailand, some internal plant

tissues were contaminated by A. flavus from silking whereas in the US the

exterior of tassels and silks were always contaminated (Siriacha, 1991;

Siriacha et al., 1991).

In the studies carried out in Thailand, it was found that the silk internal

tissues were contaminated from silk emergence (Siriacha, 1991). This was also

reported in the US (Payne, 1992). However, as mentioned above, under the

conditions observed at the corn-belt, this contamination does not appear to be

the primary route for infestation of the developing kernels but rather acts as a

source for producing inoculum for further contamination. So this fungus

spreads to the adjacent kernels through cracks in the outer layers of the kernel

and through the inoculation of the wounds in rachis. Typically, in the US corn-

belt, these contaminations from infested kernels are distributed randomly in

the ears after wound inoculation. The contaminated kernels as the sites of

initial infection have very high aflatoxin contents (Smart et al., 1990). On the

other hand, in the Thailand studies, the contaminated kernels contained little

aflatoxin. Also, A. flavus was found commonly as an internal contaminant of

the husk. A. flavus infestation of silks, husks and kernels peaked as the tissues

senesced or developed to maturity in the absence of significant internal

contamination of the rachis by this fungus (Siriacha, 1991).


Table 1. Food contamination by aflatoxins in different countries

Countries Commodity Contamination rate

Australia Peanut

Straw/silage

381 µg/Kg

17 µg/Kg

Bangladesh Maize

Groundnut

33 µg/Kg

65 µg/Kg

Botswana Peanut

Peanut butter

To 64 µg/Kg

0.3-23 µg/Kg

Brazil Corn

Peanuts and products

0.2-129 ppb

43-1099 ppb

China Corn 9-1396 ppb

Egypt Hazelnut

Soybean

Walnut

25-175 ppb

5-35 ppb

15-25 ppb

Gambia Ground nut source 162 ppb

Ghana Kernels 5.7-22168

India Pistachio nuts

Dry slices of quince

Maize

Peanut

15 to 259 µg/Kg

96 to 8164 µg/Kg

>30 ppb

275 µg/Kg

Malaysia Wheat flour

Peanut

Maize

25.6-289 µg/Kg

1-378 µg/Kg

106 µg/Kg

Nepal Peanut, Corn flakes

Peanut butter, Vegetable

oli

>30 ppb

Nigeria Yam chips

Pre harvest maize

Shelled melon

Corn and corn based

snacks

4-186 µg/Kg

3-138 µg/Kg

5-20 µg/Kg

25-770 µg/Kg

Philippines Rice bran and rice hull

Corn

0.27-11 µg/Kg

130 µg/Kg

Senegal Peanut 40 ppb

Sudan Peanut butter and peanut 87.4-197.3 µg/Kg

Thailand Corn

Peanut oil

73 µg/Kg

102 µg/Kg

Turkey Red pepper 1.1-97.5 ppb

Adopted from: Farombi, 2006; Binder et al., 2007.


The occurrence of toxigenic fungi in medicinal plants in Brazil has already

been reported by several investigations. One investigation evaluated 91

samples of medicinal plants from 65 different species. A. flavus was the

dominant isolated species (58 isolates; 23.39%). Among these, 16-27.6% was

able to produce aflatoxin B1 or B1 and B2 (Bugno, et al., 2006). Aquino and

colleagues (2010) analysed the samples of plants including Boldo (Peumus

boldus), green tea (Camellia sinensis), Espinheira-Santa (Maytenus ilicifolia),

and Senna (Cassia angustifolia). Except for three samples of P. boldus and

two samples of C. sinensis, all the samples presented fungal contamination,

with 75% above the limit established by the World Health Organization for the

Total Fungal Count of 103 UFC/g (World Health Organization, 1998; Aquino

et al., 2010). The other survey identified eight A. flavus isolates in chamomile

(Matricaria recutita), two of which were aflatoxin producers (B1 and B2). The

total fungal count reached values over 105 UFC/g in these plants (Prado et al.,

2009). In Argentina, researchers detected 52% of the genus Aspergillus in 56

species of medicinal plants. A. flavus and A. parasiticus were the prevalent

species, 50% among the 40 aflatoxin-producer isolates (Rizzo, et al., 2004).

Exposure to aflatoxins differs between countries particularly due to

consumption of different diets. A summary of various commodities and

aflatoxin contamination rates in different countries is presented in Table 1

(Farombi, 2006; Binder et al., 2007).

ENVIRONMENTAL FACTORS IN AFLATOXIN OCCURRENCE

Food contamination with Aspergillus, and its produced aflatoxins in

general, are associated with warm and dry climates (Hell et al., 2003). Several

mathematical models on climatic risk of Aspergillus species growing and their

in situ production of aflatoxins have been published (Chauhan et al., 2008,

Pitt, 1993). However, it has also been reported that aflatoxin contamination

may considerably differ from one year to another year at the same location due

to variable environmental conditions in different growing seasons and also

diverse (inconstant) management practices (Hell et al., 2003). For instances,

growing crops consecutively in the same field increases the risk of toxin

contamination year after year (Hennigen et al., 2000).

The influence of temperature on the growth of A. flavus and A.

parasiticus, and their aflatoxin production has been studied in different

commodities using artificial media. In one study, the optimum temperature for

aflatoxin production by A. flavus was defined at 25ºC on ground nuts, while

the optimum temperature for A. parasiticus production of aflatoxin was 25-


30ºC. Also, this study showed a change in the proportions of aflatoxin B1 and

G1 produced by A. parasiticus, with a reduction in aflatoxin G1 as

temperatures increased (Diener & Davis, 1967). Molina and Giannuzzi (2002)

with using laboratory media and mathematical modelling found that optimum

temperatures for aflatoxin production by A. parasiticus were 27.8ºC and

27.3ºC at pH 5.9 and 5.5, respectively. The optimum temperature for aflatoxin

production by A. bombycis and A. nomius was 25ºC (Peterson et al., 2001).

The optimum water activity (aw) for growth of A. flavus is indicated as

0.996, with the minimum supporting growth at 0.80-0.82. At higher water

activities (0.98-0.99), aflatoxins are produced in greater amount but toxin

production apparently ceases at or near aw 0.85 (Gqaleni et al., 1997; Northolt

et al., 1977). It is also reported that more than 70% of high moisture grains

(>18%) are infected with A. flavus with a positive correlation between the rate

of infection and aflatoxin development. Toxin contamination is directly

correlated with the moisture content of crops (Mora & Lacey, 1997).

According to an investigation on medicinal plants, no aflatoxin was detected

with water activity below 0.81 and temperatures of 25 ± 2ºC, 30 ± 2ºC and 40

± 2ºC. Similar observation was made when the water activity was over 0.81

and temperature below 10 ±2ºC (Kulshrestha et al., 2008).

One study concluded that locations with both dry and hot climates have a

higher probability of aflatoxin risk compared with locations having either dry

or hot conditions alone (Gnonlonfin et al., 2013). On the other hand, other

studies have shown that the relations between climate and toxin development

are complex. Climate influences contamination partly by direct effects on the

causative fungi. As the climate changes, complex fungal communities develop.

This includes changes in quantity of aflatoxin-producers as well as the

alterations to fungal community structure. Fluctuations in climate also

influence predisposition of hosts to the insects that wound the plant. This

increases the case for fungal contamination (Chauhan et al., 2008; Setamou et

al., 1998; Hell et al., 2003; Cotty & Jaime-Garcia, 2007).

Favourable conditions of temperature and water activity are crucial for

mycotoxigenic fungi. In general, the countries with cool or temperate climates

may become more liable to aflatoxins when the temperature increases. An

example is Italy during recent years (FAO, 2000). In particular, tropical

countries may become too inhospitable for fungal growth and mycotoxin

production. Countries which afford to control the storage environment may be

able to avoid postharvest contamination but at high additional cost. The lack of

awareness about the link between food safety and climate change could be


problematic in terms of aflatoxin contamination especially in Africa

(Gnonlonfin et al., 2013).

Aflatoxin production is an aerobic process. Production of aflatoxin by A.

flavus cultures grown on a groundnuts medium in oxygene-depleted

atmosphere is lower than under normal conditions (Diener & Davis, 1967;

Dobson & Sweeney, 1998). A maximum yield of 212 mg of aflatoxin per litre

of fluid culture was produced at an aeration rate of 9 L/min while a

considerable reduction in aflatoxin occurred at lower aeration rates (Heathcote

& Hibber, 1978). Diener and Davis (1967) investigated the effects of different

levels of the normal atmospheric gases, carbon dioxide, oxygen and nitrogen

on aflatoxin production under conditions of varying temperature and humidity.

It was found that aflatoxin production in sound mature peanut kernels

decreased with increasing concentrations of carbon dioxide from 0.03% to

100%. Reducing the oxygen concentration decreased aflatoxin production.

Another study reported that a significant decrease in mycotoxin production

resulted when the oxygen was reduced from 5% to 1% regardless of the

carbon dioxide concentration, so that storage under reduced oxygen or in a

modified atmosphere could be one of the options to reduce aflatoxin

biosynthesis (Magan & Aldred, 2007).

AFLATOXIN GENE CLUSTER

Aflatoxins are polyketide synthases (PKS) -derived mycotoxins,

synthesized from a large cluster near one telomere of chromosome 3 of A.

flavus (C Figure 1, Amaike & keller, 2009) (secondary metabolite cluster 54 in

the SMURF chromosome map). Mapping of A. parasiticus and A. flavus

genomic DNA has established that the genes in the aflatoxin biosynthetic

pathway are clustered. In general, the aflatoxin gene cluster in A. parasiticus

and A. flavus consists of 25 genes, approximately 70 kb. This cluster is of

critical importance since its genetics has been a model for better understanding

of fungal secondary metabolism. So it has been subject to many reviews

(Georgianna and Payne, 2009). The aflatoxin cluster is conserved to varying

degrees in several fungi including A. parasiticus, A. ochraceoroseus,

Emericella astellata, A. flavus var. parvisclerotigenus, Aspergillus toxicarius,

A. nomius, A. pseudotamarii, A. zhaoqingensis, A. bombycis, Emericella

venezuelensis, A. rambellii, A. nidulans, A. oryzae, A. sojae, and related fungi

such as Dothistroma. However, only some Aspergillus species produce

aflatoxin (Amaike & Keller, 2011). The specific pathway regulatory gene,


aflR, is located in the cluster. A Zn(II)2Cys6 type transcriptional factor

encoded by aflR regulates expression of the aflatoxin/sterigmatocystin

biosynthetic genes. Over-expressed aflR results in increasing aflatoxin

production and up-regulates other biosynthetic genes in the aflatoxin

biosynthesis pathway. While, in aflR deletants, aflatoxin/sterigmatocystin

biosynthetic genes, and their products, are not expressed. AflR is able to bind

the consensus motif 5´-TCGN5CGR-3´ found in the promoter regions of many

aflatoxin and sterigmatocystin genes (Fernandes et al., 1998). A second

binding site 5´- TTAGGCCTAA is reported as important in autoregulation of

aflR transcript in A. flavus and A. parasiticus (Chang et al., 1995). Expressed

divergently from aflR is aflS (formerly termed aflJ), whose product also

regulates aflatoxin production through binding and activating AflR in A.

parasiticus and A. flavus and presumably other Aspergilli (Du et al., 2007).



tools that enable rapid genetic analysis of individual genes within the genome.

Particularly, the genetics of aflatoxin synthesis is regarded as a model for

better understanding of fungal secondary metabolism through its role in the

identification of the secondary metabolite clusters in chromatin regulation of

such clusters through histone modifications (Amaike & Keller, 2009).

REGULATION OF AFLATOXIN BIOSYNTHESIS

Aflatoxin represents a classic polyketide produced by species of

Aspergillus (Duran et al., 2007). So aflatoxins are synthesized by polyketide

metabolic pathway. As noted above, aflR located in the cluster is the specific

pathway regulatory gene and encodes a zincfinger DNA-binding protein which

is required for transcriptional activation of most of the aflatoxin structural

genes (Bhatnagar et al., 2003; Yu et al., 2004a).

Well-designed research on production of the aflatoxin precursor

sterigmatocystin with the genetic model A. nidulans, has contributed greatly to

our understanding of the aflatoxin pathway and the global regulatory

mechanisms (Georgianna & Pyne, 2009). In some fungi distantly related to A.

flavus and A. parasiticus, sterigmatocystin is the final metabolite rather than a

precursor for aflatoxin. The biosynthetic and regulatory genes of

sterigmatocystin production in A. nidulans are clustered and also homologous

to those genes of aflatoxin production in A. flavus and A. parasiticus.

However, the organization of the genes in the A. nidulans cluster is different


from that in A. parasiticus and A. flavus (C Figure 1, Yu et al., 2004a) (Yu et

al., 2004a).

The biosynthesis of aflatoxin has many levels of regulation. Some of them

are almost specific to the pathway while others present a more global

regulation of secondary metabolism. Also many environmental factors control

aflatoxin biosynthesis, including light (Calvo et al., 2002), carbon source,

temperature, and pH (O‘Brian et al., 2007; Price et al., 2005). The

physiological state of the fungus is also a factor that affects aflatoxin

biosynthesis. It is predictable that many of the factors which regulate the

biosynthesis of aflatoxin, also regulate the synthesis of other secondary

metabolites such as proteins VeA and LaeA (Duran et al., 2007).

Two genes, aflR and aflS are in the aflatoxin cluster and they are located

divergently adjacent to each other. These genes are involved in the regulation

of aflatoxin/ sterigmatocystin gene expression (Chang et al., 1993; Price et al.,

2006). Despite clear differences in the sequence of AflR between A. nidulans

and A. flavus, its function is conserved. AflR from A. flavus is able to regulate

expression of the sterigmatocystin cluster in an A. nidulans which is an aflR

deletion strain (Yu et al., 1996). The genes aflS and aflR are divergently

transcribed, but have independent promoters. Although, the intergenic region

between them is short, they may share binding sites for transcription factors or

other regulatory elements (Ehrlich & Cotty, 2002). The exact role of AflS in

aflatoxin biosynthesis is not clear (Georgianna & Payne, 2009).

According to some studies, AflR is sufficient to initiate gene transcription

of early, mid, and late genes in the pathway, and that AflS enhances the

transcription of early and mid aflatoxin pathway genes. It has been suggested

that AflS‘s roles are as diverse as assisting in transport of pathway

intermediates to the interaction of AflS with AflR for altered aflatoxin

pathway transcription. According to the observation of binding AflS to AflR,

it has been indicated that AflS modulates aflatoxin expression through its

interaction with AflR (Chang, 2003).

Although the role for aflatoxin in the ecology of the fungus is not known,

biosynthesis of this mycotoxin is tightly regulated by environmental and

developmental cues. The complete signalling network for processes regulating

aflatoxin biosynthesis is unclear, but components of this network have been

distinguished (Yu & Keller, 2005). Several environmental and cultural

conditions modulate aflatoxin biosynthesis including light, temperature, pH,

nitrogen, carbon source and metals (Calvo et al., 2004; Luchese & Harrigan,

1993). As it is important to determine the role of aflatoxin in the ecology of

the producing organism, an understanding of how these factors impact


aflatoxin biosynthesis is critical and it may identify target sites for control of

aflatoxin formation. Unfortunately, the regulatory networks involved in

sensing and transmitting environmental and nutritional stimuli are not well

understood (Georgianna & Payne, 2009). One study has examined the effect of

four cultural and environmental conditions on gene transcription in the

aflatoxin pathway. It has been found that temperature have the most profound

effect followed by pH, nitrogen source, and then carbon source (Price et al.,

2005). Other researcher surveyed temperature and water activity in relation to

secondary metabolism genes in several fungal species, including the aflatoxin

cluster in A. parasiticus. Under suboptimal growth conditions, intermediate

environmental stress to the organism was most favourable for production of

mycotoxins (Schmidt-Heydt et al., 2008). Calvo and colleges (2004) indicated

that light affects the transcription of several genes, including aflatoxin gene

cluster and genes putatively involved in the development of sclerotia in A.

flavus (Calvo et al., 2004).

GLOBAL SECONDARY METABOLITES REGULATORS

As mentioned in the previous section, the biosynthesis of aflatoxin

involves many levels of regulation. Some are almost specific to the aflatoxin

biosynthesis pathway like aflR and aflS regulatory genes, whereas others

present a more global regulation of secondary metabolism including LaeA.

While investigating the expression of deficient aflR, a novel protein termed

LaeA (Bok & Keller, 2004) was detected in A. nidulans. In this study, a

mutagenesis screen was used to identify sterigmatocystin production mutants

(Butchko et al., 1999). Therefore, LaeA has been shown to be a global

regulator of secondary metabolism in Aspergilli and other filamentous fungi,

including Penicillium spp. and Fusarium fujikuroi. LaeA is a member of a

heterotrimeric nuclear complex, termed the Velvet complex, with two other

proteins, VeA and VelB. The Velvet complex is also conserved in filamentous

fungi (Yu et al., 2008; Xing et al., 2010). LaeA (and possibly the Velvet

complex) is hypothesized to activate secondary metabolite clusters through

histone modifications. Consequently, loss of laeA lead to accumulation of

heterochromatic marks in the sterigmatocystin gene cluster (Reyes-Dominguez

et al., 2010).

Moreover, Velvet complex is appeared as a transcriptional complex

regulating both sporulation and secondary metabolism in A. nidulans. Also

proteins LaeA and VeA are important in A. flavus colonisation and aflatoxin


contamination of seed crops. Both proteins are required for virulence. Null

mutants produce fewer conidia and less aflatoxin in seed. In addition, these

mutants are impaired in lipid degradation of host cells (Amaike & Keller,

2009; Bayrum et al., 2008).

LaeA, encodes a putative methyltransferase that affects the expression of

secondary metabolite genes in different clusters including aflatoxins,

sterigmatocystin, penicillin, emericellamide, terrequinone, gliotoxin, and

lovastatin (Bok & Keller, 2004). Well conserved LaeA in numerous fungi has

suggested that it has significant evolutionary functions in fungal physiology

(Chang et al., 2012). How LaeA regulates the expression of secondary

metabolite biosynthesis genes is still not well understood. LaeA was known as

a protein that was thought to be crucial for expression of aflR, the gene

encodes the transcriptional regulator of genes in the sterigmatocystin cluster in

A. nidulans (Bok & Keller, 2004) and the aflatoxin cluster in A. flavus (Kale et

al., 2008). In another investigation to find further characterization of LaeA‘s

function, A. flavus laeA deletion strains have been able to express low levels of

aflR, but are unable to produce aflatoxins, although they have produced small

amounts of an early precursor metabolite such as noranthrone (Chang et al.,

2012).

GENES RELATED TO ASPERGILLUS-HOST CROSSTALK

According to the recent studies, fungal pathogenesis is related to lipid-

mediated fungal-host crosstalk. As previously described, A. flavus

preferentially colonizes oilseed crops. These crops, as well as this fungus and

other Aspergilli, contain high levels of the unsaturated fatty acids including

linoleic (18:2) and oleic acid (18:1). These fatty acids are substrates for

oxygenases. Oxylipins, derived from oxygenases, are a class of oxygenated,

unsaturated fatty acids involved in signalling pathways in different kingdoms

such as fungi (filamentous fungi, yeasts and oomycetes), plants and animals.

Considerable attention has been paid to the fungal oxylipins where one fungal

oxylipins, a precocious sexual inducer (psi factor), was discovered as

extracellular signals to regulate asexual and sexual spore development

(Champe et al., 1987). Oxylipins are encoded by ppo (psi producing

oxygenase in fungi) and lox (lipoxygenases of plants, animals, and fungi),

could regulate sclerotia and conidia production and secondary metabolism

such as aflatoxin in A. flavus (Calvo et al., 1999; Tsitsigiannis et al., 2004).

Further studies confirm a global regulatory role for ppo genes in natural


product biosynthesis. For example, long chain unsaturated fatty acid mutants

in A. nidulans and the field pathogens A. parasiticus and A. flavus with

oxylipin defects negatively affected sterigmatocystin and aflatoxin production

at the level of gene regulation (Maggio-Hall et al., 2005), suggesting that

secondary metabolism may be controlled by Ppo-derived oxylipins at the

transcription level (Christensen and Kolomiets, 2011).

A. flavus contains four dioxygenases including PpoA, PpoB, PpoC, and

PpoD, as well as one lipoxygenase such as LoxA (Horowitz et al., 2009). The

fungal oxylipin structure has similarities to plant and mammalian oxylipins.

This resemblance has partly explained the oxylipin driven cross-signalling

observed in Aspergillus-host (Tsitsigiannis et al., 2004; Brodhagen et al.,

2008). It has been found that A. flavus ppoA and ppoC mutants produce less

conidia but more sclerotia, whereas the ppoD mutant shows the opposite

phenotype (Horowitz et al., 2009). A knockdown mutant of all four

dioxygenases and lox represented high levels of aflatoxin and sclerotial

production (Tsitsigiannis et al., 2004). Intercellular communications with

regards to the lox and ppo expression in both plants and Aspergilli have not

revealed an exact role for any oxylipin in cross-kingdom communication but

have shown the importance of this entire system in the Aspergillus-host

interaction (Table 2) (Amaike & Keller, 2011).

Table 2. Synopsis of major virulent factors in A. flavus

Gene

Fungal morphology

Aflatoxin Conidiaa

Sclerotiab

∆ppoA NA NA NA

∆ppoB Decreased Increased Slight increase

∆ppoC Increased Decreased Decrease on seed

∆lox Decreased Increased Slight increase

∆ppoA/B/D (IRT2) Decrease NA Slight increase

∆ppoA/B/C/D Decreased Increased Not done

∆ppoA/B/D/lox Decreased Increased Not done

∆ppoA/B/C/D/lox (IRT4) Decreased Increased Increased

∆veA Decreased No production Decreased

∆laeA Increased No production Decreased

a,b

The data indicate the relative differences in conidial, sclerotial, and aflatoxin

production compared to the wild type, NRRL3357.

Adopted from Amaike & Keller, 2011.


QUORUM SENSING MOLECULES AND SECONDARY

METABOLITE PRODUCTION IN ASPERGILLUS

Signalling mechanisms that control physiological and morphological

responses based on cell density are called quorum sensing. This phenomenon

is very common in bacteria and has also been reported in fungi, especially

yeasts. Among filamentous fungi, Aspergillus uses quorum regulation to affect

population dependant behaviour such as morphogenesis and secondary

metabolite production (Brown et al., 2009).

Several studies report the effect of certain types of quorum sensing

molecules, secreted from one group of fungi, on the growth, morphology,

sporulation (conidiation), apoptosis and metabolite production of the other

fungi. Oxylipins constitute a large family of oxidized fatty acids and

metabolites derived from them (Brodhun & Feussner, 2011). Linoleic acid-

derived oxylipins have been reported to be engaged in the quorum sensing

responses in Aspergillus cultures. A recent investigation on the impact of

linoleic acid on lovastatin (cholesterol-lowering drug) production in A. terreus

revealed that production of lovastatin was enhanced by up to 1.8-fold (Flavia

et al., 2010). Another study on A. terreus has shown the novel role for

butyrolacton I as a quorum sensing molecule. This molecule is a growth

phase-specific inducer of lovastatin production (Raina et al., 2012). In A.

nidulans, oxylipins are involved in cell density-dependent production of

asexual and sexual spores, as well as secondary metabolites such as penicillin

(Tsitsigiannis et al., 2005). Also, γ-heptalactone is a quorum sensing molecule

in A. nidulans that regulate growth and secondary metabolite (penicillin)

production (Williams et al., 2012). Moreover, in A. flavus the switch from

conidium to sclerotium and the production of aflatoxin are cell density

dependent and related to oxylipins such as linoleic, oleic and linolenic acid

(Brown et al., 2008 and 2009). Curiously, instances of small molecule

exchange between bacteria and eukaryotes have also been reported (Mullard,

2009).

According to Brown et al., (2009), the deletion of lox gene, Aflox, reduced

density-dependent development of both sclerotia and conidia significantly. It

has been shown that the lox mutant results in increasing the number of

sclerotia and decreasing the number of conidia at high cell density. All these

evidences indicate that LOX-derived metabolism may be crucial for shifting

sclerotia to conidia in a density dependent morphology. It has been shown that

at high population density, the PpoA, PpoC and Aflox products suppress


sclerotia formation but induce conidiation. At low population density, products

of PpoD apparently cause sclerotia production while they inhibit conidia

formation. All things considered, these result suggested that Ppo-and LOX-

derived oxylipins govern sexual and asexual reproduction, harmonize a

quorum sensing process and modulate density-dependent sporulation. The

survey on ppo mutants of A. flavus at high cell density illustrated the

suppression of aflatoxin production due to the ppoA, ppoC and lox mutants

effects. In the IRT4 strain (reduced in expression of all four Ppo and LOX),

there was a significant rise in aflatoxin production through all the cell density.

So the population density regulation was entirely lost. These findings

demonstrated the importance of oxygenase activity for density dependent

aflatoxin production and governing fungal-seed interactions (Brown et al.,

2009).

CONTROL OF AFLATOXIN OCCURRENCE

AND FUTURE DIRECTIONS

Aflatoxin contamination of crops remains a critical problem worldwide

with additional health threats in increasing numbers of A. flavus–induced

aspergillosis. Pre-harvest control of A. flavus has traditionally depended on

determining resistant crop-lines to help little protection under environmental

conditions (e.g., drought) which are favourable to aflatoxin contamination

(Campbell & White, 1995). In addition, irrigation is a key to avoid drought

stress (Payne, 1998). Efforts have also focused on identifying plant proteins

that are important for defence against A. flavus invasion, including pathogen-

related and drought-resistant proteins (Chen et al., 2010). Likewise, effort has

been focused on specific molecules like oxylipins which are as the

oxygenated, unsaturated fatty acids involved in signalling pathways in

different kingdoms such as fungi, plants and animals. These molecules could

regulate sclerotia and conidia production and secondary metabolism such as

aflatoxin in A. flavus. They may play important role in Aspergillus –host

interaction which could apply to control aflatoxin production as a chemical

agent or its relevant gene to make a resistant transplant. Further identification

of the quorum sensing molecules and their relevant genes that are responsible

for interactions between bacterial cells, unicellular and mycelial fungal cells or

inter-kingdom communications (bacteria and fungi /plant and fungi) would be

of critical importance in future control strategies. Moreover, the understanding


of this fungus‘ biology has progressed with the advent of the genome sequence

and improved molecular tools allowing rapid genetic analysis of individual

genes within the genome as well as specific regulators.

Other available control methods, such as ―optimal cultural practices‖ (date

of planting and harvesting, choosing the cultivar as well as a suitable region)

have reduced but have not eliminated pre-harvest aflatoxin contamination in

susceptible crops. Furthermore, in recent years public concern over pesticide

residues in the environment, food and feed, has led to a limitation and

reduction of availability of some chemical fungicides commonly used to

control plant pathogens and post-harvest diseases. Consequently, alternative

methods for controlling these pathogens and diseases are needed. Therefore,

biological control or use of microbial fungicides may be an alternative strategy

to chemical fungicides. Identification of new antifungal, quorum sensing

peptide molecules from antagonistic bacteria like Bacilli, against A. flavus has

been investigated. This ongoing survey could lead to the development of

biotechnological strategies. These strategies would facilitate aflatoxin

contamination control as well as genetic engineering of plant resistance to

fungal invasion through the use of genes related to the bacterial antifungal

peptide molecules.

All these novel knowledge will contribute to the development of inhibitors

of aflatoxin, design of the bio-competitive Aspergillus strains, application of

biocontrol bacterial strains and improvement in host- resistance against fungal

invasion or toxin production.

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Yu, J., Payne, P.A., Nierman, W.C., Machida, M., Bennett, J.W., et al. (2008).

Aspergillus flavus genomics as a tool for studying the mechanism of

aflatoxin formation. Food Addit. Contam., 15, 1–6.



Chapter 3

AFLATOXINS IN FOOD AND FEED:

CONTAMINATION EXPOSURE,

TOXICOLOGY AND CONTROL

Marta Herrera, Antonio Herrera and Agustín Ariño* University of Zaragoza, Department of Animal Production

and Food Science, Veterinary Faculty, Zaragoza, Spain

ABSTRACT

Aflatoxins (AFs) are secondary metabolites produced by various

fungal species of the genus Aspergillus such as Aspergillus flavus and

Aspergillus parasiticus. The most important compounds are aflatoxins

B1, B2, G1 and G2, as well as two metabolic products secreted in milk,

M1 and M2.

The worldwide occurrence of aflatoxins contamination in raw

agricultural products has been well documented; such contamination

occurs in a variety of food and feed, such as cereals, nuts, dried fruits,

spices and also in milk as a consequence of the ingestion of contaminated

feed. However, pistachios, peanuts and corn are the most frequently

contaminated food items reported in the Rapid Alert System for Food and

Feed (RASFF) of the European Union. The occurrence of aflatoxins is

mainly affected by environmental factors such as climatic conditions,

* Contact: [email protected] (A. Ariño), University of Zaragoza, Department of Animal

Production and Food Science, Veterinary Faculty. c/Miguel Servet 177, 50013 Zaragoza,

Spain.

Marta Herrera, Antonio Herrera and Agustín Ariño 64

geographic location, agricultural practices, and susceptibility of the

products to fungal growth during harvest, storage and processing. High

contamination levels of aflatoxins are mainly associated with post-harvest

growth of Aspergillus moulds in poorly stored commodities.

Aflatoxins can cause adverse effects to the health of animals and

humans. These toxins have been reported to be associated with acute liver

damage, liver cirrhosis, induction of tumors and teratogenic effects.

Aflatoxin B1 (AFB1) is usually predominant and the most toxic among

aflatoxins because it is responsible for hepatocarcinoma in animals and

strongly associated with the incidence of liver cancer in humans. AFB1 is

a genotoxic and mutagenic chemical, and it has been classified by the

International Agency of Research on Cancer (IARC) as human

carcinogen (group 1). The toxic effects of the ingestion of aflatoxins in

both humans and animals depend on several factors including intake

levels, duration of exposure, metabolism and defense mechanisms, and

individual susceptibility. Aflatoxins affect not only the health of humans

and animals but also the economics of agriculture and food.

Because of the multiple adverse health effects to humans and animals

caused by aflatoxin consumption, many nations worldwide have

regulatory standards on aflatoxin in food and feed. The European Union

(EU) regulation on aflatoxins in foodstuffs is among the strictest in the

world (Commission Regulation (EC) nº 1881/2006 and successive

amendments). Maximum contents of aflatoxins in feeds are also

established by Commission Regulation (EU) nº 574/2011 on undesirable

substances in animal feed.

Throughout the world there are many advisory bodies concerned

with food safety, including the World Health Organization (WHO), the

Food and Agriculture Organization of the United Nations (FAO), the

Codex Alimentarius Joint Expert Committee for Food Additives and

Contaminants (JECFA), and many others, which regularly assess the risk

from mycotoxins, advise on controls to reduce consumer exposure and

establish different regulations for these toxins in different countries.

1. INTRODUCTION

Aflatoxins (AFs) are secondary metabolites produced by various fungal

species of the genus Aspergillus, and have the highest toxicity among

mycotoxins due to its genotoxic, mutagenic and carcinogenic properties.

Chemically, these toxins are difuranocoumarin derivatives (Figure 1) produced

primarily by two species of Aspergillus fungus which are especially found in

areas with hot and humid climates. A. flavus is ubiquitous, favouring the aerial

parts of plants (leaves, flowers) and produces only B aflatoxins (aflatoxin B1

Aflatoxins in Food and Feed 65

and B2). A. parasiticus produces both B and G aflatoxins (AFB1, AFB2,

AFG1 and AFG2) and it is more adapted to a soil environment and has more

limited distribution. The highest risk of aflatoxin contamination is due to the

more frequent growth of A. flavus (Pittet, 1998). Other Aspergillus species

such us A. bombycis, A. ochraceoroseus, A. nomius, A. pseudotamarii, A.

tamarii, A. foetidus and A. oryzae are known to produce aflatoxins but their

toxicological significance is low (Goto et al., 1996; Klich et al., 2000; Peterson

et al., 2001; Rodríguez et al., 2012). Aflatoxins M1 and M2 are the

hydroxylated metabolites of aflatoxin B1 and B2, respectively, and may be

found in milk and milk products obtained from livestock that have ingested

contaminated feed (EFSA, 2007).

Figure 1. Chemical structures of the B and G aflatoxins and of aflatoxin M1 (EFSA,

2007).

These mycotoxins are found in food as a result of fungal contamination

both pre and postharvest, with the rate and degree of contamination dependent

on various factors such as temperature, humidity (and water activity), substrate


and storage conditions. Aflatoxins have been found in a variety of agricultural

commodities, but the most pronounced contamination has been encountered in

cereals such as corn and barley, nuts and oilseeds such as peanuts and

pistachios, dried fruits such as figs as well as spices. The main sources of

aflatoxins in feed are peanut, maize and cottonseed meal.

Exposure to aflatoxins in European countries is generally considered to

occur mainly from imported materials, but it is currently uncertain whether

future changes in climate would lead to increased aflatoxin contamination in

domestic food chain.

2. AFLATOXIN CONTAMINATION IN FOOD AND FEED

Aflatoxins were first identified in 1961 in animal feed responsible for the

deaths of 100,000 turkeys in the United Kingdom (Sargeant et al., 1961), also

affecting ducklings and young pheasants among other animal species. The

occurrence and production of aflatoxins differ geographically and climatically

as in years. Aflatoxin contamination can generally be sourced from countries

of origin with hot climates, poor hygienic conditions, mould growth and poor

storage conditions (Trucksess and Scott, 2008).

Aspergillus contamination and subsequent aflatoxin production can

happen in crops themselves, as with peanuts, sometimes assisted by insect

action, or it can occur during transport or storage as for example in cereals

(ICMSF, 1996). Aflatoxin contamination is also promoted by stress or damage

to the crop due to drought prior to harvest, insect activity, poor timing of

harvest, heavy rains at harvest and post-harvest, and inadequate drying of the

crop before storage. Humidity, temperature, and aeration during drying and

storage are also important factors.

The toxin can persist in food, even when the mould has disappeared. In

addition, the fact that these toxins have a great thermal stability is also a key

factor, enabling them to remain in some cooked foods, and meaning that

freezing has very little effect on their presence in foods.

The biosynthesis of aflatoxins in food depends on several environmental

factors (Table 1), such as water activity, temperature, pH, redox potential and

microbial competition.

Aflatoxins production can be found in a wide range of substrates due to

non-visible spoilage in the field (pre-harvest), storage or processing (post-

harvest). However, high contamination levels are mainly associated with post-

harvest production by Aspergillus moulds in poorly stored commodities. These


fungi grow and produce toxins during storage and are mainly influenced by

factors related to inadequate moisture and temperature, combined with long

storage in warehouses, which are conductive situations that can originate

potential toxigenic outbreaks (Dilkin, 2002). The most important factors that

help to predict the occurrence of aflatoxins in foodstuffs include weather

conditions (temperature, atmospheric humidity, drought), agronomical

practices in the field (crop rotation, crop residues removal, soil cultivation)

and internal factors of the food chain (drying and storage conditions).

Table 1. Limits of mould growth and aflatoxin production

by A. flavus and A. parasiticus (ICMSF, 1996)

Parameter Aspergillus

flavus

Aspergillus

parasiticus

Aspergillus

flavus

Aspergillus

parasiticus

Aspergillus

flavus

Aspergillus

parasiticus

GROWTH Minimum Optimum Maximum

Temperature

(ºC) 10-12 12 33 32 43 42

Water activity 0.8 0.80-0.83 0.98 0.99 >0.99 >0.99

pH 2 2 5-8 5-8 >11 >11

AFLATOXIN

PRODUCTION Minimum Optimum Maximum

Temperature

(ºC) 13 12 16-31 25 31-37 40

Water activity 0.82 0.86-0.87 0.95-0.99 0.95 >0.99 >0.99

pH - 2 - 6 - >8

Different studies developed in the European Union have reached a

consensus on the most important indicators for the risk of aflatoxins, based on

three stages in the food production chain. For cultivation stage the selected

indicators are: relative humidity, temperature, crop rotation, tillage practices

and water activity of seeds. For transportation and storage the following

factors are included: water activity, relative humidity, ventilation, temperature,

storage capacity and logistics and for the processing stage the indicators are

the fraction of grain used, the water activity of grains, implanted traceability

and system quality (Park and Bos, 2007).

Food legislation in the EU demands food businesses to be responsible for

the safety of the food and feed they sell, and not to place on the market unsafe

(including mycotoxin-contaminated) feed or food. These businesses are also

required to identify and review the risks associated with mycotoxins, and,

where practicable, put in place processes and controls to reduce these risks.

Since a zero-tolerance for mycotoxins in foods and feeds is not possible,

legislation has been set in terms of maximum contents for specific mycotoxins


in certain food and feed. These levels are set for mycotoxins with the greatest

health concern and are based on scientific advice. The aim of maximum

contents is to minimize human exposure and the risks of both acute and long-

term adverse health effects and to support international trade (Hwang et al.,

2004).

Table 2. Maximum contents of aflatoxins in foodstuffs

in the European Union

Foodstuffs Maximum levels (μg/kg)

2.1. Aflatoxins B1 Sum of B1,

B2, G1 and

G2

M1

2.1.1. Groundnuts (peanuts) and other oilseeds , to be

subjected to sorting, or other physical treatment,

before human consumption or use as an ingredient

in foodstuffs, with the exception of: — groundnuts

(peanuts) and other oilseeds for crushing for refined

vegetable oil production

8,0 15,0 —

2.1.2. Almonds, pistachios and apricot kernels to be

subjected to sorting, or other physical treatment,

before human consumption or use as an

ingredient in foodstuffs

12,0 15,0 —

2.1.3. Hazelnuts and Brazil nuts, to be subjected to

sorting, or other physical treatment, before human

consumption or use as an ingredient in foodstuffs

8,0 15,0

2.1.4. Tree nuts, other than the tree nuts listed in 2.1.2

and 2.1.3, to be subjected to sorting, or other

physical treatment, before human consumption or

use as an ingredient in foodstuffs

5,0 10,0 —

2.1.5. Groundnuts (peanuts) and other oilseeds and

processed products thereof, intended for direct

human consumption or use as an ingredient in

foodstuffs, with the exception of: — crude

vegetable oils destined for refining — refined

vegetable oils

2,0 4,0 —

2.1.6. Almonds, pistachios and apricot kernels, intended

for direct human consumption or use as an


8,0 10,0 —

2.1.7. Hazelnuts and Brazil nuts, intended for direct

human consumption or use as an ingredient in

foodstuffs

5,0 10,0

2.1.8. Tree nuts, other than the tree nuts listed in 2.1.6

and 2.1.7, and processed products thereof, intended

for direct human consumption or use as an


2,0 4,0 —


Foodstuffs Maximum levels (μg/kg)

2.1.9. Dried fruit, other than dried figs, to be subjected to

sorting, or other physical treatment, before human


5,0 10,0 —

2.1.10.

Dried fruit, other than dried figs, and processed

products thereof, intended for direct human


2,0 4,0 —

2.1.11.

All cereals and all products derived from cereals,

including processed cereal products, with the

exception of foodstuffs listed in 2.1.12, 2.1.15 and

2.1.17

2,0 4,0 —

2.1.12.

Maize and rice to be subjected to sorting or other

physical treatment before human consumption or

use as an ingredient in foodstuffs

5,0 10,0 —

2.1.13.

Raw milk, heat-treated milk and milk for the

manufacture of milk-based products

— — 0,05

2.1.14.

Following species of spices:

Capsicum spp. (dried fruits thereof, whole or

ground, including chillies, chilli powder, cayenne

and paprika)

Piper spp. (fruits thereof, including white and black

pepper)

Myristica fragrans (nutmeg)

Zingiber officinale (ginger)

Curcuma longa (turmeric)

Mixtures of spices containing one or more of the

above mentioned spices

5,0 10,0 —

2.1.15.

Processed cereal-based foods and baby foods for

infants and young children

0,10 — —

2.1.16.

Infant formulae and follow-on formulae, including

infant milk and follow-on milk

— — 0,025

2.1.17.

Dietary foods for special medical purposes

intended specifically for infants

0,10 — 0,025

2.1.18.

Dried figs 6,0 10,0 —

The foods most susceptible to aflatoxin contamination, and which are at

greater risk of exposure are included in Table 2 from Regulation (EC) nº

1881/2006 (corn, rice, cereals in general, almonds, Brazil nuts, nutmeg,

hazelnuts, pistachios, peanuts and other oilseeds, dried fruits such as raisins or

figs, spices like paprika, nutmeg, turmeric or ginger). In the European Union,

maximum levels of aflatoxin B1, aflatoxin M1, and for the sum of aflatoxins

B1, B2, G1 and G2 in foodstuffs are laid down in above mentioned

Commission Regulation (EC) nº 1881/2006, as amended by Commission

Regulation (EU) nº 165/2010 and Commission Regulation (EU) nº 1058/2012.


These maximum limits were established for certain food commodities based

on the principle of as low as reasonably achievable (ALARA) (EFSA, 2009).

By other hand, aflatoxin contamination in feeds is regulated by the

Commission Regulation nº 574/2011 of 16 June 2011 on undesirable

substances in animal feed. This regulation states that the maximum content of

AFB1, related to a feeding stuff with a moisture content of 12%, varies from 5

to 20 μg/kg .

The incidence of aflatoxins in domestic and imported food stuffs in the

European Union can be assessed using the data reported by the RASFF (Rapid

Alert System for Food and Feed). Mycotoxins, and especially aflatoxins, were

the hazardous category with the highest number of notifications in

commodities within EU in 2012 and in previous years as well (Table 3).

Table 3. RASFF notifications on mycotoxins in food and feed in the period

2003-2012 (RASFF, 2012)

Hazard 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012

Aflatoxins 762 839 946 801 705 902 638 649 585 484

Deoxynivalenol 10 4 3 2 11 4

Fumonisins 15 14 2 15 9 2 1 3 4 4

Ochratoxin A 26 27 42 54 30 20 27 34 35 32

Patulin 6 7 3

Zearalenone 1 6 2

Total

mycotoxins 803 880 996 878 760 933 669 688 635 525

The decrease in aflatoxins notification in 2012 can be explained by the

effectiveness of preventive measures and reinforced control of aflatoxins in

certain products such as pistachios and almonds from certain countries. The

most frequently affected food category was nuts, nut products and seeds with

more than 200 notifications in 2012 (Table 4).

In surveys and monitoring programs that have been carried out in several

countries attempting to obtain a general pattern of the extent of aflatoxin

contamination in nuts, pistachios showed the highest contamination incidence.

As a result, this situation not only causes uncertainty in consumer for buying

pistachios, but also leads to additional cost in the production and loss of

income for producers, distributors and other stakeholders.

The European Commission in order to control the presence of aflatoxins

in foodstuffs from different countries, published several regulations in 2009 as


regards the increased level of official controls on imports of certain feed and

food and imposes an increased frequency of controls and special conditions at

import on products from certain countries because of the presence of

aflatoxins (RASFF, 2012).

An extensive review of the levels of aflatoxins encountered in

commodities in North America, South America, Europe, Asia and Africa was

included in an early IARC monograph (IARC, 1993). Many years of research

have generated a great number of publications concerning aflatoxin

contamination in various products such as peanuts (Ding et al., 2012),

pistachios (Ariño et al., 2009), chestnuts (Pietri et al., 2012), pepper (Set and

Erkmen, 2010), paprika (Shundo et al., 2009), corn (Wagacha and Muthomi,

2008) and chilli (Russell and Paterson, 2007) among others. In most surveys

and monitoring programs that have been carried out in several countries

attempting to obtain a general pattern of the extent of aflatoxin contamination

in foodstuffs, peanuts and pistachios have shown the highest contamination

incidence (Georgiadou et al., 2012).

Thus, aflatoxin contamination of peanuts can occur in the field (pre-

harvest) when severe late-season drought stress occurs and during storage

(post-harvest) when improper conditions of moisture and temperature exist

(Cole et al., 1995; FAO, 2000). In China, during 2009, 1040 peanut samples

were analyzed and the incidence was 25%, one of them was contaminated with

720 µg/kg of total aflatoxins (Ding et al., 2012). A survey carried out in Kenya

showed that 37% of the peanut samples exceeded the 10 µg/kg regulatory limit

for aflatoxin levels. Raw peanuts had the lowest levels of aflatoxin, with 96%

having levels of less than 4 µg/kg and only 4% having more than 10 µg/kg.

The most aflatoxin-contaminated products were peanut butter and spoilt

peanuts, with 69% and 75% respectively, exceeding 10 µg/kg (Mutegi et al.,

2013). Aflatoxin levels of about 30 times higher than the legal limits (10

µg/kg) have been reported in peanut butter given to school children in Eastern

Cape, South Africa (Wagacha and Muthomi, 2008).

Likewise, moulds of the genus Aspergillus frequently decay the kernel of

pistachio nuts. Pistachio nuts are among the commodities with the highest risk

of aflatoxin contamination due to more frequent growth of A. flavus (Pittet,

1998; Freire et al., 2000). The serious problems occurring during post-harvest

handling and storage of pistachio nuts are mould spore contamination and

aflatoxins production which results in serious health hazards and economical

losses. Natural occurrence of aflatoxins in pistachio nuts has been studied in

various countries. According to a report from Mexico, 2.2% of pistachio nut

samples showed aflatoxin contents higher than 20 µg/kg (JECFA, 1998). In


Sweden, 9.5% pistachio nut samples contained AFB1 higher than 2 µg/kg

(Thuvander et al., 2001). In the Netherlands, AFB1 was found in 17 of 29

pistachio nut samples with contamination levels ranging from 0.8 to 165 µg/kg

(Scholten and Spanjer, 1996) and in Spain 50% of bulk pistachio nuts were

contaminated with AFB1 (Ariño et al., 2009).

Table 4. RASFF notifications on mycotoxins by product category in 2012

(RASFF, 2012)

Product

category

Aflatoxins Deoxynivalenol Fumonisins Ochratoxin A Zearalenone

Cereals and

bakery

products

17 4 4 6 3

Confectionery 7 1

Feed 79

Fruits and

vegetables

137 19 1

Herbs and

spices

33 4

Milk and milk

products

5

Nuts, nut

products ad

seeds

204

Prepared

dishes and

snacks

2 2

Total 484 4 4 32 4

A similar percentage of 50.5% was found for total aflatoxins and AFB1 in

95 samples of unpacked pistachio nuts with the contamination levels ranging

from 0.007 to 7.72 µg/kg in Turkey (Set and Erkmen, 2010).

Other foodstuffs are also prone to fungal attack and subsequent aflatoxin

contamination. A total of 2183 cereals and cereal products collected around

Europe between 2007 and 2012 were available for occurrence data (EFSA,

2013). For cereals and their milling products, mean aflatoxin contents ranged

from 2.21 µg/kg in unspecified grain milling products to 2.60 µg/kg in oats,

while for processed cereal products the average concentrations varied from

0.45 µg/kg in fine bakery wares and 1.87 in raw pasta (EFSA, 2013).


However, maize is probably the most sensitive cereal crop to Aspergillus

contamination and aflatoxin production worldwide. Muriuki and Siboe (1995)

reported 100% contamination incidence of three packed corn brands in Kenya

with aflatoxins B1 and B2 (0.4-2.0 μg/kg).

The incidence of aflatoxin B1 in chestnuts from Italy was 62.2 and 21.4%

in chestnut flour and dried chestnuts, respectively; in the same products, the

percentage of samples exceeding the value of 2.0 µg/kg for aflatoxin B1 was

24.3% and 7.1% (Pietri et al., 2012).

Twenty-seven aromatic herbs, 28 spices and 48 herbal infusions and

medical plants were analyzed for estimation of aflatoxins by high-performance

liquid chromatography (HPLC). Samples were randomly collected, from 2000

to 2005, from markets, shops and bonded warehouses in Italy. Of the 103

samples analyzed only 7 spices tested positive for aflatoxins: 5 chilli-peppers,

1 nutmeg and 1 cinnamon. Two samples contained the toxin at non-

permissible levels and none of the herbal samples were contaminated

(Romagnoli et al., 2007).

Also, a survey of aflatoxin contamination in 82 unpacked and packed

ground red pepper samples was conducted in Turkey from September 2008 to

February 2009. In unpacked ground red pepper the percentage of samples

exceeding maximum limits were 17.1% for total aflatoxins and 23.1 for

aflatoxin B1, respectively, while only one packed sample containing 89.99

µg/kg was over the legal limit of AFB1 (Set and Erkmen, 2010).

Aflatoxin M1 is a metabolite of aflatoxin B1 that can occur in milk and

milk products from animals consuming feed contaminated with B aflatoxins

(Asi et al., 2012), which worldwide levels were reviewed by Galvano et al.

(1996). Fallah (2010) investigated the occurrence of AFM1 in 225 commercial

liquid milk samples composed of pasteurized milk (116 samples) and UHT

milk (109 samples).

AFM1 was detected in 67.1% samples, consisting of 83 pasteurized milk

samples at a mean of 52.8 ng/L (maximum 528.5 ng/L) and 68 UHT milk

samples at a mean of 46.4 ng/L (maximum 515.9 ng/L). In Pakistan, Iqbal et

al. (2011) analyzed a total of 178 milk samples (94 of buffalo and 84 of cow)

and reported that from Punjab about 46% of buffalo‘s and 49% of cow‘s milks

were contaminated with AFM1 as compared with 52% and 51% for milk

samples from NWFP, respectively. Overall, the mean AFM1 concentration

was 46 ng/L with a maximum of 350 ng/L.

Another set of 415 buffalos and cows milk samples (213 morning milks

and 202 evening milks) were analyzed and results revealed significant


differences between morning milks (mean of 43 ng/L) and evening milks (28

ng/L).

Aflatoxin contamination has been also reported in other products of

animal origin such as liver (Mahmoud et al., 2001), spiced hamburgers (Aziz

and Youssef, 1991), and poultry meat (Bintvihok et al., 2002, Hussain et al.,

2010).

2.1. Legislation Worldwide

Due to the significant health risks associated with the presence of

aflatoxins in food, many nations worldwide have regulatory standards on

aflatoxin in food and feed (Georgiadou et al., 2012). Of all mycotoxins

regulated worldwide, aflatoxin is the most regulated, and many countries

might have only legislation with limits for aflatoxins. It is important to note

that these standards vary greatly among countries, requiring harmonization to

remove the variability (Wu, 2008). For comparison, Tables 5 and 6 shows

aflatoxins regulations in different commodities in several countries, apart from

EU regulation already mentioned in Table 2. Currently, the Codex Committee

on Food Additives and Contaminants (CCFAC) has set two aflatoxin related

standards: one for peanuts destined for further processing (15 µg/kg) and one

for aflatoxin M1 in milk (0.5 µg/kg). The European Union (EU) has some of

the strictest standards for mycotoxins in food and feed in the world.

From a practical point of view, the best approach for the elimination of

mycotoxins from foods and feed is to prevent mould growth at all levels of

production, including harvesting, transport, and storage. Thus, the occurrence

of fungi and mycotoxins can be controlled by applying a number of preventive

measures both before and after harvest, including insect control, good

harvesting, drying, storage and good manufacturing practices. If mycotoxin

contamination has occurred, it is difficult to remove them from food because

they resist high temperatures.

Milling, food processing, and regulatory control of toxins to safety levels

can also have a positive impact on food safety (Trucksess and Diaz-Amigo,

2011).


Table 5. Maximum levels of aflatoxin B1, total aflatoxins and aflatoxin

M1 in foodstuffs (and feedstuffs) in several Asian countries (EMAN, 2013;

Romerlabs, 2013)

China Maximum level (µg/kg)

Commodity B1 Sum of

B1+B2+G1+G2

M1

Corn and corn products, peanut and peanut products 20 - -

Rice, edible oil except corn and peanut oil) 10

Other grains, beans and fermented products 5

Infant food 5

Fresh milk - - 0.5

Dairy products - - 0.5

Aflatoxins in feed

Corn, peanut meal, cottonseed meal, rapeseed meal. ≤50 - -

Soybean meal. ≤30 - -

Complementary, complete and concentrated feeding

stuffs for piglets

≤10 - -


stuffs for fattening pigs

≤20 - -


stuffs for young broilers, chicks.

≤10 - -


stuffs for broilers, layers.

≤20 - -


stuffs for young ducks, ducklings.

≤10 - -


stuffs for ducks, layers.

≤15 - -


stuffs for pigeons.

≤20 - -

Supplementary feeding stuffs for dairy cattle. ≤10 - -

Supplementary feeding stuffs for beef cattle ≤50 - -

Singapore Maximum level (µg/kg)

Commodity B1 Sum of B1+B2+G1+G2 M1

Food in general (mainly nuts, corns and their

products).

5 5

Milk and dairy products. 0.5

Infant formulae and follow-up formulae (ready-to-

consume).

0.5


Table 5. (Continued)

Indonesia Maximum level (µg/kg)

Commodity B1 Sum of

B1+B2+G1+G2

M1

Corn and its products. 15 20 -

Peanut and its products. 15 20 -

Dairy products - Milk, milk drink product, fermented

milk, rennin hydrolysed milk products, concentrated milk

and its analog, cream and its related products, cheese and

analog products, dessert (pudding, yoghurt), whey and its

product.

0.5

Dried milk and related products. 5

Feed and corn (final products) 50

Malaysia Maximum level (µg/kg)

Commodity B1 Sum of

B1+B2+G1+G2

M1

Groundnuts, almonds, hazelnuts, pistachios, Brazil nuts,

shelled, for further processing.

- 15 -

Groundnuts, almonds, hazelnuts, pistachios, Brazil nuts,

shelled, ready-to-eat.

- 10 -

Cereal-based food for infants and children. 0.1 - -

Milk. - - 0.5

Infant formula and follow-up formula (ready-to-drink). - - 0.02

5

Others. - 5 -

Japan Maximum level (µg/kg)

Commodity

Food-all Food B1 Sum of

B1+B2+G1+G2

M1

- 10 -

Formula feed (for others). 20 - -

Formula feed (for suckling calf, dairy cattle, suckling

pigs, starting chicks, starting broilers).

10 - -

Korea Maximum level (µg/kg)

Commodity B1 Sum of

B1+B2+G1+G2

M1

Grain, beans, peanut, nuts & their processed food

(grinding, cutting etc.).

10 15 -

Processed cereal products & processed bean product. 10 15 -


Korea Maximum level (µg/kg)

Commodity B1 Sum of

B1+B2+G1+G2

M1

Nutmeg, turmeric, dried red pepper, dried red pepper,

dried paprika & spice products containing them.

10 15 -

Wheat flour, dried fruits. 10 15 -

Confectionaries (peanut of nut-containing food) 10 15 -

Processed corn products for popcorn 10 15 -

Soybean paste, red pepper paste, curry powder. 10 15 -

Meju (dried fermented soybeans). 10 15 -

Steamed rice. 10 15 -

Baby foods for infants and young children. 10 - -

Raw milk and milk before processing. 10 - 0.5

India Maximum level (µg/kg)

Commodity B1 Sum of

B1+B2+G1+G2

M1

Wheat, maize, jawar (sorghum) and bajra (pearl millet),

rice, whole and split pulse (dal) masur (lentil), whole

and split pulse urd (mung bean), whole and split pulse

moong (green gram), whole and split pulse chana

(gram), split pulse arhar (red gram), and other food

grain

- 30 -

Groundnut kernels (shelled) (peanuts); - 30 -

Milk - - 0.5

3. EXPOSURE AND TOXICOLOGY OF AFLATOXINS

Mycotoxicosis can be classified as acute or chronic. Acute toxicity

generally has a rapid onset and an obvious toxic response, while chronic

toxicity is characterized by low-dose exposure over a long time period (many

toxins are present in low amounts in daily food intake contributing to the risk

of cancer and other generally irreversible effects (Sforza et al., 2006). The

best-known mycotoxin episodes are manifestations related to acute effects (i.e.

turkey X disease); however, the main human and veterinary health hazard is

associated to chronic exposure (i.e. cancer induction). Diseases caused by

aflatoxin consumption are called aflatoxicosis. These toxins are highly toxic

secondary metabolic products of Aspergillus flavus and Aspergillus parasiticus

and exhibit acute and chronic toxicity including carcinogenic, mutagenic and

teratogenic effects on humans and most animals. Exposure to large doses of


aflatoxin (>6000 mg) may cause acute toxicity with lethal effects (Groopmann

and Kensler, 1999). In turn, the long-term ingestion of diets contaminated with

aflatoxin B1 has been associated with an increased risk of liver cancer.

Table 6. Maximum levels of total aflatoxins and aflatoxin M1

in foodstuffs (and feedstuffs) around the world

(Canadian Food Inspection Agency, 2013; EMAN, 2013;

Romerlabs, 2013)

Country Food Sum of

B1+B2+G1+G2

(µg/kg)

M1

(µg/kg)

Australia/New

Zealand

Peanuts

Tree nuts

15 -

Canada Nut and nut products 15 -

Animal feeding stuffs 20

Codex,

GCC (a),

Nigeria

Peanuts, almonds, shelled Brazil nuts,

hazelnuts, pistachios intended for further

processing

15 -

Nigeria Almonds, hazelnuts, pistachios, shelled

Brazil nuts, ―ready-to-eat‖

10 -

USA Brazil nuts, peanuts and peanut products,

pistachio products

20 -

Dairy products 0.5

Feedstuff ingredients 20

Cottonseed meal intended for beef cattle,

swine or mature poultry

300 -

Corn and peanut products intended for

breeding beef cattle, swine or mature

poultry

100 -

Corm and peanut products intended for

finishing swine of 100 lbs or more

200 -

Corn and peanut products intended for

finishing beef cattle.

300 -

South Africa Peanuts 15 -

Milk 0.05

Codex, GCC (a),

Kenya, USA

Milk - 0.5

Argentina Milk, liquid including milk used in the

manufacture of milk and milk products and

reconstituted milk

- 0.5

Milk, powder - 5

Mexico Milk - 0.05

(a) Members of GCC are Saudi Arabia, United Arab Emirates (UAE), Kuwait,

Bahrain, Oman, Yemen and Qatar.


Moreover, routine ingestion of aflatoxins may happen in countries where

populations are suffering starvation or where regulations are either not

enforced or non-existent, so that the incidence rates of liver cancer worldwide

are 2 to 10 times higher in developing countries than in developed ones

(Henry, 1999). The rank order of toxicity of aflatoxins is AFB1>

AFG1>AFB2>AFG2 (Erkmen and Bozoglu, 2008). AFB1, the most toxic and

widespread of aflatoxins, is a potent genotoxic carcinogen in laboratory

animals and there is strong evidence that it is a liver carcinogen in humans

(Shephard, 2008). Aflatoxins have been classified by the International Agency

for Research on Cancer (IARC) as a class 1 (human carcinogen) (IARC 1993,

2002). Consequently, a tolerable daily intake (TDI) was not set, but

contamination in food should be reduced to the lowest possible level.

In animals, aflatoxins cause liver damage and unspecific symptoms such

as decreased milk and egg production, reduced reproductivity and suppressed

immunity in animals consuming low dietary concentrations. The principal

target organ for aflatoxins is the liver. After the invasion of aflatoxins into the

liver, lipids infiltrate hepatocytes and lead to necrosis. The clinical signs in

acute toxicity include mainly gastrointestinal affections, inappetence, weight

loss, ascites, jaundice, decrease in milk and egg production, nervous

symptoms, bleeding, pulmonary edema and death. All species are susceptible

to aflatoxicosis, but outbreaks occur mostly in pigs and poultry followed by

sheep and cattle. Bovine species are generally less sensitive compared to non-

ruminants because aflatoxins B1 and B2 are turned into other components

(AFM1 and AFM2) by the rumen microbiota.

In humans, clinically, the main features of acute human aflatoxicosis are

edema of the legs and feet, abdominal pain and vomiting as well as liver

dysfunction, convulsions, gastrointestinal hemorrhage, hematemesis, fever,

diarrhea and coma. Fatty degeneration in the liver and kidneys, and cerebral

edema are the major findings in autopsy (Agag, 2004). Adult humans usually

have a high tolerance of aflatoxin, and, in the reported acute poisonings, it is

usually the children who die (Williams et al., 2004). The ingestion of 2-6

mg/day of aflatoxin for a month can cause acute hepatitis and death (Patten,

1981). Early symptoms of hepatotoxicity from aflatoxicosis can manifest as

anorexia, malaise and low-grade fever. Aflatoxicosis can progress to

potentially lethal acute hepatitis with vomiting, abdominal pain, hepatitis and

death (Etzel, 2002). Exposure to amounts less than 1000 µg/kg have been

linked to aflatoxicosis in humans. Consuming approximately 5000 µg/kg of

aflatoxin can cause acute aflatoxicosis leading to death (Chang et al., 2013).

The LD50 value of aflatoxin ranges from 0.3 to 10 mg/kg for most animal


species, and from 0.54 to 1.62 mg/kg for human beings (Wild and Gong,

2010). The individual susceptibility to aflatoxicosis depends on doses,

duration of exposure, species (according to their abilities to detoxify aflatoxins

by biochemical processes), age (young people are more susceptible than

elder), sex (levels of testosterone), weight, diet, immunologic status, and

exposure to infectious agents such as viral hepatitis or parasite infestation.

Consumption of sub-lethal quantities of aflatoxin for a long time can develop a

sub-acute (chronic) toxicity syndrome, which commonly includes moderate to

severe liver damage.

Although susceptibility of humans to aflatoxins is not well known,

epidemiological studies of human populations exposed to diets naturally

contaminated with aflatoxins, revealed an association between the high

incidence of liver cancer in Africa and elsewhere and dietary intake of

aflatoxins (Jaimez, 2000). It has been also reported that the risk of lung cancer

may increase among workers handling contaminated grain (Kelly et al., 1997).

For people who are infected with hepatitis B and C, which is common in

sub-Saharan Africa, aflatoxin consumption raises the risk of primary

hepatocellular carcinoma by more than ten-fold compared to either exposure

alone (Turner et al., 2000; Murphy et al., 2006). In addition, preliminary

evidence suggests that there may be an interaction between chronic mycotoxin

exposure and malnutrition, immuno-suppression, impaired growth, and

diseases such as malaria and HIV/AIDS (Gong et al., 2003, 2004).

3.1. Biomarkers

Each biochemical process results in derivatives (biomarkers) that have a

characteristic half-life within the body, and thus the exposure over a period of

days, weeks, or months can be studied. Thus, one of the best methods of

measuring human exposure to aflatoxins consists of the analysis of body fluids

for the presence of aflatoxin derivatives (Makarananda et al., 1998). For

epidemiologic studies, biomarkers of aflatoxins in urine and serum provide a

better estimate of dietary aflatoxin exposure than food analysis (Azziz-

Baumgartner et al., 2005; EFSA, 2007). Aflatoxin metabolites in urine reflect

recent exposure whereas the measurement of aflatoxin albumin adducts in

blood reflects exposure over a long-time period and hence is a more reliable

indicator of a person´s chronic exposure (Groopman et al., 1994; Groopman

and Kensler 2005), given that the half-life in the body of aflatoxin-albumin

adducts is 30-60 days (EFSA, 2007). Thus, recent exposure to aflatoxin is


reflected in the urine as directly excreted aflatoxin M1 and other detoxification

products, but only a small fraction of the dose is excreted in this way.

Moreover, measurements of aflatoxins and its derivatives in urine have been

found to be highly variable from day to day, which reflects the wide variability

in the contamination of food samples, and, for this reason, the measurement of

aflatoxin M1 on a single day may not be a reliable indicator of chronic

exposure (Wild and Pisani, 1998).

Over 90% of West African sera were reported to contain detectable levels

of aflatoxin albumin (AF-alb) adducts, with exposure occurring throughout

life, including in utero and via breast milk (Turner et al., 2000; Wild et al.,

2000). Additionally, aflatoxin albumin (AF-alb) adducts were detected in 99%

of children in Benin and Togo (Gong et al., 2003).

However, the detection of aflatoxin metabolites or adducts in urine and

serum indicate exposure but do not necessarily equate to adverse health

effects. The evidence of contamination in market and food samples and the

human biomarker data show that, regardless of food preparation practices, the

human populations of developing countries are widely and significantly

exposed to aflatoxins, but usually at a level less than that needed for direct

acute illness and death. The data on the temperature conditions needed for

aflatoxin synthesis, the vulnerability of staple commodities to contamination,

the systems for food production, storage, and marketing, and the regulation

enforcement failures all indicate that there is high risk of chronic aflatoxin

exposure in developing countries. Population data from the FAO database

indicate that nearly 4,500,000 people live in this zone.

Economic pressures have created a double standard for allowable

contamination of commodities destined for human and animal consumption.

As a consequence of the successful regulation of aflatoxin in developed

countries, the human medical research literature is clearly focused on the

carcinogenic aspects of aflatoxin, which reflects the concerns of North

Americans and Europeans about the consequences of long-term cumulative

exposure, which is the only concern at the low concentrations of aflatoxins

that their food systems achieve (Williams et al., 2004).

CONCLUSION

Aflatoxins are produced by moulds that are especially found in areas with

hot, humid climates. They are most likely to contaminate tree nuts, ground

nuts, figs and other dried fruits, spices, crude vegetable oils, cocoa beans and


maize. Measures to apply Good Agricultural and Storage Practices by

producing countries are required in order to reduce incidents of highly

contaminated products being consumed. However, the possible aflatoxin

contamination of domestic foods should be kept under review, particularly in

the light of potential changes in climate.

Aflatoxin B1 was found to be the dominating aflatoxin in all foods. In the

European Union the highest total aflatoxin levels have been found in peanuts,

pistachios, and Brazil nuts, so improved pre-export controls are also required

to reduce incidents of highly contaminated products imported to the EU.

Aflatoxin B1 is clearly genotoxic and carcinogenic in a variety of animal

species. Increasing evidence demonstrates that aflatoxin B1 also has the

potential to affect the immune system, nutrition and growth. Because

aflatoxins are considered to be genotoxic and carcinogenic, it is not possible to

identify an intake without risk, and many countries worldwide introduced

regulations for these toxins at levels considered to be as low as reasonably

achievable. A number of epidemiological studies have shown clear

associations between aflatoxins exposure and incidence of hepatocellular

carcinoma in areas with high prevalence of hepatitis B and C, which is itself a

risk factor for liver cancer. A biomonitoring approach using validated

biomarkers would complement food analysis and consumption data in

providing information on prevalence and level of aflatoxin exposure

worldwide.

ACKNOWLEDGMENTS

This review chapter was supported by the Government of Aragón, Spain

(Grupo de Investigación Consolidado A01) and the European Social Fund.

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102.



Chapter 4

IMMUNOSUPPRESSIVE ACTIONS

OF AFLATOXIN AND ITS ROLE

IN DISEASE SUSCEPTIBILITY

Johanna C. Bruneau,*1 Orla Hayden,

2

Christine E. Loscher2 and Richard O’Kennedy

1,3

1 Applied Biochemistry Group, School of Biotechnology,

Dublin City University, Dublin, Ireland 2 Immunomodulation Group, School of Biotechnology,

Dublin City University, Dublin, Ireland 3 Biomedical Diagnostics Institute, Dublin City University, Dublin, Ireland

ABSTRACT

Aflatoxins are secondary metabolites produced by fungi of the

Aspergillus species. They occur as contaminants in a variety of food and

feed stuffs that have been infected with the producing fungi. Aflatoxin

exposure is known to cause a number of acute and chronic effects in both

humans and animals, including immunosuppression, liver and other

cancers, and failure of vaccination regimens. The immunomodulatory

effects of the aflatoxins have been shown to affect cell-mediated

immunity more than humoral immunity. In particular, aflatoxin exposure

* Corresponding Author: Dr. Johanna Bruneau; Applied Biochemistry Group, School of

Biotechnology, Dublin City University, Dublin 9, Ireland. Email: johanna.bruneau@

gmail.com.

Johanna C. Bruneau, Orla Hayden, Christine E. Loscher et al. 92

modulates secretion of inflammatory cytokines and phagocytic function.

Decreases in phagocytosis and inflammation observed following

aflatoxin exposure may reduce the effectiveness of the host immune

response to infection, thereby increasing susceptibility to infection in

individuals exposed to these toxins. The aim of this chapter is to

summarise the immunomodulatory effects of aflatoxin exposure in order

to better understand its potential immunosuppressive effects in humans

and animals. The relationship between these immunosuppressive actions

and susceptibility to infection will also be discussed.

INTRODUCTION

Aflatoxins were first identified in the 1960s as the causative agent in

Turkey ―X‖ disease in Britain. In this incident, thousands of turkey poults died

after consuming contaminated groundnut (peanut) meal (Spensley, 1963).

Since then, aflatoxin contamination has been identified in a number of

foodstuffs, including cereals (maize, wheat, sorghum, rice and millet), nuts

(peanuts, pistachios, walnuts, brazil and coconut) spices (chilli, turmeric,

paprika, black pepper, and ginger) dried fruit, and seeds (Pitt, 2000; Williams

et al., 2004).

Aflatoxins are produced by the fungal species Aspergillus as secondary

metabolites, therefore they are not necessary for the normal growth and

function of the fungus. Their production is regulated by environmental and

developmental signals such as light, temperature and pH (Georgianna and

Payne, 2008). Structurally, the aflatoxins belong to the coumarin family of

compounds, consisting of a dihydrofuran or tetrahydrofuran moiety fused to a

coumarin ring (Keating and O'Kennedy, 1997). While there are 17 related

aflatoxin isoforms and aflatoxin metabolites, only four of these (aflatoxin B1,

B2, G1 and G2) are the main food contaminants (Figure 1). Aflatoxin B1

(AFB1) and aflatoxin B2 (AFB2) are produced by A. flavus, while A.

parasiticus can produce all four isoforms (Ogundero, 1987; Creppy, 2002).

Aflatoxin M1 (AFM1) is the hydroxylated metabolite of AFB1 which can be

found in the milk, urine and feces of humans and animals that have consumed

contaminated food (Peraica et al., 1999; Creppy, 2002). AFB1, the

predominant isoform, is a potent hepatocarcinogen in humans. The naturally

occurring aflatoxins B1, B2, G1, and G2, including mixtures of isoforms, and

the metabolite aflatoxin M1 have been designated Group 1 carcinogens

(carcinogenic to humans) by the International Agency for Research on Cancer

(IARC) (IARC, 2002).

Immunosuppressive Actions of Aflatoxin and Its Role … 93

Figure 1. Chemical Structure of Aflatoxins B1, B2, G1 and G2.

Once ingested, aflatoxin is metabolised by cytochrome P450 in the liver

(Figure 2) (McClean and Dutton, 1995; Turner et al., 1998). Cytochrome P450

converts aflatoxin into a highly reactive and mutagenic compound, AFB1-8,9-

epoxide. AFB1-8,9-epoxide forms a covalent bond with the N7 of guanine,

forming 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua)

(Bedard and Massey, 2006). This adduct causes a G T transversion that

results in DNA repair, lesions, and mutations, eventually leading to tumour

formation (Guengerich et al., 1998; Bennet and Klich, 2003). The formation of

AFB1-N7-Gua is directly proportional to the amount of AFB1 ingested.

Several human studies have used this correlation to investigate the relationship

between dietary exposure to AFB1 and hepatocellular carcinoma (HCC) by

measuring the amount of the AFB1-N7-Gua excreted in urine (Groopman et

al., 1992, Groopman et al., 1993; Groopman et al., 1996).

Aflatoxin exposure also causes toxic effects in addition to its carcinogenic

effects, which makes it a double hazard. The toxic effects of aflatoxin are

mediated by the AFB1-8,9-epoxide. The AFB1-8,9-epoxide undergoes rapid

hydrolysis to form AFB1-8,9-dihydrodiol. This product is relatively stable, but

undergoes a slow reaction to form AFB1-dialdehyde. The AFB1-dialdehyde

molecule binds to lysine residues on proteins (Guengerich, et al., 1998;


Guengerich et al., 2001). Aflatoxin-protein adducts may inhibit key cellular

functions, particularly if enzymes or signalling molecules are affected. It has

been demonstrated that AFB1 binds to a number of proteins, including albumin

(Wild et al., 2000), serine proteases (Cuccioloni et al., 2009), and histones

(Ch‘ih et al., 1993).

Figure 2. Metabolic Pathway of Aflatoxin B1. Hydroxylated metabolites include

Aflatoxin M1.

Aflatoxin has a positive association with hepatocellular carcinoma (HCC),

which is the fifth most common and the third most fatal cancer worldwide,

causing an estimated 500,000 deaths annually. Hepatitis B virus (HBV)

infection is endemic in areas where aflatoxin contamination of foodstuffs is

commonplace. Studies have shown that there is a synergistic relationship

between HBV infection, aflatoxin exposure, and the development of HCC

(Kew, 2003). Research has shown that a person is three times more likely to

develop HCC when they test positive for AFB1-N7-Gua, versus seven times

more likely when they are infected with HBV. However, when a person tests

positive for both AFB1-N7-Gua and HBV, they are 60 times more likely to

develop the disease (Smela, 2001; Smela, 2002). These studies provide

evidence to explain why HCC incidence is high in areas where HBV infection


and aflatoxin consumption are prevalent. For example, in Mozambique and

some provinces in China, HCC accounts for 65-75% of male and 30-55% of

female cancer fatalities, compared to 2% in the United States (Sell, 2003).

IMMUNOSUPPRESSIVE ACTION OF AFLATOXIN

The immunosuppressive effects of AFB1 have been studied in a number of

species both in vivo and in vitro. Many of these investigations have focused on

the effect of aflatoxin on macrophages. Macrophages are innate immune cells

which have an important role in the primary immune response and

maintenance of tissue homeostasis. Macrophages are derived from circulating

monocytes and can be found in virtually all tissues in the body, including the

lungs (peritoneal macrophages), liver (Kupffer cells) and the central nervous

system (microglia). These cells regulate the immune response and homeostasis

through the release of cytokines and chemokines. They also contribute to the

adaptive immune response by presenting foreign antigens to T and B cells

(Gordon and Taylor, 2005; Mosser and Edwards, 2008). Several groups have

investigated the effect of aflatoxin on murine-derived macrophages. Moon et

al. reported that murine peritoneal macrophages exposed to AFB1 in vitro

exhibited decreased production of the pro-inflammatory cytokines Tumour

Necrosis Factor (TNF)-α, Interleukin (IL) -1 and IL-6. Reactive intermediates,

including nitric oxide (NO), hydrogen peroxide (H2O2) and superoxide anion

(O2-), which have potent anti-microbial activity, were also decreased (Moon et

al., 1999a). In agreement with these findings, murine peritoneal macrophages

exposed to AFB1 in vivo exhibited a significant decrease in the generation of

reactive intermediates (NO, H2O2, and O2-), as well as a significant decrease in

the phagocytic capability of the cells and a significant decrease in TNF-α

production (Moon et al., 1999b). Results of an earlier study by Dugyala et al.

(1996) assessed the effect of LPS-induced cytokine production from murine

peritoneal macrophages exposed to AFB1 in vivo. AFB1 treatment increased

mRNA levels of the pro-inflammatory cytokines IL-1α, IL-6 and TNF-α,

however secretion levels of the same cytokines were significantly reduced.

This same study also found that AFB1 exposure in vivo had minimal effect on

cytokine expression and secretion from murine splenic lymphocytes stimulated

in vitro. Although there was a significant decrease in IL-2 mRNA, there was

no significant change in IL-2, IL-3 or IFN-γ secretion levels at any of the

doses tested (Dugyala and Sharma, 1996).


Studies in a mouse macrophage cell line have also demonstrated the

immunosuppressive capacity of aflatoxins. When the murine macrophage cell

line J774A.1 was pre-incubated with AFB1, AFB2 or AFG1, both singly and in

combination, altered secretion of key pro- and anti- inflammatory cytokines

was observed (Bruneau et al., 2012). In particular, AFB1 and AFB2

significantly decreased secretion of the anti- inflammatory cytokine IL-10,

which plays an important role in maintaining homeostasis (Saraiva and

O‘Garra, 2010). Interestingly, combination treatment of J774A.1 cells with

low doses of aflatoxin isoforms decreased the secretion of IL-12p40 even

though single isoform treatment did not, indicating that these molecules exert a

synergistic effect on cytokine secretion (Bruneau et al., 2012). Other

investigators also found that AFB1, AFB2 and their hydroxylated metabolites

AFM1 and AFM2, alone and in combination, reduced the production of

reactive oxygen intermediates (NO). In addition, they reported that the

cytotoxic activity of AFB1 in these cells is due to its effects on the cell cycle

rather than apoptotic pathways. In particular, AFB1 treatment of J774A.1

macrophages increased the proportion of cells in the S phase, with a

corresponding decrease in the number of cells in the G1/G0 phase (Bianco et

al., 2012).

Further investigations into the effect of AFB1 exposure on murine

macrophages have found that AFB1 treatment altered expression of CD14 on

the cell surface. CD14 is a cell surface protein which, along with Toll-like

receptor 4 (TLR4) and myeloid differentiation protein 2 (MD-2), form the

lipopolysaccharide (LPS) receptor complex. Binding of LPS, a component of

gram negative bacterial cell walls, to the receptor complex triggers an

inflammatory response, inducing secretion of inflammatory cytokines

(Pållson-McDermott and O‘Neill, 2004). Macrophages pre-treated with AFB1

followed by LPS stimulation showed a significant decrease in CD14

expression compared to cells treated with LPS only. AFB1 pre-treatment also

significantly increased the amount of CD14 released into the medium (Moon

and Pyo, 2000). Our study in a murine macrophage cell line found that

combination treatment with AFB1 and AFB2 decreased CD14 expression

levels (Bruneau et al., 2012). These results suggest that aflatoxin exposure can

decrease the ability of macrophages to mount an appropriate response to

infection.

The effect of AFB1 on immunological parameters has also been

investigated in pigs. Liu and colleagues (2002) assessed the effect of AFB1

treatment in vitro on primary swine alveolar macrophages. Their study found a

time and dose-dependent decrease in cellular viability following exposure to


AFB1. However, there was no change in TNF-α or IL-1β mRNA levels

compared to controls in cells following LPS stimulation (Liu et al., 2002). A

study of whole blood samples taken from weaning piglets fed a diet containing

aflatoxin found a significant a decrease in IL-1β and TNF-α mRNA

expression, and an increase in IL-10 mRNA expression. Th1 (IL-2) and Th2

(IL-4) cytokine mRNA expression was unaffected in these samples (Marin et

al., 2002). Meissonnier and colleagues (2008) undertook a similar

investigation into the effect of an AFB1-contaminated diet on immunological

parameters in piglets. Expression levels of TNF- α, IL-1β, IL-6, IFN- γ, and

IL-10 mRNA in spleen samples were significantly increased at the highest

dose tested compared to controls. There was also a significant decrease in

lymphocyte proliferation after stimulation with ovalbumin (OVA), indicating

that T cell activation may be impaired (Meissonnier et al., 2008).

Investigations into the immunological response in rats following AFB1

exposure have yielded varied results. One study examined the effect of chronic

or intermittent AFB1 dosing on isolated splenic lymphocytes. Hinton and

colleagues (2003) found that the percentage of T cells was significantly

increased with intermittent and chronic doses of AFB1 and the percentage of B

cells were significantly decreased compared to untreated control samples. The

effect of AFB1 treatment on cytokine secretion in this model depended on the

dosage time. There was a significant decrease in the production of IL-2, IL-1

and IL-6 with both chronic and intermittent dosage schedules at 8 weeks.

However, increased production of IL-1 and IL-6 was observed in both chronic

and intermittent dosage schedules at 12 weeks, while IL-2 production was

unaffected (Hinton et al., 2003). Another study examined the effect of AFB1

treatment in vivo and in vitro on rat spleen mononuclear cells. Spleen

mononuclear cells isolated from rats fed a diet containing 40ppb AFB1

(equivalent to 40μg/kg) for 90 days showed a significant decrease in IL-2

secretion and a significant increase in IL-4, but there was no change in IL-10

compared to controls. However, when spleen mononuclear cells were exposed

to 20μmol/l AFB1 in vitro, there was no change in IL-2, IL-4 or IL-10

secretion levels compared to controls (Theumer et al., 2003).

Bovine neutrophils, when exposed to very low doses of AFB1 (0.01-0.5

ng/ml), exhibited decrease myeloperoxidase activity, superoxide radical (O2-)

production, and phagocytic capacity. Although the function of the cells was

affected, AFB1 exposure did not affect the viability or percentage of apoptotic

cells in comparison to untreated neutrophils (Mehrzad et al., 2011).

Hematopoietic progenitor cells are also targets of aflatoxin toxicity. Roda

and colleagues (2010) investigated the effect of AFB1 on erythroid and


myeloid progenitors. They found a dose dependent decrease in the formation

of erythroid colony forming units (CFU-E) and granulocyte monocyte colony

forming units (CFU-GM) from human or murine progenitor cells incubated

with AFB1. Of note, there was a species-specific effect. The IC50 values for

human granulocyte-monocyte progenitors was four times lower than that of

mice, indicating increased sensitivity (CFU-GM IC50: 2.45 ± 1.08 in human

cells, versus 11.08 ± 2.92 in murine cells) (Roda et al., 2010).

There are several investigations into the effect of aflatoxin exposure on

immunological parameters in humans. In these studies, aflatoxin exposure is

estimated by measuring the concentration of aflatoxin-albumin (AF-albumin)

adducts in the serum of study participants. Jiang and colleagues (2008)

investigated the effect of high or low AF-albumin levels in HIV+ an HIV–

individuals. HIV+ individuals with high AF-albumin levels had significantly

lower percentages of T regulatory cells (Tregs), naïve CD4+ cells, and B cells

compared to HIV+ individuals with low AF-albumin levels. In addition, high

AF-albumin levels accentuated HIV-related changes in T and B cells in HIV+

individuals (Jiang et al., 2008). In an earlier study, Jiang and colleagues (2005)

investigated the effect of AF-albumin adduct levels on leukocyte

immunophenotypes and monocyte phagocytic function. This study found that

individuals with high AF-albumin levels had lower percentages of

CD3+CD69+ and CD19+CD69+ cells. In addition, individuals with high AF-

albumin levels showed a decrease in proportion of perforin-expressing CD8+

and granzyme A and perforin expressing CD8+ T cells compared to low AF-

albumin individuals. There was no difference in monocyte phagocytosis

between the two groups (Jiang et al., 2005). These data indicate that CD8+ T

cell function is impaired in individuals exposed to high levels of aflatoxin,

which will have consequences for cellular immunity to infectious diseases.

Further studies in human monocytes incubated with AFB1 (0.05 – 50

pg/ml) showed a decrease in secretion of IL-1α, IL-6 and TNF-α. The mRNA

levels of the three cytokines studied were also reduced (Rossano et al., 1999).

Exposure to AFB1 in vitro significantly inhibited the phagocytic and

microbicidal activity of human monocytes (Cusumano et al., 1996). Other

investigations have found that human polymorphonuclear cells showed

significantly decreased chemotaxis after AFB1 treatment (Ubagai et al., 2008).

These results indicate that the ability of these cells to respond infection may be

affected by exposure to aflatoxin.

The effect of AFB1 treatment on immune cells varies between species and

cell type. There are several plausible explanations for this variability.

Aflatoxins are metabolized by cytochrome P450 enzymes into an epoxide


(Figure 2). It is through this intermediate that AFB1 exerts its toxic and

carcinogenic effects (Guengerich, et al., 1998; Guengerich et al., 2001). The

P450 enzymes are differentially expressed between species, and also between

cell types within a species. In a recent investigation by Bahari et al. (2013),

relative gene expression of cytochrome P450 isoforms CYP1B1 and CYP3A4

was significantly increased in human monocytes treated with AFB1 compared

to human lymphocytes exposed to AFB1 (Bahari et al., 2013). Induction of

cytochrome P450 would increase the conversion of AFB1 into AFB-epoxide,

thereby increasing its toxic and carcinogenic effect. Therefore, it is plausible

that myeloid cells are more susceptible to aflatoxin toxicity due to the

difference in cytochrome P450 isoform expression compared to lymphoid

cells.

It has been well established that aflatoxin metabolites have the ability to

bind to DNA. Another mechanism through which aflatoxin may exert its

immunosuppressive effects can form an adduct with the guanine nucleotide in

DNA (Bedard and Massey, 2006). The AFB1-epoxide can also be hydrolysed

to a compound which can bind to lysine residues in proteins (Guengerich, et

al., 1998; Guengerich et al., 2001). Aflatoxin-protein conjugates may disrupt

cell signalling pathways, especially if the AFB1-epoxide interacts with

enzymes or signalling molecules. Prior research has demonstrated that AFB1

binds to key cellular proteins including serine proteases (Cuccioloni et al.,

2009), albumin (Wild et al., 2000), and histones (Ch‘ih et al., 1993).

EFFECT OF AFB1 ON HUMORAL IMMUNITY

While aflatoxin exposure primarily affects cell-mediated immunity,

studies have investigated its effect on response to vaccination regimens.

Meissonnier et al. (2008) examined the effect of a diet containing AFB1 on the

humoral response in pigs immunised with OVA. There was no significant

change in the total concentrations of IgA, IgM and IgG, or of anti-OVA IgG

between AFB1-treated and control animals (Meissonnier et al., 2008). Another

study in pigs examined the effect of AFB1-contaminated feed on response to

Mycoplasma agalacticae vaccination. Marin et al. (2002) found that while

specific antibody responses to M. agalacticae were reduced in animals fed a

diet containing AFB1 compared to controls, the decrease was not statistically

significant (Marin et al., 2002). Investigations by Turner et al. (2003) found

that aflatoxin exposure was significantly associated with decreased levels of

secretory IgA in humans. There was a weak association between AF-albumin


adduct levels and response to pneumococcal vaccine type 23. However, there

was no statistically significant difference in the antibody response to

pneumococcal serotype 1, 5 and 14 vaccinations, or rabies vaccination,

between children with detectable and undetectable levels of AF-albumin

adducts (Turner et al., 2003).

Since antibody production and immune response to vaccines are reduced

by AFB1, this undoubtedly impacts an individual‘s susceptibility to infection.

Williams et al. (2004) refers also to an increased pace of disease rate following

exposure to AFB1. HIV and AIDS have been studied in relation to AFB1

exposure, it is speculated that there is an acceleration of disease when the

individual is simultaneously exposed to AFs again stemming from reduced

immunity. Altered CD4+ T cell function and the reduction in IL-2 would lead

to increased progression of HIV according to Williams et al. (2004). In 2005,

Oswald et al. (2005) reported a reduction and alteration of CD4+ T cells and

the related interleukin IL-2, in pigs treated with AFB1.

CONCLUSION

The immunomodulatory effects of AFB1 have been investigated in a

number of species and cellular targets. While the results of these various

studies differ, these differences are most likely due to variations in the assays

used, the target cell type investigated, and the mode of exposure (i.e. in vitro

or in vivo). Other factors that could contribute to the variability include

differences in dosages and route of exposure. Regardless of this variability,

when the data are considered together, it is apparent that AFB1 has the ability

to modulate the cellular immune response, however, humoral immunity is

largely unaffected. In particular, AFB1 inhibits the ability of macrophages and

T cells to respond to an infection by decreasing pro-inflammatory cytokine

secretion. Other macrophage functions that are affected include decreased

phagocytosis and decreased release of reactive intermediates which help fight

infection. T cell function is also affected, including decreased T cell

populations, in particular reduced numbers of perforin and granzyme A cells,

which mediate lysis of infected cells.

The data available to date makes it clear that aflatoxin is able to exert an

immunosuppressive effect in a number of species, but at present the

mechanism by which this effect is mediated remains unknown. A number of

studies have shown that aflatoxin has the ability to bind to both DNA

(Guengerich et al., 1998) and protein (Chi‘ih, et al., 1993; Wild et al., 2000;


Cuccioloni et al., 2009) via reactive intermediates. It is possible that the AFB1-

8,9-epoxide has the ability to bind to signalling molecules that initiate the

inflammatory response, thereby impairing the ability of the immune system to

react to pathogen challenge. This is an area that warrants further investigation.

Several investigators have suggested that aflatoxin-induced

immunosuppression could inhibit the host response to infection, reactivate

chronic infections or decrease the efficacy of vaccination regimens (Bondy

and Pestka, 2000; Jiang et al., 2005; Oswald et al., 2005: Meissonnier et al.,

2008). This is an important area of research when considered in the context

high incidence of infectious diseases in areas where aflatoxin exposure is

common. It has already been proven that there is a strong correlation between

aflatoxin exposure and the development of HCC, particularly in individuals

with concurrent HBV infection (Kew, 2003; Sell, 2003). The high correlation

may be due in part to the immunosuppressive activity of repeated aflatoxin

exposure which would prevent the host from properly responding to chronic

HBV infection. In addition, high AF-albumin levels have been shown to

accentuate HIV-related changes in T and B cell population from HIV-infected

individuals (Jiang et al., 2008). The combination of HIV infection and

repeated aflatoxin exposure may contribute to accelerated disease progression

in infected individuals. Further research in this area is warranted to assess the

immunological impact of aflatoxin exposure on human and animal health, in

particular the synergism between aflatoxin consumption and disease

progression.

ACKNOWLEDGMENT

The financial support of Enterprise Ireland and the Biomedical

Diagnostics Institute is gratefully acknowledged.

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and ex-vivo myelotoxicity of aflatoxins B1 and M1 on haematopoietic

progenitors (BFU-E, CFU-E, and CFU-GM): species-related

susceptibility. Toxicol In Vitro. 2010, 24, 217-23.


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Catania, MR. Secondary metabolites of Aspergillus exert

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13-19.

Saraiva, M; O'Garra, A. The regulation of IL-10 production by immune cells.

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Sell, S. Mouse Models to Study the Interaction of Risk Factors for Human

Liver Cancer. Cancer Res 2003, 63, 7553-7562.

Smela, ME; Currier, SS; Bailey, EA; Essigmann, JM. The chemistry and

biology of aflatoxin B(1): from mutational spectrometry to carcinogenesis.

Carcinogenesis 2001, 22, 535-45.

Smela, ME; Hamm, ML; Henderson, PT; Harris, CM; Harris, TM; Essigmann,

JM. The aflatoxin B(1) formamidopyrimidine adduct plays a major role in

causing the types of mutations observed in human hepatocellular

carcinoma. Proc Natl Acad Sci USA 2002, 99, 6655-6660.

Spensley, PC. Aflatoxin, the active principle in turkey 'X' disease. Endeavour

1963, 22, 75-79.

Theumer, MG; Lopez, AG; Masih, DT; Chulze, SN; Rubinstein, HR.

Immunobiological effects of AFB1 and AFB1-FB1 mixture in

experimental subchronic mycotoxicoses in rats. Toxicology 2003, 186,

159-170.

Turner, PC; Dingley, KH; Coxhead, J; Russell, S; Garner, CR. Detectable

levels of serum aflatoxin B1-albumin adducts in the United Kingdom

population: implications for aflatoxin-B1 exposure in the United

Kingdom. Cancer Epidemiol Biomarkers Prev 1998, 7, 441-447.

Turner, PC; Moore, SE; Hall, AJ; Prentice, AM; Wild, CP. Modification of

immune function through exposure to dietary aflatoxin in Gambian

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Nutr 2004, 80, 1106-1122.



Chapter 5

AFLATOXINS HAZARDS AND REGULATIONS

IMPACTS ON BRAZIL NUTS TRADE

Otniel Freita-Silva1,2, Renata Galhardo Borguini

1

and Armando Venâncio2

1EMBRAPA Food Technology, Rio de Janeiro, RJ, Brazil 2IBB - Institute for Biotechnology and Bioengineering,

Center of Biological Engineering, Universidade do Minho,

Campus de Gualtar, Braga, Portugal

ABSTRACT

Brazil nut is an important non-timber forest product produced in

Amazon region. This nut is used as food with high value in the

international market, due to its high nutritional and flavor characteristic

and to their association with environmental conservation and alleviation

of poor people living from Amazonia. Annually, several hundred tons of

Brazil nuts are produced in Brazil. However, they are susceptible to

aflatoxins (AF) contamination. Because of the detection of unacceptable

level of AF in Brazil nuts consignments arriving in European Union

ports, in 2003, special conditions were imposed on Brazil nuts entering

the European Union, decreasing the acceptable levels of AF. In 2010, the

European Union revised AF regulation on nuts; these new limits are more

adequate when considering the complexity of Brazil nut chain and the

Phone: ++55 21 3622-9645; Fax: ++55 21 3622-9713; E-mail: [email protected].

O. Freita-Silva, R. G. Borguini and A. Venâncio 108

low risk related to its low consumption. This chapter points data on the

occurrence of AF in Brazil nuts, as reported by the Rapid Alert System

for Food and Feed (RASFF), and evaluates the efforts made by all sectors

involved in the agribusiness of Brazil nuts, in Brazil, in order to

contribute to protection of both domestic and international consumers

from possible health hazard caused by AF.

INTRODUCTION

Brazil nut is the main commodity from the Amazon rainforest

extractivism. The nuts are destined to national or international trade. Gatherers

pick and store the fruits; they are responsible for the initial handling and

processing, which is still done in the forest [1]. The main stages in Brazil nut

production are: production and collection in the forest (cleaning paths between

trees, gathering the fruit, opening the fruit and transporting them to the camp),

and processing (cleaning, drying and soaking, peeling the nuts, drying the

peeled nuts) and commercialization in the packing house [2].

Brazil nuts are a typical non-timber forest product (NTFPs) and, for its

characteristic flavor, high nutritional value and their association with

environmental conservation, have been increasingly valued in the market [3].

It is the only seed internationally traded that is gathered in the forest [4].

Its origin comes from the Amazon region, mostly in the north of Brazil

and neighbor countries (Peru, Guyana, Venezuela, Suriname and Bolivia), but

only Bolivia, Brazil and Peru export this nut [5].

According The International Nut and Dried Fruit Council Foundation

(INC) report [6] the world production of Brazil nuts in 2012 was estimated at

46,155 metric tons, a 94 percent increase from the previous year (32,130

metric tons). Bolivia accounts for 70% of total production with 32,130 metric

tons, followed by Brazil with 22 percent (10,200) and Peru with 8 percent

(3,825 metric tons) (Figure 1). Brazil nut is the most economically important

plant product that is harvested sustainably from Amazon rain forest. This

report also reinforces that close 70 percent of world supply comes from Pando

region, an area representing around 3percent of total Amazonian rain forest.

In 2008, Bolivia was responsible for 53% of in-shell Brazil nut world

production, compared with 39.35% and 0.37% from Brazil and Peru,

respectively [5, 6]. In recent years, producers countries have taken a series of

actions by their respective governments, research institutes, and Non-

Governmental Organizations among others, for controlling the Brazil nut

Aflatoxins Hazards and Regulations Impacts ... 109

contamination by aflatoxins (AF) with the aim to attend to the international

sanitary standards and consolidate the export markets.

Contamination by AF is a major problem for tree nuts, as well as for other

stored grains, milk and dry fruits, especially because the causal fungi,

Aspergillus flavus group, occurs as a natural contamination [7]. Industries and

producers have endeavored considerable efforts over the last 20 years to

minimize fungal growth and AF production in tree nuts, particularly in the

case of Brazil nuts, due to the hot and humid climatic conditions found in the

Amazon environment, with an average temperature of 26 °C and relative

humidity of 80–95%, which favors the production of these toxins. In addition,

the extractivism characteristics (temperature and relative humidity during

gathering and handling) are hard to be controlled, having a direct or indirect

effect on toxigenic fungi and on the production of AF. Since contamination is

usually associated with shelled nuts, proven processing/treatments that reduce

AF levels in Brazil nuts include shelling or sorting by size, specific gravity,

color or damage [8, 9, 10].

Figure 1. World production of Brazil nuts in 2012.

Background

Over the last 15 years in Brazil, Brazil nuts have experienced a significant

decrease in exports. According to a comparative assessment, one of the

explanations for the crisis caused by such decrease in the exports in the


Brazilian Amazon was the entry of Bolivia into the international market in

1996 [11], as well as a high incidence of AF in the nut recorded in Brazil.

The crash and crisis can still be justified by non-tariff barriers imposed by

the European Community since 1998, EC regulation 1525/98 and EC decision

493/2003 [12, 13]. Because of those decisions, Brazilian exports of Brazil nuts

in shell to Europe fell by almost 90% between 2000 and 2004. The first

regulation reduced the acceptable limit of total aflatoxins (AFT) in Brazil nuts

to 4 μg/kg and 2 μg/kg for aflatoxin B1 (AFB1) and rejected contaminated

consignments from Brazil [14]. While the second one imposed special

conditions on the import of Brazil nuts in shell originating in or consigned

from Brazil. Moreover, the domestic market absorbs only 10% of the Brazil

nut production, because what is left in Brazil is the lower quality product and

no marketing strategy for Brazilians consumers was established yet.

Although Brazil nuts are exported since 1800, only had a place in the

exports agenda of NTFPs after the beginning of the twentieth century [15].

After the decline of rubber production (Hevea brasiliensis), Brazil nuts

became the primary extraction product for export purposes in the Northern

Region of Brazil, and its exploitation has a key role in the socio-economic

organization of large areas of natural forest [5].

The Brazil nut industry comports with the principal objectives of

European policy on development co-operation (poverty reduction linked with

environmental protection) and forest conservation (maintaining forest cover).

However, European Regulation 1525-98 EC, which decreases acceptable

levels of AF in Brazil nuts to 4 g/kg, caused a crash in the Brazil nut trade.

Thus, European policies on food quality, development co-operation and forest

conservation are likely to operate cross-purposes. Brazil nut producer

countries had questioned the legal basis of the Regulation in terms of scientific

justification for the stricter limits on AF content and lack of conformity with

international standards set by Codex Alimentarius. The EU has countered by

invoking the precautionary principle [16].

An EU mission carried out in Brazil from 25 January to 9 February 2003,

from the Commission's Food and Veterinary Office (FVO), presented their

conclusion [13] imposing special conditions on the import of Brazil nuts in

shell originating in or consigned from Brazil, pointing out that:

1. Brazil nuts in shell originating in or consigned from Brazil have been

found, in many cases, to be contaminated with excessive levels of

AFB1 and AFT.


2. The Scientific Committee for Food has noted that AFB1, even at

extremely low levels, can cause cancer of the liver and is also

genotoxic.

3. Commission Regulation (EC) No 466/2001 of 8 March 2001, as last

amended by Regulation (EC) No 563/2002, sets maximum levels for

certain contaminants and in particular AF in foodstuffs. Those limits

have been frequently and largely exceeded in samples of Brazil nuts.

4. Such contamination constitutes a serious threat to public health within

the Community and it is therefore appropriate to adopt protective

measures at Community level.

5. To assess the control systems in place to prevent AF contamination

levels in Brazil nuts intended for export to the Community. The

mission revealed that: the national legislation provides an inadequate

sampling procedure; no adequate traceability system is in place,

neither during the process chain, nor in relation to the export

procedure and certification; control over the sample during the

dispatch to the laboratory is inadequate, some laboratories entitled to

perform analysis for the purposes of export certification do not

produce accurate or dependable results; on some AF certificates,

issued by private laboratories, lot identification is often inadequate to

enable dependable guarantees on the relationship between sample, lot

and certificate; the official controls on returned lots is inadequate. It is

therefore appropriate to subject Brazil nuts in shell originating in or

consigned from Brazil to special, strict conditions to provide a high

level of protection to public health.

6. It is necessary that Brazil nuts be collected, sorted, handled,

processed, packaged and transported following good hygiene

practices. It is also necessary to establish the levels of AFB1 and AFT

in samples taken from consignment immediately prior to their

dispatch from Brazil. The sampling and the analysis should be

performed in accordance with Commission Directive 98/53/EC of 16

July 1998 laying down the sampling methods and the methods of

analysis for the official control of the levels for certain contaminants

in foodstuffs, as amended by Directive 2002/27/EC of 13 March

2002.

7. Brazil should provide documentary evidence to accompany each

consignment of Brazil nuts, relating to the conditions of collection,

sorting, handling, processing, packaging and transport, as well as the


results of laboratory analysis of the samples taken from consignment

for levels of AFB1 and AFT.

8. From the findings of the FVO's mission, it may be concluded that

Brazil cannot ensure currently dependable analytical results or

guarantee lot integrity in respect of certification of consignments of

Brazil nuts. Therefore, any certificate issued for Brazil nuts from

Brazil raises serious doubts with regard to its reliability. Furthermore,

it may also be concluded that current official controls on returned lots

are inadequate. It is therefore appropriate to impose strict conditions

on the return of nonconforming lots. In the event that those strict

conditions are not complied with, subsequent non-conforming lots

should be destroyed.

9. It is therefore necessary in order to safeguard public health that all lots

of Brazil nuts imported into the Community, are subjected to

sampling and analysis for their AF level by the competent authority of

the importing Member State prior to release onto the market.

10. In the interests of public health, Member States should provide the

Commission with periodical reports of all analytical results of official

controls carried out in respect of consignments of Brazil nuts. Such

reports should be in addition to the notification obligations under the

Rapid Alert System for Food and Feed established under Regulation

(EC) No 178/2002.

11. The measures provided for in this Decision are in accordance with the

opinion of the Standing Committee on the Food Chain and Animal

Health.

Therefore the Commission adopted these decisions: (i) restrictions on

imports of Brazil nuts in shell originating in or consigned from Brazil, (ii)

sampling and analysis of Brazil nuts by the competent authority of Brazil, (iii)

coding at points of entry into the EU for consignments of Brazil nuts,

improving traceability (iv) obligations on member States as regards imports of

Brazil nuts from Brazil, (v) if a consignment is split, a copy of the analytical

report shall accompany each part of consignment and (vi) consignments of

Brazil nuts not complying with the maximum levels for AFB1 and AFT may be

returned to the original country [13].

Some years later, the Scientific Panel on Contaminants in the Food Chain

(CONTAM Panel) of the European Food Safety Authority (EFSA) adopted an

opinion related to the potential increase of consumer health risk by a possible

increase of the existing maximum levels for AF in almonds, hazelnuts and


pistachios and derived products in January 2007 (Question No EFSA-Q-2009-

00675).

This risk assessment was requested by the European Commission

following discussions at the Codex Committee for Food Additives and

Contaminants (CCFAC) where the setting of higher levels than the 4 μg/kg,

the current EU maximum level for AF in almonds, hazelnuts and pistachios,

had been proposed.

The CONTAM Panel concluded in its assessment that changing the

maximum levels for AF from 4 to 8 or 10 μg/kg in almonds, hazelnuts and

pistachios would have a minor impact on the estimates of dietary exposure,

cancer risk and the calculated margin of exposures [17].

Based on the information which was available in 2007 the CONTAM

Panel concluded that public health would not be adversely affected by

increasing the levels for AF from 4 μg/kg to 10 μg/kg for all tree nuts. The

CONTAM Panel, however, reiterated its previous conclusion that exposure to

AF from all sources should be as low as reasonably achievable, because AF

are genotoxic and carcinogenic, and that priority should be given to reducing

the numbers of highly contaminated foods reaching the market, irrespective of

the commodity involved [17].

Although the limits imposed by the European Union are too restrictive,

there was an attempt in recent regulations [18] to abide by the maximum

permitted limits of the Codex Alimentarius concerning tree nuts. An AF limit

of 10 and 15 μg/kg, for ready-to-eat (RTE) and destined for further processing

(DFP), respectively, was established for Brazil nuts. Under these regulations,

this relaxation in the legislation does not result in increased consumer

exposure to AF [5, 17].

Data Analysis of Imports of Brazil Nuts from 2002-2010

An analysis on the Brazil nut, the most valued NTFP from Amazon

biome, exported to Europe, is focused on in this chapter. The period of this

studied was from 2002 to 2010.

This chapter discusses the EU import restrictions for Brazil nut from

Brazil, with focus on data of Brazil nuts according the Rapid Alert System for

Food and Feed (RASFF) also it was pointed the efforts made by the sectors

involved in the agribusiness of Brazil nuts in Brazil in order to contribute to

protection of both domestic and international consumers from possible health

hazard caused by AF.


The Rapid Alert System for Food and Feed (RASFF)

and Notifications in Brazil Nuts

The Rapid Alert System for Food and Feed (RASFF) was put in place to

provide food and feed control authorities with an effective tool to exchange

information about measures taken responding to serious risks detected in

relation to food or feed. This exchange of information helps Member States of

EU to act more rapidly and in a coordinated manner in response to a health

threat caused by food or feed.

The occurrence data in the RASFF notifications concerning mycotoxins

provide basis for the analysis of the situation and evaluation of tendencies in

AF Brazil nuts levels. This Chapter analyses the data collected by RASFF in

2003, 2004, 2005, 2006, 2007, 2008 and 2009 [19, 20, 21, 22, 23, 24, 25, 26]

related to AF and nuts contamination based on the data of the rapid alert

system of the EU. Once AF is the major mycotoxin, especially in-shell Brazil

nuts, and is found at concerning levels, imported products are 100% AF

regulated by specific Commission Decision [27].

According to the European Commission Directorate General for Health

and Consumer Affairs, through the Rapid Alert System for Food and Feed

(RASFF), the number of notifications based on AF in 2009 (638 notifications)

has significantly decreased compared to 2008 (902). AF findings in nuts, nut

products and seeds generated 518 notifications (Table 1). Brazil received 16

notifications, 7 of which concerned Brazil nuts (4 notifications on in-shell nuts

and 3 notifications on Brazil nut kernels from Bolivia) [26]. The Table 1

compares the border notification on general AF contamination in all products,

in nuts and seeds, and in Brazil nuts. Freitas-Silva and Venâncio [5] pointed

that these numbers reflected a little progress on the production chain of Brazil

nuts in Brazil, on the steps represented by an implementation of good

manufacturing practices, appropriate sampling plans, favorable analysis

conditions and certification of the final product. On the other hand, the

numbers of the AF notifications in Brazil nut may suggest the option to export

shelled Brazil nuts instead of in-shell ones since the EU legislation only

requires 100% screening on import in-shell Brazil nuts from Brazil.

According to RASFF reports, since the AF control in Brazil nuts by

regulation was implemented [13], 101 lots of Brazil nuts were rejected (Figure

2). This regulation led to a greater control of the final product, with a high

reduction in the incidence of AF since 2005. An exception occurred in 2009,

which showed alarming levels of AF (Figure 2). These findings are the results


of a joint effort of governmental and nongovernmental associations to better

organization of the complex chain of Brazil nut.

Table 1. Border rejection notification on lots with aflatoxins in general

seed and nuts, and in Brazil nuts per yeara

Aflatoxins 2002 2003 2004 2005 2006 2007 2008 2009

In all

commodities

288 763 844 947 800 705 902 638

Nuts and seeds na 695 675 827 684 568 710 517

Brazil nuts 48 16 2 5 2 2 3 7b

aData adapted from RASFF Annual Report (2002-2009).

b4 samples from Brazil and 3 from Bolivia.

na: not available.

This adjustment in regulation brought the greater control as it can be

observed in Figure 3. However, if the subsequent EU legislation of 2010

would have felt implemented since the beginning of the intervention about 10

to 15% of the lots disposed of, could have been sold (Figure 3). According to

Table 2, 11 samples would have been accepted if 2010 regulation was applied

before, to the RTE situation. This percentage is statistically significant at

p<0.001 (z=-3.423). The Table 2 also shows that 12 samples would have been

accepted if 2010 regulation was applied before, to the DFP situation. This

percentage is statistically significant at p<0.001 (z=-3.575). These values

represent an economic and social impact, considering the complex chain that

Brazil nut has.

Figure 2. Brazil nuts lots contaminated with AF (μg/kg) rejected by the EU between

2001 and 2011. Number of rejected lots= 98. Data from RASFF Annual Report (2002

to 2011).


Table 2. Possibility of frequency and acceptance rate of Brazil nut

samples, before and after 2010 regulation, for RTE and DFP samples

(N=98)

Frequency Percentage Proportion Test

RTE samples

Accepted Samples with

regulation <2010 0 0 -3.423

a


regulation >2010 11 11.24%

DFP samples


regulation <2010 0 0 -3.575

a


regulation >2010 12 12.20%

asignificance at 0.001.

Data from RASFF Annual Report (2002-2009).

Iran pistachios were also regulated by EU and according to Cheraghali and

Yazdanpanah [28] the multi-approach intervention to prevent and control AF

contamination in pistachio nuts was fruitful, being observed a significantly

reduction of AF pistachios levels. Those authors also reported that it was very

difficult to convince both national authorities and private sectors to spend

more resources on research in this area, but the costs incurred because of

rejection of consignments would create a logic base for any development in

this area, since the importance of pistachio nuts for Iran economy.

This question has been addressed by Wu [29] which states that the

complex effects of regulatory limits for mycotoxins on price, trade, public

health, selling and purchasing decisions of nations affects the producing

countries that face economic losses. These losses are enhanced by other

indirect losses by far more dramatic for local populations. Since the highest

quality crop is exported to the developed countries, the lower quality one is

consumed internally, yielding to chronic to acute intoxication cases. This

author still points out its combination with the wide spread of malnutrition and

the lack of health care.


Figure 3. Estimated percentage of lots that could be commercialized in the EU from

samples discarded. Data adapted from RASFF Annual Report (2002-2009).

Another question is also addressed to a direct impact on the economy of

developing countries: due to a lack of monitoring at the export points, or – if

monitoring is present – a lack of confidence in the existing test management,

exported goods get rejected at the importing points of developed countries

leading to pricing pressure [30]. The value of RASFF system is unquestionable

and it fulfills its intended function, since that the system is a significant source

Number of lots


of valuable information. However, for risk assessment purposes, another

additional information is needed [31]. Szeits-Szabó and Szabó [31] suggested

RASFF to provide data on the ratio of all/tested/positive lots and to provide

not only the positive results, but also the exact mycotoxins level of every

analyzed sample. In this way it would be more interesting for risk assessment.

Brazilian Actions for Guarantee Brazil Nut’s Food Safety

The incidence and frequency of Brazil nut contamination by AF has been

monitored by the Ministry of Agriculture of Brazil since 1998. The data

concerning the occurrence of AF in Brazil nut samples obtained from export

batches and batches rejected by importing countries between 2005 and 2006,

analyzing only the edible portion (kernels), demonstrated that about 85% of

294 samples showed no detectable levels of AFB1. In AF-positive samples, the

lower AFT limit ranged from 0.4 to 2.42 μg/kg and only 13 samples (4.4%)

showed levels above 20 μg/kg [1]. These results are favorable, in comparison

with previous years. In 2003, Brazil was audited by the EU with regard to the

production and processing of Brazil nuts. Exports of the product were totally

restricted since the publication of Decision 2003/493/EC [13]. This publication

established criteria and conditions for Brazil in order to export in-shell Brazil

nuts to the EU. This included the implementation of good manufacturing

practices, appropriate sampling plans, favorable analysis conditions and

certification of the final product [14]. This measure represented the suspension

of Brazilian exports, which caused an enormous socioeconomic impact on the

entire production chain and led to a stoppage of Brazil nut exports to the EU

[5].

In 2004, the Programa de Alimentos Seguros (PAS) – Campo (Food

Safety Program - Field), a joint national action by SENAI / SEBRAE /

EMBRAPA, was structured as a program from farm to fork, aiming at

producing a document that would instruct producers on how to make a better

control and monitoring throughout the Brazil nut chain. The program

integrates activities of monitoring, control, inspection, and tracking of

contaminants, including mycotoxins. It should be implemented throughout the

production chain, promoting and establishing Good Agricultural Practices

(GAP) and Hazard Analysis and Critical Control Points (HACCP) principles

in order to certify their conformity with a food safe product [32].

Corrective and continuous actions to improve product quality are still

necessary for reduction AFT levels in this product [33].


To prevent and reduce contamination of the Brazil nuts by AF, the

Ministry of Agriculture, Livestock and Supply of Brazil through Instrução

Normativa (Normative Instruction) n° 11, of march 2010 [32] established

under the National Security and Quality of Plant Products and the National

Plan for Control of Residues and Contaminants in Plant Products, the criteria

and procedures to control hygiene and sanitary conditions of the Brazil nut and

its by-products intended for human consumption in the domestic market,

import and export along the production chain [34].

Nowadays, AFT limit for Brazil nuts in Brazil varies from 10 to 20 g/kg,

to RTE and DFP, respectively. This new limit was set by the Ministry of

Health of Brazil through its regulatory agency (ANVISA) which has recently

reviewed and updated the national mycotoxins regulation [35]. The current

limit for AF in Brazil nuts is in agreement with EU levels, in order to meet

food safety standards, since the consumption of contaminated Brazil nuts can

pose a risk to the consumers‘ health, both nationally and internationally, in

addition to causing serious losses to the economy and agribusinesses due to a

rejection of contaminated consignments by the market.

CONCLUSION

There is no easy or rapid solution to completely eliminate mycotoxins in

nuts or to put the commodities within the regulation limits. The impact of

regulations on Agro-food trade is not only a hindrance for exporters in

developing countries, because they cannot reach the limits or because their

products were not safe. It is mainly due the lack in infrastructure to assist all

the chain of monitoring, testing and certification. Without this infrastructure it

will be impossible to demonstrate compliance of products with the regulation

of the importing country.

With regard to AF, the management of the entire Brazil nut chain is still a

challenge. This wild commodity needs to be safely offered to consumers,

especially for the ones who expect to eat not only a nut but also a functional

food, because of its health-related appeal, and mostly because of its oil

characteristics and high selenium content. Prevention of contamination by

Aspergillus spp. through good handling practices is still the best measure to

avoid AF in Brazil nuts and to ensure the quality and safety of this product [5]

and needs to continuous investment to keep quality of the product [36, 37].

It still needs initiative to help to achieve food safety through of assisting

national governments and through working closely with regional economic


organizations to develop continuous local program for food safety which

optimize regional conditions for attaining food safety in trade policy area.

CONFLICT OF INTEREST

The authors have declared no conflict of interest.

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Chapter 6

POLYMORPHISMS OF DNA REPAIR

GENES AND TOXICOLOGICAL EFFECTS

OF AFLATOXIN B1 EXPOSURE

Xi-Dai Long1, 2,*†, Jin-Guang Yao

2*†, Qian Yang

3†,

Cen-Han Huang4†

, Pinhu Liao4†

, Le-Gen Nong4†

,

Yu-Jin Tang4†

, Xiao-Ying Huang2†

, Chao Wang4†

,

Xue-Ming Wu3, Bing-Chen Huang

3, Fu-Zhi Ban

5,

Li-Xia Zeng6, Yun Ma

7, Bo Zhai

1, Jian-Jun Zhang

1,

Feng Xue1, Cai-Xia Lu

8 and Qiang Xia

1

1 Department of Liver Surgery, Ren Ji Hospital, Shanghai Jiao Tong

University School of Medicine (RJHSJTM), P.R. China 2 Department of Pathology, the Affiliated Hospital of Youjiang Medical

College for Nationalities (AHYMCN), P.R. China 3 Department of Cell Biology and Anatomy, Louisiana State University

Health Sciences Center, Baton Rouge, LA, US 4 Department of Medicine, AHYMCN, P.R. China

* Address all correspondence to: Xi-Dai Long, Department of Liver Surgery, RJHSJTM, No.

1630, Dongfang Road, Shanghai 200127, P.R. China; or Department of Pathology,

AHYMCN, No. 98, Chengxiang Rd., Baise City, Guangxi Zhuang Autonomous Region

533000, P.R.China. Email: [email protected]. Qiang Xia, Department of Liver Surgery,

RJHSJTM, No. 1630, Dongfang Road, Shanghai 200127, P.R. China. Email:

[email protected] or [email protected]. Tel.: +86 21 68383775. Fax.: + 86 21

58737232 † These authors contributed equally to this work.

Xi-Dai Long, Jin-Guang Yao, Qian Yang et al. 126

5 Department of Test, the Affiliated Southwestern Hospital of Youjiang

Medical College for Nationalities, P.R. China 6 Department of Pathology, the Affiliated Tumor Hospital, Guangxi

Medical University (GXMU), P.R.China 7 Department of Pathology, the First Affiliated Hospital, GXMU,

P.R.China 8 Department of Medicine, Min Hang Hospital of Shanghai, P.R. China

ABSTRACT

Aflatoxin B1 (AFB1) is an important genic toxin produced by the

moulds Aspergillus parasiticus and Aspergillus flavus. AFB1 is

metabolized by cytochrome P450 enzymes to its reactive form, AFB1-

8,9-epoxide (AFB1-epoxide), which covalently binds to DNA and induces

DNA damage. DNA damage induced by AFB1, if not repaired, may cause

such genic tox toxicological Effects as DNA adducts formation, gene

mutations and hepatocellular carcinoma (HCC). During the repair process

of DNA damage produced by AFB1, DNA repair genes play a central

role, because their function determines DNA repair capacity. In this

study, we investigated the association between seven polymorphisms

(including rs25487, rs861539, rs7003908, rs28383151, rs3734091,

rs13181, and rs2228001) in DNA repair genes XPC, XRCC4, XRCC1,

XRCC4, XPD, XRCC7, and XRCC3, and toxicological effects of AFB1

using a hospital-based case-control study. Toxicological effects of AFB1

were analyzed by means of the levels of AFB1-DNA adducts, the mutant

frequency of TP53 gene, and the risk of AFB1-related HCC. We found

that the mutants of XPC, XRCC4, XRCC1, XRCC4, XPD, XRCC7, and

XRCC3 had higher AFB1-DNA adducts levels, compared with the wilds

of these genes (3.276 vs 3.640 μmol/mol DNA for rs25487, 2.990 vs

3.897 μmol/mol DNA for rs861539, 2.879 vs 3.550 μmol/mol DNA for

rs7003908, 3.308 vs 3.721 μmol/mol DNA for rs28383151, 3.229 vs

3.654 μmol/mol DNA for rs3734091, 2.926 vs 4.062 μmol/mol DNA for

rs13181, and 3.083 vs 3.666 μmol/mol DNA for rs2228001,

respectively). Furthermore, increasing risk of TP53 gene mutation and

HCC was also observed in these with the mutants of DNA repair genes.

These results suggested that polymorphisms of DNA repair genes might

modify the toxicological effects of AFB.

Keywords: Aflatoxin B1, DNA repair gene, toxicological effect,

polymorphism, hepatocellular carcinoma

Polymorphisms of DNA Repair Genes and Toxicological Effects … 127

ABBREVIATIONS

AFB1, aflatoxin B1;

HCC, hepatocellular carcinoma;

DSB, double strand break;

NHEJ, the non-homologous end joining;

OR, odds ratio;

PCR, polymerase chain reaction;

TP53M, the hot-spot mutation at codon 249 of TP53 gene;

XRCC1, x-ray repair cross-complementing group 1;

rs25487-CC, the homozygotes of XRCC1 rs25487 C alleles;

rs25487-CT, the heterozygotes of XRCC1 rs25487 C and T allele;

rs25487-TT, the homozygotes of XRCC1 rs25487 T alleles;


rs861539-GG, the homozygotes of XRCC3 rs861539 G alleles;

rs861539-GA, the heterozygotes of XRCC3 rs861539 G and A allele;

rs861539-AA, the homozygotes of XRCC3 rs861539 A alleles;


rs7003908-AA, the homozygotes of XRCC7 rs7003908 A alleles;

rs7003908-AC, the heterozygotes of XRCC7 rs7003908 A and C allele;

rs7003908-CC, the homozygote of XRCC7 rs7003908 C alleles;



rs28383151-GA, the heterozygotes of XRCC4 rs7003908 G and A allele;

rs28383151-AA, the homozygote of XRCC4 rs28383151 A alleles;


rs3734091-GT, the heterozygotes of XRCC4 rs7003908 G and T allele;

rs3734091-TT, the homozygote of XRCC4 rs3734091 T alleles;

XPD, the xeroderma pigmentosum complementation group D;

rs13181-GG, the homozygotes of XPD rs13181 G alleles;

rs13181-GA, the heterozygotes of XPD rs7003908 G and A allele;

rs13181-AA, the homozygote of XPD rs13181 A alleles;

XPC, the xeroderma pigmentosum complementation group C;

rs2228001-GG, the homozygotes of XPC rs2228001 G alleles;

rs2228001-GA, the heterozygotes of XPC rs7003908 G and A allele;

rs2228001-AA, the homozygote of XPC rs2228001 A alleles.


1. INTRODUCTION

Aflatoxin B1 (AFB1) was am important member of aflatoxin family highly

substituted coumarins containing a fused dihydrofurofuran moiety [1-3]. This

aflatoxin is mainly produced by some strains of the moulds Aspergillus

parasiticus and Aspergillus flavus, and is structurally characterized by fusion

of a cyclopentanone ring to the lactone ring of the coumarin moiety [2, 3].

AFB1 was discovered as a contaminant of human and animal food, especially

peanuts (ground nuts), core, soya sauce, and fermented soy beans in tropical

areas such as the Southeastern China as a result of fungal contamination

during growth and after harvest which under hot and humid conditions. This

type of toxin has three toxicological effects: a. genotoxicity, mainly inducing

the formation of AFB1-DNA adducts and the hot-spot mutation of p53 gene; b.

the attraction of specific organs, especially liver; and c. carcinogenicity,

primarily causing hepatocellular carcinoma (HCC) [1-9]. Increasing evidences

have shown that DNA damage by AFB1 plays the central role of

carcinogenesis of HCC-related to this toxin in the toxic studies [2, 3, 10].

Today, AFB1 has been classified as a known human carcinogen by the

International Agency for Research on Cancer [1-3]. However, more and more

epidemiological evidence has exhibited that although many people are

exposed to the same levels of AFB1, only a relatively small proportion of

exposure person feature the toxicological effects of AFB1 such as showing

gene mutations and developing HCC [2, 3]. This indicates individual DNA

repair capacity related to AFB1-induced DNA damage might be associated

with the toxicological effects of AFB1 exposure. Here, we investigated the

effects of genetic polymorphisms in DNA repair genes XRCC1, XRCC3,

XRCC4, XRCC7, XPD, and XPC (including rs25487, rs861539, rs7003908,

rs28383151 and rs3734091, rs13181, and rs2228001) on the toxicological

effects of AFB1 exposure through the analysis of AFB1-DNA adducts amount,

TP53 gene mutation frequency, and HCC risk.

2. MATERIALS AND METHODS

2.1. Study Design

Because the toxicological effects of AFB1 exposure can be elucidated by

the analysis of AFB1-DNA adducts, TP53M, and HCC risk, a hospital-based


case-control study was designed and conducted (Figure 1). In this study, we

firstly analyzed the amount of AFB1-DNA adducts and the frequency of

TP53M in the liver cancer tissue samples, next evaluated HCC risk using

individual matching case-control design.

Figure 1. Study design. This study, including 1486 hepatocellular carcinoma (HCC)

patients and 1996 controls (matching to HCC cases based to age, sex, race, HBV

status, and HCV status), was conducted in the high AFB1 exposure areas. The

corresponding samples, including peripheral blood samples for the analysis of

genotypes in the polymorphic loci of DNA repair genes and HCC risk, and cancerous

tissues samples for the analysis of AFB1-DNA adducts and TP53 mutation, were

collected and tested.

2.2. Study Subject

Cases

A total of 1486 HCC cases treated at the affiliated hospitals of Guangxi

Medical University and Youjiang Medical College for Nationalities during the

period from January 2004 through December 2012 were recruited for

participation in the study. All cases were the residents of Guangxi Zhuang


Autonomous Region, a high AFB1 exposure area. The cases included in this

study, representing a significant proportion (>90%) of HCC patients in the

Guangxi population, were identified by histopathological diagnosis in 100% of

the HCC cases. All patients gave informed consent for participation and were

interviewed uniformly before surgery by a well-trained interviewer. The

questionnaire used in the interview sought detailed information on current and

past living habits, occupational history, family disease history, dietary history

and general demographic data. Clinical pathological data (including cirrhosis,

tumor size, PVT, and tumor stage), -fetoprotein (AFP), hepatitis virus B

(HBV) and hepatitis virus C (HCV) infection information, and therapeutic data

were collected from medical records in the hospitals by a Youjiang Cancer

Institution staff member.

At the same time of interview, 4 mL of peripheral blood was obtained for

the extraction of genomic DNA for each case. Additionally, surgically

removed tumor samples of all cases were collected for analyzing AFB1-DNA

adducts amount and TP53M frequency. Tumor tissue samples were surgically

dissected into small pieces, frozen immediately in liquid nitrogen and stored at

–80 °C. In this study, those hepatitis B surface antigen (HBsAg) positive and

anti-HCV positive in their peripheral serum were defined as groups infected

with HBV and HCV. Liver cirrhosis was diagnosed by pathological

examination, and stages of tumor were confirmed according to the tumor-

nodes-metastasis (TNM) staging system.

Controls

To investigate the oncogenicity toxicological effects of AFB1 exposure,

we designed and established case-control study. During the same period of

HCC investigation, controls without any evidence of liver disease were

randomly selected from a pool of healthy volunteers who visited the general

health check-up centers of the same hospitals for their routinely scheduled

physical examinations supported by local governments. In the present study, a

total of 2032 controls were enrolled and interviewed. Because of 36 volunteers

dropped out, 1996 controls were included in final analysis. To control the

effects of confounders which were associated with the distribution of

genotypes or the exposure of AFB1, controls were individually matched (1:1 or

2:1) to cases based on ethnicity (Han, Minority), sex, age ( 5 years), and

hepatic B virus (HBV) and hepatic C virus (HCV) infection. After giving

written consent, demographic information (including age, sex, race, medical

history, family disease history, dietary history, and living history), and HBV

and HCV infection information were collected in the hospitals using a


standard interviewer-administered questionnaire. At the same time, 4 mL of

peripheral blood was obtained for the extraction of genomic DNA.

Ethical Protocol

The study protocol was been carried out in accordance with "Ethical

Principles for Medical Research Involving Human Subjects" (World Medical

Association Declaration Of Helsinki, 2004) and approved by Institutional

review boards from Guangxi Cancer Institute, and the Medical Research

Council from the corresponding hospitals.

2.3. DNA Extraction

DNA was extracted from HCC cancerous tissues from all cancer patients

in a 1.5 mL microcentrifuge tube for deparaffinization and proteinase K

digestion, as described by standard procedures (Protocol #BS474, Bio Basic,

Inc., Ontario, Canada). DNA was stored at 48 oC until additional analysis. For

peripheral blood samples from all cases and controls, DNA was isolated using

classical phenol-chloroform extraction.

2.4. AFB1-DNA Adducts Assay

DNA Samples Preparation

In this study, the amount of AFB1-DNA adducts in cancerous tissues

samples were evaluated competitive enzyme-linked immunosorbent assay

(ELISA) [11-14]. To convert any N-7 adduct to AFB1-FAPy adducts, DNA

was treated with 15 mM Na2CO3 and 30 mM NaHCO3 (pH 9.6) for 2 hours,

precipitated with 2.5 volumes of 95% ethanol, and then redissolved in 10 mM

Tris-HCl (pH 7.0). The DNA samples were reprecipitated, dissolved in 1 ×

PBS, and denatured by boiling for 5 min. After that, AFB1-FAPy adducts were

quantitated by ELISA using monoclonal antibody 6A10 (Novus Biologicals

LLC, catalog # NB600- 443).

ELISA

ELISA was accomplished as described previously [11-14]. Briefly,

Immulon 2 plates (Dynatech Laboratories, Chantilly, VA) were first coated

with 5 ng of imidazole ring-opened AFB1-DNA in 1 × PBS by drying

overnight at 37 ◦C. The test solutions contained unbound AFB1-DNA and


6A10 antibody. Goat anti-mouse IgG alkaline phosphatase (1:1500) and then

p-nitrophenyl phosphate (1 mg/mL in 1 M diethanolamine, pH 8.6) was added

to the DNA. After 90 min incubation at 37 ◦C, absorbance at 405 nm was read

on a Bio-Tek Microplate Reader (Bio-Tek Instruments, Inc., Winooski, VT).

The standard curve for different imidazole ring-opened AFB1-DNA

concentration was drawn and adducts amount in samples were calculated

according to the corresponding standard curve. For the standard curve, highly

modified imidazole ring-opened AFB1-DNA was serially diluted with non-

modified denatured calf thymus DNA (R&D Systems, Inc., catalog # 9600-5-

D) such that 50 μL contained from 0 to 1000 fmol adduct and 50 μg DNA.

These samples were mixed with an equal volume of diluted 6A10 antibody (50

μL, diluted 1:1.25×106), added to the wells, and measured by competitive

ELISA [12-14].

For the test samples, 25 μg heat-denatured DNA from cancerous tissues

samples in 50 μL hydration solution was mixed with 50 μL diluted antibody

before being added to the wells. The amount of AFB1-DNA in the test samples

was quantitated relative to a standard curve based on known concentrations of

AFB1-DNA. Each sample was measured in triplicate on the three different

assay dates. Adduct levels were ascertained according to the average of three

measures. The quality control for adduct assays was administered by blank and

positive controls.

2.5. TP53 Gene Mutation Analysis

Laboratory personnel were blinded to subject status. The hot-spot

mutation of TP53 gene (at codon 249, TP53M) was analyzed using the

TaqMan-PCR on iCycler iQ™ real-time PCR detection system (iQ5, Bio-Rad

Laboratories Inc., Hercules, CA). Primers for TP53M analysis were 5‘-TTG

GCT CTG ACT GTA CCA CCA T-3‘ and 5‘-TGG AGT CTT CCA GTG

TGA TGA TG-3‘; whereas probes were 5‘-FAM-ACC GGA GTC CCA TC-

MGB-3‘ and 5‘-VIC-AAC CGG AGG CCC AT-MGB-3‘ [15, 16]. Primers

and probes were synthesized by Introgen Bio., Ltd. (Shanghai, China) and

Applied Biosystems. PCR was carried out in a total volume of 25 μL

consisting of 1 × Premix Ex TaqTM

(catalog # DRR039A, TaKaRa

Biotechnology (Dalian) Co., Ltd., Dalian, China), 0.2 μM of each probe, 0.2

μM of each primer, and 50 – 100 ng of genomic DNA. The PCR program had

an initial denaturation step of 2 min at 95 0C followed by 50 cycles of 10 sec at

95 0C and 1 min at 60

0C. For quality control, controls were included in each


run, and repeated genotyping and sequencing of a random 10% subset yielded

100% identical results. To analysis, the information of TP53M was divided

into two groups: TP53M negative status (spot mutation at codon 249 of TP53

gene not detected), and positive status (spot mutation at codon 249 of TP53

gene detected).

2.6. Polymorphisms Selection of DNA Repair Genes

and Genotyping

In this study, we only selected these single nucleotide polymorphisms

(SNPs) in the DNA repair genes that might modify AFB1-related HCC risk.

According to our previous results, a total of 7 SNPs , including rs25487 (in the

XRCC1), rs861539 (in the XRCC3), rs7003908 (in the XRCC7), rs28383151

and rs3734091 (in the XRCC4), rs13181 (in the XPD), and rs2228001 (in the

XPC), were finally analyzed in the present study (Table 1).

The genotypes of DNA repair genes were genotyped using the previous

TaqMan-PCR methods on iCycler iQ™ real-time PCR detection system (iQ5,

Bio-Rad Laboratories Inc.). Primer and probe sets and annealing temperatures

used for TaqMan-PCR assay are shown in Table 2. For quality control,

controls were included in each run, and repeated genotyping and sequencing of

a random 5% subset yielded 100% identical genotypes.

2.7. Statistical Analysis

Genotype data were analyzed as trichotomous variables, including wild

homozygotes (wild genotype), heterozygotes (heterozygotes genotype), and

mutant homozygotes (mutant genotype). For the association analysis between

polymorphisms in the DNA repair genes and the amount of AFB1-DNA

adducts, the different distribution of groups was tested using the Student‘s t

test. In this analysis, each genotype was evaluated as a categorical variable

with three levels (homozygous low activity, heterozygous, and homozygous

high activity). The mean value (with SD and SE) of AFB1-DNA adducts levels

for each high activity group was calculated and compared with low activity

group (wild genotypes).

Table 1. The characteristics of polymorphisms in the DNA repair genes

Polymorph-

ism Gene Chr:bp

Alleles Genotypes Codon

No.

Amino

acid Geno-typing Wild Mutant Wild Heterozygotes Mutant

rs25487 XRCC1 19:44055726 C T CC CT TT 399 Arg/Gln TaqMan-PCR

rs861539 XRCC3 14:104165753 G A GG GA AA 241 Thr/Met TaqMan-PCR

rs7003908 XRCC7 8:48770702 A C AA AC CC / / TaqMan-PCR

rs28383151 XRCC4 5:82406873 G A GG GA AA 56 Ala/Thr TaqMan-PCR

rs3734091 XRCC4 5:82500734 G T GG GT TT 247 Ala/Ser TaqMan-PCR

rs13181 XPD 19:45854919 T G TT TG GG 751 Lys/Gln TaqMan-PCR

rs2228001 XPC 3:14187449 T G TT TG GG 939 Lys/Gln TaqMan-PCR

Table 2. Technical details of TaqMan-PCR analysis

Polymorphism Gene Primers Probes

rs25487 XRCC1 5'-GTGGGTGCTGGACTGTC-3'

5'-GCAGGGTTGGCGTGTGA-3'

5'-FAM-CCTCCCGGAGGTAA-MGB-3'

5'-VIC-CCCTCCCAGAGGTAA-MGB-3'

rs861539 XRCC3 5'-CCAGGGCCAGGCATCTG-3'

5'-CAGCACAGGGCTCTGGA-3'

5'-FAM-CAGCATGGCCCCCA-MGB-3'

5'-VIC-CAGCGTGGCCCCCA-MGB-3'

rs7003908 XRCC7 5'-CCTACCTCACGAACTCAGCAATT-3'

5'-GCTGCCAACGTTCTTTCCTTATAGT-3'

5'-FAM-CTAAGAGTCCGCTGTTT-MGB-3'

5'-Hex-CCTAAGAGTCAGCTGTTT-MGB-3'

rs28383151 XRCC4 C__58444701_10a C__58444701_10

rs3734091 XRCC4 5'-TGAGGAAAGTGAAAACCAAACTGATCT-3'

5'-GCCCAAATAAGATATTCAACAGAGGAGAT-3'

5'-FAM-CCTGAAGACAACCC-MGB-3'

5'-HEX-CCTGAAGCCAACCC-MGB-3'

rs13181 XPD 5'-AGTCACCAGGAACCGTTTATGG-3'

5'-TCTGTTCTCTGCAGGAGG ATC-3'

5'-HEX-CTCTATCCTCTGCAGCG-MGB-3'

5'-FAM-TATCCTCTTCAGCGTCT-MGB-3'

rs2228001 XPC 5‘-AGCAGCTTCCCACCTGTTC-3‘

5‘-GTGGGTGCCCCTCTAGTG-5‘

5‘-FAM-CACAGCTGCTCAAAT-MGB-3‘

5‘-Hex-CTCACAGCTTCTCAAAT-MGB-3‘

a From the Applied Biosystems.


Frequency tables of independent variables (including TP53M variable and

HCC risk variables) and genotype data were evaluated for statistical

significance by Pearson's χ2. To analyze the risk for gene mutation and HCC

associated with each genotype while adjusting for confounders, multivariable

logistic regression was done and odds ratios (OR) along with 95% confidence

intervals (95% CI) generated. In this type of the additive model, we treated

genotype as an ordinal variable (wild type coded as 0, heterozygote as 1, and

homozygotes variant as 2). For TP53M risk, unconditional logistic regression

model (enter method consisting of age, sex, race, AFB1 exposure, and HBV

and HCV infection status) was used for risk values. For HCC risk, based on

individually matched design of case-control study, we did conditional logistic

regression (with multivariate factors, including known causes of HCC among

the Guangxi population) to estimate ORs for risk of HCC and their 95% CIs.

In the present study, a P-value of < 0.05 was considered statistically

significant. All statistical analyses were done using SPSS version 18 (SPSS,

Inc., Chicago, IL).

3. RESULTS

3.1. The Demographic Characteristics of HCC Cases

The 1486 HCC cases were from high AFB1 exposure areas and included

in the final analysis and the demographic data of these patients is shown in

Table 3. The HBV and HCV infective rates were 72.95% (1084 of 1486) and

18.57% (276 of 1486), respectively. They were characterized by age, 49.32 ±

11.43 years, and more commonly male. Among them, 72.68% (1080 of 1486)

cases featured liver cirrhosis and about 70% patients were in the second stage

of TNM system.

3.2. DNA Repair Genes Polymorphisms and AFB1-DNA Adducts

Levels

Cancerous tissue samples from the 1486 HCC patients were examined for

AFB1-DNA adducts. A mean SD of 3.296 1.710 μmol/mol DNA was

observed in the tumor tissues samples (Table 4). To analyzed the represents of

AFB1-DNA adducts levels in cancerous tissue samples for AFB1 exposure, we


also investigated the levels of AFB1-DNA adducts in peripheral blood

leukocytes and AFB1-albumin adducts in peripheral blood serum, other two

important biomarkers for AFB1 exposure evaluation, according to previously

published methods (Table 4) [14, 16]. Results showed that AFB1-DNA

adducts levels in cancerous tissue samples were lineally related to these two

biomarkers, and higher DNA adducts levels were found in the tumor tissues

samples. Because the corresponding DNA adducts in cancerous tissues were

higher and more stable compared with peripheral blood, this adducts were

used for the following the toxicological effects of AFB1 exposure analysis.

Table 3. The characteristics of HCC cases

Characteristics

Total, n (%) 1486 (100.00)

Age (years)

Mean ± S.D. 49.32 ± 11.43

Sex

Male, n (%) 1120 (75.37)

Female, n (%) 366 (24.63)

Race

Han, n (%) 700 (47.11)

Minority, n (%) 786 (52.89)

HBV

HBsAg(-), n (%) 402 (27.05)

HBsAg(+), n (%) 1084 (72.95)

HCV

anti-HCV(-), n (%) 1210 (81.43)

anti-HCV(+), n (%) 276 (18.57)

AFP

Negative, n (%) 747 (50.27)

Positive, n (%) 739 (49.73)

Liver Cirrhosis

No, n (%) 406 (27.32)

Yes, n (%) 1080 (72.68)

TNM stage

I, n (%) 112 (7.54)

II, n (%) 1010 (67.97)

III, n (%) 326 (21.94)

IV, n (%) 38 (2.56)


Table 4. The levels of AFB1 adducts in cancerous tissues and

peripheral blood

Adducts Mean SE SD

ADATa 3.296 0.044 1.710

ADABb 1.983 0.029 1.126

AAAc 28.390 1.162 44.798

ln(AAA) d 2.980 0.021 0.796 aADAT, AFB1-DNA adducts in HCC cancerous tissues (μmol/mol DNA).

bADAB, AFB1-DNA adducts in peripheral blood leukocytes (μmol/mol DNA).

cAAA, AFB1-albumin adducts in peripheral blood (fmol/mg).

dln(AAA), logarithmical transformation of AAA (ln fmol/mg).

Table 5. Polymorphisms in DNA repair genes and AFB1-DNA

adduct levels

Adduct levela

Gene Polymorphism Genotype Mean SD Pb

XRCC1 rs25487 CC 3.276 0.063

CT 3.264 0.069 0.899

TT 3.640 0.149 0.026

XRCC3 rs861539 GG 2.990 0.078

GA 3.216 0.065 0.025

AA 3.897 0.089 4.96210-14

XRCC7 rs7003908 AA 2.879 0.084

AC 3.347 0.065 1.66310-5

CC 3.550 0.082 1.75110-8

XRCC4 rs28383151 GG 3.308 0.054

GA 3.405 0.094 0.069

AA 3.721 0.128 2.86710-4

XRCC4 rs3734091 GG 3.229 0.051

GT 3.439 0.113 0.095

TT 3.654 0.141 0.005

XPD rs13181 TT 2.926 0.066

TG 3.253 0.070 0.011

GG 4.062 0.097 4.26510-6

XPC rs2228001 TT 3.083 0.071

TG 3.332 0.067 0.001

GG 3.666 0.032 3.40410-22 a AFB1-DNA adducts levels in the HCC cancerous tissues (μmol/mol DNA).

b Calculated by genotypes with mutant alleles compared with wild homozygote.


In this study, the genotypes of DNA repair genes XRCC1, XRCC3,

XRCC4, XRCC7, XPD, and XPC were tested using TaqMan-PCR techniques.

We found the mutants of these DNA repair genes had higher levels of AFB1-

DNA adducts, compared with their wilds (Table 5). For example, these with

XRCC7 rs7003908-CC genotype, compared to those with XRCC7 rs7003908-

AA genotype (2.879 0.084 μmol/mol DNA), faced an increasing amount of

AFB1-DNA adducts (3.550 0.082 μmol/mol DNA, P < 0.01).

3.3. DNA Repair Genes Polymorphisms and TP53M

The status of hotspot mutation in codon 249 of the p53 gene (TP53M) in

cancerous tissue samples was evaluated by TaqMan-PCR among 1486 HCC

cases. One thousand one hundred-sixteen (75.10%) of the 1486 HCCs showed

TP53M. The frequency of hotspot mutation was not related with HBV

infection and HCV infection (P > 0.05, data not shown). However, individuals

with the heterozygotes of XRCC1 rs25487 (namely: rs25487-CT) or the

variant homozygotes of XRCC1 rs25487 (namely: rs25487-TT) were more

probably to have higher frequency of TP53M than those with the wild-type

homozygote of XRCC1 rs25487 (namely: rs25487-CC). Its adjusted ORs

(95% CIs) were 2.419 (1.863-3.141) for rs25487-CT and 5.028 (2.490-10.153)

for rs25487-TT, respectively (Table 6). Similar risk role was also found in the

association analysis between other 6 polymorphisms (including rs861539 (in

the XRCC3], rs7003908 (in the XRCC7], rs28383151 and rs3734091 (in the

XRCC4], rs13181 (in the XPD], and rs2228001 (in the XPC]) and TP53M

(Table 6).

3.4. DNA Repair Genes Polymorphisms and HCC Risk

To explore the correlation between these 7 polymorphisms and another

biomarker of the toxicological effect of AFB1 exposure (also called HCC risk),

we conducted a hospital-based case-control study according to our previously

published methods [14-16]. A total of 1486 HCC cases and 1996 individually-

matched (based on age, sex, race, and HBV and HCV infection status) controls

were included in the present risk analysis (Table 7). There were no significant

differences between cases and controls in terms of distribution of age, sex,

race, and HBV and HCV status as a result of individual matching (P > 0.05).


More detailed information has been reported in our previous studies. These

results suggest that HCC patient data were comparable to control data.

Higher frequency of mutants of DNA repair genes was observed in the

HCC patients than in the controls. Conditional logistic regression analysis

exhibited that mutant alleles increased about 2 to 6 fold of HCC risk value.

This risk was more noticeable under the conditions of mutant homozygotes

(Table 7). For example, HCC risk for the genotype with XRCC4 rs3734091-

GT was 1.460 (1.195-1.783); whereas risk value was 2.244 (1.676-3.003) for

XRCC4 rs3734091-TT genotype. These results suggested the risk of HCC was

associated with the number of mutant alleles of DNA repair genes XRCC1,

XRCC3, XRCC7, XRCC4, XPD, and XPC.

4. DISCUSSION

4.1. The Evaluation of Toxicological Effects of AFB1

A main toxicological effect of AFB1 is to induce DNA damage, consisting

of AFB1-DNA adducts and the hot-spot mutation of tumor suppressor gene

p53 at codon 249 (TP53M) [5, 17-19]. Thus, the toxicological effects of AFB1

exposure might be elucidated using the analysis of AFB1-DNA-adducts levels

and TP53M frequency in the liver tissues or other tissues [2]. AFB1 can induce

several DNA adducts formation, including AFB1-N7-Gua adduct, AFB1-FAPy

adduct, and so on. Among these adducts, AFB1-FAPy adduct is the imidazole

ring-opened product of AFB1-N7-Gua adduct, also the stable form of the later

adduct, and may play an important role in the development of HCC.

Moreover, the accumulation of this adduct is time-dependent and non-

enzymatic, and may have potential biological importance because of its

apparent persistence in DNA. Thus, many researchers in the relative fields

regard AFB1-FAPy adduct as a validated biomarker of AFB1 exposure [1, 2, 4,

10]. Increasing evidences have exhibited that AFB1-FAPy-adducts levels in

the liver or placenta tissues are lineally correlated with AFB1 exposure levels

and HCC risk [14, 15], suggesting this adduct should be regarded as a

toxicological elucidation biomarker of AFB1. Our previous studies have shown

that peripheral blood leukocytes' adduct levels were positively and linearly

related to AFB1-DNA adduct levels of the HCC cancerous tissue. These data

suggested that the levels of peripheral blood leukocytes' DNA adducts were

representative of the tissues' DNA-adduct levels and might be regard as a

biomarker for AFB1 exposure [14, 15].

Table 6. Polymorphisms in DNA repair genes and TP53M risk

TP53M (-) TP53M (+)

Gene Polymorphism Genotype n % n % ORa 95% CI P

XRCC1 rs25487 CC 258 69.73 519 46.51 Reference

CT 103 27.84 505 45.25 2.419 1.863-3.141 3.37110-11

TT 9 2.43 92 8.24 5.028 2.490-10.153 6.65110-6

XRCC3 rs861539 GG 150 40.54 359 32.17 Reference

GA 148 40.00 486 43.55 1.380 1.057-1.802 0.018

AA 72 19.46 271 24.28 1.524 1.102-2.108 0.011

XRCC7 rs7003908 AA 126 34.05 237 21.24 Reference

AC 149 40.27 514 46.06 1.883 1.416-2.505 1.37210-5

CC 95 25.68 365 32.71 2.089 1.526-2.861 4.36810-6


GA 58 15.68 242 21.68 1.688 1.229-2.318 0.001

AA 13 3.51 126 11.29 3.829 2.129-6.888 7.38710-6


GT 29 7.84 196 17.56 2.799 1.856-4.222 9.19110-7

TT 9 2.43 113 10.13 5.104 2.556-10.190 3.82610-6

Table 6. (Continued)

TP53M (-) TP53M (+)


XPD rs13181 TT 165 44.59 384 34.41 Reference

TG 140 37.84 467 41.85 1.458 1.120-1.898 0.005

GG 65 17.57 265 23.75 1.744 1.256-2.422 0.001

XPC rs2228001 TT 166 44.86 380 34.05 Reference

TG 157 42.43 539 48.30 1.500 1.162-1.936 0.002

GG 47 12.70 197 17.65 1.818 1.258-2.625 0.001 a Adjusted by age, sex, race, HBV status, and HCV status.

Table 7. Polymorphisms in DNA repair genes and HCC risk

Controls HCCs


XRCC1 rs25487 CC 1437 71.99 777 52.29 Reference

CT 520 26.05 608 40.92 2.155 1.861-2.495 9.91810-25

TT 39 1.95 101 6.80 4.774 3.264-6.981 7.61410-16


GA 539 27.00 634 42.66 3.321 2.848-3.872 5.67110-53

Controls HCCs


AA 27 1.35 343 23.08 5.846 3.907-13.747 3.39910-67

XRCC7 rs7003908 AA 1141 57.16 363 24.43 Reference

AC 608 30.46 663 44.62 3.434 2.921-4.037 1.66410-50

CC 247 12.37 460 30.96 5.867 4.828-7.129 7.47810-71


GA 217 10.87 300 20.19 2.248 1.857-2.722 1.03410-16

AA 62 3.11 139 9.35 3.690 2.708-5.029 1.34110-16


GT 226 11.32 225 15.14 1.460 1.195-1.783 2.06710-4

TT 81 4.06 122 8.21 2.244 1.676-3.003 5.55510-8

XPD rs13181 TT 1214 60.82 549 36.94 Reference

TG 611 30.61 607 40.85 2.193 1.884-2.551 3.21610-24

GG 171 8.57 330 22.21 4.270 3.458-5.273 1.84110-41

XPC rs2228001 TT 988 49.50 546 36.74 Reference

TG 804 40.28 696 46.84 1.570 1.357-1.817 1.38810-9

GG 204 10.22 244 16.42 2.185 1.764-2.706 8.23110-13

a Adjusted by age, sex, race, HBV status, and HCV status.


Our present study found more amount of AFB1-DNA adducts in cancerous

tissues than in the peripheral blood. Thus, AFB1-DNA adducts in tumor tissues

were furthermore analyzed in the following study.

As regard of the mutations of p53 gene, AFB1 mainly induces the

transversion of G → T in the third position at codon 249 of TP53M. The

frequent value of TP53M is more persistent biomarker and more directly

represents genic toxic effects compared with AFB1-DNA adducts [6, 18, 20,

21]. Our study also showed more than 75% of HCC cases had TP53M in the

cancerous tissues. Additionally, HCC is the most common malignant tumors

caused by AFB1 exposure. More and more epidemiological studies have

shown HCC risk is related to different the toxicological capacity of AFB1,

suggesting that tumor risk value might be regard as a selective elucidative

marker for AFB1 toxic effects [2, 15].

Because of the aforementioned reasons, the toxic effects of AFB1

exposure were evaluated through the following three biomarkers: (1) AFB1-

DNA adducts amount in HCC cancerous tissues, (2) the frequency of TP53M,

and (3) HCC risk, in this study. Our results also exhibited these biomarkers

reflected AFB1 exposure information and represented the toxicological

capacity of AFB1.

4.2. XRCC1 Polymorphism and Toxicological Effects of AFB1

XRCC1 gene also calls RCC and is one of three submits of DNA repair

complex in the SSBR pathway (Gene dbase from PubMed). This gene spans

about 32 kb on chromosome 19q13.2 and contains 17 exons and 16 introns.

Its‘ encoding protein (633 amino acids), consists of three functional domains:

N-terminal domain (NTD), central breast cancer susceptibility protein-1

homology C-terminal (BRCT I), and C-terminal breast cancer susceptibility

protein-1 homology C-terminal (BRCT II) [22-24]. This protein is directly

associated with Pol β, DNA ligase III, and PARP, via their three functional

domains and is implicated in the core processes in single-strand break repair

(SSBR) and base excision repair (BER) pathway [22-24]. More than 50 SNPs

in the coding region of XRCC1 gene that lead to amino acid substitution have

been described (SNP database). Among these polymorphisms, rs25487

polymorphism (also called codon Arg399Gln polymorphism) is of special

concern, because this polymorphism resides in functionally significant regions

(BRCT II) and may be related to decreasing DNA repair activity [22, 25-27].

In this study, to investigate the genic toxin effects of AFB1, we designed and


conducted a hospital-based HCC case study in the high AFB1 exposure areas.

Results showed that the HCC patients with XRCC1 genotypes with rs25487 T

alleles (namely: rs25487-CT or rs25487-TT) faced a significantly increasing

risk of TP53M than those with the wild-type homozygote of XRCC1 (namely,

rs25487-CC, OR = 2.155, 95% CI = 1.863-3.141 for rs25487-CT; OR = 5.028,

95% CI = 4.44-42.08 for rs25487-TT, respectively). Additionally, we also

found that rs25487 polymorphism was significantly associated with other two

toxic biomarkers: AFB1-DNA adducts and HCC risk. Supporting our results,

several studies from high AFB1 areas showed the XRCC1 rs25487 T alleles

were significantly correlated with higher levels of AFB1-DNA adducts and

higher risk of TP53M [19, 28]. As regards of risk biomarker for the

toxicological effects of AFB1 (namely AFB1-related HCC risk), sixteen studies

about XRCC1 rs25487 polymorphism were reported with the results being

contradictory in the several decades years [25-27]. Our results showed this

polymorphism could modulate HCC risk. Previous several meta-analysis

based on different AFB1 exposure levels supported our conclusion [20, 26, 27,

29]. These results suggest that the decreasing capacity of DNA repair resulting

from XRCC1 rs25487 polymorphism is contributed to the toxicological effects

of AFB1.


The protein encoded by XRCC3 gene is one of identified paralogs of the

strand-exchange protein RAD51 in human beings. This protein associates

directly with DNA breaks and facilitates of the formation of the RAD51

nucleoprotein filament, which is crucial both for homologous recombination

and HRR [30, 31]. Previous studies have shown that a common polymorphism

(rs861539) at codon 241 of XRCC3 gene (Thr to Met) modifies the function of

this gene [32-41]. Two reports from high AFB1-exposure areas all of world

supported above-mentioned conclusions [42, 43]. In the first frequent case-

control study in Guangxiese, we observed that the higher-frequency of

genotypes with XRCC3 codon 241 Met alleles (namely Thr/Met and Met/Met)

was observed in controls (33.01%) than HCC cases (61.48%, P < 0.001).

Regression analysis showed that Met alleles increases about 2- to 10-fold risk

of HCC and this running-up risk is modulated by the number of Met alleles

(adjusted OR 2.48 and 10.06 for one and two this alleles) [43]. The followed

relative size analysis and the present study not only found similar risk value of

AFB1-related HCC [42], but also found this polymorphism increased risk of


other two toxic biomarkers high AFB1-DNA adduct and TP53 gene mutation

[44]. Our present study supported aforementioned results. These data exhibits

that the polymorphism at codon 241 of XRCC3 gene is a genetic determinant

in the detoxication of AFB1.


DNA repair gene XRCC7, also called DNAPK, DNPK1, HYRC, HYRC1,

or p350) (Genbank ID. 5591), spans about 197 kb on chromosome 8q11 and

contains 85 exons and 86 introns (Gene dBase in PubMed). The protein

encoded by XRCC7 acts as DNA-dependent protein kinase catalytic subunit

(DNA-PKcs) that constitutes the large catalytic subunit of the DNA-PK

complex [45]. When DNA-PKcs is recruited to the site of DSBs by the

Ku70/Ku80 heterodimer, DNA-PK complex changes into its active form and

subsequently initiates the non-homologous end joining (NHEJ) repair, an

important DSBR pathway [46]. Murine mutants defective in the XRCC7 have

non-detectable DNA-PK activity, suggesting that XRCC7 is required for

NHEJ pathway protein [47, 48]. More than 100 polymorphisms have been

reported in the XRCC7 gene, some of which are correlated with malignant

tumors such as bladder cancer (dbSNP in NCBI Database). Of these genetic

polymorphisms in XRCC7 gene, we only investigated the relation between

rs7003908 polymorphism and the effects on AFB1 toxicological effects, and

found this polymorphism might be an important modifying factor for AFB1

toxic role. Supporting our findings, a previous study was also found that these

individuals with XRCC7 rs7003908 G alleles increased HCC risk compared

the homozygote of XRCC7 rs7003908 T alleles (XRCC7-TT), with OR value

3.45 (2.40–4.94) for XRCC7-TG and 5.04 (3.28–7.76) for XRCC7-GG,

respectively. Furthermore, this genetic mutation was correlated with higher the

levels of AFB1-DNA adducts (r = 0.142, P < 0.001) [49]. Taken together,

these results explored that genetic polymorphism of XRCC7 rs7003908 might

decrease AFB1-related DSBR capacity and result in an increasing

toxicological capacity of AFB1, inquiring more studies to support this

conclusion.


4.5. XRCC4 Polymorphisms and Toxicological Effects of AFB1

XRCC4, located on chromosome 5q14.2, is an important the

nonhomologous end-joining (NHEJ) gene [50, 51]. The encoded protein of

this gene consists of 336 amino acid residues (DDBJ/EMBL/Genbank

accession no. AAD47298) and interacts directly with Ku70/Ku80 in the NHEJ

pathway [50, 51]. It is hypothesized that XRCC4 serves as a flexible join

between Ku70/Ku80 and its associated protein, Ligase IV [50, 51]. XRCC4 is

required for precise end-joining of blunt DNA DSBs in mammalian

fibroblasts, and the mutant, XRCC4, results in more-deficient NHEJ capacity.

A gene-targeted mutation study has also shown that differentiating neurons

and lymphocytes strictly require XRCC4 end-joining proteins. The targeted

inactivation of this gene leads to late embryonic lethality accompanied by

defective neurogenesis and defective lymphogenesis. These results

demonstrate that XRCC4 is essential for the DNA repair capacity of NHEJ

[52-54]. More than 100 polymorphisms have been reported in the XRCC4

gene (SNP database), some of which are correlated with DNA adducts, gene

mutation, and malignant tumors (such as oral, gastric, liver, and bladder

cancers) [15, 16, 55-60]. In this study, we only analyzed 2 known SNPs

(rs28383151 and rs3734091) in the coding region of this gene because these

polymorphisms localize at conserved sites of this gene. They change the coded

amino acids and may be associated with a decreased DNA repair capacity and

an increased cancer risk [15, 16]. Our results exhibited that these two

polymorphisms increased AFB1-DNA adducts levels, TP53M risk, and AFB1-

related HCC risk, suggesting they should be important modified factors of

AFB1 toxicological effects.

4.6. XPD Polymorphism and Toxicological Effects of AFB1

XPD gene, also called excision repair cross-complementing rodent repair

deficiency complementation group 2 (ERCC2), COFS2, EM9, or TTD

(Genbank ID. 2068), spans about 20 kb on chromosome 19q13.3 and contains

23 exons and 22 introns [2, 61]. Its encoded- protein is one of seven central

proteins in the NER pathway and act as a DNA-dependent ATPase/helicase.

This protein is associated with the TFIIH transcription-factor complex, and

plays a role in NER pathway. During NER, XPD participates in the opening of

the DNA helix to allow the excision of the DNA fragment containing the

damaged base [61]. There are four described polymorphisms that induce


amino acid changes in the protein: at codons 199 (Ile to Met), at codon 201

(His to Tyr), at codon 312 (Asp to Asn) and at codon 751 (Lys to Gln) [62].

Among these polymorphisms, we only analyzed codon 751 polymorphism

(rs13181) in this study, mainly because our previous studies [14] found the

variant XPD codon 751 genotypes (namely Lys/Gln and Gln/Gln) detected by

TaqMan-MGB PCR was significantly different between HCC cases (35.9%

and 20.1% for Lys/Gln and Gln/Gln, respectively) and controls (26.3% for

Lys/Gln and 8.6% for Gln/Gln, P < 0.001). Individuals having variant alleles

had about 1.5- to 2.5-fold risk of developing the cancer (adjusted OR 1.75 and

95% CI 1.30-2.37 for Lys/Gln; adjusted OR 2.47 and 95% CI 1.62-3.76 for

Gln/Gln). Our present study (based on relative large sample size) suggested

that the genetic polymorphisms at conserved sequence of XPD gene such as at

codon 751 may have potential effect on AFB1-related HCC susceptibility. This

supports different AFB1-toxin capacity might be modified by genetic

polymorphisms at codon 751 in DNA repair gene XPD.

4.7. XPC Polymorphism and Toxicological Effects of AFB1

XPC gene (Genbank accession NO. AC090645) spans 33kb on

chromosome 3p25, and consists of 16 exons and 15 introns. This gene encodes

a 940-amino acid protein, an important DNA damage recognition molecule

which plays an important role in NER pathway [63-65]. XPC protein binds

tightly with another important NER protein HR23B to form a stable XPC-

HR23B complex, the first protein component that recognizes and binds to the

DNA damage sites. XPC-HR23B complex can recognize a variety of DNA

adducts formed by exogenous carcinogens such as AFB1 and binds to the

DNA damage sites [63-65]. Therefore, it may play a role in the decreasing

toxic effects of AFB1. Some studies have shown that low DNA repair capacity

resulting from the genetic mutation of XPC rs2228001 can progress the toxic

effects of AFB1 [63-65]. In the past several decade years, a total of three

studies reported XPC rs2228001 polymorphism was involved in AFB1

detoxication [66-68]. The first study is from Shunde area, Guangdong

Province which is characterized by high AFB1 exposure and high incidence

rate of HCC. In this study, researchers explored the correlation between this

polymorphism and risk of HCC via an 1-1 case-control study (including 78

HCC patients and 78 age- and sex-matching controls) method, and found the

mutation of XPC modified HCC risk (adjusted odds ratios [ORs] were 6.78

with 95% CI 2.03-22.69) [68]. Although they did not directly evaluated the


effects of XPC rs2228001 polymorphism on the toxic role of AFB1, study

population in their study is from high AFB1 exposure areas and high risk of

HCC for XPC mutant. The other two studies was conducted by our teams and

showed XPC codon 939 Gln alleles increased about 2-times risk of HCC [66,

67]. In our present study, we not only observed this mutation increased AFB1-

related HCC risk, but found more direct evidence of XPC polymorphism

modified the toxicological effects of AFB1 exposure through the analysis of

AFB1-DNA adducts the TP53M induced by AFB1 in the HCC cancerous

tissues. As a result, these data suggest that genetic polymorphism at codon 939

of XPC gene is a genetic determinant in the DNA repair process of DNA

damage induced by AFB1 exposure, and its low activity might result in

increasing strength of AFB1 toxin act.

4.8. Limitation

This study had several limitations. First, because the present study is a the

hospital-based study, potential selection bias might have occurred. Second, the

increased risk with AFB1 exposure status noted in this study was probably

underestimated, because the liver disease itself may affect the metabolism of

AFB1 and modify the levels of AFB1-DNA adducts. Third, in spite of the

status of TP53M was investigated in cases of HCC, other AFB1-related

mutations of the TP53 gene were not evaluated. Finally, we only analyzed 7

genetic mutations in DNA repair genes. Therefore, more genes deserve further

elucidation based on a large sample and the combination of genes and AFB1

exposure.

CONCLUSION

In conclusion, to the best of our knowledge, this is the first report to

investigate association between polymorphisms in DNA repair genes XRCC1,

XRCC3, XRCC4, XRCC7, XPC, and XPD and the toxicological effects of

AFB1 among Guangxi population from an high AFB1-exposure area. We find

that the genetic mutations in the DNA repair genes XRCC1, XRCC3, XRCC4,

XRCC7, XPC, and XPD might increase the amount of AFB1-DNA adducts,

the frequency of TP53M, and the risk of HCC, and the low DNA repair

capacity from genetic mutations of these genes should contribute to the

toxicological effects of AFB1. Given that AFB1 is am important genic agent


and a kind of I type carcinogen, our findings might have prevention

implications through identifying population with low DNA repair capacity,

once these findings are replicated by other studies based on a larger scale or

prospective studies.

CONFLICTS OF INTEREST AND SOURCE OF FUNDING

The authors declare no competing financial interests. This study was

supported in part by the National Natural Science Foundation of China (No.

81160255 and No. 81372639), the Innovation Program of Guangxi Municipal

Education Department (No. 201204LX674), Innovation Program of Shanghai

Municipal Education Commission (No.13YZ035), the Natural Science

Foundation of Guangxi (No. 2013GXNSFAA019251), and the Science

Foundation of Youjiang Medical College for Nationalities (No. 2005 and

2008).

ACKNOWLEDGMENT

We thank Dr. Qiu-Xiang Liang, Dr. Yun Yi, and Dr. Yuan-Feng Zhou for

sample collection and management; Dr. Hua Huang for molecular biochemical

technique. We also thank all members of Department of Medical Test and

Infective Control, Affiliated Hospital of Youjiang Medical College for

Nationalities for their help.

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Chapter 7

INCIDENCE OF ASPERGILLUS SECTION FLAVI

AND INTERRELATED MYCOFLORA IN

PEANUT AGROECOSYSTEMS IN ARGENTINA

María Alejandra Passone, Andrea Nesci,

Analía Montemarani and Miriam Etcheverry Laboratorio de Ecología Microbiana,

Departamento de Microbiología e Inmunología,

Facultad de Ciencias Exactas Físico Químicas y Naturales

Universidad Nacional de Río Cuarto,

Río Cuarto, Córdoba, Argentina

Members of the Research Career, Consejo Nacional de Investigaciones

Científicas y Técnicas (CONICET), Argentina

1. ABSTRACT

Studies in typical and new Argentinean peanut areas showed that

toxigenic Aspergillus section Flavi strains are widely distributed in soils

and seeds, with high probability of being transferred to the storage

ecosystem. Mycological analyses of soil showed that Aspergillus section

Flavi population were present in the two areas at similar counts (3.2x102

cfu g-1

). Within this section, two fungal species were frequently isolated

with isolation percentages of 73 and 90% for A. flavus and of 27 and 9%

for A. parasiticus in soil samples from traditional and new areas,

respectively. The percentages of the different A. flavus phenotypes from

M. A. Passone, A. Nesci, A. Montemarani et al. 158

both peanut-growing areas showed that L strains were recovered in the

highest percentage and represented 59 and 88% of the isolates with

variable ability to produce aflatoxins (AFs). Peanut kernels collected at

harvest time from different localities of Córdoba and Formosa provinces

showed A. flavus and A. parasiticus contamination. The 42.8 and 70%

were classified as type L and the percentages of aflatoxigenic A. flavus

strains were 68.6 and 80.0% in samples from traditional and recent

peanut-growing areas, respectively. Highly toxigenic A. flavus S strains

were isolated with major frequency from soil and kernel samples coming

from traditional peanut-growing area. Aflatoxin contamination was

detected in peanut kernels from typical peanut growing area. Harvested

peanut were stored during 5 months in three storage systems (big bags,

wagons of conditioning and drying and stockpiled warehouse) and

mycological population succession was analyzed. Fungal isolation was

greater from pod (95%) than from kernel tissues. The most common fungi

identified included Penicillium, Aspergillus, Eurotium and Fusarium spp.

Within Aspergillus genus, the section Flavi had the greatest mean counts

of 1.4x104, 9.4x10

2, 5.2x10

2 cfu g

-1 for big bags, wagon and warehouse,

respectively. A. flavus and A. parasiticus strains with variable ability to

produce AFs were isolated from peanut kernels stored in the three

systems at all sampling periods in the order of 1.5x102, 2.3x10

2 and 4.5

cfu g-1

, respectively. .A. flavus S and L strains contributed to silo

community toxigenicity during all storage period. Total AF levels

ranging from 1.1 to 200.4 ng g-1

were registered in peanuts conditioned at

the higher aW values (0.94–0.84 aW) and stored in big bags. Despite the

water stress conditions registered in the stockpiled warehouse throughout

the storage period, AFB1 levels ranging between 2.9 and 69.1 ng g-1

were

registered from the third sampling.

Therefore, the interaction between biological and abiotic factors and

substrate may promote the Aspergillus contamination and the subsequent

AF accumulation in peanut from sowing to storage, highlighting the need

to promote good practices in order to avoid the risk of these metabolites

contamination in peanut food chain.

Incidence of Aspergillus Section Flavi and Interrelated Mycoflora ... 159

2. INTRODUCTION

Peanut is an economically important crop in Argentina, its annual

production in 2013 reached 0.9 million tons. Such importance lies in its

participation in international market. Peanut exportations fluctuate between

0.44 and 0.68 million tons since 2011, ranking the first position since 2012

(SIIA, 2013). The important role of Argentinean peanuts in the world market

has strictly two reasons; the internal consumption reduced (270 g annual per

capita) and the quality that allows it access to markets, such as EU the world‘s

largest consumer market that is closed to other countries (Atayde et al., 2012;

Ding et al., 2012; Mutegi et al., 2013; SIIA, 2013). This nut is attractive

worldwide for their nutritional, sensory and health promoting attributes.

Peanuts are rich in energy and contain health beneficial nutrients, minerals,

antioxidants and vitamins giving it an exceptional nutrient profile that are

essential for optimum health (Jubeen et al., 2012).

Peanut is a dicotyledonous plant and the only species cultivated is Arachis

hypogaea L. Peanuts are annual, herbaceous, pubescent, erect or low-growing

plants. Their peculiarities are the aerial flowers and subterraneous fruits

(Ramantha Rao and Murty, 1994). In commercial plantations, once the plants

are uprooted, the pods are placed to dry in the sun in a windrow. This is still a

slow process; requiring 6-10 days under good weather conditions to reduce the

moisture content of peanut kernel from 40-50% to 20-25% (Schilling and

Misari, 1992). This is one of the most important stages of production since

poor drying can provoke a significant increase in fungal contamination

(Fonseca, 2010). The increase in the level of fungal contamination does not

only occur in the field, but also during the process of kernel formation,

harvesting, drying, transport and storage (Rossetto et al., 2005), as well as

during handling (Santos et al., 2001). The economic impact of fungal invasion

includes: reduction of seed germination rate and, more importantly,

compromise of product quality such as mold growth, discoloration, unpleasant

odor, loss of dry fabric, heating, cooking, chemical and nutritional alterations,

and mycotoxin production, particularly aflatoxins (AFs) that are strictly

regulated; all of which may make peanut products unsuitable for consumption

(Christensen, 1982; Paster and Bullerman, 1988). Aflatoxins in general and

specially aflatoxin B1 (AFB1) are a genotoxic, immunotoxic and

hepatocarcinogenic secondary metabolites (group 1) (IARC, 2002). Therefore,

the final regulations proposed by the European Union for maximum levels of

total AFs and AFB1 in peanuts was 4 and 2 ng g-1

, respectively (Commission

Regulation, 2010). The objective of this chapter is to review the bibliography


concerning to the analysis of the total mycobiota population and aflatoxigenic

contamination in soils and seeds from two peanut growing areas of Argentina

(traditional and new) and in stored kernels in three storage systems (big bags,

wagons and stockpiled warehouse). This information will help to know the

distribution of potentially toxigenic Aspergillus section Flavi strains and the

risk of AF contamination in peanut kernels from sowing to storage.

3. CHARACTERIZATION OF SOIL ASPERGILLUS

SECTION FLAVI POPULATION FROM TWO PEANUT

GROWING AREAS OF ARGENTINA

3.1. Traditional Peanut-growing Area

The population under study was isolated from field soils within the peanut

growing region (General Deheza, Río Cuarto, Charras) of Córdoba Province,

Argentina during the planting and harvest periods. The three regions evaluated

showed no significant differences in the incidence of filamentous fungi and

Aspergillus species from section Flavi (Table 1). The filamentous fungi

present in the soil samples as estimated from dilution plating ranged from

8.2x103 to 2.2x10

4 cfu g

-1 of soil (mean 1.7x10

4 cfu g

-1). Within each region,

filamentous fungi showed similar cfu g-1

at planting and harvest, except in

Charras. The same differences were observed in the isolation frequency of

Aspergillus section Flavi strains with counts ranged from 2.9x101 to 6.7x10

2

cfu g-1

(mean 3.2x102 cfu g

-1) (Barros et al., 2003). Such differences between

planting and harvest could be explained by the environmental conditions

(temperature and rainfall) and soil temperature as demonstrated in previous

studies (Hill et al., 1983; Horn et al., 1994). Out of 506 Aspergillus section

Flavi isolates, 369 were A. flavus (73%) and 137 were A. parasiticus (27%).

The differences between the percentages of the different A. flavus phenotypes

isolated are shown in Table 2. The L phenotype (diameter of sclerotia > 400

µm) was recovered in the highest percentage and represents 59% of the

isolates. In contrast, the recoveries of S (diameter of sclerotia < 400 µm) and

non-sclerotial strains were 22 and 19%, respectively. Statistical analyses

showed significant differences in AF and cyclopiazonic acid (CPA) production

among L, S and NS producer strains (p<0.05). The S strains produced the

highest AF levels (3315.8 ng ml-1

).

Table 1. Comparison of soil and peanut Aspergillus section Flavi populations

of two peanut-growing regions in Argentina

Samples Periods

Traditional region New region

Filamentous

fungi (cfu g-1)

section

Flavi

(cfu g-1)

A.

flavus

(%)

A.

parasiticus

(%)

AFs

(µg g-1)

Filamentous

fungi (cfu g-1)

section

Flavi

(cfu g-1)

A.

flavus

(%)

A.

parasiticus

(%)

AFs

(µg g-1)

Soil Planting 1.6x104 1.8x102a/

2.6x102b

76/90 19/9

Harvest 1.9x104 1.4x103/

2.3x102

87/88 13/12 3.6x104c 3.2x102 90 6

Peanut N.D. N.D. 60d 20 124.8+20.1a N.D. N.D. N.D. N.D. N.D.

N.D. not determined. aBarros et al., 2003;

bAlaniz Zanon et al., 2013;

cOrtiz et al., 2013;

dVaamonde et al., 1995.


Table 2. Production of aflatoxin B and G groups and CPA by L, S and NS

strains of A. flavus isolated from two peanut-growing regions of Argentina

Samples

A. flavus strains (%) Mycotoxin production

Traditional region New region

L S NS L S NS AFB AFG CPA

Soil 76.6 78.2 77.9 71* + n.d. +

11.1 8.0 9.9 10 n.d. n.d. +

- 10.0 - - + + +

11.3 3.8 9.1 - + n.d. n.d.

1.0 - 3.1 29 n.d. n.d. n.d.

Peanut 51.4* 89.3 33.3 44.0 + n.d. +

31.4 10.7 - 56.0 n.d. n.d. +

17.2 - 66.7 - + + +

n.d.: not detected; *Determined as the percentage of the total A. flavus strains.

Soil samples; Traditional region: L strain (n=218), S strain (n=81); NS strain (n=70)

(Barros et al., 2005); New region: L strain (n=340), S strain (n=10); NS strain

(n=37) (Ortiz et al., 2013).

Peanut samples; Traditional region: L strain (n=15), S strain (n=9); NS strain (n=11)

(Vaamonde et al., 1995); New region: L strain (n=28), S strain (n=3); NS strain

(n=9) (Pildain et al., 2004).

Similarly to the results for AFs, the S strains produced higher CPA levels

(65.3 µg ml-1

) than L and NS strains (mean levels = 54.7 and 39.6 µg ml-1

).

Only 4 out of 369 A. flavus isolates did not produce AFs or CPA, whereas

80% of the isolates produced both mycotoxins. Among the S strains, 10% of

the isolates showed an unusual pattern of mycotoxin production (AF group B

and G simultaneously with CPA) (Barros et al., 2005).

3.2. New Peanut-growing Area

To analyze the biodiversity of Aspergillus section Flavi soil population

from new peanut growing areas, soil samples were collected from Formosa, La

Pampa and south of Córdoba provinces. The mean values of total mycobiota

and Aspergillus section Flavi were 3.6x104 and 319 cfu g

-1, respectively

(Table 1). Out of 430 strains isolated within the Aspergillus section Flavi 90%

were identified as A. flavus, 6% as A. parasiticus and 4% as A. caelatus.

Among the A. flavus isolated 88% were L strains, 3% were S strains and 10%

were not able to produce sclerotia (Table 2). Seventy one percent of A. flavus

were AF producers and 81% were CPA producers. A relatively large

Incidence of Aspergillus Section Flavi and Interrelated Mycoflora … 163

proportion of A. flavus strains (n=111; 29%) isolates were not able to produce

AF. Molecular analysis of omt-A, ver-1, nor-1 and afl-R genes of 34 strains of

non aflatoxigenic A. flavus showed that 19 strains had absence of 1, 2 or 3 of

the genes analyzed. Only 1 strain showed absence of all 4 genes studied. There

were no significant differences in the mean level of AFB1 production among

strains isolated from the different new areas of peanut cultivation (Ortiz et al.,

2011; 2013).

Therefore, the data of the works conducted by Barros et al. (2003) and

Ortiz et al. (2011, 2013) showed that the total mycoflora count was higher and

the incidence of Aspergillus section Flavi was lower in samples collected from

the new peanut cultivation areas in comparison with the levels found in soil

samples collected in the main peanut growing area. Data on the incidence of

the soil native population from Las Acequias, located within the peanut

growing region of Córdoba, Argentina were recently reported by Alaniz Zanon

et al. (2013) and are consistent with those found by Barros et al. (2003) (Table

1). The inoculum level and the incidence of toxigenic isolates of native A.

flavus and A. parasiticus in soil at planting and pod maturation times were

similar among plots and A. flavus was the dominant specie from section Flavi,

showing an isolation percentage nearly to 90%. The remaining species

identified were A. parasiticus (9%) and the non aflatoxigenic specie A.

caelatus (1%). In relation to the incidence of toxigenic native isolates at

planting and harvest times, a total of 75 and 80% of A. flavus isolates were

toxigenic, whereas 98 and 100% of A. parasiticus isolates were toxigenic.

Similarly, Atayde et al. (2012) reported that A. flavus was the Aspergillus

species most frequently isolated (13.4%) from peanut soil after plant

emergence and 2 weeks prior to uprooting in four regions of São Paulo, Brazil,

with the higher frequency in samples from Jaboticabal (70.3%). The mean

frequency of A. flavus in soil varied according to sampling period and was

72% lower in the second sampling. In contrast, A. parasiticus was only

detected in 4 of the 40 samples analyzed. Although classified as a ―storage

fungus‖, the presence of A. flavus in soil samples indicates this substrate as the

primary reservoir of this fungus, a fact previously reported by Horn (2005) and

Gonçalez et al. (2008).

The importance of determine type S, L or NS strains can be explained by

these resistance structures exhibit sporogenic germination. Thereby an

eventual preharvest control of A. flavus infection in that crops where AF is a

problem, agronomic practices designed to reduce the importance of sclerotia

as primary inoculum source are required (Wicklow, 1983). The results of these

studies provide new data on the communities of A. flavus in peanut soils in


Argentina, by associating of morphological and toxigenic characteristics of

strains isolated with the possible agroecological differences of the diverse

geographical areas from which strains were isolated. Therefore, the levels of

toxin produced by A. flavus strains isolated from new peanut growing area

were lower than those produced by strains isolated from areas with long term

history of peanut cultivation. Principal component analysis showed that fields

with recent history of peanut cultivation are closely related with the isolates

belonging to A. flavus L phenotype, producers of low AF levels, while typical

groundnut regions are closely related with high percentages of A. parasiticus

and A. flavus S phenotype, producers of higher AF levels. Such differences

may be due to: 1) the areas analyzed present different macro and microclimatic

conditions, 2) in the Córdoba province, the peanut is grown for several years

and over a larger area than in the other regions where this nut was developed a

few years ago and seeding in lesser extent, 3) agroecological regions analyzed

are at different latitudes and give another possible cause of variability (Cotty,

1997), 4) history of soil, type of crop, insect levels, rainfall, temperature and

cultural practices are also different (De Fina, 1992; Orum et al., 1997).

Therefore, the high presence of A. flavus in soil samples should have

contaminated the peanut pods. As the soil is a primary reservoir for A. flavus

and A. parasiticus and peanuts are an underground fruit, their pods should be

directly exposed to the two soil species (Horn et al., 1994).

4. INCIDENCE OF AFLATOXINS AND CHARACTERIZATION

OF PEANUT ASPERGILLUS SECTION FLAVI POPULATION

FROM TWO GROWING AREAS OF ARGENTINA

4.1. Traditional Peanut-growing Area

Natural Aspergillus section Flavi infection of peanut from 21 localities

(Baigorria, Holmberg, Alcira Gigena, Las Perdices, Corralito, Río Cuarto,

Hernando, Matorrales, Oliva, Charras, Villa del Rosario, General Cabrera,

Carnerillo, Alejandro, Manfredi, Reducción, Pampayasta, Villa Ascasubi, La

Carlota, Ticino and Colazo) situated in the traditional production areas of

Córdoba, Argentina was evaluated. From 21 locations and of the 32 samples

analyzed, a total of 44 strains were isolates (Table 1). A. flavus was isolated

from 60% of the samples, while A. parasiticus was present in 20% of them.

Strains of A. flavus and / or A. parasiticus were found in peanut kernels from


all locations, except two, Corralito and Hernando. The percentage of

aflatoxigenic A. flavus strains was very high (68.6%) as well as the percentage

of strains producing CPA, representing all the strains (100%) (Table 2). The

68.6% of the strains tested (n = 24) produced sclerotia under the following

culture conditions: Czapek agar, 30 ° C, 30 days in dark. The 25.7% were

classified as type S, of which most of them produced simultaneously AFB and

AFG (Vaamonde et al., 1995).

The AF analyses of peanut kernels from three regions showed a higher

frequency of contaminated samples from the Charras region (mean level of

total AFs = 158 µg g-1

/ 8 positive samples) in comparison with the General

Deheza and Río Cuarto regions that showed mean levels of total AF

contamination of 7.4 and 209 µg g-1

with 3 and 2 positive samples,

respectively (Table 1) (Barros et al., 2003).

4.2. New Peanut-growing Area

Table 2 shows characteristics of A. flavus strains (n = 40) isolated from

peanut kernels from Formosa province. Seventy eight percent of the isolates

were sclerotia producers under culture conditions and on peanut kernels. Three

of these sclerotia producers were classified as S strains. Eighty-nine percent of

the L strain isolates produced AFB and CPA, and no isolates that produced

only AF were observed (Pildain et al., 2004).

Recently, Alaniz Zanon et al. (2013) reported the density of A. flavus, the

only specie from section Flavi isolated from harvested peanut kernels in the

typical groundnut region. Peanut was subjected to two environmental

conditions of growth; with and without drought stress; with infection

percentage in the order of 6.5 and 57%, respectively. Incidence of Aspergillus

section Flavi in peanut kernels at harvest in different growing-areas of other

countries was also reported. Studies investigating the mycobiota and

mycotoxins in Brazilian peanut kernels from sowing to harvest reported that A.

flavus was detected in the pod filling (43.9%), full pod maturity (33.6%) and

dried pod (7.6%) stages. Moreover, A. parasiticus was found more frequently

than A. flavus in dry pod stage, 10.3 and 7.6% respectively, in spite of low aW

(0.71) (Gonçalez et al., 2008). Similarly, Zorzete et al. (2011) reported that the

presence of A. flavus seems to vary according to the growth phase of the

peanut plant. For pods, all dried grain samples were positive for this fungus,

with an average contamination of 32.5%, whereas no contamination was

observed during the other phases. In kernels, increased presence of A. flavus


was observed during the stages of ripe grains (13%) and dried kernels (4%). A.

parasiticus was isolated from pod samples in low percentage (1%).

This indicate that A. flavus is the more aggressive specie and the

responsible for most of the AF contamination of peanuts.

Natural occurrence of AF and CPA contamination in peanuts coming from

the nucleus of the Argentinean peanut-growing area was investigated. Co-

occurrence of CPA and AF was detected in two of 50 samples analyzed. The

levels of these toxins found in positive samples were 4300 and 493 μg kg-1

for

CPA, 625 and 435 μg kg-1

for AFB1, and 625 and 83 μg kg-1

for AFG1,

respectively (Fernandez Pinto and Patriarca, 2001). In the Charras region the

high AF-producing potential of Aspergillus species from section Flavi at

harvest time, together with predisposing conditions for peanut infection, could

explain the higher number of samples contaminated with AFs (Barros et al.,

2003). On the one hand, Alaniz Zanon et al. (2013) demonstrated that

temperatures close or above to 30 °C during several days summed to drought

stress were conducive conditions for AF accumulation in peanut. A household

survey was carried out in Busia and Homabay districts in western Kenya,

which were chosen, based on their significance in terms of peanut production

and because they offered a contrasting environment under which peanuts are

cultivated. The levels of AFs ranged from 0 to 2687.6 μg kg-1

and from 0 to

7525.0 μg kg-1

in samples from Busia and Homabay districts, respectively.

There was a highly significant association between agroecological zones and

AF levels. While 10.70% of samples from Busia district had AF levels >20 μg

kg-1

, only 4.09% of samples from Homabay were in this category (Mutegi et

al., 2013). On the other hand, Gonçalez et al. (2008) analyzed AF content of

peanut kernels during the different crop growth stages and showed that AF

concentrations increased when the kernels aW decreased (0.71), which

occurred at dried pod stage. Dorner et al. (1989) reported that immature peanut

pods are more resistant to fungi and AF contamination because they produce

more total phytoalexins than mature peanuts, at high aW. Attack by moles also

was found to be significantly associated with AF levels. Damage by moles

predisposes pods to colonization by AF-producing fungi, and similar damage

by terrestrial arthropods has been reported (Dicko et al., 1999). Pod damage

also exposes the kernels to colonization by AF-producing and other

saprophytic fungi (Chapin et al., 2004).

Therefore, peanut kernels harvested from different peanut-growing areas

of Argentina contain mycelia and spores of aflatoxigenic fungi, which can

result in a significant decrease in grain quality when they are stored.


Table 3. Effect of storage time on total mycoflora (cfu g-1

) from peanut

pods and kernels stored in different storage systems

Time

(months)b

Log cfu g-1

+ S.D.a

Big bagc

(250 kg)

Wagond

(4,000 kg)

Warehousee

(26,000 ton)

Pods Kernels Kernels Kernels

First 3.5 + 2.8 b < b 5.2 + 4.9 b 3.6 + 0.4 b

Second 4.9 + 4.6 a 3.6 + 2.9 a 6.2 + 5.9 a 3.7 + 0.5 a

Third 4.4 + 3.4 a 3.4 + 2.5 a 5.9 + 5.4 a 4.6 + 1.1 a

Fourth 4.4 + 3.7 a 3.5 + 3.0 a 5.4 + 4.9 a 4.7 + 0.9 a

Fifth 4.8 + 3.9 a 3.3 + 2.8 a 5.7 + 5.3 a 4.5 + 1.2 a a Mean + S.D. based on 120 independent pod and seed samples.

b Storage period: July-November for big bag (0.76+0.02 aW); July-November for

wagons; May-October for warehouse. c Passone et al. (2009a).

d Doprado (2008).

e Nesci et al. (2011).

Data not sharing a common letter in the same group are significantly different

according to Tukey Test (P < 0.05).

5. INCIDENCE OF FUNGAL POPULATION, AFLATOXINS

AND CHARACTERIZATION OF ASPERGILLUS SECTION

FLAVI IN PEANUT FROM DIFFERENT STORAGE SYSTEMS

5.1. Incidence of Total Mycoflora in Stored Peanut

5.1.1. Analyses of Peanut Stored in Big Bags Conditioned at Different aW

Levels

Mycoflora analyses were conducted on peanuts artificially dried up to

0.94 + 0.01, 0.88 + 0.01, 0.84 + 0.01 and 0.76 + 0.02 aW levels by using a

continuous dryer that insufflated air at 35 °C and stored in big bags with a

capacity for 250 kg in-pod peanuts. Ten samples were monthly collected from

each big bag during a 5-month period.

Fungal populations from 200 peanut kernel samples harvested for human

consumption and stored in four big bags (250 kg) in Storage Company in the

south of Córdoba province were analyzed. Total fungal counts were higher in

peanuts conditioned at high aW levels (big bags 1 and 2). Total mean counts of


6.9x107 and 3.6x10

7 cfu g

-1 were recorded in big bags with 0.94 and 0.88

initial aW, respectively. Meanwhile, mean fungal counts in big bags 3 and 4

were estimated at 6.7x106 and 2.1x10

4 cfu g

-1, respectively. Analyses of fungal

populations from 50 peanut kernel and pod samples did not demonstrated

significant differences between the incidences in each sampling period.

Isolation from pod tissue yielded more fungi than from kernel, 85% of the

fungi isolated were from pod tissue. The mean fungal counts during the six

sampling periods for pods and kernels were 4.0x104 and 2.3x10

3 cfu g

-1,

respectively. Most of the fungi were isolated in the last sampling period. The

total counts in the first sampling were 3.1x103 and < 10

3 cfu g

-1, while after a

5-month storage period, the count increased to 6.1x104 and 2.0x10

3 cfu g

-1 in

pods and kernels, respectively (Table 3) (Passone et al., 2009a).

During monitoring, Aspergillus, Penicillium and Eurotium were the

genera commonly isolated from peanut kernels during all sampling periods;

Fusarium spp. were only detected in the first storage period (1-3 months). The

two higher initial water stress conditions assayed (0.84 and 0.76 aW) mainly

affected the development of genera such as Fusarium and filamentous fungi

group. On the contrary, Eurotium spp. counts increased within the second

storage period and the highest inoculum of this genus (mean = 7.8x105 cfu g

-1)

was found in peanut kernels conditioned at the middle aW levels (0.88 and 0.84

aW) (Table 4) (Passone et al., 2009b).

A deeper identification, at section levels were done for samples from big

bag 4 and the most frequently occurring fungi are presented in Table 5.

Ninety-eight percent of the fungal isolates were Deuteromycetes and

Ascomycetes, and the remaining were Zygomycetes. A mycological survey of

90 peanut pod and kernel samples showed the presence of three principal

genera of filamentous fungi (Penicillium, Aspergillus and Fusarium spp.). The

fungal genera that showed a relatively low frequency of isolation and that were

not important mycotoxin producers (Eurotium, Monascus, Alternaria,

Cladosporium, Byssochlamys, Rhizopus, Mucor, Absidia spp. and sterile

mycelium) were all included in the filamentous fungi group.

Penicillium spp. had the greatest mean frequency levels in pod and kernel

tissues during the research period with a count of around 1.8x104 and 1.5x10

3

cfu g-1

, respectively. Penicillium species sorted in three sections –

Divaricatum, Furcatum and Simplicia – were isolated from both tissue type

during the storage period. The highest frequency of isolation corresponded to

the Simplicia section (44.7%), followed by the Furcatum (32.7%) and

Divaricatum sections. Aspergillus spp., a common peanut contaminant, was

isolated from both pod and kernel tissues at the six sampling periods.

Table 4. Incidence of total mycoflora in kernels from in-pod peanuts

conditioned at different aW levels and stored in big bags

aW cfu g

-1a

1-3 months 4-5 months

Aspergillusb Penicillium Fusarium Eurotium Filamentous

fungic

Aspergillus Penicillium Eurotium Filamentous

fungi

Big bag 1

(0.94+0.01)

1.3x106 4.8x10

7 2.3x10

4 3.3x10

2 2.4x10

5 1.5x10

5 1.9x10

7 5.0x10

3 1.1x10

4

Big bag 2

(0.88+0.01)

4.6x106 1.9x10

7 1.0x10

3 < 2.1x10

5 9.7x10

5 1.0x10

7 1.1x10

6 3.3x10

2

Big bag 3

(0.84+0.01)

6.7x103 2.9x10

6 7.7x10

3 9.8x10

4 1.1x10

6 5.0x10

4 2.1x10

6 4.3x10

5 8.6x10

3

Big bag 4

(0.76+0.02)

7.6x101 3.7x10

3 1.6x10

2 1.3x10

1 1.9x10

3 2.5x10

1 1.4x10

4 7.0x10

2 6.0x10

1

a Mean values based on ten replicate data in DRBC and in DG18 medium.

b Includes the sections: Nigri, Circumdati and Fumigati.

c Includes the genera: Absidia, Alternaria, Cladosporium, Monascus, Paecilomyces, Rhizopus, Trichoderma, dematiaceous and non-

sporulating fungi.

Passone et al. (2009b).


Table 5. Incidence of mycobiota in peanut pod and kernel tissues stored in

different storage systems

Mycobiotab Log cfu g-1 + S.D.a

Big bagc

(250 kg)

Wagond

(4,000 kg)

Warehousee

(26,000 ton)

Pods Kernels Kernels Kernels

Sterile mycelium 4.76 + 5.06 3.91 + 4.09 3.03 + 2.99 1.40 + 1.64

Zygomycetes

Absidia 3.54 + 3.81 2.26 + 2.48 < <

Mucor 4.08 + 4.23 2.30 + 2.32 < <

Rhizopus 2.22 + 2.61 < < <

Ascomycetes

Byssochlamys < < 3.86 + 3.86 <

Eurotium 2.92 + 3.31 < 3.37 + 3.37 0.10 + 0.24

Monascus 4.69 + 4.59 3.09 + 3.25 2.48 + 2.48 <

Talaromyces < < 1.83 + 1.69 <

Deuteromycetes

Alternaria 3.87 + 4.25 2.22 + 2.14 < 0.34 + 0.78

Aspergillus

Section Flavi 3.56 + 0.30 2.29 + 2.19 3.46 + 3.16 1.72 + 1.64

Fumigati < < 1.23 + 1.23 0.18 + 0.72

Nigri 3.54 + 1.48 2.32 + 2.55 2.86 + 2.55 0.28 + 0.80

Terrei < < 1.23 + 1.23 <

Cladosporium < < < 1.08 + 1.10

Fusarium 3.33 + 3.71 2.30 + 2.80 3.23 + 2.88 0.94 + 1.24

Paecilomyces < < 2.81 + 2.81 <

Penicillium 2.72 + 1.82

Section

Aspergilloides

< < 2.18 + 2.18 <

Exilicaulis < < 1.92 + 1.81 <

Divaricatum 2.16 + 2.59 1.57 + 1.79 3.75 + 3.48 <

Furcatum 3.36 + 3.62 2.02 + 2.16 3.55 + 3.42 <

Simplicia 4.20 + 4.30 3.15 + 2.99 4.98 + 4.52 <

Trichoderma 2.22 + 2.61 1.23 + 1.61 < <

Total filamentous

fungi

6.69 5.79 5.08 4.22

a Mean + S.D. based on 120 independent pod and seed samples.

b Distribution of genera and section into group according to Pitt (2000), Klich (2002),

Samson et al. (2002). c Passone et al. (2008), Big bag (0.76+0.02 aW).

d Doprado (2008).

e Nesci et al. (2011).


The cfu counts were 7.1x103 and 4.0x10

2 cfu g

-1 in pod and kernel

samples, respectively. Two sections of Aspergillus genus were identified from

pod and kernel tissues. The Aspergillus section Flavi had the greatest mean

frequency (49.9%). Isolation frequency of Fusarium spp. was sporadic

throughout the study. The mean counts for this genus were 2.1x103 and

2.0x102 cfu g

-1 in pod and kernel tissues, respectively. Monascus spp. was

consistently isolated from pods and kernels during the last 3 months of the

assay. It is notable that Monascus spp. was isolated at numerically greater

levels from pods (97.6%) than from kernels during the study. Sterile

mycelium, Absidia, Mucor, Rhizopus, Alternaria, Eurotium, Paecilomyces and

Trichoderma spp. were all isolated in low frequency (mean: 4.5x104 cfu g

-1)

during the 5-month of storage. The Zygomycetes such as Absidia, Mucor and

Rhizopus spp. were consistently isolated from pod and kernel tissues at the

first two sampling periods, whereas the incidence of Monascus spp. and

Eurotium spp. increased at the end of the storage time. Alternaria and

Trichoderma spp. were isolated at low levels during the assay (Passone et al.,

2008).

The determination of physical properties of the samples revealed

considerable differences in aW and temperature between the first and the fifth

sampling. Water availability levels in peanut conditioned at four aW decreased

at mean level of 0.63 + 0.04 aW. Temperature values of peanut from the four

big bags increased from 12.6 + 0.6 °C to 29.3 + 0.9 °C between the first and

fifth sampling period and pH values were maintained relatively stable (mean:

6.7) (Passone et al., 2009b).

5.1.2. Analyses of Peanut Stored in Wagons of Conditioning and Drying

Mycoflora analyses were conducted on harvested peanut kernels with 0.87

+ 0.04 aW levels and stored in wagons of conditioning and drying with a

capacity for 4000 kg in-pod peanuts. Ten in-pod peanut samples (500 g) were

taken random at time zero, after filling the silo, and every 30 days over a

period of 5 months (July-November). Total fungal of all samples obtained

from DRBC agar, and counts of xerophilic fungi obtained from DG18 agar

were over 1x105 cfu g

-1, showing a high degree of contamination (Table 3).

Eight genera of filamentous fungi were isolated from peanut kernels, with

prevalence of Penicillium, Aspergillus, Fusarium and Eurotium which showed

a relative density (RD) of isolation of 86.0, 3.4, 2.0 and 1.4%, respectively.

While Monascus, Byssochlamys, Talaromyces and Paecilomyces spp., that

make up the filamentous fungi group were isolated in lower RD (0.25, 6.0,

0.05, 0.54%) over the 5 months of storage (Table 5).


Of all isolates found in the Penicillium genus, species of the following

sections were recovered: Simplicia, Aspergilloides, Exilicaulis, Furcatum,

Divaricatum and Penicillium, with a prevalence of 88.0, 0.2, 0.1, 7.7 and

0.3%, respectively. In the Aspergillus genus, species from sections Flavi,

Fumigati, Nigri, Restricti, Circumdati, Nidulantes and Terrei were isolated

with a prevalence of 54.2, 29.7, 13.8, 1.7%, respectively and 0.2% for the

three last sections.

Considering the environmental variability, storage temperatures increased

gradually during the 5 months of storage of 15 to 28 °C. In relation to records

of aW, the level medium was 0.72 aW, a reduction was observed during the first

4 months of storage from 0.87 to 0.63 (Doprado, 2008).

5.1.3. Analyses on Peanut Stored in Stockpiled Warehouse

Analyses of fungal populations in 95 peanut kernel samples did not

demonstrated a significant sampling period effect (p=0.578) on the incidence

of the total fungi isolated in peanut kernel during May, June, August,

September and October. The values were in a range of 3.6-4.7 (log cfu) (Table

3). Penicillium, Fusarium, and Aspergillus section Flavi were the principal

filamentous fungi isolated between the first and fifth sampling periods. Other

fungal communities present as minor components of the mycoflora included

Cladosporium, Aspergillus section Nigri, Alternaria, Eurotium, Aspergillus

spp. and sterile mycelia (Table 5). The great fungal diversity was observed

during the second and fourth sampling periods, Aspergillus section Flavi,

Penicillium, Aspergillus section Nigri and sterile mycelia were isolated during

all incubation period. The lowest incidence of Aspergillus section Flavi was

detected in the first sampling period, with an increase in the fourth sampling

period.

The temperature varied between 9 and 19 °C during the sampling period.

The lowest temperature was detected in June and the highest in October.

Water activity of samples ranged from 0.43 to 0.57 during 180 days of storage.

Water activity values were higher in the first and fourth months than in the

fifth month of the experiment (Nesci et al., 2011).

Also fungi were detected in all peanut samples analyzed, a low incidence

of fungal colonization was observed during five sampling period in peanut

kernels stored in big bag (0.76 + 0.02) and warehouse. Fungal counts in big

bag were higher from pods (95%) than from kernels. The low incidence of

fungal colonization in kernels supports earlier research showing the

importance of injury for invasion by microorganisms and the role of the seed

coat as a barrier for invasion (Carter, 1973). However, the level of fungal


populations from peanut kernels stored in the wagon exceeded 1x105 cfu g

-1

and was maintained throughout the storage period despite the environmental

factor variations. These results suggested a high fungal activity and count

levels exceeded the recommended limits to ensure peanut hygienic quality

(Atlanta Poland, 2013). These differences were not due to the storage system

employed if not, the conditioning of the grain before storing, because peanut

entered more wetter (initial aW = 0.87) in the wagon. Therefore total fungal

count in the wagon was similar to peanut stored in big bag 3. In a study

conducted in Indonesia by Bulaong and Dharmaputra (2002) shelled peanuts

of Gajah var. with initial moisture content of 7% were stored at 11 kg/bag in

four bag types namely: jute bag, polypropylene bag, jute bag doubled with thin

polyethylene (PE), and jute bag doubled with thick PE. Storage was done for 6

months under warehouse conditions with monitoring of relative humidity and

temperature. Statistical analyses showed that moisture content and fungal

population were significantly higher in jute (JB) (8.2%; 4.3x104 cfu g

-1) and

polypropylene (PB) bags (8.3%; 3.3x104 cfu g

-1) than in PE-doubled jute bags

(7.6%; 1.1x103 and 7.5%; 2.4x10

3 cfu g

-1).

Our studies revealed some distinct trends in the relative density of fungal

populations in peanut kernels. The mycological population succession

observed in three storage systems showed that Penicillium and Aspergillus

were the most prevalent genera throughout the storage time and that Eurotium

spp. counts increased after the third month. Similarly, Bhattacharya and Raha

(2002) observed that during harvest field fungi such as Fusarium, Alternaria,

Curvularia and Rhizopus spp. mostly induced the infection of peanut seeds,

but their numbers decreased gradually during storage probably because they

were replaced by storage fungi, mainly by different species of Aspergillus as

found by other researchers (Adebesin et al., 2001, Magnoli et al., 2006). In our

studies, disappearance of field fungi after the first month was evident due to

reduction of aW in kernels, as most of the field fungi were unable to continue

growing at seed moisture of less than 90% RH as pointed out by Lacey (1989).

Bulaong and Dharmaputra (2002) also reported differences in the peanut

fungal population stored in different bag types throughout the storage period.

For all bag types, the significant increase in fungal count was attributed to 2

fungal species, i.e. Penicillium funiculosum and Aspergillus penicillioides. In

JB, the dominant fungus was P. funiculosum from month 0 to month 4. At

month 5 and 6, the dominant species was A. penicillioides. For PB, the

dominant species from month 0 to month 4 was P. funiculosum, while A.

penicillioides became dominant in PB from month 4 to month 6. Eurotium

amstelodami was the second dominant species from month 5 to 6 in JB and


PB. In JB+1 P. funiculosum was dominant from month 0 until month 6.

Eurotium chevalieri was the second dominant species at 1.70 and 17.6% of

population for months 5 and 6, respectively. In JB+2, P. funiculosum was

dominant from month 0 to month 6. Eurotium chevalieri contributed 2.08 and

0.75% to the total population at months 5 and 6 respectively. Therefore, in the

complex system where several fungal species exist together, the dominance of

a species may not be solely due to its ability to tolerate existing moisture

conditions. Several types of interactions exist among fungi. In addition to

competition for space and nutrients, there could be inhibitory interactions due

to release of metabolites of derailing biochemical path ways (Choudhary,

1992).

The most important environmental determinants on fungal growth are aW

and temperature (Pitt, 1975; Troller and Christian, 1978). In these studies,

temperature values were not a limiting factor for the fungal development since

from around the seventh week in all storage systems, the level was the

minimum enough (15 °C) to allow the growth of mesophilic fungi. However,

aW values were low and it tended to reduce even more (mean level = 0.60) at

the end of the storage. This fact limited fungal development such as Fusarium

spp. and filamentous fungi group (Beuchat, 1983), favoring the growth of only

the xerophilic fungi that dominated in peanut samples from the three storage

systems.

5.2. Incidence of Aspergillus Section Flavi and Aflatoxins in

Stored Peanut

5.2.1. Analyses of Peanut Stored in Big Bags Conditioned at Different aw

Levels

Peanut kernels showed natural infections by members of Aspergillus from

section Flavi during the 5-month storage period, at the different aW initial

values (big bag 1=0.92±0.01, big bag 2=0.88±0.01, big bag 3=0.84±0.01 and

big bag 4=0.76±0.02) (Figure 1). Peanut samples taken at different storage

times to determine the density of Aspergillus section Flavi spp. by

conventional and molecular methods, showed correlation between the data

obtained (r=0.613; p<0.0001). However, the cfu values obtained by RT-PCR

were usually higher (0.5–1 log units) than those obtained by conventional

counts (CC). Both count methods showed that the highest cfu values were in

peanut kernels conditioned at the two highest aW and that this counts were

relatively stable during the first three months of storage (Figure 1). The mean


counts obtained between the first and fourth samplings were 4.9×106 cfu g

-1

(CC) and 2.6×107 cfu g

-1 (RT-PCR), and 1.0×10

7 cfu g

-1 (CC) and 2.6×10

7 cfu

g-1

(RT-PCR), for big bags 1 and 2 respectively. A reduction of Aspergillus

section Flavi spp. counts [2.3–4.6 log units (CC) and 1.7–2 log units (RT-

PCR)] was observed at the end of the storage period. In peanut samples from

big bag 3, Aspergillus section Flavi spp. counts were relatively constant

(7.9×105–7.1×10

6 cfu g

-1) during the whole storage period. Similar to the

results observed in big bags 1 and 2, Aspergillus section Flavi spp. counts

were reduced at about 51% at the end of the storage period. In samples taken

at the third and fourth months of storage from big bag 4, RT-PCR was able to

detect nor-1 copies estimated at 92 and 17 cfu g-1

, respectively, while the

conventional method gave <100 cfu g-1

.

Figure 1. Colony forming units per gram of peanut (log cfu g-1

) of Aspergillus section

Flavi spp. present in peanut seed samples from big bags 1, 2, 3 and 4, wagon and

warehouse during a 5-month storage period (Doprado, 2008; Nesci et al., 2011;

Passone et al., 2010).

Figure 2 shows that two species of Aspergillus section Flavi, A. flavus and

A. parasiticus, were identified from peanut pod samples, with a predominance

of A. flavus (58.6%). A. flavus was predominant in all peanut samples showing

Log c

fu g

-1

0

2

4

6

8

Big bag 1

Big bag 2

Big bag 3

Big bag 4

Wagon

Warehouse

First Second Thirth Fourth Fifth

Sampling Periods


isolation percentages of 88.9, 96.4, 100 and 89.2% for big bags 1, 2, 3 and 4,

respectively. A. parasiticus was isolated in low percentage in big bags 1, 2 and

4 ranging from 3.7 to 13.0%. A. parvisclerotigenus were sporadically isolated

from big bags 1 and 2 in a mean level of 2.2%. Only two A. caelatus and A.

bombycis isolates from big bag 1 and at the third and fourth sampling were

identified.

Figure 2. Propagules of Aspergillus section Flavi species per gram of peanut stored in

different storage systems. (*Mean of four big bags) (Doprado, 2008; Nesci et al., 2011;

Passone et al., 2010).

Two hundred and twenty seven (70%) out of 323 strains of Aspergillus

section Flavi, were AF producer in culture (Table 6). Toxin levels ranged from

1.3 to 76530.5 ng ml-1

of culture medium (mean level: 1003.9 ng ml-1

). A.

parasiticus was the specie with the highest percentage of AF-producing

isolates (100%), their AF production levels ranging from 10.8 to 76530.5 ng

ml-1

(mean level: 4537.3 ng ml-1

). Sixty eight percent of A. flavus were AF

producer with mean levels ranging from 19.9 to 3285.6 ng mL-1

. Out of 296 A.

flavus isolates, 4, 45 and 51% were classified as S, L and non sclerotial (NS)

strains, respectively. One hundred percent of S strains were AF producers,

whereas L strains produced the highest AF levels. Among S strains, 69% of

Big bags Wagons Warehouse

Pods Seeds* Seeds Seeds

Log c

fu g

-1

0

1

2

3

4

A. flavus

A. parasiticus

A. caelatus

A. bombycis

A. minisclerotium

A. parvisclerotigenus

A. spp.


the isolates showed an unusual pattern of mycotoxin production (AF group B

and G simultaneously).

Table 6. Aflatoxins production by species in the Aspergillus section Flavi

isolated from stored peanut seeds in different storage systems

Species Positive

strains a

Range (ng ml-1) b Mean levels

(ng ml-1) + SE c B aflatoxins G aflatoxins

Big bagsd

A. flavus S strains 4/13 6.4 – 45.6 – 23.4 + 9.8

9/13 117.3 – 232.8 15.5 – 150.7 212.2 + 39.1

A. flavus L strains 28/132 – – <1

66/132 1.5 – 79.9 – 20.2 + 2.8

38/132 108.0 – 30612.5 – 3285.6 + 1193.3

A. flavus NS strains 66/151 – – <1

58/151 1.3 – 95.7 – 19.9 + 3.1

27/151 100.6 – 1218.8 – 388.1 + 70.6

A. parasiticus 3/20 9.4 – 78.6 0.7 – 26.3 53.3 + 27.7

17/20 119.3 – 75468.0 16.2 – 5670.0 9021.2 + 5336.5

A. parvisclerotigenus 4/4 1.5 – 58.7 – 25.8 + 12.4

A. caelatus 2/2 – – <1

A. bombycis 1/1 – – 1.3 + 0.0

Wagone

A. flavus S strains 3/4 <

1/4 2 – 40500

A. flavus L strains 51/93 <

41/93 2 – 40500

1/93 40600 – 82000

A. flavus NS strains 25/32 <

7/32 2 – 40500

Warehousef

A. flavus 52/79

A. parasiticus 2/2 <2 – 2500.0 – 2700 + 5700

SE: standard error. Detection limit: 1 ng ml-1

medium. a Ratio of AF-producing strains vs. total strains tested.

b Range levels of AFB and AFG between 1-100 ng ml

-1 and 101- maximum production

ng ml-1

. c Mean levels of total AFs.

d Passone et al. (2010).

e Doprado (2008).

f Nesci et al. (2011).


Table 7. Aflatoxin levels in peanut kernel samples analyzed over 5 months

of storage

Samplingb

Aflatoxins concentration ng g-1 (mean + SE)a

Big bag 1c Big bag 2 Big bag 3 Warehoused

First 86.8 + 27.8 a 3.9 + 0.2 c 1.1 + 1.1 b <1

Second 158.2 + 38.1 a 50.7 + 3.8 bc 1.2 + 0.1 b <1

Third 139.6 + 28.7 a 82.6 + 21.8 ab 10.6 + 0.3 a 2.9 + 5.2

Fourth 153.4 + 47.5 a 83.6 + 41.2 ab 13.8 + 3.5 a 68.9 + 152.9

Fifth 200.4 + 69.1 a 140.9 + 14.9 a 12.5 + 3.0 a 69.1 + 152.1 a Mean levels of total aflatoxins (AFB1 + AFB2 + AFG1 + AFG2) from peanut seed

samples.

SE: standard error, n = 5. Detection limit: 1 ng g-1

. b Storage period: July-November for big bags; May-October for warehouse

c Big bag 1 0.94 + 0.01 aW; big bag 2 0.88 + 0.01 aW; big bag 3 0.84 + 0.01 aW

(Passone et al., 2010). d Nesci et al. (2011).

Values in columns with not letters in common are significantly different (p < 0.05)

according to Fisher LSD test.

Aflatoxin B1, B2, G1 and G2 concentrations of the one-hundred peanut

kernel samples collected are shown in Table 7. Aflatoxin levels in samples

from big bags 1 and 2 increased with the time of storage, although with

different time rates. When samplings of the first and fourth months were

compared, toxin increases were 57 and 97% for big bags 1 and 2, respectively.

The mean value of total AFs obtained in samples from big bag 3 at the third

sampling was significantly higher (89%) than from those obtained in the first

and second samplings (p<0.05). Aflatoxins were not detected at any analyzed

peanut sample from big bag 4. Statistical analysis showed a very poor negative

correlation between cfu and AFs accumulation (r=−0.514; p=0.05) in peanut

samples.

The determination of physical properties of the samples revealed marked

differences in aW and temperature among individual samples in the four big

bags throughout the storage period. Water activity levels in peanut samples

from big bags 1 and 2 decreased relatively faster during the first three months

of storage, from 0.92±0.01 aW to 0.76±0.00 aW and from 0.88±0.01 aW to


0.67±0.03 aW, respectively. Thereafter the aW decreased at a slower rate until

the end of storage period (mean=0.65±0.01 aW). However, aW levels in peanuts

from big bag 3 were lower during the five months of storage (from 0.84±0.01

aW to 0.60±0.04 aW). Peanut samples aW from big bag 4 significantly

decreased (p<0.05) during the second month of storage and after that remained

relatively constant until the end of storage period, reaching similar values

(0.66±0.01 aW) to those registered in big bags with the highest initial aW. Due

to the gradual increase of ambient temperature at Argentinean spring,

temperatures registered in peanuts from the four big bags showed a constant

increase during the four months of storage, from 12.7±0.45 °C to 29.2±0.6 °C.

These environmental variations significantly affected fungal density

(F=584.413; p<0.001) and total AF accumulation (F=32.475; p<0.001).

Statistical analysis carried out with data registered from big bags 1, 2 and 3

showed a negative correlation between aW and toxin accumulation (r=−0.643;

p=0.031) and a positive correlation between temperature and total AFs

(r=0.658; p=0.0225). In both cases the correlation was significant (Passone et

al., 2010).

5.2.2. Analyses on Peanut Stored in Wagons of Conditioning and Drying

Aspergillus section Flavi species were isolated at low count levels,

between 1x101 and 4.6x10

2 cfu g

-1, at all sampling carried out. Within this

section two species, A. flavus, with the highest prevalence (89.3%), and A.

parasiticus were identified (Figure 2). Throughout the five samplings carried

out between July and November, 144 isolated from Aspergillus section Flavi

were identified, of which 129 and 16 were A. flavus and A. parasiticus,

respectively. In relation to production of sclerotia, of the total A. flavus strains

isolated, 75.2% were producers and of these 95.9% were classified as L

strains, while only 4.1% as S strains (Table 7). When the toxigenic ability of

A. flavus isolates was examined, it was observed that only 1 isolate produced

AFG1 and the others produced type B AFs. L strains produced, on average,

higher AF levels than S morphotype strains. Of the 16 A. parasiticus isolates,

only 4 (25%) produced undetectable AF levels (<1 ppm). All toxigenic strains

produced AFB1, 2 of them also produced AFB2 and 4 produced AFB1 and

AFG1.

Of the 60 samples analyzed, no AFs were detected in any of them

(Doprado, 2008).


5.2.3. Analyses on Peanut Stockpiled Warehouse

The lowest incidence of Aspergillus section Flavi was detected in the first

sampling period, with an increase in the fourth sampling period (Figure 1).

Figure 2 shows the incidence of Aspergillus species that colonize the stored

grain during five sampling moments. Among the Aspergillus populations

recovered from peanut kernels, A. flavus was the most frequently isolated

(79%), followed by A. caelatus (7.6%), A. parasiticus (5.9%), A. spp. (5.1%)

and A. minisclerotium (1.7%).

Analyses of toxigenic potential of Aspergillus section Flavi strains

isolated from the peanut samples revealed the production of AFB1 by 52

strains out of 79 of A. flavus with mean levels ranging from <2 to 225000 ng

ml-1

and 2 strains of A. parasiticus with mean levels ranging from <2 to 25000

ng ml-1

(Table 6). The highest proportion of A. flavus toxigenic strains (87.5%)

was obtained from the second sampling period (June). However, the highest

number of A. flavus strains was isolated in September, as were the strains with

ability to produce the maximum AFB1 level (225000 ng ml-1

). A. parasiticus

strains were isolated during the third sampling period (August) and produced

AFB1 levels between <2 and 25000 ng ml-1

.

In samples corresponding to sampling periods of May and June, no AFB1

were detected. Aflatoxin B1 accumulation in peanut kernels during all

incubation periods was determined (Table 7). The greater levels of AFB1 were

detected in September and October with a mean of 68.86 and 69.12 µg kg-1

,

respectively, coinciding with the highest Aspergillus section Flavi count

(Nesci et al., 2011).

Stored peanuts represent a complex ecosystem in which seed spoilage by

fungi is determined by a range of factors which can be classified into four

groups; intrinsic nutritional factors, and extrinsic, processing and implicit

factors (Magan et al., 2004). Alone or in combination among them, these

factors affect the composition of the fungal population, inducing changes

throughout the storage period. In our studies, reductions of peanut aW and

increases of temperatures were registered in all big bags and wagon

throughout the storage period and these physical changes were reflected by a

reduction of Aspergillus section Flavi growth, independently of the

quantification methodology applied. When the CC method was used, count

reductions of these fungi were estimated in 35.2, 65.1, 33.6, 100 and 55.9% for

big bags 1, 2, 3, 4 and wagon, respectively. It is important to emphasize that

similar results were obtained with RT-PCR assays, whose cfu reductions were

around 36.0%. Similarly, Bulaong and Dharmaputra (2002) reported that A.

flavus, the only specie isolated from section Flavi, constituted a minor


percentage of the total population (from 0.001 to 1.45%) during month 4 to

month 6 in the four bag types, with a mean percentage of 15.6% at the

beginning of the assay. On the contrary, we observed an increase in the

frequency of Aspergillus section Flavi in peanut stored in warehouse,

estimated in 90.3% at the two last sampling periods. In their study,

Bhattacharya and Raha (2002) detected a high percentage of peanut seeds

contaminated by A. flavus (40%) at the beginning of the storage and the

incidence of this fungus increased to 78% after the tenth month. The results of

the work conducted by Nakai et al. (2008) on stored peanut in 25 kg bags

during 12-month period showed that the relative frequency of A. flavus

increased from 19.4 to 26.7% at the end of the storage. The presence of

Aspergillus and Penicillium in peanut kernels, is because the great adaptation

of these fungi to this substrate, especially during storage (Rossetto et al.,

2005).

These results indicate that Aspergillus species from section Flavi were

found at important counts from stored peanuts in the different storage system

in Argentina and demonstrated to be highly toxigenic. Among representatives

of the section Flavi, A. flavus was the prevalent specie in the three storage

system analyzed followed by A. parasiticus in big bags and wagon and by A.

caelatus in warehouse. It is noteworthy that, although the fungal composition

analyses from peanut pods were performed only in the big bag with the lowest

initial aW content (0.76+0.02), the mean counts of A. flavus and A. parasiticus

were 12.2 and 79.9% higher than the mean counts obtained from peanut

kernels of the four big bags. Tannis, waxes, amino compounds and structural

features in the peanut seed coat have been implicated in resistance to invasion

by A. flavus and A. parasiticus (Amaya-F et al., 1977; LaPrade et al., 1973;

Sanders and Mixon, 1978; Zambettakis and Bockelee-Morvan, 1976). A.

flavus have previously been reported as the prevalent specie from section Flavi

in peanut stored in different storage systems and markets. In addition to A.

flavus (21.2%), A. niger was isolated but at low frequency (0.6%) from stored

peanut kernels in Brazil (Nakai et al., 2008). Recently, Zorzete et al. (2013)

found species from sections Flavi and Nigri (mean fungal frequency= 10.1, 1.5

and 5.2% for A. flavus, A. parasiticus and Aspergillus section Nigri,

respectively) in peanut seed cultivars Runner IAC Caiapó and 886 stored in 25

kg sacks, stacked on wooden pallets during April to September 2006 in Brazil.

Bulaong and Dharmaputra (2002) also reported that A. flavus (4.6%) and A.

niger (1.9%) were isolated during all storage period between other Aspergillus

species with high occurrence but only during the first (A. fumigatus 22.8%, A.

versicolor 0.9%, A. wentii 2.1%) or the last (A. penicillioides 47.6%) two


months of storage in Phillipines. In unshelled peanuts obtained from five

fresh-produce markets in Kenya the Aspergillus species isolates were A. flavus

(28%), A. parasiticus (32%), A. niger and A. ochraceous (41%) (Gachomo et

al., 2004). In a survey conducted in Nairobi, Nyanza and Western provinces in

Kenya between March and July 2009 with 1263 peanut products sampled out

of which 705 samples were microbiologically analyzed; six Aspergillus

species were detected in the samples and were in decreasing order of cfu g-1

:

A. flavus (808), A. niger (156), A. tamari (27), A. alliaceus (21), A. parasiticus

(10), and A. caelatus (5). The combined incidence of A. flavus and A.

parasiticus was varied significantly (p<0.05) with peanut product: peanut flour

(69%), shelled raw peanuts (53%), spoilt peanuts (49%), boiled podded

peanuts (45%), podded peanuts (39%), peanut butter (31%), fried peanuts

(22%) and roasted peanuts (20%) (Wagacha et al., 2013). Main fungi present

in 20 samples of 50 g roasted and salted peanuts and 16 samples of 50 g unsalted

peanuts (pure) collected from Zanjan BAZAR (India) were A. flavus (39.1%),

Penicillium (9.2%), Rhizopus (7.2%), Mucor (2.5%), Alternaria (1.03%) and

Nigrospora (0.5%) (Rostami et al., 2009).

In our studies the low aW levels detected in peanut stored in big bag 4 and

wagon ensured the absence of AFs, however A. flavus S, L and NS strains and

A. parasiticus with different AF-producing potential were present between the

first and the fifth sampling periods. Therefore, if the aW of the grains remains

below 0.65, molds are unable to grow and the stored grain will be stable.

However, localized pockets of higher moisture may be present if temperature

and moisture gradients develop in the storage by the biological activity of

insects and rodents, opening the way for mold germination, growth and

consequently AF production (Hocking, 2003). A low percentage of peanut

samples from warehouse were contaminated with AFB1, but with values

exceeding the maximum limit of 20 µg kg-1

allowed for peanut without

shelling, shelling, raw or toasted, by the Mercosur regulations (56/94) for the

sum of AFs (B1+B2+G1+G2) (FAO, 2004). The production of AFB1 was

detected in 67% of strains. Studies conducted in Brazil showed that 33.3% of

peanut samples were contaminated with AFB1 at mean levels ranging from 7.0

to 116 µg kg-1

of which 24 samples exceeded the maximum limit established

by the aforementioned legislation and 93.8% of A. flavus strains isolated were

AF-producers (Nakai et al., 2008). Recently surveys analyzed the occurrence

of AF and the incidence of AF-producing fungi in peanuts and peanut products

traded in Kenyan markets (Wagacha et al., 2013). Seventy three percent of A.

flavus and A. parasiticus isolates produced at least one of the AF types, with

66% producing AFB1. The total AF level among peanut products ranged from


0 to 1629 mg g-1

; and there was a positive correlation (r = 0.2711; p<0.05)

between the incidence of A. flavus and A. parasiticus, and total AF level.

Thirty seven percent of the samples exceeded the 10 mg kg-1

regulatory limit

for AF levels set by the Kenya Bureau of Standards (2007). Raw podded

peanuts had the lowest AF levels, 96% having levels less than 4 mg kg-1

and

only 4% having more than 10 mg kg-1

. The most AF-contaminated products

were peanut butter and spoilt peanuts, with 69% and 75% respectively,

exceeding 10 mg kg-1

. The AF concentrations in peanuts from big bags 1, 2

and 3 increased toward the end of storage period. The increases in the second

bimonthly ranging between 15.8 and 55.1%, which is about twice lower than

those registered in the first two months. These results appear related to the

reduction of the Aspergillus section Flavi population throughout the storage

period. It is possible that a balance occurs in the closed silos environment,

between the cumulative increase in AFs concentration and the lower marginal

efficiency in their production and/or release, due to the fungus population

decrease. Despite the fungal population reduction observed at the end of the

storage period, we suggested that the environmental stress could produce an

increase in AF levels. Gunterus et al. (2007) reported that no correlation was

observed between inoculum level and AF accumulation. Aflatoxin

concentration increased with decreasing inoculum size. The lowest inoculum

(102 conidiospores g-1

of peanuts) generated highest AF levels (1.000 μg g-1

of

peanuts).

CONCLUSION

Based on these results, A. flavus and A. parasiticus strains are widely

distributed in soil and groundnut kernels coming from the traditional and new

peanut-growing areas and during the storage period in the three peanut storage

systems analyzed in Argentina. The ecophysiology of these fungi implies a

risk of AF contamination if environmental conditions became conducive,

highlighting the need to promote good practices at all stages of the peanut

value chain in order to minimize market access by non-complying products.


ACKNOWLEDGMENTS

This work was carried out through grants from the Agencia Nacional de

Promoción Científica y Tecnológica and from the Secreteraría de Ciencia y

Técnica, Universidad Nacional de Río Cuarto.

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Chapter 8

TOXICOLOGICAL EFFECTS,

RISK ASSESSMENT AND LEGISLATION

FOR AFLATOXINS

Marina Goumenou1, Dimosthenis Axiotis

2*,

Marilena Trantallidi5, Dionysios Vynias

2,

Ioannis Tsakiris3, Athanasios Alegakis

2,

Josef Dumanov4 and Aristidis Tsatsakis

2

1General Chemical State Laboratory, D Division of Athens,

Athens, Greece 2Laboratory of Toxicology, Department of Medicine,

University of Crete, Voutes, Heraklion, Greece 3TEI of Western Macedonia, Florina Branch, Department of Agricultural

Products Management and Quality Control, Terma Kontopoulou,

Florina, Greece 4Mycological Institute EU US

5Dept. of Biomedical and Clinical Sciences, University of Milan,

Milan, Italy

*

Corresponding author: Dimosthenis Axiotis, Email: [email protected], Mob: +30 693 7

656182, +39 345 6694328.

Marina Goumenou, Dimosthenis Axiotis, Marilena Trantallidi et al. 192

SUMMARY

Aflatoxins are toxic metabolites produced by the fungus Aspergillus. The

main representatives are aflatoxins B1, B2, G1, G2. Their occurrence in food

like nuts, cereals and cereal-derived products is a result of fungal

contamination before harvest and during storage. Milk can also be

contaminated by aflatoxin M1 (main metabolite of B1) as a result of animals‘

exposure to feed contaminated by the aflatoxin B1.

Aflatoxins manifest acute and chronic toxicity. Evidence of acute

aflatoxicosis in humans involving a range of symptoms from vomiting to death

has been reported mainly in Third World Countries. In relation to chronic

toxicity aflatoxins are well known for their genotoxic and carcinogenic

properties while recent studies evident a series of other possible effects like

reprotoxicity, impaired growth in children, intestinal functions, chronic fatigue

syndrome, compromise immunity and interfere with protein metabolism and

multiple micronutrients that are critical to health.

The critical step for aflatoxins‘ risk assessment is the estimation of the real

exposure. For this reason a number of surveys are conducted globally using

tools like biomarkers of exposure and modeling. In addition new parameters

like the climate change are now taken into consideration in order to predict

possible current and future changes of exposure to aflatoxins. As aflatoxins are

compounds of natural origin and their presence in food cannot be totally

eliminated the risk management is based on keeping the total exposure as low

as reasonably achievable taking into account the social-economic impact of

crop and livestock losses. Exposure reduction is achieved mainly by reducing

the number of highly contaminated foods reaching the market by regulatory

control but also applying detoxification strategies. According to the EU

regulatory framework minimization of the exposure to aflatoxins is based on

setting maximum levels of aflatoxins in different foodstuffs (4 – 10 µg/kg total

aflatoxins) and feed (EC/1881/2006, Directive 2002/32/EC). Products

exceeding the maximum levels should not be placed on the EU market.

Methods of sampling and analysis for the official control of aflatoxins, are also

set (EC/401/2006) in order to ensure common sampling criteria to the same

products and that certain performance criteria are fulfilled. The United States

Food and Drug Administration (FDA) has established the action levels for

aflatoxin present in food to the 20 µg/kg (0.5 µg/kg for milk) and up to 300

µg/kg for feed. Finally an action level of 10 µg/kg total aflatoxins is also used

from Japan authorities.

Toxicological Effects, Risk Assessment and Legislation … 193

1. INTRODUCTION

The acute effects from Aspergillus flavus have been well studied when its

primary toxins, the aflatoxins, were isolated from peanut meal in 1961 during

the investigation of an epizootic of "Turkey X" disease in England [1]. At that

time, these toxins were identified as secondary metabolites of some strains of

Aspergillus flavus and were considered as the etiologic agents of the disease in

turkeys following contamination of their feed by A. flavus. Since then,

numerous studies have identified Aflatoxins to be the causative agent of

adverse health effects with liver toxicity as the major end-point.

Initially, four major aflatoxin types were recognized, referred to,

collectively, as ―aflatoxins‖ and designated B1, B2, G1, and G2 due to their

fluorescence and retention factor values on thin-layer chromatographic plates.

Upon ingestion of aflatoxin B1 by humans and animals, production of

aflatoxin M1, as a metabolite, was discovered in mother's milk in exposure

levels of ng. Furthermore, aflatoxin M2, another potent hepatotoxic and

hepatocarcinogenic aflatoxin has been identified in milk of cows fed on meal

contaminated with aflatoxin B2 [2]. In addition to the aforementioned

aflatoxins, the aflatoxin Q1, a major metabolite of B1, was found in in vitro

liver preparations of other higher vertebrates [3].

Currently, at least 14 different types of aflatoxin are known [4]. Aflatoxin

B1 is considered as the most toxic and the most highly monitored and

regulated mycotoxin in foodstuffs produced by both Aspergillus flavus and

Aspergillus parasiticus. Aflatoxin G1 and G2 are produced exclusively by A.

parasiticus. Although Aspergilli are often present in food and feed products,

their occurrence does not consist necessarily an indicator of harmful levels of

aflatoxin as they do not all produce aflatoxins.

It is well documented that aflatoxins manifest acute and chronic toxicity.

Apart from their strongly evident acute liver toxicity and carcinogenicity

potential, recent studies manifest a series of other possible effects, such as

reprotoxicity, impaired growth in children, intestinal malfunctions,

neurotoxicity and chronic fatigue syndrome, nephrotoxicity, compromised

immunity and interference with protein metabolism and multiple

micronutrients that are critical to health. Part of aflatoxins‘toxic potential is

their very fast absorption by the small intestine, as indicated by studies in rats

[5].

Risk assessment of aflatoxins requires in addition to thorough knowledge

of health effects (hazard identification and characterization), exposure data.

About 4.5 billion people, mostly in developing countries, are at risk of chronic


exposure to aflatoxins from contaminated food crops [6]. The great majority of

studies for aflatoxins mainly focus on the contamination levels in certain food

commodities. However, since consumers may be exposed to aflatoxins

through a normal diet, dietary studies are crucial in order to accurately

estimate exposure and its variations between different groups (age,

geographical distribution, diet habits etc).

According to the classic risk assessment approach for non-genotoxic

substances, risk characterization is based on the comparison of a substance‘s

derived no-effect level, based on the evaluation of toxicological data, with the

estimated exposure levels. Such an approach is not possible to be followed for

aflatoxins as a threshold is not considered to exist. Alternatively, the As-Low-

As-Reasonably-Achievable (ALARA) approach is used by setting maximum

acceptable amounts of aflatoxins in foodstuffs. The ALARA level, reflects the

concentration of a substance that cannot be eliminated from food without

involving the discard of that food altogether or without severely compromising

the availability of major food supplies [7].

The protection of public health, consumers and workers from aflatoxins is

addressed in different aspects of legislation. Aflatoxins are subject to

regulations at national, regional and international level. A well-structured legal

framework should guarantee safe products (foodstuffs and feedstuffs) intended

for human and animal consumption, including regulatory limits, measures for

monitoring compliance with limits, guidance to the food industry, cooperation

between agencies on food safety and enforcement action. Legislation in

Europe, USA and elsewhere contributes to the global effort of controlling

aflatoxins‘ adverse health effects with respect to any social-economic

reasoning.

2. HEALTH EFFECTS

Health effects caused by aflatoxins are mainly divided into acute and

chronic toxic effects. Among chronic effects, carcinogenicity is by far the

most studied. However, a number of other chronic adverse health effects are

known nowadays, and include reproductive toxicity, impaired growth in

children, intestinal malfunctions and compromised immunity.


2.1. Mechanisms of Action

The numerous health effects caused by aflatoxins are the result of

common or different underlined mechanisms of action that can be or not

crossed in the complex net of biochemical reactions in the human body. At

molecular level, a key player in aflatoxin toxicity is the epoxide derivative of

aflatoxin B1. This epoxide has the ability to bind on DNA and to disrupt

transcription and translation activities, thus initiating carcinogenesis. Due to

the oxidative nature of this toxic derivative it can release free radicals causing

cell damage (Figure 1).

The evolution of molecular techniques like microarrays and PCR was

critical in understanding more precisely the mechanism of action of aflatoxins,

starting from molecular level, to genes, cells and organ level. Recent gene

expression studies have shown that aflatoxins can cause:

a) down regulation of carnitine palmitoyltransferase (CPT) system in

mitochondrion leading to decrease of body weight gain

b) down regulation of fatty acid metabolism pathway increasing liver

weight and causing fatty liver

c) up-regulation of cell proliferation pathway causing carcinoma, and

d) down regulation of B cell activation lowering immunity in birds fed

aflatoxin.

In addition, aflatoxins can impair protein biosynthesis by forming adducts

with DNA, RNA and protein, inhibit RNA synthesis, DNA-dependent RNA

polymerase activity, and cause degranulation of endoplasmic reticulum [9, 10].

Susceptibility to aflatoxin is higher in younger ages; it is sex-related

(testosterone-dependent) and with a great inter- and intra-species variability

depending on the biochemical detoxification abilities [11].

2.2. Acute Effects

The acute toxic effects of aflatoxins have been studied for many decades

[12]. Epidemiologic, experimental and clinical studies have shown that high

exposure to aflatoxins (above 6000 mg), through digestion, can cause severe

intoxication, which results in direct liver damage and subsequent illness or

death [13,14,15]. The mechanism of aflatoxin acute toxicity is related to

malfunction of the liver, which is the target organ of aflatoxin toxicity.


Aflatoxin metabolites, such as the aforementioned electrophilic B1, 8-9

epoxide [16] react with cell components causing serious disturbance in cell

lipid and carbohydrates metabolism. Lipids infiltration from the liver cells

causes hepatocytes death and liver cirrhosis and necrosis. The manifestation of

the cellular mechanism of aflatoxicosis at whole organism level regards

mainly the reduction of critical blood proteins, icterus, vomiting, edema and

abdominal pain [17].

No animals are immune to the acute toxic effects of aflatoxins. Animal

studies have found two orders of magnitude difference in the median lethal

dose for aflatoxin B1. Susceptible species such as rabbits and ducks have a

low (0.3 mg/kg) median lethal dose, whereas chickens (18 mg/kg) and rats

have greater tolerance. Adult humans usually have a high tolerance of

aflatoxin; in the acute poisonings reported, usually children are the ones who

die [11, 9].

Figure 1. Mechanism of cell damage in aflatoxins toxicity [8].

Outbreaks of aflatoxicosis affecting up to several hundred people at a time

have occurred sporadically, most recently in eastern Kenya in early 2004

[18,19]. The Kenya incidence was attributed to poor harvest of maize that had


been damaged and stored in warm and humid conditions making it susceptible

to mold by drought. From January to June 2004, 317 people sought hospital

treatment for symptoms of liver failure, and 125 died.

2.3. Genotoxicity and Carcinogenicity

Aflatoxins as toxins are among the most carcinogenic substances known

and the major cancerous hepatocellular carcinoma (HCC) risk factor. Cancer is

broadly defined as any disease in which normal cells are damaged and do not

undergo programmed cell death (apoptosis) as fast as they divide via mitosis.

Aflatoxin B1 is metabolized by cytochrome-P450 enzymes to the reactive

intermediate AFB1-8,9 epoxide (AFBO) which binds to liver cell DNA,

resulting in DNA adducts. DNA adducts interact with the guanine bases of

liver cell DNA6. This is thought to cause mutations at the codon 249 hotspot

in exon 7 of the p53 gene, an important gene in preventing cell cycle

progression, when there are DNA mutations, or signaling apoptosis [20].

These mutations seem to affect some base pair locations more than others —

for example, the third base of codon 249 of the p53 gene appears to be more

susceptible to aflatoxin-mediated mutations than nearby bases [21]. As a

consequence of aflatoxin genotoxicity potential, even short-term exposure can

lead to a risk of developing liver cancer.

Although this mechanism of genotoxicity is well recognized and

understood, non-genotoxic effects also exist and resulting cancers in the form

of enzymatic necrosis from the hyphae of pathogenic Aspergilli with the

production the hyphal digestive enzymes: the primary are proteases (protein),

amylases (carbohydrate), and lipases (fats).

From neoplasia (tumor) to necrosis, cancers are recognized in over 200

differing pathologies referred to by cell, tissue or organs affected. Aflatoxins,

as mentioned above, can be classified as genotoxic (p53) or non-genotoxic. In

neoplastic disease, the neoplasms may be benign, pre-malignant as a

carcinoma in situ or a malignancy as a cancer. Immunohistological

Aspergilloma, resulting from invasive Aspergillus fumigatus is the part of

immunological defensive mechanism in the formation of encapsulating fibroid

tumors known as a mycetoma or fungus ball rarely developing into a cancer.

In any case, the carcinogenic potential seems to increase due to malnutrition,

especially in cases of pyridoxine deficiency [22].


2.4. Other Health Effects

2.4.1. Reproductive Toxicity

The intensive research for aflatoxin-induced reproductive toxicity started

long after the study of acute toxicity and carcinogenicity. However, there is up

to now a considerable number of studies that provide evidence for the

association of aflatoxin exposure with testicular toxicity, infertility,

developmental toxicity and possible mechanisms involved.

According to the study of Verma and Nair [23], aflatoxins can inhibit the

stereoidogenesis in mice by reduction in 3b- and 17b-hydroxysteroid

dehydrogenases probably due to alterations in mitochondria, inhibition of

protein synthesis/enzyme activity, alteration in plasma membrane of Leydig

cell due to lipid peroxidation and/or irregular protein biosynthesis. In the study

of Ibeh and Ogonor[24], a correlation between infertility in men and aflatoxin

concentration in semen was suggested. In addition, higher frequency in sperm

count, morphology and motility was observed in the higher exposed infertile

men. In the same study,similar results were observed by intentionally exposing

rats. Additional studies in animals have shown spermatotoxic activity of

aflatoxins supporting these conclusions [25, 26, 27, 28]. Possible mechanism

involve disruption on sex hormone synthesis, protein synthesis inhibition, fat

metabolism disruption and, possibly, direct lysis of sperm cell membrane.

There is a considerable number of studies related to the association of

exposure to aflatoxins and low birth weight [29]. However, the overall result is

inconclusive as there are both positive and negative associations and in many

cases additional possible causes were not considered. In any case, the

possibility of such an association remains of high concern as birth weight

lower than 2500 g is closely associated with fetal and neonatal morbidity and

mortality, inhibited growth, poor cognitive development, and chronic diseases

later in life [30].

2.4.2. Impaired Growth in Children

Chronic, subclinical exposure does not lead to symptoms as severe as

acute aflatoxicosis. Children, however, are particularly affected by aflatoxin

exposure, which leads to delayed development [31] and impaired growth [32].

Food borne aflatoxin exposure is common in many areas of Africa and Asia

where childhood stunting and underweight are also frequent, due to a variety

of possibly interacting factors such as enteric diseases, socioeconomic status,

and suboptimal nutrition [33]. Gong et al. performed in 2002 [34] a cross

sectional study in young children from Benin and Togo in relation to the


dietary aflatoxin exposure and impaired growth revealing a striking

association between exposure to aflatoxin in children and both stunting (a

reflection of chronic malnutrition) and being underweight (an indicator of

acute malnutrition). In this study, children still partially breast-fed had lower

exposure, almost certainly reflecting lower toxin levels in milk than in

weaning and family foods. Thus, growth faltering occurs at a time of change to

solid foods, when there is co-exposure to aflatoxin and a plethora of infectious

hazards, such as malaria, diarrhea and respiratory infections. Studies in Togo,

Gambia, Ghana, Iran, Kenya, and the United Arab Emirates have shown

similar findings, outlined in a review published by Wu and colleagues in 2011

[35, 33]. However, studies with cross sectional design cannot always confirm

if the association between aflatoxin exposure and impaired growth is a direct

result of aflatoxin toxicity or reflects consumption of fungus affected food of

poor nutritional quality. Apart from any association between children exposure

to aflatoxins and after birth growth impairment, aflatoxins can cause genetic

defects at fetal stage as they can cross placental barriers [36].

2.4.3. Intestinal Malfunctions

Intestinal cells are the first cells to be exposed to aflatoxins [37]. Rapidly

dividing and activated cells and tissues with a high protein turnover are

predominant in gut epithelium; consequently, the effect of aflatoxins as

disruptors of protein synthesis is of great importance. Intestinal homeostasis

depends on the balance between nutrition, immune system and gut microbiota

and this homeostasis is crucial for the health of the GIT, the absorption of

nutrients and the functions of the immune system. For these reasons, intestinal

investigations have gained significant interest over the last decade.

Evidence on the impairment of growth after exposure to aflatoxins was

questioned as related to malnutrition of exposed populations. However, there

is evidence that such malnutrition is not just the output of socio-economic

conditions, but probably also a consequence of aflatoxin exposure which can

compromise the absorption of nutrients. Several studies in animals support the

reduction of nutrient requirements, absorption, digestibility and utilization due

to aflatoxins [40, 38, 39, 40, 41]. In addition, disturbance on digestive

enzymes has been reported [41, 43, 42] without however to be clear if this is a

cause or the output of other aflatoxin-related effects. Studies on birds revealed

effects in the morphology of the GIT, as well, while the results for other type

of effects, such as nutrients utilization and microbiota sensitivity on these

studies are rather controversial [43].


2.4.4. Neurotoxicity

The effects of aflatoxins in the nervous system are related to the action of

their metabolite AFB-8, 9-epoxide and its ability to create DNA and protein

adducts, to inhibit protein, RNA and DNA synthesis, to promote mitochondrial

directed apoptosis of the nerve cells and to produce Oxygen Reactive Species

(ROS) [44, 45, 46, 47]. Myelin depletion and disturbance of brain chemistry

by aflatoxins is also reported [50, 48, 49]. Aflatoxins have been reported to

cause tumors in both the central and peripheral nervous system [54]. Several

neurotransmitters are additionally reported to be affected by aflatoxins in

animals, such as acetylcholinesterase enzymes that may affect the memory,

learning and cognitive functions[50], dopamine, serotonin as well as tyrosine

and tryptophan, leading to neurocognitive decline and alteration of sleep cycle,

dullness, restlessness, muscle tremor, convulsions, loss of memory, epilepsy,

idiocy, loss of muscle coordination, and abnormal sensations [51, 52, 53, 54].

AFB1 has also been reported to increase the central and peripheral nervous

system Na/K ATPase, β-glucuronidase and β-galactosidase while inhibiting

the Mg-ATPse in experimental animals; this is also important in the normal

functioning of the glutamate neurotransmitter and their NMDA receptors [55,

56].

Liver malfunction caused by aflatoxicosis has as secondary effect, i.e. the

gathering of non-detoxified ammonia in the brain. Ammonia can pass the brain

barrier causing encephalopathy. Toxic encephalopathy due to aflatoxin

involves multiple symptoms such as loss of balance, recent memory decline,

headaches, light‐headedness, spaciness/disorientation, insomnia, loss of

coordination; an association with a Reye-like Syndrome in Thailand, New

Zealand, Czech Republic, Slovakia, the United States, Malaysia, Venezuela

and Europe [57] has been reported.

2.4.5. Immunosuppression

Many studies prove that aflatoxin B1 is immunosuppressive in animals,

with particularly strong effects on cell-mediated immunity and increased

susceptibility to bacterial and parasitic infections [58]. According to these

studies, performed mainly to birds and rats, aflatoxins decrease complement

activity leading to impairment of phagocytosis and reduce chemotactic ability

of leucocytes. Immunosuppression caused by exposure to aflatoxins is the

output of their interference with normal function of B and T-cells [59],

reduction of the phagocytosis by macrophages and of the activity of vitamin K

[60]. In addition, the impairment of protein synthesis caused by dietary


aflatoxin could account for the lack of humoral immunity without the

necessity of B and T cell destruction [61].

Human monocytes treated with aflatoxin B1 resulted in impaired

phagocytic and microbicidal activity and decrease in specific cytokine

secretion [61]. Studies have linked human exposure to aflatoxins to increased

prevalence of infection [62]. According to the study of Turner et al. (2003)

[63] sIgA in saliva may be reduced because of dietary levels of aflatoxin

exposure in children in rural Gambia that are frequently exposed to high levels

of aflatoxin. Aflatoxins can also cross the human placenta [68]. This finding is

also supported by the study of Pier et al. [64] where the immunosuppressive

effects of aflatoxin were also shown to be transferred across the porcine

placenta and to affect the unborn fetus. Finally, it is important to mention that

various recent studies have focused on ameliorating the effects of aflatoxin by

supplementing or amending the diet [65].

The immunosuppressive potency of aflatoxins is also responsible for the

decrease of the vaccination value reducing the antibody response in animals

like poultry, rabbits and cattles [11]. Considering that aflatoxins exposure

occurrence is mainly reported in regions of Africa and Asia, where infectious

diseases are common and vaccination is of great importance, one can easily

understand the additional implication that could be created by this aspect of

aflatoxins adversity if relevant for humans. However, existing studies in

humans do not support clearly such relevance. According to the study of

Turner et al. (2003) [64] mentioned above, antibody response to one of four

pneumococcal serotypes, but not rabies vaccine, was weakly associated with

higher levels of serum aflatoxin-albumin (AF-alb) adducts (as biomarker of

long term exposure) in children exposed to aflatoxin in Gambia. Furthermore,

no association between cell-mediated immunity responses to test antigens and

AF-alb was reported. Allen et al. (1992) [66] also observed absence of

correlation between Af-alb concentration and the malaria specific antibody.

2.4.6. Miscellaneous

Other health effects mentioned in the open literature concern the

respiratory system [17], the renal system [70] and incidences of teratogenicity.

More information about aflatoxins health effects can be found in the literature

collection ―Additional Studies on Aflatoxin and Health‖ published by the

PACA (Partnership for Aflatoxins Control in Africa) [67].


3. RISK ASSESSMENT

3.1. Exposure

Aflatoxins are mainly observed during and after post-harvest food and

feed contamination, mainly due to temperature and humidity conditions which

favor their production. Weather conditions and cultivation techniques may also

favor spoilage during field stage. The food chain is the main pathway through

which aflatoxins enter the human body. In a worldwide basis, it is difficult and

in some cases impossible to estimate the degree of exposure to aflatoxins

based on the consumption of specific food commodities and the levels of

contamination. Worldwide trade of food products and different dietary habits

may also increase the difficulty of exposure assessment to aflatoxins [68].

The most common food commodities in which aflatoxins were detected

are: a) cereals and small grains i.e. wheat and barley b) milk and dietary

products and c) nuts and dried fruits. The aforementioned foodstuffs are

consumed by all age groups and some of them constitute a crucial part of daily

diet i.e milk for children. There are several studies reporting the levels of

aflatoxins in food and, at the same time, estimating the exposure of population

through consumption. A number of them are full diet studies while the rest are

referred to a specific food commodity.

Jager et al. (2013) present an assessment of aflatoxin intake in Brazil [69].

In this study, the levels of aflatoxins were determined in peanut, corn, bean

and milk – milk products collected directly from home of residents during

2011 and 2012. A total of 240 samples were analyzed. The results point out

that peanuts and derivatives were the most important source of exposure for

the population. The estimated daily intake of aflatoxins through peanut

consumption was 13.7 ng kg-1

b.w day-1

. These results are much higher

compared to the intake levels of aflatoxins from other countries such as

France, Australia and United States (0.345 ng kg-1

b.w day-1

, 0.15 ng kg-1

b.w

day-1

and 0.26 ng kg-1

b.w day-1

, respectively). Milk represents a high portion

of the daily intake with a maximum value of 0.3 ng kg-1

b.w day-1

which, in

this case, was in accordance with values reported for French population.

In a study performed in Spain, a total of 603 samples were collected from

Catalonia supermarkets during 2008 and 2009 [70]. The samples represented

the most common food commodities consumed in Catalonia and included

peanuts, pistachios, dried figs, sweet corn, breakfast cereals, corn snacks, dried

red pepper and baby food. In this study, the levels of population exposure to

aflatoxins through diet were ranged from 0.036 up to 0.788 ng kg-1

b.w day-1

.


In another study in France a total of 577 food samples (food products based on

cereals, milk, dairy products, solid food products, beverages and breakfast

cereals) were analyzed for mycotoxins during the period 2006-2007.

According to the results the levels of exposure to aflatoxins through

consumption of selected foods ranged from 0.001 ng kg-1

b.w day-1

up to 0.198

ng kg-1

b.w day-1

for children, while for adults from 0.001 ng kg-1

b.w day-1

up

to 0.188 ng kg-1

b.w day-1

[71]. In Japan from 2004 to 2006, a total of 884

food samples (chocolate, buckwheat, cocoa, almond, peanut butter, peanut, red

pepper, pistachio and white pepper) were collected and analysed for aflatoxin

residues [72]. The estimated daily intake based on 95th values ranged from

0.003 up to 0.014 ng kg-1

b.w day-1

according to the different scenarios based

on the age of the consumers.

In some cases, studies focus on more specific food commodities, which

represent local diet habits, such as spices in China. Zhao et al. (2013)

estimated the exposure of the Chinese population to aflatoxins through the

consumption of different spices (pepper, chili prickly ash, cinnamon, aniseed,

fennel, curry powder, cumin and ginger) [73]. A total of 480 samples were

collected from retailers and analyzed during 2009. In this study, several

scenarios were followed for the estimation of aflatoxins daily intake. In a

worst-case scenario, the estimated daily intake mean values based on

consumptions of spices in New Zealand, Europe, US, India and Thailand were

0.057, 0.115, 0.461, 1.097 and 1.672 ng kg-1

b.w day-1

, respectively.

Literature provides extended data for the exposure to aflatoxins through

milk and dairy products consumption mostly based on the results from

monitoring studies. In a study that took place in Brazil a total of 125 different

milk types were collected from Sao Paulo during 2006 and analyzed for

aflatoxins [74]. The estimated daily intake of aflatoxins based on local milk

consumption was 1 ng kg-1

b.w day-1

for children and 0.188 ng kg-1

b.w day-1

for adults. In another study,176 milk samples were collected in Serbia during

2013 [75]. The highest estimated daily intake of aflatoxin through milk

consumption was 36.8 ng kg-1

b.w day-1

.

As already mentioned, peanuts are considered as a potential source of

aflatoxins. In a study that took place in Brazil, 100 samples were collected

from the market and analyzed during 2006-2007 [76]. According to the results,

the estimated daily intake varied from 0.6 up to 10.4 ng kg-1

b.w day-1

. In a

study conducted in China, a total of 1040 samples were collected from four

different cultivation areas during 2009 – 2010 and analyzed for aflatoxin

residues. According to the results and based on local consumption figures the


estimated daily intake for children was 0.218 -0.222 ng kg-1

b.w day-1

while

for adults 0.106-0.108 ng kg-1

b.w day-1

, [77] .

Studies have shown that a widely eaten staple food in West African

countries such as Ghana; ―Kenkey‖ typically contains large amounts of

aflatoxin-producing Aspergillus even after fermentation [78]. Pica (an eating

disorder among pregnant women which involves the ingestion of non-nutritive

substances such as raw maize, soil, gum, ash and other substances that may be

contaminated by Aspergillus moulds) is another source of increased aflatoxin

intake [79,80].

What is clear up to now is that the great majority of studies for aflatoxins

mainly focus on the contamination levels in certain food commodities, and on

new trends of analytical techniques. When it comes to exposure, and since it is

not clear which food commodity contributes more to exposure, there are many

variables, which may affect the estimation, such as dietary habits. Since

consumers may be exposed to aflatoxins through a normal diet, exposure

studies constitute a crucial parameter for estimating variations in exposure

between different groups (age, geographical distribution, diet habits etc). In

any case, the genotoxic potential of aflatoxins makes any level of exposure to

be a risk factor.

3.2. Risk Characterization

3.2.1. Introduction

Risk assessment is the scientific evaluation of the probability of

occurrence of known or potential adverse health effects resulting from human

exposure to (food-borne) hazards; it is the primary scientific basis for the

establishment of regulations [81]. The availability of toxicological data, as

well as the availability of data on the occurrence of contaminants in various

commodities, are the main factors required in order to perform an assessment

of risks. Usually, the methodology followed in risk assessment implies the

identification of a No-Observed-Adverse-Effect-Level (NOAEL) for the

critical effect from toxicological studies and the application of relevant

uncertainty (or assessment) factors for the derivation of limits of exposure

(e.g. tolerable intake levels). These limits of exposure are subsequently

compared with the outcome of the exposure assessment for the final step of the

risk assessment procedure, namely the risk characterization, in order to

conclude on the existence (or non-existence) of risk.


In the case of aflatoxins, however, this approach cannot be followed due

to the fact that the critical effect is carcinogenicity. No-effect concentration

limits cannot be established for genotoxic carcinogens; any small dose will

have a proportionally small probability of inducing an effect. Exposure to this

type of compounds should, in theory, be completely prevented; nevertheless,

exposure of the population to some level of aflatoxins has to be tolerated, as

these are naturally occurring food contaminants. It is, therefore, recommended

that the level of the contaminant in food should be reduced so as to be As Low

As Reasonably Achievable (ALARA). The ALARA level, which reflects the

irreducible level for a contaminant, is defined as the concentration of a

substance that cannot be eliminated from a food without involving the discard

of that food altogether or without severely compromising the availability of

major food supplies [81].

In the European Union, the opinion of the Scientific Committee (SC) of

the European Food Safety Authority (EFSA) addresses approaches beyond the

ALARA principle, allowing a level of potency assessment of specific

substances which are present in food and which are both genotoxic and

carcinogenic. Such an approach does not substitute for minimizing exposure to

all such substances. It ensures that, where resources are limited, the highest

priority is given first to those substances which present the greatest risk for

humans. The SC of EFSA recommends the application of the margin of

exposure (MOE) approach as a harmonized methodology for assessing the risk

of genotoxic and carcinogenic substances which may be found in food,

irrespective of their origin. The margin of exposure is defined as the reference

point on the dose-response curve (usually based on animal experiments in the

absence of human data) divided by the estimated intake by humans [82].

Evaluation of risks to public health arising from dietary exposure to

aflatoxins has been performed by various expert groups throughout the years.

In this section, the principal risk assessment approaches related to aflatoxin

exposure are presented.

3.2.2. The Joint FAO/WHO Expert Committee on Food Additives

(JECFA) Approach

A quantitative risk assessment was performed in 1997 by the Joint

Committee on Food Additives (JECFA) - a scientific advisory body of the

World Health Organization (WHO) and the Food and Agriculture

Organization (FAO), focusing on the increase in primary liver cancer [13].

The Committee reviewed the experimental evidence concerning the

carcinogenicity of the aflatoxins, evaluated their potencies, linked these


potencies to intake estimates, and discussed the impact of hypothetical

standards on sample populations and their overall risks. The conclusions of the

Committee regarding aflatoxin potency were contingent upon the dynamics of

hepatitis B infection (HBV) in a human population, since the said potency

appears to be significantly enhanced in carriers of hepatitis B virus - as

determined by the presence in serum of the hepatitis B surface antigen

(presence denoted HBsAg+ and absence denoted HbsAg

-). Overall, based on

the weight of scientific evidence (epidemiological data, laboratory animal

studies, in vivo and in vitro metabolism studies) the Committee considered that

aflatoxins should be treated as carcinogenic food contaminants, the intake of

which should be reduced to levels as low as reasonably achievable.

Regarding aflatoxin B1 (AFB1) potencies, the Committee reviewed the

potency estimates from the epidemiological studies which showed a positive

association between aflatoxins and liver cancer and selected separate potency

estimates and ranges for HBsAg+ and for HBsAg

- individuals. Potency values

of 0.3 cancers/year per 100 000 population per ng aflatoxin/kg of body weight

per day (uncertainty range: 0.05-0.5) in HBsAg+ individuals and of 0.01

cancers/year per 100 000 population per ng aflatoxin/kg of body weight per

day (uncertainty range: 0.002-0.03) in HBsAg- individuals were chosen.

Linking the potency estimates (risk per unit dose) to the estimates of

aflatoxin intake (dose per person), the fraction of the incidence of liver cancer

in a population attributable to aflatoxin intake could be derived (population

risk), based upon the prevalence of hepatitis B infection in various regions.

Relative estimates of mean dietary intake of aflatoxins for various regions

were provided using regional diets from the Global Environment Monitoring

System - Food Contamination Monitoring and Assessment Programme

(GEMS/Food) combined with data on levels of aflatoxin contamination.

Comparing two hypothetical standards (10 μg/kg and 20 μg/kg aflatoxin

B1 in food) and considering the limitations and assumptions inherent in this

approach, the Committee concluded that populations with a low prevalence of

HBsAg+ individuals and/or with a low mean intake (less than 1 ng/kg of body

weight per day) are unlikely to exhibit detectable differences in population

risks for standards in the range of the hypothetical cases; populations with a

high prevalence of HBsAg+ individuals and high mean intake of aflatoxins

would benefit from reductions in aflatoxin intake. Specifically, reducing the

hypothetical standard from 20 μg/kg to 10 μg/kg, would yield a drop in the

estimated population risk of approximately 2 cancers/year per 109 people in

case of a low prevalence of hepatitis B, and of 300 cancers/year per 109 people

in case of a high prevalence of hepatitis B.


Overall, it was concluded that when two alternative standards for aflatoxin

contamination in food are considered, the higher standard will yield essentially

the same risk as the lower standard if the fractions of samples excluded under

the two standards are similar. When a substantial fraction of the current food

supply is heavily contaminated with aflatoxins, reducing the levels of

contamination may result in detectable reductions in rates of liver cancer.

Conversely, when only a small fraction of the current food supply is heavily

contaminated, reducing the standard by an apparently substantial amount may

have little appreciable effect on health. FAO and WHO encourage

governments and the Codex Alimentarius Commission (CAC) to make use of

the aforementioned evaluation in deciding on the appropriate standards to

apply to aflatoxins. However, this requires a significant amount of information

at national level including monitoring data and information on dietary patterns

and the prevalence of hepatitis B in the population.

3.2.3. The European Food Safety Authority (EFSA) Approach

In 2007, the Scientific Panel on Contaminants in the Food chain

(CONTAM) of the European Food Safety Authority (EFSA) - an independent

body of the European Commission - was asked to provide a scientific opinion

on the potential increase of consumer health risk associated with a proposed

change of the EU maximum level of 4 μg/kg for total aflatoxins (sum of

aflatoxins B1, B2, G1 and G2) in ―ready-to-eat‖ almonds, hazelnuts, pistachios

and derived products to 8 or 10 μg/kg, taking into account exposure to all

aflatoxins from other food sources, including aflatoxin M1 [83]. For this

evaluation, the following were considered: (i) occurrence data provided and

uncertainties related to the heterogeneous distribution of aflatoxins; (ii)

specific consumption patterns of the relevant food commodities in the different

Member States; (iii) specific (vulnerable) population groups, including

children, hepatitis carriers and high level consumers; (iv) relative proportion of

AFB1 to total aflatoxins. Additionally, EFSA was requested to estimate the

margin of exposure (MOE) for the presence of aflatoxins (total

aflatoxins/AFB1) in food, considering vulnerable population groups.

For the current opinion, occurrence data on aflatoxins in a range of food

products were mainly collected from random and targeted monitoring and

surveillance activities in Member States over a seven year period. For those

samples where aflatoxins were detectable, AFB1 was generally the major

contributor to total aflatoxins. Data relating to concentrations of aflatoxin M1

(the major metabolite of AFB1) in commercial milk samples were generally


below 0.05 μg/kg and taking into account the lower carcinogenic potency of

M1, these were not further considered.

For the assessment of the impact of a possible change in the maximum

levels for almonds, hazelnuts and pistachios, the CONTAM Panel estimated

dietary exposure excluding occurrence data above 4, 8 and 10 μg/kg,

respectively. The estimates indicated that increasing the maximum levels for

total aflatoxins in almonds, hazelnuts and pistachios from 4 to 8 or 10 μg/kg

would result in an increase in the average total dietary exposure to aflatoxins

in the region of 1 %. In the case of consumers with the highest level of

consumption, the estimates indicated that increasing the maximum levels for

total aflatoxins from 4 to 8 or 10 μg/kg could increase total dietary exposure to

aflatoxins by up to 20 %. If, as is expected, nuts exceeding the maximum

levels are occasionally consumed, the total long term average dietary

exposures might be higher, but the relative impact of raising the maximum

level from 4 to 8 or 10 μg/kg in the three nuts would be less.

Considering liver carcinogenicity as the pivotal effect for the risk

assessment, the Panel considered dose-response modelling of experimental

data from both animal studies and epidemiological studies for the MOE

calculation, based on the risk assessment approach for genotoxic carcinogens

[82]. The available database for dose-response modelling was only sufficient

for AFB1.

Taking into account that AFB1 constituted a major proportion of total

aflatoxins in the samples analysed, for the purposes of this evaluation, the

Panel made the precautionary assumption that the potency of total aflatoxins is

equivalent to that of AFB1.

Overall, based on the information available in 2007, the CONTAM Panel

concluded that public health would not be adversely affected by changing the

maximum levels for total aflatoxins from 4 to 8 or 10 μg/kg in almonds,

hazelnuts and pistachios; minor effects would be expected on the estimates of

dietary exposure, cancer risk and the calculated MOEs.

The CONTAM Panel, however, reiterated its previous conclusion that

exposure to aflatoxins from all sources should be as low as reasonably

achievable, because aflatoxins are genotoxic and carcinogenic, and that

priority should be given to reducing the numbers of highly contaminated foods

reaching the market, irrespective of the commodity involved.


4. LEGISLATION

4.1. Introduction

Aflatoxins are subject to regulations at national, regional and international

level. The principle objective behind this action is to protect public health,

consumers and workers as well as to ensure safety on food supply (‗farm to

fork‘ approach). A well-structured legal framework should guarantee safe

products (foodstuffs and feedstuffs) intended for human and animal

consumption. Thus, an effective legal framework should typically include:

Regulatory limits;

Monitoring to ensure compliance with limits;

Guidance to the food industry;

Cooperation between agencies on food safety;

Enforcement action.

Limits for contaminants are set with the purpose to reduce contamination

in food and feed. The case of aflatoxins is particular due to the fact that these

toxins are genotoxic carcinogens. Actions on their exclusion from food and

feed would have to be imposed; however, aflatoxins are natural occurring

contaminants and exposure cannot be completely prevented. In this context a

range of regulatory limits are established by national, regional and

international authorities depending mainly on scientific and socioeconomic

grounds.

Since 1998 harmonized limits exist for aflatoxins in various foodstuffs and

feedstuffs in the EU; EU regulations regarding aflatoxins are of the most

extensive and detailed compared to other regions of the world. This is also true

for most of the developed market economies that have more stringent

standards than developing countries. The U.S. Food and Drug Administration

issued for the first time Action Levels for aflatoxins in 1969. Based on an

assessment of FAO which was carried out by the National Institute for Public

Health and the Environment (The Netherlands) 39 countries in Europe,

accounting for approximately 99% of the continent‘s population, had specific

mycotoxin regulations in 2003 [81, 84].

Improvements in food regulations have taken place constantly and reflect,

in a great extent, the increase of exports and imports of food and feed

products. This increase is a result of the growing globalization of trade.


However, the ease of moving products raises the risk of the spread of food

hazards and the need for the countries to ensure confidence in the safety of

their products.

Due to the importance of food trade and safety, EU and U.S. have

established agreements and partnerships with international organizations such

as WTO (World Trade Organization), FAO (Food and Agriculture

Organization) and WHO (World Health Organization). The EU is committed

to multilateralism and has acknowledged the fundamental importance of WTO

in the international trade system. WTO works towards multilateral rule-

making, trade liberalisation and sustainable development. FAO and WHO

approved in 1963 the establishment of the Joint FAO/WHO Food Standards

Programme with the Codex Alimentarius Commission as its principal organ,

which develops harmonised international food standards, guidelines and codes

of practice to protect the health of the consumers and ensure fair practices in

the food trade. The Commission represents the EU in the Codex Alimentarius

Commission and its bodies, and as a member of the WTO should apply the

Codex Alimentarius standards and meet its obligations under the World Trade

Organisation Agreement on Sanitary and Phyto-sanitary Measures (SPS

Agreement). Under this agreement the EU and other members have the right to

apply stricter standards than the CODEX standards, as long as those are based

on science (e.g. risk assessment) [85].

The setting of aflatoxin regulations is a complex activity that involves

many factors and interested parties. Recently, EU‘s more stringent limits have

been debated and the European Commission requested the European Food

Safety Authority (EFSA) – the EU risk assessment body for food and feed

safety - to give its opinion on possible adverse effects on humans after an

increase of limits for certain kind of nuts. The EFSA‘s conclusion permitted

the increase and limits were set higher. However, differences between

regulatory limits and in general regulations of developed and developing

countries still exist reflecting how socioeconomic criteria affect legislation.

4.2. EU - Legal Framework

Historically the European Union guided its food policy towards advanced

food safety as an integral part and contributor to food security. In the first

decades after the 2nd

World War, EU dealt with food security issues, animal

diseases and new consumer habits. As a result, new Regulations and

Directives were born to tackle animal diseases; CAP – Common Agriculture


Policy improved food security; RASFF – Rapid Alert System for Food and

Feed was established as a back-up measure to health risks from food and feed;

and the European Foundation for the Improvement of Living and Working

conditions dealt, amongst other issues, with food and nutrition. Although EU

legislation covered numerous aspects in food and feed safety, there was not in

place an integrated legal framework which could effectively address and cover

all sectors of the food chain as a whole, from primary production to retail sale,

as well as a unique authority which could provide with rigorous scientific

advice the European Commission, and clear communication for the risks[86].

The aforementioned gap was first addressed in the Commission‘s Green

Paper [87] on Food Law (1997) which was followed by the ‗White Paper on

Food Safety‘ [88], one of the most important milestones in the history of food

policy in the EU, issued in 2000. The main scope of the White Paper was to

suggest and set out actions which will eventually lead to a new integrated

policy on food safety in the EU. The actions were based on the following

strategic priorities of the White Paper:

High level of consumer health protection;

Establishment of an independent European Food Authority;

Consistent implementation of a ‗farm to table‘ approach in food

legislation;

Clear attribution of primary responsibility of safe food production to

industry, producers and suppliers;

Application of Risk analysis (Risk Assessment, Risk Management -

precautionary principle where appropriate - and Risk Communication)

in legislation. The Precautionary principle may apply when adverse

health effects have been identified but scientific uncertainty persists;

Traceability of products through the whole food chain;

Greater transparency at all levels of Food Safety policy;

Official Controls at both national and EU level (all parts of the food

production chain must be subject to official controls);

Ability to take rapid, effective safeguard measures in response to

health emergencies throughout the food chain;

Well informed consumers and re-establishment of confidence.

As a result, in 2002, the General Food Law [89] was drawn up by the

European Commission laying down the general principles and requirements of

Food Law, establishing procedures in the context of food safety, and setting up


the European Food Safety Authority. It could be described as a solid base upon

which further important food safety rules can be provided.

On this basis, and taking into account of general principles and concepts

(provided in the General Food Law) such as the high level of human life and

health; the free movement of safe food and feed, and the measures which

guarantee that unsafe food is not placed on the market, a legal framework on

contaminants in food and feed has been put into effect, in line with the existing

Council Regulation (EEC) No 315/93[90] on Community procedures for

contaminants in food. The latter regulation lies down, among other, that food

containing a contaminant in an amount which is unacceptable from the public

health viewpoint, and in particular at a toxicological level, shall not be placed

on the market. In addition, contaminant levels shall be kept as low as

reasonably achievable by following good practices at all stages (production,

processing, treatment, storage, etc). Subsequent amendments (EC No

1882/2003, EC No 596/2009) enable the Commission, where necessary, to

establish maximum tolerances for specific contaminants. Prior to provisions‘

adoption which may have an effect upon public health, consultation of the

EFSA‘s Scientific Committee on Food is obligatory.

Maximum Levels

Due to the fact that aflatoxins occur naturally in food it is not possible to

impose total ban of them. Therefore, it was necessary to take restriction

actions on their maximum concentration, minimizing any risk to human health.

Replacing the former EC No 466/2001 regulation [91], the Commission

Regulation EC No 1881/2006 - setting maximum levels for certain

contaminants in foodstuffs [92] - entered into force.

This was subsequently amended by the EC No 165/2010 regulation [93]

increasing certain levels for certain foodstuffs respecting at the same time

EU‘s agreement with WTO. The latest amendment had previous been justified

according to EFSA‘s opinion on the potential increase of consumer health risk

by a possible increase of the existing maximum levels for aflatoxins in

almonds, hazelnuts and pistachios and derived products in 2007, and by its

statement related to the effects on public health of an increase of the levels for

aflatoxin total for tree nuts other than almonds, hazelnuts and pistachios in

2009. Previous and new levels of concentration for aflatoxin in certain

foodstuffs are reported in Table 1.


Table 1. Maximum levels for aflatoxins (B1 and total) in foodstuffs before

and after the amendment of EC No 1881/2006[95] regulation

EC No

1881/

2006

EC No

165/

2010

EC No

1881/

2006

EC No

165/

2010

B1 (μg/

kg)

B1 (μg/

kg)

Total*

(μg/

kg)

Total*

(μg/

kg)

Treenuts Almonds,

Pistachios,

Apricot

kernels

Ready to eat 2 8 4 10

For further

processing

5 12 10 15

Hazelnuts,

Brazil nuts


For further

processing

5 8 10 15

Other tree

nuts


For further

processing

5 5 10 10

Oilseeds

(not for

crushing)

Peanuts Ready to eat 2 2 4 4

For further

processing

8 8 15 15

Other

oilseeds

Ready to eat - 2 - 4

For further

processing

- 8 - 15

Cereals Corn Ready to eat 2 2 4 4

For further

processing

5 5 10 10

Rice Ready to eat 2 2 4 4

For further

processing

- 5 - 10

Other

cereals


For further

processing

- - - -

* Total aflatoxins – B1, B2, G1, G2

Limits have been developed considering that aflatoxins are genotoxic

carcinogens, and are set at a level which is as low as reasonably achievable

(ALARA). Limits exist for ‗ready to eat‘ and ‗for further processing‘

foodstuffs, and apply to aflatoxin B1, which is the most potent for human

health, total aflatoxins (B1, B2, G1, G2) and aflatoxin M1.

Vulnerable groups, such as babies, infants and young children, are

protected under more stringent limits for certain food products. In other cases,


however, foodstuffs may be placed on the market when concentration levels of

aflatoxins exceed limits as soon as they are not intended for human

consumption, they do not exceed the maximum limits for these products which

are to be sorted before human consumption, and labelled clearly showing their

use.

Similar to food, feedstuffs for animals are subject to tolerance levels for

aflatoxin, as well. Commission Directive 2002/32/EC [94], amending

Commission Directive 1999/29/EC and all subsequent amendments of it, lays

down rules to ensure agricultural productivity and sustainability and to ensure

public and animal health, animal welfare and the environment.

Official Control

Maximum levels for aflatoxins set by the EU should be subject to official

control in order to ensure and enable effective enforcement. To this end,

competent authorities throughout Community should perform the same

sampling criteria and achieve the same analysis performance in accordance

with Commission Regulation EC No 401/2006 [96] amended by EC No

178/2010 regulation [97].

Official controls are necessary to ensure safe imported and exported

products. EU is a major importer and exporter of foodstuffs worldwide. To

protect public health and to ensure, amongst other, safe products at export and

import, EC has put into effect regulations and decisions which safeguard

contaminated products to aflatoxins. Commission Regulation (EC) No

1152/2009 [98] imposes special conditions governing the import of certain

foodstuffs from certain third countries due to contamination risk by aflatoxins

[99]. A Common Entry Document is used in order for the authorities to be

informed prior to the shipment of the consignment. Commission Regulation

(EC) No 669/2009 implementing regulation EC No 882/2004 dealing with the

increased level of official controls on imports of certain feed and food of non-

animal origin (also referred to as high – risk legislation) provides for the use of

Document.

In other cases, in order to further facilitate decrease of controls at import

of products into the EC, pre-export controls on aflatoxin can be applied as

described under article 23 of Commission Regulation EC No 882/2004 [100].

After inspection of the Food and Veterinary Office on USA‘s control system

and related laboratories for aflatoxin levels in peanuts, approval of pre-export

was granted by Commission Decision to USA in 2007.


Monitoring and Reporting

Monitoring and reporting of aflatoxin levels after controls are essential

actions which assist to identify potential risks to human health. For that reason,

EC has put into effect the Rapid Alert System for Food and Feed. In addition,

an EU Reference Laboratory [101] has been established for mycotoxins, and

guidance on different aspects regarding contaminants in food and feed has

been issued assisting related authorities, namely the ‗Guidance document for

competent authorities for the control of compliance with EU legislation on

aflatoxins‘ [102].

EU legislation on aflatoxins in food and feed is regularly amended and

updated in light of new scientific evidence, EFSA‘s opinions and statements,

and in line with the international Codex Alimentarius. This is essential in order

to safeguard public health, place in the market safe and wholesome products

and safeguard the important role of trade.

4.3. USA - Legal Framework

In the early 1900‘s, USA enacted the ‗Wiley act‘ (named after Harvey W.

Wiley) with the official title ‗Pure Food and Drug Act‘. Prior to the Act, a

number of products had been released in the market, and deemed legal without

the need of pre-market approval. In many cases such products provoked

adverse health effects to consumers. On the basis of the Wiley Act, interstate

commerce of adulterated and misbranded food and drugs was prohibited [103].

Certain deficiencies of the obsolete Act and a major outbreak due to an unsafe

drug gave pace to a substantial amendment. After about 30 years, in 1938, a

new act was drawn up mandating, among other, food standards, and improved

food packaging policy [104]. The Federal Food, Drug and Cosmetic Act –

FD&C Act [103] increased substantially the level of government intervention

in the food, drug and agricultural markets and established the legal framework

within which the Food and Drug Administration - FDA operates. In this

context, significant power was given to FDA (former Bureau of Chemistry), as

part (since 1968) of the Public Health Service [105]. The federal agency is

charged with the FD&C Act enforcement ensuring that food is safe, pure,

wholesome and appropriately labelled. Although FDA is responsible for the

safety of quite all food, there are other federal agencies (about 15)

administering food safety related laws, and of the most importance is Food

Safety and Inspection Service – FSIS (meat inspections) which is part of the

Department of Agriculture [106]. Together with the Centers for Disease


Control and Prevention – CDC [107], which is the link between foodborne

illness and food safety systems (governmental and food producers), these three

agencies cooperate closely to enforce the US food safety Law.

The case of aflatoxins appears in the FD&C Act under section 402(a)(1),

where food and feed containing naturally occurring contaminants are

considered to be ―adulterated‖ if they are deemed by FDA to be injurious to

human or animal health. In order for the FDA to evaluate whether adulteration

of food or feed (domestic or imported) has taken place, regulations and

guidance are developed. FDA regulations have a federal character and are

based on the laws set forth in the FD&C Act. FDA guidance is not legally

binding to the public or FDA and defines how the agency deals with a

regulatory issue. In addition to the above, FDA provides a convenient and

organized system for statements – Compliance Policy Guides Manual- of its

compliance policy including those statements which contain regulatory action

guidance information [108].

Maximum Levels

By the 1960‘s, aflatoxins as food contaminants were identified and at that

time about half of the food supply was subject to standards in the U.S. [109].

With the scope to reduce natural occurred contamination in foodstuffs and

feedstuff, FDA issues policy guidance (or enforcement pronouncements) in

one of the following three forms:

Advisory Levels - Provide guidance to the industry concerning levels

of a substance present in food or feed but enforcement is not the

fundamental purpose of an advisory level.

Action Levels - Specify a precise level of contamination at which the

agency is prepared to take regulatory action. Action levels alert the

industry that FDA believes it has the scientific data to support

regulatory and/or court action if a toxin or contaminant is present at

levels exceeding the action level if the agency chooses to do so.

Regulatory Limits – Are established after issuing valid regulations

under the public notice and comment rulemaking procedures.

For aflatoxins, the FDA has established Action Levels according to the

intended use of food (CPG sec. 555.400, 570.200, 570.500 and 570.375) and

feed (CPG sec. 683.100) [103]. Table 2 depicts such levels for a number of

products.


The Center for Food Safety and Applied Nutrition - CFSAN is the branch

of the FDA that is responsible for establishing standards of tolerance levels.

Table 2. FDA Action Levels for aflatoxins in human food,

animal feed and animal feed ingredients

Products Intended Use

Aflatoxins

Level

(ppb)

Notes

Milk Direct

consumption

0.5 Identity of aflatoxin

M1 is confirmed by

the chemical

derivative test

Foods, peanuts and

peanut products,

brazil nuts, and

pistachio nuts

Direct

consumption

20 Not raw peanut

products*. Identity

of aflatoxin B1 is

confirmed by

chemical derivative

Corn and peanut

products

Finishing beef

cattle

300 I.e. feedlot

Cottonseed meal Beef cattle,

swine, or poultry

300 Regardless of age or

breeding status

Corn and peanut

products

Finishing swine

≥ 100 pounds

200

Corn and peanut

products

Breeding beef

cattle, breeding

swine, or mature

poultry

100

Corn, peanut

products, and other

animal feeds and

feed ingredients

Immature

animals

20 Excluding

cottonseed meal

intended for

immature animals

Peanut products,

cottonseed meal, and

other animal feeds

and feed ingredients

Dairy animals,

animal not listed

above, or when

the intended use

is unknown

20

* The USDA has a comprehensive program involving raw peanuts which can be

expected to result in proper processing or destruction of any high aflatoxin raw

peanuts.


Official Control and Inspections

FDA has issued the below compliance programs for mycotoxins,

including aflatoxins, which are updated and reissued periodically due to

changes in methodology, and number of products to be collected.

Mycotoxins in Domestic Foods (CFSAN)

Mycotoxins in Imported Foods (CFSAN)

Feed Contaminants Program (CVM)

The programs‘ objectives are to control, monitor and report, among other

contaminants, aflatoxins. More specifically they aim to collect and analyse

domestic and import samples of various food products and to determine the

occurrence and levels of aflatoxins.

In addition to the above programs, prevention (pre-harvest – post-harvest)

and decontamination strategies exist for grains [110].

Recent Developments in U.S. Food Safety Policy

The Federal Food, Drug and Cosmetic Act concerning Food Safety (food

contaminants) was subject to numerous amendments through the years. In

2011, the US congress enacted the ‗FDA Food and Drug Modernization Act –

FSMA‘ [111], the most extensive amendment of the Food Safety Laws in the

last 7 decades. The amendment was deemed necessary due to incidents of

foodborne illness outbreaks and the need of a higher level of Food Safety in

general (e.g. protection against bioterrorism). In contrary to previous food

safety policy which was structured to respond in contamination of food, the

FSMA makes use of a systematic approach to food safety management and

aims to prevent contamination of food and feed.

The basic Law enhancements of SFMA are listed below:

Preventive controls: Food facilities are required to implement a

written preventive control plan; FDA‘s produce safety standards;

FDA regulations to prevent intentional contamination of food and

feed.

Inspection and Compliance: the FDA ensures compliance of

producers and processors to the preventive control standards by

establishing a mandated inspection frequency for food facilities based

on risk; accessing records of food safety plans; and assures that testing

laboratories are accredited meeting high-quality standards.


Imported Food Safety: the FDA ensures that imported products meet

U.S. standards by: (i) regulating importers‘ responsibility to verify

that their foreign suppliers have adequate preventive controls in place;

(ii) qualifying third parties to certify foreign facilities which comply

with U.S. food standards; (iii) requiring third party certification of

compliance (or other assurance) of high-risk imported foods; (iv)

establishing a voluntary qualified importer program; (v) and having

the authority to deny entry of food from a foreign facility which

refuses access.

Response: the FDA uses tools in order to respond effectively to food

contamination when preventive controls fail to. Mandatory recall of

unsafe food, expanded administrative detention of suspect food,

suspension of registration of a facility, enhanced track and trace

system for both domestic and imported foods and feeds and additional

recordkeeping for high risk foods are such tools.

Enhanced Partnerships: the FDA initiates a formal system of

collaboration with other domestic and foreign government agencies in

order to achieve public health goals.

The above enhancements, among other objectives, aim to prevent

contamination of food and feed from aflatoxins, and improve food quality of

domestic and imported products. Depending also on the considerable funding

needed, the implementation of the new Act will take time until rule making

process, driven by the FDA, issues final rules and regulations with

transparency after public consultation [112].

4.4. Other Countries

Other countries such as Turkey, Bosnia and Herzegovina and Switzerland

seem to be influenced by the EU Regulation and tend to have comprehensive

legislation controlling the level of aflatoxins. As for international markets,

China followed by Brazil and Mexico have the most comprehensive legislation

on aflatoxins. Specific maximum limits are set for aflatoxin B1 and/or total

aflatoxin in several foodstuffs in these countries. Other countries such as

Canada, Australia and New Zealand, Gulf Cooperation Council (GCC) and

Nigeria lay down specific limits for total aflatoxins mainly in nuts as indicated

in Table 3 [113].


Table 3. Total aflatoxins limits in Australia/New Zealand, Canada,

Codex, GCC, Nigeria, India, USA and South Africa

Country Foodstuffs

Total

aflatoxins

(µg/kg)

Australia/

New Zealand

Peanuts

Tree nuts

15

Canada Nut and nut products 15

Codex

GCC(a)

Nigeria

Peanuts, almonds, shelled Brazil nuts, hazelnuts

pistachios intended for further processing

15

Almonds, hazelnuts, pistachios, shelled Brazil nuts,

―ready-to-eat‖

10

India Wheat, maize, jawar (sorghum) and bajra, rice, whole

and split pulse (dal) masur (lentil), whole and split

pulse urd (mung bean), whole and split pulse moong

(green gram), whole and split pulse chana (gram),

split pulse arhar (red gram), and other food grains

30

Groundnut kernels (shelled) (peanuts); 30

USA Brazil nuts, peanuts and peanut products, pistachio

products

20

South Africa Peanuts 15

(a) Members of GCC are Saudi Arabia, United Arab Emirates (UAE), Kuwait,

Bahrain, Oman, Yemen and Qatar.

Countries such as India establish general maximum levels for total

aflatoxins for foods: 30 µg/kg and 20 µg/kg, respectively. In South Africa, a

general maximum limit for total aflatoxins is set at 10 µg/kg and additionally a

general maximum limit for aflatoxin B1 is set at 5 µg/kg for all foodstuffs.

Recently, Japan has established strict tolerance levels also for total

aflatoxins in all foodstuffs which must not exceed 10 µg/kg.

It is evident that established tolerance levels among countries vary in a

great extent. This reflects on great differences in risk perception of each

country and/or region. Developing countries, mainly located in tropical areas,

encounter greater contamination in commodities from aflatoxins. A major part

of these countries export a significant amount of such commodities to

developed countries which tend to emanate more stringent standards. In that

context, developing countries are forced to balance their policy taking into

account trade interests, food security and food safety issues; a task which is

not trivial and leads to higher tolerance levels. On the contrary, developed

countries, having in place more advanced and efficient agricultural practices


and manufacturing processes are able to achieve lower levels of aflatoxins in

commodities. Thus, they focus more on public health issues and food safety

resulting in stringent limits.

Table 4. Aflatoxin M1 limits

Country Foodstuffs Aflatoxin M1

(μg/kg)

EU

Bosnia and

Herzegovina

Turkey

Raw milk, heat-treated milk and milk for the

manufacture of milk-based products

0.050

Infant formulae and follow-on formulae,

including infant milk and follow-on milk

0.025

(products ready

to use)

Dietary foods for special medical purposes

intended specifically for infants

0.025

(products ready

to use)

China Milk and milk products (for milk powder,

calculated on a fresh milk basis)

0.5

Formulated foods for infants (milk or milk

protein based)

0.5

(calculated on

a dry powder

basis)

Formulated foods for older infants and

young children (milk or milk protein based)

0.5

(calculated on

a dry powder

basis)

Formulated foods for special medical

purposes intended for infants

0.5

(calculated on

a dry powder

basis)

Codex, GCC,

India, Kenya,

USA

Milk 0.5

Argentina

Milk, liquid including milk used in the

manufacture of milk and milk products and

reconstituted milk

0.5 [1]

Milk, powder 5.0

Milk formula ND

Mexico Pasteurised, ultrapasteurised, sterilised and

dehydrated milk, milk formula and combined

milk products

0.5 [1]

South Africa Milk 0.05

ND: Not Detectable, [1] Given in µg/l.


Significant effort dealing with above aspects is made in an international

level. In order to achieve harmonization and avoid misuse of the

‗precautionary approach‘ Codex Alimentarious sets standards to be respected

from countries under WTO international agreement. However, countries may

establish different limits upon scientifically based evidence maintaining

current differences of standards among countries.

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Chapter 9

FOOD SOURCES AND OCCURRENCE

OF AFLATOXINS:

THE EXPERIENCE IN GREECE

Ioannis N. Tsakiris,1 Elisavet Maria Renieri,

2

Maria Vlachou,2 Eleftheria Theodoropoulou,

2

Marina Goumenou2 and Aristides M. Tsatsakis,

2

1TEI of Western Macedonia, Florina Branch,

Department of Agricultural Products Marketing

and Quality Control, Terma Kontopoulou 53100 Florina, Greece 2Laboratory of Toxicology, Department of Medicine, University of Crete,

Voutes 71003, Heraklion, Greece

ABSTRACT

This paper presents a review of the occurrence of aflatoxins in

different food commodities in Greece, based both on results represented

in literature as well as results derived from monitoring programs of the

Center of Toxicology Science & Research, Medical School, University of

Crete. Aflatoxins, can pose a severe threat to food safety, since they are

characterized carcinogenic to humans, IARC Group 1. They may be

formed or developed in any stage of the agricultural production (primary

Author for correspondence: Ioannis N. Tsakiris. Email: [email protected], Mob: 0030

6977270004.

Ioannis N. Tsakiris, Elisavet Maria Renieri, Maria Vlachou et al. 234

production, processing and storage) as a result of transitional weather

conditions or of poor storage. Studies, monitoring programs and surveys,

which have been carried out in Greece, are mainly focused in milk and

dairy products. In this context, several studies have been conducted in

animal feeds as well, since there is notable evidence that they are

potential sources of aflatoxins in milk production. Additionally, both

black and green olives have been examined for possible contamination by

aflatoxins, due to the fact that they are damaged during harvest and

processing and thus providing a substrate for aflatoxin development.

Finally, a limited number of studies investigate the presence of aflatoxins

in different processed products like breakfast cereals. The above

foodstuffs have been studied on account of their high nutritional value

and the fact that they are consumed by different population groups.

Results indicate that residue levels of aflatoxins which are presented in

fresh as well as processed agricultural products, do not pose any

considerable risk for the Greek population groups. The most important

factors influencing the levels of aflatoxins in major agricultural products

appear to be the growing and cultivation techniques, as well as the food

safety parameters during harvesting, storage and processing. An

additional issue, which seems to raise concern internationally, is the fact

that climate change in combination with modifications in the cultivation

techniques may affect the frequency and severity of aflatoxin residues in

agricultural products.

LIST OF ABBREVIATIONS

AFB1 Aflatoxin B1

AFM1 Aflatoxin M1

ELISA Enzyme linked immunosorbent assays

GC Gas chromatography

GAP Good Agricultural Practice

HACCP Hazard Analysis and Critical Control Point

HPLC High performance liquid chromatography

IARC International Agency for Research on Cancer

LC-MS/MS liquid chromatography- tandem mass spectrometry

NIRS Near infrared reflectance spectroscopy

TLC Thin-layer chromatography

Food Sources and Occurrence of Aflatoxins 235

1. INTRODUCTION

Aflatoxins are a group of toxic secondary metabolites produced by certain

strains of the fungi Aspergillus flavus, Aspergillus parasiticus and the rare

Aspergillus nomius (Rahimi et al., 2010, Cano-Sancho et al., 2010), which

contaminate agricultural products both primary and processed. Aflatoxins may

subsequently enter the food chain creating serious risks to animal and human

health. The production of these compounds is highly influenced by several

environmental factors such as soil composition, insect infestation, temperature

and water availability (aw) both pre- and post- harvest (Paterson and Lima,

2010). Aflatoxins may be present in a wide range of food commodities,

particularly cereals, oilseeds, spices and tree nuts (Pitt et al., 2013). Maize,

peanuts, pistachios, brazils, black pepper, dried fruit and figs are all known to

be high-risk foods for aflatoxin contamination, but the toxin has also been

detected in many other commodities. Milk, cheese and other dairy products are

also known to be at risk of contamination by aflatoxins (Tsakiris et al., 2013a;

Tsakiris et al., 2013b ). The highest levels are usually found in commodities

from warmer regions of the world where there is a great deal of climatic

variation.

Aflatoxins are the most studied mycotoxins and mainly include aflatoxin

B1, B2, G1 and G2 with aflatoxin B1 being the most toxic of these

metabolites. A.flavus produces exclusively B aflatoxins, while A. parasiticus

and A. nomius produce B and G types (Fallah, 2010; Sidhu et al., 2009).

Aflatoxins are extremely toxic and present teratogenic, mutagenic and

carcinogenic effects that have been demonstrated in several studies (Green et

al., 1982; Wangikar et al., 2005; Mckean et al., 2006; El-Sayed and Khalil,

2009). Aflatoxin B1 (AFB1) has been classified as a Group 1 human

carcinogen by the International Agency for Research on Cancer (IARC)

(IARC, 1993). The main monohydroxylated derivative of AFB1 is aflatoxin

M1 (AFM1) which is also of great interest since it is formed in liver and

excreted into milk. Even though AFM1 is less toxic than AFB1, it has been

classified as a Group 2B possible human carcinogen by the IARC. Figure 1

presents the chemical structure of AFB1 and AFM1.

In Greece, most studies and monitoring programs which deal with the

occurrence, risk assessment and control of aflatoxins in foodstuff are mainly

focused in milk and dairy products (Roussi et al., 2013; Malissiova et al.,

2013; Tsakiris et al., 2013a ). In this context, interest has also been laid on

animal feeds and the possible hazards they represent for the food chain

(Vlachou et al., 2004).


Figure 1. Chemical Structure of Aflatoxin B1(a) and M1(b).

Occurrence of aflatoxins has also been investigated for black and green

olives originating from Greece, since it‘s a product of high nutritional value

and a major component of the Mediterranean diet (Ghitakou et al., 2006). A

more limited number of studies have been carried out on crops and processed

products such as breakfast cereals (Villa and Markaki, 2009). Other products,

less essential but considerably frequent components of the Greek diet such as

pistachio nuts and bee pollen, have been under investigation for possible

aflatoxin contamination.

Aspergillus lipases are involved in the mechanisms that follow fungal

invasion as A. flavus primarily destroys the lipid body formation. The fatty

acid contained in the lipid bodies of olive seeds is composed of palmitic, oleic

and linoleic acid which promote the Aspergillus spp. growth. Olives support

aflatoxin production and their biosynthesis is affected by lipid oxidation.

Therefore, it is possible for toxinogenesis to occur which may transfer into

olive oil (Leontopoulos et al., 2003; Ghitakou et al., 2006).

Cereal crops are exposed to fungal invasion both before harvest in the

field and post-harvest during storage and processing. Growing and cultivation

techniques seem to influence aflatoxin levels in cereal crops. Bee pollen which

is increasingly consumed due to its beneficial properties may be a substrate for

aflatogenic fungi and AFB1 production is likely even following a minor

contamination (Pitta and Markaki, 2010). High levels of AFB1 found in

pistachio nuts on the other hand, render it dominant among other types of

aflatoxins also detected in them. Pistachio nuts may be contaminated in every

stage from maturity till storage with maturity being the most critical stage for

aflatoxin contamination (Cheraghali et al., 2007; Georgiadou et al., 2012).


The aforementioned products are of great economic importance and most

of them are consumed on a daily basis by all age groups. It should be noted

that even if these products contain low levels of aflatoxins, they contribute to

the daily intake causing a cumulative effect over a period of years. Excessive

intake of products containing aflatoxins at high levels on a daily basis can

create considerable health problems. Therefore, it is necessary to establish a

critical control point system in order to monitor exposure and minimize health

risks associated with aflatoxin intake.

In Greece, the regulations concerning aflatoxins in food are conformed to

the European Commission regulation (EC, 2006), according to which the

maximum limits of AFB1 and total aflatoxins (sum of AFB1, AFB2, AFG1

and AFG2) were established in specific foodstuffs ranging from 2.0 to 12.0

μg/kg for AFB1 and 4.0–15.0 μg/kg for total aflatoxins. Considering that

imported foods are likely to be major sources of aflatoxins as well as

aflatoxigenic moulds, special food control measures have been implemented in

order to minimize risk of aflatoxin occurrence in foodstuffs imported from

other countries (EC, 2009a and EC, 2009b). Products exceeding the maximum

levels should not be placed on the markets of the EU. Directive 2002/32/EC

lays down maximum levels for aflatoxins B1 in feed materials.

The present chapter focuses on reviewing the status of aflatoxins in

Greece and carries out a basic comparison with other Mediterranean countries.

In this context, it is essential to point out the critical factors which raise

concern such as human health impact due to aflatoxin accumulation. The daily

intake of aflatoxins depends on the concentration in food, the amount

consumed and the frequency of the consumption. Risk assessment will provide

a link between hazards in the food chain and the actual risks to human health.

2. CRITICAL POINTS FOR THE DETERMINATION

OF AFLATOXIN IN AGRICULTURAL PRODUCTS

Monitoring for aflatoxins is performed by national certified laboratories

such as the General Chemical State Laboratory, which is reporting to the

Hellenic Food Authority (EFET), by private ones and by university

laboratories for research purposes. In previous years results from National

laboratories, were reported to European Food Safety Authorities as part of the

food safety and quality control program complied with the EU 1881/2006

regulation. Private laboratories perform analysis for aflatoxins in different


substrates mainly for food industries as a prerequisite for certification

purposes.

There are several analytical methods and procedures for the detection of

aflatoxins in agricultural products. Thin-layer chromatography (TLC) was one

of the first methods used to detect aflatoxins in agricultural products

(Fernández-Ibañez et al., 2009). Usually immunological methods i.e. enzyme

linked immunosorbent assays (ELISA) are used for monitoring purposes while

several methods based on high performance liquid chromatography (HPLC)

and gas chromatography (GC) are being used as well (Muscarella et al., 2007)

The great majority of proposed methods for the detection of aflatoxins suggest

two steps including isolation from the substrate with extraction procedures and

quantification using different analytical methods and techniques i.e. liquid-

liquid extraction, supercritical fluid extraction and solid-phase extraction

(Tripathi and Mishra, 2009). HPLC and ELISA are highly sensitive methods

compared to the rest of the suggested methods and are characterized by low

detection limits, a very important asset in risk assessment studies. The

disadvantage of these methods is the fact they are time consuming, require a

well trained personnel and use non-environmentally friendly solvents.

Moreover, ELISA may present pseudo-positive results and unacceptable

quantification accuracy (Wang et al., 2011). Due to the fact that aflatoxins are

not the only mycotoxins infecting agricultural products, liquid

chromatography coupled to mass spectrometry (LC-MS) or tandem MS (LC-

MS/MS) methods are developed for simultaneous detection of different

mycotoxins in foodstuffs and feeds. While conducting risk assessment the co-

occurrence of several mycotoxins in the same sample is crucial since it is

affecting their mode of action and consequently their adverse health effects

(Ibañez-Vea et al., 2012). During the last years rapid methods for the detection

of aflatoxins on site have been developed. Additionally HPLC and GC require

expensive analytical instruments and this reduces their suitability for screening

purposes. Near infrared reflectance spectroscopy (NIRS) is one of the most

promising methods mainly due to rapid and low-cost characteristics. With the

exception of NIRS all previously mentioned techniques have been used in

studies performed in Greece.

The analyzed agri-food products for aflatoxins residues may be divided

into the following categories: 1) Cereals (primary production and processed

products) and small grains such as wheat, barley and rice 2) Milk and dietary

products i.e. butter, yogurt 3) Nuts and dried fruits 4) Feeds and 5) Other i.e.

olives, olive oil, bee pollen. The most important factors affecting aflatoxin

contamination are biological and environmental while harvesting, storage and


distribution-processing conditions of agricultural products are also crucial

(Paterson and Lima, 2010). Sampling procedures are also of great importance

during aflatoxins analysis. The proposed sampling plans, techniques and

approaches should ensure that the samples are representative which in some

cases is really difficult mostly due to the quantity of initial batches i.e.

containers with cereals.

As the consumption of food is increasing and climate is changing the

detection of aflatoxins in agricultural products is a major challenge and

concern for food safety. Due to globalization, agricultural products with

aflatoxins may travel all over the world. That in combination with the fact that

there is an increasing number of new products in the food market, results in

need for less time consuming methods, but still reliable in order to monitor

food within the tight time limits set by commercial needs (Stroka and Anklam,

2002). Fast methods requiring less specialized personnel for screening

purposes as well as more precise methods might be a part of future food safety

strategy against aflatoxins.

3. AFLATOXINS IN THE GREEK PRODUCTS

3.1. Milk and Dairy Products

As already mentioned milk and dairy products are of great importance for

the agricultural economy and in the same time are characterized by high

nutritional value. Almost 32% of EU milk production is consumed as fresh

milk while cheese, and butter represent the 37% and 16% of milk used

(http://ec.europa.eu /agriculture/publi/fact/milk/2007_en.pdf). Beside cows,

ewes and goats‘ milk, feta cheese and yogurt are the most important dairy

products produced and consumed in Greece. It is well known up to know that

the Greek dairy industry is accounting for 17% of the total food and drink

production in sector (http://gain.fas.usda.gov/Recent%20GAIN%20 Publi

cations/Greece%20Dairy%20Semi-Annual%202012_Rome_Greece_6-8-20

12.pdf). Based on the recent literature, it is concluded that aflatoxin M1

(AFM1) is one the major xenobiotics affecting milk and dairy products

worldwide, in terms of safety and quality (Tsakiris et al., 2013b). Aflatoxin B1

found in animal feeds is metabolized to M1 and secreted in to milk.

Consumption of M1 contaminated milk and dairy products is of great concern

for consumers mainly because of the severe impact on human health and

especially for sensitive human groups such as neonates and children (Oveisi et


al., 2006). Greece and other Mediterranean countries are characterized by

climate conditions, which potentially favor the occurrence of AFM1 in milk

and milk products indicating a high-risk geographical area. Despite the fact of

the importance of milk and dairy products in the local economy and diet the

number of surveys in literature are limited.

Feta is one the most well known Greek cheese, daily consumed as one the

major diet components. During 1985 Penicillium and Aspergilus seem to be

the most common fungus species found on teleme cheese (similar to feta)

while no residues of aflatoxins were detected in 94 commercial teleme cheese

samples analyzed (Zefiridis, 1985). AFM1 was also not detected in a similar

study performed during 1989 in feta and teleme cheese samples

(Karaioannoglou et al., 1989). Another monitoring study was performed

during 2002, from March to June (Kaniou-Grigoriadou et al., 2005). More

specific, a total of 162 samples of ewe‘s milk, curd and feta cheese were

monitored for residues of AFM1. All milk samples presented residues lower

than 50 ng l-1

, while in curd, which is an intermediate product and is not

consumable, the levels ranged from non-detectable up to 84.1 ng kg-1

. The

concentration ratio of AFM1 from initial milk samples to corresponding curd

present values from 4.3 up to 5.6. In the final products, produced after two

months of ripening, no residues of AFM1 were detected. The detection of

AFM1 in selected samples was based on ELISA.

The surveys referring to the occurrence of AFM1 in yoghurt are even

more limited compared to milk and feta. To the best of our knowledge the

occurrence of AFM1 during the production process of yoghourt alone, has

been evaluated in Greece, using a liquid chromatographic system with a

spectrofluorometric detector (Govaris et al., 2002). In general, levels of AFM1

seem to be decreased during yoghurt production and only Streptococcus

thermophiles (from lactic acid bacteria) presented a lower increase in yoghurt

with high AFM1 concentration. Additionally pH 4.0 seems to have a negative

effect on the stability of AFM1 especially after fermentation compared to pH

4.6 in which AFM1 seems to be more stable.

Karaioannoglou in 1989 presented the first survey of AFM1 on raw and

pasteurized milk. The levels of AFM1 in raw milk varied from 100 up to 130

ng l-1

in four out of 99 samples while no residues were detected in pasteurized

milk. In another monitoring study in Greece during 1995 and 1996, 81 samples

of commercial pasteurized milk from Athens market were analyzed for the

presence of AFM1. From 81 samples analyzed, only 3 presented residues in

levels exceeding the current MRLs (160, 170 and 177 ng l-1

). The rest of the

samples, with an exception of 9 samples in which no AFM1 was detected,


were found to contain less than 5 ng l-1

(Markaki and Melissari, 1997). The

analytical methods applied for the detection of AFM1 in milk in both studies

were ELISA and HPLC (for quantification in samples contained AFM1 above

5 ng l-1

).

Another monitoring study in raw and pasteurized milk was performed

from 1999 until 2001 from December up to May for each year. A total of 114

of pasteurized, ultrahigh temperature-treated and concentrated milk (UHT)

samples were analyzed for AFM1 residues. In the same study another 52 raw

milk samples from cow, sheep and goat were monitored (Roussi et al., 2002).

More than 80% of pasteurized, UHT and concentrated milk samples had

residues of AFM1 (though lower than the MRL) while for raw milk the levels

were ranging from 40% up to 73% of the total samples. Only 2 raw cow

(3.4%), 1 raw sheep (3.7%) and 2 concentrated (13.3%) samples presented

residues of AFM1 above the limit of 50 ng l-1

. Ιn the specific study it was

concluded that during the collection of milk in milk industries the

contamination of raw milk samples in the bulk tank results in higher frequency

and lower concentration of AFM1 residues. A liquid chromatographic system

equipped with a spectrofluorometer was used for the detection of AFM1 in the

selected milk samples.

Nowadays the objective of monitoring studies is to evaluate human

exposure to AFM1 and potential risk via dietary consumption. From

November 2009 until June 2010, 196 milk samples from the Greek market

(conventional, organic and kids milk) with different lipid content were

monitored for AFM1 residues using an ELISA method. Only two milk

samples were found above MRLs while risk assessment scenarios developed

for ages 1, 3, 5, 7 and 12 presented Hazard Index (HI) ratio less than one, with

highest HI values during ages of 1-3 (Tsakiris et al., 2013). In another study

243 samples of ewe‘s and goat‘s milk, conventional and organic, where

collected from December 2010 until July 2011, directly from farms, and

monitored for AFM1 residues (Malissiova et al., 2013). Only 1.7% of milk

samples, which were organic, presented AFM1 residues above MRLs. Organic

milk samples presented higher contamination levels when compared with

conventional ones.

Comparing the reported results in milk with recent related studies

performed in other Mediterranean countries we may say that similar mean

values are presented. In Italy 316 pasteurized samples were monitored during

2003 to 2005 for Aflatoxin M1 and the reported mean value was 27 ng/L while

the reported range of detection was 10-90 ng/L (Nachtmann et al., 2007). In

Spain 72 samples were monitored for aflatoxin M1 with reported mean value


of 9.69 ng/Kg and maximum reported value of 13.61 ng/kg (Cano-Sancho et

al., 2010). In Portugal 40 samples of pasteurized milk were monitored for

AFM1 during 2011 with reported mean value 23.4 ng/L (Duarte et al., 2013).

Since feta is a traditional Greek cheese protected by domination of origin it is

not possible to compare the results with other Mediterranean countries. As far

as yogurt is concerned results for AFM1 residues are very limited. Only one

study from Portugal presented details from ninety-six samples of yogurt

monitored for AFM1 residues during 2001. AFM1 was detected in 18.8% of

samples ranging from 19 to 98 ng/kg (Martins, 2004).

Based on the presented results we may conclude the AFM1 is still present

in milk and dairy products but the severity and frequency of detection are

reduced within the last decades. Monitoring results should be followed by risk

assessment studies in order to access the potential adverse health effects in

humans (especially in sensitive groups such as children).

3.2. Olive and Olive Oils

Olive and its derivatives, especially olive oil, are key components of the

Mediterranean diet as well as one of the basic products of the Mediterranean

basin. Greece is the world‘s third larger producer of olive oil coming after

Spain and Italy. Despite having the highest annual per capita consumption in

the world (16 kg), one third of annual production (135.000 t) goes to export,

thus rendering Greece the leading exporter of extra virgin olive oil (IOC).

Greece also supports a large production of table olives, with many varieties

which include Konservolia (dual-purpose variety), Kalamon (another dual-

purpose variety) and lastly Chalkidiki. Through the last decade, table olive

production has remained above 100.000 t a year (IOC).

The Mediterranean diet features olive oil as the primary source of fat, due

to its beneficial health effects. "Extra-virgin" and "virgin" olive oils in

particular, contain a high level of polyphenols, which act like antioxidants.

The medicinal and nutritional value of olives and olive oil though, may be

significantly reduced by the development of fungi on olives, because they can

disturb the synthesis of fatty acids (El Adlouni et al., 2006) as well as lead to

the production of aflatoxins.

Mold growth and mycotoxin production are generally related to weather

extremes. As far as olives are concerned, inadequate storage conditions, such

as prolonged contact with the ground and weakly ventilated places both favor

the toxinogenic moulds‘ colonization (Ferracane et al., 2007). This may result


in the contamination of the olive, with a strong possibility to transfer to the

olive oil. Olives which are held under conditions of high humidity and

temperature are often contaminated by toxigenic fungi (Pardo et al., 2005),

although toxigenic molds do not grow equally well on all varieties of Greek

olives (Ghitakou et al., 2006). Most storage fungi belong to the genus

Aspergillus which is adapted to low moisture conditions. However, mold

growth often occurs in products due to recontamination (Leontopoulos et al.,

2003).

Nevertheless, the studies concerning aflatoxin production on olives are

few in number. Reports from several authors show that olives may be

contaminated with a wide variety of molds including Aspergillus parasiticus

(Ghitakou et al., 2006), as well as that the possible consequent presence of

aflatoxins in olives, can lead to their transfer in oil (Cavaliere et al., 2007;

Ferracane et al., 2007). Some incidences of aflatoxin occurrence in olive oil

have been reported from Greece (Daradimos et al., 2000; Leontopoulos et al.,

2003; Papachristou and Markaki, 2004; Ghitakou et al., 2006), Spain

(Cavaliere et al., 2007), Morocco (Cavaliere et al., 2007), and other

Mediterranean countries. The leading factor which promotes mold growth and

mycotoxin production in olives seems to be the inadequate storage practices

(Cavaliere et al., 2007; Papachristou and Markaki, 2004; Ferracane et al.,

2007). However, reports on aflatoxin contamination in olives and their

products are sometimes contradictory.

Daradimos et al., 2000 analyzed 50 samples of Greek olive oil for

aflatoxin B1, and found traces in 72% of samples tested, within a range

generally very low (2.8 ± 15. 7 ng/kg) with only one sample to be

contaminated with 46.3 ng/kg. The levels were lower than levels allowed even

by the strictest regulation existing for aflatoxins, such as for aflatoxin M1 in

liquid milk with the advisory level in some countries being 0.05 mg/kg or 0.5

mg/kg. Previous reports state that olive oil samples originating from Greece

and Spain were found to contain AFB1 at levels of 5±10 mg/kg (Toussaint et

al., 1997). Therefore, the study presented by Daradimos et al., 2000 indicates a

big reduction of the contamination level in the olive oil of Greek origin during

the last decades.

Other data on the aflatoxins occurrence in olive and olive oil samples from

Greece reveal that the presence of AFB1 is infrequent and rather limited. The

Papachristou and Markaki, 2004 study showed the presence of aflatoxin B1

(60 ng/kg) in only one out of 50 olive oil samples from Southern Greece.

Besides these results (2004), data from Leontopoulos et al., 2003 confirm that

olives are a feeble substrate for the biosynthesis of AFB1. Leontopoulos et al.,


2003 also stated that in black olives and olive oil produced in Greece,

occurrence of AFB1 is limited and not dangerous even though among all kinds

of olives, the black‗ ‗Greek style‘‘ is the most exposed to mold contamination

(Tantaoui-Elaraki and Mannioui, 1996; Ghitakou et al., 2006). Ghitakou et al.,

2006 revealed AFB1 presence ranging between 0.15–1.13 ng AFB1 /g in all

the tested olives from Greek retail market. AFB1 production in two different

varieties of black olives after inoculation by A. parasiticus was found to be not

significantly higher compared with control samples.

In the majority of studies conducted in Greece since the year 2000, HPLC

technique with fluorescence detector was applied following aflatoxin

extraction and purification by immunoaffinity columns clean – up step.

Recovery levels and sensitivity have proven to be quite satisfactory (Ghitakou

et al., 2006; Daradimos et al., 2000; Leontopoulos et al., 2003; Papachristou

and Markaki, 2004). More sophisticated techniques such as liquid

chromatography- tandem mass spectrometry (LC-MS/MS) have been applied

to olive oils but although suitable for confirmatory analysis the sensitivity was

less than that of HPLC-fluorescence detection. Furthermore, the equipment is

expensive and requires considerable operator expertise (Mahoney and

Molyneux, 2010).

Aflatoxin occurrence in olives and olive oils has been under investigation

in other Mediterranean countries as well, mostly Spain, Italy, Morocco and

Egypt, since olive and olive oil consumption constitute a major part of the

Mediterranean diet as aforementioned. Table black olives ―Greek style‖

produced in Egypt, found to be the most exposed to mold contamination

(Yassa 1994). In that study, a total of 40 mold strains were isolated from black

table olives produced in Egypt, whilst nine out 40 were strains of A. flavus and

five strains of A. parasiticus which were found to produce AFB1 on culture

media and olive paste. In a more recent study, presence of AFB1 has been

confirmed in four out of ten olive samples, bought at retailer and at

supermarket in Morocco and ranged between 0.5 and 5 μg/kg (El Adlouni et

al., 2006).

Aflatoxin contamination of olive oil has received the most attention but

the results obtained have been somewhat inconsistent. Total aflatoxin levels of

0.006–0.04 ppb were found in 46% of 28 Sicilian olive oil samples examined

(Finoli et al., 2005). In a later study, Cavaliere et al., 2007, out of 20

experimental and 15 commercial olive oil samples analyzed by LC-MS/MS

only three of the latter were contaminated.

Ferracane et al., 2007 analyzed 30 samples of virgin oils from southern

Italy and Morocco, and reported 0.54 to 2.50 ng/g (ppb) AFB1 in 10% of


samples. AFB1 was also found in three out of four samples from North Africa

(up to 2.4 ng/g). Contamination levels of AFB1 seem to be rather similar

among the Mediterranean countries including Greece and were generally

found to be low.

The presence of aflatoxins can be limited during the refining steps that

virgin and extra virgin olive oils are produced. Stringent conditions for seed

cleaning, extraction, high-temperature heating, degumming, bleaching and

deodorizing can lead to the elimination of aflatoxins in highly refined oils

(Bao et al., 2010). Although the refining step prior the analysis of oil partially

removes aflatoxins from contaminated oil (Le Tutour et al., 1983; Parker and

Melnick 1996), pressed olive oil has been shown to contain 18–47% of the

aflatoxins originally present in the contaminated olives (Mahjoub and

Bullerman 1990). Moreover, olive oils processed through cold-pressed

methods allow for antioxidants to be retained therefore special attention must

be given to proper harvesting and preservation of the olives so that they

remain uncontaminated.

In some other cases, limited AFB1 production in damaged olives was

supported by the presence of antimicrobial constituents such as caffeic acid,

coumarins, flavones, catechin and phenolic compounds (Ghitakou et al., 2006).

Furthermore, Aziz et al., 1998 reported that compounds extracted from olive

fruits, such as oleuropein, inhibited aflatoxin production. Another important

factor is microbial competition. Leontopoulos et al., 2003 mentioned

previously that treatment does not eliminate totally the natural microflora

present in olives. In the Ghitakou et al., 2006 study microbial competition

seems to restrain high AFB1 production in selected kinds of olives. However,

AFB1 production is different depending on the substrate. AFB1 levels

produced in all three kinds of olives were dependent a) on the variety of

substrates and b) on the time of incubation. Finally, there doesn‘t seem to be a

correlation between mycotoxin occurrence and conventional qualitative

parameters such as peroxide number, spectrophotometric evaluation and acid

values (Ferracane et al., 2007).

It is essential that olives are stored and handled properly to prevent the

growth of aflatoxigenic mold and AFB1 production. Conditions during storage

can be controlled to a greater extent than in the field. In order to achieve high

quality and safety of final products, optimization of storage conditions

(temperature, salt content, packaging) could inhibit contamination from

toxigenic molds and mycotoxin production (Ghitakou et al., 2006).

Furthermore, application of strict regulation for the AFB1 in olive oil is

essential in order to fortify the safe AFB1 daily intake. Current tolerance


levels set by the European Community for most food products are 2 ppb

aflatoxin B1 and 4 ppb total aflatoxins, although edible oils are not specifically

addressed (EC, 2006). Therefore, it is not possible to assess the risk due to the

AFB1 occurrence in olives since to our knowledge an AFB1 Tolerable Daily

Intake is not established for humans yet.

Although the levels of Aflatoxin detected in olives and olive oils

originated from aflatoxins in Greek products in general do not reach alarming

levels but instead they are well below normal levels. However, the frequent

consumption even at low levels can pose a hazard to public health.

Considering that Greece has the highest consumption per capita, the daily

consumption of olive oil containing low level aflatoxins significantly

contributes to the total daily intake of aflatoxins. The possibility of synergism

with other mycotoxins present in other food commodities must also be taken

under consideration.

3.3. Feeds

Aflatoxin contamination constitutes a major issue not only in food but also

in feeds. Measures for the contamination of feed are essential since they have

an enormous health and economic significance. There are two types of feeds.

Feeds of animal origin that are usually commercial compound feeds and feeds

of vegetable origin. Common feeds of vegetable origin used are maize grain,

barley grain, wheat grain and cottonseed meal. Feeds also often contain corn,

oat and field pea.

In order to ensure the safety of food products deriving from animals it is

important to maintain a good level of feed hygiene. The moisture content of

feeds is a crucial factor for the development of moulds and the subsequent

production of aflatoxins. The initial mould population is indicative of the

hygienic status of feedstuff. Factors promoting aflatoxins in feed are high

moisture and temperature, the type of feed and the quality of feed storage

(Vlachou et al., 2004; Malissiova et al., 2013; Oluwafemi et al., 2009). Feed

contamination with aflatoxins occurs on a global scale but the relative impact

of toxins can vary depending on the different geographical characteristics. In

Greece and the Mediterranean area, the humid conditions and warm climate

favor toxin production. Contamination can occur during processing of

products pre- and/or post- harvest whenever conditions allow the development

of spoilage fungi. Inadequate pre-harvest and post-harvest conditions and

practices (e.g. improper drying of grains after harvest), poor storage, insect


attack, non-use of mold inhibitors, are all factors that facilitate aflatoxin

contamination in feed ingredients (Boudra and Morgavi, 2005). The

application of Good Agricultural Practice (GAP) programs as well as the

establishment of Hazard Analysis and Critical Control Point (HACCP)

systems is considered essential for the control of aflatoxin contamination in

feeds. As an additional control method, feed millers are encouraged to add

toxin binders of fungal growth inhibitors to their feeds. However, in the case

of organic farming the restriction of fungicide use may affect the quality of the

feed ingredients used (Malissiova et al., 2013).

Vlachou et al., 2004 investigated the presence of AFB1 in feed samples

collected from storage areas of animal farms from all provinces of Greece. The

samples corresponded to the most common ingredients used in feeds at the

time: maize grain, barley grain, wheat grain and cottonseed meal. Samples of

commercial compound feeds were also examined. TLC technique was used to

measure AFB1. AFB1 was detected in 7 out of 183 samples containing levels

less than the maximum permitted EU limit for feeds (20-50 ppb). AFB1 was

only detected in maize grain and cottonseed meal. Maize grain presented the

highest moisture levels while cottonseed meal the lowest. At the time of the

investigation, even the maize sample that was found containing 90 ppb AFB1

could have been used as feed by diluting it with uncontaminated maize grain

according to a provision of EU law that stated that raw materials containing up

to 200 ppb of AFB1 could be used by Recognized Feed Manufacturers.

However, this directive changed in 2003 due to certain food crises scandals

and the possibility to dilute contaminated feeds was banned (EU, 2002; EU,

2003). Generally, aflatoxin B1 does not constitute a problem for feeds in

Greece.

In general, studies on the presence of aflatoxins in animal feeds are

limited. A couple of papers are reported from Morocco, which is surrounded

by the Mediterranean Sea and the Atlantic Ocean and thus presents a similar

climate with the Southern Greece as it is characterized by high temperature

and humidity. Kichou and Walser (1993) analyzed 315 samples of poultry

feeds using a semi-quantitative ELISA and TLC methods. The feed

ingredients included corn, wheat, soybean meal, sunflower meal, cottonseed

meal and sorghum. Contamination levels ranged from 20-200 μg/kg apart from

4 samples that reached AFB1 levels from 2000-5625 μg/kg. The latter highly

contaminated feed samples were linked to clinical aflatoxicosis in chickens. In

another survey, Zinedine et al. (2007b) analysed 21 samples from Rabat. The

results exposed a 66.6% of aflatoxin contamination while AFB1 contamination

levels of poultry feeds ranged between 0.05 and 5.38 μg/kg. In both studies


from Morocco, the contamination levels often exceeded the MRLs set by the

European Regulations in feedstuff. This can be attributed to the fact that food

safety and quality standards practices such as GAP, Good Manufacturing

Practices and HACCP system are often not applied by the Moroccan food

production units (Zinedine and Manes, 2009).

AFM1 as discussed earlier has been traced in the milk of sheep, cows,

goats, camels and buffalos in different proportion ranges (Battacone et al.,

2003; Battacone et al., 2005). The relationship between the ingested AFB1 and

the AFM1 excreted in milk varies according to the animal breed, the

production of milk and the frequency of the daily milking. Because of AFM1

toxicity, the European Union has set the maximum limit of detection at 50 ppt

(EC, 2006).

The environmental conditions in Greece favor AFB1 production in feeds.

Therefore the presence of AFM1 in milk is possible as it was pointed out by

Roussi et al. (2002). Since an important part of the Greek economy relies on

farming of goats, a few studies have investigated the AFM1 carryover in the

milk of Greek goats. Kourousekos et al. (2012) divided 30 greek goats into 3

groups of ten goats each. One group was used as control and the other two

groups were treated. Each goat received 50 or 100 μg of AFB1 per day for 35

days. According to the results, after AFB1 administration, AFM1 was

detectable in quite high concentrations in the milk of goats from the two

treated groups. AFM1 concentrations increased with the increase of the

administrated AFB1 dosage. 59.38% of milk samples from the group

administered with 50 μg AFB1 and 83.33% from the group administered with

100 μg AFB1 presented AFM1 concentrations >80 ppt. AFM1 concentrations

in most milk samples from the two treated groups exceeded the maximum

limit of 50 ppt set by the European Union. This practically shows that doses of

even 50 μg AFB1 included in feeds may render the milk unsafe. The excretion

of AFM1 appears to be direct and depends on the AFB1 dose.

Malissiova et al. (2013) as already reported performed another study in

Greece involving the monitoring of AFM1 levels in ewe's and goat's milk. The

results suggest the use of safe feeds by the majority of the monitored farms as

well as secure production practices. Risk factors potentially related to AFM1

contamination include winter season, the use of warehouse for feed storage

and feeding pea. Moreover differences noticed in levels of AFM1 in milk in

different countries may be attributed to the different climatic conditions and

the feed storage practices (Zinedine et al., 2007a; Montagna et al., 2008;

Magan et al., 2011).


In general, AFB1 does not constitute a problem for feeds in Greece.

However, appropriate measures to prevent contamination should never be

ignored. It is essential to follow a HACCP system as well as a GAP program.

Finally, feed manufacturers should comply with the regulations and EU

legislation to ensure feed and consequently food safety.

3.4. Aflatoxins in Minor Crops and Food Commodities

Aflatoxins can be found as well on other food commodities that are

otherwise considered beneficial for the human health. Bee pollen, pistachio

nuts and breakfast cereals are food items that could be consumed daily by the

inhabitants of Greece.

Bee pollen is known for its medical properties (antibiotic, antioxidant,

antineoplastic, antidiarhoeic) since ancient times and it is nowadays used as a

food supplement. It consists mainly of protein, but also contains vitamins,

minerals, carotenoids, sugars, lipids as well as flavonoids and phenolic acids,

which is why it has such a strong antioxidant effect (Graikou et al., 2011).

There is only one study about the AFB1 in pollen of Greek origin (Pitta and

Markaki, 2010). In this study, a routine method was developed and validated

to determine AFB1 in bee pollen, using a simple extraction step,

immunoaffinity column clean-up and finally determination by HPLC with a

fluorescence detector. The main aim of the study was to examine if the bee

pollen can act as a substrate for the AFB1 production. There were three groups

of samples (15g pollen/flask) in the study: a) Bee pollen without any treatment

containing natural microbiota., b) Treated bee pollen, in order to eliminate the

natural microbiota and then inoculated with A. parasiticus., c) Treated, non-

inoculated bee pollen (control samples). The study revealed that in the first

group, where the pollen still had its natural micro biota, there was no

production of AFB1 throughout the incubation and observation time (at 30o C

for 20 days). Moreover, no mycelial growth was apparent, which could be

explained if the microbial competition is taken into account. On the contrary,

AFB1 production was observed in the other two groups with treated bee

pollen, inoculated with A. parasiticus as well as the control group (maximum

values: 79.29 ng/flask and 32.44 ng/flask, respectively, with the inoculated

group having significantly higher values, p≤0.05). These values occurred

during the 12th day of incubation and after that peak the concentration of

AFB1 started to decline. Furthermore, it is apparent that the treatment did not

eliminate all the aflatoxigenic molds of the bee pollen, but the production of


AFB1 in the inoculated group started on the 3rd day of the incubation, whereas

the control group appeared to have a delayed effect (7th day). In a previous

study it was shown, that in ready-to-eat bee pollen produced in Spain, A.

parasiticus was isolated, which was able to produce AFB1 (González et al.,

2005). Furthermore, a study on 20 samples of Spanish bee pollen yielded no

quantifiable amounts of five different mycotoxins, including AFB1 (Medina et

al., 2004).

Another food commodity known for its beneficial health properties

(especially for the cardiovascular system) are pistachio nuts. They are a rich

source of carotenoids, selenium and flavonoids, as well as other minerals,

sugars and fibers, proteins, several vitamins and fatty acids (Tsantili et al.,

2011). However, pistachio nuts as well have been found to be a substrate for

the production of AFB1. In a study conducted on samples of Greek pistachio

nuts of the variety ―Aegina (P. vera cv Aegina), five different steps of the

pistachio nuts cultivation and processing were examined (early maturity,

maturity, harvest, drying and storage), in order to detect where the highest

production of aflatoxins occurs and which factors affect it (Georgiadou et al.

2012).

The pistachio trees were from four different orchards and producers,

where the cultivation and processing of the nuts varied. However all the trees

were of the same variety and grew under the same weather conditions. The

results showed that the aflatoxins could occur at any stage of the procedure,

with AFB1 being the most abundant aflatoxin in all stages (its value was much

higher than G1, B2, and G2). However, maturity appeared to be the most

critical stage for aflatoxin contamination in pre-harvest stages (only one

orchard appeared to have a small amount of AFB1: 3.1 μg/kg). Higher levels

of aflatoxins were observed during maturity in the case where there was high

insect infestation (1191.47±148.34 μg/kg AFB1) compared to the orchards that

were well irrigated and where plant protection was applied (6.6±2.0, 15.0±3.7

and 33.7± 9.5 μg/kg AFB1). Furthermore, improper sun drying led to higher

AFB1 values (1058±31 μg/kg AFB1) compared to mechanical drying in a hot

air dryer (0.24±0.17 μg/kg AFB1). Finally, poor storage conditions of room

temperature and relatively high humidity also contributed to the production of

aflatoxins (926±81 and 371±37 μg/kg AFB1), while storing the pistachio nuts

under controlled conditions (5-7 °C and 45-60% RH) prevented the occurrence

of aflatoxins (0 μg/kg AFB1). These findings are in accordance to other

studies in pistachios from other countries. In addition, imports of nuts in

Greece are constantly controlled through the national market surveillance


program according to which sampling of cargos in Greek ports is

implemented.

Finally breakfast cereals were a minor food commodity and only one

paper in Greek literature provides details about analytical techniques and

levels of AFB1 in selected samples from Greek market (Villa and Markaki,

2009).

An HPLC method with fluorescence detector was used for the

determination of AFB1 in 55 cereal samples during 2006 and 2007 collected

randomly from super-markets in Athens. 56.3% of samples presented residues

of AFB1 while seven samples were found to be contaminated at levels higher

than the EU limit of 2 ng/g.

CONCLUSION

Aflatoxin M1 (AFM1) is one the major xenobiotics affecting milk and

dairy products in terms of safety and quality.

AFM1 residues were not detected in feta samples during the last

years. The concentration ratio of AFM1 from initial milk samples to

corresponding curd present values from 4.3 up to 5.6.

AFM1 is still present in milk in Greece and other Mediterranean

countries but the severity and frequency of detection are reduced

within the last decades. Monitoring results should be followed by risk

assessment studies in order to access the potential adverse health

effects in humans (especially in sensitive groups such as children).

The leading factor which promotes mould growth and mycotoxin

production in olives seems to be inadequate storage practices.

Studies presented within the last decades indicating a recent reduction

of the contamination level in the olive oil of Greek origin.

Contamination levels of AFB1 seem to be rather similar among the

Mediterranean countries including Greece and were generally found

to be low.

In Greece, as far as feeds are concerned, AFB1 was only detected in

maize grain and cotton seed meal but in general, aflatoxin B1 does not

constitute a problem for feeds in Greece. However, appropriate

measures to prevent contamination should never be ignored. It is

essential to follow a HACCP system as well as a GAP programme.


In minor crops such as pistachio, breakfast cereals and bee pollen

more monitoring studies are needed for safe conclusions.

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Chapter 10

AFLATOXINS AS SERIOUS THREATS

TO ECONOMY AND HEALTH

Lipika Sharma1, Bhawana Srivastava2, Shelly Rana

2,

Anand Sagar2 and N. K. Dubey

3

1G.B. Pant Institute for Himalayan Environment and Development

Himachal Unit, Mohal- Kullu, Himachal Pradesh, India 2Mycology & Plant Pathology Laboratory, Department of Bio-Sciences,

Himachal Pradesh University, Shimla, India 3Laboratory of Herbal Pesticide, Centre of Advanced Study in Botany,

Banaras Hindu University, Varanasi, India

ABSTRACT

This review deals with the aflatoxins especially with their food

sources, wide occurrence and toxicological effects on animals and

humans. Aflatoxins are highly oxygenated, heterocyclic,

difuranocoumarin compounds and are an important group of mycotoxins

produced by the fungi. There are almost 20 different types of aflatoxins

identified till now; among these AFB1 is considered to be the most toxic.

Aflatoxins persist to some extent in food even after the inactivation of the

fungi by food processing methods, such as ultra-high temperature

Corresponding Author: Dr. Bhawana Srivastava, (Dr. D.S. Kothari postdoctoral fellow, UGC),

Mycology & Plant Pathology Laboratory, Department of Bio-Sciences, Himachal Pradesh

University, Shimla, India-171005, Tel: 91-9889206615, 91-177-2621692, e-mail:

[email protected].

Lipika Sharma, Bhawana Srivastava, Shelly Rana et al. 260

products, due to their significant chemical stability. Aflatoxins can affect

a wide range of commodities including cereals, oilseeds, spices, and tree

nuts as well as milk, meat, and dried fruits. Twenty five percent of the

world‘s crops are affected with mycotoxins. On a worldwide scale, the

aflatoxins are found in stored food commodities and oil seeds. Some of

the foods on which aflatoxin producing fungi grow well include cereals

(maize, sorghum, pearl millet, rice, wheat, corn, oats, barley), oilseeds

(peanut, soybean, sunflower, cotton), spices (chile peppers, black pepper,

coriander, turmeric, ginger), and tree nuts (almond, pistachio, walnut,

coconuts), sweet potatoes, potatoes, sesame, cacao beans, almonds, etc.,

which on consumption pose health hazards to animals, including

aquaculture species of fish, and humans. Food commodities affected by

aflatoxins are also susceptible to other types of mycotoxins and multiple

mycotoxins can co-exist in the same commodity. Various cereals affected

by aflatoxins are also susceptible to contamination by fumonisins,

trichothecenes (especially deoxynivalenol), zearalenone, ochratoxin A

and ergot alkaloids.

More than 5 billion people in developing countries worldwide are at

risk of chronic exposure to naturally occurring aflatoxins through

contaminated foods. Aflatoxin is a potent liver toxin causing

hepatocarcinogenesis, hepatocellular hyperplasia, hepatic necrosis,

cirrhosis, biliary hyperplasia, and acute liver damage in affected animals.

Effects of aflatoxins in animals depend on age, dose and length of

exposure, species, breed and nutritional status of the animal. Health

effects occur in fish, companion animals, livestock, poultry and humans

because aflatoxins are potent hepatotoxins, immunosuppressants,

mutagens, carcinogens and teratogens. Aflatoxin– B1 has been shown to

cause significant morphological alterations along with reduced

phagocytic potential in chicken and turkey macrophages. Aflatoxin- B1

exposure to chicken embryos causes significant suppression in

macrophage phagocytic potential in chicks after hatch. Aflatoxin

intercalates into DNA and alkylates the DNA bases through its epoxide

moiety resulting in liver cancer. Other effects include mutagenic and

teratogenic effects. Exposure of biological systems to harmful levels of

aflatoxin results in the formation of epoxide, which reacts with proteins

and DNA leading to DNA-adducts, thus causing liver cancer. The

primary target of aflatoxins is the hepatic system. Acute effects include

hemorrhagic necrosis of the liver and bile duct proliferation while chronic

effects include hepatocellular carcinoma (HCC). HCC is the sixth most

prevalent cancer worldwide with a higher incidence rate within

developing countries. Preliminary evidence suggests that there may be an

interaction between chronic aflatoxin exposure and malnutrition,

immunosuppression, impaired growth, and diseases such as malaria and

HIV/AIDS. Outbreaks of acute aflatoxin poisoning are a recurrent public

health problem. The discussion of this problem and its remedies must be

Aflatoxins As Serious Threats to Economy and Health 261

held in the context of the associated question of food insufficiency and

more general economic challenges in developing countries. Aflatoxin

constitutes a serious health concern to the entire food chain, necessitating

a multidisciplinary approach to analysis, action, and solution.

Keywords: Aflatoxons, Aspergillus sp., Hepatotoxins, Mycotoxins,

Occurrence, Toxicological Effects

INTRODUCTION

One of the greatest challenges of the world is to produce enough food for

the growing population. The situation is particularly critical in developing

countries, where the rate of net food production is slowing down in relation to

population growth. The world food situation is aggravated by the fact that in

spite of the use of all available means of plant protection, about one-third of

the yearly harvest of the world is destroyed by pests (Varma and Dubey,

1999). Considerable postharvest losses of food commodities are brought about

by infestations caused by different pests. International agencies that monitor

world food resources have acknowledged that one of the most feasible options

for meeting future food needs is reduction of postharvest losses (Kelman,

1984; Tripathi and Dubey, 2004). Fungi are significant destroyers of

foodstuffs during storage, rendering them unfit for human consumption by

retarding their nutritive value and sometimes by producing mycotoxins.

Approximately 25–40% of cereals world-wide are contaminated with

mycotoxins produced by different storage fungi (Kumar et al., 2007). Tropical

countries, because of their temperature and congenial environment suffer

severe losses from pests due to conducive atmosphere. Generally, conditions

of these countries such as high temperatures and moisture, unseasoned rains

during harvest and flash floods lead to fungal proliferation and mycotoxins.

Aflatoxin belongs to a group of fungal toxins known as mycotoxins; these

are highly oxygenated, heterocyclic, difuranocoumarin compounds and are an

important group of mycotoxins produced by the fungi Aspergillus flavus, A.

parasiticus and A. nomius (Diaz et al., 2008).Other species of Aspergillus such

as A. bombycis, A. ochraceoroseusand A. pseudotamari mayalso produce

aflatoxins (Bennett & Klich, 2003; Klich et al., 2000; Mishra & Das, 2003;

Akbarsha et al., 2011). These species contaminate various agricultural

commodities either before harvest or at post-harvest stages under favorable

conditions of temperature and humidity. The discovery of aflatoxins dates


back to the year 1961 following the severe outbreak of turkey ―X‖ disease, in

the England, which resulted in the deaths of more than 100.000 turkeys and

other farm animals. Aflatoxins are toxic secondary metabolites produced by

Aspergillus fungus growing in susceptible agricultural commodities. They can

result in major economic losses and can negatively affect animal and human

health. The name aflatoxins, an acronym, has been formed from the following

combination : the first letter, ―A‖ for the genus Aspergillus, the next set of

three letters, ―FLA‖, for the species flavus, and the noun ―TOXIN‖ meaning

poison (Rustom,1997; Filazi & Sireli,2013).

Characteristics and Types

Once aflatoxin is produced, it is stable. Heat, cold and light do not affect

it. It is also colorless, odorless and tasteless, and because of the low

concentrations involved and the uneven distribution in grain bins, aflatoxins

are difficult to detect. Aflatoxins persist to some extent in food even after the

inactivation of the fungi by food processing methods, such as ultra-high

temperature products, due to their significant chemical stability (De Viries,

1997; Peraica, 1999). There are almost 20 different types of aflatoxins

identified till now, among these B1, B2, G1 andG2 are more prominent while

AFB1 is considered to be the most toxic (IARC,2002; Mushtaq et al. 2012).

There are four major natural aflatoxins (AFs), AFB1, AFB2, AFG1 and

AFG2. The hierarchy of toxicity of different aflatoxins is in the order

AFB1>AFG1>AFB2>AFG2. There are two additional metabolic products of

aflatoxins B1and B2, viz., M1 and M2. Aflatoxin M1 (AFM1) is a major

metabolite of aflatoxin B1 (AFB1), which is formed when animals ingest feed

contaminated with aflatoxin B1. The AFB1, once ingested by the animal, is

rapidly absorbed by the gastrointestinal tract and is transformed into the

metaboliteAFM1, which appears in the blood after 15 minutes and is then

secreted in the milk by the mammary gland (Van Egmond, 1989; Battacone, et

al. 2003). The amount of AFM1 which is found in milk depends on several

factors, such as animal breed, lactation period, mammary infections etc… It

has, anyway, been demonstrated that up to 6% of the ingested AFB1 is

secreted into the milk as aflatoxin M1 (Van Egmond & Dragacci, 2001) and,

because AFM1is relatively resistant to heat treatments (Yousef & Marth,

1989; Galvano et al., 1996), it is almost entirely retained in pasteurized milk,

powdered milk, and infant formula. Moreover,only a limited decrease of

AFM1 content has been verified in UHT milk after long storage(Galvano et


al., 1996; Martins & Martins, 2000; Tekinsen & Eken, 2008). Aflatoxin B1

(AFB1) is the most potent and potentially lethal metabolite and is a known

human carcinogen. The hepatotoxicity and carcinogenic effects of AFB1 have

been clearly demonstrated, thus it has long been classified as a group 1 human

carcinogen by the International Agency on Research on Cancer (IARC, 2002).

Initially, the IARC classified AFM1 as a possible carcinogen for humans

(group 2b) since toxicological data was limited (IARC, 1993). However,

genotoxicity and cancerogenity of AFM1 have been observed in vivo,

although lower than those of AFB1, and its cytotoxicity has been definitively

demonstrated (Caloni et al., 2006). As a result of these and other further

investigations, the IARC moved aflatoxin M1 from group 2B to group 1

human carcinogen (IARC, 2002, Anfossi et al., 2011).

Figure 1. Structure of some major aflatoxins.

Factors That Affect Aflatoxin Contamination

Factors that affect aflatoxin contamination include the climate of the

region, the genotype of the crop planted, soil type, minimum and maximum

daily temperatures, and daily net evaporation (Wilson and Payne 1994; Ono,


Sugiura et al. 1999; Brown, Chen et al. 2001; Bankole and Mabekoje 2004;

Fandohan, Gnonlonfin et al. 2005). Aflatoxin contamination is also promoted

by stress or damage to the crop due to drought prior to harvest, insect activity,

poor timing of harvest, heavy rains at harvest and post-harvest, and inadequate

drying of the crop before storage (Hell, Cardwell et al. 2000; Ono, Sasaki et al.

2002; Hawkins, Windham et al. 2005; Turner, Sylla et al. 2005). Humidity,

temperature, and aeration during drying and storage are major factors behind

contamination.

Table 1. Chemical and physical properties of aflatoxins

Aflatoxin Molecular

formula

Molecular weight Melting point

(0C )

B1 C17 H12O6 312 268-269

B2 C17 H14O6 314 286-289

G1 C17 H12O7 328 244-246

G2 C17 H14O7 330 237-240

M1 C17 H12O7 328 299

M2 C17 H14O7 330 293

B2A C17 H14O7 330 240

G2A C17 H14O8 346 190

Food Sources and Occurrence

Well-known within the agricultural community, aflatoxins have been

studied for over forty years due to their widespread occurrence and their

significant impact on crops (Eaton and Groopman 1994; Wild and Turner

2002; Shephard 2003; Fung and Clark 2004; Williams, Phillips et al. 2004).

Aflatoxins are toxic secondary metabolites produced by Aspergillus fungi.

Aflatoxins can affect a wide range of commodities including cereals, oilseeds,

spices, and tree nuts as well as milk, meat, and dried fruit. Twenty five percent

of the world‘s crops are affected with mycotoxins. Some of the foods on which

aflatoxin producing fungi grow well include cereals (maize, sorghum, pearl

millet, rice, wheat, corn ), oilseeds (peanut, soybean, sunflower, cotton), spices

(chile peppers, black pepper, coriander, turmeric, ginger), and tree nuts

(almond, pistachio, walnut, coconuts). Many of the above mentioned

ingredients are used in human food andin various livestock and poultry feed

rations and so these species are often contaminated with aflatoxin (Mushtaq et


al. 2012). The major sources of exposure are maize and groundnuts as these

are the foods that are most susceptible to contamination and consumed in the

greatest amounts. Aflatoxins are most prevalent in areas located between

latitudes 40ºN and 40ºS of the equator. On a worldwide scale, the aflatoxins

are found in stored food commodities and oil seeds such ascorn, peanuts,

cottonseed, rice, wheat, oats, barley, sorghum, millet, sweet potatoes, potatoes,

sesame, cacao beans, almonds, etc., which on consumption pose health

hazards to animals, including aquaculture species of fish, and humans (Abdel-

Wahab et al., 2008;Hussein & Brassel, 2001; Akbarsha et al., 2011). The

greatest risk for health impact lies within developing countries located in

tropical regions, which rely on these commodities as their staple food source.

Food insufficiency and lack of diversity substantially contribute to the

susceptibility of individuals and communities to aflatoxins.

Various approaches exist for the determination of aflatoxin in food and

feed commodities. Generally, all analytical methods follow the basic protocol

of extraction, clean-up, separation, detection, identification and quantification.

However, the most widely used techniques are those which include a

chromatographic step to separate the mycotoxin of interest like minicolumn

chromatography, thin layer chromatography, high performance liquid

chromatography and gas liquid chromatography.

Figure 2. Presentation showing economic loss because of aflatoxin occurrence.


Although immunoassay-based quantitative methods are fast and

promising, for mycotoxin research they have the possibility of producing mis-

leading results because of cross-reaction and interference in the complex

matrixes (Nilu & Boyaciog, 2002; Hu, 2006; Mushtaq et al. 2012).

Toxicological Effects

The health issues related to aflatoxins are equally complex and demand

more research. The ingested aflatoxin undergoes various possible pathways

depending on different parameters like dose quantity, type of species, age,

diet, and immune system of host. Aflatoxin is a potent liver toxin causing

hepato-carcinogenesis, hepatocellular hyperplasia, hepatic necrosis, cirrhosis,

biliary hyperplasia, and acute liver damage in affected animals. Other effects

include mutagenic and teratogenic effects. Large doses of aflatoxin are lethal

and chronic exposure to low levels of aflatoxin can result in cancer and

immunosuppression (Sharma, 1993; Yarru, 2008). Exposure of biological

systems to harmful levels of aflatoxin results in the formation of epoxide,

which reacts with proteins and DNA leading to DNA-adducts, thus causing

liver cancer (Turner et al., 2009; Groopman et al., 2008). Infants are at much

higher risks of health problems compared to adults (Sergent et al., 2008).

Aflatoxins, in general, and AFB1 in particular, can induce DNA damage, gene

mutation, sister-chromatid exchanges and other chromosomal anomalies,

which account for their genotoxic, teratogenic and carcinogenic properties

(Batt et al., 1980; International Agency for Research on Cancer (IARC, 1993;

Ray-Chaudhuri et al., 1980). AFB1 can form adducts with DNA, RNA and

protein, which form the major basis of the health risks (Sun et al., 2001;

Williams et al., 2004). The maximum legal limit allowed for AFB1 in infant

food in the European Union is 0.1 μg kg−1

(European Commission, 2006). In

developing countries, the majority of the people survive largely on cereal

based diets. Consequently, nutritional deficiencies are very prevalent in

populations consuming high levels of cereals, particularly in children (Bankole

& Adebenjo, 2003). Moreover, poor diet and multiple infectious hazards are

associated with malnutrition and growth faltering in infancy and childhood

(IARC, 2002). More than 5 billion people in developing countries worldwide a

great risk of chronic exposure to naturally occurring aflatoxins through

contaminated food (Shephard, 2003; Williams et al., 2004) and more so in the

tropical regions, where the climatic conditions favour luxurious growth of

Aspergillus spp, and people rely on commodities such as cereals, oilseeds,


spices, tree nuts, milk, meat and dried fruits that are potentially contaminated

by aflatoxins (Strosnider et al., 2006). In July 2005, the United States Centers

for Disease Control and Prevention (US CDC) and the World Health

Organization (WHO) hosted a workshop to create an integrated plan intended

to generate culturally appropriate, long-term, public health strategies to reduce

aflatoxin exposure in developing countries. Participants included 40

internationally recognized scientists from diverse backgrounds (i.e. public

health, agriculture, animal health, trade and social science) and key public

health officials and stakeholders from countries heavily affected by aflatoxins.

Aflatoxins affect many species including humans, dogs, feeder pigs, dairy

cattle, and chickens.

Effects of Aflatoxins on Animal Health

The effects of aflatoxins on animal health have been observed in many

species for over forty years (Patten 1981; Miller and Wilson 1994) beginning

with the documentation of Turkey X disease in 1960. The primary target of

aflatoxins is the hepatic system. Acute effects include hemorrhagic necrosis of

the liver and bile duct proliferation while chronic effects include

hepatocellular carcinoma (HCC). In animals, suppression of immunity, growth

retardation, and increased susceptibility to infectious disease due to aflatoxin

exposure is well-documented (Patten 1981; Miller and Wilson 1994). Trout,

one of the early models for aflatoxicosis is very sensitive to aflatoxin and

develop clinical signs such as hepatoma. Swine at weaning and marketing

stages are resistant to dietary levels of aflatoxins up to300 ppb. Clinical signs

associated with aflatoxicosis in dairy cattle include reduced feed intake, milk

production, weight gain and liver damage. Aflatoxin M1, an aflatoxin

metabolite found in milk, was also found in milk products such as yogurt

(Martins M.L e tal., 2004; Yarru, 2008). Symptoms of aflatoxicosis include

feed refusal, decreased feed efficiency, and stunted growth, decreased milk

production and impaired reproductive efficiency (Diekman& Green, 1992;

Oguz&Kurtoglu, 2000; Pier, 1992; Raju & Devegowda, 2000). Numerous

studies have shown that feed intake is reduced in broiler chicks fed aflatoxin

(Osborn et al., 1992; Ledoux et al., 1998). Reduced feed intake results in

decreased average daily gain (Ledoux et al., 1998). One important thing to be

determined is the effects of aflatoxin on nutrient digestibility and

bioavailability. This can be done by measuring the activity and levels of

various digestive enzymes and by evaluating serum chemistry of affected


animals. Balachandran and Ramakrishnan (1987) studied the influence of

dietary aflatoxin on serum enzyme levels in broiler chicken. They measured

SGPT, SGOT, serum amylase and lipase levels in Cobb broiler chickens fed

3ppm aflatoxin.

Table 2. FDA action levels for aflatoxins in human and animal foods

Commodities . Total aflatoxin

action level (ppb)

Human food 20

Milk 0.5

Beef cattle 300

Swine over 100 lbs 200

Breeding beef cattle, swine, or mature poultry 100

Corn for immature animals and dairy cattle 20

Corn for breeding beef cattle, swine and mature poultry 100

Corn for finishing swine 200

Corn for finishing beef cattle 300

Cottonseed meal (as feed ingredient) 300

All feedstuff other than corn 20

Source: Wu, et al. 2011.

They observed an increase in serum lipase and SGOT levels, and a

decrease in serum amylase levels all of which resulted in alteration in nutrient

digestion, absorption and metabolism. Osborne and Hamilton, (1981) observed

decreased serum lipase, amylase, trypsin, lipase, RNase, and DNase activities

in chickens fed aflatoxin. They did not measure serum calcium and

phosphorus levels which play a role in rubbery leg syndrome, one of the

symptoms associated with aflatoxicosis. Health effects occur in fish,

companion animals, livestock, poultry and humans because aflatoxins are

potent hepatotoxins, immunosuppressants, mutagens, carcinogens and

teratogens. Public health concerns center on both primary poisoning from

aflatoxins in commodities, food and feedstuffs, and relay poisoning from

aflatoxins in milk (Coppock& Christian, 2007). The ability of the immune

system to combat infection and disease is another key component of animal

health. Aflatoxin B1 has been shown to cause significant morphological

alterations along with reduced phagocytic potential in chicken (Neldon-ortiz

and Qureshi, 1991a) and turkey (Neldon-ortiz and Qureshi, 1991b)

macrophages. Aflatoxin-B1 exposure to chicken embryos causes significant


suppression in macrophage phagocytic potential in chicks after hatch (Neldon-

ortiz and Qureshi, 1991c; Yarru, 2008).

Effects of Aflatoxins on Human Health

The effects of aflatoxins on humans, as with animals, are dependent upon

dosage and duration of exposure. Acute exposure can result in aflatoxicosis,

which manifests as severe, acute hepatotoxicity with a case fatality rate of

approximately 25% (Cullen and Newberne 1994). Early symptoms of

hepatotoxicity from aflatoxicosis can manifest as anorexia, malaise, and low-

grade fever.

Figure 3. A picture summarizing main characteristics of aflatoxins.

Acute high level exposure can progress to potentially lethal hepatitis with

vomiting, abdominal pain, jaundice, fulminant hepatic failure, and death.

Outbreaks of acute aflatoxicosis are a recurring public health problem

throughout the world (Krishnamachari, Bhat et al. 1975; Ngindu, Johnson et

al. 1982; Lye, Ghazali et al. 1995; CDC 2004).Hepatocellular carcinoma

(HCC) as a result of chronic exposure has been well documented, generally in

association with hepatitis B virus or other risk factors (Qian, Ross et al. 1994;


Wang, Hatch et al. 1996; Chen, Chen et al. 2001; Henry, Bosch et al. 2002;

Omer, Kuijsten et al. 2004). The International Agency for Research on Cancer

(IARC) first recognized aflatoxins as carcinogenic in 1976 and has

subsequently reaffirmed naturally occurring mixtures of aflatoxins and

aflatoxin B1 as Group 1 carcinogens (carcinogenic to humans) (IARC 2002).

Additional effects of chronic exposure have not been widely studied but are

thought to include immunologic suppression, impaired growth, and nutritional

interference (Patten 1981; Cullen and Newberne 1994; Fung and Clark 2004;

Williams, Phillips et al. 1994).

Health Impact and Burden of Disease

HCC is the sixth most prevalent cancer worldwide with a higher incidence

rate within developing countries (Parkin, Bray et al. 2005), however, the

burden of HCC attributable to aflatoxins when accounting for other co-

morbidities, such as hepatitis B (HBV), is not known. Several studies in China

have indicated combined exposure to HBV and aflatoxins is associated with a

much higher risk of HCC (Qian, Ross et al. 1994; Wang, Hatch et al. 1996).

This interaction has not been studied in other high risk areas such as sub-

Saharan Africa and the molecular mechanism of the interaction between HBV

and aflatoxins is not known (Turner, Sylla et al. 2002; Wild and Turner 2002).

Quantifying the proportion of HCC attributable to aflatoxin exposure, to HBV,

and to the interaction of aflatoxin exposure and HBV will help identify the

best public health strategy to reduce HCC, including the benefits and limits of

widespread HBV vaccination. Additional health effects associated with

chronic aflatoxin exposure have not been well studied. Without knowing the

relationship between chronic exposure and health, the true human health

impact and the resulting burden of disease in developing countries are not

known. Preliminary evidence suggests that there may be an interaction

between chronic aflatoxin exposure and malnutrition, immunosuppression,

impaired growth, and diseases such as malaria and HIV/AIDS. Experimental

animal evidence suggests that chronic exposure to aflatoxins may lead to

impaired immunity, reduced uptake of nutrients from the diet, and growth

retardation (Hall and Wild 1994; Miller and Wilson 1994). Several studies of

children in Benin and Togo have shown an association between aflatoxin

albumin adduct levels and impaired growth (Gong, Cardwell et al. 2002;

Gong, Egal et al. 2003; Gong, Hounsa et al. 2004). In a recent study in Ghana,

higher levels of aflatoxin B1-albumin adducts in plasma were associated with


lower percentages of certain leukocyte immune phenotypes (Jiang, Jolly et al.

2005). A study in Gambian children found an association between serum

aflatoxin-albumin levels and reduced salivatory secretory IgA levels (Turner,

Moore et al. 2003). While the effects on immunity suggest the possible

influence of aflatoxins on susceptibility to infectious disease, further

investigation is needed.

Aflatoxin associated health effects pervade the developing world despite

the fact that these effects could be mitigated or prevented with the current state

of agricultural knowledge and public health practice. The discussion of this

problem and its remedies must be held in the context of the associated

question of food insufficiency and more general economic challenges in

developing countries. Outbreaks of acute aflatoxin poisoning are a recurrent

public health problem. In 2004, one of the largest, most severe aflatoxicosis

outbreaks occurred in Kenya followed by another outbreak in 2005 (CDC

2004). Given that diseases in the developing world often go unreported, the

Kenya outbreaks are likely to be an underestimation of the problem;

furthermore, the burden of disease attributable to chronic aflatoxin exposure

(e.g. hepatocellular carcinoma, impaired growth, immune suppression)

remains undefined. These outbreaks emphasize the need to quantify and

control aflatoxin exposure in developing countries and highlight the potential

role of public health.

A broad range of signs and symptoms can be used to diagnose

aflatoxicosis based on the level of exposure. The signs and symptoms of this

condition include vomiting, abdominal pain and hemorrhaging, pulmonary

edema, acute liver damage, loss of digestive tract function, convulsions,

cerebral edema, and coma. Hepatitis B vaccinations, education through

awareness campaigns, and chemoprevention measures such as competitive

displacement, plant extract application, and methyl eugenol spray have proven

to be effective interventions in controlling and preventing the adverse health

effects of aflatoxin exposure (Right Diagnosis, 2011). Aflatoxin constitutes a

serious health concern to the entire food chain, necessitating a

multidisciplinary approach to analysis, action, and solution. To maximize

resources, a targeted monitoring and surveillance system for high-risk areas

and their populations should collect and analyze appropriate specimens

(usually food, urine, and serum) (Strosnider, 2006). Aflatoxicosis is a disease

caused by aflatoxin poisoning. The disease can be acute, meaning it is caused

by the short-term exposure to high levels of aflatoxin, or chronic, meaning that

it has been caused by long-term exposure to low to moderate levels of


aflatoxin. Symptoms differ between the acute and chronic forms of the disease

and have been outlined in this section.

Acute Aflatoxicosis

Acute aflatoxicosis, associated with extremely high doses of aflatoxin, is

characterized by hemorrhaging, acute liver damage, edema, and high mortality

rates in humans. Acute aflatoxicosis is associated with sporadic outbreaks of

the consumption of highly contaminated foods. Early symptoms of acute high

level exposure to aflatoxin include diminished appetite, malaise, and low

fever; later symptoms, which include vomiting, abdominal pain, and hepatitis,

can signal potentially fatal liver failure (Barret, 2005). Acute aflatoxicosis in

animals was first documented in 1960, after more than 100,000 turkeys died

following an outbreak in the United Kingdom (Wu et al., 2011.)

Chronic Aflatoxicosis

Chronic aflatoxicosis is associated with long-term exposure to low to

moderate levels of aflatoxin in the food supply. Chronic low-level exposure to

aflatoxin, particularly aflatoxin B1, is associated with an increased risk of

developing hepatocellular carcinoma, or liver cancer, as well as impaired

immune function and malnutrition and stunted growth in children. Aflatoxin

B1 is the most potent liver carcinogen and is found in greater concentrations

than any other naturally occurring aflatoxin (Wu et al., 2011; Bhat, &

Vasanthi, 2003). According to the World Health Organization (WHO),

hepatocellular carcinoma is the third leading cause of cancer deaths globally

(WHO., 2008). Approximately 83 percent of cancer fatalities in East Asia and

Sub-Saharan Africa are due to liver cancer (Parkin et al., 2002; Strosnider et

al., 2006; Kirk et al., 2006). Hepatocellular carcinoma, as a result of chronic

aflatoxin exposure, presents most often in persons with a chronic hepatitis B

virus and/or chronic hepatitis C virus infections (Groopman & Kensler, 2005;

Wu & Khlangwiset, 2010; Liu & Wu, 2010). This indicates that exposure to

aflatoxin and hepatitis binfection, key risk factors for liver cancer, are

particularly prevalent in developing nations in which people subsist largely on

grains (Liu & Wu, 2010). Chronic aflatoxicosis also increases the risk of

developing impaired immune function and malnutrition, a concern already

prevalent in populations consuming high levels of cereals (Wu et al., 2011;


Bhat & Vasanthi, 2003; Bankole & Adebanjp, 2003). Cancer risk assessments

and acute toxicity studies acrossspecies show that adult humans are relatively

tolerant of aflatoxin; however, data reviewed in earlier sections indicate that

there is evidence that aflatoxin exposure affects early development, as well as

some aspects of human immunity and nutritional processes (Williams et al.,

2004). Maize (Zeamayis L.) and peanuts (Arachishypogaea L.) form the staple

food of many African and Asian diets. As these two crops are highly

susceptible to aflatoxin infection, the incidence of aflatoxin exposure is closely

related to the subsistence diet of populations in developing countries (Liu &

Wu, 2010). From 2001 to2003, developing countries produced 46 percent of

the global maize crop (Wu et al., 2011). Poor harvesting and storage practices

and weak regulations of mycotoxin contamination in developing countries

exacerbate rates of aflatoxin exposure (Wild & Gong, 2010).

Links to HIV and TB

It has been suggested that the immune suppression and nutritional effects

of chronic aflatoxin exposure may be linked to the high prevalence of HIV.

This possible link, however, is not conclusive, as research targeting the cancer-

causing effects of aflatoxin has generally overshadowed research focusing on

nutrition and immunity (Shekhar et al., 2009). Aflatoxin exposure has been

shown to cause immune suppression, particularly in cell-mediated responses

(Safarac et al., 2010).

The correlation between aflatoxin-albumin levels and CD4 counts in HIV

positive individuals has recently been studied. CD4 interacts with cells that act

as the gateway for HIV infection. CD4 proteins that have been weakened by

aflatoxin exposure may correlate positively with HIV infection (Nyandieka et

al., 2009). In addition, for the first time, new research has linked high aflatoxin

levels with an increased risk of developing tuberculosis (TB) in HIV positive

individuals. TB transmission associated with aflatoxin exposure raises a new

health concern among HIV positive individuals, in addition to concerns related

to increased susceptibility to liver disease (Allameh et al., 2005). Persons who

are exposed to aflatoxin and are HIV positive have decreased plasma vitamin

A and vitamin E in the blood, although there was no interaction detected

between aflatoxin and HIV infection (Safamehr, 2008). Nevertheless, other

mechanisms have been proposed to explain the link between HIV and

aflatoxin exposure. Williams et al. hypothesized that HIV infection is likely to

increase aflatoxin exposure by two possible routes: (1) HIV infection


decreases the levels of antioxidant nutrients that promote the detoxification of

aflatoxin, or (2) the high degree of co-infection of HIV-infected people with

hepatitis B also increases the biological exposure to aflatoxin. Although no

specific studies on humans have yet been conducted, the evidence suggests a

decrease in animal immune systems as a result of aflatoxin exposure

(Dixon et al., 2008)

Co-Existence and Interactions of Multiple Mycotoxins

Food commodities affected by aflatoxins are also susceptible to other

types of mycotoxins and multiple mycotoxins can co-exist in the same

commodity (Bankole and Mabekoje 2004; Fung and Clark 2004; Speijers and

Speijers 2004).

Various cereals affected by aflatoxins are also susceptible to

contamination by fumonisins, trichothecenes (especially deoxynivalenol),

zearalenone, ochratoxin A and ergot alkaloids. Maize can be contaminated

with aflatoxins, fumonisin, trichothecenes, zearalenone and, rarely,

ochratoxin-A, while wheat can be contaminated with aflatoxins,

trichothecenes, ochratoxin-A, ergot alkaloids and zearalenone. Therefore

individuals may be exposed to various combinations of mycotoxins (CAST

2003). The health effects associated with exposure to multiple mycotoxins are

not well documented. Related mycotoxins are thought to have an additive

effect while unrelated mycotoxins may have a synergistic effect (Speijers and

Speijers 2004). A better understanding of exposure to multiple mycotoxins and

the health effects associated with the interactions of multiple mycotoxins

would clarify the true health impact of mycotoxins.

Future Prospective

As it has been mentioned before, most aflatoxicosis results from eating

contaminated foods. Unfortunately, except for supportive therapy (e.g., diet

and hydration) there are almost no treatments for aflatoxin exposure. In Figure

4, we reproduce an overview for preventing acute aflatoxicosis in developing

countries (Strosnider et. al., 2006). Methods for controlling aflatoxin exposure

are largely prophylactic. In a primary prevention trial, the goal is to reduce

exposure to aflatoxins in the diet. A range of interventions includes planting

pest-resistant varieties of staple crops, attempting to lower mold growth in


harvested crops, improving storage methods following harvest, and using

trapping agents that block the uptake of unavoidably ingested aflatoxins. In

secondary prevention trials, one goal is to modulate the metabolism of

ingested aflatoxin to enhance detoxification processes, thereby reducing

internal dose and subsequent risk (Groopman, 2008,).

Figure 4. Overview of preparedness, surveillance and response activities for preventing

acute aflatoxicosis (Strosnider et. al., 2006).

Further, more studies and researches are needed in order to develop

appropriate technology for treatment of aflatoxin exposure and to minimize

their resulting health effects. Aflatoxins are not only a big problem at crop

production level, but also it has become a global health issue because of the

consequences that the consumption of this toxin generates in animals and

human beings. Diverse worldwide established groups have the challenge of


identifying public health strategies, which complement the agricultural ones in

order to reduce aflatoxin exposure, especially in developing countries.

Although there have been documented some researches about how to prevent

and control aflatoxicosis, but a little is known about aflatoxin exposure and the

resulting health effects. Efforts to reduce aflatoxin exposure require the

commitment of sufficient resources and the collaboration between the

agriculture and public health communities as well as local, regional, national,

and international governments.

Because of the recent investigations conducted, it is important to take

actions to prevent damage and diseases; thats why, at first, governments

supported by scientific research groups should report publicly the risks that

aflatoxins consumption means by quantifying the human health impacts and

the burden of disease due to the toxin exposure; then, they should compile

inventory and worldwide statistics in order to evaluate the efficacy of the

current intervention strategies. It is also important to increase disease

surveillance, food monitoring, laboratory detection of mycotoxins and public

health response capacity of affected regions. Public health services should

offer immediate attention to aflatoxicosis diagnoses and opportunistic diseases

caused by them in order to reduce mortality rates in humans and animals.

Finally, it is important to develop response protocols to be used in an event of

an outbreak of acute aflatoxicosis, which could become in an epidemic stage.

ACKNOWLEDGMENTS

The authors are thankful to the University Grants Commission, New Delhi

for providing financial assistance in the form of Dr. D.S. Kothari postdoctoral

fellowship and to Food and Public Health Branch of the Food and

Environmental Hygiene Department of HKSAR Government for their report.

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INDEX

A

access, 159, 219, 242, 251

accounting, 209, 239, 270

acetonitrile, 16

acetylcholinesterase, 200

acid, 11, 12, 16, 23, 25, 28, 50, 51, 54, 56,

57, 59, 85, 133, 143, 160, 185, 186, 190,

195, 236, 245, 283

activity level, 178

acute aflatoxin poisoning, xv, 260, 271

adaptation, 181

additives, 83, 253

adenine, 151

adenovirus, 155

adjustment, 115

adolescents, 278

adsorption, 11, 27

adults, 203, 204, 266

adverse conditions, vii, 1

adverse effects, ix, 6, 64, 210

advisory body, 205

aetiology, 152

aflatoxicosis, xiii, 8, 26, 30, 77, 79, 88, 104,

105, 192, 196, 198, 200, 223, 247, 267,

268, 269, 271, 272, 274, 275, 276, 281,

282, 283, 285

Africa, 45, 71, 80, 88, 122, 198, 201, 227,

272, 278, 280, 284, 285, 286

agar, 23, 57, 165, 171

age, xv, 5, 80, 85, 128, 129, 135, 138, 141,

142, 147, 155, 194, 202, 203, 204, 217,

237, 260, 266

agencies, 194, 209, 215, 219, 261

agribusiness, xi, 108, 113

agricultural market, 215

agriculture, ix, 28, 30, 32, 64, 222, 239, 267,

276

AIDS, 100

albumin, 80, 81, 94, 98, 99, 101, 103, 105,

136, 137, 151, 201, 227, 270, 273, 278,

280

alkaloids, xv, 260, 274

allele, 149

almonds, xiv, 5, 69, 70, 76, 78, 84, 112,

113, 121, 207, 208, 212, 220, 229, 260,

265

alveolar macrophage, 96, 103

amines, 29, 58, 85, 226, 254, 280

amino, 143, 146, 147, 181, 184

amino acid(s), 143, 146, 147

ammonia, 12, 200, 277, 283

amylase, 268

anatomy, 189

animal disease(s), 8, 210

animal feeds, xiv, 12, 217, 222, 234, 235,

239, 247, 278

animal welfare, 214

annealing, 132

anorexia, 6, 79, 269

ANOVA, 18

Index 288

antibiotic, 249

antibody, 10, 99, 100, 131, 201

antigen, 104, 129, 206, 278

antioxidant, 249, 257, 274, 281

apoptosis, 51, 151, 197, 200

apoptotic pathways, 96

appetite, 6, 272

appropriate technology, 275

aquaculture, xv, 260, 265

aqueous solutions, 14

ARC, 254

Argentina, vi, 42, 57, 78, 157, 159, 160,

161, 162, 163, 164, 166, 181, 183, 184,

185, 186, 187, 188, 189, 221

Argentinean peanut, xi, 157, 159, 166, 184

arthropods, 166, 185

ascites, 79

Asia, viii, 35, 41, 71, 198, 201

Asian countries, 75

aspergillosis, 52

Aspergillus terreus, 57, 60

assessment, 32, 83, 89, 109, 113, 118, 193,

202, 204, 205, 208, 209, 227, 228, 237,

238, 252, 253, 257, 284

atmosphere, 45, 261

attachment, 40

attribution, 211

authority(s), xiii, 9, 112, 114, 116, 192, 209,

211, 214, 215, 219, 230

autopsy, 79

avoidance, 4

awareness, 45, 271

B

B1 (AFB1), vii, ix, xi, 1, 3, 64, 92, 110,

126, 127, 159, 206, 235, 262

Bacillus subtilis, 11

bacteria, viii, 2, 4, 11, 23, 24, 25, 27, 28, 29,

30, 32, 50, 52, 53, 59, 240

bacterial cells, 53

bacterial strains, 23, 53

Bahrain, 78, 220

ban, 212

Bangladesh, 42

barriers, 110

base, 30, 40, 116, 143, 146, 197, 212

base pair, 197

BCG immunotherapy, 156

BD, 150

beef, 75, 78, 217, 268

benefits, 120, 270

benign, 197

beverages, 203

BI, 155

bias, 148

bile duct, xv, 260, 267

bioavailability, 12, 267

biochemical processes, 80

biochemistry, 277, 283

biodiversity, 162

biological activity(s), 19, 24, 182

biological consequences, 104, 283

biological control, 31, 53

biological samples, 29

biological systems, xv, 260, 266

biomarkers, xiii, 80, 82, 85, 86, 88, 102,

136, 143, 144, 145, 192, 222, 223

biomonitoring, 82

biosynthesis, 37, 45, 46, 47, 48, 49, 50, 54,

55, 56, 57, 59, 61, 62, 66, 86, 102, 186,

195, 198, 236, 243, 280

biotic, 36

birds, 195, 199, 200

birth weight, 198, 224

bladder cancer, 145, 146, 153

bleaching, 245

bleeding, 79

blood, 80, 97, 103, 104, 136, 139, 151, 196,

225, 262, 273

body fluid, 80

body weight, 195, 206

Bolivia, 108, 110, 114, 115, 121

Bosnia, 219, 221

Botswana, 42

brain, 200, 226

brain chemistry, 200

Brazil, v, x, 42, 58, 59, 68, 69, 76, 78, 82,

84, 107, 108, 109, 110, 111, 112, 113,

114, 115, 116, 118, 119, 120, 121, 122,

Index 289

123, 163, 181, 182, 184, 202, 203, 213,

219, 220, 228, 282

Brazil nuts, x, 68, 69, 76, 78, 82, 84, 107,

108, 109, 110, 111, 112, 113, 114, 115,

118, 119, 120, 121, 122, 123, 213, 220

breakfast cereals, xiv, 202, 234, 236, 249,

251, 252, 257

breast cancer, 143, 152, 153, 154

breast milk, 37, 57, 81, 225

breeding, 78, 217, 268

Britain, 92

buffalo, 73, 86, 89

Burkina Faso, 185

businesses, 67

by-products, 11, 119

C

Ca2+, 226

cacao, xiv, 260, 265

calcium, 12, 32, 268, 281

calibration, 15, 17

Cameroon, 38, 53

campaigns, 271

cancer, xv, 77, 79, 85, 88, 94, 111, 113, 130,

143, 146, 147, 151, 153, 154, 156, 197,

208, 223, 260, 266, 270, 272, 273, 282

cancer death, 272

CAP, 210

capillary, 16

carbohydrate(s), 184, 196, 197

carbon, 45, 47, 48

carbon dioxide, 45

carcinogen, ix, 3, 9, 29, 64, 79, 127, 148,

235, 263, 272

carcinogenesis, 28, 105, 127, 195, 266, 279

carcinogenicity, 31, 127, 193, 194, 198,

205, 208

carcinoma, 126, 155, 156, 195, 197, 269,

272

cardiovascular system, 250

carotenoids, 151, 249, 250

case study, 32, 144

catalysis, 225

cattle, 4, 6, 8, 9, 25, 32, 38, 75, 76, 78, 79,

217, 267, 268

CCA, 131

CD8+, 98

CDC, 216, 267, 269, 271, 278

cell cycle, 96, 197

cell death, 197, 226

cell killing, 12

cell line, 96, 153

cell surface, 96, 102

cellular immunity, 98

central nervous system, 95

cerebral edema, 79, 271

certificate, 111, 112

certification, 111, 112, 114, 118, 119, 219,

238

CFR, 26

challenges, xv, 261, 271

cheese, 24, 76, 84, 235, 239, 240, 242, 254,

258, 284

chemical(s), ix, xiv, 2, 10, 11, 12, 14, 29,

53, 64, 85, 87, 89, 155, 159, 217, 226,

235, 260, 262, 280

chemical stability, xiv, 260, 262

chemokines, 95

chemoprevention, 271

chemotaxis, 98, 105

Chicago, 135

chicken, xv, 260, 268, 281, 282

chicken embryos, xv, 260, 268

childhood, 155, 198, 266

children, xiii, 33, 69, 71, 76, 77, 79, 81, 85,

88, 100, 105, 192, 193, 194, 196, 198,

201, 202, 203, 204, 207, 214, 221, 224,

227, 239, 242, 251, 257, 266, 270, 272,

279, 285

China, 42, 71, 75, 84, 95, 125, 126, 127,

131, 150, 152, 154, 155, 185, 203, 219,

221, 228, 270, 283

chloroform, 130

cholesterol, 51

chromatid, 266, 277

chromatographic technique, 87

chromatography, 10, 29, 37, 54, 234, 238,

265

Index 290

chromosome, 45, 143, 145, 146, 147, 277

chromosome map, 45

chronic diseases, 198

chronic fatigue syndrome, xiii, 192, 193

cirrhosis, xv, 129, 226, 260, 266

City, 91, 125, 184, 224

cleaning, 108, 245

cleanup, 9

climate(s), viii, xiii, xiv, 2, 6, 8, 18, 36, 39,

43, 44, 45, 56, 59, 64, 66, 81, 82, 192,

234, 239, 240, 246, 247, 255, 256, 263

climate change, xiii, xiv, 44, 45, 192, 234,

256

climatic factors, 7

cloning, 62

clusters, 46, 48, 49, 60

cocoa, 81, 203

coding, 112, 143, 146, 152

codon, 131, 138, 139, 143, 144, 147, 148,

149, 152, 154, 156, 197, 222

coffee, 256

cognitive development, 198

cognitive function, 200

collaboration, 219, 276

colleges, 48

colonisation, 40, 41, 49

colonization, 166, 172, 187, 189, 242

color, 11, 109

colorectal cancer, 153, 154, 156

coma, 79, 271

commerce, 215

commercial, 8, 12, 25, 30, 73, 159, 207,

239, 240, 244, 246, 247, 255, 256

commodity, xv, 108, 113, 119, 202, 204,

208, 250, 251, 260, 274

communication, 50, 211

community(s), xii, 44, 158, 163, 172, 185,

264, 265, 276, 285

compatibility, 188

competition, 11, 66, 174, 245, 249

compilation, vii

complement, 82, 200, 276

complexity, xi, 107

compliance, 119, 194, 209, 215, 216, 218,

219, 230

composition, 11, 180, 181, 188, 235

compounds, viii, ix, xiii, xiv, 2, 3, 11, 12,

26, 29, 35, 37, 63, 92, 181, 184, 192,

205, 235, 245, 259, 261

concordance, 17

conditioning, xii, 158, 171, 173

confinement, 38

conflict, 120

conflict of interest, 120

conformity, 110, 118

confounders, 129, 135

congress, 218, 231

consensus, 46, 67

consent, 129

conservation, x, 107, 108, 110

constituents, 29, 58, 85, 245, 254, 255, 280

consumers, xi, 8, 108, 110, 113, 119, 194,

203, 204, 207, 208, 209, 210, 211, 215,

239

consumption patterns, 207

containers, 239

contaminant, vii, viii, 1, 8, 35, 41, 127, 168,

205, 212, 216

contaminated food, ix, xiii, xv, 5, 11, 63, 92,

113, 192, 194, 208, 260, 266, 272, 274

control group, 249

control measures, 237

controversial, 199

conversion rate, 6

cooking, 159

cooperation, 194

coordination, 54, 58, 200

copper, 12

correlation, 93, 101, 138, 147, 174, 178,

179, 183, 198, 201, 245, 273

cost, 9, 10, 11, 45, 70, 238, 286

Costa Rica, 33, 59

cotton, viii, xiv, 5, 35, 39, 40, 56, 58, 185,

251, 260, 264

coumarins, 127, 245

covalent bond, 93

covering, 8

CPT, 195

cracks, 41

crises, 247

Index 291

Croatia, 1, 13, 18, 19, 22, 23

crop(s), viii, xiii, xiv, 3, 4, 6, 7, 31, 35, 36,

37, 38, 39, 40, 41, 44, 49, 52, 53, 59, 62,

66, 67, 73, 116, 159, 163, 164, 166, 189,

192, 194, 236, 252, 255, 256, 260, 263,

264, 273, 274, 275, 280, 282

crop production, 275

crop residue, 67

cross sectional study, 198, 224, 279

CT, 133, 137, 138, 140, 141, 144, 149, 155

cues, 47

cultivars, 181, 190, 257

cultivation, vii, xiv, 2, 4, 22, 67, 163, 164,

202, 203, 234, 236, 250

cultural conditions, 48

cultural practices, 53, 164

culture, 45, 57, 165, 176, 225, 244

culture conditions, 165

culture media, 244

culture medium, 176

current limit, 119

CV, 14

cycles, 131

cytochrome, xi, 93, 98, 126, 197

cytokines, x, 92, 95, 96, 98

cytoplasm, 22

cytotoxicity, 263, 278

Czech Republic, 200

D

dairy industry, 239

dairy products, xiv, 8, 9, 32, 38, 60, 75, 203,

234, 235, 239, 242, 251, 286

database, 81, 143, 146, 208

deaths, 57, 66, 84, 94, 262

decay, 21, 71

decontamination, 31, 32, 218

defects, 50

defence, 52

defense mechanisms, ix, 64

deficiency(s), 146, 197, 215

degradation, 4, 11, 12, 19, 49, 286

degumming, 245

demographic data, 129, 135

denaturation, 131

Department of Agriculture, 216

Department of Health and Human Services,

231

depth, 8

derivatives, 64, 80, 81, 202, 242

destruction, 19, 201, 217

detectable, 81, 100, 118, 145, 206, 207, 240,

248

detection, x, 9, 10, 13, 15, 18, 25, 29, 31,

34, 81, 86, 87, 107, 131, 132, 238, 239,

240, 241, 242, 244, 248, 251, 253, 254,

255, 257, 265, 276

detection system, 131, 132

detention, 219

detoxification, xiii, 5, 10, 11, 25, 29, 81,

192, 195, 274, 275, 283

developed countries, 81, 116, 117, 220

developing countries, xv, 2, 33, 57, 79, 81,

87, 88, 105, 117, 119, 193, 209, 210,

220, 223, 260, 261, 265, 266, 270, 271,

273, 274, 276, 279, 285

developing nations, 272

diarrhea, 79, 199

diet, 2, 80, 97, 99, 194, 201, 202, 203, 204,

222, 225, 236, 240, 242, 244, 266, 270,

273, 274

dietary habits, 202, 204

dietary intake, 80, 89, 206

digestibility, 199, 225, 267

digestion, 130, 195, 268

digestive enzymes, 197, 199, 267, 282

disease progression, 101

disease rate, 100

diseases, xv, 6, 36, 38, 53, 80, 98, 101, 198,

201, 210, 223, 224, 260, 270, 271, 276

disorder, 204

dispersion, 280

displacement, 271

disposition, 226

distillation, 11

distilled water, 22

distribution, 8, 65, 129, 132, 138, 160, 194,

204, 207, 239, 262

diversity, 172, 265

Index 292

DNA damage, xi, 102, 126, 127, 139, 147,

150, 151, 226, 266

DNA ligase, 143, 152

DNA polymerase, 152

DNA repair, xi, 93, 126, 127, 128, 132, 133,

137, 138, 139, 140, 141, 143, 145, 146,

147, 148, 150, 153, 154, 155, 156

DNA-adducts, xv, 139, 260, 266

DNase, 268

documentary evidence, 111

dogs, 5, 267

dominance, 174

dopamine, 200

dosage, 97, 248, 269

dosing, 97, 103

dough, 228

draft, 120

drought, 6, 7, 39, 40, 52, 56, 66, 67, 71, 165,

166, 185, 197, 264

drugs, 55, 215

drying, xii, 6, 11, 39, 58, 66, 67, 74, 108,

130, 158, 159, 171, 246, 250, 264, 279

E

East Asia, 272

ecology, 39, 41, 47, 61, 62

economic losses, 116, 262

economic problem, viii, 2, 10

economics, ix, 64

ecosystem, xi, 157, 180

edema, 79, 196, 272

education, 271

egg, 6, 79

Egypt, 42, 83, 244, 276

electron, 224

ELISA, vii, 2, 4, 9, 13, 14, 16, 17, 18, 21,

24, 26, 30, 34, 130, 234, 238, 240, 241,

247, 255

ELISA method, 13, 14, 16, 21, 24, 241

elucidation, 139, 148

e-mail, 259

encephalopathy, 200, 226

encoding, 143

endocrine, 6

energy, 5, 6, 159

enforcement, 81, 194, 214, 215, 216

engineering, 7

England, 3, 193, 262

environment(s), 6, 36, 45, 53, 65, 109, 166,

183, 214, 261

environmental conditions, viii, 33, 36, 39,

43, 48, 52, 60, 160, 165, 183, 248

environmental factors, ix, 5, 25, 47, 63, 66,

187, 235

environmental protection, 110

environmental stress, 48, 183

enzyme(s), xi, 10, 94, 98, 99, 126, 130, 197,

198, 200, 225, 226, 238, 268, 277

enzyme-linked immunosorbent assay, 130

epidemic, 276

epidemiologic studies, 80

epidemiology, 88, 103, 280

epilepsy, 200

epithelial cells, 283

epithelium, 199

equipment, 10, 244

ethanol, 22, 130

ethnicity, 129

eukaryote, 59

Europe, 71, 72, 102, 110, 113, 194, 200,

203, 209

European Commission, 70, 113, 114, 207,

210, 211, 229, 230, 237, 255, 266, 279

European Community, 12, 110, 246

European Parliament, 27, 28, 83, 253

European policy, 110

European Social Fund, 82

European Union, ix, x, 8, 27, 63, 64, 67, 68,

69, 70, 74, 82, 107, 113, 121, 122, 159,

185, 205, 210, 229, 248, 253, 266, 279

evaporation, 263

evidence, xiv, xv, 8, 79, 80, 81, 82, 94, 127,

129, 148, 198, 199, 205, 206, 215, 222,

234, 260, 270, 273, 274

evolution, 29, 195

examinations, 129

excision, 143, 146, 156

exclusion, 209

excretion, 34, 248

Index 293

exons, 143, 145, 146, 147

expertise, 244

exploitation, 110

export control, 82, 214

export market, 109

exporter(s), 119, 214, 231, 242

exports, 109, 110, 118, 209

expressed sequence tag, 62

extraction, 10, 11, 16, 57, 110, 129, 130,

238, 244, 245, 249, 265

extracts, 15, 26, 256

extrusion, 224

F

farms, 3, 7, 58, 241, 247, 248

FASAY, 151

fat, 2, 198, 242

fatty acids, 49, 51, 52, 55, 56, 186, 242, 250

FDA, xiii, 5, 28, 192, 215, 216, 217, 218,

219, 231, 268

feces, 92

federal agency, 215

feedstock, 5

feedstuffs, 8, 9, 75, 78, 194, 209, 214, 268

fermentation, 11, 204, 240

fetal development, 257

fetus, 201

fever, 79, 269, 272

fibers, 250

fibroblasts, 146, 155

filament, 144

financial, 101, 150, 276

financial support, 101

fish, xv, 6, 260, 265, 268

flagellum, 224

flavonoids, 249, 250, 257

flavor, x, 107, 108

floods, 261

flora, 5, 25

flotation, 11

flour, 43, 73, 77, 86, 87, 182

flowers, 64, 159

fluid, 30, 45, 238

fluid extract, 238

fluorescence, 10, 86, 193, 244, 249, 251,

255

food additive(s), 223

Food and Drug Administration, xiii, 192,

209, 215, 231

food chain, xii, xv, 28, 59, 66, 67, 84, 158,

202, 211, 235, 237, 261, 271

food industry, 194, 209

food intake, 77

food production, viii, 2, 10, 67, 81, 211,

248, 261

food products, 12, 28, 84, 202, 203, 207,

214, 218, 238, 246

food safety, vii, x, xiii, 1, 2, 8, 30, 45, 64,

74, 83, 88, 119, 122, 194, 209, 210, 211,

216, 218, 220, 224, 229, 231, 233, 237,

239, 248, 249

food security, 210, 220

foodborne illness, 216, 218

force, 8, 212

formation, viii, xi, xv, 2, 3, 18, 19, 25, 48,

52, 56, 60, 62, 93, 98, 126, 127, 139,

144, 159, 188, 197, 236, 260, 266

formula, 76, 221, 262, 264

France, 29, 58, 85, 202, 203, 254, 280

free radicals, 19, 104, 195

freezing, 66

fruits, ix, xiv, 63, 66, 69, 77, 81, 88, 108,

109, 120, 159, 202, 238, 245, 260, 267,

280, 284

Functional Analysis of Separated Alleles in

Yeast, 151

functional food, 119

funding, 219

fungal infection, 3, 57

fungus, xii, 36, 39, 40, 41, 47, 49, 53, 54,

60, 64, 92, 163, 165, 173, 181, 183, 189,

192, 197, 199, 240, 262

furan, 19

fusion, 127

G

gamma radiation, viii, 2, 26, 33, 54

gastrointestinal tract, 225, 262

Index 294

gene expression, 47, 99, 195, 286

gene regulation, 50

genes, vii, ix, xi, 36, 45, 46, 47, 48, 49, 50,

53, 61, 62, 126, 127, 128, 132, 133, 137,

138, 139, 140, 141, 148, 150, 151, 153,

155, 163, 195

genetic defect, 199

genetic engineering, 53

genetic mutations, 148

genetics, vii, ix, 36, 45, 46

genome, vii, viii, 36, 46, 53, 57, 102

genomics, 62

genotype, 61, 132, 135, 138, 139, 263

genotyping, 132

genus, vii, ix, xii, 1, 3, 18, 42, 63, 64, 71,

158, 168, 171, 172, 243, 262

Georgia, 61

Germany, 13, 14, 15, 188

germination, 19, 21, 40, 159, 163, 182, 256

gerontology, 155

ginger, xiv, 69, 92, 203, 260, 264

gland, 262

global scale, 246

globalization, 209, 239

glutamate, 200, 226

glutathione, 151, 225, 284

government intervention, 215

governments, 108, 119, 207, 276

grants, 184

Great Britain, 188, 189

Greece, vi, xiii, 191, 233, 235, 236, 237,

238, 239, 240, 242, 243, 244, 245, 246,

247, 248, 249, 250, 251, 255, 256, 257

green olives, xiv, 234, 236, 254

growth rate, 6, 21

Guangdong, 147

guanine, 93, 99, 102, 103, 151, 197

guidance, 194, 215, 216, 230

guidelines, 210

Guinea, 284

Guyana, 108

H

half-life, 21, 80

haplotypes, 154

harmful effects, viii, 2, 8, 25

harmonization, 74, 222

harvesting, xiv, 3, 4, 6, 7, 10, 53, 74, 159,

234, 238, 245, 256, 273

hazards, vii, xiv, 30, 31, 71, 199, 204, 210,

235, 237, 258, 260, 265, 266

HBV, 94, 101, 128, 129, 135, 136, 138, 141,

142, 156, 206, 270

HBV infection, 94, 101, 138

HCC, xi, xv, 93, 94, 101, 126, 127, 128,

129, 130, 132, 135, 136, 137, 138, 139,

141, 143, 144, 145, 146, 147, 148, 149,

197, 260, 267, 269, 270

health care, 37, 116

health effects, x, 64, 68, 81, 193, 194, 195,

201, 204, 211, 215, 224, 226, 238, 242,

251, 270, 271, 274, 275

health problems, 237, 266

health risks, 74, 211, 237, 266

health services, 276

hematemesis, 79

hemorrhage, 79

hepatic encephalopathy, 281

hepatic failure, 269

hepatic necrosis, xv, 260, 266

hepatitis, 6, 79, 80, 82, 103, 129, 152, 206,

207, 227, 269, 270, 272, 274, 278, 281,

282, 284

hepatitis a, 79

hepatitis b, 272

hepatitis c, 207, 282

hepatocarcinogen, 92

hepatocarcinogenesis, xv, 103, 260, 284

hepatocarcinoma, ix, 64

hepatocellular carcinoma, xi, xv, 6, 33, 57,

80, 82, 93, 94, 105, 126, 127, 128, 149,

151, 152, 154, 155, 156, 197, 260, 267,

271, 272, 276, 282, 284, 285

hepatocytes, 79, 152, 196, 222, 254

hepatoma, 267

hepatotoxicity, 79, 222, 263, 269

hepatotoxins, xv, 260, 268

herbal medicine, 186, 280

heterogeneity, 102

Index 295

heterozygote, 135

histone(s), 46, 49, 99

history, 129, 164, 211, 225

HIV, xv, 80, 98, 100, 101, 260, 270, 273

HIV/AIDS, xv, 80, 260, 270

homeostasis, 95, 96, 199

homozygote, 137, 138, 144, 145, 149

hormone, 198

host, ix, x, 36, 38, 49, 50, 53, 54, 56, 58, 92,

101, 266

human body, 195, 202

human exposure, 68, 80, 88, 201, 204, 241

human health, viii, 8, 9, 10, 35, 38, 87, 212,

214, 215, 223, 224, 235, 237, 239, 249,

258, 262, 270, 276

human immunodeficiency virus, 103

humidity, 2, 7, 8, 18, 45, 56, 65, 67, 109,

173, 202, 243, 247, 250, 261

humoral immunity, x, 91, 100, 201

hydrogen, 19, 95

hydrogen peroxide, 19, 95

hydrolysis, 93

hydrophobicity, 55

hygiene, 111, 119, 246

hyperplasia, xv, 260, 266

I

icterus, 196

ID, 103, 123, 145, 146, 231

identification, 46, 53, 62, 111, 168, 193,

204, 223, 265

IFN, 95, 97

immune function, 33, 105, 227, 272, 285

immune response, x, 92, 95, 100, 103

immune system, 6, 82, 101, 199, 266, 268,

274

immunity, x, xiii, 79, 91, 99, 100, 192, 193,

194, 195, 200, 201, 225, 267, 270, 273

immunoglobulin, 153

immunomodulatory, x, 91, 100

immunosuppressants, xv, 260, 268

immunosuppression, x, xv, 91, 101, 260,

266, 270

impaired immune function, 272

impairments, 6

import restrictions, 113

imported products, 114, 219

imports, 71, 112, 209, 214, 250

improvements, 3

in utero, 81

in vitro, 21, 23, 95, 96, 97, 98, 100, 104,

189, 193, 206, 278, 281

in vivo, 23, 95, 97, 100, 104, 206, 263

incidence, ix, xv, 2, 64, 70, 71, 73, 79, 80,

82, 94, 101, 110, 114, 118, 147, 160,

163, 171, 172, 180, 181, 182, 186, 196,

206, 260, 270, 273

income, 70

incubation period, 172, 180

incubation time, 57

independent variable, 135

India, 33, 43, 77, 182, 189, 203, 220, 221,

223, 230, 259, 281

indirect effect, 109

individuals, x, 6, 92, 98, 101, 138, 145, 206,

265, 273, 274

Indonesia, 76, 173

inducer, 49, 51, 55

induction, ix, 61, 64, 77

industry(s), 11, 25, 88, 110, 121, 186, 211,

216, 238, 241

infancy, 266

infants, 37, 69, 76, 77, 214, 221

infection, x, 7, 36, 40, 41, 44, 55, 56, 58, 88,

92, 94, 96, 98, 100, 101, 129, 135, 138,

156, 163, 164, 165, 166, 173, 201, 206,

227, 268, 272, 273

infectious agents, 80

infertility, 198, 224

infestations, 261

inflammation, x, 92

informed consent, 129

infrastructure, 119

ingest, 262

ingestion, ix, 4, 6, 63, 64, 78, 79, 193, 204,

225

ingredients, 8, 38, 54, 78, 217, 247, 264

inhibition, 19, 22, 23, 198, 255, 256

initiation, 7

Index 296

injury, 172

inoculation, 39, 41, 244

inoculum, 3, 39, 40, 41, 59, 163, 168, 183,

186

insects, 6, 36, 40, 44, 182, 187

insomnia, 200

inspections, 216

integrity, 112

inter kingdom communications, ix, 36

interference, 36, 193, 200, 266, 270

international standards, 110

international trade, 68, 108, 210

intervention, 115, 116, 276, 285

intervention strategies, 276

intoxication, 6, 116, 195

introns, 143, 145, 146, 147

investment, 119

ionization, 12, 16

ionizing radiation, 30, 33

ions, 17

Iowa, 278

Iran, 37, 60, 116, 122, 199, 252, 253, 256

Ireland, 91, 101

irradiation, 11, 12, 19, 20, 21, 27, 31

irrigation, 52, 56

ischemia, 226

isolation, xii, 41, 157, 160, 163, 168, 171,

176, 238

issues, 8, 210, 216, 219, 220, 225, 266

Italy, 28, 45, 73, 87, 191, 222, 228, 241,

242, 244, 255, 256

J

Japan, xiii, 58, 61, 76, 155, 192, 203, 220,

228

jaundice, 79, 269

justification, 110

K

Kenya, 30, 57, 58, 71, 73, 78, 83, 84, 86,

166, 182, 183, 187, 196, 199, 221, 223,

271, 278, 282

kidneys, 79

kinetic parameters, 225

Korea, 76, 77

Kuwait, 78, 84, 220

L

lack of confidence, 117

lactation, 83, 89, 262

lactic acid, viii, 2, 4, 11, 23, 24, 27, 28, 29,

32, 240

Lactobacillus, 23

laws, 216

LC-MS/MS, vii, 2, 4, 13, 15, 16, 17, 54,

123, 234, 238, 244

lead, 3, 4, 41, 49, 53, 66, 79, 100, 143, 151,

155, 197, 198, 211, 242, 243, 245, 261,

270

learning, 200

legislation, vii, 32, 67, 74, 111, 113, 114,

115, 182, 194, 210, 211, 214, 215, 219,

230, 249, 258

legs, 79

Lepidoptera, 61

lesions, 93

leukemia, 155, 156

leukocytes, 136, 137

light, 12, 47, 48, 54, 61, 82, 85, 92, 200,

215, 224, 262

linoleic acid, 51, 57, 236

lipases, 197, 236

lipid oxidation, 236

lipid peroxidation, 198

lipids, 79, 249

liquid chromatography, vii, 2, 4, 10, 34, 54,

73, 86, 234, 238, 244, 252, 255, 256,

257, 265, 280

liver cancer, ix, xv, 5, 64, 78, 80, 82, 85,

102, 128, 151, 152, 197, 205, 206, 207,

222, 223, 260, 266, 272, 279, 280, 283

liver cells, 196

liver cirrhosis, ix, 64, 135, 196

liver damage, ix, xv, 64, 79, 80, 195, 260,

266, 267, 271, 272

liver disease, 129, 148, 273

Index 297

liver failure, 197, 272

livestock, xiii, xv, 6, 65, 192, 260, 264, 268,

278

local government, 129

localization, 29

loci, 128

logistics, 67

longitudinal study, 85, 224, 279

Louisiana, 125

lovastatin, 49, 51, 57, 60

low risk, xi, 108

LSD, 178

lung cancer, 80, 153

lymph node, 155

lymphocytes, 95, 97, 99, 101, 102, 103, 146

lymphoid, 99

lysine, 93, 99

lysis, 100, 198

M

Macedonia, 191, 233

macrophages, xv, 95, 96, 100, 102, 104,

200, 260, 268, 281

magnitude, 196

majority, 194, 204, 238, 244, 248, 266

malaise, 79, 269, 272

malaria, xv, 80, 199, 201, 227, 260, 270

Malaysia, 43, 76, 200, 281

malignancy, 197

malignant tumors, 143, 145, 146

malnutrition, xv, 80, 116, 197, 199, 260,

266, 270, 272

mammalian cells, 154

mammals, 3

management, 39, 43, 57, 88, 117, 119, 150,

218

manufacturing, 3, 74, 114, 118, 221

market access, 183

marketing, 81, 110, 267

marketing strategy, 110

mass, vii, 2, 4, 10, 16, 54, 87, 234, 238, 244,

252

mass spectrometry, vii, 2, 4, 10, 16, 54, 87,

234, 238, 244, 252

materials, 7, 12, 13, 19, 21, 28, 62, 66, 237

matrix(s), 15, 266, 280

MB, 155

measurement(s), 15, 25, 31, 80, 81

meat, xiv, 4, 23, 26, 29, 30, 74, 83, 85, 86,

216, 260, 264, 267

media, 24, 28, 44, 227

median, 196

medical, 69, 73, 81, 129, 221, 249

medical history, 129

medicine, 154

Mediterranean, 236, 237, 240, 241, 242,

243, 244, 245, 246, 247, 251, 253

Mediterranean countries, 237, 240, 241,

243, 244, 245, 251

melon, 43

memory, 200

meristem, 40

meta-analysis, 144, 152, 153, 154

metabolic, 94

metabolic disorder(s), 30

metabolism, ix, xiii, 31, 36, 48, 50, 52, 64,

85, 103, 148, 192, 193, 195, 196, 198,

206, 254, 268, 275, 279, 280

metabolites, vii, ix, x, xii, 1, 3, 5, 11, 38, 47,

51, 63, 64, 80, 81, 91, 92, 94, 96, 99,

102, 105, 123, 158, 159, 174, 192, 193,

196, 235, 262, 264

metabolized, xi, 98, 126, 197, 239

metals, 48

metastasis, 129, 155

methanol, 14

methodology, 8, 180, 204, 205, 218

Mexico, 39, 58, 71, 78, 219, 221

mice, 98, 151, 198

microbiota, 60, 79, 199, 249

micronutrients, xiii, 192, 193

microorganism(s), 6, 11, 21, 37, 172

microscopy, 61

minicolumn, 265

mission, 110, 111, 112

Missouri, 40, 286

misuse, 222

mitochondria, 198

mitosis, 197

Index 298

modelling, 44, 208

models, 43, 103, 267

modifications, xiv, 46, 49, 234

moisture, 6, 7, 11, 13, 31, 36, 44, 67, 70, 71,

159, 173, 182, 186, 187, 243, 246, 247,

261, 282

moisture content, 7, 11, 13, 31, 44, 70, 159,

173, 246, 282

mold(s), 31, 60, 83, 87, 89, 159, 182, 184,

197, 226, 243, 244, 245, 247, 249, 257,

258, 274

molecular weight, 23

molecules, ix, 36, 37, 51, 52, 53, 94, 96, 99,

101

monoclonal antibody, 29, 30, 130

Moon, 95, 96, 104

morbidity, 198

Morocco, 243, 244, 247, 258

morphogenesis, 50

morphology, 51, 52, 151, 189, 198, 199,

283

mortality, 198, 272, 276

mortality rate, 272, 276

motif, 46

mould spores, 21

Mozambique, 95

MR, 101, 105, 156

mRNA, 95, 97, 98, 102, 156

multilateralism, 210

mung bean, 77, 220

mutagenesis, 48, 156

mutant, xi, 50, 52, 126, 132, 137, 139, 146,

148

mutation(s), xi, 12, 93, 102, 105, 126, 127,

128, 131, 135, 138, 139, 143, 145, 146,

147, 148, 149, 151, 152, 153, 156, 197,

222, 223, 266

mycelium, 22, 168, 170, 171

mycology, 61

myeloid cells, 99

N

naphthalene, 186

native isolates, 163

native population, 163

near infrared spectroscopy, 253

nebulizer, 16

necrosis, xv, 79, 196, 197, 260, 267

neonates, 239

Nepal, 43

nerve, 200

nervous system, 200

Netherlands, 72, 187, 189, 209

neurodegeneration, 226

neurogenesis, 146

neurons, 146

neurotoxicity, 193

neurotransmitter(s), 200

neutrophils, 97, 104

New Zealand, 78, 200, 203, 219, 220

Nigeria, 26, 43, 78, 219, 220, 224, 230, 256,

277

NIR, 257

nitric oxide, 95, 104

nitrite, 83

nitrogen, 45, 48, 56, 129

NMDA receptors, 200

nodes, 129

North Africa, 245

North America, 71, 81

nucleoprotein, 144

nucleus, 166

nutrient(s), 6, 11, 33, 36, 40, 159, 174, 199,

225, 267, 268, 270, 274

nutrition, 2, 82, 198, 199, 211, 222, 253,

273

nutritional deficiencies, 266

nutritional status, xv, 5, 260

O

officials, 267

oil, xiv, 22, 32, 43, 75, 89, 119, 242, 243,

244, 245, 246, 260, 265, 280

oil samples, 243, 244

oilseed(s), viii, xiv, 27, 35, 39, 49, 60, 66,

68, 69, 213, 230, 235, 260, 264, 266

oleic acid, 49

Index 299

olive oil, 236, 238, 242, 243, 244, 245, 246,

251, 252, 253, 254, 256, 257

operations, 3

optimization, 245

organ(s), 79, 127, 195, 197, 210, 225, 226,

283

organism, 48, 196

ox, xiv, 49, 52, 259, 261

oxidative stress, 154

oxygen, 45

oxylipins, ix, 36, 49, 50, 51, 52

ozone, 123

P

p53, 127, 138, 139, 143, 151, 152, 197, 222,

223

pain, 79, 196, 269, 271, 272

Pakistan, 58, 73, 83, 86, 87, 89, 281

parasite(s), 4, 80

parasitic infection, 200

participants, 98

pasta, 72

pasteurization, 38

pasture, 8, 38

pathogenesis, ix, 36, 49, 54, 280

pathogens, 36, 50, 53, 86

pathology, 32, 280

pathways, 49, 52, 99, 266

PCR, 87, 131, 132, 133, 134, 138, 147, 149,

174, 180, 188, 195

peanut meal, 3, 12, 19, 33, 75, 193

penicillin, 49, 51

peptide, 53

peripheral blood, 128, 129, 130, 136, 137,

139

peripheral nervous system, 200

peroxide, 14, 245

Peru, 108

pesticide, 53

pests, 61, 261

pH, 14, 23, 44, 47, 48, 59, 66, 67, 92, 130,

131, 171, 240

phagocytosis, x, 92, 98, 100, 200

phenol, 130

phenolic compounds, 245, 252

phenotype(s), xii, 50, 157, 160, 164, 271

Philippines, 39, 41, 43, 58, 60

phosphate, 14, 131

phosphorus, 268

physical properties, 171, 178, 264

physiology, 49

pigs, 5, 26, 75, 76, 79, 96, 99, 100, 227, 267

pistachios, ix, 5, 30, 63, 66, 68, 69, 70, 71,

76, 78, 82, 84, 92, 113, 116, 121, 202,

207, 208, 212, 220, 222, 229, 235, 250

placenta, 139, 151, 201

placental barrier, 199

plant growth, 40, 190

plants, viii, 4, 26, 32, 35, 36, 37, 40, 41, 42,

44, 49, 50, 53, 54, 58, 64, 73, 87, 159

plasma membrane, 198

platinum, 153

point mutation, 153

poison, 262

Poland, 173, 184

polarity, 16

policy, 210, 211, 215, 216, 218, 220

pollen, 40, 236, 238, 249, 252, 256

pollutants, 257

polymerase, 149, 195

polymerase chain reaction, 149

polymorphism(s), xi, 126, 127, 132, 133,

138, 143, 144, 145, 146, 147, 148, 150,

151, 152, 153, 154, 155, 156, 284

polyphenols, 242

polypropylene, 173

population density, 52

population group, xiv, 207, 234

population growth, 261

population structure, 188

Portugal, 107, 242, 253, 255, 281

positive correlation, 44, 179, 183

positive relationship, 40

poultry, xv, 3, 6, 74, 78, 79, 86, 201, 217,

247, 254, 256, 258, 260, 264, 268, 286

poverty, 110

poverty reduction, 110

pregnancy, 228

preparation, 13, 81

Index 300

preparedness, 275

present value, 240, 251

preservation, 245

prevention, viii, 2, 10, 19, 25, 28, 32, 33, 85,

121, 148, 152, 218, 274, 280, 284

principles, 118, 211, 212, 229

private sector, 116

probability, xi, 44, 157, 204, 205, 228

probe, 131, 132

probiotic, 23, 24, 29

producers, 36, 37, 42, 44, 70, 108, 109, 118,

162, 164, 165, 168, 176, 179, 182, 211,

216, 218, 250

productive efficiency, 26

progenitor cells, 97

pro-inflammatory, 95, 100

proliferation, xv, 21, 89, 97, 195, 260, 261,

267

promoter, 46

prophylactic, 274

prostate cancer, 152, 155

protection, xi, 22, 25, 52, 108, 111, 113,

194, 211, 218, 250, 261

protein synthesis, 6, 198, 199, 200

proteinase, 130

proteins, xv, 47, 48, 49, 52, 93, 99, 146,

196, 250, 260, 266, 273

public concern, 53

public health, xv, 32, 84, 111, 112, 113,

116, 121, 194, 205, 208, 209, 212, 214,

215, 219, 221, 246, 260, 267, 269, 270,

271, 276, 285

pulmonary edema, 79, 271

purification, 244

P-value, 135

pyridoxine, 197

Q

quality control, 122, 131, 132, 237

quality standards, 218, 248

quantification, 9, 10, 17, 87, 180, 188, 238,

241, 265

questionnaire, 129, 130

R

race, 128, 129, 135, 138, 141, 142

radiation, 4, 12, 19, 21, 25, 26, 28, 89

radicals, 19

rain forest, 108

rainfall, 38, 160, 164

rainforest, 108

raw materials, 4, 247

RE, 103

reactions, 151, 195

reactive oxygen, 96, 105

reagents, 9

reasoning, 194

recall, 219

recognition, 147

recombination, 144, 153, 154, 155

recovery, 9, 13, 14, 15, 16

recurrence, 156

regions of the world, 8, 209, 235

regression, 135, 139

regression analysis, 139

regression model, 135

regulations, vii, x, 8, 28, 64, 70, 74, 79, 82,

87, 88, 113, 119, 159, 182, 194, 204,

209, 210, 214, 216, 218, 219, 237, 249,

273

regulatory framework, xiii, 192

rejection, 115, 116, 119

relative size, 144

relaxation, 113

relevance, 201

reliability, 112

repair, xi, 102, 126, 127, 138, 139, 143, 145,

146, 148, 149, 150, 151, 152, 154, 155,

156

repression, 60

reproduction, 52, 58, 278

requirements, 10, 199, 211, 229

researchers, 42, 139, 147, 173

residues, xiv, 12, 53, 93, 99, 146, 203, 227,

234, 238, 240, 241, 242, 251, 257

resistance, 53, 55, 56, 83, 163, 181, 184,

278

resolution, 10

Index 301

resources, 116, 205, 261, 271, 276

respiration, 6

response, 77, 95, 96, 97, 99, 101, 103, 104,

114, 154, 155, 201, 205, 208, 211, 225,

275, 276

response capacity, 276

restrictions, 112

retail, 211, 244

retardation, 267, 270

reticulum, 195

retina, 226

RH, 173, 250

Rhizopus, 168, 169, 170, 171, 173, 182

risk assessment, vii, xiii, 8, 113, 118, 122,

192, 194, 204, 205, 208, 210, 228, 235,

238, 241, 242, 251, 257, 273

risk factors, 255, 269, 272

risk management, xiii, 192

risk perception, 220

RNA, 195, 200, 266

rodents, 6, 182

room temperature, 250

roots, 40

routes, 273

rubber, 110

rules, 212, 214, 219, 230

S

safety, xiv, 12, 67, 74, 119, 120, 209, 210,

211, 212, 215, 218, 221, 231, 234, 239,

245, 246, 251

saliva, 201

salts, 283

samplings, 175, 178, 179

Saudi Arabia, 25, 78, 220

schistosomiasis, 151

schizophrenia, 156

school, 71

science, 210, 223, 267

scope, 10, 211, 216

secondary metabolism, vii, ix, 36, 45, 46,

47, 48, 49, 50, 53, 54, 55, 58, 60, 62

secretion, x, 92, 95, 96, 97, 98, 100, 102,

201

security, 27, 210

seed, viii, 19, 35, 39, 40, 49, 51, 52, 54, 58,

61, 108, 115, 159, 167, 170, 172, 173,

175, 178, 180, 181, 185, 187, 189, 230,

245, 251

seeding, 164

selectivity, 186

selenium, 119, 151, 250

semen, 198

sensations, 200

sensing, ix, 36, 48, 50, 51, 52, 53, 60, 61

sensitivity, 10, 98, 199, 244

sequencing, vii, viii, 36, 46, 132

Serbia, 203, 228

serine, 94, 99, 102

serotonin, 200, 226

serum, 80, 81, 98, 105, 129, 136, 201, 206,

267, 268, 271, 277, 283

services, 231

sex, 80, 128, 129, 135, 138, 141, 142, 147,

151, 195, 198

sexual development, 55

SGOT, 268

SGPT, 268

sheep, 4, 24, 79, 241, 248, 252

showing, 127, 163, 171, 172, 175, 214, 265

sibling, 59, 87

signalling, ix, 36, 47, 49, 50, 52, 94, 99, 101

signals, 50, 92

signs, 79, 267, 271

silk, 39, 40, 41

Singapore, 75

sister chromatid exchange, 283

Slovakia, 200

small intestine, 193, 222

SNP, 143, 146, 153

socioeconomic status, 198

sodium, 12, 32, 281

software, 15

soil type, 263

solution, xv, 13, 15, 16, 19, 23, 119, 131,

261, 271

solvents, 10, 11, 238

South Africa, 71, 78, 220, 221, 228

South America, 39, 71

Index 302

sowing, xii, 158, 160, 165, 186

soy bean, 8, 127

soybeans, 77

SP, 152, 155, 189

Spain, 63, 72, 82, 88, 89, 202, 228, 241,

242, 243, 244, 250, 252, 254

specific gravity, 109

spectroscopy, 234, 238

sperm, 198, 224

spleen, 97

spore, 7, 50, 71

SS, 105, 156

stability, 84, 240, 254, 279

stakeholders, 70, 267

standard deviation, 13, 17

standard error, 177, 178

starvation, 57, 79, 84

state(s), 8, 9, 32, 47, 58, 70, 116, 184, 243,

271

statistics, 276, 282

stereospecificity, 225

sterigmatocystin, ix, 36, 38, 46, 47, 48, 49,

50, 55, 86, 280

sterile, 12, 56, 168, 172

stimulation, 96, 97

stress, xii, 39, 52, 56, 66, 71, 158, 165, 166,

168, 264

stressors, 5

structural gene, 46

structure, 10, 24, 44, 50, 62, 190, 223, 235

style, 244, 253, 257

styrene, 186, 280

sub-Saharan Africa, 80, 270

subsistence, 273

substitution, 143

substrate(s), viii, xii, xiv, 7, 10, 14, 21, 35,

39, 49, 65, 66, 158, 163, 181, 234, 236,

238, 243, 245, 249, 250, 255

succession, xii, 158, 173

sucrose, 22

Sudan, 43

sulfate, 12

sulfuric acid, 14

Sun, 153, 266, 284

supplementation, 57

suppliers, 211, 219

supply chain, 27

suppression, xv, 6, 52, 80, 260, 267, 269,

270, 271, 273

surface component, 24

surveillance, 207, 250, 271, 275, 276

survival, 22, 26, 184, 279

susceptibility, ix, x, 5, 6, 30, 64, 80, 92, 100,

104, 143, 147, 152, 153, 154, 155, 200,

265, 267, 271, 273, 284

sustainability, 122, 214

sustainable development, 210

Sweden, 13, 72

Switzerland, 33, 219

symptoms, xiii, 6, 79, 192, 197, 198, 200,

268, 269, 271, 272, 283

syndrome, 80, 268

synergistic effect, 96, 256, 274

synthesis, vii, ix, 36, 46, 47, 81, 185, 195,

198, 200, 242

T

T cell(s), 97, 98, 100, 201

T regulatory cells, 98

Taiwan, 155, 278, 284, 285

tannins, 189

target, xv, 3, 10, 48, 79, 100, 195, 260, 267

tariff, 110

TDI, 79

teams, 148

techniques, vii, viii, xiv, 2, 7, 36, 46, 138,

195, 202, 204, 234, 236, 238, 239, 244,

251, 265

technology, 25

telomere, 45

teratogenic effects, ix, xv, 64, 77, 260, 266

testing, 119, 218

testosterone, 80, 195

tetrahydrofuran, 92

TGA, 131

Thailand, 37, 39, 41, 43, 57, 60, 61, 200,

203

therapy, 274

thermal stability, 66

Index 303

Third World, xiii, 192

threats, 52

thymus, 131

tissue, 58, 95, 128, 129, 135, 138, 139, 168,

197

tissue homeostasis, 95

TLR4, 96

TNF-α, 95, 97, 98

tocopherols, 186

Togo, 81, 85, 198, 224, 270, 279

total product, 108

toxic effect, ix, 5, 38, 64, 93, 102, 143, 147,

194, 195, 196, 281, 284

toxic products, 10

toxicity, xiii, 3, 5, 19, 25, 32, 33, 37, 64, 77,

79, 80, 97, 99, 102, 151, 192, 193, 194,

195, 196, 198, 199, 248, 255, 262, 273,

285

toxicology, vii, 88, 105, 150, 223, 224, 226,

285

toxigenic fungi, viii, 35, 36, 42, 55, 60, 86,

109, 243

toxin, viii, xi, xv, 2, 3, 9, 10, 11, 22, 23, 35,

37, 38, 44, 53, 55, 66, 73, 89, 126, 127,

143, 147, 148, 164, 178, 179, 199, 216,

235, 246, 260, 266, 275, 276, 283

TP53, xi, 126, 127, 128, 131, 145, 148, 149,

151

trade, 8, 37, 110, 116, 119, 120, 202, 209,

210, 215, 220, 267

trade liberalisation, 210

trade policy, 120

traits, 86, 187

transcription, 47, 48, 50, 59, 60, 146, 195

transcription factors, 47

transduction, 104

transformation, 137, 153

translation, 195

transmission, 224, 273

transparency, 211, 219

transplant, 53

transport, 4, 7, 47, 66, 74, 111, 159

transportation, 67

traumatic brain injury, 226

treatment, 12, 68, 69, 95, 96, 97, 98, 197,

212, 224, 226, 245, 249, 257, 275

tremor, 200

trial, 257, 274

triggers, 96

trypsin, 268

tryptophan, 200, 226

tuberculosis, 273

Tukey Test, 167

tumor(s), ix, 64, 129, 135, 139, 143, 152,

155, 197, 200, 222, 223

tumor cells, 155

Turkey, 43, 72, 73, 87, 92, 193, 219, 221,

267, 284

turnover, 7, 199

tyrosine, 200, 226

U

U.S. Department of Agriculture, 231

United Kingdom (UK), 66, 103, 105, 272,

285, 286

United Nations, x, 64, 84, 186

United States, xiii, 38, 95, 185, 192, 200,

202, 231, 267, 285

urea, 12, 14

urine, 5, 27, 34, 80, 81, 92, 93, 102, 103,

271

USDA, 217

UV, 29

UV light, 37

V

vaccinations, 100, 271

vaccine, 100, 104, 201

validation, 13, 14, 15, 103, 253

variables, 132, 135, 204

variations, 5, 83, 89, 100, 173, 179, 194,

204

varieties, 190, 242, 243, 244, 274

vector, 187

vegetable oil, 27, 68, 81, 230, 256

vegetables, 72, 229, 280, 284

Index 304

Venezuela, 108, 200

ventilation, 67

vertebrates, 193

virus infection, 272, 282

viruses, 85, 152, 279, 284

visualization, 37

vitamin A, 273

vitamin E, 223, 273

vitamin K, 200

vitamins, 159, 249, 250

vomiting, xiii, 79, 192, 196, 269, 271, 272

vulnerability, 81

W

Washington, 27, 61

water, xii, 6, 7, 14, 16, 19, 21, 37, 44, 48,

57, 58, 59, 65, 66, 67, 158, 168, 184,

185, 188, 235, 253, 256

weight gain, 267

weight loss, 6, 79

welfare, 230

well-being, 37

wells, 14, 131

West Africa, 81, 85, 102, 189, 204, 224,

277, 279, 284

wheat germ, 89

White Paper, 211, 229

wild type, 51, 135

wood, 19

workers, 80, 194, 209

World Health Organization (WHO), x, 8,

12, 28, 29, 32, 33, 42, 62, 64, 85, 86,

120, 205, 207, 210, 223, 224, 226, 229,

254, 267, 272, 282, 285

World Trade Organization (WTO), 210,

212, 222

worldwide, ix, x, xiv, xv, 5, 38, 52, 63, 64,

73, 74, 79, 82, 84, 94, 159, 202, 214,

239, 260, 265, 266, 270, 275, 276

X

xeroderma pigmentosum, 149, 156

Y

yeast, 22, 26

Yemen, 78, 220

yield, 26, 45, 206, 207

young people, 80

Z

zinc, 57