Evaluation of cadmium and zinc inbiological samples of tobacco andalcohol user male mouth cancerpatients

Tasneem Gul Kazi1, Sham Kumar Wadhwa1, Hassan Imran Afridi1,Naveed Kazi2, Ghulam Abbas Kandhro1, Jameel Ahmed Baig1,Abdul Qadir Shah1, Nida Fatima Kolachi1 and Sumaira Khan1

AbstractThere is compelling evidence in support of positive associations between zinc (Zn) deficiency and highaccumulation of cadmium (Cd) in biological samples of different cancerous patients. The incidence of mouthcancer has been increased among people who possess habit of drinking alcohol, tobacco smoking and chewingwith gradients in Pakistan. In the present study, Cd and Zn contents of blood and scalp hair samples of 92 malemouth cancer patients and 78 referents of same age group (range 35–65 years), socio-economic status, local-ities and dietary habits. Among referent male subjects, 56 consumed tobacco (smoking/chewing), 22 had noneof smoking and chewing tobacco habits, while none of them admitted for alcohol drinking. The scalp hair andwhole blood samples were oxidized by 65% nitric acid and 30% hydrogen peroxide (2:1) ratio in microwaveoven. The Zn in digests was determined by flame atomic absorption while Cd was determined by electrother-mal atomic absorption spectrometry. The average Cd concentration was higher, while level of Zn was lower inthe blood and scalp hair samples of mouth cancer patients as compared to referent subjects (p < .001). Themale referents consuming tobacco (chewing and smoking) have high level of Cd in both their biological samplesas compared to those not smoking or chewing tobacco (p < .012). The Cd/Zn ratio in both biological sampleswas found to be higher in patients than in referent subjects.

Keywordscadmium, zinc, tobacco smoking/chewing, alcohol drinking, mouth cancer, scalp hair and whole blood

Introduction

Cancers of the oral cavity make up 3%–4% of all can-

cers, being in eighth place in men and eleventh in women

when the cancer is caused by smoking and alcohol mis-

use.1 The mouth cancer is the common malignancy

among people who have tobacco smoking and chewing

habits, with a worldwide incidence of over 300,000 new

cases annually.2 The disease is characterized by a high

rate of morbidity and mortality.3 Tobacco use is by far

the most common risk factor for cancers of the mouth

and throat. Both smoking (cigarettes, cigars and pipes)

and ‘smokeless’ tobacco (snuff and chewing tobacco)

have increased the risk of developing oral cancer.4,5

Tobacco smoke can cause cancer anywhere in the

mouth and throat as well as in the lungs, bladder and

many other organs in the body.6 Smokeless tobacco is

linked with cancers of the cheeks, gums and inner sur-

face of the lips.7 Many epidemiologic studies address

the risk of smokeless tobacco for the cancer of oral

cavity and adjacent sites.8,9 Cancers caused by

smokeless tobacco use often begin as leukoplakia or

1 Center of Excellence in Analytical Chemistry, University ofSindh, Jamshoro, Pakistan2 Liaquat University of Medical and Health sciences, Jamshoro,Pakistan

Corresponding author:Tasneem Gul Kazi, Center of Excellence in Analytical Chemistry,University of Sindh, Jamshoro, Pakistan, 76080.E-mail tgkazi@yahoo.com

Human and Experimental Toxicology29(3) 221–230

ª The Author(s) 2010Reprints and permission:

sagepub.co.uk/journalsPermissions.navDOI: 10.1177/0960327109360045

het.sagepub.com

erythroplakia, erythroplakia is generally more severe

than leukoplakia and has a higher chance of becoming

cancerous over time.10 The carcinogenic substances

in alcohol and tobacco chewing can cause cancer and

other problems in mouth, because all these materials

remain in contact with gums, inner lining of mouth

and tongue over long periods.11,12

The current estimate is that a nutritional deficiency

of Zn may affect over 2 billion subjects in the

developing world. Growth retardation, immune dys-

function and cognitive impairment are among major

consequences of Zn deficiency.13,14 Zinc deficiency

was associated with increased tumor size and overall

stage of the head and neck cancers.15

Cadmium (Cd) is a toxic metal with sterilizing,

teratogenic and carcinogenic effects.16 Cd exposure

due to industrial pollution or cigarette smoke is an

important risk factor for public health, which may

lead to cancer. It is shown that Cd is an inhibitor of

the enzymes with sulphydryl groups and disrupts the

pathways for the oxidative metabolism.17 In vitro

studies suggest that there is competition for transport

mechanisms between Cd and some essential trace

elements mainly Zn, Se and Cu.18

Determinations of trace metals in human tissues

and fluids (whole blood, serum, plasma and urine)

were used to obtain information on the nutritional sta-

tus for diagnosis of diseases, indication of systemic

intoxication and to obtain information on environ-

mental exposure.19,20 The importance of exploring the

depot-storage capacities of various elements, particu-

larly the toxic ones, remains a vital aspect in elemen-

tal analysis largely met by urine and hair testing.21

The determination of trace metals in biological

samples requires the use of sensitive and selective

techniques such atomic absorption spectrometer.22

This technique has need for solubilization and

enrichment of the analyte and complete or partial

decomposition of the matrix using either convective

systems or microwave ovens and dry ashing.23,24

Chewing tobacco or betel quid is very common in

different parts of Asia, especially Pakistan and India.

It is also popular in some immigrant groups in Europe,

North America and Australia. The term ‘quid’ means

a substance or mixture of substances put in the mouth

and chewed, usually for long periods. It usually con-

tains tobacco, either on its own or mixed with areca

nut (from the Areca catechu tree), slaked lime and

different additives that enhance the toxicity as well

as the psychotropic effect of tobacco.25 This mixture

is wrapped in a leaf (betel leaf), which is where

the name betel quid (also called paan in India and

Pakistan) comes from. The harmful substances in

tobacco and betel quid can cause cancer and other

problems in the mouth, because all these materials

remain in contact with gums, inner lining of the

mouth and tongue for many hours.26

As the rate of mouth cancer has increased in

Pakistan, mainly due to habit of smoking, chewing

unrefined tobacco or tobacco with betel nuts and alco-

hol consumption are considered the important causes

of mouth cancer. The betel nut and paan are not pro-

duced in Pakistan and a huge amount of foreign

exchange is spent on imports of these items. This

hospital-based study is aimed at evaluating the con-

centration of Cd and Zn in whole blood and scalp hair

among male mouth cancer patients (MCPs) and refer-

ents of same age group, from different cities of Sindh,

Pakistan. The biological samples were digested by

microwave prior to analysis of both elements by

flame/electrothermal atomic absorption spectrome-

tery (FAAS/ETAAS).

Materials and methods

Reagents and glassware

Ultra pure water obtained from a Milli-Q purifier

system Millipore Corp. (Bedford, MA, USA) was used

throughout the work. Concentrated nitric acid (65%),

hydrogen peroxide (30%) and other chemicals were

used as analytical reagent-grade E. Merck (Darmstadt,

Germany) and checked for possible trace metal con-

tamination. Working standard solutions of Cd and Zn

were prepared immediately before their use, by step-

wise dilution of certified standard solution (1000 ppm)

Fluka Kamica (Buchs, Switzerland), with 0.2 mol L–1

HNO3. The stock standard solution of modifier used

for Cd stabilization was 0.05% m/v Pd and 0.03%m/v Mg (NO3)2, which were prepared from, 3.0 g

L�1, Pd 99.999% Aldrich (Milwaukee, WI, USA) and

2.0 g L�1 Mg (NO3)2 solutions (Merck). All solutions

were stored in polyethylene bottles at 4�C. For the

accuracy of methodology, certified reference materials,

human hair BCR 397 (Commission of European com-

munities, Brussels, Belgium) and clincheck control-

lyophilized human whole blood (Recipe, Munich,

Germany) were used. All glassware and plastic materi-

als used were earlier soaked for 24 hours in 2 mol L–1

nitric acid, washed with distilled water and finally

rinsed with ultrapure water, dried and stored in class

100 laminar flow hoods.

222 Human and Experimental Toxicology 29(3)

Apparatus

The analysis of elements was carried out by means of

a double-beam Perkin-Elmer atomic absorption spec-

trometer model A. Analyst 700 (Norwalk, CT, USA),

equipped with a flame burner, graphite furnace

HGA-400 and an autosampler AS-800. The pyro-

coated graphite tubes with an integrated platform

were used. The Cd was measured under optimized

operating conditions by ETAAS. Single element hol-

low cathode lamp for Cd was operated at 4 mA with a

spectral bandwidth of 0.7 nm. The analytical wave-

length was set at 228.8 nm. The graphite furnace heat-

ing program for Cd was set for different steps, drying,

ashing, atomization and cleaning as temperature

range �C/time (sec) (80–120/15, 300–600/15, 1500–

1800/5 and 1800–2000/2), respectively. The acid

digests were transferred to auto sampler cups, and

with a chemical modifier solution introduced 20 mL

of digest and modifier (10 mL þ 10 mL) directly into

the graphite furnace. The Zn was measured under

optimized operating conditions by FAAS with air–

acetylene flame. A wavelength of 213.9 nm was used.

A PEL (PMO23, Osaka, Japan) domestic microwave

oven (maximum heating power at 900 W) was used

to digest the samples. Acid-washed polytetrafluor-

oethylene (PTFE) vessels and flasks were used for

preparing and storing solutions.

Study group

Before the start of this study, all referents and

mouth cancer patients, age range 35–65 years, were

informed through a consent form by the

administration about the aim of study, and all agreed

to participate and signed the form. A questionnaire

was also administered to them in order to collect

details concerning physical data, ethnic origin, health,

dietary habit, age and consent. All participants (refer-

ents and patients) were interviewed personally by the

authors. The standardized interview assessed

socio-demographic characteristics (Table 1). Partici-

pant’s history of cigarette smoking, tobacco chewing

with and without gradient and alcohol consumption,

including initiation age, quantities and years con-

sumed, was also collected. Among study groups,

more than 70% patient’s condition was apparently

worse in terms of chronic illnesses, malnutrition, pov-

erty and ignorance of disease for long time.

Physical examinations were performed in the can-

cer hospitals to measure participant’s weight, height,

blood pressure and biochemical data. The data of

study population (92 mouth cancer patients), admitted

in Nuclear Institute of Medicine and Radiotherapy

(NIMRA) Jamshoro and Larkana institute of nuclear

medicine and radiotherapy (LINAR), situated in dif-

ferent areas of Sindh, Pakistan, in the year 2008, was

obtained by collecting files and extracting important

information. During 1-year study period (2008), the

information department of both the hospitals recorded

>3600 cases of cancers of all types, and mouth cancer

comprised of 2.7%.

The patients were grouped according to their

habits, tobacco smoking (PST), chewing tobacco with

other gradients (PCT) and alcohol consumption with

tobacco smoking (PAST). While control group are

divided into two groups, first group comprises two

sub groups, tobacco smoker (RST) and tobacco

chewing with and without gradients (RCT), while sec-

ond group have none of these habits (RNT) as shown

in Table 1. The anatomical sites used to calculate the

total numbers of cases were gum (23), floor of the

mouth (27) and different part of the mouth (42).

Among MCPs, tobacco chewing with other gradients

(betel quid, calcium and areca nut) included 42.4%,

smokers were 45.6 %, while 12.0% of the total

patients were admitted to alcohol consumption with

tobacco smoking.

The biochemical tests of patients and referents,

haemoglobin, red blood cells, packed cell volume,

mean corpuscular haemoglobin, mean corpuscular

haemoglobin concentration, mean corpuscular

volume and transferrin iron-binding capacity in the

blood, are shown in Table 2. The histological distribu-

tion is not shown in the present study.

Table 1. Characteristics of study subjects (35–65) agegroups

Referents(n ¼ 78)

Mouth cancerpatients (n ¼ 92)

OccupationLabour 50 (64%) 46 (50.0%)Office worker 28 (36%) 33 (35.9%)Not working —— 13 (14.1%)

HabitsSmoking tobacco 29 (37.2%) 42 (45.6%)Chewing tobacco with

and without gradients(paan, areca nut and slakedlime)

27 (34.6%) 39 (42.4%)

Non-chew tobacco 22 (28.2%) 0 (0%)Smoking þ alcoholdrinkers

0 (0%) 11 (12.0%)

Kazi TG et al. 223

The criteria for the selection of patients was of

biopsy proved cancerous lesions in mouth, prior to

any treatment i.e., surgery, chemotherapy or radio-

therapy and they were not taking any mineral supple-

ment during last 3 months. The MCPs who had not

any habits of smoking or chewing tobacco with and

without gradients and alcohol drinking, were

excluded from this study. The criteria for selection

of 78 control male subjects were belonging to the

same age group, socio-economic status and dietary

habits, not suffering from any cancerous lesions and

not taking any mineral supplement. They were mostly

the healthy family members of the patients. Prior to

the biological samples collection, they have under-

gone a standard routine medical examination.27 We

excluded those control subjects who have tried

tobacco with betel quid only for a few times in their

lifetime.

Sampling

Venous blood samples (3–5 mL) were sampled by

using metal-free vacutainer EDTA tubes (Becton

Dickinson, Rutherford, NJ, USA) and was stored at

–20�C until required for analysis.28 Thoroughly

mixed the whole blood sample and transferred to the

storage tube at –4�C till further treatment. The hair

samples were taken from five different parts of the

scalp (frontal, cranial, occipital, right and left lat-

eral).29 The first 5 cm of hair from the root were used

for analysis. In the laboratory, the hair specimens

were further cut into approximately 0.2- to 0.3-cm

pieces and washed four times with a 1:200 v/v dilu-

tion of Triton X-100, then rinsed three times with

ultra-pure water and two times with acetone.30 The

samples were then dried in an oven at 80–85�C.

Microwave-assisted acid digestion method

A microwave-assisted digestion procedure was car-

ried out for decomposition of organic matrixes of bio-

logical samples. Replicate samples of scalp hair

(200 mg) and blood (0.5 mL) of each MCPs and

referents and five replicate samples of certified refer-

ence material samples were directly taken into Teflon

PFA flasks (Kartell; 25 mL in capacity). Added to

each flask was 2 mL of a freshly prepared mixture

of concentrated HNO3–H2O2 (2:1, v/v). The flasks

were kept for 10 min at room temperature and then

placed in a covered PTFE container. This was then

heated following a one-stage digestion programme

at 80% of total power (900 W), 3 min for blood sam-

ples, while 6 min was required for hair samples.

Thereafter, the digestion flasks were cooled and the

resulting solution was evaporated to a semidried mass

to remove excess acid, and then diluted upto 10.0 mL

in volumetric flasks with 0.1 mol L–1 nitric acid.

Blank extractions were carried out through the com-

plete procedure. Blanks and standard solutions were

prepared in a similar acid matrix. The concentrations

were obtained directly from calibration graphs after

correction of absorbance for the signal from an appro-

priate reagent blank. The validity and efficiency of the

microwave-assisted digestion method were also

checked with a conventional wet acid digestion

method on the same certified reference materials, as

reported in previous works.31,32

Table 2. Biochemical tests of tobacco smoking/chewing and alcohol users male mouth cancer patients and referents

Referents Patients

ParameterNormalrange RNT RCT RST PCT PST PAST

Hb (mg/dL) 13–16 15.2 + 1.1 14.6 + 0.7 14.2 + 1.2 11.6 + 0.7 11.2 + 0.9 10.2 + 1.4RBC (�1012/L) 4–6.6 6.3 + 0.2 5.5 + 0.5 5.2 + 0.6 3.45 + 0.28 3.17 + 0.34 2.85 + 0.19PCV (%) 40–54 52.5 + 1.3 48.8 + 1.55 45.6 + 1.2 32.5 + 1.2 27.5 + 2.3 25.6 + 2.6MCH (pg) 27–32 30.6 + 0.9 31.5 + 0.3 31.7 + 0.4 36.9 + 1.8 38.7 + 2.4 43.6 + 3.7MCHC (g/dL) 32–36 35.4 + 0.3 34.6 + 0.4 34.5 + 0.6 28.2 + 1.5 27.1 + 1.4 25.3 + 0.9MCV (fl) 76–94 93.4 + 0.5 95.5 + 2.1 97.4 + 1.2 129 + 3.8 135 + 4.2 146 + 6.7Serum Fe (mg/100 mL) 60–160 149 + 9.5 137 + 5.4 135 + 8.2 95.6 + 5.4 97.7 + 3.8 109 + 8.4TIBC (mg/100 mL) 280–400 365 + 35.1 345 + 14.5 335 + 19.7 263 + 26.7 254 + 36.4 217 + 15.8

Hb, haemoglobin; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; MCV, mean corpus-cular volume; PAST, smoking tobacco and alcohol drinking; PCT, patients chewing tobacco; PCV, packed cell volume; PST, smokingtobacco; RBC, red blood cells; RNT, referent not smoking/chewing tobacco; RCT, referents chewing tobacco; RST, referents smokingtobacco; TIBC, transferrin iron-binding capacity.

224 Human and Experimental Toxicology 29(3)

Statistical analysis

All statistical analyses were performed using com-

puter program Excel X State (Microsoft Corp., Red-

mond, WA, USA) and Minitab 13.2 (Minitab Inc.,

State College, PA, USA). ANOVA was used to assess

the significance of the differences between the vari-

ables investigated in cancerous and non-cancerous

persons. Stepwise multivariate linear regression anal-

ysis was performed using two models, cancerous

patients and referents.

Results

The elemental contents in the biological samples,

especially in blood samples, varied widely among

individuals; thus, a significantly large number of sam-

ples were required for statistical interpretation of the

data to achieve a meaningful correlation between phy-

siological disorders and concentrations of trace and

toxic elements. The mean concentrations with stan-

dard deviations for Zn and Cd in biological samples

are shown in Table 3. The results indicate that the

concentrations of Zn and Cd were altered in both bio-

logical samples (blood and scalp hair) of MCPs who

were chewing and smoking tobacco habits as well

as in alcohol users. In the case of RCT and RST, there

is no significant difference for Zn levels in both bio-

logical samples, while the level of Cd was signifi-

cantly higher in referent – those who have not

consumed any type of tobacco products.

The concentrations of Zn in the scalp hair samples

of male RNT, RCT and RST were significantly higher

at 95% confidence interval, (CI) (215, 222), (194,

201) and (173, 196) mg/g, respectively, as compared

with those in MCPs (PCT, PST and PAST) (CI: 151,

159), (CI: 132, 146) and (CI: 119, 130) mg/g,

respectively, with p < .01. The Zn levels in the blood

of RNT, RCT and RST, (CI: 9.89, 10.3), (9.22, 9.62) and

(CI: 8.78, 9.14) mg/L, respectively, were found to

be higher than those in PCT, PST and PAST,

(CI: 6.65, 7.02), (CI: 5.68, 6.44) and (CI: 4.96,

5.58) mg/L, respectively, (p ¼ .004–.006). It was

observed that the level of Zn did not vary signifi-

cantly in the biological samples of referent group

(RNT, RST and RCT), indicating that the alteration

of Zn in biological samples of MCPs was mainly

due to the cancerous state.

An elevated level of Cd content was observed in

the scalp hair of MCPs (PCT, PST and PAST). The

ranges of Cd in the scalp hair samples of PCT, PST

and PAST were (CI: 6.86, 7.39), (CI: 7.24, 7.67) and

(CI: 7.72, 8.22) mg/g, respectively, and were found to

be higher than RNT, RCT and RST, (CI: 1.34, 1.47)

(CI: 2.02, 2.28) and (CI: 2.36, 2.68) mg/g, respectively

(p < .003). The level of Cd in blood was statistically

significantly higher (p < .01) in PCT, PST and PAST,

(CI: 7.94, 8.56), (CI: 9.83, 10.7) and (CI: 11.4, 12.5)

mg/L, respectively, than those of referent groups. It

was observed that level of Cd was low in RNT,

whereas RST and RCT have high levels of Cd in their

blood samples, (CI: 7.99, 9.02) and (CI: 5.35, 6.16)

mg/L, respectively, but these values were still lower

than those values of MCPs.

The unpaired student t test at different degrees of

freedom between male mouth cancer patients and

referents were calculated at different probabilities.

Our calculated tvalue exceeds that of tcritical value at

95% confidence intervals, which indicated the signif-

icant differences between mean values of understudy

elements in referents and mouth cancer patients

(p < .001). The inter-elemental correlation (r) of Zn

versus Cd, in PCT and PST and referents, indicates

that the values of Zn in scalp hair and blood have low

correlation (0.39–0.53; Table 4). The Cd/Zn, ratio

was higher in mouth cancer patients as compared to

the referent subjects (Table 5).

Table 3. Concentrations of zinc and cadmium in biological samples (scalp hair and blood samples) of mouth cancerpatients and referents

Samples/elementsReferents Patients

Whole blood RNT RCT RST PCT PST PAST

Zinc (mg/L) 10.1 + 1.86 9.4 + 2.89 8.92 + 1.34 6.83 + 1.84 6.12 + 1.54 5.27 + 1.98Cadmium (mg/L) 4.16 + 1.2 5.76 + 1.60 8.5 + 2.33 8.25 + 1.34 10.2 + 2.90 12.0 + 3.62Scalp hair (mg/g)Zinc 218 + 15.5 198 + 13.4 189 + 12.6 155 + 16.30 136 + 13.6 125 + 12.8Cadmium 1.4 + 0.28 2.15 + 0.42 2.52 + 0.65 7.12 + 1.3 7.45 + 1.93 7.97 + 1.23

PAST, patients smoking tobacco and alcohol drinking; PCT, patients chewing tobacco; PST, patients smoking tobacco; RCT, referentschewing tobacco; RNT, referent not chewing/smoking tobacco; RST, referents smoking tobacco.

Kazi TG et al. 225

Discussion

This study provides data on the essential trace ele-

ment (Zn) and toxic element (Cd) in scalp hair and

blood samples obtained from mouth cancer male

patients (PST, PCT and PAST) and healthy referent

subjects (RNT, RST and RCT) of same age group.

Cancer is the most dreaded disease and it is the second

leading cause of death in many developed and under-

developed countries of the world.33 Worldwide, oral

cancer is a serious cause of morbidity and mortality

and its incidence varies widely according to geogra-

phical location. All forms of smoking are linked to

different cancers, including cigarettes, cigars and

pipes. Approximately 90% of people with mouth can-

cer are tobacco users. People who stop using tobacco,

even after many years of use, can greatly reduce their

risk of all smoking-related illnesses, including mouth

cancer.5

In developing countries, the early detection of oral

cancer – for example, as pre-malignant oral epithelial

dysplasia or early oral squamous cell carcinoma – was

not possible, probably due to poverty and illiteracy. As

observed in the present study, the under-study subjects

were referred to medical quacks (non-qualified medical

practitioners) instead of proper diagnostic centre, at the

onset of the disease.34 This study was conducted in dif-

ferent areas of Sindh, Pakistan. The resulting data as

shown in table 3, indicated that the concentration of

Zn and Cd were altered in whole blood and scalp hair

samples of biopsy confirmed mouth cancer male

patients, as compared to referent males.

The referent subjects, who are smokers or chew

tobacco, have high level of Cd in their both biological

samples as compared to those of referents who have

none of these habits, while the levels of Zn in both

samples were also found to be low, but the difference

was not significant (p ¼ .087). Poor oral cavity

hygiene and ill-fitting denture, tobacco use and alco-

hol consumption can increase the risk of developing

oral cancer.35 People chewing tobacco in betel quid

(paan) with areca nut are over five times more likely

to be at risk of oral cancer.36 The International

Agency for Research on Cancer (IARC) now regards

the betel nut itself to be a known human carcinogen.37

In making this new finding, a working group has been

investigated the epidemiological facts from India and

Pakistan, which allowed disentangle the effect of

betel quid with and without tobacco, and studies from

Table 4. Linear regression and Pearson’s coefficient for zinc versus cadmium in referents and mouth cancer patients

Samples/habits Referents Patients

Scalp hairRNT 0.0165x – 5.01; r ¼ .88 —————————CT 0.026x – 7.32; r ¼ .71 0.031x þ 2.65; r ¼ .53ST 0.039x – 9.93; r ¼ .68 0.044x – 14.3; r ¼ .51AST ————————— 0.039x þ 1.97; r ¼ .48BloodRNT 0.0011x - 0.015; r ¼ .81 —————————CT 0.0015x – 0.020; r ¼ .73 0.0004x – 0.011; r ¼ .53ST 0.0019x – 0.027; r ¼ .72 0.0018x – 0.023; r ¼ .52PAST ————————— 0.0011x – 0.018; r ¼ .39

CT, referents and patients chewing tobacco; RNT, referent not chewing or smoking tobacco; ST, referents and patients smokingtobacco; PAST, patients smoking tobacco and alcohol drinking.

Table 5. Cd/Zn mole ratio in referents versus mouth cancer patients

Specimens Referents/patients

Cd/Zn

RNT CT ST PAST

Scalp hair Referents 3.74 � 10–3 6.32 � 10–3 7.76 � 10–3 —————Patients ——— 2.67 � 10–2 3.19 � 10–2 3.71 � 10–2

Blood Referents 2.4 � 10–4 3.57 � 10–4 5.54 � 10–4 —————Patients ——— 7.03 � 10–4 9.74 � 10–4 1.32 � 10–3

CT, referents and patients chewing tobacco; RNT, referent not chewing or smoking tobacco; ST, referents and patients smokingtobacco; PAST, patients smoking tobacco and alcohol drinking.

226 Human and Experimental Toxicology 29(3)

Taiwan (China), where tobacco is not added to the

betel quid.37,38 It was reported in literature that betel

quid with areca nut produce carcinogenicity in experi-

mental animals.34 Chewing betel quid (with mixture

of tobacco leaves, areca nut and spices) is a signifi-

cant risk factor for oral cancer is also confirmed by

another study.36

Malnutrition is one of the major problems in cancer

patients. Although not prevalent in all types of can-

cers, but mouth cancer, undernourishment is a com-

mon and serious problem.34 Cancer patients usually

have inadequate energy and protein intakes, increased

metabolic rate and abnormalities in carbohydrate,

lipid and protein metabolism.39 Cancer treatment with

chemotherapy and radiation therapy is also potentially

damaging the nutritional status.40 A poor diet may

increase the risk of certain types of mouth cancer,

because of a lack of Zn, vitamins and other essential

minerals. Epidemiological studies also suggest a role

for diet in this context.41 In western countries, the

main risk factors are tobacco and alcohol consump-

tion, which have been shown to account for 75%–

90% of oral cancer, with evidence of a synergistic

effect of these two exposures.42 In industrial regions

of the world, increased consumption of tobacco has

been held responsible for the pronounced increase in

oral cancer.43 It is demonstrating that reactive oxygen

species, such as hydroxyl radical, are formed in the

human oral cavity during areca quid chewing, and that

the activity might cause oxidative DNA damage to the

surrounding tissues.44 It has been suggested that free

radicals, reactive oxygen species and reactive nitro-

gen species in the inhaled tobacco smoke and eating

raw tobacco induce lesions of the mucosa, which are

then transformed into oral cancer.45

Alcohol acts as a solvent that enhances the penetration

of carcinogenic compounds into the mucosa. Ethanol

may facilitate the uptake of environmental carcinogens,

especially from tobacco smoke, through cell membranes

that are damaged and changed in their molecular compo-

sition by the direct effect of alcohol. Furthermore,

chronic alcoholism leads to atrophy and lipomatous

metamorphosis of the parenchyma of the parotid and sub-

maxillary gland and this alteration results in a functional

impairment of saliva flow and its increased viscosity.

Thus, the mucosal surface will be insufficiently rinsed

and is, therefore, exposed to higher concentrations of

locally acting carcinogens in addition to a prolongation

of the contact time of the substances with the mucosa.46

Tobacco leaves naturally accumulate and concen-

trate relatively high levels of Cd, and therefore

smoking of tobacco is an important source of Cd

exposure for smokers.47 The total amount of carcino-

gens in cigarette smoke ranges from one to 3 mg per

cigarette.35 The country of origin and type of the

product play major roles in determining the chemical

composition of cigarette tobacco.48 It was investi-

gated that one pack of cigarettes deposits 2–4 mg Cd

into the lungs of a smoker, whereas some of the

smoke passes into the air to be inhaled by smokers and

nonsmokers alike.49 It was also consistent with

another study that smokers generally exhibit signifi-

cantly higher Cd body burdens than non-smokers,

while smoking with alcohol consumption enhance the

Cd absorption and accumulation in all the tissues.47

The results suggested that although Cd poses a hazard

to essential trace metal homeostasis of various organs,

co-exposure can pose a major threat, while consump-

tion of ethanol may absorb much more Cd than their

unexposed counterparts.50

The results of present study are consistent with the

hypothesis that exposure to Cd oxide fumes by

tobacco smoking or chewing increases the risk of

mortality from oral cancer, due to deficiency of essen-

tial elements like Zn.51 In the past few years, increas-

ing consideration has been given to interactions

occurring in the organism between toxic metals and

bioelements essential for life. These interactions are

complex and involve biometals such as zinc, copper,

iron, selenium, calcium and toxic elements, including

cadmium.52 The basis of Cd toxicity is its negative

influence on enzymatic systems of cells, resulting

from substitution of other essential metal ions (mainly

Zn, Cu and Ca) in metalloenzymes and its very strong

affinity to biological structures containing –SH

groups, such as proteins, enzymes and nucleic acids.53

The relevance of Cd–Zn interactions should be con-

sidered in the light of the general population exposure

to Cd54 and common deficiency of Zn in the world,

mainly due to nutritional factors.55

Zn was considered one of the necessary composi-

tions of many enzymes in human body, involved in

the synthesis of DNA and RNA polymeric enzymes,

took part in the nucleic acid metabolism and immuno

surveillance protection, affecting the process of can-

cer development directly or indirectly. Epidemiologi-

cal studies also indicated that content of Zn in serum

of tumor patients was lower than in healthy persons.56

Zinc is well known to induce production of metal-

lothionein, which is very rich in cysteine, and this is

an excellent scavenger of hydroxyl radical. Thus, it

is clear that Zn has multiple roles as antioxidant and

Kazi TG et al. 227

is, therefore, an excellent candidate for clinical che-

moprevention trials in humans.57

It was reported that the mechanism by which Cd

antagonizes Zn may be from its ability to substitute

for Zn in the Zn finger DNA binding domain, and this

may be the way Cd causes toxicity and cancer.

Cadmium depletes glutathione and protein-bound

sulfhydryl groups, resulting in enhanced production

of reactive oxygen species such as superoxide ion,

hydroxyl radicals and hydrogen peroxide.58 A far

more comprehensive study of the basic mechanism

for alteration of Zn level in blood, and other biologi-

cal samples, and its significance in all malignancies is

needed. Many epidemiologic studies suggest that Zn

deficiency may be associated with increased risk of

cancer.57 Zn supplementation is associated with

decreased oxidative stress and improved immune

function and might aid in the prevention and treat-

ment of cancer. The effect of Zn on the immune sys-

tem strengthens the body’s defense against abnormal

cell growth associated with cancer development.59

The profound Zn deficiency is quite rare in

humans, but mild-to-moderate Zn deficiency may be

relatively common throughout the world in both

less developed and industrial countries,60 and

Zn-deficient persons have increased susceptibility to

a variety of pathogens; although the cause in some

cases may be inadequate dietary intake, Zn absorption

is possibly the most common causative factor.55 The

effects of Zn on the initiation and progression of can-

cer are not well established; although the negative

effects of Zn deficiency on the immune system are

clear.61 Changes in blood Zn have been found in lym-

phoproliferative disorders as well as in breast, lung

and gastrointestinal tumors.62 It was investigated that

the decrease in plasma Zn concentration was observed

after stress, trauma and in several malignancies.63

A balanced diet that includes enough calcium, iron,

protein and Zn will also help to reduce the amount

of Cd that may be absorbed into the body from food

or drink.64

It was also observed in present study that the socio-

economic factors also play a role in higher mortality

rates in patients, such as poor nutrition, irregular

screening, late diagnosis and unequal access to health

care due to poverty. On other side, the cost of treat-

ment for mouth cancer and other types of cancer is

very high, which is commonly not affordable. The

local hygiene center facilities are poor in our country

and there are no routine monitoring and screening

carried out for those people living in small towns.

Conclusion

The results of this study revealed that mouth cancer

male patients have a different pattern of essential

trace (Zn) and toxic (Cd) elements in their biological

samples as compared to the healthy referents, with the

prevalence being more in smoking and chewing

tobacco patients. However, higher levels of Cd, as

well as a lower level of Zn, correlated well with the

consequences of mouth cancer. The deficiency of the

Zn, which is replaced by Cd, may result in abnormal

physiology disorders, and, in addition to other factors,

which play role in developing mouth cancer. This

study provides some support for the hypothesis that

dietary intake of essential elements and inhalation

of Cd, most probably through drinking alcohol, smok-

ing and chewing tobacco, may increase the risk of

mouth cancer and related disorders, which indicates

that the causal link may be stronger among tobacco

smokers/chewers and alcohol users. We propose that

essential and toxic elemental measurements may be

performed on patients reaching in the emergency

department, to test whether the concentration of it

may serve not only as markers of oral or mouth cancer

and its remedies but also as predictors of adverse

outcomes.

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