ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH · Determinação da toxicidade em artemias salinas ... MONICA GOMES LIMA UFPA . ... 1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL

1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH

CEBiTOR 2012

1ST INTERNATIONAL CONFERENCE ON

ENVIRONMENTAL BIOINORGANIC AND

TOXICOLOGY RESEARCH

CEBiTOR 2012

December 6-8, 2012

Federal University of São Paulo

Institute for Environmental, Chemical and Pharmaceutical Sciences Rua

São Nicolau, 210. Diadema-SP. Brazil


CEBiTOR 2012


CEBiTOR 2012

ORGANIZING COMMITTEE

President:

Raúl Bonne Hernández, PhD (ICAQF-UNIFESP, Brazil)

Vise President:

Breno Pannia Espósito, PhD (IQ-USP, Brazil)

Treasurer:

Diogo de Oliveira, PhD (ICAQF-UNIFESP, Brazil)

Other members:

Georgia C. Labuto, PhD (ICAQF-UNIFESP, Brazil)

Mary Ishimine Nishita (ICAQF-UNIFESP, Brazil)

Ísis Marques (ICAQF-UNIFESP, Brazil)

Pollyanna Ferreira de Carvalho (ICAQF-UNIFESP, Brazil)

Bianca Gomes (ICAQF-UNIFESP, Brazil)

Karina Bugan Debs (ICAQF-UNIFESP, Brazil)

Pedro Amorim (ICAQF-UNIFESP, Brazil)

SCIENTIFIC COMMITTEE

President: Fernando Barbosa Junior, PhD (FCFRP-USP, Brazil)

Vice Presidents:

Robert Gerlai, PhD (UofT, Canada)

Ciro Alberto de Oliveira, PhD (UFPR, Brazil)

FINAL PROGRAM

DECEMBER, 6th


CEBiTOR 2012

15:00 –18:00 Accreditation 5 th Floor

DECEMBER, 7th

07:30-08:10 Accreditation 5 th Floor

08:15-08:55 Opening ceremony 09:00-16:00 Metals and other substances in biomedicine and pharmacology

5 th Floor

09:00-09:40 Robert Gerlai

Department of Psychology.

University of Toronto - Canada

Using zebrafish to unravel the genetics of complex brain disorders.

09:55 - 10:30 Adelson Silva

Especialista de Produtos em Espectroscopia| Agilent Technologies - Brazil

“Transformando a técnica de ICP-MS com Triplo Quadrupolo”

10:30-10:20 Coffe break and Poster discussion 1st Floor 10:25-10:55 Adelson Silva

Especialista de Produtos em Espectroscopia| Agilent Technologies - Brazil

“Aplicações de ICP-MS acoplado a Cromatografia (GC e HPLC)”

5 th Floor 11:00-11:50 Breno Pannia Espósito

Instituto de Química. University of São Paulo. Brazil

Fluorimetric detection of redox-active metals in cells and

biological fluids. 12:00-12:40 Mohamme Idris

Centre for Cellular and Molecular Biology. India Bio-mechanism of regeneration and degeneration in

zebrafish model system. 12:50-13:55 Lunch Free 14:00-14:40 Diogo Losch de Oliveira

Department of Biochemistry Federal University of Rio Grande do Sul. Brazil

A detailed characterization of the behavioral pattern of zebrafish in the open tank paradigm: implications for

drug toxicity evaluations.

5 th Floor 14:50-15:30 Steven A. Farber

Depart.of Embryology.

Carnegie Institution for Science. USA

Why we dip our lobster in butter: Using zebrafish larvae to uncover an unexpected linkage between dietary fat and

cholesterol absorption 15:40-16:20 Marco Aurélio Zezzi Arruda

Depart.of Analytical Chemistry. University of Campina Metallomics-based systems focusing on life-science:

from plant to fish 16:30-16:50 Coffe break and Poster discussion 1st Floor 16:55-17:45 Oral short communication and evaluation

5 th Floor

16:55-17:05 Anderson Arndt

University of São Paulo - Brazil

“Toxicity of manganese(III) complexes to Danio rerio”

17:10-17:20 Heydi Noriega Guerra


“Importancia da protease ADAMTS-1 na invasão local e

sistêmica de células de fibrossarcoma”

17:25-17:35 Monica Gomes Lima

Federal University of Pará - Brazil

“Behavioral and neurochemical changes in the zebrafish

leopard strain” 17:40-17:50 Ana Carina Nogueira Vasconcelos

Federal University of Lavras

“Toxicological analysis of caffeine using embryos of

Danio rerio as an experimental model” 18:00-19:00 Raúl Bonne Hernández

ICAQF-Federal University of São Paulo - Brazil

Constitution of an International Network for

Environmental Bioinorganic and Toxicological Research

- NEBITOR: Environmental Neurotoxicology of metals

5 th Floor

Only among speakers

DECEMBER, 8th

Environmental bioinorganic chemistry and environmental health, and the zebrafish model as teaching tool

5 th Floor

09:00-09:40 Ciro Alberto Oliveira

Federal University of Paraná

09:50-10:30 Michael J. Carvan III

School of Freshwater Sciences.University of Wisconsin-

USA

Zebrafish as a Model System for Environmental Health Studies in the Classroom

10:40-11:20 José Antonio Menezes-Filho

Depart. of Clinical and Toxicological Analysis

Federal University of Bahia-Brazil

Cognitive and neurobehavioral effects of children’s exposure to airborne manganese

11:30-11:55 Poster discussion and evaluation 1st Floor 11:55-13:25 Lunch and NEBITOR discussion 13:30-14:10 Michael J. Carvan III

School of Freshwater Sciences.

University of Wisconsin-USA

The Effects of Developmental MeHg Exposure at

Environmentally-Relevant Levels

5 th Floor

14:20-15:00 Fernando Barbosa Junior

FCFRP - University of São Paulo

Oral short communication and evaluation 15:10-15:20 Caio Maximino de Oliveira

Federal University of Pará - Brazil

“Serotonergic modulation of zebrafish behavior in

anxiety tasks” 15:25-15:35 Carine Rodrigues Pereira

Federal University of Lavras “Exposure of Danio rerio embryos to Glyphosate cause

teratogenic effects” 15:40-15:50 Hector Aguilar Vitorino


“Determinação da toxicidade em artemias salinas frente a

metalofarmacos de ferro de uso comercial e humano com capacidade de gerar atividade redox ativo”

15:55-16:05 Mary Ishimine Nishita

ICAQF-Federal University of São Paulo - Brazil

Embryotoxicity induced by mixtures of metal species.

16:10-16:55 Coffe break 1st Floor 17:00–18:00 Closing ceremony 5 th Floor


CEBiTOR 2012

Students considered with stays in double room-breakfast included

Nome Instituição

Hotel Trade Hotel Diadema

Rua São Pedro, 135 - Centro,

Diadema, 09910-620

(11) 4056-8611 ‎ ·

1. ANA CARINA NOGUEIRA

VASCONCELOS

UFL

2. CAIO MAXIMINO DE

OLIVEIRA

UFPA

3. CARINE RODRIGUES

PEREIRA

UFL

4. MONICA GOMES LIMA UFPA


CEBiTOR 2012


CEBiTOR 2012

Using zebrafish to unravel the genetics of complex brain disorders

Robert Gerlai

Department of Psychology.

University of Toronto - Canada

The zebrafish has been prominently utilized in developmental biology for the past three decades and

numerous genetic tools have been developed for it. Due to the accumulated genetic knowledge the

zebrafish has now been considered an excellent research tool in other disciplines of biology too,

including behavioral neuroscience and behavior genetics. Given the complexity of the vertebrate

brain in general and the large number of human brain disorders whose mechanisms remain mainly

unmapped in particular, there is a substantial need for appropriate laboratory research organisms

that may be utilized to model such diseases and facilitate the analysis of their mechanisms. The

zebrafish may have a bright future in this research field. It offers a compromise between system

complexity (it is a vertebrate similar in many ways to our own species) and practical simplicity (it is

small, easy to keep, and it is prolific). These features have made zebrafish an excellent choice, for

example, for large scale mutation and drug screening. Such approaches may have a chance to tackle

the potentially large number of molecular targets and mechanisms involved in complex brain

disorders. However, although promising, the zebrafish is admittedly a novel research tool and only

few empirical examples exist to support this claim. In this chapter, first I briefly review some of the

rapidly evolving genetic methods available for zebrafish. Second, I discuss some promising

examples for how zebrafish have been used to model and analyze molecular mechanisms of

complex brain disorders. Last, I present some recently developed zebrafish behavioral paradigms

that may have relevance for a spectrum of complex human brain disorders including those

associated with abnormalities of learning and memory, fear and anxiety, and social behavior.

Although at this point co-application of the genetics and behavioral approaches is rare with

zebrafish, I argue that the rapid accumulation of knowledge in both of these disciplines will make

zebrafish a prominent research tool for the genetic analysis of complex brain disorders.


CEBiTOR 2012

Fluorimetric detection of redox-active metals

in cells and biological fluids

Breno Pannia Espósito Instituto de Química – Universidade de São Paulo – Brasil

Metal ions are persistent contaminants present in ever-increasing technological, medical and

nutritional applications. The properties of redox-active metal cations in biologically relevant media

(cells and plasma) are conveniently assessed by high throughput fluorimetric assays. In this

presentation, the case study of iron contamination and the clinical and environmental applications of

fluorimetric detection of iron-mediated oxidative stress will be discussed.


CEBiTOR 2012

The Effects of Developmental MeHg Exposure

at Environmentally-Relevant Levels

Michael J. Carvan III and Rebekah Klingler

School of Freshwater Sciences, University of Wisconsin-Milwaukee, USA

Children prenatally exposed to methylmercury (MeHg) display a range of effects varying from

severe cerebral palsy to subtle developmental delays. Many of the more subtle effects of

developmental MeHg exposure can be permanent, including behavioral abnormalities, nervous

system deficits, and loss of IQ. It is estimated that as many as 15% of American women of

childbearing age have total blood mercury concentrations at a level above that associated with

neurological deficits in children. The zebrafish is a well-supported model system for the study of

the mechanistic basis of human developmental disease and dysfunction. As a result, it is also a very

powerful model for the study of environmental health, especially for exploring gene-environment

interactions in developmental disease and dysfunction. Zebrafish suffer neurological deficits

(learning and memory, and visual system problems) at exposures similar to those that affect humans

and these deficits persist into adulthood and across generations. We are using a number of

approaches including knockouts, transgenics, and transcriptomics, in conjunction with

neurobehavioral analyses to explore gene-environment-dysfunction relationships. Our findings have

implications for human health and can also be used to model the impact of environmental mercury

on wild populations of fish.


CEBiTOR 2012

Metallomics-based systems focusing on life-science: from plant to fish

Marco Aurélio Zezzi Arruda

Spectrometry, Sample preparation and Mechanization Group – GEPAM, National Institute of Science and Technology -

INCT for Bioanalytics, Institute of Chemistry, Department of Analytical Chemistry, UNICAMP,

PO Box 6154, 13803-970, Campinas, São Paulo, Brazil

e-mail: [email protected]

Metalloproteomics encompasses the inorganic elements content and assemble of their complexes

with proteins [1]. Although metalloenzymes and metalloproteins are responsible for catalyzing

molecular oxygen reduction and nitrogen fixation, photosynthesis and respiration, water oxidation

[2], the information related to metals and proteins is frequently found fragmented in the literature.

Then, this hiatus contributes to remain allusive some mechanisms involving metal incorporated as a

cofactor in a cell [3], metal-sensing related to metalloproteins [4], among others. In this way, the

proposal of this conference is to demonstrate the potentialities of metallomics focusing on life-

science examples, which are being developed in our research group, with emphasis to comparative

metallomics studies involving transgenic and non-transgenic soybean seeds and plants, bipolar

disorder using blood serum samples of control and bipolar groups, and some preliminary results

regarding oxidative stress in Zebrafish.

References

[1] Garcia, J. S., Magalhães, C. S., Arruda, M. A. Z., Talanta 69 (2006)1-15

[2] Lu, Y., Yeung, N., Sierack, N., Marshall N. M., Nature 460(2009)855-862

[3] Szpunar, J., Anal. Bioanal. Chem. 378(2004)54-56

[4] Waldron, K. J., Rutherford, J. C., Ford, D., Robinson, N. J., Nature 460(2009)823-830


CEBiTOR 2012

Zebrafish as a Model System for Environmental Health Studies

in the Classroom

Michael J. Carvan III, Henry G. Tomasiewicz, Renee Hesselbach,

Craig Berg, David H. Petering

School of Freshwater Sciences, University of Wisconsin-Milwaukee, USA

We are using the zebrafish model system as an educational tool to teach the concepts of

environmental toxicology to high school students. Our program (called “Biology-Environmental

Health Science Nexus: Inquiry, Content, and Communication”) focuses on the high school General

Science and Biology teachers and students in metropolitan Milwaukee, particularly minority

students in the Milwaukee Public School District. Our general objective is to develop the skills of

inquiry in teachers and students as the basis for doing and understanding science, particularly in

relation to life science and environmental health. This goal takes form in science content

recommended by the National Science Education Standards in the areas of genetics, cellular

properties, and organismic behavior. These areas are enriched by strong links to environmental

health issues of importance to students, including exposure to lead, alcohol, and UV radiation and

conditions such as asthma. A series of fully developed and supported experiment modules have

been created that can be used to teach ninth grade General Science and tenth grade Biology content.

Each involves the use of aquatic organisms as biomedical models and some include

macromolecular models and molecular biology experimentation. The Modules set in motion

activities that together provide teachers and students with multiple encounters with authentic

scientific inquiry. Integrated with the modules are student communication about the experiments

within and between classes, writing and publishing research communications and reports, and

participation in an annual research conference. Teachers gain professional development through

workshops, yearly scientific and educational support, evaluation activities and feed-back, and doing

program activities that enhance their abilities as scientists and science mentors for their students.

(Supported by NIH-NIEHS grants ES-04184 and R25RR026299)


CEBiTOR 2012

A detailed characterization of the behavioral pattern of zebrafish in

the open tank paradigm: implications for drug toxicity evaluations.

Diogo Losch de Oliveira

Department of Biochemistry

Federal University of Rio Grande do Sul. Brazil

In order to test the toxicity of an specific chemical compound we need a complex iterative process

of biochemical and cellular assays, with final validation in an animal model, and ultimately in

humans. Mammalian models for drug toxicity evaluation are expensive, laborious and consume

large quantities of precious compounds. In addition, there is also increasing pressure to limit animal

use to situations in which they are absolutely necessary, such as in preclinical toxicity and safety

assessment. Thus, zebrafish are beginning to be used at various stages for drug discovery and drug

toxicity evaluation and can be a useful and cost-effective alternative to some mammalian models.

However, in spite of the several behavioral tasks described for mammals, behavioral tests for drug

toxicity evaluation in zebrafish are scarce. Therefore, in the present work we aimed to perform a

detailed characterization of the behavioral profile of the short-fin zebrafish behavior in the open

tank paradigm, a protocol used to evaluate several aspects of the zebrafish behavior. Fish were

divided in three groups and confined during 10 min into bright, dark or transparent tanks. After,

animals were subjected to the novel tank test and their exploratory profile was recorded during a 15-

min trial. The results demonstrated that zebrafish increased their vertical exploratory activity during

the first 6-min, where the bright group spent more time and travelled a higher distance in the top

area. All behavioral parameters measured for the dark group were similar to the transparent one.

These data were confirmed by automated analysis of track and occupancy plots and also

demonstrated that zebrafish display a classical homebase formation in the bottom area of the tank. A

detailed spatio-temporal study of zebrafish exploratory behavior and the construction of

representative ethograms showed that the experimental groups presented significant differences in

the first 3-min vs. last 3-min of test. Although the main factors involved in these behavioral

responses still remain ambiguous and require further investigation, the current report describes an

alternative methodological approach for assessing the zebrafish behavior after a forced exposure to

different environments. Additionally, the analysis of ethologically-relevant patterns across time

could be a potential phenotyping tool to evaluate the toxic effects of several chemical compounds

on exploratory profile of zebrafish in the open tank task.


CEBiTOR 2012

Why we dip our lobster in butter: Using zebrafish larvae to uncover

an unexpected linkage between dietary fat and cholesterol absorption Steven A. Farber

Depart.of Embryology.Carnegie Institution for Science. USA

There is a significant body of work linking alterations in lipid metabolism (induced through genetic

mutation and/or lifestyle choices) with cardiovascular disease, diabetes mellitus, and obesity. In the

US and Canada, approximately one in eleven adults has diabetes and one in three children is obese,

and the diseases caused by these conditions are by far the leading cause of morbidity and health

care spending. One focus of our efforts is to understand mechanisms by which the intestine absorbs

and processes lipids. The vertebrate intestine contains symbiotic organisms, bile and mucus that

make it difficult, if not impossible, to study ex vivo. This is one reason that many longstanding

questions remain unresolved in the field of intestinal lipid metabolism. In general, the goal of my

lab is to better understand lipid trafficking in digestive organs by exploiting the optical clarity of

zebrafish larvae to observe, at the whole organ and subcellular levels, the localization of a variety of

fluorescent lipids. While the zebrafish has been established as a powerful model for the study of

early development, few researchers have taken advantage of the accessibility and optical clarity of

the embryos to visualize lipid uptake and processing in vivo.

Using the larval zebrafish found an unexpected mechanism coupling long chain mono unsaturated

fatty acid (a common fat component) with cholesterol uptake by the absorptive cells of the small

intestine (enterocytes). This study utilized a variety of diets that contained either fluorescent

cholesterol or fluorescent fatty acid to visualize lipid absorption. Using this strategy, we found that

a lipid-rich meal promotes BODIPY-cholesterol absorption into an endosomal compartment

physically distinguishable from lipid droplets that are labeled with BODIPY-fatty acid. We also

establish that long chain dietary fatty acids promote intestinal cholesterol absorption by inducing

the rapid re-localization of a cholesterol transport protein (NPC1L1). The cellular dynamics of

NPC1L1 was monitored by using a hsp70-humanNPC1L1-mCherry transgenic line we created.

Using pharmacological, dietary, and transgenic approaches we show that NPC1L1 must be

displayed on the intestinal brush border for BODIPY-cholesterol uptake into enterocytes. These data

are the first of their kind to visualize cholesterol uptake in a live whole animal and to elucidate the

cell biological mechanism of the process. We hypothesize that this system may have evolved to

ensure that potentially toxic cholesterol is never imported without corresponding uptake of the fatty

acids needed for its processing and export.


CEBiTOR 2012

Bio-mechanism of regeneration and degeneration in

zebrafish model system

Mohamme Idris

Centre for Cellular and Molecular Biology. India

The epimorphic regeneration of zebrafish caudal fin is rapid and complete. We have analyzed the

molecular mechanism of zebrafish caudal fin regeneration based on transcriptomics, proteomics and

epigenetic studies. A total of 96 proteins were identified as differentially regulated at different time

points of zebrafish caudal fin regeneration. The identified proteins were further validated and

studied for its differential expression at the transcript and protein level. Based on our study we

found that Annexin family genes were differentially regulated and posttranslational modified at

different time points of regeneration. Epigenomic associations of the gene based on histone

regulation were also studied involving ChiP assay. We are also trying to understand the mechanism

of degeneration in neurodegenerative disorders such as Spino cerebellar ataxia (SCA) and

Parkinson’s disease in the zebrafish model. Neuro-degeneration, the chronic breakdown of neuronal

structures, is mostly associated with expansion of CAG triplet repeats leading to aggregation and

polymerization of proteins. Our group aims at understanding the association of expanded triplet

repeats (CAG) in the ataxin gene with neurodegeneration based on transgene approach in zebrafish.

Also we are trying to understand the molecular and functional mechanism of neurodegeneration in

the zebrafish Parkinson’s disease model.


CEBiTOR 2012

Cognitive‎and‎neurobehavioral‎effects‎of‎children’s‎exposure‎to‎

airborne manganese

José Antonio Menezes-Filho Department of Clinical and Toxicological Analysis

College of Pharmacy

Federal University of Bahia

Background: High levels of waterborne manganese have been associated with problematic

behaviors and cognitive deficits in school-aged children, however to date this has not been reported

with respect to airborne manganese.

Objectives: We report here the results of our investigations carried between 2007 and 2009 on

children’s exposure to airborne manganese from ferro-alloy plant and cognitive effects. Preliminary

results of a new study that is evaluating neurobehavioral effects on children will also be presented.

Methods: In the cross-sectional studies carried out between 2007 and 2009 we evaluated 109

children from Cotegipe Village in the Simões Filho town, metropolitan area of Salvador, Bahia,

Brazil. The community was a priori divided according to the distance from the ferro-manganese

alloy plant, whose atmospheric emissions have been causing complains from the community since

its inauguration in the early 1970’s. We measured Mn exposure by collecting air, water and crop

production samples. Children’s exposure was assessed by analyzing blood and hair Mn levels. Later

on, we reevaluated 83 school-aged children and investigated Mn association with cognitive deficits

applying WISC-III (Wechesler scale for children). Confounders like maternal intelligence,

socioeconomic status, malnutrition, lead co-exposure, etc were also evaluated. Currently, in a new

cross-sectional study, we are reevaluating school-aged children from this community and from a

neighboring and further community for cognitive, behavioral and motor effects. New biomarkers

for Mn exposure are being studied as well.

Results and Comments: The average and median Mn concentrations during the rainy period were

0.151 µg/m3 and 0.114 µg/m3, respectively (range 0.011–0.439 µg/m3). Mn concentrations in raw

and drinking water were 74.4±8.63 µg/L and 27.7±15.02 µg/L, respectively, and below the

permissible level in Brazil (100 µg/L). In 2007 the observed mean MnH level was 15.20 µg/g

(1.10–95.50 µg/g) for the exposed children and 1.37 µg/g (0.39–5.58 µg/g) for the non-exposed.

For the former, MnH concentrations were 7.95±1.40 µg/g (farthest from the plant), 11.81±1.11 µg/g

(mid-region), 34.43±8.66 µg/g (closest to the plant) and 34.22±9.15 µg/g (directly downwind).

Thus, Mn exposure was related to airborne Mn exposure and was also associated with the time of

maternal exposure previous to the child birth.

In the investigation carried out in 2008, the observed mean MnB and MnH were 8.2 µg/L (2.7–

23.4) and 5.83 µg/g (0.1–86.68), respectively. Mean maternal MnH was 3.50 mg/g (0.10–77.45)

and correlated to children’s MnH (rho=0.294, p=0.010). Children’s MnH was negatively related to

Full-Scale Intelligence Quotient (IQ) and Verbal IQ; β-coefficients for MnH were -5.78 (95%CI -

10.71to -0.21) and -6.72(-11.81to -0.63), adjusted for maternal education and nutritional status.

Currently our preliminary results are showing significant associations of Mn hair levels with

neuropsychological effects: aggressive and disruptive behaviors β=8.24 (95%CI 2.40-14.08) and

β=3.24 (95%CI 0.09-6.40), respectively; and cognitive functions (lower IQ and neuropsychological

performance in tests of executive function of planning, strategic formation, verbal working memory

and phonemic fluency).

Conclusion: Our previous results and these preliminary ones suggest that children’s high exposure

to airborne Mn may be having detrimental effects on cognitive functioning and on children’s

behavior, especially those related to externalizing anti-social behaviors.


CEBiTOR 2012


CEBiTOR 2012

Toxicity and mutagenicity of b(a)p-spiked sediments – preliminary

results

Flavia Mazzini1, Celia M. Rech1, Deborah A. Roubicek1, Raúl Bonne Hernández2

1Human Toxicology and Environm. Health Section, CETESB - Environmental Company of São Paulo, Av. Prof.

Frederico Hermann Jr, 345 São Paulo-SP. Brazil.

. 2Federal University of São Paulo.

Introduction

The use of biotests or biomarkers enables a more substantial appraisal of the impact of existing

conditions on aquatic biota. To evaluate the suitability of these tests for environmental screening,

the toxicity and the mutagenicity of two sediment samples (S1 and S2) were evaluated with the

Zebrafish Embryo Toxicity Test (ZFET) and the Salmonella/microsome test (Ames test),

respectively.

Experimental

The sediments were dried, divided in two parts, and one sub-sample of each (80 g) was spiked with

1,5 mg benzo(a)pyrene (S1+B(a)P and S2+B(a)P). The samples were extracted with

dichloromethane (DCM) in an ultrassonic bath, evaporated and ressuspended in dimethylsulfoxide

(DMSO) or ISO water, depending on the bioassay needs. For the ZFET, zebrafish embryos were

collected after approximately 2 h postfertilization, distributed in 96 well microplates and exposed

for 120 h to 7 concentrations of the different sediment organic extracts. Plates were kept at 26°C

during the experiments. Artificial ISO water and DMSO 0,2% were used as negative controls and

sodium dodecyl sulfate (SDS) as positive control. The embryos were observed every 24 hour, and

the toxicological endpoints (coagulation, no somites, undetached tail, no heartbeat, no circulation,

no movement, no pigmentation, development retardation, edema, malformation and hatch) were

evaluated. For the mutagenicity test, S. Typhimurium TA98 e TA100 tester strains were used with

metabolic activation. Seven concentrations of the different extracts were tested in triplicate.

Negative control was DMSO and the positive control used was 2AA. Revertant colonies were

counted after incubation for 66 h at 37°C.

Results and Discussion

The S1 and S2 results showed that after 96 h, all doses caused effects to the exposed embryos. The

sublethal effects observed to S1+B(a)P and S2+B(a)P began at 72 h of exposure. Death or

coagulation of embryos were only observed to S2+B(a)P. These lethal endpoints were encountered

after 24 h at the maximum dose and after 48 h for doses greater than 60 mg/mL.

The percentage of sublethal effects in the embryos exposed to non-spiked sediments were higher

than those verified to the organisms exposed to the B(a)P-spiked sediments, although they were

observed around 24 h latter.

Mutagenicity was not detected in the assays performed with the non-spiked sediment, in none of the

S. Typhimurium strains tested. Both spiked sediments showed mutagenicity around 2.700 rev/g

(TA98) and 13.000 rev/g (TA100).

Conclusions

The preliminary results show that both bioassays were able to detect the adverse effects of


CEBiTOR 2012

contaminated sediments, and could be suitable to environmental monitoring programs.

Bibliography

HOLLERT, H.; KEITER S.; KÖNIG, N.; RUDOLF, M.; ULRICH, M.; BRAUNBECK, T. A New

Sediment Contact Assay to Assess Particle-bound Pollutants Using Zebrafish (Danio rerio)

Embryos. Journal of Soils & Sediments, v. 3, n. 3, p. 197-207, 2003.

KIMMEL, C.B.; BALLARD, W. W., KIMMEL S.R.; ULLMANN B.; SCHILLING T.F. Stages of

Embryonic Development of the Zebrafish. Developmental dynamics, v. 203, p. 253-310, 1995.

MARON, D.M.; AMES, B.N. Revised methods for the Salmonella mutagenicity test. Mutation

Research, v. 113, p. 173-215, 1983.

Acknowledgements

The authors are particularly grateful for the support and assistance by Celso Fumio Suzuki, Genival

de Oliveira, Gilson Alves Quinaglia, Ísis Marques, Mary Ishimine Nishita and Pollyanna Ferreira de

Carvalho.


CEBiTOR 2012

Toxicity of manganese(III) complexes to Danio rerio

Anderson Arndt1, Maria Inês Borella2, Breno Pannia Espósito1

1Institute of Chemistry. University of São Paulo, Av. Prof. Lineu Prestes, 748, São Paulo, Brazil. [email protected] 2Institute of Biomedical Sciences. University of São Paulo.

Introduction

Manganese (Mn) is an abundant element which is present also in food and water. It is the cofactor

of important enzymes as mitochondrial superoxide dismutase, glutamine synthetase and others [1].

Mn from diet, drinking water or as inhaled particles may be assimilated and delivered to the brain

via active transport through the blood-brain barrier, olfactory nerve or cerebrospinal fluid [1].

However, workers exposed to Mn showed increased symptoms of a Parkinson-like neurological

dysfunction termed manganism [1,2].

In this work, we assessed the acute toxicity and brain damage of EUK108, Mn(dfb) and Mn(aha) to

adult Danio rerio fishes and their correlation to complex stability, lipophilicity and ability to

promote peroxide-mediated oxidative damage to organic molecules. It was found that the stable,

highly redox-active and relatively lipophilic EUK108 was the most active compound, but visual

morphological damages in the telencephalic region are not observed.

Experimental

After synthetized, complexes Mn(dfb), Mn(aha) and EUK108 had their lipophilicity described as

octanol-water partition coefficient (log P). The relative stability of the complexes was studied by

the quenching of the fluorescent probe calcein as a model of high affinity biological binding sites

[3]. Pro-oxidant activity mediated by peroxide was assessed fluorimetrically by the

dihydrorhodamine (DHR) oxidation method [4,5]}.

Acute toxicity tests were performed according to the OECD guidelines [6]. Adult zebrafish (Danio

rerio; n = 10) were exposed for 96 h at different concentrations of the complexes and, after that,

their brains were removed by surgery. Histological slices were stained with Hematoxilin-Eosin (HE)

and analyses were performed of the dorsal nucleus of the ventral telencephalon (Vd) area, a

correspondent area to the human basal ganglia [7,8] where Mn is preferentially accumulated [9].


0 1 2 3 4 5

0,2

0,4

0,6

0,8

1,0

EUK108

[Mn(aha)3]

Flu

ore

sc

en

ce

(r.

u.)

complex:calcein mol ratio

[Mn(dfb)]

(a)

0 20 40 60 80

0

30

60

90

120

150

Mn(aha)

EUK108

Flu

ore

sc

en

ce

ra

te

(m

in-1)

[H2O

2] (M)

Mn(dfb)

(b)

Figure 1: (a) Quenching of calcein fluorescence induced by the Mn metallodrugs. (b)Pro-oxidant activity of Mn

complexes (10 M) as a function of H2O2.

All complexes showed highly hydrophilicity but Mn(aha) was the more lipophilic (log P = -0,95)

mailto:[email protected]


CEBiTOR 2012

when compared to Mn(dfb) (log P = -1,73) and EUK108 ( log P = -1,13). In the other hand,

Mn(aha) was the less stable when challenged by the fluorescent chelator calcein while EUK108

remain relatively unchanged (Figure 1a).

Mn(aha) did not display any pro-oxidant activity. EUK108 induced oxidative stress even at very

low (< 5 M) peroxide levels, which are close to physiologically relevant H2O2 levels [10].

Curiously, Mn(dfb) pro-oxidant activity is high even in the absence of peroxide, indicating an

oxidation mechanism still unknown (Figure 1b).

We for the first time examined the toxicity of Mn(III) compounds to Danio rerio. EUK108 is over

the high end of the range of toxicity mentioned above (LD50 ~ 20 M), and the hydroxamate

derivatives are comparatively much less toxic. From the compounds studied here, only the stable,

relatively lipophilic and redox-active EUK108 induced death of D. rerio at low concentrations.

Mn(dfb), being a less stable, relatively large molecule of poor lipophilicity, was hypothesized to be

better tolerated by animals, which was confirmed in the acute toxicity tests.

Inspection of the dorsal telencephalic nucleus (Vd) region for animals from the control group or

treated with Mn(dfb) 1000 µM (the less toxic compound) or EUK108 20 µM (the more toxic

compound) does not reveal significant morphological changes (Figure 3), indicating that

neurological damage, if present, was either insufficient to induce morphological changes, or

occurred elsewhere in the brain. This observation suggests that the extrapolation to humans of

Danio rerio-derived neurologic damage after Mn exposure would not be direct.

Figure 3: Slices of Danio rerio brains at the ventral telencephalic (Vd) region after different treatments (a) control (b)

Mn(dfb) (c) EUK108.

Conclusions

Although showing some pro-oxidant effect and toxicity, the compounds studied here didn’t show

any observable damage to Danio rerio telencephalon probably their low lipophilicity prevent

reaching this region. EUK108 toxicity correlates with its higher lipophilicity and pro-oxidant

activity.

Bibliographic [1] Yokel, R. A. (2009). Neuromolecular Medicine 11(4): 297-310.

[2] Aschner, M., K. M. Erikson, et al. (2009). Neuromolecular Medicine 11(4): 252-266.

[3] Esposito, B. P., S. Epsztejn, et al. (2002). Analytical Biochemistry 304(1): 1-18.

[4] Esposito, B. P., W. Breuer, et al. (2003). Blood 102(7): 2670-2677.

[5] Esposito, B. P., W. Breuer, et al. (2002). Biochemical Society Transactions 30: 729-732.

[6] OECD (1992). OECD Guideline for testing of chemicals 203 - Fish Acute Toxicity Test.

[7] Rink, E. and M. F. Wullimann (2004). Brain Research 1011(2): 206-220.

[8] Roth, J. A. (2009). NeuroMolecular Medicine 11(4): 281-296.

[9] Wullimann, M. F. and T. Mueller (2004). Journal of Comparative Neurology 475(2): 143-162.

[10] Day, B. J. (2009). Biochemical Pharmacology 77(3): 285-296.

Acknowledgements

Fapesp, CNPq and Capes for the financial support


CEBiTOR 2012

Toxicological analysis of caffeine using embryos of

Danio rerio as an experimental model

Ana Carina Nogueira Vasconcelos1, Shayenne Elizianne Ramos1, Mônica Rodrigues Ferreira1,

Galileu Crovatto Veras2 , Luis David Solis Murgas1

1Departament of Veterinary Medicine. Federal University of Lavras, Campus Universitary, Post office box 3037, Zip

code 37200-000. Lavras – MG. Brazil. [email protected]

2Coastal Studies Institute. Federal University of Pará. Bragança – PA. Brazil

Introduction

Caffeine (1,3,7-trimethylxanthine) is a chemical compound produced by more than 60 species of

plants and is the most consumed stimulating substance in the world. Among many applications, this

alkaloid causes an increase in sympathetic nervous system activity and reduces the activity of the

parasympathetic nervous system (Rauh et al., 2006), beyond the ability to control the flow of

intracellular calcium by regulating the activity of ryanodine receptors (Tsai et al. 1995). The effects

of caffeine are still unknown on heart rate because there are reports of increased, decreased and

unchanged frequency. These findings indicate that caffeine has multiple applications and more

research is needed on their effects during embryonic development, since most pregnant women

ingest this substance, either through diet or medication. To obtain information about the action of

this compound in the body, the use of animal models is an important tool study. Among the

advantages of using embryos of Danio rerio (zebrafish) highlight the complexity of a vertebrate

animal, embryonic transparency, fast development and high degree of homology with humans.

Therefore, this study was conducted to evaluate the effects of exposure of zebrafish embryos at four

concentrations of caffeine (0, 50, 150 and 200 ppm) during two distinct stages of embryonic

development.

Experimental

A completely randomized delineation was used with five replicates in 4 x 2 factorial design, with

four levels of caffeine (0, 50, 150 and 200 ppm) and two times of immersion (group I - 0 to 48hpf

and group II - 24 to 72hpf). In each repetition, 400 embryos were distributed in eight incubators,

constantly oxygenated. The first group consisted of adding caffeine in water in the beggining of

embryonic development and the second, only after 24 hours. The variables evaluated at 24, 48 and

72 hours of incubation were: length of the yolk sac and embryo growth. Heart rate was measured

after 48 and 72 hours of development and the survival rate was observed at the end of the

experiment.


The addition of caffeine did not affect the survival rate (P > 0.05), however, decreased heart rate,

impaired absorption of the yolk sac and retarded embryo growth (P < 0.05). In zebrafish embryos

treated with 100 μg/ml of caffeine, Chakraborty et al. (2011) observed that the expression of

vascular endothelial growth factor (VEGF) was practically null. The linear increase of the yolk sac

lenght in a function of increasing levels of caffeine can be explained by reduced expression of

VEGF. This factor stimulates the development of blood vessels in the yolk sac and by this

expression inhibited by caffeine, there may be a decrease in the absorption of this structure,

resulting in a reduction of embryonic growth. Probably the heart rate reduction occurred by the

inhibition of the hERG potassium ion channel (Human Ether-a-go-go) present in cardiac muscle


CEBiTOR 2012

cells (Rana et al., 2010). These channels contribute to cell repolarization following an initial

depolarization and muscle contraction, making it necessary to maintain normal cardiac output and

frequency. When these channels are blocked by caffeine, there is a "Q-T interval prolongation",

better known as bradycardia, which is a heart rate reduction (Rana et al., 2010).

Conclusions

This study suggests that caffeine retards the embryonic length as a consequence of impaired

absorption of nutrients in the embryo yolk sac. The decrease in heart rate was evident only in

embryos exposed to caffeine from the beginning of development. The effect is reversible when the

embryos are again immersed in water. Thus, these data demonstrate the need for further study to

determine the mechanisms of action of caffeine and limit the exposure that affects the embryonic

development.

Bibliographic

CHAKRABORTY, C.; HSU, C. H.; WEN, Z. H.; LIN, C. S.; AGORAMOORTHY, G. Effect of

caffeine, norfloxacin and nimesulide on heartbeat and VEGF expression of zebrafish larvae. J

Environ Biol, v. 32, 32 p.179-183. 2011.

RANA, N.; MOOND, M.; MARTHI, A.; BAPATLA, S.; SARVEPALLI, T.; CHATTI, K.;

CHALLA, A. K. Caffeine-Induced Effects on Heart Rate in Zebrafish Embryos and Possible

Mechanisms of Action: An Effective System for Experiments in Chemical Biology. Zebrafish, v. 7,

n. 1, p. 69-81. 2010.

RAUH, R.; BURKERT, M.; SIEPMANN, M.; MUECK-WEYMANN, M. Acute effects of caffeine

on heart rate variability in habitual caffeine consumers. Clin Physiol Funct Imaging, v. 26, n. 3,

p.163–166, May. 2006.

TSAI, T. D.; BARISH, M. E. Imaging of caffeine-inducible release of intracellular calcium in

cultured embryonic mouse telencephalic neurons. Journal Neurobiol, v. 27, p. 252–265. 1995.

Acknowledgements

I thank Universidade Federal de Lavras and the Department of Biological Sciences and Veterinary

Medicine for improving my studies. My teacher Dr. David Luis Solis Murgas, for the confidence

and opportunity to work in the group. I thank the staff of the Universidade Federal de Lavras

Aquaculture and the study group on fish nutrition for cede the animals for this project. Anyway, to

all the people who somehow contributed to the realization of this study.


CEBiTOR 2012

Evaluation of the toxicological effects of herbicide Glyphosate on

embryonic development of Danio rerio

Ana Carina Nogueira Vasconcelos1, Carine Rodrigues Pereira1, Shayenne Elizianne Ramos1 &

Luis David Solis Murgas1



Introduction

The agricultural model adopted in Brazil is based on the use of pesticides. However, disorderly and

excessive use of pesticides has caused many impacts on the environment. Depending on the

chemical nature and concentration, the pesticide into the environment can cause several damages in

biota (Druart et al, 2010). Several studies have shown that, in different organisms, some products

can be toxic and affect the survival, fertility and genetic composition of populations. Glyphosate

acid, C3H8NO5P, is an herbicide from chemical group substituted glycines and this is one of the

most widely used in Brazil. In this context, the knowledge about the toxicity and genotoxicity of

pesticides used on brazilian crops is important. With this goal, this study examined the influence of

exposure of Danio rerio embryos (zebrafish) to different concentrations of glyphosate (0, 5, 35, 65

and 95 μL/L) during embryonic development.

Experimental

We used a completely randomized design with four replicates. In each replicate 120 embryos were

distributed individually in containers with a total capacity of 0.5 mL. Glyphosate was previously

added to water and the embryos were exposed to this environment from the early embryonic

development until complete 96 hours after fertilization. The variables evaluated were hatching rates,

survival rates and larval size. The hatching rates were checked after 72 hours of development and

the survival rates and larval size were checked at the end of the experiment.


The glyphosate addition reduced the length larval and survival rate at concentrations of 65 and

95μL/L, which can be explained by changes that this product causes in retinoic acid concentrations

(Paganelli et al, 2010). This retinoic acid regulates the functions of vitamin A required for growth

and development (Niederreither & Dollé, 2008). Any change in this acid quantity cause abnormal

development of the embryo, resulting in a reduction in the length or death.

Conclusions

These results show teratogenic effects caused by exposure to glyphosate and demonstrate the need

for deeper studies to determine the mechanisms of action of this herbicide during embryonic

development.

Bibliographic

DRUART, C.; SCHEIFLER, R.; VAUFLEURY, A. Towards the development of an embryotoxicity

bioassay with terrestrial snails: Screening approach for cadmium and pesticides. J of Hazardous

Materials, v. 184, p. 26-33. 2010.

NIEDERREITHER, K.; DOLLÉ, P. Retinoic acid in development: towards an integrated view. Nat.

Rev. Genet., v. 9, p. 541-553. 2008.


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PAGANELLI, A.; GNAZZO, V.; ACOSTA, H.; LÓPEZ, S. L.; CARRASCO, A. E. Glyphosate

Based Herbicides Produce Teratogenic Effects on Vertebrates by Impairing Retinoic Acid Signaling.

Chem. Res. Toxicol., v. 23, n. 10, p. 1586-1595. 2010.

Acknowledgements

We want to thanks to Federal University of Lavras and Veterinary Medicine Department by

execution support of this experiment. We are indebted to Dr. David Luis Solis Murgas for trusting,

teaching and giving us the opportunity to work in his group. Finally, thanks to everyone who

contributed with development of this work.


CEBiTOR 2012

Exposure of Danio rerio embryos to glyphosate

cause teratogenic effect

Carine Rodrigues Pereira1, Ana Carina Nogueira Vasconcelos1 & Luis David Solis Murgas1



Introduction

Gliphosate is an herbicide widely used on the agricultural production in many countries. The

products based on this compost are highly toxic to people and animals, and lab studies find adverses

effects as carcinogenicidde, mutageneicidade and reproductive problems both parents as in the

offspring, when they were directly or indirectly exposed to this substance (Benachour & Seralini,

2009; Gasnier et al.; 2009). Thus, it becomes necessary the use of animal models to achieve a better

understanding of the effects that this product will be able to produce. An experimental model that

has stood out in the cientific area in fields as toxicology, oncology and teratogenicity, is Zebrafish

(Danio rerio) (Kari et al., 2007). This is due to its small (porte), easy maintenance, high

reproductive rate, genomics practically sequenced and display similarity with mammals genes

(Lieschke & Currie, 2007). The aim of this paper is to find what are the teratogenic effects in

zebrafish larvas when exposed to different concentrations of glyphosate during the embryonic

development.

Experimental

We used a completely randomized design with four replications and in each of them, 120 embryos

were distributed individually in containers with a total capacity of 0.5 mL. Glyphosate was

previously added to water and the embryos were exposed to this product from the early embryonic

development until completion 96 hours after fertilization. The macroscopic variables evaluated at

the end of the experiment were: malformation and epidermal pigmentation.


The addition of glyphosate caused malformations and depigmentation of the larvae treated with

concentrations of 65 and 95 μL / L. According Paganelli et al. (2010) glyphosate causes an increase

in the activity of retinoic acid in the early hours of development. This acid controls the growth

pattern of the early stages of the animal and any change in its quantity, modify the enzymatic

metabolism, thus causing, embryonic malformation (Niederreither & Dollé, 2008). The

depigmentation can be explained by impaired antioxidative activity of some cells caused by

glyphosate (Heu et al., 2012), making this product is able to cause a discoloration of the pigment

melanin since it is susceptible to bleaching by oxidizing agents.

Conclusions

These results clearly show that at certain concentrations, glyphosate causes morphological changes

in embryos, confirming its potential risk to human health, since it is possible to extrapolate the

results. Therefore, it becomes necessary to study the limit of exposure to glyphosate that affect

embryonic development.


CEBiTOR 2012

Bibliographic

, N., SERALINI, G. E. Glyphosate formulations induce apoptosis and necrosis in human umbilical,

embryonic, and placental cells. Chem. Res. Toxicol. v. 22, 97–105. 2009.

GASNIER, C., DUMONT, C., BENACHOUR, N., CLAIR, E., CHAGNON, M. C., SERALINI, G.

E. Glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines. Toxicology.

v. 262, p. 184–191. 2009.

HEU, C.; ELIE-CAILLE, C.; MOUGEY, V.; LAUNAY, S.; NICOD, L. A step further toward

glyphosate-induced epidermal cell death: Involvement of mitochondrial and oxidative mechanisms.

Environ. toxicol. and pharmacol., v. 34, p. 144-153. 2012.

Kari, G.; Rodeck, U.; Dicker, A. P. Zebrafish: an emerging model system for human disease and

drug discovery. Clin. Pharmacol. and Therap., v. 82, n.1, p. 70-80. 2007.

LIESCHKE, J.G.; CURRIE, P.D. Animal models of human disease: Zebrafish swim into view. Nat.

Rev. Genet., v. 8, n.5, p. 353-367. 2007.

NICHOLAUS, R. A.; PIATELLI, M.; FATTORUSSO, E. 1964. The structure of melanins and

melanogenesis-IV: On some natural melanins. Tetrahedron, v. 20, n. 5, p. 1163–1172. 1964.

NIEDERREITHER, K.; DOLLÉ, P. Retinoic acid in development: towards an integrated view. Nat.

Rev. Genet., v. 9, p. 541-553. 2008.

PAGANELLI, A.; GNAZZO, V.; ACOSTA, H.; LÓPEZ, S. L.; CARRASCO, A. E. Glyphosate

Based Herbicides Produce Teratogenic Effects on Vertebrates by Impairing Retinoic Acid Signaling.

Chem. Res. Toxicol., v. xxx. 2010.

Acknowledgements

Thanks especially to Luis David Solis Murgas for the opportunity and confidence to work in his

group and Ana Carina Nogueira Vasconcelos for all help and teaching.

http://www.ncbi.nlm.nih.gov/pubmed?term=Kari%20G%5BAuthor%5D&cauthor=true&cauthor_uid=17495877

http://www.ncbi.nlm.nih.gov/pubmed?term=Rodeck%20U%5BAuthor%5D&cauthor=true&cauthor_uid=17495877

http://www.ncbi.nlm.nih.gov/pubmed?term=Dicker%20AP%5BAuthor%5D&cauthor=true&cauthor_uid=17495877


CEBiTOR 2012

Protease ADAMTS-1 role in local and systemic invasion of

fibrosarcoma cells

Heydi Noriega Guerra1, Mário Costa Cruz2, Raúl Bonne Hernández3, Vanessa Morais Freitas1

1 Department of Cell and Developmental Biology, Biomedical Sciences Institute, University of São Paulo, Brazil. 2 Center of Facilities and Support Research, Biomedical Sciences Institute, University of São Paulo, Brazil.

3 Institute for Environmental, Chemical and Pharmaceutical Sciences, Federal University of São Paulo, Brazil.

INTRODUCTION

Soft tissue sarcomas (STS) are malignant tumors of mesenchymal origin, among which

fibrosarcoma. A malignant tumor derived from fibrous connective tissue and characterized by the

presence of immature proliferating fibroblasts (1). For tumor cell disseminate in the body it must

perform several important steps, including invasion of surrounding tissues, intravasation into the

blood vessel, survival circulation, extravasation from the blood stream, and proliferation (2). It has

become increasingly recognized that the growth and malignancy of a tumor is largely dictated by

the surrounding microenvironment, i.e. tumor stroma (3). On tumor microenvironment, the

extracellular matrix is not limited to being a physical barrier against tumor invasion. It is an integral

part of the tumor microenvironment and extensively modified by proteases during malignant

progression (4). The secreted proteases ADAMTS-1, member of the ADAMTS family (a disintegrin

and metalloproteinase with thrombospondin motifs), is differentially expressed in various tumors

which regulate extracellular matrix and influence tumor biology (5). Controversial data showed that

ADAMTS-1 could be associated with anti-tumor activity (6) or pro-tumor activity and metastasis

stimulation (7). The potential role of ADAMTS-1 in tumor development and progression is still

unclear; it becomes an important research target. Furthermore, there are no reports about the role of

ADAMTS-1 in respect of tumor cells and blood vessels, i.e. intravasation and extravasation of these

tumor cells. This study aimed to evaluate the role of ADAMTS-1 within the tumor

microenvironment of fibrosarcoma. We addressed the role played by ADAMTS-1 in the local and

systemic invasion of fibrosarcoma in vivo, using zebrafish embryo model.

EXPERIMENTAL

Human fibrosarcoma HT1080 cells were seeded in culture plate containing MEM with 10% FBS,

and maintained at 37⁰ C, 5% CO2. When monolayer HT1080 cells reached 70% confluency, cells

were loaded with the vital red dye Calcein CellTrace (Invitrogen). Tumor cells were trypsinized,

suspended in cell culture and divided in two groups 1) Tumor cells without any treatment (Control

group) and 2) Tumor cells with exogenous administration of 5nM ADAMTS-1 (Treatment Group).

At 48 h.p.f., embryos were de-chorionized if necessary, anesthetized with 0.003% tricaine and

positioned on their right side on a wet agarose pad. 1,6x103 cells/50nl were injected into the

common cardinal vein (CCV) using picoinjector-1000 BTX Harvard Apparatus with a borosilicate

glass needle. Injected embryos were transferred to E3 embryonic medium with PTU and kept at

35.5°C. Embryos at 24 hours post-injection were anesthetized and immobilized in a drop of

agarose. This was mounted inside a chamber and covered with a thin layer of E3 medium and

anesthetic. The temperature was maintained at 28⁰ C for capturing images using confocal

microscope (Zeiss LSM-780 NLO). Images were taken every 15 min for a total time of 13 hours.

Some embryos were euthanized and fixed to acquire the z-stacks with confocal microscope.


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RESULTS AND DISCUSSION

Preliminary results showed tumor cell interact with the surrounding tissues, such as muscle and

venous vessels. In the context of a fibrossarcoma model, we observed tumor cells into the entering

vasculature and some of them were arrested within the ISVs and the surrounding tissue; feature

enhanced in presence of ADAMTS-1 (Fig 1). The dynamism of tumor microenvironment is crucial

for the behavior of tumor cells. This process is complex and involves dynamic interaction between

tumor cells and the stroma.

Fig. 1. Tumor cells interaction with the surrounding tissues. Control Group: Tumor cells without any treatment and;

Treatment Group: Tumor cells with exogenous administration of ADAMTS-1, injected into zebrafish embryo. Arrows

indicate tumor cell with lamellipodial protrusion.

CONCLUSIONS

Our preliminary results suggest that ADAMTS-1 induces both cell migration and proliferation. The

interaction between tumor cells and the stroma are of intense interest for further studies that will be

carried out.

BIBLIOGRAPHIC

1. JAYAMATHI, P.; KEERTHIDAA, G.; VIDYALAKHSMI, K.; BHAVANI, G.; RUKMANI DEVI, S. Anti oxidant

Property of Plumbagin on Fibrosarcoma Induced Rats. Recent Research in Science and Technology, v.2, n.11, 2010.

2. NGUYEN, D.X.; BOS, P.D.; MASSAGUE, J. Metastasis: from dissemination to organ-specific colonization. Nat

Rev Cancer. n.9, p.2927-2931, 2009.

3. TLSTY, T.D.; COUSSENS, L.M. Tumor stroma and regulation of cancer development. Annu Rev Pathol, n.1,

p.119–150, 2006.

4. VAZQUEZ, F.; HASTINGS, G.; ORTEGA, M.A.; LANE, T.F.; OIKEMUS, S.; LOMBARDO, M.; IRUELA-

ARISPE, M.L. METH-1, a human ortholog of ADAMTS-1, and METH-2 are members of a new family of proteins with

angio-inhibitory activity. J Biol Chem. n.274, p.23349-23357, 1999.

5. ROCKS, N.; PAULISSEN, G.; EL HOUR, M.; et al. Emerging roles of ADAM and ADAMTS metalloproteinases in

cancer. Biochimie, v.90, p.369-379, 2008.

6. PORTER, S.; CLARK, I.M.; KEVORKIAN, L.; EDWARDS, D.R. The ADAMTS metalloproteinases. Biochem. J.,

v.386, p.15-27, 2005.

7. LIU, Y.J.; XU, Y.; YU, Q. Full-length ADAMTS-1 and the ADAMTS-1 fragments display pro- and antimetastatic

activity, respectively. Oncogene, v.25, p.2452-2467, 2006.


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ACKNOWLEDGEMENTS

We are grateful to PhD. Raúl Bonne Hernández (ICAQF-UNIFESP, Brazil) for providing the embryos of

zebrafish for this study; and to Mário Costa Cruz (CEFAP-USP, Brazil) for expert help and assistance with

intravital confocal microscopy. This work is supported by FAPESP.


CEBiTOR 2012

Synthesis, characterization and malarial activity of

binuclear copper (II) complex

Roxana Y. Pastrana Alta1*, Alejandro Miguel Katzin2, Breno Pannia Espósito1,

Ana C. Valderrama Negrón3.

Instituto de Química, Universidade de São Paulo, São Paulo – SP, 05508-000, Brazil.

Instituto de Biociencias, Universidade de São Paulo, São Paulo

Facultad de Ciencias, Universidad Nacional de Ingenieria, Lima-Perú

Introduction

Malaria is one of the tropical neglected disease whose therapy may benefit from a bioinorganic

chemistry approach. Quinine is antibacterial agents active against various microorganisms specially

Plasmodium falciparum. However, although this neglected tropical disease still poses an important

public health problem, resulting in high morbidity and considerable mortality rates, few investments

have been allocated towards developing novel anti-malarial agents. With that in mind, our team

developed a new complex with quinine that shows more activity than quinine. Both present superior

activity in comparison with other reported antimalarial complexes [1].

Experimental

Materials; Quinine and Cu2(CH3COO)4.2H2O were purchased from Sigma-Aldrich. All solvents

were purchased from Merck.

Apparatus; Elemental analyses were performed on a ELEMENTAR ANALYZER CHN

Instruments, model 2400 Perkin-Elmer. A Perkin Elmer Lamda 10 UV–Vis spectrophotometer was

used to obtain UV–Vis spectra with a 1.00 cm quartz cell. The IR spectra were acquired on a FTIR

Perkin-Elmer, model 1750 spectrophotometer using KBr pellets. The TGA spectra were acquired on

equipo Luxx, Netzsch STA 409 PG, the temperature range varied between 25 and 900 ºC and the

heating rate was 10 ºC min-1. X-band electron paramagnetic resonance (EPR) spectra were obtained

with a Bruker ENX spectrometer with modulation frequency of 100 kHz and modulation amplitude

of 1 mT. Magnetic susceptibility measurements at 298 K of powdered samples were determined by

the Gouy method. X-ray crystallograph, a green rectangular crystal was selected and cut to give a

fragment of dimensions suitable for X-ray analysis, data were collected on a BRUKER SMART

CCD. Structure and graphical illustrations were made with SHELXS-97¤ y SHELXL-97 ф.

Synthesis of the complex

A solution of quinine (0.63 g, 1.92mmol) in hot methanol (25 cm3) was added to a solution of

Cu2(CH3COO)4.2H2O (0.35 g, 0.96 mmol) in hot methanol (25 cm3). The mixture was heated

under reflux (T=45ºC) for 6 h. After cooling the solution to room temperatura, single crystals were

grown by slow evaporation of metanol (20mL) solutions(and after 1 week). The final solution was

stored ≈ 3 °C, to accelerate the formation of green crystals of complex. [Cu(O2CCH3)(μ-Qn)]2 (1):

Green solid.Yield: 38 %. Anal.: found, C 53.18, H 6.11, N 5.16. Calc. For Cu2(CH3COO)2

(C20H24N2O2)2(H2O)2, C 52.9%, H 6.21%, N 5.61%. IR (cm-1): 3422 s ʋ (OH); 1588 s (C=O)p;

1620 m ʋ as(COO-); 1395 m ʋ s(COO-).

Malarial analysis

For the in vitro analysis on plasmodium, the parasites were incubated at 37 °C, each compound


CEBiTOR 2012

were diluted in methanol, and used at the following concentrations: 100, 50, 10nM of complex and

200, 100, 50 and 10 nM of quinine. Controls with methanol were performed in parallel. The method

of Desjardins et al. was used to determine IC50 values (drug concentration that reduces 50 % of the

number of lived parasites). Briefly, ring stage parasite cultures (5% hematocrit, 2% parasitemia)

were exposed to increasing drug concentrations.

Type 3 copper sites studies

Part of this work, was compared the two classes of type 3 Cu sites in biology. Focus is on evaluating the

different O2-binding properties of Hc/Tyr and the type 3 site in the MCOs and elucidating their origins. This

work provides molecular insight into the critical role of the mimetic new protein environment in directing the

O2 reactivity of these catalytic active sites.


In this work, we prepared a binuclear copper(II) derivative of the type [Cu(O2CCH3)(μ-Qn)]2 (Qn =

quinine). Its structure was resolved by X-ray diffraction and showed two copper metal centers each

surrounded by three oxygen atoms and one nitrogen atom (CuNO3), figure 1. The complex has been

further characterized by thermogravimetric and shows that N quinuclidic was linked with copper(II)

spectroscopic UV-Vis, showing a band . In spectro IR was found Cu-N, that confirms a new Bond

[2].

EPR analysis, shows a binuclear species Cu-Cu, and magnetic measurements is 1,88M.B

characteristic of a binuclear complex [3]. Quinine and acetate coordinate to Cu as a zwitterionic

bidentade ligand (O,N) and monodentade ligand (O), respectively. The antimalarial activity of the

complex and the starting ligand was evaluated in vitro against bloodstream schizonts of

Plasmodium falciparum. The complex (IC50 = 14.6 nM) was more active than quinine (IC50 =

60.15 nM). Both present superior activity in comparison with other reported antimalarial complexes

[3]. The complex present geometric and electronic similarities with Type 3 copper centers of the

multicopper oxidases and hemocyanin/tyrosinase, that could be beginning of a new bio-research.

Figure N°1. Crystal for [Cu(O2CCH3)(μ-Qn)]2


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Conclusions

Has been synthesized and characterized the dimeric complex [Cu(O2CCH3)(μ-Qn)]2 whose

structure was confirmed by X-ray structural analysis. The developed Cu(II) complex present a

significant antimalarial activity in vitro against P. Falciparum (schizont stage), showing

concentrations (14,59nM) smaller than those of quinine (60,15nM) or another references the

literature [1].

Bibliographic

[1] Nikhil H. G., Subhash B. P., David C. Billington ,Daniel L. Rathbone , Synthesis and

characterization of copper(II) commplexes of pyridine-2-carboxamidrazones as potent antimalarial

agents. Inorg. Chim. Acta 2003;349:23-/29.

[2] Nicolas A. Rey,Karina C. Dos Santos.Two different Modes for copper(II)Ion cordination to

Quinine-Type Ligands.J. Braz. Chem.,2006;17:497-504.

[3] Synthesis and characterization of copperibuprofenate complexes with 2,2'-bipyridineand

1,10-phenanthrolines and their hydrolytic activities in phosphate diester cleavage; A. Latif

Abuhijleh, Polvhedro,'t Vol. 16, No. 4, pp. 733 740, 1997.

Acknowledgements

The present study was supported by the Facultad de Ciencias-UNIVERSIDAD NACIONAL DE

INGENIERIA, DE Lima-PERÚ, Instituo de Quimíca , Instituto de Biociencias da

UNIVERSIDADE DE SÃO PAULO and Coordenação de Aperfeiçoamento de Pessoal de Nível

Superior.

http://www.capes.gov.br/

http://www.capes.gov.br/


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Measurement of heavy metals in electronic waste and its occupational

hazards for recyclable material collectors

Carlos Alberto Conde Regina1, Etelvino Jose Henriques Bechara1,

Nilson Antonio de Assunção1

1Institute for Environmental, Chemical and Pharmaceutical Sciences. Federal University of São Paulo (ICAQF-

UNIFESP)

Introduction

In Brazil, there is a lot of people working as waste picker in landfills and streets, they collected all

kind of recyclable materials as paper, plastics and a special different one, Waste Electric and

electronic Equipments (WEEE) up and after it is sold to the market. The WEEE when handled

incorrectly can result in risks of contamination by heavy metals, especially Lead (Pb).

The WEEE has become a worldwide problem, because its has been produced in large scales allied

with the emergence of technologies increasingly attractives resulted in huge quantities of obsolete

materials and disposable1. In this waste can be found computers, cell phones, monitors and

televisors Cathode Ray Tube (CRT), 80% being constituted only by monitors and televisors CRT2.

Lead is most heavy metal found in WEEE, it is present in alloys of the welding of printed circuit

boards and particularly in CRT tubes. The Pb in cationic form is bioaccumulative, it has not

biological functional activity and could cause negative behaviors as agressivity, QI reduction, acting

especially in Central Nervous System (CNS) 3,4

This scenery can be found in countries called BRICS (Brazil, Russian, Índia, China and South

Africa) and it must be treated carefully as a health occupational problems of these envolved

workers. Because it is the most socially and economically be excluded, has not been given due

attention to chronic exposure to toxic chemicals, potentially cumulative and could result in serious

occupational injuries. Situation frequent in these countries due to lack of public awareness as a

result of low educational level of the majority population.

Experimental

The tecniques used in this study was X- Ray Portable Fluorescence, used in situ to determine the

amount of heavy metals possible contaminated locals by heavy metals, and help from a Non

Governamental Organization, Instituto GEA, the recruitment and interview with a questionnaire,

Adulf Self Report (ASR), of these workers. ASR can reveal anti- social behaviors and this study

believes that the lead in nails, as a biomarker of continuous expose, can be correlated with

aggressivity.


A total of 19 Cooperativas were visited and had their equipments, soils and some WEEE analyzed

by X-Ray. The Table 1 shows the concentration of heavy metals found. Until this moment 15

volunters were interviewed and 6 samples of nails were collected


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Table 1. Materials Analysis by X-RAY. Materials Concentration of Heavy

Metals (emg.kg-1)

Cadmium(Cd) Chromium(Cr6+) Mercury(Hg) Lead(Pb)

Monitor CRT 972 844 453 240872

Printed Circuit Board

185 409 58 11170

Surface Mats 75 519 263 3802

Surface tables 87 689 315 9357

Bags 101 464 487 439

Conclusions

The WEEE and another materials analyzed have high and amount concern heavy metals

concentrations and if it is saved in open places, where they can receive sunlight and rains, those

heavy metals can be released to the environment in cathionics forms. These findings clearly indicate

an urgent need for better monitoring and control of the recycling and give safe conditions for this

workers.

Bibliographic 1. Ogunseitan,O. A et al; 2009 apud Chen, A.; Dietrich, K.N.; Huo, X.; Ho, S.; 119, 431-438, 2010. 2. Andreola, F. 2005 apud Mear. F.; Yot, P.; Ribes, M.; Waste Management 26, 1468-1476, 2006. 3. Papanikolaou, N.C; Hatzdaki, E.G.; Tzanakakis, G.N.; Tsatsakis, A.M.; Medical Science Monit, 2005.

4.- Olympio, K. P.; Naozuka, J.; Oliveira, P. V.; Cardoso, M. R. A.; Bechara, E. J. H.; Günther, W. M. R; Rev Saúde Pública, 44, 851, 2010;.

Acknowledgements

FAPESP, CNPq, and CAPES


CEBiTOR 2012

Embryotoxicity induced by mixtures of metal species

Mary Ishimine Nishita1 and Raúl Bonne Hernández1

1Institute for Environmental, Chemical and Pharmaceutical Sciences. Lab. Bioinorgânica e Toxicologia Ambiental –

LABITA. Federal University of São Paulo, Rua São Nicolau, 210. Diadema-SP. Brazil. [email protected].

Introduction

Manganese and their chemical species are associated with others chemical species in both

environment and physiological level. Toxicologically, these interactions can lead to synergistic,

additives or an antagonistic effect, which are not always well known and has received little

attention. Previous reports indicate that this metal can also be toxic, particularly during

development, where their physiological functions suggest a multifactorial mechanism of toxicity.

Furthermore, considering that both chemicals, aluminum (Al) and manganese (Mn), present

neurotoxicity, preliminary experiments were made to evaluate the positive or negative effects of Mn

and Al mixture in zebrafish (Danio rerio) embryos. The neurotoxicity of manganese, in low

concentrations, and it association with others and their chemical species can be associated with

mitochondrial alterations and apoptotic events, consequently leading to Alzheimer's disease. Thus,

studies of embryotoxicity induced by mixtures of different species of Mn and Al can contribute to

the assessment of risk to human health and ecotoxicological risk. Therefore, through the

characterization of in vivo toxicological impact of potential mixtures it will be determinate the

mitochondrial alterations, apoptotic events, neurobehavioral changes and toxicological potential of

mixtures during development of Danio rerio.

Experimental

MnCl2 stock solutions were prepared by dissolving manganese(II) chloride tetrahydrate

(MnCl2·4H2O, Sigma-Aldrich, Germany) in sterile ultrapure water to obtain a final concentration of

2 M. Mn(II)Cit solution was obtained by mixing MnCl2 solutions with sodium citrate solution (6

mM). A serial dilution (factor 2) was applied to obtain final concentrations of 0.375 – 6 mM.

Manganese(III) citrate was obtained by dissolution of solid manganese(III) acetate dehydrate

((CH3COO)3Mn·2H2O, Sigma-Aldrich, Germany) in a 6 mM citrate solution to obtain final

concentrations of 0.25, 0.5, 1, and 2mM. A proportion of 2:1 ligand to metal was selected in order

to allow preparation of stable Mn(III)Cit solutions. The mixtures between manganese and their

chemical were constituted by Mn(II)Cit (0-6 mM) (A) and a fixed value of Mn(III)Cit (B): Mix

0.25 (A+ 0.25 mM of B), Mix 0.5 (A+0.5 mM of B), Mix 1 (A+1 mM of B) and Mix 2 (A+2 mM

of B). Exposure media were prepared by serial dilutions in test medium (2 mM CaCl2, 0.5 mM

MgSO4, 0.75 mM NaHCO3, 0.08 mM KCl). The metal oxidation state (II) or (III) in water solutions

was confirmed by EPR (Electronic Paramagnetic Resonance, using a Varian E-109 Century Series

X-band Spectrometer (Varian, Inc., Palo Alto, CA, USA)) and UV-Visible spectrophotometric

analysis, using a used a UV-1700 Pharmaspec Spectrophotmeter (Shimadzu, Brazil). The amount of

metal in the stock solutions were determined by atomic absorption spectroscopy in a Shimadzu

AA6300 equipment. And the mixtures between manganese and aluminum were constituted by

Mn(II)Cl2 (0-6 mM) (A) and a fixed value of Al(III)Cl3 (B): Mix 0.5 (A+0.5 mM of B) and Mix 0.1

(A+ 0.1 mM of B). DarT: Danio rerio embryos (15 for each Mn concentration) were exposed to

mixtures of chemical species of manganese [Mn(II)/Mn(III)] for 48-96 hours post fertilization (hpf),

72-120 hpf, 48-120 hpf and 2-122 hpf (Fig. 1). Controls were exposed to exposure medium without

metal, and/or including 6 mM Na-citrate in case of experiments with metal complexes. Endpoints



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recorded: Lethality and developmental disorders was identified by coagulation of the embryo,

missing heart beat, failure to develop somites or a non-detached tail (Nagel, 2002), including metal

concentration that induce 50% of lethality (LC50). Statistics: LC50, Normal distribution of data

(D'Agostino & Pearson omnibus normality) and ANOVA followed by Bonferroni’s tests were

performed with GraphPad 4.0 Software.


Initially, we made a comparison of LC50 among different binary mixtures of Mn(II)Cit/Mn(III)Cit in

zebrafish embryos exposed for 120 h (2-122 hpf).

Mix 0.25 mM Mix 0.5 mM Mix 1 mM Mix 2 mM0

1

2

3

4

5

a b c d

a,b,c

Exposition from 2 to 122 hpf

Mixtures of Mn(II) and Mn(III)

LC

50 (

mM

)

Experiments results showed that the redox couple Mn(II)/Mn(III), regardless the concentrations

ratio, induces sub-lethal effects. And it’s possible to observe that mixtures treated with lower

concentrations of Mn(III)Cit present a similar toxicity (LC50), which is less ecotoxic than mixture

Mix 2 mM (A+2 mM of B). Thus, we selected the lower and higher mixture concentration and

exposed them in different developmental stages and different time exposure (obs: hatching occurs

between 48 and 72 hpf).


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Mix x time and dev stages

48-96 (48) 72-120 (48) 48-120 (72) 2-122 (120)0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0Mix 0.25 mM

Mix 2 mM

a1,b1,d1

a1,b,c,d

b1,d

a1b1 c1 d1a b c d

c1

d1

Embryo developmental stages-hpf (time exposition-hexp)

LC

50 (

mM

)

For mixture with the lower concentration, the greater toxicity is on 48-120 hpf (72 hexp), and to the

higher is on 72-120 hpf (48 hexp). Which indicate that developmental stages and toxicity increasing

appear to be depending of energy status in zebrafish: Before hatching (chorion (C) + high energy

level (E) = tolerant embryos or lower toxicity); after hatching (embryos dechorioned + decreased of

energy level = increased of developmetal toxicity) and not with the time exposure. Toxicity of these

mixtures, during special developmental stages such as hatching, appear to be following a

mechanism more associated with biological effect than additivity phenomena (Norwood et al.,

2003). These results also suggest that the stage of animal development as well as any Mn species

redox event may be related to toxicological effects observed, which can be synergistically

potentiated by mixtures of species of Mn(II)/Mn(III).


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Additionally, we realized preliminary experiments of mixtures of MnCl2 and AlCl3, which showed a

higher toxicity of mixtures comparing to metal species studied separately. It’s also possible to

observe that the manganese specie wasn’t able to decrease the aluminum toxicity.

Conclusions

The present work is one of the few works studying the role of mixtures of manganese species to the

toxicity this metal during developmental stages. In this case we found that for zebrafish a model of

aquatic organisms (ecotoxicological relevance) and of health disorder in human (environmental and

human toxicological relevance), the toxicity induced by mixtures of Mn appear to be following a

mechanism more associated with biological effects than additive phenomena. And preliminary

experiments indicate that the association of these metals can represent a major risk to human health

and to the environment.

Bibliographic

NAGEL, R. (2002). DarT: the embryo test with the Zebrafish Danio rerio-a general model in

ecotoxicology and toxicology. Altex-Alternativen Zu Tierexperimenten 19., 38-48.

NORWOOD, W.P., Borgmann, U., Dixon, D., Wallace., G.A., 2003. Effects of Metal Mixtures on

Aquatic Biota: A Review of Observations and Methods. Human and ecological risk assessment

Acknowledgements

FAPESP (2011/11921-4, 2012/04726-3)


CEBiTOR 2012

DETERMINAÇÃO DA TOXICIDADE EM ARTEMIAS SALINAS

FRENTE A METALOFARMACOS DE FERRO DE USO

COMERCIAL E HUMANO COM CAPACIDADE DE GERAR

ATIVIDADE REDOX ATIVO.

Hector Aguilar Viorino1, Flavia Zanotto2, Luca Mantovanelli2, Breno Pannia Espósito1

[email protected] 1Institute of Chemistry. University of São Paulo. São Paulo – SP, 05508-000, Brazil.

2Institute of Biosciences. Department of Physiology. University of São Paulo. São Paulo – SP, 05508-900, Brazil.

Resumo

O objetivo do trabalho é avaliar a estabilidade e capacidade de geração de atividade redox de

fármacos comerciais de uso veterinário e compostos-modelo de metalofarmacos de ferro como o

ferroceno, TMH-ferroceno e (TMH)2-ferroceno frente à calceína em meio fisiologicamente

relevante. Além disso, avaliou-se a toxicidade dos metalofármacos em Artemia salina. Os

metalofarmacos comerciais apresentam uma estabilidade similar em diferentes concentrações, assim

como a atividade redox ativa,enquanto a taxa de mortalidade o fármaco dexiron apresenta o maior

valor (30%, 1000

-oxidante (ao redor de 10%), e uma maior taxa de mortalidade em A.

Salinas.

Introdução

A sobrecarga de ferro é uma condição desfavorável tanto para humanos como, possivelmente, os

organismos aquáticos. A toxicidade do ferro em humanos é originada, principalmente, pela indução

de reservatórios de ferro lábil plasmático (LPI) [1] Metalofármacos de ferro compreendem agentes

anti-hipertensivos, antimicrobianos e suplementos minerais proposto para diversas aplicações

clínicas ou farmacológicas. Contudo, os possíveis danos por sobrecarga induzida desses compostos

não são totalmente compreendidos.

A Artemia salina é um pequeno crustáceo que habita lagoas temporárias de águas salgadas, onde

poucos animais podem sobreviver, possuindo o melhor sistema de osmorregulação do reino animal,

além de resistir em ambientes de baixos níveis de oxigênio. Com estes fatores de adaptação podem

coexistir em densidades de população muito elevadas [2]. Por estes motivos a utilização de artêmias

para testes de toxicidade tem aumentado, pois além de responder aos compostos testes sem ser

influenciada pela concentração salina do meio, sua obtenção é muito fácil e de baixo custo.

Experimental

Estabilidade frente à calceína: A estabilidade dos fármacos (Tabela 1) foram avaliadas com

auxílio da sonda fluorescente calceína, registrada a temperatura ambiente com um BMG Fluostar

Optima (λexc/λem=485/520 nm) por 24 horas [3].

Atividade pró-oxidante: A atividade pro-oxidante dos metalofármacos de ferro foi avaliada

através da mudança na velocidade de oxidação da sonda DHR (Dihidrorodamina) catalisada pela

reação entre ferro e ascorbato [4].


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Toxicidade em A. salina: Machos de A. salina adultos foram triados e acondicionados em placa de

petri para aclimatação em água do mar reconstituída. Dez animais foram acondicionados em frascos

e incubados ao abrigo da luz a 30°C por um período de 24h, na presença dos compostos de

interesse. [5].

Tabela 1: Fármacos comerciais de uso veterinário e humano.

Fármaco comercial Características

Concentração nominal de Fe

(M)2

Gleptoferril Ferro dextrano1 3,580

Biovet/Bio-Ferr Ferro-dextrano1 1,790

Dexiron Ferro-dextrano1 1,790

Ferro Dextrano Ferro-dextrano1 1,790

Fertal Ferro dextrano1 1,790

Ferrodex Ferro dextrano1 1,790

Netrofer Glicinato Férrico 2 1,790

Noripurum Ferripolimaltosa2 0,895

1 Fármacos injetável de uso veterinário.2 Fármacos oral de uso humano. 3 Concentrações que indica a bula de cada

fármaco.

Resultados e Discussão

Os fármacos comerciais apresentam uma tendência similar de estabilidades (Figura 1). Os derivados

do ferroceno apresentaram uma estabilidade dependente da estrutura química (Figura 2), sendo os

com substituintes no anel ciclopentadieno os menos estáveis, provavelmente por efeitos estéricos.

Os fármacos apresentam atividade pró-oxidante dependendo da concentração, mas a mortalidade

das artêmias não parece depender do nível de ferro redox-ativo.

Figura 1. Estabilidade dos fármacos comerciais com concentrações 2.0, 4.0 e 8.0 µM (10 µL)

frente ao quelante calceina 2µM (180 µL) a pH=7,44 e T=37°C. Para n=4, P<0.05vs cotrole ,

α=0.05


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Figura 2. Estabilidade dos compostos de ferroceno sintetizados com concentrações 2.0, 4.0 e 8.0 µM (10

µL) frente ao quelante calceina 2µM (180 µL) a pH=7,44 e T=37°C. Para n=4, P<0.05vs cotrole, α=0.05

Tabe1a 1. Atividade pro-oxidante dos compostos de ferro com concentrações 2.0, 4.0 e 8.0 µM ,a

pH=7,44 e T=37°C. Para n=4, P<0.05vs cotrole, α=0.05

Atividade redox ativo

Fármaco 2.0 µM 4.0 µM 8.0 µM

Gleptoferril 0.09 ± 0.06 0.07 ± 0.03 0.05 ± 0.01

Biovet/Bio-Ferr 0.13 ± 0.06 0.12 ± 0.03 0.14 ± 0.01

Dexiron 0.10 ± 0.07 0.09 ± 0.05 0.06 ± 0.01

Ferro Dextrano 0.09 ± 0.05 0.06 ± 0.02 0.03 ± 0.01

Fertal 0.10 ± 0.06 0.08 ± 0.03 0.07 ± 0.05

Ferrodex 0.12 ± 0.06 0.11 ± 0.04 0.10 ± 0.01

Netrofer 0.13 ± 0.09 0.15 ± 0.08 0.17 ± 0.06

Noripurum 0.14 ± 0.07 0.14 ± 0.08 0.15 ± 0.05

Sulfato Ferroso 0.21 ± 0.01 0.21 ± 0.01 0.07 ± 0.01

Ferroceno 0.05 ± 0.01 0.05 ± 0.01 0.04 ± 0.01

TMH-Ferroceno 0.17 ± 0.03 0.25 ± 0.02 0.40 ± 0.06

(TMH)2-ferroceno 0.35 ± 0.07 0.64 ± 0.08 1.31 ± 0.12


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Tabela 2: taxa de mortalidade das artemias salinas frente aos compostos de ferro com concentrações

2.0, 4.0 e 8.0 µM ,a pH=7,44 e T=37°C. Para n=4, P<0.05vs cotrole, α=0.05.

Conclusões

Os fármacos comerciais apresentaram uma tendência similar de estabilidade frente ao quelante

calceína. Já os derivados substituídos de ferroceno são menos estáveis, e também os que apresentam

maior capacidade de gerar ferro redox-ativo. A maior estabilidade do composto Ferroceno sugere

que esse fator é o responsável pela maior toxicidade dessa substância frente a A. salina.

References

[1] Breno P. Esposito, William Breuer, Pornpan Sirankapracha, Pensri Pootrakul, Chaim Hershko,

and Z. Ioav Cabantchik. Labile plasma iron in iron overload: redox activity and susceptibility to

chelation. BLOOD, 1 OCTOBER 2003 _ VOLUME 102, NUMBER 7.p-2671.

[2] (Barnabé, G. “Aquaculture – Biology and Ecology of cultured species”. Ellis Horwood Ed., Grã

Bretanha (1994).

[3] Breno P. Esposito, Silvina Epsztejn,William Breuer, and Z. Ioav Cabantchik2, A Review of

FluorescenceMethods for Assessing Labile Ironin Cells and Biological Fluids1 Analytical

Biochemistry 304, 1–18 (2002).

[4] Mayara Marinovic Baccan ,Orlando Chiarelli-Neto, Regina Mara Silva Pereira, Breno Pannia

Espósito .Quercetin as a shuttle for labile iron.Journal of Inorganic Biochemistry 107 (2012) 34–

39.

[5] Mee young Song, John d. Stark and John J. Brown. Comparative toxicity of four insecticides,

including imidacloprid and tebufenozide, to four aquatic arthropods. Environ. Toxicol. Chem. 16,

1997;p-2495.

Agradecimentos

This work was funded by CAPES and FAPESP (Brazilian government agencies).

Compostos de ferro

Taxa de Mortalidade (%)

10µM 100µM 1000µM

Gleptoferril 0 0 10

Biovet/Bio-Ferr 0 0 0

Dexiron 0 10 30

Ferro Dextrano 0 0 0

Fertal 0 0 0

Ferrodex 10 10 0

Netrofer 0 0 10

Noripurum 20 20 30

Sulfato Ferroso 0 0 40

Ferroceno 40 40 90

TMH-Ferroceno 10 10 10

(TMH)2-ferroceno 10 20 40


CEBiTOR 2012

Tin-induced developmental toxicity in zebrafish embryos

Ísis Marques da Costa;1 Geórgia Labuto2; Raúl Bonne Hernández1

1Lab. Bioinorganic and Environmental Toxicology - LABITA. Federal University of São Paulo

2AQUA (Chemical Analysis Applied Group), Federal University of São Paulo

Introduction

Organotin are organometallic compounds of tin that has aroused concern for the environment,

especially the derivatives of tributyltin chloride (TBT), which is a biocide and has been employed

as an antifouling for marine, biocide for agriculture, antifungal fabric and stabilizer in the PVC

industry. Although some countries have banned its use, there is a lot of work has been done on the

presence of organotin compounds in environmental samples, mainly marine samples[1].

Searches related to TBT in gastropods occurring phenomenon known as "imposex" which is

irreversible and causes the sterilization of animals, and may even lead to extinction, the most

sensitive species and proving its action as an endocrine disruptor. Bivalves, mollusks and oysters

have a high capacity of TBT accumulation due to limited ability to metabolize this compound, such

observation, alert to the possibility of reaching the man[2]. This job present preliminary results of the

use of zebrafish as a model to evaluate the TBT and derivatives toxicity, namely dibutyltin (DBT)

and monobutyltin (MBT).

Experimental

TBT and MBT stock solutions were prepared by diluting tributyltin chloride 96% ([CH3 (CH2) 3]

3SnCl, Sigma-Aldrich) and butyltin trichloride 95% (CH3 (CH2) 3SnCl3, Sigma-Aldrich),

respectively, in sterilized ultra pure water to give a final concentration of 1mM. The DBT stock

solution of was prepared by dissolving dibutyltin dichloride ([CH3 (CH2) 3] 2SnCl2, Sigma-

Aldrich) in DMSO 1% and sterile ultra pure water to give a final concentration of 1mM. A serial

dilution was applied to obtain final concentrations of 0.001, 0.003, 0.01, 0.03, 0.1 and 0.3 mM for

all three compounds. Danio rerio embryos (15 for each concentration of MBT and TBT and 10 of

DBT per concentration) were exposed for 2-50 hours after fertilization (hpf) and 2-122 hpf.

Controls were exposed to medium without metal exposure, LVD in the case of the controls were

exposed beyond the means of exposure without metal amid exposure with only DMSO 1%.


The records of disturbances lethality and developmental was identified by coagulation of the

embryo, missing heart beat, heart rate reduced, deformation of the body and altered spontaneous or

stimulated movement of embryos, including metal concentration inducing 50% of lethality (LC50).

Exposure during or after hatching (hpf 48-122 or 72-122 hpf) for DBT and TBT seems be

equivalent to a long exposure (2-122 hpf). Already for MBT not seem to be equivalent to a long

exposure. News assays is being conducted to solve these speculations.

Comparison of in zebrafish embryos at different development stages and with different exposure

durations and comparison between static and semi-static (changing of MBT, DBT and TBT solution


CEBiTOR 2012

each 24h) are with assays in course. The LC50 was obtained with the GraphPad Software 4.0.

Conclusions

The present work is one of the few studies on the role of chemical speciation and developmental

stage sensitivity of MBT, DBT and TBT toxicity in zebrafish a model of aquatic organisms

(ecotoxicological relevance) and of health disorder in human (environmental and human

toxicological relevance). In summary, TBT seems be more toxic than MBT which seems be more

toxic than DBT.

Bibliographic

[1] Okoro HK, Fatoki LS, Adekila FA, Ximba BJ, Snyman RG, Sources, Environmental Levels and

Toxicity of Organotin in Marine Environment-A Review, Asian Journal of Chemistry, 23,(2011),

473-482.

[2] Rudel, H. Case study: bioavailability of tin and tin compounds. Ecotoxicology and

Environmental Safety., 56 (2003) 180-189.

Acknowledgements

FAPESP (2011/11921-4)


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Serotonergic modulation of zebrafish behavior in anxiety tasks

Caio Maximino1,2, Bruna Puty1, Vanessa Miranda3, Rancés Benzecry3,

Anderson Manoel Herculano1,2

1 Laboratory of Neuroendocrinology, Institute of Biological Sciences, Federal University of

Para, r. Augusto Correa 01. Belem-PA, Brazil. [email protected]. 2 Zebrafish Neuroscience Research

Consortium. 3 Institute of Biological Sciences, Federal University of Para.

Introduction.

Defensive behavior has been shown to be modulated by the serotonergic system (Maximino, 2012).

Nonetheless, the specific role of serotonin (5-HT) on this modulation (i.e., whether 5-HT increases

or decreases defensive, anxiety-like behavior) is not clear, and seems to depend on the specificities

of the behavioral test. In zebrafish, two of the most well-validated and studied behavioral models of

anxiety – the novel tank diving test and the light/dark tests – have been proposed to model different

aspects (Maximino et al., 2012). Thus, these might represent good models to study the differential

modulation of serotonergic drugs on behavior.

Experimental

155 adult zebrafish of the longfin wildtype phenotype were used in the experiments. Animals were

acclimated to laboratory conditions (tank water held at 28―1 ﾁ‹C, pH 6.0, food dispensed once per

day at the light phase of the 14:10 photoperiod) for at least two weeks before experiments begun.

After ice-cold anesthesia, animals were injected intraperitoneally with vehicle or test drug and, 30

min later, subjected to both tests in sequence. Test presentation was random and counterbalanced

such that 50% of the animals in each group were first subjected to the novel tank test (NTT; Egan et

al., 2009) while the other half was first subjected to the light/dark test (LD; Maximino et al., 2010).

Fluoxetine, WAY 100635, buspirone and pCPA were dissolved in Cortland’s salt solution; SB

224289 was dissolved in 1% DMSO. A different group of 20 animals was injected with vehicle and

subjected to either NTT or LD; after that, their brains were dissected in ECF extracting solution,

which was later injected in a reverse phase HPLC with electrochemical detection (+0.72 V, 2 nA

full deflection; mobile phase: 70 mM phosphate buffer (pH 2.9), 0.2 mM EDTA, 34.6765 mM SDS,

10% methanol) for 5-HT quantification. 12 other brains were dissected into hindbrain, midbrain and

forebrain, homogenized in PBS, and injected in the HPLC.

On the contrary, serotonin levels were associated with increased time in the top of the novel tank (r2

= 0.7225, p = 0.01828; n = 8), with no association with the number of squares crossed (r2 = 0.001,

NS; n = 8), erratic swimming (r2 = 0.4927, NS; n = 8) or freezing duration (r2 = 0.001, NS; n = 8).

Administration of the serotonin reuptake inhibitor fluoxetine was anxiogenic at the smaller dose

(2.5 mg/kg) in the scototaxis test, decreasing the time spent in the white compartment (F[3, 39] =

9.251, p = 0.0001) and increasing the latency to enter it (χ2 = 6.417, hazard ratio = 3.909, p =

0.0113), thigmotaxis (F[3, 39] = 3.054, p = 0.0407) and risk assessment (H = 22.89, p < 0.0001; n =

10 each group). The highest dose (10 mg/kg) increased the number of entries in the white

compartment (H = 9.683, p = 0.0215; n = 10 each group) and the number of squares crossed in this

area (H = 13, p = 0.0046; n = 10 each group). No other effects were observed.

Fluoxetine increased the time spent on the top of the novel tank in all doses (F[3, 39] = 28.99, p <

0.0001), while facilitating habituation in the sixth minute at the 5.0 and 10.0 mg/kg doses (F[3,216]


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= 11.45, p < 0.0001). The highest dose produced hyperlocomotor effects (H = 10.34, p =0.0159, n =

10 each group). No effects were observed on homebase behavior (F[3, 39] < 1.29, NS). At 5.0

mg/kg, fluoxetine decreased the number of erratic swimming events (H = 14.23, p = 0.0026) and

freezing duration (F[3, 39] = 17.5, p < 0.0001); this last parameter was also decreased at 2.5 mg/kg

(q = 7.233 in relation to control).

The 5-HT1AR partial agonist buspirone decreased bottom-dwelling in the novel tank test in both doses (F[2, 28]

= 5.458, p = 0.0105), increasing habituation at the 50 mg/kg (F[10, 150] = 1.948, p = 0.0429 for the interaction

term). No effects were observed on total locomotion.

Buspirone (25 and 50 mg/kg) also increased the time spent in the white compartment in a dose-

dependent fashion (F[2, 29] = 53.94, p < 0.0001) without affecting the number of entries in the

white compartment or total locomotion while in it (H < 0.62, NS; n = 10 each group). No effect was

observed in the latency to enter the white compartment (Figure 8B; χ2 < 1.2, NS) or erratic

swimming (H = 3.574, NS; n = 10 each group). A dose-dependent effect was observed in

thigmotaxis, decreased by buspirone (F[2, 29] = 8.396, p = 0.0015), while freezing was equally

diminished by both doses (F[2, 29] = 7.439, p = 0.0027). Finally, the highest dose decreased risk

assessment in relation to both controls and animals treated with 25 mg/kg (H = 15.48, p =

0.0004; n = 10 each group).

The 5-HT1AR antagonist WAY 100635 increased the time spent on the top of the novel tank at

0.003 and 0.03 mg/kg, with a greater effect of the lower dose than the higher dose (F[2,23] = 5.406,

p = 0.0128). The lowest dose also promoted habituation (F[2, 150] = 3.507, 0.0325). Total

movement, erratic swimming and freezing were not altered by any dose (H < 3.06, NS for total

movement and erratic swimming; F[2,23] = 0.004, NS for freezing; n = 9-10). Unexpectedly,

animals treated with the lowest dose spent more time in the “homebase” (F[2,23] = 6.428, p =

0.0056) than both controls and animals treated with the highest dose (q > 3.148, Tukey's Multiple

Comparison Test); the drug had no effect on the frequency of “homebase” visit (inset; F[2,23] =

0.1268, NS).

In the light-dark test, 0.03 mg/kg of WAY 100635 increased the time spent in the white

compartment (F[2,26] = 6.962, p = 0.0041) and decreased thigmotaxis (F[2,26] = 3.845, p =

0.0356) and risk assessment (H = 12.62, p = 0.0018; n = 9 per group). No effect was observed on

latency to white or freezing (χ2 = 0.9238, NS; F[2,26] < 0.52, NS) nor in total locomotion, entries

on white or erratic swimming (H < 3.3, NS).

5-HT1BR inverse agonist SB 224289 increased the time spent on the top of a novel tank at 2.5 and

5 mg/kg, with the greatest effect with 2.5 mg/kg (F[2,25] = 8.701, p = 0.0015). Consistent with this

observation, an interaction effect between SB 224289 with time was also found (F[10, 138] = 1.99,

p = 0.0388), with decreased bottom-dwelling in the 2.5 mg/kg dose in the third minute (t = 3.043, p

< 0.05, Bonferroni posttest). No effects on total locomotion were observed (H = 4.974, NS; n = 8-

9). Again, “homebase” time was increased by this lower dose (F[2, 25] = 7.645, p = 0.0028),

without effects on homebase visits (F[2, 25] = 0.3659, NS). Both doses decreased the frequency of

erratic swimming (H = 13.8, p = 0.001; n = 8-9), but no effect was observed on freezing (F[2, 25] =

0.3237, NS).

In contrast to these observations, no effect of SB 224289 was observed on the time spent in the

white compartment, latency to white, thigmotaxis or freezing (F[2, 25] < 1.69, NS; χ2 = 0.4937,

NS), nor in total locomotion, entries on white or erratic swimming (H < 4.92, NS; n = 8-9). An

increase in frequency of risk assessment was observed with 2.5 mg/kg (H = 8.165, p = 0.0169).

Two injections of pCPA (spaced by 24 h between injections) at 300 mg/kg reduced extracellular


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serotonin levels to 10% of that observed in controls (Data not shown). This treatment increased

bottom-dwelling (t[df = 12] = 2.711, p = 0.0351) and abolished habituation (F[5, 84] = 2.799, p =

0.0218). No effects were observed on total movement (U = 13.5, NS; n = 7 on each group), erratic

swimming (U = 9.5, NS; n = 7 on each group) or freezing (t[df = 12] = 1.842, NS). pCPA also

increased the time spent on the white compartment on the light/dark test (t[df = 12] = 6.265, p <

0.0001), sparing latency and freezing (χ2 = 0.706, NS; t[df =12] < 1.4, NS), as well as total

locomotion, entries on white and erratic swimming (U < 23, NS; n = 7 on each group). pCPA also

decreased thigmotaxis (t[df = 12] = 2.528, p = 0.0265) and risk assessment (U = 6, p = 0.0212; n =

7 on each group).

Conclusions.

These results suggest that 5-HT, probably acting on forebrain structures, is anxiogenic in the LD;

acting on the midbrain, 5-HT is anxiolytic in the NTT. Moreover, the difference between 5-HT’s

effects might be moderated by receptors other than 5-HT1A, since drugs which act on this receptor

had the same effect on both tests.

Bibliography.

Egan, R. J., Bergner, C. L., Hart, P. C., Cachat, J. M., Canavello, P. R., Elegante, M. F.,

Elkhayat, S. I., Bartels, B. K., Tien, A. K., Tien, D. H., Mohnot, S., Beeson, E., Glasgow, E.,

Amri, H., Zukowska, Z., Kalueff, A. V., 2009. Understanding behavioral and physiological

phenotypes of stress and anxieyt in zebrafish. Behavioural Brain Research 205, 38-44.

Maximino, C., Brito, T. M., Dias, C. A. G. d. M., Gouveia, A., Jr., Morato, S., 2010. Scototaxis

as anxiety-like behavior in fish. Nature Protocols 5, 209-216.

Maximino, C. 2012. Serotonin and anxiety. Neuroanatomy, pharmacology and functional

aspects. New York: Springer.

Maximino, C., Benzecry, R., Oliveira, K. R. M., Batista, E. J. O., Herculano, A. M., 2012. A

comparison of the light/dark and novel tank tests in zebrafish. Behaviour 149, 1099-1123.

Acknowledgments.

The present work was financially supported by CAPES and CNPq


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Aluminum-induced developmental toxicity in zebrafish embryos

Pollyanna Ferreira de Carvalho and Raúl Bonne Hernández

Institute for Environmental, Chemical and Pharmaceutical Sciences. Federal University of São Paulo, Rua São Nicolau,

210. Diadema-SP. Brazil. [email protected].

Introduction

Epidemiological studies have suggested that aluminum (Al), historically known as an element non

toxic is a potential environmental factor in the pathogenesis of Alzheimer's disease; as well as other

reports have highlighted the developmental neurotoxicity of the Al. However, the mechanisms

involved in the events listed above are still controversial, and the role of chemical speciation hasn't

been satisfactorily clarified. Precisely, the present study aims to study the importance of chemical

speciation in neurotoxicity of Al, to developing organisms using as a model the experimental danio

rerio.

Experimental

AlCl3 stock solutions were prepared by dissolving aluminum chloride hexahydrate (AlCl3•6H2O,

Sigma-Aldrich, Germany) in sterile ultra pure water to obtain a final concentration of 2 M. AlCit

solution was obtained by mixing AlCl3 solutions with sodium citrate solution (6 mM). A serial

dilution (factor 2) was applied to obtain final concentrations of 0.01 – 6 mM. Aluminum citrate was

obtained by dissolution of solid aluminum (Sigma-Aldrich, Germany) in a 6 mM citrate solution to

obtain final concentrations of 0.01, 0.1, 1, 3 and 6mM. A proportion of 1:1 ligand to metal was

selected in order to allow preparation of stable AlCit solutions. AlLac solution was obtained by

mixing AlCl3 solutions with sodium lactate solution (6 mM). A serial dilution (factor 2) was applied

to obtain final concentrations of 0.01 – 6 mM. Aluminum lactate was obtained by dissolution of

solid aluminum (Sigma-Aldrich, Germany) in a 6 mM lactate solution to obtain final concentrations

of 0.1875, 0.375,0,75, 1,5, 3 and 6mM. A proportion of 1:3 ligand to metal was selected in order to

allow preparation of stable Allac solutions. A serial dilution (factor 10) was applied to obtain final

concentrations of 0.01 – 100 mM. Aluminum chloride was obtained by dissolution of solid

aluminum (Sigma-Aldrich, Germany) in a 100 mM aluminum solution to obtain final

concentrations of 0.01, 0.1, 1, 10 and 100mM. The mixtures were constituted by AlCit (0-6 mM),

AlLac(0-6mM) and AlCl3(0-100mM). Danio rerio embryos (15 for each Mn concentration) were

exposed to mixtures of chemical species of aluminum [AlCl3/AlLac/AlCit] for 48-96 hours post

fertilization (hpf), 72-120 hpf, 48-120 hpf, 24-120 hpf and 2-122 hpf. Controls were exposed to

exposure medium without metal, and/or including 6 mM citrate, 20mM lactate in case of

experiments with metal complexes. Endpoints recorded: Lethality and developmental disorders was

identified by coagulation of the embryo, missing heart beat, failure to develop somites or a non-

detached tail (Nagel, 2002), including metal concentration that induce 50% of lethality (LC50).

Statistics: LC50, Normal distribution of data (D'Agostino & Pearson omnibus normality) and

ANOVA followed by Bonferroni’s tests were performed with GraphPad 4.0 Software.


In the Figure 1 we see the stage 48-120 and 72-120 hours of embryonic development no significant

difference in concentrations between AlLac, AlCl3 and AlCit. During fertilization of 2 hours there

was a higher mortality in increased concentrations of AlCl3 and AlCit.



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Figure 1: Comparison of LC50 of AlLac, AlCl3, AlCit, in zebrafish embryos at different

development stages and with different exposure durations. Hatching occurs between 48 and 72 hpf.

Treatments that do not share a common letter are significantly different from each other (p<0.05).

Bars represent means of the LC50 from independent experiments ± standard deviation.

Conclusions

The present work is one of the few works studying the role of mixtures of aluminum species to the

toxicity this metal during developmental stages. In this case we found that for zebrafish a model of

aquatic organisms (ecotoxicological relevance) and of health disorder in human (environmental and

human toxicological relevance), the toxicity induced by mixtures of Al appear to be following a

mechanism more associated with biological effects than additive phenomena.

Bibliographic

1)Apostoli, P., Telišman, S., Sager, P.R., 2007. Reproductive and Developmental Toxicity of

Metals. In Nordberg, G.F., Handbook on the Toxicology of Metals. Cap 32, 645-674. 3 ed. San

Diego, California, Elservie.

2)Nordberg, G. F., Fowler, B.A., Nordberg, M., Friberg L., 2007. Handbook on the Toxicology of

Metals. Elsevier, Amsterdam, 1024 pp.

Acknowledgements

FAPESP and CAPES for the financial support.


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Behavioral and neurochemical changes in the zebrafish leopard strain

Monica Lima, Caio Maximino, Bruna Puty, Vanessa Miranda, Karen Renata Matos Oliveira,

Anderson Manoel Herculano

The literature shows differences in anxiety-related behavior between zebrafish strains. These

observations might be due to differences in systems associated with defensive behavior, such as the

serotonergic system. OBJECTIVE: This work investigated behavioral differences between wild-

type and leopard strain zebrafish and its relationship to parameters of the serotonergic system.

METHODS: 10 zebrafish wildtype longfin (lof) and 10 leopard (leo) zebrafish were used, housed in

groups of 20-30 fishes per 40L tank with reconstituted water, at 25-27°C and 14:10 photoperiod. We

evaluated behavior, as well as the effects of fluoxetine (5mg/kg, i.p.), in the light-dark test (LDT)

and in the novel tank test (NTT). 5-HT and 5-HIAA levels in the extracellular fluid of the brain

were analyzed by HPLC with electrochemical detection. Monoamine oxidase (MAO) activity was

assessed by the rate of disappearance of kynuramine, expresssed in µmol/min/mg of protein, by a

spectrophotometric metho d. Data were analyzed with t-tests, Mann-Whitney tests, or two-way

(strain X treatment) ANOVAs. Differences were considered statistically significant at p<0.05.

RESULTS: leopard showed increased anxiety-like behavior in the LDT (increased white avoidance

and risk assessment) but not in the NTT, in relation to longfin. In leopard, but not longfin,

fluoxetine was anxiolytic in both tests, impaired habituation in the NTT and improved it in the

LDT; moreover, leopard showed increased 5-HT levels and MAO activity in comparison with

longfin. CONCLUSION: These data show that leopard phenotype is more anxious than longfin,

probably due to downregulated expression and/or activity of serotonin transporters. FINANCIAL

SUPPORT: CAPES


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NOME INSTITUIÇÃO EMAIL

ADELSON SILVA AGILENT TECHNOLOGIES

AMANDA MORELLI ICAQF - UNIFESP [email protected]

ANA CLARA DOS SANTOS UNIFESP [email protected]

ANA CARINA NOGUEIRA

VASCONCELOS

UFL [email protected]

ANDERSON ARNDT IQ – USP [email protected] ANDERSON SEIJI OKADA UNIFESP [email protected]

ANDRESSA MENEGUELO UNIFESP [email protected] ANTHONY BOATENG JINLARCO VENTURES [email protected]

BRENO PANNIA ESPÓSITO IQ - USP

CAIO MAXIMINO DE

OLIVEIRA

UFPA [email protected]

CARINE RODRIGUES PEREIRA UFL [email protected] CARLOS ALBERTO CONDE REGINA UNIFESP [email protected]

CIRO ALBERTO OLIVEIRA UFPR BIANCA TACORONTE GOMES UNIFESP [email protected]

DANIELA SORIA VIRGENS UNIFESP [email protected]

DEBORAH ARNSDORFF

ROUBICEK

CETESB [email protected]

DIOGO LOSCH DE OLIVEIRA UFRGS ECHEMAZU STANLEY KOGI STATE INSTITUTE FOR

ENVIROMENTAL RESEARCH

[email protected]

ELIANA MAÍRA AGOSTINI

VALLE

UNIFESP [email protected]

ELIZABETH CARMEN

PASTRANA ALTA

IQ – UNIFESP [email protected]

ENYOBI CHUKWUNONSO NATIONAL INSTITUTE FOR

ENVIROMENTAL RESEARCH

[email protected]

EZIEVUO JULIUS CHINEDU CHARLES WHAT ESLE

NIGERIA LIMITED

[email protected]

FÁBIO KUMMROW UNIFESP [email protected]

FERNANDO BARBOSA JUNIOR FCFRP - USP

FLAVIA MAZZINI CETESB [email protected]

GUILHERME HENRIQUE DOS

SANTOS


HECTOR AGUILAR VITORINO IQ – USP [email protected]

HERON DOMINGUEZ TORRES

DA SILVA


HEYDI NORIEGA GUERRA ICB – USP [email protected] IGNACIO SIMO INSTITUTO DE BIOLOGÍA

MOLECULAR Y CELULAR DE

ROSARIO (IBR)

[email protected]

ÍSIS MARQUES DA COSTA ICAQF – UNIFESP [email protected]

JAQUELINE MOURA SANTOS UNIFESP [email protected] JOSÉ ANTONIO MENEZES-

FILHO

UFBA

KARINA BUGAN DEBS UNIFESP [email protected]

LUIS DAVID SOLIS MURGAS UFL [email protected]

MARCO AURÉLIO ZEZZI

ARRUDA

UNICAMP

MARIANA MOREIRA

GUIMARÃES


MARY ISHIMINE NISHITA ICAQF – UNIFESP [email protected]

MICHAEL J. CARVAN III UNIVERSITY OF WISCONSIN

MOHAMED EL GEDAWY SELINK INTERNATIONAL [email protected] MOHAMME IDRIS CENTRE FOR CELLULAR AND

MOLECULAR BIOLOGY

MONICA GOMES LIMA UFPA [email protected]

NKEIRUKA ARINE ONYEBUM BAMBINO WELFARE PROJECT [email protected]



CEBiTOR 2012

NZEKWE KENECHUKWU COLLINS PA - STANLEY INTEGRATED

NIGERIA LIMITED

[email protected]

ODIRI ABIODUM ANAMBRA BROADCASTING

SERVICE

[email protected]

ONYEJIAKA TOCHUKWU

FRANCIS

ANAMBRA BROADCASTING

SERVICE

[email protected]

PEDRO JOSÉ AMORIM PINTO UNIFESP [email protected]

POLLYANNA FERREIRA DE

CARVALHO

UNIFESP

RAFAEL AUGUSTO

MANTOVANI SILVA


RAÚL BONNE HERNÁNDEZ ICAQF – UNIFESP [email protected]

ROBERT GERLAI UNIVERSITY OF TORONTO

ROXANA YESENIA PASTRANA

ALTA

IQ – USP [email protected]

SERGIO RODRIGUES HONDA ICAQF – UNIFESP [email protected]

STEVEN A. FARBER CARNEGIE INSTITUTION FOR

SCIENCE

THAYS DE SOUZA LIMA ICAQF – UNIFESP [email protected]

VITOR ALVES SA DA SILVA UNIFESP [email protected]

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ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH · Determinação da toxicidade em artemias salinas ... MONICA GOMES LIMA UFPA . ... 1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL