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1STINTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH

CEBiTOR2 12

1STINTERNATIONAL CONFERENCE ONENVIRONMENTAL BIOINORGANIC AND

TOXICOLOGY RESEARCH

CEBiTOR2 12

December 6-8, 2012Federal University of So Paulo

Institute for Environmental, Chemical and Pharmaceutical Sciences RuaSo Nicolau, 210. Diadema-SP. Brazil

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ORGANIZING COMMITTEE

President:Ral Bonne Hernndez, PhD (ICAQF-UNIFESP, Brazil)

Vise President:Breno Pannia Espsito, 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)

sis 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 PROGRAMDECEMBER, 6th

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15:0018:00 Accreditation 5 th Floor

DECEMBER, 7th07:30-08:10 Accreditation

5 th Floor08:15-08:55 Opening ceremony09:00-16:00 Metals and other substances in bi omedicine and pharmacology

5 th Floor

09:00-09:40 Robert GerlaiDepartment of Psychology.

University of Toronto - Canada

Using zebrafish to unravel the genetics of complex braindisorders.

09:55 - 10:30 Adelson Silva

Especialista de Produtos em Espectroscopia|Agilent Technologies - BrazilTransformando a tcnica de ICP-MS com TriploQuadrupolo

10:30-10:20 Coffe break and Poster discussion 1stFloor10:25-10:55 Adelson Silva

Especialista de Produtos em Espectroscopia|Agilent Technologies - Brazil

Aplicaes de ICP-MS acoplado a Cromatografia (GC eHPLC)

5 th Floor11:00-11:50 Breno Pannia EspsitoInstituto de Qumica. University of So Paulo. Brazil

Fluorimetric detection of redox-active metals in cells andbiological fluids.

12:00-12:40 Mohamme IdrisCentre for Cellular and Molecular Biology. India

Bio-mechanism of regeneration and degeneration inzebrafish model system.

12:50-13:55 Lunch Free14:00-14:40 Diogo Losch de Oliveira

Department of BiochemistryFederal University of Rio Grande do Sul. Brazil

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

drug toxicity evaluations.

5 th Floor14:50-15:30 Steven A. Farber

Depart.of Embryology.Carnegie Institution for Science. USA

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

cholesterol absorption15:40-16:20 Marco Aurlio Zezzi Arruda

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

from plant to fish16:30-16:50 Coffe break and Poster discussion 1stFloor16:55-17:45 Oral short communication and evaluation

5 th Floor

16:55-17:05 Anderson ArndtUniversity of So Paulo - Brazil

Toxicity of manganese(III) complexes to Danio rerio

17:10-17:20 Heydi Noriega GuerraUniversity of So Paulo - Brazil

Importancia da protease ADAMTS-1 na invaso local esistmica de clulas de fibrossarcoma

17:25-17:35 Monica Gomes LimaFederal University of Par - Brazil

Behavioral and neurochemical changes in the zebrafish

leopard strain17:40-17:50 Ana Carina Nogueira Vasconcelos

Federal University of LavrasToxicological analysis of caffeine using embryos of

Danio rerio as an experimental model18:00-19:00 Ral Bonne Hernndez

ICAQF-Federal University of So Paulo - BrazilConstitution of an International Network for

Environmental Bioinorganic and Toxicological Research

- NEBITOR: Environmental Neurotoxicology of metals

5 th FloorOnly among speakers

DECEMBER, 8th

Envi ronmental bioinorganic chemistry and environmental health, and the zebraf ish model as teaching tool

5 th Floor

09:00-09:40 Ciro Alberto OliveiraFederal University of Paran

09:50-10:30 Michael J. Carvan IIISchool of Freshwater Sciences.University of Wisconsin-

USA

Zebrafish as a Model System for Environmental HealthStudies in the Classroom

10:40-11:20 Jos Antonio Menezes-FilhoDepart. of Clinical and Toxicological Analysis

Federal University of Bahia-Brazil

Cognitive and neurobehavioral effects of childrensexposure to airborne manganese

11:30-11:55 Poster discussion and evaluation 1stFloor11:55-13:25 Lunch and NEBITOR discussion13:30-14:10 Michael J. Carvan III

School of Freshwater Sciences.University of Wisconsin-USA

The Effects of Developmental MeHg Exposure atEnvironmentally-Relevant Levels

5 th Floor

14:20-15:00 Fernando Barbosa JuniorFCFRP - University of So Paulo

Oral short communication and evaluation15:10-15:20 Caio Maximino de Oliveira

Federal University of Par - BrazilSerotonergic modulation of zebrafish behavior in

anxiety tasks15:25-15:35 Carine Rodrigues Pereira

Federal University of LavrasExposure of Danio rerio embryos to Glyphosate cause

teratogenic effects15:40-15:50 Hector Aguilar Vitorino

University of So Paulo - BrazilDeterminao da toxicidade em artemias salinas frente ametalofarmacos de ferro de uso comercial e humano com

capacidade de gerar atividade redox ativo15:55-16:05 Mary Ishimine Nishita

ICAQF-Federal University of So Paulo - BrazilEmbryotoxicity induced by mixtures of metal species.

16:10-16:55 Coffe break 1stFloor17:0018:00 Closing ceremony 5 th Floor

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Students considered with stays in double room-breakfast includedNome Instituio

Hotel Trade Hotel Diadema

Rua So Pedro, 135 - Centro,

Diadema, 09910-620

(11) 4056-8611

1. ANA CARINA NOGUEIRAVASCONCELOS

UFL

2. CAIO MAXIMINO DEOLIVEIRA

UFPA

3. CARINE RODRIGUESPEREIRA UFL

4. MONICA GOMES LIMA UFPA

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Using zebrafish to unravel the genetics of complex brain disorders

Robert GerlaiDepartment of Psychology.

University of Toronto - Canada

The zebrafish has been prominently utilized in developmental biology for the past three decades andnumerous genetic tools have been developed for it. Due to the accumulated genetic knowledge thezebrafish 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 mainlyunmapped in particular, there is a substantial need for appropriate laboratory research organismsthat may be utilized to model such diseases and facilitate the analysis of their mechanisms. Thezebrafish may have a bright future in this research field. It offers a compromise between systemcomplexity (it is a vertebrate similar in many ways to our own species) and practical simplicity (it issmall, 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 tacklethe potentially large number of molecular targets and mechanisms involved in complex braindisorders. However, although promising, the zebrafish is admittedly a novel research tool and onlyfew empirical examples exist to support this claim. In this chapter, first I briefly review some of therapidly evolving genetic methods available for zebrafish. Second, I discuss some promisingexamples for how zebrafish have been used to model and analyze molecular mechanisms ofcomplex brain disorders. Last, I present some recently developed zebrafish behavioral paradigmsthat may have relevance for a spectrum of complex human brain disorders including thoseassociated 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 withzebrafish, I argue that the rapid accumulation of knowledge in both of these disciplines will makezebrafish a prominent research tool for the genetic analysis of complex brain disorders.

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Fluorimetric detection of redox-active metalsin cells and biological fluids

Breno Pannia EspsitoInstituto de QumicaUniversidade de So PauloBrasil

Metal ions are persistent contaminants present in ever-increasing technological, medical andnutritional 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 offluorimetric detection of iron-mediated oxidative stress will be discussed.

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The Effects of Developmental MeHg Exposureat 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 fromsevere cerebral palsy to subtle developmental delays. Many of the more subtle effects ofdevelopmental MeHg exposure can be permanent, including behavioral abnormalities, nervoussystem deficits, and loss of IQ. It is estimated that as many as 15% of American women ofchildbearing age have total blood mercury concentrations at a level above that associated withneurological deficits in children. The zebrafish is a well-supported model system for the study ofthe 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-environmentinteractions in developmental disease and dysfunction. Zebrafish suffer neurological deficits(learning and memory, and visual system problems) at exposures similar to those that affect humansand these deficits persist into adulthood and across generations. We are using a number ofapproaches including knockouts, transgenics, and transcriptomics, in conjunction withneurobehavioral analyses to explore gene-environment-dysfunction relationships. Our findings haveimplications for human health and can also be used to model the impact of environmental mercuryon wild populations of fish.

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

Marco Aurlio Zezzi Arruda

Spectrometry, Sample preparation and Mechanization GroupGEPAM, National Institute of Science and Technology -INCT for Bioanalytics, Institute of Chemistry, Department of Analytical Chemistry, UNICAMP,

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

e-mail: [email protected]

Metalloproteomics encompasses the inorganic elements content and assemble of their complexeswith proteins [1]. Although metalloenzymes and metalloproteins are responsible for catalyzingmolecular 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 acofactor 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, bipolardisorder using blood serum samples of control and bipolar groups, and some preliminary resultsregarding oxidative stress in Zebrafish.

References[1] Garcia, J. S., Magalhes, C. S., Arruda, M. A. Z., Talanta69 (2006)1-15[2] Lu, Y., Yeung, N., Sierack, N., Marshall N. M., Nature460(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

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Zebrafish as a Model System for Environmental Health Studiesin 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 ofenvironmental toxicology to high school students. Our program (called Biology-EnvironmentalHealth Science Nexus: Inquiry, Content, and Communication) focuses on the high school General

Science and Biology teachers and students in metropolitan Milwaukee, particularly minoritystudents in the Milwaukee Public School District. Our general objective is to develop the skills ofinquiry in teachers and students as the basis for doing and understanding science, particularly inrelation to life science and environmental health. This goal takes form in science contentrecommended by the National Science Education Standards in the areas of genetics, cellular

properties, and organismic behavior. These areas are enriched by strong links to environmentalhealth issues of importance to students, including exposure to lead, alcohol, and UV radiation andconditions 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 includemacromolecular models and molecular biology experimentation. The Modules set in motionactivities that together provide teachers and students with multiple encounters with authenticscientific inquiry. Integrated with the modules are student communication about the experimentswithin and between classes, writing and publishing research communications and reports, and

participation in an annual research conference. Teachers gain professional development throughworkshops, 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)

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A detailed characterization of the behavioral pattern of zebrafish inthe 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 processof biochemical and cellular assays, with final validation in an animal model, and ultimately inhumans. Mammalian models for drug toxicity evaluation are expensive, laborious and consumelarge quantities of precious compounds. In addition, there is also increasing pressure to limit animaluse to situations in which they are absolutely necessary, such as in preclinical toxicity and safetyassessment. Thus, zebrafish are beginning to be used at various stages for drug discovery and drugtoxicity 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 drugtoxicity evaluation in zebrafish are scarce. Therefore, in the present work we aimed to perform adetailed characterization of the behavioral profile of the short-fin zebrafish behavior in the opentank paradigm, a protocol used to evaluate several aspects of the zebrafish behavior. Fish weredivided 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 duringthe first 6-min, where the bright group spent more time and travelled a higher distance in the toparea. 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 alsodemonstrated that zebrafish display a classical homebase formation in the bottom area of the tank. Adetailed spatio-temporal study of zebrafish exploratory behavior and the construction of

representative ethograms showed that the experimental groups presented significant differences inthe first 3-min vs. last 3-min of test. Although the main factors involved in these behavioralresponses still remain ambiguous and require further investigation, the current report describes analternative methodological approach for assessing the zebrafish behavior after a forced exposure todifferent environments. Additionally, the analysis of ethologically-relevant patterns across timecould be a potential phenotyping tool to evaluate the toxic effects of several chemical compoundson exploratory profile of zebrafish in the open tank task.

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Why we dip our lobster in butter: Using zebrafish larvae to uncoveran 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 geneticmutation and/or lifestyle choices) with cardiovascular disease, diabetes mellitus, and obesity. In theUS 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 healthcare spending. One focus of our efforts is to understand mechanisms by which the intestine absorbsand processes lipids. The vertebrate intestine contains symbiotic organisms, bile and mucus thatmake it difficult, if not impossible, to study ex vivo. This is one reason that many longstandingquestions remain unresolved in the field of intestinal lipid metabolism. In general, the goal of mylab 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 offluorescent lipids. While the zebrafish has been established as a powerful model for the study ofearly development, few researchers have taken advantage of the accessibility and optical clarity ofthe embryos to visualize lipid uptake and processing in vivo.

Using the larval zebrafish found an unexpected mechanism coupling long chain mono unsaturatedfatty acid (a common fat component) with cholesterol uptake by the absorptive cells of the smallintestine (enterocytes). This study utilized a variety of diets that contained either fluorescentcholesterol or fluorescent fatty acid to visualize lipid absorption. Using this strategy, we found thata lipid-rich meal promotes BODIPY-cholesterol absorption into an endosomal compartment

physically distinguishable from lipid droplets that are labeled with BODIPY-fatty acid. We alsoestablish that long chain dietary fatty acids promote intestinal cholesterol absorption by inducingthe 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 bedisplayed on the intestinal brush border for BODIPY-cholesterol uptake into enterocytes. These dataare the first of their kind to visualize cholesterol uptake in a live whole animal and to elucidate thecell biological mechanism of the process. We hypothesize that this system may have evolved toensure that potentially toxic cholesterol is never imported without corresponding uptake of the fattyacids needed for its processing and export.

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Bio-mechanism of regeneration and degeneration inzebrafish 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 themolecular mechanism of zebrafish caudal fin regeneration based on transcriptomics, proteomics andepigenetic 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 andstudied for its differential expression at the transcript and protein level. Based on our study wefound that Annexin family genes were differentially regulated and posttranslational modified atdifferent time points of regeneration. Epigenomic associations of the gene based on histoneregulation 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) andParkinsons disease in the zebrafish model. Neuro-degeneration, the chronic breakdown of neuronalstructures, 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 tripletrepeats (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 inthe zebrafish Parkinsons disease model.

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Cognitive and neurobehavioral effects of childrens exposure to

airborne manganese

Jos Antonio Menezes-FilhoDepartment of Clinical and Toxicological Analysis

College of Pharmacy

Federal University of Bahia

Background: High levels of waterborne manganese have been associated with problematicbehaviors and cognitive deficits in school-aged children, however to date this has not been reportedwith respect to airborne manganese.

Objectives: We report here the results of our investigations carried between 2007 and 2009 onchildrens exposure to airborne manganese from ferro-alloy plant and cognitive effects. Preliminaryresults 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 109children from Cotegipe Village in the Simes Filho town, metropolitan area of Salvador, Bahia,

Brazil. The community was a priori divided according to the distance from the ferro-manganesealloy plant, whose atmospheric emissions have been causing complains from the community sinceits inauguration in the early 1970s. We measured Mn exposure by collecting air, water and crop

production samples. Childrens exposure was assessed by analyzing blood and hair Mn levels. Later

on, we reevaluated 83 school-aged children and investigated Mn association with cognitive deficitsapplying WISC-III (Wechesler scale for children). Confounders like maternal intelligence,socioeconomic status, malnutrition, lead co-exposure, etc were also evaluated. Currently, in a newcross-sectional study, we are reevaluating school-aged children from this community and from aneighboring and further community for cognitive, behavioral and motor effects. New biomarkersfor Mn exposure are being studied as well.

Results and Comments: The average and median Mn concentrations during the rainy period were0.151 g/m3and 0.114 g/m3, respectively (range 0.0110.439 g/m3). Mn concentrations in rawand drinking water were 74.48.63 g/L and 27.715.02 g/L, respectively, and below the

permissible level in Brazil (100 g/L). In 2007 the observed mean MnH level was 15.20 g/g(1.1095.50 g/g) for the exposed children and 1.37 g/g (0.395.58 g/g) for the non-exposed.For the former, MnH concentrations were 7.951.40 g/g (farthest from the plant), 11.811.11 g/g(mid-region), 34.438.66 g/g (closest to the plant) and 34.229.15 g/g (directly downwind).Thus, Mn exposure was related to airborne Mn exposure and was also associated with the time ofmaternal exposure previous to the child birth.

In the investigation carried out in 2008, the observed mean MnB and MnH were 8.2 g/L (2.723.4) and 5.83 g/g (0.186.68), respectively. Mean maternal MnH was 3.50 mg/g (0.1077.45)and correlated to childrens MnH (rho=0.294, p=0.010). Childrens MnH was negatively related to

Full-Scale Intelligence Quotient (IQ) and Verbal IQ; -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 withneuropsychological effects: aggressive and disruptive behaviors =8.24 (95%CI 2.40-14.08) and=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 memoryand phonemic fluency).

Conclusion:Our previous results and these preliminary ones suggest that childrens high exposureto airborne Mn may be having detrimental effects on cognitive functioning and on childrens

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

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Toxicity and mutagenicity of b(a)p-spiked sedimentspreliminaryresults

Flavia Mazzini1, Celia M. Rech1, Deborah A. Roubicek1, Ral Bonne Hernndez21Human Toxicology and Environm. Health Section, CETESB - Environmental Company of So Paulo, Av. Prof.

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

2Federal University of So Paulo.

Introduction

The use of biotests or biomarkers enables a more substantial appraisal of the impact of existingconditions 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 theZebrafish 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 with1,5 mg benzo(a)pyrene (S1+B(a)P and S2+B(a)P). The samples were extracted withdichloromethane (DCM) in an ultrassonic bath, evaporated and ressuspended in dimethylsulfoxide(DMSO) or ISO water, depending on the bioassay needs. For the ZFET, zebrafish embryos werecollected after approximately 2 h postfertilization, distributed in 96 well microplates and exposedfor 120 h to 7 concentrations of the different sediment organic extracts. Plates were kept at 26Cduring the experiments. Artificial ISO water and DMSO 0,2% were used as negative controls andsodium 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) wereevaluated. For the mutagenicity test, S. Typhimurium TA98 e TA100 tester strains were used withmetabolic activation. Seven concentrations of the different extracts were tested in triplicate.

Negative control was DMSO and the positive control used was 2AA. Revertant colonies werecounted after incubation for 66 h at 37C.

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 orcoagulation of embryos were only observed to S2+B(a)P. These lethal endpoints were encounteredafter 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 higherthan those verified to the organisms exposed to the B(a)P-spiked sediments, although they wereobserved around 24 h latter.Mutagenicity was not detected in the assays performed with the non-spiked sediment, in none of theS. 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

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

Bibliography

HOLLERT, H.; KEITER S.; KNIG, N.; RUDOLF, M.; ULRICH, M.; BRAUNBECK, T. A NewSediment 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 ofEmbryonic 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. MutationResearch, v. 113, p. 173-215, 1983.

Acknowledgements

The authors are particularly grateful for the support and assistance by Celso Fumio Suzuki, Genivalde Oliveira, Gilson Alves Quinaglia, sis Marques, Mary Ishimine Nishita and Pollyanna Ferreira deCarvalho.

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Toxicity of manganese(III) complexes to Danio rerio

Anderson Arndt1, Maria Ins Borella2, Breno Pannia Espsito1

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

Introduction

Manganese (Mn) is an abundant element which is present also in food and water. It is the cofactorof 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 brainvia 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 neurologicaldysfunction 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 topromote 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 visualmorphological damages in the telencephalic region are not observed.

ExperimentalAfter synthetized, complexes Mn(dfb), Mn(aha) and EUK108 had their lipophilicity described asoctanol-water partition coefficient (log P). The relative stability of the complexes was studied bythe 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 thedihydrorhodamine (DHR) oxidation method [4,5]}.

Acute toxicity tests were performed according to the OECD guidelines [6]. Adult zebrafish (Daniorerio; 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, acorrespondent 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]

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(r.u.)

complex:calcein mol ratio

[Mn(dfb)]

(a)

0 20 40 60 80

0

30

60

90

120

150

Mn(aha)

EUK108

Fluorescencerate(min-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]:[email protected]:[email protected]:[email protected]

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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 EUK108remain relatively unchanged (Figure 1a).

Mn(aha) did not display any pro-oxidant activity. EUK108 induced oxidative stress even at verylow (< 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 anoxidation mechanism still unknown (Figure 1b).

We for the first time examined the toxicity of Mn(III) compounds to Danio rerio. EUK108 is overthe high end of the range of toxicity mentioned above (LD50 ~ 20 M), and the hydroxamatederivatives 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 M (the less toxic compound) or EUK108 20 M (the more toxiccompound) does not reveal significant morphological changes (Figure 3), indicating thatneurological damage, if present, was either insufficient to induce morphological changes, oroccurred 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 ofDanio reriobrains at the ventral telencephalic (Vd) region after different treatments (a) control (b)Mn(dfb) (c) EUK108.

ConclusionsAlthough showing some pro-oxidant effect and toxicity, the compounds studied here didnt showany observable damage to Danio rerio telencephalon probably their low lipophilicity preventreaching this region. EUK108 toxicity correlates with its higher lipophilicity and pro-oxidantactivity.

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

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Toxicological analysis of caffeine using embryos ofDanio reri oas an experimental model

Ana Carina Nogueira Vasconcelos1, Shayenne Elizianne Ramos1, Mnica Rodrigues Ferreira1,Galileu Crovatto Veras2 , Luis David Solis Murgas1

1Departament of Veterinary Medicine. Federal University of Lavras, Campus Universitary, Post office box 3037, Zipcode 37200-000. LavrasMG. Brazil. [email protected]

2Coastal Studies Institute. Federal University of Par. BraganaPA. Brazil

Introduction

Caffeine (1,3,7-trimethylxanthine) is a chemical compound produced by more than 60 species ofplants and is the most consumed stimulating substance in the world. Among many applications, thisalkaloid 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 ofintracellular 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 andunchanged frequency. These findings indicate that caffeine has multiple applications and moreresearch is needed on their effects during embryonic development, since most pregnant womeningest this substance, either through diet or medication. To obtain information about the action ofthis compound in the body, the use of animal models is an important tool study. Among theadvantages of using embryos of Danio rerio (zebrafish) highlight the complexity of a vertebrateanimal, 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 fourconcentrations of caffeine (0, 50, 150 and 200 ppm) during two distinct stages of embryonicdevelopment.

Experimental

A completely randomized delineation was used with five replicates in 4 x 2 factorial design, withfour levels of caffeine (0, 50, 150 and 200 ppm) and two times of immersion (group I - 0 to 48hpfand 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 ofembryonic development and the second, only after 24 hours. The variables evaluated at 24, 48 and72 hours of incubation were: length of the yolk sac and embryo growth. Heart rate was measuredafter 48 and 72 hours of development and the survival rate was observed at the end of theexperiment.


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 embryostreated with 100 g/ml of caffeine, Chakraborty et al. (2011) observed that the expression ofvascular endothelial growth factor (VEGF) was practically null. The linear increase of the yolk saclenght in a function of increasing levels of caffeine can be explained by reduced expression ofVEGF. This factor stimulates the development of blood vessels in the yolk sac and by thisexpression 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

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cells (Rana et al., 2010). These channels contribute to cell repolarization following an initialdepolarization and muscle contraction, making it necessary to maintain normal cardiac output andfrequency. 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 impairedabsorption of nutrients in the embryo yolk sac. The decrease in heart rate was evident only inembryos exposed to caffeine from the beginning of development. The effect is reversible when theembryos are again immersed in water. Thus, these data demonstrate the need for further study todetermine the mechanisms of action of caffeine and limit the exposure that affects the embryonicdevelopment.

Bibliographic

CHAKRABORTY, C.; HSU, C. H.; WEN, Z. H.; LIN, C. S.; AGORAMOORTHY, G. Effect ofcaffeine, norfloxacin and nimesulide on heartbeat and VEGF expression of zebrafish larvae. JEnviron 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 PossibleMechanisms 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 caffeineon heart rate variability in habitual caffeine consumers. ClinPhysiol Funct Imaging, v. 26, n. 3,

p.163166, May. 2006.TSAI, T. D.; BARISH, M. E. Imaging of caffeine-inducible release of intracellular calcium incultured embryonic mouse telencephalic neurons. Journal Neurobiol, v. 27, p. 252265. 1995.

Acknowledgements

I thank Universidade Federal de Lavras and the Department of Biological Sciences and VeterinaryMedicine for improving my studies. My teacher Dr. David Luis Solis Murgas, for the confidenceand opportunity to work in the group. I thank the staff of the Universidade Federal de LavrasAquaculture and the study group on fish nutrition for cede the animals for this project. Anyway, toall the people who somehow contributed to the realization of this study.

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Evaluation of the toxicological effects of herbicide Glyphosate onembryonic development of Danio rer io

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 andexcessive use of pesticides has caused many impacts on the environment. Depending on thechemical 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 productscan be toxic and affect the survival, fertility and genetic composition of populations. Glyphosateacid, 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 ofpesticides used on brazilian crops is important. With this goal, this study examined the influence ofexposure ofDanio rerioembryos (zebrafish) to different concentrations of glyphosate (0, 5, 35, 65and 95 L/L) during embryonic development.

Experimental

We used a completely randomized design with four replicates. In each replicate 120 embryos weredistributed individually in containers with a total capacity of 0.5 mL. Glyphosate was previouslyadded to water and the embryos were exposed to this environment from the early embryonicdevelopment 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 andthe 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 and95L/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 growthand development (Niederreither & Doll, 2008). Any change in this acid quantity cause abnormaldevelopment 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 needfor deeper studies to determine the mechanisms of action of this herbicide during embryonicdevelopment.

Bibliographic

DRUART, C.; SCHEIFLER, R.; VAUFLEURY, A. Towards the development of an embryotoxicitybioassay with terrestrial snails: Screening approach for cadmium and pesticides. J of HazardousMaterials, v. 184, p. 26-33. 2010.

NIEDERREITHER, K.; DOLL, 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.; LPEZ, S. L.; CARRASCO, A. E. GlyphosateBased 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 byexecution 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 whocontributed with development of this work.

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Exposure of Danio rer ioembryos to glyphosatecause 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. Theproducts based on this compost are highly toxic to people and animals, and lab studies find adverseseffects as carcinogenicidde, mutageneicidade and reproductive problems both parents as in theoffspring, 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 betterunderstanding of the effects that this product will be able to produce. An experimental model thathas 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, highreproductive 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 inzebrafish larvas when exposed to different concentrations of glyphosate during the embryonicdevelopment.

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 waspreviously added to water and the embryos were exposed to this product from the early embryonicdevelopment until completion 96 hours after fertilization. The macroscopic variables evaluated atthe end of the experiment were: malformation and epidermal pigmentation.


The addition of glyphosate caused malformations and depigmentation of the larvae treated withconcentrations of 65 and 95 L / L. According Paganelli et al. (2010) glyphosate causes an increasein 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 & Doll, 2008). Thedepigmentation can be explained by impaired antioxidative activity of some cells caused byglyphosate (Heu et al., 2012), making this product is able to cause a discoloration of the pigmentmelanin since it is susceptible to bleaching by oxidizing agents.

Conclusions

These results clearly show that at certain concentrations, glyphosate causes morphological changesin embryos, confirming its potential risk to human health, since it is possible to extrapolate theresults. Therefore, it becomes necessary to study the limit of exposure to glyphosate that affectembryonic development.

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Bibliographic

, N., SERALINI, G. E. Glyphosate formulations induce apoptosis and necrosis in human umbilical,embryonic, and placental cells. Chem. Res. Toxicol. v. 22, 97105. 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. 184191. 2009.HEU, C.; ELIE-CAILLE, C.; MOUGEY, V.; LAUNAY, S.; NICOD, L. A step further towardglyphosate-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 anddrug 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. 11631172. 1964.

NIEDERREITHER, K.; DOLL, P. Retinoic acid in development: towards an integrated view. Nat.Rev. Genet., v. 9, p. 541-553. 2008.PAGANELLI, A.; GNAZZO, V.; ACOSTA, H.; LPEZ, S. L.; CARRASCO, A. E. GlyphosateBased 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=17495877http://www.ncbi.nlm.nih.gov/pubmed?term=Rodeck%20U%5BAuthor%5D&cauthor=true&cauthor_uid=17495877http://www.ncbi.nlm.nih.gov/pubmed?term=Dicker%20AP%5BAuthor%5D&cauthor=true&cauthor_uid=17495877http://www.ncbi.nlm.nih.gov/pubmed?term=Dicker%20AP%5BAuthor%5D&cauthor=true&cauthor_uid=17495877http://www.ncbi.nlm.nih.gov/pubmed?term=Rodeck%20U%5BAuthor%5D&cauthor=true&cauthor_uid=17495877http://www.ncbi.nlm.nih.gov/pubmed?term=Kari%20G%5BAuthor%5D&cauthor=true&cauthor_uid=17495877

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Protease ADAMTS-1 role in local and systemic invasion offibrosarcoma cells

Heydi Noriega Guerra1, Mrio Costa Cruz2, Ral Bonne Hernndez3, Vanessa Morais Freitas1

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

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

INTRODUCTION

Soft tissue sarcomas (STS) are malignant tumors of mesenchymal origin, among whichfibrosarcoma. 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 mustperform several important steps, including invasion of surrounding tissues, intravasation into theblood 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 bythe surrounding microenvironment, i.e. tumor stroma (3). On tumor microenvironment, theextracellular 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 malignantprogression (4). The secreted proteases ADAMTS-1, member of the ADAMTS family (a disintegrinand metalloproteinase with thrombospondin motifs), is differentially expressed in various tumorswhich regulate extracellular matrix and influence tumor biology (5). Controversial data showed thatADAMTS-1 could be associated with anti-tumor activity (6) or pro-tumor activity and metastasisstimulation (7). The potential role of ADAMTS-1 in tumor development and progression is stillunclear; it becomes an important research target. Furthermore, there are no reports about the role ofADAMTS-1 in respect of tumor cells and blood vessels, i.e. intravasation and extravasation of thesetumor cells. This study aimed to evaluate the role of ADAMTS-1 within the tumormicroenvironment of fibrosarcoma. We addressed the role played by ADAMTS-1 in the local andsystemic 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 37 C, 5% CO2. When monolayer HT1080 cells reached 70% confluency, cellswere 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 (Controlgroup) 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 thecommon cardinal vein (CCV) using picoinjector-1000 BTX Harvard Apparatus with a borosilicateglass needle. Injected embryos were transferred to E3 embryonic medium with PTU and kept at35.5C. Embryos at 24 hours post-injection were anesthetized and immobilized in a drop ofagarose. This was mounted inside a chamber and covered with a thin layer of E3 medium andanesthetic. The temperature was maintained at 28 C for capturing images using confocalmicroscope (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 andvenous vessels. In the context of a fibrossarcoma model, we observed tumor cells into the enteringvasculature and some of them were arrested within the ISVs and the surrounding tissue; featureenhanced 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 betweentumor 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. Arrowsindicate tumor cell with lamellipodial protrusion.

CONCLUSIONS

Our preliminary results suggest that ADAMTS-1 induces both cell migration and proliferation. Theinteraction between tumor cells and the stroma are of intense interest for further studies that will becarried out.

BIBLIOGRAPHIC

1. JAYAMATHI, P.; KEERTHIDAA, G.; VIDYALAKHSMI, K.; BHAVANI, G.; RUKMANI DEVI, S. Anti oxidantProperty 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. NatRev 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.119150, 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 withangio-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 incancer. 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 antimetastaticactivity, respectively. Oncogene,v.25, p.2452-2467, 2006.

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ACKNOWLEDGEMENTS

We are grateful to PhD. Ral Bonne Hernndez (ICAQF-UNIFESP, Brazil) for providing the embryos ofzebrafish for this study; and to Mrio Costa Cruz (CEFAP-USP, Brazil) for expert help and assistance withintravital confocal microscopy. This work is supported by FAPESP.

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Synthesis, characterization and malarial activity ofbinuclear copper (II) complex

Roxana Y. Pastrana Alta1*, Alejandro Miguel Katzin2, Breno Pannia Espsito1,Ana C. Valderrama Negrn3.

Instituto de Qumica, Universidade de So Paulo, So PauloSP, 05508-000, Brazil.Instituto de Biociencias, Universidade de So Paulo, So Paulo

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

Introduction

Malaria is one of the tropical neglected disease whose therapy may benefit from a bioinorganicchemistry approach. Quinine is antibacterial agents active against various microorganisms speciallyPlasmodium falciparum. However, although this neglected tropical disease still poses an important

public health problem, resulting in high morbidity and considerable mortality rates, few investmentshave been allocated towards developing novel anti-malarial agents. With that in mind, our teamdeveloped a new complex with quinine that shows more activity than quinine. Both present superioractivity in comparison with other reported antimalarial complexes [1].

Experimental

Materials; Quinine and Cu2(CH3COO)4.2H2O were purchased from Sigma-Aldrich. All solventswere purchased from Merck.Apparatus; Elemental analyses were performed on a ELEMENTAR ANALYZER CHNInstruments, model 2400 Perkin-Elmer. A Perkin Elmer Lamda 10 UVVis spectrophotometer wasused to obtain UVVis 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 onequipo Luxx, Netzsch STA 409 PG, the temperature range varied between 25 and 900 C and theheating rate was 10 C min-1. X-band electron paramagnetic resonance (EPR) spectra were obtainedwith a Bruker ENX spectrometer with modulation frequency of 100 kHz and modulation amplitudeof 1 mT. Magnetic susceptibility measurements at 298 K of powdered samples were determined bythe Gouy method. X-ray crystallograph, a green rectangular crystal was selected and cut to give afragment of dimensions suitable for X-ray analysis, data were collected on a BRUKER SMARTCCD. Structure and graphical illustrations were made with SHELXS-97y SHELXL-97 .

Synthesis of the complex

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

) was added to a solution ofCu2(CH3COO)4.2H2O (0.35 g, 0.96 mmol) in hot methanol (25 cm3). The mixture was heatedunder reflux (T=45C) for 6 h. After cooling the solution to room temperatura, single crystals weregrown by slow evaporation of metanol (20mL) solutions(and after 1 week). The final solution wasstored 3 C, to accelerate the formation of green crystals of complex. [Cu(O2CCH3)(-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 (OH); 1588 s (C=O)p;1620 m as(COO-); 1395 m s(COO-).

Malarial analysis

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

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were diluted in methanol, and used at the following concentrations: 100, 50, 10nM of complex and200, 100, 50 and 10 nM of quinine. Controls with methanol were performed in parallel. The methodof Desjardins et al. was used to determine IC50values (drug concentration that reduces 50 % of thenumber 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 thedifferent O2-binding properties of Hc/Tyr and the type 3 site in the MCOs and elucidating their origins. Thiswork provides molecular insight into the critical role of the mimetic new protein environment in directing theO2reactivity of these catalytic active sites.


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

quinine). Its structure was resolved by X-ray diffraction and showed two copper metal centers eachsurrounded by three oxygen atoms and one nitrogen atom (CuNO3), figure 1. The complex has beenfurther 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.Bcharacteristic 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 thecomplex 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 themulticopper oxidases and hemocyanin/tyrosinase, that could be beginning of a new bio-research.

Figure N1. Crystal for [Cu(O2CCH3)(-Qn)]2

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Conclusions

Has been synthesized and characterized the dimeric complex [Cu(O2CCH3)(-Qn)]2 whosestructure was confirmed by X-ray structural analysis. The developed Cu(II) complex present asignificant antimalarial activity in vitro against P. Falciparum (schizont stage), showingconcentrations (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 andcharacterization of copper(II) commplexes of pyridine-2-carboxamidrazones as potent antimalarialagents. Inorg. Chim. Acta 2003;349:23-/29.[2] Nicolas A. Rey,Karina C. Dos Santos.Two different Modes for copper(II)Ion cordination toQuinine-Type Ligands.J. Braz. Chem.,2006;17:497-504.[3] Synthesis and characterization of copperibuprofenate complexes with 2,2'-bipyridineand1,10-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 DEINGENIERIA, DE Lima-PER, Instituo de Quimca , Instituto de Biociencias daUNIVERSIDADE DE SO PAULO and Coordenao de Aperfeioamento de Pessoal de NvelSuperior.
http://www.capes.gov.br/http://www.capes.gov.br/http://www.capes.gov.br/http://www.capes.gov.br/

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Measurement of heavy metals in electronic waste and its occupationalhazards for recyclable material collectors

Carlos Alberto Conde Regina1, Etelvino Jose Henriques Bechara1,Nilson Antonio de Assuno1

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

Introduction

In Brazil, there is a lot of people working as waste picker in landfills and streets, they collected allkind of recyclable materials as paper, plastics and a special different one, Waste Electric andelectronic Equipments (WEEE) up and after it is sold to the market. The WEEE when handledincorrectly 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 alliedwith the emergence of technologies increasingly attractives resulted in huge quantities of obsoletematerials and disposable1. In this waste can be found computers, cell phones, monitors andtelevisors 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 notbiological functional activity and could cause negative behaviors as agressivity, QI reduction, actingespecially in Central Nervous System (CNS) 3,4

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

Africa) and it must be treated carefully as a health occupational problems of these envolvedworkers. Because it is the most socially and economically be excluded, has not been given dueattention to chronic exposure to toxic chemicals, potentially cumulative and could result in seriousoccupational injuries. Situation frequent in these countries due to lack of public awareness as aresult 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 theamount 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 studybelieves that the lead in nails, as a biomarker of continuous expose, can be correlated withaggressivity.


A total of 19 Cooperativas were visited and had their equipments, soils and some WEEE analyzedby X-Ray. The Table 1 shows the concentration of heavy metals found. Until this moment 15volunters 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 CircuitBoard

185 409 58 11170

Surface Mats 75 519 263 3802Surface tables 87 689 315 9357

Bags 101 464 487 439

Conclusions

The WEEE and another materials analyzed have high and amount concern heavy metalsconcentrations and if it is saved in open places, where they can receive sunlight and rains, thoseheavy metals can be released to the environment in cathionics forms. These findings clearly indicatean 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 apudChen, A.; Dietrich, K.N.; Huo, X.; Ho, S.; 119, 431-438, 2010.2. Andreola, F. 2005 apudMear. 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.; Gnther, W. M.R; Rev Sade Pblica, 44, 851, 2010;.

Acknowledgements

FAPESP, CNPq, and CAPES

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Embryotoxicity induced by mixtures of metal species

Mary Ishimine Nishita1and Ral Bonne Hernndez1

1Institute for Environmental, Chemical and Pharmaceutical Sciences. Lab. Bioinorgnica e Toxicologia AmbientalLABITA. Federal University of So Paulo,Rua So Nicolau, 210. Diadema-SP. [email protected].

Introduction

Manganese and their chemical species are associated with others chemical species in bothenvironment and physiological level. Toxicologically, these interactions can lead to synergistic,additives or an antagonistic effect, which are not always well known and has received littleattention. Previous reports indicate that this metal can also be toxic, particularly duringdevelopment, where their physiological functions suggest a multifactorial mechanism of toxicity.Furthermore, considering that both chemicals, aluminum (Al) and manganese (Mn), presentneurotoxicity, 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 lowconcentrations, and it association with others and their chemical species can be associated withmitochondrial 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 tothe assessment of risk to human health and ecotoxicological risk. Therefore, through thecharacterization of in vivo toxicological impact of potential mixtures it will be determinate themitochondrial alterations, apoptotic events, neurobehavioral changes and toxicological potential ofmixtures during development ofDanio rerio.

Experimental

MnCl2 stock solutions were prepared by dissolving manganese(II) chloride tetrahydrate(MnCl24H2O, Sigma-Aldrich, Germany) in sterile ultrapure water to obtain a final concentration of2 M. Mn(II)Cit solution was obtained by mixing MnCl2solutions with sodium citrate solution (6mM). 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)3Mn2H2O, Sigma-Aldrich, Germany) in a 6 mM citrate solution to obtain finalconcentrations of 0.25, 0.5, 1, and 2mM. A proportion of 2:1 ligand to metal was selected in orderto allow preparation of stable Mn(III)Cit solutions. The mixtures between manganese and theirchemical were constituted by Mn(II)Cit (0-6 mM) (A) and a fixed value of Mn(III)Cit (B): Mix0.25 (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 mMof B). Exposure media were prepared by serial dilutions in test medium (2 mM CaCl

2, 0.5 mM

MgSO4, 0.75 mM NaHCO3, 0.08 mM KCl). The metal oxidation state (II) or (III) in water solutionswas confirmed by EPR (Electronic Paramagnetic Resonance, using a Varian E-109 Century SeriesX-band Spectrometer (Varian, Inc., Palo Alto, CA, USA)) and UV-Visible spectrophotometricanalysis, using a used a UV-1700 Pharmaspec Spectrophotmeter (Shimadzu, Brazil). The amount ofmetal in the stock solutions were determined by atomic absorption spectroscopy in a ShimadzuAA6300 equipment. And the mixtures between manganese and aluminum were constituted byMn(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 rerioembryos (15 for each Mn concentration) were exposed tomixtures 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
mailto:[email protected]:[email protected]:[email protected]:[email protected]

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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 metalconcentration 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 LC50among different binary mixtures of Mn(II)Cit/Mn(III)Cit inzebrafish 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

Mixtures of Mn(II) and Mn(III)

LC50(mM

)

Experiments results showed that the redox couple Mn(II)/Mn(III), regardless the concentrationsratio, induces sub-lethal effects. And its possible to observe that mixtures treated with lowerconcentrations of Mn(III)Cit present a similar toxicity (LC50), which is less ecotoxic than mixtureMix 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 occursbetween 48 and 72 hpf).

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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

a1 b1 c1 d1a b c d

c1

d1

Embryo developmental stages-hpf (time exposition-hexp)

LC50(m

M)

For mixture with the lower concentration, the greater toxicity is on 48-120 hpf (72 hexp), and to thehigher is on 72-120 hpf (48 hexp). Which indicate that developmental stages and toxicity increasingappear to be depending of energy status in zebrafish: Before hatching (chorion (C) + high energylevel (E) = tolerant embryos or lower toxicity); after hatching (embryos dechorioned + decreased ofenergy level = increased of developmetal toxicity) and not with the time exposure. Toxicity of thesemixtures, 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 speciesredox 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 MnCl2and AlCl3, which showed ahigher toxicity of mixtures comparing to metal species studied separately. Its also possible toobserve that the manganese specie wasnt 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 thetoxicity this metal during developmental stages. In this case we found that for zebrafish a model ofaquatic organisms (ecotoxicological relevance) and of health disorder in human (environmental andhuman toxicological relevance), the toxicity induced by mixtures of Mn appear to be following amechanism more associated with biological effects than additive phenomena. And preliminaryexperiments indicate that the association of these metals can represent a major risk to human healthand to the environment.

Bibliographic

NAGEL, R. (2002). DarT: the embryo test with the Zebrafish Danio rerio-a general model inecotoxicology and toxicology. Altex-Alternativen Zu Tierexperimenten 19., 38-48.

NORWOOD, W.P., Borgmann, U., Dixon, D., Wallace., G.A., 2003. Effects of Metal Mixtures onAquatic Biota: A Review of Observations and Methods. Human and ecological risk assessment

Acknowledgements

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

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DETERMINAO DA TOXICIDADE EM ARTEMIAS SALINASFRENTE A METALOFARMACOS DE FERRO DE USO

COMERCIAL E HUMANO COM CAPACIDADE DE GERARATIVIDADE REDOX ATIVO.

Hector Aguilar Viorino1, Flavia Zanotto2, Luca Mantovanelli2, Breno Pannia [email protected]

1Institute of Chemistry. University of So Paulo. So PauloSP, 05508-000, Brazil.2Institute of Biosciences. Department of Physiology. University of So Paulo. So PauloSP, 05508-900, Brazil.

Resumo

O objetivo do trabalho avaliar a estabilidade e capacidade de gerao de atividade redox defrmacos comerciais de uso veterinrio e compostos-modelo de metalofarmacos de ferro como oferroceno, TMH-ferroceno e (TMH)2-ferroceno frente calcena em meio fisiologicamente

relevante. Alm disso, avaliou-se a toxicidade dos metalofrmacos em Artemia salina. Osmetalofarmacos comerciais apresentam uma estabilidade similar em diferentes concentraes, assimcomo a atividade redox ativa,enquanto a taxa de mortalidade o frmaco dexiron apresenta o maiorvalor (30%, 1000

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

Introduo

A sobrecarga de ferro uma condio desfavorvel tanto para humanos como, possivelmente, osorganismos aquticos. A toxicidade do ferro em humanos originada, principalmente, pela induode reservatrios de ferro lbil plasmtico (LPI) [1] Metalofrmacos de ferro compreendem agentesanti-hipertensivos, antimicrobianos e suplementos minerais proposto para diversas aplicaesclnicas ou farmacolgicas. Contudo, os possveis danos por sobrecarga induzida desses compostosno so totalmente compreendidos.

AArtemia salina um pequeno crustceo que habita lagoas temporrias de guas salgadas, ondepoucos animais podem sobreviver, possuindo o melhor sistema de osmorregulao do reino animal,alm de resistir em ambientes de baixos nveis de oxignio. Com estes fatores de adaptao podemcoexistir em densidades de populao muito elevadas [2]. Por estes motivos a utilizao de artmias

para testes de toxicidade tem aumentado, pois alm de responder aos compostos testes sem serinfluenciada pela concentrao salina do meio, sua obteno muito fcil e de baixo custo.

Experimental

Estabilidade frente calcena: A estabilidade dos frmacos (Tabela 1) foram avaliadas comauxlio da sonda fluorescente calcena, registrada a temperatura ambiente com um BMG FluostarOptima (exc/em=485/520 nm) por 24 horas[3].Atividade pr-oxidante: A atividade pro-oxidante dos metalofrmacos de ferro foi avaliadaatravs da mudana na velocidade de oxidao da sonda DHR (Dihidrorodamina) catalisada pela

reao entre ferro e ascorbato [4].

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Toxicidade em A. salina: Machos deA. salinaadultos foram triados e acondicionados em placa depetri para aclimatao em gua do mar reconstituda. Dez animais foram acondicionados em frascose incubados ao abrigo da luz a 30C por um perodo de 24h, na presena dos compostos deinteresse. [5].

Tabela 1: Frmacos comerciais de uso veterinrio e humano.

Frmaco comercialCaractersticas

Concentrao 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 Frrico2

1,790Noripurum Ferripolimaltosa2 0,895

1 Frmacos injetvel de uso veterinrio.2Frmacos oral de uso humano. 3 Concentraes que indica a bula de cadafrmaco.

Resultados e Discusso

Os frmacos comerciais apresentam uma tendncia similar de estabilidades (Figura 1). Os derivadosdo ferroceno apresentaram uma estabilidade dependente da estrutura qumica (Figura 2), sendo oscom substituintes no anel ciclopentadieno os menos estveis, provavelmente por efeitos estricos.

Os frmacos apresentam atividade pr-oxidante dependendo da concentrao, mas a mortalidadedas artmias no parece depender do nvel de ferro redox-ativo.

Figura 1. Estabilidade dos frmacos comerciais com concentraes 2.0, 4.0 e 8.0 M (10 L)frente ao quelante calceina 2M (180 L) a pH=7,44 e T=37C. Para n=4, P

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Figura 2. Estabilidade dos compostos de ferroceno sintetizados com concentraes 2.0, 4.0 e 8.0 M (10L) frente ao quelante calceina 2M (180 L) a pH=7,44 e T=37C. Para n=4, P

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Tabela 2: taxa de mortalidade das artemias salinas frente aos compostos de ferro com concentraes2.0, 4.0 e 8.0 M ,a pH=7,44 e T=37C. Para n=4, P

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Tin-induced developmental toxicity in zebrafish embryos

sis Marques da Costa;1Gergia Labuto2; Ral Bonne Hernndez1

1Lab. Bioinorganic and Environmental Toxicology- LABITA. Federal University of So Paulo2AQUA (Chemical Analysis Applied Group), Federal University of So 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 employedas an antifouling for marine, biocide for agriculture, antifungal fabric and stabilizer in the PVCindustry. 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 mostsensitive species and proving its action as an endocrine disruptor. Bivalves, mollusks and oystershave a high capacity of TBT accumulation due to limited ability to metabolize this compound, suchobservation, alert to the possibility of reaching the man[2]. This job present preliminary results of theuse 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 stocksolution 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 serialdilution was applied to obtain final concentrations of 0.001, 0.003, 0.01, 0.03, 0.1 and 0.3 mM forall three compounds. Danio rerio embryos (15 for each concentration of MBT and TBT and 10 ofDBT 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 wereexposed 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 theembryo, missing heart beat, heart rate reduced, deformation of the body and altered spontaneous orstimulated 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 beequivalent to a long exposure (2-122 hpf). Already for MBT not seem to be equivalent to a longexposure. News assays is being conducted to solve these speculations.

Comparison of in zebrafish embryos at different development stages and with different exposuredurations and comparison between static and semi-static (changing of MBT, DBT and TBT solution

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

(ecotoxicological relevance) and of health disorder in human (environmental and humantoxicological relevance). In summary, TBT seems be more toxic than MBT which seems be moretoxic than DBT.

Bibliographic

[1] Okoro HK, Fatoki LS, Adekila FA, Ximba BJ, Snyman RG, Sources, Environmental Levels andToxicity of Organotin in Marine Environment-A Review, Asian Journal of Chemistry, 23,(2011),473-482.[2] Rudel, H. Case study

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