1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
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
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
CEBiTOR 2012
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
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
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
15:00 –18:00
07:30-08:10
08:15-08:55
09:00-16:00
09:00-09:40
09:55 - 10:30
10:30-10:20
10:25-10:55
11:00-11:50
12:00-12:40
12:50-13:55
14:00-14:40
14:50-15:30
15:40-16:20
16:30-16:50
16:55-17:45
16:55-17:05
17:10-17:20
17:25-17:35
17:40-17:50
18:00-19:00
09:00-09:40
09:50-10:30
10:40-11:20
11:30-11:55
11:55-13:25
13:30-14:10
14:20-15:00
15:10-15:20
15:25-15:35
15:40-15:50
15:55-16:05
16:10-16:55
17:00–18:00
Accreditation
DECEMBER, 7th
Accreditation
Opening ceremony
Metals and other substances in biomedicine and pharmacology
Using zebrafish to unravel the genetics of complex brain
Robert Gerlai
Department of Psychology.
disorders.
University of Toronto - Canada
Adelson Silva
Especialista de Produtos em Espectroscopia|
“Transformando a técnica de ICP-MS com Triplo
Agilent Technologies - Brazil
Quadrupolo”
Coffe break and Poster discussion
Adelson Silva
Especialista de Produtos em Espectroscopia|
“Aplicações de ICP-MS acoplado a Cromatografia (GC e
Agilent Technologies - Brazil
HPLC)”
Fluorimetric detection of redox-active metals in cells and
Breno Pannia Espósito
Instituto de Química. University of São Paulo. Brazil
biological fluids.
Bio-mechanism of regeneration and degeneration in
Mohamme Idris
Centre for Cellular and Molecular Biology. India
zebrafish model system.
Lunch
Diogo Losch de Oliveira
A detailed characterization of the behavioral pattern of
Department of Biochemistry
zebrafish in the open tank paradigm: implications for
Federal University of Rio Grande do Sul. Brazil
drug toxicity evaluations.
Why we dip our lobster in butter: Using zebrafish larvae
Steven A. Farber
Depart.of Embryology.
to uncover an unexpected linkage between dietary fat and
Carnegie Institution for Science. USA
cholesterol absorption
Metallomics-based systems focusing on life-science:
Marco Aurélio Zezzi Arruda
Depart.of Analytical Chemistry. University of Campina
from plant to fish
Coffe break and Poster discussion
Oral short communication and evaluation
“Toxicity of manganese(III) complexes to Danio rerio”
Anderson Arndt
University of São Paulo - Brazil
“Importancia da protease ADAMTS-1 na invasão local e
Heydi Noriega Guerra
sistêmica de células de fibrossarcoma”
University of São Paulo - Brazil
“Behavioral and neurochemical changes in the zebrafish
Monica Gomes Lima
Federal University of Pará - Brazil
leopard strain”
“Toxicological analysis of caffeine using embryos of
Ana Carina Nogueira Vasconcelos
Federal University of Lavras
Danio rerio as an experimental model”
Constitution of an International Network for
Raúl Bonne Hernández
ICAQF-Federal University of São Paulo - Brazil
Environmental Bioinorganic and Toxicological Research
- NEBITOR: Environmental Neurotoxicology of metals
DECEMBER, 8th
Environmental bioinorganic chemistry and environmental health, and the zebrafish model as teaching tool
Ciro Alberto Oliveira
Federal University of Paraná
Zebrafish as a Model System for Environmental Health
Michael J. Carvan III
School of Freshwater Sciences.University of WisconsinStudies in the Classroom
USA
Cognitive and neurobehavioral effects of children’s
José Antonio Menezes-Filho
Depart. of Clinical and Toxicological Analysis
exposure to airborne manganese
Federal University of Bahia-Brazil
Poster discussion and evaluation
Lunch and NEBITOR discussion
The Effects of Developmental MeHg Exposure at
Michael J. Carvan III
School of Freshwater Sciences.
Environmentally-Relevant Levels
University of Wisconsin-USA
Fernando Barbosa Junior
FCFRP - University of São Paulo
Oral short communication and evaluation
“Serotonergic modulation of zebrafish behavior in
Caio Maximino de Oliveira
Federal University of Pará - Brazil
anxiety tasks”
“Exposure of Danio rerio embryos to Glyphosate cause
Carine Rodrigues Pereira
Federal University of Lavras
teratogenic effects”
“Determinação da toxicidade em artemias salinas frente a
Hector Aguilar Vitorino
University of São Paulo - Brazil
metalofarmacos de ferro de uso comercial e humano com
capacidade de gerar atividade redox ativo”
Embryotoxicity induced by mixtures of metal species.
Mary Ishimine Nishita
ICAQF-Federal University of São Paulo - Brazil
Coffe break
Closing ceremony
CEBiTOR 2012
5 th Floor
5 th Floor
5 th Floor
1st Floor
5 th Floor
Free
5 th Floor
1st Floor
5 th Floor
5 th Floor
Only among speakers
5 th Floor
1st Floor
5 th Floor
1st Floor
5 th Floor
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
1.
2.
3.
4.
Students considered with stays in double room-breakfast included
Nome
Instituição
Hotel Trade Hotel Diadema
ANA CARINA NOGUEIRA
UFL
VASCONCELOS
CAIO MAXIMINO DE
UFPA
Rua São Pedro, 135 - Centro,
OLIVEIRA
Diadema, 09910-620
CARINE RODRIGUES
UFL
PEREIRA
(11) 4056-8611 ‎ ·
MONICA GOMES LIMA
UFPA
CEBiTOR 2012
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
CEBiTOR 2012
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
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
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
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
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
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
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
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 lifescience 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
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
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
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
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 15min 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
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
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
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
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
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
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
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
CEBiTOR 2012
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
Toxicity and mutagenicity of b(a)p-spiked sediments – preliminary
results
Flavia Mazzini1, Celia M. Rech1, Deborah A. Roubicek1, Raúl Bonne Hernández2
1
Human Toxicology and Environm. Health Section, CETESB - Environmental Company of São Paulo, Av. Prof.
Frederico Hermann Jr, 345 São Paulo-SP. Brazil.
.
2
Federal 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
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
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
1ST INTERNATIONAL CONFERENCE ON ENVIRONMENTAL BIOINORGANIC AND TOXICOLOGY RESEARCH
Toxicity of manganese(III) complexes to Danio rerio
Anderson Arndt1, Maria Inês Borella2, Breno Pannia Espósito1
1
Institute of Chemistry. University of São Paulo, Av. Prof. Lineu Prestes, 748, São Paulo, Brazil. [email protected]
2
Institute 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].
Results and Discussion
(a)
(b)
150
1,0
Mn(dfb)
120
EUK108
-1
Fluorescence rate (min )
Fluorescence (r.u.)
EUK108
0,8
[Mn(dfb)]
0,6
0,4
90
60
30
[Mn(aha)3]
0,2
0
1
2
3
4
5
Mn(aha)
0
complex:calcein mol ratio
0
20
40
60
80
[H2O2] (M)
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)
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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 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
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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
1
Departament of Veterinary Medicine. Federal University of Lavras, Campus Universitary, Post office box 3037, Zip
code 37200-000. Lavras – MG. Brazil. [email protected]
2
Coastal 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.
Results and Discussion
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
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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.
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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
1
Departament of Veterinary Medicine. Federal University of Lavras, Campus Universitary, Post office box 3037, Zip
code 37200-000. Lavras – MG. Brazil. [email protected]
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.
Results and Discussion
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.
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Exposure of Danio rerio embryos to glyphosate
cause teratogenic effect
Carine Rodrigues Pereira1, Ana Carina Nogueira Vasconcelos1 & Luis David Solis Murgas1
1
Departament of Veterinary Medicine. Federal University of Lavras, Campus Universitary, Post office box 3037, Zip
code 37200-000. Lavras – MG. Brazil. [email protected]
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.
Results and Discussion
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.
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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, 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.
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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.; IRUELAARISPE, 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.
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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
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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.
Results and Discussion
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.
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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
1
Institute for Environmental, Chemical and Pharmaceutical Sciences. Federal University of São Paulo (ICAQFUNIFESP)
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.
Results and Discussion
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
Monitor CRT
Printed Circuit
Board
Surface Mats
Surface tables
Bags
Concentration of Heavy
Metals (emg.kg-1)
Cadmium(Cd)
972
185
Chromium(Cr6+)
844
409
Mercury(Hg)
453
58
Lead(Pb)
240872
11170
75
87
101
519
689
464
263
315
487
3802
9357
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
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Embryotoxicity induced by mixtures of metal species
Mary Ishimine Nishita1 and Raúl Bonne Hernández1
1
Institute 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.
Results and Discussion
Exposition from 2 to 122 hpf
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).
5
LC50 (mM)
4
3
2
a,b,c
1
0
a
b
c
d
Mix 0.25 mM
Mix 0.5 mM
Mix 1 mM
Mix 2 mM
Mixtures of Mn(II) and Mn(III)
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
4.0
Mix 0.25 mM
Mix 2 mM
3.5
3.0
LC50 (mM)
a1,b,c,d
2.5
2.0
a1,b1,d1
d1
1.5
1.0
0.0
c1
b1,d
0.5
a1
a
48-96 (48)
b1
b
72-120 (48)
c1
c
48-120 (72)
d1
d
2-122 (120)
Embryo developmental stages-hpf (time exposition-hexp)
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)
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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]
Institute of Chemistry. University of São Paulo. São Paulo – SP, 05508-000, Brazil.
Institute of Biosciences. Department of Physiology. University of São Paulo. São Paulo – SP, 05508-900, Brazil.
1
2
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
(M)2
Ferro dextrano1
3,580
Ferro-dextrano
1
1,790
Dexiron
Ferro-dextrano
1
1,790
Ferro Dextrano
Ferro-dextrano1
1,790
1
1,790
Gleptoferril
Biovet/Bio-Ferr
1
Concentração nominal de Fe
Fertal
Ferro dextrano
Ferrodex
Ferro dextrano1
1,790
2
Netrofer
Glicinato Férrico
Noripurum
Ferripolimaltosa2
1,790
0,895
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
0.21
±
0.01
0.21
±
0.01
0.07
±
0.01
0.05
±
0.01
0.05
±
0.01
0.04
±
0.01
0.17
±
0.03
0.25
±
0.02
0.40
±
0.06
0.35
±
0.07
0.64
±
0.08
1.31
±
0.12
Sulfato Ferroso
Ferroceno
TMH-Ferroceno
(TMH)2-ferroceno
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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.
Taxa de Mortalidade (%)
Compostos de ferro
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
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).
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Tin-induced developmental toxicity in zebrafish embryos
Ísis Marques da Costa;1 Geórgia Labuto2; Raúl Bonne Hernández1
1
Lab. Bioinorganic and Environmental Toxicology - LABITA. Federal University of São Paulo
2
AQUA (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, SigmaAldrich) 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%.
Results and Discussion
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
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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 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 dosedependent 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 = 89). 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 (AlCl 3•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 nondetached 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.
Results and Discussion
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 wildtype 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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