Aspectos histológicos de gônadas e fígado de Danio rerio como potenciais biomarcadores indicativos de atuação de interferentes endócrinos do pesticida Chlorantraniliprole
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2016-08-19
Autores
Corrêa-Souza, Cristina Elena [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O processo produtivo agrícola brasileiro está cada vez mais dependente dos agrotóxicos, tais produtos pelo uso constante e indevido atingem os corpos d’água e passam a interagir com a biota presente, podendo desencadear efeitos tóxicos agudos ou crônicos, além de atuar como interferentes endócrinos (IE). A maioria desses poluentes não apresenta na legislação vigente, limites de exposição segura, considerando-se os múltiplos usos dos recursos hídricos, destacando-se dentre eles a proteção da vida aquática. Diante do exposto, este estudo teve por objetivo verificar efeitos adversos do pesticida Chlorantraniliprole (CHLO) sobre a morfologia de gônadas e fígado de machos e fêmeas adultos de Danio rerio e o seu potencial para atuar como IE. O método consistiu na exposição de 63 machos e 36 fêmeas da espécie D. rerio por 14 e 21 dias a 0,60 μg/L de CHLO. O experimento foi realizado em regime semi-estático com troca total da solução teste a cada 48h, montados em aquários de vidro com capacidade para 2 L de solução teste, de modo a manter a proporção de 1g de peixe/L. O Controle (CC) foi apenas com água de cultivo e o Controle Acetona (CA) com 2,39 μl/L de acetona e água de cultivo seguindo-se recomendações da OECD (2000; 2010). Após os períodos experimentais os peixes foram eutanasiados em água com gelo a 4C° e destinados ao procedimento de histologia com inclusão na parafina, coloração com Hematoxilina e Eosina (HE) e análise em microscópio de luz. As análises histológicas das gônadas das fêmeas controle (CC) e controle acetona (CA), 14 dias e 21 dias revelaram padrão normal de distribuição e maturação gonadal, mostrando células em diferentes fases de desenvolvimento e ovários em diferentes estádios de maturação gonadal. Para as fêmeas expostas ao (CHLO) a 0,60μg/L por 14 dias, foi possível observar um predomínio de ovócitos com vitelogênese lipídica e protéica (IV) e ovócitos com vitelogênese completa (V), poucas ovogônias e ovócitos de reserva (II), além de alterações como um aumento da vacuolização interna de ovócitos (III e IV), com uma basofilia predominante, que nestas fases tende a ser menor. Já em 21 dias foram observados poucos ovócitos na fase II, nenhum na fase I e uma quantidade maior de ovócitos nas fases IV e V. Foi observado também a presença de muitos grânulos de vitelo acidófilos colados, compondo uma massa única homogênea e coalescente ocupando boa parte do citoplasma de alguns ovócitos na fase V. Em relação aos machos, a análise histológica dos controles (CC e CA) mostraram um padrão de desenvolvimento gonadal normal, sem alterações, apresentando todas as fases de desenvolvimento das células germinativas, Espermatogônia, Espermatócitos, Espermátides, Espermatozóides, além da presença das células de Leydig e células de Sertoli. No teste 14 dias não constatou-se a presença de machos entre os organismos estudados, mas para 21 dias de exposição ao CHLO foi notado hipertrofia e degeneração em células gaméticas, especialmente em espermatócitos. Para os tecidos de fígado, degeneração e hipertrofia dos hepatócitos estavam muitos frequentes tanto em machos como em fêmeas expostas a CHLO. De acordo com os resultados apresentados, podemos dizer que a espécie de peixe de D. rerio pode ter sofrido influência do pesticida CHLO tanto nas gônadas quanto no fígado dos organismos estudados em ambos os tempos de exposição. As alterações encontradas neste estudo são os primeiros relatos morfo-histológicos para o CHLO e corroboram as alterações encontradas em estudos de IE para ambos os tecidos (gônadas e fígado). Sendo assim, as análises histológicas demonstraram ser uma ferramenta importante para detectar alterações xenobióticas, sendo considerado nesta pesquisa um biomarcador para o CHLO.
The Brazilian agricultural production process is increasingly dependent on pesticides, such products by constant misuse reach water bodies and begin to interact with this biota and can trigger acute or chronic toxic effects, besides acting as endocrine disruptors (IE). Most of these pollutants not presented in the current legislation, the safe exposure limits, considering the multiple uses of water resources, highlighting among them the protection of aquatic life. Given the above, this study aimed to verify the adverse effects of pesticide Chlorantraniliprole (CHLO) on the morphology of gonads and liver of adult male and female zebrafish and their potential to act as IE. The method consisted of exposure of 63 males and 36 females of the species D. rerio for 14 and 21 days to 0.60 μg / L CHLO. The experiment was performed in a semi-static scheme with complete exchange of the test solution every 48 hours, mounted on glass tanks with a capacity of 2 L of the test solution, so as to maintain the proportion of fish 1 g / L. The Control (CC) was only cultivation water and Control Acetone (CA) with 2.39 μL / L and cultivation of acetone water followed recommendations OECD (2000; 2010). After the experimental period the fish were euthanized in ice water to 4C ° and intended for histology procedure with inclusion in paraffin, staining with hematoxylin and eosin (HE) staining and examination by light microscopy. Histological analysis of the gonads of female control (CC) control and acetone (CA), 14 and 21 days showed normal distribution pattern and maturation, showing cells at different stages of development and ovaries in different maturation stages. For females exposed to (CHLO) to 0,60μg / L for 14 days, we observed a prevalence of oocytes with lipid and protein vitellogenesis (IV) and oocytes with complete vitellogenesis (V), few ovogonia and reserve ovocytes (II ), and changes such as increased internal vacuolization oocytes (III and IV) with a predominant basophilia that these phases tends to be lower. As early as 21 days were observed few oocytes in stage II, no phase I and a larger amount of oocytes in stages IV and V. It was also observed the presence of many granules glued acidophilus calf, making up a homogeneous and good coalescing mass occupying only of the cytoplasm of some oocytes in phase V in regard to males, histologic analysis of the controls (CC and CA) showed a standard normal gonadal development without changes, with all the development stages of spermatogenesis, spermatogonia, spermatocytes, spermatozoa, spermatozoa, and the presence of Leydig and Sertoli cells. In 14 test days there was found the presence of males between the studied organisms, but for 21 days exposure 17 was noted CHLO hypertrophy and degeneration Gametic cells, especially in spermatocytes. For liver tissue degeneration and hypertrophy of hepatocytes were many common in both males and females exposed to CHLO. According to the results, we can say that the species of fish D. rerio may have been influenced by the pesticide CHLO both gonads and in the liver of the organisms studied in both exposure times. The alterations found in this study are the first morphological and histological reports for CHLO and corroborate the changes found in IE studies for both tissues (liver and gonads). Thus, the histological analysis demonstrated to be an important tool for detecting changes xenobiotic, this being considered a biomarker for research CHLO.
The Brazilian agricultural production process is increasingly dependent on pesticides, such products by constant misuse reach water bodies and begin to interact with this biota and can trigger acute or chronic toxic effects, besides acting as endocrine disruptors (IE). Most of these pollutants not presented in the current legislation, the safe exposure limits, considering the multiple uses of water resources, highlighting among them the protection of aquatic life. Given the above, this study aimed to verify the adverse effects of pesticide Chlorantraniliprole (CHLO) on the morphology of gonads and liver of adult male and female zebrafish and their potential to act as IE. The method consisted of exposure of 63 males and 36 females of the species D. rerio for 14 and 21 days to 0.60 μg / L CHLO. The experiment was performed in a semi-static scheme with complete exchange of the test solution every 48 hours, mounted on glass tanks with a capacity of 2 L of the test solution, so as to maintain the proportion of fish 1 g / L. The Control (CC) was only cultivation water and Control Acetone (CA) with 2.39 μL / L and cultivation of acetone water followed recommendations OECD (2000; 2010). After the experimental period the fish were euthanized in ice water to 4C ° and intended for histology procedure with inclusion in paraffin, staining with hematoxylin and eosin (HE) staining and examination by light microscopy. Histological analysis of the gonads of female control (CC) control and acetone (CA), 14 and 21 days showed normal distribution pattern and maturation, showing cells at different stages of development and ovaries in different maturation stages. For females exposed to (CHLO) to 0,60μg / L for 14 days, we observed a prevalence of oocytes with lipid and protein vitellogenesis (IV) and oocytes with complete vitellogenesis (V), few ovogonia and reserve ovocytes (II ), and changes such as increased internal vacuolization oocytes (III and IV) with a predominant basophilia that these phases tends to be lower. As early as 21 days were observed few oocytes in stage II, no phase I and a larger amount of oocytes in stages IV and V. It was also observed the presence of many granules glued acidophilus calf, making up a homogeneous and good coalescing mass occupying only of the cytoplasm of some oocytes in phase V in regard to males, histologic analysis of the controls (CC and CA) showed a standard normal gonadal development without changes, with all the development stages of spermatogenesis, spermatogonia, spermatocytes, spermatozoa, spermatozoa, and the presence of Leydig and Sertoli cells. In 14 test days there was found the presence of males between the studied organisms, but for 21 days exposure 17 was noted CHLO hypertrophy and degeneration Gametic cells, especially in spermatocytes. For liver tissue degeneration and hypertrophy of hepatocytes were many common in both males and females exposed to CHLO. According to the results, we can say that the species of fish D. rerio may have been influenced by the pesticide CHLO both gonads and in the liver of the organisms studied in both exposure times. The alterations found in this study are the first morphological and histological reports for CHLO and corroborate the changes found in IE studies for both tissues (liver and gonads). Thus, the histological analysis demonstrated to be an important tool for detecting changes xenobiotic, this being considered a biomarker for research CHLO.
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Palavras-chave
Ecotoxicologia, Peixes, Histologia, Agrotóxicos, Ovários, Testículos, Ecotoxicology, Fish, Histology, Agrochemicals, Ovaries, Testicles