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Efeitos Da Contaminação Ambiental Nas Respostas De CYP1A Em Duas Espécies De Peixes Guarús Cíntia Da Cruz Chivittz

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Efeitos Da Contaminação Ambiental Nas Respostas De CYP1A Em Duas Espécies De Peixes Guarús Cíntia Da Cruz Chivittz Universidade Federal do Rio Grande Instituto de Ciências Biológicas Pós-graduação em Biologia de Ambientes Aquáticos Continentais Efeitos da contaminação ambiental nas respostas de CYP1A em duas espécies de peixes guarús Cíntia da Cruz Chivittz Orientador: Juliano Zanette Rio Grande 2014 Universidade Federal do Rio Grande Instituto de Ciências Biológicas Pós-graduação em Biologia de Ambientes Aquáticos Continentais Efeitos da contaminação ambiental nas respostas de CYP1A em duas espécies de peixes guarús Aluna: Cíntia da Cruz Chivittz Orientador: Juliano Zanette Dissertação apresentada ao Programa de Pós-graduação em Biologia de Ambientes Aquáticos Continentais como requisito parcial para a obtenção do título de Mestre em Biologia de Ambientes Aquáticos Continentais. Rio Grande 2014 AGRADECIMENTOS Primeiramente quero agradecer a minha família, em especial aos meus pais, pelo incentivo de sempre. Pai e mãe, obrigada por toda a educação que vocês me proporcionaram até hoje e por me ensinarem o verdadeiro valor das coisas. Gostaria de agradecer também a professora Ioni Colares, exemplo de profissional e de pessoa, que me ajudou muito na fase inicial do mestrado, obrigada pelas conversas e pelos conselhos. Ao meu orientador Juliano Zanette, por ter aceitado me orientar, mesmo sabendo que eu não tinha conhecimento algum sobre peixes e sobre biologia molecular. Obrigada pelos ensinamentos e pela confiança! Aos colegas do PPG-BAC 2012, obrigada pela troca de conhecimento e pela companhia sempre agradável. As meninas do BAC, obrigada pelas conversas e por não me deixarem esmorecer nos momentos mais difíceis, vocês são demais! A minha turma da graduação, que mesmo com a distância está sempre presente de alguma forma, e que a cada encontro me faz ter orgulho de ter escolhido esta profissão. Aos técnicos Bernardo Vaz e Josencler Ferreira, sempre prestativos e dispostos a me ensinarem tudo sobre peixes, biotério e experimentos, muito obrigada. A querida Loraine Moraes, técnica do laboratório mais bonito e organizado da FURG, que com o seu bom humor contagiante fez os meus dias de laboratório muito mais agradáveis. Obrigada ao professor Cleber Palma Silva e ao técnico Leonardo Furlanetto por me ajudarem com as coletas e análise dos dados de água. Aos meus “orientadores de laboratório” Mauricio e Cássia, que me ajudaram muito no começo do mestrado, sempre fazendo tudo parecer mais fácil. Ao Mauricio, que passou dias e dias me ensinando absolutamente tudo, muito obrigada por toda a paciência e pelo apoio, e por sempre me tranquilizar quando eu fazia alguma coisa errada. A minha amiga querida Debora, minha eterna dupla, que me acompanhou desde o vestibular até aqui, obrigada por tudo! Obrigada por me ajudar no laboratório, nas saídas de campo e por me dar aquela força sempre que eu precisava. Vamos formar sempre a melhor equipe (de duas) de pescadoras, de fotógrafas, seja lá o que vier pela frente. Quero agradecer também ao Roger, o MALA mais legal que eu conheço. Obrigada pela ajuda nas saídas de campo, nas dissecções, no trabalho braçal, no laboratório e também pelos sustos noturnos na fisiologia (vai ter volta! hahaha). Deixo um agradecimento também ao grupo de biomarcadores ambientais, que me proporcionou conhecer pessoas tão queridas como a Sabrina, a Vanessa, o Guilherme e o Félix. Aos meus companheiros de aventura, que literalmente enfiaram o pé na valeta comigo, elém do Roger e da Debora; Mainara, Eduardo, Guilherme, Profº Flávio, Fernanda, Viviane, Bia e Félix, a ajuda de vocês foi fundamental e com certeza vocês tornaram as saídas muito mais divertidas. As minhas amigas de fé e de sempre Karine, Keli, Bel, Manuh e Tchana, obrigada por todo o apoio e por fazerem da nossa amizade o meu porto seguro, amo vocês! Obrigada ao tio César, pelas dicas de pescador, por me indicar os melhores pontos de coleta e por se preocupar com as minhas pescarias, muitas vezes frustradas! A Manuh e a Bel, que além de amigas, também me acompanharam nos melhores dias de coleta, durante as férias e os finais de semana, realmente quem têm amigas tem tudo! Ao meu namorado Leonardo, que esteve comigo desde o começo do mestrado, tornando os meus dias mais leves e os meus finais de semana mais completos. Obrigada por acreditar mais no meu potencial do que eu mesma, por viver repetindo que eu era capaz e que tudo iria acabar bem. Tudo é muito melhor e mais fácil do teu lado! Quero agradecer também a Michele Cristello, um exemplo de profissional, que me fazer acreditar na conclusão deste mestrado e que foi fundamental para que eu concluísse essa etapa da melhor maneira possível, com toda minha admiração, muito obrigada! “Agradecer é o reconhecimento do bem feito por alguém”, e por isso sou imensamente grata a todos que de alguma forma contribuíram com o mínimo que fosse para a conclusão desse mestrado. Sempre digo que minha turma da graduação, eterno ATBio 2011, foi a melhor parte da faculdade, agora também tenho a melhor parte do mestrado. Muito obrigada! RESUMO A poluição dos ambientes aquáticos por contaminantes orgânicos e misturas complexas, como aquelas provenientes de efluentes domésticos e industriais, tem levado certas populações de peixes a apresentarem adaptações bioquímicas e moleculares. Dentre estas, estão o aumento nos níveis do citocromo P450 1A (CYP1A), e em algumas populações, a perda da sua capacidade de indução após exposição a contaminantes agonistas do receptor de hidrocarbonetos aromáticos (AHR). Tal impossibilidade de induzir CYP1A é denominada resposta refratária, e vem sendo estudada há decadas em certas populações de peixes nativos da América do Norte. O presente estudo avaliou as respostas transcricionais de CYP1A utilizando RT-qPCR em brânquia, fígado e gonopódio dos peixes guarús Sul-Americanos Jenynsia multidentata e Phalloceros caudimaculatus coletados no entorno do estuário da Lagoa dos Patos, RS, Brasil. Os níveis de CYP1A em J. multidentata e P. caudimaculatus de um local contaminado por efluentes domésticos e outro próximo ao polo petroquímico, respectivamente, foram mais altos do que em locais referência, distantes destas fontes de contaminação. Exemplares de J. multidentata, provenientes de quatro locais foram expostos a 1 µM de β-naftoflavona (BNF) durante 24 h; e obtiveram indução de CYP1A em todos os órgãos em relação aos controles, exceto no fígado de peixes de um local contaminado, o que indica a possível existência de resposta refratária de CYP1A. Os resultados sugerem que os mecanismos responsáveis pela adaptação de J. multidentata aos locais poluídos são de alguma forma semelhantes aos encontrados em peixes da América do Norte e, possivelmente, envolve a resposta refratária de CYP1A e polimorfismos do AHR. O presente estudo forneceu informações a respeito da adaptação envolvendo respostas refratárias de CYP1A em peixes de locais poluídos e dá suporte para a utilização dos níveis transcricionais de CYP1A em guarús Sul-Americanos como biomarcador para o monitoramento da contaminação ambiental. Palavras-chave: América do Sul, CYP1A, peixes, poluição, resposta recalcitrante. ABSTRACT The water pollution caused by organic contaminants and complex mixtures, such as domestic and industrial sewage, caused some fish species to adapt at the biochemical and molecular level. This adaptation includes the increase in the level of cytochrome P450 1A (CYP1A) and an absence of induction by compounds that are agonists of the aryl hydrocarbon receptor (AHR). This impossibility to induce CYP1A is called refractory response of the CYP1A, and has been studied by decades in some native fish populations in North America. The present study evaluated the transcriptional responses of CYP1A using RT-qPCR in gills, liver and gonopodium of the South American guppies Jenynsia multidentata and Phalloceros caudimaculatus collected around the Patos Lagoon Estuary, RS, Brazil. The level of CYP1A in J. multidentata and P. caudimaculatus collected in a stream contaminated with domestic sewage discharges and from an area close to a petrochemical center, respectively, showed high levels of CYP1A expression when compared to reference locations distant from the these sources of contamination. Copies of J. multidentata, from four sites were exposed to 1 µM of β-naftoflavona (BNF) for 24 h; and obtained induction of CYP1A in all organs compared to controls, except for the liver of fish from one contaminated site, which indicates the possible existence of CYP1A refractory response. The results suggest that the mechanisms responsible for the adaptation of J. multidentata to live in polluted sites are somehow similar to those found in North American fish and possibly are also related to the CYP1A refractory response and AHR receptor polymorphisms. This study provided informations about the adaptation involving refractory responses of CYP1A in fish from polluted environments and give support for the use of CYP1A transcriptional levels in South American guppies as biomarkers for the monitoring of environment contamination. Key-words: South America, CYP1A, fish, pollution, recalcitrant response. SUMÁRIO LISTA DE FIGURAS DA INTRODUÇÃO GERAL .................................................................... 9 LISTA DE FIGURAS DO ARTIGO .............................................................................................. 9 LISTA DE TABELAS DO ARTIGO ........................................................................................... 10 1. INTRODUÇÃO GERAL .........................................................................................................
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