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Avaliação Do Biomarcador De Contaminação Ambiental CYP1A Em Duas Espécies De Peixes Guarús Cíntia Da Cruz Chivittz

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Avaliação Do Biomarcador De Contaminação Ambiental 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 Avaliação do biomarcador de contaminação ambiental 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 Avaliação do biomarcador de contaminação ambiental 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 Algumas espécies de peixes guarús Sul-Americanos habitam o entorno do Estuário da Lagoa dos Patos (RS, Brasil), incluindo regiões impactadas por contaminantes orgânicos e efluentes domésticos e industriais. As adaptações dos organismos aquáticos para viverem nestes ambientes contaminados envolvem alterações ao nível bioquímico e molecular, como já foi demonstrado em populações de peixes nativos da América do Norte. Tais adaptações incluem o aumento nos níveis da enzima citocromo P450 1A (CYP1A), e em alguns casos, a perda da sua capacidade de ser induzida ao nível transcricional, denominada resposta refratária. No presente estudo, as respostas transcricionais de CYP1A em fígado e brânquias de guarús Jenynsia multidentata e Phalloceros caudimaculatus foram avaliadas pela técnica de RT-qPCR. Foi avaliado também o uso do gonopódio como alternativa de biopsia não invasiva e a ocorrência de resposta refratária na indução de CYP1A em quatro populações de J. multidentata. Os níveis de mRNA de CYP1A em J. multidentata e P. caudimaculatus de um local próximo a descarga de efluente doméstico e outro próximo a uma refinaria de petróleo, respectivamente, foram mais altos do que em locais distantes destes locais possivelmente contaminados. Para as duas espécies, o gonopódio apresentou níveis mensuráveis do transcrito CYP1A e respostas que foram semelhantes aos outros órgãos analisados. Os peixes J. multidentata, provenientes dos quatro locais obtiveram indução de CYP1A pela exposição a 1 µM de beta-naftoflavona (BNF) por 24h em todos os órgãos em relação aos controles, exceto no fígado de peixes de um local contaminado. Esta perda da capacidade de indução indica uma possível resposta refratária de CYP1A em fígado para a população de peixes deste local. Os resultados sugerem que os mecanismos responsáveis pela adaptação de J. multidentata a locais possivelmente contaminados são de alguma forma semelhantes aos encontrados em peixes da América do Norte e, possivelmente, envolve a resposta refratária de CYP1A e polimorfismos do receptor de hidrocarboneto aromático (AHR). Além disso, os resultados de expressão e indução de CYP1A em gonopódio foram satisfatórios, e apresentam vantagens em comparação com outros órgãos, por se tratar de um órgão com capacidade de regeneração. Assim, o presente estudo forneceu informações a respeito da adaptação em peixes guarús para habitar locais contaminados e favorece o uso do biomarcador de contaminação ambiental CYP1A em peixes guarús Sul- Americanos. Palavras-chave: Adaptação, América do Sul, Cyprinodontiformes, citocromo P450, resposta recalcitrante. ABSTRACT Some species of South American guppies inhabit the vicinity of the Patos Lagoon Estuary (RS, Brazil), including regions impacted by organic contaminants and domestic and industrial effluents. The adaptations of aquatic organisms to live in this contaminated environment involve biochemical and molecular alterations, as previously demonstrated in population of North American fishes. These adaptations include the increase in the the levels of the cytochrome P450 1A (CYP1A) enzyme, and the loss of the capability to induces it mRNA levels, denoted refractory response. In the present study, the transcriptional responses of CYP1A in liver and gills of the guppies Jenynsia multidentata and Phalloceros caudimaculatus, were evaluated by RT-qPCR. The use of gonopodium was also evaluated as an alternative for noninvasive biopsy and the occurrence of CYP1A refractory response in four populations of J. multidentata. The CYP1A mRNA levels in J. multidentata and P. caudimaculatus a location near the discharge of wastewater and another near an oil refinery, respectively, were higher than in remote locations of these possible sources of contamination. For both species, gonopodium showed measurable levels of the CYP1A transcript and responses that were similar to other organs examined. The J. multidentatafish collected infour sites present CYP1A induction after exposure to 1 µM of betanaphtoflavone (BNF) for 24 h in all organs compared to controls, except in the liver of fish from a contaminated site. These loss of induction capacity indicates a possible refractory response of CYP1A induction in the liver considering the fish population from this site. The results suggest that the mechanisms responsible for the adaptation of J. multidentata are possibly polluted sites somehow similar to those found in fish from North America and possibly involve the CYP1A refractory response and polymorphisms in the aryl hydrocarbon receptor (AHR). Furthermore,
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