Caracterização transcricional dos genes CTR1 e ATP7B no peixe Poecilia vivipara: efeitos do cobre e da salinidade

Abstract

Em peixes, o cobre (Cu) é absorvido a partir da água, via branquial, e pela ingestão de água e alimento, via gastrintestinal. Para evitar reações não específicas prejudiciais e suprir proteínas dependentes de Cu, existem transportadores específicos, como as proteínas de absorção de alta afinidade ao Cu (CTR1) e as Cu-ATPases (ATP7), que auxiliam na translocação intracelular do metal. No presente estudo, os genes CTR1 e ATP7B foram identificados em Poecilia vivipara e os seus transcritos foram quantificados por RT-qPCR nas brânquias, no fígado e no intestino de guarús expostos (96 h) ao Cu (0, 5, 9 e 20 µg/L) em água doce e salgada (salinidade 24). Foram identificadas novas sequências nucleotídicas dos genes CTR1 (1560 pb, completa) e ATP7B (617 pb, parcial), as quais tiveram altos valores de identidade com as descritas para Fundulus heteroclitus (CTR1=81%) e Sparus aurata (ATP7B=81%). A análise por RT-qPCR indicou níveis de transcrição para CTR1 e ATP7B em todos os tecidos analisados. Em guarús na água doce, a maior expressão da CTR1 e da ATP7B se deu no fígado. Em guarús na água salgada, a maior expressão da CTR1 ocorreu no intestino, enquanto a da ATP7B se deu no fígado e intestino. Na água doce, a exposição ao Cu aumentou o conteúdo branquial e hepático de Cu, diminuiu os transcritos de CTR1 e ATP7B nas brânquias e aumentou os transcritos destes genes no fígado, sem alterar o conteúdo corporal de Cu. Na água salgada, a exposição ao Cu aumentou o conteúdo de Cu e diminuiu o transcrito de ATP7B no intestino, sem alterar o conteúdo corporal de Cu nos P. vivipara. Estes resultados indicam que a homeostasia do Cu em P. vivipara envolve a redução da expressão do CTR1 e ATP7B nas brânquias (água doce) e intestino (água salgada) para limitar a absorção do Cu e o aumento da expressão destes genes no fígado (água doce) para facilitar o armazenamento e desintoxicação do Cu.

In fish, copper (Cu) is absorbed directly from the water, via gills, and through the ingestion of water and food, via gastrointestinal. To avoid non-specific and harmful reactions and supply the Cu-dependent proteins, there are specific protein transporters involved in Cu metabolism, such as the high-affinity copper transporter (CTR1) and CuATPases (ATP7). They play an important role in the intracellular metal translocation. In the present study, CTR1 and ATP7B were identified in the guppy Poecilia vivipara. Transcripts of these genes were quantified by RT-qPCR in gills, liver and gut of guppies exposed (96 h) to Cu (0, 5, 9 e 20 µg/L) in freshwater and salt water (salinity 24 ppt). New nucleotidic sequences of the genes CTR1 (1560 bp, complete) and ATP7B (617 bp, partial) were identified. These sequences showed high identity values with those described for the killifish Fundulus heteroclitus (CTR1=81%) and the gilthead sea bream Sparus aurata (ATP7B=81%). The RT-qPCR data indicated transcription levels for CTR1 and ATP7B in all tissues analyzed. In freshwater guppies, the highest level expression of CTR1 and ATP7B was seen in the liver. In salt water guppies, the highest expression of CTR1 occurred in the gut, while ATP7B was more expressed in the liver and the gut. In freshwater guppies, exposure to Cu increased the Cu content in gills and liver, reduced the CTR1 and ATP7B expression in gills and increased the expression of these genes in liver, without affecting the whole-body Cu content. In saltwater guppies, Cu exposure increased Cu content and reduced ATP7B expression in gut, without change in whole-body Cu content. These findings indicate that Cu homeostasis in P. vivipara involves down-regulation of the CTR1 and ATP7B expression in gills (freshwater) and gut (salt water) to limit Cu absorption, as well as up-regulation of these genes in the liver (freshwater) to facilitate Cu accumulation and detoxification.

It is well accepted that environmental changes in salinity is a major factor influencing the metal accumulation and toxic effects in aquatic organisms. We addressed the effects of salinity (0 and 24 ppt) in the accumulation of Cu after 96 hrs waterborne exposure to 0, 5, 9 and 20 µg Cu/L and the possible regulation of key genes involved in the Cu homeostasis in the eurihaline guppy Poecilia vivipara, recently suggested as a model fish for environmental studies in South America. The doseresponse Cu accumulation in gill and liver observed only in the fresh water and the dose-response Cu accumulation in the gut only in saltwater, suggested that Cu accumulation in P. vivipara is salinity- and organ-dependent. In order to identify key genes involved in the Cu metabolism we sequenced, for the first time, transcripts that code for the high-affinity copper transporter (CTR1) and copper-transporting ATPase (ATP7B) in P. vivipara using Polymerase Chain Reaction (PCR) and sequencing. The full-length CTR1 open reading frame (1560 bp) and a partial ATP7B (690 bp) was uncovered and codes for amino acid sequence that shared high identities with the CTR1 of the killifish Fundulus heteroclitus (81%) and the ATP7B (87%) of Sparus aurata fish. Basal transcriptional levels, addressed by RT-qPCR in control fish, indicate that CTR1 and ATP7B was highly expressed in the liver of freshwater guppies and CTR1 in the gut of saltwater guppies, which could explain in part the high Cu accumulation observed in liver in freshwater and in gut in saltwater, considering that CTR1 is involved in the Cu uptake. After Cu exposure, guppies showed a reduced CTR1 expression in the gills (20 µg Cu/L in freshwater and 5 µg Cu/L in saltwater), comparing with the controls. In turn, ATP7B was mainly reduced in gut by exposure to 9 and 20 µg Cu/L in fresh water and by 20 µg Cu/L in salt water. Both, CTR1 and ATP7B were unaltered in the liver by Cu exposure. Those results suggest that CTR1 is down-regulated in gills and ATP7B in gut to limit Cu absorption, and CTR1 and ATP7B 18 are unaltered in liver, to facilitate Cu storage and detoxification. Taken altogether, these findings clearly indicate that salinity influences the Cu accumulation in P. vivipara organs and also suggests that homeostasis involves differential expression of the transcriptional regulation of the newly identified Cu transporters CTR1 and ATP7B.