Avaliação transcricional de Glutationas -transferases em PEIXE-ZEBRA (Danio rerio) e alterações causadas pela exposição á Microcistina-LR

Abstract

As Microcistinas são heptapeptídios cíclicos produzidos como metabólitos secundários por diferentes espécies de cianobactérias, sendo relevantes pelo seu potencial hepatotóxico. Peixes apresentam estratégias bioquímicas para detoxificar contaminantes ambientais, incluindo a ativação de enzimas de fase II de biotransformação, que incluem as isoformas de glutationa S-transferase (GST). As GST catalizam a conjugação de glutationa reduzida (GSH) com uma variedade de xenobióticos, incluindo as microcistinas. O presente estudo avaliou os níveis transcricionais de quinze isoformas de GST a fim de identificar isoformas possivelmente envolvidas na detoxificação de contaminantes ambientais como a microcistina-LR (MC-LR) em Danio rerio. A técnica de PCR em tempo real (RT-qPCR) foi utilizada para avaliação dos níveis transcricionais, permitindo análise das GST em diferentes órgãos, abundância e a ativação/repressão das isoformas de GST pela exposição à MC-LR. Foram avaliados os possíveis efeitos causados em brânquia e fígado após exposição por 24 hs às concentrações de 5 µg.L-1 e 50 µg.L-1 de MC-LR. Baseado nos scores de estabilidade para oito genes normalizadores, foram selecionados glicose-6-fosfato desidrogenase (g6pdh), β-actina1 e beta-2-microglobulina (b2m); b2m, alfa-tubulina 1 (tuba) e β- actin1; e tuba, b2m e g6pdh, para normalização dos níveis trancricionais de GST para distribuição órgão-específica, abundância e efeito da MC-LR em brânquia e fígado, respectivamente. A avaliação transcricional da distribuição órgão-específica revelou níveis significativos de gstal e gstk1.1 no fígado; gstp1 e gstp2 em brânquia; mgst3a, gstr1, gstm2, gstm33, gstp1, gstp2 e gstk1.1 no intestino; gstm2, gstm3 e gstal no olho e gstt1a e gsta2.1 no cérebro. Considerando os níveis de transcritos para um dado órgão, gstk1.1, gstal, gstp1 e gstt2 foram mais abundantes nos órgãos de detoxificação, tais como o fígado, brânquias e intestino, enquanto gstt1a e gsta2.1 foram mais abundantes no rim. Em brânquia, gsta2.1 e gstt1b foram reprimidas por 5 µg.L-1 de MC-LR e mgst1.1 foi reprimida em 50 µg.L-1 de MC-LR. No fígado, as isoformas gst2.2 e gstp2 foram reprimidas em ambas as concentrações, gstal foi reprimida em 5 µg.L-1, e gstt1a e gstk1.1 foram reprimidas em 50 µg.L-1 de MC-LR. As isoformas gstal, gstr1, gstp1, mgst3a, gstm1, gstm2 e gstm3 não foram alteradas pela exposição a MC-LR. Os resultados obtidos fornecem informações para a escolha de isoformas específicas de

GST possivelmente envolvidas na detoxificação/toxicidade de MC-LR, a serem melhores caracterizadas ao nível protéico e também contribui para a escolha de genes normalizadores a serem utilizados em outros estudos da mesma natureza

The microcystins are cyclic heptapeptides produced as secondary metabolites by different cyanobacteria species, being relevant for its potential hepatotoxicity. Fish possess biochemical strategies for detoxification of environmental contaminants, including the activation of phase II enzymes of biotransformation like glutathione Stransferase isoforms (GST). The GSTs catalyze the conjugation of reduced glutathione (GSH) with a variety of xenobiotics, including microcystins. The present study aimed to evaluate the transcriptional levels of fifteen GST isoforms to identify possibly isoforms involved in detoxification of environmental contaminants such as MC-LR in Danio rerio. The technique of real-time PCR (RT-qPCR) was used to assess the transcriptional level, allowing the analysis of the levels of fifteen GSTs in different organs, abundance and activation/repression of specific isoforms of GSTs by exposure to MC-LR. The possible effects caused by the exposure for 24 hours at concentrations of 5 µg.L-1 and 50 µg.L-1 of MC-LR on gill and liver transcriptional levels were evaluated. Based on the stability scores for eight housekeeping genes, were selected glucose-6-phosphate dehydrogenase (g6pdh), β-actina1 and beta-2-microglobulin (b2m); b2m, alpha-1 tubulin (tuba) and β-actin1; and tuba, b2m and g6pdh, to normalize the levels of GSTs trancriptional distribution organ-specific, abundance and effect of MC-LR in gill and in the liver, respectively. Analysis of organ-specific distribution showed significant levels of gstal and gstk1.1 in the liver; gstp1 and gstp2 in gill; mgst3a, gstr1, gstm2, gstm3, gstp1, gstp2 and gstk1.1 in intestine; gstm2, gstm3 and gstal in eye; and gstt1a and gsta2.1 in brain. Considering the levels of transcripts for a given organ, gstk1.1, gstal, gstp1 and gstt2 were the most abundant in the detoxification organs such as the liver, intestine and gill, while gstt1a and gst2.2 were the most abundant in the kidney. In gill, gsta2.1 and gstt1b were down-regulated by 5 µg.L-1 of MC-LR and mgst1.1 was downregulated by 50 µg.L-1 of MC-LR. In the liver, the isoforms gsta2.2 and gstp2 were down-regulated by both concentrations, gstt2 was down-regulated by 5 µg.L-1, and gstt1a and gstk1.1 were down-regulated by 50 µg.L-1 of MC-LR compared to the control group. Isoforms gstal, gstr1, gstp1, mgst3a, gstm1, gstm2and gstm3 were not altered by exposure to MC-LR. The results obtained provide basic information for the selection of specific GST isoforms possibly involved in the detoxification/toxicity, for better

characterization in the protein level and the results also contributed for the choice of housekeeping genes to be used in other similar studies.

The study of glutathione S-transferase (GST) isoforms is required for understanding the biotransformation of numerous environmental contaminants, such as the microcystinLR (MC-LR) produced by cyanobacterial blooms. The organ-specific distribution and abundance of fifteen GST transcripts in zebrafish Danio rerio using RT-qPCR and the possible induction or repression in liver and gills after 24-hrs waterborne exposure to 5 µg.L-1 and 50 µg.L-1 MC-LR were evaluated in the present study. The transcriptional levels for gstk1.1, gstal, gstp1 and gstt2 in important detoxification organs such liver, gill and intestine indicates a possible contribution of those isoforms in the biotransformation of contaminants. Significant levels of gstal and gstk1.1 in liver; gstp1 and gstp2 in gill; mgst3a, gstr1, gstm2, gstm3, gstp1, gstp2 and gstk1.1 in intestine; gstm2, gstm3 and gstal in eye; and gstt1a and gsta2.1 in brain, suggest an important function of the corresponding proteins in these organs. The exposure to 5 µg.L-1 and 50 µg.L-1 of MC-LR caused repression of gsta2.1, gstp2 and gstt2 (52 and 90; 76 and 71; 47 and 22 fold, respectively) in liver. The gsta2.1 in gill and gstt1b in liver were downregulated only in the lower concentration of 5 µg.L-1, and mgst1.1 in gill, and gstt1a in liver were down-regulated only in the highest concentration of 50 µg.L-1, compared with the control group. The gstal, gstr1, gstp1, mgst3a, gstm1, gstm2 and gstm3 isoforms were not altered by exposure to MC-LR. It is concluded that different isoforms have distinct abundance and organ-specific distribution, even within a certain GST class, and various GST isoforms are down-regulated at the transcriptional level by MCLR.