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Sensitive biological responses to environmental contaminants are useful as early warning signals to predict the damages by long-term exposure. Protocols standardization to quantify biochemical parameters in different fish species is required to validate its use as biomarkers. Comparative studies from different fish species and its interpretation are a challenge for the validation of its use as general biomarkers, representative of environmental impact. In this study, the protocol for liver cytochrome P450 (CYP) analysis from the native Brazilian fish Ancistrus multispinis was established. The microsome contamination by hemoglobin during the analysis of CYP in liver was detected, leading to misinterpretation of the results. The spectrophotometric method for CYP analysis was adapted in order to diminish the hemoglobin interference. Additionally, the western blotting method for CYP1A analysis was tested with success for this fish species.Respostas biológicas sensíveis aos contaminantes ambientais são úteis para prever efeitos prejudiciais devido a exposições crônicas. Padronização de protocolos para quantificar parâmetros bioquímicos em diferentes espécies de peixes é necessária para validar o uso como biomarcador. Estudos comparativos de diferentes espécies de peixe e sua interpretação são um avanço para a validação do uso de biomarcadores gerais, representativos do impacto ambiental. Neste estudo o protocolo para a análise do citocromo P450 (CYP) do peixe nativo brasileiro Ancistrus multispinis foi estabelecido. Cyp é um biomarcador de exposição principalmente de hidrocarbonetos policíclicos aromáticos (HAP), bifenilas policloradas (PCB) e dioxinas. A contaminação do microssomo pela hemoglobina durante as análises do CYP no fígado foi detectada, levando a uma interpretação errônea dos resultados. O método espectrofotométrico para análise do CYP foi adaptado para diminuir a interferência da hemoglobina. Além disso, o método de western blotting para análise de CYP1A foi testado com sucesso para essa espécie de peixe.

This study aimed to determine the effect of sublethal doses of deltamethrin, using biochemical biomarkers as activities of cholinesterase (ChE), ethoxyresorufin-O-deethylase (EROD) and the Na + K + - ATPase and levels of total cytochrome P450 (CYP450). Fishes received sublethal doses of deltamethrin and were sacrificed after 96 h of exposure. Samples of gills, heart, brain, liver and muscle were collected for enzymatic analyses. Deltamethrin inhibited the activity of the gills and heart Na + K + -ATPase, induced the liver total CYP450, as well as the liver EROD activity. The activity of the ChE was not inhibited by deltamethrin. Deltamethrin altered the hepatic metabolism and the normal ionic flux in Ancistrus multispinis . Key words: deltamethrin, cytochrome P450, fish, Ancistrus multispinis , biomarker * Author for correspondence: helassis@ufpr.br INTRODUCTION Pyrethroids are synthetic derivatives of pyrethrins, which are toxic components contained in the flowers of Chrysanthemum cinerariaefolium . Although synthetic pyrethroids are less persistent and less toxic to mammals and birds (Sayeed et al, 2003), they are highly toxic to a number of non-target organisms such as bees, freshwater fish and other aquatic organisms even at very low concentrations (Oudou et al, 2004). For this reason, these organisms are extremely sensitive to neurotoxic effects of pyrethroids when they reach surface water-courses (Bradbury and Coats 1989, Haya, 1989; Mittal et al, 1994). One of the pyrethroids that has found wide acceptability and is extremely used in agriculture and forestry because of its high activity against a broad spectrum of insect pests (Villarini et al, 1998) is deltamethrin ((S) α-cyano-3-phenoxybenzyl-(1R)-cis-3-(2.2-dibromovinyl)-2,2- dimethyl cyclopropane carboxylate). However, its effects on nervous, respiratory, and hematological systems in fishes have been reported (Ural and Sa glam, 2005, Pimpao et al, 2007). Balint et al (1995) observed 20% decrease in acetylcholinesterase activity of brain, heart, blood, liver and skeletal muscle of carp after the 3 days exposure to deltamethrin. In rats, the main reaction involved in the metabolism of deltamethrin is ester cleavage, by CYP450 and carboxyesterase action. Metabolism in fish is largely oxidative and deficient in esterases metabolization (Demoute, 1989). ATPase has been demonstrated to be one of the targets of pyrethroids. Some authors showed that ATPase including cell membrane-associated