The first demonstration of CYP1A and the ABC protein(s) gene expression and activity in European seabass (Dicentrarchus labrax) primary hepatocytes

Abstract

Primary hepatocytes are a model for studying various effects of different xenobiotics, including detoxification strategies. In this study we have isolated and cultured European seabass (Dicentrarchus labrax) primary hepatocytes and assessed gene transcription and activity of CYP1A (phase I of cellular detoxification) and ABCC1 and ABCC2 (phase III) transport proteins after exposure to benzo(a)pyrene (BaP). A dose dependent increase in Abcc2 and Cyp1a mRNA transcripts was observed in seabass primary hepatocytes upon exposure to BaP. The activity of ABC proteins, as key mediators of the multixenobiotic resistance (MXR), was further confirmed by assessing the accumulation of the model fluorescence substrate rhodamine 123 in the absence and presence of model inhibitors. A weak interaction between BaP and ABC proteins was observed. CYP1A dependent ethoxyresorufin-O-deeethylase (EROD) activity was significantly induced by the presence of BaP. After the 24h exposure period only 10% of the initial BaP was present in the incubation medium, clearly demonstrating biotransformation potential of primary seabass hepatocytes. Furthermore, the presence of the 3-hydroxybenzo(a)pyrene, a BaP metabolite, in the medium implies its active efflux. In conclusion, we showed that seabass primary hepatocytes do express important elements of the cellular detoxification machinery and may be a useful in vitro model for studying basic cellular detoxification mechanisms and their interaction with environmental contaminants.

Keywords: ABC transporters; Biotransformation; CYP1A; Detoxification; MXR.