Molecular characterization of cytochrome P450 1A (CYP1A) in Asian sea bass (Lates calcalifer Bloch) and its application as a biomarker in the Gulf of Thailand

Abstract

This study focuses on aquatic pollution in Thailand waters, using induction of the hepatic cytochrome P450 1A (CYP1A) in Asian sea bass (Lates calcalifer Bloch) as a biomarker to assess exposure to polyaromatic hydrocarbons (PAHs). Polyclonal antibodies (PAb) were raised against Asian sea bass CYP1A in mice. Protein blot analyses revealed that these antibodies recognized a CYP1A protein in Asian sea bass injected with 0.1-10 mg benzo[a]pyrene (BaP)/kg fish. The cross-reactivity with CYP1A was confirmed by comparison with PAb raised against rainbow trout CYP1A in rabbits, which have been used as biomarker for PAH exposure in fish from temperate waters (Förlin and Celander, 1993). The presence of a CYP1A orthologue in Asian sea bass liver was confirmed by isolating a partial cDNA, using a reverse transcriptase polymerase chain reaction (RT-PCR) approach. A 300 base pair sequence was isolated and it showed 79 % identity with rainbow trout CYP1A1. Quantitative RT-PCR analysis revealed that CYP1A mRNA was 2,5-fold up-regulated in Asian sea bass injected with 10 mg BaP/kg fish. The PAb raised against Asian sea bass CYP1A was next applied on 15 different tropical fish species, caught off the Chonburi coast. Three different sample sites were selected: Koh Loi, Si Racha (station 1), Ao Udom (station 2) and Laem chabang (station 3). In station 1, all three fish analyzed had detectable CYP1A levels. In station 2, six of totally eleven fish had detectable CYP1A levels and in station 3, one of totally nine fish had detectable CYP1A levels. Although, species variation in CYP1A protein levels was observed, the data suggest that Si Racha and Ao Udom were more contaminated with PAH compared with Laem chabang. This study confirm studies in Europe and North America that the hepatic CYP1A could be used as early indicators of chemical exposure in Thailand waters, and that PAb against Asian sea bass is suitable for analyses of CYP1A in a number of different tropical fish species.