Effects of prochloraz and nonylphenol diethoxylate on hepatic biotransformation enzymes in trout: a comparative in vitro/in vivo-assessment using cultured hepatocytes

Abstract

The suitability of cultured rainbow trout hepatocytes as a model system for the assessment of xenobiotic effects on hepatic biotransformation enzymes in fish was examined. Two model water pollutants, the imidazole fungicide prochloraz and the alkylphenolic compound nonylphenol diethoxylate (NP2EO), were investigated in a comparative in vitro/in vivo approach. Biotransformation enzymes were measured in cultured rainbow trout hepatocytes following exposure to xenobiotics in vitro, or in the liver of juvenile rainbow trout ( Oncorhynchus mykiss) exposed in vivo. The patterns of biochemical responses to the model pollutants were generally similar between in vitro and in vivo investigations. Levels of cytochrome P4501A (CYP1A) protein and the catalytic activity of the CYP1A-dependent enzyme 7-ethoxyresorufin- O-deethylase (EROD) were induced in vitro after 24 h of exposure to 1.0 μM prochloraz. In vitro, higher prochloraz concentrations induced only the levels of CYP1A above control levels, but not EROD activity. In vivo exposure of juvenile trout to 0.27 μM prochloraz resulted in an induction of CYP1A and EROD after 7 and 14 days, while 0.027 μM prochloraz had no effects. In vitro, the 6β- and 16β-hydroxylation of testosterone was significantly decreased by 1.0–3.0 μM prochloraz, while in vivo these variables were significantly inhibited after exposure to 0.27 μM prochloraz for 7 and 14 days. NP2EO did not affect EROD activity in vitro. In vivo, EROD activity and CYP1A remained unchanged following 7 days of exposure to 0.32 or 1.30 μM NP2EO. NP2EO (15–50 μM) inhibited the 16β-hydroxylation and glucuronidation of testosterone in vitro. In vivo, 7 days of exposure to 0.32 or 1.30 μM NP2EO resulted in a significant inhibition of the 6β- and 16β-hydroxylation of testosterone. The good qualitative correspondence between in vitro and in vivo results indicates that studies using trout hepatocytes allow the identification of biochemical targets of xenobiotic effects in fish liver. However, more research is needed before quantitative predictions, e.g. of effective concentrations, can be made from in vitro investigations.