Inhibition of cytochrome P4501A by organotins in fish hepatoma cells PLHC-1

Inhibitory effects of several organotin compounds on cytochrome P4501A (CYP1A) induction response and enzyme activity have been analyzed in fish hepatoma cells (PLHC-1). In a first set of experiments, cells were exposed for 3 d to 3-methylcholanthrene (3-MG), an inducer of CYP1A. Simultaneously, series of dilutions of the widely used organotin compounds triphenyltin (TPT), tributyltin (TBT), dibutyltin (DBT), and monobutyltin (MET) were added to the cells. Relative CYP1A protein contents were measured in an enzyme-linked immunosorbent assay (ELISA), CYP1A activities in the ethoxyresorufin-O-deerhylase (EROD) assay, and cytotoxicity in the neutral red (NR) assay. Induction of CYP1A protein and activity was found in the presence of low concentrations of organotins and 3-MG. The three assays did not show significant differences in sensitivity for TBT and TPT Concentrations that reduced control values by 50% (EC50) were between 1.6 . 10(-7) M and 3.5 . 10(-7) M, which emphasizes the high cytotoxicity of both compounds. In contrast, DBT led to inhibition of EROD activity at significantly lower concentrations (1.2 . 10(-6) M) than loss of CYP1A protein in the ELISA (9.0 . 10(-6) M) and cytotoxicity in the NR assay (8.7 . 10(-6) M). In a second set of experiments, reduction of CYP1A activity was also obtained after sequential exposure to 3-MG and organotins. In a third set of experiments, lysates of CYP1A-induced cells were exposed to organotins. Organotins caused a 50% inhibition of EROD activity at significantly higher concentrations (namely at 4.7 . 10(-5) M and 6.7 . 10(-5) M for TBT and DBT, respectively, and at 1.1 . 10(-3) M for MET) than in the first set of experiments. For TBT a noncompetitive inhibitory mechanism on CYP1A enzyme activity has been found. The experiments in this study demonstrate inhibitory capacities of TBT and TPT, but also of DBT and MET, on the CYP1A system in fish cells. The results lead to the conclusion that the effect is mainly caused by direct inhibition of enzyme activity, not by inhibition of CYP1A protein synthesis. The induction of CYP1A protein and activity in the presence of both an inducer (3-MG) and a low concentration of inhibitors (organotins) indicates that organotins do not interfere with the Ah receptor binding, but act at the CYP1A protein level.