CYTOCHROME-P4501A INDUCTION AND INHIBITION BY 3,3',4,4'-TETRACHLOROBIPHENYL IN AN AH RECEPTOR-CONTAINING FISH HEPATOMA-CELL LINE (PLHC-1)

The induction of cytochrome P4501A1 (CYP1A1) in rat hepatoma cells has been used by some investigators to determine 'dioxin equivalents' in- environmental samples, including extracts of fish tissues. However, th e relative potency of inducing compounds may vary between species, sug gesting the need for taxon-specific model systems. In this paper we pr esent an initial characterization of CYP1A induction in one such syste m, a teleost liver cell line (PLHC-1) derived from a hepatocellular ca rcinoma of Poeciliopsis lucida (Hightower, L.E. and Renfro, J.L., 1988 . J. Exp. Zool. 248, 290). Specific binding of the photoaffinity ligan d 2-azido-3-[I-125]iodo-7,8-dibromodibenzo-p-dioxin ([I-125]N3Br2DD) t o proteins in PLHC-1 cytosol indicated the presence of the Ah receptor , which is known to control CYP1A induction in mammals. 3,3',4,4'-Tetr achlorobiphenyl (TCB) induced a microsomal protein in PLHC-1 cells tha t was recognized by monoclonal antibody (MAb) 1-12-3 to scup CYPIA1 (P 450E) on immunoblots. Immunohistochemical staining of whole cells with MAb 1-12-3 showed specific recognition of CYP1A induced by TCB. No st aining was seen in untreated or vehicle-treated cells. There was an ex cellent quantitative correlation between amounts of CYPIA protein dete cted immunohistochemically and in immunoblots of cell homogenates. In a dose response experiment, maximal induction of ethoxyresorufin O-dee thylase (EROD) activity occurred at 0.1 muM TCB; at higher concentrati ons (1 and 10 muM), EROD activity was reduced as compared to the activ ity at 0.1 muM TCB. In contrast, immunoreactive CYP1A protein increase d with increasing TCB concentration up to 10 muM. The loss of EROD act ivity at high concentrations of TCB did not result from changes in cel l number or viability. The apparent inhibition or inactivation of CYPI A catalytic activity by the higher concentrations of halogenated biphe nyls has been seen, but not generally recognized, both in vivo and in cultured cells from diverse vertebrate species. PLHC-1 cells may be a good model system for studying Ah receptor-mediated regulation of gene expression, for determining the fish-specific toxic or inducing poten cy of halogenated aromatic hydrocarbon congeners, and for investigatin g the mechanism of CYP1A inhibition or inactivation by environmental c ontaminants such as TCB.