RAPID ASSESSMENT OF INDUCED CYTOCHROME P4501A PROTEIN AND CATALYTIC ACTIVITY IN FISH HEPATOMA-CELLS GROWN IN MULTIWELL PLATES - RESPONSE TOTCDD, TCDF, AND 2 PLANAR PCBS

Induction of cytochrome P450 1A1 (CYP1A1) in cultured cells can be use d to determine taxon-specific relative potencies of Ah receptor agonis ts. This report describes optimized methods for growth and treatment o f PLHC-1 fish hepatoma cells in multiwell plates, in situ analysis of ethoxyresorufin O-deethylase (EROD) activity, and measurement of CYP1A protein by immunoblotting of eel lysates. EROD activity was undetecta ble (<1 pmol min(-1) mg-l) in untreated or dimethyl sulfoxide-treated cells, but was highly induced (up to 150 pmol min(-1) mg(-1)) in cells exposed to Ah receptor agonists such as 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), or planar chlor obiphenyls (CB). Addition of exogenous, NADPH was not required for mea surement of EROD activity in PLHC-1 cells. As inducers of EROD activit y, TCDD, TCDF, 3,3'4,4',5-pentachlorobiphenyl (CB-126), and 3,3',4,4'- tetrachlorobiphenyl (CB-77) differed both in potency and in apparent e fficacy (maximal level of induced activity). In each case, EROD induct ion was biphasic, with stronger induction at lower concentrations and an attenuated response at higher concentrations. In contrast, the cont ent of immunodetectable CYP1A protein increased monotonically with dos e of CB, and the maximum level achieved was similar for all inducers. The discrepancy in results obtained for EROD activity versus CYP1A pro tein may result from inhibition or inactivation of catalytic function at high concentrations of inducer. By reducing peak EROD values, this inhibition leads to lower apparent EC50 values and thus the overestima tion of relative potencies or toxic equivalency factors (TEFs) for man y inducers. These studies demonstrate the necessity of measuring both EROD activity and immunodetectable CYP1A protein for me accurate asses sment of CYP1A induction and relative potencies in cultured cells.