Rapid assessment of induced cytochrome P4501 a protein and catalytic activity in fish hepatoma cells grown in multiwell plates: Response to TCDD, TCDF, and two planar PCBS

Abstract

Induction of cytochrome P450 1A1 (CYP1A1) in cultured cells can be used to determine taxon-specific relative potencies of Ah receptor agonists. This report describes optimized methods for growth and treatment of 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 cell lysates. EROD activity was undetectable (< 1 pmol min{sup {minus}1} mg{sup {minus}1}) in untreated or dimethyl sulfoxide-treated cells, but was highly induced (up to 150 pmol min{sup {minus}1} mg{sup {minus}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 plant chlorobiphenyls (CB). Addition of exogenous NADPH was not required for measurement of EROD activity in PLHC-1 cells. As inducers of EROD activity, TCDD, TCDF, 3,3{prime},4,4{prime},5-pentachlorobiphenyl (CB-126), and 3,3{prime},4,4{prime}-tetrachlorobiphenyl (CB-77) differed both in potency and in apparent efficacy (maximal level of induced activity). In each case, EROD induction was biphasic, with stronger induction at lower concentrations and an attenuated response at higher concentrations. In contrast, the content of immunodetectable CYP1A protein increased monotonically with dose of CB, and the maximum level achieved was similar for all inducers. The discrepancy in results obtained for EROD activity versus CYP1A protein may result frommore » 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 overestimation of relative potencies or toxic equivalency factors (TEFs) for many inducers. These studies demonstrate the necessity of measuring both EROD activity and immunodetectable CYP1A protein for the accurate assessment of CYP1A induction and relative potencies in cultured cells.« less