UROPORPHYRIN ACCUMULATION ASSOCIATED WITH CYTOCHROME P4501A INDUCTIONIN FISH HEPATOMA-CELLS EXPOSED TO ARYL-HYDROCARBON RECEPTOR AGONISTS,INCLUDING 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN AND PLANAR CHLOROBIPHENYLS

Hepatic uroporphyria is a well-known effect of halogenated aromatic hy drocarbons in mammalian and avian systems, including primary cell cult ures, but attempts to produce uroporphyria in vertebrate (mammalian) h epatoma lines have been unsuccessful. In this study, the ability of 2, 3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofu ran (TCDF), and selected chlorobiphenyl congeners to cause uroporphyri a was examined in a fish hepatoma cell line (PLHC-1) that expresses ar yl hydrocarbon (Ah) receptors and an inducible cytochrome P4501A (CYP1 A). Dose-dependent accumulation of porphyrins was observed in cells tr eated for 48 h with TCDD or 3,3',4,4'-tetrachlorobiphenyl (3,3',4,4'-T CB; IUPAC 77) when the heme precursor delta-aminolevulinic acid (ALA) was present during the last 5 h of treatment. HPLC analysis identified the porphyrins as uroporphyrin (similar to 80%) and heptacarboxylporp hyrin (similar to 20%). Uroporphyria did not occur in cells treated wi th TCDD or 3,3',4,4'-TCB in the absence of added ALA. ALA-dependent po rphyrin accumulation was also seen following treatment of PLHC-1 cells with TCDF or with the non-ortho-substituted chlorobiphenyls 3,4,4',5- tetrachlorobiphenyl (IUPAC 81) and 3,3',4,4',5-pentachlorobiphenyl (IU PAC 126). Neither of the mono-ortho-substituted chlorobiphenyls 2,3,3' ,4,4'-pentachlorobiphenyl (IUPAC 105) or 2,3',4,4',5-pentachlorobiphen yl (IUPAC 118) increased the porphyrin content of PLHC-1 cells. The ab ility of the PCB congeners to cause porphyria correlated with their ab ility to induce the CYP1A catalytic activity ethoxyresorufin O-deethyl ase (EROD) and immunodetectable CYP1A protein in these cells, suggesti ng direct or indirect regulation of porphyrin accumulation via the Ah receptor and/or the induced CYP1A. Induction of EROD activity by TCDD, TCDF, and the planar polychlorinated biphenyls was biphasic, with inc reases at lower concentrations of inducer followed by decreased induct ion at higher concentrations, as seen previously. EC(50) values for po rphyrin accumulation were similar to, or slightly higher than, the con centrations at which peak EROD activities were obtained, suggesting a relationship between the decline in EROD activity and enhanced porphyr in accumulation. alpha-Naphthoflavone inhibited TCDD-induced EROD acti vity and porphyrin accumulation, providing further evidence for the in volvement of a fish CYP1A in the mechanism of this porphyria. Addition of 3,3',4,4'-TCB to TCDD-treated cells also inhibited EROD activity, but enhanced porphyrin accumulation, suggesting that an interaction be tween the halogenated inducer and the induced CYP1A is necessary for t he porphyrogenic response. PLHC-1 cells grown in medium supplemented w ith ALA may be a useful model system for studying mechanisms of chemic al uroporphyria induced by Ah receptor agonists. (C) 1996 Academic Pre ss, Inc.