Uroporphyrin accumulation associated with cytochrome P4501A induction in 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 halo- genated aromatic hydrocarbons in mammalian and avian systems, including primary cell cultures, but attempts to produce uroporphyria in vertebrate (mammalian) hepatoma lines have been unsuccessful. In this study, the ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and selected chlorobiphenyl congeners to cause uroporphyria was examined in a fish hepatoma cell line (PLHC-1) that expresses aryl hydrocarbon (Ah) receptors and an inducible cytochrome P4501A (CYP1A). Dose-dependent accumulation of porphyrins was observed in cells treated for 48 h with TCDD or 3,3',4,4'-tetrachlorobiphenyl (3,3',4,4'-TCB; 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 (approximately 80%) and heptacarboxylporphyrin (approximately 20%). Uroporphyria did not occur in cells treated with TCDD or 3,3',4,4'-TCB in the absence of added ALA. ALA-dependent porphyrin 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 (IUPAC 126). Neither of the mono-ortho-substituted chlorobiphenyls 2,3,3',4,4'-pentachlorobiphenyl (IUPAC 105) or 2,3',4,4'5-pentachlorobiphenyl (IUPAC 118) increased the porphyrin content of PLHC-1 cells. The ability of the PCB congeners to cause porphyria correlated with their ability to induce the CYP1A catalytic activity ethoxyresorufin 0-deethylase (EROD) and immunodetectable CYP1A protein in these cells, suggesting 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 increases at lower concentrations of inducer followed by decreased induction at higher concentrations, as seen previously. EC50 values for porphyrin accumulation were similar to, or slightly higher than, the concentrations at which peak EROD activities were obtained, suggesting a relationship between the decline in EROD activity and enhanced porphyrin accumulation. alpha-Naphthoflavone inhibited TCDD-induced EROD activity and porphyrin accumulation, providing further evidence for the involvement of a fish CYP1A in the mechanism of this prophyria. Addition of 3,3',4,4'-TCB to TCDD-treated cells also inhibited EROD activity, but enhanced porphyrin accumulation, suggesting that an interaction between the halogenated inducer and the induced CYP1A is necessary for the porphyrogenic response. PLHC-1 cells grown in medium supplemented with ALA may be a useful model System for studying mechanisms of chemical uroporphyria induced by Ah receptor agonists.