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.