The mechanism of action of α-naphthoflavone as an inhibitor of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 gene expression

Treatment of rat hepatoma H-4-II E cells with α-naphthoflavone (αNF) (10^-8, 10^-7, 10^-6 m) resulted in only minimum induction of ethoxyresorufin O-deethylase (EROD) activity and cytochrome P4501A1 mRNA levels only at 10^-6 m. In contrast, 2,3,7,8-tetrachlorodi-benzo-p-dioxin (TCDD) caused maximum or near maximum induction responses at 10^-8 and 10^-9 m. In a timecourse study with TCDD (10^-9 m), and TCDD plus αNF (cotreated), αNF significantly inhibited the induction of EROD activity and cytochrome P4501A1 mRNA levels by TCDD for 6-24 h after initial exposure of the cells to the chemicals. In addition, treatment of the cells with 10^-9 m TCDD in the presence or absence of 10^-8, 10^-7, and 10^-9 m αNF showed that the latter compound inhibited the induction effects by TCDD in a concentration-dependent manner and these inhibitory effects could be overcome, in part, by a higher concentration of TCDD (10^-8 m). Treatment of the rat hepatoma H-4-II E cells with [³H]TCDD showed that within 60 min, there was an initial rapid increase in nuclear [³H]TCDD receptor complex levels (38 fmol/mg protein) which decreased to less than 10 fmol/mg protein within 4 h and remained relatively constant for up to 24 h. However, in cells treated with [³H]TCDD (10^-9m) plus αNF (10^-6 m) the levels of the nuclear [³H]TCDD receptor complex were <5 fmol/mg protein throughout the 24-h time course. These data, coupled with the results which indicate that the αNF competitively inhibits the binding of [³H]-TCDD to the cytosolic aryl hydrocarbon (Ah) receptor, suggest that αNF inhibits the TCDD-mediated induction of CYP1A1 gene transcription and translation by direct competition for cytosolic Ah receptor binding sites.