Preferential induction of CYP1B1 by benzo[ a ]pyrene in human oral epithelial cells: impact on DNA adduct formation and prevention by polyphenols

Abstract

The roles of CYP1A1 and 1B1 in tobacco smoke carcinogen, e.g. benzo[a]pyrene (BaP), induced DNA binding and their inhibition by the dietary polyphenol 5,7-dimethoxyflavone (DMF), compared with 3',4'-dimethoxyflavone (3',4'-DMF) and resveratrol, were investigated in the human oral epithelial squamous cell carcinoma (SCC)-9 cells. A low concentration of BaP (1 microM) dramatically induced BaP-DNA adduct formation (approximately 40-fold) in a time-dependent manner, while it only increased CYP1A1/1B1 activities, as measured by ethoxyresorufin O-deethylation, approximately 3-fold. Furthermore, BaP induced both CYP1B1 and CYP1A1 mRNA and protein expression, as determined by the branched DNA assay and western blot analysis, but with considerably higher levels of CYP1B1. Combined treatment of SCC-9 cells with 1 microM BaP and 20 microM DMF inhibited BaP-DNA adduct formation. The mechanism of this effect appeared to be direct inhibition of CYP1B1 enzyme with a K(i) value of 0.58 microM, a highly potent inhibition considering the high cellular uptake of DMF in the SCC-9 cells. DMF also inhibited CYP1A1, but not CYP1B1 protein, and mRNA expression in the cells. In an extension to other polyphenols, the structural analog 3',4'-DMF, in contrast to DMF, inhibited the expression of CYP1B1 both at the mRNA and protein levels. Surprisingly, in contrast to previous studies in other cell types, resveratrol had no effect on CYP1B1 in the SCC-9 cells. Based on this study, CYP1B1 mRNA may be an early biomarker of oral cancer, being a sensitive signal for tobacco-carcinogen exposure. Methoxylated dietary flavonoids, e.g. DMF and 3',4'-DMF, may be potent chemoprotectants by direct inhibition of CYP1B1/1A1 function and/or their protein expression.