Cytochrome P450 expression and related metabolism in human buccal mucosa

Constituents in food and fluids, tobacco chemicals and many drugs are candi dates for oral absorption and oxidative metabolism. On this basis, the expr ession of cytochrome P450 isozymes (CYPs) and the conversion of CYP substra tes were analysed in reference to buccal mucosa, A RT-PCR based analysis of human buccal tissue from 13 individuals demonstrated consistent expression of mRNA for the CYPs 1A1, 1A2, 2C, 2E1, 3A4/7 and 3A5, CYP 2D6 was express ed in six out of the 13 specimens, whereas all samples were negative for 2A 6 and 2B6, Serum-free monolayer cultures of the Siman virus 40 large T-anti gen-immortalized SVpgC2a and the carcinoma SqCC/Y1 buccal keratinocyte line s expressed the same CYPs as tissue except 3A4/7 and 3A5 (SVpgC2a), and 2C, 2D6 and 3A4/7 (SqCC/Y1), Dealkylation of ethoxyresorufin and methoxyresoru fin in both normal and transformed cells indicated functional 1A1 and 1A2, respectively. SVpgC2a showed similar activity as normal keratinocytes for b oth substrates, whereas SqCC/Y1 showed about 2-fold lower 7-ethoxy-resorufi n O-deethylation and 7-methoxyresorufin O-demethylation activities. SVpgC2a showed detectable and many-fold higher activity than the other cell types towards chlorzoxazone, a substrate for 2E1, Absent or minute catalytic acti vity of 2C9, 2D6 and 3A4 in the various tell types was indicated by lack of detectable diclofenac, dextromethorphan and testosterone metabolism (<0.2- 0.5 pmol/min/mg), Metabolic activation of the tobacco-specific N-nitrosamin e 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the mycotoxin af latoxin B-1 (AFB(1)) to covalently bound adducts was indicated by autoradio graphic analysis of both monolayer and organotypic cultures of SVpgC2a, In contrast, SqCC/Y1 showed lower or absent metabolic activity for these subst rates, Finally, measurements of various non-reactive AFB1 metabolites indic ated rates of formation <0.1 pmol/min/mg in both normal and transformed cel ls. The results indicate presence of several CYPs of which some may contrib ute to significant xenobiotic metabolism in human buccal epithelium. Notabl y, metabolic activation of AFB1 was not previously implicated for oral muco sa. Further, the results show that CYP-dependent metabolism can be preserve d or even activated in immortalized keratinocytes, Metabolic activity in SV pgC2a under both monolayer and organotypic culture conditions suggests that this cell line may be useful to pharmaco-toxicological and carcinogenesis studies.