Effects of cytochrome b5 expression and its genetic variant on the activity of CYP2C9, CYP2C19 and CYP3A4

Abstract

BACKGROUND: The objective of present study was to investigate the effects of cytochrome b5 (cytb5) on the drug metabolism catalyzed by CYP2C9, CYP2C19 and CYP3A4. METHODS: Expression of cytb5 protein and mRNA in human liver tissues was evaluated using Western blot and quantitative real-time PCR, respectively. Genetic polymorphisms in cytb5 gene were extensively analyzed from the HapMap and 1000 genome data. The putative splicing variant, c.288G>A (rs7238987) was identified and it was screened in 36 liver tissues by direct DNA sequencing. Activities of CYP2C9, CYP2C19, and CYP3A4 were determined by using the prototypical substrates tolbutamide, omeprazole and midazolam, respectively. RESULTS: Cytb5 protein and mRNA contents showed large inter-individual variations with 11-and 6-fold, respectively. All of three P450s showed an increased activity in proportion to the amount of cytb5 expression. Particularly, CYP3A4 showed the strongest correlation between cytb5 protein amount and the activity, followed by CYP2C9 and CYP2C19. Liver tissues having a splicing variant exhibited unexpected sizes of cytb5 mRNA and a decreased expression of cytb5 protein content compared to the wild-type (P<0.05). A decreased activity in the metabolism of the CYP2C9, CYP2C19, and CYP3A4 prototypical substrates was observed in liver tissues carrying the splicing variant when compared to the wildtype (P<0.05 for omeprazole, P=0.21 and 0.25 for tolbutamide and midazolam, respectively). CONCLUSION: The present results propose that different expression of cytb5 and a splice variant can cause variations in CYP mediated drug metabolism, which may explain, at least in part, inter-individual different drug responses in addition to the CYP genetic polymorphisms.