Effect of Cytochrome b5 Content on the Activity of Polymorphic CYP1A2, 2B6, and 2E1 in Human Liver Microsomes.

Haifeng Zhang,Jun Zhou,Qiang Wen,Hailing Qiao,Tingting Liu,Bing Qi,Na Gao,Yan Fang,Linjing Jia,Aditya Bhushan Pant

doi:10.1371/journal.pone.0128547

Abstract

Human cytochrome b5 (Cyt b5) plays important roles in cytochrome P450 (CYP)-mediated drug metabolism. However, the expression level of Cyt b5 in normal human liver remains largely unknown. The effect of Cyt b5 on overall CYP activity in human liver microsomes (HLM) has rarely been reported and the relationship between Cyt b5 and the activity of polymorphic CYP has not been systematically investigated. In this study, we found that the median value of Cyt b5 protein was 270.01 pmol/mg from 123 HLM samples, and 12- and 19-fold individual variation was observed in Cyt b5 mRNA and protein levels, respectively. Gender and smoking clearly influenced Cyt b5 content. In addition, we found that Cyt b5 protein levels significantly correlated with the overall activity of CYP1A2, 2B6, and 2E1 in HLM. However, when the CYP activities were sorted by single nucleotide polymorphisms (SNP), the effect of Cyt b5 protein on the kinetic parameters varied greatly. There were significant correlations between Cyt b5 content and Vmax and CLint of CYP1A2 wild-types (3860GG, 2159GG, and 5347CC) as well as homozygous mutants (163AA and 3113GG). In contrast to Vmax and CLint, the Km of CYP2B6 516GG and 785AA genotypes was inversely associated with Cyt b5 content. Correlations between Cyt b5 content and Vmax and CLint of CYP2E1 -1293GG, -1293GC, 7632TT, 7632TA, -333TT, and -352AA genotypes were also observed. In conclusion, Cyt b5 expression levels varied considerably in the Chinese cohort from this study. Cyt b5 had significant impact on the overall activity of CYP1A2, 2B6, and 2E1 in HLM and the effects of Cyt b5 protein on polymorphic CYP1A2, 2B6, and 2E1 activity were SNP-dependent. These findings suggest that Cyt b5 plays an important role in CYP-mediated activities in HLM and may possibly be a contributing factor for the individual variation observed in CYP enzyme activities.

Highlights

Human cytochrome b5 (Cyt b5), a small molecular protein containing one heme prothetic group, is encoded by a single gene on chromosome 18q23 that generates two isoforms throughPLOS ONE | DOI:10.1371/journal.pone.0128547 June 5, 2015Effect of Cytochrome b5 Content on cytochrome P450 (CYP) Activity
The Cyt b5 mRNA levels were determined by real-time RT-PCR and normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels (Fig 1A)
No unique characteristics were identified for this patient, and it can be inferred that individuals with extreme Cyt b5 mRNA levels exist in the Chinese population

Summary

Introduction

Human cytochrome b5 (Cyt b5), a small molecular protein containing one heme prothetic group, is encoded by a single gene on chromosome 18q23 that generates two isoforms throughPLOS ONE | DOI:10.1371/journal.pone.0128547 June 5, 2015Effect of Cytochrome b5 Content on CYP Activity. In this study we first characterized the expression variation of Cyt b5 in 123 Chinese livers and analyzed the effects of Cyt b5 content on the metabolic activities of CYP1A2, 2B6, and 2E1. Similar to Cyt b5 mRNA, the protein content of Cyt b5 in 123 liver samples was not normally distributed, with a median of 270.01 pmol/mg.

Results

Conclusion