Effect of P450 Oxidoreductase Polymorphisms on the Metabolic Activities of Ten Cytochrome P450s Varied by Polymorphic CYP Genotypes in Human Liver Microsomes

Abstract

Background/ Aims: Little is known about the effect of P450 oxidoreductase (POR) gene polymorphisms on the activities of CYPs with multiple genotypes. Methods: We genotyped 102 human livers for 18 known POR single nucleotide polymorphisms (SNPs) with allelic frequencies greater than 1% as well as for 27 known SNPs in 10 CYPs. CYP enzyme activities in microsomes prepared from these livers were determined by measuring probe substrate metabolism by high performance liquid chromatograph. Results: We found that the effects of the 18 POR SNPs on 10 CYP activities were CYP genotype-dependent. The POR mutations were significantly associated with decreased overall K_m for CYP2B6 and 2E1, and specific genotypes within CYP1A2, 2A6, 2B6, 2C8, 2D6 and 2E1 were identified as being affected by these POR SNPs. Notably, the effect of a specific POR mutation on the activity of a CYP genotype could not be predicted from other CYP genotypes of even the same CYP. When combining one POR SNP with other POR SNPs, a hitherto unrecognized effect of multiple-site POR gene polymorphisms (MSGP) on CYP activity was uncovered, which was not necessarily consistent with the effect of either single POR SNP. Conclusions: The effects of POR SNPs on CYP activities were not only CYP-dependent, but more importantly, CYP genotype-dependent. Moreover, the effect of a POR SNP alone and in combination with other POR SNPs (MSGP) was not always consistent, nor predictable. Understanding the impact of POR gene polymorphisms on drug metabolism necessitates knowing the complete SNP complement of POR and the genotype of the relevant CYPs.

NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies

“…As a cell line originating from a human hepatoblastoma this was not quite clear for HepG2, since cells could adapt their phenotype due to genetic instability typical for cancer cells. For primary-like hepatocytes, however, this observation supports the finding from earlier studies that these cell models remain functionally active over a longer cultivation period than is the case for cultivated primary human hepatocytes [12,13,17]. Interestingly, cancer cell line HepG2 cells showed significantly increased POR expression at both gene and protein levels compared to primary-like hepatocytes.…”

“…Currently, primary human hepatocytes (pHHs) are the "gold standard" for in vitro studies on hepatic metabolism, clearance, hepatotoxicity and drug-drug interaction [9]. However, this research is still restricted by pHH scarcity, donor variability and their rapid dedifferentiation in vitro [10][11][12][13][14]. An inflammatory response by endotoxin contamination [15,16] originating from bacterial collagenase preparations, loss of normal cell polarity when dissolving them from liver tissue or down-regulation of liver-specific transcription factors influencing phase I/II protein expression were discussed as possible causes [17][18][19].…”

“…Stoichiometrically, liver POR expression is up to 10 times lower compared to microsomal CYPs, suggesting that POR could be a limiting factor for the activity of CYP monooxygenases [35,36]. Indeed it was shown, that the amount of POR in human liver as well as the enzyme activity of a particular polymorphic variant impact expression and activity levels of several relevant CYP enzymes [13,37,38]. Interindividual variability in POR expression and activity can be caused by influences on gene regulation at the transcriptional level by transcription factors like PXR or CAR, by hormonal influences or polymorphism in the POR gene itself [3,4,39].…”

High CYP2E1 activity aggravates hepatofibrosis by limiting macrophage polarization towards the M2 phenotype

“…By comparing the physiological and pathological activity of CYP2E1, we previously found that CYP2E1‐mediated probe clearance increased remarkably in hepatofibrotic tissue and positively correlated with enzyme velocity for DEN metabolism. CYP2E1‐mediated probe drug clearance showed intraindividual variation in patients with hepatofibrosis, and the difference in the metabolism of DEN by CYP2E1 may be the cause of hepatofibrosis . Herein CYP2E1 activity was determined by measuring the enzyme velocity for DEN metabolism in human hepatofibrotic tissue.…”

Biological Application and Disease of Oxidoreductase Enzymes

“…Another best example is mutation of p450 oxidoreductase (POR) gene, which leads to insufficiency of P450 enzymes characterized by defective steroidogenesis. Similarly, deficiency of mitochondrial acetaldehyde dehydrogenase disturbs normal metabolism of alcohol and leads to accumulation of acetaldehyde [8,15,16]. These conditions in turn affect the normal development and reproduction.…”