Correlation of Cytochrome P450 Oxidoreductase Expression with the Expression of 10 Isoforms of Cytochrome P450 in Human Liver

Abstract

Human cytochrome P450 oxidoreductase (POR) provides electrons for all microsomal cytochromes P450 (P450s) and plays an indispensable role in drug metabolism catalyzed by this family of enzymes. We evaluated 100 human liver samples and found that POR protein content varied 12.8-fold, from 12.59 to 160.97 pmol/mg, with a median value of 67.99 pmol/mg; POR mRNA expression varied by 26.4-fold. POR activity was less variable with a median value of 56.05 nmol/min per milligram. Cigarette smoking and alcohol consumption clearly influenced POR activity. Liver samples with a 2286822 TT genotype had significantly higher POR mRNA expression than samples with CT genotype. Homozygous carriers of POR2286822C>T, 2286823G>A, and 3823884A>C had significantly lower POR protein levels compared with the corresponding heterozygous carriers. Liver samples from individuals homozygous at 286823G>A, 1135612A>G, and 10954732G>A generally had lower POR activity levels than those from heterozygous or wild-type samples, whereas the common variant POR*28 significantly increased POR activity. There was a strong association between POR and the expression of P450 isoforms at the mRNA and protein level, whereas the relationship at the activity level, as well as the effect of POR protein content on P450 activity, was less pronounced. POR transcription was strongly correlated with both hepatocyte nuclear factor 4 alpha and pregnane X receptor mRNA levels. In conclusion, we have elucidated some potentially important correlations between POR single-nucleotide polymorphisms and POR expression in the Chinese population and have developed a database that correlates POR expression with the expression and activity of 10 P450s important in drug metabolism.

Fig. 1.

Frequency distribution of POR mRNA levels (as measured by qPCR, relative to GAPDH, n = 107) (A), POR protein content (as measured by LC-MS/MS, n = 100) (B), and POR activity (as measured by the spectral method, n = 125) (C). The data are presented as the means of three independent experiments.

Fig. 2.

Correlations in POR mRNA, protein, and activity in human livers. (A) Correlation between POR protein content and POR mRNA level; (B) correlation between POR protein content and POR activity; and (C) correlation between POR activity and mRNA level. The data are presented as the means of three independent experiments.

Fig. 3.

Effect of cigarette smoking (A) and alcohol consumption (B) on POR activity in HLM. The black horizontal line represents the median value with 2.5th to 95th percentile values.

Fig. 4.

Effect of SNP on POR mRNA level in human livers. Median mRNA level of the genotype variant of POR2286822C>T was statistically significant (P < 0.05). Data are shown as box plots representing medians with 2.5th to 97.5th percentile values.

Fig. 5.

Effect of SNPs on POR protein content in HLM. Differences in the median protein levels of the different genotype variants of POR 2286822C>T (A), 286823G>A (B), and 3823884A>C (C) were statistically significant (P < 0.05). Data are shown as box plots representing medians with 2.5th to 95th percentile values.

Fig. 6.

Effects of SNPs on POR activity in HLM. Differences in the median activity level of the different genotype variants of POR 2286822C>T (A), 286823G>A (B), 1135612A>G (C), and 1057868C>T (D) were statistically significant (P < 0.05). Data are shown as box plots representing medians with 2.5th to 95th percentile values.

Fig. 7.

Correlation between POR expression and transcriptional factors. (A) Correlation between POR mRNA and HNF4α mRNA levels; (B) correlation between POR mRNA and PXR mRNA levels. The data were determined by qPCR in 107 liver samples and are presented as the means of three independent experiments.