Multifold Activation of CYP3A4 Caused by Alcohol-Induced Increase in the Content of CYP2E1 in Human Liver Microsomes

Abstract

Here we investigate the effect of alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. In these studies we used a model that implements enrichment of HLM samples with CYP2E1 through membrane incorporation of the purified protein. Enrichment of HLM with CYP2E1 considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. Incorporation of CYP2E1 also eliminates the activating effect of -Naphthoflavone (ANF) on BQ metabolism seen in some untreated HLM samples. To probe the physiological relevance of these effects we compared three pooled preparations of HLM from normal donors (HLM-N) with a preparation obtained from heavy alcohol consumers (HLM-A). The composition of the P450 pool in all four samples was characterized with mass-spectrometric determination of 11 cytochrome P450 species. The molar content of CYP2E1 in HLM-A was from 2.5 to 3.3 times higher than that found in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all four HLM samples. Despite of that, HLM-A exhibited much higher rate of metabolism and lower degree of homotropic cooperativity with BQ, similar to that observed in CYP2E1-enriched HLM-N. In order to substantiate the hypothesis on the involvement of physical interactions between CYP2E1 and CYP3A4 in the observed effects we probed hetero-association of these proteins in Supersomes containing recombinant CYP3A4 with a technique based on homo-FRET and employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments demonstrated high affinity interactions between the two enzymes and revealed an inhibitory effect of ANF on their hetero-association. Our results demonstrate that the catalytic activity and allosteric properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and imply a profound impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4.