The role of CYP3A-CYP2E1 interactions in activation of CYP3A enzymes by chronic alcohol exposure

Abstract

To examine the effect of chronic alcohol exposure on the activity of CYP3A enzymes in human liver, we studied the metabolism of CYP3A-specific substrates 7-benzyloxyquinoline (7-BQ) and ivermectin in 23 preparations of human liver microsomes (HLM) obtained from donors with documented alcohol exposure, from non-drinkers to heavy alcoholics. All HLM samples were characterized for the composition of the cytochrome P450 pool by global proteomics. Our studies revealed a significant increase in the activities of CYP3A enzymes by alcohol exposure. This effect is not associated with CYP3A enzyme levels, which do not correlate with alcohol exposure. Instead, the rates of 7-BQ and ivermectin metabolism correlate with the content of alcohol-inducible CYP2E1. However, this enzyme does not metabolize ivermectin, and its activity with 7-BQ is negligible. A significant increase in the rate of ivermectin demethylation was also observed in CYP3A4-containing Supersomes® and pooled HLM upon incorporation of purified CYP2E1 into their membrane. These results suggest that the reported acceleration of the elimination of drugs metabolized by CYP3A enzymes by alcohol exposure is due to functional effects of the interaction between CYP3A and CYP2E1. To elucidate the potential mechanism of this effect, we studied the formation of CYP2E1-CYP3A4 complexes in CYP3A4-containing Supersomes with co-incorporated CYP2E1 using tag-transfer chemical crosslinking mass spectrometry (CX-MS). These experiments confirmed physical interactions between the proteins and allowed the identification of CYP3A4 residues at the sites of contact. This information was used to build structural models of the CYP2E1-CYP3A4 complex and to propose possible mechanisms for the observed effects.