Effects of alcohol consumption and tobacco smoking on the composition of the ensemble of drug metabolizing enzymes and transporters in human liver

Abstract

We examined the effect of alcohol consumption and smoking on the abundance of drug-metabolizing enzymes and transporters (DMET) in human liver microsomes (HLM) isolated from liver tissues of 94 donors. Global proteomics analysis was performed and DMET protein levels were analyzed in relation to alcohol consumption levels, smoking history, and sex using non-parametric tests (p-value ≤ 0.05; cutoff of 1.25-fold change, FC). The examination of the alcohol-induced changes was further enforced by correlational analysis, where we used arbitrary alcohol consumption grade (ACG) scaling from 0 to 4 to establish a set of protein markers. We elaborated a provisional index of alcohol exposure (PIAE) based on a combination of relative abundances of four proteins (ER chaperone HSPA5, protein disulfide isomerases PDIA3 and P4HB, and cocaine esterase CES2) best correlating with ACG. The PIAE index was then used to find its correlations with the abundances of DMET proteins. Our results demonstrate considerable alcohol-induced changes in composition of the pool of cytochrome P450 enzymes in HLM. We observed significantly increased abundances of CYP2E1, CYP2B6, CYP2J2, and NADPH-cytochrome P450 reductase. In contrast, CYP1A2, CYP2C8, CYP2C9, CYP4A11, and cytochrome b₅ protein levels were downregulated. Significant alteration in abundances of UDP-glucuronosyltransferase (UGT) were also detected, comprising of elevated UGT1A6, UGT1A9, and UGT2A1, and reduced UGT1A3, UGT1A4, UGT2B7, UGT2B10, and UGT2B15 levels. Important alcohol-induced changes were also observed in the expression of non-CYP and non-UGT DMET. Additionally, tobacco smoke was associated with elevated CYP1A2, UGT1A6, UGT2A1, and UGT2B4 and decreased FMO3, FMO4, and FMO5 levels.

Keywords: alcohol exposure; cytochromes P450; drug metabolism; human liver microsomes; liver proteome; tobacco smoking.

Fig. 1. Global proteomics analysis of human liver microsomes prepared from 94 individuals with known alcohol intake histories.

A) Mass spectrometry-based workflow scheme of human liver microsomes (N=94, 33 females and 61 males). B) HLM specimens from individuals in each alcohol and smoking history category. C) Venn Diagram of proteins detected and overlapping in non-drinkers vs heavy drinkers and individuals who smoked > 1ppd vs non-smokers. D) Differentially expressed proteins in HLM derived from individuals with heavy alcohol intake compared to non-drinking individuals (P<0.05, Fold change cut off of 2.0. E) Differentially expressed proteins in HLM derived from individuals who smoked > 1ppd compared to non-smoking individuals (P<0.05, Fold change cut off of 1.5. F) Heat map of all DMET proteins resulting from label-free quantitative proteomic analysis of individual-derived HLM, based on the normalized mean protein levels for each DMET across the alcohol use categories. Proteins with significantly different levels of expression in the HLM from heavy drinkers as compared to non-drinkers are indicated with an asterisk (P value < 0.05). G) Differential expression of DMET proteins in individual-derived HLM from heavy drinkers as compared to non-drinkers. (P value <0.05, fold-change cutoff of 1.25).

Fig. 2. Differential DMET protein expression with heavy alcohol intake.

A) Protein levels of major drug metabolizing CYPs in HLM from heavy drinkers compared to non-drinkers. B) UGT protein levels in HLM from heavy drinkers compared to non-drinkers. C) Protein levels of major non-CYP, non-UGT enzymes in HLM of individuals with heavy alcohol intake compared to non-drinkers. D) Transporter protein levels in HLM of heavy drinkers compared to non-drinkers. Samples were analyzed for significance using the student t-test with Welch’s correction for unequal variance. P Value *<0.05, **<0.01, ***<0.001, ****<0.0001. Absolute protein concentration for each DMET protein, expressed as pmol/mg of total HLM protein, was calculated via the TPA method.

Fig 3. Shifts in overall composition of proteins related to drug metabolism and disposition with excessive alcohol intake.

A) CYP protein composition in HLM from heavy alcohol drinkers compared to non-drinkers. B) UGT protein composition in HLM of heavy drinkers as compared to non-drinkers C) Non-CYP, non-UGT enzyme composition in HLM of heavy drinkers compared to HLM from non-drinkers. D) Composition of transporters measured in HLM of heavy alcohol drinkers as compared to that of non-drinkers. Absolute protein concentration for each protein was calculated using the TPA method and samples were analyzed for significance using the student t-test with Welch’s correction for unequal variance; * P value <0.05 indicates significant changes in protein levels were observed in the HLM of individuals with heavy alcohol intake as compared to that of the non-drinking control group.

Fig 4. Approximation of the apparent alcohol exposure grade (ACG) with the provisional index of alcohol exposure (PIAE).

The left panel shows the plots of the grade of alcohol exposure (circles) and the index of alcohol exposure (solid line) for all 94 HLM samples sorted by increasing the relative abundance of HSPA5 (GRP78). The right panel shows the same data as a plot of ACG versus PIAE.

Fig. 5. Differential DMET protein expression with moderate to heavy tobacco use.

A) Protein levels of major drug metabolizing CYPs in HLM from >1 ppd smokers compared to non-smokers. B) UGT protein levels in HLM from >1 ppd smokers compared to non-smokers. C) Protein levels of major non-CYP, non-UGT enzymes in HLM of >1 ppd smokers compared to non-smokers. D) Transporter protein levels in HLM of >1 ppd smokers compared to non-smokers. Samples were analyzed for significance using the student t-test with Welch’s correction for unequal variance. P Value *<0.05, **<0.01. Absolute protein concentration for each DMET protein, expressed as pmol/mg of total HLM protein, was calculated via the TPA method.

Fig. 6. Differential DMET protein expression with sex.

A) Protein levels of major drug metabolizing CYPs in HLM from female donors compared to males. B) UGT protein levels in HLM from female compared to male donors. C) Protein levels of major non-CYP, non-UGT enzymes in HLM female donors compared to males. D) Transporter protein levels in HLM females compared to males. Samples were analyzed for significance using the student t-test with Welch’s correction for unequal variance. P Value *<0.05, **<0.01, ***<0.001, ****<0.0001. Absolute protein concentration for each DMET protein, expressed as pmol/mg of total HLM protein, was calculated via the TPA method.