Impact of alcohol consumption on CYP1A2 activity in human liver microsomes studied with a new naphthalene-based fluorogenic substrate.

We introduce 6-methoxy-2-naphthoic acid (MONA) as a novel fluorogenic substrate for the drug-metabolizing CYP1A2 enzyme. Oxidative demethylation of MONA by human liver microsomes resulted in a red shift and a significant increase in its fluorescence. Screening 14 recombinant human cytochrome P450 (P450) enzymes revealed detectable activity in MONA demethylation by CYP1A2 (kcat = 12 ± 2 min-1, KM = 578 ± 106 μM), CYP2A6 (kcat = 0.42 ± 0.09 min-1, KM = 48 ± 15 μM), and CYP1A1 (kcat = 0.19 ± 0.09 min-1, KM = 89 ± 43 μM). The high rate of CYP1A2-catalyzed reaction along with a distinct resolution of its KM value establishes MONA as a selective fluorogenic probe for CYP1A2 activity. MONA was used to investigate the effects of chronic alcohol exposure on CYP1A2 activity using a series of 23 proteomically characterized human liver microsomes from donors with various levels of alcohol consumption. Global kinetic analysis of the set of saturation profiles yielded 2 Michaelis-Menten components with KM values of 11 and 553 μM, whose combinations adequately approximate all profiles in the dataset. The amplitudes (Vmax) of both components showed a marked increase with increasing alcohol exposure. The Vmax of the minor high-affinity component was best correlated with a combination of abundances of CYP2E1, CYP2A6, and NADPH-P450 reductase, suggesting that it reflects the activity of CYP2A6 in the complex with CYP2E1. The Vmax of the predominant CYP1A2-dependent component exhibits a pronounced correlation with the content of NADPH-P450 reductase, whose increased expression in alcohol consumers appears to be the main causative factor in alcohol-induced increase in CYP1A2 activity that takes place despite an alcohol-induced decrease in CYP1A2 expression. SIGNIFICANCE STATEMENT: We introduced 6-methoxy-2-naphthoic acid as a specific fluorogenic substrate for CYP1A2. By studying its metabolism in a series of 23 proteomically characterized human liver microsome samples from donors with various levels of alcohol consumption, we demonstrated a significant increase in CYP1A2 activity with increasing alcohol exposure despite decreased CYP1A2 expression. This increase, which is best correlated with the alcohol-induced increase in the abundance of NADPH-cytochrome P450 reductase, may have a striking effect on the pharmacokinetics of CYP1A2-metabolized antidepressants and antipsychotics in alcoholics.