||Human alcohol dehydrogenase (ADH) family and aldehyde dehydrogenase (ALDH) isozymes are two principal enzyme systems responsible for alcohol metabolism in humans. Cimetidine, an H2-receptor antagonist for peptic ulcers treatment, and acetaminophen for antipyretic and analgesic medications, are two widely used over-the-counter drugs. It has been reported that cimetidine or acetaminophen can elevate blood ethanol level after ingestion of alcohol and might affect bioavailability of ethanol by inhibiting gastric ADH. The purpose of this dissertation is to (1) characterize kinetic properties of recombinant human ADH/ALDH isozymes/allozymes, (2) determine inhibition patterns and inhibition constants for ethanol/acetaldehyde oxidation by cimetidine or acetaminophen, (3) quantitatively simulate in vivo inhibition of ethanol/acetaldehyde oxidation by the drug, and (4) model the drug binding to the binary complexes of NAD+ with ADH/ALDH X-ray structure for auxiliary analysis of inhibition pattern. |
The investigations were done at near physiological pH 7.5 and with a cytoplasmic coenzyme concentration of 0.5 mM NAD+. Cimetidine acted as a competitive inhibitor against ethanol oxidation for ADH1A, ADH1B1,ADH1C1, ADH1C2 and ADH4 as well as against acetaldehyde oxidation for ALDH2 and ALDH3A1, but as a noncompetitive inhibitor against the substrate for ADH1B2, ADH1B3, ADH2, ADH3 and ALDH1A1. The inhibition constants of cimetidine for ADH isozymes/allozymes were range of Kis = 0.21−7.0 mM, Kii = 0.27−4.4 mM and that for ALDH isozymes were Kis = 0.84−12 mM, Kii = 1.1 mM. Acetaminophen acted as a noncompetitive inhibitor for all ADH enzymes and ALDH2, with inhibition constants range of Kis = 0.9−20 mM, Kii = 1.4−19 mM for ADHs and Kis = 3.0 mM, Kii = 2.2 mM for ALDH2, but as a competitive inhibitor for ALDH1A1, with Kis = 0.96 mM, and existed no inhibition for ALDH3A1. The metabolic interactions between cimetidine or acetaminophen and ethanol/acetaldehyde were assessed by computer simulation using the inhibition equations and the determined kinetic constants. At physiologically reachable drug level and relevant concentrations of ethanol and acetaldehyde in target tissues, cimetidine (0.2 mM) could inhibit the activities of ADH1B2/3 in the liver, ADH1C1/2 and ADH2 in the liver and small intestine, ADH4 in the stomach, and ALDH3A1 in all three tissues, with inhibition range of 5.1−44%, and acetaminophen (0.5 mM) also could inhibit the activities of ADH1C allozymes (12−26%), ADH2 (14−28%), ADH4 (15−31%), and ALDH1A1 (16−33%) and ALDH2 (8.3−19%) in three tissues. With the assistance of computer molecular modellings showed that the residues of amino acid of ADH2, Y-94 and F-146, and that of ADH4, F-93 and M-140, were the key residues for jostling cimetidine or acetaminophen against ethanol reaction with zinc and nicotinamide ring of NAD+. The results suggest that inhibition by cimetidine or acetaminophen of hepatic and gastrointestinal FPM (first-pass metabolism) of ethanol through ADH and ALDH pathways might become significant at higher, subtoxic levels of the drug and thus ethanol bioavailability may be significantly increased after ingesting both the drug and alcoholic beverages. Elevation of alcohol availability may enhance the potential toxicity and impair the driving performance.