Mechanisms behind the inhibitory effect of ethanol on the conjugation of morphine in rat hepatocytes.

Liver microsomes were isolated by calcium aggregation, and isolated hepatocytes from male Wistar rats were prepared according to a two-step Ca++-free collagenase perfusion method. With the hepatocytes maximal inhibition of glucuronidation (about 40%) was reached at 10 mM ethanol after incubation at 37 degrees C for 60 min. UDP-glucuronic acid concentration and energy charge in the hepatocytes also did decrease maximally (about 90 and 50%, respectively) and the amount of UDP-glucose was tripled in the presence of 10 mM and higher concentrations of ethanol. The alcohol dehydrogenase inhibitor 4-methylpyrazole abolished ethanol-induced inhibition of morphine glucuronidation in the hepatocytes. Acetaldehyde (250-50 microM) and the pH decrease induced by ethanol did not reduce morphine-3-glucuronide formation by the cells. Cellular uptake of morphine and excretion of morphine metabolites were similar in the absence and presence of ethanol. Ethanol (60 mM) did not affect the glucuronidation of morphine (1.7 mM added) during a 30-min incubation at 37 degrees C with the microsomes (UDP-glucuronic acid, 5 mM). When the concentration of UDP-glucuronic acid in the microsomes was lowered from 1 to 0.1 mM, the decrease in morphine-3-glucuronide formation was similar to that observed in cells. The data indicate that the inhibition by ethanol of morphine glucuronidation was due to decreased levels of UDP-glucuronic acid. The mechanism is likely to be inhibition of UDP-glucose dehydrogenase activity by ethanol from increased intracellular NADH/NAD ratio accompanying ethanol oxidation.