TY - JOUR T1 - Involvement of Cytochromes P-450 2E1 and 3A4 in the 5-Hydroxylation of Salicylate in Humans JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 322 LP - 326 VL - 27 IS - 3 AU - Isabelle Dupont AU - François Berthou AU - Pierre Bodenez AU - Louis Bardou AU - Caroline Guirriec AU - Nathalie Stephan AU - Yvonne Dreano AU - Danièle Lucas Y1 - 1999/03/01 UR - http://dmd.aspetjournals.org/content/27/3/322.abstract N2 - Hydroxylation of salicylate into 2,3 and 2,5-dihydroxybenzoic acids (2,3-DHBA and 2,5-DHBA) by human liver microsomal preparations was investigated. Kinetic studies demonstrated that salicylate was 5-hydroxylated with two apparent Km: one high-affinity Km of 606 μM and one low-affinity Km greater than 2 mM. Liver microsomes prepared from 15 human samples catalyzed the formation of 2,5-DHBA at metabolic rate of 21.7 ± 8.5 pmol/mg/min. The formation of 2,3-DHBA was not P-450 dependent. Formation of 2,5-DHBA was inhibited by 36 ± 14% following preincubation of microsomes with diethyldithiocarbamate, a mechanism-based selective inhibitor of P-450 2E1. Furthermore, the efficiency of inhibition was significantly correlated with four catalytic activities specific to P-450 2E1, whereas the residual activity was correlated with three P-450 3A4 catalytic activities. Troleandomycin, a mechanism-based inhibitor selective to P-450 3A4, inhibited by 30 ± 12% the 5-hydroxylation of salicylate, and this inhibition was significantly correlated with nifedipine oxidation, specific to P-450 3A4. The capability of seven recombinant human P-450s to hydroxylate salicylate demonstrated that P-450 2E1 and 3A4 contributed to 2,5-DHBA formation in approximately equal proportions. The Kmvalues of recombinant P-450 2E1 and 3A4, 280 and 513 μM, respectively, are in the same range as the high-affinityKm measured with human liver microsomes. The plasmatic metabolic ratio 2,5-DHBA/salicylate, measured 2 h after ingestion of 1 g acetylsalicylate, was increased 3-fold in 12 alcoholic patients at the beginning of their withdrawal period versus 15 control subjects. These results confirm that P-450 2E1, inducible by ethanol, is involved in the 5-hydroxylation of salicylate in humans. Furthermore, this ratio was still increased by 2-fold 1 week after ethanol withdrawal. This finding suggests that P-450 3A4, known to be also inducible by alcoholic beverages, plays an important role in this increase, because P-450 2E1 returned to normal levels in less than 3 days after ethanol withdrawal. Finally, in vivo and in vitro data demonstrated that P-450 2E1 and P-450 3A4, both inducible by alcohols, catalyzed the 5-hydroxylation of salicylate. The American Society for Pharmacology and Experimental Therapeutics ER -