Involvement of cytochromes P-450 2E1 and 3A4 in the 5-hydroxylation of salicylate in humans

Hydroxylation of salicylate into 2,3 and 2,5-dihydroxybenzoic acids (2,3-DH BA and 2,5-DHBA) by human liver microsomal preparations was investigated. K inetic studies demonstrated that salicylate was 5-hydroxylated with two app arent K-m: one high-affinity K-m of 606 mu M and one low-affinity K-m great er 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 f ormation of 2,3-DHBA was not P-450 dependent. Formation of 2,5-DHBA was inh ibited by 36 +/- 14% following preincubation of microsomes with diethyldith iocarbamate, a mechanism-based selective inhibitor of P-450 2E1. Furthermor e, the efficiency of inhibition was significantly correlated with four cata lytic activities specific to P-450 2E1, whereas the residual activity was c orrelated with three P-450 3A4 catalytic activities. Troleandomycin, a mech anism-based inhibitor selective to P-450 3A4, inhibited by 30 +/- 12% the 5 -hydroxylation of salicylate, and this inhibition was significantly correla ted with nifedipine oxidation, specific to P-450 3A4, The capability of sev en recombinant human P-450s to hydroxylate salicylate demonstrated that P-4 50 2E1 and 3A4 contributed to 2,5-DHBA formation in approximately equal pro portions. The K-m values of recombinant P-450 2E1 and 3A4, 280 and 513 mu M , respectively, are in the same range as the high-affinity K-m measured wit h human liver microsomes. The plasmatic metabolic ratio 2,5-DHBA/salicylate , measured 2 h after ingestion of 1 g acetylsalicylate ate, 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. Fu rthermore, this ratio was still increased by 2-fold 1 week after ethanol wi thdrawal. 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 withdr awal. 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 salic ylate.