Vol. 27, Issue 3, 322-326, March 1999

Involvement of Cytochromes P-450 2E1 and 3A4 in the 5-Hydroxylation of Salicylate in Humans

Isabelle Dupont, François Berthou, Pierre Bodenez, Louis Bardou, Caroline Guirriec, Nathalie Stephan, Yvonne Dreano, and Danièle Lucas

Laboratoire de biochimie-EA-948 Faculté de Médecine, BREST Cedex, France

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 K_m: one high-affinity K_m of 606 µM and one low-affinity K_m 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 K_m values of recombinant P-450 2E1 and 3A4, 280 and 513 µM, respectively, are in the same range as the high-affinity K_m 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.