Reduction and glucuronidation of naftazone by human and rat liver microsomes.
Reduction and glucuronidation of the vasoprotectant drug, naftazone, by human and rat liver microsomes and by recombinant UDP-glucuronosyltransferases (UGT) stably expressed in V79 cells were studied. The oxo group was first reduced in the presence of NADPH or NADH, and was subsequently readily glucuronidated on the phenolic moiety leading to a 1 beta-O-glucuronide, as revealed from MS and by proton and 13C-NMR. Glucuronide extracted from the urine of rats treated with the drug presented the same structure. In all enzyme systems tested, NADH was the most efficient electron donor, when compared with NADPH. The reaction was strongly inhibited by quercitrin, a specific inhibitor of carbonyl reductase. Attempts to isolate the reduced intermediate were unsuccessful because of its marked instability. In humans, a large interindividual variation for the formation of glucuronide was observed with microsomes of seven different liver samples (3.98 +/- 3.22 nmol/min.mg). In rat, glucuronidation of reduced naftazone was strongly induced (12-fold) by 3-methylcholanthrene and, to a lesser extent (2.6-fold) by phenobarbital, but was not affected by clofibrate. In addition, liver microsomes from Gunn rats, which present a genetic defect in bilirubin and phenol UGTs could not form glucuronide of reduced naftazone. The drug, after addition of NADH, was a substrate of the human liver recombinant UGT1*6 that presents a strict specificity toward planar phenolic substances, but not that of UGT2B4 and UGT2B1 expressed in V79 fibroblasts. The reducing step by the endogenous V79 cellular reductase was rate-limiting. In conclusion, the powerful inducing effect exerted by 3-methylcholanthrene, the lack of glucuronidation in the Gunn rat and the ability of UGT1*6 encoded by the UGT1 gene to glucuronidate reduced naftazone suggest that, in humans and in the rat, the compound is metabolized by a UGT isoform (UGT1*6 and the rat orthologous form) belonging to family 1, with a restricted specificity toward the drug.
Herber
R
R
Centre du Médicament, Nancy, France.
Hercelin
B
B
Van Cantfort
J
J
De Graeve
J
J
Fournel-Gigleux
S
S
Taguchi
T
T
Magdalou
J
J
D000328:Adult; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004790:Enzyme Induction / Q000187:drug effects; D005260:Female; D005965:Glucuronates / Q000037:antagonists & inhibitors / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D008958:Models, Molecular; D009285:Naphthoquinones / Q000378:metabolism*; D010084:Oxidation-Reduction; D011794:Quercetin / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D005965:Glucuronates; D007527:Isoenzymes; D009285:Naphthoquinones; D011994:Recombinant Proteins; C005272:naftazone; C012526:quercitrin; D011794:Quercetin; D014453:Glucuronosyltransferase