Phenobarbital inducible UDP-glucuronosyltransferase is responsible for glucuronidation of 3'-azido-3'-deoxythymidine: characterization of the enzyme in human and rat liver microsomes.
Glucuronidation by liver microsomes of 3'-azido-3'-deoxythymidine (AZT) was characterized in human and in various animal species. The glucuronide isolated by HPLC, was identified by mass spectrometry (fast atom bombardment, desorption in chemical ionization), and beta-glucuronidase hydrolysis. AZT glucuronidation reaction in liver microsomes of human and monkey proceeded similarly with an apparent Vmax of 0.98 nmol/min/mg protein and apparent Km of 13 mM. Oleoyl lysophosphatidylcholine activated more than twofold the formation of the glucuronide. Human kidney microsomes could also biosynthesize AZT glucuronide, although to a lower extent (six times less than the corresponding liver). Probenecid, which is administered to AIDS patients, decreased hepatic AZT glucuronidation in vitro (I50 = 1.5 mM), whereas paracetamol did not exert any effect at concentrations up to 21.5 mM. Morphine also inhibited the reaction (I50 = 2.7 mM). AZT glucuronidation presented the highest rate in human and in monkey (0.50 nmol/min/mg protein); pig and rat glucuronidated the drug two and three times less, respectively. In Gunn rat, the specific activity in liver microsomes was similar (0.18 nmol/min/mg protein) to that of the congenic normal strain; this suggests that an isozyme other than bilirubin UDP-glucuronosyltransferase catalyzed the reaction. In rats, AZT glucuronidation was stimulated fourfold by phenobarbital; 3-methylcholanthrene or clofibrate failed to increase this activity. This result was consistent with the bulkiness of the AZT molecule (thickness 6.7 A), which is a critical structural factor for glucuronidation of the drug by phenobarbital-induced isozymes. Altogether, the results strongly indicate that UDP-glucuronosyltransferase (phenobarbital inducible forms) is responsible for AZT glucuronidation.
Haumont
M
M
Centre du Médicament, U.R.A. CNRS n. 597, Faculté des Sciences Pharmaceutiques et Biologiques, Nancy, France.
Magdalou
J
J
Lafaurie
C
C
Ziegler
J M
JM
Siest
G
G
Colin
J N
JN
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005965:Glucuronates / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D007700:Kinetics; D008253:Macaca mulatta; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011911:Rats, Gunn; D011919:Rats, Inbred Strains; D013045:Species Specificity; D013552:Swine; D015215:Zidovudine / Q000378:metabolism*
D005965:Glucuronates; D007527:Isoenzymes; D015215:Zidovudine; D014453:Glucuronosyltransferase; D010634:Phenobarbital