| Three UDP-glucuronosyltransferases (UGTs) isolated from female rhesus monkey liver tissue have been cloned, sequenced, expressed and characterized. The first isoform, rhesus UGT1A01, is greater than 99 and 95% identical to cynomolgus monkey UGT1A01 and human UGT1A1, respectively. Rhesus UGT1A01 catalyzed the glucuronidation of beta-estradiol (3-glucuronide), 1-naphthol, 17alpha-ethinylestradiol (3-glucuronide) and bilirubin. The second isoform, UGT2B9*2, shares greater than 99, 93 and 88% amino acid sequence identity to the cynomolgus monkey UGT2B9, cynomolgus UGT2B23 and human UGT2B7, respectively. UGT2B9*2 catalyzed the glucuronidation of beta-estradiol (17-glucuronide), estriol (17-glucuronide), 17alpha-ethinylestradiol (3 and 17-glucuronide), 4-hydroxyestrone, 16-epiestriol, hyodeoxycholic acid, morphine (3-glucuronide), and naloxone (3-glucuronide). The third isoform, UGT2B33, shares 96, 93 and 88% amino acid sequence identity with cynomolgus monkey UGT2B23, cynomolgus UGT2B9 and human UGT2B7, respectively. UGTG2B33 catalyzed the glucuronidation of estriol (17-glucuronide), 4-hydroxyestrone, androsterone, diclofenac, hyodeoxycholic acid, morphine (3-glucuronide), naloxone (3-glucuronide) and naltrexone (3-glucuronide). Comparison of kinetic parameters for the three UGTs to kinetic parameters for liver microsomes suggests the suitability of beta-estradiol-3-glucuronidation, beta-estradiol-17-glucuronidation and estriol-17-glucuronidation for assaying UGT1A01, UGT2B9*2 and UGT2B33 activity, respectively, in rhesus liver microsomes.; During analysis of rhesus urine, enterolactone (ENL), a mammalian lignan, was detected as two O-glucuronide conjugates. In vitro incubations of ENL with rhesus liver microsomes suggested extensive metabolism with NADPH- and/or UDPGA-supplemented samples. NADPH-dependent metabolism resulted in the formation of multiple monohydroxylated products. Addition of a nucleophilic trapping agent (N-acetylcysteine, NAC) resulted in the formation of a new metabolite with a corresponding m/z 474 in the negative ion mode using LC-MS. Purification and NMR analysis of this metabolite suggests bioactivation of ENL via catechol formation, followed by further oxidation to a reactive quinone and Michael addition of NAC. Glucuronidation of ENL was more efficient than the NADPH-dependent pathway in vitro, and resulted in formation of both ENL O-glucuronides. Incubations with NADPH, NAC and UDPGA resulted only in formation of the two O-glucuronides with no detectable NAC adduct. ENL appears to be excreted mainly as glucuronide conjugates in rhesus urine, rhesus bile and human urine. In human bile, ENL appears to be excreted as the aglycone. However, based on peak area measurements, biliary excretion of ENL and its glucuronide appears to be a minor excretory pathway. Overall, the high efficiency and specificity for glucuronidation of ENL in vivo may decrease its potential toxicity via CYP450 bioactivation. |
