hymecromone and 4-phenylphenol

Fresh rat liver slices were used to demonstrate the glucuronidation of the model substrates 4-methylumbelliferone (MU) and 4-hydroxybiphenyl (HB). Both glucuronidation reactions proved to be more stable than cytochrome P450-dependent monooxygenations. After an incubation time of 48 h there was no decrease in MU glucuronidation rate, whereas HB glucuronidation was stable until 24 h, and then decreased by about 50% until 48 h. The technique of quantitative competitive RT-PCR was used to determine the expression of UDP-glucuronosyltransferase 2B12-mRNA (UGT2B12-mRNA) in precision-cut rat liver slices. Constitutive levels of UGT2B12-mRNA were measurable. Following 24 h culture of rat liver slices in the presence of phenobarbital, the level of UGT2B12-mRNA increased about twofold, which corresponds to the inducibility in vivo. The addition of beta-naphthoflavone had no influence. The results show that precision-cut liver slices are not only suitable for the detection of an in vitro induction of cytochrome P450-mRNAs, which is characterized by high induction factors, but also of poor induction effects, e.g. on UGT2B12-mRNA, provided that the respective mRNA is exactly quantified.

Topics: Animals; Animals, Outbred Strains; Biphenyl Compounds; Enzyme Induction; Gene Expression Regulation, Enzymologic; Glucuronides; Glucuronosyltransferase; Hymecromone; Liver; Male; Microtomy; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The phase I and phase II drug-metabolizing capacity of freshly isolated and cryopreserved parenchymal cells (PC) from human, rat, and mouse liver held in suspension at 37 degrees C for up to 120 min after thawing was compared. Although 7-ethoxycoumarin-O-deethylase activity was strongly reduced in freshly isolated as well as in cryopreserved PC from human, rat, and mouse liver after 120 min, 7-ethoxyresorufin-O-deethylase activity as well as the profile and formation rates of hydroxylated testosterone metabolites in general remained constant throughout the 2-h incubation period in cryopreserved PC from all three species and was similar to that measured in freshly isolated PC. The activity of glutathione S-transferase (GST) and that of UDP-glucuronosyltransferase (UDP-GT) toward 4-methylumbelliferone significantly decreased, whereas the activities of UDP-GT activity toward 4-hydroxybiphenyl and sulfotransferase in cryopreserved human PC were similar to those measured in freshly isolated PC. The activities of GST, UDP-GT toward 4-methylumbelliferone, and sulfotransferase in cryopreserved rat PC showed a significant decrease when compared with the activities in freshly isolated PC. The phase II enzyme activities in cryopreserved mouse PC proved to be far more stable, being similar to the activities of freshly isolated mouse PC at all four time points measured with the exception of GST, which showed a decay from t = 60 min onward. In conclusion, phase I drug-metabolizing enzyme activities in cryopreserved human, rat, and mouse PC are very similar to those of freshly isolated PC, whereas phase II enzyme activities are affected by cryopreservation.

Topics: Animals; Biphenyl Compounds; Cryopreservation; Glucuronosyltransferase; Glutathione Transferase; Humans; Hymecromone; In Vitro Techniques; Liver; Male; Mice; Rats; Rats, Sprague-Dawley

A UDP-glucuronyltransferase isoform glucuronizes phenolic xenobiotics such as 4-nitrophenol, and an isoform glucuronizing 4-hydroxybiphenyl has also been found in rat liver. We purified a UDP-glucuronyltransferase isoform glucuronizing 4-hydroxybiphenyl from bovine liver microsomes by solubilization with 0.7% sodium cholate followed by three column chromatographic separations using DEAE-Toyopearl 650S, UDP-hexanolamine Sepharose 4B, and hydroxyapatite. The purified bovine liver 4-hydroxybiphenyl UDP-glucuronyltransferase (named Bovine 4HBGT) had glucuronidation activities toward 4-hydroxybiphenyl and 4-methylumbelliferone but had little activity toward 4-nitrophenol and 1-naphthol. The apparent molecular mass of Bovine 4HBGT was 54,000 Da on SDS-PAGE, and this was decreased to 50,000 Da by digestion with endo-beta-N-acetylglucosaminidase H. These data suggest that Bovine 4HBGT consists of a 50,000 Da polypeptide and a high mannose type oligosaccharide chain(s) of about 4,000 Da. The NH2-terminal sequence of GT-3 was GKVLVWPVDFSXWINI. These properties of Bovine 4HBGT were very similar to those of rat UDP-glucuronyltransferase glucuronizing xenobiotics. However, the NH2-terminal sequence of Bovine 4HBGT had higher homology with that of rat liver 4-hydroxybiphenyl UDP-glucuronyltransferase than with that of rat liver 4-nitrophenol UDP-glucuronyltransferase.

Topics: Amino Acid Sequence; Animals; Biphenyl Compounds; Catalysis; Cattle; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel; Glucuronosyltransferase; Hymecromone; Male; Microsomes, Liver; Molecular Sequence Data; Naphthols; Nitrophenols; Sequence Homology, Amino Acid; Substrate Specificity

Studies on the induction of non-oxygenative detoxication enzymes in mice by anticarcinogenic thionosulfur compounds have been extended to include hepatic and pulmonary UDP-glucuronosyltransferases. Dietary administration of disulfiram and of bisethylxanthogen to female CD-1 mice enhanced microsomal glucuronidation of 4-methylumbelliferone, a characteristic GT1 substrate, and of 4-hydroxybiphenyl, a GT2 substrate. Latency of the activity toward 4-methylumbelliferone was not affected appreciably. Disulfiram also enhanced glucuronidation of 4-nitrophenol. Diethyldithiocarbamate was ineffective under the conditions used. These thionosulfur compounds caused no significant change in beta-glucuronidase activity measured in homogenates of 7 organs.

Topics: Animals; Biphenyl Compounds; Diet; Disulfides; Disulfiram; Ditiocarb; Female; Glucuronates; Glucuronidase; Glucuronosyltransferase; Hymecromone; Lung; Mice; Microsomes; Microsomes, Liver; Nitrophenols; Thiones

Six hydroxylated substrates were examined as potential inducers of UDP-glucuronosyltransferases towards their conjugation and the conjugation of other 'model' aglycones with glucuronic acid. Time-dependence (4, 14 and 28 days) and dose-dependence of treatments (from 25 mg/kg to 1 g/kg) were examined for some of these compounds. Monoterpenoid alcohols (borneol and terpineol) did not enhance the glucuronidation of the ten substrates tested. p-Hydroxybiphenyl gives typical 'substrate-induction' towards its own conjugation. Eugenol, a previously described inducer of UDP-glucuronosyltransferase activities in mouse, and 4-methylumbelliferone, give a general enhancement of all activities tested, especially those of the '3-methylcholanthrene-inducible' group of aglycones. p-Nitrophenol at low dose (2.5 mg/kg) gives a limited 'polycyclic aromatic hydrocarbon'-like increase.

Topics: Animals; Biotransformation; Biphenyl Compounds; Enzyme Induction; Eugenol; Glucuronosyltransferase; Hymecromone; Male; Microsomes, Liver; Rats; Rats, Inbred Strains; Terpenes; Umbelliferones