A novel biologically active seleno-organic compound--I. Glutathione peroxidase-like activity in vitro and antioxidant capacity of PZ 51 (Ebselen).

UDP-Glucuronosyltransferases (UGTs) are glycoproteins localized in the endoplasmic reticulum (ER) which catalyze the conjugation of a broad variety of lipophilic aglycon substrates with glucuronic acid using UDP-glucuronic acid (UDP-GIcUA) as the sugar donor. Glucuronidation is a major factor in the elimination of lipophilic compounds from the body. In this review, current information on the substrate specificities of UGT1A and 2B family isoforms is discussed. Recent findings with regard to UGT structure and topology are presented, including a dynamic topological model of UGTs in the ER. Evidence from experiments on UGT interactions with inhibitors directed at specific amino acids, photoaffinity labeling, and analysis of amino acid alignments suggest that UDP-GIcUA interacts with residues in both the N- and C-terminal domains, whereas aglycon binding sites are localized in the N-terminal domain. The amino acids identified so far as crucial for substrate binding and catalysis are arginine, lysine, histidine, proline, and residues containing carboxylic acid. Site-directed mutagenesis experiments are critical for unambiguous identification of the active-site architecture.

Structural and functional studies of udp-glucuronosyltransferases*

The glucuronidation kinetics of the prototypic substrates 4-methylumbelliferone (4MU) and 1-naphthol (1NP) by human UDP-glucuronosyltransferases (UGT) 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17 were investigated. Where activity was demonstrated, inhibitory effects of diclofenac, probenecid, and the solvents acetone, acetonitrile, dimethyl sulfoxide, ethanol, and methanol were characterized. All isoforms except UGT1A4 glucuronidated 4MU, whereas all but UGT 1A4, 2B15, and 2B17 metabolized 1NP. However, kinetic models varied with substrate (for the same isoform) and from isoform to isoform (with the same substrate). Hyperbolic (Michaelis-Menten), substrate inhibition, and sigmoidal kinetics were variably observed for both 4MU and 1NP glucuronidation by the various UGTs. K(m) or S(50) (sigmoidal kinetics) and V(max) values varied 525- (8-4204 microM) and 1386-fold, respectively, for 4MU glucuronidation, and 1360- (1.3-1768 microM) and 37-fold, respectively, for 1NP glucuronidation. The use of a two-site model proved useful for those reactions exhibiting non-Michaelis-Menten glucuronidation kinetics. The organic solvents generally had a relatively minor effect on UGT isoform activity. UGT 2B15 and 2B17 were most susceptible to the presence of solvent, although solvent-selective inhibition was occasionally observed with other isoforms. Diclofenac and probenecid inhibited all isoforms, precluding the use of these compounds for the reaction phenotyping of xenobiotic glucuronidation pathways in human tissues. Diclofenac and probenecid K(i) values, determined for selected isoforms, ranged from 11 to 52 microM and 96 to 2452 microM, respectively. Overall, the results emphasize the need for the careful design and interpretation of kinetic and inhibition studies with human UGTs.

Human udp-glucuronosyltransferases: isoform selectivity and kinetics of 4-methylumbelliferone and 1-naphthol glucuronidation, effects of organic solvents, and inhibition by diclofenac and probenecid

Biotransformation of xenobiotics in the fetus

The use of primary hepatocytes is now well established for both studies of drug metabolism and enzyme induction. Cryopreservation of primary hepatocytes decreases the need for fresh liver tissue. This is especially important for research with human hepatocytes because availability of human liver tissue is limited. In this review, we summarize our research on optimization and validation of cryopreservation techniques. The critical elements for successful cryopreservation of hepatocytes are (1) the freezing protocol, (2) the concentration of the cryoprotectant [10% dimethyl-sulfoxide (DMSO)], (3) slow addition and removal of DMSO, (4) carbogen equilibration during isolation of hepatocytes and before cryopreservation, and (5) removal of unvital hepatocytes by Percoll centrifugation after thawing. Hepatocytes of human, monkey, dog, rat, and mouse isolated and cryopreserved by our standard procedure have a viability > or = 80%. Metabolic capacity of cryopreserved hepatocytes determined by testosterone hydroxylation, 7-ethoxyresorufin-O-de-ethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), glutathione S-transferase, UDP-glucuronosyl transferase, sulfotransferase, and epoxide hydrolase activities is > or = 60% of freshly isolated cells. Cryopreserved hepatocytes in suspension were successfully applied in short-term metabolism studies and as a metabolizing system in mutagenicity investigations. For instance, the complex pattern of benzo[a]pyrene metabolites including phase II metabolites formed by freshly isolated and cryopreserved hepatocytes was almost identical. For the study of enzyme induction, a longer time period and therefore cryopreserved hepatocyte cultures are required. We present a technique with cryopreserved hepatocytes that allows the induction of testosterone metabolism with similar induction factors as for fresh cultures. However, enzyme activities of induced hepatocytes and solvent controls were smaller in the cryopreserved cells. In conclusion, cryopreserved hepatocytes held in suspension can be recommended for short-term metabolism or toxicity studies. Systems with cryopreserved hepatocyte cultures that could be applied for studies of enzyme induction are already in a state allowing practical application, but may be further optimized.

Cryopreserved primary hepatocytes as a constantly available in vitro model for the evaluation of human and animal drug metabolism and enzyme induction.

Udp-glucuronosyltransferase activities - guidelines for consistent interim terminology and assay conditions

Commentary: Control of UDP-glucuronyltransferase activity.

Evidence that D-glucaro-1,4-lactone shortens the pharmacological action of drugs being disposed via the bile as glucuronides.

1. The thio-beta-d-glucosiduronic acids (thio-beta-glucuronides) of o-aminothiophenol, diethyldithiocarbamic acid, p-nitrothiophenol and thiophenol are formed biosynthetically in broken- and intact-cell preparations of mouse liver. 2. For this biosynthesis to occur in homogenates or microsomal fractions, UDP-glucuronic acid was required during incubation; glucose, glucuronic acid or UDP could not replace it. UDP was a product of the reaction. 3. The biosynthetic mechanism linking glucuronic acid to thiol and carbodithioic groups therefore requires UDP-glucuronyltransferase activity and resembles that forming the various types of O-glucuronides. 4. An analogous enzymic mechanism employing UDP-glucose synthesizes the thio-beta-d-glucosides of diethyldithiocarbamic acid and thiophenol in gut preparations of the mollusc Arion ater; this mechanism resembles that forming the O-glucosides. The thio-beta-d-glucosides are formed also in intact cells. 5. As expected from the distribution of O-glycosides, S-glucuronides of these aglycones were not detectable with the invertebrate, nor were the S-glucosides with the vertebrate. 6. Despite their similar biosyntheses, S- and O-beta-glycosides differ in susceptibility to hydrolysis by beta-glycosidases. Rat preputial-gland beta-glucuronidase hydrolysed thioglucuronides of o-aminothiophenol, diethyldithiocarbamic acid and p-nitrothiophenol, hydrolysis being inhibited by glucarolactone; the thioglucuronide of thiophenol was not hydrolysed by preputial-gland or liver beta-glucuronidase. The two S-glucosides resisted hydrolysis by beta-glucosidase from almond emulsin.

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G. J. Dutton

Papers

Udp-glucuronosyltransferase activities - guidelines for consistent interim terminology and assay conditions

Commentary: Control of UDP-glucuronyltransferase activity.

Evidence that D-glucaro-1,4-lactone shortens the pharmacological action of drugs being disposed via the bile as glucuronides.

Mechanism of biosynthesis of thio- -D-glucuronides and thio- -D-glucosides.

Comparison between o-aminophenol glucuronidation in liver slices and homogenates from control and phenobarbital-treated Wistar and Gunn rats