Rat Liver Microsomal UDP-glucuronosyltransferase Research Articles

ATP Serves as an Endogenous Inhibitor of UDP-Glucuronosyltransferase (UGT): A New Insight into the Latency of UGT

We have suggested that adenine-related compounds are allosteric inhibitors of UGT in rat liver microsomes (RLM) treated with detergent. To clarify whether the same occurs with a pore-forming peptide, alamethicin, the effects of adenine-related compounds on 4-metylumbelliferone (4-MU) glucuronidation were examined using RLM and human liver microsomes (HLM). ATP inhibited 4-MU glucuronidation when polyoxyethylene cetyl alcohol ether (Brij-58)-treated RLM were used (IC(50) = approximately 70 μM). However, alamethicin-treated RLM exhibited a lower susceptibility (IC(50) = approximately 460 μM) than Brij-58-treated RLM. A similar phenomenon was observed when pooled HLM were used. Then, the endogenous ATP content of RLM was determined in the presence and absence of alamethicin or detergent, and although no ATP remained in the microsomal pellets after Brij-58 treatment, more than half of the microsomal ATP remained even after treatment with alamethicin. Furthermore, the V(max) in the absence of an adenine-related compound was approximately three times higher in Brij-58-treated than in alamethicin-treated RLM. The difference in the inhibitory potency observed was due to the difference in remaining endogenous ATP and the accessibility of exogenous ATP to the luminal side of the endoplasmic reticulum (ER), where the active site of UDP-glucuronosyltransferase (UGT) is located. Gefitinib (Iressa), a protein tyrosine kinase inhibitor, markedly inhibited human UGT1A9 activity. It is interesting to note that AMP antagonized Gefitinib-provoked inhibition of UGT1A9, and ATP exhibited an additive inhibitory effect at a lower concentration. Therefore, Gefitinib inhibits UGT1A9 at the common ATP-binding site shared with ATP and AMP. Releasing adenine nucleotide from the ER is suggested to be one of the mechanisms that explain the "latency" of UGT.

Effect of s-triazine and phenoxyalkanoic acid herbicides on UDP-glucuronosyltransferase in rat liver microsomes

Effect of s-triazine and phenoxyalkanoic acid herbicides on UDP-glucuronosyltransferase in rat liver microsomes

Effect ofs-triazine and phenoxyalkanoic acid herbicides on UDP-glucuronosyltransferase in rat liver microsomes

Twelve herbicides, representatives of two chemical groups, substituted phenoxyalkanoic acids and s-triazines, were tested for their inhibitory effect on the glucuronidation of 4-nitrophenol (4-NP), phenolphthalein (PPh) and 4-methylumbelliferone (4-MU) by rat liver microsomes. One millimole MCPA, ametryn and cyanazine significantly decreased PPh UDP-glucuronosyltransferase (UGT) activity, while propazine was found to be a most potent inhibitor of 4-NP glucuronidation. Concentrations of 0.1 mM dichlorprop and cyanazine were still inhibitory against PPh-UGT. The inhibition of 4-MU glucuronidation by the herbicides was low and not specific. As a whole, s-triazine derivatives were more inhibitory than the substituted phenoxyalkanoic acids. Kinetic studies with propazine revealed a non-competitive type of inhibition towards the acceptor substrate 4-NP, with an apparent Ki value of 0.540 mM. With ametryn, an uncompetitive type of inhibition against PPh and a mixed type of inhibition towards UDPGA were found, with apparent Ki values of 0.330 mM and 0.380 mM, respectively. © 1999 Society of Chemical Industry

Application of Photoaffinity Labeling with [11,12-3H]All-trans-Retinoic Acid to Characterization of Rat Liver Microsomal UDP-Glucuronosyltransferase(s) with Activity toward Retinoic Acid

[3H]All-trans-retinoic acid has been shown to be an effective photoaffinity label for microsomal UDP-glucuronosyltransferases. Labeling of rat liver microsomal proteins with [3H]all-trans-retinoic acid and [32P]5-azido-UDP-glucuronic acid has shown that at least one protein in the 50-56 kDa mass range encompassing the UDP-glucuronosyltransferases photoincorporated both probes. The fraction of solubilized microsomal protein eluted from a UDP-hexanolamine affinity column with 50 μM UDP-glucuronic acid contained two protein bands, both of which photoincorporated [3H]all-trans-retinoic acid and were detected on Western blot by anti-UDP-glucuronosyltransferase antibodies. Enzymatic glucuronidation activity toward atRA in the same fraction was enriched five-fold over that of native or solubilized microsomes.

Influence of Pesticides on UDP-glucuronosyltransferase in Rat Liver Microsomes

Fifteen pesticides, representatives of different chemical groups, were tested for their inhibitory effect on the glucuronidation of 4-nitrophenol (4-NP) and phenolphthalein (PPh) by rat liver microsomes. Three herbicides (simazine, chlorsulfuron, tribenuron-methyl), two insecticides (dioxacarb, carbaryl) and one fungicide (zineb) significantly decreased the UDP-glucuronosyltransferase (UDPGT) activity. The carbamate insecticide dioxacarb was found to be the most potent inhibitor, at 1 mM concentration suppressing 4-NP-UDPGT activity completely, and reducing by 55% the activity associated with the conjugation of PPh. One millimole simazine and carbaryl affected only 4-NP glucuronidation, while chlorsulfuron and zineb exerted a marked inhibition of both 4-NP and PPh conversion. Concentrations of 0.1 mM carbaryl, dioxacarb and zineb were still inhibitory against 4-NP-UDPGT, with zineb producing 40% inhibition of PPh glucuronidation. As a whole, UDPGT isoforms conjugating PPh were less sensitive to the agrochemicals tested. Kinetic studies with dioxacarb, chlorsulfuron and carbaryl revealed a mixed type of inhibition with respect to the acceptor substrate 4-NP, with apparent K i values of 70 μM, 120 μM and 160 μM, respectively.

Rat liver microsomal UDP-glucuronosyltransferase activity toward thyroxine: Characterization, induction, and form specificity

Glucuronidation of thyroxine (T 4) by liver microsomal UDP-glucuronosyltransferase (UDP-GT) is a predominant pathway by which T 4 is deactivated. This study was conducted to characterize in vitro T 4 UDP-GT activity in rat liver microsomal preparations, to determine if T 4 glucuronidation is mediated by a particular form of UDP-GT, and to determine if T 4 glucuronidation can be increased by microsomal enzyme inducers. Characterization of microsomal T 4 UDP-GT activity led to the establishment of optimal assay conditions. UDP-GT activity toward T 4 was determined in hepatic microsomal preparations from Wistar and Gunn rats, a mutant strain of Wistar rats deficient in several forms of UDP-GT. Hepatic microsomal preparations from Gunn rats glucuronidated T 4 at one-third the rate catalyzed by microsomal preparations from Wistar rats. To determine the effect of four inducers that each increase a separate class of UDP-GT, phenobarbital (PB), 3-methylcholanthrene (3MC), pregnenolone-16α-carbonitrile (PCN), clofibrate (CLO), saline, or corn oil was administered to male Sprague-Dawley rats ip for 4 days. T 4 UDP-GT activity was increased by PB, 3MC, PCN, and CLO 88, 150, 100, and 160%, respectively on a per-milligram-microsomal-protein basis and 138, 125, 100, and 145% on a per-kilogram-body-weight basis, respectively. Therefore, all four classes of UDP-GT inducers increase T 4 glucuronidation, suggesting that T 4 is not a selective substrate for a particular form of UDP-GT.

Glucuronide formation of various drugs in liver microsomes and in isolated hepatocytes from phenobarbital- and 3- methylcholanthrene-treated rats

Various substrates of rat liver microsomal UDP-glucuronosyltransferase were classified in vitro as preferred substrates of either 3-methylcholanthrene- or phenobarbital-inducible enzyme forms. Microsomal UDP-glucuronosyltransferase activities towards a third group of substrates (including oestrone, phenolphthalein, paracetamol and oxazepam) are not markedly altered by treatment with either 3-methylcholanthrene or phenobarbital. Some substrates of the 3-methylcholanthrene- and phenobarbital-inducible enzyme activities were selected to evaluate the importance of multiple enzyme forms for glucuronide formation in the intact cell. The metabolism of these compounds was compared in isolated hepatocytes from untreated controls and from rats treated with 3-methylcholanthrene (MC-hepatocytes) or phenobarbital (PB-hepatocytes). Glucuronidation of 1-naphthol and 3-hydroxy-benzo[ a]pyrene was chiefly enhanced in MC-hepatocytes (> 2-fold), whereas glucuronidation of chloramphenicol and bilirubin was chiefly enhanced in PB-hepatocytes. These observations are in agreement with differential induction of UDP-glucuronosyltransferase activities in vitro suggesting that, besides other factors such as cofactor supply, physiological activators, etc., the levels of the multiple enzyme forms are critically determining glucuronide formation in the intact cell.

A rapid and sensitive method for the assay of UDP-glucuronosyltransferase activity toward bile acids

A rapid and sensitive procedure is described for the assay of rat liver microsomal UDP-glucuronosyltransferase activity toward the bile acids chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and lithocholic acid using the radioactively labeled bile acids as substrates. The unreacted bile acids were separated from the bile acid glucuronides formed as products of the enzymatic reactions by extraction with chloroform, leaving the bile acid glucuronides in the aqueous phases. The bile acid glucuronides were characterized by their mobilities in thin-layer chromatography and identified by their sensitivity to hydrolysis with β-glucuronidase and inhibition of hydrolysis by the specific β-glucuronidase inhibitor d-saccharic acid-1,4-lactone. Enzyme activities were optimal at pH 6.8 and were maximally stimulated about fourfold by the addition of the nonionic detergent Brij 58 at a concentration of 0.3 mg/mg microsomal protein. The kinetic parameters for the various bile acids as substrates were determined.

Electroimmunochemical quantification of UDP-glucuronosyltransferase in rat liver microsomes.

Microsomal UDPglucuronosyltransferase(1-naphthol), an enzyme form previously shown to be selectively inducible in rat liver by 3-methylcholanthrene-type inducers, was purified to apparent homogeneity. Rabbit antibodies against this enzyme form precipitated UDPglucuronosyltransferase activities towards 1-naphthol and 4-methylumbelliferone faster and to greater extents than enzyme activities towards bilirubin, oestrone and 4-hydroxybiphenyl. Ouchterlony double-diffusion analysis showed immunochemical similarity of the rat liver enzyme with the enzymes from other organs of the rat (kidney, testes) and the mouse liver but not with the enzyme from cat and human liver. Electroimmunochemical quantification of the enzyme indicated that its level was enhanced 1.3-fold and 2.5-fold in liver microsomes from phenobarbital-treated and 3-methylcholanthrene-treated rats, respectively. The results indicate that 3-methylcholanthrene treatment increases the enzyme level of rat liver microsomal UDPglucuronosyltransferase(1-naphthol). Despite phospholipid-dependence of its catalytic activity microsomal enzyme activity appears to be a good index of the enzyme level.

Isolation and characterization of rat liver microsomal UDP-glucuronosyltransferase activity toward chenodeoxycholic acid and testosterone as a single form of enzyme.

Microsomal UDP-glucuronosyltransferase activity toward chenodeoxycholic acid and testosterone has been isolated from rat liver and appears to be homogeneous in sodium dodecyl sulfate gel electrophoresis and polyacrylamide gradient gel electrophoresis. The conjugating activities toward chenodeoxycholic acid and testosterone co-purified and showed identical mobilities in disc gel electrophoresis, indicating that chenodeoxycholic acid and testosterone are glucuronidated by a single form of enzyme. UDP-glucuronosyltransferase activities toward estrone, bilirubin, 4-nitrophenol, and morphine did not co-purify with the activity toward chenodeoxycholic acid and testosterone and were not detectable in the pure enzyme in the presence or absence of phospholipids. In addition to glucuronic acid conjugation, the enzyme is able to catalyze galacturonic acid conjugation of chenodeoxycholic acid and testosterone. The enzyme has a subunit Mr approximately 54,000 and in the presence of the stabilizing detergent chenodeoxycholic acid, it appears to exist as an aggregate with an apparent Mr = 318,000 as estimated by gel filtration and 316,000 by polyacrylamide gradient gel electrophoresis.

Differential induction of rat liver microsomal UDP-glucuronosyltransferase activities by various inducing agents

The selectivity of various inducers of UDP-glucuronosyltransferase was investigated in rat liver microsomes and compared with their effect on monooxygenase reactions. (1) Similar to 3-methylcholanthrene β-naphthoflavone selectively stimulated the glucuronidation of 1-naphthol and 4-methylumbelliferone (GT 1 substrates). (2) In contrast, DDT preferentially enhanced the glucuronidation of morphine, 4-hydroxybiphenyl (GT 2 substrates) and bilirubin, similar to phenobarbital. (3) Clofibric acid and bezafibrate selectively enhanced bilirubin glucuronidation without affecting GT 1 and GT 2 reactions. (4) Similar to ethoxyquin and Aroclor 1254, trans-stilbene oxide enhanced both GT 1 and GT 2 activities but not bilirubin glucuronidation. (5) In contrast to 3-methylcholanthrene-type inducers which induce both cytochrome P-450 MCand GT 1, probably through a common receptor protein, ethoxyquin and trans-stilbene oxide markedly induced gt 1 reactions without affecting benzo[a]pyrene monooxygenase.

Effects of detergents and organic solvents on rat liver microsomal UDP-glucuronosyltransferase activity towards phenolic substrates.

1. The effects of several detergents (Brij 58, deoxycholate and Lubrol 12A9) and ether on the initial rate of UDP-glucuronosyltransferase activity towards fixed concentrations of five phenolic acceptor substrates of widely different octanol-buffer (pH 7.4) partition coefficient have been compared with those observed in non-activated and Triton X-100-and n-pentane-activated rat liver microsomes. 2. Enzymic activity was dependent on the lipid-solubility of acceptor substrate. Each activator, except Triton X-100, enhanced enzymic activity towards all substrates by a similar factor, which was independent of the octanol-buffer partition coefficient. For Triton X-100 microsomes, the activation was also partition-dependent. 3. The highest activation factor was seen with ether. Pre-incubation of ether-activated microsomes for 30 min at 37 degrees C before assay resulted in inactivation of the enzyme towards more water-soluble substrates. Tryptic digestion (30 min at 37 degrees C) of the ether-activated microsomes resulted in marked reduction of enzyme activity towards all substrates. 4. Ether, and the two detergents, Brij 58 and Lubrol 12A9, released small amounts of protein (5-12% total present); both detergents also released some (8-12%) phospholipid. 5. The Kappm towards acceptor substrate also depended on the octanol-buffer partition coefficient, and was largely unchanged on activation by n-pentane. Vmax was not dependent on partition coefficient and was significantly increased on activation.

Lipophilicity of acceptor substrate as a factor in “late foetal” rat liver microsomal UDP-glucuronosyltransferase activity

The UDP-glucuronosyltransferase activity towards phenolic compounds, as measured by initial velocity, has been directly related to lipophilicity of acceptor substrates, as obtained by measurement of octanol-buffer or octanol-water partition. The acceptor substrates examined include 17 compounds, all probably conjugated by the “late foetal” enzyme activity. Rat liver microsomal enzyme activity towards five acceptor substrates of the “late foetal” enzyme was examined under different activation conditions. A statistically significant, partition-dependent increase in activity was observed when the effects of ageing or Triton X-100 treatment were studied. With n-pentane, phospholipase C or UDP- N-acetylglucosamine, although the enzyme activity depended on the partition coefficient of the acceptor substrate, activity towards each substrate was enhanced by a similar amount. Mild trypsin treatment (which did not itself activate the enzyme) or ageing converted the n-pentane dependent general activation into a partition related form by reducing the activity of the enzyme towards the less lipophilic substrates. Removal of phospholipid from the membrane by n-pentane or hydrolysis by phospholipase C resulted in the partition independent activation. Protein release, which by itself did not activate the enzyme, was also required for a partition-dependent effect. As enzyme activity towards the five substrates was induced by 3-methylcholanthrene, but not by phenobarbital, the “late foetal” enzyme was being studied. The induced enzyme activity appeared similar to the non-induced activity.