Hepatic Microsomal UDP-glucuronyltransferase Research Articles

Developmental changes in hepatic microsomal interactions between UDP-glucuronyltransferase and phospholipid in the rat fetus

The interaction of hepatic microsomal UDP-glucuronyltransferase (UDPGT) and phospholipid was studied using rat fetuses from the 16th to 21st day of gestation. UDPGT activity for phenol and p-phenylbenzoic acid, and phospholipid content and fluidity increased with day of gestation. Digestion of microsomes with phospholipase A 2 removed a similar ratio of phospholipids throughout the gestation days, whereas UDPGT activity decreased with day of gestation, indicating that UDPGT and phospholipids interacted strongly with day of gestation. Use of various modifiers showed a prominent decrease of UDPGT activity in the later part of gestation with changes in tryptophane residues and amino groups, and no change in activity with modification of thiol groups. These results suggest that tryptophane residues and amino groups interact with phospholipids. The results of this work will be useful in analyzing fetotoxicity associating with glucuronidation.

Responses of prepubertal female rats to environmental PCBs with high and low dioxin equivalencies.

An extract of landfill soil containing high levels of polychlorinated biphenyls (PCBs) was refined by alumina-charcoal column chromatography to remove coplanar PCBs and high relative levels of PCDFs. Both extracts were administered ip in corn oil to immature female rats on Days 21 and 22 of age. Rats were terminated on Day 23 and organ weights, enzyme activities, and serum thyroxine (T4) were measured. The change in characteristics caused by charcoal-stripping was confirmed by comparing hepatic microsomal 7-ethoxyresorufin O-deethylase (EROD), 7-pentoxyresorufin O-depentylase (PROD), 7-benzyloxyresorufin O-dearylase (BROD), and UDP glucuronyl transferase (UDPGT) activities. EROD and 4-nitrophenol (4-NP) UDPGT activities, estimates of aryl hydrocarbon (Ah) receptor agonists present, peaked at 140 mg/kg soil extract, declining at 210 mg/kg; these activities in rats treated with the charcoal-refined soil extract were not induced at low doses, but increased linearly from 70 to 210 mg/kg. Conversely, PROD and BROD activities were modestly induced at 7 to 70 mg/kg of either soil extract; however, at higher doses induction was threefold higher in rats receiving the charcoal-filtered extract. Liver weights increased in a pattern similar to EROD induction. At all doses, the weak uterotropic response was greater in rats receiving the charcoal-filtered extract, probably due to removal of antiestrogenic Ah receptor agonists by the charcoal. Serum total T4 declined similarly in rats receiving either extract to about 40% of control values at the highest doses. Short-term integrated bioassays such as this are useful in detecting multiple complex interactions and can be used to define the net effects of mixtures with changing compositions for improved risk assessment.

Diffusion characteristics of chitosan-entrapped microsomal UDP-glucuronyl transferase gel beads

Rabbit hepatic microsomal UDP-glucuronyl transferase (EC 2.4.1.17) was immobilized by entrapment in chitosan which is an ionotropic gelation agent and the resulting preparation was used as a biocatalyst for the glucuronidation of 1-naphthol in order to study the drug metabolism in vitro and obtaining artificial liver support (detoxification). In this study, depending on the porous structure of the chitosan/UDPGT gel beads, mainly the diffusion characteristics of 1-naphthol from chitosan gel beads in well stirred solutions were investigated, and the values of the effective diffusion coefficients ( D e) indicate that UDP-glucuronyl transferase in chitosan beads has no additional diffusion barrier. Using the data, the capacity of the chitosan-gel as an immobilization matrix for microsomal UDP-glucuronyl transferase is discussed from the point of view of diffusion characteristics.

Substrate specificity and the use of chitosan-entrapped rabbit hepatic microsomal UDP-glucuronyl transferase for detoxification.

Rabbit hepatic microsomal UDP-Glucuronyl transferase (EC.2.4.1.17) was immobilized in chitosan, which is an ionotropic gelatation agent, and the resulting preparation was used as a biocatalyst for the glucuronidation of toxic substances and drugs such as; 1-naphthol, phenol, 4-nitrophenol, bilirubin, testesterone, estrone, lamotrigine, imipramine and chlorpromazine. For studying the drug metabolism in vitro and obtaining artificial liver support (detoxification) chitosan-entrapped UDPGT towards 1-naphthol was tested by using two model reactor system. The conversion curves of 1-naphthol to its glucuronide indicated that the column reactor system was found to be better and seems suitable for detoxification with a high yield of glucuronide formation.

Preparation and characterization of chitosan-entrapped microsomal UDP-glucuronyl transferase.

The hepatic microsomal UDP-Glucuronyl transferase which catalyze the glucuronidation of drugs, pesticides, carcinogens and other xenobiotics, was immobilized by entrappment in chitosan. Chemical and physical characterization were made by using 1-naphthol as substrate. Thermal, storage and operational stabilities of the immobilized enzyme was also searched and found to be better in comparison with the free enzyme. In conclusion, chitosan gel beads appear to have good characteristics for use as UDPGT immobilization support suitable for large scale use and offer the prospect that immobilized UDPGT may become an important form of catalyst in medicine.

3-Trifluoromethyl-4-nitrophenol (TFM) toxicity and hepatic microsomal UDP-glucuronyltransferase activity in larval and adult bullfrogs

Effects of TFM (3-trifluoromethyl-4-nitrophenol) were observed in bullfrog ( Rana catesbeiana) larvae and adults in vivo by aqueous and intraperitoneal (i.p.) exposures. UDP-glucuronyltransferase (UDPGT) kinetics were examined in vitro from larval and adult hepatic microsomes. The effect of TFM on bullfrog egg development was also examined. Bullfrog larvae and adults showed similar sensitivities to TFM by i.p. exposure ( LD 50 s = 11.62 and 15.35 mg/kg , respectively), however, larvae were more sensitive than adults in aqueous exposures ( LC 50 s = 0.95 and 12.99 mg/l , respectively). Bullfrog egg development was arrested at the blastula stage at ≥ 1.0 mg/l TFM. No microscopic lesions were observed in tissues from TFM-exposed larvae. TFM-UDPGT kinetics in bullfrog larvae and adults differed significantly ( P < 0.05). V max for TFM-UDPGT in bullfrog larvae and adults was calculated to be 1.05 and 2.45 nmol/min · mg, respectively; the apparent K m for bullfrog larvae and adults was 38 and 64 μM, respectively. However, the enzyme V max K m ratio between the two life stages was not significantly different. Based on data from this and other studies, greater sensitivity to aqueous TFM in gill-respiring larvae (compared with adults) does not primarily appear to be a function of differences in UDPGT-mediated metabolism, but rather may reflect differences in other processes such as uptake.

Differential effects of blood insulin levels on microsomal enzyme activities from hepatic and extrahepatic tissues of male rats.

Microsomal glutathione S-transferase, UDP-glucuronyl transferase, and aniline hydroxylase activities were determined in liver, renal cortex, and small intestine of control, streptozotocin-diabetic, alloxan-diabetic, and untreated insulin-injected male Wistar rats. Renal microsomal glutathione S-transferase activity showed a direct linear relationship with insulin blood levels, in agreement with our previous report on cytosolic glutathione S-transferase. This result suggests a possible regulatory mechanism of insulin that needs to be further examined. The hepatic microsomal UDP-glucuronyl transferase was only decreased in streptozotocin-diabetic rats and was not restored by insulin treatment. Intestinal UDP-glucuronyl transferase exhibited an opposite response in streptozotocin-treated animals that was not normalized by the administration of insulin. Hepatic aniline hydroxylase showed the same behaviour as intestinal UDP-glucuronyl transferase. These results suggest that streptozotocin and (or) its metabolites have a direct effect on hepatic and intestinal UDP-glucuronyl transferase activity and on hepatic aniline hydroxylase activity. On the other hand, insulin regulation of enzyme activity varies from one organ to another.

Low-calcium diets increase both production and clearance of 1,25-dihydroxyvitamin D3 in rats

Administration of large doses of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] to animals induces 1,25(OH)2D3 side-chain oxidative pathways. This study determined if the elevated plasma 1,25(OH)2D3 seen in rats fed low-Ca diets is associated not only with an increased production rate (PR) but also with an increased metabolic clearance rate (MCR) of the hormone. In vitamin D-replete rats fed a Ca-deficient diet for 3-4 wk, the PR increased 21-fold, plasma levels 15-fold, and the MCR by 37%. The increased MCR in Ca-deficient rats was associated with a 48% increase in hepatic microsomal UDP glucuronyl transferase enzyme activity, whereas 1,25(OH)2D3 catabolism by homogenates of liver and small intestinal mucosa was unchanged. In contrast to the effects of low-Ca diets, acute (7 h) pharmacological elevation of plasma 1,25(OH)2D3 to 1.5 ng/ml in normal rats did not influence the MCR. Thus chronically elevated 1,25(OH)2D3 levels are necessary to stimulate clearance. In conclusion, 1,25(OH)2D3 clearance in rats can be stimulated not only by chronic pharmacological doses of 1,25(OH)2D3 but also by the physiological stimulus of a low-Ca diet. Hence, plasma 1,25(OH)2D3 levels can be regulated by changes in both PR and MCR.

Increase of a Form of UDP-Glucuronyltransferase Glucuronizing Various Phenolic Xenobiotics and the Corresponding Translatable mRNA in 3-Methylcholanthrene-Treated Rat Liver

Induction of hepatic microsomal UDP-glucuronyltransferase activity toward various phenolic xenobiotics by 3-methylcholanthrene treatment of rats was observed, and the process of the induction was studied. We had previously purified a form of UDP-glucuronyltransferase (called GT-1) having a catalytic activity toward phenolic xenobiotics from liver microsomes of 3-methylcholanthrene-treated rats. The antibodies against GT-1 inhibited the enzyme activity toward those xenobiotics in liver microsomes, and bound to a single protein having a molecular weight of about 54,000 Da (same value as that of GT-1) among microsomal proteins on immunoblotting analysis. The amount of GT-1 protein in hepatic microsomes was found to be increased in close correspondence with the activity increase by 3-methylcholanthrene treatment, by immunoblotting analysis using an uninducible cytochrome P-450 reductase as a negative standard. It was shown by in vitro translation assays that the protein increase described above resulted from the enhancement of the level of translatable mRNA encoding for GT-1. Increases in the amount of the protein immunochemically corresponding to GT-1 in the microsomes from liver of phenobarbital-treated rats and from extrahepatic organs, such as kidney, small intestine, and lung, of phenobarbital- or 3-methylcholanthrene-treated rats were also observed.

Effects of aging and caloric restriction on hepatic drug metabolizing enzymes in the fischer 344 rat II: Effects on conjugating enzymes

The effects of long-term caloric restriction on the hepatic phase II drug metabolizing enzymes were investigated in the male Fischer 344 rat. Rats that had been restricted to 60% of their pair-fed control's consumption from 14 weeks post-partum exhibited altered conjugating enzyme activities at 22 months. Caloric restriction significantly reduced the age-related decrease in glutathiones- S-transferase activity towards 1,2-dichloro-4-nitrobenzene, but did not significantly alter the age-related changes in UDP-glucuronyltransferase or sulfotransferase activities towards hydroxysteroids. Caloric restriction appeared to increase hepatic microsomal UDP-glucuronyltransferase activity toward bilirubin and γ-glutamyltranspeptidase activites. These observations suggest that caloric restriction has multiple effects on the hepatic phase II drug metabolizing enzymes in the rat. Such effects may alter hepatic metabolism and activatin or detoxification of drugs and carcinogens.

Hepatic microsomal 4,5,6-trichloroguaiacol glucuronidation in five species of fish

Abstract 1. 1. Hepatic microsomal UDP glucuronyl transferase activity towards 4,5,6-trichloroguaiacol was characterized in five species of fish. The simple extraction technique used to determine glucuronidation rates was sufficiently accurate, as judged by high pressure liquid chromatography. 2. 2. UDP glucuronyl transferase activities were found to vary considerably between fish species. The level of UDP glucuronyl transferase activity in rainbow trout is among the highest in vitro conjugating activities reported in fish. 3. 3. Intraperitoneal injection of β-naphthoflavone resulted in an almost doubling of the UDP glucuronyl transferase activity in rainbow trout.

Elevation of sex steroids and inhibition of PDP-glucuronyltransferase are out of phase during gonadal maturation in the common carp

1. Plasma sex steroid concentrations, onset of gonadal maturation, and hepatic microsomal UDP-glucuronyltransferase (UDPGT) activities were followed under natural temperature and photoperiod in outdoor tanks, and under controlled laboratory temperature and photoperiod regimens in common carp ( Cyprinus carpio). 2. Decreased activity of UDPGT was out of phase with elevations in plasma testosterone and 17β-estradiol during gonadal maturation. 3. Injection of pituitary extract induced final gonadal maturation and transient elevations (within 24 hr) of both plasma sex steroid concentrations and UDPGT activities. 4. There were no simple relationships between plasma sex steroid concentrations and activity of hepatic microsornal UDPGT in common carp.

Modifications of Hepatic Microsomal 9-Oxidation ofN-2-Fluorenylacetamide in Response to Gonadectomy and Treatment of Rats with Phenobarbital or Diethylnitrosamine

1. The hepatic microsomal 9-hydroxylation of N-2-fluorenylacetamide (2-FAA) is greater in the presence of male, or absence of female, hormones. Thus, 9-hydroxy-2-FAA was the major microsomal metabolite of male rats, which formed 6-fold greater amounts than did female rats. One week after gonadectomy, the amount of 9-hydroxy-2-FAA formed by male rats was decreased by 61%, whereas that formed by female rats was increased 1.4-fold. 2. Treatment of rats with phenobarbital (PB) increased 2- to 3-fold the capacities of hepatic microsomes of both sexes (normal and gonadectomized) for 9-hydroxylation of 2-FAA. 3. Hepatic microsomes of male rats also had greater capacities to form 9-oxo-2-FAA, the metabolite of 9-hydroxy-2-FAA, and 6-hydroxy-2-FAA, a newly identified microsomal metabolite of 2-FAA. These metabolites were also decreased by orchidectomy and induced by PB. 4. 9-Hydroxy-2-FAA was a poor substrate for hepatic microsomal UDP-glucuronyltransferase, and conjugation was not induced by treatment of rats with PB. This indicated retention of 9-hydroxy-2-FAA in the liver and/or further metabolism (e.g. to 9-oxo-2-FAA). 5. The formation of 9-oxo-2-FAA from 2-FAA or 9-hydroxy-2-FAA was increased (1.5-fold) two weeks after treatment of male rats with a single i.p. dose of diethylnitrosamine (200 mg/kg), an initiator of hepatocarcinogenesis. 6. Based on the data we suggest that 9-oxidized metabolites of 2-FAA, the preferential formation of which coincides with the susceptibility of the rat to hepatocarcinogenesis, are promoters.

Effect of scoparone on the hepatic microsomal UDP glucuronyltransferase activity in mice

The effect of scoparone on UDPglucuronyltransferase in mouse hepatic microsomes was studied. After treatment with scoparone, hepatic microsomal UDPglucuronyltransferase activity was increased with dose-dependent manner as compared to control. The Vmax value (control=23.2 n moles/mg protein/min, scoparone=31.2 n moles/ mg protein/min) without affecting the Km value (414 μM) for p-nitrophenol was increased by the scoparone treatment, and the pattern of kinetic studies for UDP-glucuronic acid was also similar to those of p-nitrophenol. Whereas, the hepatic microsomal UDPglucoronyltransferase was not changed by the addition of scoparone in vitro. The results obtained suggest that the characteristics of increase in the enzyme activity may include induction of enzyme proteins.

The effects of immunomodulating peptidoglycan monomer and muramyl dipeptide on hepatic microsomal UDP-glucuronyltransferase and β-glucuronidase

The effects of immunomodulating peptidoglycans, peptidoglycan monomer (PGM) and muramyl dipeptide (MDP), on hepatic microsomal UDP-glucuronyltransferase (uridine diphosphoglucuronate glucuronosyl transferase, EC 2.4.1.17) and β-glucuronidase (β- d-glucuronide glucuronohydrolase, EC 3.2.1.31) were tested in female C57Bl mice. 4-Methylumbelliferone and p-nitrophenol were used as representative substrates for one functional form of UDP-glucuronyltransferase (GT 1) and testosterone for the second functional form (GT 2) of the enzyme. Both PGM and MDP were found to transiently inhibit the activity of UDP-glucuronyltransferase. There was no significant difference in the magnitude of inhibition of the two functionally different enzyme forms. The activity of microsomal β-glucuronidase was tested using 4-methylumbelliferyl glucuronide and p-nitrophenyl glucuronide as substrates. Time dependent transient inhibition of β-glucuronidase activity was observed with both peptidoglycans. In addition, the effect of MDP on cytochrome P-450 was tested, since we have shown previously that PGM affected this system. MDP decreased the content of cytochrome P-450 and inhibited the activity of related enzymes.

Induction of liver microsomal epoxide hydrolase, UDP-glucuronyl transferase and cytosolic glutathione transferase in different rodent species by 2-acetylaminofluorene or 3-methylcholanthrene.

Control activities vary 12-fold for microsomal epoxide hydrolase, two-fold for UDP-glucuronyl transferase and five-fold for cytosolic glutathione (GSH) transferase among the different rodents (rat, hamster, guinea-pig, mouse) examined. For all three enzymes the activities in rat liver are towards the lower values. In these rodents, except for a 100% increase in microsomal epoxide hydrolase in guinea-pig liver, 2-acetylaminofluorene induces the three phase 2 enzymes only in rat. Treatment with 3-methylcholanthrene also produces the largest effects on these three enzyme activities in rat liver; exceptions are its failure to induce microsomal epoxide hydrolase in female rat and the large induction of cytosolic GSH transferase in hamster liver. Quantitatively, hepatic microsomal epoxide hydrolase, UDP-glucuronyl transferase and cytosolic GSH transferase activities, and their inducibility by 2-acetylaminofluorene or 3-methylcholanthrene, in male Sprague-Dawley rats are not representative for other rodent species or even, in all cases, for female rat.

Carbon tetrachloride modulates the rat hepatic microsomal UDP-glucuronyl transferase activity and membrane fluidity

Modulations in rat hepatic microsomal UDP-glucuronyl transferase activity have been observed during carbon tetrachloride (CCl4) poisoning, with a large decrease in the enzyme cooperatively and increase in the membrane fluidity, occurring 30 min after administration. The results strengthen the possibility that an increase in microsomal membrane fluidity may be an early key event in liver injury induced by CCl4.

Storage conditions of rainbow trout liver cytochrome P-450 and conjugating enzymes

1. 1. The stability of hepatic microsomal cytochrome P-450 monooxygenase and UDP glucuronyl transferase activities and cytosolic glutathione S-transferase activity in rainbow trout was investigated in order to develop a standardized freezing and storage procedure of liver samples that maintain the enzyme activity levels. 2. 2. Livers can be frozen and stored in liquid nitrogen a short period of time (3 days) without any significant loss of the biotransformation enzyme activities studied whereas freezing of the livers on dry ice resulted in a rapid loss of cytochrome P-450 activities. 3. 3. Cytochrome P-450 and UDP glucuronyl transferase activities in microsomes suspended in buffer containing 20% glycerol were stable for 1 year when the microsomes were stored at −80°C.

Hepatic microsomal glucuronidation of clofibric acid in the adult and neonate albino rat

Hepatic microsomal UDP glucuronyltransferase activity towards the acid substrate clofibric acid has been described in the adult and neonate albino rat. The enzyme was maximally activated, approximately 2-fold, in the presence of 0.1–0.4% (w/v) digitonin. Induction of the digitonin activated clofibric acid glucuronyltransferase was observed following phenobarbitone treatment in vivo (2.2-fold), and to a lesser extent, following β-naphthoflavone treatment (1.3-fold). Clofibrate treatment in vivo (of which clofibric acid is the ester hydrolysis product) had no effect on clofibric acid glucuronidation in vitro. The activity of clofibric acid glucuronyltransferase in the liver of rat before and at birth was low (approx. 0.08 nmoles glucuronide formed/min/mg microsomal protein). The activity increased 5-fold during the first three post-natal days. After this time, the activity increased linearly reaching adult levels by four weeks after birth. The data indicated that clofibric acid glucuronyltransferase belongs to the neonatal cluster of enzymes and clofibric acid is a group 2 substrate. Clofibric acid, a common therapeutic agent, is a useful, acid substrate for the estimation of mammalian hepatic microsomal glucuronyltransferase activity.

Differential regulation by triiodothyronine of substrate-specific uridinediphosphoglucuronate glucuronosyl transferases in rat liver

Hepatic uridinediphosphoglucroonate glucuronosyl transferase (UDPglucuronyltransferase, EC 2.4.1.17) functionally heterogeneus; 4-nitrophenol and bilirubin are representative subtrates for two separated from of the enzyme. UDPglucuronyltransferase activity for bilirubin and 4-nitrophenol was separated from solubilized rat liver microsomes by DEAE-cellulose chromatography and corresponding enzymes were purified. A radioimmunoassay was developed using a rabbit antiserum against purified rat 4-nitrophenol-specific UDPglucuronyltransferase, which precipitated enzyme activities toward both 4-nitrophenol and bilirubin. After treatment with triiodothyronine(T 3) (0.55 mg/kg body weight), hepatic microsomal UDPglucuronyltransferase activity for 4-nitropheelos was increased 400% as compared to controls; the enzyme activity for bilirubin was decreased by 80%; the changes in the substrate-specific enzyme activities were reflected in the enzymatically active fractions separated after DEAE-cellulose chromatography. The changes in enzyme activities paralleled changes in the concentrations of the two corresponing UDP glucuronyltransferase proteins in the chromatographic fractions, as measured by radioimmunoassay. The results indicate that the opposite effects of T 3 on the two forms of UDPglucuronyltransferase activity is due to its differential effect on corresponding enzyme proteins.