Microsomal UDP-glucuronyltransferase in rat liver: oxidative activation

Activation of microsomal UDP-glucuronyltransferase (UDPGT) activity by treatment of hepatic microsomes with either detergents or Fe(3+)/ascorbate pro-oxidant system has been reported; however, definite mechanisms underlying these effects have not been clarified. In this work, we characterize Fe(3+)/ascorbate-induced activation of UDPGT activity prior to solubilization with Triton X-100 and after the oxidation process provoked the solubilization of the enzyme. We observed a time-dependent increase in UDPGT activity up to 20 min. incubation of the microsomes with Fe(3+)/ascorbate (3-times); after 20 min. incubation, however, we observed a time-dependent decrease in this activity to basal levels after 4 hr incubation. Treatment of microsomes with 0.1% Triton X-100 (5 min.) lead to a similar increase in UDPGT activity; higher detergent concentrations produced a dose-dependent decrease in this activity to basal levels with 1% Triton X-100. Interestingly, UDPGT activity was susceptible to activation only when associated to microsomal membranes and the loss of activation correlated with the solubilization of this activity. UDPGT activation by either Fe(3+)/ascorbate or Triton X-100 was correlated with an increase in p-nitrophenol apparent K(m) and V(max) values. This activation was prevented or reversed by the reducing agents glutathione, cysteine or dithiothreitol when it was induced by the Fe(3+)/ascorbate. Furthermore, the latter provoked a significant decrease in microsomal thiol content, effect not observed after treatment with Triton X-100. Our results suggest that the main mechanism responsible for Fe(3+)/ascorbate-induced UDPGT activation is likely to be the promotion of protein sulfhydryl oxidation; this mechanism appears to be different from detergent-induced UDPGT activation.     

The inadequacy of perinatal glucuronidation: immunoblot analysis of the developmental expression of individual UDP-glucuronosyltransferase isoenzymes in rat and human liver microsomes

Two anti-rat UDP-glucuronosyltransferase (UDPGT) antibody preparations, exhibiting different specificity of recognition of UDPGT isoenzymes on immunoblot analysis, were used to investigate the molecular basis of the perinatal inadequacy of glucuronidation in rats and humans. Immunoblot analysis of microsomes from developing rat liver demonstrated that the deficiency in bilirubin and testosterone glucuronidation in the fetus was due to the absence of the UDPGT isoenzyme proteins responsible for these conjugations. In contrast, phenol UDPGT enzyme activity and protein was detectable in significant amounts in fetal rat liver (greater than 30% of adult levels). In human liver, only one major immunoreactive polypeptide was observed in fetal microsomes. The remaining UDPGTs present in adult human liver developed postnatally, in parallel with the appearance of enzyme activities. Therefore, there was a correlation between the development of enzyme activity and enzyme protein. The possible consequences of developmental inadequacy of conjugation reactions for the fetus is discussed.     

Photoaffinity labeling of rat liver microsomal morphine UDP-glucuronosyltransferase by [3H]flunitrazepam

Benzodiazepines have been shown to competitively inhibit morphine glucuronidation in rat and human hepatic microsomes. Flunitrazepam exerted a potent competitive inhibition of rat hepatic morphine UDP-glucuronosyltransferase (UDPGT) activity (Ki = 130 microM). It has no effect on the activity of p-nitrophenol, 17 beta-hydroxysteroid, 3 alpha-hydroxysteroid, or 4-hydroxybiphenyl UDPGTs. Because flunitrazepam is an effective photoaffinity label for benzodiazepine receptors, studied were performed in solubilized rat hepatic microsomes and with partially purified preparations of morphine UDPGT to determine the enhancement of flunitrazepam inhibition and binding to morphine UDPGT promoted by exposure to UV light. Under UV light, flunitrazepam inhibition was markedly enhanced. UV light exposure also led to a marked increase in binding of [3H]flunitrazepam to microsomal protein, which was protected substantially by preincubation with morphine. Testosterone, androsterone, and UDP-glucuronic acid did not protect against UV-enhanced flunitrazepam binding, and morphine did not reverse flunitrazepam binding once binding had occurred. As morphine UDPGT was purified, a good correlation was found between the increases in specific activity of morphine UDPGT and flunitrazepam binding to protein. Chromatofocusing chromatography showed that flunitrazepam bound only to fractions containing active morphine UDPGT, and no binding to 4-hydroxybiphenyl UDPGT was observed. Fluorography of a sodium dodecyl sulfate-polyacrylamide electrophoresis gel of solubilized hepatic microsomes that had been treated with [3H] flunitrazepam under UV light revealed a band with a monomeric molecular weight between 54,000 and 58,000. This monomeric molecular weight compares favorably with the reported monomeric molecular weight of homogeneous morphine UDPGT (56,000). These studies suggest that flunitrazepam binds rather selectively to the morphine binding site of morphine UDPGT and may prove to be a useful probe for this enzyme.     

Differential expression and induction of UDP-glucuronosyltransferase isoforms in hepatic and extrahepatic tissues of a fish, Pleuronectes platessa: immunochemical and functional characterization.

Glucuronidation of three substrates prototypical for different UDP-glucuronosyltransferase (UDPGT) isoforms in hepatic, renal, intestinal, and branchial microsomes of corn oil, 3-methylcholanthrene, Aroclor 1254, and clofibrate-pretreated plaice was investigated. The differential expression of UDPGT in the four tissues clearly demonstrated for the first time that multiple isoforms with differing substrate specificities were present in fish. The liver was quantitatively the most important site for the glucuronidation of all three compounds studied. Phenol UDPGT activity was ubiquitous to all tissues and was induced by 3-methylcholanthrene and Aroclor 1254 in hepatic tissue and by Aroclor 1254 in renal tissue. The glucuronidation of testosterone was restricted to liver and intestinal tissue, while conjugation of bilirubin was expressed solely in hepatic tissue. The biotransformation of the endogenous compounds was not induced in the xenobiotic-treated animals. The presence of immunoreactive UDPGTs in the four tissues was demonstrated by immunoblot analysis using sheep anti-plaice UDPGT antibodies. Hepatic tissue displayed a range of immunoreactive polypeptides of 52 to 57 kDa, while a 55-kDa polypeptide was detected in extrahepatic tissues. An increased intensity of the latter polypeptide species was demonstrated in liver and kidney microsomes in which there was a concomitant induction of phenol UDPGT activity in xenobiotic-treated fish. The results indicate that the 55-kDa polypeptide was the major polyaromatic hydrocarbon-inducible UDPGT isoenzyme in hepatic and renal microsomes.     

On non-specific inhibition of rat liver microsomal UDP glucuronyltransferase by some drugs

The effect of imipramine. desipratnine, harmol, harmalol and some monoamine oxidase inhibitors of the hydrazine type on rat liver UDP glucuronyltransferase (EC 2.4.1.17) activity has been investigated. Substrates of the enzyme were p-nitrophenol and bilirubin; with p-nitrophenol only Triton X-100 activated microsomes were used as enzyme source, with bilirubin both not-activated and Triton X-100 activated microsomes were used. The degree of inhibition obtained with the various inhibitors was dependant on the substrate used and on the pretreatment of the microsomes. It is concluded that most of the effects were caused by the action of the compounds on microsomal membrane structure, which affects UDP glucuronyltransferase activity, and that the physiological relevance of inhibition of the enzyme in vitro is questionable.     

St. John's wort extract induces CYP3A and CYP2E1 in the Swiss Webster mouse.

This investigation was designed to determine the ability of St. John's wort (SJW), a readily available antidepressant, to induce various hepatic drug metabolizing enzymes. SJW (140 or 280 mg/kg/day) was administered to male Swiss Webster mice for 1, 2, or 3 weeks. Enzymatic activity was analyzed in hepatic microsomes for all of the following drug metabolizing enzymes: CYP3A, CYP1A, CYP2E1, and UDP-glucuronosyltransferase (UDPGT). The catalytic activity of CYP1A was unchanged from control following any dose or duration of SJW, while both CYP3A and CYP2E1 catalytic activities were increased 2-fold by both SJW concentrations but only following 3 weeks of administration. Results from Western immunoblotting studies supported the changes in catalytic activity, as protein levels for CYP2E1 and CYP3A were increased (2.5-fold and 6-fold, respectively) following 3 weeks of SJW administration. Additionally, the catalytic activity of the conjugation enzyme UDPGT was unchanged from control following all SJW treatments. These results indicate that in the mouse moderate doses of SJW cause an increase in the catalytic activity and polypeptide levels of CYP2E1 and CYP3A but only following 21 days of administration, while the catalytic activity of CYP1A and UDPGT activity remain unaffected.     

Aromatic hydroxylation of amphetamine with rat liver microsomes, perfused liver, and isolated hepatocytes.

The hydroxylation of amphetamine to p-hydroxyamphetamine has been investigated. In 1 hr, perfused rat livers hydroxylated about 85 per cent of the administered 20 μmole dose of amphetamine. Ninety-four per cent of the p-hydroxyamphetamine was further metabolized by conjugation. Isolated hepatocytes are capable of hydroxylation of amphetamine and conjugation of hydroxyamphetamine at rates comparable to those of the perfused liver. The rate of hydroxylation with microsomes is about 50 per cent of that attained with isolated hepatocytes. With hepatocytes, the hydroxylation is inhibited by typical inhibitors of microsomal oxygenase(s) such as 2,6-dichlorophenyphenoxyethylamine (DPEA) and SKF525A, and it is inhibited by iprindole. However, the rate could not be increased by treatments which induce cytochrome P450 and increase other mono-oxygenase reactions. The results obtained with both isolated hepatocytes and microsomes suggest that amphetamine is hydroxylated by a microsomal cytochrome P450-dependent enzyme system, but that amphetamine is not a typical substrate.     

Preservation of glucuronidation in carbon tetrachloride-induced acute liver injury in the rat

The influence of carbon tetrachloride on several routes of drug metabolism has been investigated in the rat in vivo, in isolated perfused livers, and in hepatic microsomal preparations to evaluate the hypothesis that hepatic glucuronyl transferase activity is preserved when mixed-function oxidase activity is impaired by this hepatotoxin. Impaired oxidative metabolism after acute administration of carbon tetrachloride was confirmed in vivo by a 65 per cent reduction in the rate of elimination of ¹⁴CO₂ in breath after [¹⁴C]aminopyrine administration and a 58 per cent reduction in the clearance of lorazepam in the whole animal. In the isolated perfused livers of control rats, glucuronide metabolism of lorazepam accounted for 26 per cent of its overall elimination; thus, by inference, oxidative metabolism accounted for the remainder. Carbon tetrachloride pretreatment resulted in a 63 per cent reduction in total lorazepam clearance. In microsomal preparations, cytochrome P-450 concentration, cytochrome P-450 reductase activity, and activities of o-demethylation of p-nitroanisole and hydroxylation of aniline were reduced by 63, 32, 85 and 95 per cent, respectively, after exposure to carbon tetrachloride. In contrast, in the carbon tetrachloride-damage isolated perfused liver the proportion of lorazepam conjugated to lorazepam glucuronide increased from 26 to 43 per cent of the dose administered. Furthermore, in vitro microsomal glucuronidation of o-aminophenol was not decreased by carbon tetrachloride pretreatment, while glucuronidation of p-nitrophenol actually increased significantly by 49 per cent. Solubilization of microsomes with Triton X-100 resulted in a 10.4-fold increase in glucuronyl transferase activity in control microsomes, but in only a 5.7-fold increase in microsomes from carbon tetrachloride-treated rats. This suggests that carbon tetrachloride enhanced basal glucuronidating activity but decreased the total amount of enzymes present. These overall results suggest that microsomal glucuronidation activity is spared in an experimental liver injury in which oxidative metabolism is impaired.     

Altered enzyme activities of xenobiotic biotransformation in kidneys after subchronic administration of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) to rats

Activities of the xenobiotic metabolizing enzymes were measured in the liver, kidney, duodenum and lung microsomes and cytosol fractions of Wistar rats after subchronic administration of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent bacterial mutagen in chlorinated drinking water. MX was administered by gavage at the dose level of 30 mg/kg for 18 weeks (low dose), or at the dose level which was raised gradually from 45 mg/kg for 7 weeks via 60 mg/kg for 2 weeks to a clearly toxic dose of 75 mg/kg for 5 weeks (high dose). Microsomal and cytosolic preparations were made and the activities of 7-ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-dealkylase (PROD), NADPH-cytochrome-c-reductase, UDP-glucuronosyltransferase (UDPGT) and glutathione-S-transferase (GST) were measured. Kidneys were affected most. A dose-dependent decrease was observed in EROD (90% in males, 80% in females at the high dose) and in PROD (58% in females, at the high dose) in kidneys. An increase was, however, detected in kidney NADPH-cytochrome-c-reductase (66% in females at high dose), UDPGT (89% in males and 97% in females at high dose) and GST activities (56% in males and 50% in females at high dose). MX caused only a few changes in the enzyme activities of the liver. The EROD activity was decreased 25% to 37%, both in the livers of males and females, but the total content of P450s was not altered. Hepatic GST activity was elevated in females in a dose-dependent manner (31% and 44%). GST activity was elevated in duodenum in females (59%) at the high dose. There were no marked changes in the enzyme activities in the lungs. MX was a weak inhibitor of EROD activity both in the liver and kidney microsomes in vitro, decreasing the EROD activity by 53% and 43%, respectively at the concentration of 0.9 mM. The results indicate that MX decreases the activity of phase I metabolism enzymes, but induces phase II conjugation enzyme activities, particularly in kidneys in vivo. It is possible that these changes contribute to metabolism of MX in kidneys and renders them susceptible to MX in the course of repeated exposure.     

Kinetic characteristics of UDP-glucuronosyltransferases towards a dithiol metabolite of malotilate in hepatic microsomes of rats and rabbits

1. The kinetic activity of UDP-glucuronosyltransferases (UDPGT) towards a dithiol metabolite of malotilate, 2,2-di(isopropoxycarbonyl)ethylene-1,1-dithiol, was investigated using rat and rabbit hepatic microsomes. The thio-glucuronide formed was analysed by h.p.l.c. The Km values obtained using rat and rabbit UDPGT were 36.3 +/- 3.3 and 443 +/- 43 microM, respectively. The Vmax values were 7.14 +/- 0.61 and 29.2 +/- 6.4 nmol/min per mg (mean +/- SD, n = 3). 2. Phenobarbital, an inducer of the GT2 isoform of UDPGT, increased rat microsomal UDPGT activity towards the dithiol. In inhibitory studies, menthol and borneol (specific substrates for GT2a isoform) competitively inhibited glucuronidation of the dithiol. Thus it was concluded that formation of the thio-glucuronide was catalysed mainly by the GT2a isozyme of UDPGT, which is involved in glucuronidation of monoterpenoid alcohols.     

Defective induction of phenol glucuronidation by 3-methylcholanthrene in Gunn rats is due to the absence of a specific UDP-glucuronosyltransferase isoenzyme

Antiserum directed against purified rat kidney UDP-glucuronosyltransferase (UDPGT) was raised in goats. IgG prepared from this antiserum exhibited specificity for only two UDPGT isoenzymes (bilirubin and phenol) on immunoblot analysis of Wistar rat liver microsomes. Use of this antibody preparation to probe Western blots of liver microsomes prepared from Gunn rats confirmed that the defective phenol glucuronidation was due to the absence of a 53-kDa, 3-methylcholanthrene-inducible UDPGT isoenzyme. Results obtained from enzyme activity measurements and immunoblot analysis of microsomes isolated from xenobiotic-treated Wistar and Gunn rat liver are consistent with the 3-methylcholanthrene/UDPGT induction deficiency in the Gunn rat being due to the absence of this phenol UDPGT isoenzyme. The contribution of other UDPGT isoenzymes to the greatly reduced glucuronidation of planar phenols observed in the Gunn rat is discussed.     

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°C before assay resulted in inactivation of the enzyme towards more water-soluble substrates. Tryptic digestion (30?min at 37°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 K^app_m towards acceptor substrate also depended on the octanol-buffer partition coefficient, and was largely unchanged on activation by n-pentane. V_mak was not dependent on partition coefficient and was significantly increased on activation.     

Glucuronide conjugation of 6-n-propyl-2-thiouracil and other antithyroid drugs by guinea pig liver microsomes in vitro.

Guinea pig liver microsome UDP glucuronyl transferase and UDPGA were incubated with the radioactive antithyroid drugs 6-n-propyl-2-thiouracil (PTU). 1-methy1-2-mercaptoimidazole (methimazole, MMI) and 2-thiouracil (TU). Radioactive metabolites were produced with PTU and thiouracil and, in each case, were identified as the corresponding β-glucuronide conjugate. No measurable glucuronidation of MMI was observed. Kinetic studies with the microsomal preparation demonstrated a K_m rmvalue of 7.2 × 10^?4 M for PTU and 6.7 × 10^?3 M for thiouracil. Glucuronide conjugation of PTU was linear for 1 hr. declining thereafter while conjugation of phenolphthalein was linear for 2 hr. Conjugation of phenolphthalein by microsomes stored in 0.154 M KCl at ?20° for 14 days was 41 per cent higher than in fresh microsomes, whereas conjugation of PTU was 67.4 per cent lower. PTU glucuronidation did not occur in the absence of UDPGA and was essentially linear with respect to enzyme concentrations. Under the same conditions, spontaneous N-glucuronidation of PTU by glucuronate was not measurable. The pH optimum for PTU glucuronidation was 8.0 and similar to the broad optimums of 7.3 to 7.9 for UDP glucuronyl transferases from a variety of sources rather than to non-enzymatic N-glucuronidation, which has a reported pH optimum of 3–4. The conjugating enzyme for PTU was located primarily in the guinea pig liver microsomes with this fraction exhibiting 75 per cent of the total activity of whole homogenates. PTU conjugation was inhibited by MMI but not by thiouracil, thiourea or 6-methy1-2-thiouracil. The results obtained demonstrate that β-glucuronide conjugation of the antithyroid drugs PTU and thiouracil, but not MMI. is readily catalyzed by a guinea pig liver microsomal UDP glucuronyl transferase in vitro.     

In vitro glucuronidation of 3'-azido-3'-deoxythymidine by human liver. Role of UDP-glucuronosyltransferase 2 form.

The glucuronidation of 3'-azido-3'-deoxythymidine (AZT) by human liver microsomes and human hepatocytes in culture has been studied in vitro to determine the UDP-glucuronosyltransferase (UDPGT) form conceivably involved in the AZT biotransformation process. The glucuronide of AZT was preliminarily identified through hydrolysis by beta-D-glucuronidase. Brij 58 was shown to be the best activator of AZT glucuronidation by human liver microsomes, as it increased the rate of glucuronide formation 3-fold. The UDPGT activities toward AZT measured in 29 different microsomal fractions was slightly variable among samples (79 to 268 nmol/hr/mg protein). The apparent KM value for AZT glucuronidation was about 5 mM. We sought to determine if various known UDPGT activities (i.e. p-nitrophenol UDPGT, 4-hydroxybiphenyl UDPGT, and DT1-UDPGT) in 18 microsomal samples were correlated with AZT-UDPGT activity. Experiments revealed that only 4-hydroxybiphenyl UDPGT activity was strongly correlated (r = 0.815, p less than 0.001) with AZT-UDPGT activity, whereas no correlation was found for the other UDPGT activities. To determine the isozyme conceivably involved in AZT glucuronidation, we studied the effect of various compounds on AZT glucuronidation. AZT glucuronidation was inhibited by numerous substrates of the UDPGT2, form: morphine (Ki = 1.8 mM), 4-hydroxybiphenyl (Ki = 0.92 mM), and ketoprofen (Ki = 0.75 mM), but also oxazepam, codeine, and chloramphenicol. p-Nitrophenol appeared to be an inhibitor, whereas acetaminophen had no effect. Bilirubin, aspirin, cimetidine, and acyclovir did not inhibit AZT glucuronidation. Since all the inhibitors tested except p-nitrophenol are known to be glucuronidated by the UDPGT2 form, our results strongly suggest the involvement of this isozyme in AZT glucuronidation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential toxicity of aflatoxin B1 in male and female rats: relationship with hepatic drug-metabolizing enzymes

It is well known that distinct differences in the metabolism of xenobiotics exist between males and females of the same species. Male and female rats were treated with a single intraperitoneal dose of aflatoxin B₁ (AFB₁): 1 or 3 mg/kg for males, 3 or 6 mg/kg for females. Comparative changes in hepatic drug metabolizing and plasma enzymes had been studied. The obtained results show that, at the common dose of 3 mg/kg, AFB₁ induced an 18% mortality in males and none in females. In the plasma, total bilirubin concentration as well as the activity of transaminases and alkaline phosphatase (ALP), utilized as indicators of liver damage, were highly increased in both males and females due to the treatment with 3 or 6 mg AFB₁/kg. In the female, the plasma features rapidly declined. In contrast, in the male, the effect of AFB₁ was prolonged. Hepatic determinations revealed a pattern difference of drug metabolizing enzymes and cytochrome P-450 between males and females. The results also show that in the male, most of the drug metabolizing enzyme activities were decreased until the ninth day with the 3 mg/kg treatment. So, we observed a decrease in the activities of UDP-glucuronosyltransferase (UDPGT) with p-nitrophenol as substrate (PNP) and GSH S-transferase, 40 and 53% respectively; while the activity of epoxide hydrolase was increased up to 170%. In the meantime, the concentration of cytochrome P-450 decreased by 69%. By contrast, in the case of the female, these decreases were only 14, 43 and 23% for the UDPGT, GSH S-transferase and cytochrome P-450, respectively. Moreover, these decreases occurred only during the first three days after treatment. Thereafter, these enzyme activities significantly increased above the control values. This study suggests that the induction of detoxicating enzymes, more important in the female (72% increase in the activity of UDPGT, 480% in that of epoxide hydrolase and 42% for GSH S-transferase, may have a protective role against AFB₁ metabolites and could explain, partly, the lower sensitivity of the female to the toxic effects of AFB₁.     

Mestranol-induced hypertension: characterization of cytochrome P-450 dependent catechol estrogen formation in brain microsomes

An animal model of estrogen-induced hypertension was used to study the effects of chronic administration of the synthetic estrogen mestranol on cytochrome P-450 content and catechol estrogen formation in brain microsomes. Cytochrome P-450 content of brain microsomes from untreated female rats in estrus was 0.034 nmole/mg protein and the dithionite-reduced carbon monoxide absorbance peak (γ_max) was 452 nm. Catechol estrogen formation in brain microsomes was optimal in the presence of both NADPH and NADH cofactors with an apparent K_m value of 71 μM for 17β-estradiol substrate. Brain microsomes from animals in estrus and diestrus were compared, and no significant differences were observed in cytochrome P-450 content, or in the apparent K_m and V_max values of catechol estrogen formation. Administration of mestranol, 15 μg biweekly, for 3–4 weeks resulted in a significant increase in systolic blood pressure in unanesthetized female rats. Mestranol treatment was not associated with a change in microsomal cytochrome P-450 content or the spectral γ_max. At 10 μM substrate concentration, catechol estrogen formation in microsomes from mestranol-treated animals was increased 2- to 3-fold, with enzyme activity being expressed either per mg protein or per nmole cytochrome P-450. In contrast, no difference was observed between groups when enzyme activity was measured at 100 μM substrate concentration. These data suggest that chronic administration of a synthetic estrogen can regulate the enzyme system involved in formation of brain catechol estrogen metabolites, a mechanism which may alter the biological impact of the parent steroid.     

Ascorbic acid deficiency and hepatic UDP-glucuronyltransferase

The effect of dietary ascorbic acid on hepatic microsomal UDP-glucuronyltransferase (UDPGT) activity towards p-aminophenol, bilirubin, and acetaminophen was investigated. Ascorbate deficiency produced a 33% reduction in the specific activity of UDPGT towards p-aminophenol, whereas there was no difference between microsomes from ascorbate-deficient and supplemented guinea pigs in the activity towards bilirubin and acetaminophen. This suggests that the effect of the vitamin is on a specific isozyme. This reduction was correlated with the reduced quantity of hepatic microsomal cytochrome P-450, which has been previously reported for ascorbate-deficient guinea pigs. No difference was found in the apparent affinity for the substrate, p-aminophenol, or the cofactor, UDP-glucuronic acid. Differences in microsomal UDPGT activity towards p-aminophenol occurred between the two groups with membrane-perturbing processes such as sonication and Triton X-100. Sonication and magnesium chloride were found to increase activity 329% in ascorbate-supplemented animals and 138% in the ascorbate-deficient group. The addition of ascorbate acid in vitro, or its analog d-isoascorbic acid, could protect against the detrimental effects of excess substrate by maintaining a linear enzymatic rate over a 30-min time period; there was no significant effect on the initial rate of hepatic microsomal UDPGT activity in the ascorbate-supplemented animals whereas there was a significant increase in the ascorbate-deficient group. Glutathione was as effective as ascorbic acid in protecting against the detrimental effects of excess substrate whereas cysteine and dimethyltetrapteridine were only partially effective. Ascorbyl-2-sulfate and alpha-tocopherol had no significant effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of rat hepatic epoxide hydratase by dietary antioxidants.

Supplementation of rat diet with butylated hydroxytoluene (BHT), butylated hydroxyanisole, or ethoxyquin resulted in increased liver epoxide hydratase activity. The increase was obvious at 0.1% and amounted to 200–400% at 0.5%. Increased activity was accompanied by increased proportion of the epoxide hydratase band in SDS polyacrylamide gels, indicating induction of the enzyme. Ethoxycoumarin deethylase activity and cytochrome b₅ concentrations were moderately elevated while cytochrome P-450 concentrations and aryl hydrocarbon hydroxylase activity remained at control levels. Preferential inhibition of monooxygenase activity by metyrapone and not 7,8-benzoflavone, as well as increased affinity of the reduced cytochrome P-450 for metyrapone as a ligand, indicated that the cytochrome P-450 population after BHT treatment was similar to that found after phenobarbital treatment. The antioxidants used in this study had no in vitro effect on epoxide hydratase activity and inhibited monooxygenase activity only in phenobarbital-stimulated microsomes but not in 3-methylcholanthrene-stimulated microsomes. Combined treatment with dietary antioxidants and intraperitoneally administered 3-methylcholanthrene resulted in marked induction of epoxide hydratase activity while the 3-methylcholanthrene-mediated increase in aryl hydrocarbon hydroxylase activity was partially depressed. Covalent binding of tritiated benzo[a]pyrene to calf thymus DNA was less effectively catalyzed by liver microsomes from animals fed antioxidants. The depression of covalent binding was marked after combined treatment with antioxidants and 3-methylcholanthrene. The shift in the microsomal enzyme pattern caused by antioxidants may be related to their inhibitory effects on chemical carcinogenesis.     

Changes in the rat hepatic mixed function oxidase system associated with chronic ethanol vapor inhalation

Chronic ethanol vapor inhalation by rats increased hepatic microsomal aniline hydroxylase activity, increasing the turnover number and decreasing the K_m. Activity of ethanol-induced microsomes toward other substrates was also examined. The increase in aniline hydroxylase activity as a result of ethanol treatment is attributed to an increase in a form of cytochrome P-450 with a high specific activity toward aniline. Since the ethanol effect on aniline hydroxylation had disappeared 24 hr after treatment was discontinued, a high rate of turnover of this enzyme was deduced. Dimethylsulfoxide (56 mM) produced a reverse type I spectral change in ethanol-induced, but not in control, microsomes. This was interpreted as being due to a change in the spin state of the cytochrome P-450 in these microsomes. Acetone added to the incubation produced an increased rate of aniline hydroxylation by microsomes from control and ethanol-induced rats. The difference between the rate of aniline hydroxylation by control microsomes and the rate by ethanol-induced microsomes was, however, abolished at higher acetone concentrations.     

Immunochemical and functional characterization of UDP-glucuronosyltransferases from rat liver, intestine and kidney

Glucuronidation of various substrates in hepatic, intestinal and renal microsomes of control, phenobarbital (PB), 3-methylcholanthrene (3MC) and Aroclor-1254 (A1254) pretreated rats was investigated. UDPGT activities tested could be divided in four groups on the basis of their tissue distribution and induction by PB or 3MC in liver microsomes. GT1 activities (1-naphthol, benzo(a)pyrene-3,6-quinol) are induced by 3MC in liver microsomes and are present in all tissues investigated. GT2 activities (morphine, 4-hydroxybipheynl) are induced by PB in liver microsomes and appear to be restricted to the liver and the intestine. UDPGT activity towards bilirubin, although induced by PB, can be detected in hepatic, intestinal and renal microsomes. UDPGT activity towards fenoterol is restricted to the liver and intestine and is not induced by PB, 3MC or A1254. The presence of inducible immunoreactive UDPGT isoenzymes in microsomes of liver, intestine and kidney of control and induced rats was demonstrated by immunoblot analysis using rabbit anti-rat liver-GT1 antibodies. Induction of both 54 and 56 kDa polypeptides in hepatitis, intestinal and renal microsomes by 3MC or A1254 was observed. Purification of UDPGT (1-naphthol as substrate) from intestinal microsomes to apparent homogeneity yielded a polypeptide with an apparent molecular weight of 54-56 kDa. The results indicate that 54 and 56 kDa UDPGT polypeptides are the major A1254 inducible isoenzymes in intestinal and renal microsomes. An increase in immunoreactive protein is correlated with a biochemically measurable increase in glucuronidation capacity for GT1 substrates.