Liver Glucuronyltransferase Research Articles

Comparison of enzymically glucuronidated flavonoids with flavonoid aglycones in an in vitro cellular model of oxidative stress protection

This study modeled, in vitro, the potential effect of conjugative (phase II) metabolism on the cytoprotective capacity of fruit flavonoids against oxidative stress. Flavonoid aglycones were compared with their corresponding isomeric mixtures of glucuronides for their ability to enhance the survival of cultured human Jurkat T and neuroblastoma cells stressed with hydrogen peroxide. Various polyphenolic compounds were tested as substrates in vitro for an ovine liver glucuronyl transferase preparation. Flavonoids and their glycoside derivatives were found to be good substrates, whereas phenolic acids were either poor or nonsubstrates. Five common flavonoids were glucuronidated to prepare mixtures for bioassay testing. Glucuronidation generally weakened the cytoprotective capacities of flavonoids (in the presence of H(2)O(2)), but some compounds were weakened much more than others. The concentration that halved cell death was well below 0.5 microM for most flavonoids tested, but glucuronidation increased median effective concentration values to a range of 1-16 microM. This compares with the generally accepted physiological range (0.1-10 microM) for circulating dietary polyphenolics detected in the body. Therefore, some flavonoids may retain a reduced cytoprotective capacity in vitro, after glucuronidation, whereas others may be effectively inactivated.

Interindividual variability in the glucuronidation and sulphation of ethinyloestradiol in human liver.

1. Glucuronidation and sulphation of ethinyloestradiol (EE2) was studied in human liver. Microsomal glucuronyltransferase activity was measured in 110 livers whose donors were 71 women and 39 men. Enzyme activity ranged between 12.6 and 242 pmol min-1 mg-1 protein, i.e. over a 19-fold range and the mean (+/- s.d.) glucuronyltransferase activity was 96.8 +/- 47.9 pmol min-1 mg-1 protein. 2. Cytosolic sulphotransferase activity was measured in 138 livers whose donors were 90 women and 48 men. Enzyme activity ranged between 14.4 and 98.2 pmol min-1 mg-1 protein, i.e. over a 7-fold range, and the mean (+/- s.d.) sulphotransferase activity was 43.7 +/- 18.6 pmol min-1 mg-1 protein. 3. Human liver glucuronyltransferase and sulphotransferase activities showed a unimodal distribution pattern. Enzyme activities were neither sex-related nor age-dependent. Sulphotransferase activity did not correlate with glucuronyltransferase activity (n = 80) suggesting that the two enzymes are independently regulated. The ratio of specific glucuronyltransferase to sulphotransferase activity ranged between 0.15 and 8.0 (mean +/- s.d., 2.44 +/- 1.51) and was unimodally distributed.

Mechanisms of the inhibitory action of p-hydroxyacetanilide on carcinogenesis by N-2-fluorenylacetamide or N-hydroxy-N-2-fluorenylacetamide.

The metabolism of N-2-fluorenylacetamide (FAA) and N-hydroxy-2-fluorenylacetamide (N-OH-FAA) was studied in groups of rats that had been prefed the protective agent p-hydroxyacetanilide (p-OH-AA) alone or in combination with each of the carcinogens for 4 weeks. Compared with controls, pretreatment increased the percentage of metabolites in the urine, chiefly as glucuronic acid conjugates, whereas the fecal excretion of FAA metabolites was lowered. The levels of total and tissue-bound material in the liver and blood plasma were also lower after prefeeding. Liver aryl hydrocarbon hydroxylase and liver deacetylase were not affected by p-OH-AA pretreatment. However, liver glucuronyl transferase was increased by either prefeeding with p-OH-AA and/or the carcinogen. The protective effect of p-OH-AA against liver tumor induction with FAA or N-OH-FAA may in part result from a combination of the decreased binding of carcinogen to hepatic cellular macromolecules and the increased excretion as the glucuronide conjugates.

The effect of gentamicin on liver glucuronyl transferase

Summary The effect of gentamicin on the liverenzyme glucuronyl transferase activity in vitro and on salicylamide glucuronide excretion in vivo was investigated. Little or no inhibition of the enzyme activity was observed after the addition of gentamicin to the substrate in concentrations similar to those observed in blood after the therapeutic administration of this agent. Only in concentrations greater than those produced by therapeutic doses was significant inhibition of the enzyme glucuronyl transferase activity observed. A significant decrease in urinary excretion of salicylamide glucuronide was observed after the administration of gentamicin. No reduction in the binding capacity of a 4 Gm. per cent crystalline human albumin solution for HBABA was observed in any of the drug dilutions.

Studies of the activation of UDP-glucuronyltransferase

Low concentrations of the detergents Triton X-100 and deoxycholate enhanced the activity of rat liver microsomal p-nitrophenol glucuronyltransferase 3-fold without radically altering the activities of pyrophosphatase or β-glucuronidase. Similar concentrations of the detergents, however, had no significant activating effect on the glucuronyltransferase activity of guinea pig liver microsomal fractions prepared in identical manner. Triton X-100 still activated the rat liver enzyme when competition between glucuronyltransferase and pyrophosphatase for UDP-glucuronate was reduced at a higher concentration of the sugar nucleotide and when β-glucuronidase had been substantially inhibited with saccharo-1,4-lactone. It is concluded that the closely associated pyrophosphatase and β-glucuronidase activities are not involved in the activation of the rat liver enzyme by detergents. The activation of rat liver glucuronyltransferase and the differences between that enzyme and guinea pig liver glucuronyltransferase are discussed.

Effect of Uridine-Diphosphate-Glucose on Liver Glucuronyl Transferase Activity in Normal and in Fasting Rats

The authors investigated the effect of uridinediphosphate glucose (UDPG) on the behaviour of liver glucuronyl transferase (GT) activity under different experimental conditions in 24 Sprague-Dawley rats.In the first series of experiments six rats received intraperitoneally 20 mg/kg of UDPG for 1 h; the second group was treated with 20 mg/kg of UDPG for 5 days and the third one with 50 mg/kg for 5 days. It was found that after UDPG treatment GT activity increases significantly, but with 50 mg/kg of UDPG the rise of enzyme activity is less important than with 20 mg/kg. 1-hour treatment does not influence GT activity. In the second series of experiments GT activity was investigated after a 72-hour fast in six rats and in six other rats receiving 20 mg/kg of UDPG 2 days before and during the 72-hour fast. Fasting caused an important decrease in GT activity, while enzyme activities did not change significantly in fasting rats treated with UDPG. The possible mechanisms by which UDPG exerts these effects are discussed.

Some effects of 3-methylcholanthrene on uridine diphosphate glucuronyltransferase in the rat and guinea pig

Treatment of young rats and guinea pigs with 3-methylcholanthrene significantly increased liver microsomal glucuronyltransferase activity. The earliest detectable increase in rat glucuronyltransferase activity occurred between 6 and 12 hr following administration of the polycyclic hydrocarbon, and after 48 hr, glucuronyltransferase activity in treated animals was twice that in control animals. Liver glucuronyltransferase activity was not effected when rats were pretreated with nikethamide, chlorcyclizine or phenobarbital. Administration of d, l-ethionine or actinomycin D, inhibitors of protein synthesis, prevented the increase in glucuronyltransferase activity following 3-methylcholanthrene administration. Kinetic analysis of glucuronyltransferase activity indicated that the V max was significantly increased in treated rats and guinea pigs. The K m of the enzyme from 3-methylcholanthrene treated rats was significantly decreased, whereas, the K m of the enzyme from treated guinea pigs was significantly increased. These changes in V max and K m suggest that induction of glucuronyltransferase by 3- methylcholanthrene may involve qualitative as well as quantitative changes in the enzyme.

Enhanced biliary excretion of thyroxine glucuronide in rats pretreated with benzpyrene

Pretreatment of male or female rats with a single injection of benzpyrene 48 hr before cannulation of the bile duct produced a 3- and 4-fold increase in billary excretion of exogenously administered or endogenously produced labeled thyroxine. Almost all of the increase could be accounted for by enhanced excretion of thyroxine glucoronide. The effect of benzpyrene on thyroxine glucuronide excretion could not be demonstrated 4·5–9·5 hr after administration of the drug. This is in contrast to results obtained with compounds such as sodium salicylate and butyl-4-hydroxy-3,5-diiodobenzoate, which acutely promote increased biliary excretion of thyroxine by displacing it from its binding sites on plasma proteins. It also contrasts with the effect of drugs such as propylthiouracil, which increase thyroxine excretion in bile by blocking the major alternate pathway of metabolism, deiodination. Liver slices and homogenates from benzpyrene-treated rats displayed a several-fold increase in capacity to form thyroxine or o-aminopherol glucuronides. Although optimal conditions for assaying thyroxine glucuronyl transferase in liver homogenates were not achieved, the several-fold increase in glucuronyl transferase activity observed in liver homogenates from benzpyrene-treated rats seems best explained by assuming a corresponding increase in the level of the enzyme. The augmented biliary excretion of thyroxine in benzpyrene-treated rats is also best explained on the basis of an increase in liver glucuronyl transferase. Phenobarbital pretreatment produced a small increase in labeled thyroxine excretion in bile, which was not limited to the glucuronide alone. There was no effect on thyroxine conjugation in vitro, although a slight increase in o-aminophenol conjugation could be demonstrated. The slight effect which occurred in vivo with thyroxine appeared to be correlated with an increase in bile flow rather than with an increase in glucuronyl transferase.

DEVELOPMENT OF GLUCURONYL TRANSFERASE AND OTHER ENZYME SYSTEMS IN THE NEWBORN RABBIT.

Dutch rabbits were found to be suitable animals for the study of the development of liver glucuronyl transferase activity during the neonatal period, as the activity was low at birth and increased to adult levels during the first 2 weeks of life.The neonatal development of a number of enzymes, which are mainly microsomal in character, has been studied. As the development patterns vary widely from enzyme to enzyme, and some display adult or greater than adult levels at birth, it is concluded that the low glucuronyl transferase activity at birth is not due primarily to a gross lack of endoplasmic reticulum.

Alterations in tissue levels of uridine diphosphate glucose dehydrogenase, uridine diphosphate glucuronic acid pyrophosphatase and glucuronyl transferase induced by substances influencing the production of ascorbic acid

Chemical agents known to enhance the production of l-ascorbic acid in animals were studied in vivo to determine their effects on tissue levels of three enzymes which may be concerned with the biosynthesis of the vitamin. The agents were found to have differential effects on the enzymes tested. Barbital and Chloretone, but not other enhancing agents, increase the activity of UDPG dehydrogenase in liver extracts. This alternation in activity is prevented by ethionine and by hypophysectomy. The guinea-pig enzyme was similarly affected by the two drugs, although ascorbic acid is not produced. Carcinogenic hydrocarbons and aminopyrine increase the activity of glucuronyl transferase in liver. The hepatic level of UDP-glucuronic acid pyrophosphatase, on the other hand, does not respond appreciably to the enhancing agents, although ethionine and barbital administered together elevate this enzyme in liver without stimulating ascorbic acid excretion. In vitro, barbiturates have an inhibitory action on the pyrophosphatase. The alterations in enzyme activities are discussed in relation to the possibility that ascorbic acid is synthesized via a pathway involving glucuronyl transferase rather than UDP-glucuronic acid pyrophosphatase.