Activation Of UDP-glucuronyltransferase Research Articles

Melatonin protects the cytochrome P450 system through a novel antioxidant mechanism

Melatonin, an endogenous hormone, is used as an antioxidant drug in doses quite higher than the endogenous circulating levels of this hormone. Hepatic endoplasmic reticulum contains the cytochrome P450 (CYP450) system, which catalyzes one biotransformation pathway of melatonin; this organelle is also one of the main sources of reactive oxygen species in cells. Therefore, we proposed that the antioxidant activity of this hormone may have a biological relevance in the organelle where it is biotransformed. To evaluate this postulate, we used Fe(3+)/ascorbate, an oxygen free radical generating system that leads to lipid peroxidation, loss of protein-thiol content, and activation of UDP-glucuronyltransferase in rat liver microsomes. We found that mM concentrations of melatonin prevented all these oxidative phenomena. We also found that Fe(3+)/ascorbate leads to structural alterations in the CYP450 monooxygenase, the enzyme that binds the substrate in the CYP450 system catalytic cycle, probably through direct oxidation of the protein, and also inhibited p-nitroanisole O-demethylation, a reaction catalyzed by the CYP450 system. Notably, melatonin prevented both phenomena at microM concentrations. We provide evidence suggesting that melatonin may be oxidized by oxygen free radicals. Thus, we postulate that melatonin may be acting as an oxygen free radical scavenger, and Fe(3+)/ascorbate-modified melatonin would be directly protecting the CYP450 system through an additional specific mechanism. Pharmacological relevance of this phenomenon is discussed.

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.

Effects of -naphthyl isothiocyanate and a heterocyclic amine, PhIP, on cytochrome P-450, mutagenic activation of various carcinogens and glucuronidation in rat liver

To elucidate the mechanism underlying suppression by alpha-naphthyl isothiocyanate (ANIT) of mammary carcinogenesis induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), we evaluated hepatic levels of cytochrome P-450 (CYP) enzymes, mutagenic activation of environmental carcinogens and UDP-glucuronyltransferase (UDPGT) activities in female Sprague-Dawley rats fed a high fat diet. Immunoblot analyses revealed induction of CYP1A1, newly found 51 and 53 kDa proteins and constitutive CYP1A2 and 2B2 by intragastric treatment with 85 mg/kg PhIP eight times for 11 days. Although the extents of induction were not as high as in the case of PhIP, 3 weeks feeding of 400 p.p.m. ANIT induced CYP1A1 and the 51 and 53 kDa proteins. CP1A2 level was decreased by the feeding of ANIT. The mutagenicity in strain TA98 of PhIP, four other heterocyclic amines (HCAs) and benzo[a]pyrene was greatly enhanced in the presence of liver S9 mix prepared from rats pretreated with PhIP but not with ANIT. The mutagenicities of these five HCAs were significantly decreased in the presence of liver S9 from rats pretreated with a combination of PhIP and ANIT as compared with that pretreated with PhIP alone. The level of hepatic CYP1A2, which is known to be involved in the metabolic activation of PhIP, was consistently decreased in liver microsomes from rats administered PhIP plus ANIT as compared with that from rats administered PhIP alone. On the other hand, UDPGT activity towards 4-nitrophenol (4-NP) was enhanced using liver microsomes prepared from rats pretreated with a combination of PhIP and ANIT as compared with those pretreated with PhIP or ANIT alone. These results show that chemoprevention by ANIT against PhIP-induced rat mammary carcinogenesis can be explained by a dual action mechanism, i.e. a reduction in metabolic activation by hepatic CYP1A2 and an enhancement of detoxification by 4-NP UDPGT. The role of the newly found 51 and 53 kDa proteins in activation of HCAs is also discussed.

Activation and stabilization of UDP-glucuronyltransferase by lysophosphatidylcholine.

Interactions between purified UDP-glucuronyltransferase from 3-methylcholanthrene-treated rat liver microsomes (named GT-1) and lysophosphatidylcholine, which is essential for expression of GT-1 activity, were examined. Phospholipid-free GT-1, which could not express its full activity [Yokota et al. (1988) J. Biochem. 104, 531-536], was activated fully by addition of lysophosphatidylcholine (0.04 mM final concentration) into the assay medium. Lysophosphatidylcholine also protected GT-1 effectively against heat inactivation. Palmitoyllysophosphatidylcholine and stearoyllysophosphatidylcholine were most successful for the activation and stabilization of GT-1. On treatment of GT-1 with carboxypeptidase Y, the transferase was inactivated immediately, but the treatment in the presence of lysophosphatidylcholine affected the activity only a little. Lysophosphatidylcholine was also found to protect GT-1 against cleavage by carboxypeptidase Y. On treatment of GT-1 with trypsin or aminopeptidase T, the activity was lost and GT-1 protein could be digested even when lysophosphatidylcholine was present. It is suggested that UDP-glucuronyltransferase forms an active and stable conformation, in which the carboxy-terminal region is protected against protease, with lysophosphatidylcholine.

Mercury-induced UDPglucuronyltransferase (UDPGT) activity in mouse kidney

Administration of mercuric chloride to young adult mice produced a significant increase in the activity of renal UDPglucuronyltransferase (UDPGT) measured with harmol as the acceptor substrate. This was observed 10 days after a daily oral dose of HgCl 2 (6 μg Hg 2+/g body wt.). The increase in UDPGT activity was correlated with an accumulation of mercury in the renal tissues and was accompanied by an increase in the apparent V max of the glucuronidation reaction without a change in the apparent K m values for harmol or UDPGA. Parallel studies with mercuric sulfide however showed negligible retention of mercury in both the liver or kidney nor was there any change in UDPGT activity compared to control values. The difference in solubilities of the two mercuric salts may be responsible for this observation. The possible mode of activation of UDPGT by mercury treatment is discussed.

Denaturation of cytochrome P-450 by indomethacin and other non-steroidal antiinflammatory drugs: Evidence for a surfactant mechanism and a selective effect of a p-chlorophenyl moiety

Indomethacin added to rat liver microsomes in vitro resulted in the denaturation of cytochrome P-450 to cytochrome P-420. This was NADPH independent, appeared to be non-enzyme mediated, did not involve free radicals or lipid peroxidation and was prevented by glycerol, butylated hydroxytoluene or SKF-525A. Indomethacin in vitro also caused a loss of cytochrome b 5, NADH-cytochrome b 5 reductase, NADPH-cytochrome c reductase and epoxide hydrolase activities, but an activation of UDP-glucuronyltransferase. Amongst a total of 22 non-steroidal anti-inflammatory drugs and derivatives there was a highly significant correlation between the extent of their denaturation of cytochrome P-450 and their surfactant potency. The results suggest that the denaturation of cytochrome P-450 by certain non-steroidal anti-inflammatory drugs was due to a detergent-like, membrane-perturbing action of the drugs and that in most cases the denaturation also involved a specific effect of a p-chlorophenyl moiety of the drug.

Inhibition and activation of UDP-glucuronyltransferase in alloxanic-diabetic rats

1. 1. Short or long term alloxan diabetes produced activation of oestrone and morphine glucuronidation and inhibition of p-nitrophenol glucuronidation in rat liver microsomes. 2. 2. Insulin treatment restored decreased glucuronyltransferase (GT) activity for p-nitrophenol and it did not abolish diabetes activation on oestrone glucuronidation. 3. 3. Triton X-100 detergent activation reduced differences between normal, diabetic and insulin treated rats in the glucuronidation rates of the substrates assayed. 4. 4. 1,4-Benzodiazepines inhibited morphine GT activity and stimulated oestrone GT activity in normal, diabetic and insulin treated diabetic rats. 5. 5. Activation and inhibition of GT activities for oestrone and xenobiotics in diabetes mellitus appears to be related with membrane perturbations of liver microsomes.

Characteristics of renal UDP-glucuronyltransferase

Rat renal microsomes catalyzed the glucuronidation of 1-naphthol, 4-methylumbelliferone and p-nitrophenol, whereas morphine and testosterone conjugation were not detected. In contrast, all five substrates were conjugated by hepatic microsomes; the activity was typically 5–10 times greater than with renal microsomes. Renal microsomal UDP-glucuronyltransferase toward 1-naphthol was fully activated (six-fold) by 0.03% deoxycholate while the hepatic enzyme was fully activated (eight-fold) by 0.05% deoxycholate. Full activation of hepatic UDP-glucuronyltransferase occurred when microsomes had been preincubated at 0 C with deoxycholate for 20 min. This effect of preincubation was not observed with renal microsomes. The presence of 0.25M sucrose in the buffers during renal microsomal preparation resulted in a two-fold greater rate of 1-naphthol conjugation in both unactivated and activated microsomes than renal microsomes prepared in phosphate buffers alone. Preparation of hepatic microsomes with or without 0.25M sucrose had no effect on UDP-glucuronyltransferase activity. Unactivated (-deoxycholate) renal enzyme was activated when incubations were done at a low pH (5.7), whereas fully activated (0.03% deoxycholate) renal microsomal UDP-glucuronyltransferase displayed a pH optimum at 6.5. Renal microsomal UDP-glucuronyltransferase activity toward 1-naphthol, p-nitrophenol and 4-methylumbelliferone was induced by pretreatment of rats with β-naphthoflavone and trans-stilbene oxide but not by phenobarbital or 3-methylcholanthrene. These data demonstrate that renal UDP-glucuronyltransferases are different from the hepatic enzymes with regard to biochemical properties, substrate specificity and in response to chemical inducers of xenobiotic metabolism.

Substrate Specific Activation of UDP-Glucuronyl Transferase by GUT Polypeptides in Rat Lives

Substrate Specific Activation of UDP-Glucuronyl Transferase by GUT Polypeptides in Rat Lives

Effects of activation of UDP-glucuronyl transferase on metabolism of benzo[a]pyrene with rat liver microsomes.

The effects of Brij 58 on microsome-mediated benzo[a]pyrene (BP)-metabolism were investigated. At a concentration of 0.01%, which did not decompose the microsomal mixed-function oxidase system, Brij 58 increased UDP-glucuronyl transferase activity on 3-hydroxy-BP to 560% of the control level. Quantitative changes of BP-metabolites by glucuronidation were found by high performance liquid chromatography. On incubation in the presence of UDP-glucuronic acid, the amounts of phenols decreased but the amounts of dihydrodiols did not change. Brij 58 enhanced the decrease in phenol metabolites. Unexpectedly, the binding of BP-metabolites to DNA was found to be higher in the presence of UDP-glucuronic acid, and Brij 58 also enhanced the binding in the presence of UDP-glucuronic acid. The ratio of bound adducts with BP-7,8-dihydrodiol-9,10-oxide to those with 9-hydroxy-BP-4,5-oxide, which were separated by Sephadex LH-20 column chromatography, was increased by UDP-glucuronic acid and further enhanced by Brij 58. These results are discussed in relation to the role of glucuronidation in metabolism of BP.

Activation and inhibition of rat hepatic UDP-glucuronyltransferase by mercuric acetate.

1. The effects of mercuric acetate on glucuronide conjugation of bilirubin and 4-methylumbelliferone in vitro represent a new type of modifier of UDP-glucuronyltransferase activity. 2. Bilirubin UDPG-transferase was normally activated (optimally 4-fold), inhibition was seen only at high concn. of Hg2+. 4-Methylumbelliferone UDPG-transferase was normally inhibited by Hg2+, but at high concn. of UDPGA (10 mmol/l) slight activation was seen. 3. Activation of bilirubin conjugation was unaffected by Mg2+ concn., indicating that the mechanism of Hg2+ activation differs from that caused by Mg2+. 4. Glucuronidation of bilirubin in liver microsomes from control rats, or rats treated with phenobarbital, methylcholanthrene or TCDD respond similarly to Hg2+, confirming with phenobarbital, methylcholanthrene or TCDD respond similarly to Hg2+, confirming the view that bilirubin conjugation is not inducible by these inducers.

The inherited deficiency of hepatic UDP-glucuronyltransferase: Structure-activity relationships of in vitro stimulators

More than 18 compounds have been tested for their ability to stimulate defective UDP-glucuronyltransferase activity of Gunn rat liver homogenates towards 2-aminophenol, up to levels of transferase activity in similarly treated Wistar rat liver homogenates. The minimum structural requirements of an effective compound are a combination of the presence of an electron-attracting atom or group and a sparing solubility in water. The activation of defective UDP-glucuronyltransferase towards 2-aminophenol by pentan-3-one is reversible. The possible mechanism of action of alkyl ketone activators is discussed.

Activation and induction of rat liver microsomal UDP-glucuronyltransferase with 3-hydroxybenzo(a)pyrene and N-hydroxy-2-naphthylamine as substrates

Activation and induction of rat liver microsomal UDP-glucuronyltransferase with 3-hydroxybenzo(a)pyrene and N-hydroxy-2-naphthylamine as substrates

Latency of epoxide hydratase and its relationship to that of udpglucuronyltransferase

Epoxide hydratase activity in liver microsomal preparations from adult male rats is latent to a slight extent. Maximal activations with neutral or anionic detergents were 30–60% whilst UDPglucuronyltransferase was maximally activated by 160–830% by the same detergents. Activation of microsomal epoxide hydratase requires much higher amounts of neutral or anionic detergents than activation of microsomal UDPglucuronyltransferase. High concentrations of inorganic salt, sonication or freeze-thawing which activate microsomal UDPglucuronyltransferase have no influence on microsomal epoxide hydratase activity. From this it appears that the activation which may involve either removal of a permeability barrier or release from conformational restraint occurs more easily for UDPglucuronyltransferase than for epoxide hydratase and that the activation of microsomal epoxide hydratase requires breakage of some hydrophobic bonds between the enzyme and membrane components(s).

Activation of hepatic microsomal glucuronyltransferase from gunn rats by exposure to light

Exposure of hepatic microsomes from icteric and nonicteric rats to 250 foot candles of blue fluorescent light for 4 hours at 4–6°C significantly increased the activity of UDPglucuronyltransferase activity using p-nitrophenol as a substrate. The light exposure reduced serum bilirubin concentration from icteric rats or bilirubin of fortified human serum albumin by 70 per cent but there was no difference in light activation of hepatic microsomes from icteric or nonicteric rats. Light exposure also decreased the activation of UDPglucuronyltransferase produced by Triton X-100.

Regulation of microsomal enzymes by phospholipids: IX. Production of uniquely modified forms of microsomal UDP-glucuronyltransferase by treatment with phospholipase A and detergents

The kinetic parameters of microsomal UDPglucuronyltransferase (EC 2.4.1.17) were compared after treatment of microsomes with phospholipase A and Triton X-100. Treatments with phospholipase A and Triton had differential effects on activities at V (assayed in both forward and reverse directions), the affinity of the enzyme for UDPglucuronic acid and UDP, the specificity of binding of UDPsugars, and the sensitivity of the enzyme to treatment with UDP- N-acetylglucosamine. Comparisons of lysophosphatide, Triton, and cholate-activated forms indicated that different types of detergents activate by separate mechanisms. These data are taken as evidence that selective types of changes of the composition and structure of microsomal lipids lead to selective types of changes of the properties of UDPglucuronyltransferase. Activation of UDPglucuronyltransferase in response to different types of perturbations of the membrane lipids is thus a specific process. It was found that constraints on the maximal potential activity of the reverse reaction catalyzed by UDPglucuronyltransferase is more extensive in untreated microsomes than constraint on the forward reaction. Also, the “activated forms” of UDPglucuronyltransferase have less activity than the untreated enzyme if assays are carried out under conditions presumed to exist in vivo. These results indicate the complexity of lipid-protein interactions as regulators of membrane-bound enzymes. They also indicate the functional significance of this type of regulation for the activity of UDPglucuronyltransferase.

Action of n-alkanes on drug-metabolizing enzymes from guinea-pig liver

Treatment of guinea-pig liver microsomal preparations with n-pentane, n-hexane and n-heptane enhanced both p-nitrophenol and o-aminophenol glucuronidation. The apparent K m values of UDPglucuronate, for both p-nitrophenol and o-aminophenol UDPglucuronyltransferase, and of o-aminophenol were considerably increased, whereas the apparent K m value of p-nitrophenol was the same after n-pentane treatment. No solubilization of UDPglucuronyltransferase or other microsomal proteins was observed after treatment with the n-alkanes. However, a substantial release of phospholipids from microsomal membranes occurred. The relative capacity of the different alkanes to release phospholipid was the same as the relative activation of UDPglucuronyltransferase. Aniline p-hydroxylation was considerably increased by the alkanes at concentrations of about 15–30 times lower than those necessary for optimal activation of UDPglucuronyltransferase. At these concentrations no activation of the aminopyrine N-demethylation was observed, whereas at higher concentrations a gradual decrease in both N-demethylating and p-hydroxylating activities occurred. n-Heptane, at a concentration which optimally activated aniline p-hydroxylation, markedly increased the rate of NADPH-cytochrome P-450 reduction. This suggests that the increase of the p-hydroxylating rate by n-heptane is related to a reversal of the cytochrome P-450 reduction by aniline.

On the activation of microsomal UDPglucuronyltransferase by phospholipase A

Activation of rat liver microsomal UDPglucuronyltransferase (EC 2.4.1.17) by phospholipase A (EC 3.1.1.4) was reversed by micellar dispersions of several phospholipids and by serum albumin. When microsomal membranes were treated with the phospholipase in presence of albumin, which binds and removes surface-active phospholipid degradation products (lysophosphatides and unsaturated fatty acids), activation was delayed by albumin at a concentration of 1 mg/mg of microsomal protein and completely prevented by higher concentrations of albumin. The UDPglucuronyltransferase of intact microsomal membranes was activated by lysophosphatidylcholine and unsaturated fatty acids at concentrations which were produced by phospholipase treatment, and the activation by lysophosphatidylcholine most closely resembled that by phospholipase A. It is concluded that the activation of UDPglucuronyltransferase by phospholipase A is due to the detergent activity of phosphilopid hydrolysis products and that the enzyme in intact microsomal membranes probably is not constrained by interaction with a specific phospholipid.

Lipid peroxidation of rat-liver microsomes. Its effect on the microsomal membrane and some membrane-bound microsomal enzymes.

Rat liver microsomes were peroxidized in vitro and chemical, physical and morphological changes in the membrane were related to the effects on certain membrane‐bound microsomal enzymes.With increasing peroxidation, microsomal phospholipids revealed decreasing concentrations of 20:4 and 22:6 fatty acids. At a high level of peroxidation, where alterations in permeability and ultrastructural changes such as breakage and deformation of microsomal vesicles were apparent, there was also a decrease in the concentration of 18:2 fatty acids.Glucose‐6‐phosphatase, cytochrome P‐450 and uridine‐5′‐diphosphate glucuronyltransferase (UDP glucuronyltransferase) (three microsomal enzymes known to be highly dependent on the integrity of the membrane for their function) responded differently to lipid peroxidation. Glucose‐6‐phosphatase showed an initial decrease in activity as well as in Km for glucose 6‐phosphate, followed by a restoration to control values at an intermediate level of lipid peroxidation. At a high level of peroxidation, both kinetic parameters were again markedly decreased. Cytochrome P‐450 concentration, on the other hand, revealed a continuous decrease with proceeding lipid peroxidation, which was only partly accounted for by a conversion to cytochrome P‐420. The loss of cytochrome P‐450 was paralleled by decreased rates of aminopyrine demethylation and 3,4‐benzpyrene hydroxylation. Glucuronyltransferase activity, finally, was activated at low, but again normalized at high, levels of lipid peroxidation.It thus seems that although electron‐microscopically observable changes in the microsomal membrane occurs only late in lipid peroxidation, chemical, physical and enzyme alterations are early phenomena during this process. The continuous loss of cytochrome P‐450 and the final decrease in glucose‐6‐phosphatase late during peroxidation probably reflect damage to membrane lipids of vital importance for the integrity and function of these enzymes whereas the activation of UDP glucuronyltransferase early during peroxidation most likely can be ascribed to increased membrane permeability.

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.