The metabolism of foreign compounds in rats after treatment with polychlorinated biphenyls (PCBs)

Pretreatment of rats with a single dose of polychlorinated biphenyls caused an increase in the activity of UDP glucuronyltransferase (EC 2.4.1.17; acceptor unspecific) in liver microsomes towards the substrates p-nitrophenol and 4-methylumbelliferone. The results of kinetic experiments suggest enhanced enzyme activity is caused by enzyme induction. The effect was dependent on dose and chlorine content of the polychlorinated biphenyls administrated. The time course of UDP glucuronyltransferase activity after a single dose of polychlorinated biphenyls demonstrated a long lasting effect. Elevated biliary excretion of sulfobromophthalein conjugates after pretreatment with polychlorinated biphenyls was also demonstrated. In addition bile flow per g liver was increased and sulfobromophthalein concentration in serum and liver was reduced. Maximum effects were observed three days after treatment; thereafter these parameters slowly returned to normal levels.     

Nature of the enhancement of hepatic uridine diphosphate glucuronyltransferase activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats

After single low-level oral doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to rats, hepatic microsomal p-nitrophenol (PNP) glucuronyltransferase activity was elevated approximately 6-fold, whereas the hepatic glucuronyltransferase conjugating testosterone or estrone was unaffected. Solubilized and purified PNP glucuronyltransferase and steroid glucuronyltransferases from control and TCDD-treated rats exhibited the same relative activities (TCDD:control) as when the enzymes were bound to the endoplasmic reticulum. Elevation of PNP glucuronyltransferase was still evident 73 days after a single oral dose of 25 μg TCDD/kg. Female rats were more susceptible to TCDD actions on liver microsomal PNP glucuronyltransferase than males. The effects of TCDD treatment on PNP glucuronyltransferase appeared to be related to increased amounts of liver enzyme for the following reasons: (1) K_m values for PNP and UDPGA were unchanged by TCDD treatments; (2) the magnitude of the TCDD-induced increase of PNP glucuronyltransferase activity was the same whether enzyme activity was measured in the presence or absence of Mg²⁺ or Triton X-100; (3) TCDD, when added in in vitro, had no detectable effect on enzyme activity; (4) TCDD treatment of rats did not change total hepatic microsomal phospholipid or cholesterol contents: (5) pH optima were unaffected by TCDD treatment; (6) solubilization of enzyme was not accompanied by a change in the TCDD induction effect: and (7) actinomycin D appeared to block the initial phase of induction.     

Drug metabolism in Gunn rats: inability to increase bilirubin glucuronidation by phenobarbital treatment

The components of the microsomal mixed-function oxidase system in Wistar and Gunn rats were the same, as were the overall hydroxylation reactions (p-nitroanisole O-demethylase, aryl hydrocarbon hydroxylase). The Gunn rat was, however, almost totally devoid of bilirubin glucuronidation activity. The UDP glucuronosyltransferase activity towards p-nitrophenol in the Gunn rat was in liver about 60 per cent and in the small intestinal mucosa about 10 per cent of that in the Wistar rat. The mono-oxygenation step in hepatic microsomal drug metabolism was induced in a similar way by phenobarbital treatment in both Wistar and Gunn strain rats, but no significant enhancement of the mono-oxygenase activity could be observed in the gut. The trypsin digestion of hepatic microsomes increased the specific UDPglucuronosyltransferase activity. Phenobarbital treatment enhanced the p-nitrophenol glucuronidation in livers of both Wistar (3-fold) and Gunn strain (2-fold) rats, but the bilirubin glucuronidation was enhanced (2-fold) only in Wistar rats. Phenobarbital-enhanced transferase activities could be detected only if the microsomes had been digested with trypsin before determination of the enzyme activity. The UDPglucuronosyltransferase activity in the small intestinal mucosa was not enhanced by phenobarbital. The results suggest that bilirubin and p-nitrophenol are conjugated by distinct enzymes and that the enzyme activities for mono-oxygenation and glucuronidation are controlled by different mechanisms. The microenvironment of the UDP glucuronosyltransferase is most probably similar in Gunn and Wistar rats.     

INHIBITION OF CARP LIVER MITOCHONDRIAL MONOAMINE OXIDASE BY SOME COMMONLY-USED DETERGENTS

The inhibitory effects of some detergents commonly used in biochemical research on carp liver mitochondrial monoamine oxidase were examined. Sodium dodecylsulfate, octyl-β-D-glucopyranoside, sodium cholate and Triton X-100 at relatively low concentrations caused strong dose-dependent inhibition of the activity towards tyramine, but digitonin caused only weak inhibition. Sodium dodecylsulfate, octyl-β-D-glucopyranoside and sodium cholate caused almost complete Inhibition of activity in the concentration ranges tested. The extent of inhibition by Triton X-100 was greater after preincubation at 37°C for 30 min than that without preincubation, but with or without preincubation, the inhibition was not substrate-selective and was not complete at a relatively high concentration (2%) of Triton X-100. Without preincubation, the mode of inhibition by Triton X-100 was competitive and reversible with respect to the oxidations of 5-HT, tyramine and PEA, but after preincubation (37°C for 30 min), it became non-competitive and irreversible, depending on the concentration of detergent used. These findings suggest that it had different actions on the enzyme depending on preincubation. Triton X-100 also slightly changed enzyme sensitivity towards clorgyline and deprenyl, regardless of the preincubation time or the substrate used. Some possible mechanisms of the inhibitory effect of Triton X-100 are discussed.     

The rate-limiting step in the biliary elimination of some substrates of uridine diphosphate glucuronyltransferase in the rat

Phenolphthalein, 4-methylumbelliferone and 8-hydroxychinoline mutually inhibit each others enzymatic conjugation by microsomal UDP glucuronyltransferase (EC 2.4.1.17; acceptor unspecific) in vitro. After intravenous injection these UDP glucuronyltransferase substrates are excreted in the rat in bile as β-d-glucuronides. When these glucuronides were injected i.v. they were excreted partially in the bile and also in the urine. Ligation of the kidneys caused an increased biliary excretion of the i.v. injected glucuronides. To determine if these UDP glucuronyltransferase substrates also inhibited each others glucuronidation in vivo, two compounds were injected i.v. simultaneously into rats and the mutual effects on the biliary excretion of these compounds were studied. Phenolphthalein inhibited the biliary excretion of 4-methylumbelliferone in the form of its glucuronide conjugate in bile. This inhibition was not due to substrate competition for UDP glucuronyltransferase but to inhibition of excretion of the formed glucuronides from the liver cell into bile. From further experiments in which the mutual effects of the i.v. injected glucuronides on each others biliary excretion were studied it could be concluded that the rate-limiting step in the biliary elimination of phenolphthalein, 4-methylumbelliferone and 8-hydroxychinoline as glucuronides in the rat was the excretion of the products of UDP glucuronyltransferase activity in vivo into the lumen of the bile canaliculus and not conversion by UDP glucuronyltransferase.     

Non-specific inhibition of some rat liver membrane-bound enzymes by an adenylate cyclase inhibitor RMI 12330 A.

RMI 12330 A [N-(cis-2-phenlcyyclopentyl) azacyclotridecan-2-imine hydrochloride] inhibits choleratoxin-induced intestinal hypersecretion, presumably via an inhibition of the mucosal enzyme adenylate cyclase. We previously reported the inhibition by RMI 12330 A of the adenylate cyclase activity from rat liver plasma membrane preparations. We firstly extend here these results, showing the inhibition by RMI 12330 A of adenylate cyclase activity from spleen, brain, heart and kidney. Furthermore, in plasma membrane preparations, this agent inhibited the activities of Mg²⁺-ATPase and leucyl-β-naphthylamidase but had no effect upon 5′nucleotidase. In non-activated rat liver microsomes, the activities of p-nitrophenol and bilirubin UDP-glucuronosyltransferases, and of glucose-6-phosphatase, were enhanced by low concentrations of RMI 12330 A but inhibited by higher concentrations. This biphasic effect disappeared when digitonin-activated or Emulgen 911-solubilized microsomal membranes were used. Thus, RMI 12330 A does not behave as a specific inhibitor of adenylate cyclase, since it also perturbs other membrane-associated enzyme activities.     

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

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

Carbon disulphide induced activation of liver UDP glucuronosyltransferase in rats pretreated with phenobarbitone.

Carbon disulphide (CS2) exposure has been shown to activate the UDP glucuronosyltransferase of liver microsomes in rats pretreated with phenobarbitone. Now the nature of CS2 induced activation of the enzymes has been studied further. Phenobarbitone pretreated rats were exposed to 0.15% CS2 for 2 hrs on two successive days. The activity of UDP glucuronosyltransferase was measured from the liver microsomes after the enzymes was activated by incubation of the microsomes with various concentrations of the detergents Triton X-100, digitonin and cetylpyridinium chloride. The exposed animals showed an increased enzyme activity at all applied concentrations of the detergents; therefore in addition to membrane destruction by CS2 exposure, some other mechanism must also be involved in the CS2 induced activation of liver microsomal UDP glucuronosyltransferase. The changes in membrane lipid-protein interactions with l-anilino-8-naphthalene sulphonate (ANS) were also probed. The CS2 exposed animals had more high-affinity binding sites for ANS in their liver microsomal membranes, and in addition the quantum yield of ANS fluorescence was enhanced by CS2. The changes differed from those found after carbon tetrachloride exposure and suggest that, even if the two drugs have some common effects on microsomes, e.g. UDP glucuronosyltransferase activation, P-450 destruction and lipid peroxidation induction, the changes they cause in the microsomal micro-environment differ.     

Some effects of non-ionic detergent triton X-100 on rat liver monoamine oxidase

The non-ionic detergent Triton X-100, an agent used to solubilize mitochondrial membrane monoamine oxidase (EC 1.4.3.4, MAO), has been shown to inhibit markedly MAO activity. The inhibition was non-competitive in nature. Triton X-100 changed the susceptibility of MAO toward clorgyline, a specific type A MAO inhibitor, and deprenyl, a type B inhibitor. Its effect on the temperature dependence of the initial velocity revealed that the transition temperatures for p-tyramine and serotonin (22°) and β-phenylethylamine (16° and 27°) were not changed. The stability of the MAO decreased considerably, however, in the presence of Triton X-100, and its inactivation was particularly pronounced somewhat higher temperatures.     

Different effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on glucuronide conjugation of various aglycones. Studies in Wistar and Gunn rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) (20 μg/kg) was administered to homozygous Gunn and Wistar as well as heterozygous rats, and the activity of UDP-glucuronosyltransferase determined in the liver and kidney toward o-aminophenol, p-nitrophenol, 4-methylumbelliferone, and bilirubin. Diethylnitrosamine was used to activate the enzyme in vitro. TCDD did not enhance UDP-glucuronosyltransferase (UDPGT) activity in the Gunn rat, either in the liver or in the kidney. In the Wistar rat, the UDPGT activity toward methylumbelliferone was enhanced eightfold in the liver and twofold in the kidney. TCDD did not affect UDPGT activity toward bilirubin. Diethylnitrosamine activated o-aminophenol and p-nitrophenol glucuronidation in vitro in the liver only. No activation in vitro due to diethylnitrosamine treatment could be seen in the liver of the TCDD-treated Wistar rat.     

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.     

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.     

Metabolism of ketotifen by human liver microsomes. In vitro characterization of a tertiary amine glucuronidation

Biotransformation of ketotifen was investigated in vitro using human liver microsomes. Three of the four metabolic pathways observed in vivo in man were exhibited under the conditions of incubation, namely demethylation, N-oxidation, and N-glucuronidation, the absent route being the ketoreduction, which probably has a cytosolic localization. The kinetic parameters of the N-glucuronidation (KM for ketotifen and UDPGA and Vmax) were determined with native and detergent-treated microsomes. Treatment by Triton X-100 increased by about 3-fold the conjugation reaction. No sex difference was observed and N-glucuronidation did not seem to be inhibited either by bilirubin or by 4-nitrophenol. Thus, human liver microsomes are a useful and suitable in vitro model for studying metabolic routes, specific for man, as in the case of ketotifen. Obviously, the results obtained can only reflect partially the multiplicity of in vivo events and interpretation has to be complemented by investigations with other models.     

Properties of human hepatic UDP-glucuronosyltransferases. Relationship to other inducible enzymes in patients with cholestasis

Summary Glucuronidation of 4-nitrophenol, nopol (a monoterpenoid alcohol) and bilirubin, which in the rat, are catalyzed by three different enzymes, has been examined in liver biopsies from patients with various liver diseases, in particular cholestasis.These different activities were not correlated, which strongly suggests that at least three independently regulated forms of UDP-glucuronosyltransferases were present in the microsomes.Non ionic detergents (Triton X100, Emulgen 911) and deoxycholate produced similar activation (more than 2-fold) of the glucuronidation of 4-nitrophenol.Amphipathic substances, such as CHAPS (3-[3-cholamidopropyl-dimethylammonio]-1-propane sulfonate), and lysophosphatidylcholines maximally increased this UDP-glucuronosyltransferase activity, the most potent being oleoyl lysophosphatidylcholine (4-fold increase).Discriminant analysis of the data revealed no correlation between the three different UDP-glucuronosyltransferase activities and the age or sex of the patients.A good correlation was found on multidimensional analysis between form 1 of the enzyme (4-nitrophenol glucuronidation) and, in decreasing order of magnitude, epoxide hydrolase (measured with benzo(a)pyrene-4,5-oxide as substrate), cytochrome P-450, 7-ethoxycoumarin deethylase, aspartate aminotransferase and gamma-glutamyltransferase (r = 0.89); and between Form 3 of the enzyme (bilirubin glucuronidation) and NADPH cytochrome c reductase, alkaline phosphatase, (r=0.81).These relation ships may reflect the differential variation in enzymatic activities in various hepatobiliary diseases.     

Regulation of microsomal UDP-glucoronyltransferase--mechanism of activation by UDP-N-acetylglucosamine.

UDP-N-acetylglucosamine, in vitro, increases the rate of glucuronidation of p-nitrophenol by microsomal UDP-glucuronyltransferase. In contrast, UDP-N-acetylglucosamine inhibits the reverse reaction. Inhibition is competitive with respect to UDP. UDP-N-acetylglucosamine does not compete, however, with UDP-glucuronic acid in assays in the forward direction. Inhibition of the reverse reaction by UDP-N-acetylglucosamine must be due, therefore, to an allosteric effect. This was verified in studies of the extent of end-product inhibition by UDP in the presence and absence of UDP-N-acetylglucosamine. The mechanism of activation by UDP-N-acetylglucosamine is suited ideally for efficient function of this enzyme.     

Effect of metal salts on UDPglucuronosyltransferase activity of various aglycones in rat liver microsomes in vitro

The effects of aluminium, manganese, ferrous and ferric iron, cobalt, nickel, copper, zinc, cadmium, barium, mercury and lead ions on the rat hepatic UDPglucuronosyltransferase activity with regard to 4-methylumbelliferone, 4-nitrophenol, bilirubin, and 2-aminophenol were investigated in vitro. Conjugation of bilirubin was found to be activated by manganese, cobalt, nickel, cadmium, mercury and lead ions in native microsomes, but not in digitonin-treated microsomes. Aluminum, ferrous iron, cadmium, barium and lead increased the conjugation of 4-nitrophenol in native microsomes and aluminium in digitonin-treated microsomes as well. Lead slightly activated 4-methylumbelliferone conjugation, whereas none of the metal salts studied were found to activate the conjugation of 2-aminophenol. Copper and zinc salts inhibited all conjugations. In digitonin-treated microsomes, the conjugation of 4-nitrophenol was inhibited by cadmium and mercury, that of 4-methylumbelliferone by nickel and lead as well. The sensitivity of 4-methylumbelliferone conjugation to inhibition by mercury was most pronounced in microsomes prepared from rats pretreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.     

UDP-glucuronyltransferase in perfused rat liver and in microsomes—III. Effects of galactosamine and carbon tetrachloride on the glucuronidation of 1-naphthol and bilirubin

Galactosamine treatment (400 mg/kg, i.p., 4 hr) markedly decreased the level of UDP-glucuronic acid (UDPGA) and 1-naphthol glucuronidation in perfused liver. In contrast, bilirubin glucuronidation was not affected. In non-activated microsomes both 1-naphthol and bilirubin glucuronidation were dependent upon the concentration of UDPGA. In UDP-N-acetylglucosamine-activated microsomes, 1-naphthol glucuronidation remained dependent upon UDPGA whereas bilirubin glucuronidation tended to be independent of UDPGA.Carbon tetrachloride treatment (5 ml/kg, per os, 24 hr) strongly decreased 1-naphthol glucuronidation in the intact liver without altering the level of UDPGA. Bilirubin glucuronidation was affected similarly but to a lesser extent, In contrast. 1-naphthol glucuronidation in liver microsomes was increased under these conditions. In the presence of UDP-N-acetylglucosamine and UDP, however, enzyme activity in microsomes from CCl₄-treated rats was lower than in control microsomes.The results suggest a differential regulation of 1-naphthol and bilirubin glucuronidation and stress the importance of intracellular effectors for glucuronidation in the intact liver.     

Effect of triton X-100 on the conjugation of tetrahydrocortisone, in vitro

The detergent, Triton X-100, increased the conjugation of estrone, estradiol and tetrahydrocortisone (THE) by uridine diphosphate glucuronyl transferase (UDPGT) in rat liver microsomes; the maximal increase of the conjugation of these substrates was measured when the concentration of Triton in the incubation mixtures was 0.05 per cent. However, the magnitude of the increase and the effect seen with varying Triton concentration were substrate dependent, which is consistent with the hypothesis that multiple forms of UDPGT may be present in hepatic microsomes. The effects of various compounds which had previously been shown to either increase or decrease the conjugation of THE in non-activated enzyme preparations were re-examined in Triton-activated preparations. Compounds such as β-diethylaminoethyldiphenylpropylacetate (SKF-525A) and 7-hydroxychlorpromazine which inhibited conjugation in non-activated preparations also inhibited conjugation in Triton-activated preparations. Alternatively, the demethylated metabolites of chlorpromazine, which increased activity in non-treated preparations, decreased activity slightly in preparations maximally stimulated by Triton. Bisubstrate kinetic analysis of the THE conjugation of UDPGT also revealed differences between the properties of the non-treated and Triton-activated enzyme preparations. Triton activation caused an increase in the V_max of the reaction in the forward direction while having an insignificant effect on the dissociation constant for THE.     

Action of styrene and its metabolites styrene oxide and styrene glycol on activities of xenobiotic biotransformation enzymes in rat liver in vivo

The effects of the administration of styrene and its metabolites, styrene oxide and styrene glycol, on drug metabolizing enzymes of rat liver were studied. Styrene (three or six daily doses of 500 g/kg) doubled the activities of microsomal p-nitroanisole O-demethylase, epoxide hydrase (styrene oxide as substrate), and UDP glucuronosyltransferase (p-nitrophenol) when measured in microsomes pretreated in vitro with digitonin or trypsin. On the other hand, glycine conjugation, the cytochrome P-450 content, and activities of NADPH cytochrome c reductase and benzpyrene hydroxylase, were less affected by styrene. Styrene oxide was more toxic to rats than styrene or styrene glycol and it also caused (one dose of 375 mg/kg) a significant decrease in the activities of benzpyrene hydroxylase and p-nitroanisole O-demethylase and in the cytochrome P-450 content. Epoxide hydratase NADPH cytochrome c reductase, and UDP glucuronosyltransferase were more resistant towards styrene oxide. Styrene glycol did not significantly alter the activities of drug metabolizing enzymes. The current data show that styrene causes an increase in the activities of some enzymes involved in its own metabolism.     

Studies of monoamine oxidases: Effect of Triton X-100 and bile salts on monoamine oxidase in brain mitochondria

Triton X-100 and the bile salts, cholate and deoxycholate, detergents often used in the solubilization of monoamine oxidase (MAO) from mitochondria, have been found to cause an inhibition of the enzyme activity. With beef brain mitochondria, it was found that there was a differential effect of Triton X-100 on the putative MAO types A and B, with MAO-A being more susceptible to inhibition by Triton X-100. This was indicated by the greater loss of serotonin-deaminating than of phenyl ethylamine-deaminating activity in the presence of Triton X-100. Although the bile salts also caused substantial inactivation at concentrations above 0.1%, no differentiation between MAO types could be made. Kinetic studies of the inhibition by Triton X-100 indicated two different mechanisms were occurring with the two MAO types. The inhibition was competitive for MAO-A, but uncompetitive for MAO-B. Removal of Triton X-100 by co-polymer beads restored some, but not all of the activity for both MAO-A and MAO-B types. This suggests that the activity loss may have been due in part to inactivation when the enzyme was separated from the mitochondrial membrane.