Search

Microsomal preparations from the liver of female sheep can effect the transfer of glucose from UDP-glucose to the 17α-hydroxyl group of 17α-estradiol-3-glucuronide, and, in much poorer yield, to some other steroids containing a 17 α-hydroxyl group. No evidence was found for steroid galactosyl or N-acetylglucosaminyl transferase activity in sheep liver. Glucuronyl transferase with an activity of a high order to a variety of substrates was present. Preliminary work indicated that the steroid glucosyl transfer is mediated by an enzyme distinct from glucuronyl transferase.

Sex variation of drug-metabolizing enzymes was studied in liver and small intestine of Wistar rats. Hydroxylation of 3,4-benzρyrene was much lower in female than in male liver microsomes, but glucuron

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.

The glucuronic acid adducts of 1-naphthol, 2-naphthol and 4-methylumbelliferone activate microsomal UDP-glucuronyltransferase (EC 2.4.1.17) when the enzyme is assayed with p-nitrophenol as aglycone. Phenyl glucuronide and oestriol 3β-glucuronide also activate UDP-glucuronyltransferase. but to a lesser extent. Activation by glucuronides is not dependent on metal ions, but is blocked by prior treatment of microsomal fractions with p-chloromercuribenzoate. The kinetic mechanism of activation is concluded to be an increase in the affinity of the enzyme for UDP-glucuronic acid. Activation by 1-naphthyl glucuronide, at high concentrations of p-nitrophenol, is not affected by 1-naphthol. Apparently 1-naphthyl glucuronide activates the preparation by binding at a site that is separate from the site of glucuronidation of 1-naphthol. Further evidence for the existence of distinct effector sites for the glucuronides was provided by the finding that activation by glucuronides is inhibited competitively by aglycone glucosides. These glucosides do not inhibit the rate of glucuronidation of p-nitrophenol in the absence of glucuronide adducts, nor do they alter the rate of glucuronidation of 1-naphthol. When UDP-glucuronyltransferase is assayed with 1-naphthol as aglycone it is activated by p-nitrophenyl glucuronide, 4-methyl-umbelliferyl glucuronide and under appropriate conditions by its own glucuronide. These activations are similarly inhibited by aglycone glucosides. p-Nitrophenyl glucuronide also stimulates the rate of glucuronidation of o-aminophenol, o-aminobenzoate and bilirubin.

1. 1. UDP-glucuronyltransferase activity towards oestriol has been demonstrated in liver preparations from foetal and adult human and mouse, and from chick embryo and chick. It was also found in foetal human kidney and gut, but appeared absent from placenta. 2. 2. In human foetal liver and kidney levels of UDP-glucuronyltransferase activity towards oestriol were as low as those towards o-aminophenol, and developed with age. 3. 3. No UDP-glucosyltransferase activity towards oestriol was detected in human foetal preparations. 4. 4. UDP-glucuronyltransferase activity towards oestriol develops precociously in cultures of chick embryo liver; in cultures of human foetal liver the transferase activity towards oestriol declines whilst increasing towards o-aminophenol. 5. 5. Pre-treatment with phenobarbital increased UDP-glucuronyltransferase activity towards oestriol in chick embryo liver, both in ovo and in culture. Pre-treatment with phenobarbital increased this activity in maternal mouse liver but not in foetal liver, in utero or in culture, nor in cultured human foetal liver. 6. 6. UDP-glucuronyltransferase activities towards oestriol and towards o-amino phenol differ markedly in development, in culture and in response to phenobarbital pre-treatment and activation procedures; heterogeneity of the enzyme is probably responsible.

SummaryIn female rats, ligature of the common bile duct for 5 weeks caused hepatic enlargement, bile duct-proliferation and hyperplasia of the smooth endoplasmic reticulum (SER). Biochemically, however, there was a decrease of microsomal protein and cytochrome P-450 content, of NADPH cytochrome C reductase and bilirubin-UDP-glucuronyl-transferase activities, as well as of ethylmorphine N -demethylation indicating that bile duct-obstruction induced hypertrophic but hypoactive SER. Prior and continued administration of pregnenolone- 16α-carbonitrile (PCN) to ligated animals corrected all these biochemical changes showing that the hypoactivity of the SER is reversible.

After force-feeding a protein-free diet to male rats for 5–7 days a substantial (2.4-fold) increase in the specific activity of the liver microsomal enzyme UDP-glucuronyltransferase (EC 2.4.1.17) was observed. A similar activation of the enzyme occurred when rats were fed on a low-protein (5%, w/w, casein) diet for 60 days. Although both the short- and long-term protein-deficient diets decreased the contents of microsomal protein and phospholipid in liver tissue they did not significantly alter the ratio of these major membrane components. Protein deficiency profoundly altered the phospholipid composition of microsomal membranes. The most striking difference in microsomal phospholipid composition between control and protein-deficient rats was their content of lysophosphatides. Whereas microsomal membranes from protein-deficient rats contained significant proportions of lysophosphatidylcholine and lysophosphatidylethanolamine very little or no lysophosphatides were detected in control preparations. Pretreatment of microsomal fractions from normal rats with phospholipase A markedly increased their UDP-glucuronyltransferase activity as did their pretreatment with lysophosphatidylcholine. It is concluded that the quantities of lysophosphatides present in microsomal membranes from protein-deficient rats were sufficient to have caused the increased UDP-glucuronyltransferase activities of these preparations. Evidence is presented suggesting that these changes in microsomal phospholipid composition and UDP-glucuronyltransferase activity caused by protein deficiency reflect changes that occur in vivo. The possible physiological significance of these findings is discussed.

Rats bearing subcutaneous Walker 256 carcinoma or intramuscular Reuber H-35 hepatoma were tested for their ability to metabolize 14 C- p -nitrophenol into its sulfate and glucuronide. Amounts of p -nitrophenyl glucuronide (PNPGA) and p -nitrophenyl sulfate (PNPS) excreted by the Walker hosts did not differ significantly from those excreted by the controls. Hepatoma hosts, compared with controls, excreted comparable amounts of PNPS but significantly higher amounts of PNPGA. To clarify the basis for the changes in the conjugate levels in urine of the hepatoma hosts, sulfotransferase and glucuronyl transferase activities of the hepatoma and of host and control livers were assayed. The hepatoma possessed significantly higher glucuronyl transferase activity than either type of liver, but had a very low sulfotransferase activity. Microsomal glucuronyl transferase of the hepatoma obeyed Michaelis-Menten kinetics; K m value for p -nitrophenol is 0.22 mM. Liver transferase(s) gave different and irregular kinetics, suggesting qualitative differences between the transferases of the hepatoma and liver.

Hexachlorophene (HCP) at oral doses of 30–50 mg/kg causes significant increases in hexobarbital sleeping time in Wistar and Sprague-Dawley rats, with the maximum effect occurring 6 hr after administration of the bisphenol. Liver microsomal 0-demethylase activity is simultaneously reduced in rats receiving HCP. Incubation of rat liver microsomes with concentrations of HCP as low as 0.38 nmole/mg of microsomal protein in vitro inhibits the O -demethylase, nitroreductase and phenol UDP-glucuronyl transferase systems and also causes a reduction in the apparent content of cytochromes P-450 and b 5 . The concentrations of HCP required to produce a 50 per cent inhibition or reduction in apparent cytochrome contents in vitro range between 4.7 and 98 nmoles HCP/mg of microsomal protein. Some evidence for a common inhibitory mechanism, perhaps involving interaction of HCP with the microsomal membrane, was obtained for the hepatic mixed function oxidase and cytochrome systems.

There exists clinical evidence that coupling of chloramphenicol with glucuronic acid is impaired in severe liver disease. To evaluate the theoretical basis of these observations the affinity of UDP-glucuronyl transferase to chloramphenicol was estimated in comparison with other substrates of the enzyme by determining the Michaelis-Menten constants.

When a very effective glucosidurogenic compound, eugenol, was administered to the rat, the following results were obtained on the metabolic response of the glucuronidation system. 1) After a single oral administration of eugenol, a large amount of ether-type glucuronide excreted was found in the urine collected over the 0-12 or 12-24 hour period after the administration. It was reduced rapidly to a normal level. 2) After the same administration, the specific activity of UDP-glucuronyltransferase in the liver started to increase. gradually at 12 hours, reaching its maximum value at 48 hours, and then declined slowly. Liver weight showed a tendency to increase in percentage to initial body weight since 36 hours after the administration. 3) UDP-Glucuronyltransferase activity enhanced by eugenol was localized in microsome in the same manner as basal activity. These results indicate that rapid glucuronide formation and excretion of the administered glucosidurogenic foreign compound took place in the living body, and that the inductive adaptation of UDP-glucuronyltransferase occurred subsequently to the administered compound in liver microsome. It was postulated that an effective glucosidurogenic compound might act in vivo as a 'substrate inducer' on liver UDP-glucuronyltransferase.

L'administration de phenobarbital a la souris augmente l'activite de l'UDP-glucosyltransferasique dans les microsomes hepatiques. On a remarque que le traitement par la drogue, ou la conservation des microsomes a −20°C, change la qualite et la quantite de cet enzyme, ainsi que de l'UDP-glucuronyltransferase.

Glucuronidation and sulfation of the phenolic — OH group of harmol and harmalol by the rat in vivo , and by rat liver subcellular fractions in vitro have been studied. In vivo harmol was extensively excreted (59% of the dose in 3 hr) in bile and urine, mostly as harmol-sulfate (70%) but also as harmol-glucuronide (30%). Harmalol was also excreted in bile and urine (50% of the dose in 3 hr), mostly as harmalol-glucuronide (70%), with only a trace of harmalol-sulfate present (less than 3%). In vitro kinetic parameters of conjugating activities towards both substrates were determined. Harmol and harmalol were glucuronidated by UDP glucuronyltransferase at comparable rates. Phenolsulfotransferase converted harmol readily to its sulfate-conjugate, whereas harmalol was a very poor substrate of this enzyme. Thus, the excretory pattern of harmol and harmalol can be explained by different rates of conjugation of these substrates by UDP glucuronyltransferase and phenolsulfotransferase, as found in vitro .

1. The clearance of a tracer dose of [14C]bilirubin from the plasma was studied in patients with Gilbert's syndrome, congenital non-haemolytic jaundice, haemolytic jaundice and in normal subjects. Clearance was significantly impaired in the patients with Gilbert's syndrome and in those with congenital non-haemolytic jaundice when compared with the normal subjects, and was normal in the patient with haemolytic jaundice. 2. Treatment for 2 weeks with phenobarbitone (180 mg/day) lowered the plasma bilirubin concentration and improved all indices of plasma clearance of the isotope in patients with Gilbert's syndrome, so that they became indistinguishable from those of normal subjects. The improvement in plasma [14C]bilirubin clearance in these patients was associated with modest increases in hepatic bilirubin glucuronyl transferase in some subjects. 3. Phenobarbitone treatment improved plasma bilirubin concentrations and plasma [14C]bilirubin clearance in patients with congenital non-haemolytic jaundice, so that they resembled those seen in patients with untreated Gilbert's syndrome. Despite this improvement hepatic bilirubin glucuronyl transferase activity remained undetectable. 4. These results are compatible with the hypothesis that Gilbert's syndrome is a manifestation of a relative deficiency of hepatic bilirubin glucuronyl transferase, and differs from congenital non-haemolytic jaundice only in severity.

The hepatic enzyme, bilirubin UDP glucuronyltransferase (UDPglucuronate glucuronyltransferase, EC 2.4.1.17) has been solubilized in stable form by treating rat liver microsomes with 10% (by weight) dissolved digitonin at pH 7.8. All enzyme activity was associated with the rough membranes of the endoplasmic reticulum as shown by ultracentrifugation of selected density gradients in combination with electron microscopy. Proof of solubilization was determined by analytic ultracentrifugation, gel filtration, and electron microscopy which indicated that the enzyme was an homogeneous protein with a calculated molecular weight in the range of 150 000. Studies to characterize enzyme kinetics and assay requirements were performed and differ from those reported for liver hemogenates.

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.

The glucuronidation of bilirubin by UDPglucuronyltransferase (EC 2.4.1.17) was investigated using a kinetic assay. The role of albumin in the assay was studied at concentrations of bilirubin both above and below its limit of solubility. In assays saturated with respect to bilirubin, albumin was almost without affect on initial rates. At concentrations of bilirubin below its limit of solubility, albumin was inhibitory. Thus, the bilirubin-albumin complex is not a substrate for the enzyme. In assays containing saturating concentrations of bilirubin, the rate of glucuronidation was influenced by conditions which “salt in” bilirubin. Using a bisubstrate kinetic analysis, the dissociation constant for the enzyme bilirubin complex was determined to be 12–18 μM. The enzyme was shown to be stimulated by the allosteric effector UDP-N-acetylglucosamine which caused an increase in the apparent affinity of the enzyme for UPDglucuronic acid. The bilirubin conjugating form of UDPglucuronyl-transferase showed the same general behavior toward sulfhydryl reagents and perturbers of the lipid environment as previously studied forms. However, because of differences in the response of the rate of conjugation of bilirubin to metals, UDP-N-acetylglucosamine, and sulfhydryl reagents, it has been concluded that the bilirubin conjugating enzyme differs from the p-nitrophenol, o-aminophenol and o-aminobenzoate forms of UDPglucuronyltransferase.

Poor quality protein supports and even potentiates some metabolic responses in DDT + lindane-treated guinea pigs. Ascorbic acid deficiency, on the other hand, impairs induction of most of the responses measured. Animals fed soy protein and gelatin exhibit the highest O -demethylase activity, hepatic glycogen content and urinary excretion of lindane and lindane metabolites. However, only soy protein permitted significant increases in glucaric acid excretion, hepatic cytochrome P-450 content and glucuronyl transferase. Furthermore, only the guinea pigs on soy protein had statistically significant increases in the metabolic stimulation induced by DDT and lindane in the casein-fed animals. Thus, the animals on the soy protein diet excreted significantly more lindane and lindane metabolites as well as more glucaric acid than the guinea pigs receiving casein.

1. Some properties of the UDP-glucuronyltransferase synthesizing thio-β-d-glucuronides were investigated and compared with those of the enzyme synthesizing the O-glucuronides of analogous phenols. 2. Enzyme activity was generally similar for both classes of substrate in tissue distribution, intracellular location, optimum pH, perinatal development and induction by organ culture or by phenobarbital. 3. Certain differences were noted between the two types of activity in behaviour on storage and on activation, in kinetic behaviour and in distribution between Wistar and Gunn rats; the Gunn rats were not deficient in hepatic UDP-glucuronyltransferase activity towards o-aminothiophenol. 4. These differences are no greater than those exhibited in the synthesis of various O-glucuronides; therefore thiolic substrates could compete in vivo with phenolic compounds for access to the UDP-glucuronyltransferase complex as well as for UDP-glucuronic acid.

Pregnanediol, Δ 1 -17α-methyltestosterone, testosterone, estriol, 17α-methyltestosterone, progesterone, 17α-ethyl-19-nortestosterone, and 6α-methylprogesterone inhibited guinea pig liver glucuronosyl transferase using o -aminophenol, p -nitrophenol, and 4-methylumbelliferone as aglycones, while cholesterol, pregnanedione, pregnenolone, 17α-hydroxyprogesterone, cortisone, estradiol, estrone, and methylandrostenediol did not. Pregnanediol glucuronide and estriol glucuronide also were inhibitors. Microsomes prepared as described contained little or no β-glucuronidase or uridine diphosphoglucuronic acid pyrophosphatase activity. Kinetic studies gave a K m for glucuronosyl transferase of 1.92 × 10 −4 using o -aminophenol as aglycone. The kinetics of pregnanediol glucuronide and 17α-ethyl-19-nortestosterone inhibition were mainly competitive in type with K i of 3.24 × 10 −4 and 2.52 × 10 −4 , respectively.

The temporal relationships for hormonal stimulation of glycogen synthesis are not clarified. In the present study glycogen synthesis and the activation of the rate-limiting glycogen synthetase enzyme (EC 2.4.1.11) were investigated in levator ani muscles from prepubertal male rats during insulin stimulation in vitro for 5–240 min and testosterone stimulation in vivo for 4–36 h. For both hormones, glycogen content was gradually increased to a level of 8.5–9.0 mglg tissue. An activation of the synthetase enzyme by means of an increase of its I-form was found after only 5 min of insulin stimulation. The increase in I-form was continuously reduced and reached control level after 150 min of incubation. For testosterone, the I-form was steadily increased 6–16 h after injection of the hormone. The incorporation of glucose-14C into glycogen was increased in both studies even when the I-form was no longer increased. The level of glucose-6-phosphate was aiso measured and the results suggest an activation of the synthetase enzyme by changes in the concentration of this metabolite during hormonal stimulation. It is concluded that the length of stimulation is of importance for the stimulation of muscle glycogen synthesis by insulin and testosterone.

The treatment of rat liver microsomes with chaotropic agents such as NaSCN, KI, KNO 3 and urea resulted in a solubilization of a considerable amount of the membrane protein and a decrease in the absorbance of the microsomal suspension. Treatment with NaSCN and KI at 3–4 M concentrations completely abolished microsomal drug oxidation (hydroxylation of aniline and 3,4-benzpyrene); urea and KNO 3 decreased the hydroxylation of aniline and 3,4-benzpyrene by about 40%. Both NADPH- and NADH-cytochrome c reductase exhibited a biphasic response: at low concentrations these enzymes were activated by NaSCN and KI, but at higher concentrations an inhibition took place. NADPH cytochrome P-450 reductase was only inhibited by these ions. UDPglucuronyltransferase, catalyzing the conjugation step 0.5 M NaSCN or 1.0 M KI; higher concentrations were inhibitory. NaSCN treatment sensitized UDPglucuronyltransferase for trypsin digestion. Weaker chaotropic agents, urea and KNO 3 , exhibited lesser effects on drug oxidation and on UDPglucuronyltransferase. However, NADH-cytochrome c reductase was particularly activated by these agents. The results obtained support the concept that the oxidizing enzymes are more superficial in the microsomal membrane than is UDPglucuronyltransferase. The location of UDPglucuronyltransferase behind a hydrophobic barrier in the membrane of liver microsomes is supported.

A significant difference in UDP glucuronyltransferase activity (with p-nitrophenol as an acceptor) was found in the liver and kidneys of homozygous Wistar and Gunn rats. There was also a significant difference in hepatic UDP glucuronyltransferase activity between homozygous Wistar and heterozygous Gunn rats when the enzyme preparations were first activated by adding surfactants to the reaction mixture. This determination of surfactant-activated UDP glucuronyltransferase can be used to distinguish Wistar rats from heterozygous Gunn rats. Other enzymes of the glucuronic acid pathway were also studied in the liver and kidneys of homozygous Wistar and Gunn rats, but no differences were found.

These studies demonstrate that the MH(1)C(1) strain of rat hepatoma cells has the ability to take up and conjugate bilirubin and then excrete the conjugated pigment into the culture medium. On incubation with unconjugated bilirubin, the average rate of appearance of conjugated bilirubin in the medium was 4.4 +/- 0.20 microg per mg of cell protein per hour (mean +/- SE). The products formed from bilirubin by MH(1)C(1) cells were chromatographically identical to those found in normal rat bile. Assay of bilirubin UDP glucuronyl transferase activity in homogenates of MH(1)C(1) cells gave a value of 3.3 +/- 0.50 microg of conjugated pigment formed per mg protein per hour, only moderately less than the enzyme activity of liver from normal rats. Rat fibroblasts in culture did not conjugate bilirubin, nor did they contain bilirubin UDP-glucuronyl transferase activity. As in living animals, flavaspidic acid inhibited bilirubin metabolism by MH(1)C(1) cells, suggesting that the mechanism for bilirubin uptake is similar to that of normal liver. In contrast to the findings in animals, however, preincubation of MH(1)C(1) cells with phenobarbital led to only minimal enhancement of pigment conjugation. MH(1)C(1) cells represent the first example of a clonal strain of cells in culture in which many of the pathways of hepatic bilirubin metabolism remain intact. They should, therefore, serve as a useful model for studies of bile pigment metabolism which are not easily performed in the living animal.

Treatment of microsomes with EDTA abolishes the stimulation of glucuronidation produced by UDP-N-acetylglucosamine. Addition of divalent metal ions to EDTA-treated microsomes restores the sensitivity of UDP-glucuronyltransferase to UDP-N-acetylglucosamine. Regulation of the activity of this enzyme by UDP-N-acetylglucosamine depends, therefore, on the presence of divalent metal ions. In addition, divalent metals increase the rate of glucuronidation of p-nitrophenol at V max . The data indicate that metals are essential for the efficient function of UDP-glucuronyltransferase.

The present paper describes the purification and properties of a glucuronosyltransferase isolated from 13-day embryonic chick brain. The enzyme catalyzes transfer of glucuronic acid from UDP-glucuronic acid to a series of low and high molecular weight compounds which contain terminal non-reducing β-D-galactose residues. Studies utilizing enzymatically degraded chondromucoprotein as acceptor suggest that the glucuronosyltransferase terminates biosynthesis of the linkage region between protein and polysaccharide of chondromucoprotein.

Mirex (dodecachlorooctahydro-1,3,4-metheno-2H-cyclobuta[cd]pentalene) was orally administered to male and female rats at daily dosages of 5, 10, 25, and 50 mg/kg for 5 days and its effects on aniline hydroxylas,p-nitroanisoleO-demethylase, ethyl morphine-N-demethylase, and UDP-glucruonyltransferase activities in the liver were studied. Liver-to-body weight ratios show significant increases at all of the doses tested, reaching maximum of 206 and 230 per cent of controls at a dose of 25 mg/kg for males and females, respectively. Metabolism of each of the substrates is altered by each dose of mirex fed. Aniline hydroxylase activity is decreased at all levels tested and the reduction is progressive with the dose. The Mirex content of rat liver microsomes is, by analysis, 9.69, 14.75, and 36.73 µg per 0.2gram-equivalents of liver microsomes from male rats treated at 10, 25, and 50 mg/kg, respectively. Aniline hydroxylase activity is not affected by addition of up to 400 µg of mirex to the reaction mixtures, using liver preparations from untreated animals.p-Nitroanisole demethylation activity is induced to a maximum in animals treated at 5 mg/kg. Ethyl morphine demethylase is induced to a maximum by 10 mg/kg in male and 25 mg/kg in female rats. Higher doses manifest a reversal ofp-nitroanisole-and ethyl morphine demethylase activities in both the sexes. Although the UDP-glucuronyl transferase specific activity is unaffected by mirex pretreatment, total activity in the liver increases to a maximum of 173 and 149 percent of controls at 25 mg/kg in males and females, respectively. Thus, it appears that chronic low doses of mirex seriously alter hepatic mixed-function oxidase systems of exposed animals, although mirex is not a substrate for any of these enzymes.

Oral administration of two isomeric tetrachlorobiphenyls (TCBs) (2, 3, 4, 5-and 3, 5, 3′, 5′-tetrachlorobiphenyls) to rats at three dosage levels does not cause any adverse effect on their growth. However, several hepatic enzyme systems are affected, even at the lowest level (5 mg/kg). EthylmorphineN-demethylase and aniline hydroxylase activities are slightly induced by 2, 3, 4, 5-TCB but, at a medium dose level (20 mg/kg), the activity ofpara-nitroanisoleO-demethylase is induced two-fold. Also, 3, 5, 3′, 5′-TCB induces these enzyme systems in male rats but decreases the activities of aniline hydroxylase and ethyl morphineN-demethylase in female rats at higher doses. An extra polar metabolite occurs in thein vitro metabolism of oestradiol-3H when female rats are treated with 2, 3, 4, 5-TCB at a dosage level of 40 mg/kg. UDP-Glucuronyl/transferase and δ-aminolevulinic acid synthetase activities are not affected by the administration of these two TCBs.

Several steroids including hydrocortisone, estriol, and pregnanediol glucuronide are inhibitory to the enzyme glucuronyl transferase when tested in an in vitro microsome system with the use of o-aminophenol as the glucuronide acceptor. In view of the steroid hormones which are cross-placentally transferred to the newborn infant, the possible physiologic implications of these findings are discussed.

The submicrosomal distribution of a number of enzymes involved in the metabolism of xenobiotics was studied in livers of Wistar and Gunn rats. Compared with Wistar controls, ethyl morphine demethylase

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.

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 Vmax was significantly increased in treated rats and guinea pigs. The Km of the enzyme from 3-methylcholanthrene treated rats was significantly decreased, whereas, the Km of the enzyme from treated guinea pigs was significantly increased. These changes in Vmax and Km suggest that induction of glucuronyltransferase by 3- methylcholanthrene may involve qualitative as well as quantitative changes in the enzyme.

1. Conjugation of N-hydroxy-N-arylacetamides results in the metabolic activation of this class of compounds.2. This paper reviews current knowledge of these reactions with emphasis on the involvement of conjugates of N-hydroxy compounds in the mechanism of action of carcinogenic N-arylacetamides.3. The glucuronide and sulphate conjugates of the carcinogen N-hydroxy-2-acetylaminofluorene react in vitro with protein, RNA and DNA, leading to the covalent attachment of 2-acetylaminofluorene and 2-aminofluorene residues to these macromolecules.4. The most reactive groups in proteins and nucleic acids are methionine, cysteine, tryptophan, tyrosine and guanine or deoxyguanine.5. There are major differences in the reactions of the glucuronide and the sulphate conjugates of N-hydroxy-N-arylacetamides with tissue nucleophiles, mainly in the rate of reaction and in the kind of products formed.6. The structure of the aryl group in a conjugate of an N-hydroxy-N-arylacetamide also has a marked effect on the nat...

The clinicopathological syndrome of kernicterus observed in rats with congenital hypoglucuronyltransferasia (Gunn's strain of rats) is similar to the syndrome of human kernicterus. The earliest detectable changes in ganglion cells are associated with the presence of intracellular bilirubin. Sulfonamides, do not produce kernicterus in normal newborn rats, nor do they alter the blood-brain barrier. Their deleterious effect in Gunn's strain of rats and in premature infants may be explained by the displacement of bilirubin from the blood compartment into the extracellular fluid. It is suggested that a similar mechanism may be operative in kernicterus occurring under other circumstances. The toxic unconjugated bilirubin is probably the cause of central nervous system damage.

IN vertebrates many biologically-active endogenous compounds, (such as bilirubin, steroid hormones and the catecholamines) and an enormous range of drugs, carcinogens, pesticides, industrial pollutants or their metabolites are made more polar and excreted by conjugation with glucuronic acid1. This is the principal route of detoxication but only one mechanism for the biosynthesis of such ‘glucuronides’ is known: glucuronyl transfer by UDP glucuronyltransferase (GT) (EC 2.4.1.17.) to the accepting compound1.