Karl Walter Bock

Effects of phenobarbital and 3-methylcholanthrene on substrate specificity of rat liver microsomal UDP-glucuronyltransferase

Abstract 1. 1. Substrate specificity of liver microsomal UDPglucuronyltransferase (UDP-glucuronate glucuronyltransferase, EC 2.4.1.17) towards p- nitrophenol , 1-naphthol, bilirubin and chloramphenicol changes after treatment of rats with phenobarbital and 3-methylcholanthrene. Phenobarbital mainly increases the glucuronidation of chloramphenicol, whereas 3-methylcholanthrene only stimulates the glucuronidation of the two phenolic substrates. These changes have been observed using ‘native’, Triton X-100-treated, or deoxycholate-solubilized microsomes as well as partially purified enzyme preparations. 2. 2. Treatment with phenobarbital or 3-methylcholanthrene did not alter the apparent K m values for UDPglucuronic acid (0.16 mM) with p- nitrophenol (0.34 mM). 3. 3. The results suggest that several forms of UDPglucuronyltransferase exist in rat liver microsomes which are selectively induced by either phenobarbital or 3-methylcholanthrene.

Differential induction of rat liver microsomal UDP-glucuronosyltransferase activities by various inducing agents

The selectivity of various inducers of UDP-glucuronosyltransferase was investigated in rat liver microsomes and compared with their effect on monooxygenase reactions. (1) Similar to 3-methyl-cholanthrene beta-naphthoflavone selectively stimulated the glucuronidation of 1-naphthol and 4-methylumbelliferone (GT1 substrates). (2) In contrast, DDT preferentially enhanced the glucuronidation of morphine, 4-hydroxybiphenyl (GT2 substrates) and bilirubin, similar to phenobarbital. (3) Colfibric acid and bezafibrate selectively enhanced bilirubin glucuronidation without affecting GT1 and GT2 reactions. (4) Similar to ethoxyquin and Aroclor 1254, trans-stilbene oxide enhanced both GT1 and GT2 activities but not bilirubin glucuronidation. (5) In contrast to 3-methylcholanthrene-type inducers which induce both cytochrome P-450MC and GT1, probably through a common receptor protein, ethoxyquin and trans-stilbene oxide markedly induced GT1 reactions without affecting benzo[a]pyrene monooxygenase.

Functional heterogeneity of UDP-glucuronyltransferase in rat tissues

Abstract Tissue distribution of UDP-glucuronyltransferase was investigated using two substrate groups which were shown to be conjugated by two different forms of this enzyme in previous studies with rat liver. These enzyme forms were found to be differentially inducible by 3-methylcholanthrene (form 1) and phenobarbital (form 2). Group 1 substrates (conjugated by form 1) include 1-naphthol, N -hydroxy-2-naphthylamine and 3-hydroxybenzo[ a ]pyrene; group 2 substrates (conjugated by form 2) comprise 4-hydroxybiphenyl, morphine and chloramphenicol. Group 1 substrates are conjugated in a number of tissues, for example, liver, kidney, small intestinal mucosa, lung, skin, testes and spleen. However, conjugation of group 2 substrates is detectable only in liver and intestine to an appreciable extent. It is concluded that enzyme(s) efficient in the conjugation of group 1 substrates is ubiquitous in the investigated organs, whilst only liver and intestine possess enzyme(s) efficient in the conjugation of group 2 substrates. In contrast to 3-hydroxybenzo[ a ]pyrene, benzo[ a ]pyrene 7,8-dihydrodiol cannot be clearly associated with only one of the 2 substrate groups. Glucuronidation of benzo[ a ]pyrene 7.8-dihydrodiol is enhanced by both phenobarbital and 3-methylcholanthrene in liver. Conjugation of the dihydrodiol is detectable in all tissues examined. However, enzyme activity towards the dihydrodiol is much lower than that towards 3-hydroxybenzo[ a ]pyrene. It is disproportionately low with skin microsomes.

Paracetamol as a test drug to determine glucuronide formation in man. Effects of inducers and of smoking

SummaryA simple, noninvasive procedure was developed to monitor glucuronidation and sulphation in patients using paracetamol as the test drug. Urinary paracetamol and its metabolites were determined by UV absorption and electrochemical detection after separation by HPLC. The metabolite to paracetamol ratio (M/P) was used as an approximation of the partial clearance due to metabolite formation. In 14 healthy volunteers, all nonsmokers without medication, M/P was 18±5 for glucuronides and 12±4 for sulphate esters. The test was validated in patients treated with enzyme inducers. In 10 patients with epilepsy given phenytoin 0.3 g/day, and in 10 patients with tuberculosis treated with rifampicin 0.6 g/day, the M/P value for glucuronidation was significantly increased to 41±11 and 35±7, respectively. In contrast, M/P values for sulphation were not significantly different from untreated controls. In 9 heavy smokers (about 40 cigarettes/day) M/P values for glucuronidation were also significantly increased to 33±11. However, in 4 moderate smokers (about 10 cigarettes/day) no significant increase was found. The results suggest that in man glucuronidation of paracetamol is inducible both by phenobarbital-and 3-methylcholanthrene-type inducers. Monitoring the ratios of various urinary paracetamol conjugates/paracetamol may be useful as a new tool for the evaluation of factors determining glucuronide and sulphate ester formation in man.

Immunohistochemical and biochemical detection of uridine-diphosphate-glucuronyltransferase (UDP-GT) activity in putative preneoplastic liver foci

SummaryPreneoplastic liver foci were produced in female Wistar rats by the administration of 2-acetylaminofluorene (0.03% w/w) in the diet for 174 days. Increased UDP-glucuronyltransferase (UDP-GT) could be visualized immunohistochemically in the same focal areas which were ATPase-negative andγ-glutamyltranspeptidase-positive. Immunohistochemical detection was possible using rabbit anti-UDP-GT and peroxi-dase-labeled swine anti-rabbit immunoglobulins. The results of immuno-histochemistry were substantiated by enzyme determination in micro-dissected material. UDP-GT activity was 5-fold higher in focal areas in comparison with the surrounding liver tissue.Increased UDP-GT activity in conjunction with the altered pattern of other drug-metabolizing enzymes is consistent with increased resistance of preneoplastic cells to the cytotoxicity of carcinogens. Immunohistochemical detection of UDP-GT may provide a new marker for preneoplastic lesions which, in conjunction with other markers, may prove useful in analyzing the various stages of liver carcinogenesis and the remodeling of preneoplastic lesions after cessation of carcinogenic stimuli.

The mammalian aryl hydrocarbon (Ah) receptor: from mediator of dioxin toxicity toward physiological functions in skin and liver.

Abstract The mammalian Ah receptor (AhR) is a ligand-activated transcription factor with multiple functions in adaptive metabolism, development and dioxin toxicity in a variety of organs and cell systems. Phenotypes observed following sustained activation by dioxin or in AhR-null mice suggest organ-dependent physiological functions. These functions are probably deregulated following exposure to dioxin. We focus on skin and liver to facilitate discussion of mechanisms linking phenotypes and AhR-modulated genotypes. After a brief summary of currently discussed AhR ligand candidates, two groups of direct AhR target genes/proteins and associated functions are highlighted: (i) xenobiotic-metabolizing enzymes which are also involved in homeostasis of endogenous ligands and (ii) proteins controlling cell proliferation/apoptosis, differentiation and inflammation. Homeostatic feedback loops might not only include CYP1A1 but also Phase II enzymes such as UGT1A1 which controls the antioxidant AhR ligand bilirubin. The AhR is involved in extensive crosstalk with other transcription factors and multiple signaling pathways. Efforts elucidating the pathway toward identification of physiological functions of the AhR remain challenging and promising.

Metabolism of naphthalene to naphthalene dihydrodiol glucuronide in isolated hepatocytes and in liver microsomes.

Abstract The functional linkage between UDP-glucuronyltransferase (GT) and the monooxygenase-epoxide hydratase system was investigated in studies on the glucuronidation of naphthalene dihydrodiol, which was formed by epoxide hydratase during the metabolism of naphthalene. (1) Naphthalene metabolism was compared in isolated hepatocytes and in liver microsomes incubated with an NADPH regenerating system and UDP-glucuronic acid. Naphthalene dihydrodiol glucuronide was a major metabolite in isolated hepatocytes. In the liver microsomal system free dihydrodiol by far exceeded its glucuronide unless the positive allosteric effector of GT, UDP- N -acetylglucosamine, was added. (2) Treatment of rats with phenobarbital or 3-methylcholanthrene, although markedly enhancing the formation of naphthalene dihydrodiol, did not stimulate liver microsomal GT (naphthalene dihydrodiol as substrate). The results suggest that activation of GT by UDP- N -acetylglucosamine is an important factor in the coupling of glucuronidation to functionally linked microsomal enzyme reactions.

Glucuronidation of 1-naphthol in the rat intestinal loop

Abstract The glucuronidation of 1-naphthol after its instillation into the intestinal lumen was studied in closed or single pass perfused jejunal loops by measuring the appearance of 1-naphthol glucuronide in the intestinal lumen and in venous blood. Within 30 min 69% of 1-naphthol instilled into the jejunal loop was metabolized almost exclusively to the glucuronide; 49% was in venous blood and 20% in the intestinal lumen. After instillation of 1-naphthol glucuronide, 59% was left in the intestinal lumen and 20% appeared in venous blood. When the intestine was perfused with 1-naphthol, 91% of the naphthol absorbed from the intestinal lumen was glucuronidated at a rate of 6.7 nmole/min/g tissue; 82% of the glucuronide was released into the blood under steady state conditions. The results demonstrate the suitability of this system for studies on drug metabolism in intact intestinal tissue.

UDP-glucuronosyltransferase 1A6: structural, functional, and regulatory aspects.

Glucuronidation, catalyzed by two families of UDP-glucuronosyltransferases (UGTs), represents a major phase II reaction of endo- and xenobiotic biotransformation. UGT1A6 is the founding member of the rat and human UGT1 family. It is expressed in liver and extrahepatic tissues, such as intestine, kidney, testis, and brain, and conjugates planar phenols and arylamines. Serotonin has been identified as a selective endogenous substrate of the human enzyme. UGT1A6 is also involved in conjugation of the drug paracetamol (acetaminophen) and of phenolic metabolites of benzo[a]pyrene (together with rat UGT1A7 and human UGT1A9). High interindividual variability of human liver protein levels is due to a number of influences, including genetic, tissue-specific, and environmental factors. Evidence shows that homo- and heterozygotic expression of UGT1A6 alleles markedly affects enzyme activity. HNF1 may be responsible for tissue-specific UGT1A6 expression. Multiple environmental factors controlling UGT1A6 expression have been identified, including the pregnane X receptor, the constitutive androstane receptor, the aryl hydrocarbon receptor, and Nrf2, a bZIP transcription factor mediating stress responses. However, marked differences have been noted in the expression of rat and human UGT1A6. Regulatory factors have been studied in detail in the human Caco-2 colon adenocarcinoma cell model.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-mediated membrane translocation of c-Src protein kinase in liver WB-F344 cells.

Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contaminant and the most potent agonist of the aryl hydrocarbon receptor (AhR). Persistent activation of the AhR has been shown to be responsible for most TCDD-mediated toxic responses, including liver tumour promotion. However, the mechanisms responsible for these complex toxic reactions are still unknown. TCDD (1 nM) has previously been shown to reduce DNA synthesis of primary hepatocyte cultures and cell contact inhibition of confluent WB-F344 cells. The latter model was used to study early effects of TCDD on protein tyrosine kinase c-Src in confluent WB-F344 cells. It was found that TCDD decreased cytosolic c-Src (protein and tyrosine kinase activity) after 20–60 min, and increased c-Src in the membrane fraction. Membrane translocation of c-Src occurred in the presence of 100 μM cycloheximide and was observed after treatment with 1 nM TCDD or 50 nM 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin. Under these conditions epidermal growth factor (EGF) receptor tyrosine phosphorylation was also studied. As expected, its phosphorylation was low in confluent cells but was significantly enhanced by TCDD treatment. Pretreatment of WB-F344 cells for 1 h with 1 μM geldanamycin, which disrupts cytosolic heat shock protein Hsp90 complexes with AhR and Src, abolished TCDD-mediated Src translocation and TCDD-mediated reduction of cell contact inhibition. The WB-F344 cell model appears to be very useful to study TCDD effects on protein tyrosine kinases and of signaling pathways responsible for modulation of the cell cycle by TCDD.