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K W Bock

Publications and source records attributed to K W Bock.

At least 91 records · Page 5Linked to original sources
PubMed1987

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

A 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.

Acetaminophen↗
PubMed1987

Normal pathways for glucuronidation, sulphation and oxidation of paracetamol in Gilbert's syndrome.

A group of eleven subjects with Gilbert's syndrome was characterized by conventional tests and determination of bilirubin and its conjugates in plasma by alkaline methanolysis and thin layer chromatography. After a 1 g dose of paracetamol h.s. the drug and its metabolites were measured by high performance liquid chromatography (HPLC) in the overnight 8-h urine sample. The amounts of paracetamol and of its metabolites recovered in urine were almost identical with those found in the control group (n = 10). The glucuronide:paracetamol ratio, which is considered to be an index of glucuronidation, was not correlated with the fraction of bilirubin present in plasma as glucuronides. These data do not suggest that in subjects with Gilbert's syndrome therapeutic doses of paracetamol are associated with an increased risk for hepatic or systemic toxicity.

Acetaminophen↗
PubMed1987

Regioselectivity of rat liver microsomal UDP-glucuronosyltransferase activities toward phenols of benzo(a)pyrene and dibenz(a,h)anthracene.

Inducibility of rat liver microsomal UDP-glucuronosyltransferase was investigated with regard to the substrate structure using 3-, 6-, 7-, 8-, and 9-hydroxybenzo(a)pyrene, all seven phenols of dibenz(a,h)anthracene, 3-hydroxybenz(a)anthracene, and 3-hydroxyfluoranthene as substrates. Glucuronide formation of the majority of the planar phenols was preferentially inducible by 3-methylcholanthrene (4- to 8-fold, group 1). However, glucuronidation of 8-hydroxybenzo(a)pyrene, 3-hydroxybenz(a)anthracene, and 3-hydroxydibenz(a,h)anthracene was markedly inducible by phenobarbital (3- to 8-fold, group 2). Glucuronidation of 9-hydroxybenzo(a)pyrene and 3-hydroxyfluoranthene was only moderately induced by the two inducers (less than 2-fold, group 3). Glucuronidation was also determined with purified phenol UDP-glucuronosyltransferase from 3-methylcholanthrene-treated rats. A close correlation (r = 0.95) was observed between purification factors (ratio of enzyme activities in purified enzyme and microsomes) and induction factors (ratio of microsomal enzyme activities from 3-methylcholanthrene-treated rats and untreated controls). Interestingly, differences in size and shape of group 1 and 2 substrates could be recognized. Group 1 substrates were shorter (less than 1.3 nm) and broader (greater than 1.1 nm) than group 2 substrates when viewed from the hydroxy group, along the axis of the C-O bond, to the plane of the polycyclic aromatic hydrocarbon, suggesting a distinct geometry of the binding site of the 3-methylcholanthrene-inducible phenol UDP-glucuronosyltransferase.

Animals↗
PubMed1986

Immunochemical and functional characterization of UDP-glucuronosyltransferases from rat liver, intestine and kidney.

Glucuronidation of various substrates in hepatic, intestinal and renal microsomes of control, phenobarbital (PB), 3-methylcholanthrene (3MC) and Aroclor-1254 (A1254) pretreated rats was investigated. UDPGT activities tested could be divided in four groups on the basis of their tissue distribution and induction by PB or 3MC in liver microsomes. GT1 activities (1-naphthol, benzo(a)pyrene-3,6-quinol) are induced by 3MC in liver microsomes and are present in all tissues investigated. GT2 activities (morphine, 4-hydroxybipheynl) are induced by PB in liver microsomes and appear to be restricted to the liver and the intestine. UDPGT activity towards bilirubin, although induced by PB, can be detected in hepatic, intestinal and renal microsomes. UDPGT activity towards fenoterol is restricted to the liver and intestine and is not induced by PB, 3MC or A1254. The presence of inducible immunoreactive UDPGT isoenzymes in microsomes of liver, intestine and kidney of control and induced rats was demonstrated by immunoblot analysis using rabbit anti-rat liver-GT1 antibodies. Induction of both 54 and 56 kDa polypeptides in hepatitis, intestinal and renal microsomes by 3MC or A1254 was observed. Purification of UDPGT (1-naphthol as substrate) from intestinal microsomes to apparent homogeneity yielded a polypeptide with an apparent molecular weight of 54-56 kDa. The results indicate that 54 and 56 kDa UDPGT polypeptides are the major A1254 inducible isoenzymes in intestinal and renal microsomes. An increase in immunoreactive protein is correlated with a biochemically measurable increase in glucuronidation capacity for GT1 substrates.

Animals↗
PubMed1986

Induction of UDP-glucuronyltransferase and arylhydrocarbon hydroxylase activity in mouse skin and in normal and transformed skin cells in culture.

Methods have been developed which allow quantitative determination of UDP-glucuronyltransferase (UDPGT) and arylhydrocarbon hydroxylase (AHH) activities in unfractionated mouse skin. These methods were used for comparative studies of basal and induced enzyme activities in whole skin and cultured skin cells. After topical application of Aroclor 1254 to the skin UDPGT activities towards 1-naphthol, 3-hydroxybenzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol were increased 3-fold and AHH activity was increased 15-fold. Topical application of the inducer also led to a marked increase of these enzyme activities in liver. UDPGT activity towards 1-naphthol was comparable in whole skin and in cultured keratinocytes and fibroblasts. In contrast, AHH activity was higher in cultured keratinocytes than in skin. In transformed epithelial cell lines the pattern of drug metabolizing enzymes was altered: UDPGT activity was increased 4- to 6-fold whereas AHH activity was decreased. However, AHH activity was still inducible by benz[a]anthracene or 7,12-dimethylbenz[a]anthracene in cultured cells. The altered pattern of AHH and UDPGT in transformed epithelial cell lines is consistent with toxin-resistance of initiated cells, similar to the toxin-resistance phenotype characterized in liver after initiation of hepatocarcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1986

Histochemical and immunohistochemical detection of putative preneoplastic liver foci in women after long-term use of oral contraceptives.

Localized areas with altered enzyme patterns were observed in liver tissue surrounding focal nodular hyperplasia in women after long-term use of oral contraceptives. These localized lesions were of three different types. Type I lesions were characterized by glycogen storage, a reduction in ATPase and an increase in gamma-glutamyltranspeptidase (gamma-GT) and UDP-glucuronyltransferase (UDP-GT) detected immunohistochemically. Type II lesions, which were morphologically very similar to small hyperplastic nodules, showed only a decreased ATPase reaction. Type III lesions showed an increase in gamma-GT (detected histochemically) and a slight reduction in ATPase. The results indicated that in human liver from patients given oral contraceptives long-term, localized lesions with altered enzyme patterns may occur which are very similar to those observed in animal models during experimental hepatic carcinogenesis.

Adenosine Triphosphatases↗
PubMed1986

Immunohistochemical differentiation of gamma-glutamyltranspeptidase in focal lesions and in zone I of rat liver after treatment with chemical carcinogens.

gamma-Glutamyltranspeptidase (gamma-GT) is known to be increased in putative pre-neoplastic foci but also in the periportal zone I of rat liver under a variety of circumstances not directly related to carcinogenesis. To be able to distinguish between these two instances gamma-GT was studied by enzyme determination in micro-dissections obtained from the two locations and by both histochemical and immunohistochemical staining in serial sections. Altered hepatic foci and alterations in zone I were produced in three models of hepatocarcinogenesis: initiation by N-nitrosomorpholine and tumor promotion by phenobarbital, continuous administration of 2-acetylaminofluorene and continuous administration of methapyrilene hydrochloride. In micro-dissections gamma-GT activity was similarly increased in focal lesions and in zone I after feeding methapyrilene. Histochemically detectable gamma-GT, stained according to Rutenburg et al. (23), was observed both in zone I and in focal lesions. Focal lesions were also ATPase negative and UDP-glucuronyltransferase positive in all three models. gamma-GT in focal lesions could be selectively detected by immunohistochemical staining using antibodies to the rat kidney enzyme and an indirect peroxidase reaction. These findings suggest immunochemical differences between gamma-GT in focal lesions and in zone I.

2-Acetylaminofluorene↗
PubMed1985

Effects of N-nitrosomorpholine and phenobarbital on UDP-glucuronyltransferase in putative preneoplastic foci of rat liver.

Effects of initiators and promoters of hepatocarcinogenesis on UDP-glucuronyltransferase and arylhydrocarbon hydroxylase were investigated in foci of altered hepatocytes. A single administration of N-nitrosomorpholine (75 mg/kg, 24 h after partial hepatectomy) was used for initiation and chronic administration of phenobarbital (0.1% in tap water) for promotion. Histological evidence indicated that ATPase-negative, gamma-glutamyltranspeptidase-positive, and UDP-glucuronyltransferase-positive foci were highly correlated. Based on this evidence ATPase-negative foci were used as a guide to monitor early lesions and to microdissect lyophilized foci and extra-focal tissue. It was found that treatment with N-nitrosomorpholine led to a permanent increase of UDP-glucuronyltransferase activity in foci tissue (3- to 5-fold, detected 180 and 330 days after initiation). In contrast, arylhydrocarbon hydroxylase activity was decreased by 50%. Administration of phenobarbital further increased UDP-glucuronyltransferase activity in focal tissue (up to 9-fold, compared with control liver). However, this further increase of enzyme activity by phenobarbital was reversible. The results suggest that (i) initiation by chemical carcinogens leads to permanent alterations of drug metabolizing enzymes, consistent with increased toxin-resistance of initiated hepatocytes, and (ii) chronic administration of phenobarbital markedly enhances gene expression of UDP-glucuronyltransferase in initiated hepatocytes.

Adenosine Triphosphatases↗
PubMed1985

Protection against toxic redox cycles between benzo(a)pyrene-3,6-quinone and its quinol by 3-methylcholanthrene-inducible formation of the quinol mono- and diglucuronide.

Cytotoxic effects of quinones are thought to be mediated by redox cycles between quinones and quinols whereby reactive oxygen species are generated. The role of glucuronidation in preventing these toxic redox cycles was investigated by using benzo(a)pyrene-3,6-quinone and isolated rat hepatocytes or Reuber hepatoma cells (H4IIE). Inhibition of quinol glucuronidation by salicylamide enhanced quinone-dependent oxygen uptake and cytotoxicity. Conjugation of benzo(a)pyrene-3,6-quinol was shown to proceed via the 6-monoglucuronide to the diglucuronide. Diglucuronide formation was low in hepatocytes from untreated controls and phenobarbital-treated rats. However, it was highly stimulated (26-fold) in hepatocytes from 3-methylcholanthrene-treated rats and was also high in Reuber hepatoma cells. Kinetic analysis with liver microsomes indicated that 3-methylcholanthrene-stimulated glucuronidation was due to an increased Vmax of UDP-glucuronosyltransferase which was enhanced 10- and 40-fold or mono- and diglucuronide formation, respectively. These findings suggest that the investigation of quinol glucuronidation (in particular the formation of benzo(a)pyrene-3,6-quinol diglucuronide) is a most useful probe for the 3-methylcholanthrene-inducible isoenzyme(s) of UDP-glucuronosyltransferase. Moreover, this isoenzyme may be particularly suited to protect against toxic redox cycles between benzo(a)pyrene quinones and quinols.

Animals↗
PubMed1984

N-glucuronide formation of carcinogenic aromatic amines in rat and human liver microsomes.

(1) Sensitive fluorimetric assays were developed for the determination of microsomal UDP-glucuronosyltransferase activities towards 1- and 2-naphthylamine and 4-aminobiphenyl. (2) In rat liver microsomes, enzyme activity towards 1-naphthylamine was orders of magnitude higher than the activities towards 2-naphthylamine, 4-aminobiphenyl or aniline. The differences were less marked with human liver microsomes. (3) Glucuronidation of aniline and 4-aminobiphenyl was not appreciably altered in rat liver microsomes from 3-methylcholanthrene- or phenobarbital-treated rats. UDP-glucuronosyltransferase activities towards 1- and 2-naphthylamine were selectively increased (about 2-fold) by 3-methylcholanthrene-treatment. However the increases were less marked than those observed with representative substrates of the 3-methylcholanthrene-inducible enzyme form. The results suggest that the arylamines investigated are predominantly conjugated by constitutive enzyme forms in rat liver. (4) Arylamine N-glucuronides were found to be susceptible to hydrolysis by E. coli beta-glucuronidase suggesting the release of carcinogenic arylamines in the gut and their enterohepatic circulation.

1-Naphthylamine↗
PubMed1984

Glucuronide formation of various drugs in liver microsomes and in isolated hepatocytes from phenobarbital- and 3-methylcholanthrene-treated rats.

Various substrates of rat liver microsomal UDP-glucuronosyltransferase were classified in vitro as preferred substrates of either 3-methylcholanthrene- or phenobarbital-inducible enzyme forms. Microsomal UDP-glucuronosyltransferase activities towards a third group of substrates (including oestrone, phenolphthalein, paracetamol and oxazepam) are not markedly altered by treatment with either 3-methylcholanthrene or phenobarbital. Some substrates of the 3-methylcholanthrene- and phenobarbital-inducible enzyme activities were selected to evaluate the importance of multiple enzyme forms for glucuronide formation in the intact cell. The metabolism of these compounds was compared in isolated hepatocytes from untreated controls and from rats treated with 3-methylcholanthrene (MC-hepatocytes) or phenobarbital (PB-hepatocytes). Glucuronidation of 1-naphthol and 3-hydroxybenzo[a]pyrene was chiefly enhanced in MC-hepatocytes (greater than 2-fold), whereas glucuronidation of chloramphenicol and bilirubin was chiefly enhanced in PB-hepatocytes. These observations are in agreement with differential induction of UDP-glucuronosyltransferase activities in vitro suggesting that, besides other factors such as cofactor supply, physiological activators, etc., the levels of the multiple enzyme forms are critically determining glucuronide formation in the intact cell.

Animals↗
PubMed1984

Intralobular distribution of UDP-glucuronosyltransferase in livers from untreated, 3-methylcholanthrene- and phenobarbital-treated rats.

A 3-methylcholanthrene-inducible enzyme form of UDP-glucuronosyltransferase has been localized within the liver lobule both immunohistochemically and enzymatically in microdissected centrilobular and periportal liver tissue. Livers of untreated, 3-methylcholanthrene- and phenobarbital-treated rats have been compared. The enzyme was detected in hepatocytes throughout the liver. However both immunohistochemical determination of the enzyme level and biochemical determination of its activity towards 1-naphthol revealed a heterogeneous distribution of the enzyme. In untreated controls and 3-methylcholanthrene-treated rats both enzyme activity and histochemical staining was highest in centrilobular hepatocytes. However, after phenobarbital-treatment enzyme staining and activity was highest in periportal hepatocytes, suggesting that the differentially inducible enzyme activities may be localized in different zones of the liver lobule. The results demonstrate that the 3-methylcholanthrene-inducible UDP-glucuronosyltransferase is preferentially expressed in centrilobular hepatocytes.

Animals↗
PubMed1983

Electroimmunochemical quantification of UDP-glucuronosyltransferase in rat liver microsomes.

Microsomal UDPglucuronosyltransferase(1-naphthol), an enzyme form previously shown to be selectively inducible in rat liver by 3-methylcholanthrene-type inducers, was purified to apparent homogeneity. Rabbit antibodies against this enzyme form precipitated UDPglucuronosyltransferase activities towards 1-naphthol and 4-methylumbelliferone faster and to greater extents than enzyme activities towards bilirubin, oestrone and 4-hydroxybiphenyl. Ouchterlony double-diffusion analysis showed immunochemical similarity of the rat liver enzyme with the enzymes from other organs of the rat (kidney, testes) and the mouse liver but not with the enzyme from cat and human liver. Electroimmunochemical quantification of the enzyme indicated that its level was enhanced 1.3-fold and 2.5-fold in liver microsomes from phenobarbital-treated and 3-methylcholanthrene-treated rats, respectively. The results indicate that 3-methylcholanthrene treatment increases the enzyme level of rat liver microsomal UDPglucuronosyltransferase(1-naphthol). Despite phospholipid-dependence of its catalytic activity microsomal enzyme activity appears to be a good index of the enzyme level.

Animals↗