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K W Bock

Publications and source records attributed to K W Bock.

At least 37 records · Page 2Linked to original sources
PubMed1996

TCDD-inducible plasminogen activator inhibitor type 2 (PAI-2) in human hepatocytes, HepG2 and monocytic U937 cells.

Induction of PAI-2 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been studied in human primary hepatocytes, hepatoma HepG2 cells and monocytic U937 cells, extending recent findings in human keratinocytes. PAI-2 represents a serpine-type protease inhibitor with wide-ranging implications in fibrinolysis, extracellular matrix proteolysis, growth factor activation and carcinogenesis. PAI-2 was induced by >10(-9) M TCDD in hepatocytes and HepG2 cells and by >10(-10) M TCDD in U937 cells. In the latter cell line, PAI-2 induction by TCDD and by 12-O-tetradecanoyl phorbol-13-acetate (TPA) has been compared. TCDD appeared to be less efficient than TPA as an inducer of PAI-2. In contrast to induction by TPA, PAI-2 induction by TCDD was found to be biphasic, with an early peak of mRNA at 1-3 h and a late peak at 12-24 h. A biphasic response was also seen at the protein level although production of PAI-2 protein lagged behind the corresponding mRNA. PAI-2 is known to contain AP-1 sites, i.e. Jun/Fos protein-binding sites, in its promotor region. Hence, PAI-2 induction by TCDD has originally been conceived to be due to an indirect response, secondary to the induction of Jun/Fos proteins. Therefore, expression of jun/fos genes and their AP-1 activity were studied at the early phase of PAI-2 induction by TCDD. TCDD did not increase mRNA of c-fos, c-jun, junB or junD (in contrast to TPA which markedly increased the expression of c-fos and junB), nor did TCDD increase AP-1 activity. In conclusion, the findings suggest that PAI-2 induction by TCDD is not restricted to human keratinocytes but includes liver cells and monocytic U937 cells. The induction mechanism is complex but the early phase does not appear to involve Jun/Fos proteins.

Aryl Hydrocarbon Receptor Nuclear Translocator↗
PubMed1996

Phase II metabolism of benzene.

The hepatic metabolism of benzene is thought to be a prerequisite for its bony marrow toxicity. However, the complete pattern of benzene metabolites formed in the liver and their role in bone marrow toxicity are not fully understood. Therefore, benzene metabolism was studied in isolated rodent hepatocytes. Rat hepatocytes released benzene-1,2-dihydrodiol, hydroquinone (HQ), catechol (CT), phenol (PH), trans-trans-muconic acid, and a number of phase II metabolites such as PH sulfate and PH glucuronide. Pretreatment of animals with 3-methylcholantrene (3-MC) markedly increased PH glucuronide formation while PH sulfate formation was decreased. Likewise, V79 cells transfected with the 3-MC-inducible rat UGT1.6 cDNA showed a considerable rate of PH and HQ glucuronidation. In addition to inducing glucuronidation of phenols, 3-MC treatment (reported to protect rats from the myelotoxicity of benzene) resulted in a decrease of hepatic CYP2E1. In contrast, pretreatment of rats with the CYP2E1-inducer isopropanol strongly enhanced benzene metabolism and the formation of phenolic metabolites. Mouse hepatocytes formed much higher amounts of HQ than rat hepatocytes and considerable amounts of 1,2,4-trihydroxybenzene (THB) sulfate and HQ sulfate. In conclusion, the protective effect of 3-MC in rats is probably due to a shift from the labile PH sulfate to the more stable PH glucuronide, and to a decrease in hepatic CYP2E1. The higher susceptibility of mice toward benzene may be related to the high rate of formation of the myelotoxic metabolite HQ and the semistable phase II metabolites HQ sulfate and THB sulfate.

Animals↗
PubMed1995

Formation of mono- and diglucuronides and other glycosides of benzo(a)pyrene-3,6-quinol by V79 cell-expressed human phenol UDP-glucuronosyltransferases of the UGT1 gene complex.

Glucuronidation of quinols of polycyclic aromatic hydrocarbons (PAHs) represents an important detoxication pathway preventing toxic quinone/quinol redox cycles. Therefore, mono- and diglucuronide formation of benzo(a)pyrene-3,6-quinol was investigated and compared to that of structurally related 3,6-dihydroxychrysene and simple phenols (1-naphthol and 4-methylumbelliferone) using V79 cell-expressed human UGT1.6 (= P1) and human UGT1.7 (= P4). Properties of human UGT1.6 were compared to those of the rat ortholog. Cofactors related to UDP-glucuronic acid such as UDP-galacturonic acid and UDP-glucose were also studied. It was found that rat and human UGT1.6 and human UGT1.7 catalyse monoglucuronide formation of planar PAH quinols. Diglucuronide formation was only detectable with human UGT1.7. The UGT isozymes studied also formed galacturonides and, although only to a minor extent, glucosides. Rat UGT1.6 (but not the human ortholog) catalysed digalacturonide formation of benzo(a)pyrene-3,6-quinol; the in vivo significance of galacturonide formation remains to be established. The results suggest that planar PAH phenols and quinols are conjugated more efficiently by human UGT1.7 than by UGT1.6, which preferentially conjugates simple planar phenols.

Animals↗
PubMed1995

Potency of mixtures of polychlorinated biphenyls as inducers of dioxin receptor-regulated CYP1A activity in rat hepatocytes and H4IIE cells.

Among the polychlorinated biphenyls (PCBs), a family of widespread environmental pollutants, the most toxic non-ortho-substituted coplanar (non-ortho coplanar) congeners are thought to act as strong dioxin (aryl hydrocarbon) receptor agonists leading to adverse effects, such as body weight loss, immunosuppression, thymic atrophy, hepatotoxicity, tumor promotion, and disturbances of steroid hormone action. Since PCBs are present in environmental and tissue samples as complex mixtures, we investigated the possible interaction of non-ortho coplanar congeners with other major PCBs, which are less active or inactive as dioxin receptor agonists. As a parameter for dioxin receptor activation, induction of CYP1A-catalyzed 7-ethoxyresorufin O-deethylase (EROD) was determined in rat hepatocytes in primary culture and in the rat hepatoma cell line H4IIE. In rat hepatocytes, individual EC50-values and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalency factors (TEFs) for the non-ortho and mono-ortho coplanar PCBs 126, 169, 105, 118 and 156, were in good agreement with published data from in vivo experiments, while in H4IIE cells coincidence was lower. However, in both cell systems TEFs for PCB 77 were significantly higher than reported from experiments in rats. In an approximately equipotent mixture the six potent PCB congeners showed perfect additive behaviour in both cell systems. In contrast, addition of a tenfold surplus of abundant mono- and di-ortho PCBs (28, 52, 101, 138, 153 and 180) led to an almost threefold higher TEF than predicted. This finding suggests a moderate synergistic enhancement of the inducing potency of potent PCBs by less potent congeners, present in abundance in environmental and tissue samples.

Animals↗
PubMed1995

Induction of cytochrome P4501A by smoking or omeprazole in comparison with UDP-glucuronosyltransferase in biopsies of human duodenal mucosa.

Drug-metabolizing enzymes were investigated in duodenal biopsy specimens. Cytochrome P4501A (CYP1A) activity was determined by measuring 7-ethoxyresorufin O-deethylase (EROD) activity in biopsies from 20 smokers (3-30 cigarettes per day), 21 nonsmokers, and 10 nonsmokers receiving omeprazole treatment (20-60 mg/day for at least 1 week). Omeprazole is known to act as a polycyclic aromatic hydrocarbon (PAH)-type inducer in humans. EROD activity was found to be significantly induced in smokers and omeprazole-treated patients, with medians of 2.1 and 1.1 pmol.min-1.mg protein-1, respectively, compared with 0.5 pmol.min-1.mg protein-1 in nonsmokers. Immunoblot analysis substantiated that EROD activity was correlated with CYP1A protein. In contrast, UDP-glucuronosyltransferase (UGT) activity towards 4-methylumbelliferone (an overlapping substrate of several constitutive and inducible UGTs) was not significantly affected. The results demonstrate CYP1A induction by omeprazole and by constituents of cigarette smoke in the human duodenum and support the utility of duodenal biopsies to monitor CYP1A induction by PAH-type inducers.

Cytochrome P-450 CYP1A1↗
PubMed1995

Crigler-Najjar syndrome type II. New observation of possible autosomal recessive inheritance.

The inheritance of Crigler-Najjar syndrome type II (CNS II) is still unclear. Both autosomal dominant transmission with variable penetrance and autosomal recessive transmission have been reported. We describe the diagnosis of CNS II in an adult patient with unconjugated serum bilirubin levels up to 19.6 mg/dl and no detectable activity of bilirubin UDP-glucuronosyltransferase in the liver biopsy. Serum bilirubin levels decreased markedly on phenobarbital treatment. The parents of our patient are first cousins. The mother and three of the patient's five sibs were jaundiced within a few days of birth. Our patient and her jaundiced siblings have 11 children, all healthy and anicteric. We conclude from these data that the inheritance of this very rare disease follows an autosomal recessive pattern, with pseudodominance in this family.

Adult↗
PubMed1995

Role of receptors in human and rodent hepatocarcinogenesis.

The liver is a major target organ in rodent carcinogenicity assays. Amongst the agents that are effective in producing rodent liver tumours are many chemicals which are not mutagenic, but are believed to mediate their effects by promoting the clonal outgrowth of initiated cells. Some of these chemicals, such as dibenzo-p-dioxins and certain PCBs, have been demonstrated to interact with specific cellular receptors and receptor binding appears crucial for their tumourigenic activity. Enzyme-altered foci in rat liver may serve as a sensitive means to estimate the promoting activity of these agents in rodents. Mechanistic considerations are of relevance when extrapolating these data from rodents to humans.

Animals↗
PubMed1995

Role of cell proliferation at early stages of hepatocarcinogenesis.

Hepatocarcinogenesis in rodents is characterized by the early appearance of foci of enzyme-altered (initiated) cells which are believed to represent precursors on the way to malignancy. Proliferation of cells within enzyme-altered liver foci is generally increased as compared to surrounding normal hepatocytes. This may be mediated by changes in the rates of cell division and/or cell death (apoptosis). Cell proliferation is controlled by complex signaling networks and may be modulated by xenobiotics. In mouse--but not rat or human--liver, mutation of the Ha-ras gene appears to represent a critical genetic alteration which may confer a selective growth advantage to the mutated cells. Exogenous tumor-promoting agents may stimulate cell division and depress apoptosis of preneoplastic hepatocytes, thereby increasing the probability of cancer. By means of histochemical methods, data on the frequencies of both cell division and cell death can be collected separately and utilized for estimation of promoter efficacy. In addition, quantitative stereology may be applied to the analysis of the size distribution of enzyme-altered foci and used for modeling of hepatocarcinogenesis.

Animals↗
PubMed1995

Induction of CYP1A and glutathione S-transferase activities by 2,3,7,8-tetrachlorodibenzo-p-dioxin in human hepatocyte cultures.

Induction of CYP1A and glutathione S-transferase activities with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied in human hepatocytes in primary culture to investigate the variability of inducibility and the potency of TCDD. Determining induction of 7-ethoxyresorufin O-deethylase activity, preferentially catalyzed by CYP1A isozymes, we obtained concentration-response diagrams in TCDD-treated hepatocyte cultures from transplant donors and patients undergoing hepatic surgery. At a concentration of 10(-10) M TCDD approximately half-maximal induction of CYP1A was observed. Northern analysis of CYP1A gene expression showed a similar concentration-response relationship. In comparison with rat hepatocytes, human hepatocytes were about 10-fold less sensitive towards the CYP1A-inducing effect of TCDD. No pronounced interindividual differences in the inducing potency of TCDD (concentration which leads to half-maximal induction) were obvious in the six human individuals studied, whereas the efficacy of CYP1A induction was highly variable. In addition, inducibility of glutathione S-transferase (GST) activity also revealed a considerable degree of interindividual variation, i.e. a complete lack of induction in three out of six hepatocyte preparations and a highly variable efficacy of GST induction among responders which was not related to CYP1A inducibility.

Adult↗
PubMed1995

Inhibition of apoptosis during 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated tumour promotion in rat liver.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cell division and cell death (apoptosis) in glutathione S-transferase (GST-P)-positive liver foci were analyzed in diethylnitrosamine-initiated female Wistar rats that were treated with TCDD, either acutely for 3 days or chronically for 115 days. Apoptotic bodies were quantitated in liver sections simultaneously stained for GST-P expression and H&E using a novel fluorescence microscopic detection method which greatly facilitates recognition of apoptotic bodies due to their high level of eosin fluorescence. While TCDD treatment only marginally affected cell division in GST-P-positive liver foci, as estimated by 5-bromo-2'-deoxyuridine-labelling, apoptotic indices were decreased to approximately 60% and approximately 10% of control values after acute and chronic TCDD treatment, respectively. In normal liver tissue, apoptotic indices were only slightly reduced by TCDD treatment, suggesting selective inhibition of apoptosis in the enzyme-altered cell population by the dioxin. Since inhibition of apoptosis in GST-P-positive liver foci was by far more pronounced than changes in cell division, our data suggest that the promoting activity of TCDD is preferentially mediated by a decrease of apoptosis in enzyme-altered liver foci.

Animals↗
PubMed1995

Benzene metabolism in rodent hepatocytes: role of sulphate conjugation.

1. Hepatocytes isolated from the adult male NMRI mouse or Wistar rat were incubated for 1 h with 0.5 mM 14C-benzene, the supernatant was separated from the cells, and analysed for benzene metabolites. Separately, formation of sulphate conjugates during benzene metabolism was studied in hepatocytes in the presence of 35S-sulphate. In addition sulphate conjugation of the benzene metabolites hydroquinone and 1,2,4-trihydroxybenzene was investigated in mouse liver cytosol supplemented with 3'-phosphoadenosine-5'-phospho-35S-sulphate. 2. Two novel metabolites, not detectable in rat hepatocyte incubations, were found in mouse hepatocytes, and were identified as 1,2,4-trihydroxybenzene sulphate and hydroquinone sulphate. Formation of the 35S-labelled conjugates could be demonstrated in incubations of mouse liver cytosol with hydroquinone or 1,2,4-trihydroxybenzene supplemented with 3'-phosphoadenosine-5'-phospho-35S-sulphate, and in mouse hepatocytes incubated with benzene and 35S-sulphate. 3. In comparison with hepatocytes from the Wistar rat, hepatocytes from the NMRI mouse were almost three times more effective in metabolizing benzene. The higher formation of hydroquinone, and the formation of trihydroxybenzene sulphate and hydroquinone sulphate, mainly contributed to the higher rate of benzene metabolism. 4. In conclusion, qualitative and quantitative differences in benzene metabolism may contribute to the higher susceptibility of mouse towards the myelotoxic and leucaemogenic action of benzene.

Animals↗
PubMed1994

Radiation inactivation analysis of microsomal UDP-glucuronosyltransferases catalysing mono- and diglucuronide formation of 3,6-dihydroxybenzo(a)pyrene and 3,6-dihydroxychrysene.

Indirect evidence has suggested that multiple subunits of microsomal UDP-glucuronosyltransferases (UGTs) are involved in diglucuronide formation of diphenols of polycyclic aromatic hydrocarbons (Bock et al., Mol Pharmacol 42: 613-618, 1992). To substantiate this suggestion functional target sizes of UGTs catalysing these reactions were determined in microsomes in situ by radiation inactivation analysis. Target sizes of UGTs catalysing the glucuronidation of 1-naphthol and 6-hydroxychrysene were found to be 91 +/- 29 and 120 +/- 27 kDa, respectively. However, target sizes for mono- and diglucuronide formation of 3,6-dihydroxybenzo(a)pyrene were 118 +/- 33 and 218 +/- 24 kDa, respectively. Similarly, using 3,6-dihydroxychrysene as substrate target sizes of 109 +/- 21 and 101 +/- 23 kDa were found for 6-O-monoglucuronide and 3-O-monoglucuronide formation and a target size of 192 +/- 34 kDa observed for diglucuronide formation. Based on subunit molecular masses of 50-60 kDa for UGTs, these results suggest that UGTs involved in monoglucuronide formation of phenols may function as dimers. In contrast, UGTs involved in diglucuronide formation of diphenols of polycyclic aromatic hydrocarbons may function as tetramers in microsomes in situ.

Activation Analysis↗
PubMed1994

Tissue-specific constitutive and inducible expression of rat phenol UDP-glucuronosyltransferase.

To investigate constitutive and inducible expression of rat phenol UDP-glucuronosyltransferase (UGT1A1) in liver and extrahepatic tissues, a selective cDNA probe for its unique exon 1 was utilized. 6-Hydroxychrysene was used as a functional probe of UGT1A1 activity. Constitutive expression of UGT1A1 was low in liver, but high in kidney, testis, epididymis and ovary. After treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 micrograms/kg for 7 days) the UGT1A1 mRNA level was markedly increased in liver (ca. 10-fold), and only moderately enhanced (up to 2-fold) in extrahepatic tissues where constitutive enzyme expression was high. UGT activity toward 6-hydroxychrysene was strongly inducible in liver (ca. 9-fold) and only moderately inducible in extrahepatic tissues (up to 2-fold). The results suggest complex tissue-specific regulation of UGT1A1 including positive and negative transcriptional factors and marked inducibility by TCDD in liver.

Animals↗
PubMed1994

Glucuronidation and isomerization of all-trans- and 13-cis-retinoic acid by liver microsomes of phenobarbital- or 3-methylcholanthrene-treated rats.

Glucuronidation and isomerization of all-trans-retinoic acid (tr-RA) and 13-cis-retinoic acid (13-cis-RA) were investigated in an in vitro system using liver microsomes of differently pretreated rats. In agreement with their thermodynamic stability, more retinoic acid was isomerized from the 13-cis form to the all-trans form than vice versa. Also some 9-cis-retinoic acid (9-cis-RA) could be found. Isomerization was reduced, but in contrast to glucuronidation was still important if boiled microsomes were used. This supports the view that isomerization can proceed as a non-enzymatic process. 3-Methylcholanthrene (MC) pretreatment of the rats increased the microsomal glucuronidation of 13-cis-RA and tr-RA and the formation of 13-cis-retinoyl-beta-glucuronide was enhanced up to 7-fold by MC-induced rat microsomes. The rates of glucuronidation by uninduced and phenobarbital-induced rat microsomes differed only slightly. In addition to glucuronides of the applied retinoic acid isomers (13-cis-RA and tr-RA), 9-cis-RA and its glucuronide were found. Induction of retinoid glucuronidation by pretreatment with MC indicates that this metabolic reaction is catalysed by a MC-inducible UGT isozyme. After two recently described pathways (conversions of retinol to retinal and of retinyl methyl ether to retinol) this is a third step of retinoid metabolism, induced by pretreatment with MC. With human microsomes no more than traces of glucuronides were detected; also, incubations with human microsomes resulted in a lower degree of isomerization than with rat microsomal fractions.

Animals↗
PubMed1994

Drug-metabolizing enzyme activities in freshly isolated oval cells and in an established oval cell line from carcinogen-fed rats.

The activities of several different phase I and phase II drug-metabolizing enzymes were measured in freshly isolated oval cells from rats fed a choline-deficient/DL-ethionine-supplemented diet for 6 weeks and also in vitro in the established oval cell line OC/CDE 6. No cytochrome P450 was spectrophotometrically measurable in both preparations and two cytochrome P450-dependent monoxygenase activities, aminopyrine N-demethylase and ethoxyresorufin O-deethylase, could not be detected in the oval cells of both sources. However, cytosolic glutathione transferase, microsomal epoxide hydrolase and UDP-glucuronosyltransferase activities were clearly measurable in oval cells. Similar enzyme activities were found in freshly isolated and cultured oval cells. The highest activities of these three enzymes were detected during the exponential growth phase of the cultured cells; thereafter the activities decreased until the cells reached confluency. Changes in phenol UDP-glucuronosyltransferase (UGT1A1) mRNA levels paralleled the variations in UDP-glucuronosyltransferase activity, i.e. they were high in exponentially growing oval cells and low in confluent cell cultures. Taking into account that oval cells are able to proliferate in the livers of rats continuously fed a choline-deficient/DL-ethionine-supplemented diet and that none of the analyzed drug metabolizing enzymes are involved in the activation or detoxication of DL-ethionine, the described pattern might be part of a more general, nonspecific, protection mechanism enabling these cells to overcome the cytotoxic effects of a variety of carcinogens and to proliferate even in their presence. Furthermore, the expression of microsomal epoxide hydrolase, cytosolic glutathione transferase and UDP-glucuronosyltransferase appears to depend on the proliferative status of the cells.

Animals↗
PubMed1994

Aryl hydrocarbon or dioxin receptor: biologic and toxic responses.

1. The AhR represents a ligand-activated transcription factor. Receptor agonists include planar aromatic compounds, a variety of heterocyclic plant constituents, and PCDD/PCDF. The latter lead to persistent activation of the receptor due to their strong binding affinity and long biologic half-life of over 10 years in human blood and fat. Practically every person on earth is exposed to these compounds via the diet (> 90%) and by high concentrations in mother's milk. PCDD/PCDF produced toxic responses in exposed people (primarily chloracne and immunosuppression) in the past. However, the present PCDD/PCDF levels (basal levels) in the general population are below those warranting toxicologic concern. 2. The AhR has been characterized as a helix-loop-helix transcription factor related to the Drosophila developmental genes sim and per. The cytosolic form of the receptor is present as an inactive complex with two subunits of HSP90. After ligand binding HSP90 is released and the receptor enters the nucleus as a heterodimer together with a related protein ARNT. It binds with high affinity to certain enhancer elements in the upstream region of several genes such as cytochrome P4501A1 (CYP1A1). The AhR transcriptionally activates several drug-metabolizing enzymes and proteins involved in growth/differentiation, such as the plasminogen activator inhibitor PAI-2 and IL-1 beta. In addition, it modulates the action of a number of other nuclear transcription factors such as receptors of the steroid hormone receptor superfamily and of cell surface receptors such as EGF. With the exception of CYP1A1 induction, little is known about the mechanism of transcriptional activation of the AhR-controlled genes. Many AhR-modulated biologic responses (such as modulation of the estrogen and EGF receptor) appear to be indirect. 3. Persistent activation of the AhR is probably responsible for toxic responses in experimental animals and humans. They are markedly tissue and species specific. In rodents a wasting syndrome, immunosuppression, teratogenicity, chloracne, and carcinogenicity/tumor promotion have been well studied. There is good evidence for an involvement for the AhR in these responses. However, the chain of events from receptor activation to the diverse toxic endpoints is largely unknown. Alteration of growth and differentiation of epithelial tissues may underlie most of the toxic responses. A lot has already been achieved, mostly by characterizing the AhR and transcriptional activation of CYP1A1. Still more work lies ahead of us, for example, elucidation of the physiologic roles of the AhR and of the chains of events from receptor activation to the various biologic and toxic endpoints.

Animals↗