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3569.Full.Pdf [CANCER RESEARCH 50, 3569-3573, June 15, 1990] Persistently Increased Expression of a 3-Methylcholanthrene-inducible Phenol Uridine Diphosphate-Glucuronosyltransferase in Rat Hepatocyte Nodules and Hepatocellular Carcinomas1 Karl Walter Bock,2 Peter A. Münzel,Elke Röhrdanz,Dieter Schrenk, and Lennart C. Eriksson Institute of Toxicology, University of Tübingen,Wilhelmstrasse 56, D-7400 Tübingen,Federal Republic of Germany fK. W. B., P. A. M., E. R., D. S.], and Department of Pathology, Huddinge Hospital, S-14186 Huddinge, Sweden [L. C. EJ ABSTRACT to a /V-glucuronide, which is transported to the urinary bladder where it decomposes to the ultimate carcinogen (17). In the Increased UDP-glucuronosyltransferase in rat hepatocyte nodules and latter case, glucuronidation plays a role in determining the hepatocellular carcinomas produced by feeding 2-acetyIaminofluorene or A'-nitrosomorpholine was studied using isozyme-selective substrates, an target of carcinogenicity. Persistently increased UDP-GTI may tibodies, and DNA probes. UDP-glucuronosyltransferase (UDP-GT) ac explain increased excretion of glucuronides of 2AAF metabo tivities toward 4-methylumbelliferone, I-nap hthol, and benzo(a]pyrene- lites in 3MC-treated and nodule-bearing rats (18). Hence, in 3,6-quinol were reversibly increased by short term feeding of 2-acetyla- creased glucuronidation contributes to toxin resistance in the minofluorene but were persistently increased in hepatocyte nodules and Solt-Farber model, which uses 2AAF as a mitoinhibitory agent differentiated hepatocellular carcinomas. Immunoblot analysis revealed (19). Because of their toxin resistance, nodular hepatocytes that short term feeding of 2-acetylaminofluorene increased a M, 55,000 selectively proliferate in the presence of growth stimuli in this polypeptide corresponding to the previously characterized UDP-GTI or model. phenol UDP-GT. However, in some hepatocyte nodules and hepatocel In the present study, intermittent feeding of 2AAF was used lular carcinomas either the M, 55,000 or a new M, 53,000 polypeptide was preferentially increased, suggesting heterogeneous UDP-GT forms to produce hepatocyte nodules and hepatocellular carcinomas (20). The properties of these liver nodules were compared to in liver nodules and carcinomas. Northern blot hybridization with a synthetic DNA probe to phenol UDP-GT demonstrated increased levels those produced by feeding /V-nitrosomorpholine in a stop model (21). UDP-GTs were analyzed using (a) substrates for UDP- of mRNA in liver nodules. The results suggest persistently increased expression of at least two phenol UDP-GT enzyme forms in hepatocyte GTI (such as 4-methylumbelliferone, 1-naphthol, and BP-3,6- nodules, which may contribute to the toxin-resistance phenotype fre quinol) and for UDP-GTII (such as 4-hydroxybiphenyl) as well quently observed at cancer prestages. as testosterone and bilirubin for the corresponding UDP-GT isozymes (22, 23), (b) antibodies to UDP-GTI for immunoblot analysis (10), and (c) a selective DNA probe for the analysis of INTRODUCTION UDP-GTI mRNA and DNA, synthesized according to its Exposure to various carcinogens results in the appearance of cDNA (12). It was found that the levels of at least two closely hepatocyte clones showing the 'toxin-resistance phenotype' dur related UDP-GTI polypeptides (at M, 55,000 and 53,000) were ing early stages of carcinogenesis (1, 2). This phenotype in enhanced in hepatocyte nodules and hepatocellular carcinomas. cludes enhanced excretion of drugs including chemotherapeutic In addition, evidence was obtained that the enhanced enzyme agents (3, 4) and an altered pattern of drug-metabolizing en level may be due to transcriptional activation. zymes, such as decreased P450-dependent monooxygenase ac tivities (5) and increased UDP-GTs3 in hepatocyte nodules (6, 7) and hepatocyte foci (8, 9). Since UDP-GT represents an MATERIALS AND METHODS enzyme family, it is interesting that increased UDP-GT activi Chemicals. Chemicals were obtained from the following sources: ties can be ascribed to a 3MC-inducible isozyme, which has benzo[fl]pyrene-3,6-quinone and 3-hydroxybenzo(a)pyrene from the been operationally termed UDP-GTI, phenol UDP-GT (6, 10), Chemical Carcinogen Reference Standard Repository, National Insti or 4-nitrophenol UDP-GT (11). Comparative studies ascer tutes of Health (Bethesda, MD); 2AAF from Fluka (Buchs, Switzer tained the similarity of this isozyme characterized in different land); Brij 58 (a condensate of hexadecyl alcohol with 20 mol of ethylene oxide/mol) from Atlas (Essen, FRG); and UDP-glucuronic laboratories (10). Its amino acid sequence has recently been deduced from its cDNA (12). A similar UDP-GT cDNA has acid, disodium salt, from Boehringer (Mannheim, FRG). Hepatocyte Nodules and Hepatocellular Carcinomas. Male Wistar been detected in extrahepatic tissues such as kidney and in rats (120 g; Mollegaards Breeding Center, Ejby, Denmark) received a human liver (13). Substrates of UDP-GTI are various phenolic basal diet (Ewos, Södertälje,Sweden)supplemented with 0.05% (w/v) and polyphenolic metabolites of planar polycyclic aromatics. 2AAF. Rats were fed intermittently the basal diet containing the Glucuronidation of BP-3,6-quinol, for example, prevents qui- carcinogen and the basal diet alone, as suggested by Epstein et al. (20) none/quinol redox cycles which generate reactive oxygen spe and modified for Wistar rats by Eriksson el al. (24). The livers of all cies and semiquinones (14, 15). UDP-GTI is also involved in animals were perfused in situ with 0.25 Msucrose at 4°C.Large nodules the inactivation of certain aromatic amines, such as 1- and 2- (4-mm diameter) were carefully dissected from the surrounding tissue, naphthylamine (16), and converts A/-hydroxy-2-naphthylamine immediately frozen in liquid nitrogen, and stored at —80°Cuntilused for enzyme assays. Control livers from untreated animals of the same Received 12/29/89; revised 2/19/90. age were prepared and stored under identical conditions. The costs of publication of this article were defrayed in part by the payment When liver nodules were produced by feeding A'-nitrosomorpholine, of page charges. This article must therefore be hereby marked advertisement in male Wistar rats (150 g) received a basal diet (Altromin, Lage, FRG) accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by the Deutsche Forschungsgemeinschaft and the Swedish Medi and A'-nitrosomorpholine (160 mg/liter, in drinking water) for 7 weeks. cal Research Council. Then the carcinogen was withdrawn for the following 10 weeks (21). 2To whom requests for reprints should be addressed. Culture of H4IIE Cells. Rat hepatoma H4IIE cells (25) were grown 3The abbreviations used are: UDP-GT, UDP-glucuronosyltransferase (EC in 100- x 20-mm tissue culture plates, in Dulbecco's minimal essential 2.4.1.17); 2AAF, 2-acetylaminofluorene; 3MC, 3-methylcholanthrene; BP-3,6- quinol, benzo[a]pyrene-3,6-quinol; SDS, sodium dodecyl sulfate; SSC, saline medium (with L-glutamine) containing 10% fetal calf serum, penicillin sodium citrate; cDNA, complementary DNA. (100 units/ml), and streptomycin (100 ¿jg/ml),in a humidified atmos- 3569 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1990 American Association for Cancer Research. UDP-GLUCURONOSYLTRANSFERASE IN HEPATOCYTE NODULES phere of 5% CO2 in air at 37°C.Cells were harvested at a density of 2- covalently bound to the membrane by UV irradiation. The membranes 3 x IO7 cells/plate. Thereafter, they were washed with cold 0.01 M were hybridized with the "P-labeled UDP-GTI DNA probe (nucleotides phosphate-buffered 0.9% NaCl, pH 7.4, and harvested by scraping the 71-350). Prehybridization and hybridization were carried out at 44°C, cells with the same solution. Washed cells were stored at —80°C. followed by two washes in 2 x SSC (0.9% NaCl/0.03 M sodium citrate UDP-GT Assays. Published methods were used to assay UDP-GT buffer, pH 7.0), 0.1% SDS, for 20 min at 44°C.Blots were exposed to activities toward 1-naphthol (26), 4-methylumbelliferone (27), and tes Kodak XAR-5 film at —70°C,withintensifying screens, for 7 days. The tosterone (28). UDP-GT activity toward BP-3,6-quinol was determined relative amounts of mRNA were estimated by densitometric scanning as described (14), with some modifications. In brief, BP-3,6-quinone of autoradiograms of Northern blots. (0.5 mM, dissolved in hexamethyl phosphoric acid triamide) was re Digestion with Restriction Enzymes and Southern Blot Analysis. Re duced to the corresponding quinol in I M Tris/ascorbic acid buffer, pH striction enzymes were obtained from Bethesda Research Laboratories 7.4, for 10 min under nitrogen at 37°C.Afterwards, microsomes (0.1 (Bethesda, MD) and from Boehringer. Digestion of DNA samples and mg), 5 mM MgCl2, Brij 58 (0.5%, w/v), and UDP-glucuronic acid (3 Southern blot analysis were carried out as follows. DNA (20 ßg)was mM) were added, in a total volume of 0.1 ml. The reaction was started digested to completion with Mspl and Hpall (20 units/Vg DNA) or by addition of UDP-glucuronic acid. At zero time and after 1 and 2 with Beni and Neil (2 units/Vg DNA). The digested DNA was electro- min, 25-fil aliquots were removed from the incubation mixture. The phoresed on a 1% agarose gel for 18 h, at 16 V/cm, in buffer containing aliquots were added to 50 n\ methanol and 500 n\ 1.6 M glycine/NaOH 40 mM Tris-HCl, pH 8.0, 20 mM acetic acid, and 20 mM EDTA. Phage buffer, pH 10.3, in a total volume of 750 /¿I.Relativefluorescence was X DNA, digested with Hindlll, was used as a size marker during determined at excitation and emission wavelengths of 460 and 520 nm electrophoresis. The electrophoresed DNA fragments were transferred for BP-3,6-quinol monoglucuronide and 285 and 433 nm for the to nylon membranes (Hybond N) by capillary blotting and were cova corresponding diglucuronide, respectively, in a Perkin-Elmer LS-5B lently bound to the membrane by UV irradiation.
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