Identification of epoxide hydrase as the preneoplastic antigen in rat liver hyperplastic nodules.

Levin, Wayne; Lu, Anthony Y. H.; Thomas, Paul E.; Ryan, Dene E.; Kizer, Donald E.; Griffin, Michael K.

doi:10.1073/pnas.75.7.3240

Cited by 72 publications

(35 citation statements)

References 11 publications

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“…They suggested that one corresponds to epoxide hydrase as reported by Levin et al. (5) and the other may be a novel hemopolypeptide, possibly a form of cytochrome P-450.…”

Section: Resultsmentioning

confidence: 98%

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Immunocytochemical localization of epoxide hydrase in hyperplastic nodules induced in rat liver by 2-acetylaminofluorene.

Novikoff¹,

Novikoff²,

Stockert³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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A knowledge of the biological characteristics of carcinogen-induced hyperplastic nodules of rat liver may be important in the understanding of cancer development. Although its biological role remains to be elucidated, the level of microsomal epoxide hydrase (epoxide hydrolase, EC 3.3.2.3) is 5-to 7-fold greater in hyperplastic nodules induced by feeding the hepatocarcinogen 2-acetylaminofluorene than in liver of control rats. After removal of the carcinogen from the diet, the high level of the enzyme is maintained in those nodules that persist and in the hepatocellular carcinomas that subsequently develop. The availability of antibody to the epoxide hydrase made it possible to use electron microscopic immunocytochemistry to localize this enzyme in the cells of hyperplastic nodules. The immunocytochemical procedure provides direct visual evidence for the presence of this enzyme in smooth endoplasmic reticulum and also in rough endoplasmic reticulum (including the nuclear envelope) of the nodule's parenchymal cells.It is known that many chemical carcinogens are metabolized to reactive intermediates which combine with key macromolecules of cells (1, 2). The possible roles of such metabolism by the microsomal monooxygenase system and epoxide hydrase (epoxide hydrolase, EC 3.3.2.3) during liver carcinogenesis have been discussed by Levin et al. (3,4). Levin et al. (5) have shown that chronic feeding of 2-acetylaminofluorene (2-AAF) results in a 5-to 7-fold increase in epoxide hydrase activity in hyperplastic nodulesO of rat liver. This increased level is maintained after removal of the carcinogen from the diet, in the nodules and in the hepatocellular carcinomas that develop subsequently. MATERIALS AND METHODSRat liver microsomal epoxide hydrase was purified to apparent homogeneity by the procedure of Lu et al. (7).Antibody to purified epoxide hydrase was prepared by the method of Levin et al. (8). The antibody was specific as judged by Ouchterlony double-diffusion analysis using purified epoxide hydrase and detergent-solubilized rat liver microsomes.Fab fragments were prepared essentially by the methods of Porter (9) from sheep anti-rat nonspecific IgG and sheep antirat epoxide hydrase. Horseradish peroxidase (RHP) (grade 1, Boehringer Mannheim), was coupled to the Fab fragments by the method of Avrameas and Ternynck (10).The hyperplastic nodules were produced in male SpragueDawley rats by feeding four cycles of 2-AAF according to the regimen of Stout and Becker (11), followed by 4 weeks on the control diet. At this time, most nodules present in the liver would be expected to persist.The rats were anesthetized by ether, and the liver was perfused via the portal vein, at a flow rate of 8 ml/min, for 2 min with cold (40C) isotonic saline and for 15 min with a cold fixative patterned after that of Karnovsky (12). The fixative contained 4% formaldehyde (freshly prepared from paraformaldehyde), 0.05% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4, bovine serum albumin at 10 mg/ml and 5% (wt/vol) sucrose. Aft...

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“…They suggested that one corresponds to epoxide hydrase as reported by Levin et al. (5) and the other may be a novel hemopolypeptide, possibly a form of cytochrome P-450.…”

Section: Resultsmentioning

confidence: 98%

“…It is evident now (19,20), could be demonstrated in the smooth ER but not in the rough ER. Levin et al (5) reported recently that this antigen was identical with microsomal epoxide hydrase. Sharma et al (21) subsequently reported the induction of two polypeptides (molecular weights 47,000 and 48,000) in 2-AAF-induced hyperplastic nodules.…”

Section: Resultsmentioning

confidence: 99%

Immunocytochemical localization of epoxide hydrase in hyperplastic nodules induced in rat liver by 2-acetylaminofluorene.

Novikoff¹,

Novikoff²,

Stockert³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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“…Coupled with the decreases in cytochromes P450 and in activities of some drug-metabolizing microsomal enzymes are large elevations in selective microsomal enzymes as epoxide hydrolase (EH) (23,31) and in cytosolic glutathione (32), y-glutamyltransferase (y-GT) (33), some glutathione-S-transferases (34,35) and UDP-glucuronyltransferase I (34,36,37).…”

Section: Resultsmentioning

confidence: 99%

Distinctive Biochemical Pattern Associated with Resistance of Hepatocytes in Hepatocyte Nodules during Liver Carcinogenesis

Eriksson¹,

Ahluwalia²,

Spiewak³

et al. 1983

Environmental Health Perspectives

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Hepatocyte ("hyperplastic") nodules induced in the liver by initiation with diethylnitrosamine and selected by dietary 2-acetylaminofluorene plus partial hepatectomy ("resistant hepatocyte model") have a special pattern of biochemical behavior and metabolic activity different than that seen acutely with many xenobiotics including many promoting agents and carcinogens. The nodule cells show a very low uptake of 2-acetylaminofluorene, relative to surrounding and normal liver, low levels of activity in the cytochromes P-450 and aryl hydrocarbon hydroxylase, high levels of activity in y-glutamyltransferase, microsomal epoxide hydrolase, soluble glutathione-S-transferase and soluble UDP-glucuronyltransferase (UDP-GTJ) and elevated levels of glutathione. This metabolic pattern appears to maximize the resistance of the nodules to xenobiotics generally, such as 2-acetylaminofluorene, and thereby may account for the resistant behavior of nodule hepatocytes to the inhibition of cell proliferation and the cytotoxicity by 2-acetylaminofluorene and other carcinogens. The possible importance of this seemingly new metabolic program in carcinogenesis is discussed briefly.

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“…The primary hepatocellular carcinomas that develop in these livers also maintain a very high level of polypeptide PPA-80 (46) . Whether this is caused by the continued presence of peroxisome proliferator in the diet or whether the induced polypeptide PPA-80 level becomes "fixed" in these hypolipidemic drug-induced tumors, similar to that encountered in the case of the microsomal enzyme epoxide hydrase in acetylaminofluorene-induced hyperplastic liver nodules (20,30), remains to be ascertained .…”

Section: Discussionmentioning

confidence: 99%

Immunochemical identity of peroxisomal enoyl-CoA hydratase with the peroxisome-proliferation -associated 80,000 mol wt polypeptide in rat liver

Reddy¹,

Qureshi²,

Hollenberg³

et al. 1981

The Journal of Cell Biology

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Peroxisome proliferators, which induce proliferation of hepatic peroxisomes, have been shown previously to cause a marked increase in an 80,000 mol wt polypeptide predominantly in the light mitochondrial and microsomal fractions of liver of rodents. We now present evidence to show that this hepatic peroxisome-proliferation-associated polypeptide, referred to as polypeptide PPA-80, is immunochemically identical with the mutifunctional peroxisome protein displaying heat-labile enoyl-CoA hydratase activity . This conclusion is based on the following observations : (a) the purified polypeptide PPA-80 and the heat-labile enoyl-CoA hydratase from livers of rats treated with the peroxisome proliferator Wy-14,643 ([4-chloro-6(2,3-xylidino)-2-pyrimidinylthio]acetic acid) exhibit identical minimum molecular weights of 80,000 on SDS polyacrylamide gel electrophoresis; (b) these two proteins are immunochemically identical on the basis of Ouchterlony double diffusion, immunotitration, rocket immunoelectrophoresis, and crossed immunoelectrophoresis analyses ; and (c) the immunoprecipitates formed by antibodies to polypeptide PPA-80 when dissociated on a Sephadex G-200 column yield enoyl-CoA hydratase activity . Whether the polypeptide PPA-80 exhibits the activity of other enzyme(s) of the peroxisomal Q-oxidation system such as fatty acyl-CoA oxidase activity or displays immunochemical identity with such enzymes remains to be determined .The availability of antibodies to polypeptide PPA-80 and enoyl-CoA hydratase facilitated immunofluorescent and immunocytochemical localization of the polypeptide PPA-80 and enoyl-CoA hydratase in the rat liver. The indirect immunofluorescent studies with these antibodies provided direct visual evidence for the marked induction of polypeptide PPA-80 and enoyl-CoA hydratase in the livers of rats treated with Wy-14,643. The present studies also provide immunocytochemical evidence for the localization of polypeptide PPA-80 and the heat-labile enoyl-CoA hydratase in the peroxisome, but not in the mitochondria, of hepatic parenchymal cells. These studies, therefore, provide morphological evidence for the existence of fatty acyl-CoA oxidizing system in peroxisomes . An increase of polypeptide PPA-80 on SDS polyacrylamide gel electrophoretic analysis of the subcellular fractions of liver of rodents treated with lipid-lowering drugs should serve as a reliable and sensitive indicator of enhanced peroxisomal #-oxidation system . Several structurally unrelated hypolipidemic drugs and certain industrial plasticizers such as di-(2-ethylhexyl)phthalate which cause profound proliferation of peroxisomes in hepatic parenchymal cells of rodents (37,38,50,51), also cause a marked increase in the quantity of a polypeptide with an approximate 406

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Identification of epoxide hydrase as the preneoplastic antigen in rat liver hyperplastic nodules.

Cited by 72 publications

References 11 publications

Immunocytochemical localization of epoxide hydrase in hyperplastic nodules induced in rat liver by 2-acetylaminofluorene.

Immunocytochemical localization of epoxide hydrase in hyperplastic nodules induced in rat liver by 2-acetylaminofluorene.

Distinctive Biochemical Pattern Associated with Resistance of Hepatocytes in Hepatocyte Nodules during Liver Carcinogenesis

Immunochemical identity of peroxisomal enoyl-CoA hydratase with the peroxisome-proliferation -associated 80,000 mol wt polypeptide in rat liver

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