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. 1995 Jul;96(1):620–630. doi: 10.1172/JCI118077

Experimental liver cirrhosis induced by alcohol and iron.

H Tsukamoto 1, W Horne 1, S Kamimura 1, O Niemelä 1, S Parkkila 1, S Ylä-Herttuala 1, G M Brittenham 1
PMCID: PMC185237  PMID: 7615836

Abstract

To determine if alcoholic liver fibrogenesis is exacerbated by dietary iron supplementation, carbonyl iron (0.25% wt/vol) was intragastrically infused with or without ethanol to rats for 16 wk. Carbonyl iron had no effect on blood alcohol concentration, hepatic biochemical measurements, or liver histology in control animals. In both ethanol-fed and control rats, the supplementation produced a two- to threefold increase in the mean hepatic non-heme iron concentration but it remained within or near the range found in normal human subjects. As previously shown, the concentrations of liver malondialdehyde (MDA), liver 4-hydroxynonenal (4HNE), and serum aminotransferases (ALT, AST) were significantly elevated by ethanol infusion alone. The addition of iron supplementation to ethanol resulted in a further twofold increment in mean MDA, 4HNE, ALT, and AST. On histological examination, focal fibrosis was found < 30% of the rats fed ethanol alone. In animals given both ethanol and iron, fibrosis was present in all, with a diffuse central-central bridging pattern in 60%, and two animals (17%) developed micronodular cirrhosis. The iron-potentiated alcoholic liver fibrogenesis was closely associated with intense and diffuse immunostaining for MDA and 4HNE adduct epitopes in the livers. Furthermore, in these animals, accentuated increases in procollagen alpha 1(I) and TGF beta 1 mRNA levels were found in both liver tissues and freshly isolated hepatic stellate cells, perisinusoidal cells believed to be a major source of extracellular matrices in liver fibrosis. The dietary iron supplementation to intragastric ethanol infusion exacerbates hepatocyte damage, promotes liver fibrogenesis, and produces evident cirrhosis in some animals. These results provide evidence for a critical role of iron and iron-catalyzed oxidant stress in progression of alcoholic liver disease.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allen K. G., Arthur J. R. Inhibition by 5-sulphosalicylic acid of the glutathione reductase recycling assay for glutathione analysis. Clin Chim Acta. 1987 Jan 30;162(2):237–239. doi: 10.1016/0009-8981(87)90456-6. [DOI] [PubMed] [Google Scholar]
  2. BELL E. T. The relation of portal cirrhosis to hemochromatosis and to diabetes mellitus. Diabetes. 1955 Nov-Dec;4(6):435–446. doi: 10.2337/diab.4.6.435. [DOI] [PubMed] [Google Scholar]
  3. Bacon B. R., Tavill A. S., Brittenham G. M., Park C. H., Recknagel R. O. Hepatic lipid peroxidation in vivo in rats with chronic iron overload. J Clin Invest. 1983 Mar;71(3):429–439. doi: 10.1172/JCI110787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baeuerle P. A., Baltimore D. I kappa B: a specific inhibitor of the NF-kappa B transcription factor. Science. 1988 Oct 28;242(4878):540–546. doi: 10.1126/science.3140380. [DOI] [PubMed] [Google Scholar]
  5. Bassett M. L., Halliday J. W., Powell L. W. Value of hepatic iron measurements in early hemochromatosis and determination of the critical iron level associated with fibrosis. Hepatology. 1986 Jan-Feb;6(1):24–29. doi: 10.1002/hep.1840060106. [DOI] [PubMed] [Google Scholar]
  6. Benedetti A., Pompella A., Fulceri R., Romani A., Comporti M. Detection of 4-hydroxynonenal and other lipid peroxidation products in the liver of bromobenzene-poisoned mice. Biochim Biophys Acta. 1986 May 21;876(3):658–666. doi: 10.1016/0005-2760(86)90055-x. [DOI] [PubMed] [Google Scholar]
  7. Brenner D. A., Chojkier M. Acetaldehyde increases collagen gene transcription in cultured human fibroblasts. J Biol Chem. 1987 Dec 25;262(36):17690–17695. [PubMed] [Google Scholar]
  8. Casini A., Cunningham M., Rojkind M., Lieber C. S. Acetaldehyde increases procollagen type I and fibronectin gene transcription in cultured rat fat-storing cells through a protein synthesis-dependent mechanism. Hepatology. 1991 Apr;13(4):758–765. [PubMed] [Google Scholar]
  9. Castillo T., Koop D. R., Kamimura S., Triadafilopoulos G., Tsukamoto H. Role of cytochrome P-450 2E1 in ethanol-, carbon tetrachloride- and iron-dependent microsomal lipid peroxidation. Hepatology. 1992 Oct;16(4):992–996. doi: 10.1002/hep.1840160423. [DOI] [PubMed] [Google Scholar]
  10. Chojkier M., Houglum K., Solis-Herruzo J., Brenner D. A. Stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts. A role for lipid peroxidation? J Biol Chem. 1989 Oct 5;264(28):16957–16962. [PubMed] [Google Scholar]
  11. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  12. DeForge L. E., Preston A. M., Takeuchi E., Kenney J., Boxer L. A., Remick D. G. Regulation of interleukin 8 gene expression by oxidant stress. J Biol Chem. 1993 Dec 5;268(34):25568–25576. [PubMed] [Google Scholar]
  13. Derynck R., Jarrett J. A., Chen E. Y., Eaton D. H., Bell J. R., Assoian R. K., Roberts A. B., Sporn M. B., Goeddel D. V. Human transforming growth factor-beta complementary DNA sequence and expression in normal and transformed cells. Nature. 1985 Aug 22;316(6030):701–705. doi: 10.1038/316701a0. [DOI] [PubMed] [Google Scholar]
  14. Esterbauer H., Cheeseman K. H., Dianzani M. U., Poli G., Slater T. F. Separation and characterization of the aldehydic products of lipid peroxidation stimulated by ADP-Fe2+ in rat liver microsomes. Biochem J. 1982 Oct 15;208(1):129–140. doi: 10.1042/bj2080129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Esterbauer H., Jürgens G., Quehenberger O., Koller E. Autoxidation of human low density lipoprotein: loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes. J Lipid Res. 1987 May;28(5):495–509. [PubMed] [Google Scholar]
  16. French S. W., Wong K., Jui L., Albano E., Hagbjork A. L., Ingelman-Sundberg M. Effect of ethanol on cytochrome P450 2E1 (CYP2E1), lipid peroxidation, and serum protein adduct formation in relation to liver pathology pathogenesis. Exp Mol Pathol. 1993 Feb;58(1):61–75. doi: 10.1006/exmp.1993.1006. [DOI] [PubMed] [Google Scholar]
  17. Friedman S. L. Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies. N Engl J Med. 1993 Jun 24;328(25):1828–1835. doi: 10.1056/NEJM199306243282508. [DOI] [PubMed] [Google Scholar]
  18. Genovese C., Rowe D., Kream B. Construction of DNA sequences complementary to rat alpha 1 and alpha 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D. Biochemistry. 1984 Dec 4;23(25):6210–6216. doi: 10.1021/bi00320a049. [DOI] [PubMed] [Google Scholar]
  19. Griffith O. W. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem. 1980 Jul 15;106(1):207–212. doi: 10.1016/0003-2697(80)90139-6. [DOI] [PubMed] [Google Scholar]
  20. Hirano T., Kaplowitz N., Tsukamoto H., Kamimura S., Fernandez-Checa J. C. Hepatic mitochondrial glutathione depletion and progression of experimental alcoholic liver disease in rats. Hepatology. 1992 Dec;16(6):1423–1427. doi: 10.1002/hep.1840160619. [DOI] [PubMed] [Google Scholar]
  21. Houglum K., Brenner D. A., Chojkier M. d-alpha-tocopherol inhibits collagen alpha 1(I) gene expression in cultured human fibroblasts. Modulation of constitutive collagen gene expression by lipid peroxidation. J Clin Invest. 1991 Jun;87(6):2230–2235. doi: 10.1172/JCI115258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jamall I. S., Finelli V. N., Que Hee S. S. A simple method to determine nanogram levels of 4-hydroxyproline in biological tissues. Anal Biochem. 1981 Mar 15;112(1):70–75. doi: 10.1016/0003-2697(81)90261-x. [DOI] [PubMed] [Google Scholar]
  23. Kamimura S., Gaal K., Britton R. S., Bacon B. R., Triadafilopoulos G., Tsukamoto H. Increased 4-hydroxynonenal levels in experimental alcoholic liver disease: association of lipid peroxidation with liver fibrogenesis. Hepatology. 1992 Aug;16(2):448–453. doi: 10.1002/hep.1840160225. [DOI] [PubMed] [Google Scholar]
  24. Knecht K. T., Thurman R. G., Mason R. P. Role of superoxide and trace transition metals in the production of alpha-hydroxyethyl radical from ethanol by microsomes from alcohol dehydrogenase-deficient deermice. Arch Biochem Biophys. 1993 Jun;303(2):339–348. doi: 10.1006/abbi.1993.1293. [DOI] [PubMed] [Google Scholar]
  25. Livingston F. R., Lui E. M., Loeb G. A., Forman H. J. Sublethal oxidant stress induces a reversible increase in intracellular calcium dependent on NAD(P)H oxidation in rat alveolar macrophages. Arch Biochem Biophys. 1992 Nov 15;299(1):83–91. doi: 10.1016/0003-9861(92)90247-t. [DOI] [PubMed] [Google Scholar]
  26. Matsuoka M., Tsukamoto H. Stimulation of hepatic lipocyte collagen production by Kupffer cell-derived transforming growth factor beta: implication for a pathogenetic role in alcoholic liver fibrogenesis. Hepatology. 1990 Apr;11(4):599–605. doi: 10.1002/hep.1840110412. [DOI] [PubMed] [Google Scholar]
  27. Mihara M., Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem. 1978 May;86(1):271–278. doi: 10.1016/0003-2697(78)90342-1. [DOI] [PubMed] [Google Scholar]
  28. Morimoto M., Hagbjörk A. L., Nanji A. A., Ingelman-Sundberg M., Lindros K. O., Fu P. C., Albano E., French S. W. Role of cytochrome P4502E1 in alcoholic liver disease pathogenesis. Alcohol. 1993 Nov-Dec;10(6):459–464. doi: 10.1016/0741-8329(93)90065-v. [DOI] [PubMed] [Google Scholar]
  29. Nanji A. A., Zhao S., Lamb R. G., Dannenberg A. J., Sadrzadeh S. M., Waxman D. J. Changes in cytochromes P-450, 2E1, 2B1, and 4A, and phospholipases A and C in the intragastric feeding rat model for alcoholic liver disease: relationship to dietary fats and pathologic liver injury. Alcohol Clin Exp Res. 1994 Aug;18(4):902–908. doi: 10.1111/j.1530-0277.1994.tb00058.x. [DOI] [PubMed] [Google Scholar]
  30. Niemelä O., Parkkila S., Ylä-Herttuala S., Halsted C., Witztum J. L., Lanca A., Israel Y. Covalent protein adducts in the liver as a result of ethanol metabolism and lipid peroxidation. Lab Invest. 1994 Apr;70(4):537–546. [PubMed] [Google Scholar]
  31. Palinski W., Ylä-Herttuala S., Rosenfeld M. E., Butler S. W., Socher S. A., Parthasarathy S., Curtiss L. K., Witztum J. L. Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein. Arteriosclerosis. 1990 May-Jun;10(3):325–335. doi: 10.1161/01.atv.10.3.325. [DOI] [PubMed] [Google Scholar]
  32. Park C. H., Bacon B. R., Brittenham G. M., Tavill A. S. Pathology of dietary carbonyl iron overload in rats. Lab Invest. 1987 Nov;57(5):555–563. [PubMed] [Google Scholar]
  33. Parola M., Pinzani M., Casini A., Albano E., Poli G., Gentilini A., Gentilini P., Dianzani M. U. Stimulation of lipid peroxidation or 4-hydroxynonenal treatment increases procollagen alpha 1 (I) gene expression in human liver fat-storing cells. Biochem Biophys Res Commun. 1993 Aug 16;194(3):1044–1050. doi: 10.1006/bbrc.1993.1927. [DOI] [PubMed] [Google Scholar]
  34. Reinke L. A., Kotake Y., McCay P. B., Janzen E. G. Spin-trapping studies of hepatic free radicals formed following the acute administration of ethanol to rats: in vivo detection of 1-hydroxyethyl radicals with PBN. Free Radic Biol Med. 1991;11(1):31–39. doi: 10.1016/0891-5849(91)90185-6. [DOI] [PubMed] [Google Scholar]
  35. Takahashi H., Johansson I., French S. W., Ingelman-Sundberg M. Effects of dietary fat composition on activities of the microsomal ethanol oxidizing system and ethanol-inducible cytochrome P450 (CYP2E1) in the liver of rats chronically fed ethanol. Pharmacol Toxicol. 1992 May;70(5 Pt 1):347–351. doi: 10.1111/j.1600-0773.1992.tb00485.x. [DOI] [PubMed] [Google Scholar]
  36. Tsukamoto H., Gaal K., French S. W. Insights into the pathogenesis of alcoholic liver necrosis and fibrosis: status report. Hepatology. 1990 Sep;12(3 Pt 1):599–608. doi: 10.1002/hep.1840120325. [DOI] [PubMed] [Google Scholar]
  37. Tsukamoto H. Oxidative stress, antioxidants, and alcoholic liver fibrogenesis. Alcohol. 1993 Nov-Dec;10(6):465–467. doi: 10.1016/0741-8329(93)90066-w. [DOI] [PubMed] [Google Scholar]
  38. Tsukamoto H., Towner S. J., Ciofalo L. M., French S. W. Ethanol-induced liver fibrosis in rats fed high fat diet. Hepatology. 1986 Sep-Oct;6(5):814–822. doi: 10.1002/hep.1840060503. [DOI] [PubMed] [Google Scholar]
  39. WEINFELD A. STORAGE IRON IN MAN. Acta Med Scand Suppl. 1964;427:SUPPL 427–427:1+. [PubMed] [Google Scholar]
  40. Yoo J. S., Ning S. M., Pantuck C. B., Pantuck E. J., Yang C. S. Regulation of hepatic microsomal cytochrome P450IIE1 level by dietary lipids and carbohydrates in rats. J Nutr. 1991 Jul;121(7):959–965. doi: 10.1093/jn/121.7.959. [DOI] [PubMed] [Google Scholar]
  41. Zhang J. R., Sevanian A. The genotoxic effects of arachidonic acid in V79 cells are mediated by peroxidation products. Toxicol Appl Pharmacol. 1993 Aug;121(2):193–202. doi: 10.1006/taap.1993.1145. [DOI] [PubMed] [Google Scholar]

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