Steatosis is a frequent pathologic stage in alcoholic liver disease (ALD). Although the mechanisms for increased susceptibility of steatotic liver to injury have been postulated, the ability of these hepatocytes to proliferate and withstand injury is unknown. There are conflicting reports on the status of hepatocyte regeneration following chronic alcohol ingestion. Hence, the objective of this study was to investigate the temporal dynamics between the pattern of liver injury and hepatocyte proliferation during the steatosis stage of ALD. Alcoholic steatosis was induced in male Sprague—Dawley rats by feeding an ethanol (EtOH)-containing Lieber-DeCarli liquid diet for a period of 5 weeks. Microvesicular steatosis was evident in H&E sections by three weeks in the EtOH-treated rats, which further developed into panlobular macrovesicular steatosis by 5 weeks. Plasma transaminase activities indicated progressive increase in liver injury peaking at 3 weeks with significant but mild decrease at 4 and 5 weeks. CYP2E1 protein and activity was significantly increased in EtOH-fed rats as measured by Western blot and pNP hydroxylation assay. PCNA analysis of liver sections indicated that EtOH-treated rats had a significantly higher number of cells in S phase of cell division at weeks 1 (3.20 ± 0.19), 2 (7.03 ± 0.92), and 3 (4.23 ± 1.41) when compared to controls (1.5 ± 0.22). NF-κB DNA binding and Cyclin D1 proteins increased significantly in the EtOH-treated rats corresponding with enhanced hepatic proliferation. These data suggest the transient decline in liver injury during alcoholic steatosis is due to enhanced NF-κB-dependent hepatocyte proliferation.
Apte, U.M., Limaye, P.B., Ramaiah, S.K., Vaidya, V.S., Bucci, T.J., Warbritton, A., and Mehendale, H.M. (2002). Upregulated promitogenic signaling via cytokines and growth factors: potential mechanism of robust liver tissue repair in calorie-restricted rats upon toxic challenge. Toxicol Sci69, 448— 59.
2.
Armbrust, T., Batusic, D., Xia, L., and Ramadori, G. (2002). Early gene expression of hepatocyte growth factor in mononuclear phagocytes of rat liver after administration of carbon tetrachloride . Liver22, 486—94.
3.
Arteel, G.E. (2003). Oxidants and antioxidants in alcohol-induced liver disease. Gastroenterology24, 778—90.
4.
Bathgate, A.J., and Simpson, K.J. (2002). Alcoholic fatty liver. In EtOH and The Liver Mechanisms and Management (D. I. N. Sherman, V. Preedy, and R. R. Watson, eds.), pp. 3—20. Taylor and Francis, London and New York .
5.
Baykov, I., Jarvelainen, H., and Lindros, K. (2003). L-Cartinite alleviates alcohol-induced liver damage in rats: role of tumor necrosis factor-alpha. Alcohol & Alcoholism38, 400—06.
6.
Bhagwandeen, B.S., Apte, M., Manwarring, L., and Dickeson, J. (1987). Endotoxin induced hepatic necrosis in rats on an alcoholic diet. J Pathol152, 47—53.
7.
Chanda S., Mangipudy, R.S., Warbritton, A., Bucci, T.J., and Mehendale, H.M. (1995). Stimulated hepatic tissue repair underlies heteroprotection by thioacetamide against acetaminophen-induced lethality. Hepatology21, 477—86.
8.
Chen J., Ishac, E.J., Dent, P., Kunos, G., and Gao, B. (1998). Effects of ethanol on mitogen-activated protein kinase and stress-activated protein kinase cascades in normal and regenerating liver. Biochem J334, 669—76.
9.
Colell, A., Garcia-Ruiz, C., Miranda, M., Ardite, E., Mari, M., Morales, A., Kaplowitz, N., and Fernandez-Checa, J.C. (1998). Selective glutathione depletion of mitochondria by ethanol sensitizes hepatocytes to tumor necrosis factor. Gastroenterology115, 1541—51.
10.
Day, C.P., and James, O.F. (1998). Hepatic steatosis: innocent bystander or guilty party. Hepatology27, 1463—6.
11.
Deaciuc, I.V., D'Souza, N.B., Burikhanov, R., Lee, E.Y., Tarba, C.N., McClain, C.J., and de Villiers, W.J.S. (2002). Epidermal growth factors protects the liver against alcohol-induced injury and sensitization to bacterial lipopolysaccharide. Alcohol Clin Exp Res26, 864—74.
12.
Diehl, A.M. (1999). Effect of ethanol on tumor necrosis factor signaling during liver regeneration. Clin Biochem32, 571—78.
Diehl, A.M. (2002b). Liver regeneration. Front Biosci . 7, e301—314.
15.
Diehl, A.M., Yang, S.Q., Cote, P., and Wand, G.S. (1992). Chronic ethanol consumption disturbs G-protein expression and inhibits cyclic AMP-dependent signaling in regenerating rat liver. Hepatology16, 1212—9.
16.
Enomoto, N., Yamashita, S., Kono, H., Schemmer, P., Rivera, C.A., Enomoto, A.Nishiura, T., Nishimura, T., Brenner, D.A., and Thurman, R.G. (1999). Development of a new, simple rat model of early alcohol-induced liver injury based on sensitization of Kupffer cells. Hepatology29, 1680—9.
17.
Essani, N.A., McGuire, G.M., Manning, A.M., and Jaeschke, H. (1996). Endotoxin-induced activation of the nuclear transcription factor kappa B and expression of E-selectin messenger RNA in hepatocytes, Kupffer cells and endothelial cells in vivo. J Immunol156, 2956—63.
18.
Everett, L., Galli, A., and Crabb, D. (2000). The role of hepatic peroxisome proliferator-activated receptors (PPARs) in health and disease. Liver20, 191—99.
19.
Fausto, N., and Campbell, J.S. (2003). The role of hepatocytes and oval cells in liver regeneration and repopulation. Mech Dev120, 117—30.
20.
Fausto, N., Laird, A.D., and Webber, E.M. (1995). Liver regeneration. 2. Role of growth factors and cytokines in hepatic regeneration. FASEB J9, 1527—36.
21.
Fausto, N. (2000). Liver Regeneration. J Hepatol32, 19—31.
22.
Fischer, M., You, M., Matsumoto, M., and Crabb, D.W. (2003). Peroxisome proliferator-activated receptor alpha (PPARalpha) agonist treatment reverses PPARalpha dysfunction and abnormalities in hepatic lipid metabolism in ethanol-fed mice. J Biol Chem278, 27997—28004.
23.
Galli, A., Pinaire, J., Fischer, M., Dorris, R., and Crabb, D. (2001). The transcriptional and DNA binding activity of peroxisome proliferator-activated receptor alpha is inhibited by ethanol metabolism. A novel mechanism for the development of ethanol-induced fatty liver. J Biol Chem276, 68—75.
24.
Galum, E., Zeira, E., Pappo, O., Peters, M., and Rose-John, S. (2000). Liver regeneration induced by a designer human IL-6/sIL-6R fusion protein reverses severe hepatocellular injury. FASEB J14, 1979—87.
25.
Gavrieli, Y., Sherman, Y., and Ben-Sasson, S.A. (1992). Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol119, 493—501.
26.
Gouillon, Z.Q., Miyamoto, K., Donohue, T.M., Wan, Y.J., French, B.A., Nagao, Y., Fu, P., Reitz, R.C., Hagbjork, A., Yap, C., Yuan, Q.X., Ingelman-Sundberg, M., and French, F.W. (1999). Role of CYP2E1 in the pathogenesis of alcoholic liver disease: modifications by cAMP and ubiquitin-proteasome pathway. Front Biosci4, A16—25.
27.
Greenwell, A., Foley, J.F., and Maronpot, R.R. (1991). An enhancement method for immunohistochemical staining of proliferating cell nuclear antigen in archival rodent tissues . Cancer Lett59, 251—6.
28.
Hilden, M., Christoffson, P., Juhl, E., and Dalgaard, J.B. (1977). Liver histology in a `normal' population-Examination of 503 consecutive fatal traffic casualties. Scan J Gastroenterol12, 593—7.
29.
Koop, D.R. (1990). Inhibition of ethanol-inducible cytochrome P450IIE1 by 3-amino-1,2,4-triazole. Chem. Res. Toxicol3, 377—83.
30.
Koteish, A., Yang, S., Lin, H., Huang, J., and Diehl, A.M. (2002). Ethanol induces redox sensitive cell cycle inhibitors and inhibits liver regeneration after partial hepatectomy. Alcohol Clin Exp Res26, 1710—8.
31.
Leevy, C.M., and Chen, T. (1979). Ethanol inhibition of liver regeneration. Gastroenterology77, 1151—3.
McGehee, R.E. Jr., Ronis, M.J., and Badger, T.M. (1997). Regulation of the hepatic CYP 2E1 gene during chronic alcohol exposure: lack of an ethanol response element in the proximal 5'-flanking sequence. DNA Cell Biol16, 725—36.
37.
McGregor, A.H., More, L.J., Simpson, K.J., and Harrison, D.J. (2003). Liver death and regeneration in paracetamol toxicity . Hum Exp Toxicol22, 221— 27.
38.
Mehendale, H.M. (1995). Toxicodynamics of low level toxicant interactions of biological significance: inhibition of tissue repair. Toxicology105, 251— 66.
39.
Minuk, G.Y., Rockman, G.E., Gauthier, T., Markert, L., Gibson, J., and Benarroch, A. (1991). The effect of long-term, voluntary ethanol consumption on hepatic regeneration in rats. Can J Physiol Pharmacol69, 341—5.
40.
Nanji, A. (2002). Alcoholic liver disease. In Hepatology, A Text Book of Liver Disease (D. Zakim , and T. D. Boyer, eds.), pp. 839—922. W. B. Saunders, Philadelphia.
41.
Nanji, A.A., Jokelainen, K., Tipoe, G.L., Rahemtulla, A., Thomas, P., and Dannenberg, A.J. (2003). Curcumin prevents alcohol-induced liver disease in rats by inhibiting the expression of NF-κB-dependent genes. Am J Physiol Gastrointest Liver Physiol284, G321—7.
42.
Nanji, A.A., Tsukamoto, H., and French, S.W. (1989). Relationship between fatty liver and subsequent development of necrosis, inflammation and fibrosis in experimental alcoholic liver disease. Exp Mol Pathol51, 141—8.
43.
National Institute on Alcohol Abuse and Alcoholism. (2001). Alcohol Alert No. 51. NIAA , Bethesda, Md.
44.
Oneta, C.M., Lieber, C.S., Li, J., Ruttimann, S., Schmid, B., Lattmann, J., Rosman, A.S., and Seitz, H.K. (2002). Dynamics of cytochrome P4502E1 activity in man: induction by ethanol and disappearance during withdrawal phase. J Hepatol36, 47—52.
45.
Pennington, H.L., Hall, P.M., Wilce, P.A., and Worrall, S. (1997). Ethanol feeding enhances inflammatory cytokine expression in lipopolysaccharide-induced hepatitis. J Gastroenterol Hepatol12, 305—13.
46.
Phillips, M., Kashef, E., and Hughes, R. (2002). Cellular mechanisms of liver regeneration and the effect of alcohol. In Ethanol and The Liver Mechanisms and Management (D. Sherman, V. Preedy, and R. R. Watson, eds.), pp. 319—42. Taylor and Francis, London and New York.
47.
Ramaiah, S.K., Bucci, T.J., Warbritton, A., Soni, M.G., and Mehendale, H.M. (1998). Temporal changes in tissue repair permit survival of diet-restricted rats from an acute lethal dose of thioacetamide. Toxicol Sci45, 233— 41.
48.
Roberts, B.J., Shoaf, S.E., and Song, B.J. (1995). Rapid changes in cytochrome P4502E1 (CYP2E1) activity and other P450 isozymes following ethanol withdrawal in rats. Biochem Pharmacol49, 1665—73.
49.
Saso, K., Moehren, G., Higashi, K., and Hoek, J.B. (1997). Differential inhibition of epidermal growth factor signaling pathways in rat hepatocytes by long-term ethanol treatment. Gastroenterology112, 2073—88.
50.
Shayiq, R.M., Roberts, D.W., Rothstein, K., Snawder, J.E., Benson, W., Ma, X., and Black, M. (1999). Repeat exposure to incremental doses of acetaminophen provides protection against acetaminophen-induced lethality in mice: an explanation for high acetaminophen dosage in humans without hepatic injury. Hepatology29, 451—63.
51.
Soni, M.G., and Mehendale, H.M. (1998). Role of tissue repair in toxicologic interactions among hepatotoxic organics. Environ Health Perspect106, 1307—17.
52.
Sorensen, T.I., Orholm, M., Bentsen, K.D., Hoybye, G., Eghoje, K., and Christoffersen, P. (1984). Prospective evaluation of alcohol abuse and alcoholic liver injury in men as predictors of development of cirrhosis. Lancet2, 241—4.
53.
Taub, R. (1996). Liver regeneration 4: transcriptional control of liver regeneration. FASEB J10, 413—27.
54.
Teli, M.R., Day, C.P., Burt, A.D., Bennett, M.K., and James, O.F.W. (1995). Determinants of progression to cirrhosis or fibrosis in pure alcoholic fatty liver. Lancet346, 987—90.
55.
Underwood Ground, K.E. (1984). Prevalence of fatty liver in healthy male adults accidently killed. Aviat Space Environ Med55, 59—61.
56.
Wands, J.R., Carter, E.A., Bucher, N.L., and Isselbacher, K.J. (1980). Effect of acute and chronic ethanol intoxication on hepatic regeneration. Adv Exp Med Biol132, 663—70.
57.
Wang, T., Shankar, K., Ronis, M.J., and Mehendale, H.M. (2000). Potentiation of thioacetamide liver injury in diabetic rats is due to induced CYP2E1. J Pharmacol Exp Ther294, 473—79.
58.
Yang, S.Q., Lin, H.Z., Lane, M.D., Clemens, M., and Diehl, A.M. (1997). Obesity increases sensitivity to endotoxin liver injury: implications for the pathogenesis of steatohepatitis. Proc Natl Acad Sci USA94, 2557— 62.
59.
Yang, S.Q., Lin, H.Z., Yin, M., Albrecht, J.H., and Diehl, A.M. (1998). Effects of chronic ethanol consumption on cytokine regulation of liver regeneration. Am J Physiol275, G696—704.
60.
Zhang, B.H., and Farrell, G.C. (1999). Chronic ethanol consumption disrupts complexation between EGF receptor and phospholipase C-gamma1: relevance to impaired hepatocyte proliferation. Biochem Biophys Res Commun257, 89—94.
61.
Zhang, M., Gong, Y., Corbin, I., Mellon, A., Choy, P., Uhanova, J., and Minuk, G.Y. (2000). Light ethanol consumption enhances liver regeneration after partial hepatectomy in rats. Gastroenterology119, 1333—9.
62.
Zhang, M., Gong, Y.W., and Minuk, G.Y. (1998). The effects of ethanol and gamma aminobutyric acid alone and in combination on hepatic regenerative activity in the rat . J Hepatol29, 638—41.
63.
Zhang, M., Uhanova, J., Corbin, I., Bernstein, C., and Minuk, G.Y. (2003). Effects of daily, light and moderate-heavy ethanol exposure on extent of hepatic injury and recovery following toxin-induced acute hepatitis in rats. Dig Dis Sci48, 926—31.
