Does Ethanol Play a Pro-Oxidant Role during Oxidative Stress in the Liver?


Oxidative stress has been implicated in the pathophysiology of liver injury during xenobiotic and alcohol metabolism, ischemia/reperfusion injury. In this study we examined if ethanol acted as a pro-oxidant making cells become more sensitive to tert-butylhydroperoxide (tBH) killing. Cell viability was determined in a rat hepatoma cell line (FTO2B) and rat primary hepatocytes in culture in the presence or absence of ethanol pretreatment. To elucidate the contribution of labile iron, deferoxamine (DF, an iron chelator) or lipid free radicals, N,N-diphenyl-p-phenylenediamine (DPPD, a lipid scavenger) were added to the ethanol tBH co-treatment. The levels of glutathione (GSH) and glutathione disulfide (GSSG) in the hepatocytes were also measured. Ethanol treatment (both pretreatment and co-treatment during the 3-hr tBH exposure) increased cell killing dramatically in both FTO2B cells and primary rat hepatocytes. Both DF and DPPD decreased ethanol-enhanced tBH cell killing in hepatocytes. These results demonstrated that co-treatment of FTO2B cells and primary rat hepatocytes with ethanol and tBH increased cell killing. The GSH level was dramatically reduced while GSSG level rose. Both DFP and DPPD reversed or protected the cells from this insult, indicating that ethanol was a pro-oxidant.

Share and Cite:

Obih, P. , Soblosky, J. and Potter, B. (2015) Does Ethanol Play a Pro-Oxidant Role during Oxidative Stress in the Liver?. Journal of Biosciences and Medicines, 3, 1-9. doi: 10.4236/jbm.2015.38001.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Groskreutz, J.L., Bronk, S.F. and Gores, G.J. (1992) Ruthenium Red Delays the Onset of Cell Death during Oxidative Stress of Rat Hepatocytes. Gastroenterology, 102, 1030-1038.
[2] Chamulitrat, W., Carnal, J., Reed, N.M., and Spitzer. J.J. (1998) In Vivo Endotoxin Enhances Biliary Ethanol-De- pendent Free Radical Generation. American Journal of Physiology—Gastrointestinal and Liver, 274, G653-G661.
[3] Potter, B.J., Blades, B., McHugh, T.A., Nunes, R.M., Beloqui, O., Slott, P.A. and Rand J.H. (1989) Effects of Endotoxin on Iron Uptake by the Hepatocytes. American Journal of Physiology, 257, G524-G531.
[4] Cross, C.E., Halliwell, B., Borish, E.T., Pryor, W.A., Ames, B.N., Saul, R.L., McCord, J.M. and Harman, D. (1987) Oxygen Radicals and Human Disease [Clinical Conference]. Annals of Internal Medicine, 107, 526-545.
[5] Grisham, M.B. and Yamada, T. (1992) Neutrophils, Nitrogen Oxides, and Inflammatory Bowel Disease. Annals of the New York Academy of Sciences, 664, 103-115.
[6] Alzoghaibi, M.A. (2013) Concepts of Oxidative Stress and Antioxidant Defense in Crohn’s Disease. World Journal of Gastroenterology, 19, 6540-6547.
[7] Ayin, A., Orhan, H., sayal, A., Ozata, M., Sahin, G. and Isimer, A. (2001) Oxidative Stress and Nitric Oxide Related Parameters in Type II Diabetes Mellitus: Effects of Glycemic Control. Clinical Biochemistry, 34, 65-70.
[8] Cathcart, M.K., McNally, A.K., Morel, D.W. and Chisolm, G.M., (1989) Superoxide Anion Participation in Human Monocyte-Mediated Oxidation of Low-Density Lipoprotein and Conversion of Low-Density Lipoprotein to a Cytotoxin. Journal of Immunology, 142, 1963-1969.
[9] Haliwell, B (1989) Free Radicals, Reactive Oxygen Species and Human Disease: A Critical Evaluation with Special Reference to Atherosclerosis. British Journal of Pathology, 70, 737-757.
[10] Cardin, R., D’Errico, A., Fiorentino, M., Ceccehtto, A. Naccarto, R. and Farinati, F. (2002) Hepatocyte Proliferation and Apoptosis in Relation to Oxidative Damage in Alcohol-Related Liver Disease. Alcohol, 37, 43-48.
[11] Zima, T., Fialova, L., Mestek, O., Janebova, M., Crkovska, J., Malbohan, I., Tipek, S., Mikulikova, L. and Popov, P. (2001) Oxidative Stress, Metabolism of Ethanol and Alcohol-Related Diseases. Journal of Biomed Science, 8, 59-70.
[12] Wu, D. and Cederbaum, A.I. (2003) Alcohol, Oxidative Stress, and Free Radical Damage. Alcohol Research and Health, 27, 277-284.
[13] Mandrekar, P. and Ambade, A. (2012) Chapter 6: Cellular Signaling Pathways in Alcoholic Liver Disease: Trends in Alcoholic Liver Disease Research. In: Shimizu, I., Ed., Clinical and Scientific Aspects, InTech, Rijeka, 91-112.
[14] Dai, Y., Rashba-Step, J. and Cederbaum, A.I. (1993) Stable Expression of Human CYP 2E1 in HepG2 Cells: Characterization of Catalytic Activities and Production of Reactive Oxygen Intermediates. Biochemistry, 32, 6928-6937.
[15] Rashba-Step, J., Turro, N.J. and Cederbaum, A.I. (1993) Increased NADPH- and NADH-Dependent Production of Superoxide and Hydroxyl Radical after Chronic Ethanol Treatment. Archives of Biochemistry and Biophysics, 300, 401-408.
[16] Nanji, A.A., Jokelainen, K., Tipoe, G.L., Rahemtulla, A. and Dannenberg, A.J. (2001) Dietary Saturated Fatty Acids Reverse Inflammatory and Fibrotic Changes in Rat Liver despite Continued Ethanol Administration. Journal of Pharmacology and Experimental Therapeutics, 299, 638-644.
[17] Wheeler, M.D., Kono, H., Yin, M., Rusyn, I., Froh, M., Connor, H.D., et al. (2001) Delivery of the Cu/Zn-Superoxide Dismutase Gene with Adenovirus Reduces Early Alcohol-Induced Liver Injury in Rats. Gastroenterology, 120, 1241- 1250.
[18] Bellomo, G., Jewell, S.A. and Orrenius, S. (1982) Regulation of Intracellular Calcium Compartmentation: Studies with Isolated Hepatocytes and t-Butyl Hydroperoxide. Proceedings of the National Academy of Sciences of the United States of America, 79, 6842-6846.
[19] Bellomo, G., Thor, H. and Orrenius, S. (1984) Increase in Cytosolic Ca2+ Concentration during t-Butyl Hydroperoxide Metabolism by Isolated Hepatocytes Involves NADPH Oxidation and Mobilization of Intracellular Ca2+ Stores. FEBS Letters, 168, 38-42.
[20] Roberto, I., Nieminen, A., Herman, B. and Lemasters, J.L. (1992) Mitochondrial and Glycolytic Dysfunction in Lethal Injury to Hepatocytes by t-Butyl Hydroperoxide: Protection by Fructose, Cyclosporin A and Trifluperazine. The Journal of Pharmacology and Experimental Therapeutics, 265, 392-400.
[21] Kim, Y.M., Bergonia, H. and Lancaster, J.R. (1995) Nitrogen Oxide-Induced Autoprotection in Isolated Rat Hepatocytes. FEBS Letters, 374, 228-232.
[22] Griffith, O.W. (1980) Determination of Glutathione and Glutathione Disulfide Using Glutathione Reductase and 2-Vinylpyridine. Analytical Biochemistry, 106, 207-212.
[23] Ochi, T. (1990) Effects of an Organic Hydroperoxide on the Activity of Antioxidant Enzymes in Cultured Mammalian Cells. Toxicology, 61, 229-239.
[24] Masaki, N., Kyle, M.E. and Farber, J.L. (1989) Tert-Butyl Hydroperoxide Kills Cultured Hepatocytes by Peroxidizing Membrane Lipids. Archives of Biochemistry and Biophysics, 269, 390-399.
[25] Dogterom, P., Mulder, G.J. and Nagelkerke, J.F. (1989) Lipid Peroxidation-Dependent and -Independent Protein Thiol Modifications in Isolated Rat Hepatocytes: Differential Effects of Vitamin E and Disulfiram. Chemico-Biological Interactions, 71, 291-306.
[26] Roy, A. and Sil, P.C. (2012) Tertiary Butyl Hydroperoxide Induced Oxidative Damage in Mice Erythrocytes: Protection by Taurine. Pathophysiology, 19, 137-148.
[27] Venkatraman, A., Landar, A., Davis, A.J., Ulasova, E., Page, G., Murphy, M.P., Darley-Usmar, V. and Bailey, S.M. (2004) Oxidative Modification of Hepatic Mitochondria Protein Thiols: Effect of Chronic Alcohol Consumption. American Journal of Physiology, 286, G521-G527.
[28] Tsukamoto, H. and Lu, S.C. (2001) Current Concepts in Pathogenesis of Alcoholic Liver Injury. FASEB Journal, 15, 1335-1349.
[29] Joshi, M.S., Ponthier, J.L. and Lancaster, J.R. (1999) Cellular Antioxidant and Pro-Oxidant Actions of Nitric Oxide. Free Radical Biology & Medicine, 27, 1357-1366.
[30] Kohgo, Y., Ohtake, T., Ikuta, K., Suzuki, Y., Saito, H. and Kato, J. (2005) Iron Accumulation in Alcoholic Liver Diseases. Alcohol Clinical and Experimental Research, 29, 189S-193S.
[31] Do, T.H.T., Gaboriau, F., Cannie, I., Batusanski, F., Ropert, M., Moirand, R., Brissot, P., Loreal, O. and Lescoat, G. (2013) Iron-Mediated Effect of Alcohol on Hepatocyte Differentiation in HepaRG Cells. Chemical and Biological Interactions, 206, 117-125.
[32] Zhang, H. and Potter, B.J. (1991) The Effect of Ethanol Metabolism on Ferritin Uptake by Freshly Isolated Rat Hepatocytes: Is Acetaldehyde Responsible for This Action? Alcoholism: Clinical and Experimental Research, 16, 301-307.
[33] Tan, T.C., Crawford, D.H., Jaskowski, L.A., Subramaniam, V.N., Clouston, A.D., Craine, D.I., Bridle, K.R., Anderson, G.J. and Fletcher, L.M. (2013) Excess Iron Modulates Endoplasmic Reticulum Stress-Associated Pathways in a Mouse Model of Alcohol and High-Fat-Induced Liver Injury. Laboratory Investigation, 93, 1295-1312.
[34] Lu, Y. and Cederbaum, A.I. (2006) Cisplatin-Induced Hepatotoxicity Is Enhanced by Elevated Expression of Cytochrome P450 2E1. Toxicological Sciences, 89, 515-523.
[35] Comporti, M., Signorini, C., Leoncin, S., Gardi, C., Ciccoli, L., Giardini, A., Vecchio, D. and Arrezini, B. (2010) Ethanol-Induced Oxidative Stress: Basic Knowledge. Genes & Nutrition, 5, 101-109.
[36] Masters, S.B. and Trevor, A.J. (2015) Alcohols. Basic & Clinical Pharmacology, 23, 384-395.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.