Reactive oxygen and nitrogen species generation features under conditions of acute hepatotoxicity

  • I. О. Shmarakov Y. Fedkovych Chernivtsi National University
  • V. L. Borschovetska Y. Fedkovych Chernivtsi National University
  • М. М. Marchenko Y. Fedkovych Chernivtsi National University
Keywords: hepatotoxicity, superoxide, nitric oxide, vitamin A


Development of the most of pathological conditions occurs by free radical mechanism which is characterized by increased free radical production at the cellular level, especially reactive oxygen and nitrogen species (ROS/RNS). The main producers of reactive oxygen species are, first of all, membrane bound NADH-dependent mitochondrial and NADPH-dependent endoplasmic reticulum electron transport systems, cytosolic oxidoreductase enzymes and multienzyme complexes. The aim of the study was to determine the features of generation of superoxide anion radical (O2·) as the primary reactive oxygen species, and nitric oxide (NO·) under conditions of thioacetamide-induced hepatotoxicity. The features of NAD(P)H-dependent gen-eration of superoxide anion radical (O2·) as the primary reactive oxygen species, and nitric oxide (NO·) in subcellular (mitochondrial, microsomal and post-microsomal) fractions of C57BL/6J mouse liver cells isolated by the method of differential centrifugation were determined under conditions of thioacetamide-induced hepatotoxicity and supplementation with pharma-cological doses of vitamin A. It was found that the development of acute hepatotoxicity induced by single intraperitoneal ad-ministration of 500 mg/kg of thioacetamide was accompanied by increased intensity of superoxide anion radical and nitric oxide production in microsomal and cytosolic fractions of liver cells, but not in mitochondrial fraction. Consumption of the pharmacological doses of vitamin A (3000 IU) has no hepatoprotective effect, however, it enhances the production of reactive oxygen and nitrogen species in the liver during acute hepatotoxicity. 


Cantu-Medellin, N., Kelley, E.E., 2013a. Xanthine oxidoreductase-catalyzed reactive species generation: A process in critical need of reevaluation. Redox Biol. 1(1), 353–358.

Cantu-Medellin, N., Kelley, E.E., 2013b. Xanthine oxidoreductase-catalyzed reduction of nitrite to nitric oxide: Insights regarding where, when and how. Nitric Oxide 34, 19–26.

Chilakapati, J., Shankar, K., Korrapati, M.C., Hill, R.A., Mehendale, H.M., 2005. Saturation toxicokinetics of thioacetamide: Role in initiation of liver injury. Drug Metab. Dispos. 33(12), 1877–1885.

D’Alessandro, A., Rinalducci, S., Zolla, L., 2011. Redox proteomics and drug development. J. Proteomics 74(12), 2575–2595.

Guide for the care and use of laboratory animals: Eighth edition, 2011. The National Academies Press, Washington, DC.

Hajovsky, H., Hu, G., Koen, Y., Sarma, D., Cui, W., Moore, D.S., 2012. Metabolism and toxicity of thioacetamide and thioacetamide S-oxide in rat hepatocytes. Chem. Res. Toxicol. 25(9), 1955–1963.

He, S., Rehman, H., Wright, G.L., Zhong, Z., 2010. Inhibition of inducible nitric oxide synthase prevents mitochondrial damage and improves survival of steatotic partial liver grafts. Transplantation 89(3), 291–298.

Hwang, S.M., Lopez, C.A., Heck, D.E., Gardner, C.R., Laskin, D.L., Laskin, J.D., 1994. Osteopontin inhibits induction of nitric oxide synthase gene expression by inflammatory mediators in mouse kidney epithelial cells. J. Biol. Chem. 269(1), 711–715.

Jaeschke, H., Gores, G.J., Cederbaum, A.I., Hinson, J.A., Pessayre, D., Lemasters, J.J., 2002. Mechanisms of hepatotoxicity. Toxicol. Sci. 65(2), 166–176.

Kitagawa, Y., Sugimoto, E., 1980. Estimation of the in vivo translational activity of rat liver mitochondria without use of an antibiotic. J. Biochem. 88(3), 689–693.

Kostenko, V.O., Tsebrzhins’kii, O.I., 2000. [Production of superoxide anion radical and nitric oxide in renal tissues sutured with different surgical suture material]. Fiziol. Zh. 46(5), 56–62. (in Ukrainian).

Leach, J.K., Van Tuyle, G., Lin, P.S., Schmidt-Ullrich, R., Mikkelsen, R.B., 2001. Ionizing radiation-induced, mitochondria-dependent generation of reactive oxygen/nitrogen. Cancer Res. 61(10), 3894–3901.

Muriel, P., 2009. Role of free radicals in liver diseases. Hepatol. Int. 3(4), 526–536.

Novitskiy, G., Potter, J.J., Wang, L., Mezey, E., 2006. Influences of reactive oxygen species and nitric oxide on hepatic fibro-genesis. Liver Int. 26(10), 1248–1257.

Powers, S.K., Talbert, E.E., Adhihetty, P.J., 2011. Reactive oxygen and nitrogen species as intracellular signals in skeletal muscle. J. Physiol. 589(9), 2129–2138.

Poyton, R.O., Ball, K.A., Castello, P.R., 2009. Mitochondrial generation of free radicals and hypoxic signaling. Trends Endocrinol. Metab. 20(7), 332–340.

Robert, A.M., Robert, L., 2013. Xanthine oxido-reductase, free radicals and cardiovascular disease. A critical review. Pathol. Oncol. Res. 20(1), 1–10.

Schenkman, J.B., Cinti, D.L., 1978. Preparation of microsomes with calcium. Methods Enzymol. 52, 83–89.

Shirakami, Y., Lee, S.A., Clugston, R.D., Blaner, W.S., 2012. Hepatic metabolism of retinoids and disease associations. Biochim. Biophys. Acta 1821(1), 124–136.

Shmarakov, I.O., Marchenko, M.M., 2008. [Xanthine oxidase activity in the rat liver tissue in the process of oncogenesis]. Ukr. Biokhim. Zh. 80(6), 86–91 (in Ukrainian).

Stankova, P., Kucera, O., Lotkova, H., Rousar, T., Endlicher, R., Cervinkova, Z., 2010. The toxic effect of thioacetamide on rat liver in vitro. Toxicol. In Vitro 24(8), 2097–2103.

Uchi, J.O., Ryu, S.Y., Jhun, B.S., Hurst, S., Sheu, S.S., 2013. Mitochondrial ion channels/transporters as sensors and regulators of cellular redox signaling. Antioxid. Redox Signal. doi:10.1089/ars.2013.5681

Urtasun, R., Conde de la Rosa, L., Nieto, N., 2008. Oxidative and nitrosative stress and fibrogenic response. Clin. Liver Dis. 12(4), 769–790.

Wang, T., Shankar, K., Ronis, M.J., Mehendale, H.M., 2000. Potentiation of thioacetamide liver injury in diabetic rats is due to induced CYP2E1. J. Pharmacol. Exp. Ther. 294(2), 473–479.

Waterborg, J.H., Matthews, H.R., 1994. The Lowry method for protein quantitation. Methods Mol. Biol. 32, 1–4. >> doi:10.1385/0-89603-268-X:1

Zhu, H., Jia, Z., Misra, H., Li, Y.R., 2012. Oxidative stress and redox signaling mechanisms of alcoholic liver disease: Updated experimental and clinical evidence. J. Dig. Dis. 13(3), 133–142 >> doi:10.1111/j.1751-2980.2011.00569.x