Immunohistochemical characterization of hepatic malondialdehyde and 4-hydroxynonenal modified proteins during early stages of ethanol-induced liver injury.

Oxidative insult to cells is associated with the production of free radicals capable of initiating the peroxidation of membrane lipids. The autocatalytic process of lipid peroxidation results in the production of a variety of n-alkanals and alkenals derived from ω-6-polyunsaturated fatty acids such as arachidonic and linoleic acid. As noted in a recent comprehensive review (Poli and Schaur, 2000), there is significant evidence that these aldehydic products of lipid peroxidation may be involved in several chronic diseases including Parkinson’s disease, arteriosclerosis, and diabetes mellitus as well as chronic inflammatory diseases of the liver including alcoholic liver disease, hepatitis C, hepatic iron overload, and primary biliary cirrhosis. The molecular and cellular mechanisms by which the process of lipid peroxidation initiates or propagates liver injury and fibrosis are complex. At the molecular level, the production of reactive oxygen species such as superoxide anion radical (O2-.) is an essential event in the formation of hydroxyl radical (OH-.), which can then abstract a hydrogen atom from cellular membrane lipids, resulting in the autocatalytic production of lipid radicals and the peroxidative degradation of membrane lipids (Esterbauer et al., 1990, 1991). Whereas it is predictable that the destruction of membrane lipids would have a direct effect on cellular homeostasis, various lipid-derived products resulting from lipid peroxidation have been demonstrated to have multiple biological or toxicologic effects. For instance, when present in high concentrations, two major products of lipid peroxidation, 4-hydroynonenal (4-HNE) and malondialdehyde (MDA), display cytotoxic effects in several different cell types (Schaur et al., 1990). At low micromolar or submicromolar concentrations, 4-HNE has been shown to alter cellular proliferation (Cambiaggi et al., 1997), interfere with intracellular proteolysis (Okada et al., 1999), prevent nuclear factor-κB activation (Page et al., 1999), and activate activator protein-1 (Camandola et al., 1997). An involvement of both MDA and 4-HNE in the process of liver fibrosis is supported by the observation that these aldehydic products of lipid peroxidation increase procollagen type I messenger RNA and protein in cultured human stellate cells (Parola et al., 1996). The potential of these aldehydic products to modulate numerous, diverse cellular functions can be attributed to their ability to diffuse from their site of production as well as their electrophilic chemical properties that facilitate covalent interactions with nucleophilic groups of cellular proteins or nucleic acids. The potential of MDA or 4-HNE to form adducts with proteins has been documented by using purified proteins treated with 4-HNE (Uchida and Stadtman 1993), isolated cells exposed to various pro-oxidants (Hartley et al., 1997), or immunohistochemically in tissue obtained from animals treated with ethanol, iron combined with ethanol (Kono et al., 2001; Tsukamoto et al., 1995) or carbon tetrachloride (Hartley et al., 1999). Likewise, MDA- and 4HNE -protein adducts have been detected in liver samples obtained from patients with chronic liver disease such as hepatitis C (Paradis et al., 1997a) as well as in patients with hemochromatosis, Wilson’s disease, alcoholic liver disease, primary biliary cirrhosis, or cholestasis (Paradis et al., 1997b). Collectively, these studies indicate that MDA- and 4HNE-protein adducts are reliable biomarkers of hepatic lipid peroxidation associated with chronic liver injury. The involvement of lipid peroxidation in chronic liver disease is predictable given the sustained inflammatory processes associated with the progressive stages of hepatitis C, cholestasis, or alcoholic liver disease. However, the occurrence of lipid peroxidation during the early, pre-inflammatory, or sensitization stages of liver injury has not been clearly delineated. In the present study we characterized the appearance of MDA- and 4HNE-adducted proteins in livers of rats subjected to an intragastric ethanol administration regimen specifically designed to initiate early alcohol-induced changes in the liver without extensive inflammation and hepatocellular necrosis. The results of this study indicate that MDA- and 4-HNE-adducted proteins appear in hepatocytes concurrent with the development of microsteatosis in the absence of extensive cellular injury, which suggests that the process of lipid peroxidation is an early event in alcohol-induced liver injury that occurs before extensive inflammation and necrosis are manifested.