4-hydroxynonenal and malondialdehyde hepatic protein adducts in rats treated with carbon tetrachloride: Immunochemical detection and lobular localization

The metabolism of CCl4 initiates the peroxidation of polyunsaturated fatty acids producing alpha,beta-unsaturated aldehydes, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). The facile reactivity of these electroph ilic aldehydic products suggests they play a role in the toxicity of compou nds like CCl4. To determine the rate at which CCl4-initiated lipid peroxida tion results in the formation of 4-HNE and/or MDA hepatic protein adducts, rats were given an intragastric dose of CCl4 (1.0 ml/kg) and euthanized 0-7 2 h after administration. Rabbit polyclonal antisera directed toward 4-HNE- or MDA-protein epitopes were employed in immuno-histochemical and immuno-p recipitation/Western analyses to detect 4-HNE and MDA-protein adducts in pa raffin-embedded liver sections and liver homogenates. As early as 6 h post CCl4 exposure, 4-HNE and MDA adducts were detected immuno-histochemically i n hepatocytes localized to zone 2 of the hepatic acinus. Liver injury was p rogressive to 24 h as lipid peroxidation and hepatocellular necrosis increa sed. The hallmark of CCl4 hepatotoxicity, zone 3 necrosis, was observed 24 h after CCl4 administration and immune-positive hepatocytes were observed i n zone 2 as well as zone 3. Immune-positive cells were no longer visible by 36 to 72 h post CCl4 administration. From 6 to 48 h after CCl4 administrat ion, at least four adducted proteins were immune-precipitated from liver ho mogenates with the anti-MDA or anti-4HNE serum, which corresponded to molec ular weights of 80, 150, 205, and greater than 205 kDa. These results demon strate that 4-HNE and MDA alkylate specific hepatic proteins in a time-depe ndent manner, which appears to be associated with hepatocellular injury fol lowing CCl4 exposure. (C) 1999 Academic Press.