4-Hydroxynonenal and Malondialdehyde Hepatic Protein Adducts in Rats Treated with Carbon Tetrachloride: Immunochemical Detection and Lobular Localization

• Published in: Toxicology and applied pharmacology 1999-11, Vol.161 (1), p.23-33
• Main Authors: Hartley, Dylan P., Kolaja, Kyle L., Reichard, John, Petersen, Dennis R.
• Format: Article
• Language: English
• Subjects: 4-hydroxynonenal; Alanine Transaminase - metabolism; Aldehydes - analysis; Aldehydes - immunology; Aldehydes - metabolism; Alkylation - drug effects; Animals; Biological and medical sciences; Blotting, Western; carbon tetrachloride; Carbon Tetrachloride - administration & dosage; Carbon Tetrachloride - toxicity; Chemical and industrial products toxicology. Toxic occupational diseases; Fatty Acids, Unsaturated - metabolism; hepatotoxicity; Immunohistochemistry; lipid peroxidation; Lipid Peroxidation - drug effects; Liver - drug effects; Liver - enzymology; Liver - metabolism; Liver - pathology; Male; Malondialdehyde - analysis; Malondialdehyde - immunology; Malondialdehyde - metabolism; malondialdehyde-4-hydroxynonenal-protein adducts; Medical sciences; Mineral Oil; Molecular Weight; Necrosis; oxidative stress; Precipitin Tests; protein adducts; Proteins - analysis; Proteins - chemistry; Proteins - immunology; Proteins - metabolism; Rats; Solvents; Thiobarbituric Acid Reactive Substances - analysis; Thiobarbituric Acid Reactive Substances - metabolism; Time Factors; Toxicology
• ISSN: 0041-008X; 1096-0333
• DOI: 10.1006/taap.1999.8788

The metabolism of CCl4 initiates the peroxidation of polyunsaturated fatty acids producing α,β-unsaturated aldehydes, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). The facile reactivity of these electrophilic aldehydic products suggests they play a role in the toxicity of compounds like CCl4. To determine the rate at which CCl4-initiated lipid peroxidation 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 0–72 h after administration. Rabbit polyclonal antisera directed toward 4-HNE- or MDA-protein epitopes were employed in immuno-histochemical and immuno-precipitation/Western analyses to detect 4-HNE and MDA-protein adducts in paraffin-embedded liver sections and liver homogenates. As early as 6 h post CCl4 exposure, 4-HNE and MDA adducts were detected immuno-histochemically in hepatocytes localized to zone 2 of the hepatic acinus. Liver injury was progressive to 24 h as lipid peroxidation and hepatocellular necrosis increased. The hallmark of CCl4 hepatotoxicity, zone 3 necrosis, was observed 24 h after CCl4 administration and immuno-positive hepatocytes were observed in zone 2 as well as zone 3. Immuno-positive cells were no longer visible by 36 to 72 h post CCl4 administration. From 6 to 48 h after CCl4 administration, at least four adducted proteins were immuno-precipitated from liver homogenates with the anti-MDA or anti-4HNE serum, which corresponded to molecular weights of 80, 150, 205, and greater than 205 kDa. These results demonstrate that 4-HNE and MDA alkylate specific hepatic proteins in a time-dependent manner, which appears to be associated with hepatocellular injury following CCl4 exposure.