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

Toxicol Appl Pharmacol. 1999 Nov 15;161(1):23-33. doi: 10.1006/taap.1999.8788.

Abstract

The metabolism of CCl(4) 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 electrophilic aldehydic products suggests they play a role in the toxicity of compounds like CCl(4). To determine the rate at which CCl(4)-initiated lipid peroxidation results in the formation of 4-HNE and/or MDA hepatic protein adducts, rats were given an intragastric dose of CCl(4) (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 CCl(4) 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 CCl(4) hepatotoxicity, zone 3 necrosis, was observed 24 h after CCl(4) 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 CCl(4) administration. From 6 to 48 h after CCl(4) 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 CCl(4) exposure.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine Transaminase / metabolism
  • Aldehydes / analysis
  • Aldehydes / immunology
  • Aldehydes / metabolism*
  • Alkylation / drug effects
  • Animals
  • Blotting, Western
  • Carbon Tetrachloride / administration & dosage
  • Carbon Tetrachloride / toxicity*
  • Fatty Acids, Unsaturated / metabolism
  • Immunohistochemistry
  • Lipid Peroxidation / drug effects
  • Liver / drug effects*
  • Liver / enzymology
  • Liver / metabolism*
  • Liver / pathology
  • Male
  • Malondialdehyde / analysis
  • Malondialdehyde / immunology
  • Malondialdehyde / metabolism*
  • Mineral Oil
  • Molecular Weight
  • Necrosis
  • Precipitin Tests
  • Proteins / analysis
  • Proteins / chemistry
  • Proteins / immunology
  • Proteins / metabolism*
  • Rats
  • Thiobarbituric Acid Reactive Substances / analysis
  • Thiobarbituric Acid Reactive Substances / metabolism
  • Time Factors

Substances

  • Aldehydes
  • Fatty Acids, Unsaturated
  • Proteins
  • Thiobarbituric Acid Reactive Substances
  • Malondialdehyde
  • Mineral Oil
  • Carbon Tetrachloride
  • Alanine Transaminase
  • 4-hydroxy-2-nonenal