4-Hydroxynonenal and malondialdehyde hepatic protein adducts in rats treated with carbon tetrachloride: immunochemical detection and lobular localization.
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.
Hartley
D P
DP
Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas, 66160, USA.
Kolaja
K L
KL
Reichard
J
J
Petersen
D R
DR
D000410:Alanine Transaminase / Q000378:metabolism; D000447:Aldehydes / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D000478:Alkylation / Q000187:drug effects; D000818:Animals; D015153:Blotting, Western; D002251:Carbon Tetrachloride / Q000008:administration & dosage / Q000633:toxicity*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D007150:Immunohistochemistry; D015227:Lipid Peroxidation / Q000187:drug effects; D008099:Liver / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008315:Malondialdehyde / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D008899:Mineral Oil; D008970:Molecular Weight; D009336:Necrosis; D011233:Precipitin Tests; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis / Q000378:metabolism; D013997:Time Factors
D000447:Aldehydes; D005231:Fatty Acids, Unsaturated; D011506:Proteins; D017392:Thiobarbituric Acid Reactive Substances; D008315:Malondialdehyde; D008899:Mineral Oil; D002251:Carbon Tetrachloride; D000410:Alanine Transaminase; C027576:4-hydroxy-2-nonenal