Prooxidant-initiated lipid peroxidation in isolated rat hepatocytes: detection of 4-hydroxynonenal- and malondialdehyde-protein adducts.
Toxicity associated with prooxidant-mediated hepatic lipid peroxidation is postulated to originate from the interaction of the aldehydic end products of lipid peroxidation with cellular constituents. The principal alpha,beta-unsaturated aldehydic products of lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA), are known to modify proteins through covalent alkylation of lysine, histidine, and cysteine amino acid residues. To detect and characterize the formation of 4-HNE- and MDA-adducted proteins during prooxidant-initiated lipid peroxidation, rabbit polyclonal antibodies were raised to 4-HNE-sulfhydryl, dinitrophenylhydrazine (DNPH)-4-HNE-sulfhydryl, and MDA-amine conjugates of keyhole limpet hemocyanin (KLH). Each antiserum displayed high antibody titers to either 4-HNE-metallothionein, DNPH-albumin, or MDA-albumin adducts when measured by ELISA. To study the formation of 4-HNE- and MDA-protein adducts during prooxidant-initiated cellular injury, isolated hepatocytes were exposed to either carbon tetrachloride or iron/ascorbate for 2 h. Indices of hepatocellular oxidative stress (i.e., cell viability and glutathione status) and lipid peroxidation (i.e., formation of 4-HNE, protein carbonyls, and MDA) were monitored continuously. Hepatocellular viability was affected moderately by carbon tetrachloride, while cellular reduced glutathione status was moderately affected by both iron/ascorbate and carbon tetrachloride. Levels of MDA and protein carbonyls increased dramatically with both prooxidants, whereas 4-HNE levels did not change significantly over the time course studied. In addition, hepatocellular proteins were immunoprecipitated with each antiserum, and aldehyde-modified immunopositive proteins were detected by immunoblotting. Prooxidant-induced increases in MDA corresponded with increases in intensity and number of MDA-adducted proteins over the time course studied. A total of 13 MDA-modified proteins (20, 25, 28, 30, 33, 38, 41, 45, 80, 82, 85, 130, and 150 kDa) were detected with the MDA-amine antiserum. Additionally, both iron/ascorbate- and carbon tetrachloride-induced formation of DNPH-derivatizable protein carbonyls corresponded quantitatively with the ability to detect specific proteins (80, 100, 130, and 150 kDa) with the DNPH-4-HNE-cysteine antiserum. Neither CCl4 nor iron/ascorbate elicited changes in 4-HNE or induced the formation of 4-HNE-modified proteins when assessed by immunoprecipitation-immunoblot analysis with the 4-HNE-sulfhydryl antiserum. In all instances detection of aldehyde-modified proteins was not associated with cell death and may be related to the function of these proteins as aldehyde-binding proteins which sequester electrophilic molecules during oxidative liver injury.
Hartley
D P
DP
Department of Pharmaceutical Sciences, Hepatobiliary Research Center, University of Colorado Health Sciences Center, Denver 80262, USA.
Kroll
D J
DJ
Petersen
D R
DR
D000447:Aldehydes / Q000378:metabolism*; D000818:Animals; D002470:Cell Survival; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D015227:Lipid Peroxidation; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008315:Malondialdehyde / Q000378:metabolism*; D016877:Oxidants / Q000633:toxicity; D011233:Precipitin Tests; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley
D000447:Aldehydes; D007106:Immune Sera; D016877:Oxidants; D011506:Proteins; D008315:Malondialdehyde; C027576:4-hydroxy-2-nonenal