PROOXIDANT-INITIATED LIPID-PEROXIDATION IN ISOLATED RAT HEPATOCYTES -DETECTION OF 4-HYDROXYNONENAL-PROTEIN AND MALONDIALDEHYDE-PROTEIN ADDUCTS

Toxicity associated with prooxidant-mediated hepatic lipid peroxidatio n is postulated to originate from the interaction of the aldehydic end products of lipid peroxidation with cellular constituents. The princi pal a,P-unsaturated aldehydic products of lipid peroxidation, 4-hydrox y-2-nonenal (4-HNE) and malondialdehyde (MDA), are known to modify pro teins through covalent alkylation of lysine, histidine, and cysteine a mino acid residues. To detect and characterize the formation of 4-HNE- and MDA-adducted proteins during prooxidant-initiated lipid peroxidat ion, rabbit polyclonal antibodies were raised to 4-HNE-sulfhydryl, din itrophenyl-hydrazine (DNPH)-4-HNE-sulfhydryl, and MDA-amine conjugates of keyhole limpet hemocyanin (KLH). Each antiserum displayed high ant ibody titers to either 4-HNE-metallothionein, DNPH-albumin, or MDA-alb umin adducts when measured by ELISA. To study the formation of 4-HNE- and MDA-protein adducts during prooxidant-initiated cellular injury, i solated hepatocytes were exposed to either carbon tetrachloride or iro n/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 continu ously. Hepatocellular viability was affected moderately by carbon tetr achloride, while cellular reduced glutathione status was moderately af fected by both iron/ascorbate and carbon tetrachloride. Levels of MDA and protein carbonyls increased dramatically with both prooxidants, wh ereas 4-HNE levels did not change significantly over the time course s tudied. In addition, hepatocellular proteins were immunoprecipitated w ith each antiserum, and aldehyde-modified immunopositive proteins were detected by immunoblotting. Prooxidant-induced increases in MDA corre sponded with increases in intensity and number of MDA-adducted protein s over the time course studied. A total of 13 MDA-modified proteins (2 0, 25, 28, 30, 33, 38, 41, 45, 80, 82, 85, 130, and 150 kDa) were dete cted with the MDA-amine antiserum. Additionally, both iron/ascorbate-a nd carbon tetrachloride-induced formation of DNPH-derivatizable protei n carbonyls corresponded quantitatively with the ability to detect spe cific proteins (80, 100, 130, and 150 kDa) with the DNPH-4-HNE-cystein e 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 anti serum. In all instances detection of aldehyde-modified proteins was no t associated with cell death and may be related to the function of the se proteins as aldehyde-binding proteins which sequester electrophilic molecules during oxidative liver injury.