title: Diquat-dependent protein carbonyl formation. Identification of lipid-dependent and lipid-independent pathways.
abstract: In a previous report on diquat-dependent oxidative damage in rat hepatic microsomes, protein oxidation, as measured by protein carbonyl (PC) formation, was observed in addition to lipid peroxidation (LP). Both phenomena were antioxidant sensitive. Inhibition of PC formation was somewhat surprising given the proposed mechanism of metal-catalyzed protein oxidation. Studies reported here examined diquat-dependent PC formation in greater detail. In rat hepatic microsomes, diquat-dependent thiobarbituric acid-reactive substances (TBARS) and PC formation were time and concentration dependent. In this system, LP was inhibited completely by U-74006F or U-78517G, whereas PC formation was inhibited only partially by these antioxidants. In an essentially lipid-free system consisting of purified rat hepatic cytochrome P450 reductase, BSA and an NADPH-generating system, PC formation was also observed, but was not antioxidant-sensitive. Under these conditions, minimal diquat-dependent TBARS formation was observed. The observation of relative antioxidant insensitivity is consistent with H2O2 (generated during the diquat redox cycle) catalyzing protein oxidation via a site-specific, metal-catalyzed mechanism. Thus, different pathways would appear to be involved in diquat-dependent PC formation in lipid-containing and lipid-free systems. Carbon tetrachloride induces LP following reductive activation to the trichloromethyl free radical, a pathway not directly involving H2O2 generation. In the microsomal system, CCl4 induced TBARS and PC formation, both of which were completely inhibitable by antioxidants. Taken together, these data suggest that diquat induces PC formation by lipid-dependent (antioxidant-sensitive) and lipid-independent (antioxidant-insensitive) pathways. In microsomes, both pathways contribute to diquat-dependent PC formation. Data for the lipid-independent pathway are consistent with the mechanism of metal-catalyzed protein oxidation proposed by Stadtman and colleagues (reviewed in Free Radic Biol Med 9: 315-325, 1990), while the lipid-dependent pathway is likely secondary to LP itself--via a Michael-type addition reaction between hydroxyalkenals and protein sulfhydryl groups, amino groups or other protein nucleophiles. The latter pathway is also responsible for carbon tetrachloride-dependent PC formation. Additional studies are in progress to further characterize the lipid-independent mechanism.
authors.affiliation: Upjohn Laboratories, Kalamazoo, MI 49001, USA.
authors.forename: D P
authors.initials: DP
authors.lastname: Blakeman
authors.forename: T P
authors.initials: TP
authors.lastname: Ryan
authors.forename: R A
authors.initials: RA
authors.lastname: Jolly
authors.forename: T W
authors.initials: TW
authors.lastname: Petry
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002251:Carbon Tetrachloride / Q000633:toxicity; D002839:Chromans / Q000494:pharmacology; D004178:Diquat / Q000633:toxicity*; D007501:Iron / Q000737:chemistry; D015227:Lipid Peroxidation / Q000187:drug effects*; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects; D010084:Oxidation-Reduction; D010879:Piperazines / Q000494:pharmacology; D011281:Pregnatrienes / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry*; D051381:Rats; D011916:Rats, Inbred F344; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis
chemical_list: D000975:Antioxidants; D002839:Chromans; D010879:Piperazines; D011281:Pregnatrienes; D011506:Proteins; D017392:Thiobarbituric Acid Reactive Substances; C066382:U 78517F; D004178:Diquat; D002251:Carbon Tetrachloride; D007501:Iron; C053355:tirilazad