Inhibition of in vitro lipid peroxidation by 21-aminosteroids. Evidence for differential mechanisms.
In a previous report (Ryan and Petry, Arch Biochem Biophys 300: 699-704, 1993), the effects of two 21-aminosteroids (U-74500A and U-74006F) on the oxidation and reduction of iron in a buffer/organic solvent system were investigated. In those studies, U-74500A was found to be an efficient iron reductant and potential iron chelator, whereas U-74006F had little effect on iron redox chemistry. As an extension of those studies, we now report the effects of U-74006F and U-74500A on lipid peroxidation in systems that are dependent upon iron oxidation/reduction. In liposomes, U-74500A inhibited ADP:Fe(II)-dependent lipid peroxidation in a concentration-dependent manner, whereas U-74006F was minimally effective in this system. The mechanism of U-74500A-dependent inhibition probably involved interactions with iron, as iron oxidation was inhibited in the presence of this compound. No effects on iron oxidation were observed in the presence of U-74006F. Addition of Ferrozine to liposomal incubation mixtures indicated that at least two iron pools were present in samples containing U-74500A, one immediately bound by Ferrozine, and another that was bound more slowly. Furthermore, ADP:Fe(III)/ascorbate-dependent lipid peroxidation was blocked completely by U-74500A, presumably by formation of a redox inert complex upon reduction of the iron. U-74500A partially protected ADP:Fe(II) from oxidation by H2O2 and lipid hydroperoxides, indicating that the U-74500A:iron complex was stable in the presence of biologically relevant oxidants. U-74006F did not markedly affect iron oxidation or reduction when incorporated into phospholipid liposomes. In microsomal lipid peroxidation systems containing ADP:Fe(III) and NADPH, both U-74500A and U-74006F inhibited lipid peroxidation. U-74006F-dependent inhibition of microsomal lipid peroxidation was dependent on both NADPH and Fe(III). Further, it was enhanced when U-74006F was allowed to preincubate in this system prior to iron addition. Preincubation of U-74006F with microsomes, NADPH, and ADP:Fe(III) produced several metabolites detectable by HPLC. These results suggest that U-74500A inhibits lipid peroxidation by directly affecting iron redox chemistry, whereas U-74006F-mediated inhibition is enhanced by preincubation with a metabolically competent microsomal system.
Ryan
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Investigative Toxicology, Upjohn Laboratories, Kalamazoo, MI 49001.
Steenwyk
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Pearson
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Petry
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D000818:Animals; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D005290:Ferric Compounds / Q000494:pharmacology; D005296:Ferrous Compounds / Q000494:pharmacology; D005297:Ferrozine; D015227:Lipid Peroxidation / Q000187:drug effects*; D008081:Liposomes; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D009249:NADP / Q000494:pharmacology; D010084:Oxidation-Reduction; D011281:Pregnatrienes / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis; D013997:Time Factors
D005290:Ferric Compounds; D005296:Ferrous Compounds; D008081:Liposomes; D011281:Pregnatrienes; D017392:Thiobarbituric Acid Reactive Substances; D005297:Ferrozine; D009249:NADP; C053356:U 74500A; C053355:tirilazad