Cemtirestat dimerization in liposomes and erythrocytes exposed to peroxyl radicals was reverted by thiol-disulfide exchange with GSH

Abstract

In the model system of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) liposomes exposed to peroxyl radicals generated by the azoinitiator AAPH, cemtirestat (CMTI-SH) inhibited lipid peroxidation more efficiently than the natural antioxidant glutathione. In the concentrations 100 to 500 µM, both CMTI-SH and GSH induced distinct lag phases in the initial stages of lipid peroxidation yet GSH produced consistently shorter induction periods (about twice) than equimolar CMTI-SH. Moreover, concentration dependence of lipid peroxidation inhibition measured at the 80th minute, revealed about three times higher IC50 value for GSH compared to CMTI-SH. When the incubations prolonged till 180 min no further absorbance changes at 270 and 302 nm, respectively, occurred. After addition of the reducing agent tris(2-carboxyethyl)phosphine, the absorbance peak at 270 nm shifted back to 302 nm. These findings pointed to the presence of reducible CMTI-SH disulfide whose definite structure was confirmed by proving identity of TLC retention and spectral data with those of the synthesized CMTI disulfide. When CMTI-SH and GSH were present simultaneously in the liposomal incubations, the mixing effect on the induction period was synergistic rather than additive. This was explained by ability of GSH to reduce CMTI disulfide which was proved in separate experiments with an authentic CMTI disulfide prepared synthetically. This finding was also demonstrated by experiment with CMTI-disulfide to protect the erythrocytes against oxidative damage induced by peroxyl radicals. To conclude, CMTI-SH scavenges reactive oxygen species yielding CMTI disulfide while GSH maintains CMTI-SH in the reduced state. This finding was also demonstrated by experiment with CMTI-disulfide to protect the erythrocytes against oxidative damage induced by peroxyl radicals. CMTI-SH would thus represent the first line of the cellular defense against peroxyl radical mediated oxidative stress.