TEST-TUBE SIMULATED LIPOFUSCINOGENESIS - EFFECT OF OXIDATIVE STRESS ON AUTOPHAGOCYTOTIC DEGRADATION

Cysteine-stimulated oxidation of a rat liver lysosomal-mitochondrial f raction (LMF) was studied. The process would simulate oxidative stress -related events during the degradation of autophagocytosed material wi thin secondary lysosomes, which may contribute to the formation of lip ofuscin or age pigment. Millimolar concentration of cysteine was neede d to stimulate LMF lipid peroxidation, measured as thiobarbituric acid reactive substances (TBARS), The amount of endogenous LMF iron was 54 5 mu g/l anti was enough to initiate peroxidation, probably through th e reduction of ferric to ferrous iron by cysteine with induction of Fe nton chemistry. Peroxidation could be completely inhibited by the addi tion of the iron chelator desferal or the antioxidant BHT. A substanti al amount of the formed TBARS was associated with trichloroacetic acid (TCA) precipitable proteins. Elevated protein carbonyls was observed 1-2 h after the increase of TBARS. The tryptophan-tyrosine related pro tein autofluorescence (280/335 nm) decreased sharply during the first few hours of incubation, In contrast, a lipofuscin-type autofluorescen ce (345/430 nm) appeared only after a few days, suggesting that the la tter fluorophore is not an immediate product of protein oxidation. The sequential formation of TBARS, protein carbonyls and lipofuscin-type autofluorescence as well as their dependence on iron and a reducing ag ent add further support to the concept that lipofuscin forms in second ary lysosomes as a result of iron-catalyzed oxidative reactions involv ing autophagocytosed materials.