Test-tube simulated lipofuscinogenesis. Effect of oxidative stress on autophagocytotic degradation.

Cysteine-stimulated oxidation of a rat liver lysosomal-mitochondrial fraction (LMF) was studied. The process would simulate oxidative stress-related events during the degradation of autophagocytosed material within secondary lysosomes, which may contribute to the formation of lipofuscin or age pigment. Millimolar concentration of cysteine was needed to stimulate LMF lipid peroxidation, measured as thiobarbituric acid reactive substances (TBARS). The amount of endogenous LMF iron was 545 micrograms/l and was enough to initiate peroxidation, probably through the reduction of ferric to ferrous iron by cysteine with induction of Fenton chemistry. Peroxidation could be completely inhibited by the addition of the iron chelator desferal or the antioxidant BHT. A substantial 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 protein autofluorescence (280/335 nm) decreased sharply during the first few hours of incubation. In contrast, a lipofuscin-type autofluorescence (345/430 nm) appeared only after a few days, suggesting that the latter 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 reducing agent add further support to the concept that lipofuscin forms in secondary lysosomes as a result of iron-catalyzed oxidative reactions involving autophagocytosed materials.