EFFECT OF REACTIVE OXYGEN SPECIES ON LYSOSOMAL MEMBRANE INTEGRITY - ASTUDY ON A LYSOSOMAL FRACTION

Using a lysosome-enriched ''light mitochondrial'' fraction of a rat li ver homogenate, the effects of the reactive oxygen species hydrogen pe roxide, superoxide- and hydroxyl radicals were determined. Alterations in the intralysosomal pH and the release of a lysosomal marker enzyme , N-acetyl-glucosaminidase, were used as indicators of changes in the lysosomal membrane integrity. Lipid peroxidation of the fraction was a ssayed by TBARS measurement. Neither superoxide radicals, generated by hypoxanthine/xanthine oxidase, nor a bolus dose of hydrogen peroxide (0.5-1.5 mM) induced any lysosomal damage. If, however, Fe(III)ADP was included in the superoxide radical-generating system, lysosomal membr ane damage was detected, both as an increase in lysosomal pH and as a release of N-acetyl-glucosaminidase, but only after a lag phase of abo ut 7 min. Lipid peroxidation, on the other hand, proceeded gradually. Lysosomes treated with hydrogen peroxide displayed similar dose-depend ent alterations, albeit only if both Fe(III)ADP and the reducing amino acid cysteine were added. In the latter system, however, alterations of the lysosomal membrane stability occurred more rapidly, showing a l ag phase of only 2 min. Lipid peroxidation, which proceeded faster and displayed no lag phase, levelled out within 10 min. The results indic ate that neither superoxide radicals nor hydrogen peroxide are by them selves damaging to lysosomes. Available catalytically active iron in F e(II) form, however, allows reactions yielding powerful oxidative spec ies - probably hydroxyl radicals formed via Fenton reactions - to take place inducing peroxidation of the lysosomal membranes resulting in d issipation of the proton-gradient and leakage of their enzyme contents .