Apoptosis induced by exposure to a low steady-state concentration of H2O2 is a consequence of lysosomal rupture

We have re-examined the lysosomal hypothesis of oxidative-stress-induced ap optosis using a new technique for exposing cells in culture to a low steady -state concentration of H2O2. This steady-state technique mimics the situat ion in vivo better than the bolus-administration method. A key aspect of H2 O2-induced apoptosis is that the apoptosis is evident only after several ho urs, although cells may become committed within a few minutes of exposure t o this particular reactive oxygen species. In the present work, we were abl e to show, for the first time, several correlative links between the trigge ring effect of H2O2 and the later onset of apoptosis: (i) a short (15 min) exposure to H2O2 caused almost immediate, albeit limited, lysosomal rupture ; (ii) early lysosomal damage, and later apoptosis, showed a similar dose-r elated response to H2O2; (iii) both events were inhibited by pretreatment w ith iron chelators, including desferrioxamine. This compound is known to be taken up by endocytosis only and thus to become localized in the lysosomal compartment. After exposure to oxidative stress, when cells were again in standard culture conditions, a time-dependent continuous increase in lysoso mal rupture was observed, resulting in a considerably lowered number of int act lysosomes in apoptotic cells, whereas non-apoptotic cells from the same batch of oxidative-stress-exposed cells showed mainly intact lysosomes. Ta ken together, our results reinforce earlier findings and strongly suggest t hat lysosomal rupture is an early upstream initiating event, and a conseque nce of intralysosomal iron-catalysed oxidative processes, when apoptosis is induced by oxidative stress.