Extracellular reduction of alloxan results in oxygen radical-mediated attack on plasma and lysosomal membranes.

Abstract

Alloxan participation in extracellular redox processes results in the formation of the reactive oxygen species (ROS) superoxide anions (O2-), hydroxyl radical (OH.) and hydrogen peroxide (H2O2), causing cell damage through a number of complex interactions probably involving several different cellular structures. These involve the plasma membrane, and we have recently presented evidence for lysosomal interference. The present study elucidates the early (within 15 min) events in a model system of macrophage-like cells (J-774) in culture. Addition of 2 mM alloxan and 1 mM cysteine to the medium surrounding the cells (phosphate-buffered saline, PBS, 37 degrees C, pH 7.4) resulted in rapid lysosomal membrane damage with disappearance of the proton gradient as visualized by acridine orange relocalization, as well as plasma membrane alterations leading to increased leakage of fluorescein after fluorescein diacetate staining. These events were later (greater than 30 min) followed by cellular degeneration in the form of blebbing. Mitochondrial damage (rhodamine 123 relocalization) was a late event. Cells pretreated with desferrioxamine (Des) and superoxide dismutase (SOD) or Des, SOD and catalase (CAT) to induce partial (H2O2 formation only) or almost full protection (no ROS formation) showed about the same reactions as when cells were exposed to alloxan and cysteine without scavengers (O2-, H2O2 and OH. formation) or with PBS only, respectively. The results are interpreted as indicating that the cytotoxicity is a consequence mainly of H2O2 involvement and probably of lysosomal influx of H2O2 with ensuing OH.formation within secondary lysosomes containing trace amounts of reactive iron. It is suggested that the resultant lysosomal membrane damage is followed by leakage of lysosomal hydrolases and ensuing cellular degeneration.