LETHAL HYDROGEN-PEROXIDE TOXICITY INVOLVES LYSOSOMAL IRON-CATALYZED REACTIONS WITH MEMBRANE DAMAGE

Secondary lysosomes contain low-molecular weight iron-complexes as a c onsequence of normal autophagocytotic degradation of various metallo-p roteins. Thus, entry of hydrogen peroxide into these organelles may in duce iron-catalyzed oxidative reactions with ensuing damage to lysosom al membranes and leakage of destructive contents. The amount of lysoso mal reactive iron and the cellular capacity to degrade hydrogen peroxi de would then be important determining factors in cellular resistance to oxidative stress. The effects of hydrogen peroxide on cell viabilit y and, in particular, on lysosomal membrane integrity, evaluated by ac ridine orange, lucifer yellow, neutral red, and cathepsin D relocaliza tion, were investigated in a model system of cultured J-774 cells. The protective effect of the iron-chelator desferal was studied after exp osure to the drug under ordinary culture conditions and after inhibiti on of cellular endocytosis. Hydrogen peroxide-exposure (500 mu M in PB S, 37 degrees C, 5-90 min) was manifested as a time-dependent decrease in cell viability. This was preceded by a rapid reduction of the prot on gradient across the lysosomal membranes, as judged by relocalizatio n of acridine orange. Another early sign of damage was plasma membrane blebbing, found on many cells within minutes after the initiation of hydrogen peroxide-exposure. The cells also showed a partial redistribu tion of the lysosomal markers lucifer yellow, neutral red, and catheps in D, indicating lysosomal destabilization. The preexposure of cells t o desferal in culture prevented all these phenomena, unless endocytoti c uptake of the drug was prevented.