Lethal hydrogen peroxide toxicity involves lysosomal iron-catalyzed reactions with membrane damage

Abstract

Secondary lysosomes contain low-molecular weight iron-complexes as a consequence of normal autophagocytotic degradation of various metallo-proteins. Thus, entry of hydrogen peroxide into these organelles may induce ironcatalyzed oxidative reactions with ensuing damage to lysosomal membranes and leakage of destructive contents. The amount of lysosomal reactive iron and the cellular capacity to degrade hydrogen peroxide would then be important determining factors in cellular resistance to oxidative stress. The effects of hydrogen peroxide on cell viability and, in particular, on lysosomal membrane integrity, evaluated by acridine orange, lucifer yellow, neutral red, and cathepsin D relocalization, were investigated in a model system of cultured J-774 cells. The protective effect of the iron-chelator desferal was studied after exposure to the drug under ordinary culture conditions and after inhibition of cellular endocytosis. Hydrogen peroxide-exposure (500 μM in PBS, 37°C, 5-90 min) was manifested as a time-dependent decrease in cell viability. This was preceded by a rapid reduction of the proton gradient across the lysosomal membranes, as judged by relocalization 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 redistribution of the lysosomal markers lucifer yellow, neutral red, and cathepsin D, indicating lysosomal destabilization. The pre-exposure of cells to desferal in culture prevented all these phenomena, unless endocytotic uptake of the drug was prevented.

Relocalization of Cathepsin D and Cytochrome c Early in Apoptosis Revealed by Immunoelectron Microscopy

“…Cultures to be subjected to transmission electron microscopy were prepared as previously described (Brunk et al, 1995). Briefly, cardiomyocytes in situ in the plastic culture dishes were fixed by adding 2% glutaraldehyde in 0.1 M sucrose-sodium cacodylateHCl buffer (pH 7.2) and post-fixed in osmium (Johnson Matthey Chemicals, Roystone, United Kingdom).…”

Uptake of Oxidized LDL by Macrophages Results in Partial Lysosomal Enzyme Inactivation and Relocation

“…The intensities of red and green AO-induced fluorescence from 100 individual cells per coverslip were then measured using a static cytofluorometer system based on a computer-assisted MPV III (Leitz) photometer-microscope, as described previously. 19,29 The cells were also examined with an LSM 410 confocal laser scanning microscope (Carl Zeiss).…”

“…For light microscopic immunocytochemistry, the cells were fixed in 4% paraformaldehyde and then labeled with primary (polyclonal rabbit anti-human cathepsin D) and secondary (goat anti-rabbit IgG Texas Red conjugate) antibodies, as described before. 28,29 The cells were mounted in Gelvatol (Monsanto) and examined and photographed in a Microphoto-SA fluorescence microscope (Nikon). Immunocytochemical demonstration of cathepsin D at the ultrastructural level was performed as previously described.…”

Imidazoline drugs stabilize lysosomes and inhibit oxidative cytotoxicity in astrocytes

“…Obvious lysosomal breach at later stages of cell death led to overlooking the active participation of lysosomes in the process of cell death. A series of studies have shown the destabilization of the lysosomal membrane to be an early event in cellular devitalization [18,28 -30], and more recently, to be a reliable preceding event of apoptosis caused by oxidative stress, Fas activation, and growth factor deprivation [19,[31][32][33][34][35][36][37][38][39][40]. In the present study, the acridine orange relocation developed within 15 min of exposure to a lethal dose of naphthazarin, and the subsequent decreased mitochondrial potential did not appear until 2 h after exposure.…”

“…Brunk and colleagues showed that intralysosomal iron-catalyzed oxidative reactions cause lysosomal destabilization that can be prevented by allowing cells to endocytose desferrioxamine before being exposed to oxidative stress [18,28,30,[35][36][37]. We decided to determine whether the effective imidazoline drugs could be ironchelators.…”