Christina Piskernik

A novel endotoxin-induced pathway: upregulation of heme oxygenase 1, accumulation of free iron, and free iron-mediated mitochondrial dysfunction

Mitochondria are involved in the development of organ failure in critical care diseases. However, the mechanisms underlying mitochondrial dysfunction are not clear yet. Inducible hemoxygenase (HO-1), a member of the heat shock protein family, is upregulated in critical care diseases and considered to confer cytoprotection against oxidative stress. However, one of the products of HO-1 is Fe2+ which multiplies the damaging potential of reactive oxygen species catalyzing Fenton reaction. The aim of this study was to clarify the relevance of free iron metabolism to the oxidative damage of the liver in endotoxic shock and its impact on mitochondrial function. Endotoxic shock in rats was induced by injection of lipopolysaccharide (LPS) at a dose of 8 mg/kg (i.v.). We observed that the pro-inflammatory cytokine TNF-α and the liver necrosis marker aspartate aminotransferase were increased in blood, confirming inflammatory response to LPS and damage to liver tissue, respectively. The levels of free iron in the liver were significantly increased at 4 and 8 h after onset of endotoxic shock, which did not coincide with the decrease of transferrin iron levels in the blood, but rather with expression of the inducible form of heme oxygenase (HO-1). The proteins important for sequestering free iron (ferritin) and the export of iron out of the cells (ferroportin) were downregulated facilitating the accumulation of free iron in cells. The temporarily increased concentration of free iron in the liver correlated with the temporary impairment of both mitochondrial function and tissue ATP levels. Addition of exogenous iron ions to mitochondria isolated from control animals resulted in an impairment of mitochondrial respiration similar to that observed in endotoxic shock in vivo. Our data suggest that free iron released by HO-1 causes mitochondrial dysfunction in pathological situations accompanied by endotoxic shock.

Antimycin A and lipopolysaccharide cause the leakage of superoxide radicals from rat liver mitochondria

Here we show that both Antimycin A, a respiratory chain inhibitor inducing apoptosis, and endotoxic shock, a syndrome accompanied by both necrosis and apoptosis, cause not only an increase but also the leakage of superoxide radicals (O(2)(*-)) from rat heart mitochondria (RHM), while O(2)(*-) generated in intact RHM do not escape from mitochondria. This was shown by a set of O(2)(*-)-sensitive spin probes with varying hydrophobicity. The levels of O(2)(*-) detected in intact RHM gradually increase as the hydrophobicity of spin probes increases and were not sensitive to superoxide dismutase (SOD) added to the incubation medium. Both Antimycin A and endotoxic shock elevated O(2)(*-) levels. Elevated O(2)(*-) levels became sensitive to SOD but in a different manner. The determination of O(2)(*-) with water-soluble PPH was fully sensitive to SOD, while the determination of O(2)(*-) with the more hydrophobic CMH and CPH was only partially sensitive to SOD, suggesting the release of a portion of O(2)(*-) into the surrounding medium.

Proteome analysis of rat liver mitochondria reveals a possible compensatory response to endotoxic shock.

Organ failure induced by endotoxic shock has recently been associated with affected mitochondrial function. In this study, effects of in vivo lipopolysaccharide‐challenge on protein patterns of rat liver mitochondria in treated animals versus controls were studied by two‐dimensional electrophoresis (differential image gel electrophoresis). Significant upregulation was found for ATP‐synthase α chain and superoxide dismutase [Mn]. Our data suggest that endotoxic shock mediated changes in the mitochondrial proteome contribute to a compensatory reaction (adaptation to endotoxic shock) rather than to a mechanism of cell damage.

Rotenone Damages Striatal Organotypic Slice Culture

Epidemiological studies suggest the involvement of pesticides in the etiology of Parkinsons disease. Exposure to rotenone results in degeneration of the nigrostriatal pathway through inhibition of complex I. Organotypic striatal slice cultures were prepared from brains of adult mice and treated with rotenone (0.01, 0.05, 0.1, and 1 mM) for 48 h. Lactate dehydrogenase activity was elevated by 167% at 1 mM of rotenone. Using fluorescent indicators, membrane damage was up to 130% as measured by propidium iodide fluorescence, and superoxide (DHE) and nitric oxide (DAF‐FM) formation were increased by 195% and 774% at 1 mM of rotenone, respectively, compared to controls. The study concludes that formation of radicals mediated striatal degeneration by rotenone.