Effects of diabetes on oxidative and nitrosative stress in kidney mitochondria from aged rats.

Diabetes mellitus (DM) is characterized by chronic hyperglycemia resulting from defects in the secretion and/or action of insulin. Diabetic nephropathy (DN) develops in diabetic patients and is characterized by a progressive deterioration of renal function. The mitochondrial electron transport chain (ETC) produces most of the reactive oxygen species (ROS) that are involved in diabetic nephropathy. Due to the high incidence of DM in the elderly, the aim of this study was to evaluate oxidative and nitrosative stress in kidney mitochondria from aged rats. We evaluated lipid peroxidation (LPO), nitric oxide (NO(•)) production, S-nitrosylation profiles, glutathione levels, and glutathione reductase and aconitase activities under streptozotocin (STZ)-induced experimental diabetes in kidney mitochondria from aged rats. The results showed an increase in LPO, NO(•) production, and S-nitrosylated proteins in rats with STZ-induced diabetes. A decrease in glutathione (GSH) levels and glutathione reductase (GR) and aconitase activities in the rats that received the STZ-induced diabetes treatment was also observed, when compared with the age-related controls. The data suggest that oxidative and nitrosative stresses promote mitochondrial oxidative dysfunction in the more advanced age rat kidney in STZ-induced diabetes.

Malfunctioning of the iron-sulfur cluster assembly machinery in Saccharomyces cerevisiae produces oxidative stress via an iron-dependent mechanism, causing dysfunction in respiratory complexes.

Biogenesis and recycling of iron-sulfur (Fe-S) clusters play important roles in the iron homeostasis mechanisms involved in mitochondrial function. In Saccharomyces cerevisiae, the Fe-S clusters are assembled into apoproteins by the iron-sulfur cluster machinery (ISC). The aim of the present study was to determine the effects of ISC gene deletion and consequent iron release under oxidative stress conditions on mitochondrial functionality in S. cerevisiae. Reactive oxygen species (ROS) generation, caused by H2O2, menadione, or ethanol, was associated with a loss of iron homeostasis and exacerbated by ISC system dysfunction. ISC mutants showed increased free Fe2+ content, exacerbated by ROS-inducers, causing an increase in ROS, which was decreased by the addition of an iron chelator. Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe-S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe-S cluster anabolism is affected. Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe-S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants. In addition, the activity of complexes II and IV from the electron transport chain (ETC) was impaired or totally abolished in SSQ1 and ISA1 mutants. These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.

Reactive oxygen species production induced by ethanol in Saccharomyces cerevisiae increases because of a dysfunctional mitochondrial iron-sulfur cluster assembly system.

Ethanol accumulation during fermentation contributes to the toxic effects in Saccharomyces cerevisiae, impairing its viability and fermentative capabilities. The iron-sulfur (Fe-S) cluster biogenesis is encoded by the ISC genes. Reactive oxygen species (ROS) generation is associated with iron release from Fe-S-containing enzymes. We evaluated ethanol toxicity, ROS generation, antioxidant response and mitochondrial integrity in S. cerevisiae ISC mutants. These mutants showed an impaired tolerance to ethanol. ROS generation increased substantially when ethanol accumulated at toxic concentrations under the fermentation process. At the cellular and mitochondrial levels, ROS were increased in yeast treated with ethanol and increased to a higher level in the ssq1∆, isa1∆, iba57∆ and grx5∆ mutants - hydrogen peroxide and superoxide were the main molecules detected. Additionally, ethanol treatment decreased GSH/GSSG ratio and increased catalase activity in the ISC mutants. Examination of cytochrome c integrity indicated that mitochondrial apoptosis was triggered following ethanol treatment. The findings indicate that the mechanism of ethanol toxicity occurs via ROS generation dependent on ISC assembly system functionality. In addition, mutations in the ISC genes in S. cerevisiae contribute to the increase in ROS concentration at the mitochondrial and cellular level, leading to depletion of the antioxidant responses and finally to mitochondrial apoptosis.