Sterol carrier protein-2 (SCP-2) involvement in cholesterol hydroperoxide cytotoxicity as revealed by SCP-2 inhibitor effects

Sterol carrier protein-2 (SCP-2) plays an important role in cholesterol trafficking and metabolism in mammalian cells. The purpose of this study was to determine whether SCP-2, under oxidative stress conditions, might also traffic hydroperoxides of cholesterol, thereby disseminating their cytotoxic effects. Two inhibitors, SCPI-1 and SCPI-3, known to block cholesterol binding by an insect SCP-2, were used to investigate this. A mouse fibroblast transfectant clone (SC2F) overexpressing SCP-2 was found to be substantially more sensitive to apoptotic killing induced by liposomal 7α-hydroperoxycholesterol (7α-OOH) than a wild-type control. 7α-OOH uptake by SC2F cells and resulting apoptosis were both inhibited by SCPI-1 or SCPI-3 at a subtoxic concentration. Preceding cell death, reactive oxidant accumulation and loss of mitochondrial membrane potential were also strongly inhibited. Similar SCPI protection against 7α-OOH was observed with two other types of SCP-2-expressing mammalian cells. In striking contrast, neither inhibitor had any effect on H₂O₂-induced cell killing. To learn whether 7α-OOH cytotoxicity is due to uptake/transport by SCP-2, we used a fluorescence-based competitive binding assay involving recombinant SCP-2, NBD-cholesterol, and SCPI-1/SCPI-3 or 7α-OOH. The results clearly showed that 7α-OOH binds to SCP-2 in SCPI-inhibitable fashion. Our findings suggest that cellular SCP-2 not only binds and translocates cholesterol but also cholesterol hydroperoxides, thus expanding their redox toxicity and signaling ranges under oxidative stress conditions.     

Hydrogen peroxide is involved in the sclerotial differentiation of filamentous phytopathogenic fungi

Aims: The purpose of this study was to investigate the role of H₂O₂ and the related oxidative stress markers catalase (CAT) and lipid peroxidation in the sclerotial differentiation of the phytopathogenic filamentous fungi Sclerotium rolfsii, Sclerotinia minor, Sclerotinia sclerotiorum and Rhizoctonia solani. Methods and Results: Using the H₂O₂‐specific scopoletin fluorometric assay and the CAT‐dependent H₂O₂ consumption assays, it was found that the production rate of intra/extracellular H₂O₂ and CAT levels in the sclerotiogenic fungi were significantly higher and lower, respectively, than those of their nondifferentiating counterpart strains. They peaked in the transition between the undifferentiated and the differentiated state of the sclerotiogenic strains, suggesting both a cell proliferative and differentiative role. In addition, the indirect indicator of oxidative stress, lipid peroxidation, was substantially decreased in the nondifferentiating strains. Conclusions: These findings suggest that the differentiative role of H₂O₂ is expressed via induction of higher oxidative stress in the sclerotiogenic filamentous phytopathogenic fungi. Significance and Impact of the Study: This study shows that the direct marker of oxidative stress H₂O₂ is involved in the sclerotial differentiation of the phytopathogenic filamentous fungi S. rolfsii, S. minor, S. sclerotiorum and R. solani, which could have potential biotechnological implications in terms of developing antifungal strategies by regulating intracellular H₂O₂ levels.     

Oxidative stress in silicosis: Evidence for the enhanced clearance of free radicals from whole lungs

We hypothesized that reactive oxygen species (ROS) may be involved in the pathogenesis of silicosis. To investigate ROS' dependent pathophysiological processes during silicosis we studied the kinetic clearance of instilled stable nitroxide radicals (TEMPO). Antioxidant enzymes' superoxide dismutase (SOD) and glutathione peroxidase (GPx), and lipid peroxidation were also studied in whole lungs of rats exposed to crystalline silica (quartz) and sham exposed controls. Low frequency L-band electron spin resonance spectroscopy was used to measure the clearance of TEMPO in whole-rat lungs directly. The clearance of TEMPO followed first order kinetics showing significant differences in the rate for clearance between the diseased and sham exposed control lungs. Comparison of TEMPO clearance rates in the sham exposed controls and silicotic rats showed an oxidative stress in the rats exposed to quartz. Studies on the antioxidant enzymes SOD and GPx in the lungs of silicotic and sham exposed animals supported the oxidative stress and accelerated clearance of TEMPO by up regulated levels of enzymes in quartz exposed animals. Increased lipid peroxidation potential in the silicotics also supported a role for enhanced generation of ROS in the pathogenesis of silica-induced lung injury. These in vivo experiments directly demonstrate, for the first time, that silicotic lungs are in a state of oxidative stress and that increased generation of ROS is associated with enhanced levels of oxidative enzymes and lipid peroxidation. This technique offers great promise for the elucidation of ROS induced lung injury and development of therapeutic strategies for the prevention of damage.     

The role of ascorbic acid role in the differentiation of sclerotia in Sclerotinia minor

Sclerotinia minor in culture produces ascorbic acid in levels dependent on oxidative growth conditions and stage of development. During differentiation reduced/oxidized ascorbate ratio decreased by 12 and 6 fold at high and low oxidative stress, respectively. Exogenous ascorbate caused a concentration-dependent decrease of oxidative stress (lipid peroxidation), inhibition of sclerotial differentiation (up to 100%) and delay of differentiatlon (up to 10 days). Ascorbic acid may be produced to help the fungus reduce oxidative stress during growth. The data of this study support our theory proposing that oxidative stress is the inducing factor of sclerotial differentiation in fungi.This revised version was published online in October 2005 with corrections to the Cover Date.     

Antioxidant enzyme alterations in experimental and clinical diabetes

Previous studies from our laboratory have demonstrated the presence of complex alterations in the activities of antioxidant enzymes in various tissues of rats with streptozotocin (STZ)-induced diabetes. In the present investigation, it is shown that rats made diabetic with alloxan (ALX), an agent differing from STZ both chemically and in its mechanism of diabetogenesis, show virtually identical tissue antioxidant enzyme changes which, as is the case with STZ, are preventable by insulin treatment. The finding that the patterns of antioxidant enzyme alterations in chemically-induced diabetes are independent of the diabetogenic agent used and the presence of similar abnormalities in tissues of spontaneously diabetic (BB) Wistar rats (particularly when diabetic control is less than optimal) suggest that the changes observed are a characteristic feature of the uncontrolled diabetic state and that these may be responsible for (or predispose to) the development of secondary complications in clinical diabetes. Comparative studies involving red cells of diabetic rats and human diabetics revealed a number of common changes, namely an increase in glutathione reductase activity, a decreased susceptibility to oxidative glutathione depletion (which was related to the presence of hyperglycemia) and an increased production of malondialdehyde (an indirect index of lipid peroxidation) in response to in vitro challenge with hydrogen peroxide. In the diabetic patients, the extent of this increase in susceptibility of red cell lipids to oxidation paralleled the severity of diabetic complications. Our results suggest that increased (or uncontrolled) oxidative activity may play an important role in the pathogenesis of complications associated with the chronic diabetic state.This work was supported by grants from the British Columbia Health Care Research Foundation and the Canadian Diabetes Association.     

SKO1 deficiency extends chronological lifespan in Saccharomyces cerevisiae

ABSTRACT

Sko1 plays a key role in the control of gene expression by osmotic and oxidative stress in yeast. We demonstrate that the decrease in chronological lifespan (CLS) of hog1Δ cells was suppressed by SKO1 deletion. sko1Δ single mutant cells were shown to have a longer CLS, thus implicating Sko1 in the regulation of their CLS.     

Effect of aliphatic aldehydes on the lipid peroxidation and chemiluminescence of biological systems under oxidative stress

The effect of several aliphatic aldehydes on lipid peroxidation was evaluated by measuring the oxygen uptake rate, thiobarbituric acid-reactive products formation and the emitted visible chemiluminescence intensity. Measurements were carried out in brain homogenates and erythrocyte plasma membrane and liver microsomal fractions. In all systems studied, aldehydes (25 mmol/L) (e.g. acetaldehyde, 2,2-dimethylpropanal), increased the intensity of the luminescence associated with the oxidation process. In contrast, aldehyde incorporation decreased TBARS production and the rate of oxygen uptake. The increased luminescence intensity is explained in terms of secondary reactions of aldehyde derived free radicals. These results clearly indicate that extreme care must be exercized in the intepretation of chemiluminescence data in the presence of aldehydes. © 1997 John Wiley & Sons, Ltd.     

Antioxidants protect the yeast Saccharomyces cerevisiae against hypertonic stress

Yeast (Saccharomyces cerevisiae) mutants lacking CuZnSOD have been reported to be hypersensitive to hypertonic media and to show increased oxidative damage. This study demonstrates that hypertonic medium (containing 0.8?M NaCl) increases the generation of superoxide and other reactive species in yeast cells. Other sequelae of exposure to hypertonic medium include oxidation of cellular low-molecular weight thiols and decrease in total antioxidant capacity of cellular extracts. Δsod1 mutant is more sensitive than a wild-type strain to colony growth inhibition on a hypertonic medium. Anaerobic conditions, ascorbate, glutathione, cysteine and dithiothreitol are able to ameliorate this growth inhibition but a range of other antioxidants does not protect. The protective ability of the antioxidants does not correlate with the rate of their reactions with superoxide but seems to be conditioned by low redox potential for one-electron oxidation of free radicals of the antioxidants. It suggests that repair of low-redox potential targets rather than prevention of their damage by superoxide is important in the antioxidant protection against oxidative stress induced by hypertonic conditions.     

氧化胁迫环境下的酵母细胞应答调控

酿酒酵母细胞在生长过程中会不断受到内外环境的氧化攻击。活性氧族物质的累积能够损害细胞中的脂质、DNA和蛋白质,从而会影响细胞的正常功能,严重者将造成细胞死亡。为了对抗氧化胁迫,酵母细胞在不断地适应过程中,进化出了较为完整的保护机制,呈现出多水平多层次的应激应答反应。细胞在非酶水平、蛋白质水平和基因水平上协同作用,共同完成了活性氧族物质的清除和胁迫信号的传递应答。本文对酵母细胞在氧化胁迫环境下的应答调控做了简要综述。     

酿酒酵母氧化胁迫应答反应机制

氧化胁迫是生物体面对逆境时的重要反应。在与逆境和活性氧做斗争的过程中,细胞进化出一套完整的应答调控机制,通过调节体内活性氧的代谢平衡,来保护DNA、脂质和蛋白质等免受氧化攻击。本文以酿酒酵母为例,根据近年来国内外研究的进展,围绕其在氧化胁迫应答过程中的三道保护屏障,即抗氧化物质和防御酶系统、转录调节和氧化物降解以及细胞器自噬,综述了其抗氧化代谢机理,为深入认识细胞的抗氧化应答机制奠定基础。     

Arsenite as an Inducer of Lipid Peroxidation in Saccharomyces cerevisiae Cells

The ability of sodium arsenite at concentrations of 10^–2, 10^–4, and 10^–6 M to induce lipid peroxidation in Saccharomyces cerevisiae cells was studied. Arsenite at the concentrations 10^–2 and 10^–4 M enhanced lipid peroxidation and inhibited the growth of yeast cells. Enhanced lipid peroxidation likely induced oxidative damage to various cellular structures, which led to suppression of the metabolic activity of cells. Arsenite at the concentration 10^–6 M did not activate lipid peroxidation in cells. All of the tested arsenite concentrations inhibited the activity of -ketoglutarate dehydrogenase and pyruvate dehydrogenase in cells. The inference is made that the toxicity of arsenite may be related to its stimulating effect on intracellular lipid peroxidation.     

Methamphetamine and lipid peroxidation in the rat retina

BACKGROUND: The use of psychoactive drugs during adolescence and early adult life has increased in the last few decades. It is known that developmental exposure to psychostimulants affects the sensory systems, and the retina has been shown to be a target tissue. This work was conducted to evaluate the pattern of lipid peroxidation in the rat retina following prenatal exposure to methamphetamine (MA). METHODS: Pregnant female Wistar rats were given MA (5 mg/kg of body weight/day; SC, in 0.9% saline) from GD 8 to 22. Offspring were sacrificed at postnatal days (PNDs) 7, 14, and 21. The retinas were homogenized, and both the total antioxidant and superoxide dismutase (SOD) activities were measured by enzymatic-colorimetric methods. The lipid peroxidation byproducts (malondialdehyde [MDA] and MDA-like metabolites) were measured by the thiobarbituric acid test. RESULTS: Total antioxidant levels were lower in the MA group at PND 21 in both males and females. The activity of SOD was higher in PND 7 females from the MA group. MDA levels were higher in the MA group at PND 21 in both genders. CONCLUSIONS: These findings suggest that prenatal-induced MA toxicity in the retina may be related to lipid peroxidation processes and oxidative stress.