Effect of glutathione redox state on Leydig cell susceptibility to acute oxidative stress

The free radical, or oxidative stress, theory posits that imbalance in cells between prooxidants and antioxidants results in an altered redox state and, over time, an accumulation of oxidative damage. We hypothesized herein that cells with an increasingly prooxidant intracellular environment also might be particularly susceptible to acute oxidative stress. To test this hypothesis, MA-10 cells were used as a model because of their well-defined, measurable function, namely progesterone production. We first experimentally altered the redox environment of the cells by their incubation with buthionine sulfoximine (BSO) or diethyl maleate (DEM) so as to deplete glutathione (GSH), and then exposed the GSH-depleted cells acutely to the prooxidant tert-butyl hydroperoxide (t-BuOOH). Neither BSO nor DEM by themselves affected progesterone production. However, when the GSH-depleted cells subsequently were exposed acutely to t-BuOOH, intracellular reactive oxygen species concentration was significantly increased, and this was accompanied by significant reductions in progesterone production. In striking contrast, treatment of control cells with t-BuOOH had no effect. Depletion of GSH and subsequent treatment of the cells with t-BuOOH-induced the phosphorylation of each of ERK1/2, JNK and p38, members of the MAPK family. Inhibition of p38 phosphorylation largely prevented the t-BuOOH-induced down-regulation of progesterone production in GSH-depleted cells. These results suggest that, as hypothesized, alteration of the intracellular GSH redox environment results in the increased sensitivity of MA-10 cells to oxidative stress, and that this is mediated by activation of one or more redox-sensitive MAPK members.     

Calorie restriction up-regulates the plasma membrane redox system in brain cells and suppresses oxidative stress during aging

The plasma membrane (PM) contains redox enzymes that provide electrons for energy metabolism and recycling of antioxidants such as coenzyme Q and alpha-tocopherol. Brain aging and neurodegenerative disorders involve impaired energy metabolism and oxidative damage, but the involvement of the PM redox system (PMRS) in these processes is unknown. Caloric restriction (CR), a manipulation that protects the brain against aging and disease, increased activities of PMRS enzymes (NADH-ascorbate free radical reductase, NADH-quinone oxidoreductase 1, NADH-ferrocyanide reductase, NADH-coenzyme Q10 reductase, and NADH-cytochrome c reductase) and antioxidant levels (alpha-tocopherol and coenzyme Q10) in brain PM during aging. Age-related increases in PM lipid peroxidation, protein carbonyls, and nitrotyrosine were attenuated by CR, levels of PMRS enzyme activities were higher, and markers of oxidative stress were lower in cultured neuronal cells treated with CR serum compared with those treated with ad libitum serum. These findings suggest important roles for the PMRS in protecting brain cells against age-related increases in oxidative and metabolic stress.     

Mitochondria and oxidative stress in heart aging

As average lifespan of humans increases in western countries, cardiac diseases become the first cause of death. Aging is among the most important risk factors that increase susceptibility for developing cardiovascular diseases. The heart has very aerobic metabolism, and is highly dependent on mitochondrial function, since mitochondria generate more than 90 % of the intracellular ATP consumed by cardiomyocytes. In the last few decades, several investigations have supported the relevance of mitochondria and oxidative stress both in heart aging and in the development of cardiac diseases such as heart failure, cardiac hypertrophy, and diabetic cardiomyopathy. In the current review, we compile different studies corroborating this role. Increased mitochondria DNA instability, impaired bioenergetic efficiency, enhanced apoptosis, and inflammation processes are some of the events related to mitochondria that occur in aging heart, leading to reduced cellular survival and cardiac dysfunction. Knowing the mitochondrial mechanisms involved in the aging process will provide a better understanding of them and allow finding approaches to more efficiently improve this process.     

Glutathione,oxidative stress and aging

The free radical theory of aging proposes that the impairment in physiological performance associated with aging is caused by the detrimental effects of oxygen free radicals. This is interesting because it provides us with a theoretical framework to understand aging and because it suggests a rationale for intervention, i.e., antioxidant administration. Thus, the study of antioxidant systems of the cell may be very important in gerontological studies. Glutathione is one of the main nonprotein antioxidants in the cell which, together with its related enzymes, constitute the “glutathione system.” The involvement of glutathione in aging has been known since the early seventies. Several studies have reported that reduced glutathione is decreased in cells from old animals, whereas oxidized glutathione tends to be increased. Recent experiments from our laboratory have underscored the importance of cellular compartmentation of glutathione. Mitochondrial glutathione plays a key role in the protection against free radical damage associated with aging. Oxidative damage to mitochondrial DNA is directly related to an oxidation of mitochondrial glutathione. In fact, aging is associated with oxidative damage to proteins, nucleic acids, and lipids. These molecular lesions may be responsible for the low physiological performance of aged cells. Thus, antioxidant supplementation may be a rational way to partially protect against age-associated impairment in performance. Apoptosis, a programmed cell death, is an area of research which has seen an explosive growth. Glutathione is involved in apoptosis: apoptotic cells have lower levels of reduced glutathione, and administration of glutathione precursors prevent, or at least delay, apoptosis. Age-associated diseases constitute a major concern for researchers involved in aging. Free radicals are involved in many such diseases; for instance, cancer, diabetes or atherosclerosis. The key role of glutathione and other antioxidants in the pathophysiology of aging and age-associated diseases is discussed in this review.     

Peroxyl-induced oxidative stress in aging erythrocytes of rat

This study aims at determining the possible changes in intracellular calcium (Ca_i²⁺), plasma membrane calcium ATPase (PMCA) activity and phosphatidylserine (PS) along with glutathione (GSH) level in response to an oxidant challenge in vitro. Erythrocytes were isolated on Percoll and incubated with 2, 2′azobis (2-aminopropane) hydrochloride (AAPH) as well as with vitamin C preceding AAPH incubation. Membrane integrity in terms of hemolysis was negatively related to acetylcholine esterase (AChE) activity with the extent of reduction under OS being higher in the old erythrocyte than in the young. A divergent pattern was seen towards lower PMCA and higher (Ca_i²⁺) in the young and old cells. However, the PMCA activity in the stressed young cell was high when pre-treated with vitamin C. PS externalization in the young under OS is perhaps analogous to normal aging, with vitamin C preventing premature death. These findings suggest that young erythrocytes may benefit from vitamin C in therapies addressed towards the mechanisms underlying the reduced effects of OS.     

Quantifying changes in the thiol redox proteome upon oxidative stress in vivo

Antimicrobial levels of reactive oxygen species (ROS) are produced by the mammalian host defense to kill invading bacteria and limit bacterial colonization. One main in vivo target of ROS is the thiol group of proteins. We have developed a quantitative thiol trapping technique termed OxICAT to identify physiologically important target proteins of hydrogen peroxide (H(2)O(2)) and hypochlorite (NaOCl) stress in vivo. OxICAT allows the precise quantification of oxidative thiol modifications in hundreds of different proteins in a single experiment. It also identifies the affected proteins and defines their redox-sensitive cysteine(s). Using this technique, we identified a group of Escherichia coli proteins with significantly (30-90%) oxidatively modified thiol groups, which appear to be specifically sensitive to either H(2)O(2) or NaOCl stress. These results indicate that individual oxidants target distinct proteins in vivo. Conditionally essential E. coli genes encode one-third of redox-sensitive proteins, a finding that might explain the bacteriostatic effect of oxidative stress treatment. We identified a select group of redox-regulated proteins, which protect E. coli against oxidative stress conditions. These experiments illustrate that OxICAT, which can be used in a variety of different cell types and organisms, is a powerful tool to identify, quantify, and monitor oxidative thiol modifications in vivo.     

Aging and oxidative stress: studies of some genes involved both in aging and in response to oxidative stress.

Aging is a complex physiological phenomenon and several theories have been developed about its origin. Among such theories, the 'mitochondrial theory of aging' has been supported by numerous studies and reviews. Cell oxidative damage, in particular the accumulation of mtDNA mutations, is determined by the rate of reactive oxygen species production and degradation induced by the antioxidant defense systems. In this review, data from our laboratory and from the recent literature have been examined to provide arguments that reinforce the crucial role of mitochondria in aging. Various genes that affect life span have been described in numerous organisms. Some of them encode signal transduction proteins and participate in the regulation of mitochondrial metabolism.     

Alterations of antioxidant enzymes and oxidative stress markers in aging

In accordance with the present state of scientific knowledge, the excessive production of free radicals in the organism, and the imbalance between the concentrations of these and the antioxidant defenses may be related to processes such as aging and several diseases. The aging process has been described by various theories. In particular, the free radical theory of aging has received widespread attention which proposes that deleterious actions of free radicals are responsible for the functional deterioration associated with aging. Although, the relationship between lipid peroxidation and aging have been investigated extensively, the studies have produced conflicting results. To investigate the correlation between the oxidative stress and aging, we have determined the levels of lipid peroxidation expressed as thiobarbituric acid reactive substances (TBARS; MDA) and conjugated dien; oxidative protein damage as indicated by carbonyl content and activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in a sample of 100 healthy men and women ranging in age from 20 to 70years. In addition, vitamin E, C levels, reduced glutathione and sulphydryl content were determined. The oxidation end product of nitric oxide (nitrate) was also studied to investigate any role of nitrogen radicals in aging. Our data show that there is an age related increase in lipid peroxidation expressed as MDA and oxidative protein damage as indicated by carbonyl content. Aging is not linked to a decline in antioxidant enzymes except GPx. Our data suggests that the level of oxidative stress increase cannot entirely be attributed to a decrease in the activities of antioxidant defense system and probably various factors may contribute to this process.     

The plasma membrane redox system: a candidate source of aging-related oxidative stress

The plasma membrane redox system (PMRS) is an electron transport chain in the plasma membrane that transfers electrons from either intra- or extracellular donors to extracellular acceptors. Unlike the superoxide-generating NADPH oxidase of phagocytes and the homologous (but much less active) enzymes found in some other cells, the PMRS is still incompletely characterised at the molecular level. Much is known, however, concerning its function and affinity for both physiological and non-physiological substrates. A role for it in aging, the ‘reductive hotspot hypothesis’ (RHH), was proposed in 1998 as part of an explanation for the apparently indefinite survival in vivo of cells that have entirely lost mitochondrial respiratory capacity as a result of the accumulation of mitochondrial mutations. Stimulation of the PMRS might allow the cell to maintain redox homeostasis even while continuing to operate the Krebs cycle, which may be advantageous in many ways. However, the PMRS may, like the mitochondrial respiratory chain, be prone to generate superoxide when thus dysregulated – and in this case superoxide would be generated outside the cell, where antioxidant defences are more limited than inside the cell and where much highly oxidisable material is present. Cascades of peroxidation chain reactions initiated by this process may greatly amplify the oxidative stress on the organism that is caused by rare mitochondrially mutant cells. Since such cells increase in abundance with aging (though remaining rare), this is an economical hypothesis to explain the rise in oxidative stress seen in (and generally believed to contribute substantially to) mammalian aging. In an extension of previously published accounts of RHH, I propose here that the lysosomal toxicity of oxidised cholesterol derivatives (oxysterols) may contribute to the toxicity of mitochondrial mutations by affecting lysosomal function in many cell types in the same way as they have been proposed to do in arterial macrophages.     

Sirt1 regulates aging and resistance to oxidative stress in the heart

Silent information regulator (Sir)2, a class III histone deacetylase, mediates lifespan extension in model organisms and prevents apoptosis in mammalian cells. However, beneficial functions of Sir2 remain to be shown in mammals in vivo at the organ level, such as in the heart. We addressed this issue by using transgenic mice with heart-specific overexpression of Sirt1, a mammalian homolog of Sir2. Sirt1 was significantly upregulated (4- to 8-fold) in response to pressure overload and oxidative stress in nontransgenic adult mouse hearts. Low (2.5-fold) to moderate (7.5-fold) overexpression of Sirt1 in transgenic mouse hearts attenuated age-dependent increases in cardiac hypertrophy, apoptosis/fibrosis, cardiac dysfunction, and expression of senescence markers. In contrast, a high level (12.5-fold) of Sirt1 increased apoptosis and hypertrophy and decreased cardiac function, thereby stimulating the development of cardiomyopathy. Moderate overexpression of Sirt1 protected the heart from oxidative stress induced by paraquat, with increased expression of antioxidants, such as catalase, through forkhead box O (FoxO)-dependent mechanisms, whereas high levels of Sirt1 increased oxidative stress in the heart at baseline. Thus, mild to moderate expression of Sirt1 retards aging of the heart, whereas a high dose of Sirt1 induces cardiomyopathy. Furthermore, although high levels of Sirt1 increase oxidative stress, moderate expression of Sirt1 induces resistance to oxidative stress and apoptosis. These results suggest that Sirt1 could retard aging and confer stress resistance to the heart in vivo, but these beneficial effects can be observed only at low to moderate doses (up to 7.5-fold) of Sirt1.     

From the Cover: Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling

Using methodology developed herein, it is found that reactive persulfides and polysulfides are formed endogenously from both small molecule species and proteins in high amounts in mammalian cells and tissues. These reactive sulfur species were biosynthesized by two major sulfurtransferases: cystathionine β-synthase and cystathionine γ-lyase. Quantitation of these species indicates that high concentrations of glutathione persulfide (perhydropersulfide >100 μM) and other cysteine persulfide and polysulfide derivatives in peptides/proteins were endogenously produced and maintained in the plasma, cells, and tissues of mammals (rodent and human). It is expected that persulfides are especially nucleophilic and reducing. This view was found to be the case, because they quickly react with H₂O₂ and a recently described biologically generated electrophile 8-nitroguanosine 3′,5′-cyclic monophosphate. These results indicate that persulfides are potentially important signaling/effector species, and because H₂S can be generated from persulfide degradation, much of the reported biological activity associated with H₂S may actually be that of persulfides. That is, H₂S may act primarily as a marker for the biologically active of persulfide species.Hydrogen sulfide (H₂S) has been suggested to be an endogenous small molecule signaling species (1) by unknown mechanisms. Our laboratory recently showed that the presence of hydrogen sulfide anion (HS⁻) may be responsible for the regulation and metabolism of various important electrophilic species [e.g., 8-nitroguanosine 3′,5′-cyclic GMP (8-nitro-cGMP)] (2). However, these studies also indicated that reactive intermediates other than HS⁻ likely react with the electrophiles of interest. These previous studies alluded to the generation of a more reactive sulfur species capable of reacting with electrophiles, such as 8-nitro-cGMP. As reported herein, it was determined that reactive sulfur intermediates, such as hydropersulfides (RSSH) and polysulfides [RS(S)_nH and RS(S)_nSR], are formed in appreciable amounts during sulfur amino acid metabolism and possess important chemical and biological properties. Some of these sulfide species have long been known as sulfane sulfur compounds, which were suggested to exist endogenously in mammalian systems (1, 3–5). Reports also indicated that a hydropersulfide moiety with the general molecular formula RSSH may be formed on specific protein cysteine (Cys) residues, most typically of sulfur-transferring enzymes (i.e., sulfurtransferases) during enzymatic reactions (1, 5). Although such persulfide chemical reactivity is thought to be involved in the catalytic activity of particular enzymes (e.g., rhodanese, Cys desulfurases, and sulfide:quinone oxidoreductase) (6, 7), the more general physiological function and occurrence of Cys persulfides (CysSSH) and related species in cells and tissues, especially mammals, were unclear. Moreover, the exact chemical nature and physiological relevance of these biological polysulfur derivatives, especially sulfane sulfur compounds, remain uncertain.Herein, an MS-based metabolomic method for the analysis of low-molecular weight persulfides/polysulfides and a proteomic analysis combined with a Tag-Switch assay to detect S-polythiolated protein adducts have been developed. Using these techniques, the endogenous formation and presence of Cys hydropersulfide derivatives as well as their possible physiological functions were investigated. The roles of the enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in Cys hydropersulfide generation were evaluated. Moreover, the antioxidant, cytoprotective, and redox signaling properties of these persulfide species in cells were assessed. Finally, the effect of various sulfides on cellular redox/electrophilic signaling associated with the endogenous electrophile 8-nitro-cGMP was examined.     

Protective role of dietary vitamin E on oxidative stress in aging

Free radical reactions and the peroxidation of membrane lipids have been implicated in the mechanism of aging and age-associated degenerative conditions. Vitamin E appears to play a critical role in protecting the cell membrane from free radical reactions and peroxidation of polyunsaturated fatty acids (PUFA). In a series of human studies, the protective role of vitamin E supplementation against oxidative stress was investigated in young and older volunteers. Muscle biopsies taken from young (<30 y) men, following an eccentric exercise bout, exhibited a decreased level of vitamin E and increased conjugated diene status, an indication of lipid peroxidation. Older men (>55 y) supplemented with vitamin E for 48 d excreted a lower level of lipid peroxidation products in urine compared to placebo control following eccentric exercise. In conditions where the composition of membrane fatty acids changes to more PUFA, older subjects may be at a greater risk of oxidative damage. In a study of 15 young (<35 y) and 10 older (>51 y) women receiving fish oil capsules for 3 months, older women showed a greater increase in plasma levels of eicosapentaenoic (EPA) (p<0.001) and docosahexaenoic (DHA) (p<0.5) acids compared to young subjects. By substituting membrane fatty acids with potentially unstable (n-3) fatty acids of fish oil, older subjects were found to be at a greater risk of oxidative stress than young subjects. This was indicated by decreased E/EPA+DHA (4.9 fold in older and 3.6 fold in young women) and a higher increase (p<0.05) in lipid peroxides (63.1% in older vs. 29.4% in young women) in their plasma. These findings indicate that vitamin E plays an important protective antioxidant role in older subjects, particularly in conditions where oxidative stress and free radicals are potentiated. Syposium Paper: Aging and Nutrition. Presented on October 3, 1990 during the 20th Annual Meeting of AGE in New York City.     

Curcumin ameliorates arterial dysfunction and oxidative stress with aging

We tested the hypothesis that curcumin supplementation would reverse arterial dysfunction and vascular oxidative stress with aging. Young (Y, 4–6 months) and old (O, 26–28 months) male C57BL6/N mice were given normal or curcumin supplemented (0.2%) chow for 4 weeks (n = 5–10/group/measure). Large elastic artery stiffness, assessed by aortic pulse wave velocity (aPWV), was greater in O (448 ± 15 vs. 349 ± 15 cm/s) and associated with greater collagen I and advanced glycation end-products and less elastin (all P < 0.05). In O, curcumin restored aPWV (386 ± 15 cm/s), collagen I and AGEs (AGEs) to levels not different vs. Y. Ex vivo carotid artery acetylcholine (ACh)-induced endothelial-dependent dilation (EDD, 79 ± 3 vs. 94 ± 2%), nitric oxide (NO) bioavailability and protein expression of endothelial NO synthase (eNOS) were lower in O (all P < 0.05). In O, curcumin restored NO-mediated EDD (92 ± 2%) to levels of Y. Acute ex vivo administration of the superoxide dismutase (SOD) mimetic TEMPOL normalized EDD in O control mice (93 ± 3%), but had no effect in Y control or O curcumin treated animals. O had greater arterial nitrotyrosine abundance, superoxide production and NADPH oxidase p67 subunit expression, and lower manganese SOD (all P < 0.05), all of which were reversed with curcumin. Curcumin had no effects on Y. Curcumin supplementation ameliorates age-associated large elastic artery stiffening, NO-mediated vascular endothelial dysfunction, oxidative stress and increases in collagen and AGEs in mice. Curcumin may be a novel therapy for treating arterial aging in humans.