Gender differences in oxidative stress in spinal cord of rats submitted to repeated restraint stress

Behavioral and neurochemical gender-specific effects have been observed following repeated stress. The aim of this study is to verify the effects of repeated restraint stress on free radical production (evaluated by DCF test), lipoperoxidation (evaluated by TBARS levels), and total antioxidant reactivity (TAR) in the spinal cord of male and female rats. Results demonstrate no effect on lipoperoxidation; chronic stress decreased TAR both in male and female spinal cord. In addition, gender differences were observed both in TAR and in the production of free radicals, both being increased in females. These results may be relevant to the gender-specific differences observed after exposure to repeated stress.     

A review of the molecular mechanisms of hyperglycemia-induced free radical generation leading to oxidative stress

The prevalence of diabetes is growing worldwide with an increasing morbidity and mortality associated with the development of diabetes complications. Free radical production is a normal biological process that is strictly controlled and has been shown to be important in normal cellular homeostasis, and in the bodies response to pathogens. However, there are several mechanisms leading to excessive free radical production that overcome the normal protective quenching mechanisms. Studies have shown that many of the diabetes complications result from excessive free radical generation and oxidative stress, and it has been shown that chronic hyperglycemia is a potent inducer for free radical production, generated through several pathways and triggering multiple molecular mechanisms. An understanding of these processes may help us to improving our preventive or therapeutic strategies. In this review, the major molecular pathways involved in free radical generation induced by hyperglycemia are described.     

Investigation of oxidative stress during fracture healing in the rats

One of the most damaging effects of reactive oxygen species (ROS) is lipid peroxidation, the end-product of which is malondialdehyde (MDA). This study was aimed to evaluate erythrocyte MDA levels during fracture healing in rats. Thirty male rats were used and the rats were divided into two groups to serve as controls and tests. Six rats were used as a control group that was not subject to fracture. The remaining 24 rats were divided into four groups and erythrocyte MDA levels were examined on days 5, 10, 20 and 30 post fracture. The right fibulas of rats were broken by manual angulation in the experimental group. The erythrocyte malondialdehyde level was measured in the experimental and control groups. The difference between malondialdehyde levels of control and experimental groups was statistically significant (p<0.05). Oxidative stress clearly increases during fracture healing in rats.     

Gender difference in oxidative stress: a new look at the mechanisms for cardiovascular diseases

Gender differences are present in many diseases and are especially prevalent in cardiovascular disease. Males tend to suffer from myocardial infarctions earlier than females, and a woman's risk of cardiovascular disease increases after menopause, suggesting a cardio‐protective role of estrogen. However, hormone replacement therapy did not decrease the risk of cardiovascular disease in post‐menopausal women; thus, other mechanisms may be involved besides estrogen. Oxidative stress plays an important role in the development of cardiovascular diseases such as coronary artery disease. Gender is also associated with differences in oxidative stress. Under physiological conditions, females appear to be less susceptible to oxidative stress. This may be due to the antioxidant properties of estrogen, gender differences in NADPH‐oxidase activity or other mechanism(s) yet to be defined. This review strives to discuss gender differences in general terms followed by a more detailed examination of gender differences with oxidative stress and various associated diseases and the possible mechanisms underlying these differences.     

Testosterone suppresses oxidative stress in human neutrophils

The in vitro effect of testosterone on human neutrophil function was investigated. Blood neutrophils from healthy male subjects were isolated and treated with 10 nM, 0.1 and 10 µM testosterone for 24 h. As compared with untreated cells, the testosterone treatment produced a significant decrease of superoxide production as indicated by the measurement of extra‐ and intracellular superoxide content. An increment in the production of nitric oxide was observed at 0.1 and 10 µM testosterone concentrations, whereas no effect was found for 10 nM. Intracellular calcium mobilization was significantly increased at 10 nM, whereas it was reduced at 10 µM testosterone. There was an increase in phagocytic capacity at 10 nM and a decrease of microbicidal activity in neutrophils treated with testosterone at 10 µM. Glutathione reductase activity was increased by testosterone treatment, whereas no effect was observed in other antioxidant enzyme activities. An increase in the content of thiol groups was observed at all testosterone concentrations. Lipid peroxidation in neutrophils evaluated by levels of TBARS was decreased at 10 nM and 0.1 µM testosterone. These results indicate the antioxidant properties of testosterone in neutrophils as suggested by reduction of superoxide anion production, and lipid peroxidation, and by the increase in nitric oxide production, glutathione reductase activity and the content of thiol groups. Therefore, the plasma levels of testosterone are important regulators of neutrophil function and so of the inflammatory response. Copyright © 2010 John Wiley & Sons, Ltd.     

The Maillard reaction and oxidative stress during aging of soybean seeds

The chemical reactions that may lead to the loss of seed viability were investigated both during the accelerated aging and natural aging of soybeans ( Glycine max Merrill cv. Chippewa 64). Under conditions of accelerated aging (36°C and 75% RH), fluorescence of soluble proteins accumulated, which was closely correlated with the loss of seed germinability and vigor. We were able to show this correlation by using partially purified proteins for the assay. Fluorescence also increased in seeds under good storage conditions (5°C for up to 21 years), although there was a less significant correlation between seed viability and the accumulation of fluorescent products during the time of natural aging. The rise in protein fluorescence is interpreted as an increase of Maillard products. The carbonyl content of soluble proteins (a measure of the oxidative damage) did not change significantly during either accelerated aging or natural aging: however the elimination of carbonyls during germination seemed to be hindered in seeds that had poor germination. The Maillard reaction may be a consequence of the formation of reducing sugars through a gradual hydrolysis of oligosaccharides during aging. Preliminary evidence from the natural aging study showed that, when seeds were in the glassy state, the sugar hydrolysis was inhibited. These results suggest that the Maillard reaction and oxidative reaction may play an important role in seed deterioration.     

Age-related effect induced by oxidative stress on the cerebral glutathione system

In the forebrain from male Wistar rats aged 5, 15 and 25 months, age-related putative alterations in the glutathione system (reduced and oxidized glutathione; redox index) were chronically induced by the administration in drinking water of free radical generators (hydrogen peroxide, ferrous chloride) or of inhibitors of endogenous free radical defenses (diethyl-dithio-carbamate, an inhibitor of superoxide dismutase activity). In hydrogen peroxide administered rats, both reduced glutathione and the cerebral glutathione redox index markedly declined as a function of aging, whereas oxidized glutathione consistently increased. In contrast, chronic iron intake failed to modify the reduced glutathione in forebrain from the rats of the different ages tested, whereas the oxidized glutathione was increased in the older brains. The chronic intake of diethyl-dithio-carbamate enhanced the concentrations of reduced glutathione in the forebrains from the rats of the different ages tested, the oxidized glutathione being unchanged. In 15-month-old rats submitted to chronic oxidative stress, ergot alkaloids (and particularly dihydroergocriptine) interfered with cerebral glutathione system, while papaverine was always ineffective. The comprehensive analysis of the data indicates that: (a) both the type of oxidative stress and the age of the animals modulate the cerebral responsiveness to the putative modifiers in the level of tissue free radicals; (b) aging magnifies the cerebral alterations induced by oxidative stress; the (c) cerebral glutathione system may be modified by metabolic rather than by circulatory interferences; (d) a balance between the various cerebral antioxidant defenses is present, the perturbation of an antioxidant system resulting in the compensatory modified activity of component(s) of another system.     

Oxidative stress and aging: beyond correlation

The oxidative stress theory of aging has become increasingly accepted as playing a role in the aging process, based primarily on a substantial accumulation of circumstantial evidence. In recent years, the hypothesis that mitochondrially generated reactive oxygen species play a role in organismal aging has been directly tested in both invertebrate and mammalian model systems. Initial results imply that oxidative damage, specifically the level of superoxide, does play a role in limiting the lifespans of invertebrates such as Drosophila melanogaster and Caenorhabditis elegans. In mammalian model systems, the effect of oxidative stress on lifespan is less clear, but there is evidence that antioxidant treatment protects against age-related dysfunction, including cognitive decline.     

Tertiary butylhydroquinone alleviates gestational diabetes mellitus in C57BL/KsJ-Lep db/+ mice by suppression of oxidative stress

Gestational diabetes mellitus (GDM) is a common disorder characterized by abnormal glucose metabolism during pregnancy, affecting 2% to 5% of pregnant women. Currently, clinical treatment for GDM is very limited. The present study was designed to investigate the effect and underlying molecular mechanism of tertiary butylhydroquinone (TBHQ) in a pregnant C57BL/KsJ-Lep db/+ (referred to as db+) GDM mouse model. The results showed that nonpregnant db/+ mice did not show a diabetic phenotype, and TBHQ had no effect on glucose and insulin tolerance in these mice. Moreover, in db/+ pregnant mice exhibiting typical diabetes symptoms, such as hyperglycemia and hypoinsulinemia, TBHQ could remarkably decrease the blood glucose level, increase insulin level, and improve glucose and insulin intolerance. The results also revealed that TBHQ could inhibit oxidative stress in pregnant db/+ mice. Furthermore, TBHQ greatly improved offspring survival rate, glucose metabolism, and insulin tolerance. In addition, TBHQ inhibited oxidative stress by reducing malondialdehyde (MDA) and reactive oxygen species (ROS) levels and increased superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities. Moreover, we found that TBHQ activated the nuclear factor erythroid 2-related factor 2 (Nrf2), thereby increasing the levels of Nrf2, and ultimately upregulating the expression of heme oxygenase 1 (NO-1) and superoxide dismutase 2 (SOD2). In conclusion, our findings demonstrated that TBHQ alleviated GDM via Nrf2 activation.     

Effect of fish oil on bone mineral density in aging C57BL/6 female mice

Life expectancy has increased considerably over the last century in the United States. It is expected that this longevity will be accompanied by an increase in the prevalence of osteoporosis and accompanying complications in the elderly population. Age-related loss of bone mass and bone fragility are major risk factors for osteoporosis, leading to an increased risk of fractures. Therefore, nutritional strategies and lifestyle changes that prevent age-related osteoporosis and improve the quality of life for the elderly population are urgently needed. Hence, the present study compared the effects of corn oil (CO; n-6 fatty acids; commonly present in Western diets) and fish oil (FO; n-3 fatty acids) on bone mineral density (BMD) in aging C57BL/6 female mice. After 6 months of dietary treatment, we found that 18-month-old FO-fed mice maintained higher BMD in different bone regions compared to CO-fed mice. These findings were accompanied by a decreased activity of pro-inflammatory cytokines, tumor necrosis factor-alpha and interleukin-6 in stimulated splenocytes; a nonsignificant but greater increase in bone formation markers alkaline phosphatase and osteocalcin in the serum; and lower osteoclast generation in bone marrow cell cultures in FO-fed mice. In conclusion, these findings suggest that providing n-3 fatty acids may have a beneficial effect on bone mass during aging by modulating bone formation and bone resorption factors.     

Generation of free oxygen radicals in heart mitochondria: Effect of hypoxia-reoxygenation

The relationship between the rate of generation of superoxide radicals and the duration of hypoxia has been studied in isolated heart mitochondria with the use of the spin trap sodium 4,5dihydroxybenzene-1,3-disulfonate. The EPR spectra were recorded from a mitochondrial suspension placed in a gas-permeable capillary under conditions of regulated partial oxygen pressure. Earlier we have shown that the mitochondria isolated from perfused hearts after 30-min ischemia display a higher rate of superoxide generation than those from controls. However, in isolated mitochondria the EPR signal from 4,5-dihydroxybenzene-1,3-disulfonate increased already after 10-min hypoxia, but its intensity remained the same in the mitochondria subjected to 30-, 45-, and 60-min hypoxia. Thus, the isolated mitochondria in the incubation medium are less sensitive to hypoxia than the mitochondria from cardiomyocytes of an ischemic heart.     

The protective effect of melatonin against cypermethrin-induced oxidative stress damage in Spodoptera litura (Lepidoptera: Noctuidae)

Antioxidant enzymes form the first-line defense against free radicals damage in organisms. Their regulation depends mainly on the oxidant and antioxidant status of the cell, given that oxidants are their principal modulators. Therefore, the aim of the present study was to investigate the effect of melatonin on synthetic pyrethroid insecticide-induced antioxidative enzymes activity in Spodoptera litura larvae. In addition, activities of enzymatic antioxidants viz. superoxide dismutase (SOD), glutathione S-transferase (GST), catalase (CAT), glutathione reductase (GR), α, β-esterase, and acetylcholine esterase (AChE) were assessed. There was no significant change in GST levels in the melatonin-treated groups. Melatonin modulates cypermethrin-induced changes in the activities of esterase and AChE, whereas SOD, CAT, and GR activity was significantly increased in melatonin-treated samples when compared to control. In conclusion, the results of the current study revealed that SP toxicity activated oxidant systems in all antioxidant systems in some tissues of insects. Melatonin administration led to a marked increase in antioxidant activity and inhibited GST and AChE in most of the tissues studied.     

Oxidative stress in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy affecting adults. The genetic basis of DM1 consists of a mutational expansion of a repetitive trinucleotide sequence (CTG). The number of triplets expansion divides patients in four categories related to the molecular changes (E1, E2, E3, E4). The pathogenic mechanisms of multi-systemic involvement of DM1 are still unclear. DM1 has been suspected to be due to premature aging, that is known to be sustained by increased free radicals levels and/or decreased antioxidants activities in neurodegenerative disorders. Recently, the gain-of-function at RNA level hypothesis has gained great attention, but oxidative stress might act in the disease progression. We have investigated 36 DM1 patients belonging to 22 unrelated families, 10 patients with other myotonic disorders (OMD) and 22 age-matched healthy controls from the clinical, biochemical and molecular point of view. Biochemical analysis detected blood levels of superoxide dismutase (SOD), malonilaldehyde (MDA), vitamin E (Vit E), hydroxyl radicals (OH) and total antioxidant system (TAS). Results revealed that DM1 patients showed significantly higher levels of SOD (+40%; MAL (+57%; RAD 2 (+106%; and TAS (+20%; than normal controls. Our data support the hypothesis of a pathogenic role of oxidative stress in DM1 and therefore confirm the detrimental role played by free radicals in this pathology and suggest the opportunity to undertake clinical trials with antioxidants in this disorder.     

The daily rhythms of mitochondrial gene expression and oxidative stress regulation are altered by aging in the mouse liver

The circadian clock regulates many cellular processes, notably including the cell cycle, metabolism and aging. Mitochondria play essential roles in metabolism and are the major sites of reactive oxygen species (ROS) production in the cell. The clock regulates mitochondrial functions by driving daily changes in NAD⁺ levels and Sirt3 activity. In addition to this central route, in the present study, we find that the expression of some mitochondrial genes is also rhythmic in the liver, and that there rhythms are disrupted by the Clock^Δ19 mutation in young mice, suggesting that they are regulated by the core circadian oscillator. Related to this observation, we also find that the regulation of oxidative stress is rhythmic in the liver. Since mitochondria and ROS play important roles in aging, and mitochondrial functions are also disturbed by aging, these related observations prompt the compelling hypothesis that circadian oscillators influence aging by regulating ROS in mitochondria. During aging, the expression rhythms of some mitochondrial genes were altered in the liver and the temporal regulation over the dynamics of mitochondrial oxidative stress was disrupted. However, the expression of clock genes was not affected. Our results suggested that mitochondrial functions are combinatorially regulated by the clock and other age-dependent mechanism(s), and that aging disrupts mitochondrial rhythms through mechanisms downstream of the clock.     

Cadmium-induced apoptosis in C6 glioma cells: Influence of oxidative stress

Cadmium has recently been shown to induce apoptosis in C6 glioma cells via disruption of the mitochondrial membrane potential and subsequent caspase 9-activation. Here we show that both H₂O₂ and CdCl₂ induced apoptotic DNA fragmentation in C6 cells. The employment of glutathione as an antioxidant prevented the induction of apoptotic DNA fragmentation by cadmium completely and catalase strongly reduced cadmium-induced DNA fragmentation suggesting that cadmium exerts its apoptotic effects at least partly via the production of H₂O₂. Apoptosis may be induced by cadmium indirectly through formation of oxidative stress, e.g., by inhibition of antioxidant enzymes. After incubation of C6 cells with cadmium for short times (up to 4 h), we analyzed the formation of intracellular reactive oxygen species and cellular lipid peroxidation. After 1 h of incubation with inreasing concentrations of CdCl₂ (1–500 M), no increase in dichlorofluorescein fluorescence was found. At variance, lipid peroxidation was slightly elevated after 2 h incubation with cadmium (50–100 M). Furthermore, we analyzed the modulation of markers for oxidative stress after prolonged (24 h) exposure to cadmium. The intracellular glutathione content as measured using the fluorescent probe monobromobimane was decreased after incubation with CdCl₂ (0.5–10 M) for 24 h. Furthermore, we measured the effect of cadmium on the level of oxidized DNA lesions (predominantly 8-hydroxyguanine) using the bacterial Fpg-DNA-repair protein. After 24 h of incubation with 5 M CdCl₂ we found a sixfold increase in Fpg-sensitive DNA-lesions. We conclude that short time incubations with cadmium (up to 4 h) caused only slight or insignificant effects on the generation of reactive oxygen species (formation of thiobarbituric acid reactive substances, fluorescence of dichlorofluorescein), whereas incubation with this heavy metal for 24 h lead to a decrease in intracellular glutathione concentration and an increase in oxidative DNA-lesions. Our data demonstrate that cadmium as similar to H₂O₂ is a potent inducer of apoptosis in C6 cells. Even if cadmium unlike Fenton-type metals can not produce reactive oxygen species directly, the apoptotic effects of cadmium at least in part are mediated via induction of oxidative stress. Because both apoptosis and oxidative stress are thought to play important roles in neurodegenerative diseases, low concentrations of cadmium that initiate programmed cell death may lead to a selective cell death in distinct brain regions via generation of oxidative stress.     

Mitochondrial oxidative stress elicits chromosomal instability after exposure to isocyanates in human kidney epithelial cells

The role of oxidative stress is often attributed in environmental renal diseases. Isocyanates, a ubiquitous chemical group with diverse industrial applications, are known to undergo bio-transformation reactions upon accidental and occupational exposure. This study delineates the role of isocyanate-mediated mitochondrial oxidative stress in eliciting chromosomal instability in cultured human kidney epithelial cells. Cells treated with 0.005 µM concentration of methyl isocyanate displayed morphological transformation and stress-induced senescence. Along the time course, an increase in DCF fluorescence indicative of oxidative stress, depletion of superoxide dismutase (SOD) and glutathione reductase (GR) and consistent accumulation of 8-oxo-dG were noticed. Thus, endogenous oxidative stress resulted in aberrant expression of p53, p21, cyclin E and CDK2 proteins, suggestive of deregulated cell cycle, chromosomal aberrations, centromeric amplification, aneuploidy and genomic instability.     

The free-radical damage theory: Accumulating evidence against a simple link of oxidative stress to ageing and lifespan

Recent work on a small European cave salamander (Proteus anguinus) has revealed that it has exceptional longevity, yet it appears to have unexceptional defences against oxidative damage. This paper comes at the end of a string of other studies that are calling into question the free-radical damage theory of ageing. This theory rose to prominence in the 1990s as the dominant theory for why we age and die. Despite substantial correlative evidence to support it, studies in the last five years have raised doubts over its importance. In particular, these include studies of mice with the major antioxidant genes knocked out (both singly and in combination), which show the expected elevation in oxidative damage but no impact on lifespan. Combined, these findings raise fundamental questions over whether the free-radical damage theory remains useful for understanding the ageing process, and variation in lifespan and life histories.     

Fibroblasts from long-lived mutant mice exhibit increased autophagy and lower TOR activity after nutrient deprivation or oxidative stress

Previous work has shown that primary skin-derived fibroblasts from long-lived pituitary dwarf mutants resist the lethal effects of many forms of oxidative and nonoxidative stress. We hypothesized that increased autophagy may protect fibroblasts of Pit-1(dw/dw) (Snell dwarf) mice from multiple forms of stress. We found that dwarf-derived fibroblasts had higher levels of autophagy, using LC3 and p62 as markers, in response to amino acid deprivation, hydrogen peroxide, and paraquat. Fibroblasts from dwarf mice also showed diminished phosphorylation of mTOR, S6K, and 4EBP1, consistent with the higher levels of autophagy in these cells after stress. Similar results were also observed in fibroblasts from mutant mice lacking growth hormone receptor (GHRKO mice) after amino acid withdrawal. Our results suggested that increased autophagy, regulated by TOR-dependent processes, may contribute to stress resistance in fibroblasts from long-lived mutant mice.