Increased plasma peroxides as a marker of oxidative stress in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)

Background: There is evidence that myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by activation of immune, inflammatory, oxidative and nitrosative stress (IO&NS) pathways. The present study was carried out in order to examine whether ME/CFS is accompanied by increased levels of plasma peroxides and serum oxidized LDL (oxLDL) antibodies, two biomarkers of oxidative stress.

Material/Methods: Blood was collected from 56 patients with ME/CFS and 37 normal volunteers. Severity of ME/CFS was measured using the Fibromyalgia and Chronic Fatigue Syndrome (FF) Rating Scale.

Results: Plasma peroxide concentrations were significantly higher in patients with ME/CFS than in normal controls. There was a trend towards significantly higher serum oxLDL antibodies in ME/CFS than in controls. Both biomarkers contributed significantly in discriminating between patients with ME/CFS and normal controls. Plasma peroxide and serum oxLDL antibody levels were both significantly related to one of the FF symptoms.

Conclusions: The results show that ME/CFS is characterized by increased oxidative stress.

The shared role of oxidative stress and inflammation in major depressive disorder and nicotine dependence

Nicotine dependence is common in people with mood disorders; however the operative pathways are not well understood. This paper reviews the contribution of inflammation and oxidative stress pathways to the co-association of depressive disorder and nicotine dependence, including increased levels of pro-inflammatory cytokines, increased acute phase proteins, decreased levels of antioxidants and increased oxidative stress. These could be some of the potential pathophysiological mechanisms involved in neuroprogression. The shared inflammatory and oxidative stress pathways by which smoking may increase the risk for development of depressive disorders are in part mediated by increased levels of pro-inflammatory cytokines, diverse neurotransmitter systems, activation the hypothalamic-pituitary-adrenal (HPA) axis, microglial activation, increased production of oxidative stress and decreased levels of antioxidants. Depressive disorder and nicotine dependence are additionally linked imbalance between neuroprotective and neurodegenerative metabolites in the kynurenine pathway that contribute to neuroprogression. These pathways provide a mechanistic framework for understanding the interaction between nicotine dependence and depressive disorder.

Antioxidant enzyme activities of iron-saturated bovine lactoferrin (Fe-bLf) in human gut epithelial cells under oxidative stress

Chemoprevention by dietary constituents in the form of functional food has emerged as a novel approach to control inflammatory diseases and cancers. Recently we reported for the first time that iron content is a critical determinant in the anti-tumour activity of bovine milk lactoferrin (bLf). We therefore wanted to evaluate the chemo-preventative efficacy of Apo-bLF and 100% iron-saturated bLF (Fe-bLF) on hydrogen peroxide (H2O 2)-induced colon carcinogenesis, and their influence on antioxidant enzyme activities within colon carcinogenesis. This was undertaken through observing how oxidative stress induced by H2O2 alters antioxidant enzyme activity within HT29 colon cancer cells, and then observing changes in this activity by treatments with the different antioxidants ascorbic acid (AA), Apo-bLF and Fe-bLF. All antioxidant enzymes (catalase, glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-s-transferase (GsT) and superoxide dismutase (SOD)) appeared to be increased within HT29 cells, even prior to H2O2 exposure, and all enzymes showed significant decreased activity when cells were treated with the antioxidants AA, Apo-bLF or Fe-bLF, with or without H2O2 exposure. The results indicate that all three antioxidants have the ability to scavenge ROS, lower antioxidant enzyme activities within already excited states, and possibly allow colon cancer cells to be overcome by oxidative stress that would normally be prevented, perhaps leading to damage and potential apoptosis of the cancer cells. In conclusion, the anti-oxidative effects of Apo-bLF and Fe-bLf studied for the first time, show dynamic changes that may allow for necessary protection from imbalanced oxidative conditions, and potential at reducing the ability of cancer cells to protect themselves from oxidative stress states.

The effect of short-term canola oil ingestion on oxidative stress in the vasculature of stroke-prone spontaneously hypertensive rats

Background: This study aimed to determine if 25 days of canola oil intake in the absence of excess dietary salt or together with salt loading affects antioxidant and oxidative stress markers in the circulation. A further aim was to determine the mRNA expression of NADPH oxidase subunits and superoxide dismutase (SOD) isoforms in the aorta of stroke-prone spontaneously hypertensive (SHRSP) rats.

Methods: Male SHRSP rats, were fed a defatted control diet containing 10% wt/wt soybean oil or a defatted treatment diet containing 10% wt/wt canola oil, and given tap water or water containing 1% NaCl. Blood was collected at the end of study for analysis of red blood cell (RBC) antioxidant enzymes, RBC and plasma malondialdehyde (MDA), plasma 8-isoprostane and plasma lipids. The aorta was removed and the mRNA expression of NOX2, p22phox, CuZn-SOD, Mn-SOD and EC-SOD were determined.

Results: In the absence of salt, canola oil reduced RBC SOD and glutathione peroxidase, and increased total cholesterol and LDL cholesterol compared with soybean oil. RBC glutathione peroxidase activity was significantly lower in both the salt loaded groups compared to the soybean oil only group. In addition, RBC MDA and plasma HDL cholesterol were significantly higher in both the salt loaded groups compared to the no salt groups. Plasma MDA concentration was higher and LDL cholesterol concentration lower in the canola oil group loaded with salt compared to the canola oil group without salt. The mRNA expression of NADPH oxidase subunits and SOD isoforms were significantly reduced in the canola oil group with salt compared to canola oil group without salt.

Conclusion: In conclusion, these results indicate that canola oil reduces antioxidant status and increases plasma lipids, which are risk factors for cardiovascular disease. However, canola oil in combination with salt intake increased MDA, a marker of lipid peroxidation and decreased NAPDH oxidase subunits and aortic SOD gene expression.

Differential effects of dietary canola and soybean oil intake on oxidative stress in stroke-prone spontaneously hypertensive rats

Background: Canola oil shortens the life span of stroke-prone spontaneously hypertensive (SHRSP) rats compared with rats fed soybean oil when given as the sole dietary lipid source. One possible mechanism leading to the damage and deterioration of organs due to canola oil ingestion is oxidative stress. This study investigated the effect of canola oil intake on oxidative stress in this animal model.
Method: Male SHRSP rats, were fed a defatted control diet containing 10% wt/wt soybean oil or a defatted treatment diet containing 10% wt/wt canola oil, and given water containing 1% NaCl. Blood pressure was measured weekly. Blood was collected prior to beginning the diets and at the end of completion of the study for analysis of red blood cell (RBC) antioxidant enzymes, RBC and plasma malondialdehyde (MDA), plasma 8- isoprostane and plasma lipids.
Results: Canola oil ingestion significantly decreased the life span of SHRSP rats compared with soybean oil, 85.8 ± 1.1 and 98.3 ± 3.4 days, respectively. Systolic blood pressure increased over time with a significant difference between the diets at the 6th week of feeding. Canola oil ingestion significantly reduced RBC superoxide dismutase, glutathione peroxidase and catalase activities, total cholesterol and low-density lipoprotein cholesterol compared with soybean oil. There were no significant differences in RBC MDA concentration between canola oil fed and soybean oil fed rats. In contrast, plasma MDA and 8-isoprostane concentration was significantly lower in the canola oil group compared to the soybean oil group.
Conclusion: In conclusion, canola oil ingestion shortens the life span of SHRSP rats and leads to changes in oxidative status, despite an improvement in the plasma lipids.

Effect of selenium-saturated bovine lactoferrin (Se-bLF) on antioxidant enzyme activities in human gut epithelial cells under oxidative stress

Cancer and many chronic inflammatory diseases are associated with increased amounts of reactive oxygen species (ROS). The potential cellular and tissue damage created by ROS has significant impact on many disease and cancer states and natural therapeutics are becoming essential in regulating altered redox states. We have shown recently that iron content is a critical determinant in the antitumour activity of bovine milk lactoferrin (bLF). We found that 100% iron-saturated bLF (Fe-bLF) acts as a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy and thus has a broad utility in the treatment of cancer. Furthermore, we also studied the effects of iron saturated bLF's ability as an antioxidant in the human epithelial colon cancer cell line HT29, giving insights into the potential of bLF in its different states. Thus, metal saturated bLF could be implemented as anti-cancer neutraceutical. In this regard, we have recently been able to prepare a selenium (Se) saturated form of bLF, being up to 98% saturated. Therefore, the objectives of this study were to determine how oxidative stress induced by hydrogen peroxide (H₂O₂) alters antioxidant enzyme activity within HT29 epithelial colon cancer cells, and observe changes in this activity by treatments with different antioxidants ascorbic acid (AA), Apo (iron free)-bLF and selenium (Se)-bLF. The states of all antioxidant enzymes (glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-s-transferase (GsT), catalase and superoxide dismutase (SOD)) demonstrated high levels within untreated HT29 cells compared to the majority of other treatments being used, even prior to H₂O₂ exposure. All enzymes showed significant alterations in activity when cells were treated with antioxidants AA, Apo-bLF or Se-bLF, with and/or without H₂O₂ exposure. Obvious indications that the Se content of the bLF potentially interacted with the glutathione (GSH)/GPx/GR/GsT associated redox system could be observed immediately, showing capability of Se-bLF being highly beneficial in helping to maintain a balance between the oxidant/antioxidant systems within cells and tissues, especially in selenium deficient systems. In conclusion, the antioxidative defence activity of Se-bLf, investigated in this study for the first time, shows dynamic adaptations that may allow for essential protection from the imbalanced oxidative conditions. Because of its lack of toxicity and the availability of both selenium and bLF in whole milk, Se-bLF offers a promise for a prospective natural dietary supplement, in addition to being an immune system enhancement, or a potential chemopreventive agent for cancers.

The influence of genetic background on the induction of oxidative stress and impaired insulin secretion in mouse islets

Aims/hypothesis We determined whether high-glucose-induced beta cell dysfunction is associated with oxidative stress in the DBA/2 mouse, a mouse strain susceptible to islet failure.

Materials and methods Glucose- and non-glucose-mediated insulin secretion from the islets of DBA/2 and control C57BL/6 mice was determined following a 48-h exposure to high glucose. Flux via the hexosamine biosynthesis pathway was assessed by determining O-glycosylated protein levels. Oxidative stress was determined by measuring hydrogen peroxide levels and the expression of anti-oxidant enzymes.

Results Exposure to high glucose levels impaired glucose-stimulated insulin secretion in DBA/2 islets but not C57BL/6 islets, and this was associated with reduced islet insulin content and lower ATP levels than in C57BL/6 islets. Exposure of islets to glucosamine for 48 h mimicked the effects of high glucose on insulin secretion in the DBA/2 islets. High glucose exposure elevated O-glycosylated proteins; however, this occurred in islets from both strains, excluding a role for O-glycosylation in the impairment of DBA/2 insulin secretion. Additionally, both glucosamine and high glucose caused an increase in hydrogen peroxide in DBA/2 islets but not in C57BL/6 islets, an effect prevented by the antioxidant N-acetyl-l-cysteine. Interestingly, while glutathione peroxidase and catalase expression was comparable between the two strains, the antioxidant enzyme manganese superoxide dismutase, which converts superoxide to hydrogen peroxide, was increased in DBA/2 islets, possibly explaining the increase in hydrogen peroxide levels.

Conclusions/interpretation Chronic high glucose culture caused an impairment in glucose-stimulated insulin secretion in DBA/2 islets, which have a genetic predisposition to failure, and this may be the result of oxidative stress.

Oxidative stress is induced by islet amyloid formation and time-dependently mediates amyloid-induced beta cell apoptosis

Aims/hypothesis Islet amyloid in type 2 diabetes contributes to loss of beta cell mass and function. Since islets are susceptible to oxidative stress-induced toxicity, we sought to determine whether islet amyloid formation is associated with induction of oxidative stress.

Methods Human islet amyloid polypeptide transgenic and non-transgenic mouse islets were cultured for 48 or 144 h with or without the antioxidant N-acetyl-l-cysteine (NAC) or the amyloid inhibitor Congo Red. Amyloid deposition, reactive oxygen species (ROS) production, beta cell apoptosis, and insulin secretion, content and mRNA were measured.

Results After 48 h, amyloid deposition was associated with increased ROS levels and increased beta cell apoptosis, but no change in insulin secretion, content or mRNA levels. Antioxidant treatment prevented the rise in ROS, but did not prevent amyloid formation or beta cell apoptosis. In contrast, inhibition of amyloid formation prevented the induction of oxidative stress and beta cell apoptosis. After 144 h, amyloid deposition was further increased and was associated with increased ROS levels, increased beta cell apoptosis and decreased insulin content. At this time-point, antioxidant treatment and inhibition of amyloid formation were effective in reducing ROS levels, amyloid formation and beta cell apoptosis. Inhibition of amyloid formation also increased insulin content.

Conclusions/interpretation Islet amyloid formation induces oxidative stress, which in the short term does not mediate beta cell apoptosis, but in the longer term may feed back to further exacerbate amyloid formation and contribute to beta cell apoptosis.

Ascorbic acid supplementation improves skeletal muscle oxidative stress and insulin sensitivity in people with type 2 diabetes: findings of a randomized controlled study

AIM/HYPOTHESIS: Skeletal muscle insulin resistance and oxidative stress are characteristic metabolic disturbances in people with type 2 diabetes. Studies in insulin resistant rodents show an improvement in skeletal muscle insulin sensitivity and oxidative stress following antioxidant supplementation. We therefore investigated the potential ameliorative effects of antioxidant ascorbic acid (AA) supplementation on skeletal muscle insulin sensitivity and oxidative stress in people with type 2 diabetes. METHODS: Participants with stable glucose control commenced a randomized cross-over study involving four months of AA (2×500mg/day) or placebo supplementation. Insulin sensitivity was assessed using a hyperinsulinaemic, euglycaemic clamp coupled with infusion of 6,6-D2 glucose. Muscle biopsies were measured for AA concentration and oxidative stress markers that included basal measures (2',7'-dichlorofluorescin [DCFH] oxidation, ratio of reduced-to-oxidized glutathione [GSH/GSSG] and F2-Isoprostanes) and insulin-stimulated measures (DCFH oxidation). Antioxidant concentrations, citrate synthase activity and protein abundances of sodium-dependent vitamin C transporter 2 (SVCT2), total Akt and phosphorylated Akt (ser473) were also measured in muscle samples. RESULTS: AA supplementation significantly increased insulin-mediated glucose disposal (delta rate of glucose disappearance; ∆Rd) (p=0.009), peripheral insulin-sensitivity index (p=0.046), skeletal muscle AA concentration (p=0.017) and muscle SVCT2 protein expression (p=0.008); but significantly decreased skeletal muscle DCFH oxidation during hyperinsulinaemia (p=0.007) when compared with placebo. Total superoxide dismutase activity was also lower following AA supplementation when compared with placebo (p=0.006). Basal oxidative stress markers, citrate synthase activity, endogenous glucose production, HbA1C and muscle Akt expression were not significantly altered by AA supplementation. CONCLUSIONS/INTERPRETATION: In summary, oral AA supplementation ameliorates skeletal muscle oxidative stress during hyperinsulinaemia and improves insulin-mediated glucose disposal in people with type 2 diabetes. Findings implicate AA supplementation as a potentially inexpensive, convenient, and effective adjunct therapy in the treatment of insulin resistance in people with type 2 diabetes.

Regulation of redox and DNA repair genes by arsenic : low dose protection against oxidative stress?

We have evaluated the molecular responses of human epithelial cells to low dose arsenic to ascertain how target cells may respond to physiologically relevant concentrations of arsenic. Data gathered in numerous experiments in different cell types all point to the same conclusion: low dose arsenic induces what appears to be a protective response against subsequent exposure to oxidative stress or DNA damage, whereas higher doses often provoke synergistic toxicity. In particular, exposure to low, sub-toxic doses of arsenite, As(III), causes coordinate up-regulation of multiple redox and redox-related genes including thioredoxin (Trx) and glutathione reductase (GR). Glutathione peroxidase (GPx) is down-regulated in fibroblasts, but up-regulated in keratinocytes, as is glutathione S-transferase (GST). The maximum effect on these redox genes occurs after 24 h exposure to 5–10 mM As(III). This is 10-fold higher than the maximum As(III) concentrations required for induction of DNA repair genes, but within the dose region where DNA repair genes are co-ordinately down-regulated. These changes in gene regulation are brought about in part by changes in DNA binding activity of the transcription factors activating protein-1 (AP-1), nuclear factor kappa-B, and cAMP response element binding protein (CREB). Although sub-acute exposure to micromolar As(III) up-regulates transcription factor binding, chronic exposure to submicromolar As(III) causes persistent down-regulation of this response. Similar long-term exposure to micromolar concentrations of arsenate in drinking water results in a decrease in skin tumour formation in dimethylbenzanthracene (DMBA)/phorbol 12-tetradecanoate 13-acetate (TPA) treated mice. Altered response patterns after long exposure to As(III) may play a significant role in As(III) toxicology in ways that may not be predicted by experimental protocols using short-term exposures.

Red wine consumption increases antioxidant status and decreases oxidative stress in the circulation of both young and old humans

Background: Red wine contains a naturally rich source of antioxidants, which may protect the body from oxidative stress, a determinant of age-related disease. The current study set out to determine the in vivo effects of moderate red wine consumption on antioxidant status and oxidative stress in the circulation.
Methods: 20 young (18–30 yrs) and 20 older (≥ 50 yrs) volunteers were recruited. Each age group was randomly divided into treatment subjects who consumed 400 mL/day of red wine for two weeks, or control subjects who abstained from alcohol for two weeks, after which they crossed over into the other group. Blood samples were collected before and after red wine consumption and were used for analysis of whole blood glutathione (GSH), plasma malondialdehyde (MDA) and serum total antioxidant status.
Results: Results from this study show consumption of red wine induced significant increases in plasma total antioxidant status (P < 0.03), and significant decreases in plasma MDA (P < 0.001) and GSH (P < 0.004) in young and old subjects. The results show that the consumption of 400 mL/day of red wine for two weeks, significantly increases antioxidant status and decreases oxidative stress in the circulation.
Conclusion: It may be implied from this data that red wine provides general oxidative protection and to lipid systems in circulation via the increase in antioxidant status.

CoQ10 conveys protection from oxidative stress in plasma but not skeletal muscle

Coenzyme Q10 (CoQ10) is commonly consumed as an antiaging supplement at doses of 30–210 mg/day. The aim of the study was to determine if CoQ10 alters markers of antioxidant status, oxidative damage, and gene expression in aging skeletal muscle. Female guinea pigs aged 26 months were supplemented for 6 weeks with CoQ10 at a human equivalent dose of 10 mg/kg/day. Body weight, plasma CoQ10 concentration, and WBC DNA abasic sites were measured at weeks 0, 2, 4, and 6 of the supplementation period. At the end of supplementation, concentrations of skeletal muscle CoQ10, glutathione, malondialdehyde, protein carbonyls, DNA abasic sites, activities of catalase and glutathione peroxidase, and the gene expression of cyctochrome c oxidase subunits were measured. Dietary supplementation with CoQ10 elevated plasma CoQ10 levels (pre 73 ± 3 nmol/L, post 581 ± 15 nmol/L, P < 0.05) and decreased abasic sites in WBC DNA (pre 16.8 ± 0.5 Ap/100000 bp, post 9.7 ± 0.4 Ap/100000 bp, P < 0.05). In contrast, all of the measures made in skeletal muscle were not different between groups (P > 0.05). These results indicate that dietary supplementation with CoQ10 at a dose of 10 mg/kg/day may be capable of increasing antioxidant protection and reducing oxidative damage in the plasma, but may have no effect in skeletal muscle.

Oxidative stress : emerging mitochondrial and cellular themes and variations in neuronal injury

Oxidative stress plays a central role in neuronal injury and cell death in acute and chronic pathological conditions. The cellular responses to oxidative stress embrace changes in mitochondria and other organelles, notably endoplasmic reticulum, and can lead to a number of cell death paradigms, which cover a spectrum from apoptosis to necrosis and include autophagy. In Alzheimer's disease, and other pathologies including Parkinson's disease, protein aggregation provides further cellular stresses that can initiate or feed into the pathways to cell death engendered by oxidative stress. Specific attention is paid here to mitochondrial dysfunction and programmed cell death, and the diverse modes of cell death mediated by mitochondria under oxidative stress. Novel insights into cellular responses to neuronal oxidative stress from a range of different stressors can be gained by detailed transcriptomics analyses. Such studies at the cellular level provide the key for understanding the molecular and cellular pathways whereby neurons respond to oxidative stress and undergo injury and death. These considerations underpin the development of detailed knowledge in more complex integrated systems, up to the intact human bearing the neuropathology, facilitating therapeutic advances.