The effects of pre-weaning undernutrition on the expression levels of free radical deactivating enzymes in the mouse brain

A mild degree of undernutrition brought about by restricting the amount of food in the diet is known to alter the life span of an animal. It has been hypothesised that this may be related to the effects of undernutrition on an animals anti-oxidant defense system. We have therefore, used real-time PCR (rt-PCR) techniques to determine the levels of mRNA expression for manganese superoxide dismutase (MnSOD), copper/zinc superoxide dismutase (Cu/ZnSOD), glutathione peroxidase 1 (GPx 1) and catalase in the brains of Quackenbush mice undernourished from conception until 21-post-natal days of age. It was found that 21- and 61-day-old undernourished mice had a deficit in the expression of Cu/ZnSOD in both the cerebellum and forebrain regions compared to age-matched controls. The expression of MnSOD was found to be greater in the cerebellum, but not the forebrain region, of 21-day-old undernourished mice. There were no significant differences in the expression of GPx 1 and catalase between control and undernourished or previously undernourished mice. Our results confirm that undernutrition during the early life of a mouse may disrupt some of the enzymes involved in the anti-oxidant defense systems.

Cyclosporine A-induced changes to erythrocyte redox balance is time course-dependent

Cyclosporine A-treated transplant recipients develop pronounced cardiovascular disease and have increased oxidative stress and altered antioxidant capacity in erythrocytes and plasma. These experiments investigated the time-course of cyclosporine A-induced changes to redox balance in plasma and erythrocytes. Rats were randomly assigned to either a control or cyclosporine A-treated group. Treatment animals received 25 mg/kg of cyclosporine A via intraperitoneal injection for either 7 days or a single dose. Control rats were injected with the same volume of the vehicle. Three hours after the final injections, plasma was analysed for total antioxidant status, a-tocopherol, malondialdehyde, and creatinine. Erythrocytes were analysed for reduced glutathione (GSH), alpha-tocopherol, methaemoglobin, malondialdehyde, and the activities of superoxide dismutase, catalase, GSH peroxidase, and glucose-6-phosphate dehydrogenase (G6PD). Cyclosporine A administration for 7 days resulted in a significant increase (P < 0.05) in plasma malondialdehyde, methaemoglobin, and superoxide dismutase and catalase activities. There was a significant decrease (P < 0.05) in erythrocyte GSH concentration and G6PD activity in cyclosporine A animals. There were no significant differences (P > 0.05) between groups following a single dose of cyclosporine A in any of the measures. In summary, cyclosporine A alters erythrocyte redox balance after 7 days administration, but not after a single dose.

Cyclosporine A induced changes to plasma and erythrocyte antioxidant defences

Organ transplant recipients develop pronounced cardiovascular disease, and decreased antioxidant capacity in plasma and erythrocytes is associated with the pathogenesis of this disease. These experiments tested the hypothesis that the immunosuppressant cyclosporine A (CsA) alters erythrocyte redox balance and reduces plasma antioxidant capacity. Female Sprague-Dawley rats were randomly assigned to a control or CsA treated group. Treatment animals received 25 mg/kg/day of CsA via intraperitoneal injection for 18 days. Control rats were injected with the same volume of the vehicle. Three hours after the final CsA injection, rats were exsanguinated and plasma analysed for total antioxidant status (TAS), alpha-tocopherol, malondialdehyde (MDA), and creatinine. Erythrocytes were analysed for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glucose-6-phosphate dehydrogenase (G6PD) activities, alpha-tocopherol, and MDA. CsA administration resulted in a significant (P < 0.05) decrease in plasma TAS and significant increases (P < 0.05) in plasma creatinine and MDA. Erythrocyte CAT was significantly (P < 0.05) increased in CsA treated rats compared to controls. There were no significant differences (P > 0.05) in erythrocyte SOD, GPX, G6PD, alpha-tocopherol or MDA between groups. In summary, CsA alters erythrocyte antioxidant defence and decreases plasma total antioxidant capacity.

Acute exposure to cyclosporine does not increase plasma homocysteine in rats

There is interest in the postulate that cyclosporine a (CsA) contributes to the elevated homocysteine levels seen in organ transplant recipients, as hyperhomocysteinemia is now considered an independent risk factor for cardiovascular disease (CVD) and may partially explain the increased prevalence of CVD in this population. The main purpose of this investigation was to determine the effect of CsA administration on plasma homocysteine. Eighteen female Sprague Dawley rats (4 months old) were randomly assigned to either a treatment or a control group. For 18 days the treatment group received of CsA (25 mg/kg/d) while the control group received the same volume of the vehicle. Blood samples obtained following sacrifice to measure CsA, total homocysteine, and plasma creatinine. There were no significant differences in plasma homocysteine (mean values SD: treatment = 4.79 +/- 0.63 mu mol/L, control = 4.46 +/- 0.75 mu mol/L; P = .37). Homocysteine was not significantly correlated with final CsA concentrations (r = .17; P = .69). There was a significant difference in plasma creatinine values between the two groups (treatment = 60.44 +/- 7.68 mu mol/L, control = 46.33 +/- 1.66 mu mol/L; P < .001). Furthermore, plasma homocysteine and creatinine were positively correlated with the treatment group (r = .73; P < .05) but not the controls (r = -.10; P = .81). In conclusion, CsA does not influence plasma homocysteine concentrations in rats.

Protein adduct species in muscle and liver of rats following acute ethanol administration

Aims: Previous immunohistochemical studies have shown that the post-translational formation of aldehyde-protein adducts may be an important process in the aetiology of alcohol-induced muscle disease. However, other studies have shown that in a variety of tissues, alcohol induces the formation of various other adduct species, including hybrid acetaldehyde-malondialdehyde-protein adducts and adducts with free radicals themselves, e.g. hydroxyethyl radical (HER)-protein adducts. Furthermore, acetaldehyde-protein adducts may be formed in reducing or non-reducing environments resulting in distinct molecular entities, each with unique features of stability and immunogenicity. Some in vitro studies have also suggested that unreduced adducts may be converted to reduced adducts in situ. Our objective was to test the hypothesis that in muscle a variety of different adduct species are formed after acute alcohol exposure and that unreduced adducts predominate. Methods: Rabbit polyclonal antibodies were raised against unreduced and reduced aldehydes and the HER-protein adducts. These were used to assay different adduct species in soleus (type I fibre-predominant) and plantaris (type II fibre-predominant) muscles and liver in four groups of rats administered acutely with either [A] saline (control); [B] cyanamide (an aldehyde dehydrogenase inhibitor); [C] ethanol; [D] cyanamide+ethanol. Results: Amounts of unreduced acetaldehyde and malondialdehyde adducts were increased in both muscles of alcohol-dosed rats. However there was no increase in the amounts of reduced acetaldehyde adducts, as detected by both the rabbit polyclonal antibody and the RT1.1 mouse monoclonal antibody. Furthermore, there was no detectable increase in malondialdehyde-acetaldehyde and HER-protein adducts. Similar results were obtained in the liver. Conclusions: Adducts formed in skeletal muscle and liver of rats exposed acutely to ethanol are mainly unreduced acetaldehyde and malondialdehyde species.

alpha-tocopherol and alpha-lipoic acid enhance the erythrocyte antioxidant defence in cyclosporine A-treated rats

The aim of this study was to determine the effects of dietary antioxidant supplementation with alpha-tocopherol and alpha-lipoic acid on cyclosporine A (cyclosporine)-induced alterations to erythrocyte and plasma redox balance. Rats were randomly assigned to either control, antioxidant (alpha-tocopherol 1000 IU/kg diet and alpha-lipoic acid 1.6 g/kg diet), cyclosporine (25 mg/kg/day), or cyclosporine + antioxidant treatments. Cyclosporine was administered for 7 days after an 8 week feeding period. Plasma was analysed for alpha-tocopherol, total antioxidant capacity, malondialdehyde, and creatinine. Erythrocytes were analysed for glutathione, methaemoglobin, superoxide dismutase, catalase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, alpha-tocopherol and malondialdehye. Cyclosporine administration caused a significant decrease in superoxide dismutase activity (P < 0.05 control versus cyclosporine) and this was improved by antioxidant supplementation (P < 0.05 cyclosporine versus cyclosporine + antioxidant; P < 0.05 control versus cyclosporine + antioxidant). Animals receiving cyclosporine and antioxidants showed significantly increased (P < 0.05) catalase activity compared to both groups not receiving cyclosporine. Cyclosporine administration induced significant increases in plasma malondialdehyde and creatinine concentration (P < 0.05 control versus cyclosporine). Antioxidant supplementation prevented the cyclosporine induced increase in plasma creatinine (P < 0.05 cyclosporine versus cyclosporine + antioxidant; P > 0.05 control versus cyclosporine + antioxidant), however, supplementation did not alter the cyclosporine induced increase in plasma malondialdehyde concentration (P > 0.05 cyclosporine versus cyclosporine + antioxidant). Antioxidant supplementation resulted in significant increases (P < 0.05) in plasma and erythrocyte alpha-tocopherol in both of the supplemented groups compared to non-supplemented groups. In conclusion, dietary supplementation with alpha-tocopherol and alpha-lipoic acid enhanced the erythrocyte antioxidant defence and reduced nephrotoxicity in cyclosporine treated animals.

Antioxidant requirements of endurance athletes: Implications for health

This review discusses studies investigating the effects of antioxidant supplementation on exercise-induced oxidative stress with a focus on the health implications. The aim is to determine antioxidant requirements for endurance athletes. Overall, differences in methodology make it difficult to compare the relatively small number of published studies on this topic. The types of studies needed to more adequately assess the health effects of antioxidant supplements in athletes (long-term interventions with hard end points) have not been done. Therefore, there is currently insufficient evidence to recommend antioxidant supplements for endurance athletes.

Antioxidant supplementation enhances erythrocyte antioxidant status and attenuates cyclosporine-induced vascular dysfunction

The aim of this study was to determine the effects of dietary antioxidant supplementation with a-tocopherol and a-lipoic acid on cyclosporine-induced alterations to erythrocyte and plasma redox balance, and cyclosporine-induced endothelial and smooth muscle dysfunction. Rats were randomly assigned to either control, antioxidant, cyclosporine or cyclosporine + antioxidant treatments. Cyclosporine A was administered for 10 days after an 8-week feeding period. Plasma was analyzed for alpha-tocopherol, total antioxidant capacity, malondialdehyde and creatinine. Erythrocytes were analyzed for glutathione, methemoglobin, superoxide dismutase, catalase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, alpha-tocopherol and malondialdehye. Vascular endothelial and smooth muscle function was determined in vitro. Antioxidant supplementation resulted in significant increases in erythrocyte a-tocopherol concentration and glutathione peroxidase activity in both of the antioxidant-supplemented groups. Cyclosporine administration caused significant decreases in glutathione concentration, methemoglobin concentration and superoxide dismutase activity. Antioxidant supplementation attenuated the cyclosporine-induced decrease in superoxide dismutase activity. Cyclosporine therapy impaired both endothelium-independent and -dependent relaxation of the thoracic aorta, and this was attenuated by antioxidant supplementation. In summary, dietary supplementation with alpha-tocopherol and alpha-lipoic acid attenuated the cyclosporine-induced decrease in erythrocyte superoxide dismutase activity and attenuated cyclosporine-induced vascular dysfunction.

Ultra-endurance exercise and oxidative damage: Implications for cardiovascular health

At least 30 minutes of moderate-intensity physical activity accumulated on most, preferably all days is considered the minimum level necessary to reduce the risk of developing cardiovascular disease. Despite an unclear explanation, some epidemiological data paradoxically suggest that a very high volume of exercise is associated with a decrease in cardiovascular health. Although ultra-endurance exercise training has been shown to increase antioxidant defences (and therefore confer a protective effect against oxidative stress), an increase in oxidative stress may contribute to the development of atherosclerosis via oxidative modification of low-density lipoprotein (LDL). Research has also shown that ultra-endurance exercise is associated with acute cardiac dysfunction and injury, and these may also be related to an increase in free radical production. Longitudinal studies are needed to assess whether antioxidant defences are adequate to prevent LDL oxidation that may occur as a result of increased free radical production during very high volumes of exercise. In addition, this work will assist in understanding the accrued effect of repeated ultra-endurance exercise-induced myocardial damage.

Vitamin E and alpha-lipoic acid supplementation increase bleeding tendency via an intrinsic coagulation pathway

Vitamin E and a-lipoic acid are potent nutritional antioxidants, and when used together, their antioxidant capabilities are improved as a-lipoic acid recycles vitamin E. Supplementation of vitamin E has been shown to prolong platelet aggregation but the effects of vitamin E and alpha-lipoic acid supplementation on bleeding tendency have yet to be reported. Young, male rats consumed either control diet (n=5) or vitamin E and a-lipoic acid-supplemented diet (n=5) for 14 weeks. Activated partial thromboplastin time (APTT) and prothrombin time (PT) were measured as markers of intrinsic and extrinsic coagulation pathways respectively in addition to lipid peroxidation (malondialdehyde). Supplementation significantly prolonged APTT (23.8 +/- 1.5 vs 31.4 +/- 1.2s, p < 0.05) compared to the con-trol diet; however, there was no significant difference in PT (27.8 +/- 1.5 vs 26.6 +/- 0.9s, p > 0.05). While vitamin E was increased (p < 0.05), there was no significant difference in plasma levels of malondialdehyde (p > 0.05). Dietary supplementation of vitamin E and alpha-lipoic acid increases bleeding tendency via inhibition of the intrinsic coagulation pathway with no change in markers of lipid peroxidation. Such supplementation could benefit patients with cardiovascular disease who exhibit elevated levels of coagulation and oxidative stress.

The influence of antioxidant supplementation on markers of inflammation and the relationship to oxidative stress after exercise

Interest in the relationship between inflammation and oxidative stress has increased dramatically in recent years, not only within the clinical setting but also in the fields of exercise biochemistry and immunology. Inflammation and oxidative stress share a common role in the etiology of a variety Of Chronic diseases. During exercise, inflammation and oxidative stress are linked via muscle metabolism and muscle damage. Because oxidative stress and inflammation have traditionally been associated with fatigue and impaired recovery from exercise, research has focused on nutritional strategies aimed at reducing these effects. In this review, we have evaluated the findings of studies involving antioxidant supplementation on alterations in markers of inflammation (e.g., cytokines, C-reactive protein and cortisol). This review focuses predominantly on the role of reactive oxygen and nitrogen species generated from muscle metabolism and muscle damage during exercise and on the modulatory effects of antioxidant supplements. Furthermore, we have analyzed the influence of factors such as the dose, timing, supplementation period and bioavailability of antioxidant nutrients. (C) 2007 Elsevier Inc. All rights reserved.

Ceramides reduce CD36 cell surface expression and oxidised LDL uptake by monocytes and macrophages

Oxidised LDL accumulates in macrophages following scavenger receptor (SR) uptake. The expression of the SR, CD36, is increased by oxidised LDL. The signalling molecule, ceramide, can modulate intracellular peroxides and increase lipid peroxidation. Ceramide also accumulates in atherosclerotic plaques. Thus, we have examined whether ceramide can modulate CD36 expression and function in human monocyte/macrophages. Addition of synthetic short chain ceramides or the action of sphingomyelinase to generate physiological long chain ceramides in situ caused significant reductions in CD36 expression by monocytes/macrophages which was not due to inhibition of mRNA expression. Inhibition of proteasomal degradation using lactacystin had no effect on CD36 expression, however, flow cytometric analysis of permeabilised cells suggested an intracellular trafficking blockade. Ceramide treated monocytes/macrophages showed dose dependent reduction in oxidised LDL uptake. Taken together, it is suggested that ceramide blocks the transport of CD36 to the membrane of monocytes/macrophages, thereby preventing uptake of oxidised LDL. © 2006 Elsevier Inc. All rights reserved.

Biomarkers

Introduction – Why do we need ‘biomarkers? Biomarkers of protein oxidation Introduction Major issues/questions Protein carbonyl biomarkers Biochemistry Methods of measurement Storage, stability and limitations in use Protein thiol biomarkers Biochemistry Methods of measurement Storage, stability and limitations on use Aliphatic amino acid biomarkers Biochemistry Methods of measurement Storage, stability and limitations on use Oxidised Tryptophan Biomarkers Biochemistry Method of measurement Storage, stability and limitations on use Oxidised tyrosine biomarkers Biochemistry Methods of measurement Storage, stability and limitations on use Formation of neoepitopes on oxidised proteins Validation of assays for protein oxidation biomarkers Relationship of protein oxidation to disease Modulation of protein oxidation biomarkers by antioxidants Future perspectives Introduction to lipid peroxidation biomarkers Introduction: biochemistry of lipid peroxidation Malondialdehyde Methods of measurement Storage, stability and limitations on use Conjugated dienes Method of measurement Storage, stability and limitations of use LDL lag phase Method of measurement Storage, stability and limitations of use Hydrocarbon gases Biochemistry Method of measurement Storage, stability and limitations on use Lipofuscin Biochemistry Method of measurement Storage, stability and limitation on use Lipid peroxides Biochemistry Method of measurement Storage, stability and limitations on use Isoprostanes Biochemistry Method of measurement Storage, stability and limitations on use Possible new biomarkers of lipid oxidation Relationship of lipid peroxidation to disease Modulation of lipid peroxidation biomarkers by antioxidants Functional consequences of lipid peroxidation Contribution of dietary intake to lipid peroxidation products Biomarkers of DNA oxidation Introduction Confounding factors Units and terminology Nuclear and mitochondrial DNA damage Lymphocytes as surrogate tissues Measurement of DNA damage with the comet assay Practical details Storage, stability, and limitations of the assay Measurement of DNA base oxidation by HPLC Practical details Storage, stability and limitations of the method Measurement of DNA base oxidation by GC–MS Biochemistry of 8-oxoguanine, adenine and fapy derivatives Methods of measurement Storage, stability and limitations of the method Analysis of guanine oxidation products in urine Method of measurement Limitations and criticisms Immunochemical methods Methods of measurement Storage, stability, and limitations of the assay 32P post-labelling Method of measurement Limitations and criticisms Validation of assays for DNA oxidation Oxo-dGuo in lymphocyte DNA Urinary measurements DNA–aldehyde adducts Biochemistry Method of measurement Products of reactive nitrogen species Endpoints arising from oxidative DNA damage Mutations Chromosome aberrations Micronuclei Site-specific DNA damage Relationship of DNA oxidation to disease Modulation of DNA oxidation biomarkers by antioxidants Direct and indirect effects of oxidative stress: measures of total oxidant/antioxidant levels Visualisation of cellular oxidants Biochemistry: histochemical detection of ROS Method of measurement Limitations, storage and stability Measurement of hydrogen peroxide Biochemistry Methods of measurement Storage, stability and limitations on use Measurement of the ratio of antioxidant/oxidised antioxidant Biochemistry Method of measurement Storage, stability and limitations on use Total antioxidant capacity Biochemistry Terminology Methods of measurement Storage, stability and limitations on use Validation of assays for direct oxidant and antioxidant biomarkers Relationship of oxidant/antioxidant measurement to disease Modulation of oxidant/antioxidant biomarkers by dietary antioxidants Induction of genes in response to oxidative stress Background Measurement of antioxidant responsive genes and proteins Effects of antioxidant intake on the activity of antioxidant enzymes

Effects of oral vitamin C on monocyte:Endothelial cell adhesion in healthy subjects

Monocyte recruitment and retention in the vasculature is influenced by oxidative stress and is involved in cardiovascular disease (CVD). Individuals with low plasma ascorbate are at elevated risk of CVD. It is unknown whether vitamin C supplementation affects monocyte adhesion to endothelial cells (ECs) in healthy non-smokers. In a randomised double-blind crossover study the effect of vitamin C supplementation (six weeks, 250 mg/day) was determined in subjects with normal (HIC) and below average (LOC) plasma vitamin C concentration at baseline (mean = 67μM, n = 20, mean = 32μM, n = 20, respectively). LOC subjects showed 30% greater monocyte adhesion to ECs. This was significantly reduced by 37% (P < 0.02) following vitamin C supplementation to levels of HIC monocyte adhesion. No differences in plasma malondialdehyde concentrations were observed between groups or after supplementation. In conclusion, vitamin C supplementation normalises monocyte adhesion in subjects with low plasma vitamin C (LOC). This process may be related to a direct effect on monocytes, independent of lipid peroxidation. © 2002 Elsevier Science (USA). All rights reserved.