Cadmium chloride-induced oxidative stress in skeletal muscle cells in vitro

The effects of cadmium chloride (CdCl(2)) on oxidative stress in the skeletal muscle cell line C(2)C(12) were investigated. Myoblast cells that differentiated into myotubes were treated with CdCl(2) (1, 3, 5, 7.5, 10, and 12.5 microM) for 24, 48, and 72 h. Subsequent assay of cell homogenates for MTT (3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide) reduction, neutral red uptake and nucleic acid content showed that cadmium was toxic to C(2)C(12) cells in a concentration-dependent manner. Glutathione-S-transferase activity (nmol microg of protein(-1) min(-1)) was increased with 1 and 3 microM CdCl(2) (36.9 +/- 5.6 and 32.1 +/- 6.0, respectively) compared to control cells (21.8 +/- 1.5), but decreased at higher concentrations (7.5 microM = 15.9 +/- 3.3, 10 microM = 15.9 +/- 4.6, and 12.5 microM = 10.5 +/- 2.8). An increase in malondialdehyde content (nmol microg of protein(-1)), especially at high CdCl(2) concentrations (control = 7.3 +/- 0.5; CdCl(2): 7.5 microM = 11.2 +/- 3.1, 10 microM = 14.6 +/- 3.8, and 12.5 microM = 20.5 +/- 6.5) indicated that there was enhanced lipid peroxidation. Light and scanning electron microscopy showed that there was a concentration-dependent loss of adherent cells and the formation of vesicles indicative of cell death. These results indicated that CdCl(2) increased oxidative stress in C(2)C(12) cells, and this stress probably compromised cell adhesion and the cellular antioxidant defense mechanisms.     

New method to study oxidative damage and antioxidants in the human small bowel: effects of iron application

Iron may induce oxidative damage to the intestinal mucosa by its catalyzing role in the formation of highly reactive hydroxyl radicals. This study aimed to determine iron-induced oxidative damage provoked by a single clinical dosage of ferrous sulfate and to elucidate the antioxidant defense mechanisms in the human small intestine in vivo. A double-lumen perfusion tube was positioned orogastrically into a 40-cm segment of the proximal small intestine in six healthy volunteers (25 +/- 5 yr). The segment was perfused with saline and subsequently with saline containing 80 mg iron as ferrous sulfate at a rate of 10 ml/min. Intestinal fluid samples were collected at 15-min intervals. Thiobarbituric acid reactive substances concentrations as an indicator of lipid peroxidation increased significantly from 0.07 microM (range, 0-0.33 microM) during saline perfusion to 3.35 microM (range, 1.19-7.27 microM) during iron perfusion (P < 0.05). Nonprotein antioxidant capacity increased significantly from 474 microM (range, 162-748 microM) to 1,314 microM (range, 674-1,542 microM) (P < 0.05). These data show that a single dosage of ferrous sulfate induces oxidative damage and the subsequent release of an antioxidant in the small intestine in vivo in healthy volunteers.     

Molecular effects of fermented papaya preparation on oxidative damage, MAP Kinase activation and modulation of the benzo[a]pyrene mediated genotoxicity

The involvement of oxidative and nitrosative stress mechanisms in several biological and pathological processes including aging, cancer, cardiovascular and neurodegenerative diseases has continued to fuel suggestions that processes can potentially be modulated by treatment with free-radical scavengers and antioxidant. The fermented papaya preparation (FPP) derived from Carica papaya Linn was investigated for its ability to modulate oxidative DNA damage due to H2O2 in rat pheochromocytoma (PC12) cells and protection of brain oxidative damage in hypertensive rats. Cells pre-treated with FPP (50 microg/ml) prior to incubation with H2O2 had significantly increased viability and sustenance of morphology and shape. The human hepatoma (HepG2) cells exposed to H2O2 (50 microM) showed an olive tail moment of 10.56 +/- 1.44 compared to 1.37 +/- 0.29 of the solvent control. A significant reduction (P < or = 0.05) of DNA damage was observed at concentrations > or = 10 microg/ml FPP, with 50 microg/ml FPP reducing the genotoxic effect of H2O2 by about 1.5-fold compared to only H2O2 exposed cells.     

Moderate endurance training prevents doxorubicin-induced in vivo mitochondriopathy and reduces the development of cardiac apoptosis

The objective of this work was to test the hypothesis that endurance training may be protective against in vivo doxorubicin (DOX)-induced cardiomyopathy through mitochondria-mediated mechanisms. Forty adult (6-8 wk old) male Wistar rats were randomly divided into four groups (n = 10/group): nontrained, nontrained + DOX treatment (20 mg/kg), trained (14 wk of endurance treadmill running, 60-90 min/day), and trained + DOX treatment. Mitochondrial respiration, calcium tolerance, oxidative damage, heat shock proteins (HSPs), antioxidant enzyme activity, and apoptosis markers were evaluated. DOX induces mitochondrial respiratory dysfunction, oxidative damage, and histopathological lesions and triggers apoptosis (P < 0.05, n = 10). However, training limited the decrease in state 3 respiration, respiratory control ratio (RCR), uncoupled respiration, aconitase activity, and protein sulfhydryl content caused by DOX treatment and prevented the increased sensitivity to calcium in nontrained + DOX-treated rats (P < 0.05, n = 10). Moreover, training inhibited the DOX-induced increase in mitochondrial protein carbonyl groups, malondialdehyde, Bax, Bax-to-Bcl-2 ratio, and tissue caspase-3 activity (P < 0.05, n = 10). Training also increased by approximately 2-fold the expression of mitochondrial HSP-60 and tissue HSP-70 (P < 0.05, n = 10) and by approximately 1.5-fold the activity of mitochondrial and cytosolic forms of SOD (P < 0.05, n = 10). We conclude that endurance training protects heart mitochondrial respiratory function from the toxic effects of DOX, probably by improving mitochondrial and cell defense systems and reducing cell oxidative stress. In addition, endurance training limited the DOX-triggered apoptosis.     

Simvastatin restores endothelial NO-mediated vasorelaxation in large arteries after myocardial infarction

Congestive heart failure (CHF) after myocardial infarction is associated with diminished endothelial nitric oxide (NO)-mediated vasorelaxation. The 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors have been shown to modulate vascular tone independent of the effects on lipid lowering. We hypothesized that simvastatin restores NO-dependent vasorelaxation with CHF. We found that incubation of the normal rat aorta with 0.1 mM simvastatin for 24 h enhanced ACh-mediated vasorelaxation (P < 0.05). Moreover, simvastatin increased (P < 0.05) endothelial NO synthase (eNOS) protein content by >200% (82.0 +/- 14.0 vs. 21.6 +/- 7.9% II/microg). In cultured endothelial cells, simvastatin (10 and 20 microM) increased eNOS levels by 114.7 +/- 39.9 and 212.0 +/- 75.0% II/microg protein, respectively (both P < 0.05; n = 8). In the rat coronary artery ligation model, oral gavage with 20 mg. kg(-1). day(-1) simvastatin for 3 wk decreased (P < 0.05) mean arterial pressure (121 +/- 20 vs. 96.5 +/- 10.8 mmHg) and left ventricular change in pressure with time (4,500 +/- 700 vs. 4,091 +/- 1,064 mmHg/s, n = 6). Simvastatin reduced (P < 0.05) basal vasoconstriction and improved ACh-mediated vasorelaxation in CHF arterial rings. Inhibition of NO generation by N(G)-nitro-L-arginine methyl ester (100 microM) abolished the ACh-induced vasorelaxation in all rats. In conclusion, chronic treatment of CHF with simvastatin restores endothelial NO-dependent dysfunction and upregulates eNOS protein content in arterial tissue.     

Flumazenil preconditions cardiomyocytes via oxygen radicals and K(ATP) channels

We determined whether flumazenil mimics ischemic preconditioning in chick cardiomyocytes and examined the role of intracellular reactive oxygen species (ROS) and ATP-dependent potassium (K(ATP)) channels in mediating the effect. Chick ventricular myocytes were perfused with a balanced salt solution in a flow-through chamber. Cell viability was quantified using propidium iodide, and ROS generation was assessed using the reduced form of 2',7'-dichlorofluorescin (DCFH). Cells were exposed to 1 h of simulated ischemia and 3 h of reoxygenation. Preconditioning was initiated with 10 min of ischemia followed by 10 min of reoxygenation. Alternatively, flumazenil was added to the perfusate for 10 min and removed 10 min before the start of ischemia. Flumazenil (1 and 10 microM) and preconditioning reduced cell death [54 +/- 5%, n = 3; 26 +/- 4%, n = 6 (P < 0.05); and 20 +/- 2%, n = 6 (P < 0.05), respectively, vs. 57 +/- 7%, n = 10, in controls] and increased DCFH oxidation (an index of ROS production) [0.35 +/- 0.11, n = 3; 2.64 +/- 0.69, n = 8 (P < 0.05); and 2.46 +/- 0.52, n = 6 (P < 0.05), respectively, vs. 0.26 +/- 0.05, n = 9, in controls]. Protection and increased ROS signals with flumazenil (10 microM) were abolished with the thiol reductant N-(2-mercaptopropionyl)-glycine (2-MPG, 800 microM), an antioxidant (cell death: 2-MPG + flumazenil, 55 +/- 12%, n = 6; ROS signals: 2-MPG + flumazenil, 0.11 +/- 0.19, n = 6). Treatment with 5-hydroxydecanoate (1 mM), a selective mitochondrial K(ATP) channel antagonist, abolished its protection. These results demonstrate that flumazenil mimics preconditioning to reduce cell death in myocytes. ROS signals with the resultant mitochondrial K(ATP) channel activation are important components of the intracellular signaling pathway of flumazenil.     

Intrathymic and intrasplenic oxidative stress mediates thymocyte and splenocyte damage in acutely exercised mice.

Reactive oxygen species may contribute to apoptosis in lymphoid tissues observed after exercise. Thymic and splenic tissues excised from control mice (C) or mice immediately after (t0) or 24 h after (t24) a run to exhaustion (RTE) were assayed for biochemical indexes of oxidative stress [thymic and splenic membrane lipid peroxides, superoxide dismutase, catalase, plasma uric acid (UA), and ascorbic acid (AA)]. There were significant increases in membrane lipid peroxides in thymus (P < 0.001) and spleen (P < 0.001) in acutely exercised mice relative to controls (thymus: C = 2.74 +/- 0.80 microM; t0 = 7.45 +/- 0.48 microM; t24 = 9.44 +/-1.41 microM; spleen: C = 0.48 +/- 0.22 microM; t0 = 1.78 +/- 0.28 microM; t24 = 2. 81 +/- 0.34 microM). The thymic and splenic tissue antioxidant enzymes concentrations of superoxide dismutase and catalase were significantly lower in samples collected at t0 relative to C and t24 mice (P < 0.001). Plasma UA and AA levels were used to assess the impact of the RTE on the peripheral antioxidant pool. There was no significant change in UA levels and a significant reduction in plasma AA concentrations (P < 0.001); the reduction in plasma AA occurred at t24 (6.53 +/- 1.64 microM) relative to t0 (13.11 +/- 0. 71 microM) and C (13.26 +/- 1.2 microM). These results suggest that oxidative damage occurs in lymphoid tissues after RTE exercise and that such damage may contribute to lymphocyte damage observed after acute exercise.     

Characteristics of intermittent mitochondrial transport in guinea pig enteric nerve fibers

Enteric neurons controlling various gut functions are prone to oxidative insults that might damage mitochondria (e.g., intestinal inflammation). To resume local energy supply, mitochondria need to be transported. We used MitoTracker dyes and confocal microscopy to investigate basic characteristics of mitochondrial transport in guinea pig myenteric neurites. During a 10-s observation of 1 mm nerve fiber, on average, three mitochondria were transported at an average speed of 0.41 +/- 0.02 microm/s. Movement patterns were clearly erratic, and velocities were independent of mitochondrial size. The velocity oscillated periodically ( approximately 6 s) but was not consistently affected by structures such as en route boutons, bifurcations, or stationary mitochondria. Also, mitochondria transported in opposite directions did not necessarily affect each others' mobility. Transport was blocked by microtubule disruption (100 microM colchicine), and destabilization (1 microM cytochalasin-D) or stabilization (10 microM phalloidin) of actin filaments, respectively, decreased (0.22 +/- 0.02 microm/s, P < 0.05) or increased (0.53 +/- 0.02 microm/s, P < 0.05) transport speed. Transport was inhibited by TTX (1 microM), and removal of extracellular Ca(2+) (plus 2 mM EGTA) had no effect. However, depletion of intracellular stores (thapsigargin) reduced (to 33%) and slowed the transport significantly (0.18 +/- 0.02 microm/s, P < 0.05), suggesting an important role for stored Ca(2+) in mitochondrial transport. Transport was also reduced (to 21%) by the mitochondrial uncoupler FCCP (1 microM) in a time-dependent fashion and slowed by oligomycin (10 microM). We conclude that mitochondrial transport is remarkably independent of structural nerve fiber properties. We also show that mitochondrial transport is TTX sensitive and speeds up by stabilizing actin and that functional Ca(2+) stores are required for efficient transport.     

Uncoupling protein-2 participates in cellular defense against oxidative stress in clonal beta-cells

The role of uncoupling protein-2 (UCP-2) in beta-cells is presently unclear. We have tested the notion that UCP-2 participates in beta-cell defense against oxidants. Expression of the UCP-2 gene in clonal beta-cells (INS-1) was decreased by 45% after 48 h of culture with vitamin E and selenite. When INS-1 cells were exposed to 200 microM H(2)O(2) for 5 min, the cell viability (MTT assay) decreased to 85 +/- 1, 61 +/- 1, 40 +/- 2, and 39 +/- 2% of control when measured respectively 30 min, 2 h, 6 h, and 16 h after H(2)O(2) exposure. At corresponding time points UCP-2 mRNA levels were 1.01 +/- 0.09, 1.53 +/- 0.15 (P < 0.05), 1.44 +/- 0.18 (P = 0.06), and 1.12 +/- 0.09 fold of control, i.e., transiently increased. We next tested whether overexpression of UCP-2 could enhance resistance of beta-cells toward H(2)O(2) toxicity. A cotransfection method using EGFP as a suitable marker and a human cDNA UCP-2 construct was used for transient overexpression of UCP-2. Transfected cells expressed the gene about 30-fold more than normal cells. After exposure to H(2)O(2) (200 micrometer, 5 min), the survival of UCP-2 overexpressing cells was measured 30-45 min later by flow cytometry. Survival was 13 +/- 0.05% higher than control (EGFP only) cells, P < 0.004 for difference. The results indicate that oxidative stress induces UCP-2 expression in beta-cells, and that UCP-2 serves a role in beta-cell defense against oxidative stress.     

Effect of copper deficiency on oxidative DNA damage in Jurkat T-lymphocytes

The micronutrient copper is a catalytic cofactor for copper, zinc superoxide dismutase and ceruloplasmin, which are two important antioxidant enzymes. As such, a lack of copper may promote oxidative stress and damage. The purpose of this study was to determine the effect of copper deficiency on oxidative damage to DNA in Jurkat T-lymphocytes. To induce copper deficiency, cells were incubated for 48 h with 5-20 microM 2,3,2-tetraamine (2,3,2-tet), a high affinity copper chelator. Such treatment did not affect cell proliferation/viability, as assessed by measuring mitochondrial reduction of WST-1 reagent (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-ben zen e disulfonate). Furthermore, the induction of copper deficiency did not promote oxidative DNA damage as evaluated by the comet assay. Comet scores were 15 +/- 0 and 16 +/- 1 for control and copper-deficient cells, respectively. However, the copper-deficient cells sustained greater oxidative DNA damage than the control cells (comet scores of 175 +/- 15 and 50 +/- 10, respectively) when both were oxidatively challenged with 50 microM hydrogen peroxide (H(2)O(2)). Supplemental copper but not zinc or iron prevented the potentiation of the H(2)O(2)-induced oxidative DNA damage caused by 2,3,2-tet. These data suggest that copper deficiency compromises the antioxidant defense system of cells, thereby increasing their susceptibility to oxidative DNA damage.     

Exercise training decreases rat heart mitochondria free radical generation but does not prevent Ca2+-induced dysfunction.

Exercise provides cardioprotection against ischemia-reperfusion injury, a process involving mitochondrial reactive oxygen species (ROS) generation and calcium overload. This study tested the hypotheses that isolated mitochondria from hearts of endurance-trained rats have decreased ROS production and improved tolerance against Ca(2+)-induced dysfunction. Male Fischer 344 rats were either sedentary (Sed, n = 8) or endurance exercise trained (ET, n = 11) by running on a treadmill for 16 wk (5 days/wk, 60 min/day, 25 m/min, 6 degrees grade). Mitochondrial oxidative phosphorylation measures were determined with glutamate-malate or succinate as substrates, and H(2)O(2) production and permeability transition pore (PTP) opening were determined with succinate. All assays were carried out in the absence and presence of calcium. In response to 25 and 50 microM CaCl(2), Sed and ET displayed similar decreases in state 3 respiration, respiratory control ratio, and ADP:O ratio. Ca(2+)-induced PTP opening was also similar. However, H(2)O(2) production by ET was lower than Sed (P < 0.05) in the absence of calcium (323 +/- 12 vs. 362 +/- 11 pmol.min(-1).mg protein(-1)) and the presence of 50 microM CaCl(2) (154 +/- 3 vs. 197 +/- 7 pmol.min(-1).mg protein(-1)). Rotenone, which blocks electron flow from succinate to complex 1, reduced H(2)O(2) production and eliminated differences between ET and Sed. Mitochondrial superoxide dismutase and glutathione peroxidase were not affected by exercise. Catalase activity was extremely low but increased 49% in ET (P < 0.05). In conclusion, exercise reduces ROS production in myocardial mitochondria through adaptations specific to complex 1 but does not improve mitochondrial tolerance to calcium overload.     

Exercise training in normobaric hypoxia in endurance runners. II. Improvement of mitochondrial properties in skeletal muscle.

This study investigates whether adaptations of mitochondrial function accompany the improvement of endurance performance capacity observed in well-trained athletes after an intermittent hypoxic training program. Fifteen endurance-trained athletes performed two weekly training sessions on treadmill at the velocity associated with the second ventilatory threshold (VT2) with inspired O2 fraction = 14.5% [hypoxic group (Hyp), n = 8] or with inspired O2 fraction = 21% [normoxic group (Nor), n = 7], integrated into their usual training, for 6 wk. Before and after training, oxygen uptake (VO2) and speed at VT2, maximal VO2 (VO2 max), and time to exhaustion at velocity of VO2 max (minimal speed associated with VO2 max) were measured, and muscle biopsies of vastus lateralis were harvested. Muscle oxidative capacities and sensitivity of mitochondrial respiration to ADP (Km) were evaluated on permeabilized muscle fibers. Time to exhaustion, VO2 at VT2, and VO2 max were significantly improved in Hyp (+42, +8, and +5%, respectively) but not in Nor. No increase in muscle oxidative capacity was obtained with either training protocol. However, mitochondrial regulation shifted to a more oxidative profile in Hyp only as shown by the increased Km for ADP (Nor: before 476 +/- 63, after 524 +/- 62 microM, not significant; Hyp: before 441 +/- 59, after 694 +/- 51 microM, P < 0.05). Thus including hypoxia sessions into the usual training of athletes qualitatively ameliorates mitochondrial function by increasing the respiratory control by creatine, providing a tighter integration between ATP demand and supply.     

Lipid peroxidation and total antioxidant status in patients with breast cancer

Oxidative stress is considered to be involved in the pathophysiology of all cancers. The aim of this study is to examine oxidative stress and antioxidant status in patients with breast cancer by evaluation of the serum levels of total antioxidant capacity (TAC) and lipid peroxidation products as malondialdehyde (MDA) and lipid hydroperoxide and to investigate the relationship between these parameters, oxidative stress and serum lipids and lipoproteins. In our study, serum TAC, MDA, lipid hydroperoxide, HDL-cholesterol, VLDL-cholesterol, LDL-cholesterol, total cholesterol, triacylglycerol (TAG), albumin and uric acid levels of 56-breast cancer patients in different clinical stages and 18 healthy women were determined. Significantly lower-levels of TAC were detected in patients with breast cancer in comparison to controls (2.01 +/- 0.01 mmol/l and 2.07 +/- 0.03 mmol/l, respectively, p < 0.05). Serum MDA levels of the patients were higher compared to the controls (3.64 +/- 0.25 microM and 2.72 +/- 0.22 microM, respectively, p < 0.05). No significant difference between lipid hydroperoxide levels of patients and controls was found (0.33 +/- 0.05 microM and 0.32 +/- 0.01 microM, respectively, p > 0.05). These data show that lower TAC and higher MDA levels i.e. increased oxidative stress may be related to breast cancer.     

Cardioprotection initiated by reactive oxygen species is dependent on activation of PKCepsilon

To examine whether cardioprotection initiated by reactive oxygen species (ROS) is dependent on protein kinase Cepsilon (PKCepsilon), isolated buffer-perfused mouse hearts were randomized to four groups: 1) antimycin A (AA) (0.1 microg/ml) for 3 min followed by 10 min washout and then 30 min global ischemia (I) and 2 h reperfusion (R); 2) controls of I/R alone; 3) AA bracketed with 13 min of N-2-mercaptopropionyl- glycine (MPG) followed by I/R; and 4) MPG (200 microM) alone, followed by I/R. Isolated adult rat ventricular myocytes (ARVM) were exposed to AA (0.1 microg/ml), and lucigenin was used to measure ROS production. Murine hearts and ARVM were exposed to AA (0.1 microg/ml) with or without MPG, and PKCepsilon translocation was measured by cell fractionation and subsequent Western blot analysis. Finally, the dependence of AA protection on PKCepsilon was determined by the use of knockout mice (-/-) lacking PKCepsilon. AA exposure caused ROS production, which was abolished by the mitochondrial uncoupler mesoxalonitrile 4-trifluoromethoxyphenylhydrazone. In addition, AA significantly reduced the percent infarction-left ventricular volume compared with control I/R (26 +/- 4 vs. 43 +/- 2%; P < 0.05). Bracketing AA with MPG caused a loss of protection (52 +/- 7 vs. 26 +/- 4%; P < 0.05). AA caused PKCepsilon translocation only in the absence of MPG, and protection was lost on the pkcepsilon(-/-) background (38 +/- 3 vs. 15 +/- 4%; P < 0.001). AA causes ROS production, on which protection and PKCepsilon translocation depend. In addition, protection is absent in PKCepsilon null hearts. Our results imply that, in common with ischemic preconditioning, PKCepsilon is crucial to ROS-mediated protection.     

Chronic paraplegia-induced muscle atrophy downregulates the mTOR/S6K1 signaling pathway.

Ribosomal S6 kinase 1 (S6K1) is a downstream component of the mammalian target of rapamycin (mTOR) signaling pathway and plays a regulatory role in translation initiation, protein synthesis, and muscle hypertrophy. AMP-activated protein kinase (AMPK) is a cellular energy sensor, a negative regulator of mTOR, and an inhibitor of protein synthesis. The purpose of this study was to determine whether the hypertrophy/cell growth-associated mTOR pathway was downregulated during muscle atrophy associated with chronic paraplegia. Soleus muscle was collected from male Sprague-Dawley rats 10 wk following complete T(4)-T(5) spinal cord transection (paraplegic) and from sham-operated (control) rats. We utilized immunoprecipitation and Western blotting techniques to measure upstream [AMPK, Akt/protein kinase B (PKB)] and downstream components of the mTOR signaling pathway [mTOR, S6K1, SKAR, 4E-binding protein 1 (4E-BP1), and eukaryotic initiation factor (eIF) 4G and 2alpha]. Paraplegia was associated with significant soleus muscle atrophy (174 +/- 8 vs. 240 +/- 13 mg; P < 0.05). There was a reduction in phosphorylation of mTOR, S6K1, and eIF4G (P < 0.05) with no change in Akt/PKB or 4E-BP1 (P > 0.05). Total protein abundance of mTOR, S6K1, eIF2alpha, and Akt/PKB was decreased, and increased for SKAR (P < 0.05), whereas 4E-BP1 and eIF4G did not change (P > 0.05). S6K1 activity was significantly reduced in the paraplegic group (P < 0.05); however, AMPKalpha2 activity was not altered (3.5 +/- 0.4 vs. 3.7 +/- 0.5 pmol x mg(-1) x min(-1), control vs. paraplegic rats). We conclude that paraplegia-induced muscle atrophy in rats is associated with a general downregulation of the mTOR signaling pathway. Therefore, in addition to upregulation of atrophy signaling during muscle wasting, downregulation of muscle cell growth/hypertrophy-associated signaling appears to be an important component of long-term muscle loss.     

The effects of vitamin C supplementation on protein oxidation in healthy volunteers

We have investigated vitamin C supplementation effects on immunoglobulin oxidation (carbonyls) and total plasma protein sulfhydryls in healthy human volunteers. After receiving placebo, plasma ascorbate and oxidation markers were unchanged. Following 5 weeks supplementation with vitamin C (400 mg/day), plasma ascorbate increased but no significant effect on protein oxidation was observed. At 10 and 15 weeks supplementation, carbonyl levels were significantly reduced (P < 0.01) in subjects with low baseline ascorbate (29.51 +/- 5.3 microM) but not in those with normal baseline ascorbate (51.81 +/- 2.3 microM). To eliminate any effect from seasonal variation in dietary antioxidant intake, a second phase was undertaken. Subjects on vitamin C for 15 weeks were randomly assigned to receive either placebo or vitamin C. No difference in plasma sulfhydryl content was observed. Subjects withdrawn from supplementation showed an increase in immunoglobulin carbonyl content (P < 0.01). This demonstrates that dietary vitamin C supplementation can reduce certain types of oxidative protein damage in subjects with low basal antioxidant.     

Effect of dialysis on oxidative stress in uraemia

It has been postulated that dialysis of patients with chronic renal failure (CRF) is associated with increased lipid peroxidation which may contribute to vascular and other complications of the syndrome. In the present study, a specific and precise technique [ferrous oxidation in xylenol orange (FOX) assay] was used to measure plasma lipid hydroperoxides (ROOHs) in three groups of uraemic patients. Patients were either studied before starting dialysis (n = 12) or on continuous ambulatory peritoneal dialysis (CAPD, n = 12) or haemodialysis (HD, n = 36) and compared to healthy controls (n = 20). Plasma ROOHs were markedly elevated in HD patients compared with the controls (7.01 +/- 2.9 microM versus 4.25 +/- 2.05 microM; P < 0.005, Mann-Whitney test). Plasma ROOH concentrations in the CAPD patients were increased but not significantly higher than controls (5.36 +/- 3.56 microM versus 4.25 +/- 2.05 microM). By contrast, no differences in ROOH levels were found between controls and predialysis patients. There was no difference in plasma thiobarbituric acid reactive substances (TBARS) between control and the three CRF groups. Absolute and cholesterol standardised plasma alpha-tocopherol levels were lower in the patients (whether they were on dialysis or not) than in the controls (18.62 +/- 6.88 microM versus 22.73 +/- 5.33 microM; P < 0.01 and 1.99 +/- 1.88 microM/mM versus 5.25 +/- 1.0 microM/mM; P < 0.0005, respectively). This study provides direct evidence that enhanced oxidative stress in CRF patients is related to the dialysis treatment rather than the disease itself. Further studies will be necessary to establish the relationships between plasma measures of oxidative stress and cardiovascular complications in CRF patients under dialysis and whether treatment with antioxidants may reduce oxidative stress or reverse adverse effects associated with dialysis.     

Effects of high myoplasmic L-lactate concentration on E-C coupling in mammalian skeletal muscle.

The effects of high myoplasmic L-lactate concentrations (20-40 mM) at constant pH (7.1) were investigated on contractile protein function, voltage-dependent Ca(2+) release, and passive Ca(2+) leak from the sarcoplasmic reticulum (SR) in mechanically skinned fast-twitch (extensor digitorum longus; EDL) and slow-twitch (soleus) fibers of the rat. L-Lactate (20 mM) significantly reduced maximum Ca(2+)-activated force by 4 +/- 0.5% (n = 5, P < 0.05) and 5 +/- 0.4% (n = 6, P < 0.05) for EDL and soleus, respectively. The Ca(2+) sensitivity was also significantly decreased by 0.06 +/- 0. 002 (n = 5, P < 0.05) and 0.13 +/- 0.01 (n = 6, P < 0.001) pCa units, respectively. Exposure to L-lactate (20 mM) for 30 s reduced depolarization-induced force responses by ChCl substitution by 7 +/- 3% (n = 17, P < 0.05). This inhibition was not obviously affected by the presence of the lactate transport blocker quercetin (10 microM), or the chloride channel blocker anthracene-9-carboxylic acid (100 microM). L-Lactate (20 mM) increased passive Ca(2+) leak from the SR in EDL fibers (the integral of the response to caffeine was reduced by 16 +/- 5%, n = 9, P < 0.05) with no apparent effect in soleus fibers (100 +/- 2%, n = 3). These results indicate that the L-lactate ion per se has negligible effects on either voltage-dependent Ca(2+) release or SR Ca(2+) handling and exerts only a modest inhibitory effect on muscle contractility at the level of the contractile proteins.