Altered contents of tocopherols in chickens with inherited muscular dystrophy

Oxidative damage has been hypothesized as the basis for some of the changes in enzymatic functions and physical properties of membranes in inherited muscular dystrophy. The contents of alpha- and gamma-tocopherol (vitamin E) and their oxidation products, the tocopheryl quinones, were measured at 1 to 4 weeks after hatching in the muscle and other tissues of chickens with inherited muscular dystrophy. Analyses at these early ages minimized the potential influence of pathological changes on the measured parameters. The affected muscle (pectoralis major) of dystrophic birds contained significantly higher levels of alpha-tocopheryl quinone and a decreased ratio of alpha- to gamma-tocopherol. Consistent changes in these parameters were not observed in other tissues. Although their basis remains unclear, these changes in the tocopherols are suggestive of oxidative stress in dystrophic muscle membranes. Lipid extracts of tissues of normal and dystrophic birds exhibited no significant differences in the content of conjugated dienes or lipofuscins, two other indices of oxidative stress. These data do not consistently support the hypothesis that oxidative stress plays a causal role in damage to dystrophic muscle, although it remains possible that free-radical damage is involved in the secondary alterations associated with muscular dystrophy.     

Screening for increased protein thiol oxidation in oxidatively stressed muscle tissue

Elevated oxidative stress can alter the function of proteins through the reversible oxidation of the thiol groups of key cysteine residues. This study evaluated a method to scan for reversible protein thiol oxidation in tissue by measuring reduced and oxidized protein thiols. It assessed the responsiveness of protein thiols to oxidative stress in vivo using a dystrophic (mdx) mouse model and compared the changes to commonly used oxidative biomarkers. In mdx mice, protein thiol oxidation was significantly elevated in the diaphragm, gastrocnemius and quadriceps muscles. Neither malondialdehyde nor degree of glutathione oxidation was elevated in mdx muscles. Protein carbonyl content was elevated, but changes in protein carbonyl did not reflect changes in protein thiol oxidation. Collectively, these data indicate that where there is an interest in protein thiol oxidation as a mechanism to cause or exacerbate pathology, the direct measurement of protein thiols in tissue would be the most appropriate screening tool.     

Thiol redox status critically influences mitochondrial response to thyroid hormone‐induced hepatic oxidative injury: A temporal analysis

Liver is a major target organ for thyroid hormone. The objective of the present study was to investigate temporal regulation of mitochondrial glutathione and protein‐bound thiol redox status in hyperthyroid liver. Mitochondria were isolated from control and hyperthyroid rat liver tissues at different time intervals, i.e., 24, 72, and 120 h following treatment, and sub‐fractionated into sub‐mitochondrial particles (SMPs) and matrix fractions. Increased prooxidant levels were indicative of oxidative stress in hyperthyroid mitochondria. Sensitivity to membrane lipid peroxidation (LPx) was maximal after 24 h, which subsided with time. Oxidative damage to proteins was evident as high carbonylation after 72 h; thiol residue damage was an early phenomenon. Reduced and oxidized glutathione (GSH and GSSG) pools of mitochondria were progressively depleted, thereby, impairing matrix antioxidant capacity. However, adaptations to withstand oxidative challenge were elicited in both SMPs and matrix fractions over the long term. It is concluded that maintenance of appropriate intra‐mitochondrial glutathione and protein‐bound thiol redox status could be instrumental in attenuating thyroid hormone‐induced oxidative stress. Copyright © 2010 John Wiley & Sons, Ltd.     

Leucine pools in normal and dystrophic chicken skeletal muscle cells in culture

The specific radioactivity of [3H]Leu in the extracellular, intracellular, and Leu-tRNA pools of normal (white leghorn) and dystrophic (line 307) embryonic chick breast muscle cultures was analyzed as a function of equilibration time and extracellular Leu concentration (0.05-5 mM). The primary results were the following 1) [3H]Leu equilibrated to a constant specific radioactivity in the intracellular and Leu-tRNA pools within 2 min after addition to both normal and dystrophic cultures. 2) After equilibration, the extracellular [3H] Leu specific radioactivity in dystrophic cell culture medium was lower than that of medium exposed to normal cells (especially at low Leu concentrations), probably because of increased release of unlabeled Leu from the dystrophic cells as a result of faster protein breakdown. Accordingly, the specific radioactivities in the intracellular and the Leu-tRNA pools were also lower in dystrophic cells. 3) At 5 mM extracellular Leu, the specific radioactivity in the Leu-tRNA pool was approximately 40% lower than the specific radioactivity in the intracellular pool in both normal and dystrophic cells. Thus, high concentrations of extracellular Leu cannot be used to "flood out" reutilization of unlabeled Leu (released by protein degradation) during protein synthesis. 4) At 5.0 mM extracellular Leu, the specific radioactivity of [3H]Leu in the intracellular pool was comparable to that in the extracellular pool in normal and dystrophic cells; however, the specific radioactivity of Leu-tRNA (i.e. the immediate precursor to protein synthesis) was only 55-65% of the extracellular specific radioactivity in normal and dystrophic cells. In conclusion, reutilization of Leu from protein degradation is higher in dystrophic muscle cell cultures than in normal muscle cell cultures, and accurate rates of protein synthesis in cell cultures can only be obtained if specific radioactivity of amino acid in tRNA is measured.     

Synthesis of retinoids by human retinal epithelium and transfer to rod outer segments.

In order to gain an insight into the pathogenesis of mouse muscular dystrophy, we investigated the natural suppressor serine tRNA. The natural suppressor seryl-tRNA was distinguished from the other seryl-tRNAs on the basis of its specific property of being converted into phosphoseryl-tRNA by a tRNA kinase. On a wet-weight basis, the content of total tRNA in dystrophic muscles was 47% of that in normal muscles. Although the serine-accepting activities of tRNA were similar in muscles of 3-month-old dystrophic and normal mice, the ratio of [32P]phosphoseryl-tRNA (suppressor tRNA) to the total serine tRNA was significantly enhanced in dystrophic muscles compared with that in normal muscles. This high content of suppressor tRNA in dystrophic muscles was further confirmed by dot-blot hybridization experiments with the DNA probes CGTAGTCGGCAGGAT and CGCCCGAAAGGTGGAA for major tRNA(IGASer) and suppressor tRNA respectively. At the early postnatal age of 3 weeks, when only a week had elapsed since the first manifestation of the dystrophic symptom (hindleg dragging), the ratio of suppressor tRNA to major tRNAs in dystrophic hindleg muscles was abnormally increased. Thereafter it decreased with age in normal mice but remained almost unchanged in dystrophic mice. Consequently, at 3 months old, it was 1.7 times higher in dystrophic than in normal mice. The suppressor tRNA is now accepted to play a role in the synthesis of glutathione peroxidase. The present study showed that the content of this enzyme was abnormally elevated in dystrophic mice. Previously we had demonstrated that the docosahexaenoic (C22:6) acid content in phospholipids was decreased, possibly resulting from the enhanced oxidative milieu caused by the dystrophic condition. Thus far, the findings suggest that an increase in the contents of suppressor tRNA and glutathione peroxidase in dystrophic muscle may have been secondarily induced by such a highly oxidative state in the dystrophic condition. However, it is difficult to exclude the possibility that the natural suppressor tRNA plays a primary role in the pathogenesis of muscular dystrophies.     

Thiol protease and cathepsin D activities in selected tissues and cultured cells from normal and dystrophic mice

Thiol protease and cathepsin D activities were studied in extracts from hindlimb muscle of 60-day-old normal and dystrophic mice, strain 129 ReJ, and from cultured normal and dystrophic cells. Total thiol protease activity in dystrophic muscle extracts was 3.5 times higher than in normal muscle extracts, while cathepsin D, activity was 2.2 times greater in dystrophic muscle compared with normal muscle. Activation (pH 4.5, 30 degrees C) of latent thiol protease activity in extracts of muscle occurred concomitant with the inactivation or dissociation of endogenous protease inhibitors. Thiol protease assays revealed a higher ratio of active to inactive protease activity in extracts from dystrophic muscle than from normal muscle. Cultured myoblasts (L69/1) were found to contain 30-fold more thiol protease(s) and 6-fold more cathepsin D activity than whole muscle. Cells established from dystrophic muscle and grown in culture for periods up to 6 months were more responsive to thiol protease activation conditions than similar cultures derived from normal muscle. From data on the rate and extent of thiol protease activation in extracts from dystrophic cells and hindlimb muscle compared with normal tissue, it appears that cells and tissues from dystrophic mice contain a lower level of protease inhibitors than cells and tissues from normal mice.     

Detection and isolation of a 30 kDa abnormal protein in avian dystrophic muscle

We have studied the protein composition of the pectoralis superficialis muscle of genetically dystrophic (New Hampshire line 413) and normal control (line 412) chickens by one- and two-dimensional gel electrophoresis. A protein, referred to hereafter as the 30 kDa abnormal protein, was specifically detected in the affected muscle. It was purified to homogeneity, and its molecular properties were studied. It is a monomer with a molecular mass of approximately 30 kDa and an isoelectric point of about pI 8.4. We have screened by Western blotting a variety of muscles from line 412 and line 413 chickens for the presence of the 30 kDa protein. While the pattern of total protein is very similar in all cases, the 30 kDa protein was not detected in the pectoralis superficialis muscle of line 412 chickens. However, the immunoreactive bands were detected in the sartorius muscle and the tensor fasciae latae muscle from dystrophic and normal chickens. Interestingly, the immunoreactive bands of normal skeletal muscles are smaller in molecular weight than those of dystrophic skeletal muscles. To determine the early time sequence of the appearance of the abnormal protein, we studied muscles from embryos and post-hatched chickens at various ages. The abnormal protein was detected in dystrophic muscles as early as 15 days ex ovo and occurred throughout development up to six months ex ovo. Although the implication of the dystrophy-associated appearance of the 30 kDa protein in the affected muscle is not clear at present, it would be of particular interest to elucidate the biochemical functions of the 30 kDa protein in the affected muscle (pectoralis superficialis muscle) of genetically dystrophic chicken.     

Inhibition of protein carbonyl formation and lipid peroxidation by glutathione in rat liver microsomes.

The peroxidation of rat liver microsomal lipids is stimulated in the presence of iron by the addition of NADPH or ascorbate and is inhibited by the addition of glutathione (GSH). The fate of GSH and the oxidative modification of proteins under these conditions have not been well studied. Rat liver microsomes were incubated at 37 degrees C under 95% O2:5% CO2 in the presence of 10 microM ferric chloride, 400 microM ADP, and either 450 microM ascorbic acid or 400 microM NADPH. Lipid peroxidation was assessed in the presence 0, 0.2, 0.5, 1, or 5 mM GSH by measuring thiobarbituric acid reactive substance (TBARS) and oxidative modification of proteins by measuring protein thiol and carbonyl groups. GSH inhibited TBARS and protein carbonyl group formation in both ascorbate and NADPH systems in a dose-dependent manner. Heat denaturing of microsomes or treatment with trypsin resulted in the loss of this protection. The formation of protein carbonyl groups could be duplicated by incubating microsomes with 4-hydroxynonenal. Ascorbate-dependent peroxidation caused a loss of protein thiol groups which was diminished by GSH only in fresh microsomes. Both boiling and trypsin treatment significantly decreased the basal protein thiol content of microsomes and enhanced ascorbate-stimulated lipid peroxidation. Protection against protein carbonyl group formation by GSH correlated with the inhibition of lipid peroxidation and appeared not to be due to the formation of the GSH conjugate of 4-hydroxynonenal as only trace amounts of this conjugate were detected. Ninety percent of the GSH lost after 60 min of peroxidation was recoverable as borohydride reducible material in the supernatant fraction. The remaining 10% could be accounted for as GSH-bound protein mixed disulfides. However, only 75% of the GSH lost during peroxidation appeared as glutathione disulfide, suggesting that some was converted to other soluble borohydride reducible forms. These data support a role for protein thiol groups in the GSH-mediated protection of microsomes against lipid peroxidation.     

The amino acid sequence of the peptide containing the thiol group of creatine kinase from normal and dystrophic chicken breast muscle. Comparison of some of the immunological properties of the antibodies developed in rabbits against these enzymes

The major (14)C-labelled peptides from creatine kinase from normal and dystrophic chicken muscle obtained by carboxymethylating the reactive thiol groups with iodo[2-(14)C]acetic acid and digestion with trypsin were purified by ion-exchange chromatography on Dowex-50 (X2) and by paper electrophoresis. The chromatographic characteristics of the (14)C-labelled peptides, their electrophoretic mobilities at pH6.5, and their amino acid compositions were identical for the two enzymes. The sequence of amino acids around the essential thiol groups of creatine kinase from normal and dystrophic chicken muscle was shown to be Ile-Leu-Thr-CmCys-Pro-Ser-Asn-Leu-Gly-Thr-Gly-Leu-Arg (CmCys, carboxymethylcysteine). This sequence is almost identical with that for the creatine kinases in human and ox muscle and bovine brain and is very similar to that of arginine kinase from lobster muscle. Antibodies to the enzymes were raised in rabbits and their reaction with the creatine kinase from normal and dystrophic muscles in interfacial, immunodiffusion and immunoelectrophoretic experiments was studied. The cross-reaction between normal muscle creatine kinase and antisera against the dystrophic muscle enzyme (or vice versa) observed by immunodiffusion and by immunoelectrophoretic experiments further suggests that the enzymes from normal and dystrophic chicken muscle are similar in structure. The results of the present study, the identical amino acid sequence of the peptides containing the reactive thiol group from both the normal and dystrophic chicken muscle enzymes and the immunological similarities of the two enzymes are in accord with the similarity of the two enzymes observed by Roy et al. (1970).     

Ornithine and S-adenosylmethionine decarboxylase activities and polyamine contents in developing muscle tissues and primary cultures of normal and polymyopathic hamsters

Polyamine (putrescine, spermidine, and spermine) contents and ornithine (ODC) and S-adenosylmethionine (SAMDC) decarboxylase activities have been assessed in an age-dependent manner, in normal and polymyopathic (dystrophic) hamster skeletal muscle, heart, and tongue extract and in primary tongue myoblast and skin fibroblast cultures. At 2 weeks of age, polyamine contents were significantly elevated in all of the dystrophic hamster tissues studied when compared with their age-matched controls. The degree of this elevation decreased with the age of the animals, generally, to a level where no significant difference in polyamine contents could be noted between normal and dystrophic hamster tissues. ODC and SAMDC activities in whole tissue extracts were consistently highest in 2-week-old muscle extracts and also declined with age. However, no significant changes in ODC or SAMDC activities were evident in any of the dystrophic muscle tissues studied when compared with their age-matched controls. Polyamine contents in dystrophic hamster myoblast and fibroblast primary cultures were also during proliferation (1 and 2 days after the initial seeding) compared with cultures prepared from normal hamsters. ODC and SAMDC activities in primary myoblast and fibroblast cultures clearly reflected the rate of cell proliferation, with highest activities found in subconfluent cell cultures. However, in general, no significant dystrophic-related abnormality in ODC or SAMDC activity was evident in proliferating myoblast or fibroblast cultures. These results suggest that the elevated polyamine contents of dystrophic hamster tissues and primary cultures may be due to a deficiency in polyamine catabolism or transport.     

Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant DEFENSE

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.     

Elevation of calmodulin in avian muscular dystrophy

In an attempt to understand the mechanism of calcium accumulation in myopathies, changes in the major calcium-binding protein, calmodulin, was studied in genetically dystrophic chickens. Measurements by radioimmunoassay revealed an increase in the calmodulin concentration of dystrophic chicken muscles. Poly A-containing RNA(s) of fast and slow muscles from the normal and dystrophic chicks were hybridized with [32P]-labeled calmodulin cDNA probe by the dot-hybridization technique. Densitometric scan of the autoradiogram showed that the calmodulin mRNA levels of dystrophic fast muscles (pectoralis and posterior latissimus dorsi) were approximately two-fold higher than those of the corresponding normal muscles. No significant change in calmodulin and calmodulin messenger RNA of slow muscle (ALD) was found in dystrophic chickens. Our results suggest that increased calcium flux within the dystrophic muscle may be modulated by calmodulin.     

Age and sex differences in human skeletal muscle: Role of reactive oxygen species

Previous studies, conducted on experimental animals, have indicated that reactive oxygen species (ROS) are involved in the aging process. The objective of this work was to study the relationship between oxidative damage and human skeletal muscle aging, measuring the activity of the main antioxidant enzymes superoxide dismutase (total and MnSOD), glutathione peroxidase (GPx) and catalase in the skeletal muscle of men and women in the age groups: young (17–40 years), adult (41–65 years) and aged (66–91 years). We also measured glutathione and glutathione disulfide (GSH and GSSG) levels and the redox index; lipid peroxidation and protein carbonyl content. Total SOD activity was lower in the 66–91 year-old vs. the 17–40 year-old men; MnSOD activity was significantly greater in 66–91 year-old vs. 17–40 year-old women. GPx activity remained unchanged. The activity of catalase was lower in adults than in young men but higher in the aged. We observed no changes in GSH levels and significantly higher GSSG levels only in aged men vs. adult men, and a significant decrease in aged women vs. aged men. The protein carbonyl content increased significantly in the 41–65 and 66–91 year-old vs. the 17–40 year-old men. Finally, young women have lower lipid peroxidation levels than young men. Significantly higher lipid peroxidation levels were observed in aged men vs. both young and adult men, and the same trend was noticed for women. We conclude that oxidative damage may play a crucial role in the decline of functional activity in human skeletal muscle with normal aging in both sexes; and that men appear to be more subject to oxidative stress than women.     

Age and sex differences in human skeletal muscle: role of reactive oxygen species

Previous studies, conducted on experimental animals, have indicated that reactive oxygen species (ROS) are involved in the aging process. The objective of this work was to study the relationship between oxidative damage and human skeletal muscle aging, measuring the activity of the main antioxidant enzymes superoxide dismutase (total and MnSOD), glutathione peroxidase (GPx) and catalase in the skeletal muscle of men and women in the age groups: young (17-40 years), adult (41-65 years) and aged (66-91 years). We also measured glutathione and glutathione disulfide (GSH and GSSG) levels and the redox index; lipid peroxidation and protein carbonyl content. Total SOD activity was lower in the 66-91 year-old vs. the 17-40 year-old men; MnSOD activity was significantly greater in 66-91 year-old vs. 17-40 year-old women. GPx activity remained unchanged. The activity of catalase was lower in adults than in young men but higher in the aged. We observed no changes in GSH levels and significantly higher GSSG levels only in aged men vs. adult men, and a significant decrease in aged women vs. aged men. The protein carbonyl content increased significantly in the 41-65 and 66-91 year-old vs. the 17-40 year-old men. Finally, young women have lower lipid peroxidation levels than young men. Significantly higher lipid peroxidation levels were observed in aged men vs. both young and adult men, and the same trend was noticed for women. We conclude that oxidative damage may play a crucial role in the decline of functional activity in human skeletal muscle with normal aging in both sexes; and that men appear to be more subject to oxidative stress than women.     

Unexpected effect of urate on hydrogen peroxide-induced oxidative damage in embryonic chicken cardiac cells

Uric acid (UA) is a potent scavenger of oxidants in most mammalian and avian species. The aim of this study was to obtain more comprehensive information regarding the relationship between different concentrations of UA and oxidative balance in chicken cardiac cells. First, oxidative damage parameters were measured in chicken cardiac cells treated with different concentrations of UA. UA concentrations within the normal physiological range had no effect, while treatment with a high level of UA, i.e. 1200?μM, increased the malondialdehyde (MDA) and protein carbonyl contents, decreased the superoxide dismutase (SOD) and catalase (CAT) activities, and had no effect on glutathione (GSH) in cardiac muscle cells. In addition, the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway was stimulated in cells treated with 1200?μM UA. Next, the role of UA in protecting cells from oxidative damage was investigated in hydrogen peroxide (H₂O₂)-damaged chicken cardiac cells. Treatment with UA within the normal physiological range reduced the increased MDA and protein carbonyl contents and SOD enzymatic activity induced by H₂O₂ exposure to some extent and inhibited reactive oxygen species (ROS) formation, presumably as a result of the Nrf2 pathway activation in H₂O₂-damaged cells. By contrast, the MDA and protein carbonyl contents were increased, SOD enzymatic activity was depressed, and the Nrf2 pathway was further down-regulated in H₂O₂-damaged cells treated with 1200?μM UA. In conclusion, the results indicated that physiological UA concentration partially alleviated oxidative stress in chicken cardiac muscle cells treated with H₂O₂. However, supraphysiological UA concentrations promoted oxidative damages directly in primary cultured chicken cardiac muscle cells and aggravated oxidative stress in H₂O₂-damaged cells.     

Connective tissue metabolism in muscular dystrophy. Levels of collagen and mucopolysaccharides in embryonic chickens with genetic muscular dystrophy

There were marked differences between the levels of collagen (measured as hydroxyproline) and mucopolysaccharides (measured as hexosamine) found in embryonic chicks with genetic muscular dystrophy and their normal controls. The chief differences were that the dystrophic tissues (gastrocnemius muscle and tendon, pectoralis major and skin) had: (a) greater amounts of hexosamine early in embryonic development; (b) hydroxyproline levels that rose at a faster rate, yielding different slopes than their normal controls; (c) relatively greater amounts of hydroxyproline than hexosamine later in embryonic life (day 20). Connective tissue systems in muscles were preferentially affected. The connective tissue system associated with dystrophic tissues appeared to lag behind the normal rhythm pattern of embryological development. The changes in connective tissue metabolism observed in dystrophic chicks suggested that the collagen from dystrophic embryonic chicks may be of a different structure or composition than that found in the normals.     

Dynamic responses of the glutathione system to acute oxidative stress in dystrophic mouse (mdx) muscles

The precise mechanisms underlying skeletal muscle damage in Duchenne muscular dystrophy (DMD) remain ill-defined. Functional ischemia during muscle activation, with subsequent reperfusion during rest, has been documented. Therefore, one possibility is the presence of increased oxidative stress. We applied a model of acute hindlimb ischemia/reperfusion (I/R) in mdx mice (genetic homolog of DMD) to evaluate dynamic in vivo responses of dystrophic muscles to this form of oxidative stress. Before the application of I/R, mdx muscles showed: 1) decreased levels of total glutathione (GSH) with an increased oxidized (GSSG)-to-reduced (GSH) glutathione ratio; 2) greater activity of the GSH-metabolizing enzymes glutathione peroxidase (GPx) and glutathione reductase; and 3) lower activity levels of NADP-linked isocitrate dehydrogenase (ICDH) and aconitase, two metabolic enzymes that are sensitive to inactivation by oxidative stress and also implicated in GSH regeneration. Interestingly, nondystrophic muscles subjected to I/R exhibited similar changes in total glutathione, GSSG/GSH, GPx, ICDH, and aconitase. In contrast, all of the above remained stable in mdx muscles subjected to I/R. Taken together, these results suggest that mdx muscles are chronically subjected to increased oxidative stress, leading to adaptive changes that attempt to protect (although only in part) the dystrophic muscles from acute I/R-induced oxidative stress. In addition, mdx muscles show significant impairment of the redox-sensitive metabolic enzymes ICDH and aconitase, which may further contribute to contractile dysfunction in dystrophic muscles.     

Heat and chilling induced disruption of redox homeostasis and its regulation by hydrogen peroxide in germinating rice seeds (Oryza sativa L., Cultivar Ratna)

Extremes of temperature (both heat and chilling) during early inbibitional phase of germination caused disruption of redox-homeostasis by increasing accumulation of reactive oxygen species (superoxide and hydrogen peroxide) and significant reduction of antioxidative defense (assessed in terms of total thiol content and activities of superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) in germinating tissues of rice (Oryza sativa L., cultivar Ratna). Imbibitional heat and chilling stress also induced oxidative damage to newly assembled membrane system by aggravating membrane lipid peroxidation and protein oxidation [measured in terms of thiobarbituric acid reactive substances (TBARS), free carbonyl content (C = O groups) and membrane protein thiol level (MPTL)]. Treatment with standardized low titer hydrogen peroxide during early imbibitional phase of germination caused significant reversal in oxidative damages to the newly assembled membrane system imposed by heat and chilling stress [evident from the data of TBARS, C = O, MPTL, ROS accumulation, membrane permeability status, membrane injury index and oxidative stress index] in seedlings of experimental rice cultivar. Imbibitional H₂O₂ pretreatment also caused up-regulation of antioxidative defense (activities of superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and total thiol content) in the heat and chilling stress-raised rice seedlings. When the parameters of early growth performances were assessed (in terms of relative growth index, biomass accumulation, relative germination performance, mean daily germination, T₅₀ value), it clearly exhibited significant improvement of early growth performances of the experimental rice cultivar. The result proposes that an ‘inductive pulse’ of H₂O₂ is required to switch on some stress acclimatory metabolism through which plant restores redox homeostasis and prevents or repairs oxidative damages to newly assembled membrane system caused by unfavorable environmental cues during early germination to the rice cultivar Ratna. The importance of mitigating oxidative damages to membrane lipid and protein necessary for post-germinative growth under extremes of temperature is also suggested.     

Treatments with sodium selenate or doxycycline offset diabetes-induced perturbations of thioredoxin-1 levels and antioxidant capacity

Diabetes is associated with increased oxidative stress and impaired antioxidant defenses. Thioredoxin-1 (TRX-1) is a cytosolic thiol antioxidant and redox-active protein which plays a vital role in the maintenance of reduced intracellular redox state. In this study, the authors examined whether 4-week treatments with sodium selenate and doxycycline--a metalloproteinase-2 inhibitor which also has antioxidant-like effects--offset perturbations in oxidative stress and antioxidant protection in rat liver and skeletal muscle in streptozotocin-induced diabetes (SID) model. Experimental diabetes decreased TRX-1 levels in skeletal muscle and liver. On the other hand, SID increased oxidative stress marker protein carbonyl levels and decreased oxygen radical absorbance capacity (ORAC), an indicator of antioxidant capacity, in liver. A 4-week treatment of sodium selenate to diabetic rats decreased blood glucose levels moderately, while doxycycline treatment caused a reduction in weight loss of diabetic rats. Both doxycycline and sodium selenate prevented diabetes-induced decrease of TRX-1 levels in skeletal muscle, whereas only doxyxycline was effectively preventing diabetes-induced decrease of TRX-1 in liver. Furthermore, both treatments prevented diabetes-induced altered levels of protein carbonyls and ORAC in liver, and restored free and total protein thiol levels in both skeletal muscle and liver. In conclusion, the data of this study provides further evidence that sodium selenate and doxycycline treatments may control oxidative stress and improve antioxidant defense in diabetes.     

Early evolution of selenium status and oxidative stress parameters in rat models of thermal injury

The objective of the present study was to measure the relationship between selenium status and oxidative stress in two rat models of thermal injury. A non-lethal third-degree burn injury involving 20% (experiment 1) or 40% (experiment 2) of total body surface area (TBSA) was applied to male Wistar rats. Selenium level, glutathione peroxidase (GPx) activity in plasma, red blood cells (RBC) and tissues (liver, kidney, muscle, and brain), and plasma selenoalbumin (Se-alb) were measured in control rats and in burned rats respectively 6 hours after injury and daily from day 1 to day 5. In parallel, lipid and protein oxidative damages, monitored by plasma and tissue thiobarbituric acid reactive species (TBARs) levels and plasma total thiol groups were assessed.

We observed a decrease of plasma Se and Se-albumin 6 hours after burn injury. In parallel, plasma GPx activity rapidly decreased and remained significantly lower than in control rats. These alterations were enhanced by the burn injury severity. Plasma TBARs followed the same pattern as that of plasma cholesterol, with an initial decrease and an increase at day 3 in 40% TBSA burned rats. Plasma thiol groups decreased in the two experiments indicating plasma protein oxidation.

These results confirm an early oxidative stress in burn injury, and suggest an early selenium mobilization, which might counteract this oxidative stress. These data underline the crucial need of a restored selenium status in burned patients immediately after the burn injury.