Previous studies have reported that oxidizing free radical species are generated during exercise, and there has been considerable interest in the potential effects of these on exercising tissues. We hypothesized that contracting skeletal muscle was a major source of oxidizing free radical species and that untrained skeletal muscle would adapt to the oxidative stress of a single short period of contractile activity by upregulation of the activity of cytoprotective proteins in the absence of overt cellular damage. Fifteen minutes of aerobic contractile activity was found to induce a rapid release of superoxide anions from mouse skeletal muscle in vivo, and studies with contracting cultured skeletal muscle myotubes confirmed that this was due to release from myocytes rather than other cell types present within muscle tissue in vivo. This increased oxidant production caused a rapid, transient reduction in muscle protein thiol content, followed by increases in the activities of superoxide dismutase and catalase and in content of heat shock proteins. These changes occurred in the absence of overt damage to the muscle cells.

Aims: The sources of cytosolic superoxide in skeletal muscle have not been defined. This study examined the subcellular sites that contribute to cytosolic superoxide in mature single muscle fibers at rest and during contractile activity. Results: Isolated fibers from mouse flexor digitorum brevis loaded with superoxide and nitricoxide-sensitive fluorescent probes, specific pathway inhibitors and immunolocalization techniques were used to identify subcellular sites contributing to cytosolic superoxide. Treatment with the electron transport chain complex III inhibitor, antimycin A, but not the complex I inhibitor, rotenone, caused increased cytosolic superoxide through release from the mitochondrial intermembrane space via voltage-dependent anion or Bax channels, but inhibition of these channels did not affect contraction-induced increases in cytosolic superoxide. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors decreased cytosolic superoxide at rest and following contractions. Protein and mRNA expression of NADPH oxidase subunits was demonstrated in single fibers. NOX2, NOX4, and p22 phox subunits localized to the sarcolemma and transverse tubules; NOX4 was additionally expressed in mitochondria. Regulatory p40 phox and p67 phox proteins were found in the cytoplasm of resting fibers, but following contractions, p40 phox appeared to translocate to the sarcolemma. Innovation: Superoxide and other reactive oxygen species generated by skeletal muscle are important regulators of muscle force production and adaptations to contractions. This study has defined the relative contribution of mitochondrial and cytosolic sources of superoxide within the cytosol of single muscle fibers at rest and during contractions. Conclusion: Muscle mitochondria do not modulate cytosolic superoxide in skeletal muscle but NADPH oxidase is a major contributor both at rest and during contractions. Antioxid. Redox Signal. 18, 603-621.

SummaryOxidative modification of cellular components may contribute to tissue dysfunction during aging. In skeletal muscle, contractile activity increases the generation of reactive oxygen and nitrogen species (ROS). The question of whether contraction-induced ROS generation is further increased in skeletal muscle of the elderly is important since this influences recommendations on their exercise participation. Three different approaches were used to examine whether aging influences contraction-induced ROS generation. Hind limb muscles of adult and old mice underwent a 15-min period of isometric contractions and we examined ROS generation by isolated skeletal muscle mitochondria, ROS release into the muscle extracellular fluid using microdialysis techniques, and the muscle glutathione and protein thiol contents. Resting skeletal muscle of old mice compared with adult mice showed increased ROS release from isolated mitochondria, but no changes in the extracellular levels of superoxide, nitric oxide, hydrogen peroxide, hydroxyl radical activity or muscle glutathione and protein thiol contents. Skeletal muscle mitochondria isolated from both adult and old mice after contractile activity showed significant increases in hydrogen peroxide release compared with precontraction values. Contractions increased extracellular hydroxyl radical activity in adult and old mice, but had no significant effect on extracellular hydrogen peroxide or nitric oxide in either group. In adult mice only, contractile activity increased the skeletal muscle release of superoxide. A similar decrease in muscle glutathione and protein thiol contents was seen in adult and old mice following contractions. Thus, contractile activity increased skeletal muscle ROS generation in both adult and old mice with no evidence for an age-related exacerbation of ROS generation.

Investment in reproduction is costly and frequently decreases survival or future reproductive success. However, the proximate underlying causes for this are largely unknown. Oxidative stress has been suggested as a cost of reproduction and several studies have demonstrated changes in antioxidants with reproductive investment. Here, we test whether oxidative stress is a consequence of reproduction in female house mice (Mus musculus domesticus), which have extremely high energetic demands during reproduction, particularly through lactation. Assessing oxidative damage after a long period of reproductive investment, there was no evidence of increased oxidative stress, even when females were required to defend their breeding territory. Instead, in the liver, markers of oxidative damage (malonaldehyde, protein thiols and the proportion of glutathione in the oxidized form) indicated lower oxidative stress in reproducing females when compared with non-reproductive controls. Even during peak lactation, none of the markers of oxidative damage indicated higher oxidative stress than among non-reproductive females, although a positive correlation between protein oxidation and litter mass suggested that oxidative stress may increase with fecundity. Our results indicate that changes in redox status occur during reproduction in house mice, but suggest that females use mechanisms to cope with the consequences of increased energetic demands and limit oxidative stress.

Oxidative stress induces adaptations in the expression of protective enzymes and heat shock proteins (HSPs) in a variety of tissues. We have examined the possibility that supplementation of subjects with the nutritional antioxidant, vitamin C, influences the ability of lymphocytes to express protective enzymes and HSPs following exposure to an exogenous oxidant and the response of skeletal muscle to the physiological oxidative stress that occurs during exercise in vivo. Our hypothesis was that an elevation of tissue vitamin C content would reduce oxidant‐induced expression of protective enzymes and HSP content. Lymphocytes from non‐supplemented subjects responded to hydrogen peroxide with increased activity of superoxide dismutase (SOD) and catalase, and HSP60 and HSP70 content over 48 h. Vitamin C supplementation at a dose of 500 mg day−1 for 8 weeks was found to increase the serum vitamin C concentration by ∼50 %. Lymphocytes from vitamin C‐supplemented subjects had increased baseline SOD and catalase activities and an elevated HSP60 content. The SOD and catalase activities and the HSP60 and HSP70 content of lymphocytes from supplemented subjects did not increase significantly in response to hydrogen peroxide. In non‐supplemented subjects, a single period of cycle ergometry was found to significantly increase the HSP70 content of the vastus lateralis. Following vitamin C supplementation, the HSP70 content of the muscle was increased at baseline with no further increase following exercise. We conclude that, in vitamin C‐supplemented subjects, adaptive responses to oxidants are attenuated, but that this may reflect an increased baseline expression of potential protective systems against oxidative stress (SOD, catalase and HSPs).

The production of reactive oxygen and nitrogen species (RONS) by skeletal muscle is important as it (i) underlies oxidative damage in many degenerative muscle pathologies and (ii) plays multiple regulatory roles by fulfilling important cellular functions. Superoxide and nitric oxide (NO) are the primary radical species produced by skeletal muscle and studies in the early 1980s demonstrated that their generation is augmented during contractile activity. Over the past 30 years considerable research has been undertaken to identify the major sites that contribute to the increased rate of RONS generation in response to contractions. It is widely accepted that NO is regulated by the nitric oxide synthases, however the sites that modulate changes in superoxide during exercise remain unclear. Despite the initial indications that the mitochondrial electron transport chain was the predominant source of superoxide during activity, with the development of analytical methods a number of alternative potential sites have been identified including the NAD(P)H oxidases, xanthine oxidase, cyclooxygenases, and lipoxygenases linked to the activity of the phospholipase A2 enzymes. In the present review we outline the subcellular sites that modulate intracellular changes in superoxide in skeletal muscle and based on the available experimental evidence in the literature we conclude that the NAD(P)H oxidases are likely to be the major superoxide generating sources in contracting skeletal muscle.

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Background & AimsSinusoidal obstruction syndrome (SOS) following oxaliplatin based chemotherapy can have a significant impact on post-operative outcome following resection of colorectal liver metastases. To date no relevant experimental models of oxaliplatin induced SOS have been described. The aim of this project was to establish a rodent model which could be utilised to investigate mechanisms underlying SOS to aid the development of therapeutic strategies.MethodsC57Bl/6 mice, maintained on a purified diet, were treated with intra-peritoneal FOLFOX (n = 10), or vehicle (n = 10), weekly for five weeks and culled one week following final treatment. Sections of the liver and spleen were fixed in formalin and paraffin embedded for histological analysis. The role of oxidative stress on experimental-induced SOS was determined by dietary supplementation with butylated hydroxyanisole and N-acetylcysteine.ResultsFOLFOX treatment was associated with the development of sinusoidal dilatation and hepatocyte atrophy on H&E stained sections of the liver in keeping with SOS. Immunohistochemistry for p21 demonstrated the presence of replicative senescence within the sinusoidal endothelium.FOLFOX induced endothelial damage leads to a pro-thrombotic state within the liver associated with upregulation of PAI-1 (p <0.001), vWF (p <0.01) and Factor X (p <0.001), which may contribute to the propagation of liver injury.Dietary supplementation with the antioxidant BHA prevented the development of significant SOS.ConclusionsWe have developed the first reproducible model of chemotherapy induced SOS that reflects the pathogenesis of this disease in patients. It appears that the use of antioxidants alongside oxaliplatin based chemotherapy may be of value in preventing the development of SOS in patients with colorectal liver metastases.