The basic chemistry of exercise-induced DNA oxidation:oxidative damage, redox signalling and their interplay

Acute exercise increases reactive oxygen and nitrogen species generation. This phenomenon is associated with two major outcomes: (1) redox signalling and (2) macromolecule damage. Mechanistic knowledge of how exercise-induced redox signalling and macromolecule damage are interlinked is limited. This review focuses on the interplay between exercise-induced redox signalling and DNA damage, using hydroxyl radical (·OH) and hydrogen peroxide (H2O2) as exemplars. It is postulated that the biological fate of H2O2 links the two processes and thus represents a bifurcation point between redox signalling and damage. Indeed, H2O2 can participate in two electron signalling reactions but its diffusion and chemical properties permit DNA oxidation following reaction with transition metals and ·OH generation. It is also considered that the sensing of DNA oxidation by repair proteins constitutes a non-canonical redox signalling mechanism. Further layers of interaction are provided by the redox regulation of DNA repair proteins and their capacity to modulate intracellular H2O2 levels. Overall, exercise-induced redox signalling and DNA damage may be interlinked to a greater extent than was previously thought but this requires further investigation

Ten “Cheat Codes” for Measuring Oxidative Stress in Humans

Formidable and often seemingly insurmountable conceptual, technical, and methodological challenges hamper the measurement of oxidative stress in humans. For instance, fraught and flawed methods, such as the thiobarbituric acid reactive substances assay kits for lipid peroxidation, rate-limit progress. To advance translational redox research, we present ten comprehensive “cheat codes” for measuring oxidative stress in humans. The cheat codes include analytical approaches to assess reactive oxygen species, antioxidants, biomarkers of oxidative damage and redox regulation. They provide essential conceptual, technical, and methodological information inclusive of curated “do” and “don’t” guidelines. Given the biochemical complexity of oxidative stress, we present a research question-grounded decision tree guide for selecting the most appropriate cheat code (s) to implement in a prospective human experiment. Worked examples demonstrate the benefits of the decision tree-based cheat code selection tool. The ten cheat codes define an invaluable resource for measuring oxidative stress in humans

Ten “Cheat Codes” for Measuring Oxidative Stress in Humans

Formidable and often seemingly insurmountable conceptual, technical, and methodological challenges hamper the measurement of oxidative stress in humans. For instance, fraught and flawed methods, such as the thiobarbituric acid reactive substances assay kits for lipid peroxidation, rate-limit progress. To advance translational redox research, we present ten comprehensive “cheat codes” for measuring oxidative stress in humans. The cheat codes include analytical approaches to assess reactive oxygen species, antioxidants, biomarkers of oxidative damage and redox regulation. They provide essential conceptual, technical, and methodological information inclusive of curated “do” and “don’t” guidelines. Given the biochemical complexity of oxidative stress, we present a research question-grounded decision tree guide for selecting the most appropriate cheat code (s) to implement in a prospective human experiment. Worked examples demonstrate the benefits of the decision tree-based cheat code selection tool. The ten cheat codes define an invaluable resource for measuring oxidative stress in humans

Principles for integrating reactive species into in vivo biological processes:examples from exercise physiology

The equivocal role of reactive species and redox signaling in exercise responses and adaptations is an example clearly showing the inadequacy of current redox biology research to shed light on fundamental biological processes in vivo. Part of the answer probably relies on the extreme complexity of the in vivo redox biology and the limitations of the currently applied methodological and experimental tools. We propose six fundamental principles that should be considered in future studies to mechanistically link reactive species production to exercise responses or adaptations: 1) identify and quantify the reactive species, 2) determine the potential signaling properties of the reactive species, 3) detect the sources of reactive species, 4) locate the domain modified and verify the (ir)reversibility of post-translational modifications, 5) establish causality between redox and physiological measurements, 6) use selective and targeted antioxidants. Fulfilling these principles requires an idealized human experimental setting, which is certainly a utopia. Thus, researchers should choose to satisfy those principles, which, based on scientific evidence, are most critical for their specific research question

Quantitative Redox Biology of Exercise

© Georg Thieme Verlag KG Stuttgart · New York. Biology is rich in claims that reactive oxygen and nitrogen species are involved in every biological process and disease. However, many quantitative aspects of redox biology remain elusive. The important quantitative parameters you need to address the feasibility of redox reactions in vivo are: rate of formation and consumption of a reactive oxygen and nitrogen species, half-life, diffusibility and membrane permeability. In the first part, we explain the basic chemical kinetics concepts and algebraic equations required to perform "street fighting" quantitative analysis. In the second part, we provide key numbers to help thinking about sizes, concentrations, rates and other important quantities that describe the major oxidants (superoxide, hydrogen peroxide, nitric oxide) and antioxidants (vitamin C, vitamin E, glutathione). In the third part, we present the quantitative effect of exercise on superoxide, hydrogen peroxide and nitric oxide concentration in mitochondria and whole muscle and calculate how much hydrogen peroxide concentration needs to increase to transduce signalling. By taking into consideration the quantitative aspects of redox biology we can: i) refine the broad understanding of this research area, ii) design better future studies and facilitate comparisons among studies, and iii) define more efficiently the "borders" between cellular signaling and stress

Spectrophotometric assays for measuring redox biomarkers in blood and tissues: the NADPH network

Nicotinamide adenine dinucleotide (NAD+/NADH) along with its phosphorylated form (NADP+/NADPH) are two molecules ubiquitously present in all organisms, and they play key roles as cofactors in fundamental catabolic and anabolic processes, respectively. The oxidation of NADPH to NADP+ initiates a cascade of reactions, where a network of molecules is implicated. The molecules of this cascade form a network with eminent translational potential in redox metabolism. A special point of interest is that spectrophotometric assays have been developed both for NADH/NADPH and the molecules directly regulated by them. Therefore, crucial molecules of the NADPH-dependent redox network can be measured, and the results can be used to assess the bioenergetic and/or oxidative stress status. The main aim of this review is to collectively present the NADPH-related molecules, namely NADPH, NADH, NAD+ kinase, NADPH oxidase, peroxiredoxin, thioredoxin, thioredoxin reductase, and nitric oxide synthase, that can be measured in blood and tissues with the use of a spectrophotometer, which is probably the most simple, inexpensive and widely used tool in biochemistry. We are providing the researchers with reliable and valid spectrophotometric assays for the measurement of the most important biomarkers of the NADPH network in blood and other tissues, thus allowing the opportunity to follow the redox changes in response to a stimulus

Short-Term L-Citrulline Supplementation Does Not Affect Inspiratory Muscle Oxygenation and Respiratory Performance in Older Adults

In sports nutrition, nitric oxide (NO•) precursors such as L-citrulline are widely used to enhance NO• bioavailability, which is considered an ergogenic aid. Our study aimed to examine the effect of short-term L-citrulline supplementation on respiratory muscles’ performance, fatigue, and oxygenation in older adults. Fourteen healthy older males took 6 g of L-citrulline or a placebo for seven days in a double-blind crossover design. Pulmonary function via spirometry (i.e., forced expired volume in 1 s (FEV1), forced vital capacity (FVC), and their ratio)), fractional exhaled nitric oxide (NO•), maximal inspiratory pressure (MIP), rate of perceived exertion, and sternocleidomastoid muscle oxygenation (i.e., oxyhemoglobin (Δ[O2Hb]) and de-oxyhemoglobin (Δ[HHb]), total hemoglobin concentration (Δ[tHb]), and tissue saturation index (TSI%)) were evaluated at baseline, after seven days of L-citrulline supplementation, and after incremental resistive breathing to task failure of the respiratory muscles. The exhaled NO• value was only significantly increased after the supplementation (26% p •, no ergogenic aids were found on the examined parameters at rest and after resistive breathing to task failure in older adults

Redox Profile of Skeletal Muscles: Implications for Research Design and Interpretation

Mammalian skeletal muscles contain varying proportions of Type I and II fibers, which feature different structural, metabolic and functional properties. According to these properties, skeletal muscles are labeled as ‘red’ or ‘white’, ‘oxidative’ or ‘glycolytic’, ‘slow-twitch’ or ‘fast-twitch’, respectively. Redox processes (i.e., redox signaling and oxidative stress) are increasingly recognized as a fundamental part of skeletal muscle metabolism at rest, during and after exercise. The aim of the present review was to investigate the potential redox differences between slow- (composed mainly of Type I fibers) and fast-twitch (composed mainly of Type IIa and IIb fibers) muscles at rest and after a training protocol. Slow-twitch muscles were almost exclusively represented in the literature by the soleus muscle, whereas a wide variety of fast-twitch muscles were used. Based on our analysis, we argue that slow-twitch muscles exhibit higher antioxidant enzyme activity compared to fast-twitch muscles in both pre- and post-exercise training. This is also the case between heads or regions of fast-twitch muscles that belong to different subcategories, namely Type IIa (oxidative) versus Type IIb (glycolytic), in favor of the former. No safe conclusion could be drawn regarding the mRNA levels of antioxidant enzymes either pre- or post-training. Moreover, slow-twitch skeletal muscles presented higher glutathione and thiol content as well as higher lipid peroxidation levels compared to fast-twitch. Finally, mitochondrial hydrogen peroxide production was higher in fast-twitch muscles compared to slow-twitch muscles at rest. This redox heterogeneity between different muscle types may have ramifications in the analysis of muscle function and health and should be taken into account when designing exercise studies using specific muscle groups (e.g., on an isokinetic dynamometer) or isolated muscle fibers (e.g., electrical stimulation) and may deliver a plausible explanation for the conflicting results about the ergogenic potential of antioxidant supplements

The redox signal: A physiological perspective

A signal in biology is any kind of coded message sent from one place in an organism to another place. Biology is rich in claims that reactive oxygen and nitrogen species transmit signals. Therefore, we define a “redox signal as an increase/decrease in the level of reactive species”. First, as in most biology disciplines, to analyze a redox signal you need first to deconstruct it. The essential components that constitute a redox signal and should be characterized are: (i) the reactivity of the specific reactive species, (ii) the magnitude of change, (iii) the temporal pattern of change, and (iv) the antioxidant condition. Second, to be able to translate the physiological fate of a redox signal you need to apply novel and bioplausible methodological strategies. Important considerations that should be taken into account when designing an experiment is to (i) assure that redox and physiological measurements are at the same or similar level of biological organization and (ii) focus on molecules that are at the highest level of the redox hierarchy. Third, to reconstruct the redox signal and make sense of the chaotic nature of redox processes, it is essential to apply mathematical and computational modeling. The aim of the present study was to collectively present, for the first time, those elements that essentially affect the redox signal as well as to emphasize that the deconstructing, decoding and reconstructing of a redox signal should be acknowledged as central to design better studies and to advance our understanding on its physiological effects