Nikos V. Margaritelis

Reductive stress after exercise: The issue of redox individuality.

Exercise has been consistently used as an oxidant stimulus in redox biology studies. However, previous studies have focused on group differences and did not examine individual differences. As a result, it remains untested whether all individuals experience oxidative stress after acute exercise. Therefore, the main aim of the present study was to investigate whether some individuals exhibit unexpected responses after an acute eccentric (i.e., muscle-damaging) exercise session. Ninety eight (N = 98) young men performed an isokinetic eccentric exercise bout with the knee extensors. Plasma, erythrocytes and urine samples were collected immediately before and 2 days post-exercise. Three commonly used redox biomarkers (F2-isoprostanes, protein carbonyls and glutathione) were assayed. As expected, the two oxidant biomarkers (F2-isoprostanes and protein carbonyls) significantly increased 2 days after exercise (46% and 61%, respectively); whereas a significant decrease in glutathione levels (by −21%) was observed after exercise. A considerable number of the participants exhibited changes in the levels of biomarkers in the opposite, unexpected direction than the group average. More specifically, 13% of the participants exhibited a decrease in F2-isoprostanes and protein carbonyls and 10% of the participants exhibited an increase in glutathione levels. Furthermore, more than 1 out of 3 individuals exhibited either unexpected or negligible (from 0% to ± 5%) responses to exercise in at least one redox biomarker. It was also observed that the initial values of redox biomarkers are important predictors of the responses to exercise. In conclusion, although exercise induces oxidative stress in the majority of individuals, it can induce reductive stress or negligible stress in a considerable number of people. The data presented herein emphasize that the mean response to a redox stimulus can be very misleading. We believe that the wide variability (including the cases of reductive stress) described is not limited to the oxidant stimulus used and the biomarkers selected.

Blood reflects tissue oxidative stress: a systematic review

Abstract We examined whether the levels of oxidative stress biomarkers measured in blood reflect the tissue redox status. Data from studies that measured redox biomarkers in blood, heart, liver, kidney and skeletal muscle were analyzed. In seven out of nine investigated redox biomarkers (malondialdehyde, reduced glutathione, superoxide dismutase, catalase, glutathione peroxidase, vitamin C and E) there was generally good qualitative and quantitative agreement between the blood and tissues. In contrast, oxidized glutathione and the reduced to oxidized glutathione ratio showed poor agreement between the blood and tissues. This study suggests that most redox biomarkers measured in blood adequately reflect tissue redox status.

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.

Going retro: oxidative stress biomarkers in modern redox biology

The field of redox biology is inherently intertwined with oxidative stress biomarkers. Oxidative stress biomarkers have been utilized for many different objectives. Our analysis indicates that oxidative stress biomarkers have several salient applications: (1) diagnosing oxidative stress, (2) pinpointing likely redox components in a physiological or pathological process and (3) estimating the severity, progression and/or regression of a disease. On the contrary, oxidative stress biomarkers do not report on redox signaling. Alternative approaches to gain more mechanistic insights are: (1) measuring molecules that are integrated in pathways linking redox biochemistry with physiology, (2) using the exomarker approach and (3) exploiting -omics techniques. More sophisticated approaches and large trials are needed to establish oxidative stress biomarkers in the clinical setting.

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

Acute exercise increases reactive oxygen and nitrogen species generation. This phenomenon is associated with two major outcomes: (1) redox signaling and (2) macromolecule damage. Mechanistic knowledge of how exercise-induced redox signaling and macromolecule damage are interlinked is limited. This review focuses on the interplay between exercise-induced redox signaling 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 signaling and damage. Indeed, H2O2 can participate in two electron signaling 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 signaling 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 signaling and DNA damage may be interlinked to a greater extent than was previously thought but this requires further investigation.

The rat adequately reflects human responses to exercise in blood biochemical profile: a comparative study.

Animal models are widely used in biology and the findings of animal research are traditionally projected to humans. However, recent publications have raised concerns with regard to what extent animals and humans respond similar to physiological stimuli. Original data on direct in vivo comparison between animals and humans are scarce and no study has addressed this issue after exercise. We aimed to compare side by side in the same experimental setup rat and human responses to an acute exercise bout of matched intensity and duration. Rats and humans ran on a treadmill at 86% of maximal velocity until exhaustion. Pre and post exercise we measured 30 blood chemistry parameters, which evaluate iron status, lipid profile, glucose regulation, protein metabolism, liver, and renal function. ANOVA indicated that almost all biochemical parameters followed a similar alteration pattern post exercise in rats and humans. In fact, there were only 2/30 significant species × exercise interactions (in testosterone and globulins), indicating different responses to exercise between rats and humans. On the contrary, the main effect of exercise was significant in 15/30 parameters and marginally nonsignificant in other two parameters (copper, P = 0.060 and apolipoprotein B, P = 0.058). Our major finding is that the rat adequately mimics human responses to exercise in those basic blood biochemical parameters reported here. The physiological resemblance of rat and human blood responses after exercise to exhaustion on a treadmill indicates that the use of blood chemistry in rats for exercise physiology research is justified.

Adaptations to endurance training depend on exercise‐induced oxidative stress: exploiting redox interindividual variability

The aim of this study was to reveal the role of reactive oxygen and nitrogen species (RONS) in exercise adaptations under physiological in vivo conditions and without the interference from other exogenous redox agents (e.g. a pro‐oxidant or antioxidant).

N-acetylcysteine supplementation increases exercise performance and reduces oxidative stress only in individuals with low levels of glutathione

ABSTRACT Most of the evidence indicates that chronic antioxidant supplementation induces negative effects in healthy individuals. However, it is currently unknown whether specific redox deficiencies exist and whether targeted antioxidant interventions in deficient individuals can induce positive effects. We hypothesized that the effectiveness of antioxidant supplements to decrease oxidative stress and promote exercise performance depends on the redox status of the individuals that receive the antioxidant treatment. To this aim, we investigated whether N‐acetylcysteine (NAC) supplementation would enhance exercise performance by increasing glutathione concentration and by reducing oxidative stress only in individuals with low resting levels of glutathione. We screened 100 individuals for glutathione levels and formed three groups with low, moderate and high levels (N = 36, 12 per group). After by‐passing the regression to the mean artifact, by performing a second glutathione measurement, the individuals were supplemented with NAC (2 × 600 mg, twice daily, for 30 days) or placebo using a double‐blind cross‐over design. We performed three whole‐body performance tests (VO2max, time trial and Wingate), measured two systemic oxidative stress biomarkers (F2‐isoprostanes and protein carbonyls) and assessed glutathione‐dependent redox metabolism in erythrocytes (glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase and NADPH). The low glutathione group improved after NAC supplementation in VO2max, time trial and Wingate by 13.6%, 15.4% and 11.4%, respectively. Thirty days of NAC supplementation were sufficient to restore baseline glutathione concentration, reduce systemic oxidative stress and improve erythrocyte glutathione metabolism in the low glutathione group. On the contrary, the 30‐day supplementation period did not affect performance and redox state of the moderate and high glutathione groups, although few both beneficial and detrimental effects in performance were observed. In conclusion, individuals with low glutathione levels were linked with decreased physical performance, increased oxidative stress and impaired redox metabolism of erythrocytes. NAC supplementation restored both performance and redox homeostasis. Graphical abstract Figure. No caption available. HighlightsA large inter‐individual variability exists in erythrocyte GSH levels.Low GSH levels are linked to decreased performance and abnormal redox status.Chronic NAC supplementation restores both performance and redox status.GSH‐sufficient individuals do not benefit from NAC supplementation.Personalized antioxidant treatments are warranted in modern redox biology research.

Antioxidants as therapeutics in the intensive care unit: Have we ticked the redox boxes?

Critically ill patients are under oxidative stress and antioxidant administration reasonably emerged as a promising approach to combat the aberrant redox homeostasis in this patient cohort. However, the results of the antioxidant treatments in the intensive care unit are conflicting and inconclusive. The main objective of the present review is to highlight some inherent, yet widely overlooked redox-related issues about the equivocal effectiveness of antioxidants in the intensive care unit, beyond methodological considerations. In particular, the discrepancy in the literature partially stems from: (1) the largely unspecified role of reactive species in disease onset and progression, (2) our fragmentary understanding on the interplay between inflammation and oxidative stress, (3) the complex spatiotemporal specificity of in vivo redox biology, (4) the pleiotropic effects of antioxidants and (5) the divergent effects of antioxidants according to the temporal administration pattern. In addition, two novel and sophisticated practices with promising pre-clinical results are presented: (1) the selective neutralization of reactive species in key organelles after they are formed (i.e., in mitochondria) and (2) the targeted complete inhibition of dominant reactive species sources (i.e., NADPH oxidases). Finally, the reductive potential of NADPH as a key pharmacological target for redox therapies is rationalized. In light of the above, the recontextualization of knowledge from basic redox biology to translational medicine seems imperative to perform more realistic in vivo studies in the fast-growing field of critical care pharmacology.

Experimental verification of regression to the mean in redox biology: differential responses to exercise

Abstract An important methodological threat when selecting individuals based on initial values for a given trait is the “regression to the mean” artifact. This artifact appears when a group with an extreme mean value during a first measurement tends to obtain a less extreme value (i.e. tends toward the mean) on a subsequent measurement. The main aim was to experimentally confirm the presence of this artifact in the responses of the reference oxidative stress biomarker (F2-isoprostanes) after exercise. Urine samples were collected before and immediately following acute exercise in order to determine the level of exercise-induced oxidative stress. Afterwards, participants were arranged into three groups based on their levels of exercise-induced oxidative stress (low, moderate and high oxidative stress groups; n = 12 per group). In order to verify the existence of the regression to the mean artifact, the three groups were subjected to a second exercise trial one week after the first trial. This study confirmed the regression to the mean artifact in a redox biology context and showed that this artifact can be minimized by performing a duplicate pretreatment measurement after completing a nonrandom sorting based on the first assessment. This study also indicated that different individuals experience high oxidative stress or reductive stress (or no stress) to the same exercise stimulus even after adjusting for regression to the mean. This finding substantiates the methodological choice to divide individuals based on their degree of exercise-induced oxidative stress in future experiments to investigate the role of reactive species in exercise adaptations.