Consequences on aging process and human wellness of generation of nitrogen and oxygen species during strenuous exercise

Abstract

Impairment of antioxidant defense system and increase in metabolic rate and production of reactive oxygen species have been demonstrated in strenuous exercise. Both at rest and during contractile activity, skeletal muscle generates a very complex set of reactive nitrogen and oxygen species; the main generated are superoxide and nitric oxide. The nature of the contractile activity influences the pattern and the magnitude of this reactive oxygen and nitrogen species (ROS) generation. The intracellular pro-oxidant/antioxidant homeostasis undergoes alteration owing to strenuous exercise and the major identified sources of intracellular free radical generation during physical activity are the mitochondrial electron transport chain, polymorphoneutrophil, and xanthine oxidase. Reactive oxygen species increased tissue susceptibility to oxidative damage and pose a serious threat to the cellular antioxidant defense system. The possible dangerous consequences of the aging process and human wellness are emphasized in this review.

Keywords:

• Skeletal muscle
• physical activity
• oxidative stress
• aging process
• human wellness

Introduction

Physical inactivity (lack of physical activity) leads to increased incidence of a variety of diseases and it can be regarded as one of the endpoints of the exercise-associated hormesis curve. It has been identified as the fourth leading risk factor for global mortality (6% of deaths globally). Moreover, physical inactivity is estimated to be the main cause for approximately 21–25% of breast and colon cancers, 27% of diabetes and approximately 30% of ischemic heart disease burden [1,2].

On the other hand, physical activity (PA) is defined as any bodily movement produced by skeletal muscles that require energy expenditure. The benefit of exercise in promoting good health and preventing various diseases is well known. The regular exercise, with moderate intensity and duration, has a wide range of beneficial effects reducing the risk of developing heart disease, stroke, high blood pressure, some cancers, type 2 diabetes and “thinning” of the bones, called osteoporosis [3–7]. The regular physical activity also helps to control weight, body composition, and metabolic function [8,9] and may help to ease stress, decreasing the pro-inflammatory mediators. Based on the genetic predisposition, regular moderate physical exercise/activity provides systemic beneficial effects, including improved physiological function, decreased the incidence of disease and a higher quality of life. Single bouts of exercise and regular exercise decrease the oxidative challenge to the body. Furthermore, important emerging evidence has demonstrated the remarkable health benefits for cognition and well-being in persons that are regularly active: a recent study [10,11] described that PA reduced depression and anxiety. Recent systematic reviews highlighted a negative relationship between PA and the incidence of Alzheimer’s disease and dementia [12,13] or a positive effect of routine PA participation on indices of cognitive function in young to middle-aged adults [14–16].

Contrariwise, strenuous exercise bouts, excessive exercise, and overtraining lead to negative consequences on muscle strength and integrity [17] and to damaging oxidative stress. Thus, are an indication of the other endpoint of the hormetic response [18–23]. Furthermore, the moderate exercise up-regulates the immune system, while strenuous exercise increases the risk of infection. Moreover, strenuous chronic exercise also represents a form of oxidative stress to the organisms and, therefore, can alter the balance between pro-oxidants and antioxidants [24–26].

At the light of these studies, a controversy role is reserved to PA as generating of free radicals and consequently, as cancer promoter (physical inactivity or strenuous physical activity), or cancer protector (moderate physical activity).

The aim of this review is to analyze the state-of-the-art in this thematic and its relationship with aging, trying to clarify the major aspects of the PA role in the cellular antioxidant systems.

Generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS)

The ROS and RNS are free radicals with one unpaired electron derived from molecular oxygen or nitrogen. ROS and RNS are highly reactive and toxic to cellular components such as proteins, membrane lipids, and DNA, but they also act as signal transducers in Inflammatory Immune Response (IRI). ROS and RNS can be generated by nonenzymatic (ETC) and enzymatic (reduced form of nicotinamide adenine dinucleotide phosphate [NADPH] oxidase, xanthine oxidase, myeloperoxidase) pathways and have different origins during the different phases of reperfusion. It was originally thought that only phagocytic cells were responsible for ROS production as their part in host cell defense mechanisms. Recent work has demonstrated that ROS have a role in cell signaling, including apoptosis, gene expression, and the activation of cell signaling cascades [27,28].

The production of oxygen-based radicals is the bane of all aerobic species. These molecules, produced during the mitochondrial electron transport of aerobic respiration or by oxidoreductase enzymes and metal-catalyzed oxidation, have the potential to cause a number of deleterious events.

During PA there is an increase of ROS production by skeletal muscle. Moreover, a significant ROS production derives from other tissues such as heart, lungs, and white blood cells. In blood and in other cells, the excessive presence of ROS can cause lipid, DNA, and protein oxidation [29,30].

Some study reported that the increase in ROS production derives from the common metabolic changes that occur during exercise. For example, the catecholamines production contributes to increasing the ROS production [31–33].

Nowadays, it is well described that the exercise generates muscle damages and inflammatory process; both these factors are important for radical production.

In the skeletal muscle cells, the ROS production derived from numerous systems. At mitochondrial level, about 2–5% of the total oxygen consumed may concur to intracellular ROS production. Therefore, the mitochondria are not the main production in this kind of cells [30,34,35].

In the muscle cells other sites important in producing ROS, beyond mitochondria, are:

• NAD(P)H oxidase enzymes associated with the sarcoplasmic reticulum which also release superoxide to the intracellular space [36–38].

• Phospholipase A, an enzyme that produces ROS; specifically, phospho-lipase A cleaves membrane phospholipids to release arachidonic acid which is a substrate for ROS-generating enzyme systems such as the lipoxygenases [39–41]. Furthermore, the activation of phospholipase A can stimulate NAD(P)H oxidases [42].

• Xanthine oxidase, an enzyme that generates reactive oxygen species such as superoxide radicals and hydrogen peroxide when it catalyzes the oxidation of hypoxanthine to xanthine, and can further catalyze the oxidation of xanthine to uric acid [42–44]. Its role is well explained in rat skeletal muscles that contain significant levels of xanthine oxidase [45,46], while human skeletal muscle cells possessing low amounts of xanthine dehydrogenase or oxidase [47,48] additional research needed to clarify their role in human muscles.

Analyzing the RNS category, one of the most important oxidants is nitric oxide produced by NOS. Skeletal muscle normally expresses neuronal NOS (nNOS) and endothelial NOS (eNOS). nNOS is strongly expressed in “white muscle fibers” (fast-twitch), while eNOS is localized in “red muscle fibers” (slow-twitch), that are rich in mitochondria [49,50]. Nitric oxide is generated continuously by skeletal muscles and this production is enhanced by contractions [51,52].

In summary, ROS and RNS production increases during exercise and these damaging molecules can be generated at various compartments within cells and by numerous organelles and enzymes, as shown in Figure 1.

Figure 1. The principal enzymes involved in ROS and RNS production in the cell.

Cellular antioxidant defense systems

In consideration of numerous systems of the ROS/RNS production, the cells contain a network of enzymatic and non-enzymatic antioxidant defense mechanisms maintaining redox homeostasis in cells.

Enzymatic and non-enzymatic antioxidant systems are strategically located between blood, cytoplasm, and mitochondria, working together to regulate ROS and RNS.

The antioxidant enzymatic system includes superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT). Other antioxidant enzymes such as thioredoxin (TRX), glutaredoxin (GRX), and peroxiredoxin (PRX) also contribute to cellular protection against oxidation.

In addition to the enzymatic antioxidant system, there is the nonenzymatic system: for example, the glutathione (GSH) conducts an antioxidant role in a various manner [53,54]. Uric acid is another important non-enzymatic antioxidant: produced through purine metabolism is an important cell tool to destroy of peroxyl radicals, hydroxyl radicals, and singlet oxygen [55,56].

Finally, α-lipoic acid and bilirubin are two additional non-enzymatic antioxidants:

• α-lipoic acid, frequently found in numerous foods, provide antioxidant effects by recycling vitamin C [57,58];

• bilirubin, derived by heme metabolism, acts against peroxyl radicals and hydrogen peroxide [59–61], playing an important antioxidant role.

The physical activity influence on antioxidant systems

The first suggestion that physical exercise results in free radical-mediated damage to tissues appeared in 1978, and the past three decades have resulted in a large growth of knowledge regarding exercise and oxidative stress.

Skeletal muscle has been shown to generate a complex set of reactive oxygen and nitrogen species (ROS) both at rest and during contractile activity. The primary ROS generated are superoxide and nitric oxide and the pattern and magnitude of their generation is influenced by the nature of the contractile activity. It is increasingly clear that the ROS generated by skeletal muscle play an important role in influencing redox-regulated processes that control, at least some of, the adaptive responses to contractile activity. These processes are also recognized to be modified during aging and in some disease states, providing the potential that interventions affecting ROS activity may influence muscle function or viability in these situations [62].

Strenuous exercise increases oxygen consumption and causes disturbance of intracellular pro-oxidant–antioxidant homeostasis. The mitochondrial electron transport chain, polymorphoneutrophil, and xanthine oxidase have been identified as major sources of intracellular free radical generation during exercise [63,64].

Indeed, free radicals generated during or after exercise may come from several sources: (1) the mitochondria, from which oxygen radicals that have escaped scavenging enzymes present in the mitochondria may leak into the sarcoplasm; (2) the capillary endothelium, where a hypoxia or reoxygenation process is created during exercise; and (3) an oxidative burst from inflammatory cells mobilized as a result of muscle or tissue damage [23,64–68].

Reactive oxygen species pose a serious threat to the cellular antioxidant defense system, such as diminished reserve of antioxidant vitamins and glutathione, and increased tissue susceptibility to oxidative damage [69,70].

An acute bout of exercise at similar relative workload and duration enhanced muscle oxidant production in both young and old skeletal muscle, accompanied by increased oxidative stress [71–73]. This evidence has extended the relevance of the free radical theory of aging to senescent skeletal muscles that are especially vulnerable to oxidative damage caused by strenuous exercise [69,74].

Both a systemic inflammatory response as well as DNA damage has been observed following exhaustive endurance exercise. Hypothetically, exercise-induced DNA damage might either be a consequence of inflammatory processes or causally involved in inflammation and immunological alterations after strenuous prolonged exercise [67].

Exercise greatly increases the production of oxygen radicals in humans. In untrained persons, older men and women, and those with an inadequate antioxidant system, the increased rates of lipid peroxidation resulting from oxygen radical production may cause skeletal damage. The overwhelming consensus of the literature is that long- or short-term supplementation with vitamins E or C has no ergogenic effect on submaximal exercise performance, aerobic capacity, or muscle strength. However, the effects of these antioxidant vitamins may be subtle, and previous studies may not have examined appropriate endpoints. The protection against the generation of oxygen radicals and lipid peroxidation observed in untrained persons performing exercise and the enhanced acute-phase response to eccentric exercise observed in untrained older subjects indicate that vitamin E may be of some benefit in the adaptive response to exercise. In addition, the positive health benefits of using vitamins E and C may suggest an additive or synergistic effect when combined with regular exercise. Since the extent of oxidation is dependent on the exercise mode, intensity, and duration, and is specifically related to the degree of oxidant production, the programs of exercise should be very precise to avoid an induction of important damages due to physical activity [75–77].

Oxidative balance in aging

Older people have a different hormone profile than that of young people. Indeed, the former have lower serum concentrations of thyroid, adrenal and gonadal-derived hormones, and insulin like growth factor 1 (IGF1). Clinical and experimental evidences have shown that these hormones play a relevant role in the oxidant–antioxidant balance. Therefore, their decline throughout the life may expose aging people to oxidative damage.

Both free triiodothyronine (FT3) and, to a lesser extent, free thyroxine (FT4) decline throughout life [77] and both of them influence the oxidant status [78]. Indeed, SOD and CAT activities and GSH levels significantly decrease, whereas lipid peroxidation and sialic acid significantly increase in hypothyroid rats, suggesting a reduced antioxidant activity and the occurrence of a higher oxidative stress in hypothyroid animals [79]. Accordingly, an increased production of ROS (by ∼42%) and a decreased GSH transferase activity (by ∼20%) have been described in post-thyroidectomy hypothyroid patients [80]. Consistent with these findings, treatment with thyroxine decreases oxidative stress indexes in hypothyroid patients [81]. Thus, these findings suggest that the decrease of thyroid hormones in aging people may contribute to a higher oxidative stress and predispose them to oxidative damage by dipping of enzymes with a scavenger activity.

Dehydroepiandrosterone sulfate (DHEAS) can alleviate oxidative stress through the ERK 1/2 and NF-kB signaling [82]. It has been shown that DHEAS protects muscle cells from oxidative stress through the activation of the Nrf-2 pathway. In fact, it significantly decreases the loss of muscle cell death associated with H₂O₂-induced toxicity [83]. Therefore, its decrease over the lifespan, probably due to the reduction of 17,20-lyase activity [84], could be responsible for a higher susceptibility of muscle cells to oxidative stress in aging people.

Age-related hypogonadism negatively affect human health. In men, it impacts on physical performance, metabolic balance, cardiovascular function, and sexual sphere [85–87]. Accordingly, testosterone replacement therapy has been shown to induce weight loss in obese hypogonadal men [88]. Also, a worse metabolic profile influences gonadal status and sexuality [89–94]. Hypogonadism has been shown to affect the oxidative balance. Indeed, estrogens seem to protect muscle cells from oxidative stress damage in mice [95]. This protective effect of estrogens has also been shown in primary cultures of human muscle that are protected from lipid peroxidation through the AKT and p38-MAPK modulation [96]. Furthermore, estrogen replacement therapy lowers oxidative stress indexes in Winstar rats [97]. Similarly, castrated male rats showed higher levels of oxidative stress indexes [98], thus leading to the hypothesis that hypotestosteronemia, often occurring in the aging male [99,100] may also increase oxidative stress. These findings suggest gonadal hormone decrease in menopause or in patients with the so called late-onset hypogonadism might lead to a higher susceptibility to the oxidative damage in aging people.

Finally, the decline of IGF1 serum levels with aging [101] has been already shown to be responsible for the dysregulation of the nuclear factor (erythroid-derived 2-like) 2 (Nrf-2) dependent antioxidant response [102]. In fact, Nrf-2 is known to orchestrate the cellular response to oxidative stress [103]. IGF1-deficient mice show a decreased expression of Nrf-2 and its target genes and an increased oxidative stress (leading to vascular damage) compared to control animals [102]. Endothelial apoptosis induced by oxidative stress is inhibited by IGF1 receptor (IGF1R) expression. Moreover, oxidative stress is able to decrease IGF1R expression in the endothelial tissue [104], thus leading to endothelial dysfunction. Therefore, it may be hypothesized that a higher tissues susceptibility to oxidative stress may be related to the lower IGF1 serum levels in aging people.

In summary, the hormonal profile of aging people may contribute to increase the oxidative stress which the whole body and the muscle tissue are exposed to (Figure 2). Therefore, older men and women should take care in undertaking all the activities leading to a high oxidative stress production, including high-intensity or prolonged exercise.

Figure 2. Oxidative balance in aging.

Conclusions

Even if a large number of studies analyzed the effects of PA on antioxidant systems, to date, no unambiguous theory was achieved. The scientific data are frequently in contrast, on the one hand, the PA was seen as a protector factor with an antioxidant capacity, and on the other hand, it was seen with negative effects on oxidant production. Mild or moderate exercise could determine modest elevations in ROS within skeletal muscle [105,106]. Furthermore, it could have a positive action on antioxidant systems.

In contrast, during high‐intensity or prolonged exercise, ROS accumulate and/or the antioxidant defense system may not be able to buffer the excessive exercise‐induced ROS, resulting in redox imbalance which has been shown to cause impair skeletal muscle contributing to peripheral fatigue [107–109].

In conclusion, the muscle exercise can not only be positive, but also very negative for human wellness and aging processes. Aging people show a greater susceptibility to oxidative stress damage, in part due to the decline of their hormonal asset. The metabolic damages exerted by strenuous physical activity should more precisely be emphasized by the experts of muscle physiology. The identification of the optimal amount of physical activity remains one of the most important research fields of the scientific community.

Disclosure statement

No potential conflict of interest was reported by the authors.