Effect of Normobaric Hypoxia on Alterations in Redox Homeostasis, Nitrosative Stress, Inflammation, and Lysosomal Function following Acute Physical Exercise

Hypoxia is a recognized inducer of oxidative stress during prolonged physical activity. Nevertheless, previous studies have not systematically examined the effects of normoxia and hypoxia during acute physical exercise. The study is aimed at evaluating the relationship between enzymatic and nonenzymatic antioxidant barrier, total antioxidant/oxidant status, oxidative and nitrosative damage, inflammation, and lysosomal function in different acute exercise protocols under normoxia and hypoxia. Fifteen competitive athletes were recruited for the study. They were subjected to two types of acute cycling exercise with different intensities and durations: graded exercise until exhaustion (GE) and simulated 30 km individual time trial (TT). Both exercise protocols were performed under normoxic and hypoxic (FiO2 = 16.5%) conditions. The number of subjects was determined based on our previous experiment, assuming the test power = 0.8 and α = 0.05. We demonstrated enhanced enzymatic antioxidant systems during hypoxic exercise (GE: ↑ catalase (CAT), ↑ superoxide dismutase; TT: ↑ CAT) with a concomitant decrease in plasma reduced glutathione. In athletes exercising in hypoxia, redox status was shifted in favor of oxidation reactions (GE: ↑ total oxidant status, ↓ redox ratio), leading to increased oxidation/nitration of proteins (GE: ↑ advanced oxidation protein products (AOPP), ↑ ischemia-modified albumin, ↑ 3-nitrotyrosine, ↑ S-nitrosothiols; TT: ↑ AOPP) and lipids (GE: ↑ malondialdehyde). Concentrations of nitric oxide and its metabolites (peroxynitrite) were significantly higher in the plasma of hypoxic exercisers with an associated increase in inflammatory mediators (GE: ↑ myeloperoxidase, ↑ tumor necrosis factor-alpha) and lysosomal exoglycosidase activity (GE: ↑ N-acetyl-β-hexosaminidase, ↑ β-glucuronidase). Our study indicates that even a single intensive exercise session disrupts the antioxidant barrier and leads to increased oxidative and nitrosative damage at the systemic level. High-intensity exercise until exhaustion (GE) alters redox homeostasis more than the less intense exercise (TT, near the anaerobic threshold) of longer duration (20.2 ± 1.9 min vs. 61.1 ± 5.4 min—normoxia; 18.0 ± 1.9 min vs. 63.7 ± 3.0 min—hypoxia), while hypoxia significantly exacerbates oxidative stress, inflammation, and lysosomal dysfunction in athletic subjects.


Introduction
An inevitable consequence of functioning under aerobic conditions is the production of reactive oxygen (ROS) and nitrogen species (RNS). ROS and RNS are typical byproducts of oxygen metabolism and important messengers in cellular signal processing. Under physiological conditions, ROS and RNS are involved in energy metabolism, erythropoiesis, muscle contraction, and other biochemical processes [1][2][3]. The signaling activity of free radicals is based on the modulation of several transcription factors, e.g., NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and HIF-1 (hypoxia-inducible factor-1), which results in S-nitrosylation of proteins and induction of secondorder transmitter formation, as well as changes in cellular redox status [4][5][6]. However, ROS and RNS overproduction and/or insufficient antioxidant defense can cause redox imbalance, leading to cellular damage by oxidation and nitration. Such a state is defined as oxidative and nitrosative stress, which plays an essential role in many contemporary diseases, including metabolic [7,8], neurodegenerative [9,10], autoimmune [11,12], and neoplastic disorders [13,14]. Interestingly, factors inducing oxidative and nitrosative stress involve physical exercise [2,[15][16][17]. It was shown that overproduction of ROS/RNS occurs both during and after training [2,15,16]. A direct source of free radicals is the activity of mitochondrial enzymes and membrane oxidases (e.g., NADPH oxidase (NOX)), disturbances of ion homeostasis (especially iron and calcium ions), or changes in lysosomal function. The rate of ROS formation depends on the intensity and duration of exercise, the degree of training of the subjects, their age, sex, and diet [2,15,16,18,19]. It was shown that regular long-term aerobic exercise, especially at high intensity, is responsible for a significant increase in oxidative stress through intensified oxygen consumption [19][20][21]. Lipid peroxidation of muscle cells results in decreased fluidity and higher permeability of cellular membranes and enhanced oxidation of proteins and their tissue aggregation, as well as ROS-mediated DNA injury, causing an inflammatory response, delayed muscle soreness, and the release of intramuscular enzymes into the blood [15,22,23]. Nevertheless, little is known on redox homeostasis, nitrosative stress, and inflammatory response after acute physical intervention.
Nowadays, altitude/hypoxic training is becoming increasingly popular in sports [24][25][26][27]. Exposure to hypoxia leads to stimulation of HIF-1, which, apart from regulation of erythropoiesis and angiogenesis, is also a regulator of activity of glycolytic enzymes, mainly phosphofructokinase (PFK-1) [28,29]. Therefore, improvements in aerobic and anaerobic capacity may occur. However, hypoxia and subsequent reoxygenation are also responsible for ROS/RNS overproduction, during prolonged exposure to altitude, as well as during intermittent hypoxic training [29][30][31]. This is caused by disruption of the mitochondrial respiratory chain, disturbances in arachidonic acid metabolism, or migration/activation of immune cells during regular physical activity [24,25,30,31]. Nevertheless, there is a lack of studies evaluating the relationship between antioxidant systems, oxidative and nitrosative cell damage, inflammation, and lysosomal function under normoxic and hypoxic conditions. We speculate that even acute physical exercise can induce oxidative stress and inflammation, and hypoxia can exacerbate these disorders. As interest in high-altitude sports grows, it is essential to understand the differences in redox homeostasis between various protocols of acute physical intervention. Previous studies have examined only a few aspects of redox homeostasis [30,32,33] and ultimately have not systematically studied the effects of normoxia and hypoxia during acute exercise. Therefore, the present study is aimed at evaluating the relationship between (1) enzymatic and nonenzymatic antioxidant barrier, (2) total antioxidant/ oxidant status, (3) oxidative and (4) nitrosative cell damage, (5) biomarkers of inflammation, and (6) lysosomal function in different protocols (different intensity and duration) performed under normoxic and hypoxic conditions.

Materials and Methods
2.1. Participants. The investigation conformed with the principles outlined in the Declaration of Helsinki and was approved by the Bioethics Committee of the Medical University of Bialystok (approval no. R-I-002/325/2019). All subjects gave their informed consent before their inclusion in the study.

Experimental
Design. The subjects were tested on two occasions, separated by 14 days, in normoxic and hypoxic (FiO 2 = 16:5%, equivalent to 2,000 m asl) conditions applied in random order. The tests were performed in a laboratory room equipped with a normobaric hypoxia system (AirZone 25, Air Sport, Poland) allowing to freely manipulate the oxygen concentration in the room air. Temperature (19°C), humidity (50%), and CO 2 concentration (700-800 ppm) were controlled and held constant. The study participants were blinded to exercise conditions. The athletes were instructed to maintain their regular diet and supplementation throughout the experiment and avoid caffeine intake for 24 h preceding each test. All participants arrived at the camp one day before the start of each test series and consumed the same meals throughout their stay (40 kcal/kg/d, 50% carbohydrates, 20% proteins, and 30% fats).
On the first day of each stay, two hours after a light breakfast, the subjects performed graded cycling exercise beginning with a workload of 120 W, which was subsequently increased by 40 W every 3 minutes until volitional exhaustion. The total duration of exposure to hypoxia during this test was~35 min. On the second day, following 24 h of rest and two hours after a light breakfast, the athletes performed a simulated 30 km individual time trial (TT) in a 2 Oxidative Medicine and Cellular Longevity mountainous terrain. The TT was preceded by a 15 min warm-up, carried out according to the individual preferences of the athletes, under the same oxygen concentration as during the main exercise. The total duration of exposure to hypoxia during this test was~90 min. Both tests were performed on subjects' personal bicycles connected to an electromagnetic bicycle trainer (Cyclus 2, RBM Elektronik-Automation GmbH, Leipzig, Germany). During each test series, the athletes were allowed to consume water ad libitum. The oxygen saturation of arterial blood (SpO 2 ) and heart rate (HR) was measured using the WristOx2 pulse oximeter (Nonin Medical Inc., Plymouth, USA).

Blood
Collection. Blood samples were taken from the antecubital vein into 4 mL EDTA tubes at three-time points: before the exercise, immediately after its completion, and following 30 min of rest. They were kept on ice until centrifugation at 375 × g for 10 min at 4°C. Platelet-rich plasma was transferred to a fresh plastic tube, and the leukocyterich buffy coat was thoroughly removed. Separated erythrocytes were suspended in ice-cold PBS and centrifuged at 800 × g for 10 min, and the upper layer and the remaining buffy coat were discarded. Red blood cells were then resuspended in PBS and flash-frozen in liquid nitrogen. Platelet-rich plasma was centrifuged at 2000 × g for 10 min to sediment platelets. The supernatant was then transferred to a fresh plastic tube and recentrifuged at 5000 × g for 10 minutes to obtain platelet-free plasma. All samples were stored at -80°C until analysis.
2.4. Biochemical Assays. All reagents were of analytical grade and purchased (unless otherwise stated) from Sigma-Aldrich (Nümbrecht, Germany, or Saint Louis, MO, USA). Redox determinations were performed in duplicate assays: assessment of antioxidant enzymes in erythrocyte samples and assessment of nonenzymatic antioxidants, oxidative and nitrosative stress products, inflammatory mediators, and lysosomal enzymes in the plasma samples. The absorbance and fluorescence were determined using an Infinite M200 PRO multimode microplate reader (Tecan Group Ltd., Männedorf, Switzerland). The results were then standardized to 1 mg of total protein content, as reported in several other publications [34][35][36][37][38][39][40]. The total protein level was evaluated with the bicinchoninic acid (BCA) method, using a commercial kit (Thermo Scientific PIERCE BCA Protein Assay (Rockford, IL, USA)), with bovine serum albumin (BSA) as a standard. Redox determinations were performed no more than two months after the samples were frozen.
2.5. Enzymatic Antioxidant Barrier. Catalase (CAT, E.C. 1.11.1.6) activity was measured with the method developed by Aebi [41], by evaluation of hydrogen peroxide decomposition, measured spectrophotometrically at the wavelength of 240 nm. One unit of CAT was defined as the amount of the enzyme which is needed to decompose one nmol of hydrogen peroxide within 1 minute. The results are presented as nmol H 2 O 2 /min/mg protein.
The activity of glutathione peroxidase (GPx, E.C. 1.11.1.9) was determined using Paglia and Valentine's method [42] based on the conversion of NADPH (reduced nicotinamide adenine dinucleotide) to NADP + (nicotinamide adenine dinucleotide cation). The measurements were performed spectrophotometrically at 340 nm. One unit of GPx was represented as the amount of the enzyme necessary to catalyze the oxidation of 1 μmol of NADPH within 1 minute [43]. The results are presented as mU/mg protein.
Glutathione reductase (GR, E.C. 1.8.1.7) activity was evaluated spectrophotometrically with the Mize and Langdon [44] method at the wavelength of 340 nm. It was assumed that one unit of GR catalyzing oxidation of 1 μmol of NADPH within 1 minute. The results are presented as mU/mg protein.
The activity of superoxide dismutase (SOD, E.C. 1.15.1.1) was determined with the spectrophotometric method, according to Misra and Fredovich [45]. The absorbance changes accompanying adrenaline oxidation to adrenochrome were measured at the wavelength of 480 nm. One SOD unit corresponds to the amount of enzyme reducing adrenaline oxidation by 50%. The results are presented as mU/mg protein.
2.6. Nonenzymatic Antioxidant Barrier. The uric acid (UA) concentration was measured spectrophotometrically at the wavelength of 490 nm using the commercial kit (Quanti-ChromTMUric Acid Assay Kit DIUA-250; BioAssay Systems, Hayward, CA, USA) according to the manufacturer's instructions. The results are presented as μmol/mg protein.
The concentration of reduced (GSH) and oxidized (GSSG) glutathione was evaluated colorimetrically. The determination was based on the enzymatic reaction between NADPH, DTNB (5,5 ′ -Dithiobis-(2-nitrobenzoic acid), and GR. In order to determine GSSG concentration, the samples were incubated with 2-vinylpiridine to inhibit glutathione oxidation after neutralization with 1 M chlorhydrol triethanolamine to pH 6-7. The concentration of GSH was calculated as a difference in the levels of total glutathione and GSSG. The measurements were taken at the 412 nm wavelength [46,47]. The results are presented as μmol/mg protein.

Antioxidant Status.
Total antioxidant capacity (TAC) was determined by the Erel's method [49]. 2,2-Azinobis (3ethylbenzene-thiazoline-6-sulfonate) (ABTS) was mixed with potassium persulfate and incubated at room temperature for 12 hours to obtain ABTS + . In the next step, 1 mL of ABTS + was added to 10 μL of samples, and the absorbance was read 735 nm wavelength. Results of decolorization were linear with increasing Trolox concentrations. The results are presented as μmol/mg protein.
Total oxidant status (TOS) was evaluated colorimetrically by Erel's method [50], using the oxidation reaction of Fe 2+ to Fe 3+ ions. Fe 3+ ions were then detected using xylenol orange. The results are presented as nmol H 2 O 2 equivalent/ mg protein.
The spectrophotometric detection evaluated the concentration of advanced oxidation protein products (AOPP). Potassium iodide and acetic acid were added to the wells, and the absorbance was read immediately at 340 nm [55,56]. The results are presented as μmol/mg protein.
Ischemia modified albumin (IMA) concentration was determined colorimetrically at 470 nm. The determination was based on the measurement of the exogenous cobalt (Co 2+ ) binding facility of human plasma albumin [57,58]. The results are presented as μmol/mg protein.
2.9. Nitrosative Stress. Nitrate/nitrite (NOx) concentration was determined spectrofluorimetrically. Stable decomposition products of nitric oxide (NO) from the Griess reaction were evaluated by measuring absorbance at 543 nm wavelength [59]. The results are presented as μmol/mg protein.
The peroxynitrite level was determined spectrophotometrically by measurement of the absorbance of nitrophenol at the wavelength of 320 nm. The nitrophenol production resulted from the decomposition of peroxynitrite followed by nitration of glycyltyrosine and 4-hydroxyphenyloacetic acid (4-HPA) [60,61]. The results are presented as μmol/ mg protein.
3-Nitrotyrosine (3-NT) level was measured using the ELISA method. According to the manufacturer's instructions, a commercial kit (Nitrotyrosine ELISA; Immundiagnostik AG, Bensheim, Germany) was used. The results are presented as μmol/mg protein.
S-Nitrosothiol concentration was measured spectrophotometrically based on the reaction of the Cu 2+ ions with the Griess reagent [62]. The solution was shaken and incubated for 20 minutes, after which the absorbance was measured at 490 nm [63]. The results are presented as nmol/ mg protein.
The tumor necrosis factor-alpha (TNF-α) level was determined by the ELISA method using a commercially available kit (EIAab Science Inc. Wuhan; Wuhan, China) according to the manufacturer's instructions. The results are presented as pg/mL.
2.11. Statistical Analysis. Statistical analysis was performed using GraphPad Prism 8.4.3 for macOS (GraphPad Software, Inc. La Jolla, USA). The normality of the distribution was assessed using the Shapiro-Wilk test, while homogeneity of variance used the Levene test. For comparison of quantitative variables, the two-way analysis of variance (ANOVA) followed by the original FDR method of Benjamini and Hochberg was used. Multiplicity adjusted p value was also calculated. The relationship between the assessed biomarkers was evaluated using the Pearson correlation coefficient. The statistical significance level was set at p < 0:05.
The number of subjects was determined based on our previous experiment, assuming the test power = 0:8 and α = 0:05 (online ClinCalc sample size calculator). Erythrocyte GSH-Px, plasma GSH, TAC, TBARS, AOPP, and peroxynitrite were used for calculations, and the minimum number of subjects should be 13 (in one group).

Exercise
Performance. The duration of the GE under normoxic conditions was 20:2 ± 1:9 min, and the maximal work rate amounted to 5:1 ± 0:3 W/kg of body weight. The SpO 2 was 98:0 ± 0:8 and 91:9 ± 3:0% at rest and at the end of the exercise, respectively. The heart rate at the point of exhaustion was 193 ± 8 bpm. In hypoxia, the average duration of the exercise was 18:0 ± 1:9 min, whereas the maximal work rate was 4:6 ± 0:4 W/kg. The SpO 2 was 93:3 ± 3:4 and 84:3 ± 5:4% at rest and at the point of exhaustion, respectively. The heart rate at the end of the exercise was 190 ± 9 bpm.
After a 30 km TT in normoxia, the only significant change was 16.4% (p = 0:0083) increase in peroxynitrite level. In hypoxia, the level of peroxynitrite rose 13.5% (p = 0:0252) after the exercise, while the level of 3-NT increased by 13.1% (p = 0:0193) after 30 minutes of rest, both comparing to the preexercise levels. Moreover, before the exercise, the concentration of 3-NT was 12.7% higher (p = 0:0228) in hypoxia than normoxia. No other significant changes were observed (Figure 5, TT).
After 30 km TT in normoxia, the activity of GLU increased by 29.8% (p = 0:038) considering pre-TT values. In hypoxia, the level of TNF-α raised by 38.3% (p = 0:0109) subsequently to the exercise, while after 30 minutes, the activity of GLU raised by 6.4% (p = 0:0012) comparing to the activity before exercise and by 9% (p < 0:0001) comparing to the activity directly after exercise, and the activity of HEX increased by 17.2% (p = 0:043) in relation to postexercise measurement. Comparing exercising in hypoxia to exercising in normoxia, the preexercise GLU activity in hypoxia was 31.7% (p = 0:0279) higher, while the postexercise activity of MPO was 33.6% (p = 0:454) greater too. After resting, the activity of GLU was also 7.2% higher (p = 0:0002) in hypoxia ( Figure 6, TT).

Discussion
Our study is the first to evaluate the effect of different acute exercise protocols performed under normoxia and hypoxia on antioxidant status, oxidative and nitrosative damage, inflammation, and lysosomal function. We have shown that both graded exercise until exhaustion (GE) and 30 km time trial (TT) impair the efficiency of antioxidant systems and induce oxidative and nitrosative stress, with hypoxia causing more significant disruption in redox homeostasis and inflammation.
In recent years, there has been a marked increase in interest in mountain sports [67]. Apart from its undoubted advantages, this type of activity is not without health risks. Limited oxygen diffusion through the pulmonary capillaries contributes to tissue hypoxia and the overproduction of free radicals [32,67]. ROS sources under these conditions include primarily reduced partial pressure of oxygen in the air (hypobaric hypoxia), intense physical activity, and autooxidation of catecholamines. Although various adaptive mechanisms can partially compensate for tissue hypoxia (e.g., hyperventilation, tachycardia, increased cardiac output, and enhanced hemoglobin and erythrocytes content), the most effective blood response does not appear until several days later [32].
The present study generally demonstrated the strengthening of enzymatic antioxidant systems during hypoxic exercise (GE: ↑ CAT, ↑ SOD; TT: ↑ CAT vs. normoxia). Changes in the enzymatic antioxidant barrier may reflect various functional/pathophysiological states. The initial increase in enzyme activity is usually adaptation to higher production of ROS and RNS, whereas the subsequent decrease results from depletion of the antioxidant reserves.   Of particular note are the erythrocyte enzymes (GPx and CAT) that degrade hydrogen peroxide. GPx plays a key role in H 2 O 2 degradation at physiological concentrations by reducing hydrogen peroxide with the simultaneous conversion of GSH to its oxidized form (GSSG). However, under H 2 O 2 overproduction, CAT exhibits greater enzymatic activity as evidenced by the Michaelis-Menten constant (Km) for GPx (1 × 10 −6 M) and CAT (2:4 × 10 −4 M) [68,69]. Although we did not directly assess the rate of free radical formation, the increase in CAT and decrease in GPx activity (versus normoxia) indicate a higher intensity of oxidative processes during hypoxic exercise. Enhanced plasma TOS in hypoxia also supports this hypothesis. It is well known that TOS determines the total amount of oxidants in a biological system [50]. Considering that free radicals can mutually enhance their production, TOS provides more information than evaluation of individual ROS/ RNS separately. However, what could constitute an additional source of free radicals in hypoxic exercise? During tissue hypoxia (as during tissue ischemia), xanthine dehydrogenase is converted to XO, donating an electron to molecular oxygen. The reaction catalyzed by XO produces superoxide anions and hydrogen peroxide [2,30,31,70], explaining the increase in SOD and CAT activity under hypoxic exercise. However, overactivation of nitric oxide synthases (NOS), especially inducible NOS (iNOS), also occurs in these conditions [71,72]. Excess nitric oxide (NO) concentrations inhibit cytochrome oxidase activity, which in turn intensifies O 2 -• production [73,74]. If the full O 2 supply is restored, there is an increased formation of ROS referred to as the "oxygen paradox" [75,76]. Therefore, enhanced CAT activity observed in our study is not surprising (increase at each time interval versus normoxic exercise). Interestingly, the activity of antioxidant enzymes (CAT, SOD) and total oxidative capacity (TOS, OSI) were also relatively higher in high-intensity exercise until exhaustion (GE). If O 2 supply to the cells is insufficient, energy is produced in the low-efficiency process of anaerobic glycolysis, leading to an increase in H + ions and lactate concentrations. The consequence is the loss of ability to maintain ionic homeostasis, particularly an increase in the extracellular concentration of K + and the accumulation of Na + and Ca 2+ , which is responsible for ROS overproduction [77,78].
The signaling effects of hydrogen peroxide are associated with proteins recording changes in cellular redox status. The molecules responsible for transmitting the H 2 O 2 signal to the nucleus are low molecular weight thiols, of which reduced glutathione is an essential intracellular source [79,80]. Therefore, it is not surprising that GSH concentrations were significantly lower in athletes exercising in hypoxia compared to normoxia. Since GSSG concentrations and GR activity were unchanged, the decrease in GSH concentration may be due to the oxidation of enzymes responsible for glutathione synthesis or the formation of S-conjugates with glutathione and proteins. In addition to its antioxidant role, GSH participates in DNA replication and apoptosis and regulates the thiol groups of proteins in their reduced state [81,82]. Therefore, maintenance of adequate GSH levels is crucial for proper cellular function. In our study, despite strengthening the antioxidant barrier under hypoxia, there was a redox imbalance in favor of oxidative reactions (GE: ↑ TOS, ↓ redox ratio). This results in enhanced oxidation of plasma proteins (GE: ↑ AOPP, ↑ IMA; TT: ↑ AOPP) and lipids (GE: ↑ TBARS), which indicates the occurrence of systemic oxidative stress. This may be confirmed by the negative correlation between GSH concentration and TBARS and AOPP and between redox ratio and TAC, TBARS, and AOPP. Of particular note is the increase in IMA levels during hypoxic exercise. IMA is the earliest biomarker of tissue ischemia, whereas decreased oxygen saturation, ischemic reperfusion, acidosis, sodium/calcium pump dysfunction, and higher oxidative stress are factors causing conformational changes of albumin [57]. The increase in total antioxidant capacity may also be controversial (GE: ↑ TAC both after exercise and hypoxia vs. normoxia). Nevertheless, 70-80% of plasma TAC represents nonenzymatic uric acid (UA), with a robust prooxidant effect in high concentrations [83,84]. UA can generate free radicals by reacting with peroxynitrite or forming alkylated proteins, lipids, and carbohydrates [85]. Higher UA concentrations were observed in previous studies after one-time and regular high-intensity physical training [2,20,[86][87][88]. UA is the end product of purine catabolism formed in a XO-catalyzed reaction from xanthine. Under hypoxic/ischemic conditions, hypoxanthine formed from ATP decomposition is accumulated in the cell and then metabolized to xanthine with the generation of ROS/RNS upon reperfusion [89,90].
The H 2 O 2 production may also be affected by nitric oxide metabolism [91,92]. In our study, higher NO x bioavailability with a concomitant increase in CAT activity could be explained by intensified peroxynitrite (ONOO -) formation influenced by an acute hypoxic intervention. Indeed, the superoxide radicals formed in the XO-catalyzed pathway react with NO to generate the highly reactive ONOO - [92]. Peroxynitrite is a powerful oxidizing and nitrating agent that initiates lipid peroxidation and oxidation of thiols/aromatic amino acids with an efficiency of at least 1000-fold higher than hydrogen peroxide [93,94]. Tyrosine residues are particularly sensitive to ONOOdamage; hence, the increase in 3-NT concentrations (both after exercise and in hypoxic conditions) is not surprising. Interestingly, peroxynitrite formation occurs typically under increased systemic inflammation [93,94]. This may be supported by the results of our study (GE: ↑ NO, ↑ ONOO -, ↑ MPO, ↑ TNF-α). Of particular note is higher MPO activity after hypoxic exercise. Indeed, MPO is released by neutrophils and monocytes during inflammatory cell activation [95]. It is involved in hypochlorous acid production, which exacerbates oxidative stress and initiates acute inflammation [95,96]. It is well known that higher secretion of cytokines, chemokines, and growth factors is a physiological response to decreased arterial blood O 2 saturation and microdamage of muscle fibers. Activated neutrophils and macrophages can remove fragments of damaged muscle tissue induced by NO and H 2 O 2 signaling [97,98]. Simultaneously, IL-1, IL-2, IL-6, and TNF-α may stimulate white blood cells to produce significant amounts of NO through prolonged iNOS activation [99,100]. In these conditions, XO and 14 Oxidative Medicine and Cellular Longevity NOX are also induced, which, by positive feedback, enhances nitrosative cell injury (GE: ↑ NO x , ↑ ONOO -, ↑ 3-NT). A consequence of enhanced inflammatory response and oxidative/nitrosative stress can be damage to the lysosomal membrane and the release of lysosomal enzymes into the circulation (GE: ↑ HEX, ↑ GLU). Interestingly, lysosomal dysfunction, mitochondrial energy metabolism, and impaired ion homeostasis are essential sources of ROS during physical exercise [101][102][103]. However, NO signaling activity may also depend on protein S-nitrosylation, as evidenced by increased S-nitrosothiols under hypoxic conditions. It is well known that NO-mediated protein S-nitrosylation plays a vital role in the adaptation to endurance exercise/hypoxia by increasing the PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) expression [104,105]. Nevertheless, enhanced S-nitrosylation can also end in the formation of protein disulfide and a nitroxyl residue, which irreversibly alters the biological properties of proteins. Physical exercise is indicated in both health and disease. Although our study does not explain it, individuals with diseases with oxidative stress etiology (e.g., metabolic, neurodegenerative, and immune diseases) should be cautious during acute hypoxic training. This may exacerbate disturbances in redox homeostasis and inflammation. Antioxidant supplementation during acute hypoxic exercise also remains an open question.
Unfortunately, our work has numerous limitations. These include the relatively small number of participants and the evaluation of only selected biomarkers of oxidative stress, inflammation, and lysosomal function. Our study also does not explain the molecular mechanisms responsible for the observed redox disturbances. Research on nonprofessional athletes is also essential.
To summarize, our study shows that even a single session of physical exercise disrupts the enzymatic and nonenzymatic antioxidant barrier leading to enhanced oxidative and nitrosative damage at a systemic level. High-intensity exercise of short duration alters redox homeostasis more than prolonged aerobic exercise, while hypoxia significantly exacerbates oxidative stress, inflammation, and lysosomal dysfunction in athletic subjects. Although we have reported the most commonly assessed circulating redox biomarkers, further studies are needed to elucidate the molecular basis of the observed relationships. Studies on larger groups of athletes are also advisable.

Data Availability
The datasets generated for this study are available on request to the corresponding author.

Ethical Approval
The investigation conformed with the principles outlined in the Declaration of Helsinki and was approved by the Bioethics Committee of the Medical University of Bialystok (approval no. R-I-002/325/2019).

Conflicts of Interest
There is no conflict of interests.