Hypoxia/Reoxygenation modulates Oxidative Stress Level and Antioxidative Potential in Lung Mitochondria: Possible participation of P53 and NF-KB Target Proteins

Background and objective: Hypoxia/reoxygenation (H/R) is a key factor in the pathogenesis of the most lung diseases where exсessive ROS production and prooxidant/antioxidant imbalance greatly contribute to disease progression. We have used severe hypoxia in sessions of repeated H/R of different duration as a model of lung pathologic states to investigate mitochondrial oxidative stress intensity, protein expression/activity of antioxidant enzymes manganese-superoxide dismutase (MnSOD), glutathione peroxidase (GPx), and antiapoptotic Bcl-2 as well as protein expression of their upstream regulators: p53 and nuclear factorkappa B (NF-kB). Methods: A total 86 rats were divided into fi ve experimental groups and subjected to H/R [5 cycles of 10 min hypoxia (5.5 % O2 in N2) alternated with 10 min normoxia, daily]. Eight rats from each group were sacrifi ced on 1st -, 3rd day, 1st and 2nd week time points. Oxidative stress biomarkers (ROS formation, lipid peroxidation, H2O2 production, GSH/GSSG ratio, and mitochondrial aconitase activity as marker of compartment-specifi c superoxide anion production), indices of antioxidant status (MnSOD, GPx, glutathione –S-transpherase activities, and reduced glutathione level) were measured in lung mitochondria. Western blot was used to detect the protein levels of p53, Bcl-2, MnSOD, and GPx in mitochondria as well as the phosphorylated NF-kB p65 in the nucleus of lung cells. Expression of mRNA MnSOD was determined by real-time polymerase chain reaction. Results: The short(1-3 days) and long-term (1-2 wk) H/R differentially affects the oxidative stress level, p53 protein expression and its subcellular distribution as well as antioxidant capacity in lung mitochondria. The longterm H/R caused mitochondrial p53 protein translocation, a decrease in Bcl-2 protein content, and a signifi cant increase in nuclear accumulation of the phosphorylated NF κB p65 protein. We observed an increase in GPx protein content/activity, in parallel with decrease in MnSOD protein level and activity. In the dynamics of MnSOD gene expression we found a phase time point dependence. Conclusions: Long lasting H/R leads to mitochondrial prooxidant/antioxidant disbalance that resulted in redox alteration as consequence of oxidative stress propagation and apoptotic cascade activation. A close correlation between mitochondrial p53 Protein level and protein expression/activities of its targets MnSOD and GPx suggest participation of p53 in regulation of H/R-induced mitochondrial oxidative stress level. Research Article Hypoxia/Reoxygenation modulates Oxidative Stress Level and Antioxidative Potential in Lung Mitochondria: Possible participation of P53 and NF-KB Target Proteins Olga Gonchar* and Irina Mankovska Department of Hypoxic States, Bogomoletz Institute of Physiology National Academy of Sciences of Ukraine, Ukraine Dates: Received: 07 April, 2017; Accepted: 16 May, 2017; Published: 19 May, 2017 *Corresponding author: Olga Gonchar, Department of Hypoxic States, Bogomoletz Institute of Physiology NAS of Ukraine, Bogomoletz str, 4, 01024, Kyiv, Ukraine, Tel: + 38 044 2562492; E-mail:


Introduction
The repeated episodes of hypoxia and reoxygenation (H/R) are wide spread phenomena because occurs in a variety of pathological conditions. Oxygen deprivation during hypoxia and subsequent reoxygenation are thought to be the major factors contributing to ROS production and oxidative stress development [1,2]. Indeed, there is increasing evidence that ROS generation and oxidant/antioxidant imbalance play a major role in a wide range of lung pathological processes including sleep disordered breathing, chronic obstructive pulmonary disease, asthma attacks, lung ischemia/reperfusion injury, participation of P53 and NF-KB Target Proteins. Arch Pulmonol Respir Care 3(2): 035-043. etc [3][4][5][6][7]. Current fi ndings suggest that the primary sensor of hypoxia is mitochondria, which increases the ROS production at low pressures of O2 and simultaneously is subject to direct attack by ROS. Lung mitochondria are protected against these oxidants by a variety of antioxidant mechanisms among which manganese-superoxide dismutase (MnSOD) and glutathione peroxidase (GPx) constitute not only the fi rst line of defense but also are essential for maintaining the critical cellular redox balance and play a key role in modulating cellular responses to external stimuli [4,[6][7][8].
MnSOD is a homotetramer found exclusively in the mitochondrial matrix. Altered expression or enzyme activity of MnSOD has been linked to many pathologic conditions suggesting an important role for the modulation of mitochondrial ROS in the disease development [4,9,10].
The expression of MnSOD can be regulated at multiple levels from transcription and translation to posttranslational modifi cations by various extracellular and intracellular factors [4,11,12]. However, the precise mechanisms underlying MnSOD regulation in lung tissue at H/R of different duration are not clear.
The increase in the cell ROS often involves the activation of intracellular signaling pathways, which, in turn, regulates expression of a number of genes encoding antioxidant proteins, DNA repair proteins, stress-regulated chaperones, and antiapoptotic proteins [13]. The redox-sensitive transcription factors nuclear factor-kappaB (NF-kB) and p53 are considered as important stress sensors playing crucial role in determining cellular fate during oxidative stress [1,14,15]. Current data have showed that hyper-physiological and physiological levels of p53 exert different effects on cellular redox status either through directly regulating the expression of pro-oxidant and antioxidant genes or through modulating the cellular metabolic pathways [16,17]. P53 can upregulate the expression of various antioxidant enzymes such as aldehyde dehydrogenase 4, mammalian sestrin homologues that encode peroxiredoxins, and GPX1 [14]. The relationship between mitochondrial p53 protein level and both MnSOD and GPx were observed in many cell lines [16,17] supposing that p53 may regulate protein expression/activity of these antioxidant enzymes under oxygen deprivation.
NF-kB binding sites were found to be located in the regulatory regions of the SOD2 gene, which encodes MnSOD [18], providing direct evidence of the NF-kB -MnSOD connection.
In addition, oxidative mechanisms may also regulate GPX1 gene transcription via NF-kB sites in the promoter region [19]. It is well known that the ROS-dependent NF-kB activation leads to an increase in MnSOD as well as GPx expressions [11,18,19].
Recent studies have shown that acute and long-term periodic hypoxia/reoxygenation differentially affect the various metabolic processes; in some cases, prolonged H/R was found to elicit a preconditioning-like effect. The duration, frequency, and severity of hypoxic episodes are critical factors determining whether exposure to cyclic H/R has benefi cial or harmful effects on tissues [8]. Although oxidative damage associated with severe hypoxia/reoxygenation in lung tissue has been the subject of many studies [2][3][4][5][6][7], the knowledge of these effects in lung mitochondria, particularly concerning expression and activity of stress-inducible proteins, is relatively poor.
Based on previous research, the present study have been focused on the problem how the antioxidant defense system as well as p53 and NF-kB p65/RelA proteins produced by lung can be modulated by severe hypoxia in sessions of H/R of different duration. We assessed the mitochondrial oxidative stress level and redox status with an ample array of oxidative stress biomarkers and found that these changes were time-dependent, at once H/R infl uenced on the p53 and phosphorylated NF-kB p65 protein expression and distribution in cell compartments.
The modulation of the protein expression/ activity of MnSOD, GPx, and ant apoptotic Bcl-2 as well as mRNA MnSOD expression in response to varying oxygen level were analyzed.

Animals and study design
Wistar rats weighing 220-260g were used. They were housed in Plexiglas cages (4 rats per cage) and maintained in an air-fi ltered and temperature controlled (20-22°C) room.
Rats received a standard pellet diet and water ad libitum and were kept under artifi cial light-dark cycle of 12h. The

Mitochondrial fraction preparation
Rat lung mitochondria were isolated by differential centrifugation. Tissue was collected in isolation medium A

Oxidative stress biomarkers assays
The data on ROS formation were obtained from dichlorofl uorescein (DCF) fl uorescence. Mitochondria were loaded for 20 min at 37°C with membrane permeable nonfl uorescent probe (2',7'-dichlorodihydrofl uorescein diacetate, DCFHDA) which is known to be decomposed in the matrix to give dichlorofl uorescein upon oxidation by matrix ROS, primarily hydroperoxide and superoxide. DCF was excited at 504 nm, and the emission was registered at 525 nm.
Lipid peroxidation in isolated mitochondria was measured from the formation of thiobarbituric acid -reactive substances (TBARS) using the method of Buege and Aust [20].
H2O2 concentration in lung mitochondria was measured by the FOX method, based on the peroxide-mediated oxidation of Fe2+, followed by the reaction of Fe3+ with xylenol orange [21].
Manganese superoxide dismutase (MnSOD) activity was measured by the method of Misra and Fridovich [24], which is based on the inhibition of autooxidation of adrenaline to adrenochrome by SOD contained in the examined samples.
The samples were preincubated at OoC for 60 min with 6 mM KCN, which produces total inhibition of Cu, Zn-SOD activity.
The results were expressed as specifi c activity of the enzyme in units per mg protein. One unit of SOD activity being defi ned as the amount of protein causing 50% inhibition the conversion rate of adrenaline to adrenochrome under specifi ed conditions. Aconitase activity was measured spectrophotometrically by monitoring the formation of cis-aconitate from isocitrate at 240 nm in 50 mM Tris HCl buffer (pH 7.4), containing 0.6 mM MnCl2 and 20 mM isocitrate at 250C [25].

Glutathione content assays
Total glutathione -the sum of reduced glutathione and oxidized glutathione (GSH and GSSG) − was determined by a method where glutathione is extracted from the lung mitochondria with 5% ice-cold-sulfosalicytic acid and after neutralization with triethanolamine sequentially oxidized by DTNB (0.6 mM) and reduced by NADPH (0.3 mM) in the presence of glutathione reductase (2 U/ml ) [26]. For determination the GSSG alone, the GSH presented in solutions was derivatized by incubation with 2 μL 2-vinilpyridine at 40C for 1h. The rate of 2-nitro-5-thiobenzoic acid formation was monitoring at 412 nm and compared to a standard curves made with GSH and GSSG, respectively. The GSH concentration is calculated as

RNA extraction and RT-PCR for MnSOD analysis
Total RNA was isolated using a commercial kit "Trizol RNA Prep100" (Isogen, Russia) according to the  There were no signifi cant differences in intensity of GAPDH levels between experimental groups.

Statistical analysis
Data are expressed as mean ± standard deviation for each group. The differences among multiple experimental groups were detected by one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test. The correlation between pairs of variables was analysed using the bivariate Pearson method. A P value of less than 0.05 was considered as signifi cant.

Results and Discussion
In the present study, the severe hypoxia using in sessions of intermittent hypoxia caused in lung mitochondria a signifi cant increase in ROS formation as well as a rise in TBARS and H2O2 production ( Figure 1). It is known that hypoxia as well as reoxygenation can induce excessive ROS generation as a result of the univalent reduction of molecular oxygen to O2 • − by electrons that leak from the mitochondrial electron transport chain, mainly from complexes I and III [1]. Our data confi rmed that mitochondrial oxidative stress was involved already at an early phase of H/R condition -on the fi rst day of exposure and continued the whole study period for 2 weeks. The intensity of the oxidative processes reduced on 3rd day and gradually enhanced at 1 and 2 weeks of H/R exposure ( Figure 2). These data are in agreement with the results described in other model systems, which demonstrated a signifi cant increase in lipid peroxidation measured in lung homogenates [2,3,7] and mitochondria [27]. The intensifi cation of oxidative processes in mitochondria was accompanied by an increase in GSSG content and decrease in GSH concentration as well as in GSH/ GSSG ratio (P<0.05) ( Table 1), which are essential indicators of oxidative stress and mitochondrial dysfunction [6,8]. In addition, we registered a decrease in mitochondrial aconitase activity (Figure 2), which evidences an increase in superoxide anion production in mitochondria. Moreover, inactivation of aconitase may block normal electron fl ow to oxygen, leading to an accumulation of reduced metabolites such as NADH.
This condition has been termed ''reductive stress,'' and can causes an increased production of reactive oxygen species through autoxidation of the reduced metabolites, thus further increasing oxidative damage [28].
Generally, an increased production of ROS triggers an array of signal transduction pathways, resulting in stimulatory or inhibitory output signals [13]. In the present study, we have It is known that the relationship between p53 and ROS is quite complex. On the one hand, excessive ROS may cause p53 translocation to mitochondria and enhance mitochondrial oxidative stress leading to apoptosis. On the other hand, p53 can also affect ROS production and pro-/antioxidative balance in mitochondria by impact on its targets MnSOD and GPx [13,16,17].
We found that the repetitive situation of severe hypoxia followed by reoxygenation caused a disturbance of the mitochondrial pro-oxidant/antioxidant homeostasis, which manifestated in the alteration of MnSOD and GPx protein  Our experimental data showed the decrease in content of the reduced glutathione, which are in agreement with several early reports that severe hypoxia depletes GSH stores in lung mitochondria as well as in lung homogenates [2,27] and leads to a short-term fall in intracellular GSH levels in rat endothelial and alveolar cells both in vitro and in vivo [3,6]. Depletion of the reduced glutathione seems to be occurred by conjugation reactions via the GST, which activity in this study has arisen (Figure 2). GSTs are a family of dimeric proteins actually regarded as a major detoxifi cation tool against oxidative damage, as they catalyze the conjugation to GSH of a wide number of toxic elecrophilic compounds produced under oxidative stress conditions [5,6,8]. Moreover, changes in the GSH/GSSG ratio, which we found under H/R condition (Table  1) can regulate H2O2 formation, alter protein redox status and in that way modulate many signaling pathways including the activation of NF-kB [34][35].
Previously, we have demonstrated that acute severe hypoxia induces destructive changes within lung parenchyma, including the surfactant system-forming structures (delamellation of type II pneumocytes, edematous changes, damage to the alveolar lining layer, and accumulation of alveolar macrophages) [2]. In addition to cellular membrane damage and alteration in the lung lipid composition, the oxygen deprivation produced lung infl ammation due to neutrophil infl ux into the airways, which was associated with NF-kB activation and the high expression of IL-8 mRNA in alveolar macrophages and, to a lesser extent, in alveolar epithelial cells [1,7]. Emerging data indicate that chronic intermittent hypoxia as well as high altitude exposure cause NF-B activation and induce production of proinfl ammatory chemokines, cytokines, and adhesion molecules contributing to tissue injury, which, in turn, activates signaling cascades to enhance NF-B activation [1,3,36].
NF-kB is a ROS regulated and redox-sensitive factor that normally is confi ned to the cytoplasm in unstimulated cells and translocates to the nucleus in response to various stimuli including oxidative stress [13]. It is known, that under resting conditions, NF-B is bound to its co-repressor molecule IB in the cytosol. Upon stimulation, like intermittent hypoxia, IB is degraded and allows p50 and p65 to form NF-B heterodimer, which then translocates to the nucleus and activates transcription of target genes, such as infl ammatory factors and prosurvival proteins [15]. The NF-kB activation is provided by a series of redox-sensitive protein kinases that promote the dissociation of the IkB-NF-kB complex by phosphorylation of either IkB or NF-kB. It was also observed that a decrease in the intracellular pool of GSH followed by a subsequent increase in the level of GSSG, as well as a rise in H2O2 formation mediate IkBa phosphorylation and subsequent activation of NF-kB [34,35,37].
The phosphorylation of the subunit p65/RelA displays the NF-kB activation and is required for expression of a subset of NF-kB -dependent genes [38]. We measured the amount of phosphorylated NF-kB p65 by Western blot in nuclear extracts of lung cells sampled at different time points following sessions of H/R. Our results have shown the nuclear accumulation of the phosphorylated NF B p65 protein in lung cells gradually increased during the whole study period by 35, 55, 149, 135% on 1st , 3rd , 1st and 2nd weeks of H/R exposure, respectively, P<0.05) (Figure 8). Theoretically, the activated NF B p65 can be translocated to the nucleus to promote the transcription of the target genes including pro-survival MnSOD, GPx, and Bcl-2 [35]. In our experimental model the phosphorylated NF-kB p65 protein accumulation positively correlated with the ROS formation (r = 0.58) and GPx protein content (r=0.96). These fi ndings are consistent with previous studies showing that the high level of oxidative and nitrosative stresses promoted the induction of GPx mRNA transcription as well as protein expression in various cell lines [39] including via NF-kB sites in the promoter region [19]. Furthermore, a close correlation was identifi ed between phosphorylated NF-kB p65 protein content and mRNA MnSOD expression (r =0.74). All these observations as well as an increase in MnSOD protein content after 2 weeks of H/R exposure make it possible to assume that long-lasting H/R can induce MnSOD protein and mRNA expression via NF-B activation. Early, it was demonstrated that hypoxia decreased the both MnSOD activity and protein expression in rabbit lung [40] and decreased MnSOD mRNA expression in cultured rabbit alveolar type II epithelial cells and lung fi broblasts after exposed to 5% hypoxia [41]. On the other hand, some authors demonstrated an increase in the MnSOD protein expression and MnSOD mRNA content in rat lungs [42]. In our previous research, we found that prolonged moderate hypoxia/hyperoxia enhanced MnSOD protein expression without changes in mRNA MnSOD content in lung tissue [27].
It is widely known that MnSOD regulation is complex and occurs at both pre-and post-translational levels. It was reported that MnSOD mRNA levels can be up-or downregulated by several factors including VEGF, AP-2, Egr-1, Sp-1, p53, HIF-2 alpha, PKC-NF-B, PI3K-Akt-Forkhead signaling pathways [1,[11][12][13][14]18,33]. These facts indicate that the overall regulation of Mn-SOD is multifactorial and cross-talk between redoxsensitive pathways are required for synergic activation of MnSOD gene. Which of these signaling pathways is involved in the regulation of MnSOD at H/R condition is apparently dependent on the strength, duration, and type of hypoxic exposure. We consider that this problem requires attention for further researches.

Conclusion
We found that short-and long-term H/R differentially