Celecoxib decreases mitochondrial complex IV activity and induces oxidative stress in isolated rat heart mitochondria: An analysis for its cardiotoxic adverse effect

In spite of the cardiotoxic effect of selective cyclooxygenase‐2 inhibitors, they are most widely used as anti‐inflammatory and analgesic drugs. Today, valdecoxib and rofecoxib have been withdrawn in the market but celecoxib remains. In this study, we focused on an analysis of celecoxib toxic effects on isolated mitochondria. Isolated rat heart mitochondria were obtained using differential centrifugation. Using flow cytometry and biochemical assays, we searched succinate dehydrogenases, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) formation, mitochondrial swelling, ATP/ADP ratio, lipid peroxidation, and mitochondrial complexes activity in rat heart isolated mitochondria. Herein, our results indicated a significant decrease in the activity of complex IV after exposure with celecoxib (16 µg/ml). This decrease in the activity of complex IV is paralleled by the MMP collapse, ROS formation, mitochondrial swelling, depletion of ATP, and lipid peroxidation. For the first time, this introductory study has shown a significant decrease in the activity of complex IV and mitochondrial dysfunction after exposure with celecoxib in rat heart isolated mitochondria.

Multiple lines of evidence strongly suggest that the cardiotoxicity of NSAIDs direct or indirect are associated with mitochondria, which comprises inhibition of the important mitochondrial enzymes, interference with the mitochondrial respiratory chain and inhibition of the mitochondrial DNA replication which eventually leading to an increase in mitochondrial oxidative stress, loss of mitochondrial membrane potential and cell death. [12][13][14] Mitochondria play a critical role in supplying adenosine triphosphate (ATP) for the cells. Therefore, the cells and tissues with a high aerobic energy requirement are more sensitive to mitochondrial toxic agents. [15] Also, the very important enzymes such as heme synthesis, fatty acid urea synthesis, and β-oxidation reside within the mitochondrial matrix. [15] As a result, tissues like cardiac tissues that rely heavily on these processes are almost targets of mitochondrial toxins. Cardiomyocytes contain a large number of mitochondria, and when enough mitochondria are irreparably damaged and the cell cannot provide its energy requirements, in this condition, cellular injury or dysfunction will occur. [16,17] These unique functions of mitochondria in cardiomyocytes stimulate researchers to pay more attention to the interrelationships between drugs and mitochondria for both the toxic and therapeutic aspects.
Also, a published work indicated that the predictive performance of the mitochondrial assays for the evolution of cardiotoxicity is 33%. [18] In spite of significant efforts for the identification of drug-related cardiotoxicity in preclinical assessments, safety concerns still remain. This is most likely due to the lack of sufficient understanding of the molecular pathways leading to cardiotoxicity. [19] The exact mechanism by which celecoxib induces cardiotoxicity is not known.
Mitochondria have an essential role in myocardial tissue homeostasis; thus, deterioration in mitochondrial function could result in cardiomyocyte and endothelial cell death and consequently cardiovascular dysfunction. To our knowledge, there are no reports to date about the effect of celecoxib on heart isolated mitochondria and mitochondrial complexes. Thus, the present study aims to study the effect of celecoxib on mitochondrial complexes and toxicity parameters.

| Mitochondrial isolation
Cardiac mitochondria were isolated from male Wistar rats. Briefly, the animals were deeply anesthetized by a combination of ketamine (50 mg/kg) and xylazine (10 mg/kg) via intraperitoneal (i.p.) injection and were euthanized by decapitation, then the heart was dissected, chopped, cleared from blood, and minced with 10 ml glass homogenizer in the isolation buffer (225 mM D-mannitol, 75 mM sucrose, and 0.2 mM EDTA, pH 7.4) on ice. The obtained samples were centrifuged at 1000 g for 10 min and the pelletest,t was removed. The supernatant with mitochondria was poured into an ice-cold tube, followed by centrifugation at 10,000 g for 10 min. [20] The mitochondrial enriched pellets were suspended in an appropriate buffer for each test, including succinate dehydrogenases (SDH) activity, mitochondrial membrane potential (MMP) collapse, reactive oxygen species (ROS) formation, and lipid peroxidation (LP) assay. The Bradford assay was used for the protein content of mitochondria to standardize. [21] The protein concentration of the mitochondria for the reach test was adjusted to 1 mg/ml. The integrity and purity of mitochondria were tested by using SDH and lactate dehydrogenase assays. Celecoxib was freshly prepared before use and dissolved in 0.05% ethanol and isolated mitochondria were treated with celecoxib and other chemicals for one hour (60 min).

| Mitochondrial function
The SDH activity or complex II was measured as a mitochondrial function in isolated mitochondria using MTT reduction at 570 nm. [22] Briefly, after incubation of rat heart isolated mitochondria in assay buffer (3 mM HEPES, 5 mM succinate, 70 mM sucrose, 2 mM Trisphosphate, 230 mM mannitol, and 1 µM of rotenone) with celecoxib (0, 1, 10, 20, 50, and 100 µg/ml) at 37°C for 60 min. After 1 h 0.4% MTT was added to the medium and incubated at 37°C for 30 min.
The purple formazan crystals were dissolved in DMSO and the absorbance was measured at 570 nm with an ELISA reader (BioTek). [23] 2.5 | ATP/ADP ratio assay ADP/ATP ratio was assessed by ADP/ATP Ratio Assay kit (MAK135 Sigma) in isolated mitochondria using luminometer. ADP/ATP ratio was performed according to the manufacturer's instructions. [24] 2.6 | Determination of mitochondrial swelling Swelling of mitochondria as an indicator of mitochondrial permeability transition pore (mPTP) opening in the presence or absence of celecoxib was determined by monitoring the decrease in light scattering at 540 nm as described previously. [25] Rat heart isolated mi- intensities were compared between groups. [23] 2.9 | Quantification of lipid peroxidation Lipid peroxidation was measured by using the thiobarbituric acid assay and malondialdehyde (MDA) formation. The rat heart isolated mitochondria were exposed with and without celecoxib at 37°C for 60 min. Then, mitochondria were lysed in a tube containing 1 ml 0.1% (w/v) TCA and centrifugated at 10,000 g for 10 min. The obtained supernatant was transported to a new tube containing 4 ml of 20% TCA and 0.5% TBA. The mixture was boiled at 95°C for 15 min and quickly cooled on ice. The tubes were centrifugated again 10,000 g for 5 min and the optical density of supernatant measured at 532 nm. [26] 2.10 | Mitochondrial complexes estimation The reaction was started by adding 6 µl of ubiquinone 1 (10 mM), and followed the decrease of absorbance at 340 nm for 2 min (Table 1). [27] 2.10.2 | Complex-II (SDH activity) To assay the complex II activity, 10 µg of rat heart isolated mi- water. After mixing, the sample was incubated inside the spectrophotometer at 37°C for 10 min and then read the baseline activity at 600 nm for the last 2 min. The reaction was started by adding 4 µl of 12.5 mM DUB and followed the decrease in absorbance at 600 nm for 3 min. The specificity of complex II activity was checked by running the assay after the addition of 10 µl of 1 M malonate before starting the reaction (Table 1). [27] 2.  (Table 1). [27] 2.10.4 | Complex IV (cytochrome c oxidase) To assay the complex IV activity, 400 µl of distilled water, 500 µl of potassium phosphate buffer (100 mM, pH 7.0), 60 µl of reduced cytochrome c (1 mM) were added to 24-well plates and read the baseline activity at 550 nm for the last 2 min. in the following, was adjusted the volume to 995 µl with distilled water. The reaction was started by adding 5 µl of sample (2.5 µg of rat heart isolated mitochondria proteins) then after the mixing was monitored the decrease of absorbance at 550 nm for 3 min. The specificity of complex IV activity was checked by adding 30 µl of 10 mM KCN in a separate reaction prepared as described above (Table 1). [27] 2.10.5 | Complex I + III (NADH cytochrome c oxidoreductase) To assay the complex, I + III activity, 6 µg of rat heart isolated mitochondria in 700 µl of distilled water in a 4-ml well was incubated  (Table 1). [27] 2.10.6 | Complex II + III (succinate cytochrome c reductase) To assay the complex II + III activity, at 24-well plates, was added the sample (1 µg of rat heart isolated mitochondria), 800 µl of distilled water, 25 µl of succinate (400 mM), 30 µl of KCN (10 mM), 40 µl of potassium phosphate buffer (0.5 M, pH 7.5), and then was adjusted the volume to 950 µl with distilled water. After mixing, the plate was incubated for 10 min inside the spectrophotometer at 37°C. In the following, the reaction was started by adding 50 µl of oxidized cytochrome c (1 mM), after mixing the well, the increase of absorbance at 550 nm was followed for 3 min. The specificity of this assay was checked by adding 10 µl of 1 M malonate or 1 mg/ml antimycin A in a separate well prepared as described (Table 1). [27] T A B L E 1 Conditions for spectrophotometric assays of respiratory chain enzymes activities in the rat heart isolated mitochondria

| Statistical analysis
Using Graph Pad Prism (version 5, Graph Pad Software Inc.), the results were analyzed. Results were analyzed using the one-way analysis of variance (ANOVA) test, followed by the post hoc Tukey posttest and two-way ANOVA followed by the posttest Bonferroni in triplicate. Data were presented as mean ± SD. Statistical significance was set at p < 0.05. Also, the flow cytometric data was obtained with Cyflow Space-Partec and analyzed by FlowJo.

| Celecoxib decreases mitochondrial function in rat heart isolated mitochondria
MTT assay was performed for evaluation of SDH activity as an indicator of mitochondrial function. Isolated mitochondria were treated with various concentrations of celecoxib (0, 1, 10, 20, 50, and 100 µg/ml) at 60 min. As shown in Figure 1, the mitochondrial metabolism of MTT to formazan significantly (p < 0.001) decreased by celecoxib. Celecoxib at concentration 16 µg/ml decreased %50 the mitochondrial function in isolated mitochondria ( Figure 1A).

| Celecoxib decreases ATP production in rat heart isolated mitochondria
ATP/ADP ratio was assessed by ADP/ATP Ratio Assay kit (MAK135 Sigma) in isolated mitochondria using luminometer.

| Celecoxib increases mitochondrial ROS in rat heart isolated mitochondria
As shown in Figure 2

| Celecoxib induces mitochondrial swelling in rat heart isolated mitochondria
Mitochondrial swelling and mitochondrial membrane permeability were monitored at 540 nm. Decrease in absorbance at 540 nm is an indicator of mitochondrial swelling. As shown in Figure 3, celecoxib 3.7 | Celecoxib decreases complex I V activity in rat heart isolated mitochondria

| Mitochondrial complexes estimation
Conditions for spectrophotometric assays of respiratory chain enzymes activities and mitochondrial complexes enzymatic activity in the presence or absence of celecoxib in rat heart isolated mitochondria were presented in Table 2. Complex I V activity was significantly reduced by celecoxib to 47.48% of control activity. Activities of complex I, complex II, complex III, complex I + II, and complex II + III were not affected by celecoxib. In summary, these results confirm that complex IV is the principal oxidative phosphorylation (OXPHOS) target for celecoxib in rat heart isolated mitochondria.

| DISCUSSION
Cardiovascular disorder is one of the outstanding causes of death in the worldwide. [28] There are many documents that showed a strong relationship between mitochondrial dysfunctions and cardiac F I G U R E 2 Effect of celecoxib on ROS formation in isolated rat heart mitochondria. Changes in ROS generation were measured in isolated mitochondria after treatment with celecoxib (16 µg/ml) for 1 h. The fluorescence intensity of DCF for 10,000 events was significantly increased in celecoxib-treated mitochondria. DCF, 2′,7′-dichlorofluorescein; ROS, reactive oxygen species F I G U R E 3 Effect of celecoxib on mitochondrial swelling in isolated rat heart mitochondria. Mitochondrial swelling was monitored by following 540 nm absorbance decrease. Celecoxib at concentration 16 µg/ml induced mitochondrial swelling in isolated rat heart mitochondria in a time depending manner. Values were expressed as mean ± SD of three separate determinations. ***indicates p < 0.001 versus control. SD, standard deviation diseases. [29] However, understanding of cellular and molecular mechanisms of mitochondrial dysfunctions in treating cardiac disorders and the development of safe prescription drugs without cardiac toxicity is still considered a challenge. [17] Notwithstanding toxic side effects, celecoxib, as a selective COX-2 inhibitor, is usually prescribed, because of its benefits in different cases. [30] Based on such controversy and given the role of mitochondria in causing cardiac toxicity, in the current study, we analyzed the effect of celecoxib on the rat heart isolated mitochondria.
In a concentration-dependent use of celecoxib, mitochondrial function decreases considerably. In our experiments, the rat heart isolated mitochondria treated with 1-100 µg/ml of celecoxib for 1 h showed a similar pattern in cellular study by others. [31] A previous study has shown that celecoxib is rapidly absorbed and achieves peak serum concentration in approximately 1-3 h. [32] This experiment showed that 16 µg/ml of celecoxib decreases 50% of the mitochondrial function in rat heart isolated mitochondria. This preliminary result showed that concentrations of 1 µg/ml of celecoxib are relevant to clinical exposure (1.28 µg/ml), [33] although higher concentrations of the drug, such as 10-100 µg/ml are largely used in cell culture models. [31,34] Our results showed that celecoxib at a concentration of 1 µg/ml could reduce mitochondrial activity and produce ATP in isolated mitochondria, so this concentration is close to the clinical situation ( Figure 1A,B). The 16 µg/ml concentration as IC50 was kept to search the celecoxib effect in the rat heart isolated mitochondria, as in patients under high concentrations of celecoxib administration. [35] ROS have a major role in controlling the cell growth and death of cardiac cells. [36] Mitochondria are major targets for ROS damage and also, they are a major source of ROS in cardiomyocytes. [37] In clinical studies and numerous experiments have been reported that accumulation of ROS leads to the failing myocardium. In cardiac pathological conditions such as cardiac ischemia-reperfusion injury (IRI), cardiac hypertrophy, heart failure, and diabetic cardiomyopathy, dysregulated ROS formation and oxidative stress have been indicated. [38] The main source of ROS formation in the heart is related to uncoupling of mitochondrial electron transport chain at the level of complexes. [39] Our results on the rat heart isolated mitochondria indicated that celecoxib could cause ROS formation and oxidative stress. The functions of mitochondrial respiratory chain and other proteins in the mitochondria are sensitive to oxidative changes and F I G U R E 4 Effect of celecoxib on ΔΨm in isolated rat heart mitochondria. Freshly isolated mitochondria were incubated with 16 µg/ml celecoxib for 1 h. ΔΨm was measured following rhodamine 123 staining with flow cytometry. The presented data revealed that exposure to celecoxib caused a significant increase in the fluorescence intensity of rhodamine 123 and it reflects MMP collapse. MMP, mitochondrial membrane potential F I G U R E 5 Effect of celecoxib on lipid peroxidation in isolated rat heart mitochondria. Induction of lipid peroxidation in isolated mitochondria and after incubation with celecoxib. Lipid peroxidation significantly increased when isolated mitochondria were incubated with celecoxib (A). Values were expressed as mean ± SD of three separate determinations. ***indicates p < 0.001 versus control. MDA, malondialdehyde; SD, standard deviation ROS formation, particularly as a major phospholipid in the inner mitochondrial membrane, which is called cardiolipin. [40] Cardiolipin is responsible for maintaining the functional integrity of the respiratory chain complexes and other proteins in the mitochondria. [41] Also, overexpression of ROS in the cardiomyocytes can result in mitochondrial dysfunction, mitochondrial swelling, oxidative damage to lipids, proteins, and DNA and finally activation of the mitochondrial-permeability transition pore (MPTP) and cell death. [38] Increased ROS formation was found to cause the opening of mPTPs allowing the release of cytochrome c, and induction of apoptosis via the mitochondrial pathway. [42] Previous studies have reported that many NSAIDs may induce cardiomyocyte apoptosis through ROS formation and mitochondrial dysfunction and increase the progression of cardiovascular toxicity. [14,43] Hasinoff et al. reported that celecoxib treatment causes apoptosis in myocytes. This evidence demonstrates that celecoxib exerts its cytotoxicity towards myocytes through COX-2-independent mediated pathways. [34] The obtained results in this study confirmed that exposure to celecoxib leads to mitochondrial swelling, lipid peroxidation, and MMP collapse in rat heart isolated mitochondria.
The greater part of ROS in the cell is generated as a by-product of mitochondrial respiration. The main sites of superoxide formation in mitochondria are complex I and complex III. [44] However, in addition to the above complexes, complex IV has also an important role in ROS generation. [45] It is known that changes in complex IV activity lead to alteration of the electron transport chain with an increase in the generation of ROS. [39] Furthermore, alterations in the mitochondrial membrane could be related to an increase of oxidative stress and cell death. [46] The role of complex IV in the activities of complexes I and III following their assembly into supercomplexes has been demonstrated. [47] Also, there are other potential sources of ROS that are located within the mitochondria including monoamine oxidases (MAO-A and -B), NADPH oxidase 4, and p66Shc. [48] Unlike COX-1, COX-2 is not limited to endoplasmic reticulum (ER) and is reported to be located in the nucleus and other organelles. [49] COX-2 in primary cultured human cells has been reported to localize to the ER, nuclear envelope, nucleus, and other organelles such as mitochondria. Our data indicate that celecoxib decreases mitochondrial complex IV activity and probably triggers mitochondrial damage and oxidative stress in isolated mitochondria.
It has been reported that drug-induced cardiotoxicity is closely associated with mitochondrial damage and oxidative stress. [50] 5 | CONCLUSION In conclusion, the presented data in the current study showed that celecoxib induces mitochondrial dysfunction in rat heart mitochondria owing to its ability to induce ROS production and complex IV inhibition, which led to permeabilizing the mitochondrial membrane and mitochondrial swelling. However, it is suggested these findings must be verified by in vivo studies.

DATA AVAILABILITY STATEMENT
Data are available if requested from authors.