The Role of Antioxidants in Ameliorating Cyclophosphamide-Induced Cardiotoxicity

The chemotherapeutic and immunosuppressive agent cyclophosphamide has previously been shown to induce complications within the setting of bone marrow transplantation. More recently, cardiotoxicity has been shown to be a dose-limiting factor during cyclophosphamide therapy, and cardiooncology is getting wider attention. Though mechanism of cyclophosphamide-induced cardiotoxicity is not completely understood, it is thought to encompass oxidative and nitrative stress. As such, this review focuses on antioxidants and their role in preventing or ameliorating cyclophosphamide-induced cardiotoxicity. It will give special emphasis to the cardioprotective effects of natural, plant-derived antioxidants that have garnered significant interest in recent times.


Introduction
1.1. Drug-Induced Cardiotoxicity. Drug-induced cardiotoxicity poses a serious risk to human health, and cardiooncology is currently becoming an important concern [1]. Antineoplastic treatments led to increased overall and progressionfree survival in the management of an increasing number of malignancies [2]. However, as cancer survival has improved with advancing therapies, late cardiovascular adverse effects have become an important management issue, mainly in childhood cancers, leukaemia, lymphoma, and breast cancer. In patients diagnosed with early stage breast cancer, cardiovascular disease is the major cause of mortality [3]. Even though anticancer drugs are targeted against malignant cells, they are also toxic to normal cells [4].
Patients who survived cancer, when compared to their healthy counterparts, are at an increased risk of cardiovascular-related mortality, which might be due to myocardial infarction with coronary artery disease, cardiomyopathy with congestive heart failure, and cerebrovascular events [5,6]. Patients on cancer chemotherapy can be considered as a stage A heart failure group, patients with increased risk of heart failure and do not have structural heart disease [7,8]. Total dose of the anticancer agent patient received, rate of drug administration, extent of radiation of the mediastinum, age, being female, previous history of heart disease, and increased blood pressure are risk factors to develop cardiotoxicity [9,10].
Antineoplastic agents are well known to cause a wide array of toxicities including cardiac dysfunction leading to heart failure, arrhythmias, myocardial ischemia, hypertension, thromboembolism, myocarditis, and pericarditis [11]. Anthracyclines are the best known of the chemotherapeutic agents that cause cardiotoxicity. In addition, alkylating drugs, including cisplatin, cyclophosphamide, ifosfamide, carmustine, chlormethine, busulfan, and mitomycin, are also linked with cardiac toxicity [9].

Cyclophosphamide.
Cyclophosphamide is an alkylating, anticancer agent which was first characterized in experiments on rat tumors. It is an oxazaphosphorinesubstituted nitrogen mustard, with strong cytotoxic and immunosuppressive activity [12]. It is the mainstay of most preparative regimens for organ transplant and a broadly active anticancer, immunosuppressive agent used in combination chemotherapy for Hodgkin's disease, non-Hodgkin's lymphoma, leukaemia, rheumatoid arthritis, Burkitt's lymphoma, lupus erythematosus, multiple sclerosis, neuroblastoma, multiple myeloma, endometrial cancer, breast cancer, and lung cancer. At high dosages, cyclophosphamide can be used alone or in combination with bone marrow transplant in the management of solid tumors and lymphomas [9,13].
The electrophilic nature of the alkyl group enables the drug to react with nucleophilic moieties of DNA or proteins, and this leads to the covalent transfer of an alkyl group. Cyclophosphamide is a prodrug that requires an activation step by cytochromes (P450) in the liver [14]. As shown in Figure 1, the introduction of the hydroxyl group to the oxazaphosphorine ring generates 4-hydroxycyclophosphamide, which cooccurs in equilibrium with its isomer, aldophosphamide. Then, aldophosphamide is converted into two compounds, phosphoramide mustard and acrolein ( Figure 1) [15].
Phosphoramide mustard forms a highly reactive cyclic aziridinium cation, which can react with the N(7) of the guanine and with cytidine from the DNA. Due to the two reactive moieties in the molecule, intrastrand and interstrand cross-links can be formed [16]. This leads to inhibition of DNA replication and apoptosis, with the active metabolites also having cell-cycle-independent activity. The specific mechanism of action of the compound used in managing autoimmune diseases has been postulated to include apoptosis, B-cell suppression, which will lead to decreased immunoglobulin G production and decreased production of adhesion molecules and cytokines [12].
Acrolein is the cause of hemorrhagic cystitis, one of the major toxicities of cyclophosphamide therapy. Other toxicities include bone marrow suppression, cardiotoxicity, gonadal toxicity, and carcinogenesis, with cumulative doses being the principal risk factor [15]. Additionally, administration of a single, large dose of cyclophosphamide is capable of causing hemorrhagic cell death, leading to heart failure or even death [17].

Pathophysiology of Cyclophosphamide-Induced Cardiotoxicity
Cyclophosphamide-induced cardiac damage is dose dependent, and the total dose of an individual course is the best indicator of toxicity, with patients who receive greater than 150 mg/kg or 1.55 g/m 2 /day, which are at a high risk for cardiotoxicity [18]. The dose-limiting factor during cyclophosphamide therapy is cardiotoxicity [19], which is irreversible [20]. Fatal cardiomyopathy has been reported among 2-17% of patients taking cyclophosphamide. It is dependent on the regimen and the particular patient population characteristics [21]. Overall, cyclophosphamide-induced cardiotoxicity affects between 7 and 28% of patients taking the drug [13]. The pathophysiology of cyclophosphamide-induced cardiac damage is poorly understood [10], although its metabolites are thought to induce oxidative stress and direct endothelial capillary damage with resultant extravasation of proteins, erythrocytes, and toxic metabolites. In the presence of toxic metabolites, breakdown of endothelial cells contributes to direct damage to the myocardium and capillary blood vessels resulting in edema, interstitial hemorrhage, and formation of microthrombosis [22,23].
Endothelial cells are more susceptible to cyclophosphamide-induced damage than other cells ( [24]); this might be associated with their high proliferation rate [25]; cyclophosphamide-induced reactive oxygen species generation can also lead to a reduction in nitric oxide bioavailability, thus leading to compromised endothelial function [26].
Molecular mechanisms of cyclophosphamide-mediated cardiac damage are currently being postulated, potentially leading to better preventative strategies to treat cardiotoxicity. It has been shown that treatments with cyclophosphamide inhibited heart-type fatty acid-binding proteins and carnitine palmitoyltransferase-I gene expression in cardiac tissues [27]. Inhibition of these pathways leads to decreased production of adenosine triphosphate and accumulation of toxic metabolites from fatty acid oxidation, consequently leading to cardiomyopathy [28]. Heart-type fatty acid-  2 Oxidative Medicine and Cellular Longevity binding protein can be used as an early diagnostic marker of chemotherapy-induced cardiotoxicity [29]. In addition, carnitine deficiency can aggravate cardiotoxicity and it is important to monitor serum and urinary carnitine levels [30]. Carnitine supplementation showed beneficial effects in various cyclophosphamide-induced toxicities [31][32][33][34]. Cyclophosphamide administration affects the ability of the heart mitochondria to retain accumulated calcium [35]. Calcium leak from sarcoplasmic reticulum can lead to mitochondrial calcium overload, leading to reduced production of adenosine triphosphate and increased release of ROS [36]. It is reported that improving mitochondrial function through supplementation of lupeol and its ester can protect heart from cyclophosphamide-induced toxicity [37].
It has been reported that p53 expression plays an important role in apoptosis [46,47]. Reduction in apoptosis, infarct size, and hemodynamic parameter improvement can be achieved by inhibiting p53 [48]. Cyclophosphamideinduced activation of p53 protein is considered as one of the possible mechanisms for cardiomyopathy, and it is reported that probucol supplementation restored cyclophosphamideinduced upregulation of p53 and reversed apoptosis in cardiomyocytes [49].
Cyclophosphamide induces the calcineurin-mediated dephosphorylation of nuclear factor of activated T-cell (NFAT); it belongs to the family of calcium-regulated transcription factors. Unphosphorylated/active GSK-3β phosphorylates eIF2, NFAT, and c-jun and thus contributes significantly to cardiac hypertrophy inhibition/protection [13]. Cyclosporine A prevented NFAT nuclear translocation and reversed cyclophosphamide-induced cardiac damage [35,51].
In general, mechanisms of cyclophosphamide-induced cardiotoxicity encompass oxidative and nitrative stress, protein adduct formation which leads to cardiomyocyte inflammation, altered calcium homeostasis, programmed cell death, swelling of the cardiomyocytes, nuclear splitting, vacuolization, and alteration in signaling pathways. These events result in diseases of the heart muscle including heart failure, if left undiagnosed or untreated, and may result in death [13]. Further supporting a role of cyclophosphamide-induced 3 Oxidative Medicine and Cellular Longevity oxidative stress in the evident cardiotoxicity of the compound, exposure of rats to cyclophosphamide resulted in reduced pulmonary glutathione (GSH) content, GSH reductase (GRx), glucose-6-phosphate dehydrogenase, GSH peroxidase (GPx), and superoxide dismutase (SOD) activities [24].

Current Management for Cyclophosphamide-Induced Cardiotoxicity
Clinical management of cardiovascular diseases (CVDs) involves multiple drugs (angiotensin-converting enzyme inhibitors, blockers of angiotensin-II receptor, calcium channel blockers, β-blockers, aldosterone inhibitors, aspirin, statins, and warfarin), and others include diuretics, digoxin, and nitrates [52][53][54]. It is a common practice to use those medications in combination for the management of CVDs, and these lead to increased side effect and drug interactions [55]. These same preventive strategies can be considered for ischemia, heart failure, arrhythmia, hypertension, and arterial thromboembolism associated with cyclophosphamideinduced cardiotoxicity. Primary prevention may include widespread treatment of all patients who are potentially on cardiotoxic cancer treatments or early diagnosis of subclinical cardiac injury with targeted treatment.
According to the Canadian Cardiovascular Society recommendation, even though the recommendation is weak, patients believed to be at a high risk for cancer treatmentrelated left ventricular dysfunction, an angiotensinconverting enzyme inhibitor, angiotensin receptor blocker, and/or β-blocker, and/or statin can be considered to decrease the risk of cardiac damage [56]. Valsartan, an angiotensin receptor blocker, showed a strong effect in preventing acute cyclophosphamide-, doxorubicin-, vincristine-, and prednisolone-induced cardiotoxicity [57]. A nonselective βblocker, carvedilol, with antioxidant activity and nebivolol, a selective β-blocker with a nitric oxide donor capacity, were reported to have an advantageous effect on antineoplasticassociated cardiac damage [58].
Mild to moderate heart failure and small pericardial effusions generally resolve within a few days to weeks after stoppage of cyclophosphamide. In the presence of suspected hemorrhagic myocarditis, cardiac tamponade, and cardiogenic shock, timely recognition and involvement of the intensive care unit or coronary care unit are vital. These patients need aggressive monitoring and circulatory support [22].

Natural Antioxidants for the Management of Cyclophosphamide-Induced Cardiotoxicity
The use of plants and plant-based products in the treatment of ailments has been known to mankind from ancient times [59]. Various natural antioxidants have originated from medicinal plants, which are used for the treatment of different ailments throughout the world, and there has been a significant interest in finding natural antioxidants from plant sources [60]. Diseases and drug-induced toxicities with the underlying cause of oxidative stress can be effectively managed with plants having antioxidant activity. Apart from being rich sources of antioxidants, phytochemicals are also known to impede the progression of cardiac tissue damage [59]. These compounds could serve as one of the valuable sources in industrial pharmaceutical research and can be treated as a complementary and alternative medicine.
Various medicinal plants showed cardioprotective activity against cyclophosphamide-induced cardiotoxicity in different preclinical studies (Table 1). In addition, xanthineoxidase inhibitors (allopurinol and febuxostat) and nicorandil (vasodilatory drug used to treat angina) were also found to reverse cardiac damages induced by cyclophosphamide in male Wistar rats ( Table 2).

Future Hopes and Hurdles Associated with Cardioprotective Antioxidants
Antioxidants such as flavonoids, flavones, isoflavones, anthocyanin, catechins, and isocatechins are the responsible ones for the antioxidant activity of spices and herb [90]. These led supplementation of antioxidants to be a popular practice to maintain optimal body function [91]. Polyphenols may reduce cholesterol absorption and upregulate hepatic mRNA abundance for the LDL receptor, reductions in plasma TG, yielding a reduced amount of LDL in circulation, and polyphenols were found to exert anti-inflammatory effects, thereby reducing the formation of cytokines involved in cellular adhesion [92]. The production of vasodilating factors like nitric oxide, endothelium-derived hyperpolarizing factor, and prostacyclin was enhanced by plant polyphenols. These plant phenols were also found to inhibit the production of vasoconstrictor endothelin-1 in endothelial cells and inhibit the expression of two main proangiogenic factors, matrix metalloproteinase-2, and vascular endothelial growth factor in smooth muscle cells [93]. Flavonoids can also improve endothelial function, and the primary mechanism for this is that the effect is nitric oxide production [94]. Even though the results were not posted, currently, there are different agents under clinical trial, including enalapril for prevention of chemotherapy-induced cardiotoxicity in highrisk patients (NCT00292526), nutritional supplement sulforaphane on doxorubicin-associated cardiac dysfunction (NCT03934905), estimation of the effects of ACE inhibitors and β blockers in the management of cardiotoxicity in oncologic patients (NCT02818517), cardiotoxicity prevention in breast cancer patients treated with anthracyclines and/or trastuzumab using bisoprolol and ramipril (NCT02236806), carvedilol effect in preventing chemotherapy-induced cardiotoxicity (NCT01724450), prevention of chemotherapyinduced cardiotoxicity in children with bone tumors and acute myeloid leukaemia using capoten (captopril) (NCT03389724), and statins to prevent the cardiotoxicity from anthracyclines (NCT02943590), and others are under investigation. These agents might be the future hopes for the management of chemotherapy-induced cardiotoxicity.
Even though antioxidants like flavonoids have a great hope in the future clinical scenario of cardioprotection [95], the importance of antioxidants is currently in question due 4 Oxidative Medicine and Cellular Longevity Asiri [49] Male Wistar albino rats Rats were administered with the same doses of corn oil (control) and probucol (61 mg/kg/day, i.p), respectively, for one week before and one week after a single dose of CP (200 mg/kg, i.p.).
Probucol prevented the development of CPinduced cardiotoxicity by a mechanism related, at least in part, to its ability to increase mRNA expression of antioxidant genes and to decrease apoptosis in cardiac tissues with the consequent improvement in mitochondrial oxidative phosphorylation and energy production.
Avci et al. [  Vitamin E in a single oral dose failed to inhibit acute cardiotoxic activity of doxorubicin but suspended further progression of the heart muscle damage over the time. On the contrary, vitamin E did not attain cardioprotection against doxorubicin and CP in combination. Sprague-Dawley rats Carvacrol administration was started three days before the CP application and continued till the end of experiment (six days).

Oxidative Medicine and Cellular Longevity
Carvacrol at both the doses increased the GSH levels close to the control group GSH levels. Carvacrol at 5.0 and 10 mg/kg doses lowered the levels of serum ALT, AST, LDH, and CK-MB. Reduced inflammation and lipid peroxidation in the heart tissue and increase of serum GSH and total antioxidant capacity (TAS) levels were found when carvacrol was applied.
Chakraborty et al. [67] Either sex Wistar albino rats Rats were subjected to CP toxicity with the dose of (200 mg/kg i.p.) on day first.

Method and intervention
Major findings cytokines (TNF-α and nitrite/nitrate) and reduced apoptosis.
Gado et al. [71] Male Swiss albino rats Curcumin (200 mg/kg, i.p.) was administered for 8 consecutive days followed by a single dose of CP (150 mg/kg, i.p.). Serum LDH and CPK were decreased significantly with the curcumin administration. Curcumin treatment significantly decreased MDA, NO(x), and restore GSH level in the cardiac tissue. Histological alterations were also found to be improved.
Gunes et al. [72] Male Sprague Dawley rats Animals received respective selenium (Se) doses (0.5 or 1 mg/kg) for 6 days and then a single dose of CP administered on the sixth day. On day 7, the animals were sacrificed. Based on microscopic evaluation, tissue damage was noticeably lower in CP plus Se groups. Additionally, 1 mg/kg Se was more protective than 0.5 mg/kg Se.
It can be concluded that Se can be a potential candidate to ameliorate CP-induced cardiotoxicity which may be related to its antioxidant activity.
Treatment with N-acetylcysteine significantly decreased serum levels of ALT, AST, CK, and LDH.
Decrease in the NOx, MDA levels and TNF-α, SOD, catalase, GSHPx, and GST levels were increased.
Normalized lipid peroxidation and antioxidant defenses were observed in the dl-α-lipoic acid-treated rats.
Mythili et al. [76] Male Wistar albino rats Rats were injected with a single dose of CP (200 mg/kg, i.p) to induce cardiotoxicity, and then rats were treated with lipoic acid (25 mg/kg, orally for 10 days).
Treatment with lipoic acid reversed the abnormalities in the lipid levels and activities of lipid-metabolizing enzymes to near normalcy.
Lipoic acid effectively reversed abnormal biochemical changes to near normalcy. Based on the results, lipoic acid showed a protective role of lipoic acid in CP-induced cardiotoxicity. 7 Oxidative Medicine and Cellular Longevity Nagi et al. [78] Male Wistar albino rats Rats received thymoquinone (50 mg/l in drinking water) for 5 days before a single dose of CP (200 mg/kg, i.p.) and continued thereafter until day 12. On day 13, animals were sacrificed.
Thymoquinone reversed CP-induced increase in serum CK-MB and LDH.
Complete reversal of the CP-induced increase in serum cholesterol, triglycerides, urea, and creatinine to the control values.
CP-induced increase in TBARS and NO(x) and a decrease in GSH, GPx, and CAT were reversed by thymoquinone supplementation.
Thymoquinone supplementation to CP-treated rats completely reversed the increase in TNF-α induced by CP.
Kolavorin pretreatment increased food consumption, body weight, and attenuated the biochemical and histological changes.
It was reported that kolavorin inhibited oxidative stress and preserved the activity of antioxidant enzymes.
Sekeroğlu et al. [80] Male Swiss albino mice After treatment with Viscum album and quercetin for 7 days, rats were administered CP (40 mg/kg, i.p) on days 8 and 9 of the experiment. Total treatment period was 10 days.
Treatments decreased the levels of antioxidant enzymes, glutathione-S-transferases; reduced glutathione and mitotic index were observed. Quercetin completely and Viscum album partly ameliorated almost all of the examined parameters when given together with CP.
Senthilkumar et al. [81] Male albino Wistar rats Animals were cotreated with CP intraperitoneally dissolved in saline, in a dose of 150 mg/kg b.w. and different doses of squalene for the first 2 days, and squalene treatment was followed continuously, daily for 10 days up to the end of the experimental period.
Squalene oral treatment exerted protection to the heart, kidney, and liver at a dose of 0.4 ml/day/rat. Histopathological examinations also confirmed the protective efficacy of squalene.
It can be concluded that squalene may be efficacious as a cytoprotectant in CP-induced toxicities.
Results showed significant improvement in the Zingiber officinale-treated group.
Based on their conclusion, the cardiotoxic effect of CP might be prevented by Zingiber officinale supplementation.
Shanmugarajan et al. [83] Male Wistar rats Rats were treated with the methanolic leaf extract of Ficus hispida Linn. for 10 consecutive days following CP-induced oxidative myocardial injury on the first day. Treatment with Ficus hispida Linn. decreased serum cardiac biomarkers (CPK, LDH, AST, and ALT), and these were increased in the heart tissue. Ficus hispida Linn. increased the levels of enzymic antioxidants (SOD, CAT, GPx, GSH, and GRx). 8 Oxidative Medicine and Cellular Longevity Sudharsan et al. [85] Male Wistar albino rats Rats were injected with a single dose of CP (200 mg/kg, i.p) and treated with lupeol and lupeol linoleate (50 mg/kg).
Lupeol and its ester reversed alterations of serum lipoproteins and lipid fractions in both serum and cardiac tissue.
It was found that lupeol linoleate was more effective than lupeol.
Swamy et al. [86] Male Wistar albino rats Cardiotoxicity was induced by administering CP The results showed that dietary GLN decreased cardiac necrosis and maintained normal cardiac GSH levels.
GLN protected against the acute cardiotoxic effects of CP and significantly improved the short-term survival after lethal and sublethal doses of CP. 9 Oxidative Medicine and Cellular Longevity Based on the findings, ROS and XO enzymatic pathways may largely participate in the mechanism of pathogenesis of cardiac and bone marrow toxicities related to CP exposure.
Cotreatment with GP or L-NNA decreased the protective effect of NIC. 10 Oxidative Medicine and Cellular Longevity to their less effectiveness in an in vivo study. These failures of antioxidants in preventing/treating diseases have become the main obstacle in the clinical scenario [96]. As concluded by Guallar et al., known antioxidants like vitamin E, β-carotene, vitamin A and B supplements, and folic acid are ineffective for the prevention of mortality and morbidity due to chronic diseases [97].
This failure might be due to different reasons including antioxidant-related reasons including testing incorrect antioxidant or combination of antioxidants; there might be differences between synthetic and dietary source antioxidants, reductive stress (i.e., too much antioxidant capacity), and it may also be related to patient or clinical trials [98].
Unconjugated flavonoid plasma level rarely exceeds 1 μM, and metabolites of flavonoids have lower antioxidant activity. Since plasma total antioxidant capacities (TAC) are often in the range of 1 mM or more, it is difficult to picture how an additional 1 μM polyphenol could exert an in vivo antioxidant effect. Antioxidants like flavonoids and other phenols are complex molecules and have multiple potential targets/actions in addition to antioxidant activity. These may include inhibition of different enzymes including cyclooxygenase, lipoxygenase, xanthine oxidase, matrix metalloproteinases, angiotensin-converting enzyme, proteasome, and cytochrome P450, affecting signal transduction pathways. Flavonoids may also interact with cellular drug transport systems [99]. These issues need to be addressed in the future.

Conclusion
Cyclophosphamide is a known anticancer and immunosuppressive agent that becomes effective after metabolic activation in the liver. Its wider clinical application is currently limited by its toxicity. Cardiotoxicity, which is associated with oxidative and nitrative stress, is one of the toxicities limiting the clinical use of cyclophosphamide. Different natural, plant-derived antioxidants (summarized in this review) showed significant cardioprotective effects in in vivo preclinical studies. However, further investigations aimed at improving their efficacy are required. Facilitating translational clinical research on those shown to be safe and effective in the preclinical studies should also be considered, lest the evidences from the preclinical studies would only be left to be discoursed in scientific meetings and publications.

Conflicts of Interest
The authors declare no conflict of interest.