Captopril and Telmisartan Ameliorate Cisplatin-Induced Testicular Damage in Rats Via Anti-Inflammatory and Antioxidant Pathways

International Journal of Scientific and Research Publications, Volume 6, Issue 1, January 2016 408 ISSN 2250-3153

Captopril and telmisartan ameliorate cisplatin-induced testicular damage in rats via anti-inflammatory and antioxidant pathways

Ahmed H. Eid*, Noha F. Abdelkader**, Ola M. Abd El-Raouf*, Hala M. Fawzy*, Ezz-El-Din S. El-Denshary**

*Department of Pharmacology, National Organization for Drug Control and Research (NODCAR), Giza, Egypt **Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Cairo, Egypt.

Abstract- Despite the prevalent clinical applications of Male reproductive toxicity rises from targeting rapidly cisplatin, serious toxic side effects comprising reproductive growing testicular cell types such as leydig, sertoli, and germ toxicity confine its therapeutic efficacy. Thus, the current study cells (Boekelheide, 2005). Cisplatin adversely impairs non target explored the possible protective effect of captopril and tissues via increased free radical generation and depletion of telmisartan against cisplatin-induced testicular damage in rats. antioxidants; which results in inhibition of protein synthesis and Captopril (100 mg/kg) and telmisartan (10 mg/kg) were orally DNA damage (Fung and Vaughn, 2011, Ilbey et al., 2009a). administered for 15 days, whereas cisplatin (10 mg/kg; i.p.) was Growing evidence verifies that activation of the renin angiotensin injected as a single dose at the 12th day to induce testicular system (RAS) is largely implicated in cisplatin-induced tissue damage in adult male Sprague-Dawley rats. Cisplatin injury (El-Sayed et al., 2008, Saleh et al., 2009) where cisplatin prominently decreased reproductive organs weights, sperm amplifies RAS components level both experimentally (El-Sayed count, sperm motility, and increased sperm abnormalities, along et al., 2008, Okui et al., 2012) and clinically (Kurt et al., 1999). with histopathological damage of testicular tissues. In addition, it Indeed, angiotensin II, the chief component of RAS, induces resulted in a significant decline in serum testosterone as well as tissue oxidative stress and apoptosis (Cai et al., 2003, Wolf, testicular enzymatic and non-enzymatic antioxidants levels 2000). Besides, it triggers inflammation via upregulation of pro- (superoxide dismutase, catalase, glutathione peroxides, and inflammatory transcription factors, which results in a vast array reduced glutathione), parallel to a remarkable elevation in of proinflammatory cytokines that further injures the tissues testicular content of malondialdehyde, nitric oxide, tumor (Hayashi et al., 2010). necrosis factor-α, and nuclear factor-kappa B. Treatment with Therefore, the current study was conducted to investigate the captopril or telmisartan markedly attenuated cisplatin-induced efficacy of RAS blockade against cisplatin-induced testicular injury by suppression of oxidative/nitrosative stress and damage. Captopril is an angiotensin converting enzyme inhibitor inflammation, amendment of antioxidant defenses, as well as that reduces circulating and tissue levels of angiotensin II. improvement of steroidogenesis, spermatogenesis and testicular Moreover, it is a powerful thiol-containing antioxidant (Liu et histological features. This study suggests a novel therapeutic al., 2007) that possesses anti-inflammatory potential application for captopril and telmisartan as protective agents (Amirshahrokhi et al., 2010). Captopril is widely used for the against cisplatin-induced testicular toxicity through their treatment of hypertension, congestive heart failure, and promising anti-inflammatory and antioxidant capacities. diabetic nephropathy (Cohen et al., 1996). In context, telmisartan is a highly selective angiotensin II type-1 receptor Index Terms- Captopril, cisplatin, oxidative stress, blocker which is approved for the treatment of hypertension. It is telmisartan, testicular damage. endowed with potent anti-inflammatory, antioxidant and anti- apoptotic effects (Fouad et al., 2010). I. INTRODUCTION Interestingly, recent studies revealed that both drugs isplatin is an extremely effective anticancer drug for the ameliorated chemotherapy-induced cardiotoxicity and Ctreatment of diverse types of solid tumors (Amin and nephrotoxicity (Abd-Ellah, 2012, Cadeddu et al., 2010, Buratovich, 2009). Despite the improvement in quality of life of Ibrahim et al., 2009) as well as cadmium-induced testicular cancer patients; the use of cisplatin was restricted clinically by its toxicity (Fouad and Jresat, 2013). Herein, the potential major side effects including testicular toxicity (Beytur et al., protective effects of captopril and telmisartan against testicular 2012, Fung and Vaughn, 2011). In adult men, fertility can be toxicity induced by cisplatin in male rats were investigated by the preserved by spermatozoa cryopreservation and intracytoplasmic assessment of hormonal and spermatological changes, oxidative sperm injection, however, these methods are not feasible options stress parameters, inflammatory markers, and the testicular for pre-pubertal patients (Rezvanfar et al., 2013). Besides, in histopathological alterations. adults, the freezing and thawing of semen can reduce the sperm quality (Maines et al., 1990). Interestingly, growing evidences II. MATERIALS AND METHODS revealed that combination therapy can be beneficial to overcome Animals this special reproductive toxicity (Narayana et al., 2012, Adult male Sprague-Dawley rats; weighing 240-280 g, were Ahmed et al., 2011). obtained from the breeding colony of the National Organization for Drug Control and Research (NODCAR), Giza, Egypt.

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Animals were accommodated under controlled environmental oxide (NO) according to manufacturer’s prescripts using the conditions (23±2°C temperature, 60±10% humidity, 12/12 h corresponding Biodiagnostic colorimetric kits (Cairo, Egypt), light/dark cycle) and were allowed standard chow diet and water while tumor necrosis factor (TNF)-α was assayed using rat TNF- ad libitum. The investigation complies with the Guide for the α Elisa Kit (Enzo life sciences, San Diego, CA, USA). Care and Use of Laboratory Animals (NIH Publication No. 85- Histological examination 23, revised 1996) and was approved by the Ethics Committee of Light microscopic evaluation Faculty of Pharmacy, Cairo University, Cairo, Egypt (Permit The fixed testis was processed for paraffin embedding and 4 Number: PT 922). μm sections were prepared. Testicular sections were stained with Chemicals and drugs haematoxylin and eosin (H&E) and examined using a light Cisplatin was purchased from Mylan (Saint-Priest, France). microscope. Qualitative histopathological damage in the Captopril and telmisartan were obtained from Multipharma seminiferous tubules was graded according to the severity of pharmaceutical and Sigma pharmaceutical industries; degenerative findings: (−) no obvious damage, (+) fewer than respectively (Cairo, Egypt). All other chemicals were of the 25% of seminiferous tubules affected (mild), (++) 25–50% of highest purity and analytical grade. seminiferous tubules affected (moderate), and (+++) over 50% of Experimental design seminiferous tubules affected (severe) (Rezvanfar et al., 2013). Seventy rats were randomly divided into seven groups (10 Histopathological examination of testes was interpreted by an rats each). Group I served as a control. Rats of group II received experienced observer who was blind to the sample identity to orally 0.5 % carboxymethyl cellulose (10 ml/kg) for 15 days. avoid any bias. Animals of groups III & IV received orally captopril dissolved in Immunohistochemical analysis of testicular nuclear factor- water (100 mg/kg) (Fasihi et al., 2012) and telmisartan kappa B (NF-κB) suspended in 0.5 % carboxymethyl cellulose (10 mg/kg) (Fouad Paraffinized testicular sections were rehydrated in xylene and Jresat, 2013) for 15 days; respectively. Those of groups V, and graded ethanol solutions. Samples were heated in citrate VI, and VII received a single intraperitoneal injection of cisplatin buffer (pH 6) for 20 minutes, cooled, and thereafter incubated (10 mg/kg) on the 12th day to induce testicular toxicity (Longo et with primary polyclonal rabbit anti-NF-κB antibody (1:200; al., 2011); whereas, rats of groups VI and VII were treated with Invitrogen, Carlsbad, CA, USA) overnight at 4°C. Sections were captopril and telmisartan; respectively, as previously described. washed with phosphate buffered saline, and incubated for 30 min Four days after cisplatin injection, rats were weighed, at 37°C with biotinylated secondary antibody, and then with anesthetized, and blood samples were collected and centrifuged Avidin DH and biotinylated horseradish peroxidase H complex at 3000 xg for 10 minutes for serum separation to estimate according to Elite ABC kit instructions (Vector Laboratories Inc., testosterone level. Thereafter, animals were euthanized and the Burlingame, CA, USA). After another wash with phosphate reproductive organs were immediately removed, cleaned from buffered saline, the reaction was revealed by diaminobenzidine the adhering tissue, and weighed. The epididymal content for tetrahydrochloride (DAB Substrate Kit, Vector Laboratories Inc., each rat was instantly collected to examine the spermatological Burlingame, CA, USA) and the sections were counterstained parameters. Furthermore, the left testis was fixed in 10% with hematoxylin, dehydrated, and cleared in xylene then cover formalin for histological examination. Whereas, the right testis slipped for light microscopic examination. was decapsulated, homogenized in 0.05 M potassium phosphate Statistical analysis buffer (pH 7.4), and processed for the estimation of oxidative and Data were expressed as means ± S.E.M. Results were inflammatory biomarkers. All samples were stored at -80°C until analyzed using one way analysis of variance test (One-way analysis. ANOVA) followed by Tukey-Kramer multiple comparison test. Spermatological Examination Statistical analysis was performed using GraphPad Prism The epididimal content of each rat was promptly collected software version 5 (San Diego, CA, USA); a probability level of for the estimation of sperm count, motility as well as less than 0.05 was accepted as statistically significant. abnormalities as previously described (Bearden and Fluquary, 1980). III. RESULTS Estimation of serum testosterone level There was no change in any of the tested parameters Serum testosterone level was estimated according to between the control rats and those which received carboxymethyl manufacturer’s instructions using rat Testosterone EIA ELISA cellulose, captopril, and telmisartan alone (at the selected doses). Kit (Enzo life sciences, San Diego, CA, USA). Alterations in body and reproductive organs weight (Table Estimation of oxidative/nitrosative and inflammatory 1): biomarkers in testicular homogenate A significant decrease in body as well as relative Testicular homogenate was divided into 3 aliquots, where reproductive organs weight was observed as a result of cisplatin the first one was used for estimation of malondialdehyde (MDA) administration compared to control group. Cisplatin decreased (Deniz et al., 1997). The second aliquot was deproteinized with body weight to 87.7% of control rats, an effect that was not 5% sulfosalicylic acid, centrifuged at 1000 xg for 15 minutes and alleviated by captopril nor telmisartan treatment. The weight of the obtained supernatant was used for the estimation of reduced right and left testis, cauda epididymis, prostate and seminal glutathione (GSH) (Beutler et al., 1963). The third one was vesicle were significantly decreased after cisplatin exposure to centrifuged at 4000 rpm for 15 minutes at 4°C and the resultant 82.9%, 85.2%, 76.6%, 73.7% and 80.7 % of the control group; supernatant was used for the assay of superoxide dismutase respectively, while all of them were normalized by captopril (SOD), catalase (CAT), glutathione peroxidase (GPx), and nitric

www.ijsrp.org International Journal of Scientific and Research Publications, Volume 6, Issue 1, January 2016 410 ISSN 2250-3153 administration. Similarly, telmisartan restored reproductive inhibitors and angiotensin receptor blockers to platinum-based organs weight except for cauda epididymis. first-line chemotherapy may contribute to prolonged survival in Effects on spermatological parameters and serum patients with advanced lung and gastric cancer (Kim et al., 2012, testosterone level (Table 2): Wilop et al., 2009). Moreover, the combination of these drugs Cisplatin-treated rats showed a substantial decrease in sperm with chemotherapy markedly delayed tumor growth (Williams et count and motility % almost by half and an increase in sperm al., 2005, Hosseinimehr, 2014), thus much attention has been abnormalities % to 2.7 folds compared to the corresponding directed to their protective effects against chemotherapy-induced values of the control group. Furthermore, serum testosterone cardiotoxicity and nephrotoxicity (Abd-Ellah, 2012, Arozal et level was markedly declined to 73.3% of the control group. al., 2010, El-Sayed et al., 2008, Hrenak et al., 2013, Malik et Treatment with captopril alleviated the changes in motility %, al., 2015). However, to the best of our knowledge, their sperm abnormalities %, and serum testosterone level by 32.5%, protective effects against cisplatin-induced testicular toxicity 35.6%, and 19.7%, respectively as related to the cisplatin-treated have not been investigated. The current study highlights the group together with a normalization of the sperm count. protective actions of captopril and telmisartan against cisplatin- Meanwhile, telmisartan administration substantially raised sperm induced testicular toxicity in rats as verified by the restoration of count, motility %, and serum testosterone level to 1.54, 1.25 and testicular architecture, enhancement of steroidogenesis, 1.20 folds; correspondingly, and reduced sperm abnormalities by preservation of spermatogenesis, modulation of the inflammatory 33.86% versus the cisplatin group. reaction along with suppression of oxidative/nitrosative stress. Effects on testicular oxidative/nitrosative stress and In the current study, the observed reduction in relative inflammatory biomarkers (Fig. 1-3): reproductive organs weights in cisplatin-treated rats establishes Cisplatin injection markedly amplified testicular MDA, NO, the toxic potential of cisplatin on the reproductive system as and TNF-α contents by 3.6, 1.6 and 5.2 folds, respectively; previously documented by (Atessahin et al., 2006, Ilbey et al., whereas it resulted in a decline in testicular GSH content along 2009a). It was declared that this weight reduction is related to the with SOD, CAT and GPx activities by 60.47%, 56.41%, 47.10% oxidative damage induced by cisplatin (Adejuwon et al., 2015). and 65.90%, respectively as compared to the control group. Such effect was further accompanied by degeneration of However, captopril and telmisartan treatment noticeably reversed seminiferous tubules with disruption of spermatogenesis, sperm such alterations except for CAT activity which was not affected dysfunction, besides a decline in the testicular function by both drugs and SOD activity that was amended by captopril biomarker, i.e. testosterone. Many investigations confirmed our treatment only. In addition, the effect of captopril was more results (Adejuwon et al., 2015, Amin et al., 2012, Rezvanfar et pronounced than telmisartan in terms of TNF-α decline. al., 2013, Salem et al., 2012). Effects on testicular NF-κB protein expression (Fig. 4 & Herein, captopril and telmisartan treatment almost restored Table 3): the reduction in reproductive organs weights induced by The immunohistochemical examination of testicular NF-κB cisplatin, which is consistent with a previous study (Kushwaha protein expression displayed an enhanced expression confined and Jena, 2013). Moreover, cisplatin-induced reduction in within the interstitial stroma adjacent to the basement membrane testosterone level was significantly reverted by captopril and of degenerated tubules in testes of cisplatin-treated rats (Fig. 4E), telmisartan administration, which is in accordance with an effect that alleviated by management with captopril and preceding investigations (Fouad and Jresat, 2013, Kushwaha telmisartan (Fig. 4F & G). and Jena, 2013). This remarkable reduction in the hormonal Effects on histopathological damage in testicular tissues (Fig. level might be explained by cisplatin-induced severe damages on 5 & Table 4): leydig and sertoli cells resulting from increased generation of Testicular sections of the cisplatin group revealed atypical free radicals as one of the possible mechanisms (Tousson et al., morphological features manifested as massive degeneration in 2014). Actually, testosterone is essential for the normal the seminiferous tubules, shrinkage in germ cell layers and spermatogenesis as well as for the maintenance of normal disruption of spermatogenesis, interstitial edema, congestion in structure of seminiferous tumbles (Sharpe et al., 1992). blood vessels and replacement of the interstitial stroma with Noteworthy, captopril and telmisartan not only preserved the homogeneous eosinophilic material (Fig. 5E). Captopril sperm characteristics but also maintained the testicular treatment normalized these histological abnormalities and architecture by increasing the spermatogenic mass and amended spermatogenesis, though a slight congestion in blood decreasing the seminiferous tubules damage and necrosis. Such vessels of the interstitisal stroma was still observed (Fig. 5F). constructive effects may be attributed to the increased hormonal Likewise, telmisartan attenuated the histopathological changes level, decreased apoptotic cell death and suppressed oxidative and protected the seminiferous tubules albeit less pronounced stress as well as lipid peroxidation (Fouad and Jresat, 2013, than captopril (Fig. 5G). Kushwaha and Jena, 2013). It is worthy to mention that free radicals mediate reactions that are responsible for a wide range of IV. DISCUSSION cisplatin-persuaded side effects. Testicular dysfunction and infertility are serious Cisplatin disrupts the redox balance of testicular tissue via an complications that may impact the clinical effectiveness of increase in lipid peroxidation, mitochondrial dysfunction and cisplatin. Thus, it seems imperative to search for agents that can oxidative stress which result in inhibition of protein synthesis protect against testicular toxicity whenever cisplatin and DNA damage (Fung and Vaughn, 2011). Such effects were chemotherapy is employed. Interestingly, preceding clinical revealed in this work as a reduction in testicular GSH content as studies revealed that addition of angiotensin converting enzyme well as SOD, CAT and GPx activities and an elevation in MDA

www.ijsrp.org International Journal of Scientific and Research Publications, Volume 6, Issue 1, January 2016 411 ISSN 2250-3153 level. These findings are in coherence with previous 1995) with a resultant DNA fragmentation and lipid peroxidation experimental studies (Adejuwon et al., 2015, Amin et al., 2012, (Valko et al., 2007). Thereby, blockade of this cycle via Salem et al., 2012). angiotensin converting enzyme inhibition or angiotensin II type- It is well known that oxidative stress stimulate transcription 1 receptor blockade might provide a plausible justification of the factors, including NF-κB (Kundu and Surh, 2005), which is a antioxidant and anti-inflammatory effects of captopril and functional link between oxidative damage and inflammation telmisartan, respectively. (Hamza et al., 2015). Recent studies revealed that cisplatin- In addition, it is worthy to mention that captopril as a thiol- induced oxidative stress upregulated the expression of NF-κB, containing compound scavenges various reactive oxygen species which consecutively increased the expression of numerous genes and prevents lipid peroxidation; accordingly it possesses such as inducible nitric oxide synthase (iNOS) resulting in tremendous antioxidant capacity related to its sulfhydryl group apoptotic cell death in the testes (Hamza et al., 2015, Sherif et (Fouad and Jresat, 2013, Liu et al., 2007, Scribner et al., al., 2014). This is in accordance with the present results which 2003). On the other hand, telmisartan in addition to exerting its revealed that cisplatin promoted the expression of NF-κB and the effects as an angiotensin II type-1 receptor antagonist; it can act inflammatory mediators TNF-α and NO. Indeed, inflammation is as a partial agonist at peroxisome proliferator activated receptor- considered as one of the important mechanisms by which gamma (PPAR-γ) (Benson et al., 2004). PPAR-γ has a crucial cisplatin mediates testicular injury through downstream signaling role in the reproductive physiology of both rats and human pathways (Ilbey et al., 2009b). (Aquila et al., 2006, Braissant et al., 1996). It is also evident In the present study, captopril and telmisartan administration that PPAR-γ is expressed during the development of sertoli and tempered cisplatin-induced lipid peroxidation through decreased germ cells in rodents (Thomas et al., 2011). Moreover, PPAR-γ MDA level. They also amended the antioxidant status in stimulation reduces the production of inflammatory mediators testicular tissues via augmented GSH level of both reduced and and inhibit the NF-κB binding (Kushwaha and Jena, 2013). peroxidase forms. In addition, only captopril significantly V. CONCLUSION increased SOD activity in testicular tissues. Our results were in harmony with earlier reports showing that captopril and Taken together, it can be concluded that treatment with telmisartan attenuated lipid peroxidation and enhanced the captopril and telmisartan provided significant and comparable antioxidant balance in testicular tissues in cadmium and STZ- protective effects against cisplatin-induced testicular toxicity in induced testicular toxicity in rats (Fouad and Jresat, 2013, rats. The beneficial actions of these drugs are not only accredited Kushwaha and Jena, 2013). Moreover, suppression of NF-κB to their ability to block angiotensin II-dependent activation of instigated by both drugs in this work reduced the transcription of pro-inflammatory and pro-oxidant pathways, but also include downstream genes as evident by the decline in testicular TNF-α their capability to scavenge free radicles, augment antioxidants, and NO levels. In support of our results, former preceding studies and deactivate NF-κB signaling pathway. Therefore, we suggest reported that captopril and telmisartan can block the activation of that angiotensin converting enzyme inhibitors and angiotensin II NF-κB signaling pathway which promotes the transcription of type-1 receptor antagonists may be beneficial in the clinical NADPH oxidase, TNF-α, and iNOS genes (Fouad and Jresat, treatment of cancer patients with cisplatin while avoiding many of its side effects particularly reproductive system toxicity. 2013, He et al., 2007, Ilieva et al., 2006, Kushwaha and Jena, 2013, Morishima et al., 2009, Takaya et al., 2006). These CONFLICT OF INTEREST actions highlight the potential antioxidant and anti-inflammatory The authors declare that they have no financial or personal abilities of both drugs as protective agents against cisplatin- conflicts of interest to disclose. induced testicular damage in rats. Actually, angiotensin II; the dynamic component of RAS, ACKNOWLEDGMENTS induces a vicious cycle of oxidative stress and inflammation by The authors are grateful to Prof. Dr. Adel Bakeir and Prof. activating the angiotensin II type-1 receptor (Wolf, 2000). Dr. Kawkab A. Ahmed (Department of Histology, Faculty of Angiotensin II is a prominent oxidative stress initiator through Veterinary Medicine, Cairo University, Cairo, Egypt) for activating the NADPH oxidase enzyme, hence upsurges the performingthe histopathological and immunohistochemical production of superoxide anion, hydrogen peroxide, and examinations. hydroxyl radicals (Cai et al., 2003). These oxidative mediators REFERENCES sequentially trigger inflammatory cascades by upregulating the pro-inflammatory transcription factor NF-κB (Savoia and [1] Abd-Ellah, H.F., 2012. 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[48] Okui, S., H. Yamamoto, W. Li, N. Gamachi, Y. Fujita, S. Kashiwamura et [62] Wilop, S., S. von Hobe, M. Crysandt, A. Esser, R. Osieka and E. Jost, 2009. al., 2012. Cisplatin-induced acute renal failure in mice is mediated by Impact of angiotensin I converting enzyme inhibitors and angiotensin II chymase-activated angiotensin-aldosterone system and interleukin-18. Eur. type 1 receptor blockers on survival in patients with advanced non-small- J. Pharmacol., 685(1): 149-155. cell lung cancer undergoing first-line platinum-based chemotherapy. J. [49] Rezvanfar, M.A., M.A. Rezvanfar, A.R. Shahverdi et al., 2013. Protection Cancer Res. Clin. Oncol., 135(10): 1429-1435. of cisplatin-induced spermatotoxicity, DNA damage and chromatin [63] Wolf, G., 2000. Angiotensin II as a mediator of tubulointerstitial injury. abnormality by selenium nano-particles. Toxicol. Appl. Pharmacol., 266(3): Nephrol. Dial. Transplant., 15(6): 61-63. 356-365. [50] Saleh, S., A.A. Ain-Shoka, E. El-Demerdash and M.M. Khalef, 2009. AUTHORS Protective effects of the angiotensin II receptor blocker losartan on cisplatin-induced kidney injury. Chemotherapy, 55(6): 399-406. First author – Ahmed H. Eid, Assistant Researcher of [51] Salem, E.A., N.A. Salem, A.M. Maarouf, E.C. Serefoglu and W.J. pharmacology, Pharmacology Department, National Hellstrom, 2012. Selenium and lycopene attenuate cisplatin-induced Organization for Drug Control and Research (NODCAR), 6 Abo testicular toxicity associated with oxidative stress in Wistar rats. Urology, 79(5): 1181-1186. Hazem Str., Pyramids Ave., Postal Code: 12553, Giza, Egypt; E- [52] Savoia, C. and E.L. Schiffrin, 2007. Vascular inflammation in hypertension mail: [email protected]. and diabetes: molecular mechanisms and therapeutic interventions. Clin. Second author – Noha F. Abdelkader, Assistant professor of Sci., 112(7): 375-384. Pharmacology & Toxicology, Pharmacology & Toxicology [53] Scribner, A.W., J. Loscalzo and C. Napoli, 2003. The effect of angiotensin- Department, Faculty of pharmacy, Cairo University, Kasr El Aini converting enzyme inhibition on endothelial function and oxidant stress. St., Postal Code: 11562, Cairo, Egypt; E-mail: Eur. J. Pharmacol., 482(1-3): 95-99. [email protected]. [54] Sharpe, R.M., S. Maddocks, M. Millar, J.B. Kerr, P.T. Saunders and C. McKinnell, 1992. Testosterone and spermatogenesis. Identification of Third author – Ola M. Abd El-Raouf, P AssociateP professor of stage-specific, androgen-regulated proteins secreted by adult rat Physiology, Pharmacology Department, National Organization seminiferous tubules. J. Androl., 13(2): 172-184. for Drug Control and Research (NODCAR), 6 Abo Hazem Str., [55] Sherif, I.O., A. Abdel-Aziz and O.M. Sarhan, 2014. Cisplatin-induced Pyramids Ave., Postal Code: 12553, Giza, Egypt; E-mail: testicular toxicity in rats: the protective effect of arjunolic acid. J. Biochem. [email protected] .U2T Mol. Toxicol., 28(11): 515-521. Fourth author – Hala M. Fawzy,P ProfessorP of pharmacology, [56] Tak, P.P. and G.S. Firestein, 2001. NF-kappaB: a key role in inflammatory diseases. J. Clin. Invest., 107(1): 7-11. Head of Pharmacology Department, National Organization for [57] Takaya, T., S. Kawashima, M. Shinohara, T. Yamashita, R. Toh et al., Drug Control and Research (NODCAR), 6 Abo Hazem Str., 2006. Angiotensin II type 1 receptor blocker telmisartan suppresses Pyramids Ave., Postal Code: 12553, Giza, Egypt; E-mail: superoxide production and reduces atherosclerotic lesion formation in [email protected] .U2T apolipoprotein E-deficient mice. Atherosclerosis, 186(2): 402-410. Fifth author – Ezz-El-Din S. El-Denshary, Professor of [58] Thomas, K., D.Y. Sung, X. Chen, W. Thompson, Y.E. Chen et al., 2011. Developmental patterns of PPAR and RXR gene expression during Pharmacology & Toxicology, Pharmacology & Toxicology spermatogenesis. Front. Biosci., (Elite Ed) 3: 1209-1220. Department, Faculty of pharmacy, Cairo University, Kasr El Aini

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curcumin on testicular toxicity induced by Cisplatin in rats. J.Cancer Res. [email protected] .U2T Treat., 2(3): 64-68. [60] Valko, M., D. Leibfritz, J. Moncol, M.T. Cronin, M. Mazur and J. Telser, Correspondence author – Ahmed H. Eid, Department of 2007. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol., 39(1): 44-84. Pharmacology, National Organization for Drug Control and [61] Williams, R.N., S.L. Parsons, T.M. Morris, B.J. Rowlands and S.A. Research (NODCAR), 6 Abo Hazem Str., Pyramids Ave., Postal Watson, 2005. Inhibition of matrix metalloproteinase activity and growth of Code: 12553, Giza, Egypt; E-mail:

gastric adenocarcinoma cells by an angiotensin converting enzyme inhibitor [email protected] ;U2T Tel. (+2) 02-35851299 – 02- in in vitro and murine models. Eur. J. Surg. Oncol., 31(9): 1042-1050. 35851278; Fax. (+2) 02-35855582; Mobile: (+2) 01064074083.

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Figure (1): Effect of captopril and telmisartan on cisplatin-induced alterations in testicular MDA and GSH levels in rats.

Each bar represents the mean ± SEM of 10 animals in each group;vs control, @vs CIS (one-way ANOVA followed by Tukey– Kramer multiple comparisons test; p<0.05). CMC, carboxymethyl cellulose; CAP, captopril; TEL, telmisartan; CIS, cisplatin.

Figure (2): Effect of captopril and telmisartan on cisplatin-induced alterations in testicular TNF-α and NO levels in rats.

Each bar represents the mean ± SEM of 10 animals in each group;vs control, @vs CIS, #vs CIS+CAP (one-way ANOVA followed by Tukey–Kramer multiple comparisons test; p<0.05). CMC, carboxymethyl cellulose; CAP, captopril; TEL, telmisartan; CIS, cisplatin.

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Figure (3): Effect of captopril and telmisartan on cisplatin-induced alterations in testicular SOD, CAT and GPx activities in rats.

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Figure (4): Effect of captopril and telmisartan on cisplatin-induced alterations in testicular NF-κB protein expression in rats.

Photomicrographs illustrating immunohistochemical staining of NF-κB in testicular sections from (A) control, (B) CMC, (C) CAP, and (D) TEL groups showing no expression, (E) CIS group showing very extensive expression (arrows), as well as (F) CIS+CAP and (G) CIS+TEL groups displaying moderate expression (arrows) [Magnification: X 400]. CMC, carboxymethyl cellulose; CAP, captopril; TEL, telmisartan; CIS, cisplatin.

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Figure (5): Effect of captopril and telmisartan on cisplatin-induced alterations in testicular histopathological features in rats.

Photomicrographs of testicular sections from (A) control, (B) CMC, (C) CAP, and (D) TEL groups showing normal intact testicular tissues with mature seminiferous tubules (s) and complete spermatogenic series, as well as from (E) CIS group displaying marked degeneration in seminiferous tubules (ds) and sever congestion in blood vessels (v) of tunica albuginea (a) with eosinophilic infiltrationin the interstitial stroma (t), (F) CIS+CAP group showing mature active seminiferous tubules (s) associated with moderate congestion in blood vessels (v) of the interstitial stroma and (G) CIS+TEL group showing moderate degeneration in some seminiferous tubules (ds) with moderate congestion in blood vessels (v) of interstitial stroma [H&E X 40]. CMC, carboxymethyl cellulose; CAP, captopril; TEL, telmisartan; CIS, cisplatin.

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Figure (6): Diagram illustrating the alleviating action of captopril and telmisartan in cisplatin-induced testicular injury.

www.ijsrp.org International Journal of Scientific and Research Publications, Volume 6, Issue 1, January 2016 419 ISSN 2250-3153 Table (1): Effect of captopril and telmisartan on cisplatin-induced alterations in body and reproductive organs weight in rats.

Cauda Seminal Final body Right testes Left testes Prostate Groups epididymis vesicles weight (g) (g/100g BW) (g/100g BW) (g/100g BW) (g/100g BW) (g/100g BW) Control 283.0±3.86 0.498±0.005 0.493 ± 0.005 0.047±0.002 0.217±0.003 0.265±0.004

CMC 288.6±2.19 0.490±0.006 0.488± 0.005 0.045±0.001 0.223±0.004 0.278±0.005

CAP 281.6±2.92 0.489±0.005 0.492 ± 0.005 0.048±0.001 0.222±0.006 0.282±0.008

TEL 284.9±3.04 0.505±0.004 0.501 ± 0.006 0.048±0.001 0.237±0.005 0.282±0.007

CIS 248.3±2.77 0.413±0.010 0.420 ± 0.011 0.036±0.002 0.160±0.006 0.214±0.007

CIS + CAP 251.9±3.48 0.515±0.011@ 0.510 ± 0.010@ 0.045±0.002@ 0.203±0.006@ 0.266±0.008@

CIS + TEL 252.1±3.93 0.486±0.008@ 0.484 ± 0.010@ 0.041±0.001 0.196±0.009@ 0.256±0.013@

Values are expressed as mean ± SEM of 10 animals in each group. vs control, @vs CIS (one-way ANOVA followed by Tukey– Kramer multiple comparisons test; p<0.05).CMC, carboxymethyl cellulose; CAP, captopril; TEL, telmisartan; CIS, cisplatin.

Table (2): Effects of captopril and telmisartan on cisplatin-induced alterations in spermatological parameters and serum testosterone level in rats.

Sperm count Sperm motility Sperm Groups Testosterone (pg/ml) (107/ml) (%) abnormalities (%)

Control 167.0±5.60 81.88 ± 1.61 5.28 ± 0.14 5605 ± 98.7

CMC 166.1±5.03 81.25 ± 1.83 5.66 ± 0.19 5688 ± 38.5

CAP 164.1±5.69 81.88 ± 1.31 5.83 ± 0.17 5385 ± 89.9

TEL 165.0±6.42 81.25 ± 1.25 6.08 ± 0.20 5300 ± 89.0

    CIS 91.38±4.76 53.75 ± 3.62 14.29 ± 0.67 4107 ± 243.4

@ @  @ CIS + CAP 156.1±5.05 71.25 ± 2.26 9.20 ± 0.49 4920 ± 121.0

CIS + TEL 141.4±6.25@ 67.5 ± 2.11@ 9.45 ± 0.51@ 4934 ± 135.4@

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Table (3): Immunohistochemical detection of NF-κB protein expression in testes of rats

NF-κB expression Groups (in the interstitial stroma)

Control − CMC −

CAP −

TEL −

CIS ++++

CIS + CAP ++ CIS + TEL ++

- Nil, + mild, ++ moderate, +++ extensive,++++ very extensive.CMC, carboxymethyl cellulose; CAP, captopril; TEL, telmisartan; CIS, cisplatin.

Table (4): Testicular histopathological alterations in rats

Degeneration in Congestion in blood Edema in interstitial Groups seminiferous tubules vessels stromal tissue

Control − − −

CMC − − −

CAP − − −

TEL − − −

CIS ++++ ++++ +++ CIS + CAP − ++ − CIS + TEL ++ ++ −

- Nil, + mild damage, ++ moderate damage, +++ severe damage, ++++ very severe.CMC, carboxymethyl cellulose; CAP, captopril; TEL, telmisartan; CIS, cisplatin.