Yusufhan YAZIR, Melda YARDIMOĞLU YILMAZ, Kübra KAVRAM SARIHAN, Fatma Ceyla ERALDEMİR, Esra ACAR

Protective effects of apocynin on damaged testes of rats exposed to methotrexate

Background/aim: Methotrexate (MTX), widely used as a drug in cancer, has many adverse effects on tissues. Apocynin (APO) is a NADPH oxidase inhibitor and is known with many antioxidant properties. In this study, we aimed to evaluate the adverse effects of MTX on testicular tissue and the protective effects of APO at two different doses (20 mg/kg and 50 mg/kg) on MTX-induced testicular damage. Materials and methods: Fifty adult male Wistar albino rats (8 weeks old and weighing 200–250 g) were divided into five groups of 10 rats each: 1. saline control, 2. dimethyl sulfoxide (DMSO) control, 3. MTX, 4. APO-20 + MTX, and 5. APO-50 + MTX. All injections were performed intraperitoneally. At the end of day 28, all rats were sacrificed under anesthesia. The testes were evaluated histologically and the blood samples were analyzed biochemically. Results: According to histological and biochemical analyses, there was no significant difference between the DMSO and control groups. In terms of the histological findings, MTX group was significantly the worst affected group compared to the others, and in this group, apoptotic cell number (P = 0.011) was significantly increased in comparison with the control group. Except MTX, there was no significant difference in apoptotic cell number of the other groups compared to the control group. In the MTX group, malondialdehyde (MDA, P = 0.017) and myeloperoxidase (MPO, P < 0.001) levels were significantly increased in tissue and in blood (MDA P < 0.001, MPO P < 0.001), while tissue glutathione (GSH, P < 0.05) and serum testosterone levels (P < 0.01) were decreased compared with the control group. APO + MTX treatment groups exhibited better testis morphology, and apoptotic cells were also significantly decreased compared to MTX group (P < 0.001). Conclusion: Our results suggest that MTX induced defects on testis via oxidative stress and APO reversed the effects of MTX with its antioxidant properties.

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1. Liang XJ, Chen C, Zhao Y and Wang PC. Circumventing tumor resistance to chemotherapy by nanotechnology. Methods Molecular Biology 2010; 596: 467-488.
2. Madhu P, Reddy PK, Reddy SK. Role of melatonin in mitigating chemotherapy-induced testicular dysfunction in Wistar rats. Drug and Chemical Toxicology 2016; 39: (2): 137-146. doi: 10.3109/01480545.2015.1055359
3. Haghi-Aminjan H, Asghari MH, Farhood B, Rahimifard M, Hashemi Goradel N et al. The role of melatonin on chemotherapy-induced reproductive toxicity. Journal of Pharmacy and Pharmacology 2018; 70 (3): 291-306. doi: 10.1111/jphp.12855
4. Chan ES, Cronstein BN. Mechanisms of action of methotrexate. Bulletin of the Hospital for Joint Diseases 2013; 71 (Suppl. 1): 5-8.
5. Scully CJ, Anderson CJ, Cannon GW. Long-term methotrexate therapy for rheumatoid arthritis. Seminars Arthritis Rheumatism 1991; 20: 317-331.
6. Elango T, Thirupathi A, Subramanian S, Ethiraj P, Dayalan H et al. Methotrexate treatment provokes apoptosis of proliferating keratinocyte in psoriasis patients. International Journal of Clinical and Experimental Medical Sciences 2017; 17: 371-381. doi: 10.1007/s10238-016-0431-4
7. Bath RK, Brar NK, Forouhar FA, Wu GY. A review of methotrexate-associated hepatotoxicity. Journal of Digestive Diseases 2014; 15 (10): 517-524.
8. Singh NP, Muller CH, Berger RE. Effects of age on DNA double-strand breaks and apoptosis in human sperm. Fertility and Sterility 2003; 80: 1420-1430.
9. Gutierrez JC, Hwang K. The toxicity of methotrexate in male fertility and paternal teratogenicity. Expert Opinion Drug Metabolism Toxicology 2017; 13:51-58.
10. Belhan S, Comakli S, Kucukler S, Gulyuz F, Yildirim S et al. Effect of chrysin on methotrexate-induced testicular damage in rats. Andrologia 2019; 51:e13145. doi: 10.1111/and 13145
11. Yuluğ E, Türedi S, Alver A, Türedi S, Kahraman C. Effects of resveratrol on methotrexate-induced testicular damage in rats. The Scientific World Journal 2013; ID: 489659. doi: 10.1155/2013/489659
12. Wasilewski-Masker K, Seidel KD, Leisenring W, Mertens AC, Shnorhavorian M et al. Male infertility in long-term survivors of pediatric cancer: a report from the childhood cancer survivor study. Journal of Cancer Survivorship 2014; 8(3): 437-447. doi: 10.1007/s11764-014-0354-6
13. Atessahin A, Sahna E, Türk G. Chemoprotective effect of melatonin against cisplatin-induced testicular toxicity in rats. Journal of Pineal Research 2006; 41 (1): 21-27.
14. Stolk J, Hiltermann TJ, Dijkman JH, Verhoeven AJ. Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol. American Journal of Respiratory Cell and Molecular Biology 1994; 11: 95-102.
15. Zhang Y, Murugesan P, Huang K, Cai H. NADPH oxidases and oxidase crosstalk in cardiovascular diseases: novel therapeutic targets. Nature Review Cardiology 2020; (17): 170-194. doi: 10.1038/s41569-019-0260-8
16. Chirino YI, Sánchez-González DJ, Martínez-Martinez CM, Cruz C, Pedraza-Chaverri. Protective effects of APO against cisplatin-induced oxidative stress and nephrotoxicity. Toxicology 2008; 245: 18-23.
17. Şener TE, Yüksel M, Özyılmaz-Yay N, Ercan F, Akbal C et al. Apocynin attenuates testicular ischemia–reperfusion injury in rats. Journal of Pediatric Surgery 2014; 50 (8): 1382-1387.
18. Daggulli M, Dede O, Utangac MM, Bodakçı MN, Hatipoğlu NK. Protective effects of carvacrol against methotrexateinduced testicular toxicity in rats. International Journal of Clinical and Experimental Medicine 2014; 7 (12): 5511-5516.
19. Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymology 1978; 52: 302-310.
20. Li M, Liu Z, Zhuan L, Wang T, Guo S et al. Effects of Apocynin on oxidative stress and expression of apoptosis-related genes in testes of diabetic rats. Molecular Medicine Reports 2013; 7 (1): 47-52.
21. Ellman GL. Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics 1959; 82: 70-77.
22. Hartree EF. Determination of protein: A modification of the lowry method that gives a linear photometric response. Analytical Biochemistry 1972; 48 (2): 422-427.
23. Yahyazadeh, A, Altunkaynak, BZ. Protective effects of luteolin on rat testis following exposure to 900 MHz electromagnetic field. Biotechnic and Histochemistry 2019; 1-10.
24. Padmanabhan S, Tripathi DN, Vikram A, Ramarao P, Jena GB. Methotrexate-induced cytotoxicity and genotoxicity in germ cells of mice: intervention of folic and folinic acid. Mutation Research 2009; 673: 43-52.
25. Ragheb AM, Sabanegh ES. Male fertility-implications of anticancer treatment and strategies to mitigate gonadotoxicity. Anti-Cancer Agents in Medicinal Chemistry 2010; 10: 92-102.
26. Bayram M, Ozogul C, Dursun A, Ercan ZS, Isik I et al. Light and electron microscope examination of the effects of methotrexate on the endosalpinx. European Journal of Obstetrics & Gynecology and Reproductive Biology 2005; 120: 96-103. doi: 10.1016/j. ejogrb.2004.08.014
27. Türk G. Kemoterapötiklerin erkek üreme sistemi üzerindeki yan etkileri ve koruyucu stratejiler. Marmara Pharmaceutical Journal 2013; 17: 73-92 (in Turkish).
28. Jahovic N, Cevik H, Sehirli AO, Yeğen BC, Sener G. Melatonin prevents methotrexate-induced hepatorenal oxidative injury in rats. Journal of Pineal Research 2003; 34: 282-287.
29. Jahovic N, Şener G, Çevik H, Ersoy Y, Arbak S et al. Amelioration of methotrexate induced enteritis by melatonin in rats. Cell Biochemistry & Function 2004; 22 (3): 169-178.
30. Cetinkaya A1, Bulbuloglu E, Kurutas EB, Kantarceken B. N-acetylcysteine ameliorates methotrexate-induced oxidative liver damage in rats. Medical Science Monitor 2006; 12 (8): BR 274-278. Epub 2006 Jul 12.ID: 452849.
31. Armagan A, Uzar E, Uz E, Yılmaz HR, Kutluhan S et al. Caffeic acid phenethyl ester modulates methotrexate-induced oxidative stress in testes of rat. Human & Experimental Toxicology 2008; 27: 547-552.
32. Friedman AD, Shah JB, Takoudes TG, Haddad JJr. The role of free radicals in chronic rhinosinusitis. Archives Otolaryngology Head Neck Surgery 2002; 128: 1055-1057.
33. Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods in Enzymology 1990; 186: 421- 431.
34. Gokce A, Oktar S, Koc A, Yonden Z. Protective effects of thymoquinone against methotrexate-induced testicular injury. Human and Experimental Toxicology 2010; 000 (00): 1-7.
35. Köroğlu KM, Çevik Ö, Şener G, Ercan F. Apocynin alleviates cisplatin- induced testicular cytotoxicity by regulating oxidative stress and apoptosis in rats. Andrologia 2019; 51 (4): e13227. doi: 10.1111/and.13227
36. Güvenç M, Aksakal M. Ameliorating effect of kisspeptin-10 on methotrexate-induced sperm damages and testicular oxidative stress in rats. Andrologia 2018; e13057. doi: 10.1111/and.13057
37. Uyar A, Yaman T, Dogan A, Uslu S, Keles ÖF et al. Is Methotrexateinduced Testicular damage preventable using nettle seed extract: A Histopathological, immunohistochemical, biochemical and spermatological examination. Fresenius Environmental Bulletin 2018; 27 (4): 2320-2331.
38. Yüncü M, Bükücü N, Bayat N, Sancar L, Tarakçıoğlu M. The effect of vitamin E and L-carnitine against methotrexate-induced injury in rat testis. Turkish Journal of Medical Sciences 2015; 45: 517-525.
39. Vardi N, Parlakpınar H, Ates B, Çetin A, Otlu A. Antiapoptotic and antioxidant effects of b-carotene against methotrexateinduced testicular injury. Fertility and Sterility 2009; 92 (6): 2028-2033. doi: 10.1016/j.fertnstert
40. Vardı N, Parlakpınar H, Ateş B, Otlu A. The preventive effects of chlorogenic acid against to testicular damage caused by methotrexate. Türkiye Klinikleri Journal of Medical Sciences 2010; 30 (2): 507-513.
41. Nouri HS, Azarmi Y, Movahedin M. Effect of growth hormone on testicular dysfunction induced by methotrexate in rats. Andrologia 2009; 41: 105-110.
42. Belhan S, Özkaraca M, Kandemir FM, Gülyüz F, Yıldırım S et al. Effectiveness of hesperidin on methotrexate-induced testicular toxicity in rats. Kafkas Üniversitesi Veteriner Fakültesi Dergisi 2017; 23 (5): 789-796. doi: 10.9775/kvfd.2017.17752
43. Sheikhbahaei F, Khazaei M, Rabzia A, Mansouri K, Ghanbari A. Protective effects of thymoquinone against methotrexateinduced germ cell apoptosis in male mice. International Journal of Fertility and Sterility 2016; 9 (4): 541-547.
44. Maremanda KP, Jena GB. Methotrexate-induced germ cell toxicity and the important role of zinc and SOD1: Investigation of molecular mechanisms. Biochemical and Biophysical Research Communications 2017; 483 (1): 596-601. doi: 10.1016/j. bbrc.2016.12.098
45. Ozbek O, Altintas R, Polat A, Vardı N, Parlakpınar H et al. The protective effect of apocynin on testicular ischemia-reperfusion injury. The Journal of Urology 2015; 193 (4): 1417-1422.
46. Liu PG, He SQ, Zhang YH, Wu J. Protective effects of apocynin and allopurinol on ischemia/reperfusion-induced liver injury in mice. World Journal of Gastroenterology 2008; 14 (18): 2832- 2837.
47. Altıntaş R, Polat A, Vardi N, Oguz F, Beytur A et al. The protective effects of apocynin on kidney damage caused by renal ischemia/ reperfusion. Journal of Endourology 2013; 27 (5): 617-624.
48. Trottmann M, Becker AJ, Stadler T, Straub J, Soljanik I et al. Semen quality in men with malignant diseases before and after therapy and the role of cryopreservation. European Urology 2007; 52: 355-367.
49. Cagin YF, Erdogan MA, Sahin N, Parlakpınar H, Atayan Y. Protective effects of apocynin on cisplatin-induced hepatotoxicity in rats. Archives of Medical Research 2016; 46 (2015): 517-526. doi: 10.1016/j.arcmed.2015.08.005
50. Cagin YF, Parlakpınar H, Polat A, Vardi N, Atayan Y et al. The protective effects of apocynin on ionizing radiation - Induced intestinal damage in rats. Drug Development and Industrial Pharmacy 2016; 42 (2): 317-324. doi: 10.3109/03639045.2015 .1052080
51. Ilbey YO, Ozbek E, Cekmen M, Simsek A, Otunctemur A et al. Protective effect of curcumin in cisplatin-induced oxidative injury in rat testis: mitogen-activated protein kinase and nuclear factor-kappa B signaling pathways. Human Reproduction 2009; 24 (7): 1717- 1725.
52. Yalçın B, Çevik M. Erkek infertilitesinde spermatozoon DNA hasarının rolü ve önemi. Mehmet Akif Ersoy Üniversitesi Veteriner Fakültesi Dergisi 2018; 3 (2): 135-140 (in Turkish). doi: 10.24880/maeuvfd.463669
53. Agarwal A, Sekhon LH. The role of antioxidant therapy in the treatment of male infertility. Human Fertility 2010; 13: 217- 225.
54. A BL, Fu SJ, Lu JZ, Shi TK, Chen S et al. Effects and action mechanisms of apocynin in the treatment of oligoasthenozoospermia in the mouse. Zhonghua Nan Ke Xue (National Journal of Andrology) 2016; 22 (7): 649-653.
55. Lin WW, Lamb DJ, Wheeler TM, Abrams J, Lipshultz LI et al. Apoptotic frequency is increased in spermatogenic maturation arrest and hypospermatogenic states. Journal of Urology 1997; 58 (5): 1791-1793. doi: 10.1016/S0022-5347(01)64130-2
56. Takagi S, Itoh N, Kimura M, Sasao T, Tsukamoto T. Spermatogonial proliferation and apoptosis in hypospermatogenesis associated with nonobstructive azoospermia. Fertility and Sterility 2001; 76: 901-907.
57. Tesarik J, Ubaldi F, Rienzi L, Martinez F, Lacobelli M et al. Caspase-dependent and independent DNA fragmentation in Sertoli and germ cells from men with primary testicular failure: relationship with histological diagnosis. Human Reproduction 2004; 19: 254-261.
58. Gevrek F and Erdemir F. Investigation of the effects of curcumin, vitamin E and their combination in cisplatininduced testicular apoptosis using immunohistochemical technique. Turkish Journal of Urology 2018; 44 (1): 16-23. doi: 10.5152/tud.2017.95752
59. Kim JC, Kim KH, Chung MK. Testicular cytotoxicity of DA125, a new anthracycline anticancer agent, in rats. Reproductive Toxicology 1999; 13 (5): 391-397.