DOI: http://dx.doi.org/10.18203/issn.2454-2156.IntJSciRep20170356

Histological and immunohistochemical study of cyclophosphamide effect on adult rat testis

Hoda H. Anan, Nashwa S. Wahba, Maha A. Abdallah, Dalia A. Mohamed

Abstract


Background: Nowadays, cyclophosphamide is widely used as anticancer and immunosuppressive agent in various drug regimens in many diseases and in young and old age. The aim of this research is to study the possible histological changes that may occur in the testes of adult male albino rats as a result of chronic exposure to cyclophosphamide and the prognosis of this effect.

Methods: Thirty healthy adult male albino rats were used in this study.  They were equally divided into three groups; a control, an experimental and a withdrawal groups. The Animals of the experimental group were treated with daily dose of 5 mg/ kg cyclophosphamide orally for successive 28 days. Animals of the withdrawal group were left without treatment and sacrificed after 28 days from the last dose of cyclophosphamide.  At the time of sacrifice, all animals were anesthetized by ether inhalation and their testes were dissected out carefully and processed for light and electron microscope examinations.  

Results: Testes of the cyclophosphamide treated group revealed presence of many distorted shrunken seminiferous tubules which appeared with marked reduction in the thickness of the epithelium and wide lumina. Many germ cells with deeply stained nuclei, giant cells in mitosis and intracellular vacuoles were observed. Cross sections in mid pieces of sperms showed marked affection of axoneme, fibrous sheath and mitochondrial sheath. The cytoplasm of the Leydig cells contained mitochondria, dilated SER, Golgi cisternae and RER. Testes of the withdrawal group showed that the seminiferous tubules still had reduced height of their epithelium with wide intercellular spaces. Abnormal stratification and destructed germinal epithelium were evident with desquamated germ cells. Cross sections of mid pieces of the sperms showed distorted axoneme and swollen mitochondrial sheath. The cytoplasm of leydig cells contained many electron dense granules, RER, many dilated SER and mitochondria.

Conclusions: Chronic cyclophosphamide treatment not only produced serious histological changes of the testis but also in its serological parameter. These changes persisted after cessation of cyclophosphamide administration which indicates the cumulative irreversible toxic effect of cyclophosphamide. So, it is advisable to avoid the usage of cyclophosphamide as possible especially in young patients.

 


Keywords


Cyclophosphamide, Adult, Testis, Ultrastructure, Immunohistochemistry

Full Text:

PDF

References


Hellmich B, Kausch I, Doehn C, Jacham D, Holl-Ulrich K, Gross W. Urinary bladder cancer in Wagener’s granulomatosis: is more than cyclophosphamide. Ann Rheum Dis. 2004;63:1183-5.

Senthinkumar S, Yogeeta S, Subashini R, Devaki T. Attenution of cyclophosphamide induced toxicity by squalene in experimental rats. Chemico-Biol Interact. 2006;160:252-60.

Zhou D, Lu Y, Steiner M, Dalton T. Cytochrome P450 2C9 sensitizes human prostate tumour cells to cyclophosphaide via a bystander effect. Antimicrob Agents Chemother. 2000;44(10):2629-63.

Elangovan N, Chiou T, Tzeng W, Chu S. Cyclophosphamide treatment causes impairment of sperm and its fertilizing ability in mice. Toxicol. 2006;222:60.

Laura S. Regulation of male germ cell apoptosis by sphingosine-1-phosphate. Academic dissertation. Finland: University of Helsinki; 2004.

Boujbiha M, Hamden K, Guermazi F, Bouslama A, Omezzine A, Kammoun A, et al. Testicular toxicity in mercuric chloride treated rats: association with oxidative stress. Reprod. Toxicol. 2009;28(1);81-9.

Ghosh D, Das U, Misro M. Protective role of alpha-tocopherol succinate (provitamine-E) in cyclophosphamide induced testicular gamtogenic and steroidogenic disorders: A correlative approach to oxidative stress. Free rad Res. 2002;36(1):1209-18.

Kalender S, Kalender K, Ogutcu A, Uzunhisarcikli M, Durak D, Acikgoz F. Endosulfan-induced cardiotoxicity and free radical metabolism in rats: the protective effect of vitamin E. Toxicol. 2004;202:227-35.

Bancroft DJ, Gamble M. Theory and Practice of Histological Techniques. 5th edition. Churchill Livingstone; 2002: 125-134.

Kiernan JA. Histological and Histochemical Methods: Theory and Practice. 3rd edition. Oxford: Butterworth/Heinemann; 2000.

Glauert AM, Lewis PR. Biological Specimen Preparation for Transmission Electron Microscopy. London: Portland press; 1998.

Perini P, Calabrese M, Rinaldi L, Gallo P. The safety profile of cyclophosphamide in multiple sclerosis therapy. Expert Opin Drug Saf. 2007;6:183-90.

Uber W, Self S, Van Bakel A, Pereira N. Acute antibody-mediated rejection following heart transplantation. Am J Transplant. 2007;7:2064-74.

Rekhadevi P, Sailaja N, Chandrasekhar M, Mahboob M, Rahman M, Grover P. Genotoxicity assessment in oncology nurses handling anti-neoplastic drugs Mutagenesis. 2007;22:395-401.

Fukushima T, Yamamoto T, Kikkawa R, Hamada Y, Komiyama M, Mori C, et al. Effect of male reproductive toxicant on gene expression in rat testes, J Toxicol Sci. 2005;30:195-206.

El-seedy A, Taha T, El-seehy M, Maklouf A. Ultrastructure sperm defects in male mice during carcinogenicity of urethane and indoxan. Arab J Biotech. 2005;9(1):27-40.

Jedlinska -Krakowska M, Bomba G, Jakubowski K, Rotkiewicz T, Jana B, Penkowski A. Impact of oxidative stress and supplementation with vitamins E and C on testes morphology in rats. J Reprod Develop. 2006; 52(2);203-9.

Haubitz M. Acute and long- tem toxicity of cyclophosphamide. Transplantations Medizin. 2007;19:26-31.

Yamazaki M, Muguruma M, Takahashi M, Moto M, Kashida Y, Mitsumori K. Molecular pathological analysis of apoptosis induced in testicular germ cells after a single adiminstration of Mono- (2-ethylhexyl) phthalate to F344 rats. J Toxicol Pathol. 2006;19:185-90.

Sakamaki K. Physiological and pathological cell death in the reproductive organs. Cell Struct Funct. 2003;28:31-40.

Morales E, Horn R, Pastor M, Santamaria L, Pallares J, Zuasti A, et al. Involution of seminiferous tubules in hamster: An ultrastructural, immunohistochemical and quantitative morphological study. Histol Histopathol. 2004; 19:445-55.

Yan W, Jun-xing H, Anna-Stina L, Lauri P, Eeva S, Matti P, et al. Overexpression of Bcl-w in the testis disrupts spermatogenesis: Revelation of a role of Bcl-w in male germ cell cycle control. Molecular endocrinol. 2003;17(9):1868-79.

Yang S, Han K, Kim R, Sim C. Effect of alpha-tocopherol on cadmium –induced toxicity in rat testis and spermatogenesis. J of Korean-Med sci. 2006;21(3):445-51.

He D, Li X, Yue L, Wei G, Lin T, Liu J. Effect of cyclophosphamide on spermatogonial stem cells. Zhonghua Nan Ke Xue. 2006;12(5):387-90.

Yamamoto C, Hikim A, Lue Y, Portugal A, Guo T, Hus S, et al. Impairment of spertmatogenesis in transgenic mice with selective overexpression of Bcl2 in somatic cells of the testis. J Androl. 2001;22(6):981-91.

Østensen M, Khamashta M, Lockshin M, Parke A, Brucato A, Carp H, et al. Anti-inflammatory and immunosuppressive drugs and reproduction. Arthritis Res Ther. 2006;8(3):209.

Zamzami N, Brenner C, Marzo I, Susin SA, Kroemer G. Subcellular and submitochondrial mode of action of Bcl-2-like oncoproteins. Oncogene. 1998;16:2265-82.

Meeh T, Loveland K, Dekretser D, Cory S, Print C. Developmental regulation of the Bcl2 family during spermatogenesis: insights into the sterility of Bcl2-w male mice. Cell Death Diff. 2001;8:225-33.

Olderied N, Angelis P, Wiger R, Clausen O. Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis. J Mol Human Rep. 2001;7(5):403-8.

Biswabina R, Bhagath P. Histilogical and histochemical studies on the effect of sigle dose of cyclophosphamide on migration of primordial germ cells of fetal Charles foster rat-A preliminary study. Firat tip dergisi. 2007;12(4):246-50.

Krishnamoorthy G, Murugesan P, Muthuve R, Gunadharini D, Vijayababu R, Arunkumat A, et al. Effect of Arcolor 1254 on sertoli cellular antioxidant system, androgen binding protein and lactate in adult rat in vitro. Toxicol. 2005;212(2-3):195-205.

Guneli E, Tugyan K, Ozturk H, Gumustekin M, Cilaker S, Uysal N. Effect of melatonin on testicular damage in streptozotocin-induced diabetes rats. Eur Surg Res. 2008;40:354-60.

Hutson JC. Physiologic interaction between macrophages and leydig cells. Exp Biol Med (Maywood). 2006;231(1):1-7.

Baker M, Aitken R. Reactive oxygen species in spermatozoa: methods for monitoring and significance for the origins of genetic disease and infertility. Reprod Biol Endocrinol. 2005;3:67.

Rezvanafar M, Sadrkhanalou R, Ahmadi A, Shojaei-Sadee H, Rezvanfar M, Mohammadirad A, et al. Protection of cyclophosphamide-induced toxicity in reproductive tract histology, sperm characteristic and DNA damage by an herbal source evidence for role of free radical toxic stress. Hum Exp Toxicol. 2008;27(12):901-10.

Bhatia K, Ahmed F, Rashid H, Raisuddin S. Protective effect of S-allylcysteine against cyclophosphamide-induced bladder haemorrhagic cyctitis in mice. Food Chem Toxicol. 2008;46(11):3368-74.

Selvakumar E, Prahalathan C, Sudharsan P, Varalakshmi P. Protective effect of lipoic acid on cyclophosphamid induced testicular toxicity. Clin Chim Acta. 2006;367(1-2):114-9.

Murugesan P, Nuthusamy T, Balasubramanian K, Arunakaran J. studies on the protective role of vitamin C and E against polychlorinated biphenyl (aroclor 1254) induced oxidative damage in leydig cells. Free Rad res. 2005;39(11):1259-72.

Kaaij V, Heulte N, Lestamg N, Raemaekers J, Simons A, Carde P, et al. Gonadal function in males after chemotherapy for early-stage Hodgkin’s lymphoma treated in four subsequent trials by the European organization for research and treatment of cancer: EORTC lymphomia group and group d’Etude des lymphomas de L’adulte. Clin Oncol. 2007;25(19):2825-32.