INTRODUCTION:
Cisplatin is a key stone in the chemotherapy and one of the most effective and potent anticancer drugs. Even though number of platinum coordination compounds exhibit anti-viral and anti-tumor activities, but cisplatin and its direct analog carboplatin are effective anticancer drugs and currently approved for the treatment of several human carcinomas.1
Cisplatin is a platinum coordinated compound containing central atom of platinum surrounded by two chloride atoms and two ammonia molecules in its cis position. Cisplatin can bind to nucleic acids, proteins and sulfur- containing biomolecules such as glutathione. The ultimate target of cisplatin which triggers its cytotoxic activity is generally accepted to be DNA. In general cisplatin–DNA adducts inhibits DNA replication and blocks transcription by RNA polymerase, finally triggers programmed cell death or apoptosis.2
Cisplatin is cited for treatment of various cancers such as testicular, ovarian, cervical, head and neck, bladder and lung cancers. However the use of cisplatin has rendered at least one cancer such as testicular cancer which is curable and is significant in treatment of ovarian and bladder cancers. After the use of cisplatin based chemotherapy into the treatment of testicular cancer, the cancer specific five year survival rates for these patients approach 95% in the world. Despite this success, there is still a limited range of tumors sensitive to cisplatin treatment. The compound cisplatin is associated with list of side effects including severe nausea, vomiting, myelo suppression, ototoxicity, neuro toxicity and nephro toxicity. Recent reports evaluates that cisplatin based chemotherapy induces toxic effects on various body organs in animals and humans such as nephrotoxicity in the chick embryos guinea pig, ototoxity in hamsters, hepato toxicity in rats.3
In the present study platinum based anti-cancer drug i.e., cisplatin was administered to male rats in order to investigate the possible interference of cisplatin in affecting male reproduction in rats. Because these observations may be helpful to understand the causes behind the reduction of male reproductive health of cancer patients who are under treatment with cisplatin. However many reports available on cisplatin caused reproductive toxicity, but very limited studies demonstrated the use of testosterone to prevent the antifertility effects caused by cisplatin and other anti cancer drugs.
The main testicular androgen i.e., testosterone is produced by leydig cells under the stimulation of pituitary LH, which is essential for spermatogenesis, fertility and maintenance of the male phenotype. so an attempt has been made in the present study to investigate the protective role of testosterone against platinum compounds caused suppression of reproduction in male rats. Because, the pivotal role of androgens in male fertility is well established.4
MATERIALS AND METHODS:
Animals:
Healthy adult male wistar rats of same age group (70±5 Days) were selected for the present study. Animals were housed in an air conditioned animal house facility at 26±1ο C, with a relative humidity of 75%, under a controlled 12 h light/dark cycle. The rats were reared on a standard pellet diet (HLL Animal Feed, Bangalore, India) and tap water adlibitum.
Test chemicals:
Cisplatin was purchased from Sigma chemicals, St.Louis Co., MO, USA. This compound was dissolved in 0.9% normal saline to obtain the final concentration of the 3 mg/kg body wt. of the animal. Testoviron depot was obtained from the local drug suppliers.
Experimental Design:
The rats were divided into three groups consisting of eight animals in each group. The rats in the first group were served as control and received 0.9% of normal saline only. The rats in the second group were received cisplatin (3 mg/kg body wt). The rats in the third group were received cisplatin (3 mg/kg body wt) and testosterone (4.16 mg/kg body wt). Injections were given intraperitoneally to rats on 1st, 3rd and 5th day of experimentation.
On 45th day of experiment animals were sacrificed by cervical dislocation. The testes, epididymis, ventral prostate and seminal vesicles were removed and weighed. The seminal vesicle was weighed without fluid.
Collection of epididymal sperm:
The epididymal sperm were collected by cutting epididymis into small pieces and flushing the sperm in normal saline. The sperm collected was centrifuged at 225 × g for 10 min. The pellet was resuspended in 2.0 ml of normal saline. An aliquot of sperm suspension was homogenized for few seconds, centrifuged at 800 × g for 10 min and used for analysis.
Epididymal sperm count, motility and viability:
Epididymal sperm counts and evaluation of motility of epididymal sperm were done by the method of Belsey5. The epididymal sperm was obtained as described above and incubated at 37οC. The epididymal fluid was then diluted to a volume of 5.0 ml of pre-warmed (37οC) normal saline. An aliquot of this solution was placed in Neubaeur chamber and motile sperm were counted by using microscope.
Sperm motility was expressed as a percent of motile sperm of the total sperm counted. Non-motile sperm numbers were first determined followed by counting of total sperm. The ratio of live and dead spermatozoa was determined using 1% trypan blue by the method of Talbot and Chacon.6
HOS test:
The hypo osmotic swelling test (HOS test) for investigating the functional integrity of sperm membrane has been introduced as a useful assay in the diagnosis of infertile semen.7 The principle of HOS assay is based on fluid transport across the sperm tail membrane under hypo osmotic conditions until equilibrium is reached. Due to influx of fluid, the tail coils, considered as hypo osmotic response, which can be readily identified under phase-contrast microscope. The sperms were exposed to hypo osmotic medium and observed for coiled tails under the microscope and the percent of coiling was determined by the method of Jeyendran.8
Assay of testicular steroidogenic marker enzymes:
The testicular tissue was homogenized in ice-cold Tris-HCl buffer (pH 6.8). The microsomal fraction was separated and used as enzyme source. The activity levels of 3β-hydroxy steroid dehydrogenase (3β-HSD; EC.1.1.1.51) and 17β-hydroxy steroid dehydrogenase (17β-HSD; EC.1.1.1.64) were assayed by the method of Berg Meyer9. The enzyme assays were made under the conditions following zero order kinetics after preliminary standardization regarding linearity with respect to time of incubation and enzyme concentration.
The reaction mixture in a volume of 2.0 ml contained: 100 μ moles of sodium pyro phosphate buffer (pH 9.0), 0.5 μ moles of co-factor (NAD for 3β-HSD and NADPH for 17β-HSD), 0.08 μ moles of substrate (dehydro epi andro sterone for 3β-HSD and androstenedione for 17β-HSD) and 20 mg equivalent of microsomal protein as enzyme source.
The reactions were carried out in a quartz cuvette of 1.0 cm path at 23±1ο C. The absorbance at 340 nm was measured at 20s intervals for 5 min using UV- spectrophotometer (Hitachi U-2001). Protein content in the enzyme source was estimated by the method of Lowry10 using bovine serum albumin as standard. The enzyme activities were expressed in micromoles of NAD converted to NADH mg/ protein/min for 3β-HSD or micromoles of NADPH converted to NADP mg/ protein/min for 17β-HSD.
Statistical analysis:
The data were presented as mean ± SD. Statistical analysis was performed using analysis of variance (ANOVA) followed by Dunnett’s test using SPSS 10.0.
Legend for Fig− 1: Effect of cisplatin or cisplatin + testosterone on sperm parameters in rats.
Values are mean ± SD of eight individuals. Values are significantly different from control at *p<0.001, ** p<0.0001.
For calculation of % change and ‘p’ for cisplatin + testosterone injected rats, cisplatin injected rats served as controls.
Control; 
Cisplatin; 
Cisplatin + Testosterone
RESULTS:
No mortalities were observed in control or experimental groups. No behavioral abnormalities were observed in experimental animals. A significant decrease in sperm motility and viability was observed with reduction in average sperm counts in cauda epididymal tissue of cisplatin treated rats. Sperm coiling percentage was also decreased significantly in these rats. The average percentages of above mentioned sperm parameters were significantly increased in cisplatin + testosterone treated rats when compared with cisplatin alone treated rats (Fig-1).
The average sperm coiling in control rat is 60.54 % whereas the sperm coiling is 43.43 % in cisplatin treated rats. But in cisplatin + testosterone treated rats the average sperm coiling percentage is 51.12 % (Fig-1). The results presented in [Fig.2] indicate that a significant decrease in activity levels of 3β-HSD (A) and 17β-HSD (B) observed in cisplatin treated rats when compared with control rats. But cisplatin + testosterone treated rats showed significant increase in activity level of 3β-HSD (A) and 17β-HSD (B) when compared with cisplatin alone treated rats. Apart from this increased incidence of abnormal spermatozoa in morphology was observed in cisplatin treated rats. But, however control and cisplatin + testosterone treated rats were not reported any abnormalities in spermatozoa.
Legend for Fig −2: Effect of cisplatin or cisplatin+testosterone on the activity levels of 3β-HSD and 17β-HSD in the testis of rats.
Values are mean ± SD of eight individuals. Values are significantly different from control at *p<0.001, ** p<0.0001.
For calculation of % change and ‘p’ for cisplatin + testosterone injected rats, cisplatin injected rats served as controls.
Control; Cisplatin; Cisplatin + Testosterone
DISCUSSION:
The present study demonstrates the adverse effect of cisplatin on spermatogenesis and steroidogenesis and protection caused by testosterone co-administration. The significant decrease in sperm count, sperm motility, sperm viability and sperm function in cispatin injected rats indicates its toxic effects on spermatogenesis. In the present study it was observed that the exposure to platinum-based anticancer drugs caused significant decrease in sperm count, sperm motility, sperm viability and HOS sperm tail coiling in rats.
The present findings are in agreement with the earlier reports such as decrease in sperm motility in rats and in humans treated with platinum-based anticancer drugs. Other anticancer drugs such as 5-fluorouracil, methotreoxate, ifosamide and adriamycin have also been shown their effect on spermatogenesis.11
Thus a significant decrease in sperm motility, viability and sperm count in the rats exposed to platinum-based anticancer drugs indicate an inhibitory effect of cisplatin on spermatogenesis. Earlier few investigators have been reported that cisplatin treatment resulted in damage of preleptotene and leptotene spermatocytes which indicates killing of type A2-B spermatogonia. Furthermore, chemotherapy with cisplatin has been shown to have profound and long-lasting effects on spermatogenesis. At low doses cisplatin was selectively toxic for spermatogonia, whereas at higher doses it has broad toxic activity as observed by killing cells in all stages, including spermatocytes and spermatids in the adlumenal compartment. 12
Spermatogenesis is a well regulated process starting with spermatogoneal stem cells and ending with differentiated, motile spermatozoa. The significant decrease in sperm characteristics after cisplatin treatment indicates its toxic effects on seminiferous tubule epithelium and sertoli cells. Histo-pathological findings by Atenssahin suggest that cisplatin may cause decreases in diameter size and germinative cell layer thickness of seminiferous tubule which leads to spermiotoxicity. The disruption of the sertoli cell tight junctions in rats following cisplatinum administration demonstrates that sertoli cells are susceptible to this drug.13
Apart from this, administration of platinum-based anticancer drugs also caused increased incidence of abnormal spermatozoa in cisplatin treated rats, when compared with controls and cisplatin + testosterone treated rats (Exact data was not recorded). In particular, morphology of head of several sperms in experimental rats were with rod shape, arrow shape, banana shape, and pin point shape head instead of its normal hook shape (Fig. 5.3). Previous investigations supports this observation, such as adriamycin treatment reported abnormalities in epdidymal sperm in mice, carboplatin has been shown to induce high frequency of abnormal sperms at higher doses in mouse. These abnormalities however are rare in control rats.14
Thus cisplatin treatment might partially suppress spermatogenesis causing abnormalities in spermatozoa. But cisplatin + testosterone treated rats showed significant increase in epdidymal sperm count, sperm viability, sperm motility and sperm coiling percentages and decrease in abnormal spermatozoa which indicates exogenous administration of testosterone may be responsible for restoration of spermatogenesis in cisplatin + testosterone treated rats (Fig-1).
Exogenous administration of testosterone caused restoration of sperm quality and quantity in experimental rats exposed to platinum compounds. Since, spermatogenesis is well known to be dependent on androgen levels and testosterone is also thought to be the predominant androgen involved in spermatogenesis. Low levels of exogenous testosterone can partially or fully maintain spermatogenesis in rats treated with chemotherapeutic agents.15
Moreover spermatogenesis was restored using exogenous testosterone implants to normal or near normal levels in drug-stressed animals. Testosterone when administered in sufficient amount is capable of maintaining or restoring qualitatively complete spermatogenesis in hypophosectomized animals and has been proven effective in preserving spermatogenesis in animals subjected to cytotoxic insults.16
Spermatogenesis is more sensitive to testicular damage and if testosterone deficiency occurs it will always proceeded by infertility. There is increasing appreciation of the consequences of partial androgen therapy and the benefits of treatment. All features of testosterone deficiency should be reversible with appropriate testosterone replacement therapy. Moreover other studies have shown that testosterone can be used safely at higher doses for long periods.17
In cisplatin treated rats, a significant decrease in the activity levels of 3b-HSD and 17b-HSD was observed which clearly indicates the impairment of steroidogenesis. These two enzymes are having regulatory functions in the maintenance of steroidogenesis and also involves in the synthesis of testosterone. Determination of the activity levels of 3b-hydroxysteroid dehydrogenase and 17b-hydroxysteroid dehydrogenase enzymes has been used to study the testicular steroidogenesis of rats in different experimental conditions.18
Exposure to toxic substances that are not endogenous to the body can lead to chemical reactions that alter the outcome of several biochemical pathways. Alterations of the hormonal steroidogenic pathway at even one step will change the pattern of production and eventually secretion of steroid hormones. Any chemical interference to steroidogenesis viz., altering enzymatic activity, altering precursor availability, interfering with control mechanisms etc., can cause adverse effects to the reproductive system. Toxic responses to the reproductive system can result in such adverse effects as abnormal sexual and physical development, diminished fertility or sterility.19
The decreased steroidogenic enzyme activity levels may lead to decreased steroidogenesis in experimental rats which in turn may suppress the reproductive activities in the male rats. It seems platinum-based anticancer drugs acts on leydig cells and inhibits the testosterone production which was evident by decrease in the activity levels of 3b-HSD and 17b-HSD enzymes in the testes of experimental rats. Since, the enzyme 3b-HSD is localized exclusively within the leydig cells in the testes. In addition; chemotherapy may have a direct toxic effect on the leydig cells.
Previous studies also have been demonstrated the decreased activity levels of 3b-HSD and 17b-HSD in the leydig cells cultured with cisplatin and in the testis of rats treated with diphenyl ethylene diamine platinum complex. Other anticancer drugs such as cyclophosphamide and adriamycin have also been reported to cause decrease in activity levels of 3b-HSD and 17b-HSD enzymes in the testis of rats. Moreover subclinical hypoandrogenesis was also observed due to cisplatin-based chemotherapy.20
The activity levels of 3b-HSD and 17b-HSD were significantly increased in cisplatin + testosterone treated rats when compared with cisplatin treated rats. This increase in 3b-HSD and 17b-HSD activities levels in testis indicates the restoration of steroidogenesis and leads to normal fertility in cisplatin + testosterone treated rats (Fig-2). Injection of testosterone significantly increased the steroidogenic marker enzyme (3b-HSD and 17b-HSD) activity levels in the testis of platinum compounds treated rats. This may result in increased androgen production which in turn enhances the male reproductive efficiency.
CONCLUSION:
From the above results, it can be concluded that administration of platinum-based anticancer drugs suppresses the spermatogenesis and steroidogenesis by inhibiting the activity levels of testicular steroidogenic marker enzymes (3b-HSD and 17b-HSD) which are essential for production of testosterone, indicates the impairment of male reproduction in experimental animals. Supplementation of testosterone along with cisplatin could restore the deficiency of testicular testosterone contents and ameliorates the detrimental effects of cisplatin, finally preserves fertility. So it is suggested that patients under cisplatin regimen may be prescribed with testosterone during treatment period to maintain fertility.
ACKNOWLEDGEMENTS:
We are grateful to Head, Department of Biotechnology, S.V.University for providing necessary laboratory facilities. The research was conducted in accordance with the regulations in the country and approved by the Ethical Committee of Department of Biotechnology, S.V.University, Tirupati.
REFERENCES:
1. Boulikas T Vougiouka M. (2003). Cisplatin and platinum drugs at the molecular level. Oncol. Rep; 10: 1663-1682.
2. Jamieson ER, Lippard SJ. (1999). "Structure, Recognition and Processing of Cisplatin- DNA Adducts" Chem. Rev; 99: 2467-2498.
3. Church MW, Blakley BW, Burgio DL, Gupta AK (2004): WR-2721 (Amifostine) Ameliorates cisplatin-induced hearing loss but causes neuro toxicity in hamsters : Dose dependent effects. JARO, 2004, 5, 227-237.
4. Sharpe R: Regulation of Spermatogenesis. In: Knobil ENJ, ed (1994). The physiology of reproduction, Raven Press, New York, 1363-1434.
5. Belsey MA, Moshissi KS, Eliasson R, Paulsen CA, Callegos AJ, Prasad MRN (1980). Laboratory manual for the examination of human semen and semen cervical mucus interaction. Press concern.
6. Talbot P, Chacon RS (1981). A triple stain technique for evaluating normal acrosome reaction of human sperm. J Exp Zool, 215, 201-208.
7. Jeyendran RS, Van Der Van HH, Zaneveld LJ (1992). The hypo osmotic swelling test: an update. Arch Androl, 29, 105-116.
8. Jeyendran RS, VanDerVen HH, Perez-Pelaez M, Crabo BG, Zaneveld LJ (1984). Development of an assay to asses the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil, 70, 219-228.
9. Bergmeyer HU (1974) b-hydroxy steroid dehydrogenase. In: Methods of Enzymatic analysis. Edited by H.U. Bergmeyer, Academic Press, New York, 1, 447-489.
10. Lowry OH, Rosebrough MJ, Farr AL, Randall RJ (1951). Protein measurement with the folin phenol reagent. J Biol Chem, 193, 265-275.
11. Sjoblom T, West A, Lahdetie J (1998). Apoptotic response of spermatogenic Cells to the germ cell mutagens etoposide, adriamycin, and diepoxybutane. Environ Mol Mutagen. 31(2):133-48.
12. Meistrich ML, Goldstein LS and Wyrobek AJ (1985). Long term infertility and dominant lethal mutations in male mice treated with adriamycin, 152, 53-65.
13. Atessahin A, Karahan I, Turk G, Gur S, Yilmaz S, Ceribasi AO. (2006). Protective role of lycopene on cisplatin-induced changes in sperm characteristics, testicular damage and oxidative stress in rats. Rep Tox, 21, 42-47.
14. Syntin P, Robaire B (2001). Sperm structural and motility changes during aging in the Brown Norway rat. J Androl, 22, 235-244.
15. McLachlan RI, Wreford NG, O’Donnell L, de Krester DM, Robertson DM (1996). The endocrine regulation of spermatogenesis: independent roles for testosterone and FSH. Journal of Endocrinology, 148, 1-9.
16. Delic JI, Bush C, Peckham MJ (1986).: Protection from procarbazine –induced damage of spermatogenesis in rat by androgen. Cancer Res, 46, 1909-1914.
17. Cunningham GR, Silverman VE, Thornby J, Kohler PO. (1979). The potential
18. Ghosh PK, Ghosh D, Ghosh P, Chaudhuri A. (1995). Effects of prostatectomy on testicular androgenesis and serum levels of gonadotropins in mature albino rats. Prostate; 26: 19-22.
19. Gray LE, Wolf C, Lambright C, Mann P, Price M, Cooper RL, Ostby J. (1999). Administration of potentially antiandrogenic pesticides (procymidone, linuron,iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol. Ind. Health; 15: 94-118.
20. Lange PH, Narayana P, Vogelzang NJ, Shafer RB, Kennedy BJ, Fraley EL (1983). Return of fertility after treatment for non-seminitomous testicular cancer: changing concepts. J urol, 55, 548-573.
Received on 25.11.2009 Modified on 23.01.2010
Accepted on 26.02.2010 © RJPT All right reserved
Research J. Pharm. and Tech. 3(2): April- June 2010; Page 535-539