Brief Resume of the Intended Work s2

BRIEF RESUME OF THE INTENDED WORK:

6.1 Need for the study

Spermatogenesis is a process of division and differentiation by which spermatozoa are produced in seminiferous tubules. Seminiferous tubules are composed of somatic cells and germ cells (spermatogonia, spermatocytes, and spermatids). Activities of these three germ cells divide spermatogenesis into spermatocytogenesis, meiosis, and spermiogenesis, respectively. Spermatocytogenesis involves mitotic cell division to increase the yield of spermatogenesis and to produce stem cells and primary spermatocytes. Meiosis involves duplication and exchange of genetic material and two cell divisions that reduce the chromosome number to haploid and yield four spermatids. Spermiogenesis is the differentiation without division of spherical spermatids into mature spermatids which are released from the luminal free surface as spermatozoa1. The process of spermatogenesis is highly sensitive to fluctuations in the physical and chemical environment2. Dietary deficiencies (such as vitamins B, E and A), anabolic steroids, metals (cadmium and lead), x-ray exposure, dioxin, alcohol, and infectious diseases will also adversely affect the rate of spermatogenesis. In male cancer patients, surgery, radiotherapy and chemotherapy can be followed by transient or permanent infertility by affecting ejaculatory or erectile function, or by impairing spermatogenesis3. Anticancer drugs have detrimental effects on actively dividing cells specially bone marrow and gametogenesis. High rates of cellular division and metabolic activity is the basis for susceptibility to certain types of damage. During duplication of genetic material and cell division DNA is particularly vulnerable to damage. Antimetabolite anticancer drugs interfere with DNA production. Anti-metabolites masquerade as a purine (azathioprine, mercaptopurine) or a pyrimidine - which become the building blocks of DNA. They prevent these substances becoming incorporated in to DNA during the S phase (of the cell cycle), stopping normal development and division4. Gemcitabine, a new antimetabolite anticancer drug, too has detrimental effects results in predetermined myelosuppression5. Gemcitabine impacts the histological structure of mice testis and embryonic organs6. 6.2 Review of Literature

There are many studies on toxic effects of anticancer drugs on spermatogenesis both in human and animal models7. It was indicated that thiotepa affected testicular germinal epithelium by both cytotoxic effect and induction of apoptosis8. On administration of Biocarbazin (DTIC synonym) an anticancer drug, seminiferous tubule of the testis displayed dose-dependent histological alterations manifested with decrease of mitotically dividing cells and increase in the number of multinucleated spermatocytes and spermatids9. Imatinib mesylate interferes with postnatal testicular development in the rat by delaying the formation of germ-line stem cell pool, reducing proliferation of type A spermatogonia and induces germ cell apoptosis10. Miltefosine (Hexadecylphosphochlorine) a new antineoplastic drug used in treatment of breast cancer with skin metastases has shown to decrease the spermatogonial proliferation and also to exert milder effect on the structure of germinal cells11. Because of the ability of cytidine analogues, such as 5-aza-2'-deoxycytidine, to incorporate into DNA and lead to decreases in DNA methylation, spermatogenesis and progeny outcome in the mouse is interfered resulting in abnormal male germ cell development and reduced fertility.

6.3 Objective of the Study

1. To know the effect of Gemcitabine on spermatogenesis in Swiss albino mice. 2. To correlate effect of antimetabolite anticancer drugs on spermatogenesis in human. Materials and Methods:

7.1 Study design: Total 30 adult Swiss albino male mice will be subjected into the study in animal husbandry of AJIMS, dividing them into 2 experimental groups A and B, of 10 each and a control group having 10. Mice are fed on standard feed and hosed in a 12 hour night day cycle environment and allowed water without restriction. Optimum temperature is maintained throughout the experiment. Intraperitoneal Gemcitabine of 80 mg kg-1 and 160 mg kg-1 will be administered to the experimental group A and B, respectively. It has been shown that mice exposed to a single oral dose of 333 mg kg-1 or higher will lead t death of the animal indicating that these levels are lethal doses12, so a dose 1/4th of it is selected as baseline for group A mice and double of it is selected for group B mice in order to get maximum effects of the drug. Control group will be treated with intraperitoneal saline. After 7 days the mice will be sacrificed by cervical dislocation and testis will be dissected out. Gross features of testis will be noted. Paraffin wax preparation of testis will be carried out and fixation with 10 % natural formalin and standard procedures of hydration, clearing and wax embedding will follow. Sections of 3-5 m thickness will be taken and stained with hematoxylin and eosin.

7.2 Method of collection of data:

Study type: Prospective, experimental study on rats Place of study Animal maintenance – Animal husbandry, AJIMS Histological study - Department of Anatomy, AJIMS 7.3 Does the study require any investigations or interventions to be conducted on patients or other humans or animals? If so, please describe briefly.

7.4 Has the ethical clearance Been obtained from your institution in case of 7.3?

c) List of references

1. Susan Standing, Gray’s Anatomy, the anatomical basis of clinical practice, 2005, chapter 97, Testes and epididymes, p 1309-10. 2. Shalender Bhasin, J Larry Jameson, Disorders of testis and male reproductive system, Dennis L Kasper, Eugene Bruanwald, Antony S Fauci, Stephen L Hauser, Dan L Longo, Larry Jameson, Harrison’s Principles of internal medicine, 2005, 16th ed, vol 2, p 2187. 3. Norman S Williams, Chrostopher JK Bulstrode, P Ronan O’Connell, Bailey and Love’s Short practice of surgery, 25th ed, 2007, chapter 7, p 105-106. 4. Bruce A chobner, Philip C Amrein, Brain J Druller, M Dror Michaelon, Constaine S Mitsiades, Paul E Goss, David P Ryan, et al, Antineoplastic agents, Laurence L Bruton, John S Lazo, Keith L Parker, Goodman and Gilman’s the pharmacological basis of therapeutics, 11th ed, 2006, p 1340, 1346. 5. Takimoto CH, Calvo E. "Principles of Oncologic Pharmacotherapy" in Pazdur R, Wagman LD, Camphausen KA, Hoskins WJ (Eds) Cancer Management: A Multidisciplinary Approach. 11 ed. 2008. 6. Samira Omar Abu Baker, “Gemcitabine impacts histological structure of mice testis and embryonic organs”, Pakistan Journal Of Biological Sciences.2009, 12(8): 607-615.

7. Tomao F, Miele E, Spinelli GP, Tomao S. Anticancer treatment and fertility effects. Literature review. J Exp Clin Cancer Res. 2006 Dec; 25(4):475-81.

8. Nejad DM, Rad JS, Roshankar L, Karimipor M, Ghanbari AA, Aazami A, Valilou MR., A study on the effect of thiotepa on mice spermatogenesis using light and electronic microscope, Pak J Biol Sci. 2008 Aug 1;11(15):1929-34. 9. Martinova YS, Nikolova DB, Michova Z., Early effect of the anticancer drug biocarbazin (DTIC synonym) on mice spermatogenesis, Z Mikrosk Anat Forsch. 1989; 103(3):431-6. 10. Nurmio M, Toppari J, Zaman F, Andersson AM, Paranko J, Söder O, Jahnukainen K., Inhibition of tyrosine kinases PDGFR and C-Kit by imatinib mesylate interferes with postnatal testicular development in the rat., Int J Androl. 2007 Au; 30(4):366-76; discussion 376.

11. Martinova Y, Topashka-Ancheva M, Konstantinov S, Petkova S, Karaivanova M, Berger M., Miltefosine decreases the cytotoxic effect of epirubicine and cyclophosphamide on mouse spermatogenic, thymic and bone marrow cells. Arch Toxicol. 2006 Jan; 80(1):27-33. Epub 2005 Aug 4.

12. Kelly TL, Li E, Trasler JM., 5-aza-2'-deoxycytidine induces alterations in murine spermatogenesis and pregnancy outcome, J Androl. 2003 Nov-Dec;24(6):822- 30 emcitabine Hydrochloride for Injection, Eli Lilly and Company, Material Safety Data Sheet. Section 11 - Toxicological information, http://web.ncifcrf.gov/rtp/LASP/intra/forms/msds/msds_Gemcitabine.pdf , cited on 02- 09-09.