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Indian Journal of Pharmacology, Vol. 43, No. 4, July-August, 2011, pp. 389-392 Research Article Effect of imatinib on the biochemical parameters of the reproductive function in male Swiss albino mice AM Prasad1, K Ramnarayan2, K Nalini3, KL Bairy4 1 Department of Anatomy, Melaka Manipal Medical College, Manipal, Karnataka, India Correspondence Address: K L Bairy Department of Pharmacology, Kasturba Medical College, Manipal University, Manipal, Karnataka India klbairy@yahoo.com Date of Submission: 19-Jan-2010 Code Number: ph11106 Abstract Background: Treatment of cancers with cytotoxic agents such as tyrosine kinase inhibiting drugs often, but not always, result in transient to permanent testicular dysfunction. Germ cells are important targets of many chemicals. Most of the drugs are genotoxins and induce irreversible effect on genetic makeup. These mutagenic changes are proportionally related to carcinogenesis. This is alarmingly dangerous in youth and children, since these effects last longer, affecting fertility or forming basis for carcinogenesis. There is paucity of reports on planned studies of imatinib on the testicular function. Hence, the study was planned to assess the effects of imatinib on biochemical markers of testicular functions in male Swiss albino mice.
Keywords: Imatinib, lactate dehydrogenase, testosterone Introduction Treatment of cancers with cytotoxic agents such as tyrosine kinase inhibiting drugs often, but not always, results in transient to permanent testicular dysfunction. Germ cells are important targets of many chemicals. Most of the drugs are genotoxins and induce irreversible effect on genetic makeup. These mutagenic changes are proportionally related to carcinogenesis. This is alarmingly dangerous in youth and children, since these effects last longer, affecting fertility or forming basis for carcinogenesis. Many drugs are used to combat human diseases in spite of their genotoxicity. [1] Hence, the mutagenicity studies employing in vivo tests in mammals, which have close resemblance of metabolism to humans have received much attention. Over the last 10-20 years, there has been overwhelming interest in addressing this question as more people are being exposed to agents that alter fertility parameters. A number of animal studies as well as human epidemiological studies have demonstrated that exposure of males to various agents could result in abnormal reproductive, pregnancy, or progeny outcomes. [2] Adverse effects of drugs on male fertility are observed more often than they are expected. In general, they are not recognized till andrological examination for infertility. A transient or permanent inhibition of male fertility is possible by drugs [3] via the interference of one of the following functions: spermatogenesis, sperm maturation within the epididymis, sperm transport, sperm metabolism and motility, semen liquefaction, capacitation, acrosomal reaction, or ovum penetration. Imatinib mesylate is white to off-white to brownish or yellowish tinged crystalline powder. It inhibits proliferation and induces apoptosis in Bcr-Abl positive cell lines as well as fresh leukemic cells from Philadelphia chromosome positive chronic myeloid leukemia. It is indicated for the treatment of patients with chronic myeloid leukemia. Imatinib (Gleevec) (formerly STI571), Natco Pharma Pvt Ltd, Hyderabad, India has demonstrated high levels of efficacy in gastrointestinal stromal tumors (GISTs). [4] There is a report of the GIST patient with male gynaecomastia and testicular hydrocele after treatment with imatinib mesylate. [5] Also reports are available for gynaecomastia in men with chronic myeloid leukemia after imatinib. [6] But reports on effect of imatinib on reproductive function and on testicular function are scanty. Hence, this study was planned to assess the effects of imatinib on biochemical markers of testicular functions in male Swiss albino mice. Materials and Methods Male Swiss albino mice (9-12 week old) were used. Animals were bred in the central animal house of Manipal University, Manipal. Breeding and maintenance of animals were done according to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals and Animal Welfare Division, Government of India, for the use of laboratory animals. This experiment was carried out after obtaining approval from the Institutional Animal Ethics Committee. All the animals were housed in polypropylene cage using paddy husk for bedding at 28±1°C temperature and 50±5% humidity. Five animals were housed in each cage to prevent overcrowding. Animals were fed on laboratory feed (Gold Mohur Feeds; Lipton India Ltd, Hyderabad, India.) and water ad libitum. The animals were segregated into 24 groups containing six animals in each group. Eighteen groups were injected with Imatinib at the dose levels of 50, 75, and 100 mg/kg body weight intraperitonealy for a continuous period of 5 days with an interval of 24 h between two injections. Six groups were served as controls, which received distilled water. After the last dose, the animals were sacrificed on 1, 2, 4, 5, 7, and 10 weeks sample times by the overdose of anesthesia (Pentobarbital sodium, 40 mg/kg, Sigma Aldrich, Bangalore, India). These sample time (1-5 weeks) establishes the treatment of spermatozoa in the epididymis and testis, spermatids, primary spermatocytes, secondary spermatocytes, spermatogonia and 7-10 week sample time represents treatment as stem cells respectively. [7],[8],[9] Preparation of Tissue Homogenate Testes was removed and weighed. The testes were then minced in phosphate buffer solution at a ratio of 1:10 using pestle and mortar. The tissue homogenate obtained was cold centrifuged at 4°C at 1500 rpm for 30 min. The supernatant was taken for the estimation of intratesticular testosterone and intratesticular lactate dehydrogenase. Estimation of the Intratesticular Testosterone Level The testicular level of testosterone was analyzed in the homogenate by using a kit designed for ELISA (ElAgen Testosterone-Biochem immunosystem, Roche Diagnostics, Germany.). About 50 μl of calibrators and tissue homogenate sample were added into appropriate wells of strips. About 200 μl of horseradish peroxidase-testosterone conjugate was added to each well in sequence. Mixture was incubated for 2 h at 37°C without covering the plate. Following this, the solution was discarded, wells were rinsed thrice with the washing solution (Tween 20, Sigma Aldrich, Bangalore, India). and amphotericin-B (2.5 μg/ml) in citrate-borate buffer and the residual fluid was removed. Immediately, 100 μl of chromogen substrate mixture (0.26 mg/ml of 3, 3′, 5, 5′-tetramethyl benzidine and 0.01% (w/v) of hydrogen peroxide in citrate buffer) was added to the wells and incubated for 15 min at room temperature avoiding exposure to sunlight. Reaction was stopped by pipetting 100 μl of the stop solution (sulfuric acid-0.3 mol/l) into the wells. Absorption was read in ELISA at 450 nm within 1 h from the addition of the stop solution, as per manufacturer′s instructions. [10],[11] Estimation of Intratesticular Lactate Dehydrogenase Level Testicular lactate dehydrogenase (LDH) was estimated by the optimized standard kit method (Roche/Hitachi) based on the principle that LDH catalyses the conversion of pyruvate to lactate; NADH is oxidized to NAD in the process. The rate of decrease in NADH is directly proportional to the LDH activity. The LDH level was estimated by a kit using a spectrophotometer (optimized standard kit; Roche Diagnostics, Germany). The test was performed as per the procedures provided by the suppliers. [10],[11] Statistical Analysis Six animals were used for each group and mean ±SD (standard deviation) was calculated. Results obtained from the present study were correlated and analyzed by one-way Analysis of Variance (ANOVA) followed by Bonferroni′s post hoc test. Values of P<0.05 were considered statistically significant. Results The intratesticular testosterone was significantly reduced in treated groups and severe effect was observed on weeks 4 and 5. A significant decline in testosterone concentration was observed at 100 mg/kg weight on most of the sampling days. There were dose response relations for changes in testosterone concentrations except on week 1 between groups 50 mg/kg wt and 100 mg/kg wt. All the treated groups recovered and reached the normal values by the tenth week. The decline in testosterone level was highest on week 4 and thereafter it showed a progressive increase in all treated groups [Figure - 1]. Imatinib increased intratesticular LDH level up to week 5 in all treated groups, beyond which there was no effect except 100 mg/kgwt that showed a significant increase when compared to the normal up to week 7. In groups treated with 50 mg/kg weight and 75 mg/kg weight, the recovery period was similar and reached nearer to the control values by week 7 where as in 100 mg/kg weight, the recovery period was 10 weeks. The LDH value reached the highest level on week 4 and 5 and significance between the treated groups was observed during these days [Figure - 2]. Discussion Intratesticular testosterone is thought to play a very important role in spermatogenesis; however, it is very rarely measured. It has been observed that when sex hormone binding globulin (SHBG) is elevated, more of the total testosterone is bound to SHBG, and less of it remains available as free, or biologically active, testosterone. Thus, levels of total testosterone may be normal or even elevated while the concentration of free, or bioactive, testosterone is reduced. [12],[13] Imatinib was originally developed for inhibition of bcr-abl tyrosine kinase. However, it is known that imatinib can also inhibit c-KIT tyrosine kinase and platelet-derived growth factor receptor (PDGFR) tyrosine kinase. [14] PDGF signaling is important in testes organogenesis and Leydig cell differentiation. [15] Also, PDGF-A is obligatory for adult Leydig cell recruitment and spermatogenesis. [16] C-KIT activity is modulated by the stem cell factor which in combination with insulin-like growth factor has effect on the luteinizing hormone receptor and increases the expression of StAR, CYP11A, CYP17, and 3-hydroxysteroiddehydrogenase mRNA expression. [17] Therefore, imatinib can reduce testosterone biosynthesis by inhibiting c-KIT and PDGFR in the testis. [18],[19] Administration of a single dose of imatinib resulted in a decreased rate of proliferation of the mesenchymal precursor cells which affected their maturation to Leydig cells and, thereby, the size and function of the adult Leydig cell pool. [20] These changes affecting Leydig cells in their study resulted in the decreased production of testosterone. In our study, the intratesticular testosterone was significantly reduced in all treated groups of mice. The decline in the intratesticular testosterone level was highest during the fourth and fifth sampling week for all the dose levels. A high level of expression of PDGF receptors is observed in adult Leydig cells in the mature testis, a fact which may be related to the clinical observation that oral treatment with imatinib may reduce testosterone secretion. [21] The testosterone secretion may be impaired due to excessive oxidative stress and the degeneration of Leydig cells. [22] Imatinib enhanced increased reactive oxygen species (ROS) production and depolarization of the inner mitochondrial membrane. [23] So the decrease in the levels of intratesticular testosterone observed in our study may be mainly due to the effect of Imatinib on the Leydig cells and also the enhanced ROS production. The probable inhibition of PGDFR tyrosine kinase by imatinib also cannot be ignored. LDH is a very important marker of the testicular function and cytotoxicity. [24] LDH-C4 is a membrane enzyme unique to primary spermatocytes, spermatids, and spermatozoa and is the enzyme whereby the supply of lactate from the sertoli cell is utilized as an energy substrate. LDH levels provide a quantitative basis for the loss of cell viability and its application in assessing the cytotoxicity of the cell. [25],[26] In our study, the LDH level was increased in a significant manner from second to fifth week sampling time. The increase in LDH levels up to day 35 indicated that imatinib was cytotoxic to spermatogonia, hence, releasing the LDH. In our study, we have observed an increase in the LDH level and also thinning of seminiferous epithelium of testis (results not shown). Similar observations are made by Russell and Buonaguidi, who reported that increase in the LDH level is due to the induced damaged of seminiferous epithelium. [1],[27] Our study showed sloughing of seminiferous epithelium, reduced sperm count and increased percentage of abnormal sperms (results not shown) which resulted from an elevated LDH level. These findings are consistent with a previous study which stated that the increase in the LDH activity level has a direct effect on testicular functions such as sperm count, sperm production, as well as sperm morphology. [28] This indicates the cytotoxicity of imatinib. Conclusion Imatinib does affect testosterone and LDH level significantly, but this effect is reversible once the drug is withdrawn. Imatinib increased the intratesticular LDH activities indicating extensive damage to germ cells. Imatinib is not only toxic to germ cells, but also to the Leydig cells. It decreased the intratesticular testosterone level from day 7 to day 49. All these effects on the germ cells are reversible by day 70 after the last exposure indicating no effect on stem cell lines. Imatinib had demonstrated high levels of efficacy in gastrointestinal tumors and chronic myeloid leukemia. The duration of spermatogenesis in human is about 74 days. Considering that the human germ cells have same sensitivity as that of mouse germ cells, the young patients undergoing one cycle of chemotherapy with imatinib must avoid conception for the period of about 150 days. If chemotherapy is repeated in the cyclic fashion, long-term cytotoxicity, genotoxicity, and gonadotoxicity in human can be expected. This finding may help the clinicians to plan and address the fertility-related issues in young patients of reproductive age who are being treated with imatinib for gastrointestinal tumors and chronic myeloid leukemia. References
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