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Research Article

Effect of imatinib on the biochemical parameters of the reproductive function in male Swiss albino mice

A.M. Prasad, K.Ramnarayan1, K. Nalini2, K.L. Bairy3

ABSTRACT

Background: Treatment of with cytotoxic agents such as 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 Departments of Anatomy and induce irreversible effect on genetic makeup. These mutagenic changes are proportionally 1Pathology, Melaka Manipal related to carcinogenesis. This is alarmingly dangerous in youth and children, since these Medical College, Manipal, effects last longer, affecting fertility or forming basis for carcinogenesis. There is paucity Karnataka, of reports on planned studies of imatinib on the testicular function. Hence, the study was Departments of 2Biochemistry and planned to assess the effects of imatinib on biochemical markers of testicular functions 3Pharmacology, Kasturba Medical College, Manipal University, in male Swiss albino mice. Manipal, Karnataka, India. Materials and Methods: Male Swiss albino mice were treated with imatinib and sacrificed at the end of first, second, fourth, fifth, seventh, and tenth week after the last exposure Received: 19-01-2010 to imatinib. The testis were removed, weighed, and processed for biochemical analysis. Revised: 08-03-2011 Results: The intratesticular level was significantly (P<0.001) reduced in Accepted: 25-04-2011 treated groups and severe effect was observed on week 4 and 5. The intratesticular lactate dehydrogenase (LDH) level was significantly increased by imatinib in all treated groups Correspondence to: up to week 5. Dr. K.L. Bairy Conclusion: Imatinib does affect testosterone and LDH level significantly, but this effect E-mail: [email protected] is reversible once the drug is withdrawn. 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 .

KEY WORDS: Imatinib, lactate dehydrogenase, testosterone

Introduction last longer, affecting fertility or forming basis for carcinogenesis. Many drugs are used to combat human diseases in spite of their Treatment of cancers with cytotoxic agents such as tyrosine genotoxicity.[1] Hence, the mutagenicity studies employing in vivo kinase inhibiting drugs often, but not always, results in transient tests in mammals, which have close resemblance of metabolism to permanent testicular dysfunction. Germ cells are important to humans have received much attention. targets of many chemicals. Most of the drugs are genotoxins and Over the last 10–20 years, there has been overwhelming induce irreversible effect on genetic makeup. These mutagenic interest in addressing this question as more people are being changes are proportionally related to carcinogenesis. This is exposed to agents that alter fertility parameters. A number alarmingly dangerous in youth and children, since these effects of animal studies as well as human epidemiological studies have demonstrated that exposure of males to various agents could result in abnormal reproductive, , or progeny outcomes.[2] Adverse effects of drugs on male fertility are Access this article online Quick Response Code: observed more often than they are expected. In general, they Website: www.ijp-online.com are not recognized till andrological examination for infertility. DOI: 10.4103/0253-7613.83107 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.

Indian Journal of Pharmacology | August 2011 | Vol 43 | Issue 4 389 Prasad, et al.: Effect of imatinib on male reproductive parameters

Imatinib mesylate is white to off-white to brownish or added to each well in sequence. Mixture was incubated for 2 h yellowish tinged crystalline powder. It inhibits proliferation at 37°C without covering the plate. Following this, the solution and induces in Bcr-Abl positive cell lines as well as was discarded, wells were rinsed thrice with the washing fresh leukemic cells from positive solution (Tween 20, Sigma Aldrich, Bangalore, India). and chronic myeloid leukemia. It is indicated for the treatment of amphotericin–B (2.5 μg/ml) in citrate-borate buffer and the patients with chronic myeloid leukemia. Imatinib (Gleevec) residual fluid was removed. Immediately, 100 μl of chromogen (formerly STI571), Natco Pharma Pvt Ltd, Hyderabad, India mixture (0.26 mg/ml of 3, 3’, 5, 5’-tetramethyl has demonstrated high levels of efficacy in gastrointestinal benzidine and 0.01% (w/v) of hydrogen peroxide in citrate stromal tumors (GISTs).[4] There is a report of the GIST patient buffer) was added to the wells and incubated for 15 min at with male gynaecomastia and testicular hydrocele after room temperature avoiding exposure to sunlight. Reaction treatment with imatinib mesylate.[5] Also reports are available was stopped by pipetting 100 μl of the stop solution (sulfuric for gynaecomastia in men with chronic myeloid leukemia after acid-0.3 mol/l) into the wells. Absorption was read in ELISA imatinib.[6] But reports on effect of imatinib on reproductive at 450 nm within 1 h from the addition of the stop solution, as function and on testicular function are scanty. Hence, this study per manufacturer’s instructions.[10,11] was planned to assess the effects of imatinib on biochemical Estimation of Intratesticular Lactate Dehydrogenase Level markers of testicular functions in male Swiss albino mice. Testicular lactate dehydrogenase (LDH) was estimated by Materials and Methods the optimized standard method (Roche/Hitachi) based on the principle that LDH catalyses the conversion of pyruvate to Male Swiss albino mice (9-12 week old) were used. Animals lactate; NADH is oxidized to NAD in the process. The rate of were bred in the central animal house of Manipal University, decrease in NADH is directly proportional to the LDH activity. Manipal. Breeding and maintenance of animals were done The LDH level was estimated by a kit using a spectrophotometer according to the guidelines of Committee for the Purpose of (optimized standard kit; Roche Diagnostics, Germany). The Control and Supervision of Experiments on Animals and Animal test was performed as per the procedures provided by the Welfare Division, Government of India, for the use of laboratory suppliers.[10,11] animals. This experiment was carried out after obtaining Statistical Analysis approval from the Institutional Animal Ethics Committee. All Six animals were used for each group and mean ±SD the animals were housed in polypropylene cage using paddy (standard deviation) was calculated. Results obtained from husk for bedding at 28±1°C temperature and 50±5% humidity. the present study were correlated and analyzed by one-way Five animals were housed in each cage to prevent overcrowding. Analysis of Variance (ANOVA) followed by Bonferroni’s post hoc Animals were fed on laboratory feed (Gold Mohur Feeds; Lipton test. Values of P<0.05 were considered statistically significant. India Ltd, Hyderabad, India.) and water ad libitum. The animals were segregated into 24 groups containing Results six animals in each group. Eighteen groups were injected with The intratesticular testosterone was significantly reduced Imatinib at the dose levels of 50, 75, and 100 mg/kg body in treated groups and severe effect was observed on weeks 4 weight intraperitonealy for a continuous period of 5 days with an and 5. A significant decline in testosterone concentration was interval of 24 h between two injections. Six groups were served observed at 100 mg/kg weight on most of the sampling days. as controls, which received distilled water. After the last dose, There were dose response relations for changes in testosterone the animals were sacrificed on 1, 2, 4, 5, 7, and 10 weeks sample concentrations except on week 1 between groups 50 mg/kg times by the overdose of anesthesia (Pentobarbital sodium, 40 wt and 100 mg/kg wt. All the treated groups recovered and mg/kg, Sigma Aldrich, Bangalore, India). These sample time reached the normal values by the tenth week. The decline in (1-5 weeks) establishes the treatment of spermatozoa in the testosterone level was highest on week 4 and thereafter it epididymis and testis, spermatids, primary spermatocytes, showed a progressive increase in all treated groups [Figure 1]. secondary spermatocytes, spermatogonia and 7–10 week Imatinib increased intratesticular LDH level up to week 5 sample time represents treatment as stem cells respectively.[7-9] in all treated groups, beyond which there was no effect except Preparation of Tissue Homogenate 100 mg/kgwt that showed a significant increase when compared Testes was removed and weighed. The testes were then to the normal up to week 7. In groups treated with 50 mg/kg minced in buffer solution at a ratio of 1:10 using weight and 75 mg/kg weight, the recovery period was similar pestle and mortar. The tissue homogenate obtained was cold and reached nearer to the control values by week 7 where centrifuged at 4°C at 1500 rpm for 30 min. The supernatant as in 100 mg/kg weight, the recovery period was 10 weeks. was taken for the estimation of intratesticular testosterone and The LDH value reached the highest level on week 4 and 5 and intratesticular lactate dehydrogenase. significance between the treated groups was observed during Estimation of the Intratesticular Testosterone Level these days [Figure 2]. The testicular level of testosterone was analyzed in the homogenate by using a kit designed for ELISA (ElAgen Discussion Testosterone-Biochem immunosystem, Roche Diagnostics, Intratesticular testosterone is thought to play a very Germany.). About 50 μl of calibrators and tissue homogenate important role in spermatogenesis; however, it is very sample were added into appropriate wells of strips. About rarely measured. It has been observed that when sex 200 μl of horseradish peroxidase–testosterone conjugate was hormone binding globulin (SHBG) is elevated, more of the

390 Indian Journal of Pharmacology | August 2011 | Vol 43 | Issue 4 Prasad, et al.: Effect of imatinib on male reproductive parameters

Figure 1: Effect of imatinib on testosterone levels in mice (%). Each These changes affecting Leydig cells in their study resulted dose from particular time represents mean ±SD from six animals. in the decreased production of testosterone. In our study, + P values are control vs treated P < 0.001 (50 mg/kg vs 100 mg/kg), the intratesticular testosterone was significantly reduced in bbbP < 0.05, bP < 0.001 (75 mg / kg vs 100 mg/kg), cccP < 0.05 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 by imatinib also cannot be ignored. Figure 2: Effect of imatinib on lactate dehydrogenase levels (IU). Each LDH is a very important marker of the testicular function [24] dose from particular time represents mean ± SD from six animals. and cytotoxicity. LDH-C4 is a membrane enzyme unique to P values are control vs treated, +++P < 0.05, ++P < 0.01, +P < 0.001 (50 primary spermatocytes, spermatids, and spermatozoa and mg/kg vs 75 mg/kg): aP < 0.001 (50 mg / kg vs 100 mg/kg), bP < 0.001 is the enzyme whereby the supply of lactate from the sertoli cc (75 mg / kg vs 100 mg/kg), and (c) P < 0.05 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 total testosterone is bound to SHBG, and less of it remains direct effect on testicular functions such as sperm count, sperm available as free, or biologically active, testosterone. Thus, production, as well as sperm morphology.[28] This indicates the levels of total testosterone may be normal or even elevated cytotoxicity of imatinib. while the concentration of free, or bioactive, testosterone is reduced.[12,13] Imatinib was originally developed for inhibition Conclusion of bcr- tyrosine kinase. However, it is known that imatinib Imatinib does affect testosterone and LDH level significantly, can also inhibit c-KIT tyrosine kinase and platelet-derived but this effect is reversible once the drug is withdrawn. Imatinib receptor (PDGFR) tyrosine kinase.[14] PDGF increased the intratesticular LDH activities indicating extensive signaling is important in testes organogenesis and Leydig cell damage to germ cells. Imatinib is not only toxic to germ cells, differentiation.[15] Also, PDGF-A is obligatory for adult Leydig cell but also to the Leydig cells. It decreased the intratesticular recruitment and spermatogenesis.[16] C-KIT activity is modulated testosterone level from day 7 to day 49. All these effects on by the which in combination with -like the germ cells are reversible by day 70 after the last exposure growth factor has effect on the luteinizing hormone receptor indicating no effect on stem cell lines. and increases the expression of StAR, CYP11A, CYP17, and 3– Imatinib had demonstrated high levels of efficacy in hydroxysteroiddehydrogenase mRNA expression.[17] Therefore, gastrointestinal tumors and chronic myeloid leukemia. The imatinib can reduce testosterone biosynthesis by inhibiting duration of spermatogenesis in human is about 74 days. c-KIT and PDGFR in the testis.[18,19] Considering that the human germ cells have same sensitivity Administration of a single dose of imatinib resulted in a as that of mouse germ cells, the young patients undergoing one decreased rate of proliferation of the mesenchymal precursor cycle of with imatinib must avoid conception cells which affected their maturation to Leydig cells and, for the period of about 150 days. If chemotherapy is repeated thereby, the size and function of the adult Leydig cell pool.[20] in the cyclic fashion, long-term cytotoxicity, genotoxicity, and

Indian Journal of Pharmacology | August 2011 | Vol 43 | Issue 4 391 Prasad, et al.: Effect of imatinib on male reproductive parameters gonadotoxicity in human can be expected. This finding may help 15. Brennan J, Tilmann C, Capel B. Pdgfr-alpha mediates testis cord organization the clinicians to plan and address the fertility-related issues and fetal Leydig cell development in the XY gonad. Genes Dev 2003;17:800-10. in young patients of reproductive age who are being treated 16. Gnessi L, Basciani S, Mariani S, Arizzi M, Spera G, Wang C, et al. Leydig cell loss and spermatogenic arrest in platelet-derived growth factor (PDGF)-A-deficient with imatinib for gastrointestinal tumors and chronic myeloid mice. J Cell Biol 2000;149:1019-26. leukemia. 17. Huang CT, Weitsman SR, Dykes BN, Magoffin DA. Stem cell factor and insulin- like growth factor-I stimulate luteinizing hormone-independent differentiation of References rat ovarian theca cells. Biol Reprod 2001;64:451-6. 1. 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Experience Organophosphate insecticide methyl parathion (o- o- dimethyl o-4-nitrophenyl with the determination of LDH-X in seminal plasma as diagnostic and prognostic phosphorothioate) induces cytotoxic damage and tubular atrophy in the testis factor in varicocele.Arch Esp Urol 1993;46:35-9. despite elevated testosterone level in the rat. J Toxicol Sci 2006;31:177-89. 28. Pant N, Srivastava SP. Testicular and spermatotoxic effects of quinalphos in rats. 12. Isojarvi JI, Pakarinen AJ, Ylipalosaari PJ. Serum hormones in male epileptic J Appl Toxicol 2003;23:271-4. patients receiving anticonvulsant . Arch Neurol 1990;47:670-6. 13. Toone BK, Wheeler M, Nanjee M. Sex hormones, sexual activity and plasma anticonvulsant levels in male epileptics. J Neurol Neurosurg Psychiatry Cite this article as: Prasad AM, Ramnarayan K, Nalini K, Bairy KL. Effect of 1983;46:824-6. imatinib on the biochemical parameters of the reproductive function in male 14. Kim H, Chang HM, Ryu MH, Kim TW, Sohn HJ, Kim SE, et al. Concurrent male Swiss albino mice. Indian J Pharmacol 2011;43:389-92. gynecomastia and testicular hydrocele after imatinib mesylate treatment of a gastrointestinal stromal tumor. J Korean Med Sci 2005;20:512-5. Source of Support: Nil. Conflict of Interest: None declared.

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