Cytotoxic and genotoxic effects of methotrexate in germ cells of male Swiss mice

Mutation Research 655 (2008) 59–67

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Cytotoxic and genotoxic effects of methotrexate in germ cells of male Swiss mice S. Padmanabhan, D.N. Tripathi, A. Vikram, P. Ramarao, G.B. Jena ∗,1 Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, Punjab 160062, India

a r t i c l e

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Article history: Received 5 April 2008 Received in revised form 30 May 2008 Accepted 5 July 2008 Available online 17 July 2008 Keywords: Methotrexate Mice Testis Sperm count Sperm head abnormality Comet assay TUNEL assay

a b s t r a c t Methotrexate (MTX) is an anti-metabolite drug widely used in the treatment of neoplastic disorders, rheumatoid arthritis and psoriasis. Developed as an analogue of folic acid, it inhibits purine and pyrimidine synthesis that accounts for its therapeutic efficacy as well as for its toxicities. MTX has narrow therapeutic index and its toxicity has been reported in various organ systems including gastrointestinal, haematologic and central nervous system. The objective of the present study is to investigate the germ cell toxicity induced by MTX in male Swiss mice. MTX was administered intraperitoneally (ip) at the doses of 5, 10, 20 and 40 mg/kg to mice (20–25 g) weekly once (wk) for 5 and 10 weeks. The animals were sacrificed 1 week after receiving the last treatment of MTX. The germ cell toxicity was evaluated using testes weight (wt), sperm count, sperm head morphology, sperm comet assay, histology, TUNEL and halo assay in testis. MTX treatment significantly reduced the sperm count and increased the occurrence of sperm head abnormalities in a dose dependent manner. It induced the testicular toxicity as evident from the histology of testis. Sperm comet, TUNEL and halo assay in testis also revealed significant DNA damage after MTX treatment. On the basis of the present study, it can be concluded that MTX induced germ cell toxicity in mice. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Chemotherapy is one of the most effective methods for the treatment of cancer, but is often associated with several short and long-term toxicities [1]. These toxicities are either due to the nontarget specific mode of action or the manifestations in the late explicative enduring cells. MTX is a widely used anti-cancer drug and a well known immunosuppressant introduced for therapeutic use in the 1950s [2]. It is used against a broad range of neoplastic disorders including acute lymphoblastic leukaemia, non-Hodgkin’s lymphoma, breast cancer and testicular tumours [3–7]. Further, it is effective for the treatment of psoriasis, rheumatoid arthritis and different immune-suppressive conditions [8–10]. It is also one of the drug of choice in the new regimen combination treatment against rheumatoid arthritis and for several refractory/relapsed tumours [11,12]. Recent literature reports that high dose of MTX regimens can be used against primary CNS lymphomas as well as liver cholestatic disorders [13,14]. The basic principle of its therapeutic efficacy is due to the inhibition of dihydrofolate reductase,

∗ Corresponding author. Tel.: +91 172 2214682 87x2152. E-mail addresses: [email protected], [email protected] (G.B. Jena). 1 NIPER communication number: 433. 1383-5718/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2008.07.003

a key enzyme in the folic acid metabolism, which converts dihydrofolic acid to tetrahydrofolic acid. The perturbation in the folic acid metabolism leads to depletion of nucleotide precursors like thymidylates and purines, which in turn inhibits DNA, RNA and protein synthesis. MTX also inhibits thymidylate synthase and the transport of reduced folates into the cell (Fig. 1) [15,16]. Most chemotherapeutic agents act through the interaction with DNA or its precursors, thereby inhibiting the synthesis of new genetic material. Damage to the genetic materials results in the disorganization of the cellular functions, in both somatic as well as in germ cells. The toxicities pertaining to the germ cells are having greater implications due to their possible transmissions to the future generations [17–19]. Toxicity in the germ cells can result in various manifestations in the progeny like growth retardation, congenital malformations, altered endocrine functions, cancer, behavioral disorders and reproductive problems like early abortions and congenital malformations [20–22]. The toxicity of MTX in various organs like gastrointestinal, haematologic and central nervous system has already been reported [9,23,24]. The genotoxic effects of MTX have already been reported in the somatic cells employing chromosome aberration and micronucleus tests as the end points of evaluation [3,25–27]. Further, it has been reported that the long-term genotoxic effect of MTX is due to the intracellular accumulation and subsequent

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Fig. 1. Chemical structure of MTX.

enhancement of enzyme inhibition [25]. The most interesting report is that the late replicative enduring cells exhibited toxicity due to single exposure to high dose of MTX [8]. This motivated us to further explore the possible germ cell toxicities of MTX after repeated exposure in a mammalian test system. In the germ cells, decreased sperm count and increased abnormalities in the sperm head morphology were observed. Testicular toxicity was evident from the histology of testis. Further, increased DNA damage and cytotoxicity was observed by the sperm comet, halo assay and TUNEL assay in testis.

minced into small pieces to allow the sperms to swim out. The sperm suspension thus obtained was centrifuged at 1000 rpm for 5 min. After centrifugation, 1 ml of the supernatant was taken and the epididymal sperm count was determined using Neubauer’s hemocytometer. Data were expressed as the number (no.) of sperms per mg wt of epididymis. For sperm head morphology, the sperm suspension in HBSS was stained with 2% eosin solution and kept undisturbed for 1 h. Smears were prepared using the above solution, air dried and fixed with absolute methanol for 5 min. Two hundred sperms per animal were examined to determine the morphological abnormalities at 1000× magnification [32,33]. Sperm head morphology was scored under the category of normal, sperm without hook, amorphous head, banana head and triangular head essentially as described [34]. Data were shown in terms of normal to abnormal ratio of sperms.

2. Materials and methods

2.5. Testis histology

2.1. Animals

Histological slides were prepared as previously standardized in our laboratory [35]. The testes were fixed in 10% formalin, dehydrated in increasing concentrations of ethanol and embedded in paraffin. Tissue sections (5 ␮m) were mounted on glass slides coated with Mayer’s albumin and dried overnight. The sections were then deparaffinized with xylene, rehydrated with alcohol and water. The rehydrated sections were stained using H&E, mounted with DPX mounting media and examined under the microscope at both high (400×) and low (100×) magnifications (Olympus BX51, Tokyo, Japan). Testicular sections from each animal were evaluated qualitatively as well as quantitatively for structural changes. Quantification was done essentially as described with some modifications [36]. Thirty seminiferous tubules from each animal were randomly examined and scored as 0, 1, 2 and 3; depending on the extent of damage observed. The no. of seminiferous tubules under each score was multiplied with the respective scores and the sum obtained to get the final seminiferous tubule damage score.

All the animal experiment protocols were approved by the Institutional Animal Ethics Committee (IAEC) and the experimentation on animals was done in accordance with the CPCSEA (Committee for the Purpose of Control and Supervision of Experimentation on Animals) guidelines. Experiments were performed on male Swiss albino mice (6 weeks, 20 ± 2 g) procured from the central animal facility of the institute. All the animals were kept under controlled environmental conditions at room temperature (22 ± 2 ◦ C) with humidity (50 ± 10%) and a 12 h light and 12 h dark cycle. Standard laboratory animal feed (purchased from commercial supplier) and water were given ad libitum. Animals were acclimatized to experimental conditions prior to the start of dosing for a period of 1 week. 2.2. Chemicals MTX (CAS 59-05-2) was obtained as gift sample from GlaxoSmithKline Pharmaceuticals Limited, Mumbai, India. Hematoxylin and eosin (H&E), Ethidium Bromide (EtBr) (CAS 1239-45-8), Trizma (CAS 77-86-1), Dithiothreitol (CAS 3483-12-3), Proteinase-K (CAS 39450-01-6) and SYBR Green (CAS 163795-75-3) were purchased from Sigma–Aldrich Chemicals, Saint Louis, MO, USA. Dimethylsulphoxide (DMSO), normal melting point agarose, low melting point agarose, Triton X-100, ethylenediamine-tetraacetic acid (EDTA) and Hank’s balanced salt solution (HBSS) were obtained from HiMedia Laboratories Ltd, Mumbai.

2.6. Testis TUNEL assay Paraffin-embedded testis tissues were cut into thin (5 ␮m) sections with microtome and mounted on poly-l-lysine coated glass slides (Leica RM2145, Germany). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was used to assess the DNA fragmentation (Calbiochem, Oncogene Research Product, USA). The assay was conducted according to the manufacturer’s

2.3. Experimental design Kasahara et al. reported that MTX at the single dose of 10, 20, 40 and 80 mg/kg induced micronuclei in the peripheral blood reticulocytes of mice [28]. Choudhury et al. evaluated the potential transmission of the cytogenetic toxic effects of MTX at the dose of 2, 10 and 20 mg/kg in the male germline cells of Swiss mice [29]. Based on the above studies, we decided the dose of 5, 10, 20 and 40 mg/kg of MTX once in a week for 5 and 10 consecutive weeks to evaluate the germ cell toxicity in mice. MTX was dissolved in 0.1 M sodium bicarbonate and administered through ip route. The volume of administration of MTX to each animal was 10 ml per kg body wt. Animals were divided into five groups for each treatment periods (5 and 10 weeks) (n = 5). Spermatogenesis is a highly organized process and in rodents, sperms are produced from the progenitor spermatogonia after a series of meiotic and mitotic divisions, which takes approximately 6–8 weeks [21,30,31]. This explains the basis of taking the sampling after 5 and 10 weeks of exposure, which will facilitate to observe the changes in the sperms manifested during early as well as late stages of development. Group 1 received vehicle and served as the control. Groups 2, 3, 4 and 5 received MTX at the dose of 5, 10, 20 and 40 mg/kg/wk, respectively. Animals were sacrificed by cervical dislocation 1 week after the last injection of MTX in both the treatment periods. 2.4. Sperm count and sperm head morphology The cauda epididymis were removed after sacrificing the animals and placed in a Petri dish containing 2–3 ml of HBSS at room temperature. The epididymis was

Fig. 2. Effect of MTX on the sperm count after 5 and 10 consecutive weeks of treatment. All the values are expressed as Mean ± S.E.M. (n = 5), * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control.

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Table 1 Effect of 5 weeks and 10 weeks MTX treatment on the initial body wt, final body wt, paired testes wt and relative testes wt of mice Parameters

Groups Control

MTX (5 mg/kg/wk)

MTX (10 mg/kg/wk)

MTX (20 mg/kg/wk)

MTX (40 mg/kg/wk)

Initial body wt (g) Final body wt (g) 5 Weeks Paired testes wt (g) Paired testes wt (g)/100 g body wt

22 32.8 0.21 0.66

± ± ± ±

0.3 0.7 0.01 0.01

21.2 30.4 0.23 0.75

± ± ± ±

0.6 0.6 0.01 0.01

21.2 29.2 0.20 0.69

± ± ± ±

0.9 0.4 0.01 0.01

20.8 29.8 0.20 0.69

± ± ± ±

0.6 0.2 0.01 0.01

22.4 30.4 0.21 0.70

± ± ± ±

0.4 1.1 0.01 0.02

Initial body wt (g) Final body wt (g) 10 Weeks Paired testes wt (g) Paired testes wt (g)/100 g body wt

22.4 34.8 0.24 0.71

± ± ± ±

0.2 1.4 0.01 0.01

21.8 29.3 0.21 0.71

± ± ± ±

0.3 0.6** 0.01 0.05

21.9 24.8 0.20 0.81

± ± ± ±

0.5 0.2*** 0.02 0.03

21.5 27.0 0.22 0.83

± ± ± ±

0.4 0.8*** 0.01 0.05

22.2 27.5 0.20 0.75

± ± ± ±

0.3 0.9*** 0.01 0.05

All the values are expressed as Mean ± S.E.M. (n = 5). ** P < 0.01 vs. control. *** P < 0.001 vs. control.

instructions. TUNEL positive cells were counted using the image analysis software ‘Isis’ (Carl Zeiss, AxioImager M1, Germany) and images were acquired using charged coupled device (CCD) camera. The TUNEL positive cells were expressed as percentage (%) of total cells.

2.7. Sperm comet assay The sperm comet assay was performed essentially as described with some modifications [37,38]. Sperm sample (5 ␮l) containing 1–3 × 104 sperm ml−1 was suspended in 95 ␮l of 1% (w/v) low melting point agarose. From this suspension, 80 ␮l was applied to the surface of a microscope slide (pre-coated with 1% normal melting point agarose) to form a microgel and allowed to set at 4 ◦ C for 5 min. Slides were dipped in cell lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris HCl pH 10.0 containing 1% Triton X-100 and 40 mM Dithiothreitol) for 24 h at room temperature and protected from light. Following the initial lysis, proteinase K was added to the lysis solution (0.5 mg/ml) and additional lysis was performed at 37 ◦ C for 24 h. Following cell lysis, all slides were washed three times with deionized water at 10 min intervals to remove salt and detergent from the microgels. Slides were placed in a horizontal electrophoresis unit and were allowed to equilibrate for 20 min with running buffer (500 mM NaCl, 100 mM Tris HCl and 1 mM EDTA, pH 9) before electrophoresis (0.60 V/cm, 250 mA) for 30 min. After electrophoresis, slides were neutralized and the DNA fluorochrome SYBR Green (1:10,000 dilution) was applied for 1 h. Slides were rinsed briefly with double-distilled water and coverslips were placed before image analysis. The fluorescent labelled DNA was visualized (200×) using an AXIO Imager M1 fluorescence microscope (Carl Zeiss, Germany) and the resulting images were cap-

Fig. 4. Effect of MTX on sperm head abnormalities expressed as normal/abnormal ratio of sperm after 5 and 10 consecutive weeks of treatment. All the values are expressed as Mean ± S.E.M. (n = 5), * P < 0.05, ** P < 0.01, *** P < 0.001 vs control.

Fig. 3. Representative photomicrographs of sperms (magnification 1000×). Normal murine sperm (A), sperm without hook (B), banana head sperm (C), sperms with triangular heads (D and E), sperm with amorphous head (F).

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Fig. 5. Photomicrographs of H&E stained histological slides of the testis; control (A), MTX 40 mg/kg/wk (B) after 10 weeks of treatment, magnification 100×; vacuolization is indicated by arrow. Score 0—presence of all kinds of cells (C), score 1—mild vacuolization (D), score 2—moderate vacuolization (E), score 3—severe vacuolization with decreased no. of spermatids and spermatogonia (F), magnification 400×.

tured on a computer and processed with image analysis software (Metasystem software, Comet Imager V.2.0.0). The main parameters of the comet DNA damage analysis includes: tail length (TL), % DNA in comet tail (TDNA), tail moment (TM) and olive tail moment (OTM). Samples were run in duplicate and 50 cells were randomly analyzed per slide for a total of 100 cells per sample and scored for comet tail parameters as defined by Olive [39]. Comet tail length is the maximum distance that the damaged DNA migrates from the center of the cell nucleus. The percentage of tail DNA is the total DNA that migrates from the nucleus into the comet tail. Tail moment is the product of the tail length and the percentage of tail DNA, which gives a more integrated measurement of overall DNA damage in the cell. 2.8. Halo assay The halo assay was performed essentially as described with some modifications [40]. Testis was homogenized gently in PBS and 5 ␮l of the homogenate was suspended in 50 ␮l of 0.5% low melting point agarose and layered over the surface of a frosted slide (pre-coated with 1% normal melting point agarose) to form a microgel and allowed to set at 4 ◦ C for 5 min. The slides were immersed in freshly prepared lysis solution (2.5 M NaCl, 2 mM EDTA, 10 mM Tris, pH 10, 1% Triton X-100) for 2 h at 4 ◦ C. Following lysis, the slides were incubated with alkaline medium (0.3 M NaOH) for 20 min and stained using EtBr. Samples were run in duplicate and 50 cells were randomly examined per slide for a total of 100 cells per sample under the microscope (Olympus BX51, Tokyo, Japan). The damaged cells were categorized as mild, moderate and extensive as described [41].

Fig. 6. Effect of MTX on the seminiferous tubule damage score after 5 and 10 consecutive weeks of treatment. All the values are expressed as Mean ± S.E.M. (n = 5), ** P < 0.01, *** P < 0.001 vs. control.

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Fig. 7. Photomicrographs showing apoptotic spermatocytes having fragmented DNA as revealed from TUNEL assay in testis at magnification 200×. Control (A), MTX 40 mg/kg/wk for 5 weeks (B), MTX 40 mg/kg/wk for 10 weeks (C). The spermatocytes emitting green signal are TUNEL positive apoptotic cells.

2.9. Statistical analysis Results were shown as Mean ± standard error of mean (S.E.M.) for each group. Statistical analysis was performed using Jandel Sigma Stat (Version 2.03) statistical software. Significance of difference between two groups was evaluated using Student’s t-test. For multiple comparisons, One-way analysis of variance (ANOVA) was used. In case ANOVA showed significant differences, post hoc analysis was performed with Tukey’s test. P < 0.05 was considered to be statistically significant.

enhanced after MTX treatment; normal/abnormal sperm ratio decreased in a dose dependent manner (Fig. 3). The decrease in the ratio was found to be significant at MTX 20 mg/kg/wk (P < 0.05) and MTX 40 mg/kg/wk (P < 0.01) as compared with the control group after 5 weeks of treatment. The decrease in the normal/abnormal sperm ratio was significant with all the doses of MTX (5, 10, 20 and 40 mg/kg/wk) following 10 weeks of treatment (P < 0.05) (Fig. 4).

3. Results 3.1. Body weight and organ weight Significant difference in the final body wt (P < 0.001) was observed after 10 weeks of treatment with MTX at the dose of 10, 20 and 40 mg/kg/wk as compared with the control group. The relative testis wt was determined in order to eliminate the possibility of bias arising from difference in body wt of different groups. The relative testis wt showed no statistically significant difference among the groups though a marginal decrease was observed in the paired testes wt after 5 and 10 weeks of MTX treatment (Table 1).

3.3. Testis histology Morphological alterations such as disorganization, vacuolization and decreased spermatogonial and spermatid counts were induced by MTX in the seminiferous tubules of mice. The quantitative assessment of the seminiferous tubules was done based on the extent of damage induced by MTX and scored as 0, 1, 2 and 3 (Fig. 5). Significant seminiferous tubule damage was observed at all the doses of MTX (5, 10, 20 and 40 mg/kg/wk) as compared with the control group (P < 0.01) after 5 and 10 weeks of treatment (Fig. 6).

3.2. Sperm count and sperm head morphology

3.4. Testis TUNEL assay

A dose dependent decline was observed in the sperm count with MTX treatment. MTX 40 mg/kg/wk for 5 weeks led to significant decrease in sperm count as compared with the control group (P < 0.01). The groups which received MTX 20 mg/kg/wk and 40 mg/kg/wk showed significant reduction in the sperm count following 10 weeks of treatment (P < 0.001) (Fig. 2). Furthermore, the frequency of abnormalities in the sperm head was

TUNEL assay was performed in the testis to ascertain the mode of cell death by MTX. The spermatocytes emitting green signal were considered as TUNEL positive (Fig. 7). A significant increase in the % of TUNEL positive cells was observed with MTX 20 and 40 mg/kg/wk in comparison to the control group (P < 0.05). The increase was found to be significant after both 5 and 10 weeks of MTX treatment (Fig. 8).

Fig. 8. Effect of MTX on the % of TUNEL positive cells in testis after 5 and 10 consecutive weeks of treatment. All the values are expressed as Mean ± S.E.M. (n = 5), * P < 0.05, *** P < 0.001 vs. control.

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Fig. 9. Photomicrographs showing the DNA migration pattern in mice sperm nuclei after 10 weeks of treatment. The symbols “−” and “+” represents cathode and anode, respectively, during electrophoresis of negatively charged DNA. Magnification: 200×. Dye: SYBR Green. Sperm nuclei from control group (A), sperm nuclei from MTX 40 mg/kg/wk treated group (B).

Fig. 10. Representative photomicrographs of the halo assay in testis after 10 weeks of treatment. Magnification: 200×. Stain: EtBr; control (A), MTX 40 mg/kg/wk (B). Mildly damaged cell (a), moderately damaged cell (b), extensively damaged cell (c).

3.5. Sperm comet and halo assay

4. Discussion

DNA damage in the sperm was assessed using the sperm comet assay (Fig. 9). The various comet parameters viz. TL, TM, OTM and TDNA showed a significant increase at MTX 40 mg/kg/wk as compared with the control group after 5 and 10 weeks of treatment (P < 0.05) (Table 2). MTX treatment showed DNA damage in the testes as further determined by the halo assay (Fig. 10). After 5 and 10 weeks of MTX treatment, the % of moderately and extensively damaged testicular cells was found to be increased (P < 0.01) as compared with the control group (Fig. 11).

MTX is an anti-metabolite drug used against a broad range of neoplastic disorders. It has a narrow therapeutic window and its toxicity has been reported in various organ systems [42]. It inhibits the DNA synthesis by acting on the S-phase of the cell cycle [15]. MTX has been reported to have cytotoxic as well as cytostatic effects on a variety of cell systems [43,44]. Due to its structural similarity with folic acid, it binds to the enzyme dihydrofolate reductase. This enzyme is responsible for the conversion of the dihydrofolates to tetrahydrofolates, which are intermediates in the synthesis

Table 2 Effect of MTX 40 mg/kg/wk on sperm DNA damage following 5 weeks and 10 weeks treatment as revealed by comet assay Groups

Parameters TL (␮m)

5 Weeks

Control MTX (40 mg/kg/wk)

15.37 ± 2.26 24.66 ± 1.04*

10 Weeks

Control MTX (40 mg/kg/wk)

10.29 ± 0.91 24.19 ± 1.45***

All the values are expressed as Mean ± S.E.M. (n = 5). * P < 0.05 vs. control. ** P < 0.01 vs. control. *** P < 0.001 vs. control.

TM

OTM

TDNA

2.39 ± 0.46 5.41 ± 0.16***

2.56 ± 0.44 4.63 ± 0.28**

12.23 ± 1.76 19.20 ± 0.61**

1.54 ± 0.18 10.77 ± 0.47***

1.70 ± 0.18 8.30 ± 0.58***

8.81 ± 0.76 22.43 ± 1.39***

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Fig. 11. Effect of MTX on % damaged cells after 5 and 10 consecutive weeks of treatment determined by halo assay in testis. All the values are expressed as Mean ± S.E.M. (n = 5), * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control.

of the nucleotides, the thymidylates and purines [45]. Further, it has been reported by Tian and Cronstein that treatment with MTX leads to the extracellular release of adenosine, an endogenous purine nucleotide [16]. The inhibition of the synthesis of thymidylates, purines and extracellular release of adenosine causes an imbalance in the nucleotide pools, which ultimately perturb the DNA synthesis process [46]. Intracellularly, MTX gets metabolized to polyglutamates and is retained in the tissues for longer duration. The polyglutamated form of MTX is more active than MTX in inhibiting folate-dependent enzymes [16]. The increased toxic effects observed in the present study after repeated drug administration might be due to increased intracellular accumulation and subsequent enzyme inhibition. Mouse model provides a very good alternative for germ cell toxicological evaluation of anti-cancer drugs for humans [47,48]. The decrease in the body wt of the treated groups was not significant as compared with the control group at the end of 5 weeks. However, the same was found to be significant in all the treatment groups, when compared with the control group after 10 weeks of treatment, which indicated the general toxicity. MTX was found to decrease the sperm count and increase the frequency of sperm head abnormalities in a dose dependent manner. Decrease in the sperm count often results due to the interference in the spermatogenesis process and the elimination of sperm cells at different stages of development [49,50]. A single exposure study by ip route indicated the transmissibility potential of MTX, which substantiates its teratogenicity and embryo-lethality effects [29,51,52]. Sperm abnormalities can be attributed to the changes in the physiological, cytotoxic and genetic alterations in the testicular DNA [53]. However, the abnormalities in sperm head is possibly due to the interference with the DNA integrity and/or the expression of the genetic material

[34]. The gonadal toxicity of MTX was clearly evident from the histological evaluation of the testes. Increased disorganization, vacuolization, decreased spermatogonial and spermatid counts in the seminiferous tubules indicated that MTX interferes in the process of spermatogenesis. Saxena et al. reported that chronic low dose MTX treatment leads to the reduction in the size of seminiferous tubules, sertoli cells, leydig cells and vacuolization/decondensation of “chromatin mass” in the spermatocytes of rats [54]. Similar studies conducted by Russell and Russell and Shrestha et al. reported the damage in spermatogonia as well as spermatocytes after repeated treatment with MTX in rats [55,56]. The genotoxic effects of MTX have already been reported in the somatic cells employing chromosome aberration and micronucleus tests as the end points of evaluation [3,25–27]. DNA damage and the cytotoxicity of MTX in the germ cells were assessed using the sperm comet, halo and TUNEL assay in testis. The sperm comet assay revealed the genotoxicity of MTX as observed by an increase in the comet parameters viz. TL, TM, OTM and TDNA. It has been reported that strand breaks are progressively accumulated in the replicating DNA after MTX treatment as a consequence of depleted nucleotide pools and the impairment of the repair mechanisms [57]. In order to elucidate the mode of cell death, we have performed both halo as well as TUNEL assay in the testis and the percentage of cells undergoing cell death was found to increase after MTX treatment. The halo assay is a simple, sensitive and reliable “DNA diffusion” assay used for the quantification of DNA damage [39,40]. The halo assay in the testis confirms the cytotoxicity of MTX in germ cells which can be attributed to the cessation of DNA synthesis as a consequence of inhibition of nucleotide synthesis, ultimately leading to cell death [58]. While TUNEL assay detects the cells undergoing apoptosis and the enzyme terminal deoxynucleotidyl transferase

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(TdT) specifically binds with the free 3 -OH ends of DNA fragments produced in response to caspase activation [59]. It has been further reported that MTX induced apoptosis in the hepatocytes of rats, which can be attributed to the nucleotide pool imbalance or by the repression of JNK activity and up-regulation of p53 and p21 [46]. Our results clearly demonstrate that MTX treatment in mice induced germ cell toxicity as evident from the increased sperm head abnormalities, seminiferous tubule damage (disorganization and vacuolization), sperm DNA damage, TUNEL positive cells and decreased sperm count. As further efficacy of MTX is being explored for its clinical use and it is considered as one of the drug of choice for the treatment of many new pathological conditions, it warrants further attention to characterize the complete germ cell toxicities using other surrogate end points to reduce the potential risk in the patients, who are particularly in the reproductive ages. Conflict of interest There is no conflict of interest. Acknowledgements The financial assistance received from National Institute of Pharmaceutical Education and Research (NIPER), Mohali to carry out this particular study is duly acknowledged. The authors would also like to acknowledge GlaxoSmithKline Pharmaceuticals Limited, Mumbai for providing the generous gift sample of methotrexate. References [1] J. Arnon, D. Meirow, H.L. Roness, A. Ornoy, Genetic and teratogenic effects of cancer treatments on gametes and embryos, Hum. Reprod. Update 7 (2001) 394–403. [2] G.J. Peters, C.L. van der Wilt, C.J. van Moorsel, J.R. Kroep, A.M. Bergman, S.P. Ackland, Basis for effective combination cancer chemotherapy with antimetabolites, Pharmacol. Ther. 87 (2000) 227–253. [3] R.C. Choudhury, S.K. Ghosh, A.K. Palo, Cytogenetic toxicity of methotrexate in mouse bone marrow, Environ. Toxicol. Pharmacol. 8 (2000) 191–196. [4] J. Miyazaki, K. Kawai, H. Hayashi, M. Onozawa, S. Tsukamoto, N. Miyanaga, S. Hinotsu, T. Shimazui, H. Akaza, The limited efficacy of methotrexate, actinomycin D and cisplatin (MAP) for patients with advanced testicular cancer, Jpn. J. Clin. Oncol. 33 (2003) 391–395. [5] R. Seigers, S.B. Schagen, W. Beerling, W. Boogerd, O. van Tellingen, F.S.A.M. van Dam, J.M. Koolhaas, B. Buwalda, Long-lasting suppression of hippocampal cell proliferation and impaired cognitive performance by methotrexate in the rat, Behav. Brain Res. 186 (2008) 168–175. [6] U. Veronesi, G. Bonadonna, P. Valagussa, Lessons from the initial adjuvant cyclophosphamide, methotrexate, and fluorouracil studies in operable breast cancer, J. Clin. Oncol. 26 (2008) 342–344. [7] S.B. Jensen, H.T. Mouridsen, J. Reibel, N. Brunner, B. Nauntofte, Adjuvant chemotherapy in breast cancer patients induces temporary salivary gland hypofunction, Oral Oncol. 44 (2008) 162–173. [8] M. Chow, J. Koo, P. Ng, H. Rubin, Random population-wide genetic damage induced in replicating cells treated with methotrexate, Mutat. Res. 413 (1998) 251–264. [9] L.R. Belur, R.I. James, C. May, M.D. Diers, D. Swanson, R. Gunther, R.S. McIvor, Methotrexate preconditioning allows sufficient engraftment to confer drug resistance in mice transplanted with marrow expressing drug-resistant dihydrofolate reductase activity, J. Pharmacol. Exp. Ther. 314 (2005) 668–674. [10] P. Gisondi, G. Girolomoni, Biologic therapies in psoriasis: a new therapeutic approach, Autoimmun. Rev. 6 (2007) 515–519. [11] A. Potamianou, N. Ziras, N. Bountouroglou, J. Varthalitis, N. Karvounis, D. Pectasidis, A.E. Athanassiou, Methotrexate, etoposide, ifosfamide and cisplatin (MVIP): An effective salvage therapy for patients with refractory or relapsed germ-cell tumors, J. Buon. 7 (2002) 337–345. [12] F. Mielke, M. Schweigert, Safe adalimumab therapy for rheumatoid arthritis in a patient with pre-existing multiple myeloma, Nat. Clin. Pract. Rheumatol. 4 (2008) 218–221. [13] R. Liu, S.M. Chang, M. Prados, Recent advances in the treatment of central nervous system tumours, Update Cancer Ther. 3 (2008) 49–79. [14] K. Novak, M.G. Swain, Role of methotrexate in the treatment of chronic cholestatic disorders, Clin. Liver Dis. 12 (2008) 81–96. [15] T. Novakovic, O.M. Dordevic, D. Grujicic, D. Marinkovic, S. Jankovic, S. Arsenijevic, Effect of intratumoral application of methotrexate in vivo on frequency of micronuclei in peripheral blood lymphocytes, Arch. Oncol. 11 (2003) 1–4.

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