Methotrexate-induced cytotoxicity and genotoxicity in germ cells of mice: Intervention of folic and folinic acid

Mutation Research 673 (2009) 43–52

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Methotrexate-induced cytotoxicity and genotoxicity in germ cells of mice: Intervention of folic and folinic acid S. Padmanabhan, D.N. Tripathi, A. Vikram, P. Ramarao, G.B. Jena ∗ 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 16 July 2008 Received in revised form 23 November 2008 Accepted 29 November 2008 Available online 6 December 2008 Keywords: Methotrexate Folic acid Folinic acid Mice Sperm Testis

a b s t r a c t Methotrexate (MTX) is an anti-metabolite widely used in the treatment of neoplastic disorders, rheumatoid arthritis and psoriasis. The basis for its therapeutic efficacy is the inhibition of dihydrofolate reductase (DHFR), a key enzyme in the folic acid (FA) metabolism. FA is a water-soluble vitamin which is involved in the synthesis of purines and pyrimidines, the essential precursors of DNA. Folinic acid (FNA) is the reduced form of FA that circumvents the inhibition of DHFR. Folate supplementation during MTX therapy for psoriasis and inflammatory arthritis reduces both toxicity and side effects without compromising the efficacy. Further, FNA supplementation reduces the common side effects of MTX in the treatment of juvenile idiopathic arthritis. FA and FNA are reported to have protective effects on MTX-induced genotoxicity in the somatic cells; however their protective effects on the germ cells have not been much explored. Previously, we evaluated the cytotoxic and genotoxic effects of MTX in the germ cells of mice. In the present study, we have intervened FA and FNA for the protection of germ cell toxicity induced by MTX in male swiss mice. The animals were pre-treated with FA at the doses of 50, 100 and 200 ␮g/kg for 4 consecutive days per week and on day five; MTX was administered at the dose of 20 mg/kg once. FNA was administered at the doses of 2.5, 5 and 10 mg/kg, 6 h (h) after single administration of MTX at the dose of 20 mg/kg. The dosing regimen was continued up to 10 weeks. 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. The results clearly demonstrate that prior administration of FA and post-treatment with FNA reduces the germ cell toxicity induced by MTX as evident from the decreased sperm head abnormalities, seminiferous tubule damage, sperm DNA damage, TUNEL positive cells and increased sperm counts. In the present study, we report that FA and FNA ameliorate the germ cell toxicity of MTX in mice. © 2008 Elsevier B.V. All rights reserved.

1. Introduction MTX is a widely used folate antagonist and highly efficacious chemotherapeutic agent introduced for clinical use in 1950s [1]. It is used against a broad range of neoplastic disorders including acute lymphoblastic leukemia, non-Hodgkin’s lymphoma, bladder carcinoma, breast cancer and testicular tumour [2–6]. Recent literature reports that high dose of MTX regimens can be used against primary CNS lymphomas as well as liver cholestatic disorders [7,8]. The genotoxic effects of MTX have already been reported in the somatic cells employing chromosome aberration and micronucleus test as the end points of evaluation [9–11]. MTX-induced micronuclei in the peripheral blood erythrocytes of common marmoset (Callithrix jacchus) at the pediatric equivalent therapeutic dose of 2.5 mg/kg daily for 2 consecutive days [12]. Le Fevre et al. reported that MTX

∗ Corresponding author. Tel.: +91 172 2214682 87x2152. E-mail addresses: [email protected], [email protected], [email protected] (G.B. Jena). 1383-5718/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2008.11.011

at a concentration of 300 ␮g/␮L arrests cells in G1 phase of the cell cycle and perturbs the marker genes in the signal transduction and cell cycle arrest pathways as early as 4 h after exposure in human lymphoblastoid (TK6) cell culture model [13]. MTX exhibited mutagenic and recombinogenic effects in Drosophila melanogaster test system [14,15]. MTX induced significant micronuclei in CHO-WBL cells at a concentration of 270 ␮g/ml in the presence of S9 as well as exhibited clastogenic potential in deletion (DEL) recombination assay in Sacchromyces cerevisiae [16]. Further, MTX at the doses 0.5, 1, 2, and 4 mg/(kg day) via intraperitoneal injection for 3 consecutive days induced significant micronucleated-reticulocytes (MN-RETs) in the mouse peripheral blood as detected by anti CD71/flowcytometry-based technique [17]. Barclay et al. reported that MTX-induced mitochondrial DNA damage and inhibit oxidative functions at a concentration of 1 mg/well in D5 strain of S. cerevisiae [18]. MTX at a concentration of 6.25–100 nM produced significant cytogenetic damage in the culture human lymphocytes [19]. Further it induced point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cell line grown for 20 generation under non-selective conditions [20]. It has been observed that

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MTX induced population-wide random heritable damage to NIH 3T3 mouse embryo fibroblast cells [21]. The genotoxic potential of MTX was revealed by the induction of MN in the oral mucosa sweeps of rheumatoid arthritis (RA) patients as well as the sister chromatid exchanges (SCEs) in oncologic nurses [22,23]. It has been further observed that psoriasis patients treated with MTX through oral route revealed significant increase in the SCEs frequencies in the lymphocytes [24]. The most interesting report is that a single exposure to high dose of MTX, exhibited toxicity at the late replicative enduring mice germ cells [25]. In a recent study, we reported that MTX induced cytotoxic and genotoxic effects in a dose (5, 10, 20 and 40 mg/kg) and time (5 and 10 weeks) dependent manner in the germ cells of mice [26]. FA is a water-soluble vitamin found in many food sources like fresh green vegetables, legumes, whole grains and citrus fruits [27,28]. It is available commercially as folate supplement to meet the dietary requirements in pregnancy and in patients with elevated levels of plasma homocysteine. It is an essential co-factor for the enzyme DHFR involved in the de-novo synthesis of purines and pyrimidines. The protective effects of FA against MTX-induced chromosomal damage have already been reported [29,30]. It has been reported that supplementation with FA ameliorates the toxicity of MTX without compromising its therapeutic efficacy [31–34]. FNA is the 5-formyl derivative of FA that circumvents the inhibition of DHFR as a part of high dose MTX therapy and potentiates fluorouracil in the treatment of colorectal cancer [35–38]. FNA provides tetrahydrofolates intracellularly and effectively bypasses the block on DHFR induced by MTX [39,40]. The protective effects of FNA on low dose MTX-induced genotoxicity in human as well as in animal models have been reported [41]. In the present investigation, the germ cell toxicity after repeated exposure of MTX was studied in male swiss mice. The results indicate that MTX increased the sperm head abnormalities, seminiferous tubule damage (disorganization and vacuolization), sperm DNA damage, TUNEL positive cells and decreased sperm counts. In the intervention study, we have investigated the effects of pretreatment with FA and post-treatment with FNA on MTX-induced germ cell toxicity in mice. The results clearly demonstrate that prior administration of FA and post-treatment with FNA reduced the germ cell toxicity of MTX in mice as evident from decrease in sperm head abnormalities, seminiferous tubule damage, sperm DNA damage, TUNEL positive cells and partial restoration of sperm counts. FA and FNA exhibited dose-dependent chemoprotection against MTXinduced germ cell toxicity in mice.

2.3. Experimental design Experimental design describing the treatment schedule for MTX, FA and FNA is shown in Fig. 2. Animals were randomly selected and divided into eleven groups with five animals in each group. FA was dissolved in saline and administered through oral route. MTX and FNA were dissolved in 0.1 M sodium bicarbonate and administered ip. The volume of administration to each animal was 10 ml/kg in all the cases. Animals were sacrificed by cervical dislocation 1 week after the last injection, following which the testes and epididymes were isolated. 2.3.1. Rationale for the study design MTX is a structural analogue of folic acid and competes for the binding with DHFR. In our study FA was administered continuously for 4 consecutive days prior to the administration of MTX to improve the cellular reserve and plasma level. This design was adopted to demonstrate the reversal by minimizing the inhibition of DHFR by MTX. FNA is known to bypass the block of DHFR by MTX as it directly provides the tetrahydrofolates (THF) intracellularly. Keeping this in mind FNA was administered 6 h after the administration of MTX to demonstrate its effect on the toxicity of MTX. 2.3.2. Dosing schedule All the animals were treated for 10 weeks and the weekly dosing schedule for each group was as follows. Group 1 received normal saline and served as vehicle control-1 (VC-1), Group 2 received 0.1 M sodium bicarbonate and served as vehicle control-2 (VC-2). Groups 3 and 4 received FA (200 ␮g/(kg day) for 4 days in a week) and FNA (10 mg/kg, once in a week), respectively, and served as controls. Group 5 was administered MTX (20 mg/kg once in a week) and served as positive control. Groups 6–8 received FA at the dose of 50, 100 and 200 ␮g/kg (po), respectively, for 4 consecutive days in a week followed by MTX 20 mg/kg (ip) on day five. Groups 9–11 received FNA at the dose of 2.5, 5 and 10 mg/kg (po), respectively, 6 h after single administration of MTX at the dose of 20 mg/kg (ip). 2.4. Sperm count and sperm head morphology The cauda epididymis was removed after sacrificing the animals and placed in a petri dish containing 2–3 ml of HBSS at room temperature. The epididymis was 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 [42,43]. Sperm head morphology was scored under the category of normal, sperm without hook, amorphous head, banana head and triangular head essentially as described [44]. Data were shown in terms of % of abnormal sperms. 2.5. Testis histology

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 (CAF) 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.

Histological slides were prepared as previously standardized in our laboratory [45]. 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 and rehydrated with alcohol and water. The rehydrated sections were stained using H&E, mounted with DPX mounting media and examined under the microscope at 200× magnification (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 [46]. Thirty seminiferous tubules from each animal were randomly examined and each tubule was scored based on the severity of the damage. Tubules showing no damage, mild damage, moderate damage and extensive damage were scored as 0, 1, 2 and 3, respectively. 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.

2.2. Chemicals

2.6. Testis TUNEL assay

MTX (CAS 59-05-2) was obtained as gift sample from GlaxoSmithKline (GSK) Pharmaceuticals Limited, Mumbai, India (Fig. 1). FA (CAS 59-30-3) was obtained as gift sample from Bio-Drug Laboratories Pvt. Ltd., Kolkata, India. Hematoxylin and Eosin (H&E), Ethidium Bromide (EtBr) (CAS 1239-45-8), FNA (CAS 1492-18-8), 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. DMSO, Normal melting agarose (NMA), low melting agarose (LMA), Triton-X 100, ethylenediamine-tetraacetic acid (EDTA) and Hank’s balanced salt solution (HBSS) were obtained from HiMedia Laboratories Ltd., Mumbai.

Paraffin-embedded testis tissues were cut into thin (5 ␮m) sections with microtome (Leica RM2145, Germany) and mounted on poly-l-lysine coated glass slides. 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 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. Materials and methods 2.1. Animals

S. Padmanabhan et al. / Mutation Research 673 (2009) 43–52

Fig. 1. Chemical structures of MTX, FA and FNA.

Fig. 2. Schematic diagram illustrating the experimental design.

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Significant decrease in the sperm counts was observed in MTXtreated group as compared to the control groups (P < 0.001). There was a significant restoration in the sperm counts in the groups which received FA treatment (50 and 100 ␮g/(kg day)) prior to MTX (P < 0.05). The sperm count was found to significantly higher in all

0.2 0.6 0.01 0.03 ± ± ± ± 20.4 29.8 0.25 0.84 0.5 0.7 0.01 0.03 ± ± ± ±

MTX 20 + FNA 5

20.4 28.8 0.25 0.86 0.2 0.7 0.01 0.03 ± ± ± ±

MTX 20 + FNA 2.5

20.4 27.8 0.25 0.90 0.7 0.3 0.01 0.04 ± ± ± ±

FA 200 + MTX 20

22.0 28.0 0.23 0.82 0.7 0.5 0.01 0.04 ± ± ± ±

FA 100 + MTX 20

22.0 28.4 0.23 0.81 0.7 0.9 0.01 0.04 ± ± ± ±

FA 50 + MTX 20

21.2 28.6 0.24 0.84 0.4 0.7 0.01 0.03 ± ± ± ±

MTX 20

0.9 0.6 0.01 0.04 ± ± ± ± 20.8 29.2 0.25 0.87

FNA

1.2 0.8 0.01 0.03 ± ± ± ± 21.2 30.0 0.23 0.77

FA

0.7 0.8 0.01 0.02 All the values are expressed as mean ± S.E.M. (n = 5).

3.2. Sperm count and sperm head morphology

± ± ± ±

Slight decrease in the final body wt was observed after treatment with MTX as compared to the control groups. Post-treatment with FNA 10 mg/kg led to partial restoration of final body wt as compared to the group which received only MTX; however the increase in final body wt was statistically not significant. The paired testes wt in the groups which received protection with FA and FNA were not found to be significant in comparison with the MTX-treated group. The relative testis wt was found to increase slightly but not significantly in the groups administered FNA after treatment with MTX (Table 1).

VC-2

3.1. Body weight and organ weight

0.7 1.2 0.01 0.04

3. Results

± ± ± ±

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. 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.

20.8 30.4 0.23 0.77

2.9. Statistical analysis

VC-1

The halo assay was performed essentially as described with some modifications [50]. Testis was homogenized gently in PBS and 5 ␮l of the homogenate was suspended in 50 ␮l of 0.5% LMA and layered over the surface of a frosted slide (precoated with 1% NMA) 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 [51].

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

2.8. Halo assay

Table 1 Effects of FA pre-treatment and FNA post-treatment on the initial body wt, final body wt, paired testes wt and relative testes wt of mice after 10 weeks of MTX administration.

The sperm comet assay was performed essentially as described with some modifications [47,48]. Sperm sample (5 ␮l) containing (1–3) × 104 sperm ml−1 was suspended in 95 ␮l of 1% (w/v) LMA. From this suspension, 80 ␮l was applied to the surface of a microscope slide (pre-coated with 1% NMA) 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. After 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 (1:10000 dilution) was applied for 1 h. Slides were rinsed briefly with double-distilled water and coverslips were placed before image analysis. The fluorescent labeled DNA was visualized (200×) using an AXIO Imager M1 fluorescence microscope (Carl Zeiss, Germany) and the resulting images were captured 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 [49]. 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.

21.4 29.6 0.27 0.91

2.7. Sperm comet assay

20.2 28.8 0.23 0.80

MTX 20 + FNA 10

S. Padmanabhan et al. / Mutation Research 673 (2009) 43–52

Groups (parameters)

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S. Padmanabhan et al. / Mutation Research 673 (2009) 43–52

Fig. 3. Histogram showing the effects of FA and FNA administration on the sperm counts after 10 weeks of MTX treatment. All the values are expressed as mean ± S.E.M. (n = 5), *P < 0.05, ***P < 0.001, a vs. VC-1, b vs. VC-2, c vs. FA, d vs. FNA, e vs. MTX 20.

the groups post-treated with FNA as compared to the group which received MTX (P < 0.05) (Fig. 3). Sperms without hook, banana head sperms and sperms with triangular and amorphous heads were classified as sperm head abnormalities. The sperm head abnormalities were found to increase significantly in the group which received MTX for 10 weeks as compared to the control groups (P < 0.001). A significant decrease in the % of abnormal sperms was observed in the group pre-treated with the highest dose of FA in comparison with the MTX-treated group (P < 0.05). The group post-treated with FNA 10 mg/kg showed a significant reduction in the sperm head abnormalities as compared to the group which received MTX (Fig. 4).

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Fig. 4. Histogram showing the effects of FA and FNA administration on sperm head abnormalities expressed as % of abnormal sperm after 10 weeks of MTX treatment. All the values are expressed as mean ± S.E.M. (n = 5), *P < 0.05, ***P < 0.001, a vs. VC-1, b vs. VC-2, c vs. FA, d vs. FNA, e vs. MTX 20.

3.3. Testis histology Histological assessment of the testes was done on the basis of the seminiferous tubule damage score (Fig. 5). A significant increase in the testicular damage was evident with weekly once treatment of MTX 20 mg/kg as compared to the control groups (P < 0.001). Prior treatment of FA was found to significantly decrease the seminiferous tubule damage score in comparison with the group which received only MTX treatment (P < 0.001). A significant reduction in the seminiferous tubule damage score was also observed in the

Fig. 5. Photomicrographs of H&E stained histological slides of the testis after 10 weeks of MTX treatment, magnification 200×; control (A), MTX 20 (B), FA 200 + MTX 20 (C), MTX 20 + FNA 10 (D). Severe and moderate vacuolization are indicated by white and black arrows, respectively.

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Fig. 6. Histogram showing the effects of FA and FNA administration on the seminiferous tubule damage score after 10 weeks of MTX treatment. All the values are expressed as mean ± S.E.M. (n = 5), ***P < 0.001, a vs. VC-1, b vs. VC-2, c vs. FA, d vs. FNA, e vs. MTX 20.

groups post-treated with FNA in comparison with the MTX-treated group (P < 0.001) (Fig. 6). 3.4. Testis TUNEL assay A dose-dependent decrease in the % of TUNEL positive cells emitting green signals was observed in the groups pre-treated with FA and the groups post-treated with FNA (Fig. 7). The groups administered FA (100 and 200 ␮g/(kg day)) prior to MTX showed a significant reduction in the % of TUNEL positive apoptotic cells in the testis as compared to the group treated with MTX (P < 0.01). The % of TUNEL positive cells showed a significant decrease in all the groups which received FNA post-treatment in comparison with the group which received only MTX treatment (P < 0.01) (Fig. 8).

Fig. 8. Histogram showing the effects of FA and FNA administration on the % of TUNEL positive cells in testis after 10 weeks of MTX treatment. All the values are expressed as mean ± S.E.M. (n = 5), **P < 0.01, ***P < 0.001, a vs. VC-1, b vs. VC-2, c vs. FA, d vs. FNA, e vs. MTX 20.

3.5. Sperm comet assay DNA damage in the sperms was assessed using the sperm comet assay (Fig. 9). The various comet parameters viz. TL, TM, OTM and TDNA showed a significant increase in the MTX-treated group as compared to the control groups (P < 0.001). The groups which were pre-treated with FA showed significant reduction in the comet parameters in comparison with the group which received MTX (P < 0.001). Similar results were observed in the groups which received FNA treatment. The comet parameters significantly reduced as compared to the MTX-treated group (P < 0.001) (Table 2). 3.6. Halo assay DNA damage and cytotoxicity in the testis were assessed by the halo assay or the DNA diffusion assay (Fig. 10). The extent of damage

Fig. 7. Photomicrographs showing apoptotic cells having fragmented DNA as revealed from TUNEL assay in testis at magnification 200×. Control (A), MTX 20 (B), FA 200 + MTX 20 (C) and MTX 20 + FNA 10 (D). The cells emitting green signal are TUNEL positive apoptotic cells.

S. Padmanabhan et al. / Mutation Research 673 (2009) 43–52

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Fig. 9. Photomicrographs showing the DNA migration pattern in mice sperm nuclei after 10 weeks of MTX treatment. The symbols “−” and “+” represents cathode and anode, respectively, during electrophoresis of negatively charged DNA. Magnification: 200×. Dye: SYBR Green. Control (A), MTX 20 (B), FA 200 + MTX 20 (C) and MTX 20 + FNA 10 (D).

fering in the folate metabolism. The deficiency of folates is often associated with genotoxic damage like strand breaks, chromosomal abnormalities, extensive incorporation of uracil in place of thymine into the DNA, defective DNA repair, anomalous DNA methylation patterns and increased somatic mutation rates [55–59]. Intracellularly, MTX is retained in the tissues for long duration in the form of polyglutamates. The polyglutamated form is more active than MTX in inhibiting folate-dependent enzymes [60]. The increased toxic effects of MTX after repeated administration can be attributed to its intracellular accumulation and subsequent DHFR inhibition. The toxicities induced by MTX are either due to the non-target specific mode of action or the manifestations in the late replicative enduring cells. In the present investigation, MTX was found to decrease the sperm count and increase the frequency of sperms with abnormal head. Interference in the spermatogenesis process and the subsequent elimination of sperm cells at different stages of development decreases the sperm counts [61,62]. The sperm head abnormalities possibly result due to the interference with the DNA integrity and/or the expression of the genetic material [44]. FA is an essential dietary component that has an important role in the maintenance of genomic integrity [63,64]. It serves as an essential co-factor in the single-carbon transfer reactions for the

in the testicular cells was categorized as mild, moderate and extensive. The percentage of mildly and moderately damaged cells did not show any significant difference among the groups (data not shown). The percentage of extensively damaged cells was found to increase significantly in the MTX-treated group as compared to the controls (P < 0.001). The groups administered FA (100 and 200 ␮g/(kg day)) prior to MTX showed a significant decrease in the percentage of extensively damaged cells in comparison with the group treated with MTX (P < 0.05). The groups post-treated with FNA (5 and 10 mg/kg) also decreased the percentage of extensively damaged cells significantly as compared to the group which received MTX (P < 0.05) (Fig. 11). 4. Discussion MTX is a widely used folate antagonist with narrow therapeutic index [52,53]. Being a structural analogue of FA, it competes with the normal substrate FA for the binding site on DHFR which is the key enzyme involved in the synthesis of essential DNA precursors like thymidylates and purines. Inhibition of DHFR leads to an imbalance in the nucleotide pools and thereby perturb the DNA synthesis [54]. MTX also causes deficiency of folates by inter-

Table 2 Effects of FA pre-treatment and FNA post-treatment on the various sperm comet parameters following 10 weeks of MTX administration. All the values are expressed as mean ± S.E.M. (n = 5), *** P < 0.001, a vs. VC-1, b vs. VC-2, c vs. FA, d vs. FNA, e vs. MTX 20. Groups

Parameters TL (␮m)

VC-1 VC-2 FA FNA MTX 20 FA 200 + MTX 20 MTX 20 + FNA 10

10.62 10.62 10.18 9.51 20.11 11.59 13.37

± ± ± ± ± ± ±

TM 0.11 0.48 0.67 0.58 1.11***abcd 0.32***e 1.22***e

1.35 1.29 1.37 1.31 6.41 1.95 1.68

OTM ± ± ± ± ± ± ±

0.16 0.21 0.18 0.22 0.12***abcd 0.22***e 0.38***e

1.67 1.67 1.53 1.43 4.84 1.77 2.86

TDNA ± ± ± ± ± ± ±

0.60 0.16 0.19 0.09 0.14***abcd 0.45***e 0.33***e

8.65 8.32 8.15 7.75 19.03 9.68 10.34

± ± ± ± ± ± ±

0.66 0.99 0.82 1.09 1.32***abcd 0.19***e 1.15***e

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Fig. 10. Representative photomicrographs of the halo assay in testis after 10 weeks of MTX treatment. Magnification: 200×. Stain: EtBr; control (A), MTX 20 (B), FA 200 + MTX 20 (C) and MTX 20 + FNA 10 (D). Extensively damaged cells are indicated by arrow.

synthesis of nucleotides and for DNA methylation reactions [59,65]. Deficiency of FA induces aneuploidy in human lymphocytes in vitro as evident from cytokinesis-blocked cells and chromosome specific probes [66]. Further low dietary folate intake is associated with an elevated risk for carcinogenesis [67]. Morgan et al. reported that folic and folinic acid supplementation is of clinical relevance, as it prevents MTX-induced toxicity without affecting the therapeutic efficacy in rheumatoid arthritis [68]. Folinic acid supplementation reduced the side effects of MTX in the treatment of juvenile idiopathic arthritis [69]. Chemotherapy with MTX and folinic acid is highly effective and well-tolerated in patients with non-metastatic gestational trophoblastic neoplasia [70]. Keshava et al. reported that treatment with folinic acid inhibits MTX-induced genotoxicity in V79 cells [71]. It has been reported that the cytotoxicity of MTX is reversed by the intervention of FNA in human leukemic cells

Fig. 11. Histogram showing the effects of FA and FNA administration on the % extensively damaged cells after 10 weeks of MTX treatment determined by halo assay in testis. All the values are expressed as mean ± S.E.M. (n = 5), *P < 0.05, ***P < 0.001, a vs. VC-1, b vs. VC-2, c vs. FA, d vs. FNA, e vs. MTX 20.

with carrier-mediated and receptor-mediated folate uptake [72]. FA significantly decreases the incidence of chromosome breakage in human lymphocytes culture [73]. Further, FA intake has been shown to be essential to reduce the micronucleus index in humans [74]. It has been reported that the concentration of FA co-relates well with the sperm density and total sperm counts; suggesting the importance of FA in the restoration of male reproductive function [29]. However, the protective effects of FA on the germ cells of male swiss mice have not been explored. In the present study, administration of FA prior to MTX was found to restore the sperm counts and decrease the incidence of sperms with head abnormalities. Further, the intervention of FA and the reduction in the gonadal toxicity induced by MTX was clearly evident from the histological evaluation. It has already been reported that FA ameliorates chromium-induced changes in the reproductive performance in male rabbits and significantly improves the sperm parameters [75]. In the present investigation the protective effect might be due to the long term administration of FA, which abates the interference of MTX on the DNA synthesis process owing to its competition for the same binding site on DHFR. FNA is the 5-formyl derivative of tetrahydrofolates that remains unaffected by the inhibition of DHFR by MTX and supplies folates intracellularly. FNA administration 6 h following MTX treatment was also found to restore the sperm counts and significantly decrease the incidence of sperm head abnormalities. Further, posttreatment with FNA improved the gonadal damage as revealed from testis histology. The sperm comet assay indicates the genotoxicity of MTX as observed by an increase in the comet parameters viz. TL, TM, OTM and TDNA. Strand breaks are progressively accumulated following MTX treatment as a consequence of depleted nucleotide pools and the impairment of the repair mechanisms [76,77]. The protective effects on MTX-induced genotoxicity in the sperms were observed both with the intervention of FA as well as FNA. It has been reported that co-treatment with FNA prevent the DNA damage induced by

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pyrimethamine, a folate antagonist similar to MTX; in the liver, kidney, lung, brain, spleen as well as in bone marrow as determined by the comet assay [78]. FA and FNA possibly help in the synthesis of a new nucleotide pool and restore the imbalance caused by MTX [71]. The halo assay is a simple, sensitive and reliable “DNA diffusion” assay used for the quantification of DNA damage and cytotoxicity [50,51]. The intervention of FA and FNA significantly reduced the incidence of extensively damaged testicular cells induced by MTX as revealed by halo assay. The TUNEL assay was performed to ascertain the mode of cell death. It is based upon the principle that terminal deoxynucleotidyl transferase (TdT) binds to the exposed 3 -OH ends of the DNA fragments generated in response to apoptotic signals and catalyses the addition of fluorescein-labeled deoxynucleotides [79]. The frequency of TUNEL-positive cells in the testis decreased in a dose-dependent manner on treatment with both FA and FNA. Our results clearly demonstrate that intervention of FA prior to MTX and post-treatment with FNA reduces the germ cell toxicity induced by MTX in mice as evident from the decreased sperm head abnormalities, seminiferous tubule damage, sperm DNA damage, TUNEL positive cells and increased sperm counts. The beneficial effects of FA and FNA on the germ cell toxicity induced by repeated exposure of MTX need to be further explored for their chemoprotective potential in clinical settings. Conflict of interest None. Acknowledgements The financial assistance received from National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar to carry out this particular study is duly acknowledged. The authors would also like to acknowledge GlaxoSmithKline (GSK) Pharmaceuticals Limited, Mumbai and Bio-Drug Laboratories Pvt. Ltd., Kolkata for providing the generous gift samples of methotrexate and folic acid, respectively. References [1] 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. [2] R.C. Choudhury, S.K. Ghosh, A.K. Palo, Cytogenetic toxicity of methotrexate in mouse bone marrow, Environ. Toxicol. Pharmacol. 8 (2000) 191–196. [3] 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. [4] 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. [5] 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. [6] 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. [7] R. Liu, S.M. Chang, M. Prados, Recent advances in the treatment of central nervous system tumours, Update Cancer Ther. 3 (2008) 49–79. [8] K. Novak, M.G. Swain, Role of methotrexate in the treatment of chronic cholestatic disorders, Clin. Liver Dis. 12 (2008) 81–96. [9] R.C. Choudhury, A.K. Palo, Modulatory effects of caffeine on methotrexateinduced cytogenotoxicity in mouse bone marrow, Environ. Toxicol. Pharmacol. 15 (2004) 79–85. [10] Y. Kasahara, Y. Nakai, D. Miura, K. Yagi, K. Hirabayashi, T. Makita, Mechanism of induction of micronuclei and chromosome aberrations in mouse bone marrow by multiple treatments of methotrexate, Mutat. Res. 280 (1992) 117–128. [11] Y. Kasahara, A. Wakata, Y. Nakai, K. Yuno, D. Miura, K. Yagi, K. Hirabayashi, T. Makita, The micronucleus test using peripheral blood reticulocytes from methotrexate-treated mice, Mutat. Res. 278 (1992) 145–151.

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