Effectiveness Of Doxorubicin As An Anti Neoplastic Agent Biology Essay

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Medical oncology has been found to have a major impact in changing the practice of medicine in the past few decades, as curative treatments have been identified for a myriad of previously fatal malignancies. Doxorubicin (DOX), an anticancer anthracycline antibiotic derived from the fungus Streptocococcus peuceticus var. caesius; cisplatin (CIS), a divalent inorganic, water-soluble platinum containing complex and methotrexate (MTX), a folate antagonist, have been found to be potent somatic as well as germ cell toxicants [1-6]. These agents are potent chemotherapeutic agents, widely used for the treatment of various cancers. The effectiveness of DOX as an anti-neoplastic agent is due to its activity as a 'topoisomerase II poison'. DOX inhibits the negative supercoiling of DNA and intercalates into DNA and thereby inhibits the DNA and RNA polymerases resulting in the hampering of DNA transcription and replication [7]. Activity of CIS is mainly due to its binding to DNA resulting into the formation of intrastrand and interstrand cross-links between adjacent purine bases and thereby inhibiting DNA replication and transcription [8]. MTX shows its therapeutic effect due to the inhibition of dihydrofolate reductase, a key enzyme in the folic acid metabolism, which converts dihydrofolic acid to tetrahydrofolic acid. [9,10]. This has a desired effect in the treatment of many cancers, but at the same time is responsible for their toxic manifestations. As all the three agents act via interaction with the genetic material of the cell, they result into disorganization of the cellular structure in both somatic as well as germ cells. The most adverse somatic cell toxicities caused by DOX, CIS and MTX include cardiotoxicity, nephrotoxicity and hepatotoxicity respectively. The cardiotoxic effects result in cardiac dysfunction, cardiomyopathy and finally congestive heart failure. Chronic administration of DOX results into dose-dependent, late-onset and irreversible cardiomyopathy [1]. CIS treatment has been reported to induce oxidative stress in rat kidney [11]. CIS treatment is also known to induce damage in kidney genomic DNA and cause nephrotoxicity in male rats [12]. MTX treatment has been reported to cause severe hepatotoxicity in rats [13]. The germ cell toxicity of all the three agents is well reported. It has been reported that DOX treatment induces apoptosis in germ line stem cells in the immature rat testis [2]. DOX treatment results into a stage-specific inhibition of DNA and induction of apoptosis in cultured rat spermatogenetic cells [14]. It has also been reported that DOX treatment induces oxidative stress and p53-mediated mitochondrial apoptosis in rat testes [15]. CIS has been found to affect testicular function at a dose of 2 mg/kg daily for 5 days in a week and sacrificed after 10 and 30 days [16]. A single intravenous bolus administration of MTX, with necropsy 56 days later has been reported to cause testicular toxicity and sperm abnormalities [17]. Heart, kidney and liver are the major organs of the body. Patients suffering from disorders pertaining to these organs have life-threatening risks which lead to severe limitations in their daily activities thereby decreasing their acuity of living a quality lifestyle. The somatic cell toxicities caused by these chemotherapeutic agents limit their use and hence they are of great concern. But at the same time, germ cell toxicity of these agents is also well-reported and hence it should also not be overlooked. Treatment with these drugs results into a reduction in the weights of the reproductive organs, their structural disorganization, impaired fertility, thereby affecting the growth and development of the future generation. The outcomes of the impaired reproductive system may have a foremost bang on not only the physical and mental but also the social and emotional status of both men and women. Comparison of somatic cell and germ cell toxicity caused by these three drugs is enigmatic. Taking all these points into consideration, we undertook the present investigation to compare the cytotoxic and genotoxic effects of DOX, CIS and MTX on somatic as well as germ cells. The present study indicates that DOX, CIS and MTX have toxic outcomes both in somatic as well as germ cells at the same dose and duration and hence germ cell toxicity should be given an equal importance as that of somatic cell toxicity. Taking the importance of germ cell toxicity in to account, an attempt has been made to correlate sperm head abnormality and sperm DNA damage by sperm head morphological evaluation and sperm comet assay respectively, which can be used clinically for the appraisal of male infertility. The correlation between sperm head abnormality and sperm DNA damage indicates that the abnormality in the sperm head is due to impaired spermatogenesis caused as a result of damage in the genetic material of the sperm.

2. Literature review

2.1. Mechanism of action

DOX is an anticancer anthracycline antibiotic derived from the fungus Streptocococcus peuceticus var. caesius. It has several cytotoxic actions. It binds to DNA and inhibits both DNA and RNA synthesis, but its main cytotoxic action appears to be mediated through an effect on topoisomerase II, the activity of which is markedly increased in proliferating cells. It intercalates in the DNA and its effect is to stabilize the DNA topoisomerase II complex after the strands have been nicked, thus causing the process to seize up at this point [18]. CIS shows its activity because of its binding to DNA resulting into the formation of intrastrand and interstrand cross-links between adjacent purine bases. These adducts distort the DNA template thus inhibiting DNA replication and transcription, arresting cell cycle in the G2 phase and subsequent induction of apoptosis [8]. MTX results in to the inhibition of dihydrofolate reductase, a key enzyme in the folic acid metabolism, which converts dihydrofolic acid to tetrahydrofolic acid. It also directly inhibits the folate-dependent enzymes of de novo purine and thymidylate synthesis [9].

2.2. Indications and uses

DOX has been used in the patients for the treatment of acute leukaemia, lymphomas and adenosarcoma of breast, bladder, thyroid gland and prostate [19]. CIS is used for the treatment of lung cancer, adverse testicular cancer, metastatic breast cancer and colon cancer [20-23]. MTX is used against a broad range of neoplastic disorders including acute lymphoblastic leukaemia, non-Hodgkin's lymphoma, breast cancer and testicular tumours. Further, it is effective for the treatment of psoriasis, rheumatoid arthritis and different immune-suppressive conditions. It is also the drug of choice in the new regimen combination treatment against rheumatoid arthritis and for several refractory/relapsed tumours [9].

2.3. Adverse effects

Myelosuppression is a major dose-limiting complication of DOX, with leucopenia usually reaching a nadir during the second week of therapy and recovering by the further week. Other adverse effects include thrombocytopenia, anemia, stomatitis, GI disturbances and alopecia. Erythematous streaking near the site of infusion ("adriamycin flare") is a benign local allergic reaction. Cardiomyopathy is the most important long-term toxicity [19,24]. The most widely known toxicity of CIS is nephrotoxicity. Other toxicities include gastrointestinal, myelosuppression, ototoxicity, neurotoxicity and germ cell toxicity [25]. Major toxicity caused by MTX is hepatotoxicity, which may lead to cirrhosis. It is also known to cause pneumonitis, GI toxicity, cutaneous lesions, neurologic symptoms, changes in the bone metabolism and teratogenecity [26].

2.4. Pharmacokinetics

Doxorubicin is a prodrug and gets converted to an active metabolite, doxorubicinol. Its plasma half life is 29 + 16 h. Its action is prolonged when plasma bilirubin concentration is elevated. It undergoes biliary excretion [27,28]. CIS rapidly distributes throughout the body after an intravenous injection with a large volume and is highly bound to plasma protein. Monohydrated complex is also detected in biological samples after the administration of CIS. Its plasma half-life is 9-30 min [29]. MTX is readily absorbed from the GIT. Its plasma half-life is 8-10 h. Approximately 50% of MTX is bound to plasma proteins. After high doses, it gets metabolised to 7-hydroxy-methotrexate, which is potentially nephrotoxic. MTX is retained in the form of polyglutamates for long periods, for e.g., for weeks in the kidneys and for several months in the liver [19].

3. Hypothesis

DOX, an anticancer anthracycline antibiotic; CIS, a divalent inorganic, water-soluble platinum containing complex and MTX, a folate antagonist are widely used chemotherapeutic agents for the treatment of a myriad of tumours. They are used in combination for the treatment of various cancers such as metastatic osteosarcoma, retinoblastoma and muscle-invasive bladder cancer [30-32]. All the three agents lead to the disorganization of the cellular structure in both somatic as well as germ cells, because their action is mediated via interaction with the genetic material of the cell. DOX, CIS and MTX are known to adversely affect heart, kidney and liver respectively and thus cause somatic cell toxicity. They are also known to cause germ cell toxicity. The somatic and germ cell toxicities caused by these chemotherapeutic agents limit their clinical use and are of great concern, especially in the conditions of prolonged use. Comparison of somatic cell and germ cell toxicity of these agents at the same dose and duration is highly unresolved. Taking all these points into consideration, we undertook the present investigation to compare the cytotoxic and genotoxic effects of DOX, CIS and MTX on somatic and germ cells in rats. Taking in to account the importance of germ cell toxicity, an endeavour has also been made to correlate abnormality in sperm head and sperm DNA damage for the appraisal of male infertility in cancer patients while undergoing DOX, CIS and MTX therapy.

4. Objectives

To evaluate and compare the DOX, CIS and MTX induced cytotoxic and genotoxic effects in somatic cells of rats.

To evaluate and compare the DOX, CIS and MTX induced cytotoxic and genotoxic effects in germ cells of rats.

To assess the relationship between sperm head abnormality and sperm DNA damage.

To investigate the possible protective effects of hesperetin against DOX induced cytotoxicity and genotoxicity in heart and testes using rats.

5. Study design

Animals were randomized into four groups consisting of five animals in each group. Drug treatment was given weekly once for a period of five weeks and the animals were sacrificed after one week of receiving the last dose. DOX and CIS were dissolved in isotonic saline solution and MTX in 0.1 M sodium bicarbonate solution and were administered through intraperitoneal (ip) route.

Study 1: Comparative evaluation of DOX-induced cytotoxicity and genotoxicity in heart and testes of male SD rats.

Group 1: Control (Normal saline)

Group 2: DOX treated (1.25 mg/kg, ip)

Group 3: DOX treated (2.5 mg/kg, ip)

Group 4: DOX treated (5 mg/kg, ip)

Fig. 1. Schematic diagram illustrates the experimental design. Group 1 received normal saline once in a week for a period of 5 weeks and served as control. Groups 2-4 received DOX 1.25, 2.5 and 5 mg/kg/ml respectively once in a week for 5 weeks. All the animals were sacrificed 1 week after receiving the last injection of DOX.

Study 2: Comparative evaluation of CIS-induced cytotoxicity and genotoxicity in kidney and testes of male SD rats.

Group 1: Control (Normal saline)

Group 2: CIS treated (0.5 mg/kg, ip)

Group 3: CIS treated (1 mg/kg, ip)

Group 4: CIS treated (2 mg/kg, ip)

Fig. 2. Schematic diagram illustrates the experimental design. Group 1 received normal saline once in a week for a period of 5 weeks and served as control. Groups 2-4 received CIS 0.5, 1 and 2 mg/kg/ml respectively once in a week for 5 weeks. All the animals were sacrificed 1 week after receiving the last injection of CIS.

Study 3: Comparative evaluation of MTX-induced cytotoxicity and genotoxicity in liver and testes of male SD rats.

Group 1: Control (Normal saline)

Group 2: MTX treated (5 mg/kg, ip)

Group 3: MTX treated (10 mg/kg, ip)

Group 4: MTX treated (20 mg/kg, ip)

Fig. 3. Schematic diagram illustrates the experimental design. Group 1 received 0.1 M NaHCO3 once in a week for a period of 5 weeks and served as control. Groups 2-4 received MTX 5, 10 and 20 mg/kg/ml respectively once in a week for 5 weeks. All the animals were sacrificed 1 week after receiving the last injection of MTX.

6. Materials and methods

6.1. Animals

All the animal experiment protocols were approved by the Institutional Animal Ethics Committee (IAEC) and the experiments on animals were performed in accordance with the CPCSEA (Committee for the Purpose of Control and Supervision of Experimentation on Animals) guidelines. Experiments were performed on male SD rats (180-200 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 50±10 % humidity and an automatically controlled cycle of 12 h light and 12 h dark. Standard laboratory animal feed (purchased from commercial supplier) and water were provided ad libitum. Animals were acclimatized to the experimental conditions for a period of 1 week prior to the commencement of the experiment.

6.2. Chemicals

DOX (CAS no. 29042-30-6) was obtained as a gift sample from Intas Pharmaceuticals Ltd., Ahmedabad, India. MTX (CAS no. 29042-30-6) was obtained as a gift sample from GlaxoSmithKline Pharmaceuticals Ltd., Mumbai, India. CIS (CAS no. 15663-27-1), Hematoxylin and eosin (H&E), Ethidium Bromide (EtBr) (CAS no.1239-45-8), Trizma (CAS no. 77-86-1), Dithiothreitol (CAS no. 3483-12-3), Proteinase-K (CAS no. 39450-01-6) and SYBR Green 1 (CAS no. 163795-75-3) were purchased from Sigma-Aldrich Chemicals, Saint Louis, MO, USA. Dimethylsulphoxide (DMSO), normal melting point agarose (NMPA), low melting point agarose (LMPA), Triton X-100, ethylenediamine-tetraacetic acid (EDTA) and Hank's balanced salt solution (HBSS) were obtained from HiMedia Laboratories Ltd., Mumbai.

6.3. Dose selection, chemical preparation and animal treatment

The dose of DOX (1.25, 2.5 and 5 mg/kg/ml) was selected on the basis of studies conducted by Prahalathan et al. and Kato et al. [33,34] to assess male germ cell toxicity in rats. The dose of CIS (0.5, 1 and 2mg/kg/ml) was selected on the basis of studies conducted by Huang et al. [16] in rats to assess testicular toxicity after acute and chronic exposure to CIS. Further, the dose of MTX (5, 10 and 20 mg/kg/ml) was selected on the basis of studies conducted by Johnson et al. [17] to assess MTX-induced testicular cytotoxicity in rats. All the three drugs were administered to the male SD rats weekly once for a period of five weeks and sacrificed one week after receiving the last dose. DOX and CIS were dissolved in isotonic saline solution and MTX in 0.1 M sodium bicarbonate solution and were administered through ip route.

6.4. Experimental design

The detailed experimental design is shown in Fig. 1, 2 and 3. The animals were randomly divided into 4 groups for each study. For study 1, group 1 received isotonic saline solution (vehicle) and served as the control for groups receiving DOX and groups 2, 3 and 4 received DOX at the dose of 1.25, 2.5 and 5 mg/kg/wk respectively for a period of five weeks. For study 2, group 1 received isotonic saline solution (vehicle) and served as the control for groups receiving CIS and groups 2, 3 and 4 received CIS at the dose of 0.5, 1 and 2 mg/kg/wk respectively for a period of five weeks. For study 3, group 1 received 0.1 M sodium bicarbonate solution and served as the control for groups receiving MTX and groups 2, 3 and 4 received MTX at the dose of 5, 10 and 20 mg/kg/wk respectively for a period of five weeks. Animals were sacrificed by decapitation one week after the last treatment.

6.5. Measurement of lipid peroxidation

The lipid peroxide level in tissue homogenate was measured according to the method previously described [35] with some modifications. Tissues were collected and homogenized in ice cold phosphate buffer (pH 7.4) for the determination of lipid peroxidation levels. After homogenization and centrifugation, the supernatant was collected for the determination of MDA level in tissue samples. MDA level was estimated spectrophotometrically as an end product of lipid peroxidation using thiobarbituric acid reactive substance method. Lipid peroxidation was calculated from the standard curve generated using 1, 1, 3, 3 tetramethoxy propane (97%) and expressed as ηmol MDA/ mg of protein.

6.6. Measurement of glutathione (GSH) content

For determination of GSH content, an equal volume of 10% sulphosalicylic acid was added to tissue homogenate and vortexed. The mixture was kept for 30 min in ice bath. After centrifugation for 10 min, the supernatant was collected carefully without disturbing the sediment. GSH content was measured using Ellmann's reagent [5, 5'-dithiobis-2-nitrobenzoic acid (DNTB) solution] according to the method described [36]. GSH levels were calculated using a standard reference curve using reduced glutathione as a standard. Results were expressed in μmol GSH/mg protein.

6.7. Determination of protein content

Protein concentration in tissue homogenate was determined as described [37] with bovine serum albumin (CAS no. 9048-46-8, Sigma-Aldrich, USA) as the standard protein.

6.8. Histological evaluation

Histological slides were prepared as previously standardized in our laboratory [38]. The tissues were fixed in 10% formalin, dehydrated in increasing concentrations of ethanol and embedded in paraffin. Tissue sections (5μm) 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 (40x) and low (10x and 20x) magnifications (Olympus BX51 microscope, Tokyo, Japan). The sections from each animal were evaluated for structural changes.

Histological quantification was performed in the testicular sections by allotting a Johnsen score following the criteria of scoring as described [39]. Thirty seminiferous tubules from each animal were randomly examined and Johnsen score was given based on the type of the cells damaged in the seminiferous tubule. Johnsen score was calculated by dividing the sum of all scores with the total no. of seminiferous tubules examined.

6.9. Halo assay

The halo assay was performed essentially as described with some modifications [40]. Tissues were homogenized gently in 1 ml cold Hank's balanced salt solution (HBSS) containing 20 mM EDTA/ 10% DMSO, minced into fine pieces and 5μl of the homogenate was suspended in 95μl of 0.5% low melting point agarose (LMPA) and layered over the surface of a frosted slide (pre-coated with 1% normal melting point agarose (NMPA)) to form a microgel and allowed to set at 4 â-¦C for 5 min. A second layer of 1% LMPA was added and allowed to set at 4 â-¦C for 5-10 min. The slides were immersed in freshly prepared lysis solution (2.5 M NaCl, 2 mM EDTA, 10 mM Tris (pH 10.0), 1% Triton X-100) for 2 h at 4 °C. Following lysis, the slides were incubated in an alkaline solution containing 300 mM NaOH and 1 mM EDTA (pH >13.0) for 20 min and stained using EtBr. Samples were run in duplicate and 100 cells were randomly examined per slide under the microscope (Olympus BX51, Tokyo, Japan). The damaged cells were categorized as mild, moderate and extensive as described [9].

6.10. Single cell gel electrophoresis (SCGE) assay

A small piece of tissue was placed in 1 ml cold HBSS containing 20 mM EDTA/ 10% DMSO, minced into fine pieces. 5μl of this was mixed with 95μl LMPA and layered over the surface of a frosted slide (pre-coated with 1% NMPA) to form a microgel and allowed to set at 4 â-¦C for 5 min. A second layer of 1% LMPA was added and allowed to set at 4 â-¦C for 5-10 min. The slides were then immersed in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl buffer (pH 10.0), 1% sodium sarcosinate with 1% Triton X-100 and 10% DMSO) at 4°C for 24 h. After 24 h, the slides were washed with chilled water, then coded and placed in a specifically designed horizontal electrophoresis tank (Model, CSLCOM20, Cleaver Scientific Ltd., UK) and DNA was allowed to unwind for 20 min in alkaline solution containing 300 mM NaOH and 1 mM EDTA (pH >13.0). Electrophoresis was conducted at 28 V, 300 mA. After neutralization the slides were washed with chilled water and stained with SYBR Green 1 (1:10,000 dilution). Slides were rinsed briefly with double-distilled water and cover slips were placed before image analysis. The fluorescent labelled DNA was visualized (200x) 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 (Comet Imager V.2.0.0) [41,42]. Duplicate slides were prepared for each treatment and were independently coded and scored without knowledge of the code. The parameters for the DNA damage analysis include: tail length (TL, in μm), tail moment (TM), olive tail moment (OTM) and % tail DNA (% TDNA). The edges of the slides, any damaged part of the gel, any debris, superimposed comets and comets without distinct head ("hedgehogs" or "ghost" or "clouds") were not considered for the analysis.

6.11. Sperm comet assay

The sperm comet assay was performed essentially by the method standardized in our laboratory [43,44]. Sperm sample (5µl) containing 1-3Ã-104 sperms per ml were suspended in 95µl of 1% (w/v) LMPA. From this suspension, 80µl was applied to the surface of a microscope slide (pre-coated with 1% NMPA) to form a microgel and allowed to set at 4 °C for 5 min. A second layer of 1% LMPA was added and allowed to set at 4 °C for 5-10 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 then coded and placed in a specifically designed horizontal electrophoresis tank (Model, CSLCOM20, Cleaver Scientific Ltd., UK) and DNA was allowed to unwind for 20 min in an alkaline solution containing 300 mM NaOH and 1 mM EDTA (pH >13.0). Electrophoresis was conducted at 28 V, 300 mA. After electrophoresis, slides were neutralized and the DNA fluorochrome SYBR Green 1 (1:10,000 dilution) was applied for 1 h. Slides were rinsed briefly with double-distilled water and cover slips were placed before image analysis. The fluorescent labelled DNA was visualized (200x) 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 (Comet Imager V.2.0.0). Duplicate slides were prepared for each treatment and were independently coded and scored without knowledge of the code. The parameters for the DNA damage analysis include: TL, in μm, TM, OTM and % TDNA.

6.12. Sperm count and sperm head morphology

After the animal sacrifice, epididymis was removed and placed in a petri-plate containing 2 ml of HBSS medium at room temperature. The epididymis was cut into small portions to allow the sperms to swim out. The solution containing the sperms was centrifuged at 1000 rpm for 3 min. After centrifugation, 1 ml of supernatant was taken and used for sperm counting and sperm head morphology. The epididymal sperm count was determined by hemocytometer. The sperm count was expressed as number of sperms per ml of solution containing sperms. For sperm head morphology, 0.5ml of above solution containing the sperms and 0.5ml of 2% eosin solution were mixed and kept for 1 h to stain the sperm. Smear was prepared using 2-3 drops of the above solution, air dried and fixed with absolute methanol for 3 min. Two hundred sperms per animal were examined to determine the morphological abnormalities under oil immersion [45,46]. Sperm head morphology was categorised as normal, quasinormal and grossly abnormal as described by Burruel et al. [47]. Sperms missing the rostral part of the acrosome and/or the posterolateral region of the acrosome were classified as quasinormal sperms and those with collapsed, triangular and amorphous heads with highly deformed acrosomal caps and nuclei were classified as grossly abnormal sperms. Data was shown in terms of % of abnormal sperms.

6.13. Statistical analysis

Results were shown as mean ± standard error of mean (SEM) 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.

7. Results

7.1. Body weight and organ weight

DOX, CIS and MTX treatment induced significant decrease in the body weight as compared to the respective control groups at all the tested doses. A significant difference was also observed in the final weight of heart and testes at all the tested doses of DOX and that in the final weight of epididymis was observed in the groups receiving 2.5 and 5 mg/kg of DOX as compared to the control group. Final weight of kidney showed a significant decrease at all the three doses of CIS, that of testes at 1 and 2 mg/kg of CIS and that of epididymis at 2 mg/kg of CIS as compared to the control group. There was also a significant decrease in the final weight of liver at all the three doses of MTX, that of testes at 10 and 20 mg/kg of MTX and that of epididymis at 20 mg/kg of MTX as compared to the control group (Fig. 4).

Fig. 4. Effect of DOX on (A) body, (B) heart, (C) testes and (D) epididymis wt., that of CIS on (E) body, (F) kidney, (G) testes and (H) epididymis wt. and that of MTX on (I) body, (J) liver, (K) testes and (L) epididymis wt. All the values were expressed as mean ± SEM, (n=5), ***P < 0.001, **P < 0.01 and *P < 0.05 vs. control.

7.2. MDA level

DOX treatment at the dose of 2.5 and 5 mg/kg led to a significant increase in the MDA level in testes as compared to the control group. No significant difference in the MDA level in heart was observed in any of the groups receiving DOX as compared to the control group. A significant increase in the MDA level in testes was found at all the doses of CIS and that in kidney at 1 and 2 mg/kg of CIS as compared to the control group. Further, MTX showed a significant increase in the MDA level at all the three doses in testes and at 10 and 20 mg/kg in liver as compared to the control group (Fig. 5).

Fig. 5. Effect on MDA level produced by (A) DOX on (a) testes and (b) heart; (B) CIS on (a) testes and (b) kidney and (C) MTX on (a) testes and (b) liver. All the values were expressed as mean ± SEM, (n=5), ***P < 0.001 and *P < 0.05 vs. control.

7.3. GSH level

DOX resulted in a significant decrease in the GSH level as compared to control group in the group receiving 5 mg/kg of DOX, while no significant difference in the GSH level in heart was observed in any of the groups receiving DOX as compared to the control group. A significant reduction in GSH level was observed in testes at all the three doses of CIS and that in kidney at 1 and 2 mg/kg of CIS as compared to the control group. MTX resulted in to a significant decrease in GSH level in testes only at the highest dose, while that in liver at all the three doses as compared to the control group (Fig. 6).

Fig. 6. Effect on GSH level produced by (A) DOX on (a) testes and (b) heart; (B) CIS on (a) testes and (b) kidney and (C) MTX on (a) testes

and (b) liver. All the values are expressed as mean ± SEM, (n=5), ***P < 0.001, **P < 0.01 and *P < 0.05 vs. control.

7.4. Histology

Morphological alterations such as disorganization of the cellular structure and vacuolization were induced by DOX, CIS and MTX in heart, kidney and liver respectively. Moreover, all the three agents depicted damage in the seminiferous tubules of the testes in rats (Fig. 7). The quantitative assessment of the seminiferous tubules was done based on the type of cells damaged and Johnsen score was allotted from 1 to 10 accordingly. A significant damage in the seminiferous tubule was observed in the groups receiving 2.5 and 5 mg/kg of DOX, 1 and 2 mg/kg of CIS and 20 mg/kg of MTX as compared to the control group (Fig. 8).

Fig. 7. Representative photomicrographs of rat tissue sections stained with haematoxylin and eosin (H&E). (A) control heart

(B) DOX treated (5 mg/kg/wk for 5 wks) heart; (C) control kidney (D) CIS treated (2 mg/kg/wk for 5 wks) kidney; (E) control liver (F) MTX treated (20 mg/kg/wk for 5 wks) liver; (G) control testis (H) DOX treated (5 mg/kg/wk for 5 wks) testes.

Fig. 8. Testicular damage induced by (A) DOX, (B) CIS and (C) MTX depicted by Johnsen scoring. All the values are expressed as mean ± SEM, (n=5), ***P < 0.001, **P < 0.01and *P < 0.05 vs. control.

7.5. DNA damage in sperm

DOX, CIS and MTX treatment led to damage in sperm DNA as observed from a significant increase in different comet parameters such as TL, TM, OTM and % TDNA. With DOX treatment, a significant increase was found in (i) TL in the groups receiving 1.25, 2.5 and 5 mg/kg of DOX, (ii) TM and (iii) OTM in the groups receiving 5 mg/kg of DOX and (iv) % DNA in comet tail in the groups receiving 2.5 and 5 mg/kg of DOX as compared to control group (Fig 10A). CIS treatment also resulted into a significant increase in all the comet parameters at all three dose levels as compared to control group (Fig 10B). Further, MTX treatment showed a significant increase in (i) TL and (ii) TM in the groups receiving 10 and 20 mg/ kg of MTX and in (iii) OTM and (iv) % TDNA in the groups receiving 5, 10 and 20 mg/ kg of MTX as compared to the control group (Fig.10C).

Fig. 9. Photomicrographs showing the DNA migration pattern in rat sperm nuclei after 5 weeks of DOX treatment. The symbols "−" and "+" represent cathode and anode respectively during electrophoresis of negatively charged DNA. Magnification: 200x. Dye: SYBR Green. Cell nuclei from (A) Control group, (B) sperm nuclei from DOX 5 mg/kg/wk treated group.

Fig. 10. DNA damage induced by (A) DOX, (B) CIS and (C) MTX treatment in rat sperm shown by comet assay. All the values were expressed as mean ± SEM, (n=5), ***P < 0.001 and *P < 0.05 vs. control.

7.6. DNA damage in testes

In testes, DNA damage became evident from a significant increase in comet parameters like TL and %TDNA in the groups receiving 1.25, 2.5 and 5 mg/kg of DOX as compared to the control group (Fig. 11A). CIS treatment led to an increase in TL at all the three dose levels and that in TM and OTM in the group receiving 2 mg/kg of CIS as compared to the control group (Fig. 11B). There was a significant increase in OTM and %TDNA at all the three dose levels of MTX and that in TL and TM at 10 and 20 mg/kg of MTX as compared to the control group (Fig. 11C).

Fig. 11. DNA damage induced by (A) DOX, (B) CIS and (C) MTX treatment in rat testis shown by comet assay. All the values were expressed as mean ± SEM, (n=5), ***P < 0.001, **P < 0.01 and *P < 0.05 vs. control.

7.7. DNA damage in somatic cells

In heart, DNA damage was observed from a significant increase in TL in the group receiving 5 mg/kg of DOX and % TDNA in the groups receiving 2.5 and 5 mg/kg of DOX as compared to the control group (Fig. 12A). CIS treatment resulted in to a highly significant increase in all the comet parameters in kidney at all the dose levels as compared to the control group (Fig. 12B). MTX treatment led to a significant increase in TL, OTM and % TDNA at all the dose levels as compared to the control group (Fig. 12C).

Fig. 12. DNA damage induced by (A) DOX, (B) CIS and (C) MTX treatment in rat heart, kidney and liver respectively shown by comet assay. All the values were expressed as mean ± SEM, (n=5), ***P < 0.001, **P < 0.01 and *P < 0.05 vs. control.

7.8. Apoptosis

Apoptotic effect of DOX, CIS and MTX was reflected from halo assay (Fig. 14). Mild, moderate and extensive damage was observed in testes with DOX, CIS and MTX treatment. Treatment with DOX, CIS and MTX also led to cytotoxicity in heart, kidney and liver respectively.

Fig. 13. Representative photomicrographs of the halo assay in testis after 5 weeks of DOX treatment. Magnification: 200x. Stain: EtBr; (A) Control, (B) DOX treatment (5 mg/kg/wk) for 5 wks.

Fig. 14. Apoptotic effect of (A) DOX on (a) testis and (b) heart; (B) CIS on (a) testis and (b) kidney and (C) MTX on (a) testis and (b) liver shown by halo assay. All the values are expressed as mean ± SEM, (n=5), ***P < 0.001, **P < 0.01 and *P < 0.05 vs. control

7.9. Sperm count and sperm head morphology

A significant decline was observed in the sperm count in all the DOX, CIS and MTX treated groups as compared to the control group (Fig. 15). Furthermore, the frequency of abnormalities in the sperm head was enhanced after DOX, CIS and MTX treatment. % abnormality in sperm head increased significantly in groups receiving 2.5 and 5 mg/kg of DOX, 1 and 2 mg/kg of CIS and 5, 10 and 20 mg/kg of MTX as compared to the control group (Fig. 17).

Fig. 15. Effect of (A) DOX, (B) CIS and (C) MTX on the sperm count after 5 weeks of treatment. All the values were expressed as mean ± SEM, (n = 5), ***P < 0.001 vs. control.

Fig. 16. Photomicrographs showing sperm head morphology as normal, quasinormal and grossly abnormal. A represents normal, B-F represent quasinormal and G-L represent grossly abnormal sperms.

Fig. 17. Effect of (A) DOX, (B) CIS and (C) MTX on sperm head abnormalities expressed as % abnormality in sperm head after 5 weeks of treatment. All the values were expressed as mean ± SEM, (n = 5), ***P < 0.001 and **P < 0.01 and *P < 0.05 vs. control.

7.10. Correlation between sperm head morphology and sperm comet assay

A strong positive correlation was observed between sperm head morphology and sperm comet assay using all the three chemicals, i.e, DOX, CIS and MTX. Strong correlations were observed between % abnormalities in sperm head and TL (R2=0.869), TM (R2=0.999), OTM (R2=0.990) and % TDNA (R2=0.908) with DOX treatment (Fig. 18). Similar kind of correlations were observed between % abnormalities in sperm head and TL (R2=0.997), TM (R2=0.983), OTM (R2=0.997) and % TDNA (R2=0.995) with CIS treatment (Fig. 19). MTX treatment also showed positive correlations between % abnormalities in sperm head and TL (R2=0.837), TM (R2=0.868), OTM (R2=0.850) and % TDNA (R2=0.696) (Fig.20).

Fig. 18. Linear regression analysis showing the correlation between sperm head morphology and sperm comet assay in rats treated with DOX (weekly once for 5 weeks).

Fig. 19. Linear regression analysis showing the correlation between sperm head morphology and sperm comet assay in rats treated with CIS (weekly once for 5 weeks).

Fig. 20. Linear regression analysis showing the correlation between sperm head morphology and sperm comet assay in rats treated with MTX (weekly once for 5 weeks).

8. Discussion

DOX, CIS and MTX are widely used chemotherapeutic agents against a variety of neoplasms. But their long-term clinical use is limited on account of their severe somatic and germ cell toxicities. DOX has been found to induce deterioration of sperm motion and sperm content resulting into adverse effect on male fertility [34]. Impaired spermatogenesis by acute exposure of rats to DOX has been evaluated using flow-cytometry [48]. It has been reported that chronic administration of DOX leads to multifocal degeneration of cardiomyocytes [49]. CIS has been reported to impair sperm characteristics [50]. MTX has been known to cause a reduction in sperm count and induce abnormality in sperm head. In the present investigation, chronic administration of DOX, CIS and MTX resulted into a significant reduction in the body weight as well as in the weight of testes, epididymis and their respective target organs. Reduction in the weight of heart, kidney and liver by DOX, CIS and MTX respectively can be attributed to degeneration as well as vacuolization of the cells as evident from the histological evaluation. A significant decrease in testes weight by all the three agents can be attributed to severe reduction in the spermatogenic as well as leydig cells. It has been reported that oxidative stress is mainly responsible for the target organ as well as the testicular toxicity produced by these drugs [6,13,33,50-52]. Reactive oxygen species have been known to cause an increase in the concentration of lipid-peroxides and loss of membrane polyunsaturated fatty acids in spermatozoa [53].

In the present study, a significant increase in the MDA level in testes was observed in the groups receiving 2.5 mg/kg and 5 mg/kg of DOX as compared to the control group, whereas no significant difference was found in the MDA level in heart in any of the groups receiving DOX as compared to the control group. A significant increase in the MDA level in testes was found at all the doses of CIS and that in kidney at 1 and 2 mg/kg of CIS as compared to the control group. Further, MTX showed a significant increase in the MDA level at all the three doses in testes and at 10 and 20 mg/kg in liver as compared to the control group. Moreover, DOX resulted in a significant decrease in the GSH level in testes as compared to the control group in the group receiving 5 mg/kg of DOX, while no significant difference in the GSH level in heart was observed in any of the groups receiving DOX as compared to the control group. CIS and MTX resulted in to a reduction in GSH level in kidney and liver respectively as well as in testes. Higher oxidative stress produced in the testes as compared to that in the heart can be because of more lipid content in testis as compared to that in heart. Due to higher amount of polyunsaturated fatty acids present in the cellular structure of testis, it is more prone to lipid peroxidation as compared to heart. DOX, CIS and MTX have been reported to induce apoptosis in somatic as well as in germ cells [2,4,5,9,54,55]. Apoptotic effects of DOX, CIS and MTX were made evident in our study by halo assay, which substantiates the cytotoxic effect of these agents on the somatic as well as on the germ cells, which can be attributed to the inhibition of DNA synthesis due to interaction with the genetic material. Their somatic and germ cell toxicity were clearly evident from the histological evaluation of heart, kidney, liver and testis. Cellular disorganization and vacuolization were observed in the heart, kidney and liver sections. Similar cellular disorganization in heart, kidney and liver has been reported due to DOX, CIS and MTX administration respectively in rodents [3,56,57]. In testis section, increased disorganization, vacuolization, decreased spermatogonial, spermatocytes and spermatid counts were observed. Some of the seminiferous tubules were found to be completely deprieved of the cellular structure within, which indicated severe germ cell toxicity induced by these drugs. It has been reported that DOX, CIS and MTX induce genetic damage in both somatic and germ cells [9,12,58-61]. Comparable genotoxic effects of these chemicals were vividly expressed by an increase in the comet parameters like TL, TM, OTM and % TDNA in heart, kidney, liver, testes and sperm. Genotoxic effect of DOX was found to be more in germ cells as compared to heart. This can be ascribed to higher rate of cell differentiation in testes as compared to heart, as spermatogenesis is a highly organised, cyclical process by which sperms are produced from the progenitor spermatogonia. Testicular toxicity was further made evident by a reduction in the sperm count and induction of sperm head abnormality. This can be endorsed to the damage in spermatogonia and spermatocytes and hence impaired spermatogenesis leading to a reduction in sperm count and an increase in sperm head abnormality.

From the present study, a conclusion can be drawn that DOX treatment results into germ cell toxicity more that the somatic cell toxicity, which was made evident from oxidative stress and DNA damage parameters. This can be attributed to a higher rate of cell division as well as more lipid content in testes as compared to heart. Both somatic and germ cells are affected at the same dose and duration by CIS and MTX. Somatic and germ cell toxicity became apparent from the histological observation of the heart, kidney, liver and testes sections. Germ cell toxicity was further appraised using sperm count, sperm head morphology and sperm comet assay. As cardiovascular, renal and hepatic systems as well as reproductive system are key organ systems of the body, their impairment due to treatment with these drugs can deteriorate the quality of patient's life. Moreover, as testicular toxicity by DOX has been reported to be more than cardiotoxicity, it should also be paid attention to and should be looked upon as a serious problem hindering DOX application as an unbeaten chemotherapeutic agent.

Moreover, from the present study, a conclusion can be drawn that there exists a strong positive correlation between sperm head morphological evaluation and sperm comet assay, which was validated using three potent germ cell toxicants (DOX, CIS and MTX). However, it is possible that many agents not depicting sperm head abnormality may induce damage in the genetic material. Sperm head morphology gives an approximate estimation of the functional competence of spermatozoa, but does not always reveal the quality of sperm DNA. However, sperm comet assay detects damage in the DNA integrity of the sperm and hence can add further information on the quality of sperm. Sperm head abnormality evaluation alone may fail to identify certain germ cell toxicants and hence sperm comet assay should also be integrated with it for hazard identification and risk assessment of germ cell toxicants, which in turn will play a major role in reducing the potential risk and improving the quality of life of not only the present generation but also the future generation. Moreover, both these parameters will be useful in the prediction of fertilization failure and will assist the physicians to opt for appropriate therapeutic procedures. Further studies are required to validate, standardize and harmonize these techniques with different types of chemicals and different study protocols to establish these assays as accepted diagnostic tools for the detection of germ cell toxicants.

9. Future work to be done

To investigate the possible protective effects of hesperetin against DOX induced cytotoxicity and genotoxicity in heart and testes using rats.

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