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Abstract

Methotrexate (MTX) is an essential chemotherapeutic medication that had diverse side effects on different organs. This study was performed to investigate the probable protective effect of Omega-3 fatty acids and β-carotene against MTX induced testicular toxicity in adult male rats. This experiment was done in ten weeks, five groups (10 animals for each) of adult male rats were used, control group (I) without treatment, MTX group (II) and the protected groups (III, IV and V) were injected with a single dose of MTX 20 mg/kg I.P in the start of the tenth week. Protected groups regime was as follow: - Omega-3 protected group (III) which received Omega-3 fatty acids at a dose 500 mg/kg orally for ten weeks. β-carotene protected group (IV) which received β-carotene at a dose 10 mg/kg orally for ten weeks. Combination of omega-3 fatty acids & β-carotene protected group (V) which received both omega-3fatty acids (of 500 mg/kg orally) and β-carotene (10 mg/kg orally) daily at the same time for ten weeks.

Blood samples were gathered for estimation of testosterone level. Testicular specimens were subjected to morphometric, histological and immunohistochemical examination. MTX-treated group (II) displayed marked reduction in number populace of spermatocytes, atrophy and germ cell degeneration. Appearance of multinucleated giant cells and significant increase in apoptotic cells was observed. MTX -induced histopathological changes were ameliorated in the protected groups with Omega-3 fatty acids and β-carotene. Our results indicate that Omega-3fatty acids and β-carotene may be beneficial in reducing the side effects associated with MTX administration.

Key words

methotrexate, Omega-3fatty acids, β-carotene, apoptosis, testis

Introduction

Numerous investigators were documented the generous toxicity of chemotherapeutic drugs on different organs of the body. MTX, is a folic acid antagonist, is utilized as cytotoxic agent as a part of the treatment of cancer and leukemia and different malignancies and in addition in the inflammatory diseases, for example, psoriasis and rheumatoid arthritis in low doses [1-3]. There was a plenty of proof that MTX instigates histopathological changes in the proliferative and germinative cells, such as testes which may lead to infertility [4]. Many studies have demonstrated that MTX toxicity was because of the generation of reactive oxygen species (ROS) [5,6]. Many antioxidants have been accounted for to improve the MTX-induced testicular histopathological damages. Omega-3 fatty acids were proven to have valuable effect against cardiovascular illnesses, lipid perioxidation, protein cross linking, DNA mutation and homeostasis were all due to its antioxidant activity [7]. β-carotene displays the antioxidant effect by suppressing singlet oxygen, scavenging peroxide radicals, therefore reacting with peroxy radicals, thus stabilizing membrane lipids from free radical attack [8,9]. This study was performed at the histopathological and Immunohistochemical level to investigate the protective effects of Omega-3 fatty acids and β-carotene against the MTX- related testicular alterations in male rats.

Materials and methods

Animals

Experiments were achieved on fifty adults (age 6-7 months) healthy Sprague-Dawley male albino rats of body weight 180 to 200g. which were obtained from Animal House of Zoology Department, Faculty of Sciences, Suez Canal University. They were housed under standard strict hygienic conditions; fed standard laboratory feed and had free access to water. Rats were maintained in a room temperature of 20-25^◦C and exposed to12 hour light/dark cycle. Before the beginning of the experiment all animals were acclimatized for 1 week. Experimental protocol was approved in accordance with the Guidelines of Animal ethics council of the Faculty of Veterinary Medicine, Suez Canal University (Approval no.2016089).

Drugs

MTX was purchased from Ebewe Pharma Company (Egypt) and was dissolved in 0.9% saline. Omega-3 polyunsaturated fatty acids were purchased from Sedico Pharma Company (Egypt) and were obtained as gelatinous capsules. β- Carotene was purchased from Mepaco Pharma Company (Egypt) as gelatinous capsules. Both drugs were administrated by gavage.

Experimental protocol

Animals were divided equally into five groups (n=10 for each group) as follows: Control group (I) not subjected to any treatment, MTX – treated Group (П) and protective groups (III, IV and V), Rats were injected with a single dose of MTX (20 mg/kg i.p), at the start of the last tenth week of the experiment, according to the previous studies [6,10]. Omega -3fatty acids protective group (III), Rats were first given Omega-3fatty acids (orally via stomach gavage) at a dose 500 mg/kg, for ten weeks [11]. β-carotene protective group (IV), Rats were first given β- carotene orally via stomach gavage at a dose of 10 mg/kg daily for ten weeks. Combination of Omega-3 fatty acids & β-carotene group (V), at the same period, Rats were given both omega-3 (500 mg/kg orally) and β-carotene (10 mg/kg orally) daily for ten weeks.

Biochemical analysis

Estimation of serum testosterone was done at the end of tenth week of the experiment, blood samples were obtained from tail vein; each sample was collected in plain centrifuge tube and put on inclined position for 20 minute at room temperature. They were centrifuged at 3000 rpm for 10 minutes, then the clear serum of each sample was separated carefully, collected, and stored in Eppendorf tubes at 20^oC until estimation of serum testosterone. Serum testosterone concentration was determined by using enzyme-linked immunoassay (ELISA) [12].

Tissue preparation

At the end of the tenth week of the experiment, rats were anesthetized with ether and killed by cervical dislocation. Testes were isolated and fixed in 10% neutral buffered formalin for histopathological examination and immunohistochemistry. Testicular sections were washed several times in dehydrated ascending series of ethyl alcohol, cleared in xylol and then embedded in three changes of pure paraffin wax. For histopathological examination, testicular sections were subjected to routine histological technique and stained with Harri's Hematoxylin and Eosin (H&E) according to carson (1997)[13].

Immunohistochemistry

For caspase-3 immunostaining, testicular paraffin embedded sections were deparaffinized, microwaved and incubated in 3% H₂O₂, then washed in PBS. Sections were incubated in normal goat serum at 37°C for blocking of non-specific antibody binding. They were subjected to biotinylated primary antibodies (Rabbit anti-caspase-3, diluted in 1:1000, Abcam, Ltd., USA). Then, they were incubated with biotinylated secondary antibody peroxidase-conjugated goat anti-rabbit IgG (1:1000). Streptavidin-biotin complex technique (ABC kit, Vector laboratories, USA) was used. Immunostaining reaction was labeled with diaminobenzidine (DAB) as a chromogen and counterstained with Mayer's Hematoxylin. Images were inspected with light microscope and captured using a digital camera (Olympus DB25- Japan). Assessment of immunostaining expression of caspase-3 in the testicular sections was performed using image J program.

Morphometric studies

Diameters of seminiferous tubules and distances of interstitial spaces were measured using Image-J program. Five slides stained with Hematoxylin and eosin (H&E) per animal were evaluated. The diameter of 25 seminiferous tubules was calculated in 5 fields (5 seminiferous tubules per field). In a similar manner the interstitial space measured between two consecutive seminiferous tubules with Mag. X 100.

Statistical analysis

One way analysis of variance (ANOVA) test was applied for the statistical analysis for the present data of all experimental groups. The result was considered to be significant when p<0.05 and highly significant when p<0.01.

Results

Evaluation of serum level of testosterone

Testosterone level was significantly reduced in treated group with MTX-treated group (II) (p<0.05), however, testosterone level was increasing significantly in omega 3 fatty acids protected group(III) rather than β carotene protected group (IV) (Figure 1).

Figure 1. Level of testosterone in control and experimental groups (mean ± SEM)

Morphometric analysis

The diameter of seminiferous tubules was recorded to be significantly decreased (p<0.05) in with MTX treated group as compared to control and protective groups (III, IV and V) (Figure 2). Significant differences (p<0.05) were also observed increasing Interstitial spaces were measured between control and MTX treated group and other groups. Correlation was significant between different experimental groups and MTX group in changes in diameter of seminiferous tubules, interstitial space and level of testosterone (Table 1 and Figure 3-5).

Figure 2. Diameter of seminiferous tubules and interstitial spaces in both control and experimental groups

Figure 3. Shows a straight line representing the correlation between different groups changes in an interstitial space and diameter of seminiferous tubules

Figure 4. Shows a straight line representing the correlation between different groups changes in level of testosterone and diameter of seminiferous tubules

Figure 5. Shows a straight line representing the correlation between different groups changes in level of testosterone

Correlations
	Diameter of                        seminiferous tubules(mm)	Interstitial spaces(mm)	Level of Testosterone(ng/ml)
Pearson Correlation	0.612	-     0.578**	0.784**
Sig. (2-tailed)	1	0.0001	0.0001
N	64	64	40

Table 1. Correlation between different experimental groups and MTX group in changes in diameter of seminiferous tubules, interstitial space and level of testosterone. ** Correlation is significant at the 0.01 level (2-tailed). reveres relationship in correlation test

Histological results

Control group (I): Examination of H&E stained sections of control group revealed normal histological structure; seminiferous tubules were lined by sertoli cells and spermatogenic cell stages (Figure 6A1). Clusters of Leydig cells and blood capillaries were randomly scattered in the interstitial spaces (Figure 6A1 and 6A2).

MTX – treated group (II): significant decrease in the diameters of seminiferous tubules. Marked depletion in germ cell population, disorganization of germinal epithelium and degeneration of spermatogenic stages in the majority of the seminiferous tubules were demonstrated (Figure 6B1). Germ cell stages were devoid of secondary spermatocytes and spermatids (Figure 6B2). Apparent, multivesicular giant cells of different sizes were randomly distributed throughout the germinal epithelium which was associated with distinct large vacuoles (Figure 6B1 and  6B2). Seminiferous tubules showed detached basement membranes and peritubular tissue. Noticeable increase in the distances of interstitial spaces was observed which was occupied with congested blood vessels, hemorrhage, and exudates (Figure 6B1and  6B2).

Figure 6. Photomicrographs of the testis of different experimental groups, stained with H&E. (A1&A2) Control group (I) showing normal histological structure of seminiferous tubules showing spermatogenic cell stages (*) and Leydig cells (arrow). (B1&B2) MTX-treated group (II) showing disorganization, depletion of spermatogenic cell stages (*) and appearance of multinucleated giant cells (thick arrow). noticeable eosinophilic exudates in interstitial spaces. (C1&C2) Omega-3 fatty acid protected group (III) showing reorganization of germinal cell stages (*) with elongated spermatids (arrow). (D1&D2) β-carotene protected group (IV) showing nearly normal structure (*) with elongated spermatids (arrow). (E1&E2) both Omega- 3 fatty acids and β- carotene group (V) showing germinal cell stages (*) with reduction of elongated spermatids. Fig. A1, B1, C1, D1 and E1 scale bar 100 µm and Fig. A2, B2, C2, D2 and E2 scale bar 20 µm.

Omega -3 fatty acids protected group (III)

Prominent dramatic organization of spermatogenic stages was evident; spermatogonia, primary & secondary spermatocytes, elongated spermatids and spermatozoa were observed. There were no signs of degeneration of germ cell layer (Figure 6C1 and 6C2). Sperm bundles were occupying luminae of seminiferous tubules. Germinal epithelium was devoid of multinucleated giant cells (Figure 6C1 and 6C2).

 β ­-carotene protected group (IV)

Sections pretreated with β- carotene exhibited nearly normal structural architecture of the testis; all the organized spermatogenic stages were observed with apparent elongated spermatids (Figure 6D2). Germinal epithelium was devoid of multinucleated giant cells (Figure 6 D1 and  6D2). The luminae of seminiferous tubules were occupied with sperm bundles. Interstitial space showed congestion of blood vessels and exudates similar to group II (methotrexate- treated group) (Figure 6 D1and 6D2).

Combination of omega- 3 fatty acids and β- carotene group (V)

Testicular sections pretreated with both omega-3fatty acids and β-carotene showed systematized spermatogenic stages except that elongated spermatids were reduced (Figure 6 E1and 6E2). No significant differences between the protected group with both Omega- 3fatty acids and β-carotene (group V) and the protected group with either omega- 3 fatty acids (group III) or β-carotene (group IV) alone except that the reduction of elongated spermatids in group V.

Immunohistochemical result of Caspase-3 apoptotic factor

 In control group (I), immunostaining reaction of spermatogenic cells and Leydig cells was negative (Figure 7a). Strong staining reaction was apparent in methotrexate-treated group (II); it was more prominent in the spermatogenic cells, sertoli cells, multinucleated giant cells and Leydig cells (Figure 7b). Faint Immunostaining reaction of spermatogenic cells and moderate reaction of Leydig cells were observed in pretreated groups with Omega-3 fatty acids group III (Figure 7c), β-carotene group IV (Figure 7d), and both Omega-3 fatty acids & β-carotene group V (Figure 7e).

Figure 7. Photomicrographs of the testis of different experimental groups incubated with caspase-3 monoclonal antibody. (a) Control group (I) showing negative immunostaining affinity of spermatogenic stages (*) and Leydig cells (arrow). (b)  MTX-treated group (II) showing strong immunostaining of spermatogenic cells (*), multinucleated giant cells (thick arrow) and Leydig cells (arrow) different experimental groups.  (c) Omega-3 fatty acid protected group (III), (d) β-carotene protected group (IV) and (e) both Omega- 3 fatty acids and β- carotene group (V) showing faint Immunostaining reaction in the spermatogenic cells (*) Leydig cells were less intense in the(d) β-carotene protected group (IV). Scale bar 20 µm.

In studying the number of apoptotic germ cells immunostained with caspase 3, there was a significant difference between the groups as demonstrated by the ANOVA P value <0.05. There was a significant difference between control group (I) and MTX group (II) and a significant difference between MTX group (II) and rest of groups. There was no significant difference between group III, group (IV) and group (V) P value > 0.05 (Figure 8).

Figure 8. * Significantly different from control group (I) P< 0.05.

Discussion

MTX is a folic acid antagonist agent; it belongs to the antimetabolites group of cytotoxic and immunosuppressant drugs [14,15]. Many reports have been published on testicular damage caused by chemotherapeutic drugs [3,16]. They could lead to Permanent azoospermia and infertility [17].

This study demonstrated that testosterone level decreased in MTX-treated group (II) in comparison to control group (I) and protected groups with Omega-3fatty acids group (III), β-carotene group (IV) and a combination of them group (V). Some authors revealed the capacity of β- carotene to restore the testosterone level after the apparent decrease induced by titanium dioxide nanoparticles [18]. Administration of Omega-3 fatty acids for the reestablishing of serum testosterone level reduction induced by MTX was documented [19].     

In the present study, diameter of seminiferous tubules was diminished in MTX treated group compared to control and protective groups. This could be recognized with the medication effect of MTX [20,21]. Furthermore, MTX toxicity could influence cell parts, seminiferous tubules diameter and interstitial space throughout spermatogenesis [22].

 Our results showed that MTX administration causes testicular damages which demonstrated by disorganization of germinal epithelium, depletion in germ cell stages, and decrease in sperm population and also increase in apoptotic degenerated cells. This was in concurrence with [3,23] who stated that in MTX treated group, there were widespread loss of spermatozoa, disorganization of spermatogenic stages and extensive apoptotic germinal cells. MTX induced histopathological changes could be clarified by [24] who expressed that MTX and its dynamic metabolite block tetrahydrofolate synthesis through binding to the folic acid site on the catalyst dihydrofolate reductase. This activity results in the decrease of nucleotide precursors, and the suppression of DNA, RNA, protein synthesis and cellular proliferation.               

Others researchers were attributed to Sertoli cell damage induced by MTX since the nutritional and structural support of germ cells is supported by sertoli cells [25]. Some authors proposed that sloughing of germinal epithelium may be brought about by downregulation of expression of cell adhesion (e.g. cadherin) in Sertoli cells [26]. It might be because of the diverse effect of chemicals on the cytoskeleton of Sertoli cells [27].

 Moreover, MTX-treated group (II) displayed multinucleated giant cells in the germinal epithelium that had a strong immunostaining affinity to caspase-3. It is conceivable that multinucleated giant cells are a cluster of degenerated germ cells [21,28].

Besides, Oxidative stress could explain histopathogenesis of MTX-induced testicular changes and marked immunostaining intensity of apoptotic cells [29]. Oxidative stress causes diminishing in mitochondria capacities, cytochrome c release, and consequently caspase activation [30]. MTX stimulates oxidative stress by lessening the activities and the effectiveness of the antioxidant enzyme protection system [31] which reduced testicular functions and disruption of sperm development [30,32]. In the protected groups with omega 3 fatty acids, β- carotene and combination of them as an antioxidant, a significant improvement in the histopathological features of the testis was observed. Remarkable testicular histopathological damages were ameliorated that manifested through rearrangement of germinal epithelium and an increase of sperm populace. Germinal epithelium was devoid of multinucleated giant cells and degenerated cells. In addition to, noticeable reduction in the immunostaining intensity (caspase-3) of germinal epithelial cells was evident. This was in agreement with [30] and [23] who reported that β-carotene decreased testicular injury and germ cell apoptosis in the testis after testicular ischemia/reperfusion damage. The protective activity of Omega-3 fatty acids against MTX- induced testicular damage could be attributed to its antioxidant properties

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b-carotene has been accounted for attenuate tissue oxidant catalysts in various animal models [23,33-35]. b-carotene is a precursor of vitamin A which is act as potent antioxidant agent that block the free radicle formation or scavenger them [21]. The current study confirmed that pretreatment with β-carotene clearly diminishes the degenerative apoptotic alterations in germinal epithelium. [36] recommended that protection of β-carotene could owe to its antiapoptotic activity. [32] reported that nuclear structural changes as fragmentation and denaturation of DNA, and resulting apoptosis were brought on by oxidative stress. [37] revealed that β-carotene diminishes restraint of purine and pyrimidine synthesis of DNA. The protective effect of vitamin A against MTX on the small intestine might be clarified by stimulus of DNA and RNA biosynthesis [31]. In conclusion, we have presented noteworthy evidence that oral supplementation with Omega3fatty acids, β-carotene or combination of them as antioxidants could reestablish testicular histopathological alterations associated with administration of Methotrexate as chemotherapy.

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