The Expected Beneficial Effects of Watermelon Juice against Damage Induced by Gamma Radiation in the Testis of Rats

Radiation therapy (RT) is centered on delivering the best potential absorbed dose to the clinical targeted malignant growth cells while not exceptional the tolerance of close healthy tissues (Persson et al., 2011; Senel et al., 2018). Though the treatments can be advantageous, patients usually complain of azoospermia or patient becomes infertile (Jiang et al., 2013).
       
The testis is a radio-sensitive organ due to the presence of fast proliferating cells, spermatogonia. The mechanism by which radiation exposure inducing testicular cell damage may be directly by affecting the DNA molecule and cause cell destructions or indirectly by causing decomposition for water molecules and causes release of free radicals, which then lead to oxidative damage for the cell nucleus (Senel et al., 2018). Normally, the testis has defense against oxidative stress like enzymes with antioxidant activities, radical scavengers and low O₂ tension so as to support Leydig cells steroidogenic performance (Osman, 2011). However, exposure to gamma-rays or other endogenous and exogenous factors can perturb these defenses and compromise male fertility by generating free radicals in testes. Therefore, seeking for radio-protectors derived, from familiar foods and plant sources with natural antioxidant contents, is worthy to receive extra attention and special thought (Osman, 2011; Hamza et al., 2018).
       
Increasing the daily intake of vegetables and fruits wealthy in natural antioxidants has been counseled to stay a healthy standing and change the stressed physiological case of the body to unstressed one (Maotoa et al., 2019). Watermelon is incredibly well-liked fruit throughout the globe. It forms the staple diet in village community. Watermelon (Citrullus lanatus) belongs to family Cucurbitacea. It’s a vine-like climber herb; it’s a good supplier of beta carotene, another antioxidant carotenoid (Chaturvedi et al., 2014) and ascorbic acid (Vit. C) (Sevcan et al., 2011; Maotoa et al., 2019). It’s additionally wealthy supply of polyphenols and flavonoids. A cup of watermelon provides almost quarter of the daily needs of ascorbic acid and thru its beta carotene, 11.1% of the daily needs of Vit. A. The reducing function of antioxidant carotenoid (i.e. lycopene) is its ability to assist cells and alternative structures within the body from free radicals (ROS) harm and has been connected in human research about prevention of heart diseases (Georgina et al., 2011). Thus, the aim of this study was to evaluate the antioxidant capacity of watermelon juice and its protective effect against g-radiation induced inflammation and testes tissue damage in rats.

All experiments were carried out during 2018 at the Egyptian Atomic Energy Authority, Food Irradiation Department.
       
Fresh watermelon was purchased from local market (Cairo, Egypt). Chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
 
Preparation of watermelon juice (WJ)
 
Watermelon juice (WJ) was freshly prepared on a daily basis. The watermelon was cleaned with filtered tap water and peeled to obtain the red flesh. The flesh was then processed with a juice maker which automatically separates the pulp and the juice. A 50% concentration was prepared by diluting a pure watermelon juice with filtered tap water in the ratio of 1: 1 (v/v) (Mohammad et al., 2014).
 
Total phenolics: Estimation of total Phenolic Contents (TPC) of juice was carried out using Folin-Ciocalteu method as described by Singleton et al., (1999).
 
Free radical scavenging ability: Free radical scavenging activity of WJ was determined by DPPH method (Muller et al., 2011). 
 
Antioxidant activity: Extracted juice was subjected to antioxidant activity assay based on coupled oxidation of beta-carotene and linoleic acid through spectrophotometer (470 nm) by following the method of Taga et al., (1984).
 
Radiation facility
 
Whole body gamma irradiation of rats at a dose level of (6 Gy) was performed using a Canadian Gamma Cell-40, (137Cs) (Atomic Energy of Canada Ltd, Ottawa, Ontario, Canada), located at the National Center for Radiation Research and Technology (NCRRT), in Nasr City, Cairo, Egypt. The dose rate of the irradiation process was 0.43 Gy/min at the time of the experiment calculated according to the dosimeter Department in the NCRRT.
 
Experimental animals
 
Male albino rats Sprague Dawley (10±2 weeks old; 120±20 g) were purchased from the Egyptian Holding Company for Biological Products and Vaccines (Cairo, Egypt) and used for the different investigations carried out in the present study. Rats were acclimated to controlled laboratory conditions for two weeks at the animal house of the Egyptian Atomic Energy Authority, NCRRT, Nasr City. Rats were maintained on stock rodent diet and tap water that were allowed ad libitum. The rodent control diet is composed of 15% casein, 10% corn oil, 5% cellulose, 4% salt mixture, 1% vitamins mixture and starch 65% (Philip et al., 1993).
 
Experimental design
 
Animals (28 rats) were randomly divided into 4 groups (seven rats in each group) as follows:
 
Group (1): Rats fed on a balanced diet for 6 weeks, served as control group.
 
Group (2): Rats received WJ by oral gavage (100 mg/kg B.Wt./day) (Munglue et al., 2014) for 6 weeks (WJ group).
 
Group (3): Rats were exposed to whole body g-irradiation (6Gy) at the 1^st week of the experimental period (6 weeks) (Irradiated group; IRR).
Group (4): rats were exposed to whole body g-irradiation (6Gy) at the 1^st week of the experimental period and received WJ by oral gavage (100 mg/kg B. Wt./day) (IRR + WJ group).
       
At the end of the experiment, rats were fasted for 24 hours and anaesthetized with diethyl ether. Blood sample were collected through heart puncture and allowed to coagulate and centrifuged for to obtain serum for biochemical analysis.
 
Biochemical analysis
 
Estimation of testosterone hormone was performed according to the method of Wilson and Foster (1992). Follicle stimulating hormone (FSH) and luteinizing hormone (LH) were determined according to Garrett (1989). Detection of serum tumor necrotic factor-alpha (TNF-α) and interleukin-6 (IL-6) was performed by ELISA technique (BioSource International, Camarillo, CA, USA) according to the manufacturer’s instructions.
       
Moreover, the testes tissues were dissected and divided into two parts. One part for histopathological study and the other part was homogenate in saline solution. Testes homogenates were obtained using a tissue homogenizer. The homogenates (1:10 w/v) were prepared using a 100 mM KCl buffer (pH 7.0) containing EDTA 0.3 mM. Allhomo- -genates were centrifuged at 200 × g for 20 minutes at 4^oC and the supernatants were used to estimate the level of thiobarbituric acid reactive substances (TBARS) (Yoshioka et al., 1979), the activity of xanthine oxidase (XO) and xanthine dehydrogenase (XDH) (Kaminski and Jewezska, 1979), glutathione content (GSH) (Gross et al., 1967) and the activity of superoxide dismutase (SOD) (Minami and Yoshikawa, 1979) and catalase (CAT) (Aebi, 1984).
 
Histopathological examination
 
For histopathological study the tissue samples were taken rapidly from each rat and fixed in 10% formalin. All the samples were dehydrated in ascending grades of ethanol, cleared in butanol and embedded in parablast. Sections of 5-6 µm thick sections were obtained and stained with the following stains: 
1- Haematoxylin and Eosin (H&E) staining for general histological studies.
2- Masson’s Trichrome stain for collagen fibres.
 
Testes histopathological studies
 
Histological examination of rat testis in group 1 (control) as shown in Fig 1A showed the seminiferous tubules lined with stratified epithelium composed of two major cells, which are the sertoli cells and spermatogenic cells. Examination in IRR- group (6Gy) showed degeneration of the spermatogenic cells, occlusion of the lumen and hypertrophied seminiferous tubules (Fig 1B). Seminiferous tubules depict normal architecture with adequate cellularity; on the other hand histological examination in group WJ as shown in Fig 1C represented some discontinuous seminiferous epithelium. Finally, histological examination of IRR+WJ-group showed matured spermatozoa in the seminiferous tubules of rats treated. This group has a healthy testicular tissue similar to the control, but with a slight increase in the interstitial tissues. This shows that WJ administered as a treatment option may be medicinally beneficial (Fig 1D).
 

 
Statistical analysis
 
Results were presented as mean±SE (n=7). Experimental data were analyzed using one way analysis of variance (ANOVA). Duncan’s multiple range test was used to determine significant differences between means. The statistical analyses were performed using computer program Statistical Packages for Social Science (SPSS) (SPSS, 1998). Differences between means were considered significant at P<0.05.

The observed values for total phenolic contents (TPC), antioxidant activity and 2,2-diphenyl-1-picrylhydrazyl (DPPH) for watermelon juice were 23.77±1.12 (mg/100 g GAE), 48.83±2.52% and 29.38±1.85%, respectively (Table 1). These results are in agreement with Naz et al., (2013) and Reddy et al., (2010). Shofian et al., (2011) found that the total phenolic contents (29.32 mg/100 g) for fresh watermelon was comparatively higher than muskmelon.
 

       
The results revealed that whole body g-radiation (6Gy) to rats caused significant reduction in the serum levels of testosterone and LH and the activity of testicular XDH, SOD and CAT activities and GSH content with significant elevation in the serum level of FSH, TNF-α and IL-6, testicular TBARS level and the activity of XO compared to control group (Table 2,3,4 and 5). Exposure of rats to whole body g-radiation can cause alterations in DNA-single strand break, cell apoptosis, hypothalamic and pituitary gland dysfunction and induction of lipid peroxidation in testicular tissue which attack the testicular parenchyma causing damage to seminiferous tubules and Leydig cells (Ahmed and Abdel-Mageid, 2011). Inhibin is a peptide hormone produced by testicular tubules and acts by negative feedback mechanism to modulate the secretion of FSH by pituitary gland, so disruption of the inhibin production mechanism by g-irradiation may be one of the causes of FSH elevation (Green and Harris, 1978; Ahmed et al., 2017). Godbout et al., (2005) reported that exposure to g-radiation induced production of ROS that mediate the activation of transcription factor NF-κB. The activation of NF-κB up-regulates the expression of genes of the proinflammatory cytokines [IL-1β, TNF-a and IL-6]. Also, the over production of ROS due to g-irradiation enhances oxidative stress processes, which is associated with significant decrease in the oxidant status and accompanied by depleted antioxidant defensive system. Gamma-irradiation may have caused the conversion of XDH to XO resulting in an increase in XO-specific activity in both time intervals (Pereda et al., 2004). The decrease in SOD activity may result in an increased flux of super oxide in cellular compartments which may be the reason for the increased in testicular TBARS level (Abdel-Magied and Ahmed, 2011).
 

 

 

 

       
Supplementation of g-irradiated rats with WJ (100 mg/kg B. Wt./day/6 weeks) showed significant rise in the level of testosterone and LH and the activity of testicular XDH, SOD and CAT activities and GSH content with a noticeable decrease in the serum level of FSH, TNF-a and IL-6, testicular TBARS level and the activity of XO relative to g-irradiated group (Table 2,3,4 and 5). The possible ameliorative effect of the WJ against g-radiation may be attributed to its ascorbic acid content and its contents of phenols and flavonoids (Erukainure et al., 2010). Ascorbic acid content of watermelon juice may enhance the testosterone synthesis and can acts as vitaminergic transmitter activating release of LH from the anterior pituitary gland (Karanth et al., 2001). Zakaria et al., (2014) indicated that the anti-inflammatory effect of WJ against g-irradiation due to its natural source of lycopene and other antioxidants such as flavonoid and phenolic may help in down regulation of TNF-a level in rats. Ma and Kinneer (2002) reported that phenolic has the ability to inhibit signal-induced TNF transcription, thus controlling cytokine induction through its properties as anti-inflammatory. Also, decrease levels of IL 6 were observed in mice with adipose tissue inflammation when treated with lycopene (Gouranton et al., 2011). The mechanisms by which WJ protect against g-irradiation induced testicular oxidative damage may be as a result of the rich source of vitamin C, thiamine and including riboflavin which contains a high level of polyphenolic compounds present in this fruit (Godspower et al., 2015). WJ is an excellent source of lycopene, having about 40% higher lycopene content than raw tomatoes (Seif, 2014). Studies have attributed the antioxidant properties of water melon juice to its high lycopene content (Zakaria et al. 2014). Oyenihi et al., (2016) reported that the significant increase in the glutathione levels in the liver and brain of watermelon juice treated rats prior to ethanol-administration may be due to the direct ROS-scavenging effect of water melon juice or an increase in GSH synthesis.

The results of this study counsel that watermelon juice treatment boosted antioxidant status and offered some protection against whole body g-radiation evoked oxidative injury in rat’s testis. The protecting effects demonstrated by watermelon juice within the present study are also because of the antioxidant effects of a number of the active constituents or their synergism.