AMELIORATIVE EFFECT OF PUMPKIN SEED OIL AGAINST THE BISPHENOL-A ADVERSE EFFECTS IN MALE MICE . BY 1

The present study was conducted to evaluate the ameliorative role of pumpkin seed oil (PSO) against potential adverse effects of bisphenol-A (BPA) in male mice. BPA was administered to the mice orally at a dose of 50 mg/kg body weight once a day for 28 successive days. While, PSO was administered to the mice orally at 1 mL/kg b.w. either before, with or after treatment of BPA, once a day for 28 successive days. The studied parameters were DNA damage evaluation using comet assay in liver and testes cells and micronucleus test in bone marrow; and histopathological examination of liver and testes tissues. Results revealed that BPA induced DNA damage in tested cells and marked histopathological alterations in liver and testes. In contrast, PSO treatments alleviated DNA damage and improved the histopathological alterations in liver and testes tissues. Furthermore, administration of mice with the PSO before BPA treatment was the best regimen in the alleviation of the adverse effects of BPA, followed by administration of PSO after then with treatment of BPA. It can be concluded that PSO may has a protective role against BPA genotoxicity and histopathological alterations in male mice.


Introduction:
Among the various endocrine disrupting chemicals (EDCs), bisphenol-A (BPA) is the most important man-made compound with annual production exceeding 3.8 million tons (Michalowicz, 2014).It is a synthetic monomer used in production of polycarbonate plastics, epoxy resins, food packaging, dental sealant, water pipes, toys, fire retardant, plastic bottles, paints, pesticides and lacquers for food cans (Burridge, 2003;Fleisch et al., 2010).Biomonitoring studies supported the widespread exposure to BPA (Calafat et al., 2008;Fu and Kawamura, 2010); and the potential human threat from usage of these products awing to the ability of BPA to leach out of plastics into the food and beverages they contain (Berger et al., 2016).BPA causes hepatotoxic, mutagenic, reproductive and carcinogenic effects (Doherty et al., 2010;Erler and Novak, 2010;Meeker et al., 2010;Zeinab et al., 2012).Several studies confirmed the genotoxicity of BPA (Kalb et al., 2015;Xin et al., 2015).In addition, growing evidence indicated that the oxidative stress caused by BPA could be one of the mechanisms leading to this toxicity (Meeker et al., 2010;Tiwari et al., 2012).
Natural antioxidants play a main role to diminish the oxidative stress by scavenging the free radicals and by further mechanisms which can prevent a variety of diseases (Ahmad et al., 2012).The prevention of BPA-induced toxicity has been shown by using of antioxidants such as vitamin A (Aikawa et al., 2004) vitamin C (Korkmaz et al., 2010), pomegranate juice (El Ghazzawy et al., 2011) on experimental animals.
In this study, pumpkin seed oil (PSO) was used as a protective agent.Pumpkin (Cucurbita pepo) is a vegetable cultivated for human consumption and for use as functional food or medicine (Caili et al., 2006).It is rich in various antioxidants and valuable nutritional components such as essential fatty acids, amino acids, phytosterols, β-carotenes and selenium (Procida et al., 2012).Furthermore, it contains phenolic compounds such as tyrosol, vanillic acid, vanillin, ferulic acid, and luteolin (Andjelkovic et al., 2010).It has also been shown to contain high levels of tocopherol, which interpret its antioxidant activity and consequently may be able to reduce lipid peroxidation (Van Hoed et al., 2009).
Therefore, the aim of this study was to evaluate the role of PSO in alleviating the adverse effects of BPA on DNA integrity in both somatic and germ cell and histopathological changes in male mice.

Chemicals:
Bisphenol-A (BPA) (≥ 99%) was purchased from Sigma-Aldrich Company (St. Louis, MO, USA).Pumpkin seed commercial oil was purchased from EL Captin Company (Al Obour City, Cairo, Egypt).All other chemicals were of analytical grade and purchased from standard commercial suppliers.

Dose preparation:
BPA was dissolved in absolute ethyl alcohol (95%) and diluted with corn oil [1:20 alcohol : corn oil (vehicle)] to obtain a final concentration of BPA (50 mg/kg b.w.) and was always freshly prepared before use.

Animals:
Male Swiss albino mice weighing 26±5 g (10-12 week old) were purchased from the Theodor Bilharz Research Institute, Giza, Egypt and housed in polypropylene cages (43cm× 30cm × 15cm, five mice per cage) with stainless steel covers in the Animal House of Environment and Bioagriculture Department, Faculty of Agriculture, Al-Azhar University.Animals were maintained under controlled temperature (23±4 °C), 50-55% relative humidity and a photoperiod of 12 h light : 12 h dark cycle.Food and water were given ad libitum.The animals were acclimatized for 2 weeks prior to the start of the experiments.

Fatty acids analysis:
The methyl esters were prepared using the method prescribed by AOAC (2000).Fatty acid methyl esters (FAMEs) composition analysis was performed in an Thermo scientific TRACE 1310 gas chromatograph attached with ISQ LT single quadrupole mass spectrometer with DB1, 15m; 0.25mm ID (J&W scientific) capillary column.Helium was used as a carrier gas with flow rate at 1.5 mL min-1 and the injector temperature was maintained at 200 o C. Oven temperature was programmed with an initial temperature of 115 o C, hold for 1 min, then increased to 280 o C by 7.5 o C min-1, hold for 3 min.The mass spectrometer detection conditions were as follows: interface temperature, 300 o C; ionization mode, EI+; electron energy, 70 eV; single ion monitoring (SIM) mode; mass range, 40-350 amu.Compounds were identified by use of NIST-library spectra and comparison of their retention times.Quantification of 14 fatty acids was accomplished by the calibration curve of fatty acid standard mixture (Sigma).

Total phenolic content:
Total phenolic content (TPC) was determined using the Folin-Ciocalteau's reagent according to the method reported by Lin and Tang (2007) at 760 nm with a spectrophotometer (UV-1601; Shimadzu, Tokyo, Japan) and the quantification was done on the basis of the standard curve of gallic acid concentration ranging between 10 to 80 mg/mL ( r 2 = 0.99).

Determination of total tocopherol (Vitamin E):
Sample preparation was carried out based on a method described by Gimeno et al., (2000).Determination of vitamin E was carried out using HPLC system (1100 series, Agilent Technologies) consisted of a G1311A model quaternary pump, UV detector, G1329A model automatic injector system and G1316A model thermostated column compartment).Methanol 100% was used as mobile phase at flow rate of 1.8 mL min -1 .20 µL of samples were injected into an ACE5 C18 column (250×4mm, 5μm particle size, Advanced Chromatography Technologies., Aberdeen, Scotland) through an automatic injector system.Peaks were detected at 280 nm and were identified by comparing retention times of peaks for the experimental and standard sample.

Measurement of antioxidant activity:
The ability of PSO at 200 uL to scavenge 2.9 mL of 1, 1 ' -diphenyl 1-2picrylhydrazyl (DPPH) free radical was estimated by the method of Singh et al. (2002).

Evaluation of DNA damage: 1-Comet assay in cells of liver and testes:
The comet assay was performed in liver and testes cells according to Bandyopadhyaya et al. (2008).Briefly, 50 μL of cell suspension was mixed with 100 μL of 1 % low melting point (LMP) agarose and added to fully frosted slides coated with 80 μL of 1 % normal melting point (NMP) agarose.The cells were then incubated in a lysis solution (2.5 mol L -1 NaCl, 100 mmol L -1 EDTA, 10 mmol L -1 Tris-HCL, 1 % Triton X-100, pH 10) at 4 °C for at least 2 h, at which the slides were placed into an alkaline solution (300 mmol L -1 NaOH, 1 mmol L -1 EDTA, pH 13) at 4 °C for 20 min so as to allow DNA unwinding, and electrophoresed at 25 V (300 mA) for 20 min.Finally, the slides were neutralized in a 400 mmol L -1 Tris buffer (pH 7.5) for 15 min and stained with ethidium bromide (5 μg mL -1 ).Images of 50 randomly selected nuclei per experimental group were captured using a fluorescence microscope (Eclipse 800, Nikon, Tokyo, Japan) and analyzed with image analysis software (Comet Assay IV, Perceptive Instruments, Suffolk, UK).Scored parameters included tail length, DNA percentage in tail and Olive tail moment (OTM).Tail length is the maximum distance that the damaged DNA migrates from the centre of the cell nucleus.DNA Percentage in tail is the DNA content that migrates from the nucleus into the comet tail.OTM is the product of the tail length and percentage DNA, which gives a more integrated measurement of overall DNA damage in the cell.

2-Bone marrow micronucleus assay:
The micronucleus test was carried out in mice femoral bone marrow cells according to Chauhan et al. (2000).Numbers of normochromatic, polychromatic erythrocytes and micronuclei were evaluated in control and treated groups.For micronuclei evaluation, 2000 polychromatic erythrocytes were scored per animal.Both normochromatic erythrocytes (NCE) and polychromatic erythrocytes (PCE) were scored in 500 erythrocytes for determination of the PCE: NCE ratio according to the OECD No. 474 guideline of mammalian erythrocyte micronucleus test for chemicals testing (OECD, 1997).

Histopathological examination:
Liver and testes from each sacrificed mouse were dissected out and trimmed of excess fat tissues.Tissues were fixed in 10% buffered formalin and processed for paraffin sectioning by dehydration in different concentrations of alcohol, cleared with xylol and embedded in paraffin blocks.Sections of about 5µm thickness were stained with Harris haematoxylin and eosin (H&E) for histological study (Delafield, 1984).

Statistical analyses:
Statistical analyses were performed with SPSS 16 software.Experimental data were analyzed using one-way analysis of variance (ANOVA).Duncan's multiple range test was used to determine the significant differences between means.All values were expressed as mean ± SD and the significance level was set at P ≤ 0.05.

DNA damage evaluation:
Comet assay in liver and testes cells: The comet assay results of liver and testes are summarized in Table 2. BPA induced significant increase (P≤0.05) in the mean values of tail length, percentage of tail DNA and olive tail moment in liver and testes cells as compared to control.In contrast, administration of male mice with PSO at the three regimens with BPA diminished significantly (P≤0.05) the increase in the mean values of comet parameters induced by BPA in liver and testes cells.

Table (2):
Comet assay parameters in liver and testes cells of treated male mice with bisphenol-A (BPA) and/or pumpkin seed oil (PSO) for 28 consecutive days.
Data are expressed as means ± SD.Mean values in the same column within each parameter bearing the same superscript do not differ significantly (P ≤ 0.05).

Micronucleus test:
Results in Table 3 showed that BPA treatment increased (P≤0.05) the mean values of micronucleated polychromatic erythrocytes (MNPCEs) significantly compared to control.In contrary, oral administration of PSO through the three regimens of treatment decreased significantly (P≤0.05) the mean value of MNPCEs in bone marrow cells.In addition, cytotoxicity evaluation of bone marrow erythrocytes showed that BPA induced significant reduction (P≤0.05) in the ratio of PCE/NCE as compared to control.Whereas, PSO through the three regimens of treatment has protection against BPA cytotoxicity.This protection was appeared in the elevation of the PCEs/NCEs ratio as compared with those of BPA treated group.
Our results indicated that the BPA oral administration for 28 successive days showed genotoxic and cytotoxic activity in tested cells and PSO oral administration through the three different regimens of treatment with BPA significantly ameliorated the genotoxic and cytototoxic effects of BPA.These data are in accordance with the findings of several studies which demonstrated that BPA induced DNA damage via comet assay and micronucleus test in blood lymphocytes, rat spermatocytes and Chinese hamster ovary (CHO) cells (Tiwari et  The mechanism of BPA genotoxicity might work through the induction of oxidative stress and the depletion of antioxidant enzymes (Meeker et al., 2010;Jiang et al., 2011).Beside to that, BPA is an endocrine disruptor structurally similar to estrogen, exerts a weak estrogen-like effect as well as an anti-androgenic effect (Li, 2015).It was shown that estrogen causes DNA damage by estrogen-derived oxidants, DNA adducts formed by estrogen metabolites and formation of micronuclei in cell lines (Cavalieri et al., 2002;Mobley and Brueggemeier, 2004).
Concerning PSO treatments that alleviated the BPA induction of DNA damage in tested cells, our results are in agreement with those reported by Elfiky et al. (2012) who found that oral administration of PSO for ten consecutive days either before or after treatment of azathioprine was effective in the reduction of the frequencies of DNA fragmentation and MNPCEs frequencies, meanwhile, increased the percentage of PCEs/NCEs ratio in bone marrow of male mice.Therefore, it might suggest that the protective effects of PSO against BPA genotoxicity could be due to their content of varied bioactive compounds which were mentioned early.
Antioxidant compounds like phenolic acids, polyphenols and flavonoids scavenge free radicals such as peroxide, hydro-peroxide of lipid hydroxyl and thus inhibit the oxidative mechanisms that lead to degenerative diseases (Shahriar et al., 2012).The oil analysis in our study confirmed that PSO is rich in polyphenolics and vitamin E. Phenolic compounds have properties including antioxidant, anti-mutagenic, anticarcinogenic and anti-inflammatory effects that might potentially be beneficial in protecting the stability of the genome (Xie et al., 2013).In the same manner, polyphenolics ameliorate cell injury and protect oxidant induced DNA from lesion by reducing the free radical mediated oxidative damage (Urquiaga and Leighton, 2000).Meanwhile, vitamin E has an antioxidant role and can contribute to the cells protection against the deleterious effects of free radicals (Santana et al., 2015).

Histopathological examination:
Regarding the examined liver sections of BPA ' s treated mice; it revealed marked congestion of the hepatic blood vessels as well as marked vacuolar degeneration of the hepatocytes with many necrotic cells which appeared either with pyknotic nuclei or without any nuclear structure (Figure 2 B &C).On the other hand, liver histopathological examination of PSO ' s treated mice for 28 successive days before BPA treatment showed good protection against BPA ' s hepatic cellular alteration.It revealed only mild vacuolar degeneration of the hepatocytes with some necrotic cells (Fig. 2

D).
In regards to liver of PSO and BPA co-treated mice, it showed centrilobular degeneration and necrosis of the hepatocytes with appearance of increased number of binucleated cells and karyomegaly of some cells (Fig. 2 E).While, the hepatic tissue of PSO ' s treated mice for 28 successive days after BPA treatment showed moderated degree of granular degeneration and scattered necrotic hepatocytes (Fig. 2

F).
As for the examined testicular tissue of BPA treated mice, it showed an obvious defective spermatogenesis that characterized by severe necrosis and loss of the spermatogonial layers with multiple spermatid giant cells formation in most of the seminiferous tubules.Congestion of the interstitial blood vessel, destructed and abnormal spermatid formation with spermatid giant cells and loss of most of the spermatogonial cell layers were observed as well.Some seminiferous tubules showed destructed materials in the lumen with multiple spermatid giant cells formation were also observed (Figure 3 B &C). Testis of PSO ' s treated mice before BPA treatment showed marked protection of the spermatogonial cells layers with active spermatogenesis in the lumen of some seminiferous tubules (Fig. 3 D).Whereas, testis of PSO ' s treated mice in concurrently with BPA showed necrosis of some spermatogonial cells with starting of active spermatogenesis in some other seminiferous tubules (Fig. 3 E).In addition, testis of PSO ' s treated mice after BPA treatment showed degeneration and pyknosis of some spermatognial cells with active spermatogenesis in some other tubule and notice interstitial edema (Fig. 3

F).
The evaluation of histopathological alterations in liver and testes of treated male mice basically confirmed the genetic results as well.These findings are in agreement with Korkmaz et al. (2010) who noticed hepatic necrosis and congestion in the liver of male rats treated with BPA at a dose of 25 mg/kg/day three times a week for 50 day.Moreover, Kalb et al. (2015) showed that male mice exposure to BPA (3000 µg/kg b.w.) via breast milk during the breastfeeding period caused testicular degeneration and complete aplasia in some seminiferous tubules of their testes.In addition to that, Sangai and Verma, (2012) found that BPA-caused changes in the activities of ATPase in liver of mice thereby causing a reduction in ATP produced in the cell which lead to necrosis.It has been reported that BPA generate ROS that caused oxidative damage in the brain, reproductive tract and kidney of rats (Aydogan et al., 2008;Korkmaz et al., 2009).Furthermore, the great histopathological changes in testes tissues caused by BPA may be due to its estrogenic activity (Korkmaz et al., 2010).

Fig. 2 :
Fig. 2: A) Liver of control mice showing normal appearance of the central vein (C) and hepatic parenchyml cells (H).B) Liver of BPA treated mice showing congestion (C) of the hepatic blood vessels and marked vacuolar degeneration of the hepatocytes with many necrotic cells (arrow).C) Liver of BPA treated mice showing congestion of the central vein (C) and portal blood vessels (P) with marked hepatocellular vacuolar degeneration and necrosis (arrow).D) Liver of PSO treated mice before BPA treatment showing mild vacuolar degeneration of the hepatocytes with some necrotic cells (arrow).E) Liver of PSO treated mice concurrently with BPA showing centrilobular degeneration (C) and necrosis of the hepatocytes, notice the karyomegaly of some cells (arrow).F) Liver of PSO treated mice after BPA treatment showing granular degeneration and scattered necrotic hepatocytes (arrow).(H&E X400).

Fig. 3 :
Fig. 3: A) Testis of control mice showing active spermatogenesis within most of the seminiferous tubules.B) Testis of BPA treated mice showing severe necrosis and loss of the spermatogonial layers with multiple spermatide giant cells formation (arrow) in most of the seminiferous tubules.C) Testis of BPA treated mice showing congestion of the interstitial blood vessel, destructed and abnormal spermatid formation (thin arrow), spermatid giant cells (thick arrow) and loss of most of the spermatogonial cell layers.D) Testis of PSO treated mice before BPA treatment showing marked protection of the spermatogonial cells layers with active spermatogenesis (arrow)in the lumen of some seminiferous tubules.E) Testis of PSO treated mice concurrently with BPA showing necrosis of some spermatogonial cells (thin arrow) with starting of active spermatogenesis (arrow) in some other seminiferous tubules.F) Testis of PSO treated mice after BPA treatment showing degeneration and pyknosis of some spermatognial cells with active spermatogenesis in some other tubule (arrow), notice interstitial edema (thin arrow).(H&E X400).On the other side, the protective role of PSO was confirmed by Ramah et al. (2015) who found that treatment with PSO markedly alleviated lead acetate induced pathological changes in testes tissues of rats.Phenolic compounds such as polyphenols flavonoids as well as vitamins and zinc in pumpkin are attributed factors for its antioxidant activity by neutralizing free radical generation (Amara et al., 2008; Morakinyo et al., 2010).In addition, the presence of oleic acid, a monounsaturated fatty acid in pumpkin reduces the susceptibility of the testis and epididymis to lipid peroxidation (Bourre et al., 2004).

Table ( 1
): Chemical composition and antioxidant activity of pumpkin seed oil.

Table ( 3
): Frequencies of micronucleated polychromatic erythrocytes and polychromatic/normochromatic ratio in bone marrow cells of bisphenol-A (BPA) and/or pumpkin seed oil (PSO) treated male mice for 28 consecutive days.
Data are expressed as means ± SD.Mean values in the same column within each parameter bearing the same superscript do not differ significantly (P ≤ 0.05).MNPCEs: Micronucleated Polychromatic erythrocytes PCEs: Ploychromatic erythrocites NCEs: Normochromatic erythrocytes