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Journal of Environmental & Analytical Toxicology
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Protective Effects of Vitamin C against Methomyl-Induced Injures on the Testicular Antioxidant Status and Apoptosis-Related Gene Expression in Rat

Tarek M Heikal1*, Abdel-Tawab H Mossa1 and Wagdy KB Khalil2
1Environmental Toxicology Research Unit (ETRU), Pesticide Chemistry Department, National Research Centre, Cairo, Egypt
2Cell Biology Department, National Research Center, Dokki, Giza, Egypt
Corresponding Author : Tarek M Heikal
Environmental Toxicology Research Unit (ETRU)
Pesticide Chemistry Department, National Research Centre
Tahrir Street, P.O. Box 12311, Dokki, Cairo, Egypt
Tel: +2 01002591307
Fax: (202)-33370931
E-mail: [email protected]
Received November 20, 2014; Accepted December 11, 2014; Published December 12, 2014
Citation: Heikal TM, Mossa ATH, Khalil WKB (2015) Protective Effects of Vitamin C against Methomyl-Induced Injures on the Testicular Antioxidant Status and Apoptosis-Related Gene Expression in Rat. J Environ Anal Toxicol 5:255. doi: 10.4172/2161-0525.1000255
Copyright: © 2015 Heikal TM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

The present study is undertaken to evaluate the protective effects of vitamin C (VC) against methomyl-induced oxidative stress and testicular injures in Wister rat. Male rats were randomly divided into 4 groups of 6 rats each. The route of application selected for the study was oral gavage for 28 consecutive days. Group 1 was served as a control, whereas group 2 was supplemented with VC (200 mg kg-1 body weight, bw). Group 3 was administered with a dose of methomyl equivalent to 1/10 LD50 (2.034 mg kg-1 bw), whereas group 4 was co-administered VC and methomyl as the same dose of groups 2 and 3, respectively. Sub-acute exposure of rats to methomyl for 28 consecutive days resulted in a significant increase in testicular lipid peroxidation (LPO) and a decrease in glutathione (GSH) level. In addition, the testicular activities of catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferase (GST) enzymes were significantly decline in response to methomyl exposure. Furthermore, histopathological studies in testis of rats treated with methomyl exhibited incomplete spermatogenic series in seminiferous tubules and necrosis of spermatogoneal cells lining seminiferous tubules. Notably, the co-administration of VC modulated the biochemical parameters and the intensity of histopathological findings. The effect of VC on transcriptional activity of four key stress and apoptosisrelated genes (CASP3, CASP9, Tp53 and Bcl2), in response to methomyl exposure in rats, was investigated. Results revealed a significant up-regulation in the level of the expression for the tested genes, however supplementation of VC to methomyl-treated rats modulated the observed significant up-regulation in the level of the expression for those genes, indicative of an protective interfering role in the signaling transduction process of methomyl-mediated toxicity. In conclusion, these data suggested that administration of VC may partially protect against methomyl induced testicular oxidative damage and apoptosis-related genes.

Keywords
Gene expression; Methomyl; Oxidative damage; Rat; Testis; Vitamin C
Introduction
Pesticides are deliberately added to the environment for the purposes of controlling the undesired agricultural pests and insects. In fact, most pesticides are not highly selective, but are generally toxic to many non target species, including humans causing public health hazards [1-4].
Methomyl is an oxime carbamate insecticide and widely used for the control of a large variety of insects on a wide range of crops all over the world. The mechanism of toxicity of carbamates is analogous to that of OPs, in that they inhibit AChE. However, inhibition is transient and rapidly reversible, because there is a rapid reactivation of the carbamylated enzyme in the presence of water [5]. Additionally, carbamylated AChE does not undergo aging [5].
The toxicity of methomyl and other insecticides is ascribed, at least in part, to the generation of reactive oxygen species (ROS), leading to lipid peroxidation (LPO) and oxidative stress [4,6-9]. Studies from our laboratory have exhibited a state of oxidative stress induced in adult rat liver following exposure to methomyl [10].
Cell death by apoptosis is genetically controlled. Apoptosis is a part of normal development and maintenance of testicular homeostasis [11]. During various stages of spermatogenesis, an adequate amount of germ cells are eliminated via the process of apoptosis in order to maintain a precise germ cell population in compliance with the supportive capacity of the Sertoli cells [12]. Since, ROS is considered a potential signal for apoptosis so, the elevated levels of ROS can cause oxidation of the mitochondrial pores thereby disrupting the mitochondrial membrane potential, releasing cytochrome c and activating the mitochondrialmediated pathway of apoptosis. In addition, ROS have been shown to induce the expression of Fas receptor and ligand stimulating Fas/FasLmediated apoptotic signal transduction pathway. Several environmental disruptors are known to inappropriately activate apoptosis in testicular locale by increasing the levels of ROS [13-15].
Caspases are a part of highly conserved protein family that is central to the apoptotic pathway. They are proteases activated after a cell receiving an instructing signal to undergo apoptosis. They can also activate other enzymes that degrade other parts of the cellular machinery by cleaving an inhibitory sequence on these enzymes. CASP3 is an important biomarker of the apoptosis process [16].
Bcl-2 inhibits most types of cell death, implying a common mechanism of lethality. It is localized in intracellular sites of oxygenfree radical generation including mitochondria, endoplasmic reticula, and nuclear membranes [17]. The mechanism by which Bcl-2 exerts its anti-apoptotic effects is not fully resolved, although it has been speculated that Bcl-2 acts as either a regulator of an antioxidant pathway that prevents oxidative damage, or as a regulator of intracellular Ca2+ compartmentalization [18]. The anti-apoptotic activity of Bcl-2 correlates with its intracellular ratio to another recently described protein called Bax. High levels of Bax have been shown to favor apoptosis in cells subjected to growth factor deprivation, whereas high levels of Bcl-2 prolong cell survival under the same conditions [19].
Taking a lead from these reports and our previous studies, we thought to investigate whether treatment with a sub-lethal dose of methomyl would alter the antioxidant status and the levels of apoptosisrelated gene expressions (i.e. Tp53, Bcl-2, CASP3 and CASP9) in adult rat testis as well as the role of VC in attenuating such hazards.
Materials and Methods
Chemicals
Methomyl (Lannate® 90% SP, S-methyl N-[methylcarbamoyloxy] thioacetimidate), was obtained from Kafr El-Zayat Pesticides and Chemicals Company, Egypt and Vitamin C (L-Ascorbic acid) were purchased from Sigma–Aldrich Chemie (Steinheim, Germany). The assay kits used for biochemical measurements of catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST), glutathione reduced (GSH), lipid peroxidation (LPO) and total protein were purchased from Biodiagnostic Company, Dokki, Giza, Egypt. All other chemicals were of reagent grades and obtained from the local scientific distributors in Egypt.
Animals
Male albino rats of Wistar strain (Rattus norvegicus) weighing 180 ± 10 g, obtained from the Animal Breeding House, National Research Centre (NRC), Dokki, Cairo, Egypt, were kept in well-ventilated plastic cages, provided with pellets, water ad libitum and subjected to a 12:12 light-to-dark photo period. All animal procedures were approved by the National Research Centre (NRC), Cairo, Egypt Local Ethical Review Committee and were carried out in accordance with the “Guide for the Care and Use of Laboratory Animals”.
Experimental design
After acclimatization period for one week under laboratory conditions, rats were randomly divided into 4 groups each containing 6 animals. The route of application selected for the study was daily oral gavage for 28 consecutive days. Rats in Group I were served as a control. Rats in Group II (VC) were given VC in water at a dose of 200 mg/kg b.wt (0.5 ml/rat) based on our previous work [20]. Rats in Group III were administered methomyl in water at a dose of 2.034 mg kg-1 b.wt (1/10 LD50, 0.5 ml/rat) based on estimated LD50 (20.34 mg kg-1 b.wt) in our laboratory [10]. Rats in Group IV (methomyl+VC) were given the same doses of VC as in group II 30 min before the administration of methomyl (2.034 mg kg-1 b.wt.). During the experimental duration, body weights were weekly recorded and the doses of the tested compounds were modulated accordingly.
At the end of the administration, the animals were fasted overnight with water ad libitum, anesthetized with pentobarbital sodium (35mg/ kg, i.p.) and sacrificed by cervical dislocation on 29th day. Testis were dissected out immediately, washed with ice cold saline and cleaned. Testis tissues from control and experimental groups were washed with ice cold saline, homogenized in 10% (w/v) ice cold phosphate buffer (0.1M pH 7.4) and centrifugation at 10,000 ×g for 30 minutes at 4°C. The supernatants obtained were used for the assays of SOD, CAT, GST, LPO, GSH and total protein.
Oxidative stress evaluation
Lipid peroxidation (LPO) process is determined in supernatant of testicular tissue homogenate by the thiobarbituric acid (TBA) method which estimates the malondialdehyde formation (MDA) according to Esterbauer and Cheeseman [21]. Two hundred fifty microliters of tissue homogenate were added to 1.5 ml of 1% phosphoric acid (pH 2.0) and 1 ml of 0.6% of TBA in air–light tubes and were placed in a boiling water bath for 25 min. After incubation, the sample was cooled to room temperature and MDA-TBA was extracted with 2.5 ml of butanol. Organic phase was separated by centrifugation for 5 min at 2000 xg and measured at 532 nm. The concentration of MDA was calculated by the absorbance coefficient of MDA–TBA complex (1.56 x 105 M-1 cm-1). Lipid peroxidation is expressed as nmoles MDA/mg protein.
Reduced glutathione (GSH) content of supernatant estimation was performed by the method of Beutler et al. using commercial glutathione reduced kits (Biodiagnostic for diagnostic reagents: Dokki, Giza, Egypt). Determination of GSH is based on the reaction of DTNB [5, 5- dithiobis-(2-nitrobenzoic acid)] with GSH and yield a yellow colored chromophore with a maximum absorbance at 412 nm [22]. The amount of GSH present in the testicular tissue was calculated as nmoles/g tissue.
Catalase (CAT) converts H2O2 into water. In brief, 0.25 g of tissue was homogenated in 1 ml of 50 mM Tris–HCl and centrifuged at 2000 xg for 15 min. Then 10 μl of supernatant was added to a quartz cuvette containing 980 μl of distilled water, and 10 μl of 0.066 M H2O2 (dissolved in sodium phosphate buffer) was added to start the reaction. The testicular CAT activity was measured spectrophotometrically at 240 nm by calculating the rate of degradation of H2O2, the substrate of the enzyme [23]. Activity of CAT is expressed as units/mg protein.
The specific activity of testicular superoxide dismutase (SOD) was determined according to the method described by Misra and Fridovich [24]. Ten micro liters of tissue homogenate were added to 970 μl of EDTA–sodium carbonate buffer (0.05 M) at pH 10.2. The reaction was started by adding 20 μl of epinephrine (30 mM) and the activity was measured at 480 nm for 4 min. A unit of SOD is defined as the amount of enzyme that inhibits by 50% the speed of oxidation of epinephrine and the results were expressed as U/mg protein.
Glutathione-S-transferase (GST) activity of testicular was measured spectrophotometrically by the method of Habig et al. using 1-chloro- 2,4-dinitrobenzene as electrophilic substrate that binds to GSH with the participation of the enzyme and forms a colored GSH-substrate complex, detected at 340 nm [25]. The activity of GST was expressed in terms of μmol/min/mg protein.
The total protein level of supernatant was determined according the method described by Lowry et al. using bovine serum albumin (BSA) as a standard [26].
DNA fragmentation
DNA fragmentation was assessed by electrophoresis of extracted genomic DNA from testis tissues as described by Lu et al. with slight modifications. Briefly, cells were washed with PBS containing 10 mM EDTA. The pellet was lysed in 250 μL of lysis buffer (100mM NaCl, 5mM EDTA, 10mM Tris-HCl, pH 8.0, 5% Triton X-100, 0.25% SDS), containing 400 μg/mL DNase-free RNase and incubated at 37ºC for 90 minutes followed by 1 hour incubation with proteinase- K (200 μg/mL) at 50ºC. DNA was extracted with 200μL of phenol : chloroform: isoamyl alcohol (25 : 24 : 1) for 1 minute and centrifuged at 13000 × g for 3 minutes. The aqueous phase was further extracted with chloroform and centrifuged [27]. DNA was precipitated from aqueous phase with 3 volumes of chilled alcohol containing 0.3M sodium acetate at 4°C overnight. The precipitate was centrifuged at 13000 × g for 10 minutes. DNA pellet was washed with 80% alcohol, dried, dissolved in 50 μL TE buffer and electrophoresed in 1.8% agarose gel at 50V, stained with ethidium bromide, and visualized in Biometra Gel Documentation System.
Semi-quantitative RT-PCR
Total RNA (Poly(A)+ RNA) was extracted from 50 mg of testis tissues using the standard TRIzol extraction method (Invitrogen, Paisley, UK) and recovered in 100 μL diethylpyrocarbonate (DEPC)- treated water by passing the solution a few times through a pipette tip.
Total RNA was treated with one unit of RQ1 RNAse-free DNAse (Invitrogen, Karlsruhe, Germany) to digest DNA residues, re-suspended in DEPC-treated water, and quantified photospectrometrically at 260 nm. Total RNA was assessed for purity from the ratio between quantifications at 260 nm and 280 nm, and was between 1.8 and 2.1. Integrity was verified with the ethidium bromide-stain analysis of 28S and 18S bands using formaldehyde-containing agarose gel electrophoresis. Aliquots were either used immediately for reverse transcription (RT) or stored at -80°C.
To synthesise first-strand cDNA, 5 μg of complete Poly(A)+ RNA was reverse transcribed into cDNA in a total volume of 20 μL using 1 μL oligo (poly(deoxythymidine)18) primer (36). The composition of the reaction mixture was 50 mmol L-1 MgCl2, 10x RT buffer (50 mmol L-1 KCl; 10 mmol L-1 Tris-HCl; pH 8.3), 200 U μL-1 reverse transcriptase (RNase H free, Fermentas, Leon-Rot, Germany), 10 mmol L-1 of each dNTP, and 50 μmol L-1 of oligo(dT) primer. RT reaction was carried out at 25°C for 10 min, followed by 1 h at 42°C, and completed with denaturation at 99°C for 5 min. Reaction tubes containing RT preparations were then flash-cooled in an ice chamber until used for DNA amplification through polymerase chain reaction (PCR) [28].
First-strand cDNA was used as a template for semi-quantitative RT-PCR with a pair of specific primers in a 25-μL reaction volume. The sequences of specific primer and are listed in Table 1. GAPDH and β-Actin were used to normalize mRNA levels of the target genes. The reaction mixture for RT-PCR consisted of 10 mmol L-1 dNTP’s, 50 mmol L-1 MgCl2, 10x PCR buffer (50 mmol L-1 KCl; 20 mmol L-1 Tris-HCl; pH 8.3), 1 U μL-1 taq polymerase, and autoclaved water. Table 1 lists the specific gene primer sequences and PCR annealing temperature. PCR products derived from GAPDH and β-Actin were then loaded onto 2.0 % agarose gel. Each RT-PCR was repeated for each rat, generating at least ten new cDNA products per group.
Histopathological examination
For light microscopic investigations, specimens from testes were fixed in 10% phosphate buffer formalin, dehydrated in alcohols and embedded in paraffin. Five micron tissue sections were stained with hematoxylin and eosin stain (H&E) for general histopathological examination. Scoring of histopathological changes was done as follow: (-) absent; (+) mild; (++) moderate; (+++) severe, and (++++) extremely severe [29].
Statistical analysis
All results were presented as means ± SE. All data were done with the Statistical Package for Social Sciences (SPSS 17.0 for windows). The results were analyzed using one way analysis of variance (ANOVA) followed by Duncan’s test for comparison between different treatment groups. Statistical significance was set at p<0.05.
Results
Effect on oxidative stress markers
The present results revealed that administration of methomyl at a dose of 2.034 mg kg-1 b.wt for 28 days induced testicular oxidative stress as evidenced by a significant decline (p<0.05) in the content of GSH by - 44.3% as well as a significant increase (p<0.05) in the level of lipid peroxidation (LPO) by + 49%, compared with the control group. Simultaneously in methomyl-treated group, the activities of testicular antioxidant enzymes were reduced significantly (p<0.05) by -23.7, -21.3 and -20.8% for SOD, CAT and GST, respectively compared with the control group (Table 2). However, co-administration of VC to methomyl mitigated the perturbations in the oxidative stress biochemical parameters (Table 2).
Effect on DNA fragmentation
Quantitative DNA fragmentation was determined in male rats exposed to methomyl with or without VC. The DNA damage was examined in testis tissues collected from pesticides-treated rats using gel electrophoresis laddering assay (Figure 1). The results of gel electrophoresis laddering assay revealed that treatment of male rats with VC alone caused similar low DNA damage to that in control group (Figure 1). However, methomyl treated rats expressed more bands of the damaged DNA compared with the control and VC treated rats (Figure 1). In contrary, treatment of rats with VC decreased the damage in the DNA caused by methomyl in the group treated with pesticides combined with VC (Figure 1).
Effect on gene expression
Study the expression alteration of key stress and apoptosis-related genes (CASP3, CASP9, Tp53 and Bcl2) was investigated in rat testis tissues (Figure 2-5, respectively).
The expression level of CASP3, CASP9, Tp53 and Bcl2 genes was low in control group compared with other treated group. The same trend was found in the rats treated with VC alone. Where, the expression level of theses genes was similar in VC treated rat with that in control rats (Figure 2-5). On the other hand, the expression level of CASP3, CASP9, Tp53 and Bcl2 genes was significantly higher in methomyl treated rats than in control and VC treated rats (Figure 2-5).
In contrast, treatment of rats with VC decreased significantly the over-expression levels of the CASP3, CASP9 and Bcl2 genes resulted from methomyl treatment in the group treated with methomyl plus VC (Figure 2-5). Furthermore, VC was also able to decrease the overexpression level of the Tp53 gene caused by methomyl treatment in the group treated with methomyl plus VC (Figure 4), however, the decline in the expression was not significantly different.
Histopathology
While control animals exhibited normal histological structures of mature seminiferous tubules with complete spermatogenic series, methomyl treatment promoted incomplete spermatogenic series in seminiferous tubules, degenerative changes, necrosis of spermatogoneal cells lining seminiferous tubules and desquamation in the lumen (Figure 6 A-D). However, co-administration of VC to methomyl alleviated the histopathological findings and the severity of such findings in the presence and absence of VC were demonstrated in (Table 3).
Discussion
The present study was implemented to examine the protective effect of VC on both the antioxidant status and the expression level of selected apoptosis-related genes (CASP3, CASP9, Tp53 and Bcl2) during methomyl-mediated oxidative stress and testicular injuries in male rat. It is well documented that many carbamates cause genotoxicity. Genotoxicity of agrochemicals is considered one of the most serious side effects on human beings. If a chemical reacts with nuclear DNA, it may be mutagenic and carcinogenic to the exposed organisms [30,31]. Prolonged exposure to such chemicals may lead to heritable genetic diseases, carcinogenesis, reproductive dysfunction, and birth defects. Although methomyl is degraded rapidly and its metabolic residues have low toxicity, however, long-term exposure to methomyl may induce genetic damage in human beings [32]. Methomyl induced chromosome aberrations, sister-chromatid exchanges, micronuclei and DNA single strand breaks in agricultural workers peripheral blood [32,33].
The reactive oxygen species (ROS) are continuously generated inside the human body as a consequences of exposure to a lot of exogenous chemicals in our ambient environment including pesticides. Under normal circumstances, the ROS generated are eliminated by enzymatic and non-enzymatic antioxidant systems present in the body. Harmful effects caused by ROS occur as a consequence of an imbalance between the formation and elimination of these species culminates in an oxidative stress [34]. ROS readily attack and induce oxidative damage to various bio-molecules including proteins, lipids, mitochondria, lipoproteins and DNA which alter the pathways of these bio-molecules [35]. Oxidative stress affects many cellular functions by various mechanisms such as alteration in gene expression through activation of transcription [15,36].
The data of the present study confirmed an exacerbation of oxidative injury in testis tissues of methomyl-treated rat group as evidenced by an elevation in LPO and a reduction in testicular GSH, CAT, SOD and GST. These data are consistent with our previous observation in testis, liver and kidney tissues of rats and mice treated with different insecticides including methomyl [4,8,20,37]. This is supported by our histopathological results. The testicular degenerative changes induced by methomyl in intoxicated rats, as demonstrated in this study, agree with those of Mahgoub and Mednay, who reported variable degrees of degenerative changes and necrosis in the seminiferous tubules up to total cellular destruction after chronic exposure for 30 days of male rats to methomyl [2]. In this context, Pant et al. concluded that carbofuran, carbamate insecticide, induced moderate edema, congestion, damage to Sertoli cells and germ cells, along with the accumulation of cellular debris in the lumen of a few seminiferous tubules which showed disturbed spermatogenesis [38]. The role of VC as a water soluble antioxidant in biological systems is well known and can be summarized as scavenging free radical [39], restoring vitamin E [40], neutralizing the reactive oxygen metabolites, and reducing DNA damage and hence genetic mutation [20,41]. In the present study, our data remarkably suggest protective effects of co-administration of VC in attenuating the levels of biochemical parameters and histopathological observations.
The toxic effect and testicular damage induced by methomyl in this study could be possibly explained by its direct cytotoxic effect and/ or indirectly via the increased level of ROS and apoptosis-mediated genes. Many studies have reported correlation between exposure to OP insecticides and apoptosis-related genes expression in rats [42]. In this respect, we have examined the impact of methomyl treatment in the selected apoptosis-related genes, namely: Tp53, Bcl-2, CASP3 and CASP9. In our study, the results revealed that subchronic exposure of rat to methomyl for 28 days is associated with a significant upregulation of the expression of apoptosis-related genes, compared with the control.
Conclusion
These data suggested that administration of VC may partially protect against methomyl induced testicular oxidative damage and apoptosis-related genes. We propose that VC may provide a prolonged remedy against toxins-induced oxidative damage and up-regulation of genes expression without harmful side effects.
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