Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 54 issue 3 (march 2020) : 335-341

Protective effects of selenium on malathion-induced testicular toxicity in mice

Mahrous A. Ibrahim1,2, Farooq A. Wani3,*, Athar M. Khalifa3, Mina T. Kelleni4,5, Marwa M. Anwar2, Ashokkumar Thirunavukkarasu6, Mohammed U. Sayeed3
1Forensic Medicine and Clinical Toxicology, College of Medicine, Jouf University, Sakaka, Saudi Arabia.
2Forensic Medicine and Clinical Toxicology Department, Faculty of Medicine, Suez Canal University (SCU), Ismailia, Egypt.
3Pathology Department, College of Medicine, Jouf University, Sakaka, Saudi Arabia.
4Pharmacology department, Faculty of Medicine, Minia University, Egypt.
5Pharmacology Department, College of Pharmacy Jouf University, Sakaka, Saudi Arabia.
6Community Medicine Department, College of Medicine, Jouf University, Sakaka, Saudi Arabia.
Cite article:- Ibrahim A. Mahrous, Wani A. Farooq, Khalifa M. Athar, Kelleni T. Mina, Anwar M. Marwa, Thirunavukkarasu Ashokkumar, Sayeed U. Mohammed (2019). Protective effects of selenium on malathion-induced testicular toxicity in mice . Indian Journal of Animal Research. 54(3): 335-341. doi: 10.18805/ijar.B-1083.

ABSTRACT

The aim of this study was to investigate the possible protective effect of the antioxidant sodium selenite (0.1 mg/kg/day) via gavage once a day for 30 days for the first time in a model of malathion (27 mg/kg/day) via gavage once a day for 30 days induced testicular toxicity in mice. Results of this study revealed that concomitant administration of selenium has prevented the decrease in mean final and relative body weight as well as testicular and relative testicular weight as compared to malathion group. Concomitant selenium administration has also decreased malondialdehyde contents and increased serum levels of follicle stimulating hormone, luteinizing hormone, testosterone, acetylcholinesterase and testicular levels and activities of glutathione, glutathione peroxidase, catalase, superoxide dismutase and improved the testicular histopathological features as compared to malathion group. 

INTRODUCTION

Organophosphorus pesticide (OPP) self-poisoning is an important clinical problem in rural regions of the developing world and kills an estimated 200000 people every year (Eddleston et al., 2008, Kotb et al., 2016, Ibrahim et al., 2011). The unregulated use of OPPs and their application over large agricultural and urban areas, has caused severe environmental pollution and potential health hazards (Sodhi et al., 2008). Particularly, malathion [O,O-dimethyl-S-(1,2-dicarcethoxyethyl) phosphorodithioate] is a widely used OPP habitually used to eradicate ectoparasites, household insects, to conserve stored grain and to eliminate disease-inducing arthropods (Selmi et al., 2018).
       
Malathion toxicity is aggravated by its metabolites which are considered to be the primary source of malathion’s toxicity and are 40 times more toxic than malathion (Baiomy et al., 2015).
       
Briefly, malathion and its metabolites inactivate serine esterases; mostly acetylcholinesterase (AChE) and butyrylcholinesterase leading to an overstimulation of the cholinergic pathways (Selmi et al., 2018). Primarily organs like brain, pectoral muscle, heart, kidney and intestine as well as the oxidant and antioxidant system, bio-element levels, immune system, urinary system, reproductive system, hematological and biochemical parameters could be affected by malathion toxicity (Cemek et al., 2010).
       
Malathion intoxication has been previously shown to augment the oxidative stress due to over production of reactive oxygen species (ROS) and exhaustion of the endogenous antioxidant defense system (Ince et al., 2017a, href="#ali_2018"> Ali and Ibrahim, 2018). ROS can also react with biological macromolecules leading to lipid peroxidation, various enzymatic inactivation or DNA damage (Cemek et al., 2010).
       
Malathion has also been reported to interfere with reproductive and sexual development in animals. Malathion was shown to impair steroidogenesis and to induce apoptosis in germ cells with proliferation of the seminiferous epithelium (Geng et al., 2015). Moreover, it affects late stages of spermatogenic cells maturation in mice causing damaged DNA and reduced chromatin in spermatogonia and spermatids (Ojha and Srivastava, 2014). However, the full spectrum of the reproductive effects of malathion has not been fully elucidated.
 
Recently, protective effects of natural antioxidants against toxicity of various agents were the focus of interest. Selenium is an essential trace element and an integral part of many antioxidant enzymes, including glutathione peroxidase (GSH-Px), thioredoxinreductase (TR) and selenoprotein P (SeP), which contains selenium as selenocysteine. Selenium is also widely used in clinical practice to improve male fertility and several studies have demonstrated that selenium, alone or in combination with some other antioxidant, considerably attenuates oxidative damage induced by various toxicants (Milošević et al., 2017). 
       
Furthermore, selenium appears to function as an antimutagenic agent, preventing the malignant transformation of normal cells. These protective effects of selenium (as co-antioxidant) seem to be primarily associated with its presence in the seleno-enzymes, which are known to protect DNA and other cellular components from oxidative damage. For this reason, treatment with selenium and other free radical scavengers can decrease the oxidative stress and lipid peroxidation related to OPP-induced toxicity (Cemek et al., 2010, Bunglavan et al., 2018).
       
Therefore, the current study aimed at detection of the hormonal, biochemical and histopathological toxicity of malathion on the testes of male albino mice and elucidation of the possible protective role of selenium in ameliorating such toxicity.

MATERIALS AND METHODS

Animals
 
The present study was conducted on Male Balb/c mice weighing 20–30 g (5 weeks old) obtained from the Animal Facility of the National Institute for Vaccination, Helwan, Egypt. They were housed in standard cages for one week as acclimatization period before conducting the experiment. Standard mice chow diet (El-Nasr Company, Abou-Zaabal, Cairo, Egypt), ad libitum water and lighting were maintained at a 12 h cycle. Animal care and experimental procedures were in accordance with the protocols of the Research Ethics Committee of Faculty of Medicine, Suez Canal University, Egypt.
 
Drugs and Chemicals
 
The drugs used were sodium selenite and malathion powders, bought from Sigma-Aldrich, USA; thiobarbituric acid, ethylene diamine tetraacetic acid (EDTA), 5 5'-dithiobis (2-nitrobenzoic acid) (DTNB), sodium phosphate (Sigma-Aldrich Chemical Co.); trichloroacetic acid and ammonium molybdate (El-Nasr Pharmaceuticals Chemicals Co., Egypt); Hydrochloric acid (BDH Chemicals Ltd., England); pyrogallol (Zauba Technologies and Data Services Private Ltd., India).
 
Experimental design
 
The mice were randomly distributed into four groups of eight mice each as follows:
 
Control group: received 0.5 ml corn oil per animal given via gavage, once a day for 30 days.
Selenium group: received selenium as sodium selenite (Na2SeO4) dissolved in water (0.1 mg/kg/day) by gavage once a day for 30 days (Al-Othman, 2011).
 
Malathion group: received malathion at a dose of 27 mg/kg/day (1/50 LD50) dissolved in 0.5 ml corn oil and given via gavage for 30 days (Ince et al., 2017b).
Selenium/malathion group: received both selenium and malathion as described; selenium was given 30 minutes before malathion administration.       
 
Collection of blood and testis specimens
 
At the end of treatments, animals were weighed and sacrificed. The blood samples were collected and serum was separated by centrifugation to be stored at -80°C and thawed just before the biochemical analysis. The testes were excised, weighed, a part of each testis tissue was collected and fixed in 10% formalin for histopathological examination, and other parts were kept in -80°C and thawed just before homogenization in phosphate buffered saline for the biochemical assay.
 
Analytical methods
FSH and LH: Serum FSH and LH levels were measured using FSH and LH ELISA kits (Cusabio Biotech, Wuhan, Hubei, China) according to the manufacturer’s instructions. IU/L was calculated for FSH and LH.
 
MDA: Testicular malondialdehyde (MDA) level was detected biochemically. Trichloroacetic acid was added to the sample for protein precipitation and then thiobarbituric acid was added. The mixture was heated for 10 min in a boiling water bath. One molecule of MDA in the homogenized testis samples reacted with two molecules of thiobarbituric acid and the resulting chromogen was centrifuged. The intensity of the color developed in the supernatant was measured spectrophotometrically at 535 nm (Buege and Aust, 1978, Mutlu-Turkoglu et al., 2005).
 
GSH: Reduced glutathione (GSH) level was estimated following the method described by Ellman (Ellman, 1959, Bahrami et al., 2016). In this method thiols react with Ellman’s reagent (5,5’ -dithiobis-(2-nitrobenzoic acid); DTNB), cleaving the disulfide bond to give 2-nitro-5-thiobenzoate (TNB-), which ionizes to the TNB2- dianion in water; 15 μLof sample was mixed with 260 μL assay buffer (0.1 M sodium phosphate and 1 mM EDTA, pH: 8) as well as 5 μLEllman reagents; incubated for 15min at room temperature and the TNB2- formation was quantified in a spectrophotometer by measuring the absorbance of visible light at 412 nm. Absorbance values were compared with a standard curve generated from standard curve from known GSH.
 
Catalase: Catalase activity was determined spectro- photometrically by the method of Koroliuk et al., (Bahrami et al., 2016, Koroliuk et al., 1988); 10 μL of sample was incubated with 100 μmol/mL of H2O2 in 0.05 mmol/L Tris-HCl buffer pH = 7 for 10 min followed by adding 50 μL of 4% ammonium molybdate to rapidly terminate the reaction. Yellow complex of ammonium molybdate and H2O2 was measured at 410 nm. One unit of catalase activity was defined as the amount of enzyme required to decompose 1 μmol H2O2 per min.

SOD: Testicular superoxide dismutase (SOD) activity was detected biochemically. The method used to determine SOD activity in homogenized testis samples is based on the autoxidation of pyrogallol which is inhibited by SOD. One unit of SOD is generally defined as the amount of enzyme that inhibits the autoxidation of pyrogallol by 50%. The activity of SOD was monitored spectrophotometrically at 420 nm (Marklund and Marklund, 1974).
 
AChE: Acetylcholinesterase (AChE) activity as a parameter of organophosphate intoxication was assayed using mouse AChE activity ELISA kit (Sigma-Aldrich Chemical Co.).
 
Histopathology: Paraffin blocks were cut in 5µm thick sections and the sections were stained with hematoxylin and eosin.
 
Statistical evaluation: Results were shown as Mean ± SD. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test used to find statistical significance (p<0.05). Statistical analysis was done using SPSS software, version 22 for windows (SPSS, Inc., Chicago, IL, USA).

RESULTS AND DISCUSSION

In malathion treated groups, mean final body weight, relative body weight, testis weight and relative testis weight were decreased as compared to control and selenium groups. Group in which selenium was added with malathion prevented a decrease in these parameters as compared to malathion group (Table 1).
 

Table 1: Changes in body weight and testis weight of mice after thirty days of exposure to selenium (0.1 mg/kg/day), malathion (27 mg/kg) and their combination. n = 8 mice for each group.



Moreover, malathion treated mice recorded a decrease in mean serum levels of luteinizing hormone, follicle stimulation hormone, testosterone and acetylcholinesterase as compared to control and selenium groups. Adding selenium to malathion has prevented such a decrease in these serological parameters as compared to malathion treatment group (Table 2).
 

Table 2: Changes in serum levels of LH, FSH, testosterone, and acetylcholinesterase in mice after thirty days of exposure to selenium (0.1 mg/kg/day), malathion (27 mg/kg) and their combination. n = 8 mice for each group.


       
Furthermore, malathion has increased the mean level of the lipid peroxidation enzyme malondialdehyde and decreased the mean levels of the protective antioxidant enzymes catalase, superoxide dismutase, glutathione peroxidase and glutathione in testicular tissue of mice in malathion treated group as compared to control and selenium groups. Adding selenium to malathion has decreased the mean level of the oxidative stress enzyme malondialdehyde and increased the mean levels of the protective antioxidant enzymes catalase, superoxide dismutase, glutathione peroxidase and glutathione in the testicular tissue as compared to malathion treated group (Table 3).
 

Table 3: Changes in oxidative stress markers in the testis of mice after thirty days of exposure to selenium (0.1 mg/kg/day), malathion (27 mg/kg) and their combination. n = 8 mice for each group.


       
Regarding histopathology, the seminiferous tubules of the control mice and selenium-treated group were architecturally normal (Fig 1 and 2). The seminiferous tubules were uniform in size and shape and were lined by spermatogenic cells in different stages of maturation. In contrast, malathion treated group showed necrotic and degenerative changes in the seminiferous tubules and edema in interstitial tissue (Fig 3A and B). Malathion plus selenium treated group showed most of the seminiferous tubules with normal structure. Only few seminiferous tubules showed necrotic changes (Fig 4).
 

Fig 1: Photomicrograph of the testicular tissue of mice in the control group showing normal testicular architecture.


 

Fig 2: Photomicrograph of the testicular tissue of mice in selenium treated group showing normal testicular architecture.


 

Fig 3: A Photomicrograph of the testicular tissue of mice in malathion treated group showing many necrotic seminiferous tubules (shown by arrows).


 

Fig 3B: Photomicrograph of the testicular tissue of mice in malathion treated group showing seminiferous tubules depicting degenerative and necrotic changes with decrease in the number of lining cell layers.



Fig 4: Photomicrograph of the testicular tissue of mice in selenium-malathion treated group showing most of the seminiferous tubules with normal structure and very few showing necrotic changes (shown by arrows).


       
Malathion is a well-known and widely used organophosphate pesticide that was used experimentally to produce testicular and other organs toxicity in mice and rats (Al-Othman, 2011, Contreras and Bustos-Obregon, 1999). However, there are many studies in the literature, which have studied the toxic effects of malathion on different organs of the body as well as the protective effect of different anti-oxidants, but we could not find a single study, which utilized selenium as a single anti-oxidant to prevent testicular damage. Thus, the main aim of this study was to explore the potential protective effect of selenium in malathion induced testicular damage in mice.
       
The results of this present study showed that malathion produced testicular damage as revealed by necrotic and degenerative changes in the seminiferous tubules and edema in interstitial tissue whereas mice in malathion plus selenium group showed only few necrotic changes in some seminiferous tubules thereby signifying the protective effects of selenium. Furthermore, co-administration of selenium and malathion has ameliorated the reduction of the mean relative and final body weight, in addition to testicular weight as compared to malathion group. It also improved all the examined biochemical and antioxidant parameters.

Ince et al., (2017) observed reduced spermatogenic density and cell debris in testis of rats exposed to malathion. They also showed that malathion produced a significant increase in malondialdehyde levels whereas decreased glutathione levels, superoxide dismutase, catalase activities as well as serum and tissue acetylcholinesterase levels in rats (Ince et al., 2017b). Similarly, malathion has recently been showed to increase malondialdehyde and to decrease glutathione, acetylcholinesterase, superoxide dismutase, and catalase activities in the blood, liver, kidney, heart and brain tissues of rats (Akbel et al., 2018). Uzun et al., (2009) studied the protective effects of vitamin C and E in malathion induced testicular toxicity. They have observed that co-treatment of malathion-exposed rats with vitamins E and C had a protective effect on sperm counts, sperm motility and abnormal sperm numbers, but not on plasma FSH, LH or testosterone levels. Moreover, they have observed degenerative changes in the seminiferous tubules in the rats, which received malathion and supplemented with vitamins C and E. However, milder histopathological changes were observed in the interstitial tissues; results which were inferior to the results shown by selenium revealed in our study (Uzun et al., 2009). A possible explanation may be attributed to the dose needed to produce a protective effect as Vitamins C and E at concentrations that are similar to the levels found in plasma have been showed to produce no effect on malathion-induced toxicity in erythrocytes at a concentration of malathion (200 microM) that is typically used in pesticides (Durak et al., 2009). Also, selenium supplementation in Sertoli cells culture medium was recently shown to upregulate immune genes and blood-testis barrier constituent proteins of bovine Sertoli cells (Adegoke et al., 2018) and the testicular protective effect of selenium was suggested to be mediated through its anti-apoptotic in addition to its antioxidative effects (Kara et al., 2016).

CONCLUSION

The present findings revealed that selenium protects against malathion induced testicular damage in mice. Selenium improved the biochemical and antioxidant profile and improved the histopathological features when co-administered with malathion. Further researches to explore the mechanisms involved in such protection are highly recommended.

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