Background
Mercury has been utilized by humans since long and is still considered as one of the most important metalloids. Mercury is present naturally in the environment in three forms: elemental, inorganic as well as organic [
1]. Mercury enters into the atmosphere via two major sources: natural and anthropogenic. One of the most important natural sources of mercury is volcanoes, while other natural processes include outgassing of soil and water bodies, biomass burning, and geothermal processes [
2]. Mercuric chloride and methyl mercury have been declared to be highly carcinogenic by EPA [
3]. Approximately 2200 metric tons of mercury have been assessed to be released into the atmosphere annually [
4]. Elevated levels of mercury can impair the brain, nerves, kidneys, muscles of adults, and the developing fetus [
5‐
7]. Bronchitis, asthma, and temporary respiratory problems have been occurred due to inhalation or exposure to mercury vapors [
8]. Mercury exposure intoxicated reproductive activity via altering the hypothalamus-pituitary-adrenal and gonadal axis via impairing the circulating levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin, estrogen, progesterone, and the androgens [
8,
9]. Furthermore, occupational exposure to mercury vapors leads to infertility in both men and women and erectile dysfunction [
10]. The mercury-mediated pathogenesis of reproductive functions might be attributed to the abridged antioxidant defense system [
11]. Hence, treatment with antioxidants might ameliorate or reduce the adverse effects of mercury on reproductive ailments.
Chenopodium album Linn. (Family
Chenopodiaceae)
Bathua sag is a native plant of western Asia.
C. album belongs to genus Chenopodium, is a globally distributed plant, and has about 250 species. In India, it is cultivated as a vegetable.
C. album grows naturally as a weed in the fields of wheat, barley, mustard, gram, and other crops.
C. album is conventionally used as anthelmintic, cardiotonic, carminative, digestive, diuretic, and laxative [
12‐
14]. It is widely used in the treatment of peptic ulcer, dyspepsia, flatulence, strangury, pharyngopathy, splenopathy, ophthalmopathy, and general debility. Traditionally,
C. album is used for the cure of hepatic disorders, spleen enlargement, intestinal ulcers, and burns have also been recognized [
15].
C. album is a rich source of nutrients, antioxidants, and essential dietary elements [
11,
16]. Vitamin C and β-carotene are detected in the young shoots and mature plants of
C. album [
17]. Leaves contain trypsin inhibitor activity, phenols, tannins, saponin, phytic acid, phytate phosphorus, alkaloids, flavonoids, oxalates, oil, and proteins [
18]. The plant is a source of energy, proteins, carbohydrates, ascorbic acid and beta carotene, and minerals, such as potassium, sodium, calcium, phosphorous, magnesium, iron, and zinc. It has been screened for nutritional, phytochemical, antioxidant, and antibacterial activity [
19].
C. album possesses aphrodisiac activity, cures male infertility, and increases the copulatory efficiency respectively [
20]. Male rats and mice treated with ethanolic seed extract of the plant exhibited increased reproductive organs and body weight gain parallel with increased sperm count and sexual performance. There was an improvement in sexual behavior and performance and an increase in sperm count in male mice and rats [
20]. Nevertheless,
C. album seed extract (CAE) also caused sperm death due to oxidative damage induced by the generation of ROS [
21]. The DNA protective ability has also been evaluated in an animal model [
22].
Vitamin C is known for its antioxidant and DNA protecting properties. Vitamin C is an excellent radical scavenger that may neutralize the possible spermicidal and genotoxic effects of various free radicals and protect against the radicals, which are toxic by-products of many metabolic processes [
23]. Vitamin C reduces sperm agglutination and increases the fertility of men, while improving the sperm quality and male fertility [
23]. Studies have revealed the protective benefits of vitamin C against heavy metals [
24].
Methods
Chenopodium album Linn was collected from the agricultural and cultivated fields of Charsadda, Pakistan. The plant was identified by Dr. Mir Ajab (Department of Plant Sciences, Quaid-i-Azam University Islamabad) and the voucher specimen (0623451) was deposited at the Herbarium of Pakistan, Quaid-i-Azam University Islamabad. Seeds, together with the funicles, were air dried under the shade. They were stored in sealed bags until the preparation of the extract. Seeds were incubated at 25 °C, ground, and then sieved. The dried powder was then added to ethanol and placed at 4 °C in the dark for a week and was stirred every day for 30 min. The mixture was filtered with Whatman filter paper number 41 having a diameter of 150 mm. The filtrate was placed at room temperature until the solvent was evaporated. A 200 mg/kg dose of
C. album was selected based on the previous study in which the effect of the same dose was observed on sexual behavior and sperm count in male rats [
25,
26]. The ethanolic extract was suspended in 2% polyvinyl pyrrolidone and 100% stock solution was prepared.
Animals
A total of 35 adult male Sprague Dawley rats (Rattus norvegicus), allowing an average weight of 240 ± 15 g, were obtained from animal house of Quaid-i-Azam University Islamabad that were bred and kept in stainless steel cages. The animals were having free access to standard laboratory feed (Rat feed SPR 1002, Islamabad Feed Company, Pakistan) and water ad libitum. They were kept in an animal house according to the standard guideline, i.e., the temperature was maintained at 25 °C with light and dark cycle of 12 h. Before the onset of the study, the experimental protocol was approved by the ethical committee of the Department of animal sciences, Quaid-i-Azam University, Islamabad.
Experimental work
Animals were divided into five groups (n = 7). The first group received saline through a gavage (control group). The second group was treated with an i.p injection of Mercury (0.15 mg/kg b.w) dissolved in distilled water (Mercury group). The third group was the positive control group that received an i.p injection of Mercury (0.15 mg/kg b.w) dissolved in distilled water and vitamin C (200 mg/kg b.w) orally (vitamin C group). The fourth group was the co-treatment group that was treated with an i.p injection of Mercury (0.15 mg/kg b.w) dissolved in distilled water and Chenopodium album L. extract (200 mg/kg b.w) through gavage (extract treatment group). The fifth group received only Chenopodium album L. extract (200 mg/kg b.w) through gavage (extract group).
Chemical and applied dose-treatment
Mercuric chloride (Mercury) was purchased from BDH Chemicals (Ltd Pool, England). The applied dose-treatment (0.15 mg/kg) of mercuric chloride (Mercury) was carried out according to Schionning and Larsen [
27]. Vitamin C was bought from VWR Prolab (Geldena, Kseban), and 200 mg/kg dose was selected according to Uzun et al. [
28], showed same dose was effective against testicular toxicity induced by malathion in male rats. All the animals received doses for 30 days. After 24 h of the last dose administration, animals were euthanized by cervical dislocation.
Sample preparation
The blood was collected directly from the heart of each animal in heparinized syringes. Plasma was obtained by centrifugation of the blood at 3000 rpm for 10 min and was kept at – 20 °C until hormonal analysis. Testes, epididymis, and prostate were weighed. The relative organ weight was calculated using the following formula:
$$ \mathrm{Relative}\ \mathrm{organ}\ \mathrm{weight}=\mathrm{Organ}\ \mathrm{weight}/\mathrm{body}\ \mathrm{weight} $$
The left testis and epididymis were kept at − 70 °C for comet assay. The right testis and epididymis were utilized for histology. Both testis and epididymis were fixed 10% formalin and processed for histological investigation. Extra fresh-testicular tissue (180 mg) was homogenized in 10 volumes of 100 mM KH2PO4 buffer containing 1 mM EDTA, pH 7.4 and centrifuged at 12,000×g for 30 min at 4 °C. The supernatant was collected and used for the following assays.
Protein estimation
The total protein content of testicular tissue was determined by total protein kit (AMP diagnostics) using bovine serum albumin as a standard.
Hormone analysis
The plasma testosterone level was determined by ELISA kits (AmgenixInc, USA) according to the guideline provided by the manufacturer.
Biochemical analysis
Plasma urea nitrogen level (BUN), creatinine, cholesterol, triglyceride, high-density lipoprotein, and low-density lipoprotein concentrations were determined by using the AMP diagnostic kit (AMEDA labordiagnostikGmbh, Austria), and were analyzed on Picco 5 chemistry analyzer.
Assessment of oxidative stress markers
Reactive oxygen species (ROS) activity was detected in testicular tissue of control and treated animals. Absorbance was checked at 505-nm Smart Spec TM plus Spectrophotometer. The amount of TBARS as an index of lipid peroxidation was assessed by measuring the optical density of the supernatant at 535 nm using a spectrophotometer against a reagent blank using the method of Tyan and coworkers [
29]. The results were expressed as nM TBARS/min/mg tissue at 37 °C using a molar extinction coefficient of 1.56 × 10
5/Mcm.
Assessment of antioxidant status
Catalase activity was determined by the method of Afsar and colleagues [
30]. Changes in absorbance of the reaction mixture at 240 nm were determined after 1 min. One unit of CAT activity was defined as an absorbance change of 0.01 as units/min. Peroxidase activity was determined by the method described previously [
31]. Changes in absorbance of the reaction solution at 470 nm were determined. One unit of POD activity was defined as an absorbance change of 0.01 as units/min. Superoxide dismutase activity was estimated following previous protocols [
30]. Amount of chromogen formed was measured by recording the color intensity at 560 nm. Results are expressed in units/mg protein. For glutathione-S-transferase activity, changes in the absorbance of the reaction mixture were recorded at 340 nm and enzyme activity was calculated as nM CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6 × 103 M
−1 cm
−1.
Assessment of daily sperm production
Spermatids were counted by the method described previously [
32]. Shortly, frozen testis was thawed at room temperature, tunica albuginea was removed, and then parenchyma was weighed. It was homogenized in 5 ml of 0.9% NaCl solution (which included 0.5% triton X-100). Twenty microliters of the sample was placed into Neubauer chambers and quantity of late spermatids was counted under the microscope at × 400 magnification. This provides the total number of spermatids per testis. The number was used for determination of the number of spermatids per each gram (g) of testicular tissue which is the efficacy of sperm production. For the calculation of daily sperm production (DSP), the number of spermatids (per testis and per g of testis) was divided by 6.3 (DSP = Y/6.3).
DNA damage analysis
Comet assay
DNA damage was estimated by comet assay and separate spermatozoa were evaluated by using a modified basic Single Cell Gel Electrophoresis (SCGE/comet assay) according to the method of [
33]. The basic steps of comet assay include lysis of membranes to release DNA, single-strand DNA formed by contact to alkali (pH − 13), neutral electrophoresis, DNA staining and analysis, and comet scoring.
Histopathological examination
Histology of testis and epididymis was performed after removal of tissues from animals and the following steps were carried. Tissues were placed in 10% formalin for 24–48 h and then dehydrated in the ascending grades of ethyl alcohol, cleared in xylene, and mounted in molten paraffin wax 58–62 °C. Seven-micrometer histological sections were cut and stained with hematoxylin and eosin and observed under the light microscope (Nikon, 187842, Japan) at × 40 and microphotography was performed by Leica LB microscope connected to Olympus Optical Co. LTD, Japan.
Morphometric analysis
For morphometric studies, the seminiferous tubule diameter and germ cell height were measured by using Image J software (National Institute of Health, Bethesda, MD, USA) [
34]. Images were taken at × 20 and × 40 and morphometry was done using software Image J. Area of the seminiferous tubule, epididymis tubules, and interstitial space was determined by planimetry, using Image J software. An area in μm
2 was calculated by the method of Islam et al., (2010) [
34] and according to the [
35]. Shortly 25 pictures at × 20 per animal of the known area were selected and the area of seminiferous tubules, epididymis tubule, and interstitial space was determined by free selection tool of the software. The area % age was calculated by the formula:
% As = As × 100/T
where As is area covered by seminiferous tubules. T is the total area of the field. Percentage of the mean area was analyzed for comparison between treated and control groups and was reported.
Statistical analysis
Data were subjected to one-way analysis of variance (ANOVA) followed by Tukey’s test for comparison of different groups using GraphPad Prism software. All the results are shown as Mean ± SEM and the significance level was set at < 0.05.
The sample size for the current study was calculated by the resource equation method [
36] by using the following formula:
E = Total number of animals − Total number of groups
where
E is the degree of freedom of analysis of variance (ANOVA). The value of
E should lie between 10 and 20 to increase the chance of getting a more significant result. As this method is based on ANOVA, it is applicable to all animal experiments [
37].
In the present study, we made four groups with seven animals each.
$$ E=\left(7\times 5\right)-5\;E=\left(7\times 5\right)-5 $$
$$ E=35-5=30\kern0.2em E=35-5=30 $$
This sample size is adequate as chances of death of animals cannot be ignored.
Discussion
Exposure of human and animals to either inorganic or organic forms of mercury results in the creation of reactive oxygen species (ROS) and oxidative stress, which cause cell death. Oxidative damage may consequence from declined clearance of ROS by scavenging mechanisms that negatively distress male reproductive systems of humans and animals [
38]. The present study investigated the ameliorating ability of
C. album Linn. and vitamin C against oxidative stress produced by Mercury in the reproductive tissues of male rats. Also, the effect of
C. album was compared with vitamin C which served as the positive or standard control.
In previous literature, variable effects of Mercury on animal growth have been reported [
7]. Our results are in agreement with the reports of Mehboob and coworkers, who reported a decrease in body weight in Mercury-intoxicated group [
7,
39]. In the current investigation, we observed that
C. album and vitamin C ameliorated mercury-induced weight loss. The loss of body weight in Mercury-treated animals is reported in other researches as well [
40]. Improvement of body weight in
C. album and vitamin C groups indicated that might preclude Mercury intoxication and our findings are in accord with the previous findings [
41]. The testicular weight was slightly reduced in Mercury-treated animals, but overall, there was no significant change in all the groups. This is in accordance with the findings of Khan and colleagues [
42]. Also, the testicular weights of vitamin C and
C. album extract groups were almost similar to each other. Chatterjee and Pakrashi showed that the effect might be due to the greater availability of testosterone or similar steroidal compounds to gonads as
C. album is also known to contain compounds of steroidal nature [
43].
The Mercury-treated group showed a highly significant decrease in protein values. This result goes parallel with previous findings [
44]. Several reasons can account for these low levels of protein like the interaction of Mercury with thiol groups of amino acids [
45], through oxidative stress and possibly by indirectly inhibiting the protein synthesis. This inhibition might be due to disruption of the protein synthetic machinery [
6,
46], inactivation of RNA polymerase I by binding to Mercury [
47], or partly due to an autoregulatory reduction in tubulin synthesis [
48]. The vitamin C and
C. album groups have slightly higher values than the Mercury-treated group. Similar values have been observed by the previous study where higher protein values following
Chenopodium album L. administration were observed [
49].
In the current study, Mercury exposure is allied with increase oxidative stress biomarkers (ROS and TBARS) in the testis of rats accompanied by declined in antioxidant enzymes (SOD, CAT, and GST). Previous studies also validate Mercury-induced elevation in TBARS level in testicular tissue [
12]. Treatment of
C. album attenuated the Mercury-induced rise in oxidative stress markers, which indicates that
C. album may have an important role in reducing Mercury toxicity. The effect of
C. album is comparable with standard vitamin C group. Studies revealed that
C. album contains vitamin C and E which play a vital role in hampering the absorption of Mercury in the gastrointestinal tract [
11] and prevent ROS-mediated membrane damage by binding up ROS within the cell membrane [
50]. It is postulated that the antioxidant CAT, SOD, and POD depletion by mercury may be a trigger for the production of reactive oxygen species (ROS) that induce lipid, protein, and DNA oxidation. As SOD, CAT and GST are the first line of defense against free radical damage [
38,
51]. Decrease antioxidant content in testicular tissues made spermatogenic cells more susceptible to oxidative stress, especially during increased free radical production [
52]. Hence,
C. album by enhancing the endogenous antioxidants prevents the Mercury-induced oxidative stress.
Mercury treatment has been reported to produce a reduction in sperm quantity (testis and epididymis) and daily sperm production, followed by decrease in sperm motility while increase in head and tail morphologic abnormalities were observed [
53]. Similarly, Mercury-treated group showed significantly decreased values for DSP. Previous reports indicated that vitamin C supplementation significantly increased sperm count, sperm morphology, and sperm mobility in human semen [
54,
55] and/or by mitigating physiological toxicity.
C. album extract is reported to increase sperm count and sexual behavior and also its seed extract is reported as a potent sperm immobilizing agent [
20,
56].
DNA damage in sperms is assessed by comet assay. Mercury compounds have been reported to cause DNA breaks by means of free radical-mediated reactions that may lead to an increase in the occurrence of spermatozoa with anomalous heads [
38]. Protection of DNA by vitamin C in sperms against endogenous oxidative damage was evident in previous reports [
20].
C. album protects DNA damage in a manner similar to vitamin C. The antioxidant potential of
C. album might benefit in protecting against Mercury-induced sperm DNA damage.
Regarding the hormone levels, a decrease in the plasma testosterone was observed in the Mercury-treated group. This decline in testosterone level appears to be due to a drop in the activity of enzymes engaged in the biosynthesis of testosterone or due to the diminish in testicular cholesterol, a precursor of testosterone synthesis [
57]. Sperm concentration in the epididymis may decline due to miniature sperm production in the testis, which could be allied to a low level of testosterone, a chief regulator for sperm production. Similarly, growth of accessory sex glands needs testosterone [
58], and the decline in the weight of these glands (Table
1) due to mercury treatment can result from the drop in the testosterone levels (Fig.
1). Histological examination has also shown a decrease in luminal spermatozoa when exposed to mercury (Figs.
2 and
3). The reduction in epididymal sperm concentration is consistent with the histologic examination of the seminiferous tubules, which showed a decrease in luminal spermatozoa in Mercury-exposed rats (Figs.
2 and
3). In the current examination, the decline in sperm number/epididymis weight and motility was allied with a rise of sperm aberrations in rats exposed to Mercury, which showed that Mercury impedes with spermatogenesis by crossing the blood-testis barrier and gaining access to germinal cells. The unfavorable effects of Mercury on mammalian testicular tissue have been unveiled with marked testicular spermatogenic degeneration at the spermatocyte level in rats [
59]. Several active components in
C. album may forage ROS generated by Mercury, lower down lipid peroxidation, and ignite the activity of antioxidant enzymes whereby leading to protection against Mercury-induced testicular damages which are manifested by an increase in sperm abnormalities and decline in testosterone level. The improvement of sperm quality may be due to the antioxidant components of
C. album, such as α-tocopherol (vitamin E) and ascorbic acid (vitamin C) that improve testicular functions and sperm quality [
60,
61].
Limitations of the study
Before making a certain proclamation about the suitability of C. album as a protective agent against Mercury-induced testicular toxicity, further studies are essential to be undertaken in order to clarify the mechanism at the molecular level. For this, western blot analysis and immunohistochemistry would provide evidence regarding signaling pathway in the pathogenesis of testicular damage and protection afforded by the plant extract. Additionally, the effect of plant extract at different doses will provide understanding on the more appropriate prescribed amount because for natural plant investigations, the dose used, especially content of antioxidants, is always blurry, not to indicate the shift dose for humans. In animal study, antioxidants are given to animals via oral or intraperitoneal injection. Bearing in mind, ROS and oxidative stress act positively in certain circumstances and the variance between animals and humans, the effective dose and safe dose, duration of treatment, absorption, and bio-availability of antioxidants require thorough investigations. Therefore, in the future, large-scale samples and appropriate duration studies against various organ toxicities and protective influence of C. album should be performed.
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