Introduction

Mercury is a widespread environmental and industrial pollutant [1]. It is known that mercury may cause accidental and occupational exposures and poisoning can result from inhalation, ingestion, and absorption through the skin [2–4]. People can be exposed to mercury through contaminated water and food [5]. The kidney, liver, gastrointestinal system, and central nervous system are the main target sites of mercury toxicity [6]. Past studies have already documented the deleterious effects of heavy metal toxins in humans which may induce lipid peroxidation and may promote oxidative stress in tissues [7–10]. Lead and mercury exposure, air pollution, and organic compounds all have the potential to damage brain functioning yet remain understudied [6].

Thiol-containing enzymes have been recognized as the targets of inorganic Hg. Moreover, binding of mercuric ions to thiol groups may cause decreased glutathione (GSH) levels, leading to increases in levels of reactive oxygen species (ROS), such as superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals, which provoke lipid, protein, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) oxidation [1, 11, 12]. Considering that oxidative stress and endogenous thiol depletion are involved in inorganic Hg toxicity, it has been suggested that antioxidants could contribute to the treatment of Hg poisoning [13, 14]. Antioxidants like melatonin, curcumin, and vitamin E have been found to play a protective effect against mercuric chloride (HgCl2)-induced acute renal toxicity [15–17]. Similarly, a number of plant extracts with antioxidant properties have been shown to inhibit HgCl2-induced toxicity [16, 18, 19].

Another important trace element in the metabolism is selenium (Se). Se has the ability to counteract free radicals and protects the structure and function of proteins, DNA, and chromosomes against the injury of oxidation [20]. Several studies reported that selenium protects against the toxicity of heavy metals [4, 21–29]. Se can effectively counteract oxidative damage toxic agents caused by scavenging reactive oxygen radicals and protect membrane integrity. Se is considered as an essential trace mineral for living organisms, it is a structural component of several enzymes like glutathione peroxidases and thioredoxin reductase, and its role against mercury intoxication [6, 24, 30, 31]. Selenium has been found to have detoxification effects on various heavy metals [32].

It is also known that Se alters the Hg distribution in the organism, and due to this effect, Hg toxicity can be reduced [33, 34]. Moreover, Se can reduce the Hg toxicity through the prevention of oxidative damage [24, 35]. Therefore, the evaluation of toxic potentials of metals is important for the risk assessment of human beings ordinarily exposed to these substances.

Materials and Method

Maintenance of Animals

Male Wistar rats weighing approximately 180–200 g were procured. The animals were acclimatized for 7 days prior to experiments. The institutional ethics committee approved the experimental protocols. All the animals used in this study were placed in cages in an air-conditioned room maintained at a temperature of 25 ± 30 °C and 12-h light/dark schedule.

Experimental Protocol

Animal Treatments

Different groups of animals were used to study the effects of Se on mercuric-induced oxidative stress. In total, 24 animals were divided into four groups of six rats each. Group I received saline injection intraperitoneally (0.85 % NaCl) at a dose of 10 ml/kg bodyweight. Group II received a single intraperitoneal injection of mercuric chloride at a dose of 50 mg/kg bodyweight. Groups III and IV received pretreatment with Se i.p. once a day for 7 days at a dose of 0.2 mg/kg bodyweight. After the last treatment with Se, the rats of group IV received a single intraperitoneal injection of HgCl2 at a dose level of 50 mg/kg body weight. After 24 h of the last administration, the animals were euthanized under mild ether anesthesia.

The blood samples were collected in test tubes without anticoagulant. The samples were centrifuged at 3000 rpm for 15 min and the clear serum was carefully separated from all samples and stored at stored at −80 °C. The concentrations of IL-6, TNF-a, and IL-10 in the serum were measured using commercially available enzyme amplified sensitivity immunoassay kits (BioSource). All assays were conducted according to the manufacturer’s instructions. The samples, which have shown higher concentrations, were diluted and measured in duplicate.

For estimation of trace elements, the serum was separated and diluted with double-distilled water. The samples were then analyzed following established procedures for trace elements by means of a Unicam 929 atomic absorption spectrophotometer.

Malondialdehyde and Superoxide Dismutase Measurements

The oxidant–antioxidant status of the rat was assessed by determining the level of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD). Lipid peroxidation was determined by measuring the level of MDA, which is considered to be a standard marker for oxidative lipid damage. Serum MDA levels were measured by the method of Draper and Hadley [36]. The results were expressed as micromoles per liter. Serum SOD activity was measured by the method of Sun et al. [37]. The results were expressed as units per liter.

Statistical Analysis

Results were expressed as the mean ± standard error of the mean (SEM). Data for multiple variable comparisons were analyzed by one-way analysis of variance (ANOVA). For the comparison of significance between groups, Duncan’s test was used as a post hoc test according to the Statistical Package for the Social Sciences (SPSS version 17.0). All p values are two-tailed and p < 0.05 was considered significant for all statistical analysis in this study.

Results

Mercury is known to cause disturbance in immune response. In this study, its effect on the inflammatory markers TNF-α, IL-6, and IL-10 in the serum was studied (Table 1). Intraperitoneal injection of HgCl2 to rats resulted in a significant decrease in these biomarkers (compared group II with group I). However, pretreatment of HgCl2-injected rats with 0.2 mg/kg body weight selenium per day for 7 days resulted in the significant prevention of this decrease in the biomarkers (compared group IV with group II). Selenium by itself in group III did not affect the levels of these inflammatory markers when compared to group I rats. These results suggest that mercury-induced decrease in the inflammatory markers is significantly prevented with the pretreatment of selenium in these rats (p < 0.05).

Table 1 Levels of serum TNF-α, IL-6, and IL-10 in rats
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Next, the effect of mercury on the levels of trace elements in the serum of these rats was studied. Again, exposure of these rats to mercury resulted in a decrease of Cu, Zn, and Fe. The decrease in Cu was not statistically significant when compared to control rats in group I (Table 2). However, Zn and Fe were reduced significantly by mercury injection and this decrease was prevented by the treatment with selenium in group IV (p < 0.05).

Table 2 Serum trace elements concentrations in rats
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Mercury is known to increase oxidative stress and, therefore, levels of MDA and SOD in these rats were also measured. Administration of mercury chloride to rats resulted in a significant increase in the levels of MDA when compared to saline injected control rats (Fig. 1). Administration of selenium alone in group III resulted in an insignificant decrease of MDA compared to control group. Pretreatment of mercury chloride-injected rats in group IV with selenium significantly prevented the increase in MDA levels that was seen in group II rats.

Fig. 1
figure 1

Serum malondialdehyde (MDA) levels in all groups. a and b indicate statistically significant differences with those of control groups (p < 0.05)

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Also exposure of rats to mercury resulted in a significant decrease in the levels of SOD in serum and this decrease was reversed by the treatment with selenium (Fig. 2). However, there were no significant differences between the same parameters in group II (Se-alone treated) and group 1 (control). These data suggest that administration of mercury caused oxidative stress in rats which was significantly blocked by the treatment with selenium (p < 0.05).

Fig. 2
figure 2

Serum superoxide dismutase (SOD) levels in all groups. a and b indicate statistically significant differences with those of control groups (p < 0.05)

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Discussion

Several reports have suggested that all three forms of mercury (vapor, inorganic, and methyl mercury) are associated with human health hazards. However, in general, for nonclinical studies, efficacy and safety pharmacological studies are performed keeping in mind target organ toxicities, dose selection, and known lethal dose. Mercury is most toxic of all the heavy metals [22, 38–43] and is known to induce toxicity in the cardio respiratory system, reproduction system, kidneys, liver, brain, and lungs [3, 44–46]. Humans are exposed to these metals from numerous sources, including contaminated air, water, soil, and food. Selenium has been shown to alter heavy metal toxicity specially mercury which cause adverse effects on the various tissue parameters [15, 24, 31, 47–54].

Therefore, the aim of the present study is to study possible mitigating effect of Se against acute HgCl2 toxicity based on oxidative stress and inflammation induction in rats. Results show significant changes in serum Cu, Fe, and Ca levels found in the rats after mercury treatment when compared with controls. Conditioned deficiencies of trace elements may develop in states of decreased absorption or excessive excretion or utilization [55].

The present study was also designed to evaluate the protective effect of sodium selenite treatment on IL-6, TNF-α, and IL-10 in serum of mercuric chloride intoxicated rats. IL-6, IL-10, and TNF-α are effective cytokines of inflammation and endothelial functions. In this study, mercury caused decrease in the levels of IL-6 released from mononuclear phagocytes and TNF-α and IL-10 released from monocytes. The levels of these cytokines, which have different synthesis locations and functions, were decreased together as observed. Therefore, it can be said that intraperitoneal administration of mercury had an effect on the inflammatory process as mercury reduces the levels of IL-6, TNF-α, and IL-10 that are inflammatory cytokines.

Also in this study, supplementation of Se to the mercuric chloride-treated groups ameliorated malondialdehyde and SOD activities. Earlier, it has been reported that major role of sodium selenite is in inhibiting lipid peroxidation and in protecting the wholeness and functioning of tissues and cells [56]. It was observed that serum MDA levels increased while serum SOD levels decreased in the serum of the rats after HgCl2 treatment. The rise in MDA and decrease in SOD could be due to the increased generation of reactive oxygen species due to the excessive oxidative damage generated in rats. Oxidative stress refers to excessive generation of reactive oxygen species [57]. MDA content manifests the level of lipid peroxidation and indirectly represents the level of damage of the cell and tissue [30]. Results of the present study showed that the amount of MDA was very high in mercuric chloride-treated rats which were supported by the previous studies [2, 58]. The elevated level of MDA might be due to enhanced formation of free radicals.

In conclusion, the present study showed that mercuric chloride intoxication caused reactive oxygen species generation which in turn induced biochemical alterations in rats. Administration of sodium selenite proved to be beneficial in attenuating the mercuric chloride-induced oxidative toxicity.