Evaluation of nanoselenium (Nano-Se) effect on hematological and serum biochemical parameters of rat in experimentally lead poisoning

Abstract

The present study was designed to evaluate the effect of nanoselenium (Nano-Se) on hematological and biochemical parameters of rats experimentally intoxicated with lead (Pb). Thirty male rats were randomly divided into six groups as follows: the control, selenite, Nano-Se, Pb group, Pb + selenite, and Pb + Nano-Se groups. After 35 days, blood was collected from rats and hematology and serum biochemical parameters of oxidative stress were measured. The thiobarbituric acid reactive substances (TBARS) level of Pb group was significantly higher than other groups. Also, TBARS level was significantly lower in the Pb + Nano-Se group than Pb + selenite group. The serum superoxide dismutase activities were significantly lower in Pb group than the control, Pb + selenite, and Pb + Nano-Se groups. The catalase activities in the Pb group showed no significant change when compared to other groups. In the Pb group, packed cell volume was lower than the control group. A significant difference was observed between the control group and the Pb, Pb + selenite, and Pb + Nano-Se groups. In the Pb group, the numbers of white blood cell (WBC) decreased in comparison with the control group. Also, there was significant increase in WBC counts in the Pb + Nano-Se and Pb + selenite groups in comparison with Pb group. The number of lymphocytes in the Pb group decreased in comparison with the control group. By comparing the means of the Pb + Nano-Se and Pb + selenite groups together, it was determined that there were significant differences in the lymphocytes and neutrophil counts. In conclusion, usage of selenium compounds particularly Nano-Se particles inhibits the adverse effects of Pb on antioxidant activity and immune system function in the Pb poisoning.

Keywords

Nanoselenium sodium selenite lead poisoning oxidative stress

Introduction

Selenium is an essential trace element for human and animals and selenium deficiency results in many disorders in humans and animals including cardiomyopathy. Selenium has been shown to have numerous anticarcinogenic or preventative effects against carcinogen-induced breast, colon, liver, and skin cancer in humans and animals at nutritional levels.¹ It has biological role in the active center of glutathione peroxidase; the key enzyme for tissues and body fluids which plays basic defensive role in the antioxidant protection system.²

Currently, sodium selenite is the most common selenium source used in animal feeds, whereas organic forms such as selenium-enriched yeast and selenomethionine are also used in many countries.²

With the recent development of nanotechnology, nanoselenium (Nano-Se) has attracted widespread attention as a result of nanometer particulates display novel characteristics such as a large surface area, high surface activity, high catalytic efficiency, strong adsorbing ability and low toxicity.^2,3 It has been reported that Nano-Se has comparable efficiency to selenite and Se-methylselenocysteine in upregulating selenoenzymes but with dramatically decreased toxicity.³

Lead (Pb) poisoning (also known as plumbism) is a common heavy metal poisoning in domestic and wild animals as well as humans. Pb toxicity depends on its chemical form, the route of exposure, frequency, and duration of consumption. Pb causes many adverse health effects and is toxic to many organs and tissues including the heart, bones, intestines, kidneys and hematopoietic and reproductive, endocrine and nervous systems. It intervenes with the development of the nervous system and is therefore particularly toxic to children, causing potentially permanent learning and behaviour disorders. Routes of exposure to Pb include contaminated air, water, soil, food and consumer products. Environmental Pb pollution is widespread in industrialized societies and poses a number of health hazards.⁴ Based on the estimations made by the National Institute of Occupational Safety and Health, more than 3 million workers in the United States are potentially exposed to Pb in the workplace.⁵

Numerous experimental studies on the low levels of Pb exposure have been performed using the laboratory animals. These experiments have demonstrated a variety of effects such as a delay in cortical synaptogenesis, a slower cumulating of cytochrome in layers I, II and III of the parietal cortex, development delays in exploration and in locomotor activity and slowing of discriminative learning.^6
–8 The ameliorative effect of micronutrients on toxicity and carcinogenicity of heavy metals has been reported. Micronutrients can affect toxicity of heavy metals by interacting with the metal at its primary site of action. Examples of this type of interaction include the effects of calcium on Pb, phosphate on arsenate and zinc on cadmium.⁹ The purpose of this study was to evaluate the effect of Nano-Se on oxidative status and hematological parameters of rats exposed to lead acetate.

Experimental

Animals and treatments

Thirty male Wistar rats (150 ± 10 g) were used in this study. The animals were housed in stainless steel wire cages at 23 ± 1°C and exposed to 12-h light/12-h dark cycle in air-conditioned room. They had access to a standard commercial rodent laboratory diet (Javaneh Khorasan, Mashhad, Iran) and drinking water ad libitum throughout the whole experimental period. After 1-week-long acclimatization period, the animals randomly divided into six equal groups (containing each 5 rats) according to the dietary treatments applied for 35 days:

Control group; received normal ration.

Selenite group; received 100 µg/animal of sodium selenite through gastric gavage.¹⁰

Nano-Se group; received 100 µg /animal of Nano-Se through gastric gavage.¹⁰

Pb group; exposed to 1000 ppm of lead acetate (Merck, Germany) in drinking water.¹¹

Pb + selenite group; exposed to 1000 ppm of lead acetate in drinking water and 100 µg/animal of sodium selenite through gastric gavage.

Pb + Nano-Se group; exposed to 1000 ppm of lead acetate in drinking water and 100 µg/animal of Nano-Se through gastric gavage.

The animals were observed daily for signs of toxicity. Food intake and body weights were recorded daily during the experimental period. At the end of experimental period (on day 35), animals were anesthetized with ether and blood samples collected from the heart.

Biochemical assay

Serum superoxide dismutase (SOD) activity was estimated using the nitroblue tetrazolium (NBT) dye reduction test.¹² In brief, 0.1 mL serum was added to 2 mL reaction mixture, which contained 0.20 mM xanthine, 0.12 mM NBT, 0.049 IU xanthine oxidase and 0.1 M phosphate buffer (pH 7.0). The mixture was incubated for 20 min at 37°C. In the reaction mixture, the superoxide radical is produced, which reduces NBT to a blue colour dye, NBTH₂. The rate of reduction of NBT was measured at 560 nm. The results for SOD activity were expressed as the per cent inhibition of reduction of NBT by SOD.

The serum catalase activity was assayed according to the method of Goth (1991).¹³ Briefly, 0.1 mL of serum sample was incubated in 1 mL reaction mixture that contained 50 mM potassium phosphate buffer (pH 7.0) and 10.6 mM hydrogen peroxide (H₂O₂) freshly prepared at 37°C for 60 s. The reaction was terminated by adding 0.5 mL of 32.4 mM ammonium molybdate solution. A yellow complex of ammonium molybdate and H₂O₂ was formed. The absorbance of this yellow colour was measured at 405 nm with a spectrophotometer (Unico 2100) against a blank (serum was replaced with distilled water). One unit of catalase activity was defined as the amount of enzyme that catalyses the decomposition of 1 mol of H₂O₂ per minute.

Serum malondialdehyde (MD) concentrations, also known as thiobarbituric acid reactive substances (TBARS), were determined colorimetrically using the method of Buege and Aust (1978).¹⁴ In brief, 0.1 mL of serum was treated with 2 mL of TBA–TCA–HCl reagent (thiobarbituric acid 0.37%, 0.25 N HCl and 15% TCA) and placed in water bath for 15 min. After cooling, the flocculent precipitate was removed by centrifugation at 112 × g for 10 min. The absorbance of supernatant was measured against reference blank at 535 nm. Concentration was calculated using an extinction coefficient of 1.56 × 105 M⁻¹ cm⁻¹ and expressed as nanomole per litre.

Hematological assay

Hematological parameters including red blood cell count, packed cell volume (PCV) value and white blood cell (WBC) counts were measured by routine procedures.¹⁵ WBC measurement was conducted using the manual standard method. Differential leukocyte counts were performed on routinely prepared Geimsa-stained blood films using the cross-sectional technique.¹⁶

Statistical analysis

To compare differences between groups non-parametric as Kruskal–Wallis analysis of variance test and U Mann–Whitney test was used. Significance was declared at p ≤ 0.05.

Results and discussion

The effect of sodium selenite and Nano-Se supplementation during Pb poisoning on the hematological parameters and oxidative status of rats are shown in Tables 1 and 2. The percentage of protective effect of sodium selenite and Nano-Se in Pb-treated groups are shown in Figure 1.

Table 1.

The effect of Se and Nano-Se supplementation during Pb poisoning on the oxidative status in rats.^a

	Control	Selenite	Nano-Se	Pb	Pb+selenite	Pb+Nano-Se
TBARS	1.22 ± 0.14	1.5 ± 0.18^c	1.22 ± 0.13^d,g	3.25 ± 0.16^b,c,e,f	2.56 ± 0.59^b,c,d	1.94 ± 0.13^d
SOD	17.45 ± 2.01	15.05 ± 1.58	18.85 ± 1.55^d	9.22 ± 0.66^b,c,g	13.44 ± 2.11^c	16.71 ± 1.35^d
Catalase	6.9 ± 0.73	4.69 ± 0.87	5.34 ± 0.45	4.32 ± 0.73	4.94 ± 0.42	4.32 ± 0.73

TBARS: thiobarbituric acid reactive substance; SOD: superoxide dismutase; Pb: lead.

^aUnits: TBARS: nmol/mL; SOD % inhibition and catalase: U/L.

Values are mean ± SEM.

^bSignificant as compared with control group.

^cSignificant as compared with Nano-Se group.

^dSignificant as compared with Pb group.

^eSignificant as compared with selenite group.

^fSignificant as compared with Pb + selenite group.

^gSignificant as compared with Pb + Nano-Se group.

Table 2.

The effect of Se and Nano-Se supplementation during Pb poisoning on the hematological parameters in rats.^a

	Control	Selenite	Nano-Se	Pb	Pb + selenite	Pb + Nano-Se
PCV	44 ± 2.02	42 ± 5.76	42 ± 1.58	32.8 ± 0.8^b,c,e	36 ± 3.8^d	38 ± 4.21^d
WBC	8010 ± 492.29	3840 ± 222.71ac	4220 ± 414 ac	2187.5 ± 344.22 abd	3587.5 ± 963.36c	3720 ± 195.32c
Lymphocyte	6429.8 ± 499.25	2803.6 ± 151.78^b	3236 ± 535.92^b,d	1729.25 ± 260.77^b,c,g	3073.2 ± 250.59^b,d	2557.25 ± 569.08^b,d
Monocyte	122 ± 103	99 ± 13	91 ± 23^d	78 ± 0.00	100 ± 0.00	82 ± 36
Eosinophil	251 ± 5	224 ± 56	164 ± 0.00	184 ± 7.5	252 ± 0.00	177 ± 0.00
Neutrophil	1308.2 ± 79.36	946.8 ± 136.631^d	914.8 ± 168.1^d	353 ± 71.59^b,c,e,f,g	568.4 ± 81.38	1219.75 ± 325.54^d

Pb: lead.

^aValues are mean ± SEM.

^bSignificant as compared with control group.

^cSignificant as compared with Nano-Se group.

^dSignificant as compared with Pb group.

^eSignificant as compared with selenite group.

^fSignificant as compared with Pb + selenite group.

^gSignificant as compared with Pb + Nano-Se group.

Figure 1.

The percentage of protective effect of sodium selenite and Nano-Se in Pb-treated groups. Nano-Se: nanoselenium; Pb: lead.

Pb exposure of rats in the Pb group significantly (p < 0.05) increased TBARS in comparison to the control group. Also, treatment with sodium selenite of rats in the Pb + selenite group and Nano-Se of rats in the Pb + Nano-Se group decreased the levels of TBARS, that is, was statistically significant in the Pb + Nano-Se group (p < 0.05).

SOD activities were significantly (p < 0.05) lower in the Pb group in comparison to the control (17.45 ± 2.01), Nano-Se (18.85 ± 1.55) and Pb + Nano-Se (13.44 ± 2.11) groups.

Catalase activities in the Pb group showed no significant change (p > 0.05) when compared to others groups. However, the lowest activity of catalase was observed in the animals of group Pb + Nano-Se.

The percentage of TBARS levels decreased to 63.47% and 83.76% in the Pb + selenite and Pb + Nano-Se groups, respectively, in comparison to the Pb-treated group.

The percentage of SOD activities increased to 72.88% and 90.61% in the Pb + selenite and Pb + Nano-Se groups, respectively, in comparison to the Pb-treated group.

The percentage of catalase activities increased to 57.17% and 50% in the Pb + selenite and Pb + Nano-Se groups, respectively, in comparison to the Pb-treated group.

PCV in rats of the Pb group was lower than that in the rats of the control group. A significant difference (p < 0.05) was observed between the control group and Pb, Pb and selenite and Nano-Se groups, Pb + Nano-Se and Pb + selenite groups.

The number of lymphocytes in the Pb group decreased to 1729.25 ± 260.77 in comparison with the control group (6429.8 ± 499.25). By comparing the means of the Pb + Nano-Se and Pb + selenite groups together, it was determined that there were significant differences in the lymphocyte counts.

The number of eosinophils and monocytes in the lead acetate group showed no significant change (p > 0.05) when compared to other groups.

Table 2 shows that the number of neutrophils in the lead group decreased to 353 ± 71.59 in comparison with the control group (1308.2 ± 79.36). By comparing the means of the Pb + Nano-Se and Pb + selenite groups together, it was determined that there were significant differences in the neutrophil counts.

Numerous evidences indicated that cellular damage mediated by reactive oxygen species (ROS) may be involved in the pathology related with Pb intoxication.¹⁷ A potent correlation between blood Pb concentration and MD levels in blood of Pb-exposed workers has been reported.¹⁸ In erythrocytes, from the workers exposed occupationally to Pb, the activities of the antioxidant enzymes, SOD and GPx were considerably lower than the non-exposed workers. There is accumulating evidence that exposure to the heavy metal results in alteration of antioxidant enzyme activities and serum MD content.⁵

In the present study, Pb exposure of rats in the Pb group significantly increased TBARS level (p < 0.05) as compared to the control group. However, treatment of Pb-exposed rats with Se and Nano-Se in Pb + selenite and Pb + Nano-Se groups, respectively, significantly decreased the serum TBARS level (p < 0.05), showing protective effects against Pb caused oxidative stress.

Pb results in oxidative stress by inducing the generation of ROS, diminishing the antioxidant defense system of cells through evacuating glutathione, interfering with some essential metal, inhibiting sulphydryl-dependent enzymes or antioxidant enzyme activities and/or increasing susceptibility of cells to oxidative attack by shifting membrane integrity and fatty acid composition.⁵ The binding activity of Pb compounds with oxidative stress parameters and with the production of ROS, such as H₂O₂ and it’s interaction with different metals and also toxic activity of amminolevulinic acid (ALA) are reported earlier.^19,20

Increased generation of lipid peroxide (MDA) in the lead acetate-exposed group may be due to rise of ALA in blood after Pb exposure. Pb is also associated with an inhibition of the enzyme delta-aminolevulinic acid dehydratase (ALA-D), resulting in a failure of utilization of delta-aminolevulinic acid which is excreted in increased quantities in the urine. Much research, both experimental and on cell breeding, confirms that ALA may produce ROS in biological system.^21,22 It is proposed from the higher serum MDA content associated with decreased activity of SOD and catalase in the Pb group may be a part in the increased membrane lipid peroxidation.⁵

Also, there is a rupture of the lysosomal membranes, the release of lysosomal enzymes that results in necrosis of the cells and destruction of the parenchymal tissue. All these processes maximize an increase in serum MDA level. Adonaylo and Oteiza (1999) investigated oxidative damage associated with the presence of Pb in the brain from rats chronically exposed to the metal (1 g lead acetate/1 drinking water). After 8 weeks of treatment, intoxicated rats showed lower body weight and lower hematocrit and 5-aminolevulinic acid dehydratase activity as compared to controls. The content of brain 2-thiobarbituric acid-reactive substances (TBARS), an indicator of lipid oxidation, was significantly (p < 0.05) higher in the intoxicated animals than in controls. Higher activities of the antioxidant enzymes glutathione reductase and glutathione peroxidase and a lower (44%) level of ubiquinol 10 were found in the brain of the Pb-treated rats, compared to controls.²³

So, TBARS level in the Pb group increased significantly (p < 0.05) as compared to the other groups and with supplementation of sodium selenite and especially Nano-Se, TBARS level was significantly decreased in this study.

In this research, SOD activities decreased significantly (p < 0.05) in the Pb group (9.22 ± 0.66) as compared to the control, Nano-Se and Pb + Nano-Se groups.

The biological function of SOD is to dismute superoxide ion, H₂O₂, generated in this reaction is eliminated by catalase, one of the most active enzymes in the human and animal organism. The increase of lipid peroxidation levels in plasma was reported in associated with decrease erythrocyte–SOD and erythrocyte–catalase activities in the workers who were occupationally exposed to Pb.²⁴ This research is corroborated with the findings of lipid peroxide level, SOD and catalase activities of lead acetate-exposed group in the present study.

Also, the decreased SOD activity in lead acetate group is probably due to interaction of Pb with copper molecule. As SOD is a zinc–copper containing enzyme, hence Pb exposure induced copper deficiency resulted in decreased erythrocyte SOD activity.²⁵ Thus, decreasing in SOD activity in the Pb + Nano-Se group indicated that Nano-Se particles are effecting action on the inhibition of oxidative stress in Pb poisoning.

The present study indicated the decreasing of PCV in the Pb-treated group. Recent studies and kinetic behaviour of Pb in blood have implicated major Pb binding sites on erythrocyte which alters the activities of one of the cytosolic vestigial heme synthesis pathway enzyme in the cell, ALA-D.⁵

Pb is associated with a decrease in PCV by inhibiting heme synthetase, the enzyme which combines protoporphyrin and iron to form heme. Pb is also associated with a shortened erythrocyte lifespan.⁵ Thus, PCV volume was lowest significantly (p < 0.05) in the Pb group and prevented the decreasing in it with applying of selenium compositions in the Pb + Nano-Se and Pb + selenite groups.

In a study, Toplan et al. (2004) investigated the changes in hemorheological parameters due to Pb exposure in female rats. They reported that the erythrocyte count, Hb and PCV in the Pb-exposed group were significantly lower than those in the control group.²⁶

In the Pb group, the numbers of WBC decreased to 2187.5 ± 344.22 (p < 0.05), in comparison with the control group (8010 ± 492.29). Also, there was significant increase in WBC counts in the Pb + Nano-Se (3720 ± 195.32) and Pb + selenite (3587.5 ± 963.36) groups in comparison with the Pb group. The number of lymphocytes in the Pb group decreased to 1729.25 ± 260.77 in comparison with the control group (6429.8 ± 499.25). By comparing the means of the Pb + Nano-Se and Pb + selenite groups together, it was determined that there were significant differences in the lymphocytes counts. The number of eosinophils and monocytes in the Pb group showed no significant change (p > 0.05) when compared to other groups. The number of neutrophils in the Pb group decreased to 353 ± 71.59 in comparison with the control group (1308.2 ± 79.36). By comparing the means of the Pb + Nano-Se and Pb + selenite groups together, it was determined that there were significant differences in the neutrophil counts.

In vivo studies have shown that Pb is an immunotoxicant depressing humoral immunity, increasing host susceptibility to bacterial and viral infections.^27,28 The manner in which Pb affects the immune cells is not well understood. Although Pb treatment in vivo may result in immunosuppression, Moreover, Pb is a weak mitogen for lymphocytes in spleen that results in decline of lymphocyte numbers.²⁸

Rocke and Samuel (1991) investigated the effects of Pb shot ingestion on selected cells of the mallard immune system. They indicated that adult mallards, especially males, exposed to Pb pellets by ingestion and intubation in the spring of the year appeared to have lower numbers of certain immunologic cells; hence, they might be more susceptible to some infectious agents. Like other pathologic effects of Pb poisoning, immunotoxicity may be influenced by many factors, including physiologic condition, hormonal activity and seasonal changes in diet.²⁹

Sarasua et al. (2000) investigated serum immunoglobulins and lymphocyte subset distributions in children and adults living in communities assessed for Pb and cadmium exposure. They observed increase in lymphocyte counts in persons that they had higher blood Pb and urine levels than national averages.³⁰

So, in the Pb-treated group the numbers of leukocytes decreased and inhibited this adverse effect of Pb on the immune system with administration of selenium composition.

Based on the several studies about Pb poisoning, it may be suggested that oxidative stress is one of the most important mechanisms in the pathogenesis of Pb poisoning. So, sodium selenite and Nano-Se have biological role in the active centre of glutathione peroxidase; the key enzyme for tissues and body fluids which plays basic defensive role in the antioxidant protection system that result to revert the immunosuppression induced by Pb.²

Conclusion

In conclusion, alterations of peroxidant and antioxidant level in experimentally Pb poisoning in rat indicate definite oxidative stress. Also, the adverse effects of Pb on the immune system were inhibited using selenium composition especially Nano-Se particles. So, usage of supplementary antioxidant substances such as selenium may be useful in highly polluted areas. Based on this research, we can say that Nano-Se particles have more antioxidant effects in comparison to sodium selenite in Pb poisoning. The percentage of TBARS levels decreased to 63.47% and 83.76%, the percentage of SOD and catalase activities increased to 72.88% and 90.61%, 57.17% and 50% in the Pb + selenite and Pb + Nano-Se groups, respectively, in comparison to the Pb-treated group.

Also, Nano-Se particles are more efficient than sodium selenite to inhibit the immunosuppression in Pb poisoning.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was founded by Shahrekord University, Shahrekord, Iran.

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