Study on the impact of lead acetate pollutant on immunotoxicity produced by thiamethoxam pesticide.

OBJECTIVE: The curtailed knowledge about neonicotinoids that it has low affinity for vertebrate relative to insect nicotinic receptors is a major factor for its widespread use assuming that it is much safer than the previous generation insecticides. But literature regarding effect of thiamethoxam (second generation neonicotinoid)on immune system is not available. Also, there might be chances of interaction of heavy persistent metals in the water table with these pesticides. So, this study was undertaken with the objective to find immunotoxic alterations of lead acetate after exposure with thiamethoxam in animal model.
MATERIALS AND METHODS: For this albino mice were randomly divided into 6 groups (numbered I to VI) each containing 6 mice. Animals of groups I and II were administered 87.1 mg/kg b.w.(body weight) and 43.5 mg/kg b.w. respectively of thiamethoxam. Group III animals, lead acetate was administered orally and IV and V mice were administered combination of lead acetate and thiamethoxam at higher and lower dose level for 28 days. The group VI was control group. On 29(th) day and humoral and cell mediated immune responses, TLC (Total leukocyte count), DLC (Differential leukocyte count), serum total protein, globulin and albumin, and histopathological studies were conducted. RESULT: The result obtained clearly indicated that on oral administration of thiamethoxam immunotoxicity was induced in mice in dose related manner. Lead acetate when administered for 28 days showed immunotoxic potential. Thiamethoxam and lead acetate when administered together did not lead to any new altered immunotoxic response but additive toxic effects of both were observed.

Introduction

There is now overwhelming evidence that most of the chemical pesticides do pose a potential risk to human and other life forms, and unwanted side effects to the environment. No segment of the population is completely protected against exposure to pesticides, and has potentially serious health effects. Neonicotinoids are reported to have an impact on the immune system of bees[1] and is also been a great concern for some activists that neonicotinoids applied agriculturally might accumulate in aquifers.[2] Thiamethoxam is a second generation neonicotinoid insecticide. The low affinity of neonicotinoids for vertebrate relative to insect nicotinic receptors is a major factor in their favorable toxicological profile.[3] Most of the studies on the toxicity of pesticides have been focused on enzyme alteration, gross pathological effects, mutagenic and carcinogenic potential of these agents. In recent years, the effects of pesticides on the immune response have received attention. It is now clear that changes in host immunity may occur after pesticide ingestion. The most available toxicity data of the neonicotinoids is with imidacloprid. Short term toxicity results of Thiamethoxam have been reported, but no data are available regarding its immunotoxicity studies.

Thiamethoxam is a pesticide used on commonly grown crops like brinjal, mango, citrus, rice, wheat, cotton, olives, cauliflower, etc., consumed by most of the people all round the world.

As, Lead is a major pollutant present in the water table of urban areas due to heavy industrialization so, the chances of its interaction with persistent pesticide like Thiamethoxam have also increased. There are greater chances of some interactive altered immunological effects by these two compounds because of widespread use of pesticides for domestic and industrial applications the evaluation of their immunotoxic effects is of major concern to public health. Immunosuppression leads to change in length of life, increased susceptibility to infectious disease and decreased immune response to vaccination. Thus, there is urgent need to obtain more information regarding the immunotoxicity potential of Thiamethoxam alone and after interaction with Lead acetate.

Many reports are available regarding Lead toxicity and its deleterious effects in various species of animals and there has been lot of work carried out on pharmacokinetics, but very few researchers tried to correlate immunotoxic alterations of Lead acetate after exposure with pesticide in animal model. Hence, the present study was carried out with an attempt to determine the immunotoxic potential of commonly used insecticide, Thiamethoxam after 28 days of oral exposures in albino mice.

Materials and Methods

The present study was conducted on 36 healthy male albino mice that were of 4-5 weeks old. The mice were procured from Zydus Research Center, Ahmadabad and kept in cages at Animal House, Veterinary College, Anand Agricultural University, Anand. The animals were kept under constant observation for at least 10 days before commencement of the experiment. Mice were provided with standard pellet diet. Diet and deionized water were provided ad libitum. All necessary procedures were adopted to keep mice free from stress. This study was performed after the approval from Institutional Ethical Committee, and all procedures were carried out in accordance with the Guidelines laid down by the International Animal Ethics Committee (IAEC number for the experiment approval is 2011/VPT/102).

Experimental Design

Animals were randomly divided into six groups each containing six mice. The groups were numbered as Group I to VI. Considering the LD50 of Thiamethoxam 871 mg/kg body weight (b.w.) in mice,[4] calculations of different dose groups were done. Animals of Groups I and II were administered 1/10th of LD50 that is, 87.1 mg/kg b.w. and 1/20th of LD50 43.5 mg/kg b.w., respectively, of Thiamethoxam (98.40% pure) in corn oil used as a vehicle, for 28 days orally. Lead acetate was administered orally at the rate of 15 mg/kg b.w. to mice of Group III. Group IV and V mice were orally administered combination of Lead acetate at the dose rate of 15 mg/kg b.w. And Thiamethoxam at higher and lower dose level of 87.1 mg/kg b.w. and lower dose level of 43.5 mg/kg b.w., respectively, for 28 days. The Group VI mice were kept as control and were gavaged corn oil (1 ml) orally.

On 29th day of the experiment, blood samples were collected from the retro-orbital plexus with the help of the capillary tube before sacrificing the mice. Blood samples were collected in vials containing K3 ethylenediaminetetra-acetic for hematology (differential leucocyte count and total leucocyte count) and in plain vials for serum biochemical estimation (albumin, globulin and total protein) and sheep red blood cells (SRBC) antibody titer by hemagglutination. After sacrificing the mice, spleen and Thymus were collected for histopathological examination. This study was performed after the approval from Institutional Ethical Committee, and all procedures were carried out in accordance with the Guidelines laid down by the International Animal Ethics Committee. Total leukocyte count (nos/microliter) and differential leukocyte count (lymphocyte, granulocyte, and monocytes) by Autoanalyzer (Boule Medical Ab, Stockhol, Sweden).

Immunization of mice was done using SRBCs. SRBCs were collected in Alsever's solution (composed of [in g/L] 20.5 g of dextrose, 8 g of sodium citrate, 4.2 g of sodium chloride, 0.55 g of citric acid), washed in large volumes of sterile 0.9% normal saline thrice and adjusted to a concentration of 5 × 109 cells/ml, were used for immunization. Animals were immunized by injecting 0.2 ml SRBC suspension intraperitoneally 7th days prior to sacrifice (on 21st day of the experiment). Blood was collected from retro orbital plexus under ether anesthesia on 29th day, and serum was separated to determine the antibody titer by the Hemagglutination test (HA).

Antibody Titer

Antibody titer was carried out by diluting the test serum two fold times in 0.15 M phosphate buffer saline (PBS) and aliquoted in “U” bottomed microtiter plates. 1% SRBC suspended in PBS was dispensed in each well and mixed thoroughly. The plates were incubated for 4 h at 37°C and then observed visually for hemagglutination. The highest dilution of the test serum giving hemagglutination was taken as antibody titer.

Assessment of Cell-mediated Immune Response

Cell-mediated immune response was assessed by the method as described by Lagrange et al. (1974).[5] All the animals under various groups were immunized by injecting 20 μl of 5 × 109 SRBC/ml subcutaneously into the right footpad on 19th day of the treatment. Thickness of left footpad was measured using vernier callipers on 26th day of the treatment. The mice then challenged by injecting 20 μl of 5 × 109 SRBC/ml intradermally on the left hind foot pad (time 0). Foot pad thickness was measured after 24 and 48 h of challenge. The difference in mm was taken as a measure of delayed type hypersensitivity (DTH).

On 29th day of study, all the mice from each group were sacrificed by cervical dislocation. Mice that died during the experiment and mice that were sacrificed 29th day of the experiment were subjected to postmortem examination in the confined disinfected laboratory to determine the presence or absence of gross and histopathological lesions. Postmortem findings were made by systematic approach. Detailed post mortem lesions from all the mice were recorded. For gross (macroscopic) lesions thymus and spleen were collected and examined after opening the body of sacrificed and dead experimental mice. For histopathological examinations, tissues from spleen and thymus were collected in 10% formalin and preserved for processing.

The formalin fixed tissues were processed by paraffin wax embedding method of tissue sectioning. Sections were cut at 6–8 μs thickness with automatic section cutting machine (SLEE-MAINZ, Germany), and were stained with Hematoxylin and Eosin (H and E) stains. The H and E stained slides were observed under microscope and lesions were recorded.

Mean values, standard error, and analysis of variance of the tabulated data, were calculated using SPSS 12.0 (IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp) version of statistical software.

Results

Hematological Study

Results obtained in relation to hematological assessment are presented in Figures 1 and 2. There was significant dose-dependent decrease (P < 0.05) in total leukocyte count and lymphocyte count in high and low doses of Thiamethoxam-treated Group (I and II), Lead acetate-treated Group (III), and combination group of Lead acetate and Thiamethoxam (Group IV and V) in comparison to control group, that is, corn oil-treated group (VI).

Figure 1

Effect of daily oral administration of Thiamethoxam and Lead acetate alone and in combination at different dose level on mean total leukocyte count of mice

Figure 2

Effect of daily oral administration of Thiamethoxam and Lead acetate alone and in combination at different dose level on mean Lymphocyte count of mice

The group in which combination of Lead acetate and Thiamethoxam was administered (IV and V) also showed significant alteration in TLC and lymphocyte count and no alteration in monocyte count and granulocyte count. There was no significant alteration in comparison to noncombination Groups (I, II, and III) but the effect was found to be more severe showing synergistic effect of toxicity of these xenobiotics in mice.

Biochemical Study

There was significant decrease (P < 0.05) in total protein and globulin in Lead acetate-treated Group (III), high dose of Thiamethoxam-treated Group (I), and its combination group with Lead acetate (Group IV). Whereas, no significant decrease was observed in low dose of Thiamethoxam (II) alone and in combination Group (V) in comparison to negative control group, that is, corn oil-treated Group (VI). No significant effect was observed in serum albumin in all treated groups after 28 days of study. The group of mice receiving combination of metal and pesticide (Group IV and V) showed lower values of serum protein and serum globulin in comparison to their non-combination groups (Group I, II, and III). This suggested synergistic toxic effect of metal and pesticide.

Results obtained in relation to cell-mediated, and humoral-mediated immune responses are presented in Figures 3 and 4. The increase in paw thickness was significantly low after 24 and 48 h of challenge, in both doses of Thiamethoxam (I and II) in comparison to the control group (Group VI). Thiamethoxam at both concentration and Lead acetate combination Group (IV and V) also showed significantly low increase in paw thickness signifying synergistic effect of Lead acetate toxicity on Thiamethoxam induced cell-mediated immune response. Lead acetate-treated mice (III) also showed significantly lower increase in dermal thickness in comparison to pesticide control group after 24 h of challenge suggesting, alteration of cell mediated immune response by Lead at a dose administered in the present study.

Figure 3

Effect of daily oral administration of Thiamethoxam and Lead acetate alone and in combination at different dose level on mean cell-mediated immune response of mice

Figure 4

Effect of daily oral administration of Thiamethoxam and Lead acetate alone and in combination at different dose level on mean humoral-mediated immune response of mice

There was significant (P < 0.05) decrease in antibody titer against SRBC in both doses of Thiamethoxam (I and II) in comparison to negative control group.

Lead acetate-treated Group (III) showed nonsignificant decrease in titer in comparison to negative control Group (VI).

Histopathological Study

No abnormal microscopic changes were observed in spleen and thymus of mice in Group VI (control). In spleen of Group I mice, hemosiderosis or extramedullary hematopoiesis along with mild congestion and depletion of lymphocyte were observed [Figure 5]. These lesions of spleen were also observed in mice of group treated with a lower dose of Thiamethoxam (II) but these were comparatively less severe. No specific changes in thymus were observed in these two groups. No specific microscopic lesion was observed in spleen and thymus in mice of Lead acetate-treated group (Group III).

Figure 5

Section of spleen from Group I showing hemosiderosis, congestion and depletion of lymphocyte

Discussion

Toxicity on immune cells was studied in this experiment by assessing the total leukocyte count and differential leukocyte count. The results of the present study showed significant toxic effect of Thiamethoxam and Lead acetate on immune cells. This result is consistent with the report showing decrease in total leukocyte count, when imidacloprid, member of neonicotinoid, was administered to male White Leghorn chicks for 28 days.[6] It is suggested that compounds having benzene ring or other ring structure act as a hapten that combines with the protein constituent of leukocytes to form an antigen, to which the animal develops antibodies that are toxic to leukocytes, causing either lysis or agglutination. Thiamethoxam is also a ring-structured compound and thus may have caused leukocytopenia. No alteration in hematological parameters like white blood cells count, packed cell volume and hemoglobin in Sprague-Dawely rats has been reported; when Thiamethoxam is administered for 90 days at the dose of 25, 50 and 100 mg/kg b.w.[7] Variation of response in mouse might be due to species difference in metabolism of Thiamethoxam. Rats fed on Lead acetate at different dose levels has been reported to cause TLC and lymphocyte count to decrease significantly (P < 0.05).[8] Decrease in TLC is directly related with either their decreased production from the germinal center of lymphoid organs or increased lysis due to the presence of Lead in the body. Result of this study also showed no significant alteration of immune cell toxicity by combined administration of Thiamethoxam and Lead acetate but the effect was found to be more severe showing synergistic effect of toxicity of these xenobiotics in mice.

The study on total protein, globulin, and albumin suggested dose-related decrease in total protein and globulin of Thiamethoxam-treated mice. Significant decrease in serum total protein and globulin was seen in Lead acetate-treated Group (III) which was also reported by different scientists.[9] Synergistic toxic effect of metal and pesticide on these parameters was observed. Thiamethoxam[7] and avermectin[10] were reported to show dose-related significant decrease of serum total protein after feeding rats that was found consistent with results of this study. Imidacloprid does not significantly alter total protein, albumin, globulin, A/G ratio in animals exposed daily to 5 and 10 mg/kg b.w. day.[11] A decreased value of total protein may reflect liver or kidney disease.[12] The fall in total protein could be due to the stressogenic effect of Thiamethoxam, or general toxic action that also Lead to decrease in weight gain in the insecticide-treated mice. Hypoglobulinemia (decreased globulin) results due to malnutrition and immune deficiency.[8]

Results regarding cell-mediated immunotoxicity study showed Thiamethoxam and Lead acetate to be immunotoxic. There is dearth of literature on cell-mediated immunotoxic effect of Thiamethoxam, but various other pesticides are reported to cause cell-mediated immunotoxicity. Lindane[13] and malathion[14] was reported to show significant suppression of CMI response after treatment in 28 days of study. DTH responses during Lead toxicity corroborate with available literatures.[15] Decreased cell-mediated response in Lead exposed mice could be possibly due to Lead induced apoptosis of lymphocyte or altered T-cell activity.[16] DTH is antigen specific and causes erythema and induration at the site of antigen infection in immunized animals. The histology of DTH can be different for different species, but the general characteristics are an influx of immune cells at the site of injection, macrophages and basophils in mice and induction becomes apparent within 24-72 h. T-cells are required to initiate the reaction.[17]

When tested for humoral-mediated immune responses, Thiamethoxam showed immunotoxic potential. Similar suppression of the humoral immune response was observed following oral administration of pesticides like carbaryl, malathion, endosulfan, chlorpyrifos, quinalphos, alphamethrin.[1819202122] But Lead acetate at present dose level and duration of exposure failed to significantly suppress humoral immune response against SRBC. In the contrary to this finding, several researchers showed a significant reduction of humoral antibody responses by Lead treatment.[151623] The difference in results might be due to subacute exposure of Lead acetate in the present study. The combination group treated with high and low doses of Thiamethoxam and Lead acetate, that is, Group IV and V showed significant decrease in antibody titer against SRBC in comparison to pesticide control Group (VI) but this decrease was similar to that of Thiamethoxam-treated mice suggesting no additive effect of Lead acetate toxicity on humoral immune response.

The histopathological lesions found on spleen on Group I and II mice were in agreement with literature[24] where it was observed depletion of spleen lymphocytes in the periarteriolar lymphoid sheath and marginal zone in white pulp in mice treated with ethyl carbamate, which was potentiated by pretreatment with diazinon. No specific microscopic lesions were observed in spleen and thymus of Lead acetate-treated group, and this finding corroborates with the previous literature.[25]

The study, therefore, concludes that Thiamethoxam leads to leucopenia and lymphocytopenia, hypoproteinemia, humoral- and cellular-mediated immunotoxicity at higher dose level of 87.1 mg/kg b.w. but the severity of immunotoxicity was less in the lower dose of 43.5 mg/kg b.w. Extensive microscopic changes were observed in spleen by Thiamethoxam at higher dose level. Lead acetate caused cell-mediated immunotoxicity but failed to produce humoral immunotoxicity at the given dose rate in mice. There was additive immunotoxic effect due to combined dosing of Lead acetate and Thiamethoxam at both dose levels (87.1 and 43.5 mg/kg b.w.).

Finally, it is concluded from this study that Thiamethoxam is immunotoxic at the dose level of 1/10th (87.1 mg/kg b.w.) and 1/20th (43.5 mg/kg b.w.) of LD50 (871 mg/kg b.w.) in mice when exposed orally for 28 days, while Lead acetate potentiated its immunotoxicity.