http://informahealthcare.com/dct ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, Early Online: 1–9 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2014.966382
RESEARCH ARTICLE
Immunotoxicity assessment of sub-chronic oral administration of acetamiprid in Wistar rats
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R. K. Shakthi Devan, P. C. Prabu, and S. Panchapakesan Central Animal Facility, SASTRA University, Thanjavur, Tamil Nadu, India
Abstract
Keywords
Context: Neonicotinoid insecticides are synthetic analogues of nicotine that acts on the central nervous system of insects by blocking post synaptic acetylcholine receptor. Acetamiprid is one of the widely used neonicotinoid class of insecticide used to control sucking insects like aphids, bees, mosquitoes, on crops. Data on the possible immunotoxic nature of acetamiprid are lacking. Objective: The present study was conducted in Wistar rats with the objective of evaluating the immunotoxic potential of acetamiprid administered orally at the dose levels of 27.5, 55 and 110 mg/kg b.w. (equivalent to 5.5, 11 and 22 mg/kg b.w.) for a period of 90 days. Materials and methods: In experiment 1, general toxicity testing including the evaluation of clinical signs, hemato-biochemical changes, response of the lymphocytes towards T and B cell mitogens, macrophage function, gross and histopathology of the lymphoid organs (spleen, thymus, lymph nodes, etc.) were performed. In the second experiment, humoral and cellmediated responses during immunological challenges were evaluated. Results: Significant decreases were observed in the stimulation index of lymphocyte proliferation to B cell mitogen and in the nitrite production of macrophages of rats treated with 110 mg/kg of acetamiprid. Significant decrease in the lymphoproliferative response towards the B cell mitogen indicated the inability of the B lymphocytes to respond on stimulation that might increase the chances of susceptibility to infections. Acetamiprid also caused 15–28% reduction in nitrite production, an important signal for efficient inflammatory response of macrophages. The functional impairment of macrophages may involve aberrations in the enzymatic degradation of microbes, oxidative burst, generation of free radicals, phagocytosis, release of proinflammatory cytokines and thereby, may hamper host defence causing susceptibility to diseases. No significant changes over hematology, biochemistry, organ weights, histopathology of major immune organs, delayed type hypersensitivity test, response to sRBCs and lymphoproliferation assay for T cell mitogen were observed. Conclusion: In conclusion, the results demonstrate for the first time that the subchronic administration of acetamiprid (20% SP-soluble powder) cause significant decreases in the lymphocyte proliferation as well as the macrophage function at the dose level of 110 mg/kg. Considering the chronic population adjusted dose (0.023 mg/kg/day) through dietary exposure for acetamiprid, judicious use of acetamiprid is highly essential. Indiscriminate use of acetamiprid exceeding the doses advised might pose a hazard.
Cell-mediated immunity, humoral immunity, immunotoxicity, neonicotinoids, rats, safety assessment
Introduction Neonicotinoids are one of the most widely used groups of insecticides that are used to control insects observed in more than 140 crops (Lee, 2003). Neonicotinoids act as agonists of acetyl choline at the synaptic junction and bind with the acetyl choline receptors in the central nervous system of insects causing excitation through interruption of synaptic transmission (Bai et al., 1991; Lind et al., 1999; Liu & Casida, 1993; Nauen & Elbert, 1997; Zhang et al., 2000). The mode
Address for correspondence: Dr S. Panchapakesan, Professor & Coordinator, Central Animal Facility, SASTRA University, Thanjavur – 613 402, Tamil Nadu, India. Tel: +91 4362-264101-108 Extn. 680. Fax: +91 4362-264120. E-mail: [email protected]; [email protected]
History Received 14 April 2014 Revised 27 August 2014 Accepted 14 September 2014 Published online 13 October 2014
of action of neonicotinoids is distinct and cross resistance to other conventionally used insecticides is non-existent. Among neonicotinoids, acetamiprid (E)-N1-[(6-chloro-3-pyridyl) methyl]-N2-cyano-N1-methyl acetamidine is a widely used second-generation chloronicotinyl insecticide with contact and systemic activity via foliar applications. It is systemic in action and controls sucking insects like aphids, bees, mosquitoes, on crops including, but not limited to, cotton, leafy vegetables, citrus and pome fruits, cole crops, grapes and ornamental plants (EPA, 2002). It is an important insecticide in cherry farming because of its lethal action over the cherry fruit fly larvae. Its general mode of action is translaminar activity with contact and stomach action. The recognition of nicotinoids in case of mammals by nicotinic receptors involves cation–pi interactions and in case of insects, possibly a cationic subsite that interacts with
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nitro/cyano substituent of neonicotinoids (Tomizawa & Casida, 2005). There is an increasing awareness that during human exposure, insecticides might cause immune-mediated toxic effects, and therefore immunotoxicity testing of insecticides has been receiving greater attention during the past few decades. Immune system, an intensely controlled complicated network, includes lymphocytes, reticular, dendritic and epithelial cells, which interact through intercellular contacts and cytokines (Roth et al., 2006). Immunotoxicity comprises diverse harmful effects such as immuno-suppression, immuno-modulation and enhanced immune response. Adverse effect on the components and/or function of immune system may be due to either direct or indirect actions, resulting in either permanent or reversible toxicity. Long-term exposure towards chemical stressors can cause direct injury to immune system (Garg, 2004). The symptoms of acetamiprid poisoning in humans have been reported to be partially similar to acute organophosphate (OPC) intoxication. The clinical manifestations of acute poisoning of acetamiprid include vomiting, seizure, hypotension, tachycardia, coma, which mimic the symptoms reported during acute OPC poisoning (Agarwal, 1993; Eddleston et al., 2008; Imamura et al., 2010; Lee & Tai, 2001; Roberts & Aaron, 2007; Todani et al., 2008). The immunotoxic nature of OPCs over immune response including neutrophil function, macrophage, antibody production, interleukin production, serum complement, T cell proliferation, Con A, is widely reported (Li, 2007). Since acetamiprid acts over the neuro-humoral transmission similar to that of the OPC’s and further, as the symptoms of acetamiprid during human exposure mimics that of OPC’s, it might be possible that acetamiprid affects the immune system like OPC’s. However, there are no reports available on the immunotoxic potential of acetamiprid on the structure and function of immune system. The present investigation was designed to assess the immunotoxic effects of acetamiprid during sub-chronic exposure in rats. As the immune system interactions might be numerous and of varying character, a systematic approach is followed in case of immunotoxicity testing in rodents. Immunotoxicity was evaluated using two experimental animal studies, namely, (i) Experiment 1, which investigated general toxicity testing including the evaluation of clinical signs, hemato-biochemical changes, response of the lymphocytes towards T and B cell mitogens, macrophage function, gross and histopathology of the lymphoid organs (spleen, thymus, lymph nodes, etc.) and (ii) Experiment 2, which evaluated the humoral- and cellmediated response during immunological challenges.
Materials and methods Acetamiprid Acetamiprid (20% SP-soluble powder, Nagarjuna Agrichem Ltd., Hyderabad, India) was administered orally to rats for 90 consecutive days. During the pilot study, standardized at Central Animal Facility (CAF), SASTRA University, the LD50 value of acetamiprid (20% SP-soluble powder) was derived at 1100 mg/kg b.w. (equivalent to 220 mg/kg of acetamiprid). One-tenth of the LD50 was taken as highest dose and two-fold decreases were made for fixing the intermediate
Drug Chem Toxicol, Early Online: 1–9
and low dose levels. The dosing volume was maintained at 1 mL/100 g body weight. Experimental animals The present study was conducted in Wistar rats (7–8 weeks old) bred at Central Animal Facility after the clearance of Institutional Animal Ethics Committee, SASTRA University. All the rats were acclimatized for 7 days and apparently healthy rats were selected for the study. The rats were weighed and randomized using a computer-generated stratified randomization procedure based on body weights so that all groups for each sex had approximately equal mean body weights. The individual weight variation did not exceed 20% of the mean body weights. Rats were provided unique identification number by ear tag and cage cards were placed for the identification of animal number, group, sex, dose levels, etc. Rats were group-housed in polypropylene cages with wire mesh, top grill made of stainless steel, fed with standard rodent diet (M/s Provimi Animal Nutrition India Private Limited, Bangalore, India) and reverse osmosis purified water ad libitum. The temperature and relative humidity were maintained at 22 ± 3 C and 30–70%, respectively. Photoperiod of 12 h light and 12 h dark cycle was maintained by an automatic timer. The study consisted of two sets of animal experiments and were conducted as per the following procedure. Experiment 1 Forty-eight rats were segregated into 4 groups of 12 rats each and dosed orally with acetamiprid for 90 days as follows: Group No.
Dose Levels
No. of Animal
Group Group Group Group
Control Low dose Intermediate dose High dose
12 12 12 12
1 2 3 4
(6 (6 (6 (6
M+6 M+6 M+6 M+6
F) F) F) F)
M – Male, F – Female
The control rats were administered with distilled water and the low-, intermediate and high-dose group rats were administered with acetamiprid (20% SP-soluble powder) at the dose levels of 27.5, 55 and 110 mg/kg b.w., respectively (equivalent to 5.5, 11 and 22 mg/kg b.w. of acetamiprid). All the individual rats of each group were examined twice daily for the presence of any morbidity, mortality and signs of toxicity. On day 90, blood samples were collected through retro orbital plexus from all the rats before necropsy and were used for hematological, biochemical estimations. Lymphocytes were separated from the whole blood using commercially available lymphocyte separation media (Histopaque, Sigma, Bangalore, India) based on the principle of density gradient and were used for the lymphoproliferation assay. After blood collection, the rat-resident peritoneal macrophages were collected under sterile conditions in a laminar flow chamber from all the rats using sterile syringes before necropsy. Hematological and biochemical parameters. White blood cell
(WBC) count, lymphocyte (%) and neutrophil (%) were analyzed from the blood samples collected in EDTA-coated vial using a calibrated 5-part blood cell counter (Orphee,
Immunotoxicity of acetamiprid in rats
DOI: 10.3109/01480545.2014.966382
Switzerland). Similarly, plasma was separated from blood collected in heparinized vial (20 IU/mL of blood) by centrifuging at 2000 rpm for 10 min and was utilized for the estimation of total protein (TP) and albumin (ALB) using a calibrated random access clinical chemistry analyzer. Globulin was calculated by subtracting albumin from total protein.
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Lymphoproliferation assay for B and T cell mitogen. Lymphocyte proliferation was assessed using MTT
[3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye method (Mossmann, 1983; Singh et al., 2011). 100 mL of lymphocyte suspension (3 106 cells/ml) was pre-incubated for 24 h in a microtitre plate added with RPMI 1640 medium and incubated at 37 C in a humidified chamber with 5% CO2 before the addition of the appropriate mitogen, i.e. lipopolysaccharide as the B cell mitogen and concanavalin A as T cell mitogen. The cell pellets were separated by centrifuging at 1500 rpm for 10 min and were again suspended in 50 ml of MTT (5 mg/ml) in RPMI media. Following incubation in humidified 5% CO2 chamber for 4 h at 37 C, dimethyl sulphoxide was added to the medium separated by centrifuging and absorbance at 570 nm was recorded. The percentage of proliferation was calculated as follows: Proliferation % ¼ ðOD stimulated OD unstimulatedÞ 100=ðOD stimulatedÞ Macrophage function assay. The viable rat resident peritoneal
macrophages collected were calculated using trypan blue dye exclusion method. Rat peritoneal macrophages were suspended in phenol red free RPMI growth medium containing 5 mM arginine at a concentration of 2 106 cells/ml and 100 ml of this suspension was added to a 96-well plate in triplicates. The cells that were not adherent following 2 h of incubation were removed by washing with phosphate buffer saline. The final volume was made to 200 ml with LPS of 1 mg/ml concentration. Following incubation at 37 C in humidified chamber with 5% CO2, the supernatant was removed after 72 h and the nitrite production was estimated using the Griess reagent. To 50 ml of supernatant, 50 ml of Griess reagent was added, incubated for 10 min and the absorbance at 570 nm was measured (Garcia et al., 2002; Green et al., 1982). After the collection of blood samples and peritoneal macrophages, rats of all the groups were euthanized using CO2 inhalation followed by exsanguination and were examined for the presence of gross pathological changes. Weights of spleen and thymus were measured and expressed as absolute and relative organ weights (g and g/100 g of body weight). Spleen, thymus and lymph node were collected from all the groups, fixed in 10% neutral buffered formalin and processed for histopathological evaluation.
Gross and histopathological evaluation.
Experiment 2 In this experiment, 48 rats were orally administered with acetamiprid for 90 consecutive days and were evaluated for
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the cell mediated immune response and humoral immune response as follows: Group No.
Dose Levels
Cell mediated immune response Group 5 Control Group 6 Low dose Group 7 Intermediate dose Group 8 High dose Humoral immune response Group 9 Control Group 10 Low dose Group 11 Intermediate dose Group 12 High dose
No. of Animal 6 6 6 6
(3 (3 (3 (3
M+3 M+3 M+3 M+3
F) F) F) F)
6 6 6 6
(3 (3 (3 (3
M+3 M+3 M+3 M+3
F) F) F) F)
The control rats were administered with distilled water, and the low-, intermediate and high-dose group rats were administered with acetamiprid (20% SP-soluble powder) at the dose levels of 27.5, 55 and 110 mg/kg b.w., respectively (equivalent to 5.5, 11 and 22 mg/kg b.w. of acetamiprid). Cell mediated immunity – delayed-type hypersensitivity test Cell-mediated immune response was evaluated using delayedtype hypersensitivity reaction induced by ovalbumin, as described by Tamang et al. (1988) and Satheesh et al. (2005) with slight modifications. Rats were sensitized using intra-dermal injection of 100 mg of ovalbumin mixed with Freund’s Complete Adjuvant (FCA) over the sub-plantar region of the left hind limb on day 74. The sensitized rats were challenged after 14 days, i.e. on day 88, with 0.05 mL of ovalbumin. The volume of the paw edema reaction was measured using plethysmometer at 0, 3, 6, 9, 24, 36 and 48 h post challenge. After sacrifice, the reaction sites were collected, fixed, processed and sectioned for histopathological analysis. The reactions were graded as minimal (1), mild (2), moderate (3), marked (4) and severe (5) and the cumulative grading of intensity was calculated for each group. For the assessment of changes in humoral response, remaining 24 rats were divided into 4 groups of 6 rats each and were sensitized by injecting 0.5 mL of sheep red blood cells (sRBCs) containing 1.25 106 cells suspended in PBS per rat intraperitoneally on day 72. Fourteen days after sensitization, i.e. on day 86, the rats were again challenged with 0.25 mL of sRBCs. After 4 days, i.e. on day 90, blood was collected, serum separated and humoral immune response was measured by hemagglutination (HA) test following the procedure described by Beard (1980), Thakur et al. (2011) and Rajesh et al. (2011). HA test was done in a U-shaped micro-perplex plate. The serum samples were serially diluted (two-fold) in phosphate buffer saline so that the final volume in each well was 0.05 ml. In the control wells, only phosphate buffer saline was added. Further, 0.05 ml of sRBC suspension (0.5%) was added to all the wells. A known negative control was also included. The HA plate was swirled gently for ensuring uniform distribution of sRBCs and was allowed at room temperature for 45 min. The HA pattern was observed as a diffuse sheet of RBCs covering the bottom of the wells and the titre, i.e. reciprocal of the highest dilution showing
Humoral immune response – hemagglutination test.
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Drug Chem Toxicol, Early Online: 1–9
Table 1. Effect of acetamiprid on hemato-biochemical values in Wistar rats. Parameters
Sex
WBC (%)
Male Female Male Female Male Female Male Female Male Female Male Female Male Female
Lymphocyte (%) Neutrophil (%) Total Protein (g/L) Albumin (g/L) Globulin (g/L)
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Albumin Globulin ratio
Control
Low dose
Intermediate dose
High dose
9.10 ± 0.83 5.20 ± 0.15 68.63 ± 1.40 71.23 ± 2.14 29.97 ± 1.14 26.57 ± 1.39 61.76 ± 0.96 62.33 ± 1.33 32.57 ± 0.73 31.37 ± 1.33 29.19 ± 0.62 30.97 ± 0.27 1.12 ± 0.04 1.01 ± 0.04
10.20 ± 0.91 5.07 ± 0.16 69.90 ± 1.78 69.83 ± 0.89 28.77 ± 1.87 29.03 ± 0.94 62.73 ± 1.72 64.17 ± 0.65 31.63 ± 0.98 33.73 ± 0.97 31.10 ± 0.94 30.43 ± 0.72 1.02 ± 0.0 1.11 ± 0.06
9.43 ± 0.31 5.13 ± 0.28 71.33 ± 1.87 70.50 ± 0.65 27.07 ± 2.00 28.57 ± 0.43 62.17 ± 1.76 63.50 ± 0.41 31.03 ± 0.81 31.77 ± 1.02 31.13 ± 0.96 31.73 ± 1.52 1.00 ± 0.01 1.01 ± 0.41
9.17 ± 0.20 4.90 ± 0.32 71.10 ± 1.37 69.70 ± 1.63 27.63 ± 1.12 28.70 ± 1.65 64.73 ± 1.13 64.80 ± 0.80 31.70 ± 0.29 30.87 ± 1.31 33.03 ± 0.86 33.93 ± 0.62 0.96 ± 0.02 0.91 ± 0.05
Values are expressed as mean ± standard error (n ¼ 6). Means bearing * vary significantly between groups (p50.05).
complete agglutination of sRBCs, was observed and expressed as log2/0.05 mL. Statistical analysis All the quantitative data collected were subjected to statistical analysis using Graph pad Prism software (La Jolla, CA) and are presented as mean ± standard error. The mean values from organ weight, hematological, biochemical and immunological parameters were subjected to analysis of variance (ANOVA). A probability level lower than 5% (p50.05) was considered to be significant.
Results Experiment 1 Clinical signs All the rats survived throughout the study and there were no clinical signs of toxicity during the observation period. Hematology and biochemistry parameters The hematological and biochemical parameters evaluated – namely, total protein, albumin, globulin, albumin globulin ratio, WBC, lymphocyte and neutrophils values – are presented in Table 1. No significant changes were present in hematology as well as biochemical parameters of the treatment groups as compared to the control. Lymphoproliferation assay for B- and T-cell mitogen The stimulation index of the lymphocytes of the high-dose groups exposed to B-cell mitogen was significantly lower as compared to that of the controls. However, the stimulation indices of the low and intermediate dose levels were comparable to the control. The stimulation index of the lymphocytes exposed to T-cell mitogen was comparable among different groups. The stimulation indices of the lymphocytes of different groups towards B- and T-cell mitogen are presented as Table 3.
the controls. The nitrite production of stimulated macrophages in the low- and intermediate dose levels was comparable to those of the controls (Table 5). Gross pathology and histopathology There were no compound-related gross lesions observed during necropsy. No significant changes were observed in absolute and relative organ weights (Table 2). Microscopic evaluation of spleen, thymus and lymph node revealed no compound-related histopathological changes. The changes observed were either agonal or spontaneous and were observed in both control as well as treatment groups. Representative images of the organs evaluated in the control and high-dose groups are presented (Figure 1). Experiment 2 Cell-mediated immune response – delayed type hypersensitivity test Cell-mediated immune response assessed using the delayed type hypersensitivity response to ovalbumin revealed increase in paw edema volume in the treated groups that were comparable to the control group at different intervals measured (Table 3). Histopathology for DTH reaction In the skin sections, DTH reaction was observed as mild to moderate hydropic degeneration of the squamous epithelium of the epidermis, mild to moderate oedema, fibrin deposits and minimal to marked mononuclear cell infiltration in the dermis. Inflammatory reactions of different intensities were present in both controls as well as all the treated groups. The frequency and intensity of the DTH reaction of the rats from different groups are presented (Table 4). No reduction in the intensity of the inflammatory reaction was observed in the treated sections as compared to the control sections. Humoral immune response – hemagglutination test
Macrophage function assay Nitrite production of stimulated macrophages was significantly lower in the high-dose groups as compared to that of
There were no significant changes in the HA titers present in the treated groups as compared to the control on day 90 (Table 5).
Immunotoxicity of acetamiprid in rats
DOI: 10.3109/01480545.2014.966382
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Table 2. Effect of acetamiprid on absolute and relative organ weights of spleen and thymus of Wistar rats.
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Parameters
Sex
Absolute organ weight (g) Cell-mediated immunity Thymus Male Female Spleen Male Female Humoral immunity Thymus Male Female Spleen Male Female Relative organ weight (g/100 g) Cell-mediated immunity Thymus Male Female Spleen Male Female Humoral immunity Thymus Male Female Spleen Male Female
Control
Low dose
Intermediate dose
High dose
0.270 ± 0.02 0.225 ± 0.02 0.631 ± 0.04 0.474 ± 0.02
0.258 ± 0.02 0.235 ± 0.01 0.552 ± 0.02 0.414 ± 0.03
0.298 ± 0.02 0.241 ± 0.01 0.685 ± 0.02 0.429 ± 0.04
0.279 ± 0.02 0.211 ± 0.01 0.599 ± 0.08 0.516 ± 0.05
0.277 ± 0.03 0.241 ± 0.01 0.663 ± 0.04 0.564 ± 0.08
0.253 ± 0.02 0.228 ± 0.02 0.665 ± 0.06 0.533 ± 0.03
0.282 ± 0.02 0.245 ± 0.05 0.511 ± 0.03 0.484 ± 0.04
0.266 ± 0.02 0.247 ± 0.02 0.554 ± 0.05 0.478 ± 0.02
0.077 ± 0.01 0.092 ± 0.01 0.180 ± 0.02 0.194 ± 0.02
0.075 ± 0.01 0.094 ± 0.01 0.159 ± 0.01 0.166 ± 0.01
0.083 ± 0.01 0.098 ± 0.01 0.193 ± 0.01 0.173 ± 0.02
0.084 ± 0.01 0.087 ± 0.01 0.180 ± 0.02 0.213 ± 0.02
0.078 ± 0.01 0.096 ± 0.01 0.185 ± 0.02 0.225 ± 0.03
0.073 ± 0.01 0.092 ± 0.01 0.191 ± 0.02 0.213 ± 0.01
0.081 ± 0.01 0.101 ± 0.02 0.147 ± 0.01 0.198 ± 0.02
0.078 ± 0.01 0.099 ± 0.01 0.164 ± 0.02 0.192 ± 0.01
Values are expressed as mean ± standard error (n ¼ 6). Means bearing * vary significantly between groups (p50.05).
Table 3. Effect of acetamiprid on cell-mediated immune function: (i) stimulation index of lymphocytes exposed to Con A and (ii) paw edema of Wistar rats injected with ovalbumin. Group Sex Stimulation index
Control
Low dose
Male
Female
0.45 ± 0.02
0.43 ± 0.03
Hours 0 3 6 9 24 36 48
Male
Intermediate dose
Female
Male
Female
Cell-mediated immunity – Concanavalin A 0.48 ± 0.01 0.49 ± 0.01 0.41 ± 0.01 0.43 ± 0.03
High dose Male
Female
0.45 ± 0.02
0.47 ± 0.01
Paw edema volume (cm) of Wistar rats** 6.7 ± 0.7 10.9 ± 0.3 14.4 ± 0.3 15.1 ± 0.4 14.8 ± 0.3 14.1 ± 0.4 11.0 ± 0.8
4.9 ± 0.2 7.2 ± 0.4 10.1 ± 0.2 11.8 ± 0.3 13.4 ± 0.5 12.6 ± 0.4 9.8 ± 0.7
5.4 ± 0.1 9.8 ± 0.3 12.9 ± 0.5 14.8 ± 0.1 13.8 ± 0.4 13.5 ± 0.6 10.7 ± 1.0
5.8 ± 0.1 8.4 ± 0.3 9.3 ± 0.3 12.5 ± 1.0 14.2 ± 0.5 12.3 ± 0.3 8.6 ± 1.0
6.6 ± 0.7 9.8 ± 0.3 13.8 ± 0.2 15.0 ± 0.1 14.8 ± 0.1 13.9 ± 0.3 10.9 ± 0.4
5.5 ± 0.3 8.6 ± 0.5 9.6 ± 0.6 11.7 ± 0.4 13.8 ± 0.8 13.5 ± 0.3 10.6 ± 1.0
6.7 ± 0.2 10.2 ± 0.2 14.2 ± 0.4 15.5 ± 0.3 14.9 ± 0.2 14.2 ± 0.3 12.9 ± 0.4
5.6 ± 0.3 8.5 ± 0.6 10.7 ± 0.2 12.1 ± 0.8 14.6 ± 0.3 13.9 ± 0.6 11.1 ± 0.4
Values are expressed as mean ± standard error (n ¼ 6); ** n ¼ 3. Means bearing * vary significantly between groups (p50.05).
Discussion Insecticides might interact with the immune system components causing either immune suppression or immune stimulation, these adverse effects may cause a risk to the human health and are of huge concern. Prolonged exposure to acetamiprid may lead to decrease in the response of immune system and thereby, making the individual susceptible to a variety of infectious/non-infectious diseases including cancers. Human studies are limited by the ability to sample only blood and external secretions; but the majority of the immune cells are not in blood stream, only 2% of the total lymphocytes are circulating at any given time (Calder, 2007). Animal studies can provide information on the structure and functional responses of immune cells present in blood as well as spleen, thymus, lymph node, etc., and rodents are the widely used species to evaluate the effects over the immune system.
A systematic approach of testing is done to assess the effect of compounds over the immune structure and function. Initially, the changes in the immune organs and cells along with general toxicity screening are evaluated in rats. However, because of the dynamic and complex character of immune system, compound induced toxic changes needs to be reliably assessed after immunization/challenge experiments. The general opinion of toxicologists is to utilize additional assays to assess immune function for identifying the hazards (Abdel-Magied et al., 2001). This includes the second set of additional assays, i.e. functional assays that assess the humoral-/cell-mediated immune response after in vivo sensitization and in vitro lymphoproliferative response to specific antigens and macrophage function. The objective of the present investigation was to elucidate the immunotoxic potential of acetamiprid in rats using two experimental
R. K. Shakthi Devan et al.
Drug Chem Toxicol, Early Online: 1–9
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Figure 1. (A and B) Section of the sub-plantar region of the skin from the control rat and high-dose-treated rat, respectively, showing comparable inflammatory reactions characterized by marked mononuclear cell infiltration and fibrin deposits in the dermis. (C and D) Section of the mesenteric lymph node from the control rat and high dose treated rat, respectively, showing normal cortex with no significant pathological changes. (E and F) Section of the spleen from the control rat and high-dose-treated rat, respectively, showing normal periarteriolar lymphoid region with adequate T lymphocytes. (G and H) Section of the thymus from the control rat and high-dose-treated rat, respectively, showing normal cortex with adequate thymocytes exhibiting no reduction/apoptotic changes.
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Table 4. Summary of histological grading of the DTH reaction. Control
Group Sex Mild Moderate Marked Cumulative grade of Intensity
Low dose
Intermediate dose
High dose
Male
Female
Male
Female
Male
Female
Male
Female
2/3 1/3 0/3 7
1/3 1/3 1/3 9
0/3 1/3 2/3 11
1/3 0/3 2/3 10
0/3 1/3 2/3 11
1/3 1/3 1/3 9
0/3 2/3 1/3 10
1/3 1/3 1/3 9
Means bearing * vary significantly between groups (p50.05).
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Table 5. Effect of acetamiprid on stimulation indices of lymphocytes exposed to lipopolysaccharide (LPS), antibody titers against sheep RBC’s and nitrite production by macrophages in Wistar rats. Group Sex
Control Male
Low dose Female
Male
Humoral immunity – LPS Stimulation index 0.55 ± 0.02 0.51 ± 0.05 0.53 ± 0.0 Response to sheep RBCs** HA Titre (log2 values) 9.67 ± 1.33 8.67 ± 1.45 9.0 ± 1.00 Macrophage function (nitrite production in mmoles/50 ml) Unstimulated 1.28 ± 0.06 1.33 ± 0.07 1.38 ± 0.15 Stimulated 1.78 ± 0.13 1.63 ± 0.08 1.71 ± 0.16
Intermediate dose
High dose
Female
Male
Female
Male
Female
0.58 ± 0.04
0.50 ± 0.02
0.48 ± 0.04
0.45 ± 0.01*
0.39 ± 0.04*
8.0 ± 1.15
8.67 ± 1.45
8.33 ± 0.88
8.33 ± 1.20
8.0 ± 0.58
1.25 ± 0.08 1.68 ± 0.07
1.23 ± 0.07 1.48 ± 0.07
1.29 ± 0.05 1.52 ± 0.05
1.18 ± 0.05 1.28 ± 0.06*
1.14 ± 0.03 1.38 ± 0.07*
Values are expressed as mean ± standard error (n ¼ 6); and ** n ¼ 3. Means bearing * vary significantly between groups (p50.05).
animal studies that comprehensively assessed the immunotoxic endpoints. The present study is the first one that evaluated the long-term immunotoxic effects of oral administration of acetamiprid in Wistar rats. The sub-chronic administration of acetamiprid in rats did not elicit clinical signs of toxicity. However, the present study did not reveal significant effects on hematological and biochemical parameters evaluated, namely, total protein, albumin, globulin, lymphocyte, neutrophil and WBC counts after the sub-chronic administration of acetamiprid at the selected dose levels under the experimental conditions. At necropsy, there were no compound-related gross observations present in the treated groups. Absolute and relative organ weights showed no significant changes in males and females of all the dosed groups. The histopathological evaluation of organs/tissues represents one of the cornerstones in the assessment of immunotoxic effects in animals (Kuper, 2000). No significant gross/histopathological changes were present in the lymphoid organs evaluated, namely, spleen, thyroid and mesenteric lymph node indicating the absence of toxicity over the structure of the lymphoid organs. Cell-mediated immune reaction was assessed by delayed type hypersensitivity response. When challenged with an antigen that provokes cell-mediated immune response, the T lymphocytes that have been previously sensitized with the same antigen transform into lymphoblasts secreting cytokines and attract more cells to the reaction site. The cells that arrive at the reaction site get immobilized and lead to enhanced inflammatory response (Sankari et al., 2010). The increases in paw edema volume at various time points were comparable and the volumes did not decrease significantly when compared with control. Similarly, no significant changes were observed in the lymphoproliferative response to T-cell
mitogen in the treated groups. The results indicated that acetamiprid had no effect over the functional integrity of the cell-mediated immune response without causing any significant change over the structural integrity of the immune system. The humoral immune response against sheep sRBC is one of the most sensitive and frequently used endpoints in evaluating the immunotoxicity of drugs and chemicals in experimental animals (Hinton, 2000). The humoral immune responses to sRBCs are well-documented and serve as sensitive end points available to assess compound-induced changes in the immune system of rodents (Luster et al., 1992). This assay which evaluates the antibody production towards sRBC, which is a T-cell-dependent antigen, comprehensively evaluates the immune function which needs integration of several immunological processes. Any compound-induced change in antigen processing/presentation, cellular proliferation, differentiation and/or secretion will modify this response (Luster et al., 1992). The HA titers of the acetamiprid-treated groups were comparable to those of the control group, indicating that acetamiprid did not show any adverse effect on production of antibody by B-lymphocytes in response to antigen, i.e. the sheep red blood cells (sRBC). Similar observation has been reported during imidacloprid, another neonicotinoid compound by Balani et al. (2008) in birds at the dose level 50 mg/kg body wt. The results also correlated with the normal globulin and leucocyte levels. There was a significant decrease in the lymphoproliferative response of the high-dose-treated groups towards the B cell mitogen as compared to the control, indicating the inability of the B lymphocytes to respond on stimulation. Similarly, the nitrite production of the macrophages was also observed to
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R. K. Shakthi Devan et al.
be inhibited by acetamiprid at the high-dose level tested, indicating immunosuppression. Malathion, an important OPC, has been reported to cause significant reduction (24–44%) in nitrite production of the resident peritoneal macrophages (Ayub et al., 2003). In the present study, acetamiprid caused 15–28% reduction in nitrite production compared to that of malathion. The role of macrophages over immune function, inflammation, infection, tumours is well documented. The release of nitric oxide in appropriate concentrations is an important signal for efficient inflammatory response of macrophages. Macrophages liberate nitric oxide in the form of free radicals, which is highly toxic to a variety of microorganisms including bacteria, intracellular parasites, including leishmania and malaria. Nitric oxide causes DNA damage and degrades the iron sulfur centers (Green et al., 1990; Klotz et al., 1995; Li et al., 2006). Since nitrite production is one of the most important mechanism by which macrophages destroy the pathogenic organisms, the results indicate that acetamiprid can cause significant effect over the immune function. Similarly, OPCs have been reported to affect the immune response including neutrophil and macrophage function, antibody production, IL-2 production, serum complement and T cell proliferation induced by IL-2, concanavalin A and phytohemagglutinin in animals and humans (Li & Kawada, 2006). Further, the derived acute population adjusted dose for dietary exposure is 0.1 mg/kg and the chronic population adjusted dose is 0.023 mg/kg/day for acetamiprid (EPA, 2002). However, indiscriminate use of acetamiprid might pose a hazard. The hyporesponsiveness of B cell to LPS-induced proliferation in the present study might result in delayed/impaired humoral immune response. Since the principal challenge for the immune system is to recognize pathogens and to mount an immediate defense response, the decreased B lymphocyte proliferation might increase the chances of susceptibility to infections. Macrophages along with neutrophils are the predominant phagocytes and release many cytotoxic and proinflammatory substances to protect the body from wide array of pathogens and xenobiotics. The functional impairment of macrophages may involve aberrations in the enzymatic degradation of microbes, oxidative burst, generation of free radicals, phagocytosis, release of proinflammatory cytokines. All might hamper host defense causing susceptibility to diseases. Future studies involving a more robust assessment of B-lymphocytes, T-lymphocytes, and T-lymphocyte subsets (TH + TS or CD4 and CD8) by flow cytometric analysis/ immunostaining that shall provide insights into the probable mechanism of immunotoxicity are essential. Further, (i) electrophoretic analysis of serum proteins permitting the quantification of the relative percentages of albumin and the a-, b-, and -globulin fractions and quantification of -globulin fractions (IgG, IgM, IgA, and IgE), (ii) analysis of total serum complement and components of complement (such as C3) from CH-50 determinations and (iii) immunochemical assay of serum cytokines, such as IL-2, IL-1, and J-interferon and quantification of serum auto-antibodies, such as antinuclear, anti-mitochondrial and anti-parietal cell antibodies needs to be carried out to understand the immunopathogenesis of acetamiprid.
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Conclusion Based on the above findings, it is concluded that acetamiprid at the dose level of 110 mg/kg b.w. (20% SP) significantly decreased the lymphocyte proliferation and the macrophage function of rats which might contribute to immunosuppression, increasing the susceptibility to infections. However, acetamiprid caused no significant changes over relevant hematology, biochemistry, organ weights and histopathology of major immune organs and over the delayed-type hypersensitivity response, response to sRBCs and lymphoproliferation assay for T cell mitogens in Wistar rats under the experimental conditions used.
Declaration of interest The authors report no declarations of interest. The authors gratefully acknowledge the funding support provided in the form of Prof. T.R. Rajagopalan research fund by the management of SASTRA University.
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Kuper F. (2000). Histopathologic approaches to detect changes indicative of immunotoxicity. Toxicol Pathol 28:454–466. Lee AM. (2003). Characterization of the insecticidal properties of acetamiprid under field and laboratory conditions [thesis]. Raleigh: Depatment of Entomology. North Carolina State University. Lee P, Tai DY. (2001). Clinical features of patients with acute organophosphate poisoning requiring intensive care. Intensive Care Med 27:694–699. Li Q, Kawada T. (2006). The mechanism of organophosphorus pesticideinduced inhibition of cytolytic activity of killer cells. Cell Mol Immunol 3:171–178. Li Q. (2007). New mechanism of organophosphorus pesticide induced immunotoxicity. J Nippon Med Sch 74:92–105. Li C-Q, Bo P, Tanyel K, et al. (2006). Threshold effects of nitric oxideinduced toxicity and cellular responses in wild-type and p53-null human lymphoblastoid cells. Chem Res Toxicol 19:399–406. Lind RJ, Clough MS, Earley FGP, et al. (1999). Characterisation of multiple a-bungarotoxin binding sites in the aphid Myzus persicae (Hemiptera: Aphididae). Insect Biochem Mol Biol 29:979–988. Liu MY, Casida JE. (1993). High affinity binding of [3H] imidacloprid in the insect acetylcholine receptor. Pestic Biochem Physiol 46:40–46. Luster MI, Portier C, Pait DG, et al. (1992). Risk assessment in immunotoxicology. I. Sensitivity and predictability of immune tests. Fund Appl Toxicol 18:200–210. Mossmann T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63. Nauen R, Elbert A. (1997). Apparent tolerance of a field-collected strain of Myzus nicotianae to imidacloprid due to a strong antifeeding responses. Pestc Sci 49:252–258.
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Rajesh Y, Murli KD, Nita Y, Rudraprabhu S. (2011). Immunomodulatory potential of ethanol extract of Spilanthus acmella leaves. Int J Biol Med Res 2:631–635. Roberts DM, Aaron CK. (2007). Managing acute organophosphorus pesticide poisoning. Brit Med J 334:629–634. Roth DR, Roman D, Ulrich P, et al. (2006). Design and evaluation of immunotoxicity studies. Exp Toxicol Pathol 57:367–371. Sankari M, Chitra V, Jubilee R, et al. (2010). Immunosuppressive activity of aqueous extract of Lagenaria siceraria (standley) in mice. SRM Coll Pharm, Scholar Res Lib 2:291–296. Satheesh CC, Sharma AK, Dwivedi P, et al. (2005). Immunosuppressive effect of Ochratoxin A in Wistar rats. J Anim Vet Adv 4:603–609. Singh ND, Sharma AK, Dwivedi P, et al. (2011). Immunosuppressive effect of combined citrinin and endosulfan toxicity in pregnant Wistar rats. Veterinarski Arhiv 81:751–763. Tamang RK, Jha GJ, Gupta MK, et al. (1988). In vivo immunosuppression by synthetic pyrethroid (cypermethrin) pesticides in mice and goats. Vet Immunol Immunopathol 19:299–305. Thakur MK, Connellan P, Deseo MA, et al. (2011). Immunomodulatory polysaccharide from Chlorophytum borivilianum roots. Evid Based Complement Alternat Med, Article ID 598521, doi:10.1093/ecam/ neq012. Todani M, Kaneko T, Hayashida H. (2008). Acute poisoning with neonicotinoid insecticide acetamiprid. Chudoku Kenkyu 21:387–390. Tomizawa M, Casida JE. (2005). Neonicotinoid insecticide toxicology: mechanisms of selective action. Annu Rev Pharmacol Toxicol 45: 247–268. Zhang AH, Kayser M, Casida JE. (2000). Insect nicotinic acetylcholine receptor: conserved neonicotinoid specificity of [3H] imidacloprid binding site. J Neutochem 75:1294–1303.
RESEARCH ARTICLE
Immunotoxicity assessment of sub-chronic oral administration of acetamiprid in Wistar rats
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R. K. Shakthi Devan, P. C. Prabu, and S. Panchapakesan Central Animal Facility, SASTRA University, Thanjavur, Tamil Nadu, India
Abstract
Keywords
Context: Neonicotinoid insecticides are synthetic analogues of nicotine that acts on the central nervous system of insects by blocking post synaptic acetylcholine receptor. Acetamiprid is one of the widely used neonicotinoid class of insecticide used to control sucking insects like aphids, bees, mosquitoes, on crops. Data on the possible immunotoxic nature of acetamiprid are lacking. Objective: The present study was conducted in Wistar rats with the objective of evaluating the immunotoxic potential of acetamiprid administered orally at the dose levels of 27.5, 55 and 110 mg/kg b.w. (equivalent to 5.5, 11 and 22 mg/kg b.w.) for a period of 90 days. Materials and methods: In experiment 1, general toxicity testing including the evaluation of clinical signs, hemato-biochemical changes, response of the lymphocytes towards T and B cell mitogens, macrophage function, gross and histopathology of the lymphoid organs (spleen, thymus, lymph nodes, etc.) were performed. In the second experiment, humoral and cellmediated responses during immunological challenges were evaluated. Results: Significant decreases were observed in the stimulation index of lymphocyte proliferation to B cell mitogen and in the nitrite production of macrophages of rats treated with 110 mg/kg of acetamiprid. Significant decrease in the lymphoproliferative response towards the B cell mitogen indicated the inability of the B lymphocytes to respond on stimulation that might increase the chances of susceptibility to infections. Acetamiprid also caused 15–28% reduction in nitrite production, an important signal for efficient inflammatory response of macrophages. The functional impairment of macrophages may involve aberrations in the enzymatic degradation of microbes, oxidative burst, generation of free radicals, phagocytosis, release of proinflammatory cytokines and thereby, may hamper host defence causing susceptibility to diseases. No significant changes over hematology, biochemistry, organ weights, histopathology of major immune organs, delayed type hypersensitivity test, response to sRBCs and lymphoproliferation assay for T cell mitogen were observed. Conclusion: In conclusion, the results demonstrate for the first time that the subchronic administration of acetamiprid (20% SP-soluble powder) cause significant decreases in the lymphocyte proliferation as well as the macrophage function at the dose level of 110 mg/kg. Considering the chronic population adjusted dose (0.023 mg/kg/day) through dietary exposure for acetamiprid, judicious use of acetamiprid is highly essential. Indiscriminate use of acetamiprid exceeding the doses advised might pose a hazard.
Cell-mediated immunity, humoral immunity, immunotoxicity, neonicotinoids, rats, safety assessment
Introduction Neonicotinoids are one of the most widely used groups of insecticides that are used to control insects observed in more than 140 crops (Lee, 2003). Neonicotinoids act as agonists of acetyl choline at the synaptic junction and bind with the acetyl choline receptors in the central nervous system of insects causing excitation through interruption of synaptic transmission (Bai et al., 1991; Lind et al., 1999; Liu & Casida, 1993; Nauen & Elbert, 1997; Zhang et al., 2000). The mode
Address for correspondence: Dr S. Panchapakesan, Professor & Coordinator, Central Animal Facility, SASTRA University, Thanjavur – 613 402, Tamil Nadu, India. Tel: +91 4362-264101-108 Extn. 680. Fax: +91 4362-264120. E-mail: [email protected]; [email protected]
History Received 14 April 2014 Revised 27 August 2014 Accepted 14 September 2014 Published online 13 October 2014
of action of neonicotinoids is distinct and cross resistance to other conventionally used insecticides is non-existent. Among neonicotinoids, acetamiprid (E)-N1-[(6-chloro-3-pyridyl) methyl]-N2-cyano-N1-methyl acetamidine is a widely used second-generation chloronicotinyl insecticide with contact and systemic activity via foliar applications. It is systemic in action and controls sucking insects like aphids, bees, mosquitoes, on crops including, but not limited to, cotton, leafy vegetables, citrus and pome fruits, cole crops, grapes and ornamental plants (EPA, 2002). It is an important insecticide in cherry farming because of its lethal action over the cherry fruit fly larvae. Its general mode of action is translaminar activity with contact and stomach action. The recognition of nicotinoids in case of mammals by nicotinic receptors involves cation–pi interactions and in case of insects, possibly a cationic subsite that interacts with
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nitro/cyano substituent of neonicotinoids (Tomizawa & Casida, 2005). There is an increasing awareness that during human exposure, insecticides might cause immune-mediated toxic effects, and therefore immunotoxicity testing of insecticides has been receiving greater attention during the past few decades. Immune system, an intensely controlled complicated network, includes lymphocytes, reticular, dendritic and epithelial cells, which interact through intercellular contacts and cytokines (Roth et al., 2006). Immunotoxicity comprises diverse harmful effects such as immuno-suppression, immuno-modulation and enhanced immune response. Adverse effect on the components and/or function of immune system may be due to either direct or indirect actions, resulting in either permanent or reversible toxicity. Long-term exposure towards chemical stressors can cause direct injury to immune system (Garg, 2004). The symptoms of acetamiprid poisoning in humans have been reported to be partially similar to acute organophosphate (OPC) intoxication. The clinical manifestations of acute poisoning of acetamiprid include vomiting, seizure, hypotension, tachycardia, coma, which mimic the symptoms reported during acute OPC poisoning (Agarwal, 1993; Eddleston et al., 2008; Imamura et al., 2010; Lee & Tai, 2001; Roberts & Aaron, 2007; Todani et al., 2008). The immunotoxic nature of OPCs over immune response including neutrophil function, macrophage, antibody production, interleukin production, serum complement, T cell proliferation, Con A, is widely reported (Li, 2007). Since acetamiprid acts over the neuro-humoral transmission similar to that of the OPC’s and further, as the symptoms of acetamiprid during human exposure mimics that of OPC’s, it might be possible that acetamiprid affects the immune system like OPC’s. However, there are no reports available on the immunotoxic potential of acetamiprid on the structure and function of immune system. The present investigation was designed to assess the immunotoxic effects of acetamiprid during sub-chronic exposure in rats. As the immune system interactions might be numerous and of varying character, a systematic approach is followed in case of immunotoxicity testing in rodents. Immunotoxicity was evaluated using two experimental animal studies, namely, (i) Experiment 1, which investigated general toxicity testing including the evaluation of clinical signs, hemato-biochemical changes, response of the lymphocytes towards T and B cell mitogens, macrophage function, gross and histopathology of the lymphoid organs (spleen, thymus, lymph nodes, etc.) and (ii) Experiment 2, which evaluated the humoral- and cellmediated response during immunological challenges.
Materials and methods Acetamiprid Acetamiprid (20% SP-soluble powder, Nagarjuna Agrichem Ltd., Hyderabad, India) was administered orally to rats for 90 consecutive days. During the pilot study, standardized at Central Animal Facility (CAF), SASTRA University, the LD50 value of acetamiprid (20% SP-soluble powder) was derived at 1100 mg/kg b.w. (equivalent to 220 mg/kg of acetamiprid). One-tenth of the LD50 was taken as highest dose and two-fold decreases were made for fixing the intermediate
Drug Chem Toxicol, Early Online: 1–9
and low dose levels. The dosing volume was maintained at 1 mL/100 g body weight. Experimental animals The present study was conducted in Wistar rats (7–8 weeks old) bred at Central Animal Facility after the clearance of Institutional Animal Ethics Committee, SASTRA University. All the rats were acclimatized for 7 days and apparently healthy rats were selected for the study. The rats were weighed and randomized using a computer-generated stratified randomization procedure based on body weights so that all groups for each sex had approximately equal mean body weights. The individual weight variation did not exceed 20% of the mean body weights. Rats were provided unique identification number by ear tag and cage cards were placed for the identification of animal number, group, sex, dose levels, etc. Rats were group-housed in polypropylene cages with wire mesh, top grill made of stainless steel, fed with standard rodent diet (M/s Provimi Animal Nutrition India Private Limited, Bangalore, India) and reverse osmosis purified water ad libitum. The temperature and relative humidity were maintained at 22 ± 3 C and 30–70%, respectively. Photoperiod of 12 h light and 12 h dark cycle was maintained by an automatic timer. The study consisted of two sets of animal experiments and were conducted as per the following procedure. Experiment 1 Forty-eight rats were segregated into 4 groups of 12 rats each and dosed orally with acetamiprid for 90 days as follows: Group No.
Dose Levels
No. of Animal
Group Group Group Group
Control Low dose Intermediate dose High dose
12 12 12 12
1 2 3 4
(6 (6 (6 (6
M+6 M+6 M+6 M+6
F) F) F) F)
M – Male, F – Female
The control rats were administered with distilled water and the low-, intermediate and high-dose group rats were administered with acetamiprid (20% SP-soluble powder) at the dose levels of 27.5, 55 and 110 mg/kg b.w., respectively (equivalent to 5.5, 11 and 22 mg/kg b.w. of acetamiprid). All the individual rats of each group were examined twice daily for the presence of any morbidity, mortality and signs of toxicity. On day 90, blood samples were collected through retro orbital plexus from all the rats before necropsy and were used for hematological, biochemical estimations. Lymphocytes were separated from the whole blood using commercially available lymphocyte separation media (Histopaque, Sigma, Bangalore, India) based on the principle of density gradient and were used for the lymphoproliferation assay. After blood collection, the rat-resident peritoneal macrophages were collected under sterile conditions in a laminar flow chamber from all the rats using sterile syringes before necropsy. Hematological and biochemical parameters. White blood cell
(WBC) count, lymphocyte (%) and neutrophil (%) were analyzed from the blood samples collected in EDTA-coated vial using a calibrated 5-part blood cell counter (Orphee,
Immunotoxicity of acetamiprid in rats
DOI: 10.3109/01480545.2014.966382
Switzerland). Similarly, plasma was separated from blood collected in heparinized vial (20 IU/mL of blood) by centrifuging at 2000 rpm for 10 min and was utilized for the estimation of total protein (TP) and albumin (ALB) using a calibrated random access clinical chemistry analyzer. Globulin was calculated by subtracting albumin from total protein.
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Lymphoproliferation assay for B and T cell mitogen. Lymphocyte proliferation was assessed using MTT
[3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye method (Mossmann, 1983; Singh et al., 2011). 100 mL of lymphocyte suspension (3 106 cells/ml) was pre-incubated for 24 h in a microtitre plate added with RPMI 1640 medium and incubated at 37 C in a humidified chamber with 5% CO2 before the addition of the appropriate mitogen, i.e. lipopolysaccharide as the B cell mitogen and concanavalin A as T cell mitogen. The cell pellets were separated by centrifuging at 1500 rpm for 10 min and were again suspended in 50 ml of MTT (5 mg/ml) in RPMI media. Following incubation in humidified 5% CO2 chamber for 4 h at 37 C, dimethyl sulphoxide was added to the medium separated by centrifuging and absorbance at 570 nm was recorded. The percentage of proliferation was calculated as follows: Proliferation % ¼ ðOD stimulated OD unstimulatedÞ 100=ðOD stimulatedÞ Macrophage function assay. The viable rat resident peritoneal
macrophages collected were calculated using trypan blue dye exclusion method. Rat peritoneal macrophages were suspended in phenol red free RPMI growth medium containing 5 mM arginine at a concentration of 2 106 cells/ml and 100 ml of this suspension was added to a 96-well plate in triplicates. The cells that were not adherent following 2 h of incubation were removed by washing with phosphate buffer saline. The final volume was made to 200 ml with LPS of 1 mg/ml concentration. Following incubation at 37 C in humidified chamber with 5% CO2, the supernatant was removed after 72 h and the nitrite production was estimated using the Griess reagent. To 50 ml of supernatant, 50 ml of Griess reagent was added, incubated for 10 min and the absorbance at 570 nm was measured (Garcia et al., 2002; Green et al., 1982). After the collection of blood samples and peritoneal macrophages, rats of all the groups were euthanized using CO2 inhalation followed by exsanguination and were examined for the presence of gross pathological changes. Weights of spleen and thymus were measured and expressed as absolute and relative organ weights (g and g/100 g of body weight). Spleen, thymus and lymph node were collected from all the groups, fixed in 10% neutral buffered formalin and processed for histopathological evaluation.
Gross and histopathological evaluation.
Experiment 2 In this experiment, 48 rats were orally administered with acetamiprid for 90 consecutive days and were evaluated for
3
the cell mediated immune response and humoral immune response as follows: Group No.
Dose Levels
Cell mediated immune response Group 5 Control Group 6 Low dose Group 7 Intermediate dose Group 8 High dose Humoral immune response Group 9 Control Group 10 Low dose Group 11 Intermediate dose Group 12 High dose
No. of Animal 6 6 6 6
(3 (3 (3 (3
M+3 M+3 M+3 M+3
F) F) F) F)
6 6 6 6
(3 (3 (3 (3
M+3 M+3 M+3 M+3
F) F) F) F)
The control rats were administered with distilled water, and the low-, intermediate and high-dose group rats were administered with acetamiprid (20% SP-soluble powder) at the dose levels of 27.5, 55 and 110 mg/kg b.w., respectively (equivalent to 5.5, 11 and 22 mg/kg b.w. of acetamiprid). Cell mediated immunity – delayed-type hypersensitivity test Cell-mediated immune response was evaluated using delayedtype hypersensitivity reaction induced by ovalbumin, as described by Tamang et al. (1988) and Satheesh et al. (2005) with slight modifications. Rats were sensitized using intra-dermal injection of 100 mg of ovalbumin mixed with Freund’s Complete Adjuvant (FCA) over the sub-plantar region of the left hind limb on day 74. The sensitized rats were challenged after 14 days, i.e. on day 88, with 0.05 mL of ovalbumin. The volume of the paw edema reaction was measured using plethysmometer at 0, 3, 6, 9, 24, 36 and 48 h post challenge. After sacrifice, the reaction sites were collected, fixed, processed and sectioned for histopathological analysis. The reactions were graded as minimal (1), mild (2), moderate (3), marked (4) and severe (5) and the cumulative grading of intensity was calculated for each group. For the assessment of changes in humoral response, remaining 24 rats were divided into 4 groups of 6 rats each and were sensitized by injecting 0.5 mL of sheep red blood cells (sRBCs) containing 1.25 106 cells suspended in PBS per rat intraperitoneally on day 72. Fourteen days after sensitization, i.e. on day 86, the rats were again challenged with 0.25 mL of sRBCs. After 4 days, i.e. on day 90, blood was collected, serum separated and humoral immune response was measured by hemagglutination (HA) test following the procedure described by Beard (1980), Thakur et al. (2011) and Rajesh et al. (2011). HA test was done in a U-shaped micro-perplex plate. The serum samples were serially diluted (two-fold) in phosphate buffer saline so that the final volume in each well was 0.05 ml. In the control wells, only phosphate buffer saline was added. Further, 0.05 ml of sRBC suspension (0.5%) was added to all the wells. A known negative control was also included. The HA plate was swirled gently for ensuring uniform distribution of sRBCs and was allowed at room temperature for 45 min. The HA pattern was observed as a diffuse sheet of RBCs covering the bottom of the wells and the titre, i.e. reciprocal of the highest dilution showing
Humoral immune response – hemagglutination test.
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Table 1. Effect of acetamiprid on hemato-biochemical values in Wistar rats. Parameters
Sex
WBC (%)
Male Female Male Female Male Female Male Female Male Female Male Female Male Female
Lymphocyte (%) Neutrophil (%) Total Protein (g/L) Albumin (g/L) Globulin (g/L)
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Albumin Globulin ratio
Control
Low dose
Intermediate dose
High dose
9.10 ± 0.83 5.20 ± 0.15 68.63 ± 1.40 71.23 ± 2.14 29.97 ± 1.14 26.57 ± 1.39 61.76 ± 0.96 62.33 ± 1.33 32.57 ± 0.73 31.37 ± 1.33 29.19 ± 0.62 30.97 ± 0.27 1.12 ± 0.04 1.01 ± 0.04
10.20 ± 0.91 5.07 ± 0.16 69.90 ± 1.78 69.83 ± 0.89 28.77 ± 1.87 29.03 ± 0.94 62.73 ± 1.72 64.17 ± 0.65 31.63 ± 0.98 33.73 ± 0.97 31.10 ± 0.94 30.43 ± 0.72 1.02 ± 0.0 1.11 ± 0.06
9.43 ± 0.31 5.13 ± 0.28 71.33 ± 1.87 70.50 ± 0.65 27.07 ± 2.00 28.57 ± 0.43 62.17 ± 1.76 63.50 ± 0.41 31.03 ± 0.81 31.77 ± 1.02 31.13 ± 0.96 31.73 ± 1.52 1.00 ± 0.01 1.01 ± 0.41
9.17 ± 0.20 4.90 ± 0.32 71.10 ± 1.37 69.70 ± 1.63 27.63 ± 1.12 28.70 ± 1.65 64.73 ± 1.13 64.80 ± 0.80 31.70 ± 0.29 30.87 ± 1.31 33.03 ± 0.86 33.93 ± 0.62 0.96 ± 0.02 0.91 ± 0.05
Values are expressed as mean ± standard error (n ¼ 6). Means bearing * vary significantly between groups (p50.05).
complete agglutination of sRBCs, was observed and expressed as log2/0.05 mL. Statistical analysis All the quantitative data collected were subjected to statistical analysis using Graph pad Prism software (La Jolla, CA) and are presented as mean ± standard error. The mean values from organ weight, hematological, biochemical and immunological parameters were subjected to analysis of variance (ANOVA). A probability level lower than 5% (p50.05) was considered to be significant.
Results Experiment 1 Clinical signs All the rats survived throughout the study and there were no clinical signs of toxicity during the observation period. Hematology and biochemistry parameters The hematological and biochemical parameters evaluated – namely, total protein, albumin, globulin, albumin globulin ratio, WBC, lymphocyte and neutrophils values – are presented in Table 1. No significant changes were present in hematology as well as biochemical parameters of the treatment groups as compared to the control. Lymphoproliferation assay for B- and T-cell mitogen The stimulation index of the lymphocytes of the high-dose groups exposed to B-cell mitogen was significantly lower as compared to that of the controls. However, the stimulation indices of the low and intermediate dose levels were comparable to the control. The stimulation index of the lymphocytes exposed to T-cell mitogen was comparable among different groups. The stimulation indices of the lymphocytes of different groups towards B- and T-cell mitogen are presented as Table 3.
the controls. The nitrite production of stimulated macrophages in the low- and intermediate dose levels was comparable to those of the controls (Table 5). Gross pathology and histopathology There were no compound-related gross lesions observed during necropsy. No significant changes were observed in absolute and relative organ weights (Table 2). Microscopic evaluation of spleen, thymus and lymph node revealed no compound-related histopathological changes. The changes observed were either agonal or spontaneous and were observed in both control as well as treatment groups. Representative images of the organs evaluated in the control and high-dose groups are presented (Figure 1). Experiment 2 Cell-mediated immune response – delayed type hypersensitivity test Cell-mediated immune response assessed using the delayed type hypersensitivity response to ovalbumin revealed increase in paw edema volume in the treated groups that were comparable to the control group at different intervals measured (Table 3). Histopathology for DTH reaction In the skin sections, DTH reaction was observed as mild to moderate hydropic degeneration of the squamous epithelium of the epidermis, mild to moderate oedema, fibrin deposits and minimal to marked mononuclear cell infiltration in the dermis. Inflammatory reactions of different intensities were present in both controls as well as all the treated groups. The frequency and intensity of the DTH reaction of the rats from different groups are presented (Table 4). No reduction in the intensity of the inflammatory reaction was observed in the treated sections as compared to the control sections. Humoral immune response – hemagglutination test
Macrophage function assay Nitrite production of stimulated macrophages was significantly lower in the high-dose groups as compared to that of
There were no significant changes in the HA titers present in the treated groups as compared to the control on day 90 (Table 5).
Immunotoxicity of acetamiprid in rats
DOI: 10.3109/01480545.2014.966382
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Table 2. Effect of acetamiprid on absolute and relative organ weights of spleen and thymus of Wistar rats.
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Parameters
Sex
Absolute organ weight (g) Cell-mediated immunity Thymus Male Female Spleen Male Female Humoral immunity Thymus Male Female Spleen Male Female Relative organ weight (g/100 g) Cell-mediated immunity Thymus Male Female Spleen Male Female Humoral immunity Thymus Male Female Spleen Male Female
Control
Low dose
Intermediate dose
High dose
0.270 ± 0.02 0.225 ± 0.02 0.631 ± 0.04 0.474 ± 0.02
0.258 ± 0.02 0.235 ± 0.01 0.552 ± 0.02 0.414 ± 0.03
0.298 ± 0.02 0.241 ± 0.01 0.685 ± 0.02 0.429 ± 0.04
0.279 ± 0.02 0.211 ± 0.01 0.599 ± 0.08 0.516 ± 0.05
0.277 ± 0.03 0.241 ± 0.01 0.663 ± 0.04 0.564 ± 0.08
0.253 ± 0.02 0.228 ± 0.02 0.665 ± 0.06 0.533 ± 0.03
0.282 ± 0.02 0.245 ± 0.05 0.511 ± 0.03 0.484 ± 0.04
0.266 ± 0.02 0.247 ± 0.02 0.554 ± 0.05 0.478 ± 0.02
0.077 ± 0.01 0.092 ± 0.01 0.180 ± 0.02 0.194 ± 0.02
0.075 ± 0.01 0.094 ± 0.01 0.159 ± 0.01 0.166 ± 0.01
0.083 ± 0.01 0.098 ± 0.01 0.193 ± 0.01 0.173 ± 0.02
0.084 ± 0.01 0.087 ± 0.01 0.180 ± 0.02 0.213 ± 0.02
0.078 ± 0.01 0.096 ± 0.01 0.185 ± 0.02 0.225 ± 0.03
0.073 ± 0.01 0.092 ± 0.01 0.191 ± 0.02 0.213 ± 0.01
0.081 ± 0.01 0.101 ± 0.02 0.147 ± 0.01 0.198 ± 0.02
0.078 ± 0.01 0.099 ± 0.01 0.164 ± 0.02 0.192 ± 0.01
Values are expressed as mean ± standard error (n ¼ 6). Means bearing * vary significantly between groups (p50.05).
Table 3. Effect of acetamiprid on cell-mediated immune function: (i) stimulation index of lymphocytes exposed to Con A and (ii) paw edema of Wistar rats injected with ovalbumin. Group Sex Stimulation index
Control
Low dose
Male
Female
0.45 ± 0.02
0.43 ± 0.03
Hours 0 3 6 9 24 36 48
Male
Intermediate dose
Female
Male
Female
Cell-mediated immunity – Concanavalin A 0.48 ± 0.01 0.49 ± 0.01 0.41 ± 0.01 0.43 ± 0.03
High dose Male
Female
0.45 ± 0.02
0.47 ± 0.01
Paw edema volume (cm) of Wistar rats** 6.7 ± 0.7 10.9 ± 0.3 14.4 ± 0.3 15.1 ± 0.4 14.8 ± 0.3 14.1 ± 0.4 11.0 ± 0.8
4.9 ± 0.2 7.2 ± 0.4 10.1 ± 0.2 11.8 ± 0.3 13.4 ± 0.5 12.6 ± 0.4 9.8 ± 0.7
5.4 ± 0.1 9.8 ± 0.3 12.9 ± 0.5 14.8 ± 0.1 13.8 ± 0.4 13.5 ± 0.6 10.7 ± 1.0
5.8 ± 0.1 8.4 ± 0.3 9.3 ± 0.3 12.5 ± 1.0 14.2 ± 0.5 12.3 ± 0.3 8.6 ± 1.0
6.6 ± 0.7 9.8 ± 0.3 13.8 ± 0.2 15.0 ± 0.1 14.8 ± 0.1 13.9 ± 0.3 10.9 ± 0.4
5.5 ± 0.3 8.6 ± 0.5 9.6 ± 0.6 11.7 ± 0.4 13.8 ± 0.8 13.5 ± 0.3 10.6 ± 1.0
6.7 ± 0.2 10.2 ± 0.2 14.2 ± 0.4 15.5 ± 0.3 14.9 ± 0.2 14.2 ± 0.3 12.9 ± 0.4
5.6 ± 0.3 8.5 ± 0.6 10.7 ± 0.2 12.1 ± 0.8 14.6 ± 0.3 13.9 ± 0.6 11.1 ± 0.4
Values are expressed as mean ± standard error (n ¼ 6); ** n ¼ 3. Means bearing * vary significantly between groups (p50.05).
Discussion Insecticides might interact with the immune system components causing either immune suppression or immune stimulation, these adverse effects may cause a risk to the human health and are of huge concern. Prolonged exposure to acetamiprid may lead to decrease in the response of immune system and thereby, making the individual susceptible to a variety of infectious/non-infectious diseases including cancers. Human studies are limited by the ability to sample only blood and external secretions; but the majority of the immune cells are not in blood stream, only 2% of the total lymphocytes are circulating at any given time (Calder, 2007). Animal studies can provide information on the structure and functional responses of immune cells present in blood as well as spleen, thymus, lymph node, etc., and rodents are the widely used species to evaluate the effects over the immune system.
A systematic approach of testing is done to assess the effect of compounds over the immune structure and function. Initially, the changes in the immune organs and cells along with general toxicity screening are evaluated in rats. However, because of the dynamic and complex character of immune system, compound induced toxic changes needs to be reliably assessed after immunization/challenge experiments. The general opinion of toxicologists is to utilize additional assays to assess immune function for identifying the hazards (Abdel-Magied et al., 2001). This includes the second set of additional assays, i.e. functional assays that assess the humoral-/cell-mediated immune response after in vivo sensitization and in vitro lymphoproliferative response to specific antigens and macrophage function. The objective of the present investigation was to elucidate the immunotoxic potential of acetamiprid in rats using two experimental
R. K. Shakthi Devan et al.
Drug Chem Toxicol, Early Online: 1–9
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Figure 1. (A and B) Section of the sub-plantar region of the skin from the control rat and high-dose-treated rat, respectively, showing comparable inflammatory reactions characterized by marked mononuclear cell infiltration and fibrin deposits in the dermis. (C and D) Section of the mesenteric lymph node from the control rat and high dose treated rat, respectively, showing normal cortex with no significant pathological changes. (E and F) Section of the spleen from the control rat and high-dose-treated rat, respectively, showing normal periarteriolar lymphoid region with adequate T lymphocytes. (G and H) Section of the thymus from the control rat and high-dose-treated rat, respectively, showing normal cortex with adequate thymocytes exhibiting no reduction/apoptotic changes.
Immunotoxicity of acetamiprid in rats
DOI: 10.3109/01480545.2014.966382
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Table 4. Summary of histological grading of the DTH reaction. Control
Group Sex Mild Moderate Marked Cumulative grade of Intensity
Low dose
Intermediate dose
High dose
Male
Female
Male
Female
Male
Female
Male
Female
2/3 1/3 0/3 7
1/3 1/3 1/3 9
0/3 1/3 2/3 11
1/3 0/3 2/3 10
0/3 1/3 2/3 11
1/3 1/3 1/3 9
0/3 2/3 1/3 10
1/3 1/3 1/3 9
Means bearing * vary significantly between groups (p50.05).
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Table 5. Effect of acetamiprid on stimulation indices of lymphocytes exposed to lipopolysaccharide (LPS), antibody titers against sheep RBC’s and nitrite production by macrophages in Wistar rats. Group Sex
Control Male
Low dose Female
Male
Humoral immunity – LPS Stimulation index 0.55 ± 0.02 0.51 ± 0.05 0.53 ± 0.0 Response to sheep RBCs** HA Titre (log2 values) 9.67 ± 1.33 8.67 ± 1.45 9.0 ± 1.00 Macrophage function (nitrite production in mmoles/50 ml) Unstimulated 1.28 ± 0.06 1.33 ± 0.07 1.38 ± 0.15 Stimulated 1.78 ± 0.13 1.63 ± 0.08 1.71 ± 0.16
Intermediate dose
High dose
Female
Male
Female
Male
Female
0.58 ± 0.04
0.50 ± 0.02
0.48 ± 0.04
0.45 ± 0.01*
0.39 ± 0.04*
8.0 ± 1.15
8.67 ± 1.45
8.33 ± 0.88
8.33 ± 1.20
8.0 ± 0.58
1.25 ± 0.08 1.68 ± 0.07
1.23 ± 0.07 1.48 ± 0.07
1.29 ± 0.05 1.52 ± 0.05
1.18 ± 0.05 1.28 ± 0.06*
1.14 ± 0.03 1.38 ± 0.07*
Values are expressed as mean ± standard error (n ¼ 6); and ** n ¼ 3. Means bearing * vary significantly between groups (p50.05).
animal studies that comprehensively assessed the immunotoxic endpoints. The present study is the first one that evaluated the long-term immunotoxic effects of oral administration of acetamiprid in Wistar rats. The sub-chronic administration of acetamiprid in rats did not elicit clinical signs of toxicity. However, the present study did not reveal significant effects on hematological and biochemical parameters evaluated, namely, total protein, albumin, globulin, lymphocyte, neutrophil and WBC counts after the sub-chronic administration of acetamiprid at the selected dose levels under the experimental conditions. At necropsy, there were no compound-related gross observations present in the treated groups. Absolute and relative organ weights showed no significant changes in males and females of all the dosed groups. The histopathological evaluation of organs/tissues represents one of the cornerstones in the assessment of immunotoxic effects in animals (Kuper, 2000). No significant gross/histopathological changes were present in the lymphoid organs evaluated, namely, spleen, thyroid and mesenteric lymph node indicating the absence of toxicity over the structure of the lymphoid organs. Cell-mediated immune reaction was assessed by delayed type hypersensitivity response. When challenged with an antigen that provokes cell-mediated immune response, the T lymphocytes that have been previously sensitized with the same antigen transform into lymphoblasts secreting cytokines and attract more cells to the reaction site. The cells that arrive at the reaction site get immobilized and lead to enhanced inflammatory response (Sankari et al., 2010). The increases in paw edema volume at various time points were comparable and the volumes did not decrease significantly when compared with control. Similarly, no significant changes were observed in the lymphoproliferative response to T-cell
mitogen in the treated groups. The results indicated that acetamiprid had no effect over the functional integrity of the cell-mediated immune response without causing any significant change over the structural integrity of the immune system. The humoral immune response against sheep sRBC is one of the most sensitive and frequently used endpoints in evaluating the immunotoxicity of drugs and chemicals in experimental animals (Hinton, 2000). The humoral immune responses to sRBCs are well-documented and serve as sensitive end points available to assess compound-induced changes in the immune system of rodents (Luster et al., 1992). This assay which evaluates the antibody production towards sRBC, which is a T-cell-dependent antigen, comprehensively evaluates the immune function which needs integration of several immunological processes. Any compound-induced change in antigen processing/presentation, cellular proliferation, differentiation and/or secretion will modify this response (Luster et al., 1992). The HA titers of the acetamiprid-treated groups were comparable to those of the control group, indicating that acetamiprid did not show any adverse effect on production of antibody by B-lymphocytes in response to antigen, i.e. the sheep red blood cells (sRBC). Similar observation has been reported during imidacloprid, another neonicotinoid compound by Balani et al. (2008) in birds at the dose level 50 mg/kg body wt. The results also correlated with the normal globulin and leucocyte levels. There was a significant decrease in the lymphoproliferative response of the high-dose-treated groups towards the B cell mitogen as compared to the control, indicating the inability of the B lymphocytes to respond on stimulation. Similarly, the nitrite production of the macrophages was also observed to
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R. K. Shakthi Devan et al.
be inhibited by acetamiprid at the high-dose level tested, indicating immunosuppression. Malathion, an important OPC, has been reported to cause significant reduction (24–44%) in nitrite production of the resident peritoneal macrophages (Ayub et al., 2003). In the present study, acetamiprid caused 15–28% reduction in nitrite production compared to that of malathion. The role of macrophages over immune function, inflammation, infection, tumours is well documented. The release of nitric oxide in appropriate concentrations is an important signal for efficient inflammatory response of macrophages. Macrophages liberate nitric oxide in the form of free radicals, which is highly toxic to a variety of microorganisms including bacteria, intracellular parasites, including leishmania and malaria. Nitric oxide causes DNA damage and degrades the iron sulfur centers (Green et al., 1990; Klotz et al., 1995; Li et al., 2006). Since nitrite production is one of the most important mechanism by which macrophages destroy the pathogenic organisms, the results indicate that acetamiprid can cause significant effect over the immune function. Similarly, OPCs have been reported to affect the immune response including neutrophil and macrophage function, antibody production, IL-2 production, serum complement and T cell proliferation induced by IL-2, concanavalin A and phytohemagglutinin in animals and humans (Li & Kawada, 2006). Further, the derived acute population adjusted dose for dietary exposure is 0.1 mg/kg and the chronic population adjusted dose is 0.023 mg/kg/day for acetamiprid (EPA, 2002). However, indiscriminate use of acetamiprid might pose a hazard. The hyporesponsiveness of B cell to LPS-induced proliferation in the present study might result in delayed/impaired humoral immune response. Since the principal challenge for the immune system is to recognize pathogens and to mount an immediate defense response, the decreased B lymphocyte proliferation might increase the chances of susceptibility to infections. Macrophages along with neutrophils are the predominant phagocytes and release many cytotoxic and proinflammatory substances to protect the body from wide array of pathogens and xenobiotics. The functional impairment of macrophages may involve aberrations in the enzymatic degradation of microbes, oxidative burst, generation of free radicals, phagocytosis, release of proinflammatory cytokines. All might hamper host defense causing susceptibility to diseases. Future studies involving a more robust assessment of B-lymphocytes, T-lymphocytes, and T-lymphocyte subsets (TH + TS or CD4 and CD8) by flow cytometric analysis/ immunostaining that shall provide insights into the probable mechanism of immunotoxicity are essential. Further, (i) electrophoretic analysis of serum proteins permitting the quantification of the relative percentages of albumin and the a-, b-, and -globulin fractions and quantification of -globulin fractions (IgG, IgM, IgA, and IgE), (ii) analysis of total serum complement and components of complement (such as C3) from CH-50 determinations and (iii) immunochemical assay of serum cytokines, such as IL-2, IL-1, and J-interferon and quantification of serum auto-antibodies, such as antinuclear, anti-mitochondrial and anti-parietal cell antibodies needs to be carried out to understand the immunopathogenesis of acetamiprid.
Drug Chem Toxicol, Early Online: 1–9
Conclusion Based on the above findings, it is concluded that acetamiprid at the dose level of 110 mg/kg b.w. (20% SP) significantly decreased the lymphocyte proliferation and the macrophage function of rats which might contribute to immunosuppression, increasing the susceptibility to infections. However, acetamiprid caused no significant changes over relevant hematology, biochemistry, organ weights and histopathology of major immune organs and over the delayed-type hypersensitivity response, response to sRBCs and lymphoproliferation assay for T cell mitogens in Wistar rats under the experimental conditions used.
Declaration of interest The authors report no declarations of interest. The authors gratefully acknowledge the funding support provided in the form of Prof. T.R. Rajagopalan research fund by the management of SASTRA University.
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