Hematological Alterations on Sub-acute Exposure to Flubendiamide in Sprague Dawley Rats.

BACKGROUND: Pesticide poisoning is a common occurrence around the world. Pesticides can act on various body systems resulting in toxicity. Flubendiamide is a new generation pesticide, reported to have better activity against Lepidopteran insects. The present study was carried out with an objective to analyze the effects of flubendiamide sub-acute exposure on hematology of rats.
MATERIALS AND METHODS: Male and female Sprague Dawley (SD) rats (9-11 weeks) were divided into five groups with six animals in each group. First group served as control, while the rest were exposed to ascending oral doses of flubendiamide (125, 250, 500 and 1000 mg/kg) for 28 days. After the trial period, blood was collected in heparinized vials and analyzed using Siemens ADVIA 2120(®) autoanalyzer. Various erythrocytic, platelet and leukocyte parameters were measured and analyzed using statistical tests by one-way analysis of variance (ANOVA) and t-test using Statistical Package for Social Sciences (SPSS)(®) 20 software.
RESULTS: After processing the data through statistical analysis, it was observed that the effect of flubendiamide exposure on female rats was negligible. The only significant change observed in the female rats was that in total erythrocytic count, while rest of the parameters showed non-significant bidirectional changes. In males, many parameters viz., total leukocyte count (TLC), total erythrocyte count (TEC), packed cell volume (PCV), mean corpuscular volume (MCV), platelet count (PC), mean platelet volume (MPV), platelet distribution width (PDW), hemoglobin distribution width (HDW), large platelets (LPT) and plateletcrit (PCT) expressed significant difference when compared to control.
CONCLUSION: Many of the changes were dose independent, but sex specific. This lead to the hypothesis that saturation toxicokinetics might be one of the reasons for this varied response, which can only be evaluated after further testing.

INTRODUCTION

Pesticides and fertilizers form an integral part of the agriculture system in India. Due to the increasing demand in food production, there is an increase in pesticide use in intensive agrarian states such as Punjab, Haryana, Andhra Pradesh, Gujarat etc.[1] Pesticide toxicity in animals has become a matter of serious concern in the last few decades due to rapid industrial expansion and adoption of intensive farming practices in India.[2]

Flubendiamide (N2-[1,1-dimethyl-2-(methylsulfonyl) ethyl]-3-iodo-N1-[2-methyl-4-[1,2,2,2-tetrafluoro-1-(trifluoromethyl) ethyl] phenyl]-1,2-benzene dicarboxamide) belongs to phthalic acid diamide group with potent, broad-spectrum activity against the insects of order Lepidoptera.[3] The novelty of this compound lies in its selectivity to activate insect ryanodine receptors (RyRs) present on neurons and skeletal muscle; thereby leading to massive intracellular release of calcium ions.[4] This target is attractive for insecticides with no cross-resistance to other known modes of action.[5] The diamides have been classified under new group (28: Ryanodine receptor modulators) by the Insecticide Resistance Action Committee (IRAC, www.irac-online.org). Flubendiamide was registered in India in the year 2007 under the trademark Fame.[6] Major uses of flubendiamide as per the guidance document of the Government of India include application in pigeon pea, black gram, chilli, tomato, cabbage, cotton and rice crops.[7]

The reported median lethal dose (LD50) was more than 2,000 mg/kg for both male and female rats indicating that the pesticide is safe even after accidental consumption. Females were reported to be highly sensitive to flubendiamide when compared to males.[8] Though, no mammalian toxicity has been published so far, there were reports of flubendiamide poisoning in buffaloes upon consumption of fodder recently sprayed with the pesticide (unpublished data). Acute toxicity of agricultural pesticides (flubendiamide, endosulfan, and spirotetramat) to embryo, larval, and juvenile stages of African catfish, Clarias gariepinus, has been carried out recently. It was found that flubendiamide was toxic with LC50 48 h at 2 ppm (2 μg/mL). All stages of fish life, that is, egg, larva, juvenile, and adults were susceptible to flubendiamide.[9] Chronic studies of flubendiamide exposure in rodents have been reported to have a negative impact on growth.[10] Due to the scarcity of data on effect of flubendiamide exposure on hematology, the present study was planned with an objective to test the pesticide sub-acute exposure on hematology in Sprague Dawley (SD) rats.

MATERIALS AND METHODS

Thirty male and female SD rats of 9–11 weeks of age were used in the present study. Rats were obtained from National Institute of Pharmaceutical Education and Research, Mohali, India. They were maintained under optimal conditions of 12:12 h light-dark cycle, ad libitum feed (Rat Pellet Feed, Ashirwad Industries®, Chandigarh, India), and water supply. Study was carried out after obtaining permission from Institutional Animal Ethics Committee (IAEC; Lr. No. VMC/14/2413-43 dated 10.06.2014). After acclimatization for a week, rats were dosed orally with flubendiamide (technical grade) for 28 days. Rats were divided into five groups with three male and three female rats in each group with a coefficient of variation of less than 10% for body weight. Group I acted as control and received only vehicle (0.4% Tween 80 (Merck, India)), whereas Groups II, III, IV and V received flubendiamide at a dose rate of 125, 250, 500 and 1,000 mg/kg body weight. Pesticide was prepared as suspension and the concentrations were prepared so that the dosing volume in each group was between 1–2 ml/100 g body weight as per the Organization for Economic Co-operation and Development (OECD) guidelines. After completion of the trial, whole blood samples were collected using heparin sodium (at 0.5–2 IU per ml of blood) as anticoagulant. Sampling period covering all animals was completed in 2-h duration (7.00 A. M.–9.00 A. M.). Samples were instantly analyzed for various hematological parameters using Siemens ADVIA 2120® hemoautoanalyzer following manufacturer's instructions. Hematological parameters analyzed include total leukocyte count (TLC), total erythrocyte count (TEC), hemoglobin (Hb), packed cell volume (PCV), total platelet count (TPC), mean platelet volume (MPV), red cell hemoglobin concentration mean (CHCM), cellular hemoglobin concentration (CM), platelet distribution width (PDW), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), hemoglobin distribution width (HDW), plateletcrit (PCT), mean platelet component (MPC) and large platelets (LPTs). Table 1 describes in detail the parameters measured along with units giving a reference value of hematological indices in male and female SD rats of 13 weeks of age.[11] All the results have been analyzed using Statistical Package for Social Sciences® version 20 through one-way analysis of variance followed by Tukey's post-hoc test and unpaired t-test to compare between male and female rats. All the values are expressed as mean ± standard error (SE).

Table 1

Reference values along with brief description for various hematological indices in Sprague Dawley rats

RESULTS

Results of the study are presented in Tables 2 and 3. In female rats, significant (P < 0.05) difference in TEC was observed. There was an overall decrease in the RBC counts with respect to dose rate. Rest of the parameters exhibited non-significant decrease. In males, parameters viz., WBC, RBC, PCV, MCV, PLT, MPV, PDW, HDW, LPT and PCT expressed significant (P < 0.05) difference. There was significant (P < 0.05) decrease in WBC, LPT, PCT, PDW, MPV and PCs; while significant increase was observed in parameters viz., RBC, PCV and HDW. The results in the present study indicated a bidirectional response in some parameters, wherein there was increase/decrease till exposure of 500 or sometimes 250 mg/kg; while any further increase in dose resulted in returning of the values comparable to control and seldom beyond that of control. Significant differences were observed between male and female rats of the same group for parameters viz., RBC, Hb, PCV, RDW, MCHC, CHCM, CH and LPTs. Of these parameters, sex-dependent differences were consistent across most groups in Hb and PCV. There was dose-dependent increase in RBC, Hb, and PCV in males; whereas dose-dependent decrease was observed in female rats. MCH, MCHC and RDW showed a vice versa response upon increase in exposure dose rate. In case of platelet parameters, similar response was observed in PC, PDW as well as LPTs. There was significantly high number of LPTs in control group of males. Results indicated a significant effect of hematology parameters, especially in males.

Table 2

Hematological parameters (RBC, Hb, PCV (HCT), CHCM, CM, MCV, MCH, MCHC, RDW and HDW) measured after sub-acute exposure to flubendiamide in SD rats

Table 3

Hematological parameters (WBC, PCT, MPC, LPT, PLT, MPV and PDW) measured after sub-acute exposure to flubendiamide in SD

DISCUSSION

Flubendiamide stabilizes insect RyRs in open state, evoking massive calcium release from intracellular stores leading to sustained muscle contraction followed by death. However, flubendiamide has a high affinity for insect RyRs.[12] In order to test if there are any effects on hematopoiesis, which may either be related or unrelated to the pesticides mechanism of insecticidal activity, the present study was carried out in male and female SD rats. Alterations in hematological parameters were analyzed using unpaired t-test for sex-dependent changes, whereas dose-dependent effects were analyzed using one-way analysis of variance (ANOVA).

Any mutations in the protein structure of RyRs would have led to adverse effects in animals; especially domesticated herbivores that are being maintained specially on green fodder which might have resulted in isolated events of flubendiamide toxicity as already mentioned (unpublished data). These events were characterized by high body temperature due to persistent muscular contraction similar to the pesticides mechanism of action in insects. Alterations in the values of erythrocytic parameters would indicate effect on the mechanism of the genesis or termination of erythrocytes. In concurrence to the previous reports, flubendiamide resulted in marginal alterations in hematological parameters.[13] Platelets play an important role in coagulation and any deficit in the structure, numbers and stability would lead to bleeding or thrombic disorders; ultimately resulting in death. White blood cells play an important role in defense against invading bacteria, virus, fungi, etc., Any alteration in their numbers would result in deregulation of immune status. Due to the major role of calcium in most of the cellular activities from adenosine triphosphate (ATP) synthesis to regulation of hematopoiesis, any compound that alters its levels in body would affect any of these parameters.[14]

Great variation in the number of leukocytes due to excitable nature of rats has been previously reported. Platelets have played an important role in assessing the risk of cardiovascular health using parameters such as PDW for thrombocytosis[15] and MPV for overall vascular mortality, ischemic heart disease,[16] myocardial infarction, unstable angina, etc.[17] The temporal increase of MPV during storage of blood samples is usually attributed to platelet activation as it is combined with a decrease in platelet component concentration (MPC).[18] However, platelet degranulation as well as simple platelet swelling can be the cause of decrease in MPC. The high numbers of LPTs in the control group might be due to sampling time, as the sacrifice and sampling started with the control group followed by others resulting in a difference between sampling and analyzing time (more than 2 h) of platelets. It has been reported that upon storage, platelets get activated leading to increase in their volume resulting in LPTs.[18]

After analysis of differences between male and female rats among all the parameters analyzed, negligible differences have been observed. Most of the sex-dependent differences were observed in Group IV, especially in erythrocytic parameters, which might indicate sensitivity of males more than females for the pesticide used, which is in contrary to the previous reports.[8]

Interestingly, an increase in dose rate beyond 500 mg/kg exhibited reversal of response in multiple parameters as can be seen in Tables 2 and 3. The reason for this type of response might be due to saturation in the toxicokinetics of pesticide at higher dose, thereby resulting in zero order kinetics in absorption phase. Though non-significant, results indicate a stimuluslike effect on erythrocytic indices in males; but inhibitory in females basing on the changes in parameters in Groups I–V. Reason for these effects should further be verified using immunological and molecular methods to detect the mechanism for the cause as well as variability across different exposure levels.

All the parameters were in the range of reported normal values of SD rats, independent of dose rate rendering it difficult to interpret the changes in the present study. In order to be accurate in estimation, the adverse effects of the present study, a 0-day sample should have been analyzed, but this would have resulted in additional stress in animals and is not recommended. It has also been reported that the site of sample (blood) collection plays a vital role in the resulting parameters.[19]

From the present study, hemotoxicity have been observed in Group IV (500 mg/kg), wherein significant decrease and increase in erythrocyte and platelet parameters, respectively, have been observed. There were no previous reports indicating effect of flubendiamide on platelets if any. It can be concluded from the present study that flubendiamide has resulted in negligible toxicity leading to variable effects on the hematological parameters.