Assessment of hemato-biochemical parameters on exposure to low level of deltamethrin in mouse model

Aim: In this study, sub-acute toxicity of deltamethrin on hematological and biochemical blood parameters of male albino Swiss mice was evaluated. Materials and Methods: Generally, the maximum permissible residue level (MRL) of deltamethrin for food products lies between 0.01 to 0.5 mg/kg body weight. So the mice were exposed orally with two doses of pesticide i.e. 0.1 and 0.5 mg/kg body weight. The doses were given on a daily basis for a period of 15 days and 30 days respectively. Ground nut oil was used as control treatment. Samples of blood were collected at the end of the treatment. Hepatotoxicity was evaluated by quantitative analysis of the serum enzymes alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALKP), total bilirubin (TBIL) and serum urea. Alterations of hematological parameters were analysed by total leukocyte, differential leukocyte count and hemoglobin levels. Results: Significant increase in the levels of hepatic enzymes (ALT, AST, ALKP) were observed for both doses, but no considerable differences were found by histological analysis. The hematological parameters showed significant alterations for 0.5 mg/kg body weight dose which is indicated by leukocytosis, lymphocytosis and neutropenia in long duration study. Conclusions: The results indicated that even very low dose of deltamethrin can promote hematological and hepatic alterations. Thus it is imperative to do further studies on the detrimental effect of the low levels of pyrethroid commonly present in our food, which further necessitate the reduction of maximum permissible levels of residual synthetic pyrethroid levels in foods and feed.


Introduction
reportedly used annually for vector control at the global level during 2006-2007 [6].Deltamethrin is one Deltamethrin is a broad-spectrum synthetic of the insecticides recommended by the World Health pyrethroid insecticide that is registered in the U.S. for Organization (WHO) for indoor spraying [7] and is one direct application to a wide variety of food/feed crops, of the insecticides being used for treatment of mosquito for use on stored grains, and for use in food/feed nets [8].Among all pyrethroids, deltamethrin also has handling establishments [1].It is commonly used found wide acceptability for agricultural purposes.pesticide in industry, agriculture and veterinary as well Even though it is already known that this insecticide is as for household activities.It is a synthetic derivative of highly toxic to fish and various other aquatic organisms pyrethrins which are obtained from the flowers of [9] a number of studies on the side effects of this insecticide Chrysanthemum cinerariaefolium [2].The neurotoxic have been reported, including neurotoxicity, immunomechanisms of deltamethrin include prolonging the suppression and allergy, hypertension and decreased opening of the voltage-sensitive sodium channels and testosterone levels [10].According to some reports the inhibition of voltage gated chloride channels and liver was found to accumulate a greater concentration GABAA (gamma amino butyric acid) receptors [3].It of metabolites since it is the major site of pyrethroid has been employed as a substitute for organochlorines, metabolism [10].Deltamethrin was reported most organophosphates and carbamate insecticides.
significantly in the milk samples [11], vegetables [12] Because of its low persistence in the environment, low and even in soft drinks [13].

frequency of resistance development in insects and
The oral LD of deltamethrin is 50 mg/kg in mice, 50 comparatively lower toxicity to humans and other nonso the concentration range reported to cause toxicity target animals [4,5], it has become an insecticide of [14] is much higher than the concentrations (0.1 and 0.5 choice in most countries.mg/kg/d) used in the current study.The doses given in On average, 229 tonnes of pyrethroids were the present study are equal to the maximum residue limits (MRLs) for deltamethrin permitted legally in animal food and feed (MRL: 0.5 mg/kg for fat, meat and liver and 0.1 mg/kg for chicken) [1].Human beings living near the areas where agricultural and pest control Count (TLC), lymphocyte and neutrophil count were is operated would be subjected to continuous exposure estimated by following the standard protocols [16].to deltamethrin due to its high lipophilic accumulation Biochemical parameters: 7 biochemical parameters in tissues [15].Due to the extensive use of deltamethrin were estimated using an auto analyzer (BIOTRAN for public health purposes in developing countries and to BTR-830) and diagnostic reagent kits (Johnson & check the given MRLs of deltamethrin in various animal Johnson) supplied by Siemens Healthcare Diagnostics products, it is important to investigate the subacute Ltd, Gujarat, India.The sera samples were used for effects of deltamethrin on blood parameters.These liver function assessment by estimating the concentrations parameters can serve as a useful tool for evaluation of of the enzymes aspartate aminotransferase (AST), the effects of pesticides on the cellular components of alanine aminotransferase (ALT), alkaline phosphatase blood and vital organs of the body.
(ALKP), and total bilirubin (TBIL), total protein (TP) Therefore this study analysis can contribute to the and cholesterol (CHOL).Renal function was evaluated assessment of deltamethrin on human health and also from sera samples by estimating the urea concentration.the habitat conditions.The mouse model is the one Histopathological analysis: Samples of hepatic tissue commonly used for studies of human toxicology, this were obtained from the animals by surgical excision study was aimed to evaluate the subtoxic effects of following euthanasia.In all cases, a standardized 0.5 deltamethrin on hematological and biochemical paracm section of sample was removed from the same meters in mice.
hepatic lobe.The samples were fixed using 10% neutral

Materials and Methods
buffered formalin (pH 7.4), washed, dehydrated in alcohol, clarified using xylene, and mounted in Ethical approval: The experiment protocol used in this paraffin blocks.The tissues were sectioned into 5 µm study was approved by Animal Ethical Committee.
slices, stained with hematoxylin-eosin, and evaluated Chemicals: The animals were treated using technical by light microscopy.grade deltamethrin with 98.50 % purity, obtained from Statistical analysis: The comparison of the control Kilpest India Ltd, Bhopal, India.All other chemicals group with the treated group was made by the one way used in the study were of the highest grade available ANOVA followed by Tukey test for post hoc multiple commercially.
comparisons.The value of P < 0.05 was considered Animals: 36 Swiss albino male mice of 4 months age significant.Comparisons were done both on the basis were used in this study.The animals were housed in of level of exposure i.e. dose of deltamethrin as well as 0 special cages at an ambient temperature of 20 C. The duration of exposure i.e. number of days of exposure.animals were provided with ad libitum water and pellet

feed.
It was observed that deltamethrin can affect hepatic Treatment and control groups: The treatment groups metabolism and can cause important hematological were randomly divided into six groups of six animals alterations based upon concentration of pesticide and each.Desired concentration of deltamethrin was duration of exposure.Changes in the blood profiles in formulated in ground nut oil and was given orally to both control and deltamethrin-exposed Swiss albino each mouse.The grouping of mice was done as, Group mice for 15 days (Group A) and 30 days (Group B) A: Dose 1-deltamethrin (0.1 mg/kg/day) administered exposure are depicted in the Table-1 and Table-2 for 15 days; Group A: Dose 2-deltamethrin (0.5 respectively.mg/kg/day) administered for 15 days; Group B: Dose On comparisons between levels of exposure for 1-deltamethrin (0.1 mg/kg/day) administered for 30 15 day exposed mice groups showed a statistically days; Group B: Dose 2-deltamethrin (0.5 mg/kg/day) significant increase for ALT, AST and ALKP activity.administered for 30 days.Control groups i.e.Group C A Control, dose 1 (0.1 mg/kg) and dose 2 (0.5 mg/kg) and C were given only vehicle (ground nut oil) for 15 B groups differed significantly with each other for AST and 30 days, respectively.level.The dose 2 (0.5 mg/kg) group showed significantly higher ALKP level in comparison to the control Collection of blood samples: Mice from all groups group.Other parameters in Group A showed insignifiwere anaesthetized by intraperitoneal administration cant alteration in their concentrations.In Group B, ALT of anesthetics at 10 mg xylazine and 100 mg ketamine/ th st level was found significantly higher for both doses.kg body weight on 16 and 31 day of respective ALKP and CHOL activity were found to be significantly experiment.Blood samples were collected from the higher in dose 1 (0.1 mg/kg) group as compared to control group and the experimental groups.Blood control group in 30 day experiment, whereas for dose 2 samples were analyzed for hematological parameters.
(0.5 mg/kg) group, the difference was insignificant.The remaining blood samples were centrifuged at 3000 There was also significant increase in the TLC for dose rpm and the separated sera were transferred into Eppendorf 2 (0.5 mg/kg) group mice as compared to dose 1 (0.1 tubes and analyzed for biochemical parameters.mg/kg) group.Hematology: Hemoglobin (Hb), Total Leukocyte On comparison between different exposure group durations for the same dose level, it was observed that potential source of human exposure through foodstuffs there was significant increase in the ALT level and [18].Liver is the first organ to face any foreign decrease in serum urea [Group A: Dose 1 (0.1 mg/kg) molecules that is carried through portal circulation and mice compared to Group B: dose 1 (0.1 mg/kg)].While it is the one subjected to most damage.Transaminases a statistically significant decrease in AST, TBIL, (AST and ALT), ALKP and TBIL are important liver CHOL and neutrophil count was observed in Group B: enzymes and responsible for detoxification processes Dose 1 (0.5 mg/kg) mice as compared to Group B: [19].These enzymes are secreted into the blood Dose 2 (0.5 mg/kg) mice.TLC and lymphocyte count circulation during hepatocellular injury and their levels were significantly higher in Dose 2 (0.5 mg/kg) group constantly increase in the blood.Changes in these mice exposed for 30 days.
enzyme levels might differ depending on the exposure There was no significant variation in TP and Hb time and dose.The increase in the activities of these levels in different exposure level groups as well as enzymes in serum is indicative of liver, kidney and different duration of exposure groups.Histological muscle damage and thus causes alteration in the organ analysis of the hepatic tissue did not reveal any functions [20,21].significant differences between control group and for During 15 day exposure period, deltamethrin both doses and duration treatment groups (Figure -1 [23] and Rao [24].There was statistically significant of deltamethrin selected in this experiment was to increase in serum ALKP level in high dose group as mimic the permissible level of deltamethrin residue compared to control group.Yousef et al. [21] reported present in various foods and feed [17].Pyrethroids tend that the increase in the phosphatases and transaminases to bio-accumulate in lipid compartments, becoming a  < activities in the blood might be due to the necrosis of to the percentage of peripheral blood lymphocytes and liver, kidney and lung and the leakage of these enzymes neutrophils in either case. in the blood stream corresponding to the stress levels of When comparisons were made between duration the treated animals.Similar results have been indicated of exposure i.e. 15 days against 30 days exposure for by studies showing increased activities of transami-low pesticide dose, ALT level was found to be nases in human and animals after exposure to pesticide significantly higher in 30 day group, which may [25,26].Other hematological and biochemical parameters indicate long duration daily exposure of even low level showed non-significant alterations in their activity.The of deltamethrin can have detrimental effect on the liver.probable reason might be due to the low dose of But serum urea level was significantly reduced in low deltamethrin used and also given for a short period of dose group of 30 day mice group, which was also seen time (15 day), which mainly effected liver, as liver is in caeses where reduced BUN level seen in high the first organ to encounter any foreign molecule.
carbohydrate/low protein diet resulted in increased In 30 day exposure group, significantly increased anabolic demand, malabsorption state and acute liver level of serum ALT enzyme activity found in both the damage [28].In case of 30 day high dose group, there dose groups compared to control group indicates liver was statistically significant decrease in activity of TBIL.damage.Serum AST activity was also elevated but The liver processes bilirubin, it has been alterations were insignificant.The increase in ALKP suggested that bilirubin is not only a waste end-product and CHOL level at lower dose was found to be but also an antioxidant [29,30] that may protect against significant in relation to control group, whereas the diseases associated with oxidative stress [31].In high dose group showed non-significant changes for several prospective studies, an inverse relationship has both the enzymes.The significant elevation in the been reported between bilirubin and the following ALKP activity indicated damage to one or more of the disease conditions: cardiovascular disease, coronary organs producing this enzyme, such as the liver, heart disease, myocardial infarction, ischemic heart kidneys, muscle and intestinal mucosa [27].
disease and cancer [32].The above findings support For both 15 and 30 day exposure groups, deltame-that the sub-acute exposure to pesticides can still have a thrin caused a significantly elevated leukocytes in mice detrimental effect on liver and other organs when an administered with the high pesticide dose, whereas animal or human is continuously exposed to these there was no significant change observed with respect chemicals.There was also statistically significant decrease in hematological and hepatic alterations.The cumulative activity of AST and CHOL for 30 day exposure experi-effect of these pyrethroids can change the function of ment.These findings agreed with those of Bernard certain targets following exposure of organisms to low Lloyd et al. [33] and Ambali et al. [27] who reported doses which tends to accumulate during life.Thus it is decrease in the activity of AST and ALT in prolong imperative to do further studies on the detrimental effect of the low levels of pyrethroid usually present in exposure of pesticide.The reason for the low level of our food and food products.AST is not known and besides, the toxicological significance of this remains obscure.The deviation of a a,b,c Means with common superscript differ significantly (p<0.05).* Means differ significantly for Dose 1 between Group A and B (p 0.05).** Means differ significantly for Dose 2 between Group A and B (p<0.05).< a,b,c Means with common superscript differ significantly (p<0.05).* Means differ significantly for Dose 1 between Group A and B (p 0.05).** Means differ significantly for Dose 2 between Group A and B (p<0.05).

Figure- 1 .
Figure-1.Histological analysis (at 40X and 100X magnification) of the liver tissues of male mice given oral treatment with deltamethrin for 15 days at a dose rate of 0.1 and 0.5 mg/kg body weight and for 30 days at a dose rate of 0.1 and 0.5 mg/kg body weight.(A) Control (40X), (B) Control (100X), (C) 0.1 mg/kg dose for 15 days, (D) 0.5 mg/kg dose for 15 days, (E) 0.1 mg/kg dose for 30 days, and (F) 0.5 mg/kg dose for 30 days.
in the blood serum is The present study was part of AT's PhD disserconsidered as symptoms of liver diseases [34].In the tation.JPSG approved the experimentation protocol.present study the decreased cholesterol level implies AT conducted the experiment, drafted the manuscript.liver damage, which is in accordance with increased JPSG critically reviewed the manuscript.Both authors alkaline phosphatase level discussed earlier.El-Sayed read and approved the final manuscript.and Saad, [35] found increased level of CHOL after treatment with deltamethrin.This study indicated that even very low dose and and Kidney of Female Rats Exposed to Low Doses of Deltamethrin (DM): A Molecular Assessment.Biomed.short duration of exposure of deltamethrin can cause

Table - 1
. Changes in the blood profiles of control and deltamethrin-exposed Swiss albino mice group with 15 days exposure (n = 17).

Table - 2
. Changes in the blood profiles of control and deltamethrin-exposed Swiss albino mice group with 30 days exposure (n = 19).