Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 37, Num. 3, 2005, pp. 160-164

Indian Journal of Pharmacology, Vol. 37, No. 3, May-June, 2005, pp. 160-164

Research Paper

Repeated dose toxicity of deltamethrin in rats

Manna S, Bhattacharyya D, Mandal TK, Das S

Department of Pharmacology and Toxicology, West Bengal University of Animal and Fishery Sciences, Kolkata
Correspondence Address:Department of Pharmacology and Toxicology, West Bengal University of Animal and Fishery Sciences, Kolkata Email:skmv2@rediffmail.com

Date of Submission: 05-May-2004
Date of Decision: 09-Sep-2004
Date of Acceptance: 17-Oct-2004

Code Number: ph05040

Abstract

Keywords: Biochemical parameters; LD₅₀; tissue residue

Introduction

Materials and methods

Pesticide
Deltamethrin (>99% pure) was obtained from Gharda Chemicals Ltd., Bombay.

Animals and experimental design
Fifty-four adult Wistar rats of both sexes (equal sex ratio; weighing about 200±20 g) were divided into nine equal groups (I-IX) each consisting six animals. All rats were kept under controlled conditions of temperature (22±1 °C) and humidity (60±5%). They were given pellet food (Amrut feeds Ltd., Pune, India) and drinking water ad libitum . A twelve-hour day and night cycle was maintained. The experimental protocol met the national guidelines on the proper care and use of animals in the laboratory research. The institutional animal ethics committee (IAEC) approved the experimental protocol.
The animals were grouped as follows:[Table - 3]

Groups I-VI were used for determination of LD₅₀ of deltamethrin. Group VII served as control for Groups I-VI. The animals were fasted overnight and deltamethrin was administered orally after dissolving in DMSO (1 ml) as stated above. The animals were observed for respiratory and CNS symptoms, behavioural changes and death and then LD₅₀ was determined.[2] Group VIII was used for repeated dose toxicity study. Group IX served as control for Group VIII. Deltamethrin was administered orally to the animals of Group VIII at 15-mg/kg, body weight (b.w.) and Group IX animals were dosed equal volume of DMSO only (1 ml/day) for 30 days. On the 31st day, the animals were killed under halothane anesthesia by severing the neck vessels aseptically. Blood was collected in three sets of test tubes from each animal. Blood smears were prepared for differential leukocyte count. One set was kept under refrigeration (4 °C) for separation of serum and utilized for estimation of activities of aspartate transaminase (AST),[3] alanine transaminase (ALT),[3] lactate dehydrogenase (LDH),[4] alkaline phosphatase (ALP)[5] and total protein (TP),[6] globulin and albumin (GLB, ALB).[7] The blood of another set of test tubes having mixture of potassium oxalate and sodium fluoride as anticoagulant was used for estimation of glucose.[8] Blood in the third set of test tubes was heparinized and used for RBC and WBC counts and measuring PCV and hemoglobin level. All the absolute values were calculated according to Dacie and Lewis.[9]

Tissue
Portions of lungs, liver, stomach, kidney, stomach, testes and cerebellum were collected in 10% formalin solution for histopathology. One portion of liver was washed in physiological saline, homogenized and the homogenate was kept for estimation of catalase activity (CAT),[10] levels of reduced glutathione (GSH),[11] malonyldialdehyde (MDA),[12] glycogen[13] and tissue protein.[14] Another portion of liver was collected in ice-cold 1.15% KCl, homogenized within 10 min, centrifuged, microsomal pellets separated and used for estimation of superoxide dismutase (SOD),[15] cytochrome P₄₅₀ and b₅[16] contents by DB-UV-Vis spectrophotometer.

Preparation of liver microsomal fraction
Animal was killed and the liver was perfused in situ with homogenizing buffer A (Tris-HCL + EDTA + BHT) by single pass injection through the portal vein and dissected out, placed in ice-cold KCl (1.15%). All the subsequent steps in the preparation of microsomal fraction were carried out at 0-4°C. Then the liver was minced and mixed with four volumes of buffer A and homogenized in a mechanically driven Teflon glass homogenizer (Remi RQ 127 A). The homogenate was centrifuged at 10000 g in an automatic high-speed cold centrifuge (Hitachi-SCR 20B) using the rotor RPR 20-2 for 30 min. The supernatant was recentrifuged at 105000 g for 1 h in an automatic preparative ultracentrifuge (Hitachi 70 P-72, Japan) using rotor RP-65T to yield microsomal pellet. Microsomal pellet was suspended in buffer B (potassium pyrophosphate + EDTA + BHT) and homogenized with four passes of mechanically driven Teflon glass homogenizer (Remi RQ 127A) and again centrifuged at 104000 g for 1 h. The supernatant fraction was decanted and the microsomal pellet was resuspended in a minimum volume of buffer C (Tris-HCl + EDTA + glycerol) and stored at -20 °C till further use. The pellet was used for estimating SOD activity and cytochrome P₄₅₀ and b₅ levels.

Residue level
The tissue residue levels of a-CP in brain, lungs, liver, heart, kidney and testes were estimated by the method of Marie et al .,[17] modified by Mandal et al .[18]

Tissue sample preparation
Tissues (2 g) were extracted for 4 min with acetonitrile (25 ml) and anhydrous sodium sulphate (0.5 g) using a homogenizer. The extract was filtered through anhydrous sodium sulphate (0.5 g) and the tissues were re-extracted twice with acetonitrile (first by 25 and then by 12 ml). The extract was clarified by centrifugation and filtered through anhydrous sodium sulphate. The combined acetonitrile extracts were concentrated to 20 ml and partitioned with hexane (2 x 10 ml). The hexane phases were discarded and the acetonitrile phase was evaporated to dryness using a rotary vacuum evaporator at 40 °C. The volume was finally made up to 5 ml with acetone for GLC estimation.

Calibration
A stock solution of 1 mg/l of deltamethrin (analytical grade >99%) was prepared as an external standard. The retention time of deltamethrin was 5.25 min. The data were recorded in a HP 3392A integrator.

Apparatus
A Hewlett Packard (USA) model 5890A gas chromatograph coupled with a 3392 A (HP) integrator and equipped with a 63Ni electron capture detector was used for analysis of deltamethrin.

Histopathology
Small pieces of lungs, liver, stomach, kidney and cerebellum were fixed in 10% neutral buffered formalin and testis in Bouin′s fluid. Sections of 3-5 mm thickness were cut and stained with hematoxylin and eosin (H&E) for observation under light microscope.

Statistical analysis
All values are expressed as mean±SEM. Statistical analysis was done using SPSS 10.1. Statistical significance between two means was assessed by Student′s ′t′ test at P <0.05.

Results

Deltamethrin did not produce any gross effect at 100 mg/kg. However, at higher doses ranging from 125 to 225 mg/kg, it produced signs of CNS stimulation followed by prolonged depression. Initially the intoxicated animals exhibited chewing, licking and salivation, which was followed by CNS depression. A variable sequence of motor symptoms developed that involved occasional pawing, or burrowing, coarse whole body tremor associated with movement, gradual development of hind limb extensor tone. Finally, choreoathetosis (sinuous writhing) developed and the animals exhibited slow twisting or writhing movement of neck and tail. Violently twisting movements sometimes lifted the body from the floor in severely affected animals, which were cases of severe athetosis. At the terminal stage, animals showed laboured breathing, gasping and death. The acute oral LD₅₀ value of deltamethrin was calculated as 150 mg/kg body weights.

Biochemical and hematological profile
Effect of deltamethrin on certain blood and liver biochemical and antioxidants parameters are summarized in [Table - 1] and [Table - 2], respectively. Deltamethrin significantly ( P <0.05) increased the activities of serum AST, ALT, ALP and LDH while the liver cytochrome P₄₅₀ content and activities of CAT, SOD and GSH level were decreased and MDA level was increased significantly ( P <0.05) without any significant alteration of cytochrome b₅ content in liver. Blood glucose level was significantly ( P <0.05) increased and liver glycogen was significantly ( P <0.05) decreased. Serum ALB, GLB and total protein levels were not altered significantly. Deltamethrin decreased PCV, Hb level and counts of lymphocyte, monocyte and eosinophil, whereas basophil count was increased significantly [Table - 1].

Tissue residue
The tissue residual concentration of deltamethrin following repeated oral administration for 30 days were 0.92±0.01, 0.74±0.05, 0.10±0.01, 0.09±0.01, 3.00±0.12 and 0.15±0.02 ppm in liver, brain, testis, kidney, lungs and heart, respectively. The concentration of deltamethrin was maximum in the lungs.

Gross pathology
At post-mortem, rats showed bloated stomach with severe hemorrhages in both stomach and intestine. Hemorrhages were also seen in lungs. No gross changes were discernible in other visceral organs.

Histopathology
Deltamethrin produced congestion and emphysema in lungs [Figure - 1]. Congestion and fatty changes were found in liver [Figure - 2]. In stomach, it produced desquamation and necrosis of the epithelium. Kidneys showed congestion of the blood vessels [Figure - 3]. The section of testis showed edematous fluid accumulation between the tubules and vacuole formation within the tubules [Figure - 4]. Congestion and hemorrhages were also seen in intracerebellar vessels of the cerebellum [Figure - 5].

Discussion

Acknowledgments

We acknowledge Prof. A. Chowdhury and Dr. A. Bhattacharya, Pesticide Residual Laboratory, Department of Agricultural Chemicals, Bidhan Chandra Krishi Viswa Vidyalaya, Nadia, West Bengal for providing GLC-ECD for tissue residual analysis and M/S. Gharda Chemical Ltd., Mumbai, India for gifting us analytical grade deltamethrin.

References

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