Response of Carbon Tetrachloride Pretreated Rats to Endosulfan, Carbaryl and Phosphamidon

Industrial Health, 1983, 21, 263-271.

T.S.S. DIKSHITH and R.B. RAIZADA

Industrial Toxicology Research Centre, Mahatma Gandhi Marg, Lucknow-226001, India.

(Received July 11, 1983 and revised form September 14, 1983)

Abstract : Daily oral administration of carbon tetrachloride (0.5 ml/kg/day) for a period of 15 days, followed by endosulfan (2.0 and 4.0 mg/kg/d), carbaryl (50 and 100 mg/kg/d) and phosphamidon (0.5 and 1.0 mg/kg/d) for 15 days to different groups of male rats triggered significant enzymatic changes. Whereas treatment of CCl4 alone for a period of 15 days (group 5) elicited altered morphology of hepatocytes and a highly significant increase in the activity of serum transaminases, alkaline phosphatase, bilirubin content and severe hypoglycemia, CCl4-insecticide treatment did not suggest a synergistic effect of the two groups of agro industrial chemicals in experimental animals. On the contrary, the values of enzymatic activity and blood sugar in CCl4-insecticide exposed animals were nearer to those of normal. A significant inhibition of acetylcholinesterase activity of brain and red blood cell was seen after phosphamidon alone (group 4) and after CCl4-phosphamidon treatments (groups 10 and 11) which also support lack of synergistic effect of CCl4- phosphamidon.

Key words : Carbon tetrachloride—Endosulfan—Carbaryl—Phosphamidon-Inter- action-Synergism-Rat

INTRODUCTION

Various factors such as physical, chemical and nutritional are known to influence the toxicity of pesticides. Reports have shown that interaction of one pesticide with another results in additive as well as synergistic effect of the compounds.1-5,16) Organic solvents employed in the formulation of pesticides have been implicated as synergists.6-10) Carbon tetrachloride is a well known organic solvent extensively used in Industry. A wealth of information is available on the hepatic, renal, pancreatic and neural effects of CCl4 in animals and humans.11-18) Application of CCl4 as a disinfectant of food grains and particularly of its mixing along with ethylene dibromide to reduce fire hazard is of great relevance and of toxicological significance. This has prompted us to know the response of CCl4 pretreated animals to repeated doses 264 T.S.S. DIKSHITH AND R.B. RAIZADA of endosulfan, carbaryl and phosphamidon and to identify the manner of interaction and synergistic effect if any, of the two groups of agro-industrial chemicals.

MATERIALS AND METHODS

Ninety four albino rats (R. norvigicus, Wistar strain) of Industrial Toxicology

Research Centre's Colony and with an average body weight of 250 g were housed in an air conditioned room (75 •}2•‹F) of the animal house. The animals were fed pellet diet (Hindustan Lever Animal Feed, India) and water ad libitum throughout the experiment. The schedule of treatment of animals with CCl4 alone

and along with endosulfan, carbaryl and phosphamidon is given in Table 1. Body

weight of each animal was recorded before decapitation. Blood was collected

directly from the jugular vein into tubes containing oxalate.

Organ/body weight ratio The liver, kidney, adrenal, brain, spleen, testes, and epididymis were removed quickly and weighed. The organ/body weight ratio was calculated.

Biochemical studies Liver and brain were washed free of extraneous material using chilled saline solution and homogenized in 0.25 M ice cold sucrose solution (10% W/V) in

Table 1. Treatment Schedule of male rats INTERACTION OF CARBON TETRACHLORIDE AND INSECTICIDES IN RATS 265

a Potter-Elvehjem type homogenizer. The liver homogenate was centrifuged at

700 •~ g for 10 min. The liver supernatant and serum were used for the estimation of glutamic oxaloacetic transaminase (GOT, EC. 2.6.1.1.), glutamic pyruvic transaminase (GPT, EC. 2.6.1.2.) and alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC. 3.1.3.1.), following the procedure of Wootton.19)

Protein was assayed using the method of Lowry, et al.,20) and serum bilirubin by

Mallary and Evelyn.21)

Brain homogenate and red blood cells (separated by centrifuging the blood at

2500 •~ g for 5 min.) were used for estimation of acetylcholinesterase (AchE,

EC. 3.1.1.7.) by the method of Hestrin.22)

Histopathological studies Section of liver, kidney, brain, spleen, testes, epididymis and whole of the adrenal were fixed in formaline-saline solution. After routine processing of each tissue, paraffin sections were cut (6/um thickness) and stained with haematoxylin- eosin.

Haematological studies Blood collected in the tubes containing oxalate was used for estimation of haemoglobin by the procedure of Kolmer et al.,23) and total RBC and WBC counts were determined by the method of Wintrobe and Landsberg.24) Assay of blood sugar was made by the method of Nelson and Somogyi.25) Statistical significance of the values of the control and experimental group was calculated according to Fisher's student 't' test.26)

RESULTS

Mortality and Morbidity Rats treated with peanut oil, endosulfan, carbaryl and phosphamidon (groups 1 to 4) exhibited no significant signs of toxicity. The remaining number of 62 animals were pretreated with CCl4 (0.5 ml/kg/day) for a period of 15 days. During CCl4 treatment six animals died between day 1 to 15 and the rest of 56 animals survived. There was no overt indication of CCl4 toxicity in them. While 8 animals of group 5 were left as they were for 15 days animals of groups 6 to 11 were subsequently given either endosulfan or carbaryl or phosphamidon in two different doses. None of the animals of groups 6 to 11 also did not reveal any overt signs of combined toxicity of the two groups of chemicals.

Comparison of treatments A comparison of organ body weight ratios and biochemical changes (Table 2-4) of animals of group 1 with those of groups 2 to 4 suggested no significant difference. All evaluations therefore, have been made us under: group 1 with group 5 and 266 T.S.S. DIKSHITH AND R.B. RAIZADA

group 5 with groups 6 to 11.

Organ/body weight ratio A significant increase in the relative organ weights of liver and brain (group 5) and a slight reduction in the weight of brain (groups 6-11), were observed (Table 2).

Biochemical changes A significant increase in the activity of serum GOT, GPT, alkaline phosphatase,

Table 2. Relative organ weight in response to endosulfan (E) carbaryl (C) and phosphamidon (P) of Male Rats pretreated with CCl4

Table 3. Biochemical changes in liver and serum in response to endosulfan (E), carbaryl (C) and Phosphamidon (P) of male rats pretreated with CCl4 INTERACTION OF CARBON TETRACHLORIDE AND INSECTICIDES IN RATS 267

as well as high content of bilirubin and severe hypoglycemia was seen in CCl4 pretreated animals. In contrast, pretreatment of CCl, and subsequent administration of either endosulfan, carbaryl and phosphamidon triggered mild to slightly marked changes in the activity of liver and serum GOT, GPT, alkaline phosphatase, bilirubin and protein. The blood sugar level showed a marginal depletion in group 5 animals given CCl4 alone (Table 3). The AchE activity in RBC and brain cf animals exposed to both doses of endosulfan (groups 6 and 7) and carbaryl (groups 8 and 9) did not reveal any significant inhibition. Whereas animals dosed with phosphamidon alone (group 4) as well as CCl4-phosphamidon (groups 10 and 11) caused a highly significant inhibition in the activity of AchE of brain and RBC. Between brain and RBC, the former showed greater inhibition of the AchE activity (Table 4). There was no significant haematological change in all the treated animals. RBC, WBC counts and haemoglobin contents were comparable with those of normal animals.

Histopathology Daily doses of CCl4 for 2 weeks produced marked liver damage in animals. The liver of these animals appeared creamy in colour and enlarged. Microscopic examination revealed swollen and vacuolated hepatocytes with nuclei pushed to a side of the cell. Infiltration of mononuclear cells and decreased fibroblasts were seen in the parenchyma. Focal necrosis of the liver was also observed in a few animals.29) Liver of animals exposed to CCl4-insecticides revealed similar but less significant morphological changes. There was no significant morphological changes in kidney, spleen, testes, epididymis adrenal and brain of CCl4 alone and CCl4-insecticides treated animals (groups 5 to 11).

carbaryl (C) and phosphamidon (P) of male rats pretreated with CCl4 268 T.S.S. DIKSHITH AND R.B. RAIZADA

Table 4. Acetyl cholinesterase* in response to Endosulfan (E), Carbaryl (C) and Phosphamidon (P) of male rats prereated with CCl4

DISCUSSION

The study has indicated the manner of response and induction of toxicological effects in male rats after a brief exposure of CCl4 followed by daily doses of endosulfan, carbaryl and phosphamidon. It is of importance to note here that the animals were deliberately exposed to a low dose of CCl4 for a brief period only so as to disturb the liver function and not to induce cirrhosis. This was to simulate a situation where workers would be exposed to different pesticides during application and transportation of agrochemicals. The enzymatic and morphological changes along with high level of serum bilirubin in the liver of CCl4 treated animals were obvious. However it is of significance to note that the prereatment of CCl4 followed by insecticides did not produce synergistic effects. The activity of transaminases in liver and serum of CC14-insecticide treated animals suggested mild to slightly marked changes. The hepatic damage triggered by the pretreatment of CCl4 was not augumented further by the insecticides. Instead the enzymatic changes as well as serum bilirubin levels showed transformation towards normalcy. This indicates an adaptive compensatory mechanism and homeostatic regulation of the internal milieu. Similarities of this kind have been reported elsewhere.27,28) Our earlier studies suggested the susceptibility of CCl4 treated animals to quinalphos (Organo phosphorus) insecticide but not to benzene hexachloride.29) In another study no synergistic effect of solvent benzene and phosphamidon was seen in rabbits." Inhibition of AchE activity in RBC and brain of CCl4-carbaryl treated male rats could not elicit a severe change. However, phosphamidon alone and with CCl4 produced a highly significant cholinergic activity in animals. There was no significant difference in percent inhibition of AchE activity between phosphamidon alone (group 4) and CCl4-phosphamidon (group 10 and 11) which suggests the lack of synergistic effects of the two chemicals, Phosphamidon (2.16 mg/kg) INTERACTION OF CARBON TETRACHLORIDE AND INSECTICIDES IN RATS 269 alone also produced 72 to 84 percent of inhibition of AchE activity in RBC and brain of female rabbits.30) Quinalphos, produced highly significant inhibition of AchE activity in female guinea pigs and in male goats.31,32) It is of interest to observe here that quinalphos alone and along with CCl4, produced highly significant inhibition of AchE activity in male rats.29) The present study suggests that pretreatment of CCl4 in low doses for a brief period although disturbed the enzyme profile in animals, further exposure of animals to insecticides could not aggravate or potentiate CCl1 induced effects. On the contrary, a gradual reversal of the CCl4 induced toxicity was evident. Earlier study of Cornish and Block33) with low doses of CCl4, produced transient elevation of GOT which returned to normalcy within 24 hrs. in experimental rats. Exposure of animals to organic solvents and the manner of response of enzyme systems to metabolise pesticides deserves greater attention to understand the manner of intoxication/synergistic effects and the problems of pollution by agro industrial chemicals.

ACKNOWLEDGEMENT

Grateful thanks are due to Dr. C. R. Krishna Murti for his keen interest and encouragement. Thanks are also due to R. A. Kaushal, Sikandar Ali and R. P. Singh for technical assistance.

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