Comparative Effect of “Pestox” Powdered Insecticide on both Serum Aspartate Aminotransferase and Alanine Aminotransferase of Male and Female Albino Rats

Ubani Chibuike Samuel and Joshua Parker Elijah

Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria

ABSTRACT:

Pestox insecticide, whose active ingredient is cypermethrin – a synthetic pyrethroid that belongs to a group of insecticidal used widely as an industrial and agricultural pesticide is a product of Three Point Industry Limited, Lagos State, Nigeria. The present study was designed to investigate the toxicity of pestox on freshly collected blood from male and female rats. The different experimental group of rats were exposed to different concentrations of the test sample (1%, 5%, 10% and 15%) for 21 days. The activities of some liver enzyme markers such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) from hepatocyte to serum were determined throughout the analysis. The activities of AST and ALT did not significantly increase (P>0.05) in female rats during the first and second weeks of the experimental analysis. Whereas at the third week, only ALT activity in serum was observed to increase significantly (P<0.05) at 5%, 10% and 15% concentration. For the male rats, the activities of AST and ALT significantly increased (P<0.05) in serum at 5%, 10% and 15% concentrations in the first week but only the activities of ALT increased significantly (P<0.05) in serum at both second and third week at sublethal dose of 15% concentration. The results obtained suggest that the test sample has some toxic effect on both male and female rats hepatocytes using a contaminated-diet and the toxicity effect of the test sample is both concentration and time-dependent. However, the male rats showed higher level of toxicity effect compared with the female rat group at different concentration (1%, 5%, 10% and 15%).

KEYWORDS: Pestox; Aspartate aminotransferase (AST); Alanine aminotransferase (ALT).

INTRODUCTION

Insecticides are widely used in agriculture, household and in Medicine (WHO, 1989). In a petition on May 23, 1995, the Parliamentary Group of German SPD called upon the Federal Government to prohibit the use of insecticides in textiles and to give advice on non-toxic methods of fighting pests (Drucksache, 1995). Cypermethrin can be defined as a synthetic chemical similar to the pyrethins in pyrethrum extract which comes from chrysanthemum plant (Gammum et al., 1981). Cypermethrin as an insecticide is a moderately toxic material by dermal absorption or ingestion. Symptoms of high dermal exposure include numbness, tingling, itching, burning sensation, loss of bladder control incordination, seizures and possibly death (Contalamessa, 1993). Cypermethrin may adversely affect the central nervous system (Leachy, 1985). Cypermethrin is a slight skin or eye irritant, and may cause allergic skin reactions (Cantalmessa, 1993).

The oral LD₅₀ for cypermethrin in rats is 250 mg/kg body weight (in corn oil) or 413 mg/kg body weight (in water) (NPTN, 1993). The oral LD₅₀ varies from 367 to 2000 mg/kg body weight in female rats, and from 82 to 779 mg/kg in mice, depending on the ratio of cis/trans-isomers present (NPTN, 1998). The wide variation in toxicity may reflect different mixtures of isomers in the materials tested. The dermal LD₅₀in rats is 1600 mg/kg and in rabbits is greater than 2000 mg/kg (NPTN, 1998).

However, the view of World Health Organisation (WHO) is that there was no evidence of caranogeneicity in male mice as the results of mutagenic studies have been mainly negative. It was concluded that there is no evidence for the carcinogenic potential of cypermethrin (WHO, 1989). When cypermethrin was administered to pregnant and nursing rats, it led to a functional delay in the brain maturation of the pulps. The toxicity to young rats were higher also because the pathway for degrading cypermethrin is not readily developed in young rats (Leahey, 1985). Despite early findings, the microbial population of soil is affected by cypermethrin the ammonification and nitrification in treated soil is enhanced, a sign of the environmental impact of cypermethrin (Rangaswamy, 1993).

The mechanism of toxicity is common to all pyrethroid with specific effects. It causes depolarization of myelanited nerve membranes without repetitive discharges (Dorman et al., 1997). It is associated with a decrease in action potential amplitude (He, 1994). It may act post-syneptically by interacting with nicotine acetylcholins and GABA receptors (Dorma, 1991). It produces effects on cultured neurons that are largely irreversible after washing cells with a pythrin free solution (Song et al., 1996).

The application of insecticides for insect control may turn out to have negative consequences especially in developing countries where there are no regulations. This study is aimed at investigating the toxic effects of a locally manufactured insecticide labeled “Pestox” (containing 2% cypermethrin as active ingredient), using albino rats. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein and lipid peroxidation. Faecal and urine analyses will be used as the biochemical markers to determine the level of toxicity. The results obtained will aid in making recommendations on how best to apply the insecticide.

The application of pesticides for pest control may turn out to harm or have negative consequence on human being especially in developing countries where there are less/no constraints or regulation. The study is therefore aimed at investigating the potential toxic effects of a locally manufactured insecticide labeled as “pestox”. The findings of this study may help the government to formulate/regulate policies on the use of locally made pesticides.

MATERIALS AND METHODS:

Test Sample: The test sample for this study was a commercially-produced pesticide called pestox which is composed of 97.80% talc, 2% cypermethrin and 0.2% fragrance. Pestox insecticide is produced by Three-Point Industry Limited, Block C1/C2 Lagos State Small Scale Estate, Tatai Atere Way, Matori, Lagos, Nigeria.

Experimental Design: Sixty (60) healthy albino rats were purchased at the animal house of the Faculty of Biological Sciences, University of Nigeria, Nsukka. Thirty (30) of the animals were males while the other thirty (30) were females. The weight of the rats ranged between 100 – 220g. The rats were divided into 5 groups; each group consists of 6 rats in both male and female. The rats were monitored and allowed to acclimatize for seven (7) days. The animals were grouped into five (5) groups of six (6) per group for male rats and marked M₁, M₂, M₃, M₄ and M control and 6 per group for female market F₁, F₂, F₃, F₄ and F Control. Each group was fed with different concentrations of the pestox-contaminated diet {(1%, 5%, 10% and 15% (w/v)}. The control groups were fed with the normal diets and water ad libitum for 21 days. The animals were given sufficient feed throughout the experiment. Their body weights were taken in the morning two times on a weekly basis before they were given any feed.

Sample Collection: Blood (3ml) was drawn from the media canthus vein in the eyes of the rats with the aid of a capillary tube and transferred into EDTA coated plastic tubes. This was centrifuged and the plasma samples were collected into separate test tubes.

Formulation of Contaminated of Diet: Different concentrations of 1%, 5%, 10% and 15% (w/w) of the insecticide powder in the diet were prepared by weighing out the definite amount of growers marsh (feed) and then mixed with “pestox” insect powder. The concentrations of the active ingredient of the “pestox” insecticide (Cypermethrin) in the feed were 1.5g, 7.5g, 15g and 22.55g for the 1%, 5%, 10% and 15% (w/w) formulation respectively. The feed for the control group contains no “pestox” powder.

Determination of Serum Aspartate aminotransferase (AST) using Randox Kit:

Aspartate aminotransferase assay, according to this method is based on the principle that oxaloacetic acid (oxaloacetate) is formed from the reaction below:

AST

a-Oxoglutarate + L-aspartate L-glutamate + Oxaloacetate

Aspartate aminotransferase activity was measured by monitoring the following information of oxaloacetate hydrazone with 2, 4-dinitrophenylhydrazine.

Table 1: Mean food intake of pestox contaminated diet

Diet	Male (g)			Female (g)
Diet	7 days*	14 days**	21 days**	7 days*	14 days**	21 days**
Normal Feed (Control)	142.30±6.20	85.20±4.20	81.80±6.20	132.40±4.80	73.80±4.20	72.10±5.50
1% Contaminated	140.80±5.60	81.60±5.80	83.70±6.00	125.20±8.40	81.20±6.40	80.00±4.30
5% Contaminated	141.20±5.80	86.70±3.40	70.00±6.30	119.40±2.30	78.00±3.90	74.80±6.00
10% Contaminated	135.20±5.10	79.60±5.20	82.00±7.40	122.50±4.80	72.50±8.00	69.60±9.40
15% Contaminated	130.50±4.50	83.30±8.40	75.00±5.40	121.70±6.10	70.80±5.90	66.70±4.30

* = Weight of daily contaminated diet = 150g. ** = Weight of daily contaminated diet = 100g, Mean ± SD

Table 2: Mean body weight of rats (2 per group) fed pestox contaminated diet before 1^st week sacrifice

Diet	Male (g)			Female (g)
Diet	Initial Mean Weight	Final Mean Weight	Difference	Initial Mean Weight	Final Mean Weight	Difference
Normal Control (Group 1)	156.60±8.20	161.20±6.50	4.60	148.30±7.30	151.80±5.80	3.50
1% Contaminated (Group 2)	172.90±6.40	178.40±5.80	5.50	158.60±6.60	162.70±5.10	4.10
5% Contaminated (Group 3)	181.40±7.30	184.20±6.10	3.20	168.80±6.90	173.20±5.30	4.40
10% Contaminated (Group 4)	158.70±5.30	165.20±4.40	6.50	138.40±5.40	141.80±5.10	3.40
15% Contaminated (Group 5)	161.80±6.30	168.30±5.40	3.50	142.90±4.90	145.50±5.30	2.60

Mean ± SD, n = 2, P<0.05

Table 3: Mean body weight of rats (2 per group) fed pestox contaminated diet before 2^nd week sacrifice

Diet	Male (g)			Female (g)
Diet	Initial Mean Weight	Final Mean Weight	Difference	Initial Mean Weight	Final Mean Weight	Difference
Normal Control (Group 1)	168.80±7.50	177.60±6.30	8.80	151.50±4.30	158.70±5.20	7.20
1% Contaminated (Group 2)	175.40±5.50	183.50±6.10	8.10	146.80±6.40	157.20±5.80	10.40
5% Contaminated (Group 3)	186.30±4.90	194.70±6.20	8.40	162.30±4.60	170.10±5.20	7.80
10% Contaminated (Group 4)	153.60±6.80	151.30±7.20	7.50	158.60±6.80	165.90±6.50	7.30
15% Contaminated (Group 5)	177.60±5.60	185.60±6.40	8.00	152.40±5.50	158.50±3.40	6.10

Mean ± SD, n = 2, P<0.05

The AST substrate phosphate buffer of 0.5ml each was pipetted into the sample blank (B) and sample test (T) test tubes respectively. The serum sample of 0.1ml was added to the sample test (T) only and mixed immediately; then incubated in a water bath for exactly 30 minutes at 37°C. A volume of 0.5ml of 2,4-dinitrophenyldydrazine was added to both sample blank (B) and sample test (T) test tubes immediately after incubation. Also, 0.1ml of the sample was added to the sample blank (B) only. The medium was mixed and allowed to stand for exactly 20 minutes at 25°C. Finally, 5.0ml of Sodium Hydroxide solution (NaOH) was added to both the sample blank (B) and sample test (T) test tubes and mixed thoroughly.

Absorbance of the sample (A_sample) was read at a wavelength of 550nm against the sample blank after 5 minutes.

Determination of Serum Alanine aminotransferase (ALT) using Randox Kit:

ALT

Alanine aminotransferase assay, according to this method, is based on the principle that pyruvate is formed from the reaction below:

a-oxoglutarate + L-alanine L-glutamate + pyruvate

Alanine aminotransferase is measured by monitoring the concentration of pyruvate hydrazone formed with 2,4-dinitrophenylhydrazine.

The ALT substrate phosphate buffer of 0.5ml each was pipetted into two sets of test tubes labelled B (sample blank) and T (sample test) respectively. The serum (0.1ml) sample was added to the sample test (T) only and mixed properly: then incubated for exactly 30 minutes in a water bath at a temperature of 37°C.

A volume of 0.5ml each of 2,4-dinitrophenylhydrazine was added to both test tubes labelled T (sample test) and B (sample blank) immediately after the incubation. Also, 0.1ml of serum sample was added to the sample blank (B) only. The entire medium was mixed thoroughly and allowed to stand for exactly 20 minutes at 25°C.

After which, 5.0ml each of sodium hydroxide (NaOH) solution was added to the both test tubes and also mixed thoroughly. Absorbance of the Sample (A_sample) against the sample blank was read at a wavelength of 550nm after 5 minutes.

RESULTS AND DISCUSSION:

Mean Food Consumption of Pestox Contaminated Diet by Wistar Albino Rats:

In Table 1, the mean weekly consumptions of the contaminated diet by the various grouping of rats are shown. The food intake during the 7^th, 14^th and 21^st day of exposure was not significantly different (P>0.05) within the male and female groups (5%, 10% and 15%) contaminated diet. Although a general increase in the amount of food consumption by the male rats was observed throughout the period of feeding. During the first week, 150g of contaminated diet (w/w) was administered to the animals (6 rats per group). After the 1^st sacrifice (2 rats from each group, the food quantity was reduced to 100g for the remaining period of 14 days.

Mean Body Weight of Rats Fed Pestox Contaminated Diet:

The mean body weights of rats at various days of exposure to contaminated diet were shown in the Table 2, 3 and 4. After each week of exposure, the mean body weights of two rats to be sacrificed were determined to ascertain the mean body changes of body weight. The result showed that animals gained weight irrespective of the dosage of contaminant throughout the 1^st, 2^nd and 3^rd weeks of experiment.

Result of Aspartate aminotransferase (AST) Alanine aminotransferase (ALT) activities of male rats exposed to different concentrations of pestox contaminated diet for weeks 1, 2 and 3:

The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of male rats exposed to different contaminated diet are shown in Table 5a, 5b and 5c. In the first week, both AST and ALT significantly increased (P<0.05) in the serum at 5%, 10% and 15% concentrations whereas in the second and third. Only AST increased significantly (P<0.05) in the serum at 5%, 10% and 15% and 10% and 15% concentrations respectively when compared with control.

Table 5: AST and ALT activities of male rats exposed to different concentrations of pestox contaminated diet for weeks 1, 2 and 3

Week 1

Groups	AST (IU/L)	ALT (IU/L)
Normal Control (Group 1)	13.00±0.41	37.50±1.54
1% Contaminated (Group 2)	15.50±0.71	41.50±1.12
5% Contaminated (Group 3)	19.00±0.41	49.50±1.54
10% Contaminated (Group 4)	21.50±0.12	60.50±2.95
15% Contaminated (Group 5)	26.50±0.71	56.00±2.66

Week 2

Groups	AST (IU/L)	ALT (IU/L)
Normal Control (Group 1)	16.50±0.12	44.00±1.24
1% Contaminated (Group 2)	17.50±0.71	44.00±1.41
5% Contaminated (Group 3)	21.50±1.12	56.00±2.49
10% Contaminated (Group 4)	24.00±1.02	55.00±2.07
15% Contaminated (Group 5)	28.50±1.12	70.00±2.31

Week 3

Groups	AST (IU/L)	ALT (IU/L)
Normal Control (Group 1)	16.50±0.71	47.50±1.36
1% Contaminated (Group 2)	15.00±1.41	51.50±2.19
5% Contaminated (Group 3)	17.00±1.41	63.00±2.56
10% Contaminated (Group 4)	21.00±1.41	65.00±2.07
15% Contaminated (Group 5)	26.50±1.12	72.00±2.83

Result of Aspartate aminotransferase (AST) Alanine aminotransferase (ALT) activities of female rats exposed to different concentrations of pestox contaminated diet for weeks 1, 2 and 3

The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of female rats exposed to different contaminated diet are shown in Table 6a, 6b and 6c. Both AST and ALT activities in various rats were not significantly different (P>0.05) in the serum when compared with the control group in the 1^st and 2^nd weeks at various concentrations (1%, 5%, 10% and 15%). Moreover, in the 3^rd week, only ALT were significantly increased (P<0.05) in the serum at 5%, 10% and 15% concentrations when compared with the control.

Table 6: AST and ALT activities of female rats exposed to different concentrations of pestox contaminated diet for weeks 1, 2 and 3

Week 1

Groups	AST (IU/L)	ALT (IU/L)
Normal Control (Group 1)	9.00±0.41	35.50±1.78
1% Contaminated (Group 2)	9.00±0.71	43.50±1.44
5% Contaminated (Group 3)	10.00±0.41	58.00±2.83
10% Contaminated (Group 4)	10.50±0.41	58.00±2.31
15% Contaminated (Group 5)	12.50±0.71	60.00±2.66

Week 2

Groups	AST (IU/L)	ALT (IU/L)
Normal Control (Group 1)	26.50±0.83	47.50±1.54
1% Contaminated (Group 2)	26.00±0.95	55.00±2.21
5% Contaminated (Group 3)	27.00±0.83	53.50±2.19
10% Contaminated (Group 4)	31.50±1.36	65.00±2.10
15% Contaminated (Group 5)	32.00±1.41	74.00±2.83

Week 3

Groups	AST (IU/L)	ALT (IU/L)
Normal Control (Group 1)	30.00±1.41	45.50±1.36
1% Contaminated (Group 2)	36.00±1.07	49.50±2.54
5% Contaminated (Group 3)	41.50±1.78	59.00±2.24
10% Contaminated (Group 4)	50.00±2.73	64.00±2.83
15% Contaminated (Group 5)	52.00±1.12	69.00±2.07

Environmental toxicity test and laboratory experiments that measure the biological effects of pestox used to control insects in agriculture, industries and our various homes, and the effects on non-targeted organisms were predetermined by exposure to various group of rats at different concentrations (1%, 5%, 10% and 15%) and responses in the test organism are used to estimate the toxic effect.

In this study, after acclimatization, the rats showed gradual sign of toxicity when exposed to the toxicant (pestox insecticide). The consumption of the contaminated diet by the various groups of the rats at 1%, 5%, 10% and 15% concentrations did not vary with the control groups, thus, shows that the toxicant had no problems of palatability. The dose levels in the rats studied ranged up to 1500mg/kg body weight in which no was observed on 150mg/kg body weight . The activities of the liver enzymes such as AST and ALT represent the functional states of the liver. The increase or decrease of the enzymes’ activities were related to the intensity of cellular damage (Manna et al., 2004).

The activities of AST and ALT in the serum on 7^th and 14^th day of the experiment were not significant (P<0.05), whereas on the 21^st day, only ALT activity in the serum were significant (P<0.05) at 5%, 10% and 15% concentrations when compared with the control in the female rats. This suggested that the activities of AST and ALT in the blood were increased significantly (P<0.05) in conditions in which hepatocytes are damaged. The destruction of hepatic cells causes ALT (GPT) to leak out into the blood stream (Worman, 2002). The slowly toxic effect of cypermethrin may be due to the presence of suspended solids (feed) decrease the toxicity by at least a factor of 2 because of absorption of cypermethrin to the solid (Crossland et al., 1982).

Moreover, it was observed that on the 7^th day (1^st week), the concentration of both AST and ALT in the serum were significantly increased (P<0.05) at 5%, 10% and 15% in male rats exposed to the toxicant when compared with the control. But on the 14^th (2^nd week) and 21^st day (3^rd week), only the level of AST significantly increased (P<0.05) in the serum at 5%, 10% and 15%, and 10% and 15% respectively. It has been reported that liver enzymes, AST and ALT, activities fluctuate in response to the extent cellular necrosis (cell death) and therefore may be temporarily and minimally elevated at the early stage of the disease process, and extremely elevated during the most acute phase. It depends on when the initial sample was drawn, AST or ALT activities can rise indicating increasing disease severity and tissue damage or fall indicating disease resolution and tissue repair; hence, vitamin B deficiency may be one instance when the enzyme activity may be decreased (Joregen and Hedegaard, 2002).

However, from the results obtained, it suggested that female rats were less sensitive to pestox-powdered insecticide than the male rats at different concentrations (1%, 5%, 10% and 15%). This may be due to their ability to ingest the test sample; hence the male groups fed more than the female groups, or may be due to hormonal fluctuation or differences.

In conclusion, although the active ingredient of pestox insecticide (cypermethrin) has been reported by several authors to have little or no toxicological effects on mammals, but the result of this study suggested that more attention should be given to insecticides or pesticides considering its effects on the hepatic enzymes.

REFERENCES:

1. Cantalamessa, F. (1993). Acute toxicity of two pyrethroids, permethrin and cypermethrin, in neonatal and adult rats. Archives of Toxicology, 67: 510 – 513.

2. Crossland, N. O., Shires, S. W. and Benneh, D. (1982). Aquatic toxicology of cypermethrin: Fate and biological effects of spray drift deposits in freshwater adjacent to agricultural land. Aqua. Toxicol., 2: 253 – 270.

3. He, F. (1994). Synthetic Pyrethroids, Toxicol., P. 43.

4. Joregen, J. J. and Hedegaard, D. H. (2002). Common laboratory test in liver function diseases. Clin. Chem. Acta., 31: 87 – 91.

5. Manna, S., Bhattacharyya, D., Basak, D. K. and Mandal, T. K. (2004). Single oral dose toxicity of α-cypermethrin in rats. Department of Pharmacology, College of Medicine, Calcutta University, Kolkata-700020, India. Also available online: skmv2@rediffmail.com.

6. National Pesticide Telecommunication Network (NPTN) (1998). Oregeun State University, 333 Weniger Hall, Corvaliis. http://ace.orst.edu/info/nptn.

7. Rangaswany, V. (1993). Ammonification and nitrification in soils and nitrogen fixation by Azospirillum spp. as influenced by cypermethrin and fenvaterate agricult. Ecosyst, Environment, 45: 311 – 317.

8. Worman, J. H. (2002). Method for quantitative estimation of liver function disease in serum. Clin. Chem. Acta: 11: 478–479.

9. Zimmermen, H. J. (2005). West MA serum enzymatic levels in the diagnosis of hepatic disease. Am J. Gastroenterol., 40: 387.

Received on 10.04.2010

Accepted on 10.07.2010

Research J. Pharmacology and Pharmacodynamics. 2(4): July-August 2010, 309-313

Diet	Male (g)			Female (g)
Diet	Initial Mean Weight	Final Mean Weight	Difference	Initial Mean Weight	Final Mean Weight	Difference
Normal Control (Group 1)	178.60±6.50	191.40±7.30	12.80	144.80±5.30	152.90±4.60	8.10
1% Contaminated (Group 2)	169.40±5.20	180.20±4.80	10.80	152.70±3.60	161.90±4.20	9.20
5% Contaminated (Group 3)	184.50±4.10	194.20±3.40	9.70	136.80±5.60	146.70±2.90	9.90
10% Contaminated (Group 4)	154.70±3.80	166.90±4.10	12.20	148.30±3.20	156.70±3.80	8.40
15% Contaminated (Group 5)	167.10±2.90	177.40±3.60	10.30	132.80±4.30	140.00±4.10	7.20