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 carcinogeneicity 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 which is 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 were determined.

Oral exposure of cypermethrin for a long time in mammals may cause liver changes, pathological changes in the cortex of thymus, adrenal glands, lungs and skins (Hayes et al., 1990), but showed no sign of adverse effect on reproduction and birth defects.

The aim of the research work was to investigate the toxicity effects of pestox insecticides on biological subjects. The results obtained will aid in making recommendations on how best to apply the insecticide.

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.

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.

Contamination of Feed: 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 Lipid Peroxidation (Wallin et al., 1993)

Lipid peroxidation was assayed as thiobarbituric acid reacting substance (TBARS) using a method described by Wallin et al. (1992). From the test tubes containing 0.55 ml of the incubated plasma protein sample. Sample (1ml) was transferred to a clean test tube. To the tube was added 0.5 ml of 25% (w/w) TAC and 0.5 ml of 1% (w/w) TBA dissolved in 0.3% (w/w) NaOh. The tubes were incubated between 90 – 95⁰C in a water bath for 40 minutes and cooled in the ice water to reduce turbidity 0.01 ml of 20% (w/v) sodium dodecylsulphate (SDS) was added to each test tube and the tubes were mixed. Absorbance at 532nm minus the absorbance at 600nm was read in a spectrophotometer against the balnk. TBARS were quantified as malondialdehyde (MDA) equivalents generated by acid hydrolysis of 1,1,3,3-tetrathoxypropane (TEP).

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 à L-glutamate + Oxaloacetate

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

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^OC. 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^OC. 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

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

ALT

a-oxoglutarate + L-alanine à 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^OC.

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^OC.

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.

Total Protein Determination (Biuret Commercial Kit Method)

This method is based on the principle that cupric ions, in an alkaline medium, interact with peptide bonds of proteins resulting in the formation of a coloured complex.

Distilled water (0.02 ml) was pipetted into reagent blank (B) test tubes only. Standard solution (0.02 ml) was added to another set of test tubes labelled ST (standard) only. After which 0.02ml of the sera from the different rats were added to different test tubes labelled SA (sample) only. Biuret reagent (1.0ml) was added to all the three sets of test tubes. The content was mixed thoroughly and incubated for 30 minutes at 25^OC.

Absorbance of the Sample (A_sample) and of the Standard (A_standard) against the reagent blank was read at a wavelength of 530nm.

The Total Protein concentration was calculated as follows:

Total Protein Conc.=	A _sample	x Standard Conc.
	A _standard

Where:

A _sample = Absorbance of the Sample

A _standard = Absorbance of the Standard

Urinalysis Using Medi-Combi 9 Kit

The urinalysis was used for the rapid determination of blood, urobilinogen, protein, nitrite, ketoses, ascorbic acid, glucose and pH value in urine. The test strip was dipped for approximately one (1) second into the fresh urine collected in a clean test tube. The strip was drawn across the nm of the container to remove excess urine and after 60 seconds the test strip was compared with the colour scale.

Faecal Using Medi-Combi 9 Kit for Urine Sample

Some faeces were collected in a container and dissolved in a little amount of distilled water. The test strip was dipped for approximately one (1) second into the dissolved faeces and after 60 seconds the strip was compared with the colour scale.

Statistical Analysis

The results were expressed as mean ± SD and test of statistical significance were carried out using one-way ANOVA and correlation. The statistical package used was statistical package for social sciences (SPSS) version 15.0

RESULTS:

Mean Food Consumption of Pestox Insect Powder Contaminated Diet by Male and Female Albino Rats

Table 1 shows the mean weekly consumption of the contaminated diet by the different groups of both male and female rats for 3 weeks. The food intake during the 7^th, 14^th and 21 day of exposure did not show any significant different (P>0.05) each groups of different concentrations given to the rats (1%, 5%, 10% and 15 w/w). From the results, it shows that there was no significant changes (P>0.05) compared with the control in the consumption of the contaminated diet with pestox insect powder indicating that the increased concentration of pestox insect powder did not significantly affect the palatability of the diet.

Mean Body Weight of Male and Female Albino Rats Fed Contaminated Diet

Tables 2a, 2b and 2c shows the mean body weight of the different groups of both male and female rats on the 7^th, 14^th and 21^st day of exposure to contaminated diet. Weight of 2 rats from each group for both male and female rats were taken for 3 weeks to determine the weight changes. The results (Tables 2a, 2b and 2c) showed that the animals (rats) gained weight, the contamination of the diet with pestox insect powder notwithstanding.

Table 1: Mean food intake of 6 rats per group (g)

WEEK GROUP	MALE			FEMALE
WEEK GROUP	Week 1	Week 2	Week 3	Week 1	Week 2	Week 3
Control	142.30±6.20	85.20±4.20	81.80±6.20	131.40±4.20	73.80±4.20	72.10±5.50
1% Contamination	140.80±5.80	81.60±5.80	83.70±6.20	125.20±8.40	81.20±6.40	80.00±4.30
5% Contamination	141.20±6.20	86.70±3.40	78.40±6.30	119.40±2.30	78.50±3.90	74.50±6.00
10% Contamination	132.20±4.50	79.60±5.20	82.40±7.40	122.50±4.80	72.50±8.00	69.60±9.40
15% Contamination	130.50±4.50	83.30±8.40	75.80±5.40	121.70±6.10	70.80±5.90	66.70±4.50

n=6

Table 2a: Mean body weight of 2 rats per group for Day 7

WEEK GROUP	MALE			FEMALE
WEEK GROUP	Initial Mean Weight (g)	Final Mean Weight (g)	Difference in Weight (g)	Initial Mean Weight (g)	Final Mean Weight (g)	Difference in Weight (g)
Control	156.60±8.20	161.20±6.50	4.60	148.30±7.30	151.80±5.80	3.50
1% Contamination	172.90±6.40	178.40±5.50	5.50	158.60±6.60	162.70±5.10	4.10
5% Contamination	181.40±7.30	184.20±3.20	3.20	168.80±6.90	173.20±5.30	4.40
10% Contamination	156.70±5.30	165.20±4.40	6.50	134.40±5.40	141.80±5.10	3.40
15% Contamination	164.80±6.70	168.30±5.40	3.50	142.90±4.90	145.50±5.30	2.60

n=2

Table 2b: Mean body weight of 2 rats per group for Day 14

WEEK GROUP	MALE			FEMALE
WEEK GROUP	Initial Mean Weight (g)	Final Mean Weight (g)	Difference in Weight (g)	Initial Mean Weight (g)	Final Mean Weight (g)	Difference in Weight (g)
Control	168.80±7.50	177.60±6.30	8.80	151.50±4.30	158.70±5.20	7.2
1% Contamination	175.40±4.40	183.50±6.10	8.10	146.80±6.40	157.20±5.80	10.2
5% Contamination	185.30±4.90	194.70±6.20	8.40	162.30±4.60	170.10±5.20	7.8
10% Contamination	153.00±6.80	161.30±7.30	7.70	158.60±6.80	165.90±6.50	7.3
15% Contamination	177.60±5.60	185.60±6.40	8.00	152.40±5.50	158.50±3.40	6.1

n=2

Table 2c: Mean body weight of 2 rats per group for Day 21

WEEK GROUP	MALE			FEMALE
WEEK GROUP	Initial Mean Weight (g)	Final Mean Weight (g)	Difference in Weight (g)	Initial Mean Weight (g)	Final Mean Weight (g)	Difference in Weight (g)
Control	178.60±6.50	191.40±7.30	12.80	144.80±5.30	152.90±4.60	8.1
1% Contamination	169.40±5.20	180.20±4.80	10.80	152.70±3.60	161.90±4.20	9.2
5% Contamination	184.50±4.20	194.20±3.40	9.70	136.80±5.60	161.90±4.20	9.9
10% Contamination	154.70±3.80	166.90±4.10	12.20	188.40±3.20	140.00±4.10	8.4
15% Contamination	164.10±2.90	177.40±5.60	10.30	132.80±4.30	140.00±4.10	7.2

n=2

DISCUSSION:

One of the physiological properties of AST is the transfer of amino groups between α-keto acids by the process of deamination while ALT catalyses the transfer of an amino group from alanine to α-ketoglutarate with the formation of glutamate and pyruvate. Results obtained indicated that pestox insect powder was significant in the level of AST activity in the serum with significantly increased (P<0.05) contamination at 5%, 10% and 15% in the 2^nd week and 3^rd week compared with the control while that of ALT was more significant in the 3^rd week.

A report by Walker (1994) suggested that the elevation of AST was found in pulmonary embolism, following congestive out failure and probably reflecting liver involvement due to inadequate blood supply to that organ.

ALT higher elevations are found in hepatocellular disorders than in extra or intrahepatic obstruction disorder. In acute inflammatory conditions of the liver, AST will frequently be higher than those of ALT owning to the higher half-life of ALT in the serum (Walker, 1994).

The results obtained from total protein showed there was a level of significance (P<0.05) for total protein concentration from 1^st week to 3^rd week at higher diet contamination of 5%, 10% and 15% pestox insect powder compared with the

control group. The decrease in serum protein damages the hepatic cells of the liver from the work done by Schiff et al. (1982).

There was no significance difference (P>0.05) in the level of MDA as an index of lipid peroxidation except on the 3^rd week for male and female rats at higher concentrations of 5%, 10% and 15%. It suggests that an increase in the level of MDA level (biochemical marker for the testing the level of lipid peroxidation) in the liver causes damages through free radicals by oxidation pathways of cytochrome P₄₅₀ microsomal enzyme system; resulting in oxidative stress in the liver (Zimmermam, 2005).

It should be noted that the 2% claim composition of cypermethrin in pestox insect powder does not imply a level of safety when used. The degree of toxicity depends on the level of exposure in terms of use, concentration and time-dependent.

The results generally conclude that the comparative physiological and biochemical effects of pestox insecticide between the male and female albino rats had almost the same level of effect on basic physiological and biochemical parameters in both dose and time-dependent manner.

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. Halliwell, B. and Gutteridge, M. C. (1990). Methods of Enzymology, 83: 186 – 190.

3. Hayes, W. J. and Lawis, E. R. (1990). Handbook of pesticide toxicology, classes of pesticides. Toxicology, 3. Academic Press.

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

5. Kruiepert, E. and Seffner, W. (1989). Histological and histochemical changes on liver damage in rats after treatment with synthetic prethrenoid. 185 – 188.

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. Reitman, S. and Frakel, S. (1957). Amer. J. Clin. Path., 28: 56.

9. Schiff, L. and Schiff, E. R. (1982). Disease of the liver, 5^th Ed. Philadelphia, JB, Lippincott.

10. Schmidt, E. and Schmidt, J. W. (1963). Enzyme. Biol Clin., 3:1.

11. United State Environmental Protection Agency (USEPA) (1989). Cypermethrin pesticide fact sheet. Washington, D. C.

12. United State Environmental Protection Agency (USEPA) (1993). Office of pesticide programmes reference dose tracking report. Washington, D. C.

13. Walker, C. O., Shenker, S. (1994). Pathogenesis of hepatic encephalopathy with special reference to the role of ammonia. An J. Chin. Nutr., 23: 619.

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

Received on 16.07.2010 Modified on 25.07.2010

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 945-950

WEEK GROUP	MALE			FEMALE
WEEK GROUP	Week 1	Week 2	Week 3	Week 1	Week 2	Week 3
Control	9.00±1.40	26.00±2.80	30.00±1.40	13.00±1.40	16.50±2.10	16.50±0.70
1% Contamination	9.00±0.00	27.50±4.90	36.00±7.10	15.50±0.70	14.50±0.70	15.00±1.40
5% Contamination	10.00±0.00	27.00±2.80	41.50±7.80**	19.00±1.40**	21.50±2.10**	17.00±1.40
10% Contamination	10.00±1.40	31.50±6.40	50.00±12.70**	21.50±2.10**	24.00±0.00**	21.00±1.40*
15% Contamination	12.50±0.70**	32.00±1.40	52.00±0.00**	26.50±0.78**	28.50±2.18**	26.50±2.10*

WEEK GROUP	MALE			FEMALE
WEEK GROUP	Week 1	Week 2	Week 3	Week 1	Week 2	Week 3
Control	35.50±7.80	47.50±3.50	45.50±6.40	37.50±3.50	44.00±4.20	47.50±6.40
1% Contamination	43.50±13.40	55.00±21.20	44.50±3.50	41.50±2.10	44.00±4.20	51.50±5.00
5% Contamination	58.50±2.80	53.50±9.20	59.00±4.20*	44.50±3.50*	55.00±7.10	63.00±15.60
10% Contamination	58.00±2.80	65.00±9.90	64.00±2.80**	55.00±4.90*	55.00±7.10	65.00±7.10
15% Contamination	60.50±11.30	44.00±2.80	69.00±7.10**	56.00±5.70*	40.00±11.30*	72.00±2.80*

WEEK GROUP	MALE			FEMALE
WEEK GROUP	Week 1	Week 2	Week 3	Week 1	Week 2	Week 3
Control	3.71±0.10	5.36±0.00	5.51±0.50	4.75±0.30	6.00±0.10	6.08±0.30
1% Contamination	4.56±0.30	5.61±0.10	5.75±1.50	5.20±0.40	5.70±0.50	6.18±0.10
5% Contamination	4.44±0.50	5.65±1.10**	7.89±0.70	5.60±0.40*	7.32±0.10	7.89±0.10*
10% Contamination	5.32±1.10*	6.94±1.20**	8.08±0.40*	6.27±0.30**	7.65±0.40**	8.56±7.0.3*
15% Contamination	7.51±0.40*	7.66±0.70*	9.41±0.40*	6.27±0.00*	8.46±0.90**	10.83±0.80

WEEK GROUP	MALE			FEMALE
WEEK GROUP	Week 1	Week 2	Week 3	Week 1	Week 2	Week 3
Control	0.36±0.00	0.39±0.00	0.44±0.00	0.44±0.00	0.44±0.00	0.44±0.00
1% Contamination	0.42±0.00	0.42±0.00	0.50±0.10	0.50±0.10	0.56±0.10	0.56±0.10
5% Contamination	0.41±0.10	0.48±0.18	0.43±0.10	0.43±0.10	0.65±0.10	0.71±0.10**
10% Contamination	0.39±0.00	0.44±0.10*	0.67±0.10	0.67±0.10	0.67±0.10*	0.65±0.10**
15% Contamination	0.46±0.00	0.48±0.10*	0.78±0.00	0.78±0.00	0.64±0.10	0.79±0.00**

Comparative Effects of Pestox Insect Powder on Some Biochemical Parameters in Both Male and Female Albino Rats

¹Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria ²Department of Biochemistry, University of Port Harcourt, Choba, Rivers State, University of Port Harcourt, Choba, Rivers State of Nigeria.

*Corresponding Author E-mail: chkubani@yahoo.com; parkeselisco@yahoo.co.uk

OBJECTIVE: Pestox, insecticide, insect powder, contaminated diet.

Comparative Effects of Pestox Insect Powder on Some Biochemical Parameters in Both Male and Female Albino Rats

1Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria 2Department of Biochemistry, University of Port Harcourt, Choba, Rivers State, University of Port Harcourt, Choba, Rivers State of Nigeria.

*Corresponding Author E-mail: chkubani@yahoo.com; parkeselisco@yahoo.co.uk

OBJECTIVE: Pestox, insecticide, insect powder, contaminated diet.

¹Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria ²Department of Biochemistry, University of Port Harcourt, Choba, Rivers State, University of Port Harcourt, Choba, Rivers State of Nigeria.