Australian Journal of Basic and Applied Sciences, 3(4): 4190-4198, 2009 ISSN 1991-8178

The Ability of Vitamins A, C and E as Antioxidants Against The Genotoxic Potential of Tefluthrin

11Abdou H.S., Salah. S.H. and 2 Abdel Rahim E.A.

1Department of cell biology, National Research Center, Dokki, Egypt. 2Department of Biochemistry, Faculty of Agriculture, Cairo University, Giza, Egypt.

Abstract: Antioxidants are believed to be beneficial for a whole host of medical conditions and for good health. Vitamins C,A and E as antioxidants protect the cells from endogenous oxidative DNA damage. Tefluthuin, a pyrethroid insecticide, is widely used in agriculture because of its high activity against a broad spectrum of insect pests. The aim of this study was to investigate the modulatory effects of vitamins A, C and E against the toxicity induced by tefluthrin in male albino rats. Results

showed that tefluthrin (1/20of the LD50 ) oral administration to male rats sub-acutely caused strong toxic effects. This toxicity includes significant increase in chromosomal aberrations, sperm abnormalities mitotic activity and sperm counts. Also, the results of biochemical analysis showed that the immunoglobulins (IgG, IgA and IgM) content in serum was decreased significantly in tefluthrin intoxicated rats. In case of nucleic acids system; (total soluble protein, RNA and DNA content) of liver and testes tissues was decreased. Also the activity of RNAase and DNAase was inhibited. The Hb content and RBCs count were significantly decreased in contrast, the LDH activity was stimulated, but WBCs count was significantly changed under the teflurthrin toxicity conditions. Concurrently, administration of vitamin C,A and E with tefluthrin to rats for 15 consecutive days modulated the cytotoxicity induced by tefluthrin and markedly decreased the number of chromosome aberrations and sperm abnormalities. It also induced significant alleviations of the toxic effects in all biochemical parameters (immunosystem, nucleic acid system, RBCs, Hb and LDH activity). It can conclude that vitamins C,A and E can protected the animals from tefluthrin toxicity but not completely.

Key words:

INTRODUCTION

Pyrethroid pesticides are used preferably over organochlorines and organophosphates due to their high effectiveness, low toxicity to non-target organisms and easy biodegradability (Kale et al, 1999). The synthetic pyrethroid are a diverse class of more than 1000 powerful broad spectrum insecticides that are environmentally compatible by virtue of their moderate persistence, low volatility and poor aqueous mobility in soil (Erstfeld, 1999). Due to their high efficacy, easy biodegradability and low toxicity to birds and mammals (Kale et al, 1999), synthetic pyrethroids are chosen over orgnochlorine, organophosphorus and carbamate insecticides (Fotoui et al, 2008). Tefluthrin used to control a wide range of soil insect pests particularly those of the orders Coleoptera,

Lepidoptera and Diptera in maise, sugar beet, wheat and other crops. Oral and percutaneous LD50 values of tefluthrin depend on such factors are carrier, the test species, its sex, age and degree of fasting, values reported sometimes differ markedly. Acute oral LD50 for male and femal rats are 22 and 35 mg/kg b.w. respectively in corn oil carrier (Pesticide Manuel, 2000). Several biological defence mechanisms against intracellular oxidative stress are presented in the organism such as antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase and glutathione transferase) and non-enzymatic antioxidants such as caratenoids, vitamin E, vitamin C and glutathione, can also act to overcome the oxidative stress of the pesticides (Evans and Halliwell, 2001). The antioxidant vitamins are the most important free radical scavengers in extra-cellular fluids, trapping radicals in the aqueous phase and protects biomembranes from peroxidative damage (Yavuz et al, 2004 and Sulak et al, 2005). However, no detailed study of the comperative disposition of technical tefluthrin as well as the protective effects of antioxidant against the toxic effects of the tefluthrin in mammals was reported in the literature. Therefore, the

Corresponding Author: Abdou H.S., Department of cell biology, National Research Center, Dokki, Egypt.

4190 Aust. J. Basic & Appl. Sci., 3(4): 4190-4198, 2009 present study aimed to evidence the comparative effect of oral ingestion with sublethal dose of technical tefluthrin as well as the protective influences of vitamins C,A and E as antioxidant agents against the pesticide toxicity on nucleic acids system, immuno-system, RBCs, and WBCs counts, Hb content and LDH activity of blood, chromosomal aberration and sperm-shape abnormalities of male albino rats.

MATERIALS AND METHODS

Animals: Adult male albino rats weighing 150-200g obtained from a closed random-bred colony at the National Research Centre, were used for the experiment. They were maintained in a well ventilated animal house. They were housed in large polypropylene cages with free access to food and water ad libitum during the course of the experiment. Males were orally administrated with the different treatments once daily for two weeks.

Chemicals: The test substance, tefluthrin, was obtained from "Central Agricultural Pesticides Lab. Dokki, Egypt". The purity of tefluthrin was 92%. It is colourless solid, its chemical name :2,3,5,6-tetrafluoro-4-mehtylbenzyl (z)- (1RS,3RS)-3-(2chloro-3,3,3-trifluoroprop-=1-enyl)-2,2-dimethylcyclopropanecarboxylate. (Pesticide Manuel, 2000)

The insecticide technical tefluthrin was dissolved in corn oil. Administration was effected in a volume of 0.5ml / animal. Vitamins C, A and E manufactured by Memphis Company for Pharm and Chemical Industry. Cairo. A.R.E. The doses used were 200 mg/kg of vitamin C or E (Fotoui et al, 2008 and, Jalili et al, 2007) and 20 mg/kg vitamin A (Verma et al, 2007).

Dosage and Treatment: For cytogenetic and biochemical analysis, tefluthrin (dissolved in corn oil) was administered by gavage

1.1 mg/kg (1/20 LD50 ). The mentioned dose was administered orally (7 times) through 15 consecutive days. The experimental animals were divided to 5 groups (10 rats of each). The first control group was administered 0.5 ml/kg corn oil alone. The second group was orally administered with 1.1 mg/kg technical tefluthrin in 0.5 ml/kg corn oil. Intoxicated animals of groups 3,4 and 5 were administered orally with 200,20 and 200 mg/kg of vitamins C,A and E respectively secutive days. The rats were killed 24 h after the last treatment. For sperm-shape abnormalities, four groups of animals (5per group) were administrated by gavage with technical tefluthrin, for three days. Three groups of them were treated by vitamins C,A and E respectively for 5 consecutive days. The fifth group was administered 0.5 ml/kg corn oil for 5days. Animals in all groups were killed 35 days after the first administration. The protocol adopted for this study was recommended by Wyrobek and Bruce (1978) and Wyrobek et al (1984).

Slide Preparation and Scoring: For studying chromosomal aberrations, each of tefluthrin treated animals, vitamins and control animals were injected i.p. with 1ml of 0.5 mg/kg colchicines 2h before killing in order to arrest mitosis. Bone marrow metaphases were prepared according to Yosida et al, (1977) and stained with phosphate buffered Giemsa. Around 50 metaphases were studied, scoring the different types of chromosomal aberrations such as chromosomal gaps, breaks, deletions and centromeric attenuations. The mitotic activity of bone marrow cells was determined for each treated and control animal. It is expressed by the mitotic index (MI: number of dividing cells in 1000 cells). For sperm- shape abnormalities, rats were killed by cervical dislocation, the epididmids excised and minced in 10 ml of physiological saline, dispersed and filtered to exclude large tissue fragments. Smears were prepared after staining the sperms with Eosin y (aqueous). At least 800 sperm per animal were assessed for

4191 Aust. J. Basic & Appl. Sci., 3(4): 4190-4198, 2009 morphological abnormalities. Epididymal sperm count was determined by hemocytaometer. The weight of the animal and both testes were recorded. For the biochemical analysis, total hemoglobin (Hb) in blood was determined as the method described by Decra and Lewis (1975). RBCs and WBCs were counted in blood as demonstrated by Frankel and Reitman (1963). Liver and testes RNA and DNA contents were determined according to procedure described by Dische (1955) and Scheneider (1957) respectively. Total soluble protein values were carried out according to Lawary et al (1951) method. The activity of DNAase and RNAase were determined according to the methods described by Bergmeyer (1974). Immunoglobulins (IgG, IgA and IgM) were determined using immunodiffusion plate according to Fahey and Meckelvey (1965). Lactate dehydrogenase (LDH) activity was determined according to the method described by Tietz (1987).

Statistical Analysis: Intergroup difference of cytogenetic studies were analyzed statistically by using one – way analysis of variance test (ANOVA). Gaps were recorded, but not evaluated as chromosomal aberrations as refered to (Gupta 1995). To find the differences between groups dunken's test was used. P value of # 0.05 was considered to be significant. For spermatogenic damage, Wilcoxon's signed – rank sum test was used for the statistical analysis of the results (Sokal and Rohlf, 1969). The data of biochemical analysis obtained in the present work were subjected to the proper analysis of variance at 5% level of significance (t-test) according to the procedure outlined by Snedecor and Cochran (1967). RESULTS AND DISCUSSIONS

Sperm Abnormality: Various morphological sperm abnormalities (Fig. 1) were recorded in control and treated animals. The percentages of different abnormalities are listed in table 1. Tefluthrin caused an increase in abnormal sperm heads and tails. Treatment with different vitamins caused a decrease in the number of abnormal sperms of the intoxicated animals, but their frequencies exceeding significantly that of the control group (oil).

Table 1: Sperm abnormalities in tefluthrin - treated rats and the protective effect of vitamins C,A and E. Treatment Number Number Total abnormal Types of sperm head abnormalities of rats of sperms sperm ------examined ------Amorphous Banana Without Big Small total N % shape hook ------N % Control (0.5ml/kg oil) 5 4000 75 1.88 20 17 19 - 12 68 1.7 Tefluthrin (1.1 mg/kg) 5 4000 1150 28.75 400 249 100 132 100 981 24.53*** Tefluthrin +Vit. C 5 4000 413 10.33 114 47 54 31 136 382 9.55* Tefluthrin +Vit. A 5 4000 670 16.75 158 145 163 35 69 570 14.25* Tefluthrin +Vit. E 5 4000 692 17.3 130 139 161 23 123 576 14.40* *p<0.05, *** p<0.001

Fig. 1: Sperm-head abnormalities: (a) normal; (b) small head; (c) big head; (d) without hook; (e) banana shape; (f – h) amorphous; (I, j) abnormal tails.

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Testes Weight and Sperm Count: Table 2 presents the effect of tefluthrin on the testes weight and sperm count. Body weights were also recorded. The testes weight in animals treated with tefluthrin was slightly lower than that of healthy control and vitamins treated animals. The mean percentage of sperm count was decreased significantly to 56.69% after treatment with tefluthrin. Treatment with the different three vitamins increase the number of sperm count near to the normal (healthy control group) in the intoxicated animals. Vitamins C showed the most improvement in the sperm abnormalities and spermatogenic damage caused by the gavage of tefluthrin.

Table 2: Spermatogenic damage in tefluthrin-treated rats and the protective effect of vitamins C,A and E. Treatment Number of rats Body weight (g) Testes weight (g) Sperm Count (× 106 ) Mean ±S.E Mean ±S.E Mean ±S.E Control (0.5ml/kg oil) 5 316 ±1.83 3.732±0.402 52.312±0.819 Tefluthrin (1.1mg/kg) 5 186±1.64 2.00±0.263 29.655±1.021*** Tefluthrin +Vit. C 5 241±1.500 3.04±0.183 39.484±0.977 Tefluthrin +Vit. A 5 240±2.378 2.376±0.275 33.653±0.528 Tefluthrin +Vit. E 5 262±1.891 2.308±0.223 35.319±0.814 ***P> 0.001

Chromosomal Aberrations and Mitotic Activity: Table 3 presents the chromosomal aberrations and mitotic activity in bone marrow cells after subacute treatments with tefluthrin and the modulatory role of vitamins C, A and E of all experimental groups. Oral administration of tefluthrin to rats significantly increased the frequencies of individuals and total chromosomal aberrations in bone marrow cells when compared with control. This increase was found to be statistically significant at (p# 0.01). Gaps are recorded but not included in the total aberrations. In tefluthrin-treated animals chromosomal aberrations were mainly of the chromatid type and usually only one aberration was observed per nucleus. Administration of vitamin C, A and E concurrently with tefluthrin to rats indicated that these vitamins statistically significantly decreased the frequencies of chromosome aberrations induced by tefluthrin, but it can not completely protect cells from damage. Also the results showed that oral administration of vitamin C alone to rats decrease the frequency of total chromosome aberrations than vitamins A and E. Generally, the mitotic frequencies were decreased in tefluthrin- treated animals significantly (p < 0.001). Administration of vitamins C, A and E increased the mitotic frequencies but not as the control.

Table 3: Frequencies of different chromosomal aberrations induced in male bone marrow cells of all experimental groups Groups No. of Structural chromosomal aberrations Mitotic activity animals ------Gap Deletion Break Centromeric Total Total No. of MI attenuation chromosomal Chromosomal examined aberration aberration cells gaps Control (oil) 5 M ± SE 4.80b ±0. 80 0.80c ±0.20 0.60b ±0.24 0.60b ±0.24 2.00d ±0.32 6.80d ±0.80 10000 16.7 Tefluthrin (1.1 mg/kg) 5 M ± SE 11.20a ±1.28 3.20a ±0.58 2.60a ±0.51 2.60a ±0.51 8.40a ±1.03 19.60a ±0.51 10000 9.5 Tefluthrin + Vitamin C 5 M ± SE 5.80b ± 0.66 1.60bc ±0.51 1.40bc ±0.40 1.60ab ± 0.24 4.60c ±0.68 10.40c ±0.40 10000 13.6 Tefluthrin + Vitamin A 5 M ± SE 6.20b ±0.73 2.20ab ±0.20 2.00ab ±0.32 1.80a ±0.20 6.60bc ±0.55 12.20bc ±0.66 10000 11.9 Tefluthrin + Vitamin E 5 M ± SE 5.40b ±0.68 2.40ab ±0.24 2.80a ±0.84 2.60a ±0.51 7.80ab ±0.37 13.20b ±0.97 10000 11.4 Different letter (a,b,c,d) in the same column are significantly different (p#0.05)

Tissue Nucleic Acids and Blood Analysis: Table 4 showed the levels of total soluble protein, RNA and DNA contents in liver and testes tissues of the experimental animals. Data indicated that the ingestion of technical tefluthrin (1/20 of LD50 ) orally significantly decreased the values of total soluble protein, RNA and DNA either in liver or testes tissues relative to those of the normal healthy control animals. Whereas, the ingestion of antioxidant vitamins (C, A and E) into tefluthrin intoxicated rats alleviated and improved the nucleic acids and total soluble protein contents of liver and testes which readjusted their levels or normalized them, but these values were still slightly lower than those of normal animals. The results of DNA (tables 4) were confirmed by the present results of chromosomal aberration (table 3). From the results of (table 5) of nucleases activity (RNAase and DNAase) in liver and testes tissues of the experimental rats ingested by technical tefluthrin orally, it can be observed that the RNAase and DNAase activities were inhibited relative to the normal control. Table 5 also showed that the ingestion of vitamins C, A and E into intoxicated rats might be caused an alleviated effect on the toxicity of tefluthrin. Table 6 showed the influences of the experimental treatments on immuno-system and blood fractions as well as the variation in blood Ig G, Ig A and Ig M contents of tefluthrin intoxicated rats and those treated with vitamin C, A and E as anti-oxidative agents. The results observed that the serum immunoglobulins contents

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Table 4: The effect of vitamins A,C and E as antioxidant agents on protein, RNA and DNA contents of liver of tefluthrin intoxicated rats. Treatments Protein (mean± SD) RNA(mean ± SD) DNA(mean ± SD) Protein(mean ±SD) RNA(mean± SD) DNA(mean± SD) ------mg/g tissue % mg/g tissue % mg/g tissue % mg/g tissue % mg/g tissue % mg/g tissue % Normal control 190±11 100 0.210±0.013 100 0.400±0.031 100 100±7.70 100 0.185±0.008 100 0.320±0.021 100 Intoxicated control 142±10* 75 0.151±0.011* 72 0.301±0.022* 75 76±5.61* 76 0.141±0.007* 76 0.252±0.014* 79 Intox. rats+ vit.A 163±11 86 0.170±0.012 81 0.344±0.021 86 90±6.66 90 0.160±009 86 0.286±0.017 89 Intox. rats+ vit.C 179±12 94 0.189±0.014 90 0.382±0.019 96 97±7.37 97 0.177±0.008 96 0.301±0.016 94 Intox. rats+ vit.E 175±11 92 0.186±0.012 89 0.376±0.018 94 93±8.12 93 0.168±0.007 91 0.297±0.017 93 * p < 0.05

Table 5: The effect of vitamins A,C and E as antioxidant agents on RNAase and DNAase activity of liver and testes of tefluthrin intoxicated rats. Treatments RNAase (IU/g tissue) DNAase (IU/g tissue) ------Liver Testes Liver testes ------Mean ± SD % Mean ± SD % Mean ± SD % Mean ± SD % Normal Control 41.11± 3.33 100 40.46±3.26 100 58.31 ±4.01 100 79.19 ±3.47 100 Intox. Control 25.21±1.92* 61 23.11±1.96* 57 35.11 ±2.93* 60 54.12 ±2.79* 68 Intox. rats +v.t. A 34.82±2.13 85 35.00±2.21 87 48.99 ±2.94 84 68.88 ±4.00 87 Intox. rats + v.t. C 39.01±2.22 95 39.00±2.06 96 52.91 ±3.33 91 71.22 ±3.97 90 Intox. rats + v.t. E 37.91±1.99 92 38.01±2.94 94 53.11 ±3.42 91 71.00 ±4.01 90 * p < 0.05

Table 6: The effect of vitamins A,C and E as antioxidant agents on blood immunoglobulins (IgG, IgA and IgM) RBCs, WBCs, Hb and LDH of tefluthrin intoxicated rats: Treatments IgG (mg/dl) IgA (mg/dl) IgM (mg/dl) RBCs WBCs Hb content LDH activity ------(mean SD) % (mean SD) % (mean SD) % Count ×1063 % Count ×10 % g/dl % u/g Hb % Normal control 2501 ± 199 100 333±27 100 225±19 100 7.81± 0.30 100 7.11±0.45 100 15.62±0.32 100 122±10 100 Intox. Control 2200±171* 88 201±14* 60 171±11* 76 5.99±0.27* 77 6.87±0.42 97 11.11±0.71* 71 143±13* 117 Intox. rats+ vit. A 2301±200 92 260±17 78 190±10 84 7.01±0.37 90 7.00±0.38 98 14.01±0.82 90 136±10 111 Intox. rats + vit. C 2441±198 98 301±20 90 221±19 98 7.49±0.40 96 7.06±0.28 99 15.00±0.80 96 130 ±12 107 Intox. rats + vit. E 2400±201 96 289±21 87 210±18 93 7.22±0.29 92 7.10±0.35 100 14.82±0.90 95 134±11 110 * p< 0.05 were significantly decreased by tefluthrin ingestion, but the three antioxidant vitamins significantly alleviated the effect of tefluthrin in intoxicated animals and improved their values to about normal values. The intoxicated rats ingested with vitamin C or E gave higher levels of the immunoglobulins than that of vitamin A ingestion. In case of blood fractions and LDH activity, Hb content and RBCs count were significantly decreased but the LDH activity was stimulated under the effects of tefluthrin induction. WBCs count was unchanged at the same conditions. The treatments with vitamins C, A and E alleviated the toxicity of the pesticide and improved these blood parameters. Generally nucleic acids system of liver and testes tissues ammuno-system, blood fractions and LDH activity of blood of intoxicated rats subjected to the three vitamins as antioxidant agents treatments can be arranged in the following ascending order : vitamin C > vitamin E > vitamin A.

Discussion: Tefluthrin, a pyrethroid insecticide, has been widely used to control a wide range of soil insect pests, particularly those of the Lepidoptera, Diptera and Coleoptera, in maize, sugar beat, wheat and other crops. There are no published reports could be found on the potential cytogenetic effects of tefluthrin. All experiments assessed in the present study indicated that tefluthrin is a mutagenic agent and the studied vitamins have the ability to reduce its mutagenic effect partly. The dose of tefluthrin tested through post orally (p.o.) by gavage induced significant increase in the frequency of chromosomal aberration (CA), as well as increasing the number of abnormal sperms. It also decrease the mitotic activity of somatic cells and the number of sperm count. The significantly higher frequency of CA observed after substance treatment for 15 consecutive days could be due to residual effect or involvement of secondary metabolites. Wyrobek et al (1983) emphasized that pesticides that are mutagenic to somatic cells, could affect germinal cells also. The increase in the frequency of abnormal sperms (Table 1, fig. 1) suggest that tefluthrin has definite effect on the differentiation of male germ cells, and may be regarded as a potential germ cell mutagen. Further, tefluthrin significantly decrease the total sperm count (Table 2). Several compounds which induce cytogenic effects and micronucleus formation in the bone marrow cells of mice are also known to cause morphological abnormalities in sperms(Chauhan et al, 2000; Wyrobek et al, 1978; Jayashree et al, 1994; Hemavathi et al, 1993). Also induced sperm abnormalities indicate point mutation in germ cells (Narayna et al, 2002 and Wyrobek et al, 1983), which should have triggered structural changes in cell organelles involved in head and tail formation, leading to sperm abnormalities. Concurrently

4194 Aust. J. Basic & Appl. Sci., 3(4): 4190-4198, 2009 administration of vitamins C,A and E with tefluthrin to rats markedly decreased the number of CA induced by the present insecticide. But it can not completely protect cells from damage. However, the number of these aberrations did not reduced to the control number. Vitamin C decreased the number of CA induced by tefluthrin than vitamin A and E. Ascorbic acid is hydrophilic and functions as a most important free radical scavenger, trapping free- radicals in the aqueous phase, thus protecting boimembranes from oxidative damage (Altuntas et al, 2002). Ascorbic acid also prevent free-radicals- induced DNA damage (Dawson et al, 1990). The present results indicated the protective role of vitamin C, while it reduced the number of abnormal sperms caused by tefluthrin than vitamins A and E. It also suggested that vitamin C has some antioxidative effects on damage induced by free radicals generated during the metabolic activity. As an antioxidant, vitamin C may also have potential anticancer activity, but its possible role and its mechanism of action in cancer prevention has not been clearly established. Decreased ascorbic acid concentration in the testes was well correlated with decreased sperm count and increased sperm abnormalities, indicating a close relation between them (Narayna et al.,2005). So it can partly protect the cells from the oxidative damage in the testis which could have affected the normal spermatogenesis. The effects of technical tefluthrin as well as antioxidant agents (vitamins C, A and E) against the toxicity of the pesticide on body metabolism were also studied in the present work. Pesticides including synthetic pyrethroids, elicited a number of effects including hepatic dysfunctions, immunological abnormalities, embryotoxicity, genotoxicity and teratogenicity (Thrasher et al, 1993 and Bagchi et al, 1995). Being lipophilic in nature, most of the pesticides interact with living organisms through the lipid rich biomembranes (Antunus- Madeira and Madeira, 1979). Evidences suggest that the formation of oxygen free radical can be a major factor in the toxicity of pesticides (Banerjee et al, 2001). Also, pesticides have been show to damage DNA (Balsiak and Stankowska, 2001). The present study has shown that tefluthrin causes significant inhibition of nucleic acid in liver and testes and also serum immuno-systems as well as hematological parameters. In general, the biochemical data obtained with tefluthrin ingestion showed significant decreases for total soluble protein, RNA and DNA as well as inhibiton for RNAase and DNAase activity of liver and testes. Also, serum immunoglobulins (Ig A, Ig G and Ig M) were reduced by the induction of the pesticide. When the same tefluthrin treatment given to rats with the antioxidative vitamins (A, C and E individually) the toxicity action of tefluthrin was reduced and the nucleic acids system and immuno-system from the inhibitory effect of the pesticide were protected to a significant extent. The basis of pesticide toxicity in the production of reactive oxygen species (ROS) may be due to :- Their "redox-cycling" activity – they readily accept an electron to form free radicals and then transfer them to oxygen to generate superoxide anions and hence hydrogen peroxide through dismutation (Jalili et al, 2007). Generation of free radical probably because of the alteration in the normal homeostasis of the body resulting in oxidative stress, if the requirement of continuous antioxidants is not maintained (Ryrfeldt et al, 1992). The effects of the endogenous antioxidant to remove the continuously generated free radicals initially increase due to an induction but antioxidant depletion results, resulting in oxidative cell damage (Kalra et al, 1994 and Jalili et al, 2007). Exposure of rats to tefluthrin decreased hematological parameters (RBCs and Hb) but unchanged (WBCs) and developed animia in rats. The present results are in line with Fotoui et al, (2008) report which demonstrated that pyrethroid insecticide induction reduced hematological parameters in rats. The reduction in RBCs and Hb might be due to the inhibition of erythropoiesis and hemosynthesis and to an increase in the rate of erythrocyte destruction in hemopoietic organs. In addition, Hb in erythocytes is a major source of radical production when it interacts with xenobiotics (French et al, 1978) giving rise to superoxide radicals,

H22 O and in certain cases peroxy radicals leading to membrane lipid peroxidation and hemolysis (Clemens and Waller 1987). The stimulation of lactate dehydrogenase (LDH) activity is marker of hemolysis (Kato et al, 2006). Similar results have been found in rat erythrocytes exposed to cypermethrin (Gabbianelli et al, 2002) and Lambda cyhalothrin (Fotoui et al, 2008). On the other hand, Nasuti et al (2003) and Prsanthi et al (2005) reported that oxidative damage, induced by pyrethroids might be due to their lipophilieity, whereby they could penetrate easily to the cell membrane and caused membrane lipid peroxidation. The ingestion of antioxidant vitamins (C, A and E) attenuatd the in vivo effect of tefluthrin by scavenging ROS. These results indicated that the antioxidative vitamins might have a beneficial role in lowering pyrethroids toxicity probably due to its free radical scavenging property (Grajeda- Cota et al, 2004 and Giray et al, 2001). The present data confirmed each other where the increasing in RNA,DNA and protein contents were paralled with the simulation in nuclease (RNAase and DNAase) activity also the serum Ig A, Ig G and Ig M as well as RBCs and Hb levels were also elevated in intoxicated rats by the treatment with vitamin A, C and

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E. These also confirmed by the results of chromosomal aberrations and sperm shape abnormalities of the present work which may be due to the elevation in the biosynthesis processes of protein. The results of the present studies are supported with those obtained by Verma et al (2007) and Kimmick et al. (1997) who reported that the antioxidant vitamins have immune stimulation and an alteration in metabolic activities of carcinogens. These vitamins can prevent genetic changes by inhibiting DNA damage induced by the reactive oxygen metabolites such as ROS. B-carotene, one of the important carotenoids, has been thought of value to humans and other species not only as a precursor of vitamin A but also for having excellent antioxidant properties. The antioxidative mechanism of vitamin A has been suggested to be singlet oxygen quenching, free radical scavenging and chain breaking during lipid peroxidation (Terao, 1989). Vitamin A plays a role in traping epoxy radicals in tissues at low partial pressure of oxygen. Looking at the role of the antioxidant protection against oxidative stress and oxidative injuries, the present study used vitamin A which can prevent the uncontrolled formation of free radical and activated oxygen species or inhibit their reaction with biological structure (Varma and Srivastava, 2003 and Verma et al, 2007). Thus vitamin C appeared to be a promising agent for protection against tefluthrin- induced oxidative damage. Ingestion of vitamin C with tefluthrin improved the antioxidative enzymes and non-oxidative enzymes levels to reach normal values, and ameliorated the antioxidant defence system. These data are inagreement with Sae-Yong et al (2002) and Fotoui et al (2008) who reported that vitamin C increased the total antioxidant status in plasma and it could be useful as a free radical scavenger in patients with paraquat intoxication. Furthermore, Grajeda–Cota et al. (2004) have reported that extracellular vitamin C levels scavenge the ROS derived by the oxidation – reduction cycle, and acted as chemoprotector against cypermethrin that caused lipid peroxidation. In case of vitamin E (á – tocopherol), the present results with tefluthrin plus á-tocopherol treatment showed no statistical differences when compared with the control animals, but upon comparing the regressions between the treatments of tefluthrin and teflutrin + vitamin E, the data obtained showed a significant statistical difference. These may be due to that á-tocopherol is not distributed uniformly in the cell membrane, it forms small aggregations in the lipid bilayar making it possible to induce a protective effect (Aldana et al,2001). Other studies carried out in rat have shown that á – tocopherol can prevent lipid peroxidation in vivo. Vitamin E does not form a co-valent bond with free radicals, it is suggested that vitamin E acts by blocking the formation of lipoperoxides (Numan et al,1990). These observations may be due to the fact that largest amounts of vitamin E in cell are located in the membrane in conjunction with saturated fatty acids (although not in a uniform way). Therefore, the protective doses of á-tocopherol could have acted by blocking free radical attack (Rock et al, 1996 and Karaoz et al, 2002). The results of the current study suggested that the antioxidative vitamins (C, A and E) may be useful in controlling the toxic effects of tefluthrin. Nevertheless other studies are required with aim to promote a mixture of them use among animals and people exposed to this type of pollutant.

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