Zagazig J. Agric. Res., Vol. 38 No. (3) 2011 775-792

INDUCING TOXIC EFFECTS OF SUBLETHAL DOSES OF LAMBDA-CYHALOTHRIN ON ALBINO RATS,

RATTUS NORVEGICUS BORK

Mohamed G. Mahmoud*, E.A. El-Sheikh, M.M. Aamir and M-B.A. Ashour

Plant Protection Dept., Fac. Agric., Zagazig Univ., Egypt

ABSTRACT The toxic effects of daily oral administration of sub-lethal doses of

lambda-cyhalothrin pyrethroid insecticide (7.8, 3.9 and 2.6 mg/kg b wt.) for 30, 60 and 90 days were studied in male and female albino rats. Thirty six animals of each sex were divided into four groups of nine animals each. The first three groups of animals were treated separately with the previous doses which represents 1/10, 1/20 and 1/30 of LD50 that didn't give any acute toxic effects. The last group was reserved as a control. Results obtained indicated that changes in glucose, creatinine and urea levels in rat serum according to the periods of treatment were recorded. Reduction in Alkaline phosphatase, AST and ALT activity were observed in male and female rats. In the same way, decrease in general esterases was noted in rats serum when determined colormetrically, while high in intensity and size of band profiles were found when esterases investigated using native gel electrophoresis. The activity of glutathione-S-transferase was high in most cases of treatments indicating that both enzymes may be associated in pesticide toxicity elimination in treated rats. These data indicate the hazard effects of lambda-cyhalothrin exposure for long periods especially with high doses.

Keywords: Lambda-cyhalothrin, rats, biochemical parameters, esterases, glutathione-S-transferase, pyrethroids, insecticides.

* Corresponding author: Mohamed G. Mahmoud , Tel.: +20167843669

E-mail address: Mohamed_gamal6051@yahoo.com

Mahmoud, et al.

INTRODUCTION Synthetic pyrethroids were

extensively used in agricultural and home formulations for more than 40 years to prevent pest infestation and in public health purposes (Ross et al., 2006). They account for approximately one-fourth of the world-wide insecticide market (Casida and Quistad, 1998). The toxicity to vertebrates does not increase at the same rate as the toxicity to invertebrates, making the synthetic pyrethroids generally better and more selective pesticides than the natural pyrethroids (Stenersen, 2004). These compounds cause neurotoxic effects for mammals or insects, which show a pattern of toxic action typical of a strongly excitant effect on the nervous system.

Lambda-cyhalothrin is a synthetic type II pyrethroid insecticide (Meister, 1992) with a high level of activity against numerous species of harmful arthropods (WHO, 1990). It is extensively used in public health and veterinary application to control harmful insects of human and ectoparasites of animals, including cockroaches, flies, lice, mosquitoes, and ticks, as well as in agricultural formulations to control numerous

insect pests on fruits, vegetables, and field crops (Davies et al., 2000 and Kroeger et al., 2003). Due to their lipophilicity, pyrethroid insecticides favor absorption through the gastrointestinal and respiratory tracts and also confer preferential distribution into lipid-rich internal tissues, including body fat, skin, liver, kidney, ovaries and the central and peripheral nervous systems (Soderlund et al., 2002). Liver is the major site of pesticides metabolism. This soft tissue accumulates a greatest concentration of its metabolites (Rickard and Brodie, 1985). Several reports have shown that pyrethroid type II insecticide exposure inhibited cell proliferation in rodent bone marrow (Celik et al., 2005), and affected sodium and calcium channels (Clark and Symington, 2007).

This work aimed to test the long-term toxic effects of lambda-cyhalothrin, one of the most important pyrethroid insecticides used in public health for controlling mosquitoes in Egypt. Animal administration was done with sub-lethal doses through drinking water. Serum biochemical parameters of male and female albino rats, Rattus norvegicus, were assessed.

Zagazig J. Agric. Res., Vol. 38 No. (3) 2011 775-792

MATERIALS AND METHODS

Chemicals and Reagents Lambda-cyhalothrin, a

commercial formulation named Lambda 5% EC was obtained from a local formulator, Kafr El-Zayat for pesticides and chemical Company, Kafr El-Zayat City, Gharbia Governorate, Egypt. The CAS chemical name is [α-cyano-3-phenoxybenzyl-3-(2-chloro- 3,3,3-trifluoro-1-propenyl)-2,2 dimethyl-cyclopropanecarboxylate.

All other chemicals used in the study were purchased from Sigma-Aldrich (St. Louis, MO). Reagents, and other chemicals used in this study were of technical grade with the highest purity available.

Animal Maintenance Adult male and female albino

rats, Rattus norvegicus Bork, of initial body weight 140±10 g (male), 120±10 g (female) were used in the this study. The rats were obtained from the breeding animal house, Faculty of Veterinary Medicine, Zagazig University. Animals were maintained under standard laboratory conditions (temperature 25±3ºC; 12:12 h, light:dark cycle). They had free access to water and commercial standard food (pellet ration).

Experimental Design and Application

Male and female rats were left under the previously mentioned laboratory conditions for 1 week of acclimatization period. Thirty six animals of each sex were randomly divided into four groups of nine animals each. The first group of each sex received daily untreated tap water and used as a control. The second group of each sex received a daily dose of 7.8 mg kg−1 body weight of lambda-cyhalothrin (equals to 1/10 of the published LD50 (US-EPA, 1988) through drinking tap water. Animals of the third and fourth groups received daily doses of 3.9 and 2.6 mg kg−1 body weight (equal to 1/20 and 1/30 of LD50, respectively). Administration of the insecticide was continued for periods of 30, 60 and 90 days. Doses were adjusted weekly according to the changes in the body weight.

All experimental procedures were carried out according to the Local Experimental Animal Care Committee, and approved by the ethics of our institutional committee. Collection of Blood Samples

Blood samples were collected at 30, 60 and 90 days from 3 animals of different groups by cervical decapitation (to avoid animal

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stress). Collected samples were taken into 10 mL glass centrifuge tubes, left at room temperature for clotting, then centrifuged at 2500 rpm for 10 min. Serum samples were transferred in aliquots into 1.5 mL eppendorf tubes and stored at -20ºC until use.

Determination of some Serum Parameters and Enzyme Activity

Colorimetric activity assays were performed using either spectrophotometer (Spectronic 20, Bausch and Lomb, USA) or 96-well microplate using Microplate Autoreader, EL311S (Bio-TEK Instrument, Highland Park, Winooski, VT).

Total protein Total protein concentrations in

rats serum were determined using a commercial available kit according to the manufacturer’s method (Diamond Diagnostic, Heliopolis, Cairo, Egypt) according to Koller (1984).

Glucose, urea and creatinine

Glucose, urea and creatinine levels in rats serum were determined using commercial available kits purchased from the same company previously mentioned according to the manufacturer’s protocol.

AST, ALT and ALP Aspartate amino transferase

(AST), alanine amino transferase (ALT) and alkaline phosphatase (ALP) activities in rats serum were determined using commercial available kits purchased from Diamond Diagnostic for AST and ALT and from Teco-Diagnostics (Anaheim, CA) for ALP. The determination methods were done according to the manufacturers’ protocols.

General esterases

Colorimetric esterase activity assays were determined using the general substrates α- and β-naphthyl acetate as described by Gomori (1953). The optical density (OD) was measured at 450 ηm during the first 5 min of the reaction, and rates were converted to ηmoL min-1 mg-1 using the extension coefficients of 9.25 mM-1 300 µL-1 for 1-naphthol (Grant et al., 1989). Activities were corrected for non-enzymatic hydrolysis using reactions without protein as controls.

Glutathion-S-transferase GST activity assays were done

by the methods of Grant and Matsumura (1988). Three replicates were used for each measurement,

Zagazig J. Agric. Res., Vol. 38 No. (3) 2011 775-792 and activities were corrected for non-enzymatic hydrolysis using reactions without protein as controls. The conjugation of CDNB to glutathione is accompanied by an increase in absorbance at 340 ηm. The rate of increase was directly proportional to the GST activity in sample. These measurements were done using spectrophotometer (Spectronic 20, Bausch and Lomb, USA) in the same day of getting serum samples from treatments by measuring absorbance at 340 ηm at 30ºC for 15 minutes after the addition of glutathione. Rates were converted to ηmol min-1 mg-1 using the extinction coefficients of 8.5 O.D. mM-1 1000 µL-1 for CDNB

(Grant et al., 1989).

Native Gel Electrophoresis for General Esterase Profile

Electrophoresis of non-denaturing protein samples was performed using 7.5% native polyacrylamide gels according to the method of Davis (1964). Esterases were visualized by incubation of gels with 0.04% (w/v) α- and β-naphthyl actetate, 0.1% (w/v) Fast Blue B salt in 100 mM phosphate buffer, pH 7.4.

Statistical Analysis The SPSS 10.0 for Windows

software package was used for statistical analysis. Data were

given in the form of arithmetic mean values and standard deviations. One-way analysis of variance was performed and variant groups were determined by means of the Duncan test (Duncan, 1955).

RESULTS Effects of Lambda-Cyhalothrin on

Animal body weight Table 1 indicates the changes

in the body weight of male and female rats treated with lambda-cyhalothrin. Treated animals were affected with insecticide administration. 1/10 of LD50 was the most effective dose especially in the first 30 days of treatment which were 5.3 and 2.2% increase in the body weight of male and female rats, respectively, compared with 42.3 and 22.7% increase in control groups. The percentage of increase in the body weight during all periods of treatments was less than those recorded in the animals of control groups.

Glucose, urea and creatinine levels

A significant increase in glucose level was found in male and female rats treated with 1/10 and 1/30 of LD50 for 30 days. On the other hand, significant decrease

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Table 1. The changes in the body weight of treated and untreated rats during experimental periodsa

male female Days post- treatment control 1/10 of

LD50 1/20 of LD50

1/30 of LD50

control 1/10of LD50

1/20 of LD50

1/30 of LD50

0 days 156.5±6.0 159.3±9.2 140.3±9.1 161.5±5.8 118.0±9.1116.2±10.1 111.3±2.1 112.0±7.9

30 days 222.7±17.3

(42.3)*

167.8±26.0

(5.3)

181.0±31.8

(29.0)

232.0±29.8

(43.65)

144.8±9.7

(22.7)

118.7±11.6

(2.2)

144.5±11.1

(29.8)

126.8±5.1

(13.2)

60 days 257.0±18.9

(64.2)

205.5±30.6

(29.0)

192.5±36.1

(37.2)

261.0±31.9

(61.6)

152.8±5.6

(29.5)

135.0±16.5

(16.2)

158.3±19.6

(42.3)

138.3±4.8

(23.5)

90 days 276.0±33.9

(76.4)

197.0±17.0

(23.6)

174.5±36.1

(24.4)

243.5±54.4

(50.8)

167.0±4.2

(41.0)

150.5±6.3

(29.5)

139.5±2.1

(25.3)

148.5±10.6

(32.6) a values are presented as mean±SD of at least 3 rats form each sex. * values in paranthesis are calculated as % of increase in weight compared with

original weight (0 day) (weight of treated periods – original weight) x 100 / original weight.

in male rats treated with 1/10, 1/20 and 1/30 of LD50 for 60 and 90 days was recorded. In the same way, a significant decrease was noted in females treated with 1/10 of LD50 for 30 and 60 days, while at 90 days this decrease was recorded with all doses tested (Fig. 1).

A significant increase in creatinine level was occurred in female rats treated with 1/10 of LD50 for 30 days, and in male rats treated with 1/10 and 1/20 of LD50 for 90 days (Fig. 2), while significant decrease was observed in female rats treated with 1/10 and

1/30 of LD50 for 60 days. The treatment of rats with 1/10 of lambda-cyhalothrin LD50 for 30 days significantly increased urea level in male rats (Fig. 3).

Enzymes activities Alkaline phosphatase activity

was significantly decreased in male rats treated for 30 days and in both sexes treated for 90 days with all tested doses of lambda-cyhalothrin (Fig. 4). The effect of the tested insecticide on the activity of alanine amino transferase and aspartate amino transferase in both sexes of rats was the same as shown in Table (2).

Zagazig J. Agric. Res., Vol. 38 No. (3) 2011 775-792

Fig. 1. Effect of lambda-cyhalothrin administration for 30, 60 and 90 days on glucose level in male and female rats. * differences are significant at P≤0.05 compared with control groups

Fig. 2. Effect of lambda-cyhalothrin administration for 30, 60 and 90 days on creatinine level in male and female rats. * differences are significant at P≤0.05 compared with control groups

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Fig. 3. Effect of lambda-cyhalothrin administration for 30, 60 and 90 days on urea level in male and female rats. * differences are significant at P≤0.05 compared with control groups

Fig. 4. Effect of lambda-cyhalothrin administration for 30, 60 and 90 days on alkaline phosphatase activity in male and female rats. * differences are significant at P≤0.05 compared with control groups

Zagazig J. Agric. Res., Vol. 38 No. (3) 2011 775-792 Table 2. Effect of lambda-cyhalothrin administration at different

sub-lethal doses for 30, 60 and 90 days on alanine and aspartate amino transferases of albino rats

ALT (U/L) AST (U/L) Treatments sex 30 day 60 day 90 day 30 day 60 day 90 day

male 13.9±0.8 13.3±1.6 13.9±1.1 30.2±1.9 29.0±3.9 28.9±2.2 control female 11.7±1.3 11.6±0.6 10.9±1.2 24.1±2.2 24.1±1.4 23.1±2.8

male 8.1±2.0* 5.7±0.7* 7.5±0.8* 16.1±3.8* 11.6±1.4* 15.0±1.4* 1/10 LD50 female 10.1±0.8 6.8±0.5* 6.3±0.8* 20.0±2.0 13.7±1.0* 12.8±1.5*

male 7.1±0.7* 4.2±0.7* 6.4±0.2* 12.7±1.5* 9.6±1.1* 11.2±3.1* 1/20 LD50 female 6.8±1.0* 5.7±1.1* 4.2±0.3* 13.8±1.8* 11.2±2.9* 7.3±0.4*

male 4.2±0.5* 6.7±1.4* 6.1±1.1* 8.6±1.2* 13.6±2.6* 12.5±2.0* 1/30 LD50 female 9.8±1.4 11.1±0.5 5.9±0.4* 19.8±3.1 22.9±1.2 12.0±0.8*

values are presented as mean±SD of 3 rats form each sex. * differences are significant at P≤0.05 compared with control groups.

A significant decrease in the activity of both enzymes was noted for most treatments comparing with control groups. Male rats were influenced by all the tested doses when administrated for 30, 60 and 90 days. However, female rats didn’t affect by the treatments when they treated for 30 days with 1/10 and 1/30 of LD50 and for 60 days with 1/30 of LD50.

General esterase activity determined with general substrates α- and β-NA are presented in Table 3. A significant reduction was observed in general esterase activity when determined using α-NA in male rats treated with 1/10 of LD50 for 30 days. When female rats treated with 1/30 of LD50 for 60 days, a significant reduction

was noted. Using β-NA as a substrate for general esterase determination indicated that a significant decrease was found in male and female rats treated with 1/10 of LD50 for 30 days. General esterases visualized on native gel electrophoresis (Fig. 5) and stained with α- and β-NA indicated that the intensity and thickness of bands of the male group treated with 1/10 of LD50 for 30 days was high comparing with the control group. However, bands of the rest groups treated for the same period of time were less. At 60 and 90 days of treatments, bands of male and female groups were more in intensity and thickness compared with control groups in all treatments except that in a female group treated with 1/30 of LD50.

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Table 3. General esterase activity determination using α- and β-naphthyl acetate in rat serum after different periods of lambda-cyalothrin administration

activity (α -NA) (nmol/min/mg protein)

activity (β -NA) (nmol/min/mg protein) Treatments sex

30 day 60 day 90 day 30 day 60 day 90 day male 3.42±0.77 3.81±1.07 3.72±1.16 4.26±0.21 4.89±0.17 4.21±1.17 control

female 2.87±0.47 4.08±1.06 3.23±0.78 3.00±0.30 3.32±0.15 3.55±0.85

male 2.06±0.26* 4.26±0.76 3.90±0.82 2.44±0.34* 4.57±0.96 4.24±0.76 1/10 LD50 female 2.56±0.28 3.22±0.83 2.77±0.72 3.07±0.35 3.07±0.08 3.14±0.80

male 3.69±0.41 3.93±0.15 3.36±1.03 3.78±1.26 4.03±1.04 3.85±1.04 1/20 LD50 female 3.17±0.94 3.75±0.81 3.63±0.55 3.55±1.08 4.11±0.84 4.08±0.57

male 2.92±0.82 3.90±0.78 3.36±0.36 3.27±0.89 4.21±0.81 3.79±0.32 1/30 LD50 female 2.59±0.61 2.34±0.08* 2.66±0.59 2.54±0.03 2.72±0.10 3.08±0.57

values are presented as mean±SD of 3 rats form each sex. * differences are significant at P≤0.05 compared with control groups.

Fig. 5. Activity of esterases detected in male and female rats serum using 7.5%

nondenaturating polyacrylmide gel electrophoresis. Arrows indicate esterase band profiles detected by running the same amount of protein (100 µg/lane) and incubating gel in α- and β–NA substrates for 1 hr at 37ºC in the dark. A, B and C are representing rats serum after 30, 60 and 90 days, respectively. Lanes 1, 3, 5 and 7 represent of control, 1/10, 1/20, and 1/30 of LD50 male groups, respectively. Lanes 2, 4, 6 and 8 represent of control, 1/10, 1/20, and 1/30 of LD50 female groups, respectively.

Zagazig J. Agric. Res., Vol. 38 No. (3) 2011 775-792 Table 4. Glutathion-S-transferase activity determination in rat serum

after different periods of lambda-cyalothrin administration

activity (µmol/min/mg protein)a Treatments sex 30 day 60 day 90 day

male 203±70 210±31 201±21 control female 202±29 147±30 125±37

male 220±94 381±64* 271±76 1/10 LD50 female 286±17* 234±26* 192±14*

male 138±35 257±15* 207±34 1/20 LD50 female 187±51 265±42* 185±25*

male 145±53 238±70 204±27 1/30 LD50 female 168±17 168±15 162±22

a values are presented as mean±SD of 3 rats form each sex. * differences are significant at P≤0.05 compared with control groups.

Table 4 shows glutathione-S-transferase activity in male and female rats serum treated with 1/10, 1/20 and 1/30 of LD50 for 30, 60 and 90 days. After 30 days of treatment, a significant increase in enzyme activity was noted in female rats treated with 1/10 of LD50. At 60 days of treatment, a significant increase was observed in both male and female rats treated with 1/10 and 1/20 of LD50, and a similar increase was noted in females treated for 90 days with the same doses.

DISCUSSION Regard the sub-lethal doses'

administration of lambda-cyhalothrin for 30, 60 and 90 days

for male and female rats (as mammals) were resulted in adverse effects on some serum biochemical parameters, which bears significance with respect to the evaluation of the effects of pesticides on the organs and therefore their toxicity (Choudhary et al., 2003 and Kalender et al., 2005).

The changes in the body weight was measured which could affect the clinical pathology outcomes. The percentage of increase in the body weight in treated animal groups was less compared with control ones. These reductions in weight gain may due to the effect of insecticide on the ability of animals for up-take food. Blood glucose levels were found to be

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increased in the first 30 days for some used doses, and then decreased as the period of treatment was prolonged to 60 and 90 days in both male and female rats. The reason for increasing glucose level is reported by Eraslan et al. (2007) who indicated that the stress caused by deltamethrin resulted in increase of the synthesis of adrenocorticotropic hormone and glucagon in the suprarenal glands and decrease in the synthesis of insulin. Due to the indicated increase, hepatic glycogen is rapidly converted into glucose and passes into systemic circulation, lead to a rapid increase in blood glucose level (Data and Kaviraj, 2003). Our data is agreed with what Eraslan et al. (2007) found in rats treated with deltamethrin. They indicated that the increase in the first period of treatment was replaced with decrease in the group administered the lower and higher dose of deltamethrin in the last two periods. The reason for this effect may be due to increase or decrease in the synthesis of the above mentioned hormones in subsequent periods. Also, levels of glucose are reported to increase in case of exposure to pyrethroid insecticides (El-Demerdash et al., 2003 & 2004 and Manna et al., 2004).

Creatinine and urea levels were determined in male and female

treated rats with lambda-cyhalothrin as indicators for dysfunction and damage of the kidneys (Mohssen, 2001; Choudhary et al., 2003 and Manna et al., 2004). Creatinine levels were found to be increased after lamda-cyhalothrin administration in female and male rats treated with 1/10 (female and male) and 1/20 (male) for 30 and 90 days. On the other hand, females treated for 60 days with all tested doses exhibited a decrease in creatinine level. These changes may be an indicator of degeneration in the kidneys. On the same way, significant increase was found in a male group treated with 1/10 of LD50 for 30 days. The increase in urea was found to be associated with the increase in protein catabolism and in the synthesis of the enzyme that leads to synthesis of urea from ammonium in mammals (Eraslan et al., 2007). In addition, increase in protein catabolism in case of liver dysfunction also leads to increase in the level of the final product of this reaction, namely urea (El-Demerdash et al., 2003 & 2004 and Yousef et al., 2003).

ALP is a hepatic enzyme that changed in its activity. This may be related to severe damage of hepatic cells and obstruction of the bile ducts due to proliferation (El-Demerdash et al., 2003 & 2004).

Zagazig J. Agric. Res., Vol. 38 No. (3) 2011 775-792 This enzyme activity was decreased in both sexes due to treatments for all tested periods compared with control groups. A reduction in AST and ALT activities was found in rats treated with lambda-cyhalothrin. These changes in the enzyme activities due to exposure to pyrethroids are related to damage in the liver and alteration in liver functions (Manna et al., 2004 and Fetoui et al., 2009). The decreases in enzyme activities were found not only related to lambda-cyhalothrin treatment, but also to other pyrethroid insecticides such as deltamethrin (Eraslan et al., 2007). Liver and kidney enzymes go up when there are damage in their cells. In our case, parameters related to liver and kidneys go down in most cases. This decrease may be due to the effect of insecticide on the cell function rather than inducing damage in cell wall to let more amounts of enzymes release in the blood.

Esterases are a major enzyme system associated in pyrethroids metabolic pathways (Yu, 2008). Insecticides such as organophosphates, carbamates, pyrethroids and some juvenoids, which contain ester linkages, are susceptible to hydrolysis. Esterases are hydrolases that split ester compounds by the addition of

water to yield an acid and an alcohol (Yu, 2008). General esterases in rats serum after different times of exposure to lambda-cyhalothrin were determined colorimetrically and electrophoretically. The activity of esterases were estimated using two general substrates, α- and β-NA, which showed a significant decrease in enzyme activity in some cases compared with control groups. Glutathion-S-transferase is a multifunctional enzymes group that performs glutathione conjugation for xenobiotics. This enzyme catalyzes the conjugation of reduced glutathione (GSH) with electrophilic substrates. Because of their broad substrate specificities, glutathione-S-transferases are responsible for the detoxification of numerous toxicants (Salinas & Wong, 1999 and Yu, 1996 and 2006). The activity of this enzyme significantly increased in both male and female rats treated with lambda-cyhalothrin for 60 days with 1/10 and 1/20 of LD50 and some other treatments in other periods. In contrast with these findings, Fetoui et al. (2010) indicated that GST was significantly decreased due to lambda-cyhalothrin exposure. They also indicate that the decrease in the activities of GST due to failure of antioxidant defense system to overcome the influx of reactive oxygen species induced by

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lambda-cyhalothrin exposure. According to the decrease in esterases activity and increase in GST activity in treated rats compared with control groups, they seemed to be associated with insecticide toxicity elimination in male and female rats. In cases that have been studied in more detail, resistance to insecticides has been attributed to increases in the amount of one or more GST enzymes. This attributed to either as a result of gene amplification or more commonly through increases in transcriptional rate, rather than qualitative changes in individual enzymes (Grant & Hammock, 1992 and Ranson et al., 2001). Conclusion

Exposure of male and female albino rats, R. norvegicus, to sub-lethal doses of lambda cyalothrin for more than 30 successive days have been lead to changes in some important biochemical parameters as well as general esterases and glutathione-S-transferases. Theses findings revealed that precautions must be taken in consideration when lambda-cyhalothrin applied in agriculture, veterinary and human health applications.

Acknowledgments Authors would like to thank

Kafr El-Zayat Company; Gharbia Governorate, Egypt for kindly

providing lambda-cyhalothrin. This work was supported, in part, by a grant from Zagazig University (Project #27F8 /P3).

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