Lipid peroxidation, oxidative stress and acetylcholinesterase in rat brain exposed to organophosphate and pyrethroid insecticides

Food and Chemical Toxicology 49 (2011) 1346–1352

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Lipid peroxidation, oxidative stress and acetylcholinesterase in rat brain exposed to organophosphate and pyrethroid insecticides Fatma M. El-Demerdash ⇑ Department of Environmental Studies, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt

a r t i c l e

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Article history: Received 3 December 2010 Accepted 14 March 2011 Available online 17 March 2011 Keywords: Insecticides Oxidative stress Enzymes Brain Rats

a b s t r a c t Oxidative stress by increased production of reactive oxygen species has been implicated in the toxicity of many pesticides. Therefore, the aim of the present study was to investigate the effect of a broad spectrum insecticide, composed of a mixture of organophosphate plus pyrethroids (fenitrothion 25%, lambda cyhalothrin 2.5% and piperonyl butoxide 6%), on antioxidant status and oxidative stress biomarkers in rat brain. Different insecticide concentrations (0, 0.1, 1, 10, 100 and 1000 mM) were incubated with brain homogenate at 37 °C for time intervals (0, 30, 60, 120, 180 and 240 min). Exposure to insecticide mixture resulted in a significant increase (p < 0.05) in thiobarbituric acid reactive substances (TBARS), which might be associated with decreased levels of reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and acetylcholinesterase activities and beside protein content in rat brain. However, a significant induction of lactate dehydrogenase (LDH) activities was observed. The response was concentration and time dependent. Results showed that the used insecticides had the propensity to cause significant oxidative damage in rat brain, which is associated with marked perturbations in antioxidant defense system in addition to antioxidant enzymes can be used as potential biomarkers of toxicity associated with pesticides exposure. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Humans are potentially exposed to pesticides either directly, as workers in green-houses and in agriculture, or indirectly, via food consumption. In addition, it is likely that a significant amount of these pesticides and their metabolites reach rivers and estuaries via run-off from farmland that are potentially toxic to wildlife (El-Shenawy, 2010). The pesticides featured in this study belong to tow different classes of insecticides, namely organophosphate (fenitrothion; FNT) and pyrethroids (lambda-cyhalothrin; LC). The FNT [O,O-dimethyl-O-(3-methyl-4-nitrophenyl) phosphorothioate] is a contact insecticide and selective acaricide; and also used as a vector control agent for malaria in public health programs. FNT is widely used against insect pests and mites on cereals, cotton, orchard fruits, rice and vegetables (Uygun et al., 2005). It is a contact-acting organophosphorus pesticide which inhibits acetyl cholinesterase activity, thus disrupting the nervous system (Sarikaya et al., 2004). Fenitrothion have been reported to exhibits low mammalian toxicity, biochemicals, morphological and functional alterations in animal tissues (Khan et al., 1990). ⇑ Address: University of Alexandria, Institute of Graduate Studies and Research, Department of Environmental Studies, 163, Horreya Avenue, P.O. Box. 832, Alexandria 21526, Egypt. Tel.: +20 34 29 50 07; fax: +20 34 28 57 92. E-mail address: [email protected] 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.03.018

The toxic effects of fenitrothion probably occur through the generation of reactive oxygen species (ROS) causing damage to various membranous components of the cell (Goel et al., 2005). Pyrethroid compounds are synthetic analogs and derivatives of natural pyrethrins. Synthetic pyrethroids are used as insecticides on a worldwide scale. They are increasingly applied in agricultural and household settings for the prevention and treatment of ectoparasites (Hossain et al., 2005) and for control of mosquitoes and fleas in kitchens and bedrooms (Llewllyn et al., 1996). These halogenated and lipophilic compounds are generally recognized as potent neurotoxicants, characterized by high insecticidal properties and low mammalian toxicity. Lambda-cyhalothrin is a third generation pyrethroid. It has been used to control a wide range of arthropod pests found on field and vegetable crops (Bostanian and Racette, 1997; El-Demerdash, 2007). Pyrethroids are more hydrophobic than other classes of insecticides (Michelangeli et al., 1990) and therefore their general site of action is biological membranes. Insecticide used in the present study is a mixture of organophosphorus insecticide, fenitrothion and pyrethroid insecticide, lambda-cyhalothrin. It is developed to control a wide variety of pests. These chemicals have been used indiscriminately in large amounts, and have also been largely involved in progressive pollution. Therefore, the present experiment was carried out to investigate the cytotoxicity of organophosphate insecticide (Fenitrothione; FNT) plus pyrethroid insecticide (Lambda-Cyhalothrin; LC) at different concentrations

F.M. El-Demerdash / Food and Chemical Toxicology 49 (2011) 1346–1352

and various incubation times on lipid peroxidation and antioxidant enzyme activities and acetylcholinesterase in brain homogenate of male rats. 2. Materials and methods

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2.9. Determination of catalase (CAT) activity in rat brain The enzyme catalase (CAT; EC 1.11.1.6) converts H2O2 into water. The CAT activity in brain supernatant was measured spectrophotometrically at 240 nm by calculating the rate of degradation of H2O2, the substrate of the enzyme (Aebi, 1984). One unit of CAT activity is defined as the amount of enzyme, which reduces 1 lmol of H2O2 per minute.

2.1. Chemicals Formulated concentrated broad spectrum insecticide (25% Fenitrothion; 2.5% Lambda-cyhalothrin; 6% piperonyl Butoxide) was purchased from Chema Industries Company, Egypt. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents used were of analytical grade.

2.10. Determination of Acetylcholinesterase (AChE) activity in rat brain Acetylcholinesterase (AChE; EC 3.1.1.7) activity was estimated in brain using acetylcholine iodide as a substrate according to the method of Ellman et al. (1961).

2.2. Animals and care

2.11. Determination of lactate dehydrogenase (LDH) activity in rat brain

Male Sprague–Dawley rats weighting 200–235 g were used in the present experiment. Animals were housed in cages at room temperature (25 ± 2 °C) with a relative humidity of 50–60% and on a 12 h light–darkness cycle. The animals had free access to commercial pellet diet and water ad libitum. The local committee approved the design of the experiments, and the protocol conforms to the guidelines of the National Institutes of Health (NIH).

Rat brain lactate dehydrogenase (LDH; EC 1.1.1.27) was determined by the method of Cabaud and Wroblewski (1958).

2.3. Tissue preparation After decapitation, brain was immediately removed; weighed and washed using chilled saline solution. Brain tissues were minced, cut into small pieces and then dried on a filter paper and homogenized (10% w/v), separately, in ice-cold 1.15% KCl-0.01 M sodium, potassium phosphate buffer (pH 7.4) in a Potter–Elvehjem type homogenizer. The homogenate was centrifuged at 18,000g for 20 min at 4 °C, and the resultant supernatant was used for the determination of different enzyme assays and TBARS and glutathione content. 2.4. Tissue treatment Rat brain homogenates were incubated with the tested insecticide at varying concentrations in the range of 0.1–1000 mM and incubated at 37 °C for 0, 30, 60, 120, 180 and 240 min in order to differentiate between the effect of various incubation times and different insecticide concentrations. After incubation, the mixtures were stored at 20 °C. All the in vitro experiments were repeated five times under the same conditions. 2.5. Determination of thiobarbituric acid reactive substances in rat brain According to the method of Esterbauer and Cheeseman (1990), the extent of lipid peroxidation in terms of thiobarbituric acid reactive substances (TBARS) formation was measured. Brain supernatant was mixed with 1 ml TCA (20%), 2 ml TBA (0.67%) and heated for 1 h at 100 °C. After cooling, the precipitate was removed by centrifugation. The absorbance of the sample was measured at 535 nm using a blank containing all the reagents except the sample. As 99% TBARS are malondialdehyde (MDA), so TBARS concentrations of the samples were calculated using the extinction co-efficient of MDA, which is 1.56  105 M1 cm1. 2.6. Estimation of glutathione content in rat brain GSH content was quantified using Ellman’s reagent (Ellman, 1959). The assay mixture consisted of 0.2 M phosphate buffer (pH 8.0), 0.01% 5,50 -dithiobis-2-nitro benzoic acid (DTNB) and the rat brain homogenate. The reaction was monitored at 412 nm and the amount of GSH was expressed as mmol.

2.12. Protein estimation The protein content of the tissue homogenates mentioned earlier was determined by following the method described by Lowry et al. (1951) using bovine serum albumin as a standard.

2.13. Statistical analysis Data were analyzed according to Steel and Torrie (1981). Statistical significance of the difference in values of control and treated samples was calculated by (F) test at 5% significance level. Data of the present study were statistically analyzed by using Duncan’s Multiple Range Test (SAS, 1986).

3. Results 3.1. Lipid peroxidation and glutathione content Data of TBARS measured in rat brain is presented in Table 1. A significant increase (P < 0.05) in TBARS concentrations was evident in rat brain exposed to FNT plus LC insecticide. Effect of FNT plus LC insecticide on GSH content in rat brain homogenate was presented in Table 2. A significant concentration- and time-dependent decrease was observed in GSH content exposed to FNT plus LC insecticide (P < 0.05). 3.2. Antioxidant enzyme activities Data concerning brain antioxidant enzyme activities (GST, SOD and CAT) are presented in Tables 3–5. The enzyme activities were measured in rat brain exposed to FNT plus LC insecticide. A significant reduction in the activity of the measured antioxidant enzymes was observed at all concentrations except the lowest one (0.1 mM). A significant concentration- and time-dependent decrease was observed in the activity of GST, SOD and CAT exposed to tested insecticide (P < 0.05).

2.7. Determination of glutathione S-transferase(GST) activity in rat brain Glutathione S-transferase (GST; EC 2.5.1.18) catalyzes the conjugation reaction with glutathione in the first step of mercapturic acid synthesis. The activity of GST was measured according to the method of Habig et al. (1974). P-nitrobenzylchloride was used as substrate. The absorbance was measured spectrophotometrically at 310 nm using UV-Double Beam spectrophotometer. One unit of GST activity is defined as 1 lmol product formation per minute. 2.8. Determination of superoxide dismutase (SOD) activity in rat brain Superoxide dismutase (SOD; EC 1.15.1.1) was assayed according to Misra and Fridovich (1972). The assay procedure involves the inhibition of epinephrine auto-oxidation in an alkaline medium (pH 10.2) to adrenochrome, which is markedly inhibited by the presence of SOD. Epinephrine was added to the assay mixture, containing tissue supernatant and the change in extinction coefficient was followed at 480 nm in a Spectrophotometer. The unit of enzyme activity is defined as the enzyme required for 50% inhibition of auto-oxidation of epinephrine.

3.3. Lactate dehydrogenase, acetylcholinesterase activities and protein content Results indicated that LDH activity was significantly (P < 0.05) increased in rat brain treated with different insecticide concentrations for various incubation times (Table 6) as compared to control. The effect was concentration and time dependent at most. On the other hand, significant inhibition of AChE activity (P < 0.05) in brain homogenate was observed with increasing insecticide concentration and prolonging exposure time after in vitro treatment of FNT + LC (Table 7) as compared with control. Also, incubation of rat brain homogenate with different concentrations of insecticide resulted in a significant decrease in the protein content (Table 8) in concentration and time dependent manner.

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Table 1 In vitro effect of FNT and LC mixture on rat brain TBARS levels (nmol/g tissue) following incubation for different concentrations and time intervals. Time (min) Insecticide Conc. (mM)

0 min

30 min

60 min

120 min

180 min

240 min

0 0.1 1 10 100 1000

23.3 ± 0.38a,A 22.9 ± 0.49a,A 22.8 ± 0.47a,A 23.2 ± 0.39a,A 23.3 ± 0.73a,A 23.4 ± 0.63a,A

22.7 ± 0.73a,A 24.5 ± 0.58ab,AB 25.4 ± 0.91b,BC 25.9 ± 0.84b,BC 26.3 ± 0.82b,BC 27.0 ± 0.78b,C

22.9 ± 0.48a,A 25.4 ± 0.49bc,B 27.2 ± 0.58bc,BCD 28.0 ± 0.67c,CD 28.8 ± 0.69c,CD 29.3 ± 0.76bc,D

23.0 ± 0.39a,A 27.4 ± 0.53cd,B 28.9 ± 0.73cd,BC 29.1 ± 0.92cd,BC 29.9 ± 0.79cd,BC 30.1 ± 0.68c,C

23.1 ± 0.56a,A 28.2 ± 0.61d,B 29.7 ± 0.58cd,BC 30.7 ± 0.78d,C 31.1 ± 0.99d,C 31.8 ± 1.02cd,C

22.9 ± 0.59a,A 28.9 ± 0.84d,B 30.1 ± 0.94d,C 31.3 ± 0.85d,CD 32.4 ± 0.94d,CD 32.9 ± 0.99d,D

Values are expressed as mean ± standard error. abcd TBARS levels in rows with different letters were significantly differ (P < 0.05). ABCD TBARS levels in columns with different letters were significantly different (P < 0.05).

Table 2 In vitro effect of FNT and LC mixture on rat brain GSH levels (mmol) following incubation for different concentrations and time intervals. Time (min) Insecticide Conc. (mM)

0 min

30 min

60 min

120 min

180 min

240 min

0 0.1 1 10 100 1000

1.31 ± 0.031a,A 1.35 ± 0.029a,A 1.37 ± 0.032a,A 1.38 ± 0.039a,A 1.35 ± 0.290a,A 1.39 ± 0.033a,A

1.34 ± 0.038a,A 1.30 ± 0.023ab,AB 1.24 ± 0.020ab,AB 1.19 ± 0.019b,BC 1.14 ± 0.024b,C 1.09 ± 0.012b,C

1.38 ± 0.033a,A 1.33 ± 0.029ab,A 1.26 ± 0.043abc,AB 1.16 ± 0.019bc,BC 1.13 ± 0.014b,C 1.10 ± 0.009bc,C

1.39 ± 0.046a,A 1.30 ± 0.031ab,AB 1.22 ± 0.021bc,BC 1.13 ± 0.042bc,CD 1.10 ± 0.025bc,D 1.07 ± 0.020bc,D

1.36 ± 0.046a,A 1.23 ± 0.029ab,B 1.14 ± 0.023bc,BC 1.07 ± 0.030c,C 1.04 ± 0.013bc,C 0.99 ± 0.025c,C

1.38 ± 0.031a,A 1.21 ± 0.022b,B 1.13 ± 0.013c,BC 1.07 ± 0.040c,CD 1.01 ± 0.018c,DE 0.95 ± 0.019c,E

Values are expressed as mean ± standard error. abc GSH levels in rows with different letters is significantly differ (P < 0.05). ABCDE GSH levels in columns with different letters is significantly different (P < 0.05).

Table 3 In vitro effect of FNT and LC mixture on rat brain GST activities (lmol/h/mg protein) following incubation for different concentrations and time intervals. Time (min) Insecticide conc. (mM)

0 min

30 min

60 min

120 min

180 min

240 min

0 0.1 1 10 100 1000

0.47 ± 0.011a,A 0.45 ± 0.010a,A 0.46 ± 0.018a,A 0.44 ± 0.016a,A 0.45 ± 0.010a,A 0.46 ± 0.012a,A

0.45 ± 0.020a,A 0.44 ± 0.010ab,AB 0.42 ± 0.013ab,AB 0.41 ± 0.007ab,AB 0.38 ± 0.005b,BC 0.37 ± 0.011b,C

0.44 ± 0.010a,A 0.42 ± 0.009ab,AB 0.39 ± 0.009bc,BC 0.38 ± 0.007bc,C 0.36 ± 0.011bc,C 0.35 ± 0.009bc,C

0.46 ± 0.015a,A 0.41 ± 0.009abc,B 0.40 ± 0.013bc,B 0.38 ± 0.005bc,BC 0.36 ± 0.009bc,C 0.35 ± 0.015bc,C

0.45 ± 0.016a,A 0.40 ± 0.007bc,B 0.38 ± 0.010c,BC 0.36 ± 0.007c,CD 0.34 ± 0.013c,D 0.33 ± 0.007cd,D

0.44 ± 0.009a,A 0.38 ± 0.009c,B 0.36 ± 0.008c,BC 0.35 ± 0.008c,BC 0.33 ± 0.008c,CD 0.31 ± 0.010d,D

Values are expressed as mean ± standard error. abcd GST activity in rows with different letters is significantly differ (P < 0.05). ABCD GST activity in columns with different letters is significantly different (P < 0.05).

Table 4 In vitro effect of FNT and LC mixture on rat brain SOD activities (Units/mg protein) following incubation for different concentrations and time intervals. Time (min) Insecticide conc. (mM)

0 min

30 min

60 min

120 min

180 min

240 min

0 0.1 1 10 100 1000

22.7 ± 0.78a,A 22.5 ± 0.84a,A 22.5 ± 0.76a,A 21.9 ± 0.69a,A 22.8 ± 0.89a,A 22.6 ± 0.64a,A

22.6 ± 0.50a,A 22.1 ± 0.73ab,AB 21.1 ± 0.56ab,ABC 20.4 ± 0.51ab,BC 19.4 ± 0.51b,CD 18.4 ± 0.51b,D

22.6 ± 0.68a,A 21.5 ± 0.56abc,AB 20.7 ± 0.60ab,ABC 19.6 ± 0.40bc,BCD 19.0 ± 0.45bc,CD 18.0 ± 0.71bc,D

22.3 ± 0.68a,A 20.6 ± 0.63abc,AB 20.2 ± 0.39b,AB 19.0 ± 0.30bc,BC 18.0 ± 0.55bcd,CD 17.0 ± 0.71bcd,D

22.4 ± 0.93a,A 20.2 ± 0.49bc,B 19.8 ± 0.75b,B 18.4 ± 0.24c,BC 17.4 ± 0.51cd,CD 16.6 ± 0.51cd,D

22.4 ± 0.51a,A 19.8 ± 0.68c,B 19.6 ± 0.66b,B 18.2 ± 0.58c,BC 16.8 ± 0.66d,CD 16.0 ± 0.71d,D

Values are expressed as mean ± standard error. abcd SOD activity in rows with different letters is significantly differ (P < 0.05). ABCD SOD activity in columns with different letters is significantly different (P < 0.05).

4. Discussion Extensive application of pesticides is usually accompanied with serious problems of pollution and health hazards. It is established that many pesticides, in common use, can produce some toxic and adverse effects on the liver, kidney and other biological systems when tested on various types of experimental animals through their

mode of action or by production of free radicals that damage all cell components (Khan, 2006). Pesticides act as pro-oxidants and elicit effects in multiple organs (Limon-Pacheco and Gonsebatt, 2009). Organophosphorus and pyrethroid group of pesticides are the most commonly used in agriculture today. Both are efficiently absorbed and rapidly redistributed to various organs as part of their disposal mechanism. Organophosphate insecticides have been shown to

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F.M. El-Demerdash / Food and Chemical Toxicology 49 (2011) 1346–1352 Table 5 In vitro effect of FNT and LC mixture on rat brain CAT activities (Units/mg protein) following incubation for different concentrations and time intervals. Time (min) Insecticide conc. (mM)

0 min

30 min

60 min

120 min

180 min

240 min

0 0.1 1 10 100 1000

25.5 ± 1.02a,A 25.7 ± 1.00a,A 25.4 ± 0.94a,A 25.6 ± 0.83a,A 25.7 ± 0.92a,A 25.8 ± 0.89a,A

25.2 ± 0.86a,A 23.9 ± 0.84ab,AB 22.9 ± 0.87b,ABC 22.1 ± 0.74b,BCD 21.4 ± 0.68b,CD 20.7 ± 0.76b,D

25.1 ± 0.79a,A 24.0 ± 1.29ab,AB 22.5 ± 1.05b,BC 21.4 ± 0.87b,CD 20.5 ± 0.83bc,CD 19.6 ± 0.75bc,D

25.8 ± 1.02a,A 23.8 ± 0.66abc,AB 22.6 ± 0.75b,BC 21.4 ± 0.68b,CD 20.2 ± 0.87bc,D 19.4 ± 0.60bc,D

25.2 ± 1.07a,A 22.0 ± 0.92bc,B 21.5 ± 0.88b,BC 20.3 ± 0.93c,CD 19.2 ± 0.67cd,D 18.2 ± 1.02a,b

25.5 ± 0.98a,A 21.7 ± 0.97c,B 21.1 ± 0.63b,B 20.4 ± 0.87b,BC 19.0 ± 0.55c,C 17.6 ± 1.03d,C

Values are expressed as mean ± standard error. a CAT activity in rows with different letters is significantly differ (P < 0.05). b CAT activity in columns with different letters is significantly different (P < 0.05).

Table 6 In vitro effect of FNT and LC mixture on rat brain LDH (IU/mg) following incubation for different concentrations and time intervals*. Time (min) Conc. (mM)

0 min

30 min

60 min

120 min

180 min

240 min

0 0.1 1 10 100 1000

1009 ± 39a,A 1012 ± 41a,A 1005 ± 38a,A 999 ± 32a,A 1001 ± 35a,A 996 ± 41a,A

996 ± 37a,A 1040 ± 44a,AB 1061 ± 29a,AB 1107 ± 47b,B 1146 ± 30B,b 1131 ± 51b,B

1006 ± 32a,A 1079 ± 35ab,AB 1094 ± 40ab,ABC 1130 ± 42b,BCD 1194 ± 51B,cd 1213 ± 43bc,D

988 ± 36a,A 1096 ± 45ab,B 1113 ± 36b,BC 1147 ± 49b,BC 1193 ± 39B,bc 1215 ± 42bc,C

1017 ± 35a,A 1149 ± 38b,B 1191 ± 46b,B 1194 ± 33b,B 1256 ± 47B,bc 1315 ± 37c,C

995 ± 34a,A 1132 ± 61b,B 1185 ± 35b,B 1191 ± 35b,BC 1229 ± 53B,bc 1302±35c,C

Values are expressed as mean ± standard error. * IU/mg: international unit, the amount of the enzyme that under defined assay conditions will catalyze one mol of substrate/min/mg protein. abc LDH activity in rows with different letters is significantly differ (P < 0.05). ABCD LDH activity in columns with different letters is significantly different (P < 0.05).

Table 7 In vitro effect of FNT and LC mixture on rat brain AChE activities (lmole/min/mg protein) following incubation for different concentrations and time intervals. Time (min) Conc. (mM)

0 min

30 min

60 min

120 min

180 min

240 min

0 0.1 1 10 100 1000

13.63 ± 0.24a,A 13.43 ± 0.21a,A 13.47 ± 0.31a,A 13.23 ± 0.29a,A 13.09 ± 0.28a,A 13.00 ± 0.19a,A

13.59 ± 0.35a,A 12.81 ± 0.13a,AB 11.81 ± 0.30b,BC 11.24 ± 0.33b,BC 10.89 ± 0.16b,CD 9.86 ± 0.15b,E

13.01 ± 0.13a,A 12.01 ± 0.34ab,A 10.73 ± 0.14c,B 10.40 ± 0.24bc,B 10.11 ± 0.32bc,B 9.03 ± 0.14bc,C

13.42 ± 0.24a,A 12.14 ± 0.19ab,B 10.67 ± 0.52c,C 10.32 ± 0.47bc,C 10.26 ± 0.44bc,C 8.88 ± 0.17bc,D

13.27 ± 0.43a,A 11.90 ± 0.31ab,B 10.12 ± 0.18c,C 9.87 ± 0.38cd,C 9.73 ± 0.23c,C 8.23 ± 0.23cd,D

13.21 ± 0.29a,A 11.62 ± 0.21bc,B 9.65 ± 0.22c,C 9.53 ± 0.22cd,C 9.24 ± 0.11c,C 7.44 ± 0.24e,D

Values are expressed as mean ± standard error. abcde AChE activity in rows with different letters significantly differ (P < 0.05). ABCDE AChE activity in columns with different letters significantly differ (P < 0.05).

Table 8 In vitro effect of FNT and LC mixture on rat brain protein content (mg/g tissue) following incubation for different concentrations and time intervals. Time (min) Conc. (mM)

0 min

30 min

60 min

120 min

180 min

240 min

0 0.1 1 10 100 1000

51.4 ± 1.55a,A 52.3 ± 1.31a,A 51.5 ± 1.28a,A 51.9 ± 1.32a,A 51.4 ± 1.35a,A 51.7 ± 2.99a,A

51.6 ± 1.33a,A 49.4 ± 1.12ab,AB 46.6 ± 1.25b,BC 44.6 ± 1.80b,BC 42.4 ± 0.66b,C 40.6 ± 0.97b,C

51.4 ± 1.29a,A 47.9 ± 1.61ab,B 44.4 ± 1.67bc,BC 42.1 ± 1.02bc,CD 40.9 ± 1.04bc,CD 39.1 ± 0.74bc,D

52.5 ± 1.37a,A 47.9 ± 0.77ab,B 43.8 ± 1.34bc,BC 41.7 ± 1.75bc,CD 40.1 ± 0.87bc,CD 38.2 ± 0.85bc,D

52.2 ± 0.97a,A 46.2 ± 0.76b,B 41.4 ± 1.59bc,C 39.8 ± 0.82cd,CD 37.8 ± 1.10cd,CD 36.8 ± 0.97c,D

52.4 ± 0.93a,A 45.9 ± 1.56b,B 40.6 ± 1.55c,C 37.6 ± 1.61d,CD 36.9 ± 1.10d,CD 35.9 ± 1.36c,D

Values are expressed as mean ± standard error. abcd protein contents in rows with different letters significantly differ (P < 0.05). ABCD Protein contents in columns with different letters significantly differ (P < 0.05).

interfere with membrane dependent processes, including nerve conductance and plasma membrane and organelle enzyme activities (Karaoz et al., 2002). Oxidative damage primarily occurs through production of reactive oxygen species and can damage lipids, proteins and DNA. Therefore, oxidative damage may contribute to loss of enzymatic activity and structural integrity of enzymes and acti-

vate inflammatory processes (Ozyurt et al., 2004). The pyrethroid group of synthetic pesticides also seems to exert their tissue damaging effect by altering the oxidative stress in target organs (Kale et al., 1999). The extent of LPO is determined by the balance between the production of oxidants and the removal and scavenging of those

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oxidants by antioxidants (Halliwell and Gutteridge, 1999). TBARS is a major oxidation product of peroxidized polyunsaturated fatty acids, and increased TBARS content is an important indicator of lipid peroxidation (Celik and Suzek, 2009). Increased TBARS level in the brain of rats treated with different concentrations of FNT plus LC at various times (Table 1) is in agreement with the findings of Nasuti et al. (2003), Prasanthi et al. (2005) and El-Demerdash (2007) who reported that oxidative damage induced by pyrethroids may be due to their lipophilicity, whereby they could penetrate the cell membrane easily. pyrethroids indirectly generates various radicals as superoxide radical, nitrogen species such as peroxynitrite, nitric oxide and hydroxyl radical thus causing damage consistent with oxidative stress (World Health Organization, 1990; Kale et al., 1999). These radicals attack the cell membrane and lead to destabilization and disintegration of cell membrane as a result of lipid peroxidation (Stajn et al., 1997). In addition, organophosphate causes increase of lipid peroxidation through its interference with membrane dependent processes (Kalender et al., 2007, 2010; Elhalwagy et al., 2008; Uzun et al., 2010). The second line of defense includes the non-enzymatic radical scavenger GSH, which scavenges residual free radicals resulting from oxidative metabolism and escaping decomposition by the antioxidant enzymes (Leve De and Kaplowitz, 1991). During the metabolic action of GSH, its sulfhydryl group becomes oxidized resulting with the formation of the corresponding disulfide compound, GSSG (oxidized form) (Meister and Anderson, 1983). A significant depletion of GSH was noted in the present study in concentration and time dependent manner (Table 2). The decrease in GSH levels could be due to the presence of free radicals produced by insecticides. These effects have been previously observed by other authors in vitro and in vivo (Banerjee et al., 1999; Maran et al., 2009; Thompson et al., 2002). In addition, GSH also participates in the detoxification of xenobiotics as a substrate for the enzyme GST. Glutathione and other thiol containing proteins play a crucial key role in cellular defense against pesticides toxicity (Halliwell and Gutteridge, 1999). Recently, Fetoui et al. (2009) have demonstrated that the depletion of intracellular sulfhydryl groups (SH groups) by insecticides is the prerequisite for ROS generation. In agreement with this finding, the significant decrease of GSH content in FNT plus LC-treated organs could lead to increased susceptibility to free radical damage. GST are detoxifying enzymes that catalyze the conjugation of a variety of electrophilic substrates to the thiol group of GSH, producing less toxic forms ; and also reduces lipid peroxides (Mosialou et al., 1993; Mansour and Mossa, 2009). The decreased GSH contents (Table 2) in FNT plus LC treated rat tissues may probably be due to the decrease in the activity of GST (Table 3). Similarly, GST inhibition has been documented to occur under other oxidative stress conditions (Goel et al., 2005; Mansour and Mossa, 2009). The marked inhibitions in GST activity in FNT plus LC treated tissue indicate insufficient conjugation of electrophiles and detoxication of these species. The reduction in GST activity was concomitant to decrease in glutathione content in different organs. GST is one of enzyme system involved in the detoxification of organophosphorus insecticides to non-toxic products or by rapidly binding and very slowly turning over the insecticide (Ranjbar et al., 2005). In consistent with the present results, Kale et al. (1999) and El-Demerdash (2007) reported a significant decrease in GST activity after in vivo and in vitro treatment of cypermethrin and lambda-cyhalothrin. The present study demonstrates that GST is a part of adaptive response of rat organ cells to stress after FNT plus LC insecticide exposure. This can be understood in view of the fact that organophosphorus and pyrethroid insecticides consume GSH through a detoxification reaction and/or that GST catalyzes this reaction between GSH and xenobiotics, regulating possible harm (Mosialou et al., 1993).

SOD and CAT are the most important defense mechanisms against toxic effects of oxygen metabolism. SOD catalyzes the conversion of superoxide radicals to hydrogen peroxide, while CAT converts hydrogen peroxide into water. These antioxidant enzymes can, therefore, alleviate the toxic effects of ROS (Mansour and Mossa, 2009). SOD and CAT activities were significantly reduced by different concentrations of FNT plus LC in treated tissues (Tables 4 and 5). In agreement with our results, Prasanthi et al. (2005) found that fenvalerate-induced oxidative damage in terms of alterations in the enzymatic defense system in liver and erythrocytes. Changes in phospholipids, fatty acids and in the cholesterol content modulate the membrane fluidity, which influences the enzymatic activity, and the functionality of receptors and channels present at the plasma membrane level (Nasuti et al., 2003). Since FNT plus LC insecticides produced excessive ROS (Table 1) either directly or indirectly, the counter balancing effect of the antioxidant enzymes is lost (Banerjee et al., 1999; Seth et al., 2001). The present results are coincident with Abdollahi et al. (2004) who reported that the decrease in the SOD and GSH-Px activities and increase in LPO could explain the induction of free radicals in chlorpyrifos-treated rats. The decline of SOD activity, in our study, supported earlier findings (Sinha et al., 2006) which demonstrated that rats exposed to pyrethroids showed a decrease in brain SOD activity. The decrease in CAT activity could be due to the flux of superoxide radicals (Kono and Fridovich, 1982). In our previous investigation involving lambda-cyhalothrin a similar observation was made (El-Demerdash, 2007) where activities of antioxidant enzymes were decreased in rabbit erythrocytes. Thus, the inhibition of enzymes involved in free radical removal led to the accumulation of H2O2, which promoted lipid peroxidation and modulation of DNA, altered gene expression and cell death (Calviello et al., 2006). The enzyme LDH can be used as an indicator for cellular damage, clinical practice, and cytotoxicity of pollutants. LDH activity indicates the switching over of anaerobic glycolysis to aerobic respiration. The changes in the dehydrogenase activity in pesticide-treated homogenate (Table 6) may be due to severe cellular damage, leading to increased release of dehydrogenase that impaired carbohydrate and protein metabolism (Sivakumari et al., 1997). The elevation of lactate also indicated metabolic disorders and a clear response against energy depletion. Sancho et al. (1998) observed the same response when European eels were exposed to fenitrothion. AChE activity, a standard biomarker of organophosphate pesticide. AChE activities were significantly reduced by different concentrations of tested insecticide (FNT + LC) at various time of incubation with brain homogenate (Table 7). In the present study, the degree of enzyme inhibition followed a positive correlation with the different insecticide concentrations and time of exposure. In agreement with the present results, Okahashi et al. (2005) reported that the inhibition in AChE activity occurs when animals intoxicated with different doses of fenitrothion for different periods. Inhibition of AChE, an enzyme that restricts the activity of acetylcholine (ACh) in space and time, causes an increase in ACh content at sites of cholinergic transmission in the body. The inhibition of AChE is the most plausible explanation for much of the symptomatology following OP intoxication (Yamashita et al., 1997). Also, Feng et al., 2008, reported that treatment with trichlorfon an organophosphate insecticide caused a significant concentration-dependent and time-related inhibition of AChE activity at all treatment concentrations and times since trichlorfon is a cholinesterase inhibitor. Actually, the use of AChE inhibition as biomarker to assess the toxic effects of organophosphates has been studied for a wide range of species and many different xenobiotics and is a well accepted index of organophosphates toxicity both in vivo and in vitro (Sanchez-Hernandez and Walker, 2000).

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Pyrethroids have been demonstrated to cause a decrease in AChE erythrocytes and brain of living organisms (El-Demerdash, 2007; Kale et al., 1999). The decreased AChE might be referred to the increase in lipid peroxidation (Table 7). The inhibition of AChE activity decreases the cellular metabolism, induces deformities of cell membrane, and disturbs metabolic and nervous activity (Suresh et al., 1992). Also, the decrease in AChE activity could lead to ionic refluxes and differential membrane permeability (Tolosa et al., 1996). Shaw and Panigrahi (1990) have also suggested inactivation of AChE enzyme as a result of the occupation of its active sites by pollutants. In agreement with the present results (Table 8), Odland et al. (1994) reported that the rate of protein synthesis was decreased in a concentration-dependent manner in response to insecticide exposure. The decrease in protein contents in insecticide-exposed tissue indicates excessive break down of tissue proteins (Chatterjea and Shinde, 2002). 5. Conclusions In conclusion, organophosphate plus pyrethroid insecticides had cytotoxic effects on rat brain. It has the capability to induce oxidative damage as evidenced in terms of increased lipid peroxidation and perturbations in various antioxidant enzymes, and the effect was pronounced with the high doses and long time exposure. The results indicated a risk of organ damage during exposure to a combination of insecticides. Conflict of Interest None declared. References Abdollahi, M., Ranjbar, A., Shadnia, S., Nikfar, S., Rezaie, A., 2004. Pesticides and oxidative stress: a review. Med. Sci. Monitor 10, RA141–RA147. Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. 3rd Ed, Lippincott-Raven publishers, Philadelphia. Banerjee, B.D., Seth, V., Bhattacharya, A., Pasha, S.T., Chakraborty, A.K., 1999. Biochemical effects of some pesticides on lipid peroxidation and free-radical scavengers. Toxicol. Lett. 107, 33–47. Bostanian, N.J., Racette, G., 1997. Residual toxicity of lambda-cyhalothrin on apple foliage to Amblyseius fallacis and the tarnished plant bug, Lygus lineolaris. Phytoparasitica 25, 193–198. Cabaud, P.C., Wroblewski, F., 1958. Calorimetric measurement of lactate dehydrogenase activity of body fluids. J. Clin. Pathol. 30, 234–236. Calviello, G., Piccioni, E., Boninsegna, A., Tedesco, B., Maggiano, N., Serini, S., Wolf, F.I., Palozza, P., 2006. DNA damage and apoptosis induction by the pesticide Mancozeb in rat cells: Involvement of the oxidative mechanism. Toxicol. Appl. Pharmacol. 211, 87–96. Celik, I., Suzek, H., 2009. Effects of subacute exposure of dichlorvos at sublethal dosages on erythrocyte and tissue antioxidant defense systems and lipid peroxidation in rats. Ecotoxicol. Environ. Saf. 72, 905–908. Chatterjea, M.N., Shinde, R., 2002. Text Book of Medical Biochemistry, 5th ed. Jaypee Brothers, Medical Publishers Ltd., New Delhi. p. 317. El-Demerdash, F.M., 2007. Lambda-cyhalothrin-induced changes in oxidative stress biomarkers in rabbit erythrocytes and alleviation effect of some antioxidants. Toxicol. In Vitro 21, 392–397. Elhalwagy, M.E.A., Darwish, E.M., Zaher, N.S., 2008. Prophylactic effect of green tea polyphenols against liver and kidney injury induced by fenitrothion insecticide Pest. Biochem. Physiol. 91, 81–89. Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77. Ellman, G.L., Courtney, K.D., Anders, V.J.R., Featherstone, R.M., 1961. A new rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 7, 88–95. El-Shenawy, N.S., 2010. Effects of insecticides fenitrothion, endosulfan and abamectin on antioxidant parameters of isolated rat hepatocytes. Toxicol. In Vitro 24, 1148–1157. Esterbauer, H., Cheeseman, K.H., 1990. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol. 186, 407–421. Feng, T., Li, Z.B., Guo, X.Q., Guo, J.P., 2008. Effects of trichlorfon and sodium dodecyl sulphate on antioxidant defense system and acetylcholinesterase of Tilapia nilotica in vitro. Pest. Biochem. Physiol. 92, 107–113.

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