Redox imbalance in rat tissues exposed with organophosphate pesticides and therapeutic potential of antioxidant vitamins

Ecotoxicology and Environmental Safety 75 (2012) 230–241

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Redox imbalance in rat tissues exposed with organophosphate pesticides and therapeutic potential of antioxidant vitamins Anupama Ojha, Nalini Srivastava n School of Studies in Biochemistry, Jiwaji University, Gwalior 474011, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 June 2011 Received in revised form 30 July 2011 Accepted 8 August 2011 Available online 23 August 2011

Organophosphate pesticides are among the most widely used synthetic chemicals for controlling domestic and agricultural pests. Present study was aimed to evaluate the potential of chlorpyrifos, parathion and malathion, to disturb glutathione homeostasis in rat tissues and to find out whether the pre-feeding of antioxidant vitamins has some ameliorating effect on the pesticide-induced alterations. The results showed that these pesticides, alone or in combination, caused decrease in the levels of GSH and the corresponding increase in the levels of GSSG, decreasing the GSH/GSSG ratio. The results also showed NADPH/NADP þ and NADH/NAD þ ratios were also decreased in the rat tissues on pesticide exposure. These pesticides, alone or in combination, caused increase in the activities of glutathione reductase and glucose-6-phosphate dehydrogenase in all the rat tissues studied. The findings show that these pesticides generate oxidative stress and prior feeding of mixture of antioxidant vitamins tend to reduce the toxicities of these pesticides. & 2011 Elsevier Inc. All rights reserved.

Keywords: Chlorpyrifos Methyl parathion Malathion Redox status Glutathione Glutathione recycling system Glutathione reductase Antioxidant vitamins

1. Introduction Pesticides are heterogeneous group of chemicals developed to control a variety of pests. Organophosphate (OP) pesticides are among the most widely used synthetic chemicals used for agricultural and domestic pest control. Nowadays, the extensive use of OP insecticides in agriculture and public health results in environmental pollution and a large number of acute and chronic poisoning events. For this reason, there is a growing public concern about the accumulation of these insecticides in food products and water supply. The primary mode of action of these insecticides is irreversible inhibition of acetylcholinesterase (AChE) that hydrolyzes acetylcholine in cholinergic synapses and in neuromuscular junctions where this enzyme plays a key role in cell to cell communication. The resulting accumulation of acetylcholine in synapses overstimulates muscarinic and nicotinic receptors (Kwonq, 2002; Milatovic et al., 2006). Besides being potent anticholinesterase compounds, OPs elicit number of other effects including generation of oxidative stress by disturbing the activities of antioxidant enzymes and causing enhancement of lipid peroxidation (Verma et al., 2007; Soltaninejad and Abdollahi, 2009; Lukaszewicz-Hussain, 2010).

n

Corresponding author. Fax: þ91 751 2340245. E-mail addresses: [email protected], [email protected] (N. Srivastava). 0147-6513/$ - see front matter & 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2011.08.013

Xenobiotics comprise an important source of reactive oxygen species (ROS), which are produced in cells during normal metabolic processes involving oxygen. However, the levels of ROS are significantly increased on exposure to different environmental toxins produced from the industry, agriculture, tobacco smoke, or pollution accidents. The cells are equipped with both the enzymatic and nonenzymatic antioxidants for combating oxidative stress, which may be either due to increased production of ROS or impaired antioxidant defense or both. The primary defense is offered by antioxidant enzymes, namely, catalase, superoxide dismutase and glutathione peroxidase. The activities of these enzymes have been shown to be significantly decreased on exposure with OP pesticides (Verma et al., 2007; Lukaszewicz-Hussain, 2008; Ojha et al., 2011). Antioxidants belonging to the second line of defense include glutathione (GSH), vitamin C, vitamin E and b carotene (Frei, 1994; Irshad and Chaudhri, 2002). Cysteinyl residue of glutathione offers a nucleophilic thiol, which is important in the detoxification of electrophilic metabolites and metabolically produced oxidizing agents. Glutathione plays important roles in antioxidant defense, nutrient metabolism and regulation of cellular events including gene expression, DNA and protein synthesis, cell proliferation and apoptosis, signal transduction, cytokine production and immune response (Rana et al., 2002). Glutathione deficiency contributes to oxidative stress, which plays a key role in aging and the pathogenesis of many diseases including seizures, Alzheimer’s disease, Parkinson’s disease, liver disease, cystic fibrosis, sickle cell anemia, AIDS, cancer, heart attack, stroke and diabetes (Rana et al., 2002). GSH is also a substrate of enzymes, glutathione peroxidase and

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glutathione-S-transferase. A cell contains many biological redox couples, such as NAD(P) þ /NAD(P)H, GSSG/GSH, cystine/cysteine and TrxSS/Trx(SH)2, which allow the cell to maintain redox homeostasis. GSH/GSSG is the main redox buffer of the cells and is found in all the cellular compartments. GSH/GSSG ratio is a very important indicator of redox status of the cell. GSH is converted to GSSG after imparting its antioxidant role. To regenerate GSH from GSSG, the cells utilize NADPH and the enzyme glutathione reductase (http://en.wikipedia.org/wiki/Glutathione; Wu et al., 2004). The first reaction of pentose phosphate pathway catalyzed by glucse-6phosphate dehydrogenase (G6PDH) is the main site of NADPH production. Alteration in the activity of this enzyme affects the rate of generation of NADPH and thus affecting the redox status of the cell. Redox status has been proven to be an important tool in the toxicological evaluation, mostly providing the cellular and biochemical mechanisms of toxicity of chemicals and drugs. The redox status of the cells and tissues is an important indicator of oxidative stress. Oxidative stress reduces glutathione pool and decreases the GSH/GSSG ratio (Pompella et al., 2003; Zasadowski et al., 2004). The GSH/GSSG ratio is used to evaluate oxidative stress status in biological systems, and alterations in this ratio have been demonstrated in aging, cancer, HIV replication, and cardiovascular diseases (Hernanz et al., 2000; Lang et al., 2000). ROS-mediated toxicity by oxidation of GSH to GSSG in response to pesticide exposure has been demonstrated (Dorval and Hontela, 2003; Verma et al., 2009; Ajiboye, 2010; Singh et al., 2011). Chlorpyrifos (CPF, O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate) is a widely used, broad spectrum, OP insecticide. Recent concerns about the health effects of CPF have led to its ban on domestic and agricultural applications (EPA, 2000), yet it continues to be one of the most commonly used OP insecticides. Methyl parathion (MPT, O,O-dimethyl O-4-nitrophenyl phosphorothioate) on the other hand, is a highly toxic, restricted-use OP insecticide (ATSDR, 2001). MPT has been banned for indoor use as well as on several food crops; however, illegal application of MPT for several years inside homes in the US led to great concern about its health hazards (Rubin et al., 2002). Malathion (MLT, S-1,2-dicarbethoxy ethyl O,O-dimethylphosphorodithioate) is one of the most widely used OP insecticides in agriculture and public health programs (Altuntas et al., 2002). CPF, MPT and MLT are phosphorothioate insecticides that undergo cytochrome P-450-mediated bioactivation to their respective oxons (chlorpyrifos oxon, methyl paraoxon and malaoxon), which are more potent inhibitors of AChE. Most of the pesticide toxicity studies have been performed in acute, sub chronic and chronic intoxication models with exposure of only one pesticide at high and lower doses. Very few reports are available in literature examining the effect of these pesticides when they are given in mixture. Studies on the potentiation of toxicity of pesticides in mixtures were reported for atrazine, chlorpyrifos and chlorothalonil (DeLorenzo and Serrano, 2003; Denton et al., 2003) as well as cypermethrin, quinolphos and linuron (Chauhan et al., 2005). Combined exposure of non-toxic doses of dimethoate, dichlorovos and malathion induced significant reproductive dysfunction in the offspring of rats (Yu et al., in press). Combined exposure of non-toxic doses of atrazine and alachlor induced significant frequencies of chromatid breaks and fragments in mouse bone marrow cells, whereas independent exposure of similar doses failed to induce any significant effect (Meisner et al., 1992). These studies indicate that pesticide mixtures show an increase in the frequency of chromosomal aberrations, DNA adducts formation, and reactive oxygen species that can disrupt the genetic integrity and alter the biochemistry of metabolic pathways. Therefore, looking at the widespread and overlapping applications of CPF, MPT and MLT for agricultural purposes, present study was aimed to evaluate the adverse effects of CPF, MPT and MLT on redox status by monitoring ratios of NADPH/NADP þ , NADH/NAD þ and GSH/GSSG in the rat

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tissues when given alone or in combination. The activities of G6PDH and GR were estimated as the indicator of oxidative stress. Recoveries in redox balance on prior feeding with natural antioxidants have also been monitored in the present study.

2. Material and methods 2.1. Chemicals and experimental animals Chemicals used in the present study were of highest purity grade. Tris, potassium chloride, sodium carbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, phosphoric acid, sodium hydroxide, magnesium chloride were purchased from Merck, Germany. o-phthalaldehyde, nicotinamide, alcohol dehydrogenase, glutathione reduced, glutathione oxidized, ethanol, ethylene diamine tetra acetic acid, glucose-6-phosphate dehydrogenase, glucose-6-phosphate, reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate, N-ethyldibenzopyrazine ethyl sulfate (PES), 3-(4,5-dimethythiazolyl)-2,5-dipheny-2H-tetrazolium bromide (thiazolyl blue or MTT) were purchased from Sigma chemicals Co., St. Louis, USA. Methyl parathion (MPT), chlorpyrifos (CPF) and malathion (MLT) were kind gift from Devidayal (Sales) Limited, Mumbai, India. Adult male albino rats of Wistar strain (Rattus norvegicus) weighing about 1207 10 g were used in the present study. Rats were obtained from the animal facilities of Defense Research and Development Establishment, Gwalior, India, and were maintained in a light (light–dark cycle of 12 h each) and temperature (257 2 1C) controlled animal room of our department on standard pellet diet (obtained from Amrut Rat and Mice Feed, New Delhi, India) and tap water ad libitium. Rats were acclimatized for 1 week prior to the start of the experiment. 2.2. Study design Rats were divided into four groups, two groups receiving pesticides individually and in mixture for one day and two days, respectively, while the other groups were given mixture of antioxidant vitamins A, E and C for fifteen days followed by pesticide exposure individually and in mixture for one day and two days. Animals were killed 24 h after the last treatment and tissues were collected for various estimations. 2.3. Treatments Rats were randomly divided into four groups of thirty animals each. Each group is again divided into five subgroups of six animals each. The rats of I, II, III and IV subgroups received 38.8 mg CPF/kg body weight (dose equivalent to 0.25 LD50 as the reported LD50 of CPF is 155 mg/kg body weight, The Pesticide Manual, 1987), 3.3 mg MPT/kg body weight (dose equivalent to 0.25 LD50 as the reported LD50 of MPT is 13 mg/kg body weight, Gaines, 1969), 343.8 mg MLT/kg body weight, (doses equivalent to 0.25 LD50 as the reported LD50 of MLT is 1375 mg/kg body weight, Gaines, 1969) and 0.25 LD50 equivalent of pesticides mixture (each pesticide was present equivalent to 1/12 LD50) for one (Group I) and two consecutive days (Group II) orally dissolved in 0.4 ml corn oil, respectively. The rats of V subgroup from each group served as the control and received 0.4 ml corn oil orally for one and two days, respectively. The animals of third and forth groups were given a combination of vitamin A (2.0 mg), vitamin E (20.0 mg) and vitamin C (120 mg)/kg body weight (vitamin A and vitamin E were dissolved in corn oil while vitamin C was dissolved in water, these two mixtures of vitamins were mixed prior to use) for fifteen days followed by pesticide treatment as described above for one day (group 3) and two days (group 4) while the fourth subgroup received a mixture of equivalent volume of pesticides for one day and two days. The animals of fifth subgroup received 0.4 ml of corn oil orally, and served as the control. These doses are selected on the basis of recommended allowances for human beings, which are 1000 mg vitamin A, 10 mg vitamin E (a-tocopherol) and 60 mg vitamin C/day. Rats were humanly killed 24 h after the last treatment by cervical dislocation; different tissues were excised off, washed with 0.9 percent NaCl and used for different estimations. Animals were handled, ethically treated, and humanly killed as per the rules and instructions of the Ethical Committee of Animal Care of Jiwaji University, Gwalior, India, in accordance with the Indian National Law on animal care and use. 2.4. Estimation of NADP þ , NADPH, NAD þ and NADH Oxidized and reduced pyridine nucleotides (NAD þ , NADH, NADP þ and NADPH) were assayed by the method of Zerez et al. (1987). A 10 percent homogenate was prepared in 1 M Tris–HCl buffer (pH 8.0) and centrifuged at 9500g for 20 min in a refrigerated centrifuge. To 0.1 ml of the resulting supernatant, 1.9 ml extraction buffer (containing 100 mM Na2CO3 and 10 mM nicotinamide) was added and kept at 0 1C for 30 min. The frozen mixture was thawed quickly at room temperature water bath and divided into two equal parts. One part was used for estimation of NAD þ þ NADH or NADP þ þNADPH and the other

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part was incubated at 60 1C for 30 min, quickly chilled and used for the estimation of only reduced form of pyridine nucleotides. For assay of total NAD (NAD þ þNADH) or NADH, the reaction mixture containing 2.0 mmole N-ethyldibenzopyrazine ethyl sulfate (PES), 0.5 mmole 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (thiazolyl blue or MTT), 0.25 U of alcohol dehydrogenase, 600 mmole ethanol and 0.1 ml extract (for NAD þ þNADH) or 0.1 ml heat treated extract (for NADH estimation) in the total volume of 1.0 ml. Absorbance change was recorded at 570 nm for 5 min. For total NADP (NADP þ þNADPH) or NADPH assay, the reaction mixture containing 5.0 mmole Na4EDTA, 2.0 mmole PES and 0.5 mmole MTT, 1.3 U glucose6-phosphate dehydrogenase, 1.0 mmole glucose-6-phosphate and 0.1 ml extract (or 0.1 ml heat treated extract) in the total volume of 1.0 ml. Absorbance change was recorded at 570 nm for 5 min. For standard curve varying concentration of NAD þ , NADH, NADP þ and NADPH ranging from 1 pmole to 4 pmole in a total volume of 0.5 ml were taken. The rest of the procedure remains similar to the estimation of NAD or NADP. The results were expressed as pmole NAD(P) þ or NAD(P)H present g  1 tissue.

To this mixture 4.3 ml of 0.1 N NaOH was added. About 0.1 ml portion of this mixture was used for GSSG assay in the manner identical to GSH except that 0.1 N NaOH was employed as diluents in place of phosphate EDTA buffer. Both GSH and GSSG standard curves were prepared in similar manner. The levels in tissues were expressed as mg GSH or GSSG g  1 tissue.

2.6. Glutathione reductase Glutathione reductase activity in tissue homogenate was determined by measuring the rate of conversion of NADPH to NADP þ by the enzyme (Tayarani et al., 1989). A 10 percent (w/v) homogenate was prepared in 1.15 percent KCl and centrifuged at 5000g for 10 min. The resulting supernatant obtained was used for enzyme assay. A typical reaction mixture containing 0.7 ml phosphate buffer (0.1 M, pH 7.5), 0.1 ml GSSG (0.2 mM) and 0.1 ml suitably diluted homogenate was incubated at room temperature for 10 min. Reaction was initiated by the addition 0.1 ml NADPH (0.12 mM) and absorbance change was recorded for 5 min at 340 nm. Specific activity was expressed as mmole NADPH oxidized min  1 mg  1 protein taking molar coefficient of NADPH as 6300 M  1 cm  1.

2.5. Estimation of GSH and GSSG The levels of reduced and oxidized glutathione (GSH and GSSG) were estimated as described by Hissin and Hilf (1976). Approximately 250 mg of tissue was homogenized in solution consisted of 3.75 ml phosphate EDTA buffer (0.1 M sodium phosphate buffer containing 5.0 mM EDTA, pH 8.0) and 1.0 ml of 25 percent HPO3, which were used as protein precipitant and GSH or GSSG was assayed in the supernatant obtained after centrifugation at 10,000g for 30 min. To 0.5 ml of the 10,000 g supernatant, 4.5 ml of phosphate EDTA buffer, pH 8.0, was added for GSH estimation. The final mixture contained 0.1 ml diluted tissue supernatant, 1.8 ml phosphate EDTA buffer and 0.1 ml o-phthaldialdehyde (1 mg/ml solution of OPT) and fluorescence at 412 nm was determined with activation at 350 nm. To 0.5 ml of 10,000 g supernatant, 0.2 ml of N-ethylmaleimide (NEM, 0.04 M) was added and incubated at room temperature for 30 min to interact with GSH present in the tissue.

2.7. Glucose-6-phosphate dehydrogenase Glucose-6-phosphate dehydrogenase activity was estimated by the method of Askar et al. (1996). A 10 percent homogenate was prepared in 1.15 percent KCl and centrifuged at 5000g for 10 min. The resulting supernatant obtained was used for enzyme assay. Reaction mixture containing 1.5 ml Tris–HCl buffer (0.1 M, pH 7.8), 0.1 ml MgCl2 (0.1 M), 0.1 ml glucose-6-posphate (0.1 M), 1.0 ml distilled water and 0.1 ml suitably diluted tissue supernatant were incubated at room temperature for 10 min. Reaction was initiated by the addition of 0.2 ml NADP þ (2.7 mM) and absorbance change was recorded for 5 min at 340 nm. Specific activity was expressed as mmole NADPH formed min  1 mg  1 protein using molar extinction of NADPH as 6300 M  1 cm  1.

Table 1 Effect of oral exposure of chlorpyrifos, methyl parathion and malathion, individually and in mixture, for one day and two days on the levels of NADPH, NADP þ and NADPH/NADP þ ratio in rat tissues. Tissues

Treatment

Liver

1 day NADPH NADP þ NADPH/NADP þ 2 days NADPH NADP þ NADPH/NADP þ

Brain

Kidney

Spleen

1 day NADPH NADP þ NADPH/NADP þ 2 days NADPH NADP þ NADPH/NADP þ 1 day NADPH NADP þ NADPH/NADP þ 2 days NADPH NADP þ NADPH/NADP þ 1 day NADPH NADP þ NADPH/NADP þ 2 days NADPH NADP þ NADPH/NADP þ

Control

Chlorpyrifos

Parathion

Malathion

Pesticide mixture

5.59 7 0.33 2.97 7 0.13 1.88

1.66 7 0.06nnn 4.36 7 0.13nnn 0.38

1.02 7 0.01nnn 5.61 7 0.12nnn 0.18

3.86 7 0.13nn 3.34 7 0.05n 1.16

3.14 7 0.09nnn,bennn,cennn,denn 4.08 70.22n,be#,cennn,den 0.77

4.84 7 0.07 2.82 7 0.05 1.72

1.50 7 0.07nnn 7.02 7 0.42nnn 0.21

0.63 7 0.06nnn 5.04 7 0.11nnn 0.13

3.18 7 0.05nnn 3.33 7 0.14n 0.96

3.10 70.15nnn,bennn,cennn,de# 4.04 70.11nnn,bennn,cennn,den 0.76

0.92 7 0.07 0.87 7 0.16 1.10

1.18 7 0.13n 2.27 7 0.31n 0.52

0.65 7 0.03nnn 2.38 7 0.04nnn 0.27

1.15 7 0.03nnn 1.22 7 0.06# 0.94

1.25 7 0.03nbe#,cennn,dennn 1.97 7 0.01nnn,be#,cennn,dennn 0.63

1.42 7 0.09 1.74 7 0.03 0.82

0.51 7 0.02nnn 1.47 7 0.07n 0.35

0.35 7 0.03nnn 2.33 7 0.23n 0.15

1.01 70.14n 1.47 7 0.10n 0.69

1.10 70.14#,ben,cen,de# 2.08 70.24#,ben,ce#,den 0.53

1.80 7 0.01 1.17 7 0.04 1.54

0.72 7 0.09nnn 1.68 7 0.10nn 0.43

0.91 7 0.05nnn 3.10 7 0.05nnn 0.29

1.70 70.04n 2.00 70.07nnn 0.85

1.34 7 0.07nnn,benn,cenn,denn 1.73 7 0.06nnn,be#,cennn,den 0.78

2.27 7 0.04 1.31 7 0.02 1.73

1.93 7 0.08n 1.40 7 0.08# 1.38

0.57 7 0.08nnn 2.11 7 0.06nnn 0.27

1.28 7 0.02nnn 1.67 7 0.06nnn 0.77

1.56 7 0.03nnn,ben,cennn,dennn 2.45 7 0.02nnn,bennn,cenn.dennn 0.64

2.41 7 0.43 1.56 7 0.28 1.55

0.86 7 0.03n 1.51 7 0.04# 0.57

0.55 7 0.01n 2.05 7 0.10# 0.27

1.05 70.06n 1.31 7 0.04# 0.80

0.91 70.10n,be#,cen,de# 1.44 7 0.05#,be#,cenn,de# 0.63

2.21 7 0.12 1.52 7 0.03 1.45

1.11 7 0.14nnn 2.84 7 0.27nnn 0.39

0.56 7 0.08nnn 2.98 7 0.36nnn 0.19

1.11 7 0.15nnn 1.67 7 0.18# 0.66

1.04 70.12nnn,be#,cennn,de# 1.81 7 0.17#,benn,cennn,de# 0.57

Results are mean 7S.E. of six set of observations taken on different days. NADP þ and NADPH levels are expressed as mmole g  1 tissues. Rats were given 0.25 LD50 equivalent of pesticides orally, dissolved in 0.4 ml corn oil for one and two consecutive days. n P o 0.05, nnPo 0.001, nnnPo 0.0001 and #P 40.05 when compared with respective control, bewhen PM is compared with CPF, compared with MPT and dewhen PM is compared with MLT.

ce

when PM is

A. Ojha, N. Srivastava / Ecotoxicology and Environmental Safety 75 (2012) 230–241

2.8. Protein estimation Protein in tissue samples was estimated by the method of Lowry et al. (1951) using bovine serum albumin as standard.

2.9. Statistical analyses Results are expressed as mean 7 S.E. of six sets of observation taken on different days. Statistical analyses were performed using SigmaStat statistical software, Version 2.0. All the statistical analyses were performed using one-way analysis of variance with post hoc Bonferroni’s multiple comparison test applied across the treatment groups. Significance was based on P value o 0.05.

3. Results 3.1. Effect on pesticide exposure on the levels of NADP þ , NADPH and NADPH/NADP þ ratio Results of the present study showed that pesticide exposure to rats for one day and two days caused significant decrease in the levels of NADPH and consequent increase in the levels of NADP þ resulting in the decrease in NADPH/NADP þ ratio. It was observed that one day oral exposure with 0.25 LD50 equivalent of CPF caused 80 percent decrease in the NADPH/NADP þ ratio while exposure for two consecutive days decreased this ratio by 88 percent in the liver of exposed rats (Table 1). MPT exposure

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caused 90 percent and 92 percent decrease, MLT caused 38 percent and 44 percent decrease and pesticide mixture caused 59 percent and 56 percent decrease in the ratio of NADPH/NADP þ in the liver, on one day and two days oral exposure, respectively. In the brain, the same doses for one day and two days caused 53 percent and 57 percent decrease by CPF, 76 percent and 82 percent decrease by MPT, 15 percent and 16 percent decrease by MLT and 43 percent and 35 percent decrease by pesticide mixture for one day and two days, respectively. Similarly one day and two days exposure with CPF caused 72 percent and 20 percent decrease in kidney and 63 percent and 73 percent decrease in spleen, MPT exposure for one day and two days caused 81 percent and 84 percent decrease in kidney and 83 percent and 87 percent decrease in spleen; MLT exposure for one and two days caused 45 percent and 56 percent decrease in kidney and 48 percent and 55 percent decrease in spleen and combined exposure with all these three pesticides caused 49 percent and 63 percent decrease in kidney and 59 percent and 61 percent decrease in spleen of rats receiving one day and two days exposure, respectively (Table 1). Highest decrease in NADPH/ NADP þ ratio was observed in tissues of rat given MPT exposure. Prior feeding of mixture of vitamin A, E and C for 15 days followed by one day and two days exposure with 0.25 LD50 equivalent of CPF, MPT, MLT and their mixture, offered some protection and the decrease observed in NADPH/NADP þ ratio was lesser when compared with vitamin-untreated pesticide exposed

Table 2 Effect of oral exposure of chlorpyrifos, methyl parathion and malathion, individually and in mixture, for one day and two days on the levels of NADPH, NADP þ and NADPH/NADP þ ratio in tissues of rats prefed with mixture of antioxidant vitamins. Tissues

Treatment

Liver

1 day NADPH NADP þ NADPH/NADP þ 2 days NADPH NADP þ NADPH/NADP þ

Brain

Kidney

Spleen

1 day NADPH NADP þ NADPH/NADP þ 2 days NADPH NADP þ NADPH/NADP þ 1 day NADPH NADP þ NADPH/NADP þ 2 days NADPH NADP þ NADPH/NADP þ 1 day NADPH NADP þ NADPH/NADP þ 2 days NADPH NADP þ NADPH/NADP þ

Control

Chlorpyrifos

Parathion

Malathion

Pesticide mixture

4.047 0.12 2.227 0.11 1.82

3.96 70.09# 2.59 70.10n 1.53

2.15 7 0.10nnn 4.35 7 0.06nnn 0.49

3.267 0.05nnn 3.187 0.26n 1.03

4.44 70.20#,be#,cennn,denn 3.82 70.21nnn,benn,cen,de# 1.16

3.597 0.02 2.047 0.02 1.76

3.407 0.09# 2.96 70.20n 1.15

1.93 7 0.22nnn 5.07 70.06nnn 0.38

3.607 0.06# 2.977 0.04nnn 1.21

3.36 70.03nnn,be#,cennn,den 3.67 70.04nnn,ben,cennn,dennn 0.92

2.417 0.09 1.377 0.07 1.76

1.56 70.04nnn 1.37 70.02# 1.14

1.48 7 0.06nnn 2.36 7 0.05nnn 0.63

2.007 0.04n 1.487 0.06# 1.35

1.37 70.13nnn,be#,ce#,den 1.18 70.06#,be#,cennn,den 1.16

2.967 0.09 1.567 0.04 1.89

1.67 70.04nnn 1.307 0.08n 1.29

1.37 7 0.10nnn 2.19 7 0.05nnn 0.63

1.337 0.07nnn 1.007 0.04nnn 1.33

1.22 70.07nnn,benn,ce#,de# 1.15 70.02nnn,be#,cennn,den 1.06

2.597 0.06 1.377 0.05 1.89

1.89 70.08nnn 2.077 0.05nnn 0.91

1.48 7 0.06nnn 2.15 7 0.06nnn 0.69

1.767 0.26n 1.287 0.21# 1.38

1.30 70.02nnn,bennn,cen,de# 1.41 70.06#,bennn,cennn,de# 0.92

2.567 0.11 1.527 0.01 1.68

1.63 70.07nnn 1.78 70.07n 0.92

1.32 7 0.02nnn 2.32 7 0.04nnn 0.57

1.897 0.16n 1.437 0.03n 1.32

1.18 70.06nnn,benn,ce#,den 1.33 70.04n,benn,cennn,de# 0.89

2.267 0.13 1.267 0.09 1.79

1.707 0.05n 1.59 70.05n 1.07

1.11 7 0.04nnn 1.52 7 0.06n 0.73

2.527 0.13# 1.657 0.16# 1.53

1.63 70.06n,be#,cennn,denn 1.59 70.06n,be#,ce#,de# 1.03

2.707 0.05 1.637 0.06 1.66

1.82 70.02nnn 1.59 70.02# 1.15

1.37 7 0.30nnn 2.18 7 0.02nnn 0.63

1.487 0.0nnn 1.157 0.06n 1.29

1.22 70.01nnn,bennn,cen,den 1.67 70.04#,be#,cennn,dennn 0.73

Results are mean 7 S.E. of six set of observations taken on different days. NADP þ and NADPH levels are expressed as mmole g  1 tissues. Rats were given a mixture of vitamin A (2.0 mg/kg body weight), vitamin E (20.0 mg/kg body weight) and vitamin C (120 mg/kg body weight), for 15 days followed by 0.25 LD50 equivalent of pesticides orally, dissolved in 0.4 ml corn oil for one and two consecutive days. n P o 0.05, nnPo 0.001, nnnP o0.0001 and #P 40.05 when compared with respective control, bewhen PM is compared with CPF, cewhen PM is compared with MPT and dewhen PM is compared with MLT.

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rats tissues. The decrease observed in liver were 16 percent, 73 percent, 43 percent and 36 percent on one day exposure and 35 percent, 78 percent, 31 percent and 48 percent on two days exposure with CPF, MPT, MLT and pesticide mixture, respectively (Table 2). Similarly one day and two days exposure with different pesticides to antioxidant pre-fed rats showed lesser decrease in NADPH/NADP þ ratio in brain, the decrease observed were 35 percent and 32 percent on CPF exposure; 64 percent and 67 percent on MPT exposure; 23 percent and 30 percent on MLT exposure and 34 percent and 44 percent on pesticide mixture exposure, for one day and two days, respectively. Similarly in kidney, the decrease observed was 52 percent and 45 percent on CPF exposure, 64 percent and 66 percent on MPT exposure, 27 percent and 21 percent on MLT exposure and 51 percent and 47 percent on pesticide mixture exposure, for one day and two days, respectively. In spleen, the decrease observed was 40 percent and 31 percent on CPF exposure; 59 percent and 62 percent on MPT exposure, 15 percent and 22 percent on MLT exposure and 43 percent and 56 percent on pesticide mixture exposure, for one day and two days, respectively (Table 2). Highest recovery was observed in brain of rats followed by liver on prior feeding of antioxidant vitamins for two weeks. 3.2. Effect on levels of NAD þ , NADH and NADH/NAD þ ratio Data obtained showed that pesticide exposure singly or in mixture for one day and two days caused significantly marked

decrease in the levels of NADH and corresponding increase in the levels of NAD þ resulting in the decreased NADH/NAD þ ratio in liver, brain, kidney and spleen of rats. The decrease in hepatic NADH/NAD þ ratio was 68 percent and 57 percent on CPF exposure; 59 percent and 77 percent on MPT exposure; 24 percent and 29 percent on MLT exposure and 39 percent and 37 percent on combined exposure with all the three pesticides, for one day and two days, respectively (Table 3). The decrease observed in brain was 74 percent and 75 percent on CPF exposure; 79 percent and 80 percent on MPT exposure; 31 percent and 39 percent on MLT exposure and 64 percent and 49 percent on combined exposure for one day and two days, respectively. In kidney of rats, the NADH/NAD þ ratio was decreased by 60 percent and 66 percent in CPF treated rats; 73 percent and 77 percent in MPT; 31 percent and 67 percent in MLT and 44 percent and 64 percent on exposure with pesticide mixture for one day and two days, respectively. Almost same level of decrease in NADH/NAD þ ratio was also observed in spleen of rats exposed with CPF, MPT, MLT singly or in mixture for one and two days (Table 3). Highest decrease in NADH/NAD þ ratio in tissues was observed on MPT treatment to rats. Prior feeding of antioxidant vitamins for 15 days followed by pesticide exposure offered some protection against their toxicity and the ratio of NADH/NAD þ was increased to some extent when compared with vitamin-unfed pesticide treated group. The NADH/NAD þ ratio was decreased in the liver to 60 percent and 64 percent on CPF treatment; 31 percent and 53 percent on MPT

Table 3 Effect of oral exposure of chlorpyrifos, methyl parathion and malathion, individually and in mixture, for one day and two days on the levels of NADH, NAD þ and NADH/NAD þ ratio in rat tissues. Tissues

Treatment

Liver

1 day NADH NAD þ NADH/NAD þ 2 days NADH NAD þ NADH/NAD þ

Brain

Kidney

Spleen

1 day NADH NAD þ NADH/NAD þ 2 days NAD þ NAD þ H NADH/NAD þ 1 day NADH NAD þ NADH/NAD þ 2 days NADH NAD þ NADPH/NADP þ 1 day NADH NAD þ NADH/NAD þ 2 days NADH NAD þ NADH/NAD þ

Control

Chlorpyrifos

Parathion

Malathion

Pesticide mixture

7.23 70.65 7.46 70.43 0.97

3.23 70.13nnn 10.27 70.26nn 0.31

4.16 7 0.15nn 10.41 7 0.47n 0.40

7.53 70.51# 10.17 70.62nn 0.74

5.52 7 0.07n 9.42 7 0.35n,be#,ce#,de# 0.59

7.23 70.65 7.46 70.43 0.97

5.38 70.11n 12.95 7 0.22nnn 0.42

2.58 7 0.20nnn 11.87 7 0.67nn 0.22

6.83 70.32# 9.907 0.40nn 0.69

3.81 7 0.11nn,bennn,cenn,dennn 6.26 7 0.51#,bennn,cennn,denn 0.61

6.087 0.09 4.45 70.08 1.37

2.017 0.13nnn 5.58 70.13nnn 0.34

1.70 7 0.12nnn 5.81 7 0.06nnn 0.29

6.037 0.18# 6.44 70.74n 0.94

3.36 7 0.42nnn,ben,cen,denn 6.72 7 0.16nnn,benn,cenn,de# 0.50

6.087 0.09 4.45 70.08 1.37

2.047 0.02nnn 6.087 0.22nnn 0.33

1.63 7 0.08nnn 5.82 7 0.09nnn 0.28

6.26 70.14# 7.43 70.19nnn 0.84

3.89 7 0.07nnn,bennn,cennn,dennn 5.59 7 0.10nnn,be#,ce#,dennn 0.70

7.86 70.18 6.24 70.21 1.26

3.49 70.24nnn 6.94 70.38# 0.50

2.60 7 0.05nnn 7.60 7 0.26n 0.34

5.77 70.11nnn 6.63 70.30# 0.87

4.36 7 0.07nnn,ben,cennn,dennn 6.20 7 0.29#,be#,cen,de# 0.70

7.86 70.18 6.24 70.21 1.26

3.43 70.10nnn 7.94 70.11nnn 0.43

2.77 7 0.06nnn 9.47 7 0.34nnn 0.29

3.82 70.15nnn 9.18 70.09nnn 0.42

4.41 7 0.22nnn,ben,cennn,de# 9.91 7 0.38nnn,bennn,ce#,den 0.45

8.29 70.04 6.63 70.01 1.25

4.44 70.31nnn 6.49 70.44# 0.68

3.23 7 0.20nnn 7.89 7 0.31n 0.41

6.42 70.24nnn 8.407 0.16nnn 0.76

4.88 7 0.24nnn,be#,cen,den 6.62 7 0.15#,be#,cen,dennn 0.74

8.29 70.04 6.63 70.01 1.25

3.46 70.16nnn 7.72 70.17nnn 0.45

3.27 7 0.09nnn 9.40 7 0.76n 0.35

5.27 70.01nnn 8.49 70.02nnn 0.62

4.14 7 0.04nnn,ben,cennn,dennn 7.08 7 0.09nn,ben,cen,dennn 0.59

Results are mean 7S.E. of six set of observations taken on different days. NAD þ and NADH levels are expressed as mmole g  1 tissues. Rats were given 0.25 LD50 equivalent of pesticides orally, dissolved in 0.4 ml corn oil for one and two consecutive days. n P o 0.05, nnPo 0.001, nnnPo 0.0001 and #P 40.05 when compared with respective control, bewhen PM is compared with CPF, compared with MPT and dewhen PM is compared with MLT.

ce

when PM is

A. Ojha, N. Srivastava / Ecotoxicology and Environmental Safety 75 (2012) 230–241

treatment; 16 percent and 26 percent on MLT treatment and 43 percent and 56 percent on treatment of pesticide mixture for one day and two days, respectively (Table 4). In the brain, the protection was also observed in vitamin pre-fed group and the significant decrease in ratio were 24 percent and 33 percent on CPF exposure; 13 percent and 47 percent on MPT exposure; 17 percent and 7 percent on MLT exposure and 17 percent and 23 percent on exposure with pesticide mixture for one and two days, respectively. Prior feeding of antioxidant vitamins also showed protection against CPF, MPT and MLT induced decrease in NADH/ NAD þ ratio in kidney and spleen also (Table 4). Highest recovery was observed in brain of rats followed by kidney on prior feeding of antioxidant vitamins for two weeks.

3.3. Effect on levels of GSH, GSSG and GSH/GSSG ratio GSH, an important cellular nonenzymatic antioxidant, is a vital substance in detoxification and also provides antioxidant protection in the aqueous phase of the cellular system. GSH plays an important role in protecting the cells against radical and oxy radical induced damage. Relationship between extent of lipid peroxidation and glutathione status of the liver has a significant inverse relation. The results of the present study showed that levels of GSH were significantly decreased and GSSG were significantly increased after CPF, MPT or MLT exposure either

235

singly or in mixture, in all the tissues of rats. Due to decrease in GSH and increase in GSSG, GSH/GSSG ratio was also decreased. The decrease in ratio of GSH/GSSG in the liver were 54 percent, 54 percent, 31 percent and 35 percent on one day exposure with 0.25 LD50 equivalent of CPF, MPT, MLT and pesticide mixture, respectively, while two days exposure with these pesticide caused 54 percent, 59 percent, 42 percent and 50 percent decrease (Table 5). The decrease in GSH/GSSG ratio in the brain on exposure with 0.25 LD50 equivalent of CPF, MPT, MLT and pesticide mixture were 44 percent, 61 percent, 20 percent and 39 percent on one day exposure, and 58 percent, 70 percent, 23 percent and 39 percent on two days exposure, respectively. Similarly pesticide exposure for one day and two days caused significantly marked decrease in GSH/GSSG ratio in the kidney and the spleen also. The decrease observed in the kidney were 54 percent, 56 percent, 31 percent and 46 percent on one day exposure and 68 percent, 68 percent, 40 percent and 45 percent on two days exposure with 0.25 LD50 equivalent of CPF, MPT, MLT and pesticide mixture, respectively (Table 5). The decrease observed in the spleen were 62 percent, 59 percent, 31 percent and 43 percent on one day exposure and 69 percent, 74 percent, 38 percent and 49 percent on two days exposure with 0.25 LD50 equivalent of CPF, MPT, MLT and pesticide mixture, respectively (Table 5). Pre-feeding of mixture of vitamin A, C and E for 15 days, followed by pesticide exposure for one day and two days caused lesser alteration in levels of GSH and GSSG and thus the ratio of

Table 4 Effect of oral exposure of chlorpyrifos, methyl parathion and malathion, individually and in mixture, for one day and two days on the levels of NADH, NAD þ and NADH/NAD þ ratio in tissues of rats prefed with mixture of antioxidant vitamins. Tissues

Treatment

Liver

1 day NADH NAD þ NADH/NAD þ 2 days NADH NAD þ NADH/NAD þ

Brain

Kidney

Spleen

1 day NADH NAD þ NADH/NAD þ 2 days NAD þ NAD þ H NADH/NAD þ 1 day NADH NAD þ NADH/NAD þ 2 days NADH NAD þ NADPH/NADP þ 1 day NADH NAD þ NADH/NAD þ 2 days NADH NAD þ NADH/NAD þ

Control

Chlorpyrifos

Parathion

Malathion

Pesticide mixture

9.78 70.05 8.46 70.05 1.16

4.72 7 0.04nnn 10.20 70.43n 0.46

5.67 7 0.06nnn 7.08 70.25nn 0.80

8.317 0.10nnn 8.467 0.13# 0.96

4.84 7 0.01nnn,ben,cennn,dennn 7.33 7 0.25n,benn,ce#,den 0.66

9.78 70.05 8.46 70.05 1.16

5.38 7 0.14nnn 12.95 7 0.31nnn 0.42

4.62 7 0.11nnn 8.62 7 0.32# 0.54

6.237 0.08nnn 7.237 0.13nnn 0.86

3.49 7 0.11nnn,bennn,cennn,dennn 6.83 7 0.05nnn,bennn,cenn,den 0.51

4.46 70.05 4.23 70.05 1.05

3.90 70.07nnn 4.85 7 0.15n 0.80

4.97 7 0.23# 5.49 7 0.06nnn 0.91

5.967 0.42n 6.827 0.34nnn 0.87

5.85 7 0.19nnn,bennn,cennn,de# 6.72 7 0.09nnn,bennn,cennn,de# 0.87

4.46 70.05 4.23 70.05 1.05

3.12 7 0.17nnn 4.46 7 0.13# 0.70

3.22 7 0.12nnn 5.78 7 0.05nnn 0.56

6.967 0.19nnn 7.097 0.15nnn 0.98

5.72 7 0.33n,bennn,cennn,den 7.08 7 0.05nnn,bennn,cennn,de# 0.81

5.69 70.18 5.51 70.12 1.03

5.69 7 0.21# 6.46 7 0.17n 0.88

7.02 70.09nnn 8.54 7 0.44nnn 0.82

6.387 0.29# 6.977 0.11nnn 0.92

5.23 7 0.10n,be#,cennn,den 5.38 7 0.05#,bennn,cennn,dennn 0.97

5.69 70.18 5.51 70.12 1.03

5.69 7 0.40# 6.75 7 0.11nnn 0.84

4.21 7 0.12nnn 6.92 7 0.29n 0.61

2.977 0.60n 3.547 0.13nnn 0.84

6.39 7 0.25n,be#,cennn,denn 8.38 7 0.35nnn,bennn,cen,dennn 0.76

7.62 70.08 6.057 0.03 1.26

6.74 7 0.35n 6.33 7 0.03nnn 1.06

6.08 70.16nnn 7.92 7 0.18nnn 0.77

6.927 0.01nnn 7.857 0.17nnn 0.88

5.59 7 0.14nnn,ben,cen,dennn 6.95 7 0.04nnn,bennn,cenn,denn 0.80

7.62 70.08 6.057 0.03 1.26

4.72 7 0.33nnn 5.90 70.58# 0.80

3.23 7 0.08nnn 6.54 7 0.21n 0.49

4.097 0.07nnn 4.857 0.39n 0.84

5.64 7 0.16nnn,ben,cennn,dennn 7.46 7 0.07nnn,ben,cen,dennn 0.76

Results are mean 7 S.E. of six set of observations taken on different days. NAD þ and NADH levels are expressed as mmole g  1 tissues. Rats were given a mixture of vitamin A (2.0 mg//kg body weight), vitamin E (20.0 mg/kg body weight) and vitamin C (120 mg/kg body weight), for 15 days followed by 0.25 LD50 equivalent of pesticides orally, dissolved in 0.4 ml corn oil for one and two consecutive days. n P o 0.05, nnPo 0.001, nnnP o0.0001 and #P 40.05 when compared with respective control, bewhen PM is compared with CPF, cewhen PM is compared with MPT and dewhen PM is compared with MLT.

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Table 5 Effect of oral exposure of chlorpyrifos, methyl parathion and malathion, individually and in mixture, for one day and two days on the levels of GSH, GSSG and GSH/GSSG ratio in rat tissues. Tissues

Treatment

Liver

1 day GSH GSSG GSH/GSSG 2 days GSH GSSG GSH/GSSG

Brain

Kidney

Spleen

1 day GSH GSSG GSH/GSSG 2 days GSH GSSG GSH/GSSG 1 day GSH GSSG GSH/GSSG 2 days GSH GSSG GSH/GSSG 1 day GSH GSSG GSH/GSSG 2 days GSH GSSG GSH/GSSG

Control

Chlorpyrifos

Parathion

Malathion

Pesticide mixture

506.62 7 8.39 309.31 7 2.88 1.64

334.967 4.53nnn 447.07 71.15nnn 0.75

320.38 714.75nnn 426.747 2.20nnn 0.75

376.637 3.96nnn 334.447 4.54nnn 1.13

384.03 73.71nnn,ben,ce#,den 360.75 72.06nnn,bennn,cennn,denn 1.06

532.157 5.49 328.287 1.82 1.62

346.577 2.59nnn 457.187 11.30nnn 0.75

326.947 3.18nnn 485.07 71.30nnn 0.67

386.957 9.24nnn 412.847 7.39nnn 0.94

367.567 6.86nnn,ben,cenn,de# 451.827 1.03nnn,be#,cennn,denn 0.81

325.30 7 0.54 205.98 7 4.51 1.58

247.197 1.77nnn 277.40 71.39nnn 0.89

229.357 2.06nnn 369.617 2.67nnn 0.62

319.777 5.61# 251.02 74.06nnn 1.27

261.317 5.28nnn,ben,cenn,dennn 268.767 5.65nnn,be#,cennn,den 0.97

327.817 1.58 211.257 3.60 1.55

235.547 4.67nnn 361.327 9.68nnn 0.65

229.457 0.95nnn 501.71 71.50nnn 0.46

263.757 6.22nnn 219.437 2.89# 1.20

260.65 73.51nnn,ben,cennn,de# 278.737 0.83nnn,bennn,cennn,dennn 0.94

456.447 3.18 218.567 1.89 2.09

344.447 1.91nnn 359.80 72.31nnn 0.96

351.317 2.62nnn 378.257 0.70nnn 0.93

381.537 2.28nnn 265.50 710.52n 1.44

380.93 72.97nnn,bennn,cennn,de# 341.317 2.59nnn,benn,cennn,dennn 1.12

484.637 1.73 230.89 7 2.27 2.10

252.40 72.05nnn 375.04 72.81nnn 0.67

238.387 1.90nnn 354.247 2.32nnn 0.67

409.23 72.03nnn 325.647 2.41nnn 1.26

369.537 13.03nnn,bennn,cennn,den 320.97 72.35nnn,bennn,cennn,de# 1.15

551.10 7 8.14 263.387 3.39 2.09

277.297 3.58nnn 353.20 710.04nnn 0.79

370.24 72.43nnn 432.827 0.94nnn 0.86

381.937 1.75nnn 263.797 3.76# 1.45

381.817 7.58nnn,bennn,ce#,de# 322.977 1.02nnn,ben,cennn,dennn 1.18

531.967 6.72 253.437 2.58 2.10

305.79 75.37nnn 472.08 71.06nnn 0.65

277.657 6.32nnn 500.777 2.42nnn 0.55

351.60 73.10nnn 270.24 72.33nn 1.30

308.007 7.42nnn,be#,cen,dennn 288.857 7.03nn,bennn,cennn,den 1.07

Results are mean 7S.E. of six set of observations taken on different days. GS H and GSSG levels are expressed as mmole g  1 tissues. Rats were given 0.25 LD50 equivalent of pesticides orally, dissolved in 0.4 ml corn oil for one and two consecutive days. n P o 0.05, nnPo 0.001, nnnPo 0.0001 and #P 40.05 when compared with respective control, bewhen PM is compared with CPF, compared with MPT and dewhen PM is compared with MLT.

GSH/GSSG is less decreased when compared with the group given same doses of pesticides. In the liver decrease in GSH/GSSG ratio remained 30 percent, 38 percent, 16 percent and 18 percent on one day exposure and 42 percent, 47 percent, 8 percent and 15 percent on two days exposure with CPF, MPT, MLT and pesticide mixture, respectively (Table 6). In the brain, decrease of GSH/ GSSG ratio remained 41 percent, 56 percent, 34 percent and 13 percent on one day exposure and 40 percent, 49 percent, 30 percent and 26 percent on two days exposure with CPF, MPT, MLT and pesticide mixture, respectively. One day exposure of CPF, MPT, MLT and pesticide mixture caused 45 percent, 63 percent, 26 percent and 42 percent decrease in the kidney, respectively, and 58 percent, 58 percent, 36 percent and 43 percent decrease in the spleen, respectively, while two days exposure caused 53 percent, 57 percent, 46 percent and 35 percent in the kidney, and 61 percent, 63 percent, 31 percent and 47 percent decrease in GSH/GSSG ratio in the spleen, respectively (Table 6). Highest recovery was observed in the spleen followed by the brain, kidney and liver of rats receiving one day exposure of these OP pestiocides singly or mixture. 3.4. Effect on activity of glucose-6-phosphate dehydrogenase Glucose-6-phosphate dehydrogenase (G6PDH), the first enzyme of pentose phosphate pathway, is the main source of NADPH, which is used in the reduction of GSSG to GSH in the presence of enzyme glutathione reductase. It is observed that OP

ce

when PM is

pesticides exposure induces the activity of G6PDH in tissues of rats. The increase in hepatic G6PDH activity were 81 percent, 133 percent, 25 percent and 58 percent on one day exposure, and 137 percent, 166 percent, 36 percent and 82 percent on two consecutive days exposure with CPF, MPT, MLT and their mixture, respectively (Table 7). In the brain, the G6PDH activity increased by 46 percent, 131 percent, 12 percent and 32 percent on one day exposure, and 85 percent, 148 percent, 51 percent and 64 percent on two days exposure with CPF, MPT, MLT and their mixture, respectively. G6PDH activities were increased in the kidney and the spleen also on one day and two days exposure with these pesticides singly or in combination (Table 7). Highest increase in G6PDH activity was observed in the liver followed by the spleen, brain and kidney, on exposure with these OP pesticides singly or in combination. MPT caused maximum induction of G6PDH activity in all the tissues of rats. The effect of OP pesticides on the activity of G6PDH was markedly reduced when pesticide exposure was given to rats prefed with mixture of vitamin A, E and C, however, the activity remained significantly increased in all the tissues when compared with corresponding control (Table 8). The increase observed in the liver were 47 percent, 69 percent, 5 percent and 30 percent on one day exposure and 27 percent, 125 percent, 12 percent and 22 percent on two days exposure with CPF, MPT, MLT and pesticide mixture, respectively. In the brain, the increase observed were 26 percent, 63 percent, 4 percent and 9 percent on one day exposure and 43 percent, 103 percent, 24 percent and 21 percent on two days

A. Ojha, N. Srivastava / Ecotoxicology and Environmental Safety 75 (2012) 230–241

237

Table 6 Effect of oral exposure of chlorpyrifos, methyl parathion and malathion, individually and in mixture, for one day and two days on the levels of GSH, GSSG and GSH/GSSG ratio in tissues of rats prefed with mixture of antioxidant vitamins. Tissues

Treatment

Liver

1 day GSH GSSG GSH/GSSG 2 days GSH GSSG GSH/GSSG

Brain

Kidney

Spleen

1 day GSH GSSG GSH/GSSG 2 days GSH GSSG GSH/GSSG 1 day GSH GSSG GSH/GSSG 2 days GSH GSSG GSH/GSSG 1 day GSH GSSG GSH/GSSG 2 days GSH GSSG GSH/GSSG

Control

Chlorpyrifos

Parathion

Malathion

Pesticide mixture

503.19 74.39 356.8273.89 1.41

312.07 7 3.43nnn 315.977 1.53nnn 0.99

288.02 7 11.35nnn 327.997 2.02nnn 0.88

356.787 3.92nnn 303.28 75.97nnn 1.18

333.08 77.06nnn,ben,cen,den 289.617 11.92nn,be#,cen,de# 1.15

521.80 70.55 347.10 71.05 1.50

359.00 7 6.42nnn 413.50 7 6.05nnn 0.87

314.427 10.85nnn 393.627 2.41nnn 0.79

473.537 6.69nnn 342.06 71.31n 1.38

446.937 4.73nnn,ben,cennn 349.70 72.36#,bennn,cennn,den 1.28

366.8879.81 223.007 1.82 1.65

247.80 7 0.82nnn 252.587 1.59nnn 0.98

233.06 7 6.61nnn 321.357 2.30nnn 0.73

265.09 73.53nnn 243.157 0.92nnn 1.09

325.647 1.74n,bennn,cennn,dennn 225.697 6.35#,ben,cennn,den 1.44

357.6879.63 234.7373.67 1.52

245.137 4.20nnn 266.477 3.86nnn 0.92

248.317 2.39nnn 322.147 1.07nnn 0.77

258.727 4.55nnn 244.477 11.85# 1.06

269.777 2.49nnn,ben,cennn,de# 241.20 75.26#,ben,cennn,de# 1.12

471.4878.55 191.6472.70 2.46

369.327 4.99nnn 273.877 4.45nnn 1.35

316.757 0.89nnn 344.227 0.24nnn 0.92

426.567 1.86nn 234.997 4.38nnn 1.82

370.83 73.89nnn,be#,cennn,dennn 259.157 5.55nnn,be#,cennn,denn 1.43

380.29 711.35 170.91 72.60 2.23

335.617 2.03n 321.667 3.47nnn 1.04

331.517 4.54n 344.297 3.50nnn 0.96

334.727 0.79n 276.687 8.62nnn 1.21

353.667 1.62n,bennn,cen,dennn 243.637 4.25nnn,bennn,cennn,den 1.45

542.3772.18 232.3271.39 2.33

331.01 7 2.37nnn 341.00 7 5.66nnn 0.97

252.817 5.54nnn 254.09 7 2.77nnn 0.99

408.99 75.18nnn 274.167 8.41nnn 1.49

363.947 1.45nnn,bennn,cennn,dennn 273.687 5.23nnn,bennn,cen,de# 1.33

552.2472.30 230.49 78.47 2.40

312.367 3.25nnn 333.357 3.25nnn 0.94

284.947 1.90nnn 317.267 14.45nn 0.90

425.03 71.77nnn 291.967 4.60nnn 1.46

392.847 1.84nnn,bennn,cennn,dennn 307.15 79.68nnn,ben,ce#,den 1.28

Results are mean 7 S.E. of six set of observations taken on different days. GSH and GSSG levels are expressed as mmole g  1 tissues. Rats were given a mixture of vitamin A (2.0 mg//kg body weight), vitamin E (20.0 mg//kg body weight) and vitamin C (120 mg/kg body weight), for 15 days followed by 0.25 LD50 equivalent of pesticides orally, dissolved in 0.4 ml corn oil for one and two consecutive days. n P o 0.05, nnPo 0.001, nnnP o0.0001 and #P 40.05 when compared with respective control, bewhen PM is compared with CPF, cewhen PM is compared with MPT and dewhen PM is compared with MLT.

Table 7 Effect of oral exposure of chlorpyrifos, methyl parathion and malathion, individually and in mixture, for one day and two days on the activity of glucose-6-phosphate dehydrogenase in rat tissues. Tissues

Treatment

Control

Chlorpyrifos

Liver

1 day 2 days

Brain

Parathion

Malathion

Pesticide mixture

15.10 7 0.15 17.22 7 1.02

nnn

27.31 7 0.17 40.84 7 0.35nnn

35.13 7 0.62 45.88 7 0.79nnn

18.86 70.10 23.41 70.55nn

23.92 7 0.49nnn,bennn,cen 31.33 7 0.55nnn,bennn.cennn

1 day 2 days

14.78 7 0.15 15.10 7 0.31

21.58 7 0.67nnn 27.98 7 0.53nnn

34.16 7 0.38nnn 37.43 7 0.22nnn

16.62 70.19nnn 22.80 70.64nn

19.50 7 0.11nnn,ben,cennn,dennn 24.79 7 0.44nnn,ben,cennn,den

Kidney

1 day 2 days

15.49 7 0.21 16.74 7 0.12

22.78 7 0.22nnn 24.55 7 0.49nnn

23.50 7 0.22nnn 29.19 7 0.46nnn

17.09 70.15nnn 21.06 70.44nnn

19.86 7 0.03nnn,bennn,cennn,dennn 21.46 7 0.51nnn,ben,cennn,de#

Spleen

1 day 2 days

21.73 7 0.27 22.15 7 0.45

41.14 7 0.41nnn 45.74 7 0.59nnn

44.29 7 0.47nnn 55.34 7 1.89nnn

37.43 70.36nnn 39.64 70.66nnn

40.077 0.85nnn,be#,cen,den 41.24 7 0.39nnn,bennn,cennn,den

nnn

nnn

Results are mean 7 S.E. of six set of observations taken on different days. Specific activity of G6PDH is expressed as m mole of NADPH oxidized min  1 mg  1 protein. Rats were given 0.25 LD50 equivalent of pesticides orally, dissolved in 0.4 ml corn oil for one and two consecutive days. n P o 0.05, nnPo 0.001, nnnP o0.0001 and #P 40.05 when compared with respective control, bewhen PM is compared with CPF, compared with MPT and dewhen PM is compared with MLT.

exposure, with CPF, MPT, MLT and pesticide mixture, respectively. In the kidney, the activity of G6PDH remained increased on one day and two days exposure with pesticides and the increase observed were 39 percent, 81 percent, 5 percent and 4 percent on one day exposure and 51 percent, 72 percent, 20 percent and 25 percent on two days exposure with CPF, MPT, MLT and pesticide mixture,

ce

when PM is

respectively. The increase in G6PDH activity in the spleen ranged from 21 percent to 53 percent on one day exposure and 35 percent to 71 percent on two days exposure with CPF, MPT, MLT either singly or in combination (Table 8). Highest recovery was observed in the brain followed by the liver, spleen and kidney on prior feeding of antioxidant mixture for 15 days.

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Table 8 Effect of oral exposure of chlorpyrifos, methyl parathion and malathion, individually and in mixture, for one day and two days on the activity of glucose-6-phosphate dehydrogenase in tissues of rats prefed with mixture of antioxidant vitamins. Tissues

Treatment

Control

Chlorpyrifos

Parathion

Malathion

Pesticides mixture

Liver

1 day 2 days

14.43 7 0.17 16.64 7 0.31

21.17 7 0.27nnn 21.14 7 0.62nnn

24.38 7 0.16nnn 37.36 7 0.18nnn

15.10 70.35# 18.62 71.09n

18.76 70.80nn,ben,cennn,den 20.32 70.35nnn,be#,cennn,de

Brain

1 day 2 days

13.96 7 0.29 14.09 7 0.06

17.52 7 0.20nnn 20.12 7 0.64nnn

22.78 7 0.51nnn 28.57 7 0.39nnn

14.51 70.09# 17.40 70.80n

15.15 70.19n,bennn,cennn,den 17.06 70.07nnn,benn,cennn,de#

Kidney

1 day 2 days

15.51 7 0.82 16.16 7 0.27

21.55 7 0.16nnn 24.39 7 0.42nnn

28.07 7 0.91nnn 27.84 7 1.21nnn

16.22 70.32# 19.37 70.68n

16.09 70.57#,bennn,cennn,de# 20.26 70.41nnn,bennn,cennn,de#

Spleen

1 day 2 days

22.25 7 0.14 22.56 7 0.71

31.83 7 0.53nnn 32.69 7 0.09nnn

33.94 7 0.26nnn 38.63 7 0.21nnn

26.96 70.46nnn 31.10 70.33nnn

27.25 70.93nnn,bennn,cennn,de# 30.53 70.29nnn,bennn,cennn,de#

Results are mean 7S.E. of six set of observations taken on different days. Specific activity of G6PDH is expressed as m mole of NADPH oxidized min  1 mg  1 protein. Rats were given a mixture of vitamin A (2.0 mg/kg body weight), vitamin E (20.0 mg/kg body weight) and vitamin C (120 mg/kg body weight), for 15 days followed by 0.25 LD50 equivalent of pesticides orally, dissolved in 0.4 ml corn oil for one and two consecutive days. n P o 0.05, nnPo 0.001, nnnPo 0.0001 and #P 40.05 when compared with respective control, bewhen PM is compared with CPF, cewhen PM is compared with MPT and dewhen PM is compared with MLT.

Table 9 Effect of oral exposure of chlorpyrifos, methyl parathion and malathion, individually and in mixture, for one day and two days on the activity of glutathione reductase in rat tissues. Tissues

Treatment

Control

Chlorpyrifos

Parathion

Malathion

Pesticide mixture

Liver

1 day 2 days

1.097 0.01 1.067 0.06

1.69 7 0.04nnn 1.76 7 0.06nnn

1.86 7 0.04nnn 1.98 7 0.03nnn

1.407 0.04nnn 1.44 7 0.04nn

1.46 7 0.02nnn,benn,cennn,de# 1.39 7 0.05n,bennn,cennn,de#

Brain

1 day 2 days

2.12 7 0.02 2.29 7 0.02

3.44 7 0.02nnn 3.49 7 0.06nnn

3.62 7 0.01nnn 4.22 7 0.05nnn

2.53 7 0.02nnn 2.61 7 0.03nnn

2.77 7 0.04nnn,ben,cennn,dennn 3.28 7 0.03nnn,bennn,cennn,denn

Kidney

1 day 2 days

1.24 7 0.03 1.18 7 0.07

1.92 7 0.03nnn 2.047 0.05nnn

2.29 7 0.06nnn 3.14 7 0.04nnn

1.307 0.01# 1.34 7 0.06#

1.407 0.03n,bennn,cennn,den 1.63 7 0.05nn,benn,cennn,den

Spleen

1 day 2 days

1.307 0.04 1.34 7 0.05

2.17 7 0.05nnn 2.75 7 0.05nnn

2.42 7 0.02nnn 3.28 7 0.03nnn

1.65 7 0.02nnn 2.17 7 0.01nnn

1.99 7 0.06nnn,ben,cennn,denn 2.37 7 0.02nnn,bennn,cennn,dennn

Results are mean 7S.E. of six set of observations taken on different days. Specific activity of G6PDH is expressed as m mole of NADPH oxidized min  1 mg  1 protein. Rats were given 0.25 LD50 equivalent of pesticides orally, dissolved in 0.4 ml corn oil for one and two consecutive days. n P o Po 0.05, nnPo 0.001, nnnPo 0.0001 and #P4 0.05 when compared with respective control, bewhen PM is compared with CPF, compared with MPT and dewhen PM is compared with MLT.

3.5. Effect on activity of glutathione reductase Oxidized glutathione is reduced by the enzyme, glutathione reductase (GR) at the expense of NADPH. This enzyme plays an important role in glutathione recycling. It was observed in the present study that acute exposure with CPF, MPT and MLT singly or in combination caused induction of this enzyme in rat tissues. It was observed that the increase in the activity of hepatic GR were 55 percent, 71 percent, 28 percent and 34 percent on one day exposure and 66 percent, 87 percent, 36 percent and 31 percent on two days exposure with CPF, MPT, MLT and their mixture, respectively (Table 9). The increase in GR activity in the brain were 62 percent, 71 percent, 19 percent and 31 percent on one day exposure and 52 percent, 84 percent, 14 percent and 43 percent on two days exposure with CPF, MPT, MLT and their mixture, respectively. In the kidney and the spleen, almost same pattern of GR activity was observed in response to these pesticides exposure. In the kidney the increase were 55 percent, 85 percent, 5 percent and 13 percent while in the spleen increase were 67 percent, 86 percent, 27 percent and 53 percent on exposure with CPF, MPT, MLT and their mixture, respectively, for one day. Two days exposure caused 73 percent, 166 percent, 14 percent and 38 percent increase in the kidney and 105 percent, 145 percent, 62 percent and 77 percent increase in activity of GR

ce

when PM is

in the spleen of CPF, MPT, MLT and mixture treated rats, respectively (Table 9). MPT treatment for one and two days, caused highest increase in GR activity followed by CPF, mixture and MLT in rat tissues. Prior feeding of mixture of vitamin A, E and C offered some protection against pesticides induced alteration in the activity of GR. The increase in hepatic GR activity were 20 percent, 52 percent, 15 percent and 14 percent on one day exposure and 99 percent, 113 percent, 19 percent and 34 percent on two days exposure with CPF, MPT, MLT and mixture treated rats, respectively, in vitamin pre-fed group (Table 10). Similarly the increase in GR activity in the brain were 201 percent, 266 percent, 38 percent and 86 percent on one day exposure, and 202 percent, 160 percent, 72 percent and 119 percent on two days exposure with CPF, MPT, MLT and mixture treated rat, respectively. One day exposure caused 60 percent, 87 percent, 13 percent and 19 percent increase in the kidney and 113 percent, 149 percent, 12 percent and 25 percent increase in the spleen of rats exposed with CPF, MPT, MLT and their mixture, respectively, while two days exposure caused 41 percent, 122 percent, 19 percent and 21 percent increase in the kidney and 115 percent, 170 percent, 55 percent and 104 percent increase in GR activity in the spleen of rats, respectively (Table 10). Highest recovery was observed in the brain of rats on prior feeding of antioxidant vitamins for two weeks.

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239

Table 10 Effect of oral exposure of chlorpyrifos, methyl parathion and malathion, individually and in mixture, for one day and two days on the activity of glutathione reductase in tissues of rats prefed with mixture of antioxidant vitamins. Tissues

Treatment

Control

Chlorpyrifos

Parathion

Malathion

Pesticide mixture

Liver

1 day 2 days

1.13 7 0.08 0.91 7 0.07

1.36 7 0.01n 1.81 7 0.03nnn

1.727 0.01nnn 1.947 0.04nnn

1.30 70.02# 1.08 70.06#

1.29 7 0.01#,ben,cennn,de# 1.22 7 0.05n,bennn,cennn,de#

Brain

1 day 2 days

1.24 7 0.04 1.37 7 0.01

3.73 7 0.13nnn 4.14 7 0.05nnn

4.547 0.09nnn 3.567 0.02nnn

1.71 70.02nnn 2.36 70.07nnn

2.307 0.03nnn,bennn,cennn,dennn 3.007 0.07nnn,bennn,cennn,dennn

Kidney

1 day 2 days

1.05 7 0.02 1.16 7 0.01

1.68 7 0.02nnn 1.63 7 0.03nnn

1.967 0.01nnn 2.577 0.06nnn

1.19 70.04n 1.38 70.01nnn

1.25 7 0.02nnn,bennn,cennn,de# 1.407 0.01nnn,bennn,cennn,de#

Spleen

1 day 2 days

1.15 7 0.02 1.13 7 0.05

2.45 7 0.02nnn 2.43 7 0.09nnn

2.867 0.04nnn 3.057 0.05nnn

1.29 70.02nn 1.75 70.01nnn

1.44 7 0.01nnn,bennn,cennn,dennn 2.307 0.06nnn,be#,cennn,dennn

Results are mean 7 S.E. of six set of observations taken on different days. Specific activity of G6PDH is expressed as m mole of NADPH oxidized min  1 mg  1 protein. Rats were given a mixture of vitamin A (2.0 mg/kg body weight), vitamin E (20.0 mg/kg body weight) and vitamin C (120 mg/kg body weight), for 15 days followed by 0.25 LD50 equivalent of pesticides orally, dissolved in 0.4 ml corn oil for one and two consecutive days. n P o 0.05, nnPo 0.001, nnnP o0.0001 and #P 40.05 when compared with respective control, bewhen PM is compared with CPF, cewhen PM is compared with MPT and dewhen PM is compared with MLT.

4. Discussion Oxidative stress represents an imbalance between the production and manifestation of reactive oxygen species and a biological system’s ability to readily detoxify the reactive intermediates or to repair the resulting damage. Disturbances in the normal redox state of tissues can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids and DNA. In humans, oxidative stress is involved in many diseases including atherosclerosis, Parkinson’s disease, heart failure, myocardial infarction, Alzheimer’s disease, schizophrenia, bipolar disorder, fragile X syndrome, etc. (de Diego-Otero et al., 2009). Several drugs, xenobiotics and environmental pollutants are known to cause this imbalance between formation and removal of free radicals. Biological antioxidants including vitamins can prevent this uncontrolled formation of free radicals and activated oxygen species or inhibit their reaction with biological molecules. The destruction of most free radicals and ROS rely on the oxidation of endogenous antioxidants mainly scavenging and reducing molecules. GSH acts as reducing agent and a vital substance in detoxification, also provides antioxidant protection in the aqueous phase of the cellular systems; its antioxidant activity is through the thiol group of its cysteine residue. Like ascorbic acid, another important water-soluble antioxidant, GSH can directly reduce a number of ROS and is oxidized to GSSG in this process. Decrease in the levels of GSH in the liver, brain, kidney and spleen of rats in response to pesticide exposure either singly or in combination, observed in the present study, clearly shows the oxidizing conditions in these tissues. Lowered GSH/GSSG ratio in tissues of pesticides exposed rats, is indicative of oxidative stress. Liver is viewed as a glutathione generating organ, which supplies the kidney and intestine with other constituents for glutathione resynthesis. Intrahepatic glutathione is reported to offer protection against liver dysfunction by at least two ways: (i) as a substrate of GPx, GSH serves to reduce large variety of hydroperoxides before they attack unsaturated lipids or convert already formed lipid hydroperoxides to the corresponding hydroxy compounds; and (ii) as a substrate of glutathione-S-transferase, it enables the liver to detoxify foreign compounds or their metabolites and to excrete the products, preferably into the bile. Role of glutathione in protecting endothelial cells against hydrogen peroxide oxidant injury, protecting alveolar type II cells against paraquat induced injury and protection of intestinal epithelial cells from t-butylhydroperoxide or manadione-induced injury are reported.

Relatively high ratios of GSH/GSSG are maintained intracellularly through the action of GR and NADPH dependent reaction (Schafer and Buettner, 2001). GSH/GSSG ratio is the indicator of redox status of the tissues. Results of the present study showed significant decrease in this ratio in the liver, brain, kidney and spleen of rats on exposure with CPF, MPT and MLT singly or in combination indicating the induction of oxidative stress. These pesticides are reported to inhibit the enzymatic defense also in rat tissues (Ojha et al., 2011). Thus it can be concluded that these OP pesticides generate oxidative stress by inhibiting both enzymatic and nonenzymatic antioxidant defenses. The generated oxidative stress may contribute substantially in the overall toxicity of OP pesticides. Reduced and oxidized nicotinamide adenine dinucleotide (NADH, NAD þ ) and nicotinamide adenine dinucleotide phosphate (NADPH, NADP þ ) are essential coenzymes throughout the metabolism, and they participate in a number of enzymes in oxidant defense and xenobiotic detoxification pathways. In addition, it has been shown that NADH and NADPH are able to directly scavenge oxidants like hypohalous acids produced during inflammation (Prutz et al., 2000). However, the most significant role for NADPH in oxidant defense is through the regeneration of reduced glutathione (GSH), which is required for the metabolism of hydrogen peroxide and fatty acid hydroperoxides by GSH peroxidase. In addition to controlling the activity of redox sensitive enzymes, the NAD þ /NADH ratio can regulate the activity of various transcription factors, leading to changes in gene expression. This includes transcription factors that regulate circadian rhythms (Rutter et al., 2001) and repressor proteins involved in the control of cell growth and differentiation (Zhang et al., 2002). Fluctuations in NADPH/NADP þ or NADH/NAD þ ratios would affect crucial cellular metabolic processes. In the present study, abnormal activation of G6PD in all the pesticide exposed rat tissues cause an increase in cellular levels of NADPH, NADH and the activity of glutathione reductase. The enzyme G6PDH is the regulatory enzyme of hexose monophosphate shunt and catalyzes the reaction that generates reducing equivalents, e.g., NADPH from NADP. GR at the expense of this NADPH reduces GSSG to GSH. Decrease in the levels of NADH, NADPH and GSH, despite increase in G6PDH and GR, confirms the induction of oxidative stress on exposure with these pesticides. Increased generation of NADPH and increased conversion of GSSG to GSH by GR is not sufficient to maintain the normal redox state. Besides serving as a substrate in the GPx reaction, it also acts as a free radical scavenger and helps in the regulation of thiol disulfide concentration of a number of glycolytic enzymes

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and Ca2 þ -adenosine triphosphatases, thus indirectly maintaining intracellular Ca2 þ homeostasis. Moreover, GSH regenerates other scavengers and antioxidants such as ascorbic acid and a-tocopherol. The GSH/GSSG couple is very influential in controlling the configuration of cellular proteins through the reduction state of protein thiols (cysteine side chains). This includes the activity of several important transcription factors that regulate a variety of stress responses. Numerous reports are available in the literature showing protective effect of antioxidants against the pesticide-induced toxicity. In this context, both water-soluble (vitamin C or ascorbic acid) and lipid-soluble (vitamin A and E) vitamins comprise an important aspect on the antioxidant defense system (Zaidi and Banu, 2004). Each of these antioxidative systems has a specific activity/concentration, but they work synergistically to enhance the antioxidant capacity of the organism. Vitamin E or a-tocopherol has been shown to promote protection to cells exposed to oxidative stress by scavenging free radicals, stabilizing membranes and blocking the cascade of biochemical routs involved on cellular necrosis. a-Tocopherol is converted to tocopheryl radical, requiring ascorbate for its regeneration to reduced tocopherol. This combination of a-tocopherol and ascorbic acid has proven to be effective in preventing biochemical and behavioral deficits produced in animal models of metabolic diseases, as well as in age-related motor and memory deficit in rats. The present study has revealed a predominant effect of vitamin A, E and C in restoring the redox status of pesticide exposed rat tissues. The prior treatment of rats with vitamin A, E and C in combination resulted in a decrease in the ratio of NADPH/NADP þ , NADH/ NAD þ , GSH/GSSG, G6PDH and GR when compared with vitamin unfed groups. Our results are in accordance with the finding of Salah et al. (2010), where the ingestion of vitamin mixtures (vit A, E and C) readjusted and normalized the hematologic parameters around those of normal healthy control. In another finding vitamin E treatment ameliorated the effects of atrazine suggesting it as potential antioxidant against atrazine-induced oxidative stress (Singh et al., 2011). Vitamins A, E and C are reported to act as effective antioxidants of major importance for protection against diseases and degenerative processes caused by oxidative stress (Olas and Wachowiej, 2002). The present study concludes that selected OP pesticides have no interactive toxicity and they cause alteration in the redox status of rat tissues namely, liver, brain, kidney and spleen. Vitamins A, E and C have strong antioxidant activity and pre-feeding with these vitamins may improve the redox status of individual and protect from injuries caused by oxidative stress.

5. Conclusion Present study clearly indicates that the CPF, MPT and MLT exposure to rats cause induction of oxidative stress and alterations in redox status of the tissues by decreasing ratios of GSH/ GSSG, NADPH/NADP þ and NADH/NAD þ . Despite increased activities of glucose-6-phosphate dehydrogenase and glutathione reductase, the levels of GSH remained declined in the rat tissues. The results of the present study also suggested that prior feeding with mixture of antioxidant vitamins tend to reduce the toxicities of these pesticides. These pesticides when given in mixture do not potentiate the toxic effects of each other.

Acknowledgments The financial supports of Department of Science and Technology, New Delhi, India in the form of FIST Grant to the School, and

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