Organophosphate Pesticides-Induced Changes in the Redox Status of Rat Tissues and Protective Effects of Antioxidant Vitamins Vibhuti Mishra, Nalini Srivastava School of Studies in Biochemistry, Jiwaji University, Gwalior 474 011, India

Received 24 January 2013; revised 31 October 2013; accepted 3 November 2013 ABSTACT: Organophosphates (OPs) pesticides are among the most toxic synthetic chemicals purposefully added in the environment. The common use of OP insecticides in public health and agriculture results in an environmental pollution and a number of acute and chronic poisoning events. Present study was aimed to evaluate the potential of monocrotophos and quinalphos to effect the redox status and glutathione (GSH) homeostasis in rat tissues and find out whether antioxidant vitamins have some protection 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 that NADPH/NADP1 and NADH/NAD1 ratios were decreased in the liver and brain of rats on exposure with mococrotophos, quinalphos, and their mixture. These pesticides, alone or in combination, caused alterations in the activities of GSH reductase and glucose-6-phosphate dehydrogenase in the rat tissues. However, the expression of the GSH recycling enzymes did not show significant alterations as compared to control. From the results, it can be concluded that these pesticides generate oxidative stress but their effects were not synergistic when given C 2013 together and prior feeding of antioxidant vitamins tend to reduce the toxicities of these pesticides. V Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2013.

Keywords: quinalphos; monocrotophos; redox imbalancer; GSH/GSSG ratio; antioxidant vitamins

INTRODUCTION Organophosphate (OP) pesticides are the most widely used synthetic chemicals throughout the world. The main target of OP pesticides is acetylcholinesterase (AChE) which hydrolyses acetylcholine (ACh) in the cholinergic synapses and in neuromuscular junctions where this enzyme plays a key role in cell-to-cell communication (Kwong, 2002). The resulting accumulation of ACh eventually causes cholinergic crisis due to continuous stimulaCorrespondence to: N. Srivastava; e-mail: [email protected] Contract grant sponsor: Department of Science and Technology, New Delhi, India Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.21924

tion of muscarinic and nicotinic receptors. Besides being potent anticholinesterase compounds, pesticides are known to cause number of other adverse health effects including generation of oxidative stress (Soltaninejad and Abdollahi, 2009; Lukaszewicz-Hussain 2010). Due to inhibition of oxidative phosphorylation by OPs coupled with high-energy consumption and their metabolism by cytochrome P450s, reactive oxygen species (ROSs) are generated in large amount, which eventually leads to oxidative stress. Excessive production of ROS may cause damage to the vital macromolecules, namely, lipids, proteins, and nucleic acids. Oxidative stress is generated by imbalance between the rate of production of ROS and their removal by antioxidant defense system. Cells are equipped with both enzymatic and nonenzymatic antioxidant defense systems. OP pesticides have been reported to

C 2013 Wiley Periodicals, Inc. V

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inhibit the activities of antioxidant enzymes namely, superoxide dismutase, catalase, and glutathione (GSH) peroxidase (Verma and Srivastava, 2003; Soltaninejad and, Abdollahi, 2009; Lukaszewicz-Hussain 2010; Karami-Mohajeri and Abdollahi, 2011; Ojha and Yaduvanshi, 2011). OP pesticides have also been reported to effect the expression of stress-proteins (Gupta et al., 2005; Woo et al., 2009; Ceyhun et al., 2010; Kapka-Skrzypczak et al., 2011). Iprobenfos, a commonly used OP pesticide, exposure has been shown to cause alterations in the expression of a set of genes encoding antioxidant enzymes and stress-responsive proteins investigated by real-time quantitative PCR in different tissues of Oryzias javanicus (Woo et al., 2009). GSH is one of the most important components of the nonenzymatic antioxidant defense and a vital substance in detoxification, cell physiology, 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; Townsend et al., 2003). GSH is also a substrate of enzymes, GSH peroxidase, and glutathione-S-transferase. GSH/GSSG ratio is a very important indicator of the redox status of the cell and a decreased GSH/GSSG ratio is a consequence of oxidative stress. GSH is converted to GSSG after imparting its antioxidant role. The fact that intracellular levels of GSH are very much higher than GSSG is consistent with the presence of very active enzyme that catalyzes reduction of GSSG at the expense of NADPH. GSH is present inside cells mainly in its reduced form. In the healthy cells GSSG, rarely exceeds 10% of total cell GSH. The formidable reducing power of the GSH/GSSG couple is a profound physicochemical asset for the aerobic organism. The excessive free radicals generated due to oxidative stress deplete GSH from the cells rendering the tissue more vulnerable to toxic damage (Forman et al., 2009). To recycle GSSG to GSH, the cells utilize NADPH and the enzyme gluatathione reductase (Wu et. al., 2004). The first reaction of pentose phosphate pathway catalyzed by glucose-6-phosphate 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 toxicological evaluation, mostly providing the cellular and biochemical mechanism of toxicity of chemicals and drugs. Redox status of the cells and tissues is an important indicator of oxidative stress. Oxidative stress reduces GSH pool and decreases GSH/GSSG ratio (Gerard-Monier and Chaudiere, 1996; Pompella et al., 2003; Zasadowski et al., 2004). ROS-mediated toxicity by oxidation of GSH to GSSG in response to pesticide exposure has been demonstrated by many workers (Doraval and Hontela, 2003; Pertejo et al., 2008; Verma et al., 2009; Ajiboye, 2010; Singh et al., 2011).

Environmental Toxicology DOI 10.1002/tox

Accumulating evidences have suggested that NAD (including NAD1 and NADH) and NADP (including NADP1 and NADPH) belong to the fundamental common mediators of various biological processes, including energy metabolism, mitochondrial functions, calcium homeostasis, antioxidation/generation of oxidative stress, gene expression, immunological functions, aging, and cell death (Ying et al., 2008). NADPH/NADP1 are directly involved in oxidative stress defense, redox regulation and GSH/GSSG redox balance. In addition to their classical role in miotochondrial energy production, novel roles of NADH/NAD1 in oxidative stress and apoptosis are underscored by their function in modulating activities of sirtuin proteins, a class of NAD1dependent deacetylases and mono-ADP ribosyl transferases (Circu et al., 2010). Antioxidant compounds including vitamins are known to reduce oxidative stress resulting from or enhanced by a large variety of conditions, including nutritional imbalance, exposure to chemicals and physical agents in the environment, strenuous physical activities, injury, and hereditary disorders. While many enzymes and compounds are involved in protecting cells from the adverse effects of oxidative stress, vitamin A, E and C occupy an important and unique position in the overall antioxidant defense (Chow 1991; Gershoff 1993; Omaye et al., 1997). Vitamin C, as a water-soluble antioxidant is reported to neutralize oxidative DNA damage and hence genetic mutations (Yu et al., 2008). Vitamin E is a family of lipid-soluble vitamins, of which a-tocopherol is the most active form and is a powerful biological antioxidant, may effectively minimize oxidative stress, lipid peroxidation, and toxic effects of ROS in biological systems. Previous studies have reported the protective effect of vitamin C, vitamin E, or vitamin C 1 vitamin E with different class of OP such as chlorpyrifos, methyl parathion, methidathion, phosalone, dimethoate, malathion, and diazinon (Ogutcu et al., 2006; Ajiboye 2010). Monocrotophos [dimethyl(E)-1-methyl-2-(methyl carbamoyl)vinyl phosphate, MCP] and quinalphos [O,O-diethylO-quinoxalin-2-yl phosphorothioate, QNP] are broad spectrum, systemic OP pesticides. They are extensively used in agriculture throughout the world including India. These pesticides are highly toxic to nontarget organisms including mammals. Studies suggest that these pesticides induce both acute and chronic toxicity. Although these pesticides alter the activities of antioxidant- and tissue-specific enzymes and affect homeostasis of different organs such as liver, brain, spleen and kidney, little information is available in literature on the effect of these pesticides on the status of GSH/GSSG redox couple and enzymes and coenzymes involved in recycling of GSSG. The present study is aimed to analyze the effect of acute exposure of MCP and QNP, individually and in mixture, on the GSH recycling system, expression of recycling enzymes and the levels of NADH/NAD1 and NADPH/NADP1 in rat tissues. The study will be carried out to evaluate the protective effect of antioxidant vitamins on

REDOX IMBALANCE ON ORGANOPHOSPHATE PESTICIDE EXPOSURE

the pesticide-induced alterations in GSH/GSSG, NADH/ NAD1 and NADPH/NADP1 ratios and the enzymes of GSH recycling system.

MATERIALS AND METHODS Chemicals Chemicals used in the present study were of highest purity grade. Tris, potassium chloride, sodium carbonate, sodium bicarbonate disodium hydrogen phosphate, sodium dihydrogen phosphate, phosphoric acid, sodium hydroxide, ethylenediamine tetraacetic acid, nicotinamide, ethanol, MOPS (3-[Nmorphlino] propanesulfonic acid) and magnesium chloride were purchased from HIMEDIA Chemicals, India. O-Phthalaldehyde, Folin-Ciocalteu’s reagent, alcohol dehydrogenase, oxidized GSH, G6PDH, glucose-6-phosphate, nicotinamide adenine dinucleotide phosphate, reduced nicotinamide adenine dinucleotide phosphate, phenazine ethosulfate, 3-(4,5dimethythiazolyl)-2,5-dipheny-2H-tetrazolium bromide (MTT), TRI reagent, formamide, trisodium citrate, sodium acetate, and ethidium bromide were purchased from Sigma Aldrich Chemicals Private Limited, MO. One step RT PCR kit and DNA molecular weight markers (100–600 bp) were obtained from Qiagen India Private Limited, New Delhi, and PCR primers were obtained from Eurofins Genomics India Private Limited. Monocrotophos and Quinalphos were kind gift from Gujarat Insecticides Limited, Ankleshwar, India.

Experimental Animals Adult male albino rats of Wistar strain (Rattus norvegicus) weighing about 120 6 10 g were used in the present study. Rats were obtained from the animal facilities of Defence Research and Development Establishment, Gwalior, India, and were maintained in a light (light-dark cycle of 12 h each) and temperature (25 6 2 C) controlled animal room of our department on standard pellet diet (obtained from Amrut Rat and Mice Feed, New Delhi, India) and tap water ad libitum. Rats were acclimatized for one week prior to the start of the experiment.

Experimental Design The animals were randomly divided into two groups of 24 animals each. Each group was further divided into four subgroups of six animals. The rats of first subgroup received 4.5 mg MCP/kg body weight dissolved in 0.4 mL corn oil orally for 2 consecutive days [dose equivalent to 0.25 LD50 of MCP is 18 mg/kg body weight, Gains, 1969], the second group received 5 mg QNP/kg body weight dissolved in 0.4 mL corn oil orally for 2 consecutive days [dose equivalent to 0.25 LD50 as the reported LD50 of QNP is 20 mg/kg body weight, Raizada et al., 1993] and the rats of the third subgroup received 0.25 LD50 equivalent mixture of MCP and

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QNP (each pesticide was present in a dose of 1/8 LD50 equivalent), orally dissolved in 0.4 mL corn oil, for 2 consecutive days, respectively. The rats of IV subgroup served as control and received 0.4 mL corn oil orally for 2 consecutive days. The animals of second group were given a mixture of vitamin A (2.0 mg), vitamin E (20.0 mg), and vitamin C (120 mg) per 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) daily for 15 days. The rats were divided into four subgroups; and were given treatment of MCP, QNP, their mixture, and corn oil as the rats of the first group. The doses of vitamins 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% NaCl, and used for different estimations. Animals were handled, ethically treated, and humanly killed as per the rules and instructions of Ethical Committee of Animal Care of Jiwaji University, Gwalior, India, in accordance with the Indian National law on animal care and use.

Estimations The levels of reduced and oxidized GSH (and GSSG) were estimated as described by Hssin and Hilf (1976). Oxidized and reduced pyridine nucleotides (NAD1, NADH, NADP1, and NADPH) were assayed by the method of Zerez et al. (1987). GSH reductase (GR, E.C. No. 1.6.4.2) activity in tissue homogenate was determined by measuring the rate of conversion of NADPH to NADP1 by the enzyme (Tayarani et al., 1989) and G6PDH (E.C. No. 1.1.1.49) activity in tissues was estimated by the method of Askar et al. (1996). Protein in tissue samples was estimated by the method of Lowry et al. (1951) using bovine serum albumin as standard.

Studies on Expression Total RNA from the tissues was separated by the method described by Sambrook and Russell (2001). Briefly, 100 mg of desired tissue was isolated and homogenized in 1.0 mL ice-cold monophasic lysis reagent (TRI reagent). The homogenate was incubated for 5 min at room temperature followed by addition of 0.2 mL chloroform with vigorous shaking. The mixture was centrifuged at 3360 3 g for 15 min in cold and the upper aqueous layer was separated. The RNA was precipitated 0.25 mL isopropanol and 0.25 mL RNA precipitation solution (1.2 M NaCl and 0.8 M trisodium citrate) by adding, mixed thoroughly, and incubated at room temperature for 10 min. The solution was centrifuged at 3360 3 g for 10 min, and the pellet containing RNA was separated. The pellet was washed twice with 75% ethanol and dissolved in 50 mL DEPC-treated water. The reverse transcriptase (RT) PCR was performed using Qiagen one-

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step RT PCR kit. A master mix was prepared containing RT PCR buffer, dNTP mix, Q solution, primers, reverse transcriptase enzyme, and template RNA as per manufacturer’s protocol. The primers used for GR mRNA and glucose-6phoshate mRNA were: Glutathionereductase

[Left Primer: GTTAATCC GGGGTTGGTTTT] [Right Primer: ATGAGG CTGTAGCTGGAGGA]

Glucose-6-phosphate dehydrogenase

[Left Primer: AGCCTCCTA CAAGCACCTCA] [Right Primer: TGGTTCG ACAGTTGATTGGA] [Left Primer: CGCGGTTCT ATTTTGTTGGT] [Right Primer: AGTCGGCAT CGTTTATGGTC]

18S RNA

18S RNA was used as housekeeping gene. First strand of cDNA was synthesized using Omniscript reverse transcriptase at 50 C for 30 min. Thirty-five cycles of PCR were done with a profile of 94 C for 10 , 59 C for 10 , and 72 C for 10 . The amplicons were analyzed on 1.5% TAE-Agarose gel. Results are expressed as mean 6 SE of six sets of observations taken on different days. Statistical analyses were performed using Sigma Stat Statistical software version 2.0. All the statistical analyses were performed using one-way analysis of variance post hoc Bonferroni’s multiple comparison test applied across the treatment groups. Significance was based on P value 0.05 when compared with respective control.

group, respectively, was observed. The hepatic GSH/GSSG ratio was decreased 34, 47, and 60% while, in brain, the decrease observed was 57, 24, and 48% in MCP, QNP, and mixture-treated group, respectively (Table II).

Effect of Pesticide Exposure on the Levels of NAD1, NADH, NADH/NAD1, NADP1, NADPH, and NADPH/NADP1 Ratio NAD(P)1 and NAD(P)H form an important pair whose ratio is very sensitive to the redox status of the organism. Results of the present study showed that MCP and QNP exposure either singly or in mixture, caused decrease in the levels of NADH in both liver and brain of rats while the levels of NAD1 are correspondingly increased. The decrease in the levels of NADH was 44, 30, and 32% in the liver while 80, 59, and 43% in the brain on exposure with MCP, QNP, and mixture, respectively (Table III). The increase observed in NAD1 levels was 184, 340, and 133% in the liver and 146, 68, and 64% in the brain of rats given 0.25 LD50 equivalent of MCP, QNP, and their mixture orally for 2 consecutive days, respectively. Decreased levels of NADH and increased levels of NAD1 caused decrease

in the ratio of NADH/NAD1 in rat tissues on exposure with these pesticides. The decrease observed in the ratio of NADH/NAD1 was 80, 84, and 71% in the liver and 92, 76, and 66% in the brain of rats on exposure with MCP, QNP, and their mixture, respectively (Table III). The changes in the levels of NADPH and NADP1 showed similar pattern on exposure with MCP and QNP either singly or in combination for two days. The decrease in NADPH levels ranged from 4 to 68% while the increase in the levels of NADP1 ranged from 57 to 427% in the liver and the brain of rats given exposure with these pesticides. The ratio of NADPH/ NADP1 was decreased by 62, 87, and 80% in the liver and 58, 84, and 54% in the brain on exposure with MCP, QNP, and their mixture, respectively (Table V). Prior feeding of mixture of antioxidant vitamins for 15 days followed by exposure with pesticides offered some protection against pesticide-induced alterations in the levels of reduced and oxidized pyridine nucleotides. Although the levels of NADH and NADPH remained decreased on exposure with pesticides either singly or in mixture, when compared with control but the decrease was marginal. The decrease in NADH levels ranged from 12 to 42% in the liver and 10– 18% in the brain (Table IV) while increase ranging from 28

TABLE III. Effect of oral exposure of monocrotophos and quinalphos individually and in mixture, for 2 days, on the levels of reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD1) and their ratio (NADH/NAD1) in rat tissues Tissue Liver NADH NAD1 NADH/NAD1 Brain NADH NAD1 NADH/NAD1

Control

Monocrotophos

Quinalphos

Mixture

7.97 6 1.49 5.70 6 1.60 1.40

4.48 6 0.05# 16.16 6 2.37*** 0.28

5.62 6 1.35* 25.12 6 3.07** 0.22

5.40 6 0.29# 13.28 6 1.80* 0.41

2.45 6 0.60 3.67 6 0.96 0.67

0.50 6 0.01* 9.04 6 0.20* 0.055

1.00 6 0.15** 6.15 6 0.22** 0.16

1.41 6 0.16# 6.02 6 0.23* 0.23

NADH and NAD1 levels are expressed as nmoles NADH or NAD1 g21 tissue. Results of mean 6 SE of six set of observations taken on different days. *P < 0.05, **P < 0.01, ***P < 0.001 when compared with respective control.

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TABLE IV. Effect of prior feeding of mixture of antioxidant vitamins on the monocrotophos and quinalphos induced alterations in the levels of NADH, NAD1, and their ratio (NADH/NAD1) in rat tissues Tissue Liver NADH NAD1 NADH/NAD1 Brain NADH NAD1 NADH/NAD1

Control

Monocrotophos

Quinalphos

Mixture

5.22 6 0.311 1.97 6 0.10 2.64

4.62 6 0.14# 2.52 6 0.14** 1.83

3.10 6 0.04*** 2.88 6 0.022** 1.07

3.04 6 0.02*** 2.70 6 0.14** 1.12

5.67 6 0.14 6.21 6 0.05 0.913

5.08 6 0.26*** 8.54 6 0.16*** 0.594

4.74 6 0.04*** 8.57 6 0.18*** 0.55

4.60 6 0.23*** 7.82 6 0.06*** 0.59

NADH and NAD1 levels are expressed as nmoles NADH or NAD1 g21 tissue. Results of mean 6 SE of six set of observations taken on different days. *P < 0.05, **P < 0.01, ***P < 0.001 and #P > 0.05 when compared with respective control.

TABLE V. Effect of oral exposure of monocrotophos and quinalphos individually and in mixture, for 2 days, on the levels of reduced and oxidized nicotinamide adenine dinucleotide (NADPH and NADP1) and their ratio (NADPH/NADP1) in rat tissues Tissue Liver NADPH NADP1 NADPH/NADP1 Brain NADPH NADP1 NADPH/NADP1

Control

Monocrotophos

Quinalphos

Mixture

3.81 6 0.10 1.11 6 0.43 3.43

3.00 6 0.01*** 2.27 6 0.32*** 1.32

2.54 6 0.16*** 5.85 6 0.95# 0.45

3.21 6 0.14** 4.61 6 0.30*** 0.70

1.14 6 0.18 0.82 6 0.17 1.39

0.75 6 0.05** 1.29 6 0.12** 0.58

0.36 6 0.07# 1.64 6 0.12* 0.22

1.10 6 0.06# 1.72 6 0.07*** 0.64

NADPH and NADP1 levels are expressed as nmoles NADPH or NADP1 g21 tissue. Results of mean 6 SE of six set of observations taken on different days. *P < 0.05, **P < 0.01, ***P < 0.001 when compared with respective control.

to 37% in the hepatic NAD1 levels and 26–38% in the brain NAD1 levels was observed in the group received mixture of antioxidant vitamins prior to pesticide exposure. The decrease observed in the ratio of NADH/NAD1 was 31, 59, and 58% in the liver and 35, 40, and 36% in the brain of rats

on exposure with MCP, QNP, and mixture, respectively. Prior feeding of mixture of antioxidant vitamins also caused improvement in the pesticide-induced alterations in the levels of NADPH, NADP1, and their ratio when compared with vitamin unfed pesticide-treated group. NADPH levels

TABLE VI. Effect of prior feeding of mixture of antioxidant vitamins on the monocrotophos and quinalphos induced alterations in the levels of NADPH, NADP1, and their ratio (NADPH/NADP1) in rat tissues Tissue Liver NADPH NADP1 NADPH/NADP1 Brain NADPH NADP1 NADPH/NADP1

Control

Monocrotophos

Quinalphos

Mixture

3.90 6 0.11 1.10 6 0.14 3.5

2.70 6 0.10*** 1.90 6 0.09*** 1.42

2.19 6 0.05*** 1.93 6 0.06*** 1.15

2.98 6 0.05*** 2.32 6 0.14*** 1.28

1.20 6 0.04 0.76 6 0.02 1.57

0.73 6 0.05*** 1.08 6 0.03*** 0.67

0.60 6 0.02** 1.00 6 0.03*** 0.60

0.80 6 0.03* 1.01 6 0.03*** 0.79

NADPH and NADP1 levels are expressed as as nmoles NADPH or NADP1 g21 tissue. Results of mean 6 SE of six set of observations taken on different days. *P < 0.05, **P < 0.01, ***P < 0.001 and #P > 0.05 when compared with respective control.

Environmental Toxicology DOI 10.1002/tox

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TABLE VII. Effect of oral exposure of monocrotophos and quinalphos individually and in mixture, for 2 days, on the activities of glutathione reductase and glucose-6-phosphate dehydrogenase in rat tissues Tissue

Control

Glutathione reductase Liver 7.05 6 0.27 Brain 21.00 6 0.01 Glucose-6-phosphate dehydrogenase Liver 37.67 61.33 Brain 36.00 61.26

Monocrotophos

Quinalphos

Mixture

4.48 6 0.30*** 5.68 6 1.67***

4.20 6 0.28*** 16.83 6 0.87

4.80 6 0.30*** ***

24.00 6 0.73*** 17.50 6 1.91***

62.00 6 4.80** 23.33 6 1.20***

25.17 6 1.19*** 18.83 6 0.87***

7.10 6 0.77***

Activity of glutathione reductase and glucose-6-phosphate dehydrogenase is expressed as mmol of NADPH oxidized min21 mg21 protein. Results of mean 6 SE of six set of observations taken on different days. *P < 0.05, **P < 0.01, ***P < 0.001, when compared with respective control.

were decreased and NADP1 levels were increased on exposure with MCP, QNP, and their mixture in the liver and the brain of rats decreasing the ratio of NADPH/NADP1. The decrease observed in the ratio of NADPH/NADP1 was 59, 67, and 63% in the liver and 57, 62, and 50% in the brain of rats on exposure with MCP, QNP, and their mixture, respectively, to vitamin fed rats (Table VI).

Effect of Pesticide Exposure on the Activities of Glutathione Reductase and Glucose-6-phosphate Dehydrogenase and Their Expression The results of the study showed that the exposure of MCP and QNP either singly or in mixture caused decrease in the activity of GR in both the liver and the brain. The decrease in the activity of hepatic enzyme was 36, 40, and 32% and in the brain was 73, 20, and 66% on exposure with MCP, QNP, and their mixture, respectively (Table VII). The activity of G6PDH was decreased by 36 and 52% in the liver and the brain on exposure with MCP while QNP exposure caused 65% increase in the liver and 35% decrease in the activity of the enzyme in brain, respectively. The group receiving mixture of these pesticides showed 33 and 48% decrease in the activity of G6PDH in the liver and brain, respectively (Table VII). Prior feeding of mixture of antioxidant vitamins showed some protection against pesticide toxicity and the decreased

activities of GR and G6PDH were restored to some extent. The decrease in the activity of GR remained 3, 15, and 22% in the liver and 16, 6, and 28% in the brain on exposure with MCP, QNP, and their mixture, respectively (Table VIII). The activity of G6PDH was also restored to some extent, and the decrease remained 22, 21, and 11% in the liver and 37, 19, and 30% in the brain of the MCP, QNP, and mixture exposed group (Table VIII). It was observed that 2 days exposure with MCP and QNP, singly or in combination altered the expression of G6PDH and GR genes in the liver and the brain of rats also. MCP exposure caused inhibition of the transcription of GR gene in the rat liver when compared with the control while the transcription of GR gene in the liver of QNP-exposed rats did not seem to be altered Fig. 1. When combined exposure of these two pesticides was given to the rats, the transcription of GR gene was marginally inhibited. In the brain, MCP and QNP exposure has not caused any marked change in the expression of GR gene while combined exposure caused slight decrease in the transcription (Fig. 1[A]). The QNP and combined exposure caused an increase in the expression of G6PDH gene in the rat liver, though the increase in the transcription of G6PDH gene was more pronounced on QNP exposure. MCP exposure did not show any change in the transcription of G6PDH gene in the liver (Fig. 1[B]). In the brain, no marked change in the expression of G6PDH gene was observed in the MCP, QNP, or mixture exposed rats when compared with the control (Fig. 1[B]).

TABLE VIII. Effect of prior feeding of mixture of antioxidant vitamins on the monocrotophos and quinalphos induced alterations in the activities of glutathione reductase and glucose-6-phosphate dehydrogenase in rat tissues Tissue

Control

Glutathione reductase Liver 6.90 6 0.43 Brain 21.00 6 1.26 Glucose-6-phosphate dehydrogenase Liver 39.36 6 0.24 Brain 42.14 6 0.49

Monocrotophos

Quinalphos

Mixture

7.12 6 0.12# 20.67 6 1.02#

5.85 6 0.02* 22.17 6 1.87#

5.35 6 0.07** 15.17 6 0.31***

30.90 6 0.40*** 26.42 6 0.79***

31.28 6 1.55*** 34.05 6 0.48***

34.87 6 1.43*** 29.76 6 1.61***

Activity of glutathione reductase and glucose-6-phosphate dehydrogenase is expressed as mmol of NADPH oxidized min21 mg21 protein. Results of mean 6 SE of six set of observations taken on different days. *P < 0.05, **P < 0.01, ***P < 0.001, and #P > 0.05 when compared with respective control.

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Fig. 1. Effect of acute expoure of MCP, QNP, singly, and in combination on the levels of glutathione reductase (A) and glucose-6-phosphate dehydrogenase (B) mRNA in rat liver and brain.

DISCUSSION Oxidative stress represents an imbalance between the production and manifestation of ROSs 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. (Alfadda and Sallam, 2012). 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 (Frei, 1994; Zaidi and Banu, 2004; Salah et al., 2010). The destruction of most free radicals and ROS rely on the oxidation of endogenous antioxidants mainly scavenging and reducing molecules. Both

Environmental Toxicology DOI 10.1002/tox

enzymatic and nonenzymatic antioxidant systems work synergistically to protect the cells from the oxidative injuries (Irshad and Chaudhuri, 2002). The tripeptide, GSH is the major antioxidant and redox regulator in cells that is important in combating oxidation of cellular constituents. Cells spend a great deal of energy to maintain high levels of GSH, which in turn helps to keep proteins in a reduced state (Forman et al., 2009). Drugs, pollutants, chemicals, infections, and inflammation in the body can increase ROS generation and/or decrease GSH levels and cause a shift in the cellular redox status resulting in more oxidative damaged macromolecules. An alteration of the normal redox balance can alter the activities of various enzymes and cell signaling pathways in tissues and may thus be an important mechanism in exerting toxicity of various xenobiotics and of mediating the pathogenesis of many diseases. Decrease in the levels of GSH in liver and brain of rats on exposure with monocrotophos and quinalphos either singly or in mixture, observed in the present study clearly demonstrate the oxidizing conditions in the tissues. Decrease in GSH/GSSG is indicative of oxidative stress as quite high GSH/GSSG ratios are maintained under normal

REDOX IMBALANCE ON ORGANOPHOSPHATE PESTICIDE EXPOSURE

circumstances (Ojha and Srivastava, 2012). Liver is viewed as GSH generating organ, which supplies the kidney, intestine, and other tissues with other constituents of GSH resynthesis. Intrahepatic GSH is reported to afford 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; (ii) as a substrate of glutathione-Stransferase, it enables the liver to detoxify foreign compounds or their metabolites and to excrete the products, preferably into the bile. Role of GSH 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 menadione induced injury are reported (Rana et al., 2002). Relatively high GSH/GSSG ratios are maintained by the enzyme GSSG reductase at the expense of NADPH. 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 and the brain of rats on exposure with MCP and QNP singly or in combination, indicating the induction of oxidative stress. OP pesticides have been shown to induce oxidative stress and affect the redox status of the tissues (Pertejo et al., 2008; Soltaninejad and Abdollahi, 2009; Ojha and Srivastava, 2012) which may be the major contributor of OP pesticide toxicity. The results of the present study also showed the decrease in the activity of GR in the liver and the brain of rats on exposure with these pesticides either singly or in combination, may be one of the reasons of decreased GSH levels in these tissues. The supply of NADPH is also decreased as the activity of G6PDH is decreased in the liver on MCP exposure either singly or in combination with QNP and in the brain on exposure of these pesticides either singly or in combination. The activity of G6PDH was significantly increased in the liver of rats on exposure with QNP. However, despite the increased activity of G6PDH in the liver of QNP-treated rats, the levels of NADPH were decreased. Besides serving as a substrate in the GPx reaction, GSH also acts as a free radical scavenger and helps in the regulation of thiol disulfide concentration of a number of glycolytic enzymes and Ca11-adenosine triphosphatases, thus indirectly maintaining intracellular Ca11 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. Pyridine nucleotides collectively comprise reduced and oxidized nicotinamide adenine dinucleotide (NADH/NAD1) and reduced and oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP1, NADP1), which are

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classically associated with ATP production and reductive biosynthesis, respectively. NADPH/NADP1 are also linked to defense against oxidative stress and redox regulation. However, the most significant role for NADPH in oxidant defense is through the regeneration of reduced GSH, which is required for the metabolism of hydrogen peroxide and fatty acid hydroperoxides by GSH peroxidase. NAD(P)H quinone-oxidoreductase may exert a protective effect against redox cycling of xenobiotics and oxidative stress (Zhang et al., 2002; Circu et al., 2010). In addition to controlling the activity of redox sensitive enzymes, the NAD1/NADH ratio can regulate the activity of various transcription factors, leading to changes in gene expression. This includes transcription factors that regulate circadian rhythms and repressor proteins involved in the control of cell growth and differentiation (Circu et al., 2010; Ghosh et al., 2010). The changes in the transcription of G6PDH and GR observed in the present study may be due to oxidative stress and/or altered redox status in the tissues of pesticide exposed rats. Fluctuations in NADPH/NADP1 or NADH/NAD1 ratios would affect crucial cellular metabolic processes. Antioxidant vitamins have long been considered to have protective role against free radicals-induced cell damage. Numerous reports are available in literature on the protective effects of antioxidants against the pesticide-induced toxicity. In this respect, both water-soluble (vitamin C) and lipidsoluble (vitamin A and E) vitamins comprise an important aspect of antioxidant defense system (Zaidi and Sahu, 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 atocopherol 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 atocopherol 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 agerelated motor and memory deficit in rats. The present study has revealed a predominant effect of vitamins A, E, and C in restoring the redox status of pesticide exposed rat tissues. The prior treatment of rats with vitamins A, E, and C in combination, resulted in restoration of ratios of NADPH/ NADP1, NADH/NAD1, GSH/GSSG, G6PDH, and GR when compared with vitamin unfed groups. Highest recovery of GSH/GSSSG ratio was seen in the liver of MCP and the brain of QNP-treated rats; of NADH/NAD1 ratio in liver and brain of MCP-treated rats while moderate recovery was observed in NADPH/NADP1 ratio of both the tissues of rats. The activity of GR was restored maximally in both the tissues of QNP- and MCP-treated rats while G6PDH activity was recovered moderately in both the tissues of rats pre fed with antioxidant vitamins. Our results are in accordance with

Environmental Toxicology DOI 10.1002/tox

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MISHRA AND SRIVASTAVA

the finding of Salah et al. (2010), where the ingestion of vitamin mixtures (vitamins 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-I-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. 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 and brain. Vitamins A, E, and C have strong antioxidant activity and prefeeding with these vitamins may improve the redox status of individual and protect from injuries caused by oxidative stress.

Gupta SC, Siddique HR, Saxena DK, Chowdhuri DK. 2005. Hazardous effect of organophosphate compound, dichlorvos, in transgenic Drosophila melanogaster (hsp70-lacZ): Induction of hsp70, anti-oxidant enzymes and inhibition of acetylcholinesterase. Biochim Biophys Acta 1725:81–92.

REFERENCES

Karami-Mohajeri S, Abdollahi M. 2011. Toxic influence of organophosphate, carbamate, and organochlorine pesticides on cellular metabolism of lipids, proteins, and carbohydrates: A systematic review. Hum Exp Toxicol 30:1119–1140.

Ajiboye TO. 2010. Redox status of the liver and kidney of 2,2dichlorovinyl dimethyl phosphate (DDVP) treated rats. Chem Biol Interact 185:202–207. Alfadda AA, Sallam RM. 2012. Reactive oxygen species in health and disease. J Biomed Biotechnol 936486. doi:10.1155/2012/ 936486. Askar MA, Sumathy K, Baquer NJ. 1996. Regulation and properties of glucose-6-phosphate dehydrogenase from rat brain. Indian J Biochem Biophys 33:512–518. Ceyhun SB, Sentuk M, Ebinci D, Erdogan D, Ciltas A, Kocaman EM. 2010. Deltamethrin attenuates antioxidant defense system and induces the expression of heat shock protein 70 in rainbow trout. Comp Biochem Physiol 152:215–223. Chow CK. 1991. Vitamin E and oxidative stress. Free Rad Biol Med 11:215–232. Circu ML, Aw TY. 2010. Reactive oxygen species, cellular redox systems, and apoptosis. Free Rad Biol Med 48:749–762. Doraval J, Hontela A. 2003. Role of glutathione redox cycle and catalase against defense oxidative stress induced by endosulfan in adrenocortical cells in rainbow trout (Oncorhychus mykiss). Toxicol Appl Pharmacol 192:191–200. Forman HJ, Zhang H, Rinna A. 2009. Glutathione: Overview of its protective roles, measurement, and biosynthesis. Mol Aspects Med 30:1–12. Frei B. 1994. Reactive oxygen species and antioxidant vitamin: Mechanism of action. Am J Med 97:58–138. Gaines TB. 1969. Acute toxicity of pesticides. Toxicol Appl Pharmacol 14:515–534. Gerard-Monier D, Chaudiere J. 1996. Metabolism and antioxidant function of glutathione. Path Biol 44: E209–E214.

Han D, Hanawa N, Saberi B, Kaplowitz N. 2005. Mechanism of liver injury. III Role of glutathione redox status in liver injury. Am J Physiol Gastrointest Liver Physiol 291: G1–G7. Hissin PJ, Hilf R. 1976. Fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem 74: 214–226. http://en.wikipedia.org/wiki/Glutathione. Irshad M, Chaudhuri PS. 2002. Oxidant-antioxidant system: Role and significance in human body. Indian J Exp Biol 40:1233–1239. Kapka-Skrzypczak L, Cyranka, Skrzypczak M, Kruszewski M. 2011. Biomonitoring and biomarkers of organophosphate pesticides exposure—State of the art. Ann Agric Environ Med 18: 294–303.

Kwong TC. 2002. Organophosphate pesticides: Biochemistry and clinical toxicology. Ther Drug Monit 24:144–149. Lang CA, Mills BJ, Mastropaolo W, Liu MC. 2000. Blood glutathione decreases in chronic diseases. J Lab Clin Med 135:402–405. Lowry OH, Rosenbrough NJ, Farr AL, Randall R. 1951. Protein measurement with folin phenol reagent. J Biol Chem 193:365–370. Lukaszewicz-Hussain A. 2010. Role of oxidative stress in organophosphate toxicity. Pestic Biochem Physiol 98:145–150. Ogutcu A, Uzunhisarcikli M, Kalenderm S, Durak D, Bayrakdar F, Kalender Y. 2006. The effects of organophosphate insecticide diazinon on malondialdehyde levels and myocardial cells in rat heart tissue and protective role of vitamin E. Pestic Biochem Physiol 86:93–98. Ojha A, Srivastava N. 2012. Redox imbalance in tissues exposed with organophosphate pesticides and therapeutic potential of antioxidant vitamins. Ecotoxicol Environ Saf 75:230–241. Ojha A, Yaduvanshi SK, Srivastava N. 2011 Effect of combined exposure of commonly used organophosphate pesticides on lipid peroxidation and antioxidant enzymes in rat tissues. Pestic Biochem Physiol 99:148–156. Omaye ST, Krinsky NI, Kagon VE, Mayne ST, Liebler DC, Bidlack WR. 1997. b carotene: Friend or foe? Fundamen Appl Toxicol 40:163–174. Pertejo YP, Reguera RM, Ordonez D, Fouce RB. 2008. Alterations in the glutathione redox balance induced by bioinsecticide, Spinosad in CHO-K1 and Vero cells. Ecotoxicol Environ Saf 70:251–258.

Gershoff SN. (1993). Vitamin C (ascorbic acid): New role, new requirements? Nutrition Rev 51:313–326.

Pompella A, Visvikis A, Paolicchi A, De Tata V, Casini AF. 2003. The changing faces of glutathione, a cellular protagonist. Biochem Pharmacol 66:1499–1503.

Ghosh S, George S, Roy U, Ramachandran D, Kolthur-Seetharam U. 2010. NAD: A master regulator of transcription. Biochim Biophys Acta 1799:6871–693.

Raizada RB, Srivastava MK, Singh RP, Kaushal RA, Gupta KP, Dikshit TS. 1993. Acute and subchronic oral toxicity of technical quinalphos in rats. Vet Hum Toxicol 35:223–225.

Environmental Toxicology DOI 10.1002/tox

REDOX IMBALANCE ON ORGANOPHOSPHATE PESTICIDE EXPOSURE

Rana SVS, Allen T, Singh R. 2002. Inevitable glutathione, then and now. Indian J Exp Biol 40:706–716. Salah SH, Abdou HS, Rahim EAA. 2010. Modulatory effect of vitamins A, C and E mixtures against tefluthrin pesticide genotoxicity in rats. Pestic Biochem Physiol 98, 191–197. Sambrook J, Russell DW. 2001. Molecular Cloning: A Laboratory Manual, Volume I, II & III. New York: Cold Spring Harbor Laboratory Press. Singh M, Sandhir R, Kiran R. 2011. Effects of antioxidant status of liver following atrazine exposure and its attenuation by vitamin E. Exp Toxicol Pathol 63:269–276. Soltaninejad K, Abdollahi M. 2009. Current opinion on the science of organophosphate pesticides and toxic stress: A systematic review. Med Sci Monit 15:75–90. Tayarani I, Cioez I, Clement M, Bourre JL. 1989. Antioxidant enzymes and related trace elements in aging brain capillaries and choroid plexes. J Neurochem 53:817–824. Townsend DM, Tew KD, Tapiero H. 2003. The importance of glutathione in human disease. Biomed Pharmacother 57:145–155. Verma RS, Mehta A, Srivastava N. 2007. In vivo chlorpyrifos induced oxidative stress: Attenuation by antioxidant vitamins. Pestic Biochem Physiol 88:191–196. Verma RS, Srivastava N. 2003. Effect of chlorpyrifos on thiobarbituric acid reactive substances, scavenging enzymes and glutathione in rat tissues. Indian J Biochem Biophys 40:423–428.

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Woo S, Yum S, Kim DW, Park HS. 2009. Transcripts level response in a marine medaka (Oryzias javanicus) exposed to organophosphate pesticide. Comp Biochem Physiol Toxicol Pharmacol 149:427–432. Wu G, Fang YZ, Yang S, Lupton JR, Turner N. 2004. Glutathione metabolism and its implications for health. J Nutr 134:489–492. Ying Y. 2008. NAD1/NADH and NADP1/NADPH in cellular functions and cell death: Regulation and biological consequences. Antioxid Redox Signal 10:179–206. Yu F, Wang Z, Ju B, Wang Y, Wang J, Bai D. 2008. Apoptotic effect of organophosphorous insecticide, chlorpyrifos on mouse retina in vivo via oxidative stress and protection of combination of vitamin C and E. Exp Toxicol Pathol 59:415–423. Zaidi SM, Banu N. 2004. Antioxidant potential of vitamins A, E and C in modulating oxidative stress in rat brain. Clin Chim Acta 340:229–333. Zasadowski A, Wysoki A, Barski D, Spodniewska A. 2004. Some aspects of reactive oxygen species (ROS) and antioxidative system agent’s action: Short review. Acta Toxicol 12:5–21. Zerez CR, Lee SJ, Tanaka KR. 1987. Spectrophotometric determination of oxidized and reduced pyridine nucleotides in erythrocytes using a single extraction procedure. Anal Biochem 164: 367–373. Zhang Q, Piston DW, Goodman RH. 2002. Regulation of corepressor function by nuclear NADH. Science 295:1895–1897.

Environmental Toxicology DOI 10.1002/tox

Organophosphate pesticides-induced changes in the redox status of rat tissues and protective effects of antioxidant vitamins.

Organophosphates (OPs) pesticides are among the most toxic synthetic chemicals purposefully added in the environment. The common use of OP insecticide...
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