In vitro studies on organophosphate pesticides induced oxidative DNA damage in rat lymphocytes

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In vitro studies on organophosphate pesticides induced oxidative DNA damage in rat lymphocytes

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A. Ojha, N. Srivastava ∗ School of Studies in Biochemistry, Jiwaji University, Gwalior 474011, India

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a r t i c l e

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Article history: Received 25 August 2012 Received in revised form 8 January 2014 Accepted 13 January 2014 Available online xxx

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Keywords: Chlorpyrifos Malathion Methyl parathion Oxidative DNA damage Superoxide anion generation In vitro studies

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1. Introduction

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Organophosphate (OP) pesticides are the most widely used synthetic chemicals purposefully added in the environment for agricultural and household pest control. Chlorpyrifos (CPF), methyl parathion (MPT) and malathion (MLT) are among the most widely used OP pesticides. The aim of present study is to determine the genotoxicity of these commonly used OP pesticides individually and in mixture, in in vitro system, using isolated lymphocytes from peripheral blood of healthy Wistar rats. Genotoxicity was estimated by measuring DNA single strand breaks and double strand breaks by single cell gel electrophoresis (SCGE). To find out whether DNA lesions were caused due to oxidative stress, combination of bacterial DNA repair enzymes namely formamidoaminopyrimidine glycosylase (Fpg) and endonuclese (Endo III) which convert base damage to breaks are used. Significant increase in DNA strand breaks measured by increase in tail length, % tail DNA and tail moment, were observed in rat lymphocytes treated with pesticides in modified comet assay. The levels of reactive oxygen species (ROS) namely, superoxide anion and hydrogen peroxide, were also increased in rat lymphocytes on treatment with CPF, MPT and MLT individually and in combination of different period of time, which further shows the involvement of oxidative stress in pesticide induced DNA damage. MPT exposure caused maximum DNA damage followed by CPF and MLT which has also correlated with ROS production. The results of the study show genotoxic potential of these selected OP pesticides. © 2014 Published by Elsevier B.V.

Pesticides are chemical compounds used extensively in modern day agriculture and household for the control of pests. Pesticides also play a role in preventing the spread of infectious diseases. Despite the beneficial effects, many of these chemicals pose potential hazards to humans and to the nature. The widespread use of pesticides for preventing, destroying, repelling, or mitigating pests, had led to anxiety about the possible hazards to public health [1]. Since organochlorine pesticides such as DDT, BHC, HCH, etc. were banned or came in restricted use, because of their high persistence and greater toxicity, the second line of pesticides i.e. organophosphates (OPs) and pyrethroids have become the most common groups available to the world. Though there can be benefits using pesticides, inappropriate use can counter productively, increase

Abbreviations: CPF, chlorpyrifos; MPT, methyl parathion; MLT, malathion; ROS, reactive oxygen species; OP, organophosphorus; Fpg, formamidoaminopyrimidine glycosylase; Endo III, endonuclease III; SCGE, single cell gel electrophoresis; PM, pesticide mixture. ∗ Corresponding author. Tel.: +91 751 2442796; fax: +91 751 2341450. E-mail addresses: [email protected], [email protected] (N. Srivastava).

pest resistance and kill the natural enemies of the pests. Some pesticides are found to be highly persistent in nature, thereby causing contamination of soil, ground and surface water. Their toxic effects are manifested in different ways such as bioaccumulation, biomagnification, chronic toxicity, acute immune response, allergic reaction, and mutagenic, teratogenic and carcinogenic effects [2,3]. Such contamination with low level of pesticides has resulted in serious environmental concern and some of the pesticides, though not showing an immediate effect in vivo, may pose long term health hazard to human beings. The environmental impact of pesticides is often greater than what is intended by those who use them. Over 98% of sprayed insecticides and 95% of herbicides reach a destination other than their target species, including nontarget species, air, water, bottom sediments, and food [4,5]. The organophosphate (OP) pesticides like chlorpyrifos (CPF), methyl parathion (MPT), and malathion (MLT) are widely used in agriculture, in public health and in some countries, large quantities are applied for hygiene purposes. OP compounds including pesticides are anticholinesterase agents. Studies in animals have shown changes in neurotransmitter levels and alterations in neurobehavioral processes after exposure to OPs like monochrotophos, chlorpyrifos, methyl parathion, and malathion [6,7]. These pesticides are known to produce oxidative stress and extensive data suggest that oxygen free radical are involved in the toxicity of

1383-5718/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.mrgentox.2014.01.007

Please cite this article in press as: A. Ojha, N. Srivastava, In vitro studies on organophosphate pesticides induced oxidative DNA damage in rat lymphocytes, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2014), http://dx.doi.org/10.1016/j.mrgentox.2014.01.007

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pesticides, including OP, in animal studies [8], in in vitro experiments [9] and in pesticide manufacturing workers [10] or pesticide sprayers [11]. DNA damage and oxidative stress have been proposed as mechanism that could mechanistically link pesticide exposure to a number of health outcomes observed in epidemiological studies [12,13]. Diseases such as hepatitis, atopic dermatitis, autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, etc.) which are associated with inflammatory response include the release of reactive oxygen species (ROS) [13,14]. ROS can play a critical role in defective sperm function and male infertility [15,16] and also cause aging by the gradual accumulation of free radical damage to biomolecules [17]. The high levels of oxidized bases present in patients infected with the human immunodeficiency virus (HIV) might influence the progression of the infection into acquired immunodeficiency syndrome (AIDS) [13]. DNA oxidative damage has been linked to other diseases, notably Alzheimer’s disease, Huntington’s disease and Parkinson’s disease [14]. Reactive oxygen species (ROS), which include free radicals and other highly reactive forms of oxygen (e.g. hydrogen peroxide, superoxide anion radical, singlet oxygen, hydroxyl radical, etc.) cause DNA oxidation which is known to be one of the most common kinds of damage to DNA. Superoxide anion and H2 O2 are known to be capable of inducing strand-breaks and base oxidation in intracellular DNA. The primary oxidant responsible for DNA damage is hydroxyl radicals (OH• ), as it reacts directly with DNA molecule [18]. Oxidative DNA damage can be measured by employing endonucleases specific for oxidized bases, which recognize oxidized purines and pyrimidines [19–21]. The two enzymes in general use are formamidopyrimidine DNA glycosylase (Fpg), which detects primarily 8-oxo-7,8-dihydroguanine (8-oxoGua), and endonuclease III (Endo III) which recognizes oxidized pyrimidines [19] and introduce breaks in the cellular DNA that are then most commonly measured using the comet assay (alkaline single cell gel electrophoresis). The alkaline single cell gel electrophoresis (SCGE) or comet assay, is now a well-established genotoxicity test for the estimation of DNA damage at the individual cell level in both in vivo and in vitro studies [22]. Thus, very low, physiologically relevant levels of oxidative DNA damage can be investigated. Besides being potent source of ROS, OP compounds also show alkylating properties and alkylating agents are known to cause DNA damage [23,24]. Among the studied pesticides most toxic OP is MPT (O,O-dimethyl-O-4-p-nitrophenyl phosphorothioate), that has been shown to induce genotoxic effects and sister chromatid exchange in human lymphocytes and demonstrated ability to interact directly with double-stranded DNA disturbing its stability and conformation [25,26]. Technical grade malathion appeared to have a potential to produce chromosomal changes including chromosomal aberrations and micronuclei in somatic and germ cells of mice [27]. The toxicity of malathion might be attributed to its metabolite maloxon, which unlike its parent compound damage DNA [28]. Chlorpyrifos has also been shown to induce DNA damage in rat brain and liver [29]. Malathion used as commercial product, i.e. containing malaoxon and isomalathion, can be considered as a genotoxic substance in vitro and may also produce DNA disturbances in vivo, such as DNA breakage at sites of oncogenes or tumor suppressor genes, thus playing a role in the induction of malignancies in exposed individuals. Therefore, malathion can be regarded as a potential mutagen/carcinogen [28]. As a range of insecticides is extensively used in pest management, the chances of exposure to multiple OP compounds simultaneously are high, especially among agricultural and public health workers. Furthermore, from dietary and other sources, there may be separate but closely timed exposures to such insecticides. Although health hazards of individual OP insecticides have been relatively well characterized, there is lesser information on the interactive toxicity of multiple OP insecticides. The data suggest

that exposure to multiple OP-containing pesticide formulations may lead to synergistic neurotoxicity by a direct mechanism at the cellular level [30]. It is reported that the herbicide, terbutryn, had DNA damaging capability, as evaluated by the alkaline single-cell microgel-electrophoresis (comet) assay [31]. In vitro study of terbutryn was found to induce primary DNA damage, even though in the absence of a clear trend for dose-dependence and in the presence of a concomitant mild cytotoxic effect observed [34]. There is lack of reports which throw light on comparative and synergistic or antagonistic genotoxic potential of CPF, MPT, MLT and their mixture in in vitro systems. Literature survey suggests that exposure to mixture of pirimiphos methyl, chlorpyrifos, temephos and malathion may induce DNA damage, decrease in AChE activity, hepatotoxicity as well as nephrotoxicity in occupational workers [33]. Chlorpyrifos, methyl parathion and malathion are widely used pesticides, however, reports of cytogenetic and genotoxic potential of these pesticides either individually or in mixture, in in vitro system are lacking. Therefore, the present study is designed to assess the genotoxic potential of CPF, MPT and MLT individually, and in mixture, by measuring DNA single and double strand breaks in isolated rat lymphocytes incubated with CPF, MPT and MLT individually and in mixture for different period of time. The study was also aimed to unravel the mechanism of this DNA damage by standard comet assay in combination with Fpg–Endo III modified comet assay/SCGE to find out whether DNA lesions were caused due to oxidative stress. The levels of H2 O2 and superoxide anions in cultured rat lymphocytes incubated with pesticides for different periods, were also measured to find out any correlation with the strand breaks.

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2. Materials and methods

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2.1. Chemicals

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Normal melting agarose (NMA), low melting agarose (LMA), Triton X-100, ethylenediamine tetraacetic acid (EDTA), tris (hydroxymethyl)-aminomethane, ethidium bromide, endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), oxidized cytochrome c, phorbol 12–13 myristate acetate (PMA), phenol red and hydrogen peroxidase were purchased from Sigma Chemicals Inc., St. Louis, MO. Sodium chloride, di-sodium hydrogen phosphate, sodium di-hydrogen phosphate, sodium hydroxide, Hisep, dimethylsulfoxide, RPMI 1640, Trypan Blue, 4-(2-hydroxyethyl)-piperazine-1-ethanesulphonic acid (HEPES) buffer, potassium hydroxide, bovine serum albumin, and potassium chloride were purchased from Merck, Germany. Methyl parathion, chlorpyrifos and malathion were kind gift from Devidayal (Sales) Limited, Mumbai, India.

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2.2. Experimental animals

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Adult male albino rats of Wistar strain (Rattus norvegicus), weighing 120 ± 10 g were used in all experiments. Rats were obtained from Defence Research and Development Establishment, Gwalior, India, and were maintained in a light (light–dark cycle of 12 h each) and temperature (25 ± 2 ◦ C) controlled animal room of our department on standard pellet diet and tap water ad libitium. Rats were acclimatized for one week prior to the start of experiment. The care and maintenance of animals were as per the approved guidelines of the ‘Committee for the Purpose of Control and Supervision of Experiments on Animals’ (CPCSEA, India).

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2.3. Lymphocytes isolation

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Peripheral blood was collected from healthy male Wistar rats (weighing about 120 ± 10 g) from eye orbital in vacutainer test tubes containing EDTA (BectonDickinson, Cedex, France). Blood samples were layered on the top of a Ficoll solution (1.077 g/ml) and the supernatant containing the leukocytes was removed after sedimentation of erythrocytes at 1000 g for 10 min at room temperature. Lymphocytes sedimented at the interface of the Ficoll layers were collected and washed twice with PBS, pH 7.4 at 20 ◦ C. The cell viability was checked by Trypan Blue exclusion test [34] and was found to be about 95%. The final concentration of the lymphocytes was adjusted to 1–3 × 105 cells/ml by adding RPMI 1640 to the single cell suspension.

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2.4. Pesticide treatment

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Chlorpyrifos [4 h LC50 0.2 mg/L, 35], methyl parathion [4 h LC50 0.135 mg/L, 36], malathion [4 h LC50 >5.2 mg/L, 37] individually and in mixture were taken from

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DMSO stock solutions (LC50 ) and added to the lymphocyte suspension to give final concentrations of 1/20, 1/10, 1/8, 1/6 and 1/4 LC50 individually and in mixture to perform time– and dose–response relationship study. The control cells were treated with DMSO without the organophosphates, which did not affect the processes under study. To examine modified bases, double strand DNA breaks, hydrogen peroxide, and superoxide anion production, the lymphocytes were incubated with pesticides for 2 h and 4 h at 37 ◦ C in CO2 incubator and results were compared with control lymphocyte cells. The positive control for DNA damage was hydrogen peroxide [38], and medium alone was used as the negative control. At the end of each incubation, cell viability was determined again by Trypan Blue dye exclusion.

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2.5. Single-cell gel electrophoresis or comet assay

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Single strand breaks were measured by alkaline comet assay as described by Sasaki et al. [39]. 75 ␮L of normal melting agarose (1% prepared in 0.1 M sodium phosphate buffer, pH 7.2 containing 0.9% NaCl) was quickly layered on an end frosted slide, and the precoated slide was coated with 100 ␮L of mixture containing equal volumes of sample (lymphocytes treated with different concentration of H2 O2 , CPF, MPT, MLT and PM) and low melting agarose (2% in phosphate buffer saline). Slides were then immersed in the lysis buffer (containing 0.25 M NaCl, 100 mM EDTA, 10 mM Trizma base, 1% sarcosine, pH 10.0 adjusted with 10 N NaOH. 5% DMSO and 1% Triton X-100 was added just before use) for 1 h at 4 ◦ C in the dark. After lysis, the slides were rinsed with chilled distilled water, transferred on a horizontal electrophoresis platform and immersed in electrophoresis buffer (300 mM sodium hydroxide and 1 mM EDTA, pH 13.0) for 20 min for unwinding of DNA. Electrophoresis was performed for 15 min at constant voltage (1 V/cm and 300 mA). After electrophoresis, the slides were washed thrice with neutralizing buffer (0.4 M Tris–HCl, pH 7.4) for 5 min each. Slides were dehydrated in absolute methanol for 10 min and left at room temperature to dry. The whole procedure was performed in dim light to minimize artifactual DNA damage. Just before visualization, each slide was stained with 50 ␮L of ethidium bromide (20 ␮g/mL), rinsed with water, and covered with a cover slip. Slides were viewed and scored under fluorescence microscope (Leica 4000B Digital Microscope) equipped with a digital camera and software for the capture, processing and image analysis (TriTek CometScoreTM , Free ware ver 1.5). Analyses were performed by measuring % tail DNA (%T DNA), tail length (TL) and tail moment (TM) of 50 nuclei in two replicate slides and taking the average. Total six set of observations were taken (Total 300 nuclei). Analyses were performed at 100× magnification, with a Leica Optiphase microscope equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm.

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2.6. Superoxide anion estimation

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Superoxide-anion release was measured by superoxide dismutase inhibitable reduction of ferricytochrome c [40]. PMA (polymorphonucleocytes) (3 × 105 ) were incubated in PBS-EDTA buffer (pH 7.4) with phorbol-12,13-dibutyrate (PDBu) at 37 ◦ C for 15 min. The total assay volume was 1 ml. The final concentrations of ferricytochrome c and PDBu were 50 ␮mol/L and 100 nmol/L, respectively. The change in absorbance was measured spectrophotometrically at 550 nm for 10 min with a double beam Shimadzu UV-160A spectrophotometer (Shimadzu Seisakusho Ltd., Kyoto, Japan) at room temperature. The amount of superoxide-anion secreted into the medium was calculated on the basis of the molar extinction coefficient of reduced cytochrome c 2.1 × 104 M−1 cm−1 [41] and values are expressed as n mole O2 • − /106 cells/min.

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2.7. H2 O2 estimation

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Hydrogen peroxide in lymphocytes was measured by the method of Pick [42]. For assay of H2 O2 , 100 ␮L of pesticide treated lymphocytes, 100 ␮L of assay solution (containing 0.2 ml phenol red, 0.2 g/l and 0.2 ml of horseradish peroxidase, 20 U/ml in potassium phosphate buffer, 0.05 M, pH 7.0 and 9.6 ml of 0.9% NaCl), was taken and reaction was started by the addition of 10 ␮L of 1.0 N NaOH. Absorbance was recorded at 600 nm by using microplate reader. Hydrogen peroxide standard curve was plotted by taking different concentrations of H2 O2 , ranging from 20 to 100 ␮mol in a total volume of 100 ␮L and processed in the same way. Results are expressed as ␮mol H2 O2 formed/ml preparation.

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2.8. Modified base estimation and DNA double strand break measurement

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Modified bases were estimated by Fpg–Endo enzyme treatment in conjunction with the comet assay based primarily on Collins’ protocols [43,44]. After exposures, lymphocytes were mixed with low melting agarose (LMA, 2% in PBS). Then, 100 ␮L of the agarose and cell mixture was pipetted onto a microscope slide pre-coated with normal melting agarose (1% in PBS). The slides were placed in cold lysis buffer (2.5 M NaCl, 0.1 M Na2 EDTA, 0.01 M Tris–HCl, 1% Triton X-100, 10% DMSO, pH 10) overnight at 4 ◦ C (minimum of 12 h). For the enzyme treatment, four slides were prepared from each cell treated sample: “lysis”, “buffer”, “Fpg” and “Endo III”. After lysis, slides “buffer”, “Fpg” and “Endo III” were washed 3 times at 4 ◦ C, 5 min each time, with enzyme buffer (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml BSA,

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adjusted to pH 8 with KOH). Then 50 ␮l of enzyme buffer, Fpg solution or Endo III (1 ␮g/ml of Endo III or Fpg) solution are placed onto the gels of the corresponding slides, covered with coverslips and incubated at 37 ◦ C for 30 min in a moist box. The slide labeled as “lysis” remained in the lysis solution during the incubation of the rest of the slides. At the end of the incubation period, the coverslips were removed and all slides, including the “lysis” slides, were placed in an electrophoresis chamber containing electrophoresis solution (0.3 M NaOH, 1 mM EDTA). Electrophoresis was carried out for 30 min at constant voltage of 0.8 V/cm (measured between the electrodes across the platform carrying the slides) and the current around 300 mA. DNA double strand break was measured by neutral version of comet assay according to protocol of Fracasso et al. [45]. After lysis, the slides were washed three times with the electrophoresis buffer (300 mM sodium acetate, 100 mM Tris–HCl, pH 8.5) and left in a fresh portion of the electrophoresis buffer for 1 h and then placed in a horizontal gel electrophoresis unit, filled with a fresh electrophoresis buffer. The slides were electrophoresed for 1 h at 14 V and 100 mA at 4◦ C using a power supply. After electrophoresis, slides were neutralized by adding Tris–HCl buffer (0.4 M, pH 7.5) in a drop-wise fashion onto the slides. The slides were rinsed in ethanol and dried for later analysis. The whole procedure was performed in dim light to minimize artifactual DNA damage. Analyses were performed at described earlier (Section 2.5). 2.9. Statistical analyses Results are expressed as mean ± S.E. of six set of observation. Statistical analyses were performed using Sigma Stat 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 <0.05.

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3. Results

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3.1. % viability estimation

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Exposure of isolated rat lymphocytes with different concentrations of CPF, MPT, MLT and their mixture caused decrease in % viability. Results of the present study showed that exposure of rat lymphocytes with 1/20, 1/10,1/8, 1/6 and 1/4 LC50 dose of H2 O2 (100 ␮mol/L), CPF, MPT MLT and their mixture for 2 h and 4 h caused time and dose dependent decrease in % viability of rat peripheral blood lymphocytes (Table 1). Data showed 20–29% and 23–34% dose dependent decrease in viability observed after exposure of rat lymphocytes with H2 O2 for 2 h and 4 h, respectively, as compared to control. Similarly 3–10% and 9–17%, 13–25% and 16–25%, 3–10% and 3–11%, and 4–11% and 4–12% dose dependent decrease in viability was observed after exposure of rat lymphocytes with CPF, MPT, MLT and their mixture for 2 h and 4 h, respectively, as compared to control (Table 1). Similarly lymphocytes exposed with 1/4 LC50 equivalents of CPF, MPT, MLT and their mixture caused time dependent decrease in the viability of rat peripheral blood lymphocytes. Data show 23%, 27% 17% and 16% decrease in cell viability after 2 h exposure of rat lymphocytes with CPF, MPT, MLT and their mixture, respectively, when compared with control. Incubation of rat lymphocytes with CPF, MPT, MLT and their mixture for 4 h caused 34%, 33%, 22% and 24% decrease in cell viability, respectively, as compared with control (Table 2). 3.2. Effect of pesticide exposure on H2 O2 generation Production of H2 O2 in rat lymphocytes following in vitro pesticide exposure of 1/4 LC50 equivalents of CPF, MPT, MLT and their mixture for 2 h and 4 h was analyzed spectrophotometrically. It was observed that H2 O2 generation by rat lymphocytes treated with OP pesticides for different time periods showed progressive increase. There was significant increase of 65%, 106%, 21% and 20% in H2 O2 production in cells exposed with CPF, MPT, MLT and PM, respectively, for 2 h. Exposure of lymphocytes with same concentration of CPF, MPT, MLT and their mixture for 4 h caused 86%, 140%, 43% and 48% increase in H2 O2 concentration. The pesticide mixture does

Please cite this article in press as: A. Ojha, N. Srivastava, In vitro studies on organophosphate pesticides induced oxidative DNA damage in rat lymphocytes, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2014), http://dx.doi.org/10.1016/j.mrgentox.2014.01.007

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Table 1 Estimation of % viability in rats lymphocyte after exposure of different doses of H2 O2 , chlopyrifos, methyl parathion, malathion singly and in mixture for 2 h and 4 h Results are expressed as mean ± S.E. of six set of observation. %Viability 91.94 ± 0.23 2h

90.73 ± 0.03 4h

Dose

Control

H2 O2

1/20 LC50 1/10 LC50 1/8 LC50 1/6 LC50 1/4 LC50

– – – – –

73.91 71.96 70.08 68.01 65.02

± ± ± ± ±

0.20*** 0.22*** 0.22*** 0.25*** 0.29***

88.90 87.60 87.62 85.21 82.50

± ± ± ± ±

0.20*** 0.07*** 0.38*** 0.2*** 0.07***

80.28 76.00 71.76 70.54 69.06

± ± ± ± ±

0.20*** 0.31*** 0.18*** 0.05*** 0.30***

89.01 88.18 86.69 85.56 83.22

± ± ± ± ±

0.32*** 0.15*** 0.09*** 0.06*** 0.20***

88.08 87.02 85.52 84.61 82.20

1/20 LC50 1/10 LC50 1/8 LC50 1/6 LC50 1/4 LC50

– – – – –

70.14 68.29 65.98 59.91 63.60

± ± ± ± ±

0.19*** 0.14*** 0.27*** 5.11*** 0.02***

78.09 75.32 79.98 82.35 75.08

± ± ± ± ±

0.30*** 0.06*** 0.23*** 0.05*** 0.18***

76.19 73.67 70.22 68.33 68.40

± ± ± ± ±

0.37*** 0.01*** 0.26*** 0.25*** 0.55***

87.87 87.22 85.44 84.10 81.00

± ± ± ± ±

0.07*** 0.38*** 0.57*** 0.16*** 0.26***

87.24 ± 0.26*** 86.40 ± 1.19** 82.71 ± 0.1*** 83.56 ± 0.19*** 79.88 ± 0.29***

CPF

MPT

MLT

PM ± ± ± ± ±

0.29*** 0.25*** 0.04*** 0.03*** 0.20***

Rat lymphocytes were given 01/20, 1/10, 1/8, 1/6 and 1/4 LC50 equivalent of H2 O2 , individual pesticide and their mixture suspended in DMSO for 2 h and 4 h and after incubation viability is calculated. Q4 *P < 0.05 when compared with respective control. ** P < 0.001 when compared with respective control. *** P < 0.0001 when compared with respective control. # P > 0.05 when compared with respective control.

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not invoke significant increase in H2 O2 production by the cells than did either pesticide individually (Table 2).

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3.3. Effect of pesticide exposure on superoxide anion generation

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Superoxide anion (O2 •− ) production in pesticide-treated rat lymphocytes was monitored by the superoxide dismutase inhibitable cytochrome c reduction assay. Data presented in Table 2 showed the amount of O2 •− generated on exposure of rat lymphocytes with CPF, MPT, MLT and their mixture as a function of time. Rat lymphocytes treated with 1/4 LC50 concentrations of CPF, MPT, MLT and PM for 2 h and 4 h showed a significant increase in O2 •− production when compared with control (Table 2). Experiments using a mixture of CPF, MPT and MLT were performed to examine if there was an additive or synergistic effect of these pesticides. Treatment of rat lymphocytes with CPF, MPT, MLT and their mixture for 2 h caused 129%, 193%, 36% and 21% increase in superoxide anion production, respectively, as compared to control while exposure for 4 h caused 157%, 236%, 93% and 50% increase in superoxide anion production, respectively, as compared to control. The pesticide mixture caused no significant augmentation of superoxide anion production by the rat lymphocytes over levels detected in control or individual pesticide-treated cells (Table 2).

3.4. Study of time– and dose–response relationship of toxicity induced by H2 O2 , CPF, MPT, MLT individually or in mixture in terms of %T DNA, TM and TL in rat lymphocytes The DNA damage comprising the % DNA in tail (%T DNA), the tail length (TL), and the tail moment (TM) were evaluated in the current work to identify the most toxic pesticide using rat lymphocytes. Substantial dose and time dependent changes were observed in these parameters in pesticide treated lymphocytes when compared with control. Results of the present study showed that in vitro exposure of rat lymphocytes with H2 O2 and pesticides, CPF, MPT, MLT, alone or in combination for 2 h and 4 h caused increase in DNA damage, as evidenced by the increase in %T DNA, TL and TM. In in vitro study, the control lymphocytes showed round and condensed shaped nuclei having 6–13% T DNA, 0.49–0.71 ␮M TL and TM between 10 and 18 after 2 h and 4 h. The data showed that in vitro exposure of rat lymphocytes with 1/20, 1/10, 1/8, 1/6 and 1/4 LC50 dose of H2 O2 caused time and dose dependent increase of 255–841% and 79–377% in % TDNA, 65–174% and 59–794% increase in TL and 19–729% and 1–595% increase in TM was observed after 2 h and 4 h exposure, respectively, as compared to control. Similarly in vitro exposure of rat lymphocytes with 1/20, 1/10, 1/8, 1/6 and 1/4 LC50 dose of CPF caused 415–705% and 155–305% dose dependent increase in %T DNA, 31–108% and 27–83% dose dependent increase

Table 2 Estimation of % viability and ROS (hydrogen peroxide and superoxide anion) in rats lymphocyte after exposure of 1/4 LC50 doses of chlopyrifos, methyl parathion, malathion singly and in mixture for 2 h and 4 h. Incubation time

Control

Chlorpyrifos

Parathion

% Viability

2h 4h

91.66 ± 0.43 91.24 ± 0.66

70.57 ± 1.89 60.56 ± 1.68***

H2 O2

2h 4h

475.62 ± 3.29 464.62 ± 8.91

785.7 ± 18.79*** 864.88 ± 6.10***

Superoxide anion

2h 4h

0.14 ± 0.01 0.14 ± 0.01

***

0.32 ± 0.01*** 0.36 ± 0.01***

Malathion

Pesticide mixture

***

66.84 ± 1.92 61.55 ± 1.55***

76.48 ± 0.39 71.50 ± 0.59***

76.87 ± 1.72*** ,be * ,ce ** ,de # 69.18 ± 0.37*** ,be ** ,ce ** ,de *

981.25 ± 8.84*** 1116.75 ± 31.9***

577.25 ± 8.48*** 666.62 ± 5.71***

570.00 ± 4.06*** ,be *** ,ce *** ,de # 687.88 ± 2.76*** ,be *** ,ce *** ,de *

0.41 ± 0.02*** 0.47 ± 0.03***

0.19 ± 0.01** 0.27 ± 0.01***

***

0.17 ± 0.01* ,be *** ,ce *** ,de * 0.21 ± 0.01*** ,be *** ,ce *** ,de ***

Results are expressed in terms of mean ± S.E. of six set of observations taken on different days. Value of H2 O2 is expressed in terms of ␮mol/ml. Superoxide anion is expressed in terms of n mole O2 • − /106 cells/min. be When pesticide mixture is compared with chlorpyrifos, ce when pesticide mixture is compared with parathion and de when pesticide mixture is compared with malathion treated lymphocytes. * P < 0.05 when compared with respective control. ** P < 0.001 when compared with respective control. *** P < 0.0001 when compared with respective control. # P > 0.05 when compared with respective control.

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Table 3 Estimations of DNA single strand breaks in rats lymphocyte after exposure of H2 O2 , chlorpyrifos, methyl parathion, malathion singly and in mixture for 2 h and 4 h. Treatment

Dose

2h

4h

%T DNA

TL (␮M)

TM

%T DNA

TL (␮M)

TM

Control H2 O2

– 1/20 LC50 1/10 LC50 1/8 LC50 1/6 LC50 1/4 LC50

6.33 22.49 31.38 38.61 56.24 59.61

± ± ± ± ± ±

0.53 0.12*** 0.09*** 0.11*** 0.21*** 0.15***

0.49 0.81 0.81 0.93 1.14 1.34

± ± ± ± ± ±

0.04 0.10** 0.03*** 0.08*** 0.12*** 0.13***

10.16 12.05 28.67 35.10 60.04 84.20

± ± ± ± ± ±

1.79 0.88# 0.20*** 2.57*** 3.19*** 8.79***

13.02 23.34 32.11 40.88 57.28 62.08

± ± ± ± ± ±

1.77 0.19*** 0.08*** 1.06*** 0.04*** 0.41***

0.71 1.13 1.27 3.34 3.92 6.35

± ± ± ± ± ±

0.03 0.03*** 0.03*** 0.72*** 1.00*** 1.06***

18.09 18.31 37.12 51.05 122.89 125.79

± ± ± ± ± ±

0.44 1.85# 1.05*** 3.10*** 5.04*** 2.69***

CPF

1/20 LC50 1/10 LC50 1/8 LC50 1/6 LC50 1/4 LC50

32.58 33.48 35.69 37.20 50.93

± ± ± ± ±

0.14*** 0.06*** 0.07*** 0.10*** 0.27***

0.64 0.77 0.57 0.76 1.02

± ± ± ± ±

0.03** 0.13# 0.02# 0.03*** 0.03***

21.87 22.80 27.37 54.35 55.52

± ± ± ± ±

0.28*** 1.35** 1.06*** 1.81*** 3.36***

33.22 35.10 36.31 38.06 52.75

± ± ± ± ±

0.15*** 0.13*** 0.13*** 0.20*** 0.43***

0.90 1.06 1.11 1.22 1.30

± ± ± ± ±

0.12** 0.05*** 0.14# 0.02*** 0.04***

42.04 48.28 32.30 44.46 69.13

± ± ± ± ±

4.34*** 1.22*** 6.14# 2.11*** 1.00***

MPT

1/20 LC50 1/10 LC50 1/8 LC50 1/6 LC50 1/4 LC50

39.89 40.85 43.76 46.18 52.75

± ± ± ± ±

0.07*** 0.06*** 0.07*** 0.05*** 0.20***

0.91 0.94 0.86 1.00 1.08

± ± ± ± ±

0.13** 0.07* 0.14** 0.01*** 0.03***

36.30 37.52 40.91 55.21 59.63

± ± ± ± ±

5.13*** 6.52*** 3.39*** 0.23*** 2.06***

40.60 42.58 44.64 46.97 53.99

± ± ± ± ±

0.12*** 0.15*** 0.34*** 0.28*** 0.34***

1.27 1.29 1.34 1.43 1.63

± ± ± ± ±

0.03*** 0.05*** 0.02*** 0.05*** 0.05***

53.96 56.53 63.84 68.19 73.12

± ± ± ± ±

0.92*** 1.76*** 1.09*** 4.83*** 1.59***

MLT

1/20 LC50 1/10 LC50 1/8 LC50 1/6 LC50 1/4 LC50

15.21 17.66 19.15 20.73 47.64

± ± ± ± ±

0.23*** 0.50*** 0.18*** 0.12*** 0.07***

0.52 0.54 0.76 0.93 1.09

± ± ± ± ±

0.04# 0.06# 0.03*** 0.01*** 0.02***

15.21 16.32 18.33 16.79 46.94

± ± ± ± ±

0.23** 0.41* 1.14# 0.92# 7.48***

16.60 19.38 19.90 22.53 51.74

± ± ± ± ±

0.11* 0.12*** 0.11*** 0.07*** 1.10***

0.78 0.95 1.07 1.27 1.30

± ± ± ± ±

0.02* 0.02*** 0.02*** 0.02*** 0.03***

19.06 19.20 26.60 26.82 65.55

± ± ± ± ±

0.36*** 1.29# 0.11# 2.47*** 0.84***

PM

1/20 LC50 1/10 LC50 1/8 LC50 1/6 LC50 1/4 LC50

25.65 26.34 27.81 30.28 48.62

± ± ± ± ±

1.44*** 0.18*** 0.11*** 0.13*** 0.30***

0.48 0.56 0.69 0.88 1.14

± ± ± ± ±

0.07# 0.05# 0.01*** 0.04* 0.02***

13.16 14.22 32.17 22.99 54.35

± ± ± ± ±

1.94# 1.58# 1.34*** 2.68*** 1.81***

25.22 27.59 29.23 30.97 50.08

± ± ± ± ±

0.07*** 0.20*** 0.27*** 0.05*** 0.18***

1.16 1.08 1.21 1.22 1.45

± ± ± ± ±

0.15** 0.04*** 0.05*** 0.02*** 0.07***

31.02 31.40 38.49 39.83 68.68

± ± ± ± ±

3.41# 1.76# 0.20*** 2.00*** 2.67***

Results are expressed as %T DNA, tail length (TL) and tail moment (TM). Results are expressed as mean ± S.E. of 50 nuclei in six set of observations. Rat lymphocytes were given 1/20, 1/10, 1/8, 1/6 and 1/4 LC50 equivalent of H2 O2 , individual pesticide and their mixture suspended in DMSO for 2 h and 4 h and after incubation comet is visualized. * P < 0.05 when compared with respective control. ** P < 0.001 when compared with respective control. *** P < 0.0001 when compared with respective control. # P > 0.05 when compared with respective control.

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in TL and 115–447% and 132–282% dose dependent increase in TM was observed after 2 and 4 h exposure, respectively, as compared to control. Likewise in vitro exposure of rat lymphocytes with 1/20, 1/10, 1/8, 1/6 and 1/4 LC50 dose of MPT, MLT and their mixture caused 140–733% and 28–315% dose dependent increase in %T DNA, 2–133% and 10–130% dose dependent increase in TL and 50–487% and 5–304% dose dependent increase in TM was observed after 2 and 4 h exposure respectively as compared to control (Table 3).

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3.5. Estimation of damaged bases and DNA double strand breaks

369 370 371 372 373 374 375

378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395

Results of the present study showed that in vitro exposure of rat lymphocytes with OP pesticides, CPF, MPT and MLT, alone or in combination for 2 h and 4 h caused significantly marked increase in DNA damage, as evidenced by the increase % TDNA, TL and TM. When the lesion-specific enzymes, Fpg and Endo III, were added during the comet assay procedure, DNA damage was significantly increased in all the OP treated lymphocytes. In in vitro study, the control lymphocytes showed almost all the nuclei of type 0, with round, condensed shape indicating intact DNA. The data showed that in vitro exposure of rat lymphocytes with 1/4 LC50 concentrations of CPF, MPT, MLT and their mixture for 2 h caused 989–1477% increase in %T DNA, 126–187% increase in TL and 562–952% increase in TM, when compared with untreated control. When the same cells were treated with buffer for 2 h, 723–1360% increase in %T DNA, 97–197% increase in TL and 240–436% increase in TM was observed as compared with buffer treated control lymphocytes. Treatment of pesticide exposed lymphocytes with Endo III and Fpg showed pronounced increase in %T DNA, TL and TM as compared to both buffer

treated and control lymphocytes. Similarly lymphocytes treated with Endo III after 2 h incubation with 1/4 LC50 concentration of CPF, MPT, MLT and their mixture, showed 274–578% increase in %T DNA, 68–183% increase in TL and 279–438% increase in TM, when compared with Endo III treated control lymphocytes while treatment with Fpg caused 263–562% increase in %T DNA, 63–141% increase in TL and 283–421% increase in TM, as compared to Fpg treated control lymphocytes. When the lymphocytes were incubated with the same concentration of pesticides for 4 h, 718–1218% increase in %T DNA, 115–164% increase in TL and 274–449% increase in TM was observed as compared with untreated control lymphocytes. Treatment of pesticide exposed lymphocytes with buffer caused 846–1146% increase in %T DNA, 172–242% increase in TL and 262–403% increase in TM as compared to buffer treated control lymphocytes. Exposure of lymphocytes with pesticides for 4 h followed by treatment with Endo III caused 623–1073% increase in %T DNA, 67–112% increase in TL and 327–496% increase in TM when compared with Endo III treated control lymphocytes while these lymphocytes when treated with Fpg, caused 496–746% increase in %T DNA, 26–55% increase in TL and 276–429% increase in TM when compared with Fpg treated control lymphocytes (Table 4). Results of the neutral comet assay showed that there was no difference in %T DNA, TL and TM in the alkaline and neutral comet assay, which indicates that strand breaks contributed to the observed increase in the damage index (%T DNA, TL and TM) in the presence of individual and combined OP exposure. The data showed that In vitro exposure of rat lymphocytes with 1/4 LC50 concentrations of CPF, MPT, MLT and their mixture for 2 h caused 321–816% increase in %T DNA, 125–322% increase in TL and 140–345% increase

Please cite this article in press as: A. Ojha, N. Srivastava, In vitro studies on organophosphate pesticides induced oxidative DNA damage in rat lymphocytes, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2014), http://dx.doi.org/10.1016/j.mrgentox.2014.01.007

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Table 4 Estimations of oxidized base and DNA single and double strand breaks in rats lymphocyte after exposure of chlopyrifos, methyl parathion, malathion singly and in mixture for 2 h and 4 h. Pesticide

Treatment

2h

4h

%T DNA

TL (␮M)

TM

%T DNA

Single strand break Control Control Buffer Endo Fpg

2.6 3.5 7.7 8.2

± ± ± ±

0.4 0.4 0.1 0.4

0.31 0.33 0.40 0.54

± ± ± ±

0.07 0.04 0.06 0.06

5.39 10.95 12.44 13.12

± ± ± ±

1.09 3.60 3.80 4.80

CPF

Control Buffer Endo Fpg

37.7 37.6 40.8 41.5

± ± ± ±

0.2*** 0.5*** 0.9*** 0.8***

0.89 0.95 1.09 1.04

± ± ± ±

0.01*** 0.04*** 0.03*** 0.02***

56.69 58.66 60.36 64.27

± ± ± ±

MPT

Control Buffer Endo Fpg

41.0 51.1 52.2 54.3

± ± ± ±

0.8*** 0.6*** 0.5*** 0.3

0.89 0.98 1.13 1.30

± ± ± ±

0.04*** 0.03*** 0.07*** 0.05***

50.86 51.74 66.95 68.29

MLT

Control Buffer Endo Fpg

28.3 28.8 28.8 29.8

± ± ± ±

0.6*** 1.4*** 0.2*** 0.3***

0.70 0.65 0.67 0.88

± ± ± ±

0.01*** 0.05*** 0.04*** 0.04***

PM

Control Buffer Endo Fpg

30.3 ± 0.3*** 30.4 ± 0.2*** 31.1 ± 0.7*** 36.1 ± 0.4***

0.76 0.78 0.84 0.93

± ± ± ±

± ± ± ± ±

0.32 1.04 1.35 0.72 0.95

± ± ± ± ±

Double strand break Control CPF MPT MLT PM

5.7 45.1 52.2 24.0 34.8

0.7 0.6*** 3.6*** 0.1*** 1.0***

TL (␮M)

TM

3.4 4.1 5.6 8.0

± ± ± ±

0.4 0.4 0.1 0.6

0.33 0.36 0.61 0.96

± ± ± ±

0.15 0.10 0.06 0.05

13.46 14.90 15.20 17.91

± ± ± ±

2.70 4.50 5.70 5.70

0.90*** 2.39*** 2.80*** 2.60***

42.3 42.8 62.4 61.6

± ± ± ±

0.05*** 0.05*** 0.1*** 1.1***

0.87 1.02 1.02 1.49

± ± ± ±

0.06* 0.03# 0.08* 0.05***

66.06 68.73 76.12 94.71

± ± ± ±

3.70*** 3.10*** 4.90*** 4.40***

± ± ± ±

2.20*** 2.70*** 4.30*** 2.90***

44.8 51.1 65.7 67.7

± ± ± ±

0.1*** 0.7*** 0.3*** 0.1***

0.73 1.23 1.29 1.36

± ± ± ±

0.03# 0.08** 0.03** 0.02**

73.94 74.97 90.53 94.67

± ± ± ±

3.30*** 3.71*** 2.80*** 10.30***

43.55 45.11 47.17 50.19

± ± ± ±

1.60*** 1.48*** 2.50*** 1.40***

27.8 38.8 40.5 47.7

± ± ± ±

0.4*** 0.4*** 0.2*** 0.6***

0.71 1.00 1.09 1.23

± ± ± ±

0.06# 0.07* 0.05* 0.04**

53.55 54.06 65.02 69.34

± ± ± ±

2.50*** 1.50*** 2.30*** 2.30***

0.04*** 0.07*** 0.05*** 0.04***

35.68 37.23 48.95 53.89

± ± ± ±

2.60*** 2.60*** 2.30*** 2.60***

39.3 49.5 49.7 49.7

± ± ± ±

0.8*** 0.5*** 1.3*** 0.7***

0.87 0.98 1.03 1.21

± ± ± ±

0.04** 0.06** 0.02# 0.04#

50.40 53.98 64.86 67.28

± ± ± ±

2.30*** 2.90*** 4.00*** 4.20***

0.06 0.12*** 0.11*** 0.16* 0.29***

13.70 50.72 60.92 35.79 32.93

± ± ± ± ±

1.79 4.70*** 7.60*** 2.80*** 7.70***

5.7 61.8 71.6 33.0 56.1

± ± ± ± ±

0.7 2.4** 0.3** 0.8** 3.4**

0.47 1.17 1.36 0.82 1.07

± ± ± ± ±

0.10 0.08*** 0.06*** 0.04* 0.01**

24.40 64.70 95.47 36.70 60.22

± ± ± ± ±

42.70 7.9*** 3.70*** 1.10*** 5.10***

Results are expressed as %T DNA, tail length (TL) and tail moment (TM). Results are expressed as mean ± S.E. of 50 nuclei in six set of observations. Rat lymphocytes were given 1/4 LC50 of individual pesticide and their mixture suspended in DMSO for 2 h and 4 h and after incubation comet is visualized. * P < 0.05 when compared with respective control. ** P < 0.001 when compared with respective control. *** P < 0.0001 when compared with respective control. # P > 0.05 when compared with respective control.

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in TM, when compared with control. When the lymphocytes were incubated with same concentration of pesticides for 4 h, 479–1156% increase in %T DNA, 75–189% increase in TL and 50–291% increase in TM was observed, as compared to control lymphocytes (Table 4).

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4. Discussion

425 426 427

430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449

Reactive oxygen species (ROS) are involved in the toxicity of various pesticides including OP pesticides. It is known that ROS induce several types of lesions in DNA including single and double and breaks, alkali labile sites, and various species of oxidized purines and pyrimidines which are easily detected by comet assay [43,44]. OP pesticides are known to produce oxidative stress by inhibiting antioxidant enzymes as well as by inducing generation of ROS [8]. There have been several attempts to establish the mechanism of OP pesticide induced DNA damage but results have been inconsistent. The comet assay can be used not only for measuring strand breaks but also DNA nicks associated with repair activities occurring through base excision repair (BER) and nucleotide excision repair (NER) [46,47]. In order to elucidate the mechanism of OP pesticides induced DNA damage, the Fpg and Endo III enzymes are included in the modified comet assay which can measure oxidized purines and pyrimidines, respectively. An in vitro study was carried out on rat peripheral blood lymphocytes taking CPF, MPT and MLT singly and in combination, in order to establish the mechanism of DNA damage and to establish the correlation between ROS and DNA damage, if any, the levels of ROS were also estimated.

Results of the present study clearly showed that CPF, MPT and MLT exposure caused significant increase in the levels of H2 O2 and superoxide anion in the cultured peripheral blood lymphocytes of rats. The increase in the levels of these ROS was exposure time dependent. The results also showed that MPT exposure for 2 h and 4 h caused maximum increase in the levels of H2 O2 and superoxide anion in cultured rat lymphocytes followed by CPF and MPT. When lymphocytes were given combined exposure of mixture of these pesticides, the increase in ROS was less than caused by any of the pesticide singly. The study confirms the induction of oxidative stress in rat lymphocytes on exposure with these pesticides. Previous studies from our lab have also shown that CPF, MPT and MLT exposure singly and in mixture caused inhibition of antioxidant defense and disturbed the redox balance in rat tissues [48,49]. Several reports on the OP pesticides induced oxidative stress are available in literature. The dose-dependent decrease in the viability of lymphocytes on exposure with these pesticides may also be due to increased production of ROS. Involvement of ROS has already been reported in cytotoxicity of many OP pesticides in cultured cells [50,51]. Results of the present study showed that in vitro exposure of rat lymphocytes with CPF, MPT and MLT, singly and in mixture, caused dose-dependent increase in the DNA damage measured by single cell gel electrophoresis. The % tail DNA, tail length and tail moment were increased on exposure of rat lymphocytes with these OP pesticides in dose-dependent manner. DNA oxidation is known to be the most common type of DNA damage to human

Please cite this article in press as: A. Ojha, N. Srivastava, In vitro studies on organophosphate pesticides induced oxidative DNA damage in rat lymphocytes, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2014), http://dx.doi.org/10.1016/j.mrgentox.2014.01.007

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and other species. The role of ROS in production of DNA single strand and double strand breaks and oxidative damage to nitrogenous bases is well known [19]. The damaged purine and pyrimidine bases were identified by modified comet assay using lesion-specific bacterial repair enzymes, Fpg and Endo III, to convert base damage to breaks. Fpg acts on damaged purines namely 8-oxoGua and also on ring opened purines, formamidopyrimidines (FaPyGua) and 4,6-diaminoformamidopyrimidine (FaPyAde) while oxidized pyrimidines including thymine glycol and uracil glycol are recognized by Endo III. Thus on treatment with these enzymes, the number of strand breaks increased which are compared with buffer treated group. It was observed that enzymes treatment revealed significantly high levels of Fpg–Endo sensitive sites, an indicator of oxidative damaged DNA bases. Significantly marked increase in the double strand breaks was also observed in lymphocyte DNA treated with these pesticides. In the present study, the difference in DNA damage parameters in the presence and absence of Fpg and Endo III enzymes suggest that oxidative stress is responsible for OP pesticides induced DNA damage. It is interesting to note that Fpg–Endo III treatment caused more breaks in the DNA of control lymphocytes thus fold increase in DNA damage parameters, %T DNA, TL and TM, was more than observed in the pesticide treated lymphocytes given similar Fpg–Endo III treatment. This may be due to the background DNA damage on pesticide treatment was so high compared with the control lymphocytes (about 11–16 folds higher) that not very large number of Fpg–Endo III sensitive sites were left for cleavage by these enzymes in pesticide treated lymphocytes. Similar findings were reported by Kushwaha et al. [52] during study of alkaline, Endo III and Fpg modified comet assay as biomarkers for the detection of oxidative DNA damage in rat tissues and lymphocytes with experimentally induced diabetes. There have been several investigations in vivo and in vitro on the correlation between toxicant induced DNA damage and oxidative stress. These studies include the effect of monocrotophos in tissues of rats [53], malathion on human liver carcinoma cells [54], chlorpyrifos in rat tissues and lymphocytes of mice [29,55], cypermethrin, pendimethalin and diclorovos on CHO cells [56], DDT on blood cells of humans chronically exposed to insecticides [38], diazinon in mouse sperm cells [57] as well as several other OP pesticides [23,24]. Amongst the three pesticides, CPF, MPT and MLT, tested in the present study, MPT exposure generated highest concentration of ROS, both H2 O2 and super oxide anion, and caused highest level of DNA damage in the form of single and double strand breaks. Besides being potent source of ROS, OP compounds also show alkylating properties and alkylating agents are known to cause DNA damage [23,58]. Alkylation of DNA bases either directly or indirectly via protein alkylation may probably be involved in DNA damage. Most genotoxins are electrophilic by themselves or activated to electrophilic intermediates that bind to critical macromolecules. Alkyl groups are more labile in electrophilic compounds and thus can easily alkylate DNA. Dichlorovos and malaoxon are electrophilic and are known to methylate DNA [55,58]. The study clearly showed that OP pesticide, CPF, MPT and MLT, induced oxidative stress in cultured peripheral blood lymphocytes of rats by enhancing the production of ROS and excessive ROS are responsible for DNA oxidation. Methyl parathion was most toxic among the three pesticides tested and caused highest level of DNA damage in lymphocytes. The study also showed that when these pesticides are given together, they do not have synergistic effect.

5. Conclusion Present study conclusively demonstrates that OP pesticides CPF, MPT and MLT, induced oxidative stress in rat lymphocytes by causing increased generation of ROS. The pesticides also caused DNA

7

damage by oxidation of purines and pyramiding bases, and inducing single and double strand breaks in DNA. The generation of ROS and DNA damage caused by these pesticides is well correlated. Since these pesticides are widely used in agriculture and household, caution should be exercised in their handling as prolonged exposure may lead to adverse health effects including cancer or organ damage.

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Conflict of interest statement

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None. Uncited reference

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Q2

[32]. Acknowledgements The financial supports of Department of Science and Technology, New Delhi, India, in the form of FIST Grant to the School, and University Grants Commission, New Delhi, India, in the form of individual research project to Nalini Srivastava, are thankfully acknowledged. References [1] D.J. Ecobichon, Pesticide use in developing countries, Toxicology 7 (2001) 27–33. [2] D.J. Ecobichon, Toxic effects of pesticides, in: C.D. Klaassen, J. Doull (Eds.), Casarett and Doull’s Toxicology: The Basic Science of Poisons, 5th ed., Macmillan, New York, 1996, pp. 643–689. [3] http://en.wikipedia.org/wiki/Health effects of pesticides [4] C.A. Damalas, I.G. Eleftherohorinos, Pesticide exposure, safety issues, and risk assessment indicators, Int. J. Environ. Res. Pub. Health 6 (2011) (web of science). [5] http://en.wikipedia.org/wiki/Environmental impact of pesticides [6] T. Satoh, R.C. Gupta, Anticholinesterase pesticides: metabolism, neurotoxicity, Q3 and epidemiology, in: Anticholinesterase Pesticides, Wiley, 2011. [7] F. Kamel, J.A. Hoppin, Association of pesticide exposure with neurologic dysfunction and disease, Environ. Health. Perspec. 112 (2004) 950–958. [8] A. Lukaszewicz-Hussain, Role of oxidative stress in organophosphate toxicity—short review, Pestic. Biochem. Physiol. 98 (2010) 145–150. [9] F. Gultekin, N. Delibas, S. Yasar, I. Kilinc, In vivo changes in antioxidant systems and protective role of melatonin and a combination of vitamin C and vitamin E on oxidative damage in erythrocytes induced by chlorpyrifos-ethyl in rats, Arch. Toxicol. 75 (2001) 88–96. [10] A. Ranjabar, P. Pasalar, M. Abdollahi, Induction of oxidative stress and acetylcholinesterase inhibition in organophosphorus pesticide manufacturing workers, Hum. Exp. Toxicol. 21 (2002) 179–182. [11] O. Lopez, A.F. Hernadez, L. Rodrigo, F. Gil, G. Pena, J.L. Serrano, Changes in antioxidant enzymes in humans with long-term exposure to pesticides, Toxicol. Lett. 171 (2007) 146–153. [12] J.F. Muniz, L. McCauley, J. Scherer, M. Lasarev, M. Koshy, Y.W. Kow, V. Naz- Stewart, G.E. Kisby, Biomarkers of oxidative stress and DNA damage in agricultural workers: a pilot study, Toxicol. Appl. Pharmacol. 227 (2008) 97–107. [13] R. Olinski, D. Gackowski, M. Foksinski, R. Rozalski, K. Roszkowski, P. Jaruga, Oxidative DNA damage: assessment of the role in carcinogenesis, atherosclerosis, and acquired immunodeficiency syndrome, Free Rad. Biol. Med. 33 (2002) 192–200. [14] M.S. Cooke, M.D. Evans, M. Dizdaroglu, J. Lunec, Oxidative DNA damage: mechanisms, mutation, and disease, FASEB J. 17 (2003) 1195–1214. [15] H.M. Shen, C.N. Ong, Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility, Free Rad. Biol. Med. 28 (2000) 529–536. [16] E. Tvrda, Z. Knazicka, L. Bardos, P. Massanyi, N. Lukac, Impact of oxidative stress on male fertility: a review, Acta Vet. Hung. 59 (2011) 465–484. [17] http://www.senescence.info/causes of aging.html. [18] E.C. Friedberg, G.C. Walker, W. Siede, DNA Repair and Mutagenesis, ASM Press, Washington, DC, 1995. [19] A. Azqueta, S. Shaposhnikov, A.R. Collins, DNA oxidation: investigating its key role in environmental mutagenesis with the comet assay, Mutat. Res. 674 (2009) 101–108. [20] A.R. Collins, M. Dusinska, A. Horska, Detection of alkylation damage in human lymphocyte DNA with the comet assay, Acta Biochem. Pol. 48 (2001) 611–614. [21] A.R. Collins, M. Dusinska, E. Horvathova, E. Munro, M. Savio, R. Stetina, Interindividual differences in repair of DNA base oxidation, measured in vitro with the comet assay, Mutagen 16 (2001) 297–301. [22] N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988) 184–191.

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Please cite this article in press as: A. Ojha, N. Srivastava, In vitro studies on organophosphate pesticides induced oxidative DNA damage in rat lymphocytes, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2014), http://dx.doi.org/10.1016/j.mrgentox.2014.01.007

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