APPENDIX II-AK: Shushila Patel, et al, Cypermethrin-Induced DNA Damage in Organs and Tissues of the Mouse: Evidence from the Comet Assay, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Volume 607, Issue 2, 5 September 2006, Pages 176-183

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doi:10.1016/j.mrgentox.2006.04.010
Copyright © 2006 Elsevier B.V. All rights reserved.

Cypermethrin-induced DNA damage in organs and tissues of the mouse: Evidence from the comet assay

Sushila Patel^a, Alok K. Pandey^a, Mahima Bajpayee^a, Devendra Parmar^a and Alok Dhawan^,^a^,^,
^aDevelopmental Toxicology Section, Predictive Toxicology Group, Industrial Toxicology Research Centre, PO Box 80, M.G. Marg, Lucknow 226001, Uttar Pradesh, India
Received 25 October 2005; revised 13 February 2006; accepted 10 April 2006. Available online 12 June 2006.

Abstract

Cypermethrin is the most widely used Type II pyrethroid pesticide because of its high effectiveness against target species and its low mammalian toxicity reported so far. It is a fast-acting neurotoxin and is known to cause free radical-mediated tissue damage.

The present study investigates the genotoxic effects of cypermethrin in multiple organs (brain, kidney, liver, spleen) and tissues (bone marrow, lymphocytes) of the mouse, using the alkaline comet assay. Male Swiss albino mice were given 12.5, 25, 50, 100, 200 mg/kg BW of cypermethrin intraperitoneally, daily for 5 consecutive days. A statistically significant (p < 0.05) dose-dependent increase in DNA damage was observed in all the organs assessed, as evident from the comet-assay parameters, viz., Olive tail moment (OTM; arbitrary unit), tail DNA (%) and tail length (μm). Brain showed maximum DNA damage followed by spleen > kidney > bone marrow > liver > lymphocytes, as evident by the OTM.

Our data demonstrate that cypermethrin induces systemic genotoxicity in mammals as it causes DNA damage in vital organs like brain, liver, kidney, apart from that in the hematopoietic system.

Keywords: Multiple-organ genotoxicity; Swiss mice; Cypermethrin; DNA damage; Comet assay

Article Outline

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Animals and treatment

2.3. Single-cell preparation from tissues

2.4. Single-cell preparation from organs

2.5. Cell count and viability assay

2.6. Single-cell gel electrophoresis/comet assay

2.6.1. Preparation of slides and lysis

2.6.2. Electrophoresis and staining of slides

2.7. Scoring of slides

2.8. Statistics

3. Results

4. Discussion

Acknowledgements

References

1. Introduction

An increase in global food demand has resulted in a significant increase in the use of pesticides in agriculture. This has caused great concern among health and environmental scientists, since some of these chemicals induce mutations (somatic as well as germ-line) in experimental systems [1]. In humans, exposure to pesticides has been associated with cancer [2].

Synthetic pyrethroid pesticides account for over 30% of the global pesticide use [3]. Two distinct classes of pyrethroids have been identified, based on different behavioral, neuropsychological and biochemical profiles. Type I pyrethroids mainly cause hyper-excitation and fine tremors, while Type II pyrethroids possess a cyano-group and produce a more complex syndrome, including clonic seizures [4]. These compounds have gained popularity over organochlorine and organophosphate pesticides due to their high effectiveness against target species [5], their relatively low mammalian toxicity [6] and rapid biodegradability [7]. Cypermethrin, the alpha-cyano-3-phenoxybenzyl ester of 2,2-dimethyl-3-(2,2-dichlorovinyl) cyclopropane carboxylic acid, is the most widely used Type II pyrethroid pesticide. It is a composite synthetic pyrethroid, a broad spectrum, bio-degradable insecticide, and a fast-acting neurotoxin with good contact and stomach action. It is used to control many pests, including moths, pests of cotton, fruit, and vegetable crops. Consistent with its lipophilic nature, cypermethrin has been found to accumulate in body fat, skin, liver, kidneys, adrenal glands, ovaries, and brain [8].

Cypermethrin exerts its neurotoxic effect through voltage-dependent sodium channel [9] and integral protein ATPase in the neuronal membrane [10]. In vitro and in vivo studies have also shown that it causes free radical-mediated tissue damage in brain, liver [11] and erythrocytes [12].

Cypermethrin has been classified by the US Environmental Protection Agency [13] (EPA, 1989) as a possible carcinogen. The pesticide has been shown to induce chromosomal aberrations and micronucleus formation in mouse bone marrow as well as in spleen [14] and [15]. It also increases the frequency of sister chromatid exchange in bone marrow cells of mice [16]. DNA damage was observed in lymphocytes of workers occupationally exposed to pesticides such as cypermethrin [17].

Conventional cytogenetic techniques like the micronucleus assay, the chromosomal aberration test and the sister chromatid exchange assay, assess genotoxicity in cells exhibiting mitotic activity, such as those in the hematopoietic system. In spite of the blood–organ barriers, several chemicals reach these organs and elicit their toxic response including genotoxicity. However, the genotoxicity in organs cannot be assessed using conventional cytogenetic techniques. Therefore, Tsuda et al. [18] devised a method to assess multi-organ genotoxicity in the mouse using alkaline comet assay. This is a rapid and sensitive procedure to measure DNA lesions in any organ regardless of its mitotic activity.

An attempt was, therefore, made in the present study to examine the genotoxic effect of cypermethrin in major organs and tissues of mouse.

2. Materials and methods

2.1. Materials

Technical grade cypermethrin (purity 98.5%, CAS no.: 52315-07-8) was a gift from Aimco Pesticides Ltd., Mumbai, India. Normal melting agarose (NMA) and ethylenediaminetetraacetic acid disodium salt (Na₂EDTA) were purchased from Hi Media Pvt. Ltd., Mumbai, India. Phosphate-buffered saline (PBS; Ca²⁺- and Mg²⁺-free) and fetal bovine serum (FBS) were purchased from Life Technologies (India) Pvt. Ltd., New Delhi, India. Low melting-point agarose (LMA), ethidium bromide (EtBr), Histopaque 1077, Hank's balanced salt solution (HBSS), 5,6-carboxyflourescein, Triton X-100, ethylmethane sulfonate (CAS no.: 62-50-0) and trypan blue were purchased from Sigma Chemicals Co. Ltd., St. Louis, MO, USA. Ethanol and dimethyl sulfoxide (DMSO) were obtained from Qualigens Fine Chemicals, Mumbai, India. Sodium chloride (NaCl) was purchased from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Sodium hydroxide (NaOH) was obtained from Merck Pvt. Ltd., Mumbai, India and Tris Buffer from Spectrochem Pvt. Ltd., Mumbai, India, respectively. All other chemicals were obtained locally and were of analytical reagent grade.

2.2. Animals and treatment

Male Swiss albino mice (6-week-old, 20 ± 2 g) were obtained from the Industrial Toxicology Research Centre (Lucknow, India) breeding colony and raised on a commercial pellet diet (Dayal Industries, Lucknow, India) and water ad libitum. Animals were cared for in accordance with the policy laid down by the Animal Ethics Committee of Industrial Toxicology Research Centre, Lucknow. Experiments were planned according to comet assay guidelines [19] and were approved by the Animal Ethics Committee of the Centre.

The study design included seven groups of four mice each caged separately, which were treated intraperitoneally, daily for five consecutive days with 12.5, 25, 50, 100, 200 mg/kg BW dose of cypermethrin as follows:

Group 1 (vehicle control)—corn oil (10 ml/kg BW, 5 consecutive days).

Group 2 (positive control)—ethylmethane sulfonate (100 mg/kg BW, 24 h before sacrifice).

Groups 3–7 were given cypermethrin, intraperitoneally and daily for 5 consecutive days with an interval of 24 h between treatments. The last dose was given 6 h before sacrifice, as follows: Group 3, 12.5 mg/kg BW; Group 4, 25 mg/kg BW; Group 5, 50 mg/kg BW; Group 6, 100 mg/kg BW; Group 7, 200 mg/kg BW.

Animals were sacrificed by cervical dislocation after withdrawing blood from the orbital sinus. Organs were taken out immediately for isolation of single cells.

2.3. Single-cell preparation from tissues

Lymphocytes were isolated from whole blood using Histopaque-1077 by the method of Boyum [20] with slight modifications. Briefly, 20 μl of blood was added to 1 ml RPMI-1640 (Gaithersburg, MD, USA) and layered over 100 μl Histopaque. This was centrifuged at 500 × g for 3 min. The interface of media/Histopaque containing the lymphocytes was taken and added to 1 ml media (RPMI-1640). It was then centrifuged at 500 × g for 3 min to pellet the lymphocytes, which were resuspended in PBS for further studies.

Both the femurs were dissected out and cleaned thoroughly to remove muscles and other tissue. Bone marrow cells were flushed in 1 ml FBS using a syringe.

2.4. Single-cell preparation from organs

Preparation of a single-cell suspension from organs was done according to the method of Tice et al. [21]. Briefly, 0.2 g of each organ was placed in 1 ml chilled mincing solution (Hank's balanced salt solution, with 20 mM EDTA and 10% DMSO) in a petridish and chopped into pieces with a pair of scissors. The pieces were allowed to settle and the supernatant containing the single cells was taken.

2.5. Cell count and viability assay

Cells from organs and tissues were counted using a hemocytometer and diluted with PBS to achieve a concentration of 0.2 × 10⁶ cells/ml. The viability of cells isolated from liver, spleen, kidney and brain was checked by 5,6-carboxyflourescein dye [22], while trypan blue was used for lymphocytes and bone marrow cells [23].

2.6. Single-cell gel electrophoresis/comet assay

2.6.1. Preparation of slides and lysis

Conventional slides (size 75 mm × 25 mm, Blue Label Scientifics Pvt. Ltd., Mumbai, India) with 19-mm frosted ends were used. The slides were dipped in methanol and burnt on a blue flame to remove traces of machine oil. NMA (1.0%) was prepared in Milli-Q water, microwaved and kept at 60 °C. Slides were then dipped into molten NMA up to two-thirds of their frosted end, the lower surface was wiped clean and the slides were left to dry. On this first layer, 80 μl of diluted sample (100 μl cell suspension mixed with 100 μl of 1% LMA) was added to form the second layer. A cover slip (size 24 mm × 60 mm, No. 1, Blue Label Scientifics Pvt. Ltd., Mumbai, India) was placed gently to evenly spread the cells in the agarose. The slides were kept on ice for 5 min to allow the gel to solidify. The cover slips were removed and a third layer of 0.5% LMA (90 μl) was added onto the slide and placed over ice for 10 min. Finally the cover slips were removed and the slides immersed in freshly prepared chilled lysing solution containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris (pH 10) with 10% DMSO and 1% Triton X-100 being added just before use. The slides remained in the lysing solution for at least 4 h at 4 °C.

2.6.2. Electrophoresis and staining of slides

Electrophoresis was carried out according to the method of Singh et al. [24]. The slides were placed in a horizontal gel electrophoresis tank (Life Technologies, Gaithersburg, USA) side-by-side and avoiding spaces, with agarose ends nearest to the anode. Fresh and chilled electrophoresis buffer (1 mM Na₂EDTA and 300 mM NaOH, pH > 13) was poured into the tank to a level of approximately 2.5 mm above the slides. The slides were left in this solution for 25 min to allow DNA unwinding and expression of alkali-labile sites as DNA strand breaks. Electrophoresis was conducted at 24 V (0.7 V/cm) and a current of 330 mA using a power supply (Electra Comet III from Techno Source India Pvt. Ltd., Mumbai, India) for 30 min at 4 °C. All these steps were performed under dimmed light and the electrophoresis tank was covered with black paper to avoid additional DNA damage due to stray light. After electrophoresis, the slides were drained and placed horizontally in a tray. Tris buffer (0.4 M; pH 7.5) was added drop-wise and left for 5 min to neutralize excess alkali. Neutralizing of slides was repeated thrice.

Each slide was stained with 75 μl EtBr (20 μg/ml) for 5 min and dipped in chilled distilled water to wash off excess EtBr, and cover slip placed over it. Slides were placed in a dark humidified chamber to prevent drying of the gel. The slides were scored within 24 h.

2.7. Scoring of slides

Slides were scored using an image-analysis system (Kinetic Imaging, Liverpool, UK) attached to a fluorescence microscope (Leica, Germany) equipped with appropriate filters (N2.1, excitation wavelength of 515–560 nm and emission wavelength of 590 nm). The microscope was connected to a computer through a charge-coupled device (CCD) camera to transport images to software (Komet 3.1) for analysis. The final magnification was 400×. The comet parameters recorded were Olive tail moment (OTM, arbitrary units), tail DNA (%) and tail length (migration of the DNA from the nucleus, μm). Images from 50 cells (25 from each replicate slide) were analyzed.

2.8. Statistics

The data of comet parameters were tested for homogeneity of variance and normality, and were found normally distributed. The data were, therefore, analyzed by use of one-way analysis of variance (ANOVA). In all cases p < 0.05 was considered significant compared with the respective controls.

3. Results

Cell viability for all the samples was found to be more than 90% in every experiment (data not shown).

A statistically significant (p < 0.05) dose-dependent increase in DNA damage was observed in all organs and tissues of mice exposed to cypermethrin, as was evident from an increase in the Olive tail moment (arbitrary units; Table 1). A similar pattern was observed for tail DNA (%) and tail length (μm) (Table 2 and Table 3). However, no significant DNA damage was observed in the mice administered the lowest dose of cypermethrin, i.e., 12.5 mg/kg BW (Group-3) as was evident from the OTM values (Table 1).

Table 1.

Effect of cypermethrin on nuclear DNA of various organs and tissues in mice with respect to Olive tail moment (arbitrary units)

Groups	Bone marrow	Brain	Kidney	Liver	Lymphocytes	Spleen
Control^a	3.28 ± 0.38	3.91 ± 0.38	3.20 ± 0.16	2.90 ± 0.18	3.23 ± 0.21	3.10 ± 0.15

CYP^b—12.5 mg/kg BW	3.93 ± 0.33^ns	9.41 ± 0.28^ns	3.58 ± 0.21^ns	3.05 ± 0.14^ns	3.66 ± 0.08^ns	3.86 ± 0.35 ^ns
CYP—25 mg/kg BW	5.40 ± 0.09^***	9.84 ± 1.64^*	6.45 ± 0.32^***	4.25 ± 0.63^*	4.40 ± 0.16^**	6.15 ± 0.55^***
CYP—50 mg/kg BW	6.13 ± 0.27^***	11.24 ± 1.89^*	6.92 ± 0.43^***	5.97 ± 0.29^***	5.64 ± 0.10^***	7.24 ± 0.21^***
CYP—100 mg/kg BW	6.59 ± 0.22^***	12.63 ± 1.21^**	7.32 ± 0.53^***	7.18 ± 0.42^***	5.84 ± 0.39^***	9.20 ± 0.43^***
CYP—200 mg/kg BW	7.52 ± 0.38^***	23.45 ± 3.87^***	13.65 ± 1.18^***	11.21 ± .52^***	8.62 ± 0.23^***	11.31 ± 0.40^***

EMS^c 100 mg/kg BW	22.08 ± 1.95^***	29.75 ± 1.85^***	24.09 ± 3.31^***	23.76 ± 3.11^***	19.15 ± 2.19^***	27.20 ± 2.93^***

Values represent mean ± S.E. of four animals in each group. ns: not significant, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 when compared with control.
^a Corn oil-vehicle control (10 ml/kg BW).
^b CYP—cypermethrin.
^c EMS—ethylmethane sulfonate—positive control (100 mg/kg BW).

Table 2.

Effect of cypermethrin on nuclear DNA of various organs and tissues in mice with respect to tail DNA (%)

Groups	Bone marrow	Brain	Kidney	Liver	Lymphocytes	Spleen
Control^a	9.97 ± 0.77	11.12 ± 0.47	10.16 ± 0.29	9.25 ± 0.68	10.44 ± 0.64	10.07 ± 0.40

CYP^b—12.5 mg/kg BW	11.64 ± 0.76^ns	19.81 ± 0.33^**	11.26 ± 0.56^ns	9.86 ± 0.52^ns	11.45 ± 0.26^ns	12.01 ± 1.09^ns
CYP—25 mg/kg BW	14.55 ± 0.34^***	21.61 ± 2.80^**	17.78 ± 0.57^***	12.73 ± 2.14^*	14.51 ± 0.29^***	17.42 ± 0.93^***
CYP—50 mg/kg BW	16.25 ± 0.68^***	22.01 ± 2.20^**	18.74 ± 1.64^***	16.17 ± 0.59^***	15.27 ± 1.27^***	17.59 ± 1.39^***
CYP—100 mg/kg BW	16.40 ± 0.69^***	23.96 ± 1.74^**	18.86 ± 0.89^***	17.25 ± 0.70^***	17.26 ± 0.29^***	19.32 ± 0.22^***
CYP—200 mg/kg BW	19.37 ± 0.83^***	34.53 ± 3.27^***	27.08 ± 2.15^***	20.98 ± 0.58^***	21.68 ± 0.75^***	23.22 ± 0.75^***

EMS^c 100 mg/kg BW	33.65 ± 1.20^***	42.95 ± 0.89^***	37.33 ± 2.38^***	37.51 ± 2.38^***	36.63 ± 2.71^***	37.40 ± 0.85^***

Table 3.

Effect of cypermethrin on nuclear DNA of various organs and tissues in mice with respect to tail length (μm)

Groups	Bone marrow	Brain	Kidney	Liver	Lymphocytes	Spleen
Control^a	61.73 ± 7.70	72.06 ± 10.15	70.24 ± 9.53	67.33 ± 7.94	58.60 ± 5.14	63.01 ± 6.98

CYP^b—12.5 mg/kg BW	97.28 ± 5.57^**	135.61 ± 16.23^**	83.96 ± 3.28^ns	92.67 ± 2.07^**	87.58 ± 0.46^***	82.58 ± 4.16^*
CYP—25 mg/kg BW	109.71 ± 1.28^***	138.67 ± 6.97^***	111.52 ± 1.51^***	104.80 ± 2.39^***	90.37 ± 2.08^***	109.53 ± 3.32^***
CYP—50 mg/kg BW	113.50 ± 7.26^***	164.43 ± 12.17^***	124.95 ± 4.10^***	137.46 ± 6.10^***	97.94 ± 4.50^***	107.44 ± 5.44^***
CYP—100 mg/kg BW	122.45 ± 7.85^***	175.25 ± 14.05^***	140.92 ± 11.33^***	147.05 ± 10.00^***	121.72 ± 3.71^***	146.95 ± 9.28^***
CYP—200 mg/kg BW	123.03 ± 6.69^***	182.15 ± 5.43^***	145.22 ± 3.38^***	191.72 ± 5.31^***	145.05 ± 14.84^***	141.66 ± 4.42^***

EMS^c 100 mg/kg BW	186.12 ± 11.55^***	195.93 ± 7.53^***	183.92 ± 11.14^***	191.72 ± 5.31^***	85.05 ± 1.25^***	205.85 ± 13.98^***

Values represent mean ± S.E. of four animals in each group. ns = not significant, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 when compared with control.
^a Corn oil-vehicle control (10 ml/kg BW).
^b CYP—cypermethrin.
^c EMS—ethylmethane sulfonate—positive control (100 mg/kg BW).

When OTM values were compared at different doses, brain was found to exhibit the highest level of DNA damage followed by spleen > kidney > bone marrow > liver > lymphocytes (Table 1 and Fig. 1). An almost similar pattern was observed for tail DNA (%) and tail length (μm) (Table 2 and Table 3).

Display Full Size version of this image (166K)

Fig. 1. Effect of cypermethrin on percentage distribution of cells with respect to Olive tail moment in: (A) Lymphocytes, (B) Bone marrow, (C) Brain, (D) Liver, (E) Spleen, and (F) Kidney.

The OTM data were further analyzed in terms of percentage distribution of cells. With increasing dose of cypermethrin a shift from less damaged cells in control (OTM category <2) to highly damaged cells (OTM category >10) in all the organs was observed (Fig. 1).

4. Discussion

The molecular mechanisms of the genotoxicity of cypermethrin are not yet elucidated and require further studies. Due to the hydrophobic nature and small molecular size, cypermethrin passes through the cell membrane and reaches the nucleus. It is suggested that within the nucleus cypermethrin binds to DNA through the reactive groups of its acid moiety, leading to destabilization as well as unwinding of the DNA, which could be a possible mechanism for its genotoxicity [25]. Cypermethrin has been shown to induce oxidative stress and generation of reactive oxygen species (ROS) in experimental systems [11] and [26]. It has been demonstrated that ROS may cause DNA damage, which could lead to single-strand breaks and mutation [27].

Our data clearly show that cypermethrin induces DNA strand breaks in different organs and tissues of mice, with the brain showing the highest level of damage. The results are consistent with our previous findings where the insecticide caused DNA damage in brain ganglia of Drosophila melanogaster [28]. Cypermethrin induces free radical-mediated lipid peroxidation in rat brain [11]. This provides evidence that DNA damage observed after cypermethrin exposure could be a consequence of free radical attack to DNA.

Brain is one of the target organs of cypermethrin as it acts on the sodium channels and induces neurotoxicity [9]. It is known that after absorption, pyrethroids rapidly distribute through the body [29] and readily enter the brain because of their high lipophilicity and lack of exclusion by the multi-drug transporter glycoprotein [30] at the blood–brain barrier. Previous studies from our laboratory have shown that deltamethrin, a Type II pyrethroid belonging to the same group as cypermethrin, is metabolized by cytochrome P450s in rat brain and has a greater neurotoxic potential than the parent compound [31], [32] and [33] and probably does not get cleared from the brain due to the blood–brain barrier [34]. The fact that metabolites of cypermethrin are generally hydrophilic, which hampers their clearance from the brain due to the blood–brain barrier, and the high metabolic activity and low DNA-repair capacity in the brain [35] could be the reason that the highest level of genotoxicity was observed in the brain.

Cypermethrin-induced DNA damage in other vital organs like liver and kidney. This could be attributed to the fact that cypermethrin exposure induces free radical-mediated tissue damage in rat liver and kidney [26].

An induction of DNA damage in the hematopoietic system, viz., spleen, bone marrow and lymphocytes was observed in the present study, which is consistent with the study of Bhunya and Pati [36] showing that cypermethrin induces chromosomal aberrations and micronucleus formation in mouse bone marrow. Amer et al. [15] have reported an increased incidence of sister chromatid exchange and chromosomal aberrations in mouse spleen cells after in vitro exposure to cypermethrin. Their data were supported by an epidemiological study showing DNA damage in human lymphocytes by use of the comet assay [17]. These studies support the findings of the present study where we have observed DNA damage in different tissues using the comet assay.

The differences in DNA damage observed among the organs in the present study could be explained by the fact that there is a differential expression of basal levels of DNA repair genes in various tissues of the mouse as shown by Tomascik-Cheeseman et al. [37] using DNA microarrays. Moreover, the variation in constitutive expression of cytochrome P450 across the various organs in mice has been demonstrated [38]. For example, CYP1A1 is shown to be maximally expressed in spleen followed by liver and kidney. Thus, the fact that tissues differ considerably in their capacity to biotransform xenobiotics has important toxicological implications, e.g., with respect to DNA damage formation.

The fact that cypermethrin induces DNA damage in various organs and tissues of mice bears significance, as cypermethrin exposure can lead to adverse effect on humans via systemic genotoxicity. Differential DNA damage as well as repair in various organs could lead to an increase in the susceptibility towards disease and disorders.

Acknowledgments

Financial assistance from Council of Scientific and Industrial Research, New Delhi, to S.P. (Junior Research Fellowship), M.B. (Senior Research Fellowship) and A.D. (HRD-YSA-99/project/99) is gratefully acknowledged. The authors wish to acknowledge the valuable contribution of Mr. Akhilesh Banerjee, BITS Pilani, as part of his M.Tech. (Biotechnology) summer project. The technical assistance of Mr. B.S. Pandey and Mr. Rajesh Mishra is also acknowledged.

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Corresponding author. Tel.: +91 522 2613786x320; fax: +91 522 2628227/2611547.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis
Volume 607, Issue 2, 5 September 2006, Pages 176-183