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Protective effects of vitamin E against atrazine-induced genotoxicity in rats
Mutation Research 654 (2008) 145–149
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Protective effects of vitamin E against atrazine-induced genotoxicity in rats Mohan Singh a,∗ , Pushpindar Kaur b , Rajat Sandhir a , Ravi Kiran a a b
Department of Biochemistry, Panjab University, Chandigarh 160014, India Department of Zoology, Panjab University, Chandigarh 160014, India
a r t i c l e
i n f o
Article history: Received 29 September 2007 Received in revised form 9 May 2008 Accepted 24 May 2008 Available online 5 June 2008 Keywords: Atrazine Comet assay Genotoxicity Micronucleus assay Vitamin E
a b s t r a c t Atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine) is one of the most commonly used herbicides to control grasses and weeds. The widespread contamination and persistence of atrazine residues in the environment has resulted in human exposure. Vitamin E is a primary antioxidant that plays an important role in protecting cells against toxicity by inactivating free radicals generated following pesticides exposure. The present study was undertaken to investigate the protective effect of vitamin E against atrazine-induced genotoxicity. Three different methods: gel electrophoresis, comet assay and micronucleus test were used to assess the atrazine-induced genotoxicity and to evaluate the protective effects of vitamin E. Atrazine was administered to male rats at a dose of 300 mg/kg body weight for a period of 7, 14 and 21 days. There was a significant increase (P < 0.001) in tail length of comets from blood and liver cells treated with atrazine as compared to controls. Co-administration of vitamin E (100 mg/kg body weight) along with atrazine resulted in decrease in tail length of comets as compared to the group treated with atrazine alone. Micronucleus assay revealed a significant increase (P < 0.001) in the frequency of micronucleated cells (MNCs) following atrazine administration. In the animals administrated vitamin E along with atrazine there was a significant decrease in percentage of micronuclei as compared to atrazine treated rats. The increase in frequency of micronuclei in liver cells and tail length of comets confirm genotoxicity induced by atrazine in blood and liver cells. In addition, the findings clearly demonstrate protective effect of vitamin E in attenuating atrazine-induced DNA damage. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Triazine herbicides constitute one of the largest groups of herbicides used throughout the world. Among the triazines, atrazine is one of the most commonly used herbicides to control dicotyledonous weed plants in maize, cereals, lucerne and sugarcane [1]. As a result of its widespread use atrazine residues have contaminated not only plants, soil, water and cultivated ground but also agricultural products like fruits, milk, butter, and sugar beet [2]. Deleterious effects of pesticides on human and animal health have been well documented in literature [3–6]. Studies have also indicated that pesticide exposure is associated with chronic health problems or health symptoms such as respiratory problems, memory disorders, dermatologic conditions, cancer, depression, neurologic deficits, miscarriages, and birth defects [7–13]. Various in vitro studies have shown the ability of atrazine to induce genetic damage in human and animal cells
∗ Corresponding author. Tel.: +91 172 2534133; fax: +91 172 2541022. E-mail address: [email protected] (M. Singh). 1383-5718/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2008.05.010
[14–17]. Further, it has been reported that pesticides such as alachlor, acephate, chlorpyriphos and atrazine induce DNA damage [18–20] and chromosomal aberrations among farm workers [21–23]. Antioxidant vitamins are able to inactivate highly reactive molecules, such as free radicals, that are generated during various biochemical processes in the cells [24]. A large number of antioxidants like vitamin C, E and plant derivatives have been tested in the experimental animals in reducing the clastogenicity induced by drugs [25,26]. The protective effects of vitamin E against pesticide-induced toxicity have been reported [27–29]. In addition, vitamin E has been shown to be effective in reducing genotoxic effects of various genotoxic compounds [30,31]. Of the antioxidants tested, vitamin E shows most promising effect in reducing genotoxic effects induced by chemicals. However, no information is available concerning the ameliorating effect of vitamin E against atrazine-induced genotoxicity. Therefore, in the present study we evaluated the genotoxicity induced by atrazine and the possible beneficial effect of vitamin E against atrazine-induced genotoxicity using comet assay, which is a rapid, sensitive and inexpensive method for
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measuring the DNA damage [32], and micronucleus test along with assessment of DNA damage by agarose gel electrophoresis. 2. Materials and methods 2.1. Chemicals Atrazine (Technical grade, 97.83%) was a gift from Meghmani Industries Ltd. (India). Vitamin E (˛-tocopheryl acetate, trade name-Evion) was procured from Merck Pharmaceuticals (India). Agarose (normal and low melting) was procured from Sigma Chemical Co. (USA). Triton X-100 was purchased from SISCO Research Laboratories (India). Tris, ethidium bromide, May Grunwald’s stain and Giemsa stain were procured from Himedia Laboratories (India). EDTA was purchased from Merck Ltd. (India). All other chemicals used were obtained from Qualigens Fine Chemicals (India) and were of analytical reagent grade. 2.2. Animals and treatment Male rats (Wistar strain), weighing about 100–120 g, were used in the study as males have been shown to be more susceptible than females to genotoxic effects of various chemicals [33]. The animals were housed in polypropylene cages, given water ad libitum and fed standard pellet diet. The rats were segregated into four groups, each group having 18 animals. Animals in each group were further subdivided into three sub-groups and were treated with atrazine and/or vitamin E daily for a period of 7, 14 and 21 days as described below. Animals in the control group received 1 ml of corn oil, orally. Atrazine treated group were given atrazine (300-mg/kg body weight) dissolved in corn oil. Vitamin E treated animals were administered vitamin E (100-mg/kg body weight) dissolved in corn oil. Animals in the atrazine + vitamin E group received a mixture of atrazine (300 mg/kg body weight) and vitamin E (100 mg/kg body weight) dissolved in corn oil, orally. Atrazine was given at a dose of 300-mg/kg body weight as it was the maximum tolerated dose based on the pilot study conducted with various doses of atrazine and also based on the doses reported in literature [34]. The dose of vitamin E used was based on the dose that was seen to be most effective in lowering toxicity induced by various xenobiotics [35]. 2.3. Collection of blood samples Blood samples (1 ml) were collected from supra orbital sinus using glass capillaries into heparinized vacutainers (Becton-Dickinson, UK). 2.4. Preparation of tissue homogenate for micronucleus test and comet assay At the end of the respective treatments rats were sacrificed under light ether anesthesia followed by cervical dislocation and their livers removed. The livers were perfused with ice-cold saline till all the traces of blood were removed. The final washing was given with chilled homogenizing buffer (pH 7.5) containing 0.024 M EDTA, 0.075 M NaCl and DMSO 10%. After weighing, the livers were minced, suspended in chilled homogenization buffer at a concentration of 1 g/ml and were homogenized on ice using a Potter-type homogenizator at 500–800 rpm. The homogenate was then centrifuged at 700 rpm for 10 min at 4 ◦ C. Supernatant was removed and the pellet was resuspended in a volume of 1 ml/g of the homogenization buffer. The pellet was stored at −20 ◦ C for further analyses.
The parameter used to assess DNA damage was tail length (migration of DNA from nucleus) and tail moment which was automatically generated by CometScore software. The control and treated slides were randomized and were not run separately or at different times to avoid variability. 2.6. Micronucleus test Micronucleus test was performed according to the method of Igarashi and Shimada [36]. The pellet obtained from the liver homogenate was re-suspended in the homogenization buffer and was allowed to settle down for 1–2 min. A small drop of the suspension was placed at one end of a pre-cleaned, grease free, microscopic slide. The drop was carefully spread into a single cell layered film without damaging the cell morphology using a polished cover glass held at an angle of 45 degree. The slides were then air dried in a dust free environment for 12 h and subsequently stained by the method described by Schmid [37]. Briefly, air dried slides were first stained for 1–2 min in concentrated May-Grunwald stain (0.25% in methanol) followed by 10% Giemsa stain solution for 10 min. The slides were then rinsed twice with distilled water, dried and rinsed with methanol. The slides were placed in xylene for clearing, mounted in DPX and analyzed for the presence of micronuclei (a total of 1000 cells were scored for each animal). 2.7. DNA isolation and electrophoresis DNA was extracted using kit DNeasy tissue kit (Qiagen GmbH, Germany), which utilizes the silica gel membrane technology for rapid and efficient purification of total cellular DNA without organic extraction or ethanol precipitation followed by agarose gel electrophoresis according to the method of Sambrook and Russel [38]. 2.8. Statistical analysis All values were expressed as mean ± S.D. of six animals per group. Data were analyzed using one way analysis of variance (ANOVA) followed by Newman–Keuls test for multiple pair wise comparison between the various groups. Values with P ≤ 0.05 were considered as significant.
3. Results and discussion DNA is a target for mutagens and carcinogens, which induce changes in DNA structure of giving rise to mutations and/or cell death [39]. Free radical generated following pesticide exposure may lead to extensive DNA damage. Atrazine is capable of inducing structural changes in chromosomes [40–43]. In the present study DNA damage was evaluated by electrophoresis of DNA isolated from rat liver. Administration of atrazine resulted in DNA damage as is evident from Fig. 1. Lanes 3, 5, 7 correspond to DNA from animals exposed to atrazine for 7, 14 and 21 days respectively. It is evident that exposure to atrazine resulted in shearing of DNA as compared
2.5. Comet assay Comet assay was performed under alkaline conditions according to the method of Singh et al. [32] with slight modifications. Microscopic slides (frosted) were covered by a thin layer of 1.0% normal melting agarose at about 50 ◦ C (dissolved in Ca2+ and Mg2+ free PBS). A cover slip was placed on the slide to promote even and firm attachment. Upon solidification of agarose, cover slip was gently removed and 30 l of sample suspension mixed with 75 l of low melting point agarose (LMPA) at 37 ◦ C was added. The cover slip was replaced and agarose was again allowed to solidify for 10 min at 4 ◦ C. The cover slip was again removed and the third layer of 85 l of 0.5% LMPA was added to the slide and was allowed to solidify on ice for 5 min. After solidification of third layer of agarose, cover slips were removed and slides were immersed in cold lysis solution containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris (pH 10) and 1% Triton X-100 was added prior to use for 1 h at 4 ◦ C. Overnight treatment in lysis buffer at 4 ◦ C is tolerated but prolonged lysis results in precipitation of the buffer. After lysis DNA was allowed to unwind for 30 min in alkaline electrophoretic solution consisting of 300 mM NaOH and 1 mM EDTA (pH > 13). Electrophoresis was performed at 4 ◦ C in field strength of 0.7 V/cm and current of 300 mA. The slides were then neutralized with cold 0.4 M Tris–HCl, pH 7.5, stained with 75 l ethidium bromide (20 g/ml) and then cover slipped. The slides were placed in a humidified chamber to prevent drying of gel and were analyzed on the same day using fluorescent microscope (NIKON ECLIPSE 80i) equipped with a 510–560 nm excitation and 590 barrier filters and integrated digital camera. Slides were scored using CometScore software (TriTek Corp., USA) and 50 cells were analyzed per sample.
Fig. 1. Agarose gel electrophoresis of DNA isolated from liver of rat after various treatments. Lane 1: DNA of control group after 21 days; lane 2: DNA of vitamin E treated group after 21 days; lane 3: DNA of atrazine treated group for 7 days; lane 4: DNA of atrazine and vitamin E treated for 7 days; lane 5: DNA of atrazine treated group for 14 days; lane 6: DNA of atrazine and vitamin E treated for 14 days; lane 7: DNA of atrazine treated group for 21days; lane 8: DNA of atrazine and vitamin E treated for 21 days.
M. Singh et al. / Mutation Research 654 (2008) 145–149
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Table 1 Effects of various treatments on tail length and tail moment in liver and blood cells Treatment
Organ
7 days
Control
Liver Blood
2.81 ± 0.29 3.17 ± 1.25
1.10 ± 0.76 3.09 ± 1.74
3.23 ± 0.70 3.29 ± 1.16
1.75 ± 1.02 2.87 ± 1.36
3.39 ± 1.06 3.19 ± 1.22
1.89 ± 0.94 3.44 ± 1.28
Atrazine
Liver Blood
9.86 ± 1.49* 14.24 ± 2.43*
4.46 ± 1.46* 13.36 ± 4.68*
17.14 ± 2.13* 22.91 ± 2.63*
15.04 ± 3.53* 14.63 ± 3.46*
22.64 ± 2.96* 25.07 ± 2.75*
12.46 ± 3.08* 14.38 ± 2.96*
Vitamin E
Liver Blood
Atrazine + vitamin E
Liver Blood
Tail length (m)
2.75 ± 0.88NS 2.35 ± 0.84NS 4.33 ± 0.69$ 11.00 ± 1.81$
14 days Tail moment
Tail length (m)
21 days Tail moment
0.89 ± 0.62NS 3.80 ± 2.26NS
2.85 ± 1.21NS 2.387 ± 1.17NS
1.69 ± 0.98NS 2.42 ± 1.01NS
4.39 ± 2.32NS 7.05 ± 1.95$
10.14 ± 1.31$ 16.05 ± 2.07$
4.09 ± 1.54$ 9.88 ± 2.85$
Tail length (m)
2.91 ± 0.92NS 2.36 ± 0.95NS 13.27 ± 2.32$ 17.72 ± 2.58$
Tail moment
1.53 ± 1.00NS 3.07 ± 1.24NS 9.09 ± 3.22$ 11.38 ± 2.68$
Values are expressed as mean ± S.D., n = 6. *Significant difference from control group (*P < 0.001). $ Significant difference from atrazine treated group ($ P < 0.001). NS: nonsignificant.
to control (lane 1) and vitamin E treated (lane 2). DNA damage was more pronounced after 14 and 21 days of atrazine treatment. It is clear from the data that extent of DNA damage is dependent on duration of atrazine exposure. Although atrazine did not induce classical DNA ladder pattern seen in apoptosis, but, shearing of DNA suggests DNA damage in the atrazine treated liver. Eldadah et al. [44] have also observed shearing of DNA instead of DNA laddering because of a fewer number of apoptotic cells or the possibility of laddering being masked by or necrotic cell death. Pino et al. [16] have also reported DNA breaks (and/or alkali-labile lesions) in cell suspensions obtained from stomach, kidney and liver of rats treated with atrazine. In addition, Liu et al. [45] have detected DNA fragmentation by atrazine in grass carp by TUNEL reaction and agarose gel electrophoresis. Vitamin E in combination with atrazine treatment reduced atrazine-induced shearing of DNA (lanes 4, 6 and 8). These results suggest that vitamin E has a protective effect on atrazine-induced DNA damage which is in agreement with the findings of Abid-Essefi et al. [46] showing that vitamin E can prevent genotoxicity induced by chemicals. These observations were supported by more sensitive assays like single cell gel electrophoresis (comet assay) and micronucleus test. Table 1 depicts the values of tail length and moment obtained from comet assay after treatment with atazine and/or vitamin E. It
is clear that there was a significant (P < 0.001) increase in tail length in blood and liver cells of rats treated with atrazine as compared to the control rats, which was also illustrated by the representative comets (Fig. 2). A significant increase in the tail length was observed at all the time points following atrazine exposure in both blood and liver cells. Using comet assay Tennant et al. [40] also showed that atrazine at concentrations of 250 and 500 mg/kg significantly increased DNA migration in the leukocytes of mice treated in vivo. Clements et al. [47] also had similar findings with comet assay in tadpoles exposed to atrazine. On the contrary, in an in vitro study on human lymphocytes, atrazine was not found to be genotoxic [43]. Data in Table 1 shows that vitamin E had a significant protective effect against atrazine-induced DNA damage at all the time points studied (P < 0.001). The protective effect of vitamin E against genotoxic effects of various chemicals has previously been reported [30,31,46]. Based on the results it is clear that atrazine might be inducing DNA damage by increased generation of reactive free radicals which are scavenged by vitamin E. The effects of vitamin E observed on atrazine-induced DNA damage might involve its antioxidant action and may not due to its interaction with atrazine as there are no documented reports about their interaction. This is the first report showing protective effect of vitamin E against atrazine-induced genotoxicity.
Fig. 2. Single cell gel electrophoresis of rat liver cells showing comets (40× magnification). Control after 21 days (A), vitamin E treated for 21 days (B), atrazine treated for 21 days (C), atrazine + vitamin E treated for 21 days (D).
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Table 2 Frequencies of micronucleated cells in liver after various treatments Time
Group
Total cells scored
Micronucleated cells
% Mean ± S.D.
7 days
Control Atrazine Vitamin E Atrazine + vitamin E
6000 6000 6000 6000
28 216 22 183
0.47 3.98 0.37 3.05
± ± ± ±
0.16 1.10* 0.16NS 0.51$
14 days
Control Atrazine Vitamin E Atrazine + vitamin E
6000 6000 6000 6000
26 360 23 314
0.43 6.00 0.38 5.23
± ± ± ±
0.27 1.15* 0.13NS 1.21NS
21 days
Control Atrazine Vitamin E Atrazine + vitamin E
6000 6000 6000 6000
24 392 23 336
0.40 6.53 0.38 5.60
± ± ± ±
0.17 0.90* 0.15NS 0.51$
Values are expressed as mean ± S.D., n = 6. *Significant difference from control group (*P < 0.001). $ Significant difference from atrazine treated group ($ P < 0.001). NS: nonsignificant.
The quantitative assessment of micronucleus containing cells serves as a good indicator for the induction of structural and numerical chromosomal aberrations. The genotoxic potency of atrazine was assessed by scoring micronuclei in liver cells. The results of micronucleus assay are shown in Table 2. The data of micronucleus test in liver cells corroborates the findings of comet assay. A significant (P < 0.001) increase in the percentage of micronuclei was observed after treatment with atrazine as compared to the control and vitamin E treated groups (Table 2). The results further confirm that atrazine exposure induces DNA damage. Using micronucleus assay, atrazine treatment to female mice at dose of 1400 mg/kg body weight has been shown to result in a significant increase in the number of micronuclei [48]. However, Ribas et al. [14] found that this herbicide was ineffective in inducing clastogenic and aneugenic damage in cultured human lymphocytes, whereas, a significant increase in the frequency of chromosome aberrations in mouse bone cells was observed. However, GarajVrhovac and Zeljezic [49] found genome damage in population of Croation workers exposed to atrazine and other pesticides assessed by micronucleus and comet assay. Administration of atrazine in combination with vitamin E showed decrease in the percentage of micronuclei as compared to atrazine treated rats. There was a significant (P < 0.001) decrease in the appearance of micronuclei in animals exposed to atrazine for 7 and 21 days of treatment. It is clear that vitamin E might be reducing the micronuclei formation by scavenging the DNA damaging free radicals generated following atrazine exposure. Another possible mechanism of vitamin E might involve selective removal of cells with DNA damage by apoptosis. Rickmann et al. [50] have reported that the suppressive effect of vitamin E on growth of tumor cells is due to the ability of vitamin E to induce cell cycle arrest and/or apoptosis of transformed cells. These results demonstrate that administration of vitamin E along with atrazine decreased the DNA damage and thus protected the cells against genotoxic effect of atrazine. The intactness of DNA is an important part of the normal cellular process. Any damage to the DNA in the form of breaks leads to changes in the integrity, which in turn can lead to abnormal cellular activity leading to toxicity and ultimately cell death [51]. The metabolic pathways for biotransformation of atrazine and the mechanisms of DNA damage remain to be elucidated. Cova et al. [52] demonstrated that metabolic activation of atrazine is independent from the liver and occurs possibly in the acidic part of the stomach. Studies have shown that atrazine is converted in stomach to nitrosamine [53]. It is believed that atrazine and many other pesticides owe their mutagenic effect due to their conversion to nitrosamines. Another mechanism that might be involved in genotoxic effect of atrazine might be due to increase in oxidative stress
as we observed that vitamin E administration had protective effect on atrazine genotoxicity. It is known that different toxic compounds induce oxidative stress in organisms and the reduction of oxidative stress involves antioxidants/antioxidant enzymes [54]. Moreover, Elia et al. [55] observed increased oxidative stress and antioxidant enzyme activity following atrazine exposure. The increase in activity of antioxidant enzyme might be a possible defense mechanism to reduce the free radicals generated by atrazine. Based on the results obtained in the present study the observed genotoxic effects of atrazine might be due to enhanced formation of free radicals. Antioxidants like vitamin E are capable of deactivating highly bioactive molecules such as free radicals that are generated during oxidative stress. Reversal of genotoxicity by antioxidant vitamins C and E, -carotene and flavonoids has been well documented in literature [24,56]. In summary, our findings demonstrate atrazine is genotoxic as assessed by DNA electrophoresis, micronucleus and comet assays. Vitamin E, on the other hand, was observed to reverse the genotoxicity induced by atrazine. It can be concluded that vitamin E is protective against atrazine induced genotoxicity and supplementation of vitamin E might be beneficial to atrazine exposed population. Conflict of Interest None. References [1] L.R. Goldman, Atrazine, simazine and cyanazine: notice of initiation of special review, Fed. Regist. (1994) 60412–60443. [2] M. Purcell, J.F. Neault, H. Malonga, H. Arakawa, R. Carpentier, H.A. Tajmir-Riahi, Interactions of atrazine and 2,4-D with human serum albumin studied by gel and capillary electrophoresis, and FTIR spectroscopy, Biochem. Biophys. Acta 1548 (2001) 129–138. [3] M. Singh, R. Sandhir, R. Kiran, Erythrocyte antioxidant enzymes in toxicological evaluation of commonly used organophosphate pesticides, Indian J. Exp. Biol. 44 (2006) 580–583. [4] C.P. Waring, A. Moore, The effect of atrazine on Atlantic salmon (Salmo salar) smolts in fresh water and after sea water transfer, Aquat. Toxicol. 66 (2004) 93–104. [5] M.C. Alavanja, J.A. Hoppin, F. Kamel, Health effects of chronic pesticide exposure: cancer and neurotoxicity, Annu. Rev. Public Health 25 (2004) 155–197. [6] L.A. McCauley, W.K. Anger, M. Keifer, R. Langley, M.G. Robson, D. Rohlman, Studying health outcomes in farm worker populations exposed to pesticides, Environ. Health Perspect. 114 (2006) 953–960. [7] T.A. Arcury, S.A. Quandt, B.G. Mellen, An exploratory analysis of occupational skin disease among Latino migrant and seasonal farm workers in North Carolina, J. Agric. Saf. Health 9 (2003) 221–232. [8] R. Das, A. Steege, S. Baron, J. Beckman, R. Harrison, Pesticide-related illness among migrant farm workers in the United States, Int. J. Occup. Environ. Health 7 (2001) 303–312.
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Protective effects of vitamin E against atrazine-induced genotoxicity in rats Mohan Singh a,∗ , Pushpindar Kaur b , Rajat Sandhir a , Ravi Kiran a a b
Department of Biochemistry, Panjab University, Chandigarh 160014, India Department of Zoology, Panjab University, Chandigarh 160014, India
a r t i c l e
i n f o
Article history: Received 29 September 2007 Received in revised form 9 May 2008 Accepted 24 May 2008 Available online 5 June 2008 Keywords: Atrazine Comet assay Genotoxicity Micronucleus assay Vitamin E
a b s t r a c t Atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine) is one of the most commonly used herbicides to control grasses and weeds. The widespread contamination and persistence of atrazine residues in the environment has resulted in human exposure. Vitamin E is a primary antioxidant that plays an important role in protecting cells against toxicity by inactivating free radicals generated following pesticides exposure. The present study was undertaken to investigate the protective effect of vitamin E against atrazine-induced genotoxicity. Three different methods: gel electrophoresis, comet assay and micronucleus test were used to assess the atrazine-induced genotoxicity and to evaluate the protective effects of vitamin E. Atrazine was administered to male rats at a dose of 300 mg/kg body weight for a period of 7, 14 and 21 days. There was a significant increase (P < 0.001) in tail length of comets from blood and liver cells treated with atrazine as compared to controls. Co-administration of vitamin E (100 mg/kg body weight) along with atrazine resulted in decrease in tail length of comets as compared to the group treated with atrazine alone. Micronucleus assay revealed a significant increase (P < 0.001) in the frequency of micronucleated cells (MNCs) following atrazine administration. In the animals administrated vitamin E along with atrazine there was a significant decrease in percentage of micronuclei as compared to atrazine treated rats. The increase in frequency of micronuclei in liver cells and tail length of comets confirm genotoxicity induced by atrazine in blood and liver cells. In addition, the findings clearly demonstrate protective effect of vitamin E in attenuating atrazine-induced DNA damage. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Triazine herbicides constitute one of the largest groups of herbicides used throughout the world. Among the triazines, atrazine is one of the most commonly used herbicides to control dicotyledonous weed plants in maize, cereals, lucerne and sugarcane [1]. As a result of its widespread use atrazine residues have contaminated not only plants, soil, water and cultivated ground but also agricultural products like fruits, milk, butter, and sugar beet [2]. Deleterious effects of pesticides on human and animal health have been well documented in literature [3–6]. Studies have also indicated that pesticide exposure is associated with chronic health problems or health symptoms such as respiratory problems, memory disorders, dermatologic conditions, cancer, depression, neurologic deficits, miscarriages, and birth defects [7–13]. Various in vitro studies have shown the ability of atrazine to induce genetic damage in human and animal cells
∗ Corresponding author. Tel.: +91 172 2534133; fax: +91 172 2541022. E-mail address: [email protected] (M. Singh). 1383-5718/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2008.05.010
[14–17]. Further, it has been reported that pesticides such as alachlor, acephate, chlorpyriphos and atrazine induce DNA damage [18–20] and chromosomal aberrations among farm workers [21–23]. Antioxidant vitamins are able to inactivate highly reactive molecules, such as free radicals, that are generated during various biochemical processes in the cells [24]. A large number of antioxidants like vitamin C, E and plant derivatives have been tested in the experimental animals in reducing the clastogenicity induced by drugs [25,26]. The protective effects of vitamin E against pesticide-induced toxicity have been reported [27–29]. In addition, vitamin E has been shown to be effective in reducing genotoxic effects of various genotoxic compounds [30,31]. Of the antioxidants tested, vitamin E shows most promising effect in reducing genotoxic effects induced by chemicals. However, no information is available concerning the ameliorating effect of vitamin E against atrazine-induced genotoxicity. Therefore, in the present study we evaluated the genotoxicity induced by atrazine and the possible beneficial effect of vitamin E against atrazine-induced genotoxicity using comet assay, which is a rapid, sensitive and inexpensive method for
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measuring the DNA damage [32], and micronucleus test along with assessment of DNA damage by agarose gel electrophoresis. 2. Materials and methods 2.1. Chemicals Atrazine (Technical grade, 97.83%) was a gift from Meghmani Industries Ltd. (India). Vitamin E (˛-tocopheryl acetate, trade name-Evion) was procured from Merck Pharmaceuticals (India). Agarose (normal and low melting) was procured from Sigma Chemical Co. (USA). Triton X-100 was purchased from SISCO Research Laboratories (India). Tris, ethidium bromide, May Grunwald’s stain and Giemsa stain were procured from Himedia Laboratories (India). EDTA was purchased from Merck Ltd. (India). All other chemicals used were obtained from Qualigens Fine Chemicals (India) and were of analytical reagent grade. 2.2. Animals and treatment Male rats (Wistar strain), weighing about 100–120 g, were used in the study as males have been shown to be more susceptible than females to genotoxic effects of various chemicals [33]. The animals were housed in polypropylene cages, given water ad libitum and fed standard pellet diet. The rats were segregated into four groups, each group having 18 animals. Animals in each group were further subdivided into three sub-groups and were treated with atrazine and/or vitamin E daily for a period of 7, 14 and 21 days as described below. Animals in the control group received 1 ml of corn oil, orally. Atrazine treated group were given atrazine (300-mg/kg body weight) dissolved in corn oil. Vitamin E treated animals were administered vitamin E (100-mg/kg body weight) dissolved in corn oil. Animals in the atrazine + vitamin E group received a mixture of atrazine (300 mg/kg body weight) and vitamin E (100 mg/kg body weight) dissolved in corn oil, orally. Atrazine was given at a dose of 300-mg/kg body weight as it was the maximum tolerated dose based on the pilot study conducted with various doses of atrazine and also based on the doses reported in literature [34]. The dose of vitamin E used was based on the dose that was seen to be most effective in lowering toxicity induced by various xenobiotics [35]. 2.3. Collection of blood samples Blood samples (1 ml) were collected from supra orbital sinus using glass capillaries into heparinized vacutainers (Becton-Dickinson, UK). 2.4. Preparation of tissue homogenate for micronucleus test and comet assay At the end of the respective treatments rats were sacrificed under light ether anesthesia followed by cervical dislocation and their livers removed. The livers were perfused with ice-cold saline till all the traces of blood were removed. The final washing was given with chilled homogenizing buffer (pH 7.5) containing 0.024 M EDTA, 0.075 M NaCl and DMSO 10%. After weighing, the livers were minced, suspended in chilled homogenization buffer at a concentration of 1 g/ml and were homogenized on ice using a Potter-type homogenizator at 500–800 rpm. The homogenate was then centrifuged at 700 rpm for 10 min at 4 ◦ C. Supernatant was removed and the pellet was resuspended in a volume of 1 ml/g of the homogenization buffer. The pellet was stored at −20 ◦ C for further analyses.
The parameter used to assess DNA damage was tail length (migration of DNA from nucleus) and tail moment which was automatically generated by CometScore software. The control and treated slides were randomized and were not run separately or at different times to avoid variability. 2.6. Micronucleus test Micronucleus test was performed according to the method of Igarashi and Shimada [36]. The pellet obtained from the liver homogenate was re-suspended in the homogenization buffer and was allowed to settle down for 1–2 min. A small drop of the suspension was placed at one end of a pre-cleaned, grease free, microscopic slide. The drop was carefully spread into a single cell layered film without damaging the cell morphology using a polished cover glass held at an angle of 45 degree. The slides were then air dried in a dust free environment for 12 h and subsequently stained by the method described by Schmid [37]. Briefly, air dried slides were first stained for 1–2 min in concentrated May-Grunwald stain (0.25% in methanol) followed by 10% Giemsa stain solution for 10 min. The slides were then rinsed twice with distilled water, dried and rinsed with methanol. The slides were placed in xylene for clearing, mounted in DPX and analyzed for the presence of micronuclei (a total of 1000 cells were scored for each animal). 2.7. DNA isolation and electrophoresis DNA was extracted using kit DNeasy tissue kit (Qiagen GmbH, Germany), which utilizes the silica gel membrane technology for rapid and efficient purification of total cellular DNA without organic extraction or ethanol precipitation followed by agarose gel electrophoresis according to the method of Sambrook and Russel [38]. 2.8. Statistical analysis All values were expressed as mean ± S.D. of six animals per group. Data were analyzed using one way analysis of variance (ANOVA) followed by Newman–Keuls test for multiple pair wise comparison between the various groups. Values with P ≤ 0.05 were considered as significant.
3. Results and discussion DNA is a target for mutagens and carcinogens, which induce changes in DNA structure of giving rise to mutations and/or cell death [39]. Free radical generated following pesticide exposure may lead to extensive DNA damage. Atrazine is capable of inducing structural changes in chromosomes [40–43]. In the present study DNA damage was evaluated by electrophoresis of DNA isolated from rat liver. Administration of atrazine resulted in DNA damage as is evident from Fig. 1. Lanes 3, 5, 7 correspond to DNA from animals exposed to atrazine for 7, 14 and 21 days respectively. It is evident that exposure to atrazine resulted in shearing of DNA as compared
2.5. Comet assay Comet assay was performed under alkaline conditions according to the method of Singh et al. [32] with slight modifications. Microscopic slides (frosted) were covered by a thin layer of 1.0% normal melting agarose at about 50 ◦ C (dissolved in Ca2+ and Mg2+ free PBS). A cover slip was placed on the slide to promote even and firm attachment. Upon solidification of agarose, cover slip was gently removed and 30 l of sample suspension mixed with 75 l of low melting point agarose (LMPA) at 37 ◦ C was added. The cover slip was replaced and agarose was again allowed to solidify for 10 min at 4 ◦ C. The cover slip was again removed and the third layer of 85 l of 0.5% LMPA was added to the slide and was allowed to solidify on ice for 5 min. After solidification of third layer of agarose, cover slips were removed and slides were immersed in cold lysis solution containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris (pH 10) and 1% Triton X-100 was added prior to use for 1 h at 4 ◦ C. Overnight treatment in lysis buffer at 4 ◦ C is tolerated but prolonged lysis results in precipitation of the buffer. After lysis DNA was allowed to unwind for 30 min in alkaline electrophoretic solution consisting of 300 mM NaOH and 1 mM EDTA (pH > 13). Electrophoresis was performed at 4 ◦ C in field strength of 0.7 V/cm and current of 300 mA. The slides were then neutralized with cold 0.4 M Tris–HCl, pH 7.5, stained with 75 l ethidium bromide (20 g/ml) and then cover slipped. The slides were placed in a humidified chamber to prevent drying of gel and were analyzed on the same day using fluorescent microscope (NIKON ECLIPSE 80i) equipped with a 510–560 nm excitation and 590 barrier filters and integrated digital camera. Slides were scored using CometScore software (TriTek Corp., USA) and 50 cells were analyzed per sample.
Fig. 1. Agarose gel electrophoresis of DNA isolated from liver of rat after various treatments. Lane 1: DNA of control group after 21 days; lane 2: DNA of vitamin E treated group after 21 days; lane 3: DNA of atrazine treated group for 7 days; lane 4: DNA of atrazine and vitamin E treated for 7 days; lane 5: DNA of atrazine treated group for 14 days; lane 6: DNA of atrazine and vitamin E treated for 14 days; lane 7: DNA of atrazine treated group for 21days; lane 8: DNA of atrazine and vitamin E treated for 21 days.
M. Singh et al. / Mutation Research 654 (2008) 145–149
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Table 1 Effects of various treatments on tail length and tail moment in liver and blood cells Treatment
Organ
7 days
Control
Liver Blood
2.81 ± 0.29 3.17 ± 1.25
1.10 ± 0.76 3.09 ± 1.74
3.23 ± 0.70 3.29 ± 1.16
1.75 ± 1.02 2.87 ± 1.36
3.39 ± 1.06 3.19 ± 1.22
1.89 ± 0.94 3.44 ± 1.28
Atrazine
Liver Blood
9.86 ± 1.49* 14.24 ± 2.43*
4.46 ± 1.46* 13.36 ± 4.68*
17.14 ± 2.13* 22.91 ± 2.63*
15.04 ± 3.53* 14.63 ± 3.46*
22.64 ± 2.96* 25.07 ± 2.75*
12.46 ± 3.08* 14.38 ± 2.96*
Vitamin E
Liver Blood
Atrazine + vitamin E
Liver Blood
Tail length (m)
2.75 ± 0.88NS 2.35 ± 0.84NS 4.33 ± 0.69$ 11.00 ± 1.81$
14 days Tail moment
Tail length (m)
21 days Tail moment
0.89 ± 0.62NS 3.80 ± 2.26NS
2.85 ± 1.21NS 2.387 ± 1.17NS
1.69 ± 0.98NS 2.42 ± 1.01NS
4.39 ± 2.32NS 7.05 ± 1.95$
10.14 ± 1.31$ 16.05 ± 2.07$
4.09 ± 1.54$ 9.88 ± 2.85$
Tail length (m)
2.91 ± 0.92NS 2.36 ± 0.95NS 13.27 ± 2.32$ 17.72 ± 2.58$
Tail moment
1.53 ± 1.00NS 3.07 ± 1.24NS 9.09 ± 3.22$ 11.38 ± 2.68$
Values are expressed as mean ± S.D., n = 6. *Significant difference from control group (*P < 0.001). $ Significant difference from atrazine treated group ($ P < 0.001). NS: nonsignificant.
to control (lane 1) and vitamin E treated (lane 2). DNA damage was more pronounced after 14 and 21 days of atrazine treatment. It is clear from the data that extent of DNA damage is dependent on duration of atrazine exposure. Although atrazine did not induce classical DNA ladder pattern seen in apoptosis, but, shearing of DNA suggests DNA damage in the atrazine treated liver. Eldadah et al. [44] have also observed shearing of DNA instead of DNA laddering because of a fewer number of apoptotic cells or the possibility of laddering being masked by or necrotic cell death. Pino et al. [16] have also reported DNA breaks (and/or alkali-labile lesions) in cell suspensions obtained from stomach, kidney and liver of rats treated with atrazine. In addition, Liu et al. [45] have detected DNA fragmentation by atrazine in grass carp by TUNEL reaction and agarose gel electrophoresis. Vitamin E in combination with atrazine treatment reduced atrazine-induced shearing of DNA (lanes 4, 6 and 8). These results suggest that vitamin E has a protective effect on atrazine-induced DNA damage which is in agreement with the findings of Abid-Essefi et al. [46] showing that vitamin E can prevent genotoxicity induced by chemicals. These observations were supported by more sensitive assays like single cell gel electrophoresis (comet assay) and micronucleus test. Table 1 depicts the values of tail length and moment obtained from comet assay after treatment with atazine and/or vitamin E. It
is clear that there was a significant (P < 0.001) increase in tail length in blood and liver cells of rats treated with atrazine as compared to the control rats, which was also illustrated by the representative comets (Fig. 2). A significant increase in the tail length was observed at all the time points following atrazine exposure in both blood and liver cells. Using comet assay Tennant et al. [40] also showed that atrazine at concentrations of 250 and 500 mg/kg significantly increased DNA migration in the leukocytes of mice treated in vivo. Clements et al. [47] also had similar findings with comet assay in tadpoles exposed to atrazine. On the contrary, in an in vitro study on human lymphocytes, atrazine was not found to be genotoxic [43]. Data in Table 1 shows that vitamin E had a significant protective effect against atrazine-induced DNA damage at all the time points studied (P < 0.001). The protective effect of vitamin E against genotoxic effects of various chemicals has previously been reported [30,31,46]. Based on the results it is clear that atrazine might be inducing DNA damage by increased generation of reactive free radicals which are scavenged by vitamin E. The effects of vitamin E observed on atrazine-induced DNA damage might involve its antioxidant action and may not due to its interaction with atrazine as there are no documented reports about their interaction. This is the first report showing protective effect of vitamin E against atrazine-induced genotoxicity.
Fig. 2. Single cell gel electrophoresis of rat liver cells showing comets (40× magnification). Control after 21 days (A), vitamin E treated for 21 days (B), atrazine treated for 21 days (C), atrazine + vitamin E treated for 21 days (D).
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Table 2 Frequencies of micronucleated cells in liver after various treatments Time
Group
Total cells scored
Micronucleated cells
% Mean ± S.D.
7 days
Control Atrazine Vitamin E Atrazine + vitamin E
6000 6000 6000 6000
28 216 22 183
0.47 3.98 0.37 3.05
± ± ± ±
0.16 1.10* 0.16NS 0.51$
14 days
Control Atrazine Vitamin E Atrazine + vitamin E
6000 6000 6000 6000
26 360 23 314
0.43 6.00 0.38 5.23
± ± ± ±
0.27 1.15* 0.13NS 1.21NS
21 days
Control Atrazine Vitamin E Atrazine + vitamin E
6000 6000 6000 6000
24 392 23 336
0.40 6.53 0.38 5.60
± ± ± ±
0.17 0.90* 0.15NS 0.51$
Values are expressed as mean ± S.D., n = 6. *Significant difference from control group (*P < 0.001). $ Significant difference from atrazine treated group ($ P < 0.001). NS: nonsignificant.
The quantitative assessment of micronucleus containing cells serves as a good indicator for the induction of structural and numerical chromosomal aberrations. The genotoxic potency of atrazine was assessed by scoring micronuclei in liver cells. The results of micronucleus assay are shown in Table 2. The data of micronucleus test in liver cells corroborates the findings of comet assay. A significant (P < 0.001) increase in the percentage of micronuclei was observed after treatment with atrazine as compared to the control and vitamin E treated groups (Table 2). The results further confirm that atrazine exposure induces DNA damage. Using micronucleus assay, atrazine treatment to female mice at dose of 1400 mg/kg body weight has been shown to result in a significant increase in the number of micronuclei [48]. However, Ribas et al. [14] found that this herbicide was ineffective in inducing clastogenic and aneugenic damage in cultured human lymphocytes, whereas, a significant increase in the frequency of chromosome aberrations in mouse bone cells was observed. However, GarajVrhovac and Zeljezic [49] found genome damage in population of Croation workers exposed to atrazine and other pesticides assessed by micronucleus and comet assay. Administration of atrazine in combination with vitamin E showed decrease in the percentage of micronuclei as compared to atrazine treated rats. There was a significant (P < 0.001) decrease in the appearance of micronuclei in animals exposed to atrazine for 7 and 21 days of treatment. It is clear that vitamin E might be reducing the micronuclei formation by scavenging the DNA damaging free radicals generated following atrazine exposure. Another possible mechanism of vitamin E might involve selective removal of cells with DNA damage by apoptosis. Rickmann et al. [50] have reported that the suppressive effect of vitamin E on growth of tumor cells is due to the ability of vitamin E to induce cell cycle arrest and/or apoptosis of transformed cells. These results demonstrate that administration of vitamin E along with atrazine decreased the DNA damage and thus protected the cells against genotoxic effect of atrazine. The intactness of DNA is an important part of the normal cellular process. Any damage to the DNA in the form of breaks leads to changes in the integrity, which in turn can lead to abnormal cellular activity leading to toxicity and ultimately cell death [51]. The metabolic pathways for biotransformation of atrazine and the mechanisms of DNA damage remain to be elucidated. Cova et al. [52] demonstrated that metabolic activation of atrazine is independent from the liver and occurs possibly in the acidic part of the stomach. Studies have shown that atrazine is converted in stomach to nitrosamine [53]. It is believed that atrazine and many other pesticides owe their mutagenic effect due to their conversion to nitrosamines. Another mechanism that might be involved in genotoxic effect of atrazine might be due to increase in oxidative stress
as we observed that vitamin E administration had protective effect on atrazine genotoxicity. It is known that different toxic compounds induce oxidative stress in organisms and the reduction of oxidative stress involves antioxidants/antioxidant enzymes [54]. Moreover, Elia et al. [55] observed increased oxidative stress and antioxidant enzyme activity following atrazine exposure. The increase in activity of antioxidant enzyme might be a possible defense mechanism to reduce the free radicals generated by atrazine. Based on the results obtained in the present study the observed genotoxic effects of atrazine might be due to enhanced formation of free radicals. Antioxidants like vitamin E are capable of deactivating highly bioactive molecules such as free radicals that are generated during oxidative stress. Reversal of genotoxicity by antioxidant vitamins C and E, -carotene and flavonoids has been well documented in literature [24,56]. In summary, our findings demonstrate atrazine is genotoxic as assessed by DNA electrophoresis, micronucleus and comet assays. Vitamin E, on the other hand, was observed to reverse the genotoxicity induced by atrazine. It can be concluded that vitamin E is protective against atrazine induced genotoxicity and supplementation of vitamin E might be beneficial to atrazine exposed population. Conflict of Interest None. References [1] L.R. Goldman, Atrazine, simazine and cyanazine: notice of initiation of special review, Fed. Regist. (1994) 60412–60443. [2] M. Purcell, J.F. Neault, H. Malonga, H. Arakawa, R. Carpentier, H.A. Tajmir-Riahi, Interactions of atrazine and 2,4-D with human serum albumin studied by gel and capillary electrophoresis, and FTIR spectroscopy, Biochem. Biophys. Acta 1548 (2001) 129–138. [3] M. Singh, R. Sandhir, R. Kiran, Erythrocyte antioxidant enzymes in toxicological evaluation of commonly used organophosphate pesticides, Indian J. Exp. Biol. 44 (2006) 580–583. [4] C.P. Waring, A. Moore, The effect of atrazine on Atlantic salmon (Salmo salar) smolts in fresh water and after sea water transfer, Aquat. Toxicol. 66 (2004) 93–104. [5] M.C. Alavanja, J.A. Hoppin, F. Kamel, Health effects of chronic pesticide exposure: cancer and neurotoxicity, Annu. Rev. Public Health 25 (2004) 155–197. [6] L.A. McCauley, W.K. Anger, M. Keifer, R. Langley, M.G. Robson, D. Rohlman, Studying health outcomes in farm worker populations exposed to pesticides, Environ. Health Perspect. 114 (2006) 953–960. [7] T.A. Arcury, S.A. Quandt, B.G. Mellen, An exploratory analysis of occupational skin disease among Latino migrant and seasonal farm workers in North Carolina, J. Agric. Saf. Health 9 (2003) 221–232. [8] R. Das, A. Steege, S. Baron, J. Beckman, R. Harrison, Pesticide-related illness among migrant farm workers in the United States, Int. J. Occup. Environ. Health 7 (2001) 303–312.
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