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Genotoxicity evaluation of chlorpyrifos to amphibian Chinese toad (Amphibian: Anura) by Comet assay and Micronucleus test
Mutation Research 680 (2009) 2–6
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Genotoxicity evaluation of chlorpyrifos to amphibian Chinese toad (Amphibian: Anura) by Comet assay and Micronucleus test XiaoHui Yin b , GuoNian Zhu a,b,∗ , Xian Bing Li a , ShaoYing Liu a a b
Institutes of Pesticides and Environmental Toxicology, Zhejiang University, HangZhou 310029, China School of Agriculture and Food Science, Zhejiang Forestry University, Lin’an, HangZhou 311300, China
a r t i c l e
i n f o
Article history: Received 1 August 2008 Received in revised form 5 May 2009 Accepted 24 May 2009 Available online 12 June 2009 Keywords: Insecticides Toxicity DNA damage Comet assay Micronucleus test
a b s t r a c t In the present study, the genotoxicity of chlorpyrifos was evaluated in the Chinese toad by using Comet assay and Micronucleus test (MN), as the potential tools for the assessment of genotoxicity. The first step was determined by the acute toxicity of chlorpyrifos. Tadpoles were exposed to the series of relatively high concentrations of chlorpyrifos for 96 h. LC50 values at 24, 48, 72, and 96 h were 3.63, 1.17, 0.82, and 0.80 mg l−1 , respectively. Secondly, the Micronucleus test was used for detecting chromosome damage in Chinese toad tadpoles exposed to the sublethal concentrations of chlorpyrifos and methyl methane sulfonate (MMS), which indicated that they induced chromosomal lesion in erythrocytes of Bufo bufo gargarizans tadpoles. Thirdly, the significant (P < 0.05 concentration-dependent increase in DNA damage (as indicated by Tail DNA%, Tail length, Olive tail moment)) were observed in erythrocytes and liver cells of tadpoles exposed to the sublethal concentrations of chlorpyrifos and MMS by Comet assay. To our knowledge, this is the first report to describe the use of B. bufo gargarizans for genotoxicity assessment of chlorpyrifos. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Chlorpyrifos (o,o-diethyl o-(3,5,6-trichloro-2-pyridyl)phosphorothioate) is a broad spectrum organophosphorus insecticide which acts as a cholinesterase inhibitor. Recently, with the prohibition of those high toxic organophosphorus insecticides in the production of vegetables and other food crops, the usage of chlorpyrifos rapidly increased, and estimated for more than 499,000 kg recently in China [1]. Most of these compounds have low persistence in aquatic ecosystems, but relative lack of target specificity has raised concerns about their potential to cause adverse effects on non-target wildlife populations [2]. Several studies have documented an apparent connection between the presence of chlorpyrifos residues and reductions in amphibian populations at both local [3] and landscape scales [4,5]. Singh et al. [6] found that the residue level of chlorpyrifos in blood maximal in fish is 0.15 mg l−1 . Either Chlorpyrifos or its metabolites have also been discovered in the diet of preschoolers [7] in the cord blood of infants born to minority women living in urban settings [8]. Besides, chlorpyrifos elicited a number of other effects including hepatic dysfunction, immunological abnormalities, embryotoxicity, genotoxicity, teratogenicity, neurochemical, and
∗ Corresponding author at: Institutes of Pesticides and Environmental Toxicology, Zhejiang University, HangZhou 310029, China. Tel.: +86 0571 86430193. E-mail address: [email protected] (G. Zhu). 1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2009.05.018
neurobehavioral changes [9–11]. Chlorpyrifos and other organic pesticides had also been shown to induce genotoxicity [12–14]. Among techniques to detect genetic and genotoxic effects, the Micronucleus (MN) test and Comet assay are often used since they allow for convenient and easy application, particularly in aquatic organism and amphibians [15–18]. Amphibians have been regarded as bio-indicators, since they live in the aquatic environment in their larval stage [19], and are broadly used as test animals in evaluating effects of chemicals on aquatic and agricultural ecosystems [20]. Due to widespread application and its classification as highly toxic to freshwater fish [21], examination of the effects of chlorpyrifos on amphibians is warranted. In contrast to plenty of experimental studies on micronucleus and DNA damage of rodent and fish induced by pesticides, relatively scarce information existed in amphibians like toads and frogs [18,22]. It is important to note that red blood cells (RBCs) in amphibians are nucleated and undergo cell division in the circulation, particularly during developmental stages [23]. Liver is the major site for the metabolism of exogenous chemicals (pesticides, drugs, and metal), and the supplement formation of metabolites may be more or less toxic than the parent compound [24]. In the present study, the acute toxicity of chlorpyrifos was tested on Chinese toad (Bufo bufo gargarizans) tadpoles, a species which is widely distributed in southeast and southwest of China, and the genotoxicity of chlorpyrifos was also investigated using Comet assay for detecting the DNA damage of erythrocytes and liver cells and Micronucleus test of erythrocytes under laboratory conditions.
X. Yin et al. / Mutation Research 680 (2009) 2–6 2. Materials and methods 2.1. Test organism and chemicals Healthy specimens (lively, moved rapidly) of Chinese toad tadpoles were selected and procured from the local ponds of Pingban village in Linan Hangzhou China and acclimated to the laboratory environment for 10 d before the tests began on March 3, 2007. Prometamorphic tadpoles (from stage 26 up to 36) [25] were used for the bioassay. The average total size (snout-tail) was 24.5 ± 1.15 mm and the average weight was 100 ± 4.5 mg. The tadpoles were maintained under normal day–night light durations and the feeding was discontinued 48 h prior to exposure. No tadpole mortality occurred during the acclimatization period. For the present study, chlorpyrifos (40% emulsifiable concentrate, EC) was afforded by Zhejiang XinNong Chemical Co. Ltd. since the chlorpyrifos of this grade is mostly employed in field practices. Half-life of chlorpyrifos degradation in water with pH 7.0 is 25.6 d [26]. Methyl methane sulfonate (MMS) was purchased from Sigma–Aldrich Company. 2.2. Acute toxicity bioassay The tap water had been filtrated (to achieve animal drinking standard) and exposed in air for 3 d before test. Then it was analyzed for physico-chemical properties by standard methods [27]. The stock solution for the chlorpyrifos and MMS were prepared before treatment. For acute toxicity test with Chinese toad tadpoles, six concentration levels were individually prepared in water. The nominal test concentrations were 0.32, 0.64, 0.72, 1.08 and 2.56 mg l−1 for chlorpyrifos. Ten tadpoles (per replicate 10, total 30 of triplicates) were transferred to each 15 l-capacity glass tank containing 10 l of chlorpyrifos solution or controls. The solution was replaced every 24 h with freshly prepared solution of the same concentration. All treatments in triplicates were carried out and visually examined once every 8 h. When they did not respond to gentle touching, tadpoles were considered dead and removed immediately. The dead tadpoles were counted after 24, 48, 72 and 96 h. The data were analyzed with the statistical program SPSS® (Version 12.0). 2.3. Micronucleus test (MN) The tadpoles were exposed to chlorpyrifos, MMS (as positive controls), and the controls in a volume of 10 l for 96 h. Each level of exposure was prepared in triplicates. The sublethal concentrations of chlorpyrifos (0.08, 0.16, 0.32, and 0.64 mg l−1 ) were selected as exposure concentrations after median lethal concentration (LC50 ) being calculated by probit analysis [28]. The concentration of MMS was 0.1, 0.2, 0.4, and 0.8 mg l−1 being referred to previous study [18]. At the end of the 96 h exposure, a single smear of blood was prepared from the heart of each tadpole (n = 10). The smears were fixed in methanol for 10 min, left to air-dry at room temperature, and finally stained with 6% Giemsa in Sorenson buffer (pH 6.9) for 20 min. A total of about 2000 erythrocytes were examined for each specimen under the light microscope (Nikon, Type TE 2000; oil immersion lens, 100/1.25). The criteria for identification of Micronuclei (MN) used were to have no connection with the main nucleus, to share same color and intensity as the main nucleus, and with area smaller than one-third of the main nucleus. MN frequency was calculated as follows: of cells containing micronucleus × 1000 MN ‰= number total number of cells counted 2.4. Comet assay or alkaline single-cell gel electrophoresis The treatment concentration of chlorpyrifos and MMS were the same to MN. At the end of the exposure period (96 h), the DNA damage was assessed in the liver and the blood by using Comet assay in the tadpoles. Firstly the erythrocytes (Heart Puncture for Collecting Blood by glass capillary) were collected by centrifugation. Referring Carrera’s sample preparation, the liver tissue (about 50 mg) was washed three times with chilled phosphate buffered saline (Ca2+ Mg2+ -free, PBS) to remove the blood cells and transferred to ice-cold homogenization buffer (1× hanks’ balanced salt solution, 20 mM EDTA, 10% dimethylsulfoxide (DMSO), pH 7.0–7.5) [29]. The liver tissue was cut into small pieces using scissors on ice and homogenized to obtain a single-cell suspension. Secondly the survival rate of erythrocytes and liver cells density and viability (>90%) were measured by tryphan blue exclusion test [30]. Cells were diluted in cold PBS at 105 cells/ml. Comet assay was based on the protocol of Singh et al. [31]. The procedure was slightly modified according to the report of Zhang et al. [32]. Twenty l erythrocytes mixed with 0.8% LMP agarose were placed on normal 0.9% agarose microscope slides. After being held at 4 ◦ C, a third layer of 0.8% LMP agarose was added and left to solidify as described. The essential steps of Comet assay involved at least 2 h of cell lysis by detergent at a high salt concentration (2.5 mM NaCl, 100 mM Na2 EDTA, 10 mM Tris, 10% dimethylsulfoxide (DMSO), 1% Triton X-100, 1% Nasarcosinate pH = 10; at 4 ◦ C), and electrophoresis under alkaline conditions (300 mM NaOH, 1 mM EDTA, pH > 13, 30 min unwinding, 20 min electrophoresis at 300 mA and 18 V, at 4 ◦ C). Nucleoids were stained with 20 g ml−1 ethidium bromide (EB) and examined the following day using Zeiss Axioplan epifluorescence microscope (Carl Zeiss, Germany). Approximately 100 cells per slide (400 cells per concentration) were examined. Microphotographs were taken with 40× objective on XP-2 color film (400 ASA). The extent of DNA migration was determined using an image
3
´ analysis system (CASP, Krzysztof Konca, Poland). The parameters selected for the quantification of DNA damage were the frequency of DNA damage, Tail DNA (%), tail length, and Olive tail moment (OTM) (calculated by SPSS® ). The one-way ANOVA was employed to compare differences for the various indexes among the different concentrations of chlorpyrifos and MMS.
3. Results 3.1. Acute toxicity bioassay The physico-chemical characteristics of the test water were detected as follows: the dissolved oxygen concentration ranged from 7.5 to 8.2 mg l−1 , water temperature varied from 20.5 to 22.5 ◦ C, the pH values ranged from 7.18 to 7.67, total hardness ranged from 198 to 210 mg l−1 as counted by CaCO3 concentration, and total alkalinity was mean 301 mg l−1 as counted by CaCO3 concentration during the experimental period. The LC50 values of chlorpyrifos for Chinese toad tadpoles were found to be 3.63, 1.17, 0.819, and 0.800 mg l−1 for 24 h, 48 h, 72 h, and 96 h, respectively. The LC50 values (95% confidence limits) are shown in Table 1. In addition, negative effects on the behavior and morphology of the tadpoles were observed throughout the 96 h experimental period, for example the larvae suffered from morphological abnormalities (visible changes: fins shrunken, tail deformities, and head edema). 3.2. Micronucleus test (MN) The first evident result was significant (P < 0.01) greater induction of MN in Chinese toad tadpole specimens due to exposure to different concentrations of chlorpyrifos than the control group (Table 2, Fig. 1A and B). Further, a significant (P < 0.01) effect of durations on induction of MN was observed for all the concentrations of chlorpyrifos, except 0.08 mg l−1 . 3.3. Alkaline single-cell gel electrophoresis or Comet assay Erythrocytes and liver cells from the Chinese toad tadpoles were used to evaluate DNA damage using Comet assay. The Tail DNA (%), Tail length (m), and OTM (arbitrary units) were measured (Table 3, Fig. 2A–D). Significantly higher levels of DNA damage were detected in tadpoles exposed to sublethal concentrations of chlorpyrifos compared with the negative and positive controls. In summary, chlorpyrifos and MMS induced significantly (P < 0.05) concentration-dependent increase in DNA damage of exposed tadpoles (Fig. 2A–D). Significant positive correlations (correlation coefficient (r) at 5% level) were found between the concentrations of two agents and DNA damage measured by OTM. 4. Discussion In present study, the LC50 (96 h) of chlorpyrifos was found between 0.1 and 1.0 mg l−1 . The results indicated that the acute lethality of the chlorpyrifos for the Chinese toad tadpoles was very Table 1 Acute toxicity response (96 h LC50 ) of Chinese toad (B. bufo gargarizans) tadpoles exposed to chlorpyrifos (n = 30). Time
24 h 48 h 72 h 96 h
a (intercept)
6.06 6.53 6.78 6.93
b (slope)
5.17 6.5 6.66 6.82
c (LC50 , mg l−1 )
3.63 1.17 0.819 0.800
95% confidence limits Lower
Upper
1.92 0.929 0.684 0.67
23.25 1.62 1.02 0.989
b, regression coefficient; c, all LC50 values are based on initial measured concentrations.
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Table 2 Frequencies of micronuclei and abnormalities in erythrocytes of Chinese toad (B. bufo gargarizans) exposed to chlorpyrifos and MMS for 96 h. Total cells scored
Number of normalities
0
6106
6006
86
Chlorpyrifos
0.08 0.16 0.32 0.64
6026 6082 6077 6005
5876 5814 5934 5663
109 197 106 249
MMS
0.1 0.2 0.4 0.8
6015 6031 6042 6031
5880 5836 5758 5624
109 158 215 306
Exposure agents
Exposure dosage (mg l−1 )
Control
Number of abnormalities
Frequency of abnormalities (‰)
Number of MN
Frequency of MN (‰) ±SE
14.1 ± 2.21
27
18.1 17.4 32.4 41.5
± ± ± ±
1.97 2.17 1.84** 0.26**
41 37 71 93
6.81 8.17 11.55 22.46
4.33 ± 0.49 ± ± ± ±
0.60 0.96* 0.68** 2.05**
18.1 26.2 35.6 50.7
± ± ± ±
1.76 1.64** 1.83** 2.16**
26 37 69 101
4.32 6.13 11.5 16.8
± ± ± ±
0.77 0.73* 0.99** 0.83*
Note: All data are shown as average median values (n = 3) ± SD; (separately for each mutagen). *LSD (least-significant difference) = P < 0.05; **LSD = P < 0.01.
Table 3 DNA damage of erythrocytes and liver cells in Chinese toad tadpoles (Bufo bufo gargarizans) exposed to chlorpyrifos and MMS for 96 h. Exposure agents
Exposure dosage (mg l−1 )
Number of Tadpoles
Tail DNA%
Tail length (m)
Erythrocytes
Liver cells
Erythrocytes
10.2 ± 0.40
42.8 ± 1.24
Liver cells
Control
0
10
14.7 ± 0.40
Chlorpyrifos
0.08 0.16 0.32 0.64
10 10 10 10
33.0 35.5 42.0 48.1
± ± ± ±
3.50*b 3.12*b 2.19**ab 0.41**a
18.2 19.9 20.1 34.5
± ± ± ±
0.73*b 0.89*b 0.79*b 2.01**a
50.2 73.0 87.1 98.6
± ± ± ±
3.47d 5.52**c 7.12**b 7.16**a
30.6 58.4 60.6 58.1
19.8 ± 8.22 ± ± ± ±
2.42b 6.3**a 7.8**a 7.12**a
MMS
0.1 0.2 0.4 0.8
10 10 10 10
17.8 27.9 35.9 43.9
± ± ± ±
0.66c 5.25*b 2.02*b 5.16*a
20.5 26.9 27.8 25.2
± ± ± ±
1.05*b 0.83*a 1.34*a 1.27*a
52.7 65.7 79.0 92.0
± ± ± ±
5.51cd 2.52**bc 4.36**ab 15.1**a
51.2 67.0 70.7 86.7
± ± ± ±
2.56**c 7.55**b 8.33**b 4.62**a
Note: All data are shown as average median values (n = 3) ± SD; (separately for each mutagen). Letters indicate statistically significant differences between the various doses *LSD (least-significant difference) = P < 0.05; **LSD = P < 0.01. Letters indicate statistically significant differences between the various doses, LSD = P < 0.05.
high. Richards and Kendall [33] pointed out that Xenopus laevis larvae exposed to chlorpyrifos during metamorphs were more sensitive than larvae exposed during premetamorphs, and the LC50 for metamorphs was 0.56 mg l−1 . Pan and Liang reported the LC50 (48 h) of chlorpyrifos to Rana limnocharis tadpoles to be moderately toxic (2.40 mg l−1 ) [34]. They did not mention the developmental stage of the tadpoles, but it seemed that Chinese toad tadpoles were more sensitive than Rana limnocharis tadpoles. There is limited information describing the acute toxic effects of chlorpyrifos on amphibians such as Chinese toad. The Micronucleus test has been performed to evaluate mutagenicity in toxicology and bio-indicators of ecosystem. To evaluate the genotoxic effects of pollutants, it has been carried out in several organisms, such as mussels [35], fish [36] and, especially, amphibians [18,22]. Recently, the presence of nuclear abnormalities besides
micronuclei, has been reported by several authors, either in humans [37] or in tadpoles [22]. In this study, erythrocytes displaying cell nuclearpycnosis, binucleated erythrocytes, and alteration in cell morphology of tadpoles’ erythrocytes were observed, and their incidence seemed to increase with the incidence of MN in high treatment (Table 2). Comet assay is commonly used as a biomarker for DNA damage, and increased Comets (presented either as DNA tail moments, Olive tail moment or percent DNA in the tail) are interpreted as compromising animal health [38–40]. The genotoxicity of chlorpyrifos has been studied in a variety of assays in the past. Gollapudi et al. [41] reported that there was no indication of genotoxic activity for chlorpyrifos in any of these assays (Ames test, CHO/HGPRT assay, cytogenetic abnormalities, rat lymphocyte chromosomal aberration test, mouse bone marrow Micronucleus test), but Rahman et al.
Fig. 1. Micronuclei and nuclear abnormalities from erythrocytes of B. bufo gargarizans tadpoles after 96 h exposure to chlorpyrifos (magnifier 400×). (A) Normal nuclear; (B) nuclear abnormalities. (a) Micronucleus, (b) nuclear protrusion, (c) non-nucleus, (d) double nucleus, (e) nuclear pycnosis, (f) nuclear concavity, and (j) nuclear cracking.
X. Yin et al. / Mutation Research 680 (2009) 2–6
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Fig. 2. Effect of a 96 h exposure to chlorpyrifos and methyl methanesulfonate (MMS, positive control) on Olive tail moment (OTM) in erythrocytes (A and B) and liver cells (C and D) of Chinese toad tadpoles (B. bufo gargarizans). Note: all data are shown as average median values (n = 3) ±SD; letters indicate statistically significant differences between the various doses, LSD = P < 0.05.
[13] indicated that it was controversial comparing with the results of numerous mutagenicity in short term assays, and found chlorpyrifos induce in vivo genotoxic effect in leucocytes of Swiss albino mice by using Comet assay. It has also been reported as genotoxic in root meristem cells of Crepis capillaries [42]. Our result demonstrated an increase of DNA damage in erythrocytes and liver cells of Chinese toad tadpoles exposed to chlorpyrifos, and induced DNA damage at a very low dose. As mentioned above, highly fragmented nuclei which showed completely disintegrated head region and images with nearly all the DNA in the tail or a much more wide tail were observed at the high concentrations of the chlorpyrifos exposure (0.32 and 0.64 mg l−1 ). This suggested that apoptosis was occurring, as apoptotic cells are known to display nucleosomal fragmentation [43]. This prediction is further supported by Fu et al. [44], who reported that chlorpyrifos was able to induce cell apoptosis in mouse retina. Saulsbury et al. [45] reported that chlorpyrifos-induced apoptosis in placental cells. Some studies indicated that chlorpyrifos was mutagenic at the cytological level in a concentration-dependent manner [46,13]. In the present study, tadpole erythrocytes appeared nuclearpycnosis, and Comet images showed some fragmented nuclei as well as images with nearly all DNA being in the tail or tails being very wide at high concentrations of chlorpyrifos. We could not estimate the contribution of apoptosis to the effects observed. So the images observed needs to be further analyzed to assess whether Chinese toad tadpoles exposed to relatively high concentrations of chlorpyrifos may induce apoptosis or necrosis. Taken together, the present findings indicated that chlorpyrifos could induce the MN of erythrocytes and DNA damage of erythrocytes and liver cells in Chinese toad tadpoles. On the assumption
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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
Genotoxicity evaluation of chlorpyrifos to amphibian Chinese toad (Amphibian: Anura) by Comet assay and Micronucleus test XiaoHui Yin b , GuoNian Zhu a,b,∗ , Xian Bing Li a , ShaoYing Liu a a b
Institutes of Pesticides and Environmental Toxicology, Zhejiang University, HangZhou 310029, China School of Agriculture and Food Science, Zhejiang Forestry University, Lin’an, HangZhou 311300, China
a r t i c l e
i n f o
Article history: Received 1 August 2008 Received in revised form 5 May 2009 Accepted 24 May 2009 Available online 12 June 2009 Keywords: Insecticides Toxicity DNA damage Comet assay Micronucleus test
a b s t r a c t In the present study, the genotoxicity of chlorpyrifos was evaluated in the Chinese toad by using Comet assay and Micronucleus test (MN), as the potential tools for the assessment of genotoxicity. The first step was determined by the acute toxicity of chlorpyrifos. Tadpoles were exposed to the series of relatively high concentrations of chlorpyrifos for 96 h. LC50 values at 24, 48, 72, and 96 h were 3.63, 1.17, 0.82, and 0.80 mg l−1 , respectively. Secondly, the Micronucleus test was used for detecting chromosome damage in Chinese toad tadpoles exposed to the sublethal concentrations of chlorpyrifos and methyl methane sulfonate (MMS), which indicated that they induced chromosomal lesion in erythrocytes of Bufo bufo gargarizans tadpoles. Thirdly, the significant (P < 0.05 concentration-dependent increase in DNA damage (as indicated by Tail DNA%, Tail length, Olive tail moment)) were observed in erythrocytes and liver cells of tadpoles exposed to the sublethal concentrations of chlorpyrifos and MMS by Comet assay. To our knowledge, this is the first report to describe the use of B. bufo gargarizans for genotoxicity assessment of chlorpyrifos. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Chlorpyrifos (o,o-diethyl o-(3,5,6-trichloro-2-pyridyl)phosphorothioate) is a broad spectrum organophosphorus insecticide which acts as a cholinesterase inhibitor. Recently, with the prohibition of those high toxic organophosphorus insecticides in the production of vegetables and other food crops, the usage of chlorpyrifos rapidly increased, and estimated for more than 499,000 kg recently in China [1]. Most of these compounds have low persistence in aquatic ecosystems, but relative lack of target specificity has raised concerns about their potential to cause adverse effects on non-target wildlife populations [2]. Several studies have documented an apparent connection between the presence of chlorpyrifos residues and reductions in amphibian populations at both local [3] and landscape scales [4,5]. Singh et al. [6] found that the residue level of chlorpyrifos in blood maximal in fish is 0.15 mg l−1 . Either Chlorpyrifos or its metabolites have also been discovered in the diet of preschoolers [7] in the cord blood of infants born to minority women living in urban settings [8]. Besides, chlorpyrifos elicited a number of other effects including hepatic dysfunction, immunological abnormalities, embryotoxicity, genotoxicity, teratogenicity, neurochemical, and
∗ Corresponding author at: Institutes of Pesticides and Environmental Toxicology, Zhejiang University, HangZhou 310029, China. Tel.: +86 0571 86430193. E-mail address: [email protected] (G. Zhu). 1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2009.05.018
neurobehavioral changes [9–11]. Chlorpyrifos and other organic pesticides had also been shown to induce genotoxicity [12–14]. Among techniques to detect genetic and genotoxic effects, the Micronucleus (MN) test and Comet assay are often used since they allow for convenient and easy application, particularly in aquatic organism and amphibians [15–18]. Amphibians have been regarded as bio-indicators, since they live in the aquatic environment in their larval stage [19], and are broadly used as test animals in evaluating effects of chemicals on aquatic and agricultural ecosystems [20]. Due to widespread application and its classification as highly toxic to freshwater fish [21], examination of the effects of chlorpyrifos on amphibians is warranted. In contrast to plenty of experimental studies on micronucleus and DNA damage of rodent and fish induced by pesticides, relatively scarce information existed in amphibians like toads and frogs [18,22]. It is important to note that red blood cells (RBCs) in amphibians are nucleated and undergo cell division in the circulation, particularly during developmental stages [23]. Liver is the major site for the metabolism of exogenous chemicals (pesticides, drugs, and metal), and the supplement formation of metabolites may be more or less toxic than the parent compound [24]. In the present study, the acute toxicity of chlorpyrifos was tested on Chinese toad (Bufo bufo gargarizans) tadpoles, a species which is widely distributed in southeast and southwest of China, and the genotoxicity of chlorpyrifos was also investigated using Comet assay for detecting the DNA damage of erythrocytes and liver cells and Micronucleus test of erythrocytes under laboratory conditions.
X. Yin et al. / Mutation Research 680 (2009) 2–6 2. Materials and methods 2.1. Test organism and chemicals Healthy specimens (lively, moved rapidly) of Chinese toad tadpoles were selected and procured from the local ponds of Pingban village in Linan Hangzhou China and acclimated to the laboratory environment for 10 d before the tests began on March 3, 2007. Prometamorphic tadpoles (from stage 26 up to 36) [25] were used for the bioassay. The average total size (snout-tail) was 24.5 ± 1.15 mm and the average weight was 100 ± 4.5 mg. The tadpoles were maintained under normal day–night light durations and the feeding was discontinued 48 h prior to exposure. No tadpole mortality occurred during the acclimatization period. For the present study, chlorpyrifos (40% emulsifiable concentrate, EC) was afforded by Zhejiang XinNong Chemical Co. Ltd. since the chlorpyrifos of this grade is mostly employed in field practices. Half-life of chlorpyrifos degradation in water with pH 7.0 is 25.6 d [26]. Methyl methane sulfonate (MMS) was purchased from Sigma–Aldrich Company. 2.2. Acute toxicity bioassay The tap water had been filtrated (to achieve animal drinking standard) and exposed in air for 3 d before test. Then it was analyzed for physico-chemical properties by standard methods [27]. The stock solution for the chlorpyrifos and MMS were prepared before treatment. For acute toxicity test with Chinese toad tadpoles, six concentration levels were individually prepared in water. The nominal test concentrations were 0.32, 0.64, 0.72, 1.08 and 2.56 mg l−1 for chlorpyrifos. Ten tadpoles (per replicate 10, total 30 of triplicates) were transferred to each 15 l-capacity glass tank containing 10 l of chlorpyrifos solution or controls. The solution was replaced every 24 h with freshly prepared solution of the same concentration. All treatments in triplicates were carried out and visually examined once every 8 h. When they did not respond to gentle touching, tadpoles were considered dead and removed immediately. The dead tadpoles were counted after 24, 48, 72 and 96 h. The data were analyzed with the statistical program SPSS® (Version 12.0). 2.3. Micronucleus test (MN) The tadpoles were exposed to chlorpyrifos, MMS (as positive controls), and the controls in a volume of 10 l for 96 h. Each level of exposure was prepared in triplicates. The sublethal concentrations of chlorpyrifos (0.08, 0.16, 0.32, and 0.64 mg l−1 ) were selected as exposure concentrations after median lethal concentration (LC50 ) being calculated by probit analysis [28]. The concentration of MMS was 0.1, 0.2, 0.4, and 0.8 mg l−1 being referred to previous study [18]. At the end of the 96 h exposure, a single smear of blood was prepared from the heart of each tadpole (n = 10). The smears were fixed in methanol for 10 min, left to air-dry at room temperature, and finally stained with 6% Giemsa in Sorenson buffer (pH 6.9) for 20 min. A total of about 2000 erythrocytes were examined for each specimen under the light microscope (Nikon, Type TE 2000; oil immersion lens, 100/1.25). The criteria for identification of Micronuclei (MN) used were to have no connection with the main nucleus, to share same color and intensity as the main nucleus, and with area smaller than one-third of the main nucleus. MN frequency was calculated as follows: of cells containing micronucleus × 1000 MN ‰= number total number of cells counted 2.4. Comet assay or alkaline single-cell gel electrophoresis The treatment concentration of chlorpyrifos and MMS were the same to MN. At the end of the exposure period (96 h), the DNA damage was assessed in the liver and the blood by using Comet assay in the tadpoles. Firstly the erythrocytes (Heart Puncture for Collecting Blood by glass capillary) were collected by centrifugation. Referring Carrera’s sample preparation, the liver tissue (about 50 mg) was washed three times with chilled phosphate buffered saline (Ca2+ Mg2+ -free, PBS) to remove the blood cells and transferred to ice-cold homogenization buffer (1× hanks’ balanced salt solution, 20 mM EDTA, 10% dimethylsulfoxide (DMSO), pH 7.0–7.5) [29]. The liver tissue was cut into small pieces using scissors on ice and homogenized to obtain a single-cell suspension. Secondly the survival rate of erythrocytes and liver cells density and viability (>90%) were measured by tryphan blue exclusion test [30]. Cells were diluted in cold PBS at 105 cells/ml. Comet assay was based on the protocol of Singh et al. [31]. The procedure was slightly modified according to the report of Zhang et al. [32]. Twenty l erythrocytes mixed with 0.8% LMP agarose were placed on normal 0.9% agarose microscope slides. After being held at 4 ◦ C, a third layer of 0.8% LMP agarose was added and left to solidify as described. The essential steps of Comet assay involved at least 2 h of cell lysis by detergent at a high salt concentration (2.5 mM NaCl, 100 mM Na2 EDTA, 10 mM Tris, 10% dimethylsulfoxide (DMSO), 1% Triton X-100, 1% Nasarcosinate pH = 10; at 4 ◦ C), and electrophoresis under alkaline conditions (300 mM NaOH, 1 mM EDTA, pH > 13, 30 min unwinding, 20 min electrophoresis at 300 mA and 18 V, at 4 ◦ C). Nucleoids were stained with 20 g ml−1 ethidium bromide (EB) and examined the following day using Zeiss Axioplan epifluorescence microscope (Carl Zeiss, Germany). Approximately 100 cells per slide (400 cells per concentration) were examined. Microphotographs were taken with 40× objective on XP-2 color film (400 ASA). The extent of DNA migration was determined using an image
3
´ analysis system (CASP, Krzysztof Konca, Poland). The parameters selected for the quantification of DNA damage were the frequency of DNA damage, Tail DNA (%), tail length, and Olive tail moment (OTM) (calculated by SPSS® ). The one-way ANOVA was employed to compare differences for the various indexes among the different concentrations of chlorpyrifos and MMS.
3. Results 3.1. Acute toxicity bioassay The physico-chemical characteristics of the test water were detected as follows: the dissolved oxygen concentration ranged from 7.5 to 8.2 mg l−1 , water temperature varied from 20.5 to 22.5 ◦ C, the pH values ranged from 7.18 to 7.67, total hardness ranged from 198 to 210 mg l−1 as counted by CaCO3 concentration, and total alkalinity was mean 301 mg l−1 as counted by CaCO3 concentration during the experimental period. The LC50 values of chlorpyrifos for Chinese toad tadpoles were found to be 3.63, 1.17, 0.819, and 0.800 mg l−1 for 24 h, 48 h, 72 h, and 96 h, respectively. The LC50 values (95% confidence limits) are shown in Table 1. In addition, negative effects on the behavior and morphology of the tadpoles were observed throughout the 96 h experimental period, for example the larvae suffered from morphological abnormalities (visible changes: fins shrunken, tail deformities, and head edema). 3.2. Micronucleus test (MN) The first evident result was significant (P < 0.01) greater induction of MN in Chinese toad tadpole specimens due to exposure to different concentrations of chlorpyrifos than the control group (Table 2, Fig. 1A and B). Further, a significant (P < 0.01) effect of durations on induction of MN was observed for all the concentrations of chlorpyrifos, except 0.08 mg l−1 . 3.3. Alkaline single-cell gel electrophoresis or Comet assay Erythrocytes and liver cells from the Chinese toad tadpoles were used to evaluate DNA damage using Comet assay. The Tail DNA (%), Tail length (m), and OTM (arbitrary units) were measured (Table 3, Fig. 2A–D). Significantly higher levels of DNA damage were detected in tadpoles exposed to sublethal concentrations of chlorpyrifos compared with the negative and positive controls. In summary, chlorpyrifos and MMS induced significantly (P < 0.05) concentration-dependent increase in DNA damage of exposed tadpoles (Fig. 2A–D). Significant positive correlations (correlation coefficient (r) at 5% level) were found between the concentrations of two agents and DNA damage measured by OTM. 4. Discussion In present study, the LC50 (96 h) of chlorpyrifos was found between 0.1 and 1.0 mg l−1 . The results indicated that the acute lethality of the chlorpyrifos for the Chinese toad tadpoles was very Table 1 Acute toxicity response (96 h LC50 ) of Chinese toad (B. bufo gargarizans) tadpoles exposed to chlorpyrifos (n = 30). Time
24 h 48 h 72 h 96 h
a (intercept)
6.06 6.53 6.78 6.93
b (slope)
5.17 6.5 6.66 6.82
c (LC50 , mg l−1 )
3.63 1.17 0.819 0.800
95% confidence limits Lower
Upper
1.92 0.929 0.684 0.67
23.25 1.62 1.02 0.989
b, regression coefficient; c, all LC50 values are based on initial measured concentrations.
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X. Yin et al. / Mutation Research 680 (2009) 2–6
Table 2 Frequencies of micronuclei and abnormalities in erythrocytes of Chinese toad (B. bufo gargarizans) exposed to chlorpyrifos and MMS for 96 h. Total cells scored
Number of normalities
0
6106
6006
86
Chlorpyrifos
0.08 0.16 0.32 0.64
6026 6082 6077 6005
5876 5814 5934 5663
109 197 106 249
MMS
0.1 0.2 0.4 0.8
6015 6031 6042 6031
5880 5836 5758 5624
109 158 215 306
Exposure agents
Exposure dosage (mg l−1 )
Control
Number of abnormalities
Frequency of abnormalities (‰)
Number of MN
Frequency of MN (‰) ±SE
14.1 ± 2.21
27
18.1 17.4 32.4 41.5
± ± ± ±
1.97 2.17 1.84** 0.26**
41 37 71 93
6.81 8.17 11.55 22.46
4.33 ± 0.49 ± ± ± ±
0.60 0.96* 0.68** 2.05**
18.1 26.2 35.6 50.7
± ± ± ±
1.76 1.64** 1.83** 2.16**
26 37 69 101
4.32 6.13 11.5 16.8
± ± ± ±
0.77 0.73* 0.99** 0.83*
Note: All data are shown as average median values (n = 3) ± SD; (separately for each mutagen). *LSD (least-significant difference) = P < 0.05; **LSD = P < 0.01.
Table 3 DNA damage of erythrocytes and liver cells in Chinese toad tadpoles (Bufo bufo gargarizans) exposed to chlorpyrifos and MMS for 96 h. Exposure agents
Exposure dosage (mg l−1 )
Number of Tadpoles
Tail DNA%
Tail length (m)
Erythrocytes
Liver cells
Erythrocytes
10.2 ± 0.40
42.8 ± 1.24
Liver cells
Control
0
10
14.7 ± 0.40
Chlorpyrifos
0.08 0.16 0.32 0.64
10 10 10 10
33.0 35.5 42.0 48.1
± ± ± ±
3.50*b 3.12*b 2.19**ab 0.41**a
18.2 19.9 20.1 34.5
± ± ± ±
0.73*b 0.89*b 0.79*b 2.01**a
50.2 73.0 87.1 98.6
± ± ± ±
3.47d 5.52**c 7.12**b 7.16**a
30.6 58.4 60.6 58.1
19.8 ± 8.22 ± ± ± ±
2.42b 6.3**a 7.8**a 7.12**a
MMS
0.1 0.2 0.4 0.8
10 10 10 10
17.8 27.9 35.9 43.9
± ± ± ±
0.66c 5.25*b 2.02*b 5.16*a
20.5 26.9 27.8 25.2
± ± ± ±
1.05*b 0.83*a 1.34*a 1.27*a
52.7 65.7 79.0 92.0
± ± ± ±
5.51cd 2.52**bc 4.36**ab 15.1**a
51.2 67.0 70.7 86.7
± ± ± ±
2.56**c 7.55**b 8.33**b 4.62**a
Note: All data are shown as average median values (n = 3) ± SD; (separately for each mutagen). Letters indicate statistically significant differences between the various doses *LSD (least-significant difference) = P < 0.05; **LSD = P < 0.01. Letters indicate statistically significant differences between the various doses, LSD = P < 0.05.
high. Richards and Kendall [33] pointed out that Xenopus laevis larvae exposed to chlorpyrifos during metamorphs were more sensitive than larvae exposed during premetamorphs, and the LC50 for metamorphs was 0.56 mg l−1 . Pan and Liang reported the LC50 (48 h) of chlorpyrifos to Rana limnocharis tadpoles to be moderately toxic (2.40 mg l−1 ) [34]. They did not mention the developmental stage of the tadpoles, but it seemed that Chinese toad tadpoles were more sensitive than Rana limnocharis tadpoles. There is limited information describing the acute toxic effects of chlorpyrifos on amphibians such as Chinese toad. The Micronucleus test has been performed to evaluate mutagenicity in toxicology and bio-indicators of ecosystem. To evaluate the genotoxic effects of pollutants, it has been carried out in several organisms, such as mussels [35], fish [36] and, especially, amphibians [18,22]. Recently, the presence of nuclear abnormalities besides
micronuclei, has been reported by several authors, either in humans [37] or in tadpoles [22]. In this study, erythrocytes displaying cell nuclearpycnosis, binucleated erythrocytes, and alteration in cell morphology of tadpoles’ erythrocytes were observed, and their incidence seemed to increase with the incidence of MN in high treatment (Table 2). Comet assay is commonly used as a biomarker for DNA damage, and increased Comets (presented either as DNA tail moments, Olive tail moment or percent DNA in the tail) are interpreted as compromising animal health [38–40]. The genotoxicity of chlorpyrifos has been studied in a variety of assays in the past. Gollapudi et al. [41] reported that there was no indication of genotoxic activity for chlorpyrifos in any of these assays (Ames test, CHO/HGPRT assay, cytogenetic abnormalities, rat lymphocyte chromosomal aberration test, mouse bone marrow Micronucleus test), but Rahman et al.
Fig. 1. Micronuclei and nuclear abnormalities from erythrocytes of B. bufo gargarizans tadpoles after 96 h exposure to chlorpyrifos (magnifier 400×). (A) Normal nuclear; (B) nuclear abnormalities. (a) Micronucleus, (b) nuclear protrusion, (c) non-nucleus, (d) double nucleus, (e) nuclear pycnosis, (f) nuclear concavity, and (j) nuclear cracking.
X. Yin et al. / Mutation Research 680 (2009) 2–6
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Fig. 2. Effect of a 96 h exposure to chlorpyrifos and methyl methanesulfonate (MMS, positive control) on Olive tail moment (OTM) in erythrocytes (A and B) and liver cells (C and D) of Chinese toad tadpoles (B. bufo gargarizans). Note: all data are shown as average median values (n = 3) ±SD; letters indicate statistically significant differences between the various doses, LSD = P < 0.05.
[13] indicated that it was controversial comparing with the results of numerous mutagenicity in short term assays, and found chlorpyrifos induce in vivo genotoxic effect in leucocytes of Swiss albino mice by using Comet assay. It has also been reported as genotoxic in root meristem cells of Crepis capillaries [42]. Our result demonstrated an increase of DNA damage in erythrocytes and liver cells of Chinese toad tadpoles exposed to chlorpyrifos, and induced DNA damage at a very low dose. As mentioned above, highly fragmented nuclei which showed completely disintegrated head region and images with nearly all the DNA in the tail or a much more wide tail were observed at the high concentrations of the chlorpyrifos exposure (0.32 and 0.64 mg l−1 ). This suggested that apoptosis was occurring, as apoptotic cells are known to display nucleosomal fragmentation [43]. This prediction is further supported by Fu et al. [44], who reported that chlorpyrifos was able to induce cell apoptosis in mouse retina. Saulsbury et al. [45] reported that chlorpyrifos-induced apoptosis in placental cells. Some studies indicated that chlorpyrifos was mutagenic at the cytological level in a concentration-dependent manner [46,13]. In the present study, tadpole erythrocytes appeared nuclearpycnosis, and Comet images showed some fragmented nuclei as well as images with nearly all DNA being in the tail or tails being very wide at high concentrations of chlorpyrifos. We could not estimate the contribution of apoptosis to the effects observed. So the images observed needs to be further analyzed to assess whether Chinese toad tadpoles exposed to relatively high concentrations of chlorpyrifos may induce apoptosis or necrosis. Taken together, the present findings indicated that chlorpyrifos could induce the MN of erythrocytes and DNA damage of erythrocytes and liver cells in Chinese toad tadpoles. On the assumption
that the MN and DNA damage observed in Chinese toad tadpoles from agriculture areas, it may be attributed to the application of pesticides within rice fields. It is necessary to study further the relationship between the concentration of chlorpyrifos and germ cells in Chinese toad, and chlorpyrifos-induced apoptosis in erythrocytes and liver cells. Conflict of interest None declared. Acknowledgements The authors gratefully acknowledge the support of National Natural Science Foundation of China (30771423) and the support of National Science Foundation of ZheJiang province (Y3090435). We thank Mr. Zheng Xiaojun (Senior Experimentalist) for helping with operating the instrument used. References [1] S.P. Zhou, C.Q. Duan, H. Fu, Y.H. Chen, X.H. Wang, Z.F. Yu, Toxicity assessment for chlorpyrifos-contaminated soil with three different earthworm test methods, J. Environ. Sci. 19 (2007) 854–858. [2] M.F. Frasco, D. Fournier, F. Carvalho, L. Guilhermino, Cholinesterase from the common prawn (Palaemon serratus) eyes: catalytic properties and sensitivity to organophosphate and carbarnate compounds, Aquat. Toxicol. 77 (2006) 412–421. [3] G.M. Fellers, L.L. McConnell, D. Pratt, S. Datta, Pesticides in mountain yellowlegged frogs (Rana muscosa) from the Sierra Nevada mountains of California, USA, Environ. Toxicol. Chem. 23 (2004) 2170–2177. [4] D.W. Sparling, G.M. Fellers, L.L. McConnell, Pesticides and amphibian population declines in California, USA, Environ. Toxicol. Chem. 20 (2001) 59–1595.
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