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Abstract

The present study investigated the genotoxic effects of flumorph in various organs (brain, liver, spleen, kidney and sperm) of mice. The DNA damage, measured as comet tail length (µm), was determined using the alkaline comet assay. The comet assay is a sensitive assay for the detection of genotoxicity caused by flumorph using mice as a model. Statistically significant increases in comet assay for both dose-dependent and duration-dependent DNA damage were observed in all the organs assessed. The organs exhibited the maximum DNA damage in 96 h at 54 mg/kg body weight. Brain showed maximum DNA damage followed by spleen > kidney > liver > sperm. Our data demonstrated that flumorph had induced systemic genotoxicity in mammals as it caused DNA damage in all tested vital organs, especially in brain and spleen.

Keywords

flumorph alkaline comet assay genotoxicity DNA damage

Introduction

During the past 50 years, the abuse use of synthetic agrochemicals, which were widely applied to increase the food production throughout the world, had caused great conflicts to environment and human safety. Therefore, high-efficiency, low-toxic and pollution-free agricultural chemicals, such as flumorph were developed and used widely. Flumorph (CAS No. 211867-47-9; [E,Z]-3-[3,4-dimethoxyphenyl]-3-[4-fluorophenyl]-1-morpholinopropenone) is a carboxylic acid amide fungicide which has two isomers (50% [E]-isomer, 50% [Z]-isomer). Both the isomers have good fungicidal activities against Peronospora and Phytophthora diseases. The fungicide was developed by Shenyang Research Institute of Chemical Industry in 1994, and commercially produced in 1999. It had been granted patents in China,¹ the United States² and Europe.³ As the use of flumorph has become increasingly widespread, studies are needed to evaluate the potential genotoxic risk of flumorph to the health of animals and humans.

The single-cell gel electrophoresis (SCGE) assay, which is also referred to as the comet assay, is a rapid, simple, sensitive and reliable technique for measuring DNA damage originated by strand breakage in individual cells.^4,5 This method was first described by Ostling and Johanson.⁶ Thereafter, the alkaline single-cell electrophoresis was reported by other research groups.^7,8 Due to its high sensitivity to detect genotoxic effect on the individual cell, the assay has quickly been adopted in short-term genotoxic and human biomonitoring studies.⁴ The technique is based on the fact that broken DNA migrates more easily in an electric field than intact molecules. It is becoming an important tool for evaluating the genotoxic potential of compounds in vitro and in vivo.^4,9

–13 Under alkaline conditions (pH > 13), the assay is able to evaluate DNA damage and other lesions, such as alkali-labile sites, DNA-protein and DNA-DNA cross-links ^14,15 and incomplete excision repair events.¹⁶ The genotoxic effect caused by pesticides could be evaluated using a very small sample of cells.¹⁷

As far as we were aware, there was no published study on the genotoxic evaluation of flumorph. Genotoxicity is one of the most important toxicity effects of a compound, and it is widely used to estimate the long-term effects such as cancer and reproductive diseases. The purpose of our study was to investigate the DNA damage caused by flumorph in organs of mice using comet assay and provide further knowledge to estimate the potential risk of this fungicide.

Materials and methods

Chemical

Flumorph standard (purity 99.5%) was obtained from Shenyang Research Institute of Chemical Industry and used without further purification.

Normal melting agarose (NMA), low-melting agarose (LMA), dimethyl sulfoxide (DMSO), Triton X-100, N-lauroyl sarcosine sodium, ethidium bromide (EtBr), Tris, ethylmethane sulfonate and trypan blue were purchased from Amresco Inc. Ethylenediaminetetraacetic acid disodium salt (Na₂EDTA) and ethanol were purchased from Beijing Chemical Factory. Camellia oil was purchased from Longyou County Tianyushan Tea Oil Development Corporation Ltd. Cyclophosphamide (CP) was obtained from the Academy of Military Medical Sciences. All other chemicals were obtained locally and were of analytical reagent grade.

Animal and treatments

Male Kunming mice (~4-week-old, 25.0 ± 2.0 g) were provided by the Peking University Health Science Center (Beijing, China). They were housed in polypropylene cages in an air-conditioned room. The animal room was maintained at approximately 25°C and 50–70% relative humidity with a 12-h light–dark cycle. They were bred on a laboratory diet, with drinking distilled water provided ad libitum.

Groups of experimental mice were treated with flumorph (n = 3 mice per group), which was dissolved in camellia oil. They were administered flumorph by intraperitoneal injection, to produce animal model of axonopathy. The dose selection was based on the value of lethal dosage 50 (LD₅₀), which for flumorph was 2700 mg/kg. The mice used in the comet assay were divided into seven groups as follows: Group 1, negative control (20 ml/kg, camellia oil, 5 consecutive days); Group 2, positive control (100 mg/kg, CP, 24 h before killing); mice in Groups 3–7 were treated with 3.375, 6.75, 13.5, 27 and 54 mg/kg flumorph, respectively, for 5 consecutive days, with an interval of 24 h. The last dose was given 6 h before killing.

Cell count and viability assay

On completion of the flumorph administration, mice were killed immediately under ether anesthesia and the organs were quickly dissected. Preparation of a single-cell suspension from organs was done as follows. Briefly, 0.2 g of each organ was placed in 1 mL chilled mincing solution (phosphate-buffered saline [PBS], pH 7.4) in a petri dish, chopped into pieces with a pair of scissors, collected through a cell strainer and blended with 4 mL PBS. The mixture was separated by centrifugation at 1,200 rpm for 5 min. The supernatant was decanted and the cells were resuspended. The supernatant containing the single cells was taken.

Cells isolated from brain, liver, kidney, spleen and sperm were counted using a hemocytometer and diluted with PBS to achieve a concentration of 10⁶ cells per 1 mL. Cell viability for the flumorph was determined by the trypan blue.

Comet assay

The alkaline single-cell gel electrophoresis was performed as a three-layer procedure with slight modifications.^7,18 The frosted ends of conventional slides were used. The frosted end was covered with 100 µL of 0.7% NMA. After application of a coverslip, the slides were allowed to gel at 4°C for 10 min. Meanwhile, 50 µL of cell suspension was added to 100 µL of 0.65% LMA. After carefully removing the coverslips, the second layer of 100 µL of sample mixture was pipetted out on the precoated slides and allowed to solidify at 4°C for 10 min. The coverslips were removed and the third layer of 100 µL of 0.6% LMA was spread onto the slides and allowed to gel at 4°C for 10 min. Finally, the coverslips were removed and the slides were immersed in freshly prepared, chilled lysing solution (2.5 M NaCl, 100 mM Na₂EDTA, 10 mM Tris, 1% sodium N-lauroyl sarcosinate, pH 10, with 1% Triton X-100 and 10% DMSO added just before use). The slides were left in the lysing solution overnight at 4°C. Slides were then placed in a horizontal gel electrophoresis tank with fresh and chilled alkaline buffer (1 mM Na₂EDTA and 300 M sodium hydroxide [NaOH], pH 13). Then, the slides were immersed into the buffer solution for 30 min to allow DNA unwinding and expressing of alkali-labile sites as the DNA strand breaks. Electrophoresis was conducted at 25 V (0.66 V/cm) adjusted to 300 mA by raising or lowering the buffer level in the tank. After electrophoresis, the slides were drained and placed horizontally in a tray and washed slowly with three changes of neutralization buffer of 5 min each (0.4 M Tris-HCl, pH 7.5). DNA was precipitated and the slides were dehydrated in absolute methanol for 10 min and then dried at room temperature. The whole procedure was carried out in dim light to avoid additional DNA damage.

Slides were stained with 50 µL EtBr (20 mg/L) and examined using an Olympus BX51 fluorescence microscope equipped with a wide band excitation filter of 515–560 nm and a barrier filter of 590 nm. For the visualizing of DNA damage, slides were examined at ×400 magnification. Randomly selected 150 cells (50 cells from each of the 3 replicate slides) were analysed per sample. Imaging was performed using the analytical software to determine comet tail length, which was correlated with the degree of DNA damage. From the image, damaged cell had the appearance of a comet; on the contrary, undamaged cells resembled an intact nucleus without a tail. The length of the DNA migrated in the comet tail was selected to estimate DNA damage. Quantification of the DNA damage for each cell was calculated as comet tail length (µm) = maximum total length − head diameter.

Statistical analysis

The results were reported as means ± standard errors. One-way analysis of variance (ANOVA) was employed to compare the mean differences in the tail length between tissues within durations and doses of flumorph. In all cases, p < 0.05 was considered statistically significant compared with the controls.

Results

During the study, mortality was not observed, as the dose selected was nontoxic to the mice. The cell viability by the trypan blue exclusion technique was relatively stable and ranged from 92 to 96% throughout the experiments. The viability of brain cell suffered in various doses of flumorph at each time interval is shown in Figure 1 .

Figure 1.

Effect of various doses of flumorph on cell viability (brain cell): cell viability was checked at each time interval by the trypan blue exclusion method.

It was the first time that the potential genotoxicity caused by flumorph was evaluated using the comet assay. The level of DNA damage effects caused by flumorph was measured by comet tail length (µm) in the cells of brain, liver, kidney, spleen and sperm in the control as well as exposed groups (Table 1 , Figure 2 ). Our study also showed statistically significant (p < 0.05) dose-dependent increase in DNA damage in all organs of mice exposed to flumorph. Assay was repeated three times and the results were reproducible.

Table 1.

Mean comet tail length (μm) of various organs of mice exposed to different concentrations of flumorph at 24 h.^a

Groups	Brain	Liver	Kidney	Spleen	Sperm
Camellia oil^c	18.13 ± 1.60	17.24 ± 1.01	19.27 ± 0.97	15.12 ± 0.60	21.16 ± 1.05
3.325 mg/kg	19.12 ± 0.96	18.73 ± 0.72	17.67 ± 0.61	18.47 ± 0.52	20.60 ± 0.97
6.75 mg/kg	23.00 ± 0.73	19.20 ± 0.41	20.27 ± 0.91	21.40 ± 0.77	18.80 ± 0.92
13.5 mg/kg	27.93 ± 0.86^b	20.27 ± 0.96	23.07 ± 0.88	23.67 ± 0.84^b	23.00 ± 0.66
27 mg/kg	27.13 ± 0.94^b	23.20 ± 1.15	24.80 ± 1.17	28.32 ± 1.32^b	23.80 ± 1.18
54 mg/kg	42.20 ± 1.59^b	30.00 ± 1.14^b	35.00 ± 1.57^b	33.07 ± 1.28^b	25.47 ± 1.52^b
CP^d	108.51 ± 9.51	79.65 ± 8.61	97.65 ± 6.21	110.27 ± 7.31	74.12 ± 9.20

^aValues represent mean ± SE of three animals in each group.

^b p < 0.01, when compared with negative control.

^cCamellia oil-negative control (20 ml/kg body weight).

^dCyclophosphamide (CP)-positive control (100 mg/kg body weight).

Figure 2.

Mean comet tail lengths in different organs of mice exposed to a series of flumorph doses at various time intervals. Camellia oil (20 ml/kg, body weight) was used as a negative control. A statistically significant difference from the control was at p < 0.05. (a) Brain, (b) Liver, (c) Kidney, (d) Spleen and (e) Sperm.

Mean comet tail lengths of various organs of mice exposed to different doses of flumorph at 24 h were presented in Table 1. It was evident from Table 1 that flumorph evoked a significant increase in the comet lengths for studied organs from 3.375 to 54 mg/kg body weight (at the same duration). At a range of dose from 3.375 to 54 mg/kg body weight, the mean comet tail lengths of brain increased from 19.12 ± 0.96 µm to 42.20 ± 1.59 µm (p < 0.01), which is 2.3 times more when compared to control. The DNA damage for other organs is also indicated in Table 1 and Figure 2.

The specimens exposed to different doses of flumorph exhibit significantly higher DNA damage (p < 0.01) in their organs than those of the negative control groups. The CP as the positive control induced a significantly higher damage in the DNA of mice cells than that of negative controls (p < 0.01; Table 1, Figure 2).

With regard to the variation in DNA damage between organs, the brain exhibited comparatively higher DNA damage than other organs at all doses. From Table 1, brain was found to exhibit higher DNA damage followed by spleen > kidney > liver > sperm. The highest DNA damage was observed at 96 h in brain, 49.08 ± 1.93 µm, followed by kidney, 44.27 ± 1.68 µm, at 54 mg/kg body weight (Figure 2).

Discussion

Most of the pesticides are considered as potential chemical mutagens. Experimental data revealed that various agrochemical ingredients possessed mutagenic properties inducing mutations, chromosomal alterations, or DNA damages,¹⁹ which led to adverse effects on exposed animals and humans. Abundant research had proved that agrochemicals are an important source of DNA damage.^19
–21

Numerous studies had proved that the comet assay was successfully employed for the evaluation of DNA damage caused by agrochemicals, and the comet assay was widely used for environmental biomonitoring.^17,22
–24 Since it could detect low levels of DNA damage in individual cells and required relatively short period, the comet assay had been popularly employed.¹⁶

During our experiment, the alkaline SCGE assay was employed to analyse the DNA damage induced by flumorph in different organs of mice. Our results demonstrated the potential DNA damages caused by flumorph in cells of different mice tissues in vivo. Compared with the other organs, the brain cells were more prone to injury caused by flumorph, whereas lowest DNA damage was observed in the sperms that received the flumorph molecules after a course. Brain was found to exhibit the highest level of DNA damage followed by spleen >kidney > liver > sperm, according to tail lengths. The research of Daoud Ali showed that the observed tissue-specific responses might be due to the physiological activities distinctive to their organs, the repair of the different types of strand breaks, and the different stages of studied cells.²⁵ The same explanation was shown by Steinert in 1996 that cells might be in the early stages of apoptosis, contributing to the different levels of DNA damage.²⁶ Tomascik-Cheeseman showed that the differences in DNA damage observed among the organs could be explained by a differential expression of basal levels of DNA repair genes in various tissues of the mice.²⁷

A statistically significant dose-dependent and duration-dependent increase in DNA damage in all organs was observed from our results. Many researchers had reported the similar phenomena. Blasiak reported the DNA-damaging effect of malathion and both its major metabolites malaoxon and isomalathion in human lymphocytes in vitro. They observed that while malathion did not cause any significant DNA damage of the lymphocytes, its metabolites induced breakage of DNA in a dose-dependent manner.²⁸ Sushila Patel reported that cypermethrin induced systemic genotoxicity in mammal as it caused DNA damage in vital organs like brain, bone marrow, spleen and so forth. And a statistically significant (p < 0.05) dose-dependent increase in DNA damage was observed, which was evident from comet assay parameters.²⁴

One of the conceivable mechanisms for DNA damage caused by agrochemicals was the breaks in DNA single strand, because the hydrogen bonds of the DNA double chain were the most sensitive structures to chromatin-damaging agents.²³ The DNA damage could have originated from DNA single-strand breaks, DNA double-strand breaks, DNA adducts formation and DNA-DNA and DNA-protein cross-links,²⁹ resulting from the interaction of pesticides or their metabolites with DNA.³⁰ Zegura had demonstrated that reactive oxygen species (ROS) may cause DNA damage originated from single-strand breaks.³¹ Another mechanism that may be involved in the generation of DNA lesions was oxidative stress.^32,33 This oxidative stress was reported as the significant mechanism of DNA damage caused by nerve agents, such as organochlorine pesticide and organophosphorous pesticide.^34
–36 However, flumorph was a kind of bactericide that did not affect the nerve system, so we presume the mechanism of DNA damage caused by flumorph was not due to oxidative stress.

Based on our results, it could be concluded that the comet assay was highly sensitive and accurate for the detection of DNA damage of flumorph in various organs of mice. Our present study could be useful in supplying more experimental data on the potential genotoxic risk of flumorph.

Footnotes

Funding

This work was supported by the Major State Basic Research Development Program of China (No. 2006CB101907 and No. 2003CB114400), National Natural Science Foundation of China (No. 20777078) and the 863 high-tech key project of China (2006AA10A203).

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Assessment of genotoxic effects of flumorph by the comet assay in mice organs

Abstract

Keywords

Introduction

Materials and methods

Chemical

Animal and treatments

Cell count and viability assay

Comet assay

Statistical analysis

Results

Discussion

Footnotes

Funding

References