Accepted Manuscript Title: Determination of genotoxic effects of Imazethapyr herbicide in Allium cepa root cells by mitotic activity, chromosome aberration, and comet assay Author: Recep Liman, İbrahim Hakkı Ciğerci, Nur Serap Öztürk PII: DOI: Reference:

S0048-3575(14)00220-X http://dx.doi.org/doi: 10.1016/j.pestbp.2014.11.007 YPEST 3738

To appear in:

Pesticide Biochemistry and Physiology

Received date: Accepted date:

18-6-2014 17-11-2014

Please cite this article as: Recep Liman, İbrahim Hakkı Ciğerci, Nur Serap Öztürk, Determination of genotoxic effects of Imazethapyr herbicide in Allium cepa root cells by mitotic activity, chromosome aberration, and comet assay, Pesticide Biochemistry and Physiology (2014), http://dx.doi.org/doi: 10.1016/j.pestbp.2014.11.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Determination of genotoxic effects of Imazethapyr herbicide in Allium cepa root cells by mitotic activity, chromosome aberration, and comet assay

Recep Liman1*, İbrahim Hakkı Ciğerci2, Nur Serap Öztürk2

1

Uşak University Faculty of Arts and Sciences, Molecular Biology and Genetics Department,

64300-Uşak, TURKEY 2

Afyon Kocatepe University, Faculty of Science and Literatures, Molecular Biology and

Genetics Department, 03200-Afyonkarahisar, TURKEY

*Recep Liman Uşak University Faculty of Arts and Sciences Molecular Biology and Genetics Department 1 Eylül Campus 64300-UŞAK TURKEY E-mails: [email protected]; [email protected] Tel.:

+90 5556214251

Fax:

+90 276 221 21 35

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Graphical Abstract Highlights  Genotoxic effects of Imazethapyr were investigated. 

Allium anaphase-telophase test and Comet assays were used.



Imazethapyr decreased mitotic index.



Imazethapyr also increased DNA damage.



The use of this herbicide should be under control in agricultural fields.

Abstract

Imazethapyr (IM) is an imidazolinone herbicide that is currently used for broad-spectrum weed control in soybean and other legume crops. In this study, cytotoxic and genotoxic effects of IM were investigated by using mitotic index (MI), mitotic phases, chromosomal abnormalities (CAs) and DNA damage on the root meristem cells of Allium cepa. In Allium root growth inhibition test, EC50 value was determined as 20 ppm, and 0.5xEC50, EC50 and 2xEC50 concentrations of IM herbicide were introduced to onion tuber roots. Distilled water and methyl methane sulfonate (MMS, 10 mg/L) were used as a negative and positive control, respectively. As A. cepa cell cycle is 24 hours, so, application process was carried out for 24, 48, 72 and 96 hours. All the applied doses decreased MIs compared to control group and these declines were found to be statistically meaningful. Analysis of the chromosomes showed that 10 ppm IM except for 48 h induced CAs but 40 ppm IM except for 72 h decreased CAs. DNA damage was found significantly higher in 20 and 40 ppm of IM compared to the control in comet assay. These results indicated that IM herbicide exhibits cytotoxic activity but not

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genotoxic activity (except 10 ppm) and induced DNA damage in a dose dependent manner in A. cepa root meristematic cells.

Keywords: Allium test, chromosome aberration, Comet assay, DNA damage

1. Introduction The

imidazolinone

herbicide

IM

{5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-4,5

dihydroimidazol-1H-2-yl)nicotinic acid}, is a common broad-spectrum herbicide, which is

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used to control weeds in soybean and other legume crops [1-2]. IM and other imidazolinone herbicides inhibit the biosynthesis of branched chain amino acids (valine, leucine and isoleucine) by competitively interacting with acetohydroxyacid synthase (AHAS, EC 2.2.1.6), first common pathway enzyme, also referred to as acetolactate synthase (ALS, EC 4.1.3.18), which stops plant growth and eventually kills the plant [3-4]. It has gained popularity recent years because of its high weed control efficacy at low use rates, flexibility in timing of application, low mammalian toxicity and highly selectivity [5]. Higher plants (Vicia faba, Tradescantia paludosa, Pisum sativum, Hordeum vulgare, Crepis capillaries, Nicotiana tabacum, and A. cepa etc.) are suitable organisms for general toxicity studies due to the possibility of assessing several genetic endpoints ranging from point mutation to CAs in cells. Among them, A. cepa anaphase-telophase test is routinely used to evaluate the genotoxic potential of pesticides due to its sensitivity and good correlation with other test systems [6-10]. Single cell gel electrophoresis (SCGE) or comet assay is a well-established, simple, cheap and sensitive method to detect strand breaks in the DNA of single cells [11-16]. Apart from Allium test, root meristem cells of A. cepa are also used in the comet assay because of its properties such as simple, fast, economical and independent of mitosis in different laboratories [9, 16-22]. The purpose of this study was to investigate the effects of IM herbicide on the root meristem cells of A. cepa using MI, mitotic phases, CAs and DNA damage by Allium anaphasetelophase and Comet assay, respectively. 2. Materials and methods 2.1. Organism

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A. cepa (2n=16) onion bulbs, 25-30 mm diameter, without any treatment, were obtained from a local supermarket. 2.2. Chemicals IM

(CAS No:138261-41-3), MMS (CAS No:67-27-3), normal melting point agarose

(NMPA), low melting point agarose (LMPA), di-sodium salt of ethylene diamine tetra acetic acid (EDTA), Tris buffer, ethidium bromide (EtBr), Trizma base, Tris HCl, Triton X-100 and SDS were obtained from Sigma Aldrich (Munich, Germany). Some chemical properties of the IM are given in Table 1. 2.3. A. cepa anaphase-telophase test 2.3.1. EC50 determination EC50 of IM against the A. cepa root growth was determined according to the method described in earlier studies [19]. We treated root meristem of A. cepa with different concentrations of IM (5, 10, 20, 40, 60 and 80 ppm) from stock solution (100 ppm) at room temperature (~21°C ± 4 °C). A set of bulbs was also exposed to distilled water and considered as negative control. On the 5th day, root lengths (ten roots from each bulb and five replicates) were measured from both IM exposed bulbs and control group. EC50 value was considered as the concentration which retards the growth of root 50% when compared to the control.

2.3.2. Mitotic activity and Chromosome Aberrations Allium test was performed by cutting 2 mm of the root tips of each onion after 24, 48, 72, and 96 h of cultivation in different concentrations of IM (10, 20 and 40 ppm). Five root tips from each individual were used. Fixation and staining of the root tip cells were carried out as reported earlier [20]. Slides were randomly coded and scored blindly. The MI and the

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frequencies of CAs were carried out according to Saxena et al. [23]. In order to obtain MI, 5000-6000 cells (1000 cells in each of the five slides) were observed for each sample. MI is calculated as: MI =total number of dividing cells/total cell number × 100. CAs (disturbed anaphase-telophase, chromosome laggards, stickiness and anaphase bridge) were scored in 100 ana/telophases per slide, if it was present. 2.4. Comet assay (single cell gel electrophoresis) Root meristem cells of A. cepa were exposed to similar concentrations of IM as used for cytogenetic analysis. 20 to 30 seedlings were placed in a petri dish kept on ice and spread with 500 µL of ice-cold Tris-MgCl2 buffer (0.2 M Tris, pH 7.5; 4 mM MgCl2-6H2O; 0.5% w/v Triton X-100). The roots were immediately chopped with a fresh razor blade and isolated root nuclei collected in the buffer. Each microscope slide was pre-coated with a layer of 1% NMPA and thoroughly dried at room temperature. Then, 100 µL of 0.8% LMPA at 37 ºC was mixed with 20 µL of the nuclear suspension and dropped on top of the first layer. The slides were allowed to solidify for 5 min on an ice-cooled tray and then immersed in ice-cold lysing solution (1 M NaCl; 30 mM NaOH, 0.5% w/v SDS, pH 12.3) for 1 h. Subsequent to lysing, the slides were placed in a horizontal gel electrophoresis chamber and the DNA was allowed to unwind for 1 h in the electrophoretic buffer, containing 30 mM NaOH and 1.5 mM EDTA at pH >12.3. Electrophoresis was then conducted for 20 min at 25 V (1 V cm-1) in the chamber cooled on ice. Following electrophoresis, the slides were rinsed three times with cold distilled water for neutralization and stained with 60 µL EtBr (20 µg ml −1) and covered with a cover slip. 25 comets (25 comets/slide) were scored visually as belonging to one of five classes (0 - undamaged, 1- mild damage, 2 – moderate damage, 3- severe damage, 4 – complete damage) using a fluorescence microscope [14]. Thus, the total score for 25 comets could range from 0 (all undamaged) to 100 (all damaged). Three slides were evaluated for

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treatment and each treatment was repeated at least twice. Arbitrary Unit used to express the extent of DNA damage was calculated by following Equation 1.

(1) Where Ni =Number of cells in i degree; i = degree of damage (0, 1, 2, 3, 4) 2.6. Statistical analysis For MI, mitotic phases and CAs (expressed as percents) and comet scores, Duncan multiple range tests by using SPSS 15.0 version for Windows software was performed. The level of significance was established at P < 0.05. 3. Results and Discussion The Allium test was carried out for detecting genotoxic effects and DNA damage of IM. Data on the root lengths and growth inhibition are presented in Table 2. The EC50 was found approximately as 20 ppm. The acute oral LD50, acute inhalation LC50 of IM for rats and dermal LD50 of IM for rabbits were found >5000 mgkg-1, 3.27 mgL-1 and >2000 mgkg-1, respectively [24]. There was statistically significant decrease in the root length of A. cepa in all concentrations by IM. The decreasing root length was in a dose dependent manner (r=0,892 p = 0.01). The inhibition of root growth generally related to apical meristematic activity [25] and to cell elongation during differentiation [26]. Over 10 ppm concentration of IM, slightly yellow and brownish coloration in roots was observed. These roots may indicate the retardation of growth and cytotoxicity [27]. After exposure to IM, inhibition in root lengths was also found in rice [28, 29], Zea mays [30] and in Arabidopsis thaliana [31]. IM also decreased plant growth through chlorophyll synthesis in A. thaliana [32]. Zhang et al. [33] suggested that application of the herbicide IM in soybean fields changed the soil microbial biomass and shifted the microbial community structure.

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The effect of IM on MI and mitotic phases in the root meristematic cells of A. cepa treated for 24-96 h is shown in Table 3. The rate of cell division in the root tips concluded that the IM had led to a significant reduction in the MI compared to negative control and MMS. The highest value was obtained from negative control in 24 h (31.57±0.58), and the lowest one was obtained from 20 ppm of IM in 96 h (9.65±0.27%). Decreasing MI values after exposure of IM were found lower than MMS. The decreasing MI was in a dose dependent manner for 24 h (r=-0,904 p=0.001), for 48 h (r=-0,907 p=0.01) and for 72 h (r=-0,391 p=0.14). A positive correlation was found between inhibition of root growth and decrease of MI except 96 h application. The cytotoxicity levels of an agent can be determined by the increase or decrease in the MI [34]. Significant decrease of MI may be due to the mitodepressive action of the IM, indicating thereby the IM interfere in the normal cell cycle resulting in decrease in number of dividing cells. Other studies have been reported to cause such effects with IM on Solanum tuberosum [35], Vicia faba [36] and Triticum durum [37]. Sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinylthio (or oxy)-benzoates and sulfonylaminocarbonyltriazolinones are AHAS-inhibiting herbicides [38-40]. It was suggested that AHASinhibiting herbicides block the synthesis of valine, leucine, and isoleucine in vivo and the branched chain amino acids deficiencies can cause a decrease in protein synthesis which in turn could slow down the rate of cell division and eventually lead to the cell death [41, 42]. An exogenous supply of the branched chain amino acids reversed the inhibition of mitosis and DNA synthesis caused by Imidazolinones and sulfonylureas [43, 44]. Moraes et al. [45] showed that the protein and carbohydrate metabolism in liver and muscle tissues and blood of Cyprinus carpio were also disrupted after exposure to IM and imazapic herbicides. IM enantioselectively inhibited plant vegetative development via affecting the biosynthesis of branched chain amino acid, carbohydrate use, chlorophyll synthesis and acetolactate synthase activity [28, 31, 32] and it caused early flowering by affecting the clock gene pathway's but

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decreased the seeding ratio in A. thaliana [46]. Incubation of roots exposed to IM, showed changes in the percentage of particular phases’ distribution in comparison to the control. The characteristic effect caused by IM was a statistically increase of prophase index except in 12 h at 10 ppm and simultaneous statistically decrease of metaphase index except in 96 h at 10 ppm, anaphase index except in 48 h at 20 ppm and in 96 h at 40 ppm, and also telophase index when compared to control. Similar result has been reported after treatment of T. durum with IM except metaphase index [37]. The accumulation of dividing cells at prophase with IM might be an indication of the blockage of the process at the end of prophase. Scolnic and Halazonetis [47] reported chfr checkpoint that delays entry into metaphase in response to mitotic stress. The CAs found in anaphase-telophase cells exposure to IM between 24 and 96 h included the disturbed anaphase-telophase, chromosome laggards, stickiness and anaphase bridge (as shown in Table 4). Analysis of the chromosomes showed that 10 ppm IM except for 48 h induced CAs but 40 ppm IM except for 72 h decreased CAs. No aberration was recorded in the chromosome of A. cepa exposed to 40 ppm of IM in 96 h. Decreased anaphase-telophase anomalies at 48 h applications and increased one at 10 pmm of IM in 96 h (37.07±1.97%) showed statistically significant results (p