ABSTRACT. In this study, we evaluated the genotoxic and cytotoxic effects of dodine, a fungicide extensively used to control scab on apples, pears and pecans, ...
© by PSP Volume 24 – No 12a. 2015
Fresenius Environmental Bulletin
EVALUATION OF GENOTOXICITY AND CYTOTOXICITY OF DODINE (1-dodecylguanidium acetate) BY Allium TEST Nurşen Çördük1, Nihan Akıncı1*, Gülru Yücel2, Nergis Kaya1, and Cüneyt Akı1 1 Çanakkale Onsekiz Mart University, Faculty of Science and Arts Department of Biology Molecular Biology Subdivision Terzioglu Campus, 17020, Canakkale,Turkey. 2 Namık Kemal University, Faculty of Arts and Sciences, Department of Biology, 59030, Tekirdag, Turkey
ABSTRACT In this study, we evaluated the genotoxic and cytotoxic effects of dodine, a fungicide extensively used to control scab on apples, pears and pecans, brown rot on peaches and several foliar diseases of cherries, strawberries, peaches and black walnuts. For this purpose the Allium cepa test was carried out exposing roots to dodine for 24, 48 and 72 h at the concentrations of EC50/2, EC50 and 2xEC50. The mitotic index was calculated as the number of dividing cells per number of 3000-4000 observed cells and the mitotic aberrations also were scored at each concentration. The results showed that dodine induced significant increases of mitotic aberrations such as C-mitosis, polar shifting, laggard chromosome and chromosome fragments. In addition, mitotic index decreased significantly with increasing of concentration and the exposure time as compared to their controls. Hence dodine should be used under control in agricultural fields due to its possible toxic effects.
KEYWORDS: Allium cepa, dodine, cytotoxic, genotoxic, mitotic aberrations.
higher plants for the assessment of chromosomal damages and disorders in mitosis [7,8]. This test is highly sensitive, reliable and capable of detecting mutagens, carcinogens and clastogens [9,10]. Dodine (1-dodecylguanidinium acetate) is an aliphatic fungicide and bactericide used to control scab on apples, pears and pecans, brown rot on peaches and several foliar diseases of cherries, strawberries, peaches, sycamore trees and black walnuts [11]. In vitro, dodine severely affects the metabolism of fungal cells. Low concentrations of dodine inhibit growth, respiration on glucose and acetate and active transport of phosphor, glucose, acetate, and phenylalanine in fungal cells [12-16]. Low concentrations of dodine also resulted in leakage of potassium and cell death in Pseudomonas syringae [17]. Dodine has been generally used at 100 mL/100 L concentration for quince and pistachio, at 80 mL/100 L concentration for apricot and apple, at 175 mL/100 L concentration for peach in the fields. There has been no study, to our knowledge, on the genotoxic and cytotoxic effects of dodine in plant test systems. The aim of this study is to evaluate genotoxic and cytotoxic effects of dodine (1-dodecylguanidium acetate) for the first time.
2. MATERIALS AND METHODS 1. INTRODUCTION Modern agriculture and industry base on a wide variety of synthetically produced pesticides including fungicides, insecticide and herbicides. Especially fungicides are most commonly used to improve crop yields against diseases of crops in many countries [1-3]. But these chemicals or their derivatives can accumulate in the organisms and cause risk of mutagenicity, carcinogenicity or teratogenicity [4-6]. The increased use of pesticides have paid attention to develop several bioassays by several researchers in order to evaluate the genotoxic effects induced by these agents in living organism. Short-term genotoxicity tests are widely employed to evaluate the genotoxicity of many chemicals, including pesticides, to people, animals and plants. Allium cepa has been considered as an encouraging system among * Corresponding author
Equal-sized bulbs (25–30 mm in diameter) of a commercial variety of A. cepa L. (2n=16) were used as the test plant and they were purchased from a local supermarket. The length of the roots of 5 bulbs at each concentration (0.02, 0.03, 0.04, 0.06, 0.08, 0.12, 0.16, 0.24 and 0.32 mL/L) of dodine were measured after 72 hours. The mean value of 10 measurements of each onion bulbs, i.e. 50 roots for each concentration, was expressed as a percent of the control value. This measurement was used to calculate the EC50 value, which is the concentration where root growth is reduced by 50% compared with the control [18]. Bulbs were placed over the test tubes filled with distilled water at room temperature (20±2°C). When the roots reached 1.5-2 cm in five days, they were treated with 0.04 mL/L (EC50/2), 0.08 mL/L (EC50) and 0.16 mL/L (2xEC50) concentration of dodine for 24, 48 and 72 hours. Ten bulbs were used for each concentration, treatment period and control groups.
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© by PSP Volume 24 – No 12a. 2015
Fresenius Environmental Bulletin
Root tips were cut after treatment and immediately placed overnight at 4°C in the dark in the Farmer fixation solution containing ethanol and glacial acetic acid (3:1), freshly prepared before use. Root tips were rinsed with distilled water and hydrolyzed with 1N HCl at 60°C for 10 min as described by Souguir et al. [19]. The root cap was removed before squashing root tissues, and samples were stained with 2% (w/v) aceto-carmine. To determine the effect of dodine on mitotic index (MI) and chromosomal aberration index (CAI), at least 1000 cells were scored in each slide, for both control and treatment groups. During slide preparation, only one root tip was used per slide. Totally at least three slides (at least 3000 cells) were prepared per treatment and control. The slides were assessed by observing the cells in interphase, prophase, metaphase, anaphase, telophase and aberrations by light microscopy at 1000x. Cytotoxicity was assessed based on the MI, which was characterised by the total number of dividing cells in the cell cycle and was calculated by the formula MI = (number of dividing cells/total number of observed cells)x100 as the number of dividing cells per number of 3000-4000 observed cells and compared with the control. Genotoxicity was evaluated based the CAI which was calculated by the formula CAI = (number of cells with CA/total number of observed cells) x100 at each concentration and compared with the control. Statistically significant differences between the groups were compared using Student’s -t test for mitotic index and mitotic aberrations. The data are displayed as means ± standard error (SE) and p values less than 0.05 is considered statistically significant.
3. RESULTS AND DISCUSSION 3.1 Effects of dodine on mitotic index
The cytotoxic effect of dodine was investigated in A. cepa root tip cells in this study. The mitotic index was calculated in A. cepa root tip cells after expose to dodine to 0.04 mL/L, 0.08 mL/L and 0.16 mL/L after 24, 48 and 72 h and compared with negative control. We found that while more than 45% of cells in division were in prophase over all the different treatments, including controls, up to 20% of dividing cells were observed in metaphase, anaphase or telophase. Our data have showed that the mitotic index significantly decreased with the increase of concentration and duration of time as compared to their controls (p
