Cytotoxicity and genotoxicity Potential of thiocyclam in root-tip cells of Allium cepa.
The attitude of using pesticides worldwide as an agents to control or kill unnecessary pests, such as insects, weeds, rodents, fungi, bacteria or other organisms has increased dramatically among the agricultural establishments and farmers' . Huge quantities of these chemicals are released into the environment and a lot of them affect other organisms, and become a possible danger to human health . Unfortunately, pesticides were not considered a problem causing agents, on the contrary, utilizing these compounds was considered as a sign of progress and modernization in increasing agricultural yields . The increase use of pesticides called the attention of several researchers to develop several bioassays in order to evaluate the genotoxicity and mutagenicity effects induced by these agents in microorganisms and mammalian cells . Bioassays using microorganisms facilitate the detection of agents that induce gene mutation and primer damages occurs in DNA. On the other hand, using mammalian cells or other eukaryotes permit the detection of a greater damage levels, which range from gene mutations to chromosome damages and aneuploidies .
Higher plants are good genetic models for the assessment of the environmental pollutants, not only due to the sensitivity in evaluation of the genotoxicity of dangerous/harmful chemicals in different environments for years, but also to the potential of assessing numerous genetic endpoints, varying from point mutations to chromosome aberrations and micronucleus formation in cells of different organs and tissues, such as leaves, roots and pollen [5-7]. Allium cepa, Vicia faba, Zea mays, are among the most common higher plants used for the evaluation of environmental pollution . Allium cepa has been considered as encouraging higher plant for the assessment of chromosomal damages and disorders in mitosis, because of low chromosomal number (2n-16) and large chromosomes, understanding the duration of its cell cycle and its response in the existence of many known mutagenic agents [8-10]. Moreover, root tip cells of Allium cepa can be used as toxicity markers by evaluating several morphological and cytogenetic factors, such as root morphology and growth, mitotic index determination, and the induction of micronuclei and aberrant metaphases and anaphases [6,10]
The micronucleus assay, as a simple and sensitive short-term screening method performed in a number of organisms for determining the mutagenicity of chemical substances [11-14] Micronuclei formed by the condensation of acentric fragments, or whole chromosomes, that fail to integrate into the daughter nuclei during mitosis. Consequently, enumeration of micronuclei in the cytoplasm of the interphase daughter cell has been used to quantitate clastogenic or aneugenic chromosome DNA damage [11,14].
Currently, A lot of pesticides are used in Asser region (South west region of Saudi Arabia) among them is Thiocyclam Hydrogen Oxalate (The trade name is Evisect[R]; Figure 1). Thiocyclam is a nereistoxin analogue insecticides, selective stomach insecticide with contact action for lepidopterous and coleopterous pests; some dipterous and thysanopterous pests. Evisect was reported to have many side effects, including irritating to skin and eyes, may cause sensitization by skin contact and it is harmful if swallowed [15,16]. At low concentrations thiocyclam act as acetyl choline receptor agonists and as channel blockers at higher concentrations . There are few agricultural studies examining the effects of thiocyclam [17,18], and few studies of its genotoxic effect in literature [19,20]. Because of the potential environmental and human health impact connected with the heavy use of pesticides, the aim of this study was to use the in vivo Allium cepa root tip cell test to investigate the cytotoxic and clastogenic potential of thiocyclam.
[FIGURE 1 OMITTED]
Material and Methods
The Allium cepa root-tip cell assay was performed according to the method described by Fiskesjo  and Rank, et al. . Briefly, three Allium cepa (ordinary onion) bulbs were peeled and used for each concentration sample. The bulbs were grown in distilled water for first 36 hours onto 15-ml-Falcon tubes (the base of the onion must reach the medium surface). The tubes were covered with aluminum foil to keep the onion roots in dark during growth and kept in the lab with light cycle. After 36 hours, the bulbs removed from the distilled water and incubated in different doses of thiocyclam solution (0.073, 0.146, 0.29, 0.59, 1.17 g/ml) at the same conditions described before, but for 24, 48 and 72 hours. The doses represented the LC50 (dose that inhibited 50% of roots from growth), 1/2 LC50, 1/4 LC50, 1/8 LC50 and 1/16 LC50 for thiocyclam that were determined in a preliminary dose selection experiment. In this study, distilled water was used as a negative control and 300mM aqueous sodium azide (Merck) as a positive control mutagen.
At the end of incubation time, the newly-formed root tips were cut from each bulb and examined for any morphological visible abnormalities. The apparently normal root tips (length 2-3 cm) of the three bulbs were removed and washed with distilled water. After washing root tips were fixed in acetic alcohol (ethanol:glacial acetic acid in 3:1 ratio) and kept at 4 [degrees]C for 3 hours. The root tips were washed twice with distilled water for 10 minutes each and allowed to dry. Thereafter, the root tips were hydrolyzed in 1N HCl at 60 [degrees]C for 10 min, followed by washing with distilled water to remove HCl. One root tip from each bulb was transferred to a clean microscope slide and cutted 2 mm from the growing tip, and then spread evenly to form a monolayer by gently tapping the cover glass to facilitate the scoring process. The root tips were stained with 5% solution of Giemsa in 0.01M phosphate buffer at pH 7.4 according to the method described by Schmid  with slight modifications by Agarwal and Chauhan . Three slides were prepared per bulb.
The slides were examined using compound light microscope (Olympus) using the 100X objective with oil immersion. Genotoxicity was determined by counting the number of micronucleated meristametic cells from at least 2000 meristametic per preparation evaluated by the number of micronuclei in 2000 interphase nuclei per bulb, the micronucleus frequency (expressed as percent micronucleated cells) . The unit of scoring was the micronucleated cell, not the micronucleus; thus, the occasional cell with more than one micronucleus was counted as one micronucleated meristametic. The normal meristametic: micronucleated meristametic ratio was calculated to evaluate the cytotoxic effect of thiocyclam by scoring the number of micronucleated meristametic cells in 2000 cells per onion bulb .
Allium cepa roots exposed to distilled water for 72 h (negative control) had an average length of 3.4 cm and all showed normal morphology whereas roots exposed to thiocyclam showed a reduction in length. In addition, roots exposed to thiocyclam showed an increase in abnormal morphology, they lost their structure and became very soft and transparent. There was an increase in the mitotic index of the root-tip cells from roots exposed to distilled water. Root-tip cells from roots exposed to thiocyclam showed no increase in the frequency of chromosome damage.
The genotoxic effects of thiocyclam was determined by comparing the number of aberrant cells in division stages for each dose of each pesticide with those of the concurrent negative control are presented in Tables 1, 2, and 3. The genotoxic effects that were observed in the present study included, multipolar anaphase, fragments, and deformed nucleus.
Bioindicators offer several types of unique information not available from other methods: (1) early warning of environmental damage; (2) the integrated effect of a variety of environmental stresses on the health of an organism, the population, community, and ecosystem; (3) relationships between the individual responses of exposed organisms to pollution and the effects at the population level; (4) early warning of potential harm to human health based on the responses of wildlife to pollution; and (5) the effectiveness of remediation efforts in decontaminating waterways 
The use of higher plants (Allium cepa), as a bioindicator for the evaluation of genotoxic and mutagenic effects of various contaminants becoming common practice, because plants are direct recipients of agrotoxics, so they are important material for genetic test and for environmental monitoring of places affected by such pollutants, and also they found to be sensitive and an efficient model [5,25-27].
In this study, relative low frequencies of MN in the meristematic cells of Allium cepa treated with thiocyclam. One explanation for the relative low values observed is that the cytotoxic effect was stronger which could weaken the genotoxic effects, since meristematic cells were lost their structure and the root tip was almost deformed in all.
The results presented in this study supports the previous findings in studies with rat bone marrow , and with human lymphocytes , in which thiocyclam exhibited a cytotoxic rather than genotoxic effects. The concentrations studied were not mutagenic on the in vivo onion root-tip cells as witnessed by the fact that despite the large number of cells assessed there were no visible chromosome aberrations in either the treated or negative control cells. The positive control (sodium azide; 300mM), was observed to be genotoxic but not cytotoxic to the onion root tip cells in the present study which consistence with the sodium azide effects.
The present study has, therefore, demonstrated that thiocyclam is not genotoxic but cytotoxic also this study showed the usefulness of the Allium cepa root tip assay as a tool in assessing the genotoxicity of environmental chemicals. In the light of these results and conclusion its very necessary to confirm that the using of thiocyclam should be under control because it may has a cytotoxic effects on farmers and humans consumed the plants.
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Mohammed A.A. Sarhan *
Department of Biology, College of Science, King Khalid University, 61413 Abha P.O.Box 9004, Saudi Arabia
* Correspondence author E-mail: firstname.lastname@example.org
Table 1: Analysis of Allium cepa root tips treated with various concentrations of thiocyclam for 24 hours. MNC Sample No. Treatment Time(h.) Meristematic A B C Cell 1 T Negative 24 2000 0 0 0 control 2 0.071 g/ml 24 2000 0 0 0 3 0.142 g/ml 24 2000 0 0 0 4 0.285 g/ml 24 2000 0 0 0 5 0.57 g/ml 24 2000 0 0 0 6 1.17g/ml 24 2000 0 0 0 7 Positive 24 2000 8 1 0 Control Sample No. Normal Multipolar fragments Anaphase Anaphase 1 + - - 2 + - - 3 + - - 4 + - - 5 + - - 6 + - - 7 + - - Sample No. Polykaryocytes Deformed Nucleus 1 - - 2 - - 3 - - 4 - - 5 - - 6 - - 7 + + Table 2: Analysis of Allium cepa root tips treated with various concentrations of thiocyclam for 48 hours. MNC Sample No. Treatment Time(h.) Meristematic A B C Cell 1 Negative 48 2000 0 0 0 control 2 0.071 g/ml 48 2000 0 0 0 3 0.142 g/ml 48 2000 0 0 0 4 0.285 g/ml 48 2000 0 0 0 5 0.57 g/ml 48 2000 1 0 0 6 1.17g/ml 48 2000 2 0 0 7 Positive 48 2000 12 2 1 Control Sample No. Normal Multipolar fragments Anaphase Anaphase 1 + - - 2 + - - 3 + - - 4 + - - 5 + - - 6 + - - 7 + - - Sample No. Polykaryocytes Deformed Nucleus 1 - - 2 - - 3 - - 4 - - 5 - - 6 - - 7 + + Table 3: Analysis of Allium cepa root tips treated with various concentrations of thiocyclam for 72 hours. MNC Sample No. Treatment Time(h.) Meristematic A B C Cell 1 Negative 72 2000 0 0 0 control 2 0.071 g/ml 72 2000 0 0 0 3 0.142 g/ml 72 2000 0 0 0 4 0.285 g/ml 72 2000 2 * 0 0 5 0.57 g/ml 72 2000 1 * 0 0 6 1.17g/ml 72 2000 3 * 0 0 7 Positive 72 2000 14 * 5 * 2 * Control Sample No. Normal Multipolar fragments Anaphase Anaphase 1 + - - 2 + - - 3 + * - * - * 4 + * - * - * 5 + * - * - * 6 + * - * - * 7 + - - Sample No. Polykaryocytes Deformed Nucleus 1 - - 2 - - 3 - * - * 4 - * - * 5 - * - * 6 - * - * 7 + + * Counting was not applicable because the root tips were deformed
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|Author:||Sarhan, Mohammed A.A.|
|Publication:||International Journal of Biotechnology & Biochemistry|
|Date:||Oct 1, 2010|
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