South Indian Journal Of Biological Sciences 2016; 2(1); 35 40 ONLINE ISSN: 2454 4787 Research Article Evaluation of cytotoxic effects of synthetic pesticide attack on root meristems of Allium cepa L. Soumya KR 1, Teena MT 1, Sudha S 1, * 1 Post Graduate Department of Botany and Research Centre, Sree Krishna College, Ariyannoor P.O. Guruvayoor 680102, Kerala, India * Corresponding author: Sudha S; E mail: sudha.soman@gmail.com Received 21 August 2015; Revised 28 September 2015; Accepted 30 September 2015; Published 2 January 2016 Abstract Generally in agriculture farmers use pesticides to increase their yield by reducing crop losses by pests. Some chemical compounds used pesticides are mutagens. Present study evaluated the cytotoxic effect of synthetic pesticide Attack which is used instead of Furadan on root meristems of Allium cepa L. as a test system. The root meristems of A. cepa L. were treated with five concentrations (1%, 2%, 3%, 4% and 5%) of the chemical pesticide Attack for 6 h duration. Exposure to different concentrations of pesticide showed an inhibitory effect on cell division in root tip cells of A. cepa and caused a general decline in Mitotic Index values. Additionally, a wide range of abnormal mitotic stages, were detected in treated cells when compared to control. The total percentage of aberrations generally increased in a dose dependant manner. Among these, frequently observed chromosomal abnormalities were despiralized prophase, disoriented prophase, vagrant prophase, diagonal metaphase, C metaphase, vagrant metaphase, clumped metaphase, star anaphase, diagonal anaphase, anaphase with two bridges and with multiple bridges, aticky anaphase, telophase stickiness, hyperchromasia, bi nucleate and 4 nucleate cells and micronucleus. The results of the present study clearly proved the sensitivity of Allium test and hence substantiate its use as a cytogenetic assay to assess the cytogenetic effect of chemical widely used as pesticides. The cytotoxic pesticides have potential to cause adverse environmental and health effects by inducing chromosomal abnormalities leading to mutations. Keywords: Allium cepa, Cytotoxicity, Attack, Chromosomal Aberrations, Mitotic Index, Root mersistem Cells. 1. Introduction Pollution is a major problem which lowers the quality of life in various aspects. A pollutant is any substance that may present in the nature in quantities greater than natural abundance, chiefly due to human activities and ultimately harm the environment along with its living system (Khopkar 2002). Environmental pollutions may be mutagenic or toxic to all living organisms. Use and constant exposure to these chemicals may result in change in the genetic constitution of an organism.. Mutagenic activities 35
at cellular level can be induced by air pollutants, water pollutants, food additives, drugs, beverages, pesticides and industrial products. Radiomimetic chemicals are those chemicals that mimic ionizing radiations in the way which they cause lesions in the nucleic acid. A wide variety of radiomimetic chemicals are now known that are capable of causing cell damage and cell death of varying magnitude. Exposure to agrochemicals at the time of use in the field has been associated with an increase in cancer incidence (Settimi et al., 2003). Many genotoxic studies have been carried out to detect the harmful effects of different pesticides which reveal their hazardous effects in addition to benefits. Their undesirable residues in water, food, and environment may cause some serious health problems. Chromosomal abnormalities induced by some of these compounds were found to be linked with their capacity to induce mutations (Panday et al., 1994). Chromosomal anomalies produced by pesticides therefore can be regarded as a reliable evidence for the evaluation of genotoxicity (Grant 1982). Plant genotoxicity assays are relatively inexpensive, fast and gives reliable results and chemicals which cause chromosomal alterations in plant cells also produces chromosomal abnormalities in cultured animal cells that are frequently identical (Grant 1978). The plants for being direct recipients of agro toxics, becomes important material for genetic test and for environmental monitoring of cases affected by such products (Sharma and Paneerselvan 1990). Allium cepa (Onion) has been considered as a most efficient test organism to indicate the presence of mutagenic chemicals due to its kinetic characteristics of proliferation and possession of chromosomes suitable for cytotoxic study. Different parameters of Allium cepa such as root shape, growth, mitotic index, chromosomal aberrations etc can be used to estimate the cytotoxicity and mutagenicity of environmental pollutants ʹʹAttackʹʹ is a commonly used insecticide in agricultural fields instead of Furadan. It is an emulsifiable concentrated liquid containing Fenvalerate 20 % as an active principle. It acts as a contact and stomach poison and is best suited to control pest affecting field crops such as Cotton, Ladyʹs finger, Cauliflower, Brinjal, Tomato and Cabbage and other vegetable crops. When applied to soil, it retains its effectiveness for a considerable period of time. So, the present study was designed to investigate the effects of Attack on mitotic index and frequencies of abnormalities in root tip cells of A.cepa L. with a view to detect their mutagenic potential. 2. Materials and methods Commercially available bulbs of onion (Allium cepa L.) were utilized for the study. Acetocarmine squash preparation was used for mitotic studies. Two slides were prepared for each treatment and scoring was done from 5 different sites that were randomly selected. The resulting data were pooled to calculate the Mitotic index (M.I), and percentage of abnormality. The mitotic index was calculated for each treatment as number of dividing cells / Total number of cells counted and expressed as percentage. The cells were also scored for cytological abnormalities and the percentage of aberrant cells to total number of dividing cells observed. The relative frequency of the different aberrations at each dose was calculated. 3. Results Exposure to different concentrations(1%, 2%, 3%, 4% and 5%) of pesticide significantly and dose dependently inhibited the mitotic index (Table 1) in the root tip cells of A. cepa L. there were significant differences in the M.I values as compared to control at all treatments right from the lowest dosage (Table 2). It shows the exponential relationship between the percentage of aberrations and concentrations of the test compounds. It could significantly increase the percentages of chromosomal aberrations at all concentrations more or less in a dose dependant manner. The highest number of 36
mitotic aberrations was recorded in root tip cells subjected to 6 hrs treatments in 5% Attack concentrations. A wide spectrum of aberrations (Fig. 1 and Fig.2) was observed in the treated meristematic cells. The cytological aberrations observed in onion root tip cells wee despiralized prophase, disoriented prophase, vagrant prophase, metaphase, C metaphase, vagrant metaphase, clumped metaphase, star anaphase, vagrant anaphase, diagonal anaphase, anaphase with two bridges and multiple bridges, sticky anaphase, telophase stickiness, nuclear lesion, binucleate and tetra nucleate cells, hyperchromasia and micronucleus (Fig. 3). Table 1. M.I in root tip cells of Allium cepa L. exposed to different concentrations of pesticide Attack. Treatment Conc. (%) No of dividing cells Total no of abnormal cells Percentage (%)of number abnormality 1 Control 1136 ± 5.54 390 ± 4.49 34.3 ± 3.02 2 1 1029 ± 1.82 280 ± 3.58 27.2 ± 1.39 3 2 1030 ± 1.58 242 ± 4.09 23.5 ± 1.48 4 3 1120 ± 2.70 295 ± 2.30 18.3 ± 1.19 5 4 1133 ± 3.69 118 ± 1.58 10.4 ± 0.54 6 5 1125 ± 4.15 104 ± 3.39 9.2 ± 2.03 Table 2. Percentage (%) of abnormality caused by pesticide Attack on root tip cells of Allium cepa L. Treatment number Conc. (%) No of dividing cells Total no of abnormal cells Percentage (%)of abnormality 1 Control 390 ± 4.49 3 ± 0.60 0.8 ± 0.13 2 1 280 ± 3.58 16 ± 0.89 5.7 ± 0.08 3 2 242 ± 4.09 26 ± 1.38 10.7 ± 0.10 4 3 295 ± 2.30 33 ± 2.10 11.2 ± 1.19 5 4 118 ± 1.58 41 ± 1.82 34.7 ± 0.51 6 5 104 ± 3.39 56 ± 2.14 53.8 ± 0.92 frequency 80 70 60 50 40 30 20 10 0 Stickiness Vagrants Disorientation Star chromosome c- Mitosis Despiralisation Binucleate cell 4 - nuucleate cell Hypochromasia Micronucleus nuclear Lesion Bridges Att ack Type of aberration Fig. 1. Percentage of abnormality caused by pesticide Attack on root tip cells of Allium cepa L. 37
40 M IT O T IC IN D E X 35 30 25 ATTACK 20 15 10 5 0 CONTROL 1 2 3 4 5 CONCENTRATION Fig. 2. Mitotic Index in root tip cellsof Allium cepa L. exposed to different concentrations of pesticide Attack. Fig.3. Different chromosomal aberrations induced by synthetic pesticide Attack on root tip cells of Allium cepa L. 38
4. Discussion The result of the present study reveals clear evidences of the cytotoxic effects of tested synthetic pesticide Attack. Wide spectrum abnormalities exhibited by the treatment proved that even exposure to relatively smaller concentrations have significant effects on mitotic index and structure of chromosome and disturbs mitotic spindle formation. Lowering of M.I in treated root meristems could be due to inhibition of DNA synthesis( Sudhakar et al., 2001), arrest of one or more mitotic phases( Kabarity and Mallalah 1980) or blocking of G2 phase in the cell cycle preventing the cell from entering mitosis. Reduction in MI with increasing concentrations clearly demonstrates the ability of chemical to inhibit DNA synthesis. Significant genotoxicity observed at high concentrations can be another probable reason for the lower mitotic index scores noted at these dosages. The decrease in mitotic activity was accompanied by several chromosomal abnormalities. Most noticeable abnormalities noted with treatments were stickiness, despiralization, disorientation, bridges, vagrants, micronucleus etc. Stickiness is one of the major abnormality noted in the present study. Stickiness may be produced by the physical adhesion of chromosomal proteins (Patil and Bhat 1992) or due to disturbances in the nucleic acid metabolism of the cell or the dissolution of protein covering the DNA in chromosomes (Mercykutty and Stephen 1980). Appearance of Bridges at various stages may be due to general stickiness of chromosomes (Nahla and Soliman 1980 ) and it may also arise because of breakage and reunion of chromosomes. Occurrence of lagging chromosomes might be due to the hindrance of pro metaphase movement of chromosomes accompanied by adhesion of centromere to the nuclear membrane (Nagpal and Grover 1994). Diagonal orientation was due to a slight tilt in the spindle apparatus (Renjana et al., 2013). Star chromosomes was less frequently found even with increased concentrations, which is believed to be a fore step of complete disturbances of spindle (Anthony and Hussey 1999). C Metaphase was the evidence of the action of pesticides on mitotic spindle (Grover and Malhi 1988). A considerable proportion of nuclear lesions can be recorded most of which may be due to the dis integration of a portion of nuclear material by the action of pesticides (Omanakumari et al., 2006). Hyperchromasia is one of the most distinguished states of aberration which is an extremely condensed and deeply staining stage of nucleus caused by the influence of toxic chemicals or during incompatible conditions. Gernand et al., (2005) suggested this to be caused by the gradual heterochromatinization in response to stress. The occurrence of binucleate and tetra nucleate cells arise as a consequence of the inhibition of cell plate formation. Failure of cell plate formation in already binucleate cells may give rise to multinucleate condition. Micronuclei may be a result of acentric fragments or entire chromosomes not incorporated into the main nucleus during the cell cycle. Later fragments may come together and form one or more micronuclei which have get only limited chance for survival (Fenech and Crott 2002). The significant reduction in mitotic index and increased percentage of abnormality reported in the present study indicates that this pesticide has clear antimitotic activity and should be regarded as a strong cytotoxic agent. According to Fiskesjo (1985), a positive result in Allium test should be taken as an indication about the potential hazards and the occurrence of wide spectrum of abnormalities is an indication of the high mutagenic potential of the pesticide tested. 4. Conclusion The present study reveals the mitodepressive effects of Pesticide Attack on root meristems of Allium cepa L.. The treatments showed a wide array of cytological abnormalities like stickiness, disorientation, despiralization, vagrants, bridges, micronucleus, bi nucleate and tetranucleate nucleate cells, hyperchromasia. This clearly indicates that these chemical is a strong clastogen having severe effects on chromosomes and on their mitotic spindle. The outcome of the present study reveals that care and attention should be paid to estimate the toxic potential of the pesticides which are constantly used in 39
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