International Journal of Current Research and Review
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IJCRR - 6(11), June, 2014

Pages: 79-86

Date of Publication: 13-Jun-2014


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STUDIES ON THE TOXICOGENOMIC EFFECTS OF ORGANOPHOSPHATE PESTICIDE DIMETHOATE [O,O-DIMETHYL S-(N-METHYLCARBAMOYLMETHYL) PHOSPHORODITHIOATE] IN CYPRINUS CARPIO L.

Author: Md. Niamat Ali, Hilal Ahmad Ganaie, ZeenatNisar

Category: Healthcare

Abstract:Aim: Since 1940s, chemical pesticides of one form or another have become a dominant and essential form of pest control throughout much of the world. Because of the widespread use of pesticides for domestic and industrial applications, the evaluation of their toxic effects is of major concern to public health. The aim of the present study was to investigate the toxicogenomics effects of dimethoate (DM), an organophosphorus pesticide, to target the chromosomes of fish. Methodology: In the present investigation, two species of Cyprinuscarpio L. i.e. Cyprinuscarpiospecularis and Cyprinuscarpiocommunis were treated with three sub-lethal concentrations of DM (5 ppm, 10 ppm, & 15 ppm.). After treatment, the frequency of micronuclei was examined at the duration of 24, 48 and 72h. The micronucleus test (MNT) was used for assessing the genotoxicity of the fishes by the preparation of slides and scoring of micronuclei. Results: The pesticide treatment caused significant changes in the frequencies of micronuclei in erythrocytes of Cyprinuscarpiospecularis and Cyprinuscarpiocommunis. Micronuclei were non-refractive small nuclei lying near the main nucleus displaying the same pattern as the main nucleus. Nuclear abnormalities were like cells with binuclei, blebbed nuclei, lobed nuclei and notched nuclei. Conclusion: It was concluded that the pesticide dimethoate possess mutagenic potential at varying extent. A time and dose dependent increase in the frequency of micro-nucleated erythrocytes was found.

Keywords: Toxicogenomics, dimethoate, micronucleus, Cyprinuscarpio

Full Text:

INTRODUCTION
At present, the pesticide manual includes 3,100 main entries and list over 10,400 products (1-3). Many of them are suspected to have mutagenic and carcinogenic activities (4). Organophosphate pesticides are finding increasing use in recent years since they are biodegradable and therefore persist in the environment only for a short time. Because of their low persistence, repeated applications of these pesticides for the control of pests in agricultural fields and thereby large quantities find way into water bodies (5, 6). Their extensive application may affect fish population as they enter the water through irrigation or rain. The organophosphate compounds are esterase inhibitor of neurotoxicants (7) with acute cholinergic effect preceded by inhibition of acetyl cholinesterase (8). Being neurotoxicants, organophosphate compounds interfere with many of the vital physiological functions (9, 10) and consequently alter the levels of various body constituents (11) Though the application of pesticides is based on their toxicity to selective pests, it is not specific, resulting in very hazardous effect, particularly on aquatic organisms since pesticides eventually reach aquatic ecosystems in considerable amounts as agricultural run-off and outputs from municipal water treatment and manufacturing plants. Many contaminants present in aquatic environment not only endanger the survival and physiology of the organisms but also induce genetic alterations, which may lead to mutation and cancer (12-16). DM, most widely used insecticide, is a particular concern to those exposed occupationally during manufacture, formulation and use. DM exerts toxic effects on many tissues and organs including pancreas (17- 20). It is acutely toxic, has possible links to cancer and is suspected of causing birth defects (21). DM was found to be mutagenic in E. Coli (22). Although data on acute, subchronic and chronic toxicity of DM in laboratory animals are well documented, it’s potential to induce genotoxicity remains unclear. Therefore, the present study is designed to study the genotoxic effects of DM on the two species of fishes i.e. Cyprinus carpio specularis and Cyprinus carpio communis.

MATERIALS AND METHODS
Chemical Mutagen
The commercial grade of DM was obtained from Premier Sales Agency (Srinagar, India), manufactured from Isagro (Asia) Agrochemicals Pvt. Ltd. (Bathinda, India). CAS Reg. no: 60-51- 5, Chemical formula: C5H12NO3PS2 Experimental Animal The present study is carried out by using two species of Cyprinus carpio L. (family: Cyprinidae), i.e. Cyprinus carpio specularis and Cyprinus carpio communis. These fishes were identified by the presence of heavy and strongly serrate spines in the anterior portion of its dorsal and anal fins and by the presence of two rather long, fleshy barbles on each side of its upper jaw (23). The mouth is terminal in the adult and sub terminal in the young (24). Cyprinus carpio communis (scale carp) has regular concentric scales and Cyprinus carpio specularis (mirror carp) has large scales running along the side of the body in several rows with the rest of the body naked (25). Average age of fishes was below one year, weight was 30-40 g and length was 10-12 cm. After collection of fish specimens were acclimated for 45 days at 280C prior to trials. Specimens were kept in polypropylene troughs each with 8-10 individuals/50 L of water. Water was kept O2 saturated by aeration.

Treatment of DM and selection of the dose
In the present study fishes were divided into two groups i.e. the control and the experimental group. The experimental group for each fish species were divided into three subgroups based on the selected dose of DM. On the basis of the literature data (LC50 values for each insecticide), three sub-lethal concentrations: 5, 10 and 15 ppm of DM were selected (26). After treatment with each insecticide, the frequency of micronuclei in all experimental groups were examined at three durations of 24, 48 and 72h. Ten fish specimens were used for at each duration and at each concentration.

Micronucleus Test The micronucleus test was performed on peripheral blood according to the standard protocols with slight modifications (10, 13, 15, 27) Slide Preparation Fishes were killed with a slight blow on the head region. Chemically treated and control fishes were cut in the caudal region and smears of peripheral blood made on grease free clean slides. After fixation the slides were stained with Mayer’s haematoxylin, rinsed in Scott’s tap water substitute followed by another staining of eosin (13, 28) Scoring of Micronucleus For each concentration and duration ten fish specimen were used and from each fish ten slides were studied and 1200 cells were scored under 1000X magnification. Small non-refractive, circular or avoid chromatin bodies, displaying the same staining and focussing pattern as the main nuclei, were scored. Other nuclear abnormalities were also studied and classified as binuclei, blebbed nuclei and notched nuclei (16, 29) Photomicrography The slides were carefully studied and various morphological peculiarities of nuclear material were examined under light microscope for accurate scoring of micronuclei. Later photomicrography was conducted with the help of Trinocular microscope (Leica DMLS2) to keep record of all the details observed under microscope.

Statistical analysis
Statistical analysis of data to verify the significant difference in the incidence of micronucleus between treated and control groups at 5% level of significance was performed using non-parametric criteria, Mann-Whitney U test to analyse the frequency of micronuclei. To ensure statistical accuracy, only cells with one micronucleus were considered, while rarely occurred two micronuclei and other nuclear abnormalities were eliminated from the counts.

RESULTS
The pesticide treatment caused significant changes in the frequencies of micronucleus in erythrocytes of Cyprinus carpio specularis and Cyprinus carpio communis. In these two species of fishes mature erythrocytes are large structures with least differences in size and shape of nucleus. The micronuclei observed were clearly structured with well-defined boundary, which facilitated the identification of fragments in their cytoplasm. Micronuclei were non-refractive small nuclei lying near the main nucleus displaying the same staining pattern as the main nucleus. The number of micronuclei was found restricted to one, or occasionally two, the latter being a very rare phenomenon. Other nuclear abnormalities were also observed like cells with binuclei, blebbed nuclei, lobed nuclei and notched nuclei. But all these nuclear abnormalities were rarely seen and therefore were not scored. The peak frequency of micronucleated peripheral erythrocytes was observed after longer periods of exposure and higher dose treatment. Examples of normal erythrocytes of control fish, micronucleated erythrocytes and other nuclear abnormalities of treated fish are presented (Figure. 1, A-D) The fishes were exposed to three sub-lethal concentrations of 5 ppm, 10 ppm and 15 ppm. The percentage of single micronuclei in Cyprinus carpio specularis (0.03 ± 0.01 of control) increased to 0.32 ± 0.03 from low to high concentrations after 24h and continued to increase by 0.74 ± 0.17 and 1.37 ± 0.16 after 72 h exposure respectively is shown in Table.1. In Cyprinus carpio communis the percentage of single micronuclei increased from 0.03 ± 0.01 of control to 0.39 ± 0.06 (24h), 0.76 ± 0.14 (48h) and 2.23 ± 0.49 (72h).

DISCUSSION
The use of fish biomarkers as indices of the effects of pollution are of increasing importance and can permit early detection of aquatic environmental problems (30). The micronucleus test in fish has been applied for both laboratory treatments of in-vivo and in-situ exposure to environmental pollution. Induction of micronuclei by several well-known clastogenic/mutagenic agents such as cyclophosphamide, mictomycine-C, bleomycine, colchicines, ethyl methane sulphonate and vinblastin was assessed in freshwater and marine fish species (31-33). The efficacy of the micronucleus test as an indicator of cytogenetic damage has already been proven and the studies of micronucleus formation have been successfully used as bioassay to measure the impacts after fish treatment with surface water disinfectants (34), herbicides (12-14, 35, 36) insecticides (37, 38), benzo (x) pyrene and other polyaromatic hydrocarbon compounds (39). In the present study, positive genotoxic effects, measured as micronucleus frequency in erythrocytes from both fish species (Cyprinus carpio specularis and Cyprinus carpio communis) exposed to different insecticides were observed. The results of the present study revealed a significant induction of micronuclei in peripheral erythrocytes (P< 0.01 and P<0.05) of Cyprinus carpio specularis and Cyprinus carpio communis. The appearance of inter-specific differences observed in the present study could be attributed to the specificity of DNA repair, cell turnover time, physiological peculiarities, contaminant uptake or biotransformation in the fish species studied. In the present study a significant difference in the micronucleus incidence among treated and control groups was observed. The peak frequency of micronucleated erythrocytes was observed at 72 h after exposure (Figure.2). A time dependent increase in the incidence of micronuclei in the peripheral erythrocytes of Cyprinus carpio specularis and Cyprinus carpio communis was established and confirms other observations (31, 34, 40). Mutagenic effects due to DM exposure were also reported in mice. They were more prominent in male mice given a single high dose of DM than in male mice given one twelfth of the same dose daily for 30 days (41). Several other studies have reported toxic effects of DM on metabolism and enzyme system of various fish species (42- 44).

CONCLUSION
In the present study DM was found to induce micronuclei in fish erythrocytes. Therefore, the present study confirms the genotoxicity of dimethoate in aquatic organisms. Genotoxicity induced by DM could be attributed to the parent compound itself as well as to its oxygen analog omethoate, which was considerably more toxic than DM (45). These effects can also be attributed to active oxygen species generated by DM (17, 46, 47). Thus, further investigations are needed to elucidate the mechanism by which DM induced DNA damage.

ACKNOWLEDGEMENTS
Special thanks are due to the Director, Centre of Research for Development (CORD) and P. G. Department of Environmental Science, University of Kashmir, Srinagar, J & K, India, who besides encouragement provided us unrestricted laboratory facilities, during the entire experimental period. Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors/ editors/ publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.

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A Study by Purushottam S. G. et al. entitled "Development of Fenofibrate Solid Dispersions for the Plausible Aqueous Solubility Augmentation of this BCS Class-II Drug" is awarded Best article for Vol 13 issue 10
A Study by Kumar S. et al. entitled "A Study on Clinical Spectrum, Laboratory Profile, Complications and Outcome of Pediatric Scrub Typhus Patients Admitted to an Intensive Care Unit from a Tertiary Care Hospital from Eastern India" is awarded Best Article for Vol 13 issue 09
A Study by Mardhiah Kamaruddin et al. entitled "The Pattern of Creatinine Clearance in Gestational and Chronic Hypertension Women from the Third Trimester to 12 Weeks Postpartum" is awarded Best Article for Vol 13 issue 08
A Study by Sarmila G. B. et al. entitled "Study to Compare the Efficacy of Orally Administered Melatonin and Clonidine for Attenuation of Hemodynamic Response During Laryngoscopy and Endotracheal Intubation in Gastrointestinal Surgeries" is awarded Best Article for Vol 13 issue 07
A Study by M. Muthu Uma Maheswari et al. entitled "A Study on C-reactive Protein and Liver Function Tests in Laboratory RT-PCR Positive Covid-19 Patients in a Tertiary Care Centre – A Retrospective Study" is awarded Best Article of Vol 13 issue 06 Special issue Modern approaches for diagnosis of COVID-19 and current status of awareness
A Study by Gainneos PD et al. entitled "A Comparative Evaluation of the Levels of Salivary IgA in HIV Affected Children and the Children of the General Population within the Age Group of 9 – 12 Years – A Cross-Sectional Study" is awarded Best Article of Vol 13 issue 05 Special issue on Recent Advances in Dentistry for better Oral Health
A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" is awarded Best Article of Vol 13 issue 06
A Study by Chen YY and Ghazali SRB entitled "Lifetime Trauma, posttraumatic stress disorder Symptoms and Early Adolescence Risk Factors for Poor Physical Health Outcome Among Malaysian Adolescents" is awarded Best Article of Vol 13 issue 04 Special issue on Current Updates in Plant Biology to Medicine to Healthcare Awareness in Malaysia
A Study by Kumari PM et al. entitled "Study to Evaluate the Adverse Drug Reactions in a Tertiary Care Teaching Hospital in Tamilnadu - A Cross-Sectional Study" is awarded Best Article for Vol 13 issue 05
A Study by Anu et al. entitled "Effectiveness of Cytological Scoring Systems for Evaluation of Breast Lesion Cytology with its Histopathological Correlation" is awarded Best Article of Vol 13 issue 04
A Study by Sharipov R. Kh. et al. entitled "Interaction of Correction of Lipid Peroxidation Disorders with Oxibral" is awarded Best Article of Vol 13 issue 03
A Study by Tarek Elwakil et al. entitled "Led Light Photobiomodulation Effect on Wound Healing Combined with Phenytoin in Mice Model" is awarded Best Article of Vol 13 issue 02
A Study by Mohita Ray et al. entitled "Accuracy of Intra-Operative Frozen Section Consultation of Gastrointestinal Biopsy Samples in Correlation with the Final Histopathological Diagnosis" is awarded Best Article for Vol 13 issue 01
A Study by Badritdinova MN et al. entitled "Peculiarities of a Pain in Patients with Ischemic Heart Disease in the Presence of Individual Combines of the Metabolic Syndrome" is awarded Best Article for Vol 12 issue 24
A Study by Sindhu Priya E S et al. entitled "Neuroprotective activity of Pyrazolone Derivatives Against Paraquat-induced Oxidative Stress and Locomotor Impairment in Drosophila melanogaster" is awarded Best Article for Vol 12 issue 23
A Study by Habiba Suhail et al. entitled "Effect of Majoon Murmakki in Dysmenorrhoea (Usre Tams): A Standard Controlled Clinical Study" is awarded Best Article for Vol 12 issue 22
A Study by Ghaffar UB et al. entitled "Correlation between Height and Foot Length in Saudi Population in Majmaah, Saudi Arabia" is awarded Best Article for Vol 12 issue 21
A Study by Siti Sarah Binti Maidin entitled "Sleep Well: Mobile Application to Address Sleeping Problems" is awarded Best Article for Vol 12 issue 20
A Study by Avijit Singh"Comparison of Post Operative Clinical Outcomes Between “Made in India” TTK Chitra Mechanical Heart Valve Versus St Jude Mechanical Heart Valve in Valve Replacement Surgery" is awarded Best Article for Vol 12 issue 19
A Study by Sonali Banerjee and Mary Mathews N. entitled "Exploring Quality of Life and Perceived Experiences Among Couples Undergoing Fertility Treatment in Western India: A Mixed Methodology" is awarded Best Article for Vol 12 issue 18
A Study by Jabbar Desai et al. entitled "Prevalence of Obstructive Airway Disease in Patients with Ischemic Heart Disease and Hypertension" is awarded Best Article for Vol 12 issue 17
A Study by Juna Byun et al. entitled "Study on Difference in Coronavirus-19 Related Anxiety between Face-to-face and Non-face-to-face Classes among University Students in South Korea" is awarded Best Article for Vol 12 issue 16
A Study by Sudha Ramachandra & Vinay Chavan entitled "Enhanced-Hybrid-Age Layered Population Structure (E-Hybrid-ALPS): A Genetic Algorithm with Adaptive Crossover for Molecular Docking Studies of Drug Discovery Process" is awarded Best article for Vol 12 issue 15
A Study by Varsha M. Shindhe et al. entitled "A Study on Effect of Smokeless Tobacco on Pulmonary Function Tests in Class IV Workers of USM-KLE (Universiti Sains Malaysia-Karnataka Lingayat Education Society) International Medical Programme, Belagavi" is awarded Best article of Vol 12 issue 14, July 2020
A study by Amruta Choudhary et al. entitled "Family Planning Knowledge, Attitude and Practice Among Women of Reproductive Age from Rural Area of Central India" is awarded Best Article for special issue "Modern Therapeutics Applications"
A study by Raunak Das entitled "Study of Cardiovascular Dysfunctions in Interstitial Lung Diseas epatients by Correlating the Levels of Serum NT PRO BNP and Microalbuminuria (Biomarkers of Cardiovascular Dysfunction) with Echocardiographic, Bronchoscopic and HighResolution Computed Tomography Findings of These ILD Patients" is awarded Best Article of Vol 12 issue 13 
A Study by Kannamani Ramasamy et al. entitled "COVID-19 Situation at Chennai City – Forecasting for the Better Pandemic Management" is awarded best article for  Vol 12 issue 12
A Study by Muhammet Lutfi SELCUK and Fatma entitled "Distinction of Gray and White Matter for Some Histological Staining Methods in New Zealand Rabbit's Brain" is awarded best article for  Vol 12 issue 11
A Study by Anamul Haq et al. entitled "Etiology of Abnormal Uterine Bleeding in Adolescents – Emphasis Upon Polycystic Ovarian Syndrome" is awarded best article for  Vol 12 issue 10
A Study by entitled "Estimation of Reference Interval of Serum Progesterone During Three Trimesters of Normal Pregnancy in a Tertiary Care Hospital of Kolkata" is awarded best article for  Vol 12 issue 09
A Study by Ilona Gracie De Souza & Pavan Kumar G. entitled "Effect of Releasing Myofascial Chain in Patients with Patellofemoral Pain Syndrome - A Randomized Clinical Trial" is awarded best article for  Vol 12 issue 08
A Study by Virendra Atam et. al. entitled "Clinical Profile and Short - Term Mortality Predictors in Acute Stroke with Emphasis on Stress Hyperglycemia and THRIVE Score : An Observational Study" is awarded best article for  Vol 12 issue 07
A Study by K. Krupashree et. al. entitled "Protective Effects of Picrorhizakurroa Against Fumonisin B1 Induced Hepatotoxicity in Mice" is awarded best article for issue Vol 10 issue 20
A study by Mithun K.P. et al "Larvicidal Activity of Crude Solanum Nigrum Leaf and Berries Extract Against Dengue Vector-Aedesaegypti" is awarded Best Article for Vol 10 issue 14 of IJCRR
A study by Asha Menon "Women in Child Care and Early Education: Truly Nontraditional Work" is awarded Best Article for Vol 10 issue 13
A study by Deep J. M. "Prevalence of Molar-Incisor Hypomineralization in 7-13 Years Old Children of Biratnagar, Nepal: A Cross Sectional Study" is awarded Best Article for Vol 10 issue 11 of IJCRR
A review by Chitra et al to analyse relation between Obesity and Type 2 diabetes is awarded 'Best Article' for Vol 10 issue 10 by IJCRR. 
A study by Karanpreet et al "Pregnancy Induced Hypertension: A Study on Its Multisystem Involvement" is given Best Paper Award for Vol 10 issue 09

List of Awardees

A Study by Ese Anibor et al. "Evaluation of Temporomandibular Joint Disorders Among Delta State University Students in Abraka, Nigeria" from Vol 13 issue 16 received Emerging Researcher Award


A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" from Vol 13 issue 06 received Emerging Researcher Award


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International Journal of Current Research and Review (IJCRR) provides platform for researchers to publish and discuss their original research and review work. IJCRR can not be held responsible for views, opinions and written statements of researchers published in this journal

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