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Geno Toxicity Presented by Subin Joy Koikkara Senior Reserch Guide, A.I.M.S Reserch Centre

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Geno Toxicity of carbamate pesticides-Confirmation with Comet Assay-Presented by Mr. Subin Joy Koikkara , Senior Reserch Guide, Dept of Analytical Toxicology A.I.M.S Reserch Centre

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Geno Toxicity Presented by Subin Joy Koikkara Senior Reserch Guide, A.I.M.S Reserch Centre

  1. 1. External Guide Dr. V.V. Pillay Head, Dept of Analytical Toxicology AIMS, Kerala. Co-Guide Internal Guide Mr.Subin joy Dr. E. G. WESELY HEAD & READER Senior Scientific Guide, Centre for Dept of Analytical Toxicology Biotechnology, AIMS, Kerala. MCAS, Rasipuram M. ARUL, II-M,Sc, Biotechnology
  2. 2. I ntroduction  Modern chemicals, leads to increased pollution of ecosystems.  Insecticides – Organochlorine, Organophosphate, Carbamates.  Carbamates – derivatives of carbamic acid • Mode of action – inhibit cholinesterase enzyme AChE overstimulation of neuromuscular junctions leads to spasms • Symptoms – Headache, Dizziness, weakness , excessive salivation , vomiting. • Genetic toxicology – effects of chemicals on the hereditary material . Comet assay – versatile, novel ,rapid but highly sensitive and well validated tool for detecting DNA damage in single cells.
  3. 3. Aim& Obj ective Aim Detection of DNA damage by Alkaline comet assay in peripheral leukocytes. Objective  To induce the blood with different carbamate pesticides.  To check whether carbamate pesticide will cause genotoxicity in human peripheral leukocytes  Detection of DNA damage by Fluorescent microscope.
  4. 4. Methomyl Human blood sample Carbaryl Carbendazim
  5. 5. Experimental Set-Up •Cold slide tray •Coupling jar
  6. 6. Electrophoresis unit •Fluorescent microscope
  7. 7. Comet Assay: The word comet means (originated from Greek – kometes) -‘long haired star’. •N.P. SINGH This single cell gel electrophoresis technique •Department of Bioengineering University of Washington has been modified and extensively validated Health Sciences Complex, G-103 Box 357962 over the years, and is now commonly referred Seattle, WA 98195 , U.S.A. narendra@u.washington.edu to as the ‘comet’ assay
  8. 8. Preparation of carbamate stock solutions 4 mg of three carbamates are dissolved in 1ml of 1%DMSO Methomyl: 1600 ppm/ml Carbaryl: 2000 ppm/ml Carbendazim:2000 ppm /ml Carbamates Stocksolution 1%DMSO Totoal (ml) (ml) concentration (ppm) Methomyl 0.5 0.5 800 Carbaryl 0.5 0.5 1000 Carbendazim 0.5 0.5 1000
  9. 9. Blood induced with Methomyl Particulars Blank Positive Negative M-1 M-2 M-3 M-4 control control Blood (μl) 20 20 20 20 20 20 20 HBSS (ml) 1 1 1 1 1 1 1 D .H2O (μl) - - - - - - 100 5%H2O2 - - - - - (μl) - 10 1% DMSO - - 100 - - - (μl) - Methomyl - - - 25 50 75 100 (μl)
  10. 10. Blood induced with Carbaryl Particulars Cyl-1 Cyl-2 Cyl-3 Cyl-4 Blood (μl) 20 20 20 20 HBSS (ml) 1 1 1 1 D.H2o (μl) - - - - 5%H2o2 - - - - (μl) 1% DMSO - - - - (μl) Carbaryl (μl) 25 50 75 100
  11. 11. Blood induced with Carbendazim Particulars Cbn-1 Cbn-2 Cbn-3 Cbn-4 Blood (μl) 20 20 20 20 HBSS (ml) 1 1 1 1 D.H2o (μl) - - - - 5%H2o2 - - - - (μl) 1% DMSO - - - - (μl) Carbendazim (μl) 25 50 75 100
  12. 12. RESULTS
  13. 13. 5%H2o2 Head Tail Distilled.Water 1%DMSO
  14. 14. Effects of Methomyl μM 20 ppm 40ppm 60 ppm 80ppm
  15. 15. Effects of carbaryl 25 ppm 50 ppm 100 ppm 75 ppm
  16. 16. Effects of carbendazim 25 ppm 50 ppm 75 ppm 100 ppm
  17. 17. CONCLUSION suggest that pesticide in long term exposure could acts as clastogens on the DNA of somatic cells. (Methomyl & carbaryl) and one fungicides (Carbendazim) showed some genotoxic effects at very low concentration in leukocytes . So these carbamate pesticides are consider as non-target specificity.
  18. 18. •Thank u....

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