Published on July 16, 2009
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
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.
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.
Methomyl Human blood sample Carbaryl Carbendazim
Experimental Set-Up •Cold slide tray •Coupling jar
Electrophoresis unit •Fluorescent microscope
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. firstname.lastname@example.org to as the ‘comet’ assay
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
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)
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
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
5%H2o2 Head Tail Distilled.Water 1%DMSO
Effects of Methomyl μM 20 ppm 40ppm 60 ppm 80ppm
Effects of carbaryl 25 ppm 50 ppm 100 ppm 75 ppm
Effects of carbendazim 25 ppm 50 ppm 75 ppm 100 ppm
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.
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