genotoxicty presented by subin joy koikkara
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Genotoxicity of Carbamate Pesticides by Comet Assay/(SGCE)
Internal Guide
Dr. E.G. WESELYHEAD & READER
Centre for Biotechnology,
MCAS, Rasipuram
Co-Guide
Mr.Subin joy
Research Co-Guide,
Dept of Analytical Toxicology
AIMS, Kerala.
M. ARUL,II-M,Sc, Biotechnology
External Guide
Dr. V.V. PillayHead, Dept of Analytical ToxicologyAIMS, Kerala.
Introduction
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 & ObjectiveAim
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
Carbaryl
Carbendazim
Human blood sample
Experimental Set-Up
• Cold slide tray
• Coupling jar
Electrophoresis unit
• Fluorescent microscope
• N.P. SINGH• Department of Bioengineering
University of WashingtonHealth Sciences Complex,
G-103 Box 357962Seattle, WA 98195 , U.S.A.narendra@u.washington.edu
The word comet means (originated from
Greek – kometes) -‘long haired star’.
This single cell gel electrophoresis technique
has been modified and extensively validated
over the years, and is now commonly referred
to as the ‘comet’ assay
Comet Assay:
Preparation of carbamate stock solutions
4 mg of three carbamates are dissolved in 1ml of 1%DMSOMethomyl: 1600 ppm/ml
Carbaryl: 2000 ppm/ml
Carbendazim:2000 ppm /ml Carbamates Stocksolution
(ml)1%DMSO (ml)
Totoal concentration (ppm)
Methomyl 0.5 0.5 800
Carbaryl 0.5 0.5 1000
Carbendazim 0.5 0.5 1000
Particulars Blank Positivecontrol
Negative control
M-1 M-2 M-3 M-4
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 (μl)
- - 100 - - - -
Methomyl (μl)
- - - 25 50 75 100
Blood induced with Methomyl
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 Carbaryl
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
Blood induced with Carbendazim
RESULTS
1%DMSO
Head Tail
5%H2o2
Distilled.Water
20 ppm
μM
40ppm
60 ppm
Effects of Methomyl
80ppm
25 ppm 50 ppm
100 ppm 75 ppm
Effects of carbaryl
Effects of carbendazim 50 ppm
100 ppm
25 ppm
75 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.
.
•Thank u . . . .
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