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

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. 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. 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. Methomyl
Human blood sample Carbaryl
Carbendazim

5. Experimental Set-Up
•Cold slide tray
•Coupling jar

6. Electrophoresis
unit
•Fluorescent microscope

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. 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. 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. 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. 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

13. RESULTS

14. 5%H2o2
Head
Tail
Distilled.Water
1%DMSO

15. Effects of Methomyl μM
20 ppm
40ppm
60 ppm 80ppm

16. Effects of carbaryl
25 ppm 50 ppm
100 ppm
75 ppm

17. Effects of carbendazim
25 ppm
50 ppm
75 ppm 100 ppm

18. 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.

19. •Thank u....