Subin Joy Koikkara -Research Guide in Forensic Toxicology Kerala

1. Internal Guide
Dr. E.G. WESELY
HEAD & 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. Pillay
Head,
Dept of Analytical Toxicology
AIMS, Kerala.

2. 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.

3. Aim & Objective
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
Carbaryl
Carbendazim
Human blood sample

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

6. Electrophoresis
unit
•Fluorescent microscope

7. •N.P. SINGH
•Department of Bioengineering
University of Washington
Health Sciences Complex,
G-103 Box 357962
Seattle, 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:

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
(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

9. Particulars Blank Positive
control
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

10. 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

11. 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

13. RESULTS

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

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

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

17. Effects of carbendazim
50 ppm
100 ppm
25 ppm
75 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. •T hank u....