Internal Guide

. . . Dr E G WESELY HEAD & READER

Centre for,Biotechnology

, MCAS Rasipuram

Co-Guide

Mr.Subin joy

Senior Scientific Guide,

Dept of Analytical Toxicology

AIMS, Kerala.

M. ARUL,

II-M,Sc, Biotechnology

External Guide

Dr. V.V. PillayHead, Dept of Analytical ToxicologyAIMS, Kerala.

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 & 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 BioengineeringUniversity 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....Thank u....