GENERAL OVERVIEW OF THE COMET ASSAY AND ITS APPLICATIONS Diana Anderson University of Bradford...

GENERAL OVERVIEW OF THE COMET ASSAY AND

ITS APPLICATIONS

Diana Anderson

University of Bradford

United Kingdom

http://internal.bradford.ac.uk/

COMET ASSAY IS ONE OF THE MOST IMPORTANT ASSAYS IN TOXICOLOGY TODAY

Standard method for measuring DNA strand breaks in single somatic and germ cells in vitro and in vivo.

Cells embedded in agarose on a microscope slide are lysed with detergent and high salt.

Electrophoresis at high pHs results in structures resembling Comets, observed by fluorescence microscopy.

Comet tail is formed by DNA fragments moving towards the anode.

ASSAY CAN BE USED FOR:

- Genotoxicity testing of novel compounds and ecotoxins.

- Human biomonitoring and molecular epidemiology.

- Basic research into DNA damage and repair.


GENOTOXICITY TESTING OF NOVEL COMPOUNDS AND ECOTOXINS

• This can be illustrated by work done in human lymphocytes and sperm with oestrogens.

• In CHO cells with halogenated acetic acids.

• Also by work in rodents with CP, EMS, EGME, BLM with bone marrow and testicular cells


Table 2. THE EFFECT IN THE COMET ASSAY OF VARIOUS OESTROGENS IN HUMAN SPERM (SP) AND LYMPHOCYTES (LY) AND THEIR RESPONSE TO TREATMENT WITH OESTROGENS IN COMBINATION WITH CATALASE (CAT),

SUPEROXIDE DISMUTASE (SOD) AND VITAMIN C (VIT C)

Effect/Result +

Catalase *

Superoxide Dismutase *

Vitamin C *

Compound/ DoseM Expt

SP

LY

DOSE

SP

LY

SP

LY

SP

LY

CAT + / + + / + 1 - / - - / - - / - - / - - / - - / - SOD + / + + / +

Equol / 250

VIT C + / + + / + 2 - / - 0 / - 0 / - 0 / - - / 0 - / -

CAT + / + + / + 1 0 / 0 - / - - / 0 0 / - 0 / - - / - SOD + / + + / +

Genistein/ 250

VIT C + / + + / + 2 - / - - / - - / - - / 0 0 / 0 0 / -

CAT + / + + / + 1 0 / - - / - - / - - / - 0 / 0 0 / 0 SOD + / + + / +

Daidzein/250

VIT C + / + + / + 2 - / - - / - - / - - / - 0 / 0 0 / 0

CAT + / + + / 0 1 - / - - / - 0 / 0 0 / 0 - / - - / 0 SOD + / + + / +

Diethylstilboestrol/ 100 VIT C + / + + / +

2 - / - 0 / 0 0 / 0 0 / 0 0 / - - / 0

CAT + / + + / + 1 - / - 0 / - 0 / - 0 / 0 - / - 0 / 0 SOD + / + + / +

-oestradiol/50

VIT C + / + + / + 2 - / - - / - - / - 0 / 0 - / - 0 / 0

CAT + / + + / + 1 0 / 0 - / - 0 / 0 0 / 0 0 / 0 0 / 0 SOD + / + + / +

Nonylphenyl/50

VIT C + / + + / + 2 0 / 0 0 / - 0 / 0 0 / 0 0 / 0 0 / 0

CAT + / + + / + 1 - / - - / - - / - 0 / 0 - / - 0 / 0 SOD + / + + / +

Hydrogen peroxide/ 80 VIT C + / + + / +

2 - / - - / - - / - 0 / 0 - / - 0 / 0

Key + = positive response with oestrogen (p<0.05) * = by comparison with oestrogen response - = reduced response with oestrogen (p<0.05) + = by comparison with negative control 0 = no significant response, 1= lower dose (100 units/ml CAT; 50 units/ml SOD; 0.5 mM Vit C). 2 = higher dose (500 units/ml CAT; 150 units/ml SOD; 1.00 mM Vit C).


HUMAN BIOMONITORING AND MOLECULAR EPIDEMIOLOGY

We have examined:

• NaNO2 in Diabetic patients because Type 2 diabetics, in an Epidemiology study, were linked to increased nitrate levels in drinking water

• Mothers and babies for differential sensitivity to the alkylating agent EMS

• Individuals exposed to food mutagens MeIQx and PhIP

• Diabetics exposed to Hydrogen Peroxide


Mean tail-moment values (± SE) of lymphocytes from mothers and babies (cordblood) in the Comet assay, with 1–40 mM of EMS (and negative control)

n 0 EMS 1mM EMS 10mM EMS 20 mM EMS 30 mM EMS 40 mM

EMS

Mothers 22 6.7 ± 0.89* 10.12 ±1.12 14.87 ± 1.35 19.40 ± 1.38 28.96 ±2.13 46.60± 5.40

Babies 22 4.90 ±0.90 8.76 ±1.07 12.72 ±1.50 15.99 ± 1.38 24.90 ± 2.58 39.46 ± 3.92


DNA damage in lymphocytes from subjects below and above 45 years of age treated with MeIQx


BASIC RESEARCH INTO DNA DAMAGE AND REPAIR

We investigated PBL from non-Hodgkins lymphoma patients.

• Those resistant to their standard chemotherapy regimen, CHOP, were found to be overexpressing mutant p53 protein (p53+ patient) and did not repair.

• Those who responded did not overexpress mutant p53 protein (p53- patient) and did repair.

• Data could be used to select resistant patients for another treatment regimen involving verapamil (chemosensitizer and p-glycoprotein antagonist) and doxorubicin and cells from all patients showed increased sensitivity to treatment.


Immunocytochemical staining for p53 protein: positive population show intense brown nuclear staining. (A) cytospin of Raji cells used as p53 positive control. (B) cytospin

of normal peripheral lymphocytes used as p53 negative control (C) cytospin of transformed AML in which more than 75% of cells were p53 positive. (D) cytospin of

CLL case showing both p53 positive and p53 negative cells.

A B

C D


Kinetics of repair with EMS

0

50

100

0 3 6 12 20Time (h)

Tai

l Mom

ents

(%)

Wild typep53+p53-


0

5

10

15

20

25

30

C C + V D D + V

Tai

l Mom

ents

c

p1

p2

p3

p4***

***

***

***

**

***

******

(***)(***)

***

(***)

(***)

(***)

***

0

5

10

15

20

25

30

35

40

45

50

Nc 10 30 50 Pc

Concentrations in µg/ /ml

OT

M &

% t

ail D

NA

healthy controls OTMhealthy controls % tail DNApatients OTMpatients % tail DNA

ns

ns

nsns

nsns

* **

**

**

***

***

***

******

***

(A)

0

5

10

15

20

25

30

35

40

45

50

Nc 10 30 50 Pc

Concentrations in µg/ /ml

OT

M &

% t

ail D

NA

healthy controls OTMhealthy controls % tail DNApatients OTMpatients % tail DNA

ns

ns

nsns

nsns

* **

**

**

***

***

***

******

***

(A)

Histogram showing the means of Olive tail moment and % tail DNA in lymphocytes of healthy controls, lung cancer, COPD and asthma patient groups in the Comet assay after treatment with different TiO2 concentrations (10, 30 and 50 µg/ml), as well as the negative control of untreated lymphocytes (Nc) and the positive control of 80 µM (2.72 µg/ml) H2O2 (Pc) for 30 minutes. Bars indicate standard errors. Not significant: ns, *p < 0.05, ** p < 0.01 and ***p < 0.001


0

5

10

15

20

25

30

35

40

45

50

Nc 10 30 50 Pc Nc 10 30 50 Pc Nc 10 30 50 Pc Nc 10 30 50 Pc

Concentrations in µg/ml

OTM

& %

tail

DNA

OTM % tail DNA

AsthmaHealthy controls Lung cancer COPD

(B)

ns

ns ns nsns

ns

nsns

nsns

**** **

* * * *

******

****** ***

***

*** ***

***

***

***

***

**

***

0

5

10

15

20

25

30

35

40

45

50



OTM

& %

tail

DNA

OTM % tail DNA

AsthmaHealthy controls Lung cancer COPD

(B)

ns

ns ns nsns

ns

nsns

nsns

**** **

* * * *

******

****** ***

***

*** ***

***

***

***

***

**

***

Histogram showing the means of Olive tail moment and % tail DNA in lymphocytes of healthy controls, lung cancer, COPD and asthma patient groups in the Comet assay after treatment with different TiO2 concentrations (10, 30 and 50 µg/ml), as well as the negative control of untreated lymphocytes (Nc) and the positive control of 80 µM (2.72 µg/ml) H2O2 (Pc) for 30 minutes. Bars indicate standard errors. Not significant: ns, *p < 0.05, ** p < 0.01 and ***p < 0.001

0

5

10

15

20

25

30

35

40

45

50



OT

M

& %

ta

il D

NA

OTM % tail DNA

Male controls Female controls Male patients Female patients

nsns

(C)

nsns

ns

nsns

ns

ns

ns

ns

ns

ns

* *

** **

** **

**

***

***

***

***

*** *** *** ***

***

***

***

ns

0

5

10

15

20

25

30

35

40

45

50



OT

M

& %

ta

il D

NA

OTM % tail DNA


nsns

(C)

nsns

ns

nsns

ns

ns

ns

ns

ns

ns

* *

** **

** **

**

***

***

***

***

*** *** *** ***

***

***

***

ns


nsns

(C)

nsns

ns

nsns

ns

ns

ns

ns

ns

ns

* *

** **

** **

**

***

***

***

***

*** *** *** ***

***

***

***

ns

Histogram showing the means of Olive tail moment and % tail DNA in lymphocytes of male and female healthy controls, and male and female patient groups in the Comet assay after treatment with different TiO2 concentrations (10, 30 and 50 µg/ml), as well as the negative control of untreated lymphocytes (Nc) and the positive control of 80 µM (2.72 µg/ml) H2O2 (Pc) for 30 minutes. Bars indicate standard errors. Not significant: ns, *p < 0.05, ** p < 0.01 and ***p < 0.001

0

5

10

15

20

25

30

35

40

45

50

Nc 10 30 50 Pc Nc 10 30 50 Pc Nc 10 30 50 Pc Nc 10 30 50 Pc Nc 10 30 50 Pc


OT

M &

% t

ail

DN

A

OTM % tail DNA

Patients >65

(D)

Controls < 50 Controls 50-65 Patients < 50 Patients 50-65

nsns ns ns

ns ns

ns

ns

ns

ns

ns ns

ns

ns

ns

*

*

* *

**

**

**

**

*** ***

***

*** ***

***

***

***

*** ***

*** ***

*** ***

***

***

***

0

5

10

15

20

25

30

35

40

45

50

Nc 10 30 50 Pc Nc 10 30 50 Pc Nc 10 30 50 Pc Nc 10 30 50 Pc Nc 10 30 50 Pc


OT

M &

% t

ail

DN

A

OTM % tail DNA

Patients >65

(D)

Controls < 50 Controls 50-65 Patients < 50 Patients 50-65

nsns ns ns

ns ns

ns

ns

ns

ns

ns ns

ns

ns

ns

*

*

* *

**

**

**

**

*** ***

***

*** ***

***

***

***

*** ***

*** ***

*** ***

***

***

***

Histogram showing the means of Olive tail moment and % tail DNA in lymphocytes of healthy controls, aged < 50 and 50 -65 patients aged <50, 50 -65 years and >65 years in the Comet assay after treatment with different TiO2 concentrations (10, 30 and 50 µg/ml), as well as the negative control of untreated lymphocytes (Nc) and the positive control of 80 µM (2.72 µg/ml) H2O2 (Pc) for 30 minutes. Bars indicate standard errors. Not significant: ns, *p < 0.05, ** p < 0.01 and ***p < 0.001

Histogram showing the means of Olive tail moment and % tail DNA in lymphocytes of non smoker healthy controls and non smoker and smoker patient groups in the Comet assay after treatment with different TiO2 concentrations (10, 30 and 50 µg/ml), as well as the negative control of untreated lymphocytes (Nc) and the positive control of 80 µM (2.72 µg/ml) H2O2 (Pc) for 30 minutes. Bars indicate standard errors. Not significant: ns, *p < 0.05, ** p < 0.01 and ***p < 0.001

0

5

10

15

20

25

30

35

40

45

50

Nc 10 30 50 Pc Nc 10 30 50 Pc Nc 10 30 50 Pc

Concentrations in µg /ml

OT

M &

% t

ail D

NA

OTM % tail DNA

Non-smoker controls Non-smoker patients smoker patients

(E)

***

***

******

*********

*

***

*

***

***

***

****

**

ns

*

ns

nsns

nsns ns

0

5

10

15

20

25

30

35

40

45

50

Nc 10 30 50 Pc Nc 10 30 50 Pc Nc 10 30 50 Pc

Concentrations in µg /ml

OT

M &

% t

ail D

NA

OTM % tail DNA


(E)

***

***

******

*********

*

***

*

***

***

***

****

**

ns

*

ns

nsns

nsns ns


(E)

***

***

******

*********

*

***

*

***

***

***

****

**

ns

*

ns

nsns

nsns ns

0

5

10

15

20

25

30

35

40

45

50



OT

M &

% t

ail D

NA

OTM % tail DNA

Caucasian controls Caucasian patients Asian patients Asian controls

(F)

ns

ns

ns

ns

ns

ns

ns

ns ns

ns

ns

ns *

**

**

**

** **

** **

**

***

*** ***

*** ***

*** ***

*** ***

***

***

Histogram showing the means of Olive tail moment and % tail DNA in lymphocytes of Caucasian controls and patients and Asian controls andpatient groups in the Comet assay after treatment with different TiO2 concentrations (10, 30 and 50 µg/ml), as well as the negative control of untreated lymphocytes (Nc) and the positive control of 80 µM (2.72 µg/ml) H2O2 (Pc) for 30 minutes. Bars indicate standard errors. Not significant: ns, *p < 0.05, ** p < 0.01 and ***p < 0.001

.51

1.5

22

.5M

ean

Oliv

e T

ail

Mom

ent (m

od

el e

stim

ate

s)

control 200 300 400100Depth of Lymphocytes in the Agar Gel (µm)

control precancer 95% CI

ANALYSIS of RESULTS (cont)

Also,to determine if differences could have a cancer diagnostic value via analysis of Receiver Operating Characteristic (ROC) curves in 208 people.

0.0

00.

25

0.5

00.

75

1.0

0S

ensi

tivity

0.00 0.25 0.50 0.75 1.001 - Specificity

Area under ROC curve = 0.8676

a. cancer plus pre-cancer v. control

a

0.0

00.

25

0.5

00.

75

1.0

0S

ensi

tivity

0.00 0.25 0.50 0.75 1.001 - Specificity


b. cancer v. pre-cancer plus control

b

0.0

00.

25

0.5

00.

75

1.0

0S

ensi

tivity

0.00 0.25 0.50 0.75 1.001 - Specificity


c. cancer v. control

ANALYSIS of RESULTS (cont.)

Sensitivity = TP X 100 Specificity = TN X 100 TP + FN FP + TN Where: TP = true positives, TN= true negatives,

FN = false negatives, FP = false positives.

The sensitivity and specificity of the assay was modelled using different threshold levels enabling optimum identification of only cancer patients and only healthy individuals using the above formula.

Use as stand alone assay or in conjunction with other assays.

0.0

00

.25

0.5

00

.75

1.0

0S

ensi

tivity

/Sp

eci

ficity

0.00 0.25 0.50 0.75 1.00Probability cutoff

Sensitivity Specificity

Regulatory Concerns

• The Comet assay is recommended for use in Regulatory guidelines by the UK in 2000m by the Committee on Mutagenicity of Chemicals in Food, Consumer Products ...

• Also in - Mutagenicity testing for Chemical Risk Assessment: update of the WHO/IPCS Harmonised Scheme (Eastmond et al., 2009,

Mutagenesis 24 341-349)

~It is used as an Indicator Test

Individual Guidelines to be found in:

Mutation Research 463 (2000) 111-172

Whole issue is available free of charge:

http://mutationresearch.com/mutat/29/43/25/23/article.pdf

• Hartmann et al, 2003, Mutagenesis,18, 45-51

• Tice et al, 2000, Environ Mol Mutagen, 35, 206-221

• Speit and Hartmann, 2005, Methods Mol Biol, 291, 85-95

• Burlinson et al, 2007, Mutat Res, 627, 31-35

• Smith et al, 2008, Mutagenesis, 23, 233-240

• Frotschi, 2015, Mutagenesis, 30 51-57 USEFUL REFERENCES to help meet regulatory requirements

GENERAL OVERVIEW OF THE COMET ASSAY AND ITS APPLICATIONS Diana Anderson University of Bradford...

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