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The comet assay for DNA damage and repair Applications in genotoxicity testing and human biomonitoring

Andrew Collins Department of Nutrition, University of Oslo

Introduction – measuring DNA damage

Increasing throughput

Genotoxicity testing

DNA repair

Applications in human biomonitoring

The comet assay –

(single cell gel electrophoresis) – a simple and versatile method

for measuring DNA breaks. Widely used in genotoxicity testing,

human biomonitoring, ecogenotoxicology, and fundamental

research into mechanisms of DNA damage and repair.

Introduction

The comet assay is a quantitative method for measuring

DNA damage - primarily strand breaks. Few genotoxic

agents break DNA directly, but many lesions can be

converted to breaks, and DNA repair pathways involve

breaks as intermediates.

DNA damage is a precursor of cancer, but the link is a

weak one, as almost all damage is repaired

DNA is readily oxidised – most commonly by endogenous reactive oxygen (free radicals) released from mitochondria or as part of the inflammatory response.

8-oxoguanine – potentially mutagenic, easily measured; a popular biomarker.

8-oxoguanine

H2NC

N

N

C

N

N

OH

OHOH

2C

O

C

OH

For an index of antioxidant status, cells are treated with H2O2 and resulting strand breaks are measured.

DNA damage can arise from exposure of cells to environmental mutagens/carcinogens. It can also occur as a consequence of endogenous processes – notably oxidation.

Lysis: Triton X-100, 2.5 M NaCl

Nucleoids; supercoiled DNA Alkaline incubation:

0.3 M NaOH, 10 mM EDTA

Electrophoresis: 0.8 V/cm, 30 min

The comet assay (single cell gel electrophoresis)

Neutralisation, DAPI stain, fluorescence microscopy

% of DNA in tail depends on

DNA break frequency

Cells embedded in agarose on microscope slide

% D

NA in t

ail

Vis

ual sc

ore

(arb

. units)

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

DNA breaks per 109 daltons

0

15

30

45

60

75

0

100

200

300

400

% DNA in tail

Visual score (arbitrary units)

The comet assay –

(single cell gel electrophoresis) – a simple and versatile method

for measuring DNA breaks. Widely used in genotoxicity testing,

human biomonitoring, ecogenotoxicology, and fundamental

research into mechanisms of DNA damage and repair.

The assay is made more sensitive and specific by incorporation

of DNA repair enzymes that convert damaged bases to breaks

(endonuclease III for oxidised pyrimidines, formamidopyrimidine

DNA glycosylase [FPG] for oxidised purines, T4endoV for UV

damage, AlkA for alkylated purines)

Lysis: Triton X-100, 2.5 M NaCl

Nucleoids; supercoiled DNA

Alkaline incubation: 0.3 M NaOH, 10 mM EDTA

Electrophoresis: 0.8 V/cm, 30 min

The comet assay (modified – with lesion-specific enzymes)

Neutralisation, DAPI stain, fluorescence microscopy

The frequency of damaged bases is given by the increase in DNA

breaks in the presence of the specific endonuclease

Cells embedded in agarose on microscope slide

Digestion with lesion-specific endonuclease

This assay allows us to estimate the level of base damage in cells (e.g. lymphocytes)

0 32

Subj ects

0

40

80

120

160

200

DN

A b

reak

s (a

rbit

rary

uni

ts)

pl us FPG

no FPG

Lymphocytes from ~30 healthy subjects: measurement of strand breaks ± FPG-sensitive sites

Net FPG-sensitive sites are calculated from the difference (± enzyme)

0 32

Subj ects

0

40

80

120

160

DN

A b

reak

s (a

rbit

rary

uni

ts)

net FPG - sensi t i ve si t es

The frequency of damaged bases is given by the increase in DNA breaks in the presence of the specific endonuclease

Increasing throughput

A limitation of the standard comet assay is the number of

samples that can be analysed in a single experiment;

around 40 gels in a standard tank.

High throughput methods were developed in the recent ‘COMICS’ project:

Twelve mini-gels on a standard

glass slide…

…or a GelBond film

with up to 96 mini-gels.

For the 12-gel system, we developed a chamber device (now

commercially available) that allows individual treatment of gels

with reagents or enzymes:

The slide is clamped under a gasket

with holes matching the gel positions.

Genotoxicity testing

Enhancing sensitivity

FPG is widely used in human biomonitoring to measure oxidised

purines. However, the enzyme recognises other kinds of damage -

either directly, or via secondary oxidative damage.

It can greatly increase the sensitivity of detection of various lesions

in genotoxicity testing.

We carried out a trial with TK-6 cells treated with cytotoxic and

non-cytotoxic chemicals, and with known genotoxins (+ S9 if

necessary) at ~non-cytotoxic concentrations.

[Azqueta et al. (2013) Mutagenesis 28, 271-277]

Non-cytotoxic:

No strand breaks, and no FPG-sensitive sites

Broken line: proliferation index

Strand breaks (lysis only)

FPG-sites (net)

Cytotoxic:

DNA strand breaks, but at cytotoxic concentrations



FPG-sites (net)

Genotoxic:



FPG-sites (net)

Genotoxic:

The use of FPG in the comet assay should significantly reduce the risk of ’false negatives’ when testing novel chemicals



FPG-sites (net)

DNA repair

Oxidised bases in DNA

antioxidants Input (free radicals)

DNA repair

apoptosis †

mutation ?

DNA damage as measured in cells represents a dynamic steady

state – a balance between input of damage and its repair.

DNA repair

So DNA repair is an important factor in defining an individual’s

genetic stability status.

DNA damage is the first step in carcinogenesis; but almost all

damage that occurs is repaired.

DNA repair capacity is regarded as an intrinsic biomarker of

individual susceptibility; the higher the repair rate, the lower the

cancer risk.

It is likely that repair rates are influenced by genetic polymorphisms,

but also by environmental factors – exposure to DNA-damaging

agents (inducing appropriate DNA repair pathways), and perhaps

micronutrients….

DNA repair

DNA repair pathways that can be studied with the

comet assay:

• Strand break (SB) rejoining – insertion of missing nucleotide

and ligation.

• Base excision repair (BER) (oxidised or alkylated DNA) – a

specific glycosylase removes the base, leaving an AP site,

which is then cleaved. A DNA polymerase fills the gap; finally,

ligation.

• Nucleotide excision repair (NER) (bulky adducts, UV-induced

pyrimidine dimers) – a repair protein complex removes an

oligonucleotide containing the damage; gap-filling by DNA

polymerase is followed by ligation.

The simplest approach to measuring DNA repair is to treat cells with

a DNA-damaging agent, incubate the cells, and measure remaining

damage at intervals.

To measure SB rejoining,

cells were treated with

H2O2. For BER of oxidised

bases, cells were irradiated

with visible light in the

presence of photosensitiser

Ro 19-8022 to induce 8-

oxoGua. Remaining

damage was detected with

FPG.

Blue: HeLa cells

Red: Caco2 cells

SB rejoining

BER of 8-oxoGua

For BER and NER, we developed an alternative in vitro assay,

suitable for assaying repair capacity in samples of lymphocytes

from human trials.

A cell extract is incubated with nucleoids containing specific

DNA damage - 8-oxoGua for BER, and UV-induced pyrimidine

dimers for NER.

Strand breaks (incision events – incomplete repair sites)

accumulate and are measured with the comet assay.

Cells damaged with Ro 19-8022

(photosensitiser) + light, to

induce 8-oxoGua, provide

substrate nucleoids to measure

activity of 8-oxoGua DNA

glycosylase (OGG).

Lymphocytes Cell-free extract

Incubation with extract:

DNA substrate incised.

Alkaline treatment

Electrophoresis

DNA damage to substrate cells

In vitro assay for BER

% DNA in comet tail

indicates break frequency.

No extract Plus extract

Rate of accumulation of

breaks is a measure of

OGG repair activity.

DNA repair measured in humans

Lymphocyte samples taken from

~30 subjects on two occasions.

Assayed for BER…

Correlation between 2 samples;

i.e. subjects have characteristic

repair rates.

Wide range of values for different

subjects; plenty of scope for

investigating environmental and

genetic influences.

…and NER

A promising biomarker assay

[Gaivão et al. (2009) Cell Biol Toxicol 25, 45-52]

Applying the comet assay in human biomonitoring

An example – nutritional effects on DNA damage and repair

14 volunteers, in 3 groups. Crossover design, different doses, with washout periods between.

Kiwifruit – 3 weeks of supplementation

2

2

2

1

1

1

3

3

3

Samples: * * * * * *

I

III II

Kiwifruit intervention trial

[Collins et al. (2003) Carcinogenesis, 24, 511]

Ex vivo H2O2 challenge

H2O2-induced DNA breaks decreased

1 2 3

Kiwifruits per day

0

50

100

150

200

250

H2O2-

induced breaks (arbitrary units)

P=0.05

P=0.004

P<0.001

H2O

2-induce

d b

reaks

(arb

. units)

Oxidised pyrimidines – decreased

Oxidised purines – decreased

Endogenous damage

1 2 3

Kiwifruits per day

0

50

100

Endo III sites (arbitrary units)

P=0.04P<0.01 P<0.01

EndoII

I si

tes

(arb

itra

ry u

nits)

1 2 3

Kiwifruits per day

0

30

60

90

120

FPG sites (arbitrary units)

P=0.04

FPG

sites

(arb

itra

ry u

nits)

1 2 3

Kiwifruits per day

0

40

80

120

160

DNA repair rate (arbitrary units)

P=0.001 P=0.03P<0.001

Before

After

DN

A r

epair r

ate

(arb

. units)

mRN

A r

atio

DNA repair (BER)

Base excision repair (OGG) – enhanced…

…but no effect at level of gene expression (APE1, OGG1)

A recent application in ophthalmology

Lens epithelium from

11 cataract patients.

FPG-sites were

relatively high and

endoIII-sites

relatively low,

compared with

lymphocytes.

[Osnes-Ringen et al. (2013) Acta Ophthalm. 91, 652-656]

Leukocytes from frozen blood

Till now, human biomonitoring has depended on isolation of

peripheral blood mononuclear cells (‘lymphocytes’). But it

was recently shown that the comet assay could be

performed on whole blood after freezing at -20°C (in small

volumes). We improved this procedure by isolating

leukocytes from the frozen/thawed blood. After thorough

washing, the cells perform well in the comet assay, with or

without FPG.

This procedure has substantial advantages for biomonitoring:

quick freezing, no need for -70°C freezer, small volumes of

blood…. How reliable is it?


Ten subjects, whole blood frozen for ~1 year, then leukocytes isolated.

SBs, FPG-sites, and H2O2-induced breaks measured.

SBs

FPG-sites

H2O2-breaks

[Akor-Dewu, MB et al. (2014) Cell Biochem. Funct. 32, 299-302]


There was a good correlation between FPG-sites and H2O2-induced breaks – both reflecting individual redox status. This concordance suggests that what we are measuring has real biological significance.

Summary: the comet assay in 2015

• The assay of choice for specific DNA damage

• High throughput, miniaturisation

• Genotoxicity testing with extra sensitivity

• DNA repair - useful in vitro biomarker assays

• Ongoing applications, in nutrition, occupational and environmental exposure, ecogenotoxicology etc.

• Assays possible on leukocytes from frozen blood

• Novel clinical applications, e.g. in ophthalmology

Thanks to: Amaya Azqueta, Sergey Shaposhnikov, Isabel Gaivão, Torgrim Langleite, Yolanda Lorenzo, Maryam Dewu, Lena Bilyk, Naouale El-Yamani (Nutrition, University of Oslo) Bjørn Nicolaissen, Øyvind Ringen (Ophthalmology, University Hospital, Oslo) Maria Dusinska (NILU, Norway) Former colleagues in Aberdeen (Rowett Research Institute, Aberdeen, UK)

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