SECTION III:

APPLICATIONS OF COMET ASSAY

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CHAPTER 8

Clinical Applications of theComet Assay

S. M. PIPERAKIS*, K. KONTOGIANNI,G. KARANASTASI AND M. M. PIPERAKIS

Biology Unit, Department of Pre-School Education, Faculty of HumanSciences, University of Thessaly, Volos, Greece

8.1 Introduction

The Single Cell Gel Electrophoresis (SCGE) or Comet assay is a very sensitivemethod for measuring DNA-strand breaks in individual cells. It is widely usedamong others in environmental toxicology, radiation toxicology and cancerresearch to assess DNA damage and repair. This technique was developed in1984 when Ostling and Johanson1 proposed a method for detecting genotoxicdamage in single cells.In recent years the Comet assay has become a new tool in the area of

assessing genetic damage in vitro and in vivo in a variety of cells.Although most of the reports on the Comet assay measure the genotoxic

effects induced in human, in animal and in plant cells in vitro, the number ofhuman studies in vivo has also increased. In particular, reports on the appli-cation of Comet assay in monitoring DNA damage from disease conditions ortreatment with genotoxic drugs, environmental pollution, occupational expo-sure and dietary studies have increased substantially.In the present review we focus on studies investigating the clinical applica-

tions of the Comet assay. In particular, we attempted to analyse the uses of theComet assay in clinical medicine.

Issues in Toxicology No 5

The Comet Assay in Toxicology

Edited by Alok Dhawan and Diana Andersonr Royal Society of Chemistry 2009

Published by the Royal Society of Chemistry, www.rsc.org

*Corresponding author

173

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8.2 The Comet Assay Methodology

In 1976 Cook et al.2 published a paper investigating the nuclear structure basedon the lysis of cells with nonionic detergent and high-molarity sodium chloride.The earliest attempt to quantify DNA-strand breaks directly was made byRydberg and Johanson3 in 1978 with cells embedded in agarose on slides andlysed under mild alkaline conditions. Staining of the nucleoid with acridineorange showed a higher ratio of red fluorescence, indicating single-strandedDNA, to green, indicating double-stranded DNA, in cells with DNA damage.In 1984 Ostling and Johanson1 based on the approach described above

developed the Comet assay, also called Single Cell Gel Electrophoresis (SCGE).This was an assay in which the lysis and electrophoresis were performed

under neutral conditions. The staining of the DNA was done with acridineorange. The image obtained looked like a ‘‘comet’’ with a distinct head,comprising of intact DNA and a tail, consisting of damaged DNA. As aconsequence the name ‘‘Comet assay’’ was given. The amount of DNA liber-ated from the head of the comet depends on the dose of mutagen used.However, in this procedure, only double-strand breaks could be analysed.This assay was later modified by two groups, Singh et al.4 in 1988 and Olive

et al.5 in 1990.The first group performed electrophoresis under highly alkaline conditions

(pH413). This enables the DNA supercoils to relax and unwind and makespossible the detection of alkali-labile sites and single-strand breaks in DNAduring electrophoresis. This method measures low levels of strand breaks withhigh sensitivity.The second group conducted electrophoresis under neutral or mild alkaline

conditions to detect single-strand breaks. This method was optimised to detecta subpopulation of cells with varying sensitivity to drugs or radiation.The version of the Comet assay developed by Singh et al.4 was found to be up

to two orders of magnitude more sensitive.The simplest types of DNA damage detected by the Comet assay are double-

strand breaks (DSBs). DSBs result in DNA fragments and can be detected bymerely subjecting them to electrophoretic mobility at neutral pH. Single-strandbreaks (SSBs) do not produce DNA fragments unless the two strands of theDNA are separated/denatured. This is accomplished by unwinding the DNA atpH12.1. It is also possible that single-strand breaks can relax the DNA andhence can also be detected with the Comet assay at neutral pH. Other types ofDNA damage broadly termed alkali-labile sites (ALS) are expressed when theDNA is treated with alkali at pH greater than pH 13. Breaks can also beintroduced at the sites of DNA base modifications by treating the DNA withlesion-specific glycosylases/endonucleases and the fragments thus produced canalso be detected by the Comet assay. By controlling the conditions that producenicks at the sites of specific DNA lesions the Comet assay can be used to detectvarious classes of DNA damage. While breaks increase DNA migration, DNAbinding and crosslinks can retard DNA migration and can also be detected bythe Comet assay.

174 Chapter 8

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Therefore, increased migration in the Comet assay can be attributed tostrand breaks, alkali-labile sites and incomplete excision repair sites, whiledecreased DNA migration could be attributed to crosslinks, DNA–DNA orDNA–protein interactions.

8.3 Clinical Studies

The Comet assay has been applied in a large number of clinical studies toinvestigate mainly the consequence of therapeutical exposure to certain chemi-cals or to study certain pathological conditions at the cellular level (Table 8.1).Elevated levels of DNA damage were found in irradiated cells isolated from

ataxia telangiectasia patients. In addition, the DNA-repair process was foundto be almost three times slower than the controls.6 In another study with fourxeroderma pigmentosum donors Green et al.7 found that few strand breaksappear after UV-irradiation if compared to controls.Burger et al.8 in a study of six Nijmegen breakage syndrome families using

peripheral blood mononuclear cells found that two out of the six families weremore sensitive to X-irradiation than the controls. DNA repair was alsoreported to take longer in four out of the six families, while cells from fivefamilies with the syndrome had significantly increased residual DNA damagefollowing repair. In an assessment of genome instability between 30 Down’sSyndrome and 14 Fanconi anaemia individuals9 increased DNA damage wasobserved.In a study of the level of primary and oxidative DNA damage in a group of

mild cognitive impairment (MCI) and another of Alzheimer’s disease (AD) asignificantly higher level of primary DNA damage in leukocytes of AD and alsoMCI patients if compared to controls, was found. Moreover, the amount ofoxidised DNA bases (both purines and pyrimidines) was significantly higher inthese two groups of patients.10

Malnourished infected children treated with antibiotics11 revealed that theobserved DNA damage in lymphocytes was associated with malnourishmentand antibiotic therapy. Elevated DNA damage was found in persons subjectedto abortion12 and malnutrition and parasite infection.13

The daily use of substances for personal hygiene, i.e. chlorhexidine diglu-conate, a mouth rinse, used as an antiplaque agent, caused a significant increasein DNA damage in buccal and peripheral blood cells.14 Higher level of DNAdamage was also reported in leprosy patients undergoing treatment,15 chronicrenal failure patients on haemodialysis16 and patients who underwent anaes-thesia.17 Sardas et al.18 reported the induction of DNA damage in lymphocytesof operation room personnel occupationally exposed to the anaesthetic agentisofurane.Biri et al.19 reported that women who used oral contraceptives had higher

levels of DNA-strand breakage than the control group.Investigations with lymphocytes of head and neck squamous cell carcinoma

patients20 and urothelial cells of urinary bladder cancer patients21 revealed that

175Clinical Applications of the Comet Assay

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Table

8.1

ClinicalapplicationoftheSingle

CellGel

Electrophoresisassay.

Studysubject

Alkaliunwindingand

electrophoresis

Parameter

ofDNA

damage/indicator

cells

Clinicalcase

Genotoxic

factor

Resultsandremarks

Ref.

i)N¼10healthydonors

ii)N¼10patients

with

breast

cancershowinga

norm

alreactionto

radiotherapy

iii)

N¼20Ataxia

Telangiectasiacarriers

and

iv)N¼4Ataxia

Telangiectasia

homozygotes

pH¼13.5;20min;25V

(0.83Vcm

�1)

Meancomet

length

(mm)

Ataxia

telangiectasia

patients

Radiosensitivity

IncreasedDNA

damage(about3

times

high)in

patients.

6

N¼4healthycontrols

N¼4xeroderma

pigmentosum

patients

Alkaline,

20V

(3000mA),(1V/cm),

24min

Meancomet

length

(mm)

Xeroderma

pigmentosum

UVC

sensitivity

Few

strandbreaks

appearafter

UV-irradiationif

comparedto

controls.

7

i)N¼10controls

ii)N¼13Nijmegen

breakagesyndrome

(NBS)patients

iii)

N¼10breast

cancer

patients

withnorm

al

sensitivityto

radiotherapy

pH¼13.5,22V

(0.83V/cm),20min

Tailmoment

NBSpatients

and

breast

cancer

patients

Radiosensitivity

DNA

damagewas

higher

inpatients.

8

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N¼14FanconyAnem

iapatients;8M;6F;age

range¼

3–16years,

N¼30DownSyndrome

patients;20M;10F;age

range¼

0–10years

and

N¼30controls;18M;12F

agerange¼

0–17years

pH413,20min,25V,

300A

Visualgradingof

comets(0,without

detectable

tail;4,

highestdamage/

lymphocytes)

Downsyndromeand

Fanconyanem

iapatients

Sensitivityto

DNA

damage

DNA

damagewas

significantlyhigher

incellsforDS

patients

thatin

controls.TheFA

grouppresented

higher

damagewhen

comparedto

controls.

9

i)N¼20patients

affected

byAlzheimer’sDisease

(AD),14F;6M

age

range¼

53–82years

ii)N¼15MildCognitive

Impariment(M

CI),6F;

9M

agerange¼

55–76

years

and

iii)

N¼15controls,9F;6M.

pH413,20min,25V,

300mA.

Taillength

(percentageDNA

damage)

Mildcognitive

impairmentand

Alzheimer’sDisease

Oxidativestress

Resultsgivean

indicationthat

oxidativestress,at

least

attheDNA

level,isanearly

eventin

the

pathogenesisofAD.

10

177Clinical Applications of the Comet Assay

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Table

8.1

(continued

).

Studysubject

Alkaliunwindingand

electrophoresis

Parameter

ofDNA

damage/indicator

cells

Clinicalcase

Genotoxic

factor

Resultsandremarks

Ref.

N¼12,5F,7M;

6malnourished

severely

infected

children,age

range¼

6–29months;

6well-nourished

severely

infected

children,age

range¼

7–36months

pH

notgiven,20min;

E¼25V,300mA,20min

TL/lymphocytes

Malnourished

children

Treatm

entwith

antibiotics/

malnutrition.

Sulfatrim

etroprim,

salbutamol,ambroxol,

amikacin,cefotaxim

e,dicloxacillin,

metronidazole,

penicillin,ampicillin,

cefalexin;malnutrition

Increase

inDNA

damagein

malnourished

childrencompared

towell-nourished

children;

significantly

increasedcomet

tail

length

inboth

well-

nourished

and

malnourished

childrenafter

treatm

entwith

antibiotics;theeff

ect

inmalnourished

childrenwastw

iceas

much

asthatin

well-

nourished

children.

Inboth

malnourished

and

well-nourished

children,therewasa

cellpopulation

resistantto

drug-

inducedDNA

damage;

however,

theproportionof

resistantcellswas

higher

inthelatter

group.

11

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N¼31couples;

18couplespatients,

13couplescontrols;age¼

ns

pH¼13,20min:E¼25V

and300mA,20min

VS/L

Coupleswitha

history

ofmore

than

fatalloss

Uknown

Greatincrease

inDNA

damagein

patientscomparedto

controls;frequency

ofcellswithlimited

andextensive

migrationvaried

within

thesame

group.Great

increase

indamaged

cellsin

smokersof

both

controland

patientgroups.

12

i)N¼10well-nourished

children:without

infectionandtreatm

ent

(WN);3F;7M.

ii)N¼4Fmildly

infected

children:wellnourished,

withouttreatm

ent(M

I).

iii)

N¼

8,6F;2M

severely

infected

children:well

nourished,without

treatm

ent(SEI).

iv)N¼5,2F;3M

severely

infected

children:well

nourished,with

treatm

ent(SEI-T).

v)N¼4,3F;1M

malnourished

children

withinfection,without

treatm

ent(M

N).

vi)

N¼5,2F;3M

malnourished

children

withinfection,with

treatm

ent(M

N-T).

Alkalne,

25V,300mA,

20min

Meantaillength

Bronhopneumonia,

pharyngitis,

rhonopharyngitis,

sepsis,tuberculosis

meningea,

gastroenteritis

typhoid,

Uknown

Severeinfectionis

associatedwitha

significantincrease

inDNA

damage.

13

179Clinical Applications of the Comet Assay

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Table

8.1

(continued

).

Studysubject

Alkaliunwindingand

electrophoresis

Parameter

ofDNA

damage/indicator

cells

Clinicalcase

Genotoxic

factor

Resultsandremarks

Ref.

N¼13,9F,4M;age

range¼

21–29years

pH¼13,40min;

E¼19V,300mA,40min

Visualgradingof

DNA

damage/

lymphocytes

Buccalcellsand

peripheralblood

lymphocytesof

antiplaqueusers

Use

ofantiplaqueagent

(chlorhexidine

digluconate)

Increase

inthe

number

ofdamaged

buccalcellsand

peripheralblood

lymphocytesof

antiplaqueusers

(0.12%

CHX

solutionfor18days)

ascomparedto

controls;meangrade

ofdamagein

buccal

cellswassignificantly

higher

thanthatin

lymphocytes.No

inform

ationonthe

effectofconfounding

factors

wasgiven

although

questionnaires

were

filled

out.

14

N¼100,26F,74M;50

leprosy

patients

undergoing

therapy,averageage¼

35

years;

50healthycontrols,average

age¼

31years

pH

413,30min;

E¼25V

(0.8

V/cm),300

mA,30min

Tailed

cell

percentage;

length/

width

ratiosofDNA

mass/lymphocytes

Leprosy/m

ultidrug

treatm

entagainst

leprosy

Leprosy

status/thedrugs

dapsone,

rifampicin,

clofazimine,

ofloxacin

IncreasedDNA

damagein

treated

leprosy

patients

as

comparedto

controls;also,

disease

factoralone

significantly

influencedDNA

damage.

15

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N¼36uremic

patients,

12F;24M,agerange¼

21–75years,undergoing

maintenance

hem

odialysis

andN¼36controls,12F;

24M,agerange¼

20–75

years

pH¼13,24V,300mA,

20min

Visualgradingof

damage

Peripheralblood

lymphocytesfrom

36

dialysispatients

before

andafter

Vitamin

E

Antioxidant

supplementationof

Vitamin

E

TheDNA

breakage

observed

inthe

lymphocytesof

patients

before

Vitamin

Esupplementationwas

significantlyhigher

thanin

thecontrols

butaclearprotective

effectofVitamin

Esupplementationwas

observed

after

14

weeksoftherapy.

16

N¼36,7M,29F;24

exposed,agerange¼

20–66

years;12unexposed,mean

age43years

pH¼13,40min;E¼19V

(1.6V/cm),300mA,

40min

Visualgradingof

damage(notail,

short

tail,longtail)

inlymphocytes

Medicaluse

of

anaestheticsin

patients

Sevoflurane,

isoflurane

Increase

ofDNA

damageat1and

2hafter

anaesthesia

as

comparedto

controls;DNA

repairwas

observed

3days

after

anaesthesia

andwascompleted

onthe5th

day.

Elevatedcomet

responsesafter

120

min

ofexposure

andbeganto

decrease

onthe

firstdayafter

operation;mean

comet

responses

observed

onthe

thirddayandfifth

dayafter

operation

werenotstatistically

differentcompared

tocontrolgroup

andbefore

operation.

17

181Clinical Applications of the Comet Assay

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Table

8.1

(continued

).

Studysubject

Alkaliunwindingand

electrophoresis

Parameter

ofDNA

damage/indicator

cells

Clinicalcase

Genotoxic

factor

Resultsandremarks

Ref.

N¼24;4M,20F;12

patients

and12controls;

agerange¼

22–66years

pH¼13,40min;E¼19V

and300mA,20min

VS/L

Patients

anesthesized

withisofluran

Isoflurane(exposure

for

60and120min)

Increase

inthe

proportionof

moderately

and

severelydamaged

cellsonthefirstday

after

beingexposed

totheanaesthetic

agent.Damage

decreased3days

after

exposure

and

wasalm

ost

identical

tocontrolvalues

5dayslater.

18

i)N¼36F;18exposedto

oralcontraceptives,mean

age24years;18controls.

ii)17women

inthelast

trim

esterofpregnancy,

meanage28years,17

controls

pH¼13,20min;

E¼1.6V/cm,300mA,

20min

Visualgradingof

damage(notail,

short

tail,longtail)/

lymphocytes

Use

oforal

contraceptives/

pregnancy

Pregnancy

horm

ones

(estroneandestradiol);

oralcontraceptives

(ethinylestradiol)

Increasedscoresof

comet

parametersin

oralcontraceptive

users

(150g

desogestreland20or

30gethinylestradiol

for24months);s.

higher

comet

scores

wereobserved

for

pregnantwomen

comparedto

controls.No

statisticallys.

differencesbetween

oralcontraceptive

users

andpregnant

women

inregard

tothelevel

ofDNA

damage;

smoking

did

notaffectthe

level

ofDNA

damagein

oral

contraceptiveusers.

19

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N¼82,51M,31F,44

healthycontrolsage

range¼

44–78years

and38

patients

withsquamouscell

carcinomaoftheheadand

neck,agerange¼

13–78

years

pH¼13,20min;E¼1V/

cm,20min

Visualgradingof

comets(0,without

detectable

tail;4,

highestdamage/

lymphocytes)

Squamouscell

carcinomaofthe

headandneck

Cancerrelated

pathologicalchanges

Increased

backgroundDNA

damagein

the

tumourpatient

group;following

irradiation,

lymphocytesof

tumourpatients

showed

higher

DNA

damage,

slower

repairandhigher

residualunrepaired

damagethanthose

ofhealthysubjects.

20

N¼62patients;34withno

history

ofUCC;28witha

history

ofUrothelialCell

Carcinoma(U

CC).

pH¼13,0.66V/cm,

20min

Mediantailmoment

andpercenttail

DNA

UrothelialCell

Carcinomapatients

Smoking

Thebackground

DNA

damagewas

significantly

increasedin

UCC

patientsascompared

tocontrols.

21

183Clinical Applications of the Comet Assay

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Table

8.1

(continued

).

Studysubject

Alkaliunwindingand

electrophoresis

Parameter

ofDNA

damage/indicator

cells

Clinicalcase

Genotoxic

factor

Resultsandremarks

Ref.

N¼140F;70breast

cancer

patients,meanage53years;

70controls,meanage53

years

pH

alkali,1h;

E¼B0.8V/cm,20min

TM/lymphocytes

Breast

cancer

Cancerrelated

pathologicalchanges

andin

vitro

exposure

toionisingradiation

Increased

backgroundDNA

damagein

thebreast

cancerpatientgroup;

DNA

damagein

lymphocytesafter

invitro

exposure

toionisingradiation

wassignificantly

higher

inpatients;

significantlylower

repaircapacity

after

treatm

entwith

radiationin

patients.

Level

ofDNA

damagewasnot

influencedbyage;

level

ofDNA

damageincreasedin

patients

with

increasingBMI;level

ofDNA

damage

decreasedin

controls

withincreasingBMI.

22

N¼40breastcancerpatients

(clinicalstageIII)

and

N¼60controls.Allnon

smokers

pH¼13,0,78V/cm,

20min

PercentageandTail

Moment

Breast

cancer

patients

andcontrols

OxidativeDNA

damage

PBLofcancer

patients

show

agreaternumber

(percentage)

anda

higher

degreeTM

of

DNA

strandbreaks

inPBLanddouble

strandbreaksare

distinctivelyhigher

inbreast

cancer

patients

thanin

controls

23

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N¼11;F;agerange¼

29–55

years

pH

ns,20min;E¼25V

and300mA,20min

Totalim

agelength/L

Breast

cancer

patients

treated

withhighdoses

ofCP

CP(l875mg/m2/day)

andcisplatin(165mg/

m2/day)

Greatincrease

but

variable

increase

inDNA

damage;

level

ofdamagedid

not

correlate

withserum

levelsof

chem

otherapeu

tic

agents

orwith

lymphocyte

toxicity.

Meanlevel

ofDNA

migrationwasnot

affectedby

cryopreservation.

24

i)N¼50unselected

BC

patients

ii)cancerpatients

withan

adverse

earlyskin

reactionto

RT,and

included

seven

patients

withBC,onewith

tonguecarcinomaand

onewithplasm

acytoma

iii)

16controls.

pH¼13,5;20min;25V

(0,83Vcm

�1)

TM

Breast

cancer

patients

and

controls

Radiosensitivity

TheComet

assaydid

notrevealany

differencesamong

thethreegroupsin

term

softheirinitial

andresiduallevelsof

DNA

damage.

25

N¼397F;188first-degree

female

relatives;

88new

lydiagnosed

untreatedpatients,121

controls;

agerange¼

18–70years;

non-smokers

pH¼13,20min;E¼20V

(0.8V/cm),300mA,

25min

TM/lymphocytes

Breast

cancer

Cancerrelated

pathologicalchanges

andin

vitro

exposure

toN-m

ethyl-N-nitro-N

-nitrosoguanidine

(MNNG)

Greatincrease

inthe

frequency

ofbasal

TLlevel

offirst-

degreefemale

relatives

as

comparedto

controlsandin

patients

as

comparedto

their

first-degreefemale

relatives;similar

patternofeff

ect

wasobserved

upon

exposing

lymphocytesofthe

threegroupsto

MNNG

invitro

orin

thestudyofthe

repaircapacity

ofthe

cells.

26

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Table

8.1

(continued

).

Studysubject

Alkaliunwindingand

electrophoresis

Parameter

ofDNA

damage/indicator

cells

Clinicalcase

Genotoxic

factor

Resultsandremarks

Ref.

N¼8F;agerange¼

25–50

years

pH¼13.5,30min;

E¼22V,30min

TM/lymphocytes,

tumourcellsfrom

breast

Treatm

entforbreast

cancer

Ifosfamide,

doxorubicin

Increase

ofDNA

damagein

lymphocytes1day

after

treatm

entand

theeff

ectlasted

or

even

increasedat48

h;DNA

damagefell

back

topre-

treatm

entvalues

27

3weeksafter

treatm

ent;in

tumour

cells,ashifttowards

increasedDNA

damagewasseen

3weeksafter

treatm

ent.Amount

ofDNA

damagein

either

tissuedid

not

significantly

correlate

with

clinicalresponse

or

toxicity.

N¼10,8F,2M,allnon-

smokers;agerange¼

37–58

years

pH¼13,20min;

E¼25V,300mA,20min

TL/TM,leukocytes

Polychem

otherapy

ofvariouskindsof

solidtumours

5-Fluorouracil,

adriamycin,

cyclophosphamide,

metho-trexate,

leucovorincalcium,cis-

platin

Increasedcomet

parametersin

cellsof

allcancerpatients

after

administration

ofvarious

antineoplastic

drugs

(amountof

administereddrugs

specified).s.pre-

treatm

entandpost-

treatm

entinter-

individualvariations

inthelevel

ofDNA

damagewerefound.

28

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N¼113breast

cancer

patients;agerange¼

36–80

years

pH¼13,20min;

E¼25V,300mA,20min

TM/lymphocytes

Radiationtreated

breast

cancer

patients

Totaldose

of50Gyto

thewhole

breast

DNA

repaircapacity

showed

large

differencesamong

patients;11

patients

showed

considerably

enhanced

inductionofDNA

damageand7

patients

had

severelyim

paired

DNA

repair

capacity.No

apparent

correlation

betweenacute

skin

reactionsupon

radiotherapyandin

vitro

radiation

effects.

29

N¼72,24F,48M,36

uremic

patients

undergoing

haem

odialysis,meanage49

years;

36healthycontrols,mean

age49years

pH¼13,20min;

E¼24V,300mA,20min

Visualgradingof

DNA

damage/

lymphocytes

Haem

odialysisdue

tochronic

renal

failure/uraem

ia

Oxidativestress

Greatincrease

DNA

damageof

lymphocytesfrom

patients

undergoing

dialysisascompared

tocontrols.Patients

treatedwithVitamin

E(600mg/dayfor14

weeks)

hada

significantdecrease

inDNA

strand

breakage;

no

significant

associationbetween

level

ofDNA

damageand

durationofdialysis.

29

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Table

8.1

(continued

).

Studysubject

Alkaliunwindingand

electrophoresis

Parameter

ofDNA

damage/indicator

cells

Clinicalcase

Genotoxic

factor

Resultsandremarks

Ref.

N¼22patients

undergoing

palliativeradiotherapyfor

treatm

entofaccessible

metastatictumours

were

exposedto

twodosesof

radiation.Onthesecond

dayofradiation,13patients

weregiven

80mg/kg

nicotinamidepost-

operativelyonanem

pty

stomach

2hbefore

treatm

ent;theremaining

ninepatients

acted

as

controls.

alkaline,

0.6V/cm,

25min

DNA

contentand

meantailmoment

Metastatictumours

Radiation,nicotinamide

Decrease

inhypoxic

fraction

30

N¼28;8M,20F;

agerange¼

39–45years

pH¼13,20min;E¼25V

and300mA,20min

TL,%

F/W

BC

Thyroid

cancer

patients

treated

with

131Isodium

iodide

131Isodium

iodide

(3700-5550MBg)

Smallincrease

inTL

butnotsignificant.

31

StudyI,N¼46;24M,22F;

11thyroid

carcinoma

patients,35controls;age

range¼

25–80years

pH¼13,60min;Es25V

(0.8V/cm)and300mA,

30min

TM/W

BC

Patients

subjected

toradiotherapyand

chronicallyexposed

toirradiationfrom

Chernobylarea

StudyI¼

131Isodium

iodide(5,55GBq)

Both

groupsshowed

decrease,in

repair

capacity;in

the

groupresidingin

Chernobylarea,

TM

after

invitro

irradiationwas

decreased

comparedto

the

controlgroup.No

adaptiveresponse

wasseen

since

the

pre-exposure

dose

washighand

equaldoseswere

usedeverytime.

32

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StudyII,N¼

48;11exposed

childrenand2exposed

adults;Sex

andage¼

ns35

controls(20M

and15F)

seeabove

seeabove

seeabove

StudyII¼environmental

radiationfrom

Chernobylarea

(120–8,170Bq)

seeabove

32

N¼6;allF;age

range¼

44–81years

PH

ns,60min;

E¼0.67V/cm

and

300mA,25min

TM/tumorcells

Breast

cancer

patients

treatedwith

radiotherapy

Radiation(5-10Gy)

Radiobiologically

hypoxic

cells

showed

adecrease

inTM.

33

N¼24;8M;16F;age

range¼

35–79years

and

N¼23controls;7M;16F;

agerange¼

27–85years

pH413,20min,25V,

300A

MTLandMTM

Varioustypes

of

cancer

Radiation10,30and50

Gy

Nosignificant

difference

between

untreatedcancer

patients

and

controls.TheMTLs

andMTMsat10,30

and50Gywere

significantlyhigher

thantheMTLand

MTM

at0Gyfor

each

patient.

34

N¼33;14M;19F;age

range¼

35–79years

and

N¼33controls14M;19F;

agerange¼

32–85years

pH413,20min,25V,

300A

TLandTM

Differentkindsof

cancer(9

mastocarcinoma

patients,3lung

cancerpatients,2

esophaguscancer

patients,3

nasopharyngeal

carcinoma)

UVC,bleomycin

Lower

DNA

repair

capacity.

35

N¼3invasivetransitional

cellbladder

carcinoma

(HT1376,UMUC-3,

RT112)

pH¼13,5;20min;25V

(0,83V/cm)

TM

Invasivetransitional

cellbladder

carcinoma

Radiosensitivity

TheUMUC-3

shows

thegreatest

DNA

damageandHT1376

displaystheleast

damageateach

dose

examined.

36

N¼13;N¼12controlsage

range¼

29–74years

pH413,20min,25V,

300A

%TDNA

Mitochondrial

disease’spatients

Ubidecarenone

Notanysignificant

difference

inprimary

DNA

damage

betweenpatients

and

control.

37

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Table

8.1

(continued

).

Studysubject

Alkaliunwindingand

electrophoresis

Parameter

ofDNA

damage/indicator

cells

Clinicalcase

Genotoxic

factor

Resultsandremarks

Ref.

N¼32;15M,17F;16

patients,16controls;age

range¼

22–60years

pH¼13,40min;E¼25V

(0.86V/cm)and300mA,

20min

Totalim

agelength/

WBC

Vasculitis/

collagen

disease

patients

CP(a

totaldose

of

50–200mgdaily)

Increase

inDNA

damagein

CP-

treatedpatients

comparedto

controlsorpatients

without

chem

otherapy;

increasedin

DNA

damagewasvariable

andnotclearly

relatedto

CPdose;

CP-inducedDNA

effects

persisted

invivoforaperiodof

severaldays,butfor

less

than2weeks.

38

N¼33withcancer;allM;

N¼14;allM

withfertility

pH

alkaline;

10min,

25V

(0,714Vcm

�1;

300mA

Percentagehead

DNA

Cancer(testicular

cancer,lymphoma

andleukaem

ia)

patients

DNA

integrity

DNA

integrity

was

reducedbycancer.

39

N¼24;17M,7F;9patients

and15controls;age

range¼

57–71years

pH¼13,20min;E¼25V

(0.66V/cm)and300mA,

20min

TM,%F/U

TPatients

with

transitionalcell

carcinoma(TCC)

DNA

baselinedamage

Increase

indamage

insamplesfrom

TCC

patients

comparedto

controls.No

significantdifference

betweenTCC

grades.

40

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N¼41;M;13fertileand28

infertile;

age¼

ns

pH¼13,20min;E¼25V

(0.66V/cm)and300mA,

20min

%DNA

headarea

Fertile

andinfertile

males

DNA

baselinedamage

Baselinelevelsand

damagein

sperm

samplesfrom

fertile

andinfertilegroups

werefoundto

be

similar.Background

DNA

damagein

sperm

cellwas

increasedcompared

tosomaticcells.

40

N¼50patients

pH¼12.5,2V

(0.714V/

cm);300mA,10min

Percentagehead

DNA

Fertile

andinfertile

males

X-rayirradiation

DNA

damage

increaseswith

X-raysdose.

41

N¼20;M;10patients

and

10controls;age

range¼

33–59years

pH

13,40min;E¼25V

(0.8V/cm)and300mA,

30min

%F/L

Insulin-dependent

diabetes

patients

Reactiveoxygen

species

Meanvalues

of

strandbreaksand

oxidized

pyrimidines

weresignificantly

increasedin

diabetics;strong

correlationbetween

altered

purinesites

andserum

glucose

concentration.

Significant

correlationbetween

bodymass

index

and

strandbreaksonly

indiabetics.

42

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Table

8.1

(continued

).

Studysubject

Alkaliunwindingand

electrophoresis

Parameter

ofDNA

damage/indicator

cells

Clinicalcase

Genotoxic

factor

Resultsandremarks

Ref.

N¼14type2diabetic

patients

(9M;5F)and

N¼14controls(7M;7F)

pH

alkaline;

20min;

0,8V/cm;300mA

%DNA

intail

Peripheralblood

cellsfrom

patients

withType2diabetes

mellitus

OxidativeDNA

damage

Norm

alhealthy

subjectsexhibited

lower

levelsofboth

DNA

breaksand

FPG-sensitive

oxidativeDNA

damagethan

diabetics,the

difference

wasmore

relevantand

statistically

significantfor

oxidativedamage.

43

N¼17

24min

at20V

Taillength

Islets

humanheart

beatingdonors

Cytokines

Prolonged

exposure

ofhumanpancreatic

islets

toamixture

of

cytokines

induces

DNA

strandbreaks.

44

N¼25Rhem

atoid

Arthritis

patients;17F;8M,

N¼26control;16F;10M

pH¼13,25min;25V/

300mA

Visualgradingof

comets(0,without

detectable

tail;4,

highestdamage/

lymphocytes)

Rheumatoid

arthritis

patiens

Oxydativestress

Lymphocyte

DNA

damagelevel

increasesin

patients

withRA.

45

N¼20;allM

(9ofwhom

haveheritable

predispositionto

schizophrenia);age

range¼

37–41years.

N¼20controls

pH413,25V

(1V/cm,

300mA),30min

Visualgradingof

comets(0,without

detectable

tail;4,

highestdamage/

lymphocytes)

Schizophrenic

populations

Oxidativestress

NotincreasedDNA

damagefrom

controls.

46

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N¼30;15M;15Fage

range¼

35–41years,stressed

exposedpopulation.

N¼30controls15M;15F

pH413,25V

(1V/cm,

300mA),30min

Visualgradingof

comets(0,without

detectable

tail;4,

highestdamage/

lymphocytes)

Chronic

psychogenic

stressed

population

Oxidativestress

Cellsfrom

the

stressed

population

weremore

sensitive

totheinductionof

DNA

damageand

hadhigher

level

of

residualdamage.

Stressconditions

maycause

the

affectedindividuals

tobesusceptible

toenvironmental

mutagenic

agents.

47

N¼35;agerange¼

50–70

years,obstructivesleep

apnea

population;

N¼35controls

pH413,25V

(1V/cm,

300mA),30min

Visualgradingof

comets(0,without

detectable

tail;4,

highestdamage/

lymphocytes)

Obstructivesleep

apnea

Oxidativestress

ObstructiveSleep

Apnea

patients

had

higher

basallevelsof

DNA

damageand

weremore

sensitive

totheeff

ects

ofthe

DNA-damaging

agents

than

lymphocytesfrom

controls.OSA

patients

hada

reducedcapacity

torepairtheDNA

damageinducedby

thethreeagents.

48

Abbreviationsused:CP:cyclophosphamide,E:electrophoresis,%

F:percentagefluorescence

inthetail,F:female,L:lymphocytes,M:male,N¼number,ns:not

specified,s.:significant,SCGE:single

cellgel

electrophoresis,TL:taillength,TM:tailmoment,WBC:whitebloodcells,UT:urothelialcells,VS:visualscoring

193Clinical Applications of the Comet Assay

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the background DNA damage was significantly increased in patients as com-pared to controls.In studies of newly diagnosed untreated breast cancer patients and patients

free of cancer/treatment for at least 6 months significantly higher DNA damagewas found in lymphocytes if compared to controls.22,23 A higher susceptibilityto in vitro treatment of lymphocytes with N-methyl-N-nitro-N-nitrosoguani-dine or ionising radiation and a decrease in the DNA-repair capacity was alsodemonstrated. Sanchez et al.24 using both alkaline and neutral Comet assaysfound that blood lymphocytes from breast cancer patients exhibited highersingle- and double-strand breaks by comparison with healthy individuals. Highdoses of cyclophosphamide and cisplatin administered to breast cancer patientsresulted in a significant increase in DNA damage of lymphocytes from thesepatients.25 Spontaneous and radiation-induced genetic instability of peripheralblood mononuclear cells derived from unselected breast cancer was examinedusing the single-cell gel electrophoresis.26 No differences in the background orradiation-induced DNA damage were observed when compared with controls.Rajeswari et al.27 showed that lymphocytes from first-degree female

relatives of breast cancer patients showed an increased DNA damage uponexposure to mutagens in vitro. The repair capacity of first-degree relatives wasalso decreased.Three studies28–30 have assessed the risk of DNA damage in lymphocytes of

cancer patients who received treatment against breast cancer or various kindsof solid tumours. In all three studies, the level of genetic damage in lymphocyteswas significantly increased through treatment.Twenty-two patients undergoing palliative radiotherapy for treatment of

accessible metastatic tumours were given nicotinamide. Both nicotinamide-treated tumours and controls demonstrated a significant increase in the per-centage of cells containing heavily damaged DNA.31 Nicotinamide, however,was found to reduce hypoxia in the tumour cells.Monitoring of genetic damage induced by therapeutic exposure to 131I was

undertaken in a group of thyroid cancer patients.32,33

The single cell gel electrophoresis was also employed to assess the effects ofradio- and chemotherapy.34 Jianlin et al.35 in their study of cancer patientsduring radiotherapy found no significant differences in the amount of single-strand DNA damage between untreated cancer patients and controls. How-ever, a dose–response relationship between radiation dose and genetic damagein patients during radiotherapy was found, as well as interindividual variance inresponse to radiation.35

Wei et al.36 investigated the repair capacity of lymphocytes from varioustypes of cancer patients after being exposed to bleomycin and UVC irradiation.The results indicate a reduced DNA-repair capacity for the lymphocytes of allthe examined cancer patients when compared to healthy controls.Approximately 50% of patients with invasive transitional cell bladder car-

cinoma fail to respond to radiotherapy. These patients are disadvantaged bythe absence of predictive information regarding their radiosensitivity, thusallowing the tumour to gain additional time for metastatic spread before

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cystectomy is performed. In this case, the Comet assay can be used in order toinvestigate the response of this malignancy to radiotherapy.37

The Comet assay was used to quantify primary and oxidative DNA damagein leukocytes of mitochondrial disease (MD) patients. The assay indicated aslightly higher level of primary DNA damage in patients compared with con-trols. A difference in oxidative DNA damage was also observed, this, however,was not statistically significant.38 Treatment of patients with vasculitis/collagendisease with cyclophosphamide resulted in a significant increase in DNAdamage compared to controls.39

O’Donovan40 evaluated the DNA integrity in spermatozoa of men withdifferent types of cancer before and after therapy. His results indicated areduced DNA integrity if compared to controls. No significant difference inDNA damage between the different cancer groups was observed. A marginallysignificant difference in comet values in spermatozoa of cancer patients beforeand after therapy was, however, found. McKelvey-Martin et al.41 compared thebaseline DNA damage in sperm cells of fertile and infertile males and did notfind a significant difference between the two groups. Hughes et al.42 comparedthe sensitivity of the Comet assay technique and enzyme-linked immunosor-bent assay (ELISA) for the assessment of human sperm integrity.Diabetic patients43 were found to have increased DNA damage when the

Comet assay was employed in order to measure the amount of DNA breaks.Evaluation of DNA base oxidation measured as formamidopyrimidineDNA glycosylase (FPG) sensitive sites in peripheral blood cells from type2 diabetes patients revealed an oxidative DNA damage increase in diabeticcompared to normal subjects. Delaney et al.44 reported that prolonged expo-sure of human pancreatic islets to a mixture of cytokines induces DNA-strandbreaks.Rheumatoid arthritis patients were found to have increased DNA-damage

levels when compared to controls.45 In addition, the DNA damage was foundto be related to the severity of the disease in the patients.The Comet assay was also used to study DNA damage and repair efficiency

in schizophrenic populations46 as well as on populations exposed to chronicpsychogenic stress.47 The results revealed that lymphocytes from schizophrenicpopulations showed response similar to the controls. In the case of the stressedpopulation, however, the results indicated that this population was more sen-sitive to the induction of DNA damage and had a higher level of residual DNAdamage than the controls.It was found that lymphocytes from patients with obstructive sleep apnoea

syndrome had higher basal levels of DNA damage and were more sensitive tothe effects of external DNA-damaging agents than the controls.48

8.4 Discussion and Conclusions

The Comet assay has already demonstrated its sensitivity as a technique for theevaluation of DNA damage among a variety of cell types.

195Clinical Applications of the Comet Assay

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The great usefulness of the Comet assay and its rapid spread amongresearches are due to several advantages in comparison with other techniques,such as:

1) It has been applied in a wide variety of eukaryotic cells in any organ,with successful results.

2) Sample size is very small (from 10 000 to 50 000 cells).3) It detects damage at the single-cell level.4) Cell lines are suitable.5) Highly sensitive (50–15 000 breaks/cell).6) Results are obtained on the same day.7) Damage can be detected in cycling as well as in noncycling cells.8) Noninvasive technique.9) Fresh or frozen samples are suitable.

10) It is fast, simple and inexpensive.

The technique is now being widely used in many studies for measuring DNA-strand breaks in single cells. However, the studies listed in this review had someflaws due to inconsistent Comet protocols and study design problems.The most common shortcomings found in the design of the studies were:

i. Use of small study groups weaken the statistical power.ii. Gender distribution between the groups was usually unequal.iii. Data on external or internal exposure to agents were not always

provided.iv. Size of the study group and the control usually was unequal, making the

comparison of the two groups rather difficult.v. No data were always provided on profession, disease status or ethnicity.

All these factors should be seriously taken into account in forthcoming clinicalstudies in order to make possible interlaboratory comparisons of the findings.

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