1. health and fitness
2. disease
3. cholesterol

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Availableonline at www.sciencedirect.com
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Toxicology
Letters
ToxicologyLetters158 (2005) I 0-19
www.elsevier.com/locate/toxlet
Differencesin DNA-damagein non-smokingmen and women
exposedto environmental
tobaccosmoke(ETS)
Abby C. Collier8,c,SachinD. Dandgeb,JamesE. Woodrowa,c,
ChrisA. Pritsos&'c'*
u Department of Nutrilion,
University of Nevada, 1664 N l4rginia/MS I42, Reno, NV Bg557, USA
b Department of Biochemistry,
University of Nevada, Reno, NV gg557, USA
c Environmental Sciencesand
Health Program,lJniversity of Nevatla, Reno, NV BgSS7,USA
Received29 December 2004; receivedin revisedform l0 February2005; acceptedI I February 2005
Available online 24Mav 2005
Abstract
There is much data implicating environmentaltobaccosmoke(ETS) in the developmentand progressionof disease,notably
cancer,yet the mechanismsfor this remain unclear.As ETS is both a pro-oxidant stressorand carcinogen,we investigatedthe
relationshipof ETS exposureto intracellular and serum levels of DNA-damage, both oxidative 8-hydroxy-2-deoxyguanosine
(8OHdG) and general,in non-smokersfrom non-smokinghouseholds,occupationallyexposedto ETS. GeneralDNA-damage
consistingof singleand doublestrandbreaks,alkali-labilesitesand incompletebase-excision
repair,increasedsignificantlyin a
d o s e - d e p e n d e n t m a n n e r w i t h E T S e x p o s u r e i n m e n ( P : 0 .n0:135l ,,P e a r s o n ) b u t n o t w o m e n( P : 0 . 7 3 6 , n = 1 7 ) . l n t r a c e l l u l a r
SOHdG-DNA-damage
and generalDNA-damagewere both greaterin men than women (P:0.0005 and 0.016,respectively)
but SOHdG seru-nlevels did not differ betweenthe genders.Neither 8OHdG-DNA-damagenor serum levels correlatedwith
increasingETS exposure.This is the first studyto demonstrate
dose-dependent
increasesin DNA-damagefrom workplaceETS
exposure.Perhapsmost interestingwas that despiteequivalentETS exposure,significantlygreaterDNA-damageotcurred in
men thanwomen. Thesedatamay begin to providea mechanisticrationalefor the generallyhigher incidenceof some
diseases
in malesdue to tobaccosmokeand/orothergenotoxicstressors.
O 2005 ElsevierIrelandLtd. All rights reserved.
Keyv'ords:Environmental
tobaccosmoke;DNA-damage;
DNA repair;8-Hydroxy-2-deoxyguanosine
Abbreviatiorts.' 8olJdG, B-hydroxy-2-deoxyguanosine;ELISA, enzyme-limited immuno sorbent assay;ETS,
environmentaltobacco smoke;
Fpg, formamidopyrimidine DNA glycosylase;HPLC/ECD, high pressureliquid chromatographywith electro
coulometric detection;hoGGl,
human8-oxoguanineDNA glycosylase
" C o r r e s p o n d i nagu t h o r T
. e l . :+ l 7 7 5 3 2 7 5 0 9 5 ;f a x : + l 7 7 5 7 9 4 l l 4 Z .
E-rttailoddresses.
[email protected]
(A.C. Collier), [email protected]
(C.A. pritsos).
0378-4214ts seefront mattero 2005 Elsevierlreland Ltd. All rishts reserved.
d o i :I 0 .I 0 I 6 / i . t o x 1 e t . 2 0 0 5 . 0 2 . 0 0 5
A.C. Collier et al. / Toxicologt Letters I 5S (2005) t 0-t 9
1. Introduction
Recently,non-smoking areas in bars and sports
clubshavebeenshownto providelittle or no protection
for patrons(Cains et al., 2004), corroboratinganecdotal medicalreportsand previousstudiescontending
that legislationis themosteffectivetool to reduceenvironmentaltobaccosmoke (ETS) exposureand smoking behaviour(l{eloma et a1.,2001).While ETS exposurehas been linked to a number of disorderssuch
as asthma,intra-uterinegrowth restriction,suddeninfant deathsyndrorneand coronaryheartdisease,it has
beenmost consistentlyand strongly implicatedin the
initiationandprogressionof cancer(Anon, 1996).Despitethis, the temporalrelationshipsand mechanisms
by which cigarettesmoke causesinitiation and progressionof diseaserernainsrelatively unknown. Furthermore,althoughsocial ETS exposureis relatively
controllable(an individual may choosenot to enter
premisesw,heresmokersarepresent),employeeshave
little or no inffuenceover ETS and may be exposedfor
a largepart of their working day.
Havingpreviouslyidentifiedsignificantlyhigher8hydroxy-2-deoxyguanosine
(8OHdG) levels in nonsmokingworkersoccupationallyexposedto ETS compared to non-smokingworkers who are not exposed
to ETS (Howarcict al., 1998),the goal of this study
wasto assess
a quantitativerelationshipbetweenDNAdamageand workplaceETS exposure,and to investigate the contributionof the oxidative DNA lesion
8-hydroxy-2-deoxyguanosine
(8OHdG) to total ETSinducedDNA-darnagein the blood of non-smokers.
Differencesin DNA-darnagewith respectto SOHdG
werealsoassessed
in relationto ETS exposureand demographicvariables.
2. llethods
2.L Par"ticipcutts
rurclrecruitment
Non-smokers,living in non-smokinghouseholds
who work at casiuosand bars in Reno and Las Vegas,NV were recrLlitedfor the NevadaEnvironmental
TobaccoSrnokeStudy(NETSS).Fifty baselineblood
samplesfiorn NETSS were randomly selectedfor the
currentstudy.All parlicipantsprovidedinformedconsentandthestudywereapprovedby theBiomedicalIn-
ll
stitutional Review Board at the University of Nevada,
Reno. Participantdemographicsand baselinecharacteristicsare shownin Table l.
2.2. Cotinine analvsis
Cotinine levels were determined to verify selfreportednon-smokingstatusand to quantitateETS exposure.Plasmasamples,I mL from NETSS filtered
throughOasis@MCX (Waters,Milford, MA) cationexchangesolid phase extractioncartridgesand cotinine elutedprior to GC/lvIS analysis,as describedin
the Product Insert. Briefly, acidified plasma samples
(0.05meq HCI) were filtered through separateprepared cation-exchangecartridges,washed with 2 mL
0.1M HCI then 2mL methanol and cotinine eluted
with 1.5mL ammoniumhydroxide(5% in methanol).
Eluateswere vacuumedto dryness,60"C (l h), redissolvedin 0.2 mL ethyl acetateand filteredthrough
0.45 pm membranefilters into autosampler
vials.Samples were chromatographed
on an Agilent 6890 plus
gas chromatograph(San Femando, CA) and detected
with an Agilent 5973N mass spectrometeroperating
in selectedion mode (EI: 70 eV). An aliquot (2 p"L)
of each sample was injected onto a 30 x 0.25mm
(i.d.) DB5-MS fusedsilica opentubularcolumn(J&W
Scientific) and chromatogramsdevelopedusing the
following GC/MSD program: 60"C (lmin), rhen
25"lmrn to 300"C (hold I min); a post-runhold of
l0min at 300oC was imposedafter eachsample(total run time : 21.6min). Cleansolventinjectionswere
made betweenstandardand sampleinjections to minimize carry over of the cotinine analyte.The fragment
at mlz:98.1 was used for quantitation,and retention
time for the cotinine analytewas 7.68min. Averagerecovery from clean, spiked plasma (0.3-4 ng/ml,) was
100.7+ .1.6%w i th a C V of 8.0+ 4.4% .The concentration range for the standardcurves was l-80 nglmL
with limits of detectionand quantitationof 0.I nglmL
and 0.3 nglmL, respectively.
2.3. Single cell gel electrophoresis(Comet)assay
DNA-damage was assessedby the Comet assay
in whole blood sampleswith or without inclusionof
human 8-oxoguanineglycosylase(hOGGl) enzyme,
which cleaves8OHdG-DNA lesionsand allows them
to run under electrophoresisand thus be detected.This
ll
A.C. Collier et al. / Tbxicolog,,Letters I Sg (2005)
l0_t g
fable I
Baselinedenrographics
of study participantsfor the whole studypopulation
and for male and femaleparticipants
-
,Mv a
r ol e, Lsr
33
nla
17
q88+es
(2s44)
nla
28'9 + 4'3 (20'4-37 '8)
29.6+.4.0 (2334;.8)
Females
Mea..,ro
i-
ctor.l.-,J
,1^,,:^+:.--
/.
- ,,
ffi;;::,;T,x',ilii';':,:::jfi"*)
x a stanoaro
deviatiorr(range)
Ethnicity
Caucasian
Larino
Asian
Black/AfricanArnerican
A m e r i c a nI n d i a no r A l a s k an a t i v e
N a t i v eH a r v a i i a n
o r o t h e rp a c i f i cI s l a n d e r
other
Education
S o n e h i g h s c h o o t( 9 - l l y e a r s )
H i g h S c h o o dr i p r o r n o
ar G'E'D'
"Ti::ilij,::ffi:ilffij;:"
Sornecollegeor associatedegree
C o l l e g eg r a d u a t eo r B a c c a l a u r e a d
r ee g r e e
S o m ec o l l e g eo r 1 r r - o f e s s i o n a l
schoolaftercollegegraduation
M a s t e r ' sd e g r e e
26 (52%)
n e2%)
2 (4%)
3 (6%)
4 $%)
2 (4%)
2 g%)
| e%)
n Q6%)
482t102(2s-64) 5'+ 8.4(2s_.62)
g
67.6%)
7 el.z%)
| Q'0%)
2 g.o%)
3 eJ%)
",
0.'''
2 6.0%)
| (3.0%)
6 (r8.r%)
widorvecl
Presently.narried
Lir''ingin a marliage-like
rike relationship
rerarionqhin
2g.0+ 4.g (20.4-37.7)
7 gt.2%)
4e3.5%)
| 6.9%)
| 69%)
| 6.9%)
z (ll.g%)
0
0
7 @r.2%)
602%)
3e%)
3(,76%)
22 g4%)
6 (12%)
I e
\-'%-/
)
n 64.s%)
4 (12.1%)
0
u
4 e3'%)
2 (ll.g%)
I (5.9%)
I e%)
| G%)
o
8(16%)
s(ts.2%)
Marital status
ilffiil':T::pararcd
u
nJa
ila
nla
12(24%)
3(17.6%)
o
o
8 Q4'2%)
4 e3.s%)
24 (48%)
6 (12%)
l5 g5.5%)
S (15.2%)
g
o
62.9%)
| 6.9%)
}:':':::';1*['Hffij',',:u'.''n.characteristicsofage,BMI,ethnicbackground,educationleve|andmarita|.ffi
wasperfbnnedr-rsing
a commerciallyavairableFlareTM
assaykit (Trevigen,Gaithersburg,
MD) asper the manufacturer'sinstructions.
Briefly,ceilswereplatedin low
meltingpoint agarose,slideswere lysedand DNA
un_
windingoccurrcclfbr l h at pH >13. Slideswere
elec_
trophoresedfor 30 min (0.g5V/cm) and dried
down
beforestainingwith SyBR greenTMand visualization
usinga ffuorescent
microscopeat 400x magnification.
Threeblocldsamplesper personwere assessed
and 50
cells werc scoredranclornlyby eye in eachsample,
on
a scaleof 0-4, basedon fluorescencebeyond
th. nucleusandwerepreviouslydescribedby Kobayashi
et al.
( 1995).Thescaleusedwasasfollowsr0:
no cometing;
I : Cornet< 0.5 times the width of nucleus;2:
Co.Ji
equalto width ofnucleus;3: Cometgreaterthan
width
of nucleus;4 : Comet> two times the width of
the nu_
cleus.Scoringcellsin this mannerhasbeenshown
to be
as.accurate
and preciseas using computerimageanal_
ysis (Kobayashiet al., 1995).The individual
sclring of
theslideswasblindedto anydemographicor biochJmi_
calaspectsofthe bloodsamples.GeneralDNA_damage
was calculatedfrom the sampleswith no hOGGI
ei_
zymeadded,while specificgOHdG-DNA-damage
was
calculatedby subtractinggeneral DNA_du-ugJ
frorn
in correspondingsamplescontainingnbCCf
.
9-.T".g:
Variabilitycausedspecificallyby the Cometissay
pro_
cedurewas controlled by running one loading.ontrot
sampleforeveryparticipant.Loading controls
werenot
lysedand unwound,but were electrophoresed
with ex_
perimentalsamplesand developedwith
ethidium bro_
mide. Every loading control was checked
visually on
a microscopeand a picture was taken with
a Geidoc
A.C. Collier et al. / Toxicologt Letters I58 (2005) 10-19
(Biorad,Hercules,CA) to ensureloading of a similar
numberof white blood cellsand therebycontrolfor retardationin DNA rnigration due to over-loading.Sampleswherewhite blood cellswere overloadedwere repeated.Loadingcontrolsalsoservedto measurelevels
of arlifactr"ral
DNA-damagecausedby electrophoresis
alone.
thawed,appliedto spinfilters(10,000Mw cut-off,Millipore,Billerica,MA) and centrifugedat 12,000x g in
a benchtopcentrifugefor 30 min. Flow-throughwasapplied to OXYcheckhigh sensitivitycompetitiveELISA
plates(Genox,Baltimore, MD) and quantitationproceededas per the manufacturer'sinstructions.
2.5. Statistical analysesand data manipulation
t,
r rrr "rninat i ott oJ'BOH dG concentratio ns in
/,.r,l
All analyseswere performed with GraphPadprism
3.0 (GraphPadSoftware,San Diego, CA). Data analysis showedthat DNA-damageresultsapproximateda
Gaussiandistribution;thus; unpairedStudent's/-tests
with Welch's correction for unequal varianceswhere
appropriate,linear regressionand Pearson'scorrelation and one way ANOVA with Tukey's post test were
usedas detailedin Figs. I and 2 andTable2. Statistical
analysiswas performed in a post hoc manner after all
biochemicalanalyseswere completed.
Concentrationsof SOHdG were determined in
serum sarnplesdrawn from participantsat the same
time as plasma and whole blood samples.Blood
was drawn into a SST vacutainer,allowed to clot at
room temperaturefor 45 min-l h and centrifuged at
1000rprnfor l0min. Serum was aliquotedinto cryovialsandstoredat -70 until use.Before 8OHdGconcentrationswere determinedin serum, sampleswere
*
1'4i
a
b 1'o'i
.
P
fi o'si
r
A
I
l
r
n ^-i A
{ 0.6j I
z
:
l
nri
r8QH'JGDartageP=0.s779
A Gen€ral
DNAdamage
P- 0.00a!
a
H r"j
(E "- i
t
O
a
I
$ "''l I ra
U
t-^* i
Io.ai
r
I
I
n
raaa
l
r
S:{*ri'",*': 3l
$ r";
0
1
2
3
(A)
t,
o
A
t,4
{r 1,0
$t o's
* 0.6
a 0.4
d n c
(B)
r r
tra
.a^
1.4
1.2
1.0
female
I BOtjdG O;rmageP * 0.310
A 0eneral DNA clamageP= 0.716
I
a '
I
z ",*
cl o.o
aS0HctGDarrageP-0.70t
A 6eneral OFiAclanragep= t).001S
I
r
4
s
Hlosd cotinine concentration {ug/mL)
male
1.4
l3
A
c
ItrA
I
0
1
2
3
4
5
Blood cantinlne concentration
(ug/ml)
0.8
0.6
0.4
0.2
0.0
0
1
2
3
4
5
Blood continine concentration
(c)
(us/ml)
Fig. I Gencralbut not SOHdG-DNA-damageis significantlycorrelatedto workplaceETS exposurein the blood
of male non-smokers.(A)
Oxidative8OHdG (I) and generalDNA-damage(A) in the srudypopulation
@:a\.(B) OxidativegOHdG lesions(l) andtotal DNA_damage
(A) in malesonly (n:32). (C) Oxidative 8OHdG lesions(l) and total DNA-damage(A) in females
only (n: l7). p-valuespresentedare
Pearson'scorrelationcoefficients.
A.C. Collier et al. / Toxicologt Letters I 5S (2005) t O-19
2g
I
c l S
.g
rA
Eai-
80H2dc
r = 0.9359
I
s8 El;
o I
I
I
eE 6
$8;
0
(A)
.b:16
I
E€ t,l
39'l
k r
c 2 0
.g ls
r 80HdG
r = 0.8557
0
r UOHdG
r = 0.4473
E :irs
fiE gll
9to
I
I
i sI
t st
6
fie :
U"
{B)
Fernale
Male
E 2 0
fr -:18
UPra
1
2
3
4
Gontinine concentralion (ng/mL)
t r
I
{ l ) 0
0
1
2
3
4
5
Contininaconcentration{ng/mL)
(C)
0
1
2
3
4
5
Continine concenrration (ng/mL)
Fig. 2' 80HdG levelsin the serumof non-smokersarenot correlatedto workplaceETS exposure.(A) Serum8OHdG concentrations
(l) in the
whole studypopulation@ - a9).(B) Serum8OHdG concentrations
(I) in malesonly (n : 32). (C) Serum80HdG concentrations
(I) in females
o n l y( r : l 7 ) .
3. Results
One participantwas excludedbecauseits cotinine
value(l37ngAnl-) represented
an activesmoker;subsequentstatisticalanalysis was performed with 49
individuals(32 rnenand l7 women).Analysisofthe rernainingparticipantsindicatedthat cotininelevelsdid
not differ significantly between or within any demographic sub-group(Table 2).
General DNA-damage in the Comet assaycorrelated significantly with increasingcotinine levels in
the whole population(P:0.0042, Pearson,Fig. lA)
but 8OHdG damagedid not. Further sub-groupanalyses showed that general DNA-damage increased
Table2
Relationships
of gencralDNA-damage,SOHdG-DNA-damage,
serum8OHdG concentrations
and cotinineto demographicsub-groupsin the
studypopulation
Non-oxidativeDNA-daurage
8OHdG-DNA-darnage
8OHdG serumconcentration
Cotinine
Gender
(t-test)
Caucasianvs.
other (l-test)
Latino vs.
other (l-test)
BMI
(Pearson)
Age
(Pearson)
Marital status
(ANOVA)
**
0.0163
0.0005"
0.0707
0.4230
Education
(ANOVA)
0.7531
0.7467
0.1138
0.309r
0.5282
0.98',72
0.6102
0.7028
0.6477
0.s282
0.8092
0.9276
0.8587
0.9s06
0.0289*
0s602
0.9203
0.4398
0.6002
0.6863
0.6221
0.8512
0.4846
0.3977
Note. tlte lack of sigrtificaltceof cotinine levels betu'eenor within any demographicparameters,indicating equivalent
ETS exposurein this
population.
* P < 0.05.
"'P<0.01.
A.C. Collier et al. / ToxicologyLetters l5S (2005) l0-19
significantlywith ETS exposurein males but not fem a l e s( P : 0 . 0 15 , n : 3 2 , F i g . l B a n dP : 0 . 7 3 6 , n : 1 ' ,l
Fig. lC respectively).The average general DNAdamagescorefor males(0.27+ 0.47)was significantly
higherat t han i n fe ma l e s(0 .0 6 + 0 .0 8 , P:0 .016, rtest).In contrast,SOHdG-DNA-damagedid not correlateto increasingETS exposurein the whole population
nor in eithergender(P:0.79, male; P:0.31 female,
Fig. lA-C). Despitethis, the averagedamagescores
for SOHdG-DNA-damagewere significantly higher in
males (0.31+ 0.34) than females(0.059+ 0.I l, respectively,
P: 0.0005,r-test).
Concentrations
of SOHdG in serumdid not correlate to ETS exposure(Fig. 2A-C) and did not differ
significantlybetweenmen than women (P:0.0756).
The 8OHdG levelsin serumdid correlatesignificantly
with age(P:0.0289) but did not differ with any other
demographicvariable(Table2). No other demographics showedsignificantdifferencesor corelations with
DNA-damage(oxidativeor otherwise),SOHdGserum
concentrations
or cotininelevels(Table2).
To eliminateconfoundingof our resultscausedby
baselinemi smatching,analysisof baselinedemographics (age, body mass index, educationlevel, marital
status,and ethnic background)was performed. There
were no statisticallysignificantdifferencesin baseline
demographicsbetween male and female participants
(Table 1), strengtheningthe apparentgender-specific
differencesin DNA-damage.Furthermore,althoughno
samplewas thawed more than once prior to theseexperiments,we assessed
whetheror not the numberof
freeze-thawcyclesaffectedthe averageDNA-damage
scorefrom the Cometassay.No significantrelationbetween the numbersof freeze-thaw cycles and general
DNA-damageor SOHdG-DNA-damage
was observed
(P:0.778 and P :0.97, respectively,
Pearson).In addition, although actual numbers were not recorded.
DNA-damageattributedto electrophoresis
in the control slideswas checkedat random and was very low
with few cellsexhibitingany cometing(2-3 Comets/50
c ells ) .
4. Discussion
GeneralDNA-damagein the blood of non-smoking
men increasedin a dose-dependent
mannerwith exposureto ETS in the workplace(P:0.015) and was
l5
significantlyhigher than in women (P:0.0163). In
contrast,neither oxidative 8OHdG-DNA-damagenor
SOHdG concentrationsin serum correlatedwith ETS
exposure.In addition,althoughSOHdG-DNA-damage
levels were significantlyhigher in men than women
(P:0.0005), serumconcentrations
of SOHdGdid not
differ betweengenders.
One of the most important aspectsof this study
is that cotinine levels were the same between males
and females (Table 2). Cotinine also did not differ
between or within any other demographic group or
stratification,which is further evidencefor equivalent
ETS exposureamong all the participants.Different
cotinine levels may be ascribedto differentialtimes
since exposure and/or genetic polymorphism in
CYP2A6, althoughthis polymorphismdoesnot exist
at significantlevelsin the populationsampled(yang et
aL,200l). In addition,confoundingvariablesfor foodrelated nicotine levels in experimentalparticipants
have been previously investigatedand cauliflower,
eggplant,tomatoes,potatoes and black tea contain
measurablelevelsof cotinine(Benowitz,1996).While
it hasbeensuggested
that the dietaryintakeof nicotine
throughthesesourcescould confoundstudiesof ETS,
especiallywith respectto genderbecauseof fruit and
vegetablesintake (Glynn et al., 2005); using pharmacokinetic analysisand typical American vegetable
consumptiondata,Benowitz(1996)placesthe average
dietary nicotine intake at around to 0.7 pg daily
(Benowitz,1996).Additionally,thereareno othersexrelateddifferencesin cotinine clearanceswith gender
(Gries et a1.,1996).Up to 80% of a dose of nicotine
is convertedto cotinine via a C-oxidation pathway,
and the resultingcotininemetabolitehas a half-life of
l5-I7 h. In comparison,nicotine has a much shorter
half-life of 2.6h and has different clearanceratesin
smokersandnon-smokers(Benowitzand.Iacob,1993;
Kyerematenet al., 1990). Hence, due to its longer
half-life, cotinine is readily identified in samples,
which may have been drawn relatively long periods
of time after ETS exposureand the methodsusedto
detectand quantify it (generallyGC/MS or LCIMS)
are rapid, sensitive, precise and accurate (Tricker,
2003).
Another issue relating to the timing of procuring blood samplesis the rate of SOHdG-DNA lesion
repair. The kinetics of hOGGI for 8OH2dG follow Michaelis-Mentenkinetics and have a V^u* of
l6
A.C. Collier et al. / Toxicologi Leuers l 5B (2005) I0-19
0.51+ 0.17nM/min 8OH2dG(Asagoshiet al., 2000).
Thus,at high 8OH2dGlevels,the enzymemay become
saturated,indicating that the length of time from ETS
exposureto DNA repair may also be dependenton the
levelof exposure.Despitethis,the ftss1
forhOGGl with
respectto8OH2dGis 0.034min, which is relativelyfast
(Asagoshiet al.,2000).This is complicatedby a distinct
sequenceaffinity of hOGGI for 8OH2dG in certain
portions of the genetic code, whereby sometimesthe
rateand efficiencyof hOGG I for SOHdG is alteredby
thepositionon the lesion.The sameis truefor the other
enzymethat removesSOH2dG,formamidopyrimidine
DNA glycosylase(Fpg). This enzymehas a V^u* of
2.1+ 0.7nM and a ksil of 1.8min (resultingin similar
intrinsicclearances
to hOGGI) and also demonstrates
a DNA-sequence
specificaffinity for lesions(Asagoshi
et al. ,2000) .
This study was designed to determine whether
a dose-dependent
relationshipbetweenDNA-damage
and increasingoccupationalETS exposureexists.Previous researchfrom our laboratory has demonstrated
significantly higher 8OHdG levels in non-smoking
workersoccupationallyexposedto ETS comparedto
non-smokingworkerswho are not exposed(Howard et
al., 1998),andwe wishedto follow on from this finding
and investigatedose-responserelationshipsbetween
levels of ETS exposureand levels of DNA-damage.
It has also been shown that self-reportedETS exposure significantlyunderestimates
the actual exposure
of studyparticipantsto ETS (Delorenze et a1.,2002)
and that ethnicity, marital status, education and age
(Iribanen et al., 2004; Kurtz et al., 2003; Reynolds et
al.,2004;SolimanetaL.,2004)canaffectexposurelevels.In ordertoaddressthis,thecurrentstudyusedblood
cotininelevelsas a marker of ETS exposurenot selfreporting,andthe datapresentedin Table2 demonstrate
that no significantdifferencesin cotinine levelswere
observedbetweenor within these demographicsubgroups.This indicatesthat our findingsarenot skewed
by mismatchingbetweenmales and females and further,that the measuredETS exposureis likely to comprise almost exclusivelyof workplace ETS exposure
and not exposurethrough the home (often correlated
with marital status(Kurtz et al. 2003; Reynolds et al.
2004)) or social exposure(often correlatedwith age
(Reynoldset al., 2004)).
Although previous studies have reported higher
8OHdGin smokersthannon-smokers(Loft et al., IggZ)
and for workers in smoking comparedto smoke-free
workplaces(Howard et al., l99B), dose-dependent
effectsof ETS on either 8OHdG lesionsor other forms
of DNA-damagehave not been reportedin humans.It
hasbeenshownby HPLC with electro-chemical
detection methodthat SOHdG-DNAlesionsaresignificantly
higher in pooled male blood samplesthan pooled female blood samples,and that large geographicaldifferencesin damagelevelsoccur (Collins et al., 1998).
Our study is in agreementwith thesefindings regarding the gender-differencesin SOHdG-DNA adducts.
althoughall of our sampleswere from a similar geographicregion.Becausewe employeda Comet assay
approach,individual blood samplescould be assessed
(blood volume is the limiting factor for HPLCiECD
analysis),therebyincreasingthe power of the experiment.The advantages
of usingthe Cometassayinclude
the small number of cells required,the high sensitivity of the procedure(DNA-damagecausedby <5 cGy
gammarays canbe observed)and the economical,fast
and simplenatureof the assay(Rojaset al., 1999).The
disadvantages
of the Comet assayas it was usedhere
mostly relate to technicalvariability in scoringcells
and interpretationof results and the sample sizesper
assay,which havebeencriticisedfor causingsampling
bias.Theseconcernsare somewhatmitigatedby having all of the cell scoringperformedby onepersonand
scoring large numbersof cells (50 cells per determination,in triplicate)(Kobayashiet al., 1995;Rojaset
al., 1999).In addition,samplingbias relatesmore to
solid tissueswhere resultsmay be skewedby regionspecificdifferencesin DNA-damage.In a circulating
tissuesuchasblood,regio-specificityin DNA-damage
is lesslikely to be observedand unlikely to havebeen
a factor in this study. Finally, our manner of scoring
cells usingthe 0-4 scaleand assessing
damageby eye
has beenshownby previousinvestigatorsto be as accurate,more sensitiveand fasterthan computerimage
analysisand to correlatealmostexactlywith computertzed analysesof ratio (DNA% in tail) and tail moment
(productofratio andtail distance)whengradedconcentrations of known DNA damaging agentswere tested
and scoredby doubleblinded investigators(Kobayashi
et al ., 1995).
Sincemost ofthe damagecausedby cigarettesmoke
is believedto occur through DNA adducts,the power
of the Comet assayin detectingDNA-damagecaused
by smokingand/orETS hasbeenquestioneddueto the
A.C. Collier et al. / ToxicologyLetters l5B (2005) l0-19
fact that DNA crosslinks and adductsretardthe migration in the assay(Sram et al., 1998).Theseproperties
of DNA migrationunder electrophoresis
are likely to
be responsible,
at leastin paft, for many conflictingreportson DNA-damagecausedby cigarettesmokewhen
the Comet assaywas used as the only form of analysis and may be accountablefor the resultsof the lone
studywhere Comet assaysreportedlessDNA-damage
in smokersthan non-smokers(Speit et a1.,2003).The
pH of DNA unwinding and electrophoresisand the
lengthof time unwinding and electrophoresis
are performed are also critical to the amount of cometing observed.For example,as pH increasesso do the amount
and typesof DNA-damagedetected.Therefore,differencesin DNA-damagereportedby different laboratories could be due to variancein the pH of DNA unwinding and/orrunning buffers. In the presentstudies,
as well as using an enzymeto cleaveDNA at 8OHdGadductssites and comparing theseresultsto assaysin
the same participantswhere enzyme was not added,
unwindingwas carriedout at pH >13 where the assay
detectsthe most possibleforms of DNA-damage.Additionally,as discussedabove,our use of loadingand
running controlsenabledchangesin DNA migration
dueto the numberof cellsloadedand cometingcaused
by themethoditselftobe eliminatedasvariables.Moreover,becauseSYBR greenTMwas usedas the detector
fluorophore,the assayis extremelysensitive.This is
importantas a recent investigationof differencesin
DNA-damagebetweensmokersand non-smokersusing ethidiumbromideasa fluorophorereportedthatdetectionof smoke-induced
DNA-damagewas limited in
the Cometassay(Sramet al., 1998).When comparing
fluorophores,
ethidiumbromideshows3O-foldfluorescenceenhancement
uponbindingto DNA while SYBR
greenrMexhibits1000-foldfluorescence
enhancement
(MolecularProbes,2004\.
Despite equivalentworkplace ETS exposure,both
intracellular 8OHdG and general DNA-damage were
greaterin males than females,but there were no significantdifferencesbetweenmen and women in serum
levelsof extracellular(repaired)8OHdG. Some caution is neededin interpretingtheseresultsbecauseit is
possiblethat if theseexperimentswere expandedto a
largerpopulation,differencesin extracellularSOHdG
serum concentrationsbetween the men and women
would becomesignificant,asthe P-valuebetweenmen
and women in the current study was low (0.07). In
t7
addition,when discussingaspectsof DNA repair in
this study,it is important to note that DNA repair was
not measureddirectly, thus attributing differencesbetween DNA-bound 8OHdG lesions and free plasma
SOHdG to repair remainsa hypothesismeaningthat
concreteconclusionsregarding gender-relateddifferencesin DNA repair are difficult to substantiatewithin
the sizeandpower of this study.Despitethis, thesedata
may indicate gender-differencesin DNA-damage and
repair capacityand speculativelyprovide a rationale
for the higher incidencesof somesyndromes(notably
most cancersand heartdisease)in malescomparedto
females(Manton, 2000). Although women are more
susceptibleto certain forms of cancersuch as cancers
of the breast,these cancersare predominantlyhormonal and not mechanisticallygenotoxic(Wingo et
a1.,2003).Inaddition,althoughwomen haveovertaken
men with respectto incidenceof lung cancerssincethe
1980s,this has largely been attributedto behavioural
and social factorsnot biochemicalor cellular events
(Levi et al., 2004; Wingo et al., 2003). For cancers
where initiation is predominantlythrough genotoxic
and mutagenicmeans,such as liver, gastro-intestinal,
bladderand colo-rectalcancers,epidemiologicalstudies have uniformly reportedgreaterincidencesin men
thanwomenin both EuropeandUSA overthe lastcentury (Levi et al., 2004;Manton, 2000 p. I l; Wingo et
al., 2003).Recentresearchhas demonstrated
a constitutive polymorphismof the hOGGI genethat results
in reducedability to repair SOHdGlesions.This polymorphism is also associatedwith higher lung cancer
risk (Park et al., 2004).Also, Gackowskiet al. (2003)
have reportedthat levelsof 8OHdG in DNA isolated
blood of lung cancerpatientswas significantlyhigher
than that in DNA isolated from healthy smokersand
non-smokers(Gackowskiet a1.,2003).Itis, therefore,
possiblethat data presentedshowing higher BOHdG
lesionsin the blood of men exposedto ETS may be
associated
with lung cancerrisk.
The distinction between excisedbasesand intracellular,bound DNA-damage is particularly important
in terms of long-termaffectson homeostasis,
cell cycling and the developmentof disease.For example,
incompletebase-excisionrepair sites,which are detected in nuclear DNA by the Comet assayand likely
make up some of the generalDNA-damage observed
here, frequently leads to inappropriatetranscription,
cell cycling and metastasis(Gantt et al., l986). It is
l8
A.C. Collier et al. / TbxicologyLetters 155(2005) l0-19
difficult to say preciselyhow detrimentalthe general
DNA-damagedetectedhereinwill be in the long-term
becausedouble and single strandbreaks,incomplete
base-excision
repair sitesand alkali-labilesitesare all
detectedby the Comet assay,but their relativefrequenciesin the presentstudy are unknown. In contrast,a direct measureof the amount of 8OHdG-DNA-damage
was measuredand the amount of SOHdG in serum
(which is more indicativeof DNA repair capacity)was
also measuredproviding a direct gaugeof repair capacity.
ETS is known to be a carcinogen,pro-oxidantstressor and genotoxicagent.Here we haveshown a significant,positivecorrelationof greaterDNA-damagewith
increasingETS exposure.A directcorrelationbetween
ETS exposureandnon-8OHdGdamagewould be even
more conclusivein the presenceof either a control
group(suchasnon-smokers
not exposedto ETS) showing a lack of suchcorrelationor in a longitudinalgroup
showing increasingDNA-damage levels with time.
However,perhapsthe most interestingfinding was the
gender-specific
differencesin DNA-damageand potential differencesin repair observed.These results
provide a "snapshot" of the interplay betweenDNAdamageand ETS exposure,and along with other recent findings, further evidencethat gene-environment
interactionsof DNA repair enzymes may be important in cancerrisk and mutagensensitivity(Ito et al.,
2004; Tuimala et al., 2002). The dose-dependency
observedfor generalDNA-damage and ETS, suggestsa
dynamic equilibrium betweendamageand repair with
respectto ETS exposureovertime, while discrepancies
betweenhigher levels of intracellularDNA-damage
(both general and 8OHdG) in men than women but
comparableamountsof (repaired)serum8OHdG, may
imply genderdisparitiesin the expressionand activity of DNA repair enzymes.This study may provide
insight into the genotoxic, mutagenic and carcinogenicmechanismsof ETS and also may also help explain gender-differences
in the susceptibilityto cancer
and other diseasescausedor exacerbatedby tobacco
smoke.
Acknowledgement
Funding for this project was supplied by NIEHS
grantnumberES09520.
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