Effects of environmental air pollution on endogenousoxidative DNA damage in humans
Rajinder Singh a,∗, Balvinder Kaur a, Ivan Kalina b, Todor A. Popov c,Tzveta Georgieva c, Seymour Garte d,e, Blanka Binkova f,
Radim J. Sram f, Emanuela Taioli d, Peter B. Farmer a
a Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester, University Road, Leicester LE1 7RH, UKb Department of Medical Biology, Medical Faculty University P.J. Safarik, Kosice, Slovak Republic
c National Center of Public Health Protection, Sofia, Bulgariad University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA
e Genetics Research Institute (ONLUS), Milan, Italyf Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine,
AS CR, Prague, Czech Republic
Available online 3 March 2007
bstract
Epidemiological studies conducted in metropolitan areas have demonstrated that exposure to environmental air pollution is associ-ted with increases in mortality. Carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) are the major source of genotoxic activitiesf organic mixtures associated with respirable particulate matter, which is a constituent of environmental air pollution. In this study,weanted to evaluate the relationship between exposure to these genotoxic compounds present in the air and endogenous oxidativeNA damage in three different human populations exposed to varying levels of environmental air pollution. As measures of oxida-
ive DNA damage we have determined 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) by liquid chromatography–tandem masspectrometry (LC–MS/MS) and cyclic pyrimidopurinone N-1,N2 malondialdehyde-2′-deoxyguanosine (M1dG) by the immunoslotlot assay from lymphocyte DNA of participating individuals. The level of endogenous oxidative DNA damage was significantlyncreased in individuals exposed to environmental air pollution compared to unexposed individuals from Kosice (8-oxodG adducts)nd Sofia (M1dG adducts). However, there was no significant difference in the level of endogenous oxidative DNA and exposureo environmental air pollution in individuals from Prague (8-oxodG and M1dG adducts) and Kosice (M1dG adducts). The average
evel of M1dG adducts was significantly lower in unexposed and exposed individuals from Kosice compared to those from Praguend Sofia. The average level of 8-oxodG adducts was significantly higher in unexposed and exposed individuals from Kosice com-
rding to the interaction of c-PAHs exposure and smoking status was
ared to those from Prague. A significant increasing trend acco bserved in levels of 8-oxodG adducts in individuals from Kosice. However, no other relationship was observed for M1dG and-oxodG adduct levels with regard to the smoking status and c-PAH exposure status of the individuals. The conclusion that cane made from this study is that environmental air pollution may alter the endogenous oxidative DNA damage levels in humansut the effect appears to be related to the country where the individuals reside. Genetic polymorphisms of the genes involved in
Abbreviations: 8-oxodG, 8-oxo-7,8-dihydro-2′-deoxyguanosine; M1dG, cyclic pyrimidopurinone N-1,N2 malondialdehyde-2′-deoxyguanosine;c-PAHs, carcinogenic polycyclic aromatic hydrocarbons; LC–MS/MS, liquid chromatography–tandem mass spectrometry; SRM, selected reactionmonitoring; GLM, general linear model
Keywords: Oxidative DNA damage; Polycyclic aromatic hydrocarbon
1. Introduction
Epidemiological studies show that exposure of humanpopulations in metropolitan areas to environmental airpollution leads to an increased risk of developing lungcancer as well as cardiovascular diseases [1–5]. A majorconstituent of environmental air pollution is respirableparticulate matter of aerodynamic diameter <2.5 �m(PM2.5) as well as <10 �m (PM10). Carcinogenic poly-cyclic aromatic hydrocarbons (c-PAHs, (B[a]A, benzo[a]anthracene; CHRY, chrysene; B[b]F, benzo[b]fluoranthene; B[k]F, benzo[k]fluoranthene; B[a]P,benzo[a]pyrene; DB[a,h]A, dibenzo[a,h] anthracene;B[g,h,i]P, benzo[g,h,i]perylene; I[c,d]P, indeno[1,2,3-c,d]pyrene)) as well as nitrated c-PAHs such as 1-nitro-pyrene and 3-nitrobenzanthrone are a major genotoxiccomponent associated with this particulate matter,although other components include transition metalsand salts [6–11].
Formation of these components results from theincomplete combustion of fossil fuels and they are foundin vehicle exhaust emissions [6,12]. Extensive epidemi-ological studies have linked c-PAHs with adverse healtheffects in human populations. Studies in Poland and theCzech Republic have shown a link between increasedlevels of c-PAH DNA adducts in populations exposedto environmental air pollution with cytogenetic effects[13–15].
The mechanism of initiation of cancer by exposure toexogenous agents such as c-PAHs has been demonstratedin animal models and involves the covalent interactionof reactive metabolites with DNA leading to the for-mation of DNA adducts [16]. However, the mechanismof the adverse effects of environmental air pollution onhuman health has not been clearly elucidated as well asthe effect of exposure to exogenous agents on endoge-nous oxidative DNA damage. The amount of backgroundendogenously derived oxidative DNA damage is muchhigher than that caused by exposure to exogenous agentssuch as c-PAHs [17]. Endogenous DNA damage canarise from oxidative stress, which leads to the genera-
tion of reactive oxygen species that can interact directlywith DNA, in the case of hydroxyl radicals to form DNAadducts such as 8-oxo-7,8-dihydro-2′-deoxyguanosine(8-oxodG), or indirectly by causing lipid peroxidation
o-7,8-dihydro-2′-deoxyguanosine; Malondialdehyde
leading to the generation of malondialdehyde to formthe cyclic pyrimidopurinone N-1,N2 malondialdehyde-2′-deoxyguanosine (M1dG) DNA adduct (Fig. 1) [18].Formation of M1dG adducts can also occur indepen-dently from lipid peroxidation by base propenals, whichare generated by the hydroxyl radical mediated removalof the deoxyribose 4′-hydrogen in DNA [19]. Further-more malondialdehyde can be generated as a by-productof prostaglandin biosynthesis, which could potentiallybe another source for the formation of M1dG adducts[20]. The formation of 8-oxodG adducts can lead to theinduction of mutations, which mainly involve GC to TAtransversions and M1dG adducts have also been shown tobe mutagenic in bacterial and mammalian cells [21,22].
Evidence suggests that exposure to respirable partic-ulate matter associated with environmental air pollutionleads to adverse health effects resulting from the gen-eration of reactive oxygen species following oxidativestress [23–26]. Thus both 8-oxodG and M1dG adductsmay have a role in human carcinogenesis resulting fromexposure to environmental air pollution. The formationof 8-oxodG adducts was found to be positively cor-related with lung tumour incidence in mice that hadbeen exposed to diesel exhaust particles [27]. The mon-itoring of personal PM2.5 exposure correlated with thelevel of 8-oxodG adducts in human lymphocytes [28].An increased urinary excretion of 8-oxodG adducts wasobserved in bus drivers from central Copenhagen com-pared to drivers from suburban areas of the city [29].The mechanism of generation of reactive oxygen speciesleading to the formation of 8-oxodG may involve Fentonchemistry following inflammation caused by exposure torespirable particulate matter, which are associated withtransition metals such as iron [30,31]. Inflammation inlung tissue following exposure to respirable particulatematter can result in the influx of alveolar macrophages,which in turn can generate free radicals leading to oxida-tive stress [32]. It has also been proposed that respirableparticulate matter induced oxidative stress may alter cellsignalling pathways within the cell resulting in alteredgene expression and carcinogenesis [33]. An alternate
mechanism may involve the metabolism of c-PAHs asso-ciated with respirable particulate matter such as B[a]Pto a catechol, which can undergo redox cycling to aquinone resulting in the generation of reactive oxygen
R. Singh et al. / Mutation Research 620 (2007) 71–82 73
F odG) ad( ′-deoxyg
sot
ilmGtamatrDdfee
taifp((dca
ig. 1. (A) Formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxB) formation of cyclic pyrimidopurinone N-1,N2 malondialdehyde-2uanine in DNA.
pecies [34,35]. There is also evidence for the presencef semiquinone radicals in respirable particulate matterhat can induce oxidative DNA damage [36].
The factors that may affect the susceptibility of anndividual to the adverse effects of environmental air pol-ution are genetic polymorphisms in genes involved in
etabolism and detoxification such as CYP1A1, GSTM1,STT1 and NAT2 [26,37,38]. The antioxidant status of
he individual will also influence the development ofdverse health effects following exposure to environ-ental air pollution. An in vitro study concluded that
ntioxidants present in lung lining fluid provided pro-ection against the oxidative DNA damaging effects ofespirable particulate matter [39]. The importance ofNA repair in environmental air pollution induced DNAamage was highlighted by a recent study in mice, whichound that there was an up-regulation in DNA repairnzymes for oxidative damage in the lung followingxposure to diesel exhaust particles [40].
The aim of the work presented here was to evaluatehe relationship between exposure to environmentalir pollution and endogenous oxidative DNA damagen different human populations using data obtainedrom a large multicentric study (EXPAH) [41]. Theopulations monitored were city police from PragueCzech Republic), Kosice (Slovak Republic) and Sofia
Bulgaria). The Sofia population also included busrivers. A group of unexposed individuals such as officelerks were used as controls for each city. Particles in their were collected by stationary and personal monitors
ducts by the reaction of hydroxyl radicals with guanine in DNA andguanosine (M1dG) adducts by the reaction of malondialdehyde with
for determination of c-PAHs at each location and bloodsamples were obtained from participating individualsfor DNA adduct analysis. As measures of oxidativeDNA damage we determined 8-oxodG adducts by liquidchromatography–tandem mass spectrometry selectedreaction monitoring (LC–MS/MS SRM) and M1dGadducts by the immunoslot blot assay.
2. Materials and methods
2.1. Chemicals
8-oxodG, deoxyribonuclease I (from bovine pancreas) andsnake venom phosphodiesterase I (from Crotalus atrox) werepurchased from Sigma (Poole, UK). Shrimp alkaline phos-phatase was purchased from Amersham Pharmacia Biotech(Uppsala, Sweden). HPLC (electrochemical) grade methanoland HPLC grade acetic acid were purchased from Fisher Scien-tific (Loughborough, UK). HPLC grade water, 18.2 M� outputquality was obtained from Maxima purification equipment(Elga, High Wycombe, UK).
2.2. The study population
A total of 356 men were subjected to the study, and thecharacteristics of this population are shown in Taioli et al.[42]. Three groups of highly exposed individuals were selected
among city policemen from Prague (Czech Republic), Kosice(Slovak Republic) and Sofia (Bulgaria), who were usuallyworking through busy streets in 8–10 h shifts. In the caseof Sofia, bus drivers were also selected as highly exposedindividuals. Unexposed controls (non occupationally exposed
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74 R. Singh et al. / Mutatio
to traffic pollutants) such as office clerks were selected andmatched for age. The populations were followed in win-ter owing to the highest exposure during this season at theselected locations. In the period of sampling personal expo-sure monitoring (PM2.5 in which were determined c-PAHs)was carried out in all individuals during two successive shiftswith blood samples being collected at the end of the secondshift. Each individual also completed a questionnaire provid-ing demographic, smoking and dietary information. Ambientair exposure was measured by stationary monitoring during the12 months of the study.
2.3. DNA extraction
Lymphocytes were isolated from blood on a Ficoll-Paquegradient. The cells were washed twice in phosphate-bufferedsaline and then resuspended in 1.5 mL of extraction buffer (1%SDS, 10 mM EDTA, 20 mM Tris) with 30 �L of RNA-mix(10 mg/mL RNase A, 5000 U/mL RNase T1). After incubationat 37 ◦C for 1.5 h, 30 �L of proteinase K (20 mg/mL) was addedand suspension was incubated for a further 2 h. The DNA wasextracted using standard phenol/chloroform/isoamylalcoholprocedure. 8-hydroxyquinoline (used as an antioxidant) wasadded to extraction solutions to a final concentration of0.01%. The DNA was precipitated with cold (−20 ◦C)ethanol, air-dried and dissolved in appropriate amount ofHPLC grade water. DNA concentration was estimated spec-trophotometrically by measuring the UV absorbance at260 nm.
2.4. Enzymatic digestion of DNA
DNA samples (20 �g) to each of which was added4.0 pmol of the stable isotope internal standard [15N5]8-oxodG (1 pmol/�L) [43] were evaporated to dryness usinga centrifugal vacuum evaporator (Speedvac, Savant, Farm-ingdale, NY). The dried samples were dissolved in 20 �Lof 40 mM Tris–HCl, 10 mM MgCl2, pH 8.5 and incu-bated with 7.75 �L of snake venom phosphodiesterase I(0.0032 U/�L), 1.25 �L deoxyribonuclease I (4 U/�L) and1.0 �L shrimp alkaline phosphatase (1 U/�L) at 37 ◦C for 2 h.The digested DNA was centrifuged at 14,000 rpm for 10 min.The supernatant was injected onto the HPLC system describedbelow.
2.5. HPLC-UV enrichment of 8-oxodG adducts
The 8-oxodG was purified from the digested DNA byinjecting a 15 �L aliquot of the supernatant (equivalent to10 �g of digested DNA) onto a Waters HPLC system con-sisting of a Alliance 2690 attached to a 2487 UV detector
connected to a Hypersil (ThermoQuest Hypersil, Runcorn,UK) C18 BDS (250 mm × 4.6 mm, 5 �m) column plus HypersilC18 BDS, (10 mm × 4.6 mm, 5 �m) guard column. The columnwas eluted using a gradient at a flow rate of 0.8 mL/min withmobile phase A (methanol/HPLC grade water (5:95, v/v) and
arch 620 (2007) 71–82
mobile phase B (methanol). The following gradient was used:0 min–0%B, 30 min–0%B, 40 min–20%B and 45 min–0%B.The UV absorbance was monitored at 254 nm. The fractioncorresponding to where 8-oxodG should elute was collectedand evaporated to dryness using a centrifugal vacuum evapora-tor and redissolved in 20 �L of HPLC grade water for analysisby LC–MS/MS.
2.6. Determination of 8-oxodG adducts in DNA samplesusing LC–MS/MS SRM
The LC–MS/MS consisted of a Waters Alliance 2695 sep-arations module with a 100 �L injection loop connected toa Micromass Quattro Ultima Pt. (Micromass, Manchester,UK) tandem quadrupole mass spectrometer with an electro-spray interface. The temperature of the electrospray sourcewas maintained at 110 ◦C and the desolvation temperature at350 ◦C. Nitrogen gas was used as the desolvation gas (550 L/h)and the cone gas (30 L/h). The capillary voltage was set at3.20 kV. The cone and RF1 lens voltages were 42 and 20 V,respectively. The mass spectrometer was tuned by using a8-oxodG (10 pmol/�L) standard solution dissolved in 0.1%acetic acid/methanol (90:10, v/v) introduced by continuousinfusion at a flow rate of 10 �L/min with a Harvard model22 syringe pump (Havard Apparatus Ltd., Edenbridge, UK).A 10 �L aliquot (equivalent to 5 �g of digested DNA contain-ing 1 pmol of [15N5]8-oxodG) of the 8-oxodG purified fractionwas injected onto a HyPurity (ThermoQuest Hypersil, Run-corn, UK), C18, (1.0 mm × 150 mm, 3 �m) column connectedto a Uniguard C18 (1.0 mm × 10 mm, 3 �m) guard cartridgeattached to a KrudKatcher disposable pre-column (0.5 �m)filter. The column was eluted isocratically with 0.1% aceticacid/methanol (90:10, v/v) at a flow rate of 50 �L/min. The col-lision gas was argon (indicated cell pressure 2.0 × 10−3 mbar)and the collision energy set at 12 eV. The dwell time was setto 200 ms and the resolution was one m/z unit at peak base.The samples were analysed in positive electrospray ionizationMS/MS SRM mode for the [M + H]+ ion to base [B + H2]+
transitions of 8-oxodG (m/z 284–168) and [15N5]8-oxodG (m/z289–173). The level of 8-oxodG was determined in each sam-ple from the ratio of the peak area of 8-oxodG to that of theinternal standard [15N5]8-oxodG. The level 8-oxodG was nor-malised to the amount of 2′-deoxyguanosine (dG) in nmolobserved for each sample following DNA digestion as deter-mined from the HPLC-UV purification step, using calibrationlines of authentic dG standards. A linear response was obtainedfor a calibration line constructed by spiking calf thymus DNA(20 �g) with [15N5]8-oxodG (final amount on column rang-ing from 12.5 to 4000 fmol per 5 �g digested DNA) that hadbeen subjected to the whole procedure (correlation coefficientof 0.998). The average coefficient of variation of the methodwas 11.9%, which was obtained by the analysis of a series
of calf thymus DNA samples. The limit of detection for theLC–MS/MS was 12.5 fmol on column (with a signal to noiseratio of 3.4), which was obtained by spiking calf thymus DNAwith the internal standard [15N5]8-oxodG and subjecting it tothe entire procedure.
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MPdDaDffi(let
2
(a(hbtcopomtwcdosv(pitm
bt0sp
3
3
a
HPLC-UV chromatogram obtained for a digested DNAsample and Fig. 2B shows the typical ion chromatogramsobtained following LC–MS/MS SRM analysis. Fig. 2Cshows a typical image of the immunoslot blot filter fol-
Fig. 2. (A) Typical HPLC-UV chromatogram obtained for the enrich-ment of 8-oxodG adducts from a digested DNA sample. The arrowindicates the fraction collected corresponding to where the 8-oxodGadduct elutes (dC, 2′-deoxycytidine, dG, 2′-deoxyguanosine, T, thymi-dine and dA, 2′-deoxyadenosine) and (B) determination of 8-oxodGadducts in lymphocyte DNA by LC–MS/MS SRM following HPLC-UV enrichment. Typical LC–MS/MS SRM ion chromatogram for8-oxodG (transition m/z 284–168) and the stable isotope internal stan-dard [15N5]8-oxodG (transition m/z 289–173) obtained from 5 �gof digested DNA. (C) Determination of M1dG adducts in lympho-cyte DNA samples by immunoslot blot analysis. An image of theimmunoslot blot filter following incubation with chemiluminescent
R. Singh et al. / Mutatio
.7. Determination of M1dG adducts in DNA samplessing the immunoslot blot assay
The immunoslot blot assay was carried out using a murine1dG monoclonal primary antibody (D10A1), provided by
rof. Lawrence Marnett (Vanderbilt University, TN, USA), asescribed previously [44]. The level of M1dG adducts in theNA samples was determined from the calibration line gener-
ted by the dilution (with control DNA) of standard calf thymusNA containing known amounts of M1dG adduct (ranging
rom 0 to 10 fmol M1dG per �g DNA) pipetted onto the samelter. The Syngene ChemiGenius2 image acquisition systemSynoptics Ltd., Cambridge, UK) was used to capture a chemi-uminescencent image of the filter. The level of the adduct inach sample was corrected for the amount of DNA bound tohe filter as determined by propidium iodide staining.
.8. Statistical analysis
Data are presented as means and standard deviationsS.D.) for continuous variables (DNA adducts levels, age),nd as frequencies and percentages for categorical variablessmoking status, country, exposure to c-PAH). Smoking statusas been defined according to cotinine levels: individuals haveeen defined as smokers when cotinine levels (adjusted byhe creatinine levels) were greater than 500 ng cotinine/mg ofreatinine. Some analyses were stratified according to countryf origin; some other analyses were conducted on the overallopulation. In this latter case, the adjustment for country ofrigin of the subjects created problems of collinearity, sinceost of the highest adducts levels were measured in one coun-
ry, irrespectively of the exposure status of the subjects, andere highest than in both exposed and non exposed from other
ountries. To avoid this problem, adducts values were stan-ardized by dividing each value for the average adducts levelsf the respective country. When necessary, adducts levels werequare root transformed in order to have normally distributedalues. Comparison of DNA adducts levels between groupsnamely country, smoking status, and exposure to c-PAH) waserformed using t-test and analysis of variance (ANOVA) des-gnated by p*. To analyze the independent factors contributingo DNA adducts levels, a multivariate analysis (general linear
odel, GLM) was performed, and the significance designated
y p§. The model included country, smoking status, exposureo c-PAH and was adjusted by age. p-Values lower than.05 have been considered as statistically significant. All thetatistical analyses have been performed using SAS statisticalackage (8.1 Version, SAS Institute Inc., Cary, NC).
. Results
.1. Determination of 8-oxodG and M1dG adducts
Previously we have reported the development ofsensitive immunoaffinity column purification and
arch 620 (2007) 71–82 75
LC–MS/MS SRM method for the determination of 8-oxodG adducts in DNA samples [43]. However, to speedup the analysis time of the DNA samples we devel-oped a HPLC-UV enrichment method to replace theimmunoaffinity column purification step. The new pro-cedure again involved the enzymatic digestion of DNAto 2′-deoxynucleosides followed by removal of unmod-ified 2′-deoxynucleosides from 8-oxodG adducts byHPLC-UV allowing the enriched 8-oxodG adducts tobe determined by LC–MS/MS. Fig. 2A shows a typical
reagents. The intensity of each band is directly proportional to thelevel of M1dG adducts present. The DNA samples (1 �g) were appliedin triplicate to the filter (QC represents quality control DNA, whichwas used ensure that results are consistent from one blot to anotherover time).
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lowing incubation with chemiluminescent reagents forthe determination of M1dG adducts in DNA samples.The intensity of each band was directly proportional tothe level of M1dG adducts present.
3.2. Endogenous oxidative DNA damage accordingto country, exposure to c-PAHs and smoking status
The relationship of endogenous oxidative DNA dam-age levels to country of origin, smoking status andexposure to c-PAHs is summarised in Table 1. A highlysignificant difference (p* < 0.0001) was observed forthe average level of M1dG adducts in individuals fromKosice (18.9 adducts per 108 nucleotides) compared tothose from Prague and Sofia (34.8 and 37.9 adductsper 108 nucleotides, respectively). A significant dif-ference (p* = 0.02) was observed for the average levelof 8-oxodG adducts in individuals from Prague (47.8adducts per 106 nucleotides) compared to those fromKosice (59.4 adducts per 106 nucleotides). Total M1dGand 8-oxodG adduct levels did not differ for the threecountries according to smoking or c-PAH exposurestatus of the individuals. However, levels of 8-oxodGadducts (58.3 versus 49.2 adducts per 106 nucleotides,p = 0.06) and M1dG adducts (33.0 versus 29.2 adductsper 108 nucleotides) were higher in exposed individu-als compared to unexposed individuals. Similarly levels
of 8-oxodG adducts (66.6 versus 50.9 adducts per106 nucleotides) and M1dG adducts (32.8 versus 30.4adducts per 108 nucleotides) were higher in smokerscompared to non smokers.
Table 1Endogenous oxidative DNA damage according to country, exposure to c-PAH
Smoking status n.s.Smokers 137 32.8 30.5Non smokers 212 30.4 23.8
–, not determined due to problems encountered with the enzymatic digestiona Adducts per 108 nucleotides.b Adducts per 106 nucleotides.c Results not obtained for two samples.* Univariate analysis (t-test or ANOVA).§ Multivariate analysis (GLM). The model included all the covariates (coun
arch 620 (2007) 71–82
Since the geographic origin of the subjects was play-ing a major role in the observed differences in biomarkerlevels, we wanted to further investigate if the differ-ences were still present independently from where thestudy was conducted. In order to do this, the results werestandardised across countries by dividing each value bythe average adduct level of the corresponding country(Table 2). In this analysis of the data, c-PAH exposurestatus was also defined according to the personal moni-tor measurements: individuals were defined as exposedwhen personal exposure levels were greater or equalthan 7.55 ng/m3 (median of the personal exposure lev-els in controls), and unexposed when personal exposurelevels were lower than 7.55 ng/m3. No significant differ-ences were observed in the levels of M1dG or 8-oxodGadducts across countries and according to c-PAHs expo-sure and to smoking status both at the univariate and atthe multivariate analysis.
3.3. Association between endogenous oxidativeDNA damage, c-PAH exposure and smoking statusaccording to country
The relationship between endogenous oxidative DNAdamage levels, smoking status and exposure to c-PAHsaccording to country is summarised in Table 3. Exposed
individuals from Kosice had significantly higher lev-els of 8-oxodG adducts than unexposed individuals(73.3 adducts per 106 nucleotides and 47.8 adducts per106 nucleotides, respectively, p* = 0.0005, p§ = 0.0004).
s and smoking status
p§ 8-oxodGb p* p§
N Mean S.D.
203 53.6 34.2
0001 n.s. 0.02 n.s.102 47.8 30.8101 59.4 36.6
– – –
n.s. 0.06 n.s.98 58.3 37.5
105 49.2 30.3
n.s. n.s. n.s.75 66.6 36.5
128 50.9 32.6
of the DNA; n.s., not significant.
try, smoking and c-PAH exposure) and has been adjusted by age.
R. Singh et al. / Mutation Research 620 (2007) 71–82 77
Table 2Endogenous oxidative DNA damage according exposure to c-PAHs and smoking status—standardised data
n.s., not significant.* Univariate analysis (t-test or ANOVA).§ Multivariate analysis (GLM). The model included all the covariates (smoking and c-PAH exposure) and has been adjusted by age.
Table 3Association between endogenous oxidative DNA damage, c-PAH exposure and smoking status according to country
M1dGa p* p§ 8-oxodGb p* p§
N Mean S.D. N Mean S.D.
Prague (Czech Republic)Occupational exposure to c-PAHs n.s. n.s. n.s. n.s.
–, not determined due to problems encountered with the enzymatic digestion of the DNA; n.s., not significant.a Adducts per 108 nucleotides.b Adducts per 106 nucleotides.* Univariate analysis (t-test).§ Multivariate analysis (GLM) adjusted by age.
78 R. Singh et al. / Mutation Research 620 (2007) 71–82
Table 4Effect on the endogenous oxidative DNA damage of the interaction between smoking status and occupational exposure to c-PAHs
Prague (Czech Republic) Kosice (Slovakia) Sofia (Bulgaria)
M1dGa 8-oxodGb M1dGa 8-oxodGb M1dGa
N Mean S.D. N Mean S.D. N Mean S.D. N Mean S.D. N Mean S.D.
a Adducts per 108 nucleotides.b Adducts per 106 nucleotides.* Results from the general linear model (GLM). The model include
Significant higher levels of M1dG adducts were alsoobserved in exposed individuals compared to unexposedindividuals from Sofia (41.5 adducts per 108 nucleotidesand 31.2 adducts per 108 nucleotides respectively,p* = 0.035, p§ = 0.04). No significant difference in thelevel of M1dG adducts between unexposed and exposedindividuals from Prague and Kosice and the level of8-oxodG adducts between unexposed and exposed indi-viduals from Prague was observed.
3.4. Effect on endogenous oxidative DNA damageof the interaction between smoking status andoccupational exposure to c-PAHs
The effect on endogenous oxidative DNA damagelevels of the interaction between smoking status andexposure to c-PAHs according to country is sum-marised in Table 4. There was a significant difference(p* = 0.002) observed for 8-oxodG adducts betweennon smokers and unexposed (41.5 adducts per 108
nucleotides)/exposed (54.2 adducts per 108 nucleotides)for Kosice individuals and smokers and unexposed (71.4adducts per 108 nucleotides)/exposed (75.3 adducts per108 nucleotides). However, no other relationship wasobserved for M1dG and 8-oxodG adduct levels withthe smoking status and c-PAH exposure status of theindividuals from the three countries.
4. Discussion
All healthy individuals have a background levelof endogenously-derived oxidative DNA damage. The
ng status, c-PAH exposure, country and age.
extent of this background DNA damage is much higherthan the DNA damage caused by exposure to exoge-nous agents such as c-PAHs found in environmental airpollution. There are conflicting reports concerning thespecificity of using adducts measured in the DNA of totalwhite blood cells as surrogates for DNA adduct levels inthe target tissue following exposure to genotoxic chem-icals. However, analysis of DNA obtained followingfractionation of total white blood cells into lymphocytesand monocytes significantly improves this association[45]. Numerous studies have been published showingthat the levels of 8-oxodG adducts are increased in thecellular DNA as well as in the urine of cancer patients[46]. There are also numerous reports of the associa-tion of increased levels of 8-oxodG adducts in humansfor different non cancerous pathological conditions suchas diabetes, cardiovascular and neurodegenerative dis-eases [47]. Similarly M1dG adducts have been detectedin DNA from a variety of human tissues such as liver,pancreas breast, colorectal mucosa and gastric mucosa aswell as from white blood cells but as yet an obvious linkbetween elevated levels of the adduct to a particular dis-ease state has not been clearly established [48–51]. How-ever, it was found that malondialdehyde-derived DNAadduct levels were elevated in normal breast tissue frombreast cancer patients compared to tissues from non can-cer patients undergoing reduction mammoplasty [52].
Both M1dG and 8-oxodG adducts serve as biomark-ers of oxidative DNA damage but mechanistically their
formation appears to be quite distinct. M1dG adductsare formed indirectly by free radical mediated lipid per-oxidation generation of malondialdehyde, which reactswith guanine in DNA. In contrast 8-oxodG adducts are
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R. Singh et al. / Mutatio
ormed by the direct interaction of hydroxyl radicalsith the C-8 position of guanine in DNA [18]. There
s evidence that M1dG adducts may also be formed byase propenals reacting with guanine in DNA, whichesult from hydroxyl radical attack on the deoxyribose19]. The main problem encountered for the measure-ent of 8-oxodG adducts in DNA samples is artefactual
eneration of the adduct during sample manipulations part of the detection method, which may ultimatelyive rise to misleading results [53]. To minimise anyrtefactual formation of 8-oxodG adducts, we added thentioxidant 8-hydroxyquinoline to the buffers used forxtraction of the DNA from the blood samples [54]. Alsohe enrichment of the 8-oxodG adducts using HPLC fromhe unmodified deoxynucleosides in particular removalf 2′-deoxyguanosine should have helped to diminishny artefactual formation of 8-oxodG adducts prior tonalysis by LC–MS/MS. In contrast, no evidence forhe artefactual formation of M1dG adducts has beeneported, so in theory it should represent a more reliableiomarker for the assessment of endogenous oxida-ive DNA damage when compared to 8-oxodG adducts48,55].
The c-PAH components of environmental traffic airollution have the potential to generate free radicalpecies by causing inflammation or undergoing redoxycling once absorbed by the human body [32,34]. In thistudy, we wanted to assess the modulation of endoge-ous oxidative DNA damage in city policemen fromrague (Czech Republic), Kosice (Slovak Republic) andofia (Bulgaria) following exposure to c-PAHs present
n traffic air pollution by determining the level of 8-xodG and M1dG adducts in DNA obtained from theymphocytes of participating individuals. In the case ofofia the levels of oxidative DNA damage was alsoetermined in bus drivers. The results obtained wereompared to individuals that were non occupationallyxposed to traffic pollutants such as office clerks, whoere matched for age. Overall, there were large inter
ndividual variations observed in the levels of M1dGnd 8-oxodG adducts for all the groups from the threeountries. The results obtained showed that there was aignificant higher level of 8-oxodG adducts in exposedndividuals from Kosice compared to unexposed indi-iduals. These findings were similar to those observedy Loft et al. [29] who showed that there was an increasen urinary excretion of 8-oxodG adducts in bus driversrom central Copenhagen compared to bus divers from
ural/suburban greater Copenhagen. A similar obser-ation of elevated levels of 8-oxodG adducts in therine was made for taxi drivers in Taiwan [56]. Theevels of 8-oxodG adducts in white blood cells were
arch 620 (2007) 71–82 79
reported to be higher in coke oven (1.38 times) andgraphite electrode producing workers (2.15 times) whencompared to individuals that were not occupationallyexposed to c-PAHs [57]. A significantly higher level ofM1dG adducts was also observed in exposed individualsfrom Sofia compared to unexposed individuals. How-ever, there was no significant difference in the level ofM1dG adducts between unexposed and exposed individ-uals from Prague and Kosice and the level of 8-oxodGadducts between unexposed and exposed individualsfrom Prague. No significant differences were observedin the levels of M1dG and 8-oxodG adducts accordingto the smoking status (i.e. smokers and non smokers) ofindividuals for all three countries. These findings werein contrast to those obtained by Lodovici et al. [58] whoobserved a significant increase in the level of 8-oxodGadducts in white blood cell DNA from smokers com-pared to non smokers and former smokers. Individualswho have been exposed to environmental tobacco smokein the workplace also have increased levels of 8-oxodGadducts in white blood cell DNA compared to unexposedindividuals [59]. However, the findings from our studywere in agreement with those obtained by Zhang et al.[60] who showed there was no effect of smoking statuson 8-oxodG adduct levels in white blood cell DNA fromcoke-oven workers. A similar observation was made byHarman et al. [61], who showed that there was no corre-lation between smoking status and the urinary excretionof 8-oxodG adducts in healthy individuals. Van Zeelandet al. [62] found that there was an inverse relationshipbetween the level of 8-oxodG adducts in white bloodcell DNA from healthy individuals and lifetime smok-ing. A significant increase was observed in this studyfor the level of 8-oxodG adducts in smokers and unex-posed/exposed individuals from Kosice compared to nonsmokers and unexposed/exposed individuals. No othersignificant relationship was observed for M1dG and 8-oxodG adduct levels with the interaction of smokingstatus and c-PAH exposure status of the individuals fromthe three countries. M1dG adducts have been detected inDNA from a variety of human tissues as well as fromwhite blood cells but as yet the consequences of smok-ing status on M1dG adduct levels has not been clearlyascertained. No difference in the levels of M1dG and 8-oxdG adducts was observed by Kadlubar et al. [54] inpancreatic DNA obtained from non smokers and smok-ers. However, Zhang et al. [63] have shown using animmunohistochemical detection method that the levels
of M1dG adducts were elevated in oral mucosa cells fromsmokers compared to those from non smokers.
An interesting finding from this study was the dif-ference in oxidative DNA damage between countries
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for individuals. A highly significant lower average levelof M1dG adducts in unexposed and exposed individu-als from Kosice was observed, compared to those fromPrague and Sofia. This observation may be explainedby differences in diet between the populations, resultsobtained by Fang et al. [64] showed that the levelof M1dG adducts was 3.6 times higher in individ-uals who were given a diet rich in polyunsaturatedfatty acids compared to those individuals on a dietrich in monounsaturated fatty acids. The results of theEXPAH study on diet will be presented elsewhere. Incontrast, the average level of 8-oxodG adducts was sig-nificantly higher in unexposed and exposed individualsfrom Kosice compared to those from Prague. Similarfindings were reported by Collins et al. [65] when theymeasured 8-oxodG adducts in lymphocyte DNA, whichwere about three-fold higher in men from France andSpain compared to those from Ireland and the UnitedKingdom. These results may be explained by differencesin the regulation of cellular defense mechanisms againstgenotoxic damage between populations. Experiments inmice, exposing them to diesel particles lead to the con-clusion that there may be thresholds for inflammationassociated genotoxic effects of diesel particles due tothe presence of defence mechanisms against inflamma-tion [66]. Microarray gene expression studies with ratalveolar macrophages have shown that there is an upregulation of antioxidant enzymes following exposureto organic extracts of diesel particles [67].
In conclusion, the results from this study suggestthat environmental air pollution may alter the endoge-nous oxidative DNA damage levels in humans but theeffect appears to be related to the country where theindividuals reside. The variation observed in the levelof endogenous oxidative DNA damage for the differentpopulations may be explained by genetic polymorphismsof the genes involved in metabolism and detoxificationand also differences in the DNA repair capacity andantioxidant status of the individuals, as well as by thedifferent spectrum of c-PAH constituents in the pollutedair [68].
Acknowledgements
The European Commission “Quality of life andmanagement of living resources” program (QLK4-2000-
00091) and the Medical Research Council (G0100873)are gratefully acknowledged for financial support. Eliza-beth Wright and Friederike Teichert are thanked for helpwith analysis of samples. Prof. L.J. Marnett is thanked forsupplying the monoclonal primary antibody for M1dG.
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