[CANCER RESEARCH 58. 4122-4126. September 15. 1998]

Serum Antibodies to Benzo(a)pyrene Diol Epoxide-DNA Adducts in the General

Population: Effects of Air Pollution, Tobacco Smoking, and Family Historyof Lung Diseases1

Stefano Petruzzelli,2 Alessandro Celi, Nolita Fulera, Filomena Baliva, Giovanni Viegi, Laura Carrozzi,

Gigliola Ciacchini, Matteo Bottai, Francesco Di Pede, Paolo Paoletti, and Carlo Giuntini

Respiratory Pathophysiology Unit, Cardiopulmonary Department, University of Pisa [S. P., A. C., N. P.. F. B., L. C., F. D. P., P. P., C. C./: Institute of Clinical Physiology.National Research Council ¡C.V.. M. B.¡:and Environmental Protection Unii ¡C.C.¡,Pisa, Italy

ABSTRACT

Polycyclic aromatic hydrocarbons (PAHs) are almost ubiquitous pollutants that may interact with metabolic systems in human tissues andeventually cause cancer. PAH-adducted DNA becomes antigenic and antibodies anti-benzo(a)pyrene diol epoxide (BPDE)-DNA may be found inserum of PAH-exposed subjects. The presence of serum antibodies anti-BPDE-DNA adduct was investigated in 1345 individuals from family

clusters of the general population of a small area in central Italy in whominformation about smoking habits, site of residence, and personal andfamily history of lung diseases, including cancer, were obtained. Anti-BPDE-DNA antibodies in the sera were detected with a direct ELISA andthe association of anti-BPDE-DNA antibodies with subjects' data from a

standardized respiratory questionnaire including age, occupation, tobaccosmoking habits, respiratory symptoms, and family history of respiratorydiseases was subsequently tested by multivariate logistic regression analysis. The overall prevalence of subjects with anti-BPDE-DNA antibodieswas 21.0% at = 283), with no differences between males and females.Anti-BPDE-DNA positivity was associated with living in the urban area[odds ratio (OR), 1.49; 95% confidence interval (CI), 1.16-1.92], withactive tobacco smoking (OR, 1.25; 95% CI, 1.06-1.48), and with familyhistory of lung cancer (OR, 1.30; 95% CI, 0.90-1.88), and positivity

increased with the number of members in the family cluster positive toanti-BPDE-DNA antibodies (OR, 1.30; 95% CI, 1.03-1.65). This study ona large general population sample indicates that serum anti-BPDE-DNA

antibodies may be considered as biomarkers of exposure to environmentalcarcinogens and of DNA damage. The genetic and familial components oftheir association with tobacco smoking lend further support to the argument about the familial predisposition to lung cancer.

INTRODUCTION

Airborne pollutants produce a wide variety of harmful effects onhuman health, with the lungs as the primary target (1). PAHs3 are

almost ubiquitous pollutants due to coal combustion, gasificationemissions, and tobacco smoke that may interact with different metabolic systems in human tissues and cells and eventually cause cancer(2-4). Identification of genetic polymorphisms in carcinogen-metab

olizing enzymes by means of modern molecular epidemiology techniques has been proposed for cancer risk assessment in individuals(5). Most of the more recent studies focused on molecular and geneticend points in environmental epidemiology studies, although they havebeen necessarily restricted to relatively small groups of individuals.

Received 2/26/98; accepted 7/17/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by funds from CNR Special Project "Applicazioni Clinichedella Ricerca Oncologica" and CNR-ENEL Project "Interactions of the Energy Systemwith Human Health and Environment," Rome. Italy.

2 To whom requests for reprints should be addressed, at Respiratory PathophysiologyUnit. Cardiopulmonary Department. University of Pisa, Ospedale "Cisanello", via Para

disa 2, 56124 Pisa, Italy. Phone: 39-50-996946; Fax: 39-50-580126; E-mail: slepetru@nsifc.ifc.pi.cnr.it.

' The abbreviations used are: PAH. polycyclic aromatic hydrocarbon; BPDE, benzo-

(«(pyrene diol epoxide: NMHC. nonmethanic hydrocarbon; OR, odds ratio; Cl, confidence interval.

either from the general population or exposed at the workplace,because of the complexity of the laboratory techniques (6, 7). Someyears ago, our group contributed to organize and carry out in Italy ajoint project on the effects of air pollution on human health (8).Among the end points of this epidemiológica! study on a generalpopulation living in the urban-suburban area of Pisa, we proposed to

detect in each individual the presence of serum antibodies against theDNA adduct of BPDE, the ultimate carcinogenic metabolite of ben-

zo(a)pyrene (taken as representative of the PAH family), which havebeen detected previously in coke oven workers and in 43% of a smallgroup of healthy volunteers (9-11). At the same level of immune

response, these autoantibodies may be considered as the ultimatebalance between the biologically effective dose of the carcinogen andthe DNA repair mechanisms. Because they can be determined moreeasily than DNA adducts as indicators of DNA damage (12), evenafter the exposure period and the possible DNA repair processes, theycould be used in relatively large numbers of individuals for environmental and personal monitoring (13). In the present study, circulatinganti-BPDE-DNA antibodies were determined for the first time, to our

knowledge, in an epidemiological investigation on a large generalpopulation sample. The study design can be defined as "transitional"

according to a recent classification based on a matrix of biologicalmarkers and study designs (14). Specifically, in transitional studies abiomarker is used as outcome variable in studies of generally healthypeople: to this purpose, it is taken from the laboratory and tested infield settings to determine correspondence with exposure, disease, orsusceptibility. The design of our study allowed us to test the validityof the use of anti-BPDE-DNA antibodies as indicators of PAH-

derived genotoxic damage in a very large sample of subjects from thegeneral population of a small area in central Italy in whom information about smoking habits, site of residence, and personal and familyhistory of lung diseases, including cancer, all of which have beenassociated with DNA damage or higher risk for lung cancer (6,15-17), was obtained.

MATERIALS AND METHODS

Study Subjects. The original population sample was a family-based, mul

tistage, stratified, cluster of 3866 individuals living in the urban area of Pisaand in the suburban zone of Cascina, under investigation in 1985-1987 for the

health effects of outdoor air pollution, mainly from heavily used motorwaysand industrial settings. Details on subject selection and methods of subjects'

data recording have been reponed previously (18, 19). Of the 3866 people ofthe first survey, 2841 were followed-up 8 years later. The other subjects had

either moved out, died, or refused to participate in the second survey. Thosewho dropped out were not significantly different from those who came thesecond time for age. weight, height, smoking habits, prevalence of cough, orphlegm. From this sample, 1688 individuals were randomly assayed for thepresence of circulating antibodies and anti-BPDE-DNA adducts, and 1345

individuals with at least another member of the family cluster in whom theassay could be performed were finally tested for the associations of anti-BPDE-DNA antibodies with questionnaire variables. Sera were obtained from

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10-ml samples of peripheral blood and stored at -70°C until assayed. Sera

were tested blindly (a complete lack of awareness of any other variable).Questionnaire. Before blood sampling, each individual completed an on

line, computer-guided questionnaire for collection of personal data, residence,

occupation, socioeconomic status, smoking habit, respiratory symptoms, andpersonal and family history of respiratory diseases (19). For the statisticalanalysis, we defined as urban residence the residence within the territoriallimits of the city of Pisa and suburban residence the residence outside of theselimits (actually eastwards). Nonsmokers were those subjects who have neversmoked; ex-smokers those who have formerly smoked regularly until 6 months

or more prior to the examination, and smokers were those subjects who havebeen smoking at least one cigarette a day within the last 6 months. Respiratorysymptoms were considered when chronic cough, phlegm, or dyspnea were

reported. Familial chronic bronchitis, emphysema, or lung cancer was presentwhen reported in one or more relatives (first to fourth degree of relationshipwith the index subject); family history of primary lung cancer was consideredas prevailing in hierarchy in comparison with familial chronic bronchitisand/or emphysema, as suggested previously (16). The administered questionnaire included neither general questions about diet type nor specific questionsabout consumption of cooked foods (charcoal-broiled beef, smoked meat/fish)

and of vegetables and fruit (as an estimate of vitamins and micronutrientsintake). However, unpublished data from the local authorities on the dietarypatterns did not reveal substantial differences between urban and suburbandwellers.

Monitoring of Air Pollutants. During the period of data collection, monitoring of air pollutants was also carried out at two fixed sites, one in the urbanarea and one in the suburban area. Gaseous pollutants, such as nitrogendioxide, sulfur dioxide, total nonmethanic hydrocarbons (NMHCs), weremeasured hourly by automatic instrumentation, and total suspended particu-

lates were measured daily by a beta counter.Detection of BPDE-DNA Antibodies. Sera were assayed by a modified

version of the ELISA published previously (11). Briefly, BPDE-DNA was

synthesized by adding 1 mg of BPDE (Carcinogen Reference Standard Repository, Bethesda, MD) to 10 mg of calf thymus DNA (Sigma-Aldrich.

Milano, Italy). The level of DNA modification after enzymatic digestion todeoxyribonucleotides and HPLC and UV spectrometry was 1.7% (20). Thenonmodified and BPDE-modified DNA were used as the standard antigens.

They were attached to Polyvinylchloride microtiter plates (Costar, Cambridge.MA) by drying at 37°Cat the concentration of 20 ng/well in 50 /xl of 20X SSC

buffer (3 M NaCl, 0.3 M sodium citrate, pH 7.0). Wells coated with thenonmodified DNA or without DNA (SSC buffer alone) were used as controls.DNA-coated plates were repeatedly washed to remove salt crystals and incu

bated for 60 min at room temperature with 100 /j.1of PBS containing 3% (v/v)normal goat serum (PBS-normal goat serum). The presence of the test antigenin the solid phase was further ascertained by the direct ELISA with a hyper-immune rabbit anti-BPDE-DNA antiserum (generous gift from Dr. Curtis C.

Harris, Laboratory of Human Carcinogenesis, National Cancer Institute, NIH,Bethesda, MD) at 1:5,000 to 1:2,560,000 serum dilutions. Selected serumsamples were further tested by competitive ELISA to confirm the presence ofBPDE-DNA antibodies. This was performed by competing the binding ofantibodies to BPDE-DNA of a given test serum with modified and nonmodi

fied DNA at various concentrations. The test serum was diluted to the concentration at which binding was maximal. These antigen/antibody mixtureswere then tested as described above for the direct binding ELISA againstsolid-phase modified and nonmodified DNA. The results have been expressed

as percentage of inhibition as reported (11). The use of competitive ELISA ona routine basis for each single serum was considered not mandatory andexceedingly cumbersome in a large epidemiological study. Sera were tested intriplicate (50 jiil/well) after 60 min incubation at 1:25, 1:125, and 1:625dilutions with PBS-normal goat serum solution. The binding of human immu-noglobulins was detected using a biotinylated goat anti-human immunoglob-ulin and the avidin-biotin horseradish-peroxidase system (Sigma-Aldrich). Theenzyme reaction was developed by addition to each well of 50 ;u.l of O-

phenylendiamine/0.05 M citrate buffer at pH 4.0 containing 0.5 (xl/ml H^O,30%. The enzymatic reaction was stopped after a 20-min room-temperature

incubation by adding 50 ftl of 2 M H2SO4 to each well. Plates were read usingan automatic ELISA reader (Titertek Multiskan), and the absorbance at 492 nmwas recorded. The reaction was defined as positive if, in one dilution at least,m, > m,. + 2 VS.,2 + S,2, where ms is the mean value of the sample, m^ is the

mean value of the control, 5S2 is the sample variance, and 5L.2is the control

variance. A random sample (n = 285) of the tested sera was re-assayed for

reproducibility throughout the study period, and discordant cases were analyzed again, giving the final outcome on the basis of the best-of-three result.

Statistical Analysis. The 1345 individuals that were analyzed come from537 families randomly selected from 25 strata (mean family size, 2.5 members;family size range. 2-5 members). The ratio of the number of sampled indi

viduals in any stratum to the total number of subjects in that stratum wasconstant. The analyses were carried out taking the complex sampling designinto account. Logistic regression models have been applied to relate theprobability of being positive for anti-BPDE-DNA antibodies to the prevalence

of residence in the urban area, tobacco smoking, passive smoking, familyhistory for chronic bronchitis, emphysema, and lung cancer, and of otherfamily members with anti-BPDE-DNA antibodies. The prevalence of otherfamily members positive for anti-BPDE-DNA antibodies was computed foreach subject as the ratio between the number of members positive for anti-BPDE-DNA antibodies (other than the subject) and the total number of familymembers for whom anti-BPDE-DNA antibodies were measured (other than the

subject). Given the sampling method based on independent family cluster,robust estimates for the variance of the regression parameters were provided byHuber's formula, which uses the so-called "sandwich estimator" for the variance of the coefficients' estimates. All second-order interactions were tested

and dropped out of the model when not significant. Urban residence, passivesmoking, family history of chronic bronchitis, of emphysema, and of lungcancer were entered into the model as dichotomous categorical variables.Tobacco smoking and the presence of other family members with serumanti-BPDE-DNA antibodies categorical variables were initially introduced in

the logistic regression models by dummy variables. The coefficients were thentested for linear trend and score variables (tobacco smoking: 0. nonsmoker; 1,ex-smoker; 2, smoker; other family members with anti-BPDE-DNA antibod

ies: 0, no; 1, £50%: and 2, >50%) were introduced to gain power for the test

of significance for the estimates of the regression coefficients.All of the variables listed in Table 1 were considered in the building phase

of the multivariate logistic regression models. Gender and respiratory symptoms were not included in the final model because they did not appear to be

Table I Prevalences of gender, sire of residence, smoking Malus, passive smokingexposure, respirator,' s\niptoms, ftnnilv history of chronic obstructive piilmonitr\

diseases and lung cancer and serum imii-BPDE-DNA adduci antibodies in 1345

subjects from a general population .sample of Italy

No. of subjects%GenderMalesFemalesResidenceUrbanSuburbanSmoking

statusNonsmokerEx-smokerSmokerPassive

smokingNoYesRespiratory

symptomsNoYesFamily

history of chronicbronchitis"NoYesFamily

history ofemphysema"NoYesFamily

history of lungcancer"NoYesSerum

anti-BPDE-DNAantibodiesNoYesOther

members of the family cluster withserumanti-BPDE-DNAantibodiesNoYes,

up to 50% of theclusterYes,

more than 50% of the cluster6646815211825388399358580765910435USI15212101231156177106228391723619249.450.638.761.343.729.726.643.156.967.732.387.811.390.09.185.913.279.021.068.217.514.3

"Twelve patients did not complete this part of the questionnaire.

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Table 2 Prevalences of ariti-BPDE-DNA adduci antibodies according to questionnaire

variables

No. of positive subjects%GenderMalesFemalesResidenceUrbanSuburbanSmoking

statusNonsmokerEx-smokerSmokerPassive

smokingNoYesRespiratory

symptomsNoYesFamily

history of chronic bronchitisNoYesFamily

history of emphysemaNoYesFamily

history of lung cancerNoYesOther

members of the family clusterwith serum anti-BPDE-DNAantibodiesNoYes,

up to 50% of the clusterYes, more than 50% of the cluster1511321351481058692119164181102251302562323546172545722.719.426.017.917.821.525.720.521.419.923.421.219.721.120.320.326.018.722.929.7

effect modifiers or confounders of the relationship between serum anti-BPDE-

DNA antibodies and factors of interest.

RESULTS

The population sample under investigation (n = 1345; meanage ±SD, 43.2 ±17.2 years; range, 8-83) consisted of 664 males

(49.4%) and 681 females (50.6%). According to their residence, 825subjects were classified as living in the suburban area (61.3%), and520 were classified as living in the urban area (38.7%). As to thesmoking status, 358 were classified as smokers (26.6%), 399 asex-smokers (29.7%) and 588 as nonsmokers (43.7%; Table 1). Prevalences of smokers, ex-smokers, and nonsmokers in urban and subur

ban residents were not statistically different. Annual means of airpollutants were within the national air quality guidelines in the twoareas, but concentrations of some pollutants in the urban area werehigher than in the suburban area (NMHC: 237 ±209 versus 126 ±50jug/m3, P < 0.001; NO2: 107 ± 51 versus 71 ± 26 |U.g/m3,

P < 0.001). However, there was no difference for total suspendedparticulates (82 ±27 versus 83 ±27 (xg/m3) nor for SO2 (24 ±11versus 22 ±10 jug/m3).

The overall prevalence of subjects with anti-BPDE-DNA antibodies was 21.0% (n = 283), with no differences between males(n = 151, 22.7%) and females (n = 132, 19.4%; Table 2). When

normalized to unmodified DNA, reactivity of positive sera was1.72 ±0.62 (range, 1.05-4.66), with a mode of 1.45, i.e., 50% ofthese sera showed >45% increased reactivity to BPDE-DNA relative

to unmodified DNA. Positive cases were slightly older (44.9 ±16.4years) than negative cases (42.7 ±17.3 years), although this difference was not significant. A clear association with the subjects' smok

ing status was evident; positivity steeply increased from 17.8% innonsmokers through 21.5% in ex-smokers to 25.7% in smokers.Anti-BPDE-DNA antibodies positivity was also associated with subjects' residence; 26.0% of subjects living in the urban area were

positive, compared with 17.9% of those living in the suburban area.No associations were found between anti-BPDE-DNA antibodies

positivity and passive smoking or respiratory symptoms (chroniccough, phlegm, and dyspnea). Of the whole sample, 38 individuals(2.8%) reported an occupational exposure to PAH during the yearbefore sampling; 9 of them were anti-BPDE-DNA antibody positive(5 of 15 smokers, 3 of 12 ex-smokers, and 1 of 11 nonsmokers),

suggesting that the role of occupational PAH exposure as contributorto the presence of circulating anti-BPDE-DNA antibodies is, at most,

minimal. When we looked at the family history of respiratory diseases, we did not find significant relationships with the relatives'

history of chronic bronchitis or emphysema. Conversely, subjectswith family history of lung cancer tended to have higher prevalencesof anti-BPDE-DNA antibodies (26.0%) than subjects without that

history (20.3%; Table 2).When we tested by multiple regression logistic model the associ

ations of personal and laboratory variables with the subject's positiv

ity for anti-BPDE-DNA antibodies, we observed an excess prevalenceof anti-BPDE-DNA antibody positivity in people living in the urbanarea (OR, 1.49; 95% CI, 1.16-1.92); an association with the smokingstatus, because the OR was 1.25 (95% CI, 1.06-1.48) for ex-smokers(score variable, 1) and 1.57 (95% CI, 1.33-1.85) for smokers (score

variable, 2) compared with nonsmokers; an excess prevalence (justmissing the statistical significance) of anti-BPDE-DNA antibodies in

subjects with a family history of lung cancer (OR, 1.30; 95% CI,0.90-1.88); and a familial aggregation for anti-BPDE-DNA antibod

ies positivity, because the positivity increased with the number ofmembers in the family cluster positive for anti-BPDE-DNA antibodies (Table 3). The intracluster correlation for anti-BPDE-DNA anti

bodies was 0.062. When we tested for interactions among variables,tobacco smoking and family lung cancer showed a clear synergism indetermining the prevalence of circulating anti-BPDE-DNA antibodies(P = 0.02); the effect of smoking was amplified in subjects with

familial lung cancer (Fig. 1).

DISCUSSION

PAHs are established lung carcinogens from many different sourcesthat have been extensively studied at the level of exposure, pulmonaryretention, lung metabolism, and DNA damage in experimental conditions or selected groups of exposed individuals (9, 21-25). DNA

adducts due to PAH exposure may induce specific antibody production both in experimental animals and in humans (9, 10, 26, 27).Substantial information on the efficiency and mechanisms of development of the immune response to these antibodies would be helpful.Indeed, after the first study in coke oven workers (9), the wideinterindividual variation in the immune response and the dose-

response effect between sera reactivity and PAHs administered inexperimental animals have been reported (28). More recently, wedescribed the release from the suppressive effect of tobacco smokeexposure on these specific antibodies with smoking cessation inhumans (29). Nevertheless, the lack of information on the immuno-

logical mechanisms underlying the development of these antibodieshas not prevented a number of informative studies on PAH-exposed

Table 3 Multiple regression logistic analysis of the presence of serum anti-BPDE-DNAantibodies with questionnairevariablesORUrban

residence 1.49Tobaccosmoking 1.25Passive

smoking0.97Familyhistory of chronic bronchitis 1.02Familyhistory of emphysema0.99Familyhistory of lung cancer1.30Other

members of the family cluster with1.30serumanti-BPDE-DNA antibodies95%

CI1.16-1.921.06-1.480.74-1.270.65-1.600.61-1.600.90-1.881.03-1.65

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o

iSooDtu

Bo

NONSMOKERS EX-SMOKERS SMOKERS

Fig. 1. Adjusted ORs (with 95% CIs, Bars) for the presence of serum anti-BPDE-DNA

antibodies in subjects with (ffl) or without (D) family history of lung cancer according tothe different smoking status of individuals.

individuals (10, 30) and on a small number of normal healthy individuals from being carried out ( 11). In the present study, circulatingBPDE-DNA antibodies were determined in a transitional epidemio-

logical investigation on a large general population sample. The firstresult pointing to anti-BPDE-DNA antibodies as putative biomarkersof exposure was the evidence of an urban-suburban gradient in antibody positivity. The observed excess prevalence of anti-BPDE-DNA

antibody positivity in people living in the urban area could not beattributed to the effect of other confounders, such as tobacco smoking,which was considered in the multivariate model, or diet (31 ), becauselarge diet variations in our population living in a relatively small areaof central Italy are not likely. This result is in accordance with theobserved greater DNA damage and CYP1A1 gene induction in environmentally polluted regions compared with suburban areas of Poland(6) and is consistent with the reported 1.5 smoking-adjusted relative

risk for lung cancer in urban areas compared with countryside areas(32). Data of continuous ambient air monitoring for NMHCs in theareas under investigation actually showed higher annual mean concentrations in the urban area than in the suburban area. These figuresparalleled those obtained for NO2, supporting the hypothesis of higherair pollution due to auto vehicle traffic in the urban area than in thesuburban area.

The second result pointing to anti-BPDE-DNA antibodies as puta

tive biomarkers of exposure was the association with the smokingstatus; the positivity increasing from ex-smokers to smokers com

pared with nonsmokers. These figures are in accordance with published data on the greater extent of DNA damage in smokers than innonsmokers (33), but they disagree with the even prevalences ofserum anti-BPDE-DNA antibodies in healthy individuals with differ

ent smoking habits (11). Wide interindividual variations in carcinogenmetabolism, DNA damage and repair (34), as well as in the immuneresponse (28) may have contributed to mask differences betweensmokers and nonsmokers in Newman's study (performed upon 99

subjects), whereas the much greater statistical power of our studyshould have controlled better for variability and let the expecteddifferences according to the smoking status to be revealed. Likely, thedifferences in the prevalence of anti-BPDE-DNA positivity with the

smoking status in our series would have been even greater if we couldhave accounted for the immunosuppressive effect of active smokingobserved in heavy smokers (29). The 18% prevalence of anti-BPDE-

DNA antibodies in nonsmokers may be due to different sources ofPAHs other than active tobacco smoking, such as environmentaltobacco smoke, engine exhausts, coal occupational exposure, carbon

ization processes (e.g., at the workplace), and diet (35). However,passive smoking did not influence the positivity for anti-BPDE-DNAantibodies at all. The lack of association of anti-BPDE-DNA positiv

ity with passive smoking does not lessen the impact of passive smokeexposure in lung carcinogenesis, which may be due to other tobaccosmoke carcinogens, such as tobacco-specific ¿V-nitrosamines(36).Prevalences in ex-smokers, which intermediate those in the other two

groups, may reflect the decline of DNA damage after smoking cessation (37).

The other two main results of this study regard the potential geneticand familial component of serum anti-BPDE-DNA antibody positivity

in the general population. Actually, we observed an excess prevalenceof anti-BPDE-DNA antibodies in subjects with family history of lung

cancer while taking into account the other confounders. Moreover,this effect became greater from nonsmokers through ex-smokers to

smokers (Fig. 1), suggesting the need for an interaction betweengenetic and environmental factors (in particular tobacco smoking) toreveal the individual's susceptibility to DNA damage (38). These

results might be considered as an extension of those published byTokuhata more than three decades ago (39), and our interpretationagrees with the recent report that biomarkers of PAH exposure such asDNA adducts may have the potential to be indicative of cancer riskwhen used in the appropriate epidemiological set (14). On the otherhand, we did not find associations with the family history of chronicbronchitis or emphysema. This is in contrast with previous observations on lung cancer risk ( 16), but it fits with the lack of associationbetween the high activity alíeleof the CYPJAl gene and panacinaremphysema and chronic obstructive airway disease (40). BecauseBPDE-DNA adducts come out from CYP1A/-driven pathways, our

results might be interpreted under the light of this latter report. This,of course, does not rule out the possibility for the contribution of othergenes to the common susceptibility for lung cancer and chronicobstructive pulmonary disease.

Finally, anti-BPDE-DNA antibody positivity showed a familial

aggregation (after adjusting for the other confounders); the positivityincreased with the number of members in the family cluster positivefor anti-BPDE-DNA antibodies. Familial aggregation has been described as a result of gene-environment interaction for common dis

eases (41), and studies based upon segregation analysis have postulated the possibility of a Mendelian codominant inheritance of a raremajor autosomal gene accounting for the increased family risk of lungcancer (42). A genetic control of carcinogen-metabolizing enzymesand BPDE-DNA adduci formation has been observed (17), and thefamilial aggregation of anti-BPDE-DNA antibodies positivity may

reflect this predisposition to the genotoxic effects of PAHs.Diet should not have affected the relationship between anti-BPDE-

DNA antibodies and active tobacco smoking (which actually depresses the immune response) and family history of lung cancer (noreport links family history of lung cancer to the diet). However, theeffect of shared dietary habits within the family group on the presenceof anti-BPDE-DNA antibodies in members of the same family cannot

be ruled out.This study on a large general population sample shows that serum

anti-BPDE-DNA antibodies may be used as markers of exposure to

environmental carcinogens and of DNA damage and supports thehypothesis about the familial susceptibility to lung cancer.

ACKNOWLEDGMENTS

We acknowledge the invaluable help of Dr. R. Puntoni for the preparationand maintenance of the serological bank. We are also indebted to Dr. U.Mammini and M. Martinelli for data banking and recording.

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1998;58:4122-4126. Cancer Res   Stefano Petruzzelli, Alessandro Celi, Nolita Pulerà, et al.   Tobacco Smoking, and Family History of Lung DiseasesAdducts in the General Population: Effects of Air Pollution,

)pyrene Diol Epoxide-DNAaSerum Antibodies to Benzo(

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