Targeted Hypermutation as a Survival Strategy: A Theoretical Approach.

Targeted hypermutation has proven to be a useful survival strategy for bacteria under severe stress and is also used by multicellular organisms in specific instances such as the mammalian immune system. This might appear surprising, given the generally observed deleterious effects of poor replication fidelity/high mutation rate. A previous theoretical model designed to explore the role of replication fidelity in the origin of life was applied to a simulated hypermutation scenario. The results confirmed that the same model is useful for analyzing hypermutation and can predict the effects of the same parameters (survival probability, replication fidelity, mutation effect, and others) on the survival of cellular populations undergoing hypermutation as a result of severe stress.

The Continuity Principle and the Evolution of Replication Fidelity.

Evolution in modern life requires high replication fidelity to allow for natural selection. A simulation model utilizing simulated phenotype data on cellular probability of survival was developed to determine how self-replication fidelity could evolve in early life. The results indicate that initial survivability and replication fidelity both contribute to overall fitness as measured by growth rates of the cell population. Survival probability was the more dominant feature, and evolution was possible even with zero replication fidelity. A derived formula for the relationship of survival probability and replication fidelity with growth rate was consistent with the simulated empirical data. Quantitative assessment of continuity and other evidence was obtained for a saltation (non-continuous) evolutionary process starting from low to moderate levels of survival probability and self-replication fidelity to reach the high levels seen in modern life forms.

Accurate phenotypic self-replication as a necessary cause for biological evolution.

Since the Origin of Species, it has been known that evolution depends on what Darwin called the "strong principle of inheritance." Highly accurate replication of cellular phenotype is a universal phenomenon in all of life since LUCA and is often taken for granted as a constant in evolutionary theory. It is not known how self-replication arose during the origin of life. In this report I use the simple mathematics of evolutionary theory to investigate the dynamics of self-replication accuracy and allelic selection. Results indicate that the degree of self-replication accuracy must be greater than a threshold related to the selection coefficients of the alleles in a population in order for evolution to occur. Accurate replication of cellular phenotype and of the molecules involved in genotype/phenotype linkage is necessary for the origin of evolution and may be considered the fundamental principle of life.

Comparison of estrogens and estrogen metabolites in human breast tissue and urine.

BACKGROUND: An important aspect of the link between estrogen and breast cancer is whether urinary estrogen levels are representative of the intra-tissue levels of bioavailable estrogens. METHODS: This study compares 15 estrogen and estrogen metabolite levels in breast tissue and urine of 9 women with primary breast cancer using a quantitative liquid chromatography-mass spectrometry method. RESULTS: The average levels of estrogens (estrone, 17 beta-estradiol) were significantly higher in breast tissue than in urine. Both the 2 and the 16-hydroxylation pathways were less represented in breast tissue than urine; no components of the 4-hydroxypathway were detected in breast tissue, while 4-hydroxyestrone was measured in urine. However, the 2/16 ratio was similar in urine and breast tissue. Women carrying the variant CYP1B1 genotype (Leu/Val and Val/Val) showed significantly lower overall estrogen metabolite, estrogen, and 16-hydroxylation pathway levels in breast tissue in comparison to women carrying the wild type genotype. No effect of the CYP1B1 polymorphism was observed in urinary metabolites. CONCLUSIONS: The urinary 2/16 ratio seems a good approximation of the ratio observed in breast tissue. Metabolic genes may have an important role in the estrogen metabolism locally in tissues where the gene is expressed, a role that is not readily observable when urinary measurements are performed.

Pooled analysis of studies on DNA adducts and dietary vitamins.

OBJECTIVES: There is some evidence that dietary components that are rich in antioxidant and vitamins are inversely associated with DNA adduct levels induced by environmental carcinogens such as polycyclic aromatic hydrocarbons, although the epidemiologic data are inconsistent. This study addresses the association between vitamins, DNA adducts and smoking. METHODS: A combined analysis of individual data on the association between bulky DNA adducts and dietary vitamins was conducted. A Medline search was performed to identify studies on healthy subjects in which smoking and vitamins intake information were available, and bulky DNA adducts were measured in peripheral blood with 32P-postlabelling. Eight published studies met the eligibility criteria, and individual data from 7 data sets including 2758 subjects were obtained. GSTM1 and GSTT1 were also available on all the subjects. RESULTS: Vitamin E was inversely significantly associated with DNA adducts after adjustment for possible confounding factors. Vitamins A and C were not independent predictors of DNA adducts. A stratified analysis showed that vitamin A had a significant inverse association with DNA adducts in ever smokers only. CONCLUSIONS: This result is relevant to planning any future chemo-preventive interventions directed to high risk subgroups of the population, for cancer prevention.

Human population genetic diversity as a function of SNP type from HapMap data.

Data from the international HapMap project were mined to determine if the degree of genetic differentiation (Fst) is dependent on single nucleotide polymorphism (SNP) category. The Fst statistic was evaluated across all SNPs for each of 30 genes and for each of five chromosomes. A consistent decrease in diversity between Europeans and Africans was seen for nonsynonymous coding region SNPs compared to the three other SNP categories: synonymous SNPs, UTR, and intronic SNPs. This suggests an effect of balancing selection in reducing interpopulation genetic diversity at sites that would be expected to influence phenotype and therefore be subject to selection. This result is inconsistent with the concept of large population specific genetic differences that could have applications in "racialized medicine."

Urinary estrogen metabolites in women at high risk for breast cancer.

OBJECTIVE: This study explored whether average urinary estrogen metabolites in breast cancer high-risk women can be used to identify a subgroup of women at particularly high risk to develop breast cancer, to which prevention strategies should be addressed. METHODS: The population consisted of 77 high-risk women, 30 breast cancer patients and 41 controls. All subjects answered a standardized questionnaire; height and weight and spot urine samples were also obtained. Urine hydroxyestrogen metabolites were measured in triplicate by enzyme immunoassay, and the estrogen metabolite ratios for each individual were calculated. RESULTS: The 2:16 OHE ratio (2-hydroxyestrone/16-alpha-hydroxyestrone) in women at high risk for breast cancer was similar to that observed in the breast cancer group (1.76 +/- 2.33 versus 1.29 +/- 0.80) and lower than in controls (2.47 +/- 1.14; P = 0.00). At the multivariate linear regression model, the 2:16 OHE ratio was significantly associated with diagnosis (P = 0.000 for both the high risk and breast cancer group versus the controls) and body mass index (P = 0.005), but not with age (P = 0.604), or smoking history (P = 0.478). CONCLUSIONS: This study suggests that lower urinary 2:16 OHE ratios are predictors of breast cancer risk. Profiling estrogen metabolites may identify women who are more probably to develop breast cancer within a population of women with known risk factors and may help to further elucidate the clinical relevance of urinary 2:16 OHE ratios as clinical markers and prognostic indicators in this population.

Recurrence in oral and pharyngeal cancer is associated with quantitative MGMT promoter methylation.

BACKGROUND: Biomarkers that predict clinical response, tumor recurrence or patient survival are severely lacking for most cancers, particularly for oral and pharyngeal cancer. This study examines whether gene-promoter methylation of tumor DNA correlates with survival and recurrence rates in a population of patients with oral or pharyngeal cancer. METHODS: The promoter methylation status of the DNA repair gene MGMT and the tumor suppressor genes CDKN2A and RASSF1 were evaluated by methylation-specific PCR in 88 primary oral and pharyngeal tumors and correlated with survival and tumor recurrence. Quantitative MGMT methylation was also assessed. RESULTS: 29.6% of the tumors presented with MGMT methylation, 11.5% with CDKN2A methylation and 12.1% with RASSF1 methylation. MGMT promoter methylation was significantly associated with poorer overall and disease-free survival. No differences in methylation status of MGMT and RASSF1 with HPV infection, smoking or drinking habits were observed. A significant inverse trend with the amount of MGMT methylation and overall and disease-free survival was observed (ptrend = 0.002 and 0.001 respectively). CONCLUSION: These results implicate MGMT promoter methylation as a possible biomarker for oral and pharyngeal cancer prognosis. The critical role of MGMT in DNA repair suggests that defective DNA repair may be correlative in the observed association between MGMT promoter methylation and tumor recurrence. Follow-up studies should include further quantitative MSP-PCR measurement, global methylation profiling and detailed analysis of downstream DNA repair genes regulated by promoter methylation.

Leptin receptor Gln223Arg polymorphism and breast cancer risk in Nigerian women: a case control study.

BACKGROUND: Leptin, a 16 kDa polypeptide hormone, implicated in various physiological processes, exerts its action through the leptin receptor, a member of the class I cytokine receptor family. Both leptin and leptin receptor have recently been implicated in processes leading to breast cancer initiation and progression in animal models and humans. An A to G transition mutation in codon 223 in exon 6 of the leptin receptor gene, resulting in glutamine to arginine substitution (Gln223Arg), lies within the first of two putative leptin-binding regions and may be associated with impaired signaling capacity of the leptin receptor. This study was designed to assess the role of this polymorphism in breast cancer susceptibility in Nigerian women. METHODS: We utilized a polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) assay to evaluate the association between the Gln223Arg polymorphism of the leptin receptor gene and breast risk in Nigeria in a case control study involving 209 women with breast cancer and 209 controls without the disease. Study participants were recruited from surgical outpatient clinics and surgical wards of four University Teaching Hospitals located in Midwestern and southeastern Nigeria between September 2002 and April 2004. RESULTS: Premenopausal women carrying at least one LEPR 223Arg allele were at a modestly increased risk of breast cancer after adjusting for confounders (OR = 1.8, 95% confidence interval [CI] 1.0-3.2, p = 0.07). There was no association with postmenopausal breast cancer risk (OR = 0.9, 95% CI 0.4-1.8, p = 0.68). CONCLUSION: Our results suggest that the LEPR Gln223Arg polymorphism in the extracellular domain of the LEPR receptor gene is associated with a modestly increased risk of premenopausal breast cancer in Nigerian women.

Bulky DNA adducts, 4-aminobiphenyl-haemoglobin adducts and diet in the European Prospective Investigation into Cancer and Nutrition (EPIC) prospective study.

In contrast to some extensively examined food mutagens, for example, aflatoxins, N-nitrosamines and heterocyclic amines, some other food contaminants, in particular polycyclic aromatic hydrocarbons (PAH) and other aromatic compounds, have received less attention. Therefore, exploring the relationships between dietary habits and the levels of biomarkers related to exposure to aromatic compounds is highly relevant. We have investigated in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort the association between dietary items (food groups and nutrients) and aromatic DNA adducts and 4-aminobiphenyl-Hb adducts. Both types of adducts are biomarkers of carcinogen exposure and possibly of cancer risk, and were measured, respectively, in leucocytes and erythrocytes of 1086 (DNA adducts) and 190 (Hb adducts) non-smokers. An inverse, statistically significant, association has been found between DNA adduct levels and dietary fibre intake (P = 0.02), vitamin E (P = 0.04) and alcohol (P = 0.03) but not with other nutrients or food groups. Also, an inverse association between fibre and fruit intake, and BMI and 4-aminobiphenyl-Hb adducts (P = 0.03, 0.04, and 0.03 respectively) was observed. After multivariate regression analysis these inverse correlations remained statistically significant, except for the correlation adducts v. fruit intake. The present study suggests that fibre intake in the usual range can modify the level of DNA or Hb aromatic adducts, but such role seems to be quantitatively modest. Fibres could reduce the formation of DNA adducts in different manners, by diluting potential food mutagens and carcinogens in the gastrointestinal tract, by speeding their transit through the colon and by binding carcinogenic substances.

NQO1, MPO, CYP2E1, GSTT1 and GSTM1 polymorphisms and biological effects of benzene exposure--a literature review.

BACKGROUND: Benzene is a ubiquitous toxic environmental pollutant. Biological effects have been detected as a result of low-level environmental exposures, suggesting that a large proportion of the population may potentially suffer ill health effects. Polymorphisms in genes involved in benzene metabolism are thought to influence individual susceptibility to various levels of benzene exposure. METHODS: Medline literature database search for articles relating to benzene exposure and polymorphisms in genes known to be involved in benzene metabolism (NQO1, CYP2E1, GSTT1, GSTM1 and MPO). Twenty-two reports were included in this review. RESULTS: A modest effect of the studied gene polymorphisms on the analyzed biomarkers was observed. GSTM1 and GSTT1 showed some consistent associations with both biomarkers of exposure and effect. CONCLUSION: Genetic polymorphisms on the benzene metabolism pathway should be taken into account when studying the biological effects of benzene exposure. Unique combinations of genetic polymorphisms may increase susceptibility of individuals and/or population subgroups. However, gene-gene interactions, and the biological effects of long-term and low-level exposure to benzene are not yet analyzed with well-designed studies that incorporate multiple biological end-points and multiple genes.

Genetic susceptibility to benzene toxicity in humans.

Human metabolism of benzene involves pathways coded for by polymorphic genes. To determine whether the genotype at these loci might influence susceptibility to the adverse effects of benzene exposure, 208 Bulgarian petrochemical workers and controls, whose exposure to benzene was determined by active personal sampling, were studied. The frequency of DNA single-strand breaks (DNA-SSB) was determined by alkaline elution, and genotype analysis was performed for five metabolic loci. Individuals carrying the NAD(P)H:quinone oxidoreductase 1 (NQO1) variant had significantly twofold increased DNA-SSB levels compared to wild-type individuals. The same result was observed for subjects with microsomal epoxide hydrolase (EPHX) genotypes that predict the fast catalytic phenotype. Deletion of the glutathione S-transferase T1 (GSTT1) gene also showed a consistent quantitative 35-40% rise in DNA-SSB levels. Neither glutathione S-transferase M1 (GSTM1) nor myeloperoxidase (MPO) genetic variants exerted any effect on DNA-SSB levels. Combinations of two genetic polymorphisms showed the same effects on DNA-SSB as expected from the data on single genotypes. The three locus genotype predicted to produce the highest level of toxicity, based on metabolic pathways, produced a significant 5.5-fold higher level of DNA-SSB than did the genotype predicted to yield the least genotoxicity.

TP53 and KRAS2 mutations in plasma DNA of healthy subjects and subsequent cancer occurrence: a prospective study.

In cancer patients, plasma often contains mutant DNA released by cancer cells. We have assessed the significance of plasma DNA mutations for subsequent cancer development in healthy subjects in a large longitudinal prospective study. The European Prospective Investigation into Cancer and Nutrition study was analyzed with a nested case-control design. Cases were nonsmokers or ex-smokers for >10 years and newly diagnosed with lung, bladder, or upper aerodigestive tract cancers or leukemia accrued after a median follow-up of 6.3 years. Controls were matched 2:1 for follow-up, age, sex, area of recruitment, and smoking status. KRAS2 mutations were detected by mutant-enriched PCR and sequencing (n = 1,098). TP53 mutations were detected by denaturing high-performance liquid chromatography, temporal temperature gradient electrophoresis, and sequencing (n = 550). KRAS2 or TP53 mutations were detected in 13 of 1,098 (1.2%) and 20 of 550 (3.6%) subjects, respectively, 16 of whom developed cancer on average after 18.3 months of follow-up. Among 137 subjects who developed bladder cancer, 5 had KRAS2 mutations [odds ratio (OR), 4.25; 95% confidence interval (95% CI), 1.27-14.15] and 7 had TP53 mutations (OR, 1.81; 95% CI, 0.66-4.97). There was a nonsignificant trend for association between TP53 mutations and bulky adducts in lymphocyte DNA (OR, 2.78; 95% CI, 0.64-12.17). This is the first report of TP53 or KRAS2 mutations in the plasma of healthy subjects in a prospective study, suggesting that KRAS2 mutation is detectable ahead of bladder cancer diagnosis. TP53 mutation may be associated with environmental exposures. These observations have implications for monitoring early steps of carcinogenesis.

Effects of polycyclic aromatic hydrocarbons (PAHs) in environmental pollution on exogenous and oxidative DNA damage (EXPAH project): description of the population under study.

The EXPAH project was a molecular epidemiology study whose aims were to evaluate the hypothesis that polycyclic aromatic hydrocarbons (PAHs) are a major source of genotoxic activities of organic mixtures associated with air pollution. Biomarkers of exposure, effects and susceptibility, and oxidative DNA damage were measured in three PAH-exposed populations from Prague (Czech Republic), Kosice (Slovakia) and Sofia (Bulgaria). Control populations were included from each city. In total 356 individuals were enrolled. A questionnaire was used to determine life style/dietary factors. Ambient air exposure was measured by stationary monitoring, and personal exposure monitoring was also carried out. The characteristics of the population are described in this paper together with their personal exposure to carcinogenic PAHs (c-PAHs). The dose of c-PAH exposure was found to vary between the occupationally exposed (e.g. policemen and bus drivers) and the control populations in each country, and also varied from country to country.

Biomarkers of exposure to carcinogenic PAHs and their relationship with environmental factors.

The EXPAH project is a multicentre European study in which biomarkers of exposure, biomarkers of effect, genetic susceptibility and environmental factors were studied in populations exposed to differing levels of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs). We describe here the relationships between the levels of DNA adducts (as biomarkers of exposure), the exposure to air pollution and smoking status. Lymphocyte bulky DNA adducts were significantly correlated with exposure when subjects were classified either by job description or by personal monitor measurements, and both bulky and benzo(a)pyrene (B[a]P) DNA adducts were also correlated with smoking status. These associations varied across the countries studied (Czech Republic, Slovakia, Bulgaria). Results from a multivariate analysis show that factors mainly contributing to bulky and B[a]P DNA adducts are age, smoking habit, country of origin and environmental exposure to c-PAHs. The B[a]P DNA adducts were more strongly associated with smoking status than with the environmental exposure to c-PAHs.

Effects of metabolic genotypes on intermediary biomarkers in subjects exposed to PAHS: results from the EXPAH study.

Data from the EXPAH project on PAH exposure and intermediary biomarkers were analyzed with respect to individual genotypes at seven metabolic gene loci. The GSTM1 null allele was associated with significantly higher levels of two biomarkers, malondialdehyde-2'-deoxyguanosine and benzo[a]pyrene DNA adducts in the total population from three Central and Eastern European countries. The CYP1B1 Leu/Val variant demonstrated effects on both markers of oxidative DNA damage in opposite directions, producing a higher level of M(1)dG with a trend from wild type (Leu/Leu) to heterozygotes to homozygous (Val/Val) variants, whereas the effects of these variants were reversed for 8-oxodG. Cluster Analysis was used to group composite genotypes in order to determine if combined genotypes of multiple loci could explain some of the variation seen with the biomarkers, expressed per unit of exposure, referred to as a sensitivity index. This analysis revealed two closely related genotypes each involving four of the loci (GSTM1*0/*0, CYP1A1*1*1, CYP1B1*1/*2, GSTP1*1/*1 and GSTT1*0/*0, CYP1A1*1*1, CYP1B1*1/*2, GSTP1*1/*1.) that conferred significant resistance to the DNA damaging effects of benzo[a]pyrene, measured as the level of a benzo[a]pyrene-like adduct per unit of benzo[a]pyrene exposed.

Effects of environmental air pollution on endogenous oxidative DNA damage in humans.

Epidemiological studies conducted in metropolitan areas have demonstrated that exposure to environmental air pollution is associated with increases in mortality. Carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) are the major source of genotoxic activities of organic mixtures associated with respirable particulate matter, which is a constituent of environmental air pollution. In this study,we wanted to evaluate the relationship between exposure to these genotoxic compounds present in the air and endogenous oxidative DNA damage in three different human populations exposed to varying levels of environmental air pollution. As measures of oxidative DNA damage we have determined 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and cyclic pyrimidopurinone N-1,N(2) malondialdehyde-2'-deoxyguanosine (M(1)dG) by the immunoslot blot assay from lymphocyte DNA of participating individuals. The level of endogenous oxidative DNA damage was significantly increased in individuals exposed to environmental air pollution compared to unexposed individuals from Kosice (8-oxodG adducts) and Sofia (M(1)dG adducts). However, there was no significant difference in the level of endogenous oxidative DNA and exposure to environmental air pollution in individuals from Prague (8-oxodG and M(1)dG adducts) and Kosice (M(1)dG adducts). The average level of M(1)dG adducts was significantly lower in unexposed and exposed individuals from Kosice compared to those from Prague and Sofia. The average level of 8-oxodG adducts was significantly higher in unexposed and exposed individuals from Kosice compared to those from Prague. A significant increasing trend according to the interaction of c-PAHs exposure and smoking status was observed in levels of 8-oxodG adducts in individuals from Kosice. However, no other relationship was observed for M(1)dG and 8-oxodG adduct levels with regard to the smoking status and c-PAH exposure status of the individuals. The conclusion that can be made from this study is that environmental air pollution may alter the endogenous oxidative DNA damage levels in humans but the effect appears to be related to the country where the individuals reside. Genetic polymorphisms of the genes involved in metabolism and detoxification and also differences in the DNA repair capacity and antioxidant status of the individuals could be possible explanations for the variation observed in the level of endogenous oxidative DNA damage for the different populations.

The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans.

Polycyclic aromatic hydrocarbons (PAHs) appear to be significant contributors to the genotoxicity and carcinogenicity of air pollution present in the urban environment for humans. Populations exposed to environmental air pollution show increased levels of PAH DNA adducts and it has been postulated that another contributing cause of carcinogenicity by environmental air pollution may be the production of reactive oxygen species following oxidative stress leading to oxidative DNA damage. The antioxidant status as well as the genetic profile of an individual should in theory govern the amount of protection afforded against the deleterious effects associated with exposure to environmental air pollution. In this study we investigated the formation of total PAH (bulky) and B[a]P DNA adducts following exposure of individuals to environmental air pollution in three metropolitan cities and the effect on endogenously derived oxidative DNA damage. Furthermore, the influence of antioxidant status (vitamin levels) and genetic susceptibility of individuals with regard to DNA damage was also investigated. There was no significant correlation for individuals between the levels of vitamin A, vitamin E, vitamin C and folate with M(1)dG and 8-oxodG adducts as well as M(1)dG adducts with total PAH (bulky) or B[a]P DNA adducts. The interesting finding from this study was the significant negative correlation between the level of 8-oxodG adducts and the level of total PAH (bulky) and B[a]P DNA adducts implying that the repair of oxidative DNA damage may be enhanced. This correlation was most significant for those individuals that were non smokers or those unexposed to environmental air pollution. Furthermore the significant inverse correlation between 8-oxodG and B[a]P DNA adducts was confined to individuals carrying the wild type genotype for both the GSTM1 and the GSTT1 gene (separately and interacting). This effect was not observed for individuals carrying the null variant.

Effects of diet on biomarkers of exposure and effects, and on oxidative damage.

Modification of DNA is believed to be a key step in carcinogenesis, and therefore DNA adducts have been proposed as predictive biomarkers of human cancer. Smoked and grilled foods are important contributors of PAH-DNA adduct levels, while the consumption of flavonoids and other antioxidants seems to decrease the level of DNA adducts. The aim of this study was to assess the effect of each group of foods and of different dietary profiles on the DNA adducts levels and on oxidative damage to DNA. Occupationally exposed men were recruited in Czech Republic, Slovak Republic and Bulgaria. Non-occupationally exposed subjects were matched on age and gender to PAH-exposed workers. Three hundred and fifty-six subjects who completed the questionnaire for dietary information and had a measurement of DNA adduct levels and oxidative damage to DNA were included in this study. No food item seemed to be individually associated with markers of exposure or DNA damage. Total DNA adducts levels were significantly higher for subjects who had eaten, in the previous 24h, smoked or fried food. A Principal Components Analysis was performed to identify groups of subjects with similar dietary profiles: no significant differences in biomarker levels were observed among the groups defined according to dietary profiles. In conclusion, this study did not show any significant association between diet and biomarkers of DNA damage, oxidative damage to DNA and chromosomal aberrations, neither when each food was considered separately, nor when the effect of different dietary profiles was tested. The recent consumption of smoked or fried food was associated with an increase in total DNA adducts levels.

DNA adducts and lung cancer risk: a prospective study.

Objectives were to investigate prospectively the ability of DNA adducts to predict cancer and to study the determinants of adducts, especially air pollutants. DNA adducts were measured in a case-control study nested in the European Prospective Investigation into Cancer and Nutrition (EPIC) investigation. Cases included newly diagnosed lung cancer (n = 115), upper respiratory cancers (pharynx and larynx; n = 82), bladder cancer (n = 124), leukemia (n = 166), and chronic obstructive pulmonary disease or emphysema deaths (n = 77) accrued after a median follow-up of 7 years among the EPIC former smokers and never-smokers. Three controls per case were matched for questionnaire analyses and two controls per case for laboratory analyses. Matching criteria were gender, age, smoking status, country of recruitment, and follow-up time. Individual exposure to air pollution was assessed using concentration data from monitoring stations in routine air quality monitoring networks. Leukocyte DNA adducts were analyzed blindly using 32P postlabeling technique. Adducts were associated with the subsequent risk of lung cancer, with an odds ratio (OR) of 1.86 [95% confidence interval (95% CI), 0.88-3.93] when comparing detectable versus nondetectable adducts. The association with lung cancer was stronger in never-smokers (OR, 4.04; 95% CI, 1.06-15.42) and among the younger age groups. After exclusion of the cancers occurring in the first 36 months of follow-up, the OR was 4.16 (95% CI, 1.24-13.88). A positive association was found between DNA adducts and ozone (O3) concentration. Our prospective study suggests that leukocyte DNA adducts may predict lung cancer risk of never-smokers. Besides, the association of DNA adduct levels with O3 indicates a possible role for photochemical smog in determining DNA damage.