Search

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. (Cancer Res 2006; 66(13): 6871-6)

Supplementary Table 1 from TP53 and KRAS2 Mutations in Plasma DNA of Healthy Subjects and Subsequent Cancer Occurrence: A Prospective Study

Facing the SARS-CoV-2 epidemic requires intensive testing on the population to early identify and isolate infected subjects. During the first emergency phase of the epidemic, RT-qPCR on nasopharyngeal (NP) swabs, which is the most reliable technique to detect ongoing infections, exhibited limitations due to availability of reagents and budget constraints. This stressed the need to develop screening procedures that require fewer resources and are suitable to be extended to larger portions of the population. RT-qPCR on pooled samples from individual NP swabs seems to be a promising technique to improve surveillance. We performed preliminary experimental analyses aimed to investigate the performance of pool testing on samples with low viral load and we evaluated through Monte Carlo (MC) simulations alternative screening protocols based on sample pooling, tailored to contexts characterized by different infection prevalence. We focused on the role of pool size and the opportunity to develop strategies that take advantage of natural clustering structures in the population, e.g. families, school classes, hospital rooms. Despite the use of a limited number of specimens, our results suggest that, while high viral load samples seem to be detectable even in a pool with 29 negative samples, positive specimens with low viral load may be masked by the negative samples, unless smaller pools are used. The results of MC simulations confirm that pool testing is useful in contexts where the infection prevalence is low. The gain of pool testing in saving resources can be very high, and can be optimized by selecting appropriate group sizes. Exploiting natural groups makes the definition of larger pools convenient and potentially overcomes the issue of low viral load samples by increasing the probability of identifying more than one positive in the same pool.

Wood dust is one of the most common occupational exposures, with about 3.6 million of workers in the wood industry in Europe. Wood particles can deposit in the nose and the respiratory tract and cause adverse health effects. Occupational exposure to wood dust has been associated with malignant tumors of the nasal cavity and paranasal sinuses. The induction of oxidative stress and the generation of reactive oxygen species through activation of inflammatory cells could have a role in the carcinogenicity of respirable wood dust. Therefore, we conducted a cross-sectional study to evaluate the prevalence of urinary 15-F2t isoprostane (15-F2t-IsoP), a biomarker of oxidative stress and peroxidation of lipids, in 123 wood workers compared to 57 unexposed controls living in Tuscany region, Italy. 15-F2t-IsoP generation was measured by ELISA. The main result of the present study showed that a statistically significant excess of this biomarker occurred in the workers exposed to 1.48 mg/m3 of airborne wood dust with respect to the unexposed controls. The overall mean ratio (MR) between the workers exposed to wood dust and the controls was 1.36, 95% Confidence Interval (C.I.) 1.18–1.57, after correction for age and smoking habits. A significant increment of 15-F2t-IsoP (43%) was observed in the smokers as compared to the non-smokers. The urinary excretion of 15-F2t-IsoP was significantly associated with co-exposure to organic solvents, i.e., MR of 1.41, 95% C.I. 1.17–1.70, after adjustment for age and smoking habits. A 41% excess was observed in long-term wood workers, 95% C.I. 1.14–1.75. Multivariate regression analysis showed that the level of 15-F2t-IsoP was linearly correlated to the length of exposure, regression coefficient (β) = 0.244 ± 0.002 (SE). The overall increment by exposure group persisted after stratification for smoking habits. For instance, in smokers, a 53% excess was detected in the wood workers as compared to the controls, 95% C.I. 1.23–1.91. Our data support the hypothesis that oxidative stress and lipid peroxidation can have a role in the toxicity of wood dust F2-IsoP measure can be a tool for the evaluation of the effectiveness of targeted interventions aimed to reduce exposures to environmental carcinogens.

Molecular mechanisms underlying Hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) pathogenesis are still unclear. Therefore, we analyzed the levels of 8-oxo-7,8-dihydro-2&prime, deoxyguanosine (8-oxodG) and other oxidative lesions at codon 176 of the p53 gene, as well as the generation of 3-(2-deoxy-&beta, d-erythro-pentafuranosyl)pyrimido[1,2-&alpha, ]purin-10(3H)-one deoxyguanosine (M1dG), in a cohort of HCV-related HCC patients from Italy. Detection of 8-oxodG and 5-hydroxycytosine (5-OHC) was performed by ligation mediated-polymerase chain reaction assay, whereas the levels of M1dG were measured by chromatography and mass-spectrometry. Results indicated a significant 130% excess of 8-oxodG at &ndash, TGC&ndash, position of p53 codon 176 in HCV-HCC cases as compared to controls, after correction for age and gender, whereas a not significant increment of 5-OHC at &ndash, position was found. Then, regression models showed an 87% significant excess of M1dG in HCV-HCC cases relative to controls. Our study provides evidence that increased adduct binding does not occur randomly on the sequence of the p53 gene but at specific sequence context in HCV-HCC patients. By-products of lipid peroxidation could also yield a role in HCV-HCC development. Results emphasize the importance of active oxygen species in inducing nucleotide lesions at a p53 mutational hotspot in HCV-HCC patients living in geographical areas without dietary exposure to aflatoxin B1.

Asbestos fibers include a group of silicate minerals that occur in the environment and are widely employed in occupational settings. Asbestos exposure has been associated to various chronic diseases, such as pulmonary fibrosis, mesothelioma, and lung cancer, often characterized by a long period of latency. Underlying mechanisms that are behind the carcinogenic effect of asbestos have not been fully clarified. Therefore, we have conducted an epidemiological study to evaluate the relationship between 8-hydroxy-2&prime, deoxyguanosine (8-oxodG), one of the most reliable biomarkers of oxidative stress and oxidative DNA damage, and asbestos exposure in the peripheral blood of residents in Tuscany and Liguria regions, Italy, stratified by occupational exposure to this carcinogen. Levels of 8-oxodG were expressed such as relative adduct labeling (RAL), the frequency of 8-oxodG per 105 deoxyguanosine was significantly higher among exposed workers with respect to the controls, i.e., 3.0 &plusmn, 0.2 Standard Error (SE) in asbestos workers versus a value of 1.3 &plusmn, 0.1 (SE) in unexposed controls (p &lt, 0.001). When the relationship with occupational history was investigated, significant higher levels of 8-oxodG were measured in current and former asbestos workers vs. healthy controls, 3.1 &plusmn, 0.3 (SE) and 2.9 &plusmn, 0.2 (SE), respectively. After stratification for occupational history, a significant 194% excess of adducts was found in workers with 10 or more years of past asbestos exposure (p &lt, 0.001). 8-oxodG can be used for medical surveillance programs of cohorts of workers with past and recent exposures to carcinogens for the identification of subjects requiring a more intense clinical surveillance.

Objectives Despite an asbestos ban in the European Union, exposure to asbestos still represents an occupational risk. Biomarkers of DNA damage and genomic instability in groups exposed to asbestos may contribute to the identification of subgroups/subjects at higher risk. Methods A cross-sectional study was conducted on 468 male individuals (80 working in occupational settings with potential exposure to asbestos fibers, 202 retired workers with past exposure, and 186 non-exposed controls) to compare genomic instability, cell proliferation and differentiation level using the non-invasive micronucleus buccal cytome assay. Data on demographic variables, lifestyle, and occupational history were collected with a standardized questionnaire. Micronuclei (MN) and other biomarkers of DNA damage and genomic instability were scored in a minimum of 2000/1000 cells per individual, respectively. Results Univariate and multivariate analysis showed opposite associations of MN frequency with current and former exposure. Compared to unexposed controls, workers with current potential exposure to asbestos had 55% lower MN frequency [95% confidence interval (CI) 71–29%, P

Worldwide, lung cancer is the leading cause of cancer death. DNA adducts are considered a reliable biomarker that reflects carcinogen exposure to tobacco smoke, but the central question is what is the relationship of DNA adducts and cancer? Therefore, we investigated this relationship by a meta-analysis of twenty-two studies with bronchial adducts for a total of 1091 subjects, 887 lung cancer cases and 204 apparently healthy individuals with no evidence of lung cancer. Our study shows that these adducts are significantly associated to increase lung cancer risk. The value of Mean Ratiolung-cancer (MR) of bronchial adducts resulting from the random effects model was 2.64, 95% C.I. 2.00-3.50, in overall lung cancer cases as compared to controls. The significant difference, with lung cancer patients having significant higher levels of bronchial adducts than controls, persisted after stratification for smoking habits. The MRlung-cancer value between lung cancer patients and controls for smokers was 2.03, 95% C.I. 1.42-2.91, for ex-smokers 3.27, 95% C.I. 1.49-7.18, and for non-smokers was 3.81, 95% C.I. 1.85-7.85. Next, we found that the generation of bronchial adducts is significantly related to inhalation exposure to tobacco smoke carcinogens confirming its association with volatile carcinogens. The MRsmoking estimate of bronchial adducts resulting from meta-regression was 2.28, 95% Confidence Interval (C.I.) 1.10-4.73, in overall smokers in respect to non-smokers. The present work provides strengthening of the hypothesis that bronchial adducts are not simply relate to exposure, but are a cause of chemical-induced lung cancer.

Asbestos is the commercial name for a group of silicate minerals naturally occurring in the environment and widely used in the industry. Asbestos exposure has been associated with pulmonary fibrosis, mesothelioma, and malignancies, which may appear after a period of latency of 20–40 years. Mechanisms involved in the carcinogenic effects of asbestos are still not fully elucidated, although the oxidative stress theory suggests that phagocytic cells produce large amounts of reactive oxygen species, due to their inability to digest asbestos fiber. We have conducted a mechanistic study to evaluate the association between 3-(2-deoxy-β-d-erythro- pentafuranosyl)pyrimido[1, 2-α]purin-10(3H)-one deoxyguanosine (M1dG) adducts, a biomarker of oxidative stress and lipid peroxidation, and asbestos exposure in the peripheral blood of 327 subjects living in Tuscany and Liguria, Italy, stratified by occupational exposure to asbestos. Adduct frequency was significantly greater into exposed subjects with respect to the controls. M1dG per 108 normal nucleotides were 4.0 ± 0.5 (SE) in 156 asbestos workers, employed in mechanic, naval, petrochemical, building industries, and in pottery and ceramic plants, versus a value of 2.3 ± 0.1 (SE) in 171 controls (p < 0.001). After stratification for occupational history, the effects persisted in 54 current asbestos workers, mainly employed in building renovation industry (2.9 ± 0.3 (SE)), and in 102 former asbestos workers (4.5 ± 0.7 (SE)), with p-values of 0.033, and

Wood dust is one of the most common occupational exposures, with about 3.6 million of workers in the wood industry in Europe. Wood particles can deposit in the nose and the respiratory tract and cause adverse health effects. Occupational exposure to wood dust has been associated with malignant tumors of the nasal cavity and paranasal sinuses. The induction of oxidative stress and the generation of reactive oxygen species through activation of inflammatory cells could have a role in the carcinogenicity of respirable wood dust. Therefore, we conducted a cross-sectional study to evaluate the prevalence of urinary 15-F

The hair-dyeing ingredient, p-phenylenediamine (PPD), was previously reported to be mutagenic, possibly by inducing oxidative stress. However, the exact mechanism of PPD in inducing oxidative stress upon skin exposure during hair-dyeing in human keratinocytes remains unknown. The aim of our studies was therefore to investigate the toxicity of PPD and its by-products in human immortalized keratinocytes (HaCaT) after auto-oxidation and after reaction with hydrogen peroxide (H2O2). We found that the PPD half maximal effective cytotoxic concentration (EC50) to HaCaT is 39.37 and 35.63 μg/mL after 24 and 48 h, respectively, without addition of H2O2 to induce oxidation. When PPD (10 or 100 μg/mL) is combined with 10.5 μg/mL of H2O2, intracellular ROS production by HaCaT after 1 h was significantly increased and enhanced levels of DNA damage were observed after 4 h of exposure. After 24 h incubations, 20 μg/mL of PPD increased the level of DNA oxidation in HaCaT. Also, we found that the in vitro reaction between PPD and H2O2, even below the maximum allowance by cosmetic industries, released hydroxyl radicals which can damage DNA. Taken together, we conclude that PPD alone and when combined with H2O2 increases the formation of reactive oxygen species in human keratinocytes, leading to oxidative stress and subsequent DNA damage. These alterations suggest that the mechanism by which PPD exposure, alone or combined with H2O2, damages keratinocytes by the formation of the high reactive HO∙ radicals.

Preconceptional paternal exposures may affect offspring's health, which cannot be explained by mutations in germ cells, but by persistent changes in the regulation of gene expression. Therefore, we investigated whether pre-conceptional paternal exposure to benzo[a] pyrene (B[a]P) could alter the offspring's phenotype. Male C57BL/6 mice were exposed to B[a] P by gavage for 6 weeks, 3 x per week, and were crossed with unexposed BALB-c females 6 weeks after the final exposure. The offspring was kept under normal feeding conditions and was sacrificed at 3 weeks of age. Analysis of the liver proteome by 2D-gel electrophoresis and mass spectrometry indicated that proteins involved in mitochondrial function were significantly downregulated in the offspring of exposed fathers. This down-regulation of mitochondrial proteins was paralleled by a reduction in mitochondrial DNA copy number and reduced activity of citrate synthase and b-hydroxyacyl-CoA dehydrogenase, but in male offspring only. Surprisingly, analysis of hepatic mRNA expression revealed a male-specific up-regulation of the genes, whose proteins were downregulated, including Aldh2 and Ogg1. This discrepancy could be related to several selected microRNA (miRNA)'s that regulate the translation of these proteins; miRNA-122, miRNA-129-2-5p, and miRNA-1941 were upregulated in a gender-specific manner. Since mitochondria are thought to be a source of intracellular reactive oxygen species, we additionally assessed oxidatively-induced DNA damage. Both 8-hydroxy-deoxyguanosine and malondialdehyde-dG adduct levels were significantly reduced in male offspring of exposed fathers. In conclusion, we show that paternal exposure to B[a]P can regulate mitochondrial metabolism in offspring, which may have profound implications for our understanding of health and disease risk inherited from fathers.

Chronic silica exposure has been associated to cancer and silicosis. Furthermore, the induction of oxidative stress and the generation of reactive oxygen species have been indicated to play a main role in the carcinogenicity of respirable silica. Therefore, we conducted a cross-sectional study to evaluate the prevalence of 3-(2-deoxy-β-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M 1 dG) adducts, a biomarker of oxidative stress and peroxidation of lipids, in the nasal epithelium of 135 silica-exposed workers, employed in pottery, ceramic and marble manufacturing plants as well as in a stone quarry, in respect to 118 controls living in Tuscany region, Italy. The M 1 dG generation was measured by the 32 P-postlabelling assay. Significant higher levels of M 1 dG adducts per 10 8 normal nucleotides were observed in the nasal epithelium of smokers, 77.9 ± 9.8 (SE), and in those of former smokers, 80.7 ± 9.7 (SE), as compared to non-smokers, 57.1 ± 6.2 (SE), P = 0.001 and P = 0.004, respectively. Significant increments of M 1 dG adducts were found in the nasal epithelium of workers that handle artificial marble conglomerates, 184 ± 36.4 (SE), and in those of quarry workers, 120 ± 34.7 (SE), with respect to controls, 50.6 ± 2.7 (SE), P = 0.014 and P

The mechanisms by which fluoride produces its toxic effects are still not clear. Therefore, we conducted a cross-sectional study to evaluate the fluoride-induced toxicity on randomly selected sheep with skeletal fluorosis resident near the large non-ferrous metallurgy Portoscuso-Portovesme industrial estate and the Carbonia and Gonnessa towns (control district) with respect to animals from a remote site without industrial settlements, on Sardinia Island, Italy. We measured the prevalence of 3-(2-deoxy-β-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) adducts, a biomarker of oxidative stress and lipid peroxidation, in the soft organs of the study animals using the 32P-postlabelling assay. Then, we analysed the association between M1dG adducts and sheep-farm localization. In addition, cadmium and lead levels were measured in the same matrices by mass spectrometry. The histopathology analysis showed that the sheep resident near the industrial site and in the control district presented typical manifestations of fluorosis. The statistical analyses using log-normal regression models indicated that there was a significant association between exocyclic DNA adducts and skeletal fluorosis. The mean ratios, adjusted for age, of renal and hepatic M1dG for the sheep with fluorosis were 5.09, 95% C.I. 1.67–15.53 and 2.04, 95% C.I. 0.91–4.57, p = 0.009 and p = 0.078, respectively. After stratification for sheep-farm localization, the renal and hepatic levels of M1dG adducts were significantly higher in the sheep resident near the industrial estate as compared to controls. Intermediate amounts of DNA damage were observed in the kidney of the sheep living in the control district. As expected, the levels of cadmium and lead were significantly increased in the sheep with fluorosis as compared to controls. Also, the concentrations of heavy metals were driven from the distance of the sheep-farms to the industrial site. Our results broaden knowledge about the role of exocyclic DNA adducts in the etiology of skeletal fluorosis. Fluoride generated from the non-ferrous metallurgy complexes may increase the intracellular amounts of oxidative stress and ROS within the soft organs. A continuous attack to DNA may contribute to the general decline of cellular functions, ranging from disturbance of DNA metabolism, triggering cell-cycle arrest and apoptosis, to inducing necrosis and cellular degenerative changes.

The generation of bulky DNA adducts consists of conjugates formed between large reactive electrophiles and DNA-binding sites. The term "bulky DNA adducts" comes from early experiments that employed a

Nanotechnology is addressing major urgent needs for cancer treatment. We conducted a study to compare the frequency of 3-(2-deoxy-β-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) adducts, biomarkers of oxidative stress and/or lipid peroxidation, on human hepatocarcinoma HepG2 cells exposed to increasing levels of Fe3O4-nanoparticles (NPs) versus untreated cells at different lengths of incubations, and in the presence of increasing exposures to an alternating magnetic field (AMF) of 186 kHz using 32P-postlabeling. The levels of oxidative damage tended to increase significantly after ≥24 h of incubations compared to controls. The oxidative DNA damage tended to reach a steady-state after treatment with 60 μg/mL of Fe3O4-NPs. Significant dose–response relationships were observed. A greater adduct production was observed after magnetic hyperthermia, with the highest amounts of oxidative lesions after 40 min exposure to AMF. The effects of magnetic hyperthermia were significantly increased with exposure and incubation times. Most important, the levels of oxidative lesions in AMF exposed NP treated cells were up to 20-fold greater relative to those observed in nonexposed NP treated cells. Generation of oxidative lesions may be a mechanism by which magnetic hyperthermia induces cancer cell death.

Epidemiologic evidence linking environmental exposure to polycyclic aromatic hydrocarbons (PAH) with breast cancer is limited. Measurement of DNA adducts formed by aromatic compounds, including PAH, has been carried in breast tissue samples and white blood cells from women with breast cancer and different kinds of controls. However, these studies provide inconsistent results and bias cannot be ruled out. During the 7-year follow-up period, 305 women were diagnosed with first primary breast cancer in the EPIC-Spain cohort, and were compared with a sample of 149 women without breast cancer at recruitment, using a case-cohort approach. Aromatic adducts to DNA from leukocytes collected at recruitment were measured by means of the 32P-post-labelling technique. The relative risk and 95% confidence interval (CI), adjusted by relevant confounders, were estimated by a modified version of Cox proportional hazards model. There was a significant increased risk for developing breast cancer when DNA adduct concentrations were doubled, with adjusted RR of 1.61 (95% CI 1.29-2.01). The increase in breast cancer risk was observed both for pre- and post-menopausal women. There was a significant interaction with tobacco smoking and body mass index, with higher effect of DNA adducts on breast cancer risk among smokers and women with normal weight. The results from our study support the hypothesis that factors leading to higher levels of aromatic DNA adducts in white blood cells may be involved in development of breast cancer.

The generation of bulky DNA adducts consists of conjugates formed between large reactive electrophiles and DNA-binding sites. The term "bulky DNA adducts" comes from early experiments that employed a 32P-DNA postlabeling approach. This technique has long been used to elucidate the association between adducts and carcinogen exposure in tobacco smoke studies and assess the predictive value of adducts in cancer risk. Molecular data showed increased DNA adducts in respiratory tracts of smokers vs nonsmokers. Experimental studies and meta-analysis demonstrated that the relationship between adducts and carcinogens was linear at low doses, but reached steady state at high exposure, possibly due to metabolic and DNA repair pathway saturation and increased apoptosis. Polymorphisms of metabolic and DNA repair genes can increase the effects of environmental factors and confer greater likelihood of adduct formation. Nevertheless, the central question remains as to whether bulky adducts cause human cancer. If so, lowering them would reduce cancer incidence. Pooled and meta-analysis has shown that smokers with increased adducts have increased risk of lung cancer. Adduct excess in smokers, especially in prospective longitudinal studies, supports their use as biomarkers predictive of lung cancer.

Tobacco smoking is a leading cause of death and disability. Interindividual variability in DNA adducts has been shown in subjects exposed to similar amounts of environmental carcinogens, including tobacco smoke. We have investigated the effects of smoking on DNA adducts in the nasal epithelium instead of peripheral blood in 42 volunteers, considering a panel of at-risk alleles by 32P-postlabeling and PCR. In detail, we have studied the association of DNA damage with tobacco smoke habits considering genes involved in DNA repair, including X-ray repair cross complementing protein 1 (XRCC1) Arg194Trp, XRCC protein 3 Thr241Met, and excision repair cross complementation group 2/xeroderma pigmentosum D (ERCC2/XPD) Lys751Gln polymorphisms. Then, we have analysed the combinations of the variant alleles of XRCC1 and ERCC2/XPD together with the wild type allele of XRCC3 by calculating the sum of at-risk alleles for lung cancer. DNA adducts were significantly higher in smokers with respect to nonsmokers (P < 0.001). An overall significant increase in adducts in heavy and long term smokers was found (P-values for trend

Formaldehyde is the most commonly used fixative for the preparation of formalin-fixed paraffin-embedded tissues (FFPETs) in the “reduction rooms” of pathology wards. Therefore, we analysed for the generation of 3-(2-deoxy-β-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one adducts (M1dG), an exocyclic DNA adduct considered to be a biomarker of oxidative stress and lipid peroxidation, in DNA extracted from the FFPETs of liver and lung samples of six C57BL/6 control mice and from the FFPETs of four cancer patients with respect to paired flash-frozen tissues by 32P-postlabeling. When the experimental animals were examined, the percentage of M1dG adducts was about 4–6 fold greater with the FFPET mouse samples as compared to flash-frozen mouse samples. Specifically, 4.75 M1dG ± 0.21 (SE) per 106 normal nucleotides (nn) were detected in the FFPET liver samples, and 1.07 M1dG ± 0.08 (SE) per 106 nn in the flash-frozen liver samples (p = 0.02). Then, 3.80 M1dG ± 0.73 (SE) per 106 nn were measured in the FFPET lung samples, and 1.02 M1dG ± 0.07 (SE) per 106 nn in the flash-frozen lung samples (p = 0.02). Also, significantly increased levels of oxidatively damaged DNA were detected in the human colon DNA from the FFPETs with respect to the flash-frozen tissues. There were 30.2 M1dG ± 7.7 (SE) per 108 nn in the colon mucosa DNA from the FFPETs and 4.4 M1dG ± 0.7 (SE) per 108 nn in the corresponding flash-frozen human tissues (p = 0.016). Formalin penetration through cell membrane components induces excessive oxidative stress, causing both direct oxidation of DNA and increased lipid peroxidation, which in turn produce M1dG adducts, a kind of DNA damage that can partially block DNA synthesis and induce error prone translesion synthesis. An excess of exocyclic DNA adducts in formalin-fixed specimens can stall DNA polymerases and contribute to the induction of artefactual sequence alterations during PRC amplification.

Tobacco smoke has been shown to produce both DNA damage and epigenetic alterations. However, the potential role of DNA damage in generating epigenetic changes is largely underinvestigated in human studies. We examined the effects of smoking on the levels of DNA methylation in genes for tumor protein p53, cyclin-dependent kinase inhibitor2A, hypermethylated-in-cancer-1 (HIC1), interleukin-6, Long Interspersed Nuclear Element type1, and Alu retrotransposons in blood of 177 residents in Thailand using bisulfite-PCR andpyrosequencing. Then, we analyzed the relationship of this methylation with the oxidative DNA adduct, M1dG (a malondialdehyde adduct), measured by 32P-postlabeling. Multivariate statistical analyses showed that HIC1 methylation levels were significantly increased in smokers compared with nonsmokers (p =.05). A dose response was observed, with the highest HIC1 methylation levels in smokers of = 10 cigarettes/ day relative to nonsmokers and intermediate values in smokers of 1-9 cigarettes/day (p for trend =.001). No additional relationships were observed. We also evaluated correlations between M1dG and the methylation changes at each HIC1 CpG site individually. The levels of this adduct in smokers showed a significant linear correlation with methylation at one of the 3 CpGs evaluated in HIC1: hypermethylation at position 1904864340 was significantly correlated with the adduct M1dG (covariate-adjusted regression coefficient (ß) =.224 ±.101 [SE], p =.05). No other correlations were detected. Our study extends prior work by others associating hypermethylation of HIC1 with smoking; shows that a very specific hypermethylation event can arise from smoking; and encourages future studies that explore a possible role for M1dG in connecting smoking to this latter hypermethylation.© The Author 2013. Published by Oxford University Press on behalf of the Society of Toxicology.

Interindividual variation in DNA adduct levels in individuals exposed to similar amounts of environmental carcinogens may be due to genetic variability. We analysed the influence of genes involved in determining/modifying DNA damage, including microsomal epoxide hydrolase1 (EPHX1) His139Arg, N-acetyl-transferase, NAD(P)H:quinone oxidoreductase1 (NQO1) Pro187Ser, manganese superoxide dismutase2 (MnSOD2) Val16Ala, and apurinic/apyrimidinic endonuclease1 (APE1) Asp148Glu polymorphisms in blood of 120 smokers. Subsequently, we examined the effects of the combinations of the variant alleles of EPHX, NQO1 and MnSOD2 together with the wild type allele of APE1 on DNA damage by calculating the “sum of at-risk alleles.” We reviewed the studies examining the relationships of DNA adducts with at-risk alleles in environmentally exposed subjects. Our findings showed that smokers carrying the EPHX1–139Arg and the NQO1–187Ser variants were significantly more likely to have higher adduct levels. Null associations were found with the other variants. Nevertheless, DNA adduct levels in smokers with ≥5 at-risk alleles were significantly different from those with fewer than two alleles. A similar picture emerged from studies of DNA adducts and at-risk alleles in environmentally exposed and smoking subjects. Certain at-risk allele combinations may confer a greater likelihood of increased levels of adducts after environmental insults. The increase in DNA adduct levels in susceptible subjects exposed to environmental carcinogens may reflect changes in the mechanisms that protect cells from the accumulation of genetic damage. Alterations of the physiological processes designed to maintain homeostasis may reduce the individual “genotoxic tolerance” to environmental challenges and result in phenotypes characterized by high levels of DNA adducts. Environ. Mol. Mutagen. 54:375–383, 2013. © 2013 Wiley Periodicals, Inc.

Air quality is a primary environmental concern in highly industrialised areas, with potential health effects in children residing nearby. The Sarroch industrial estate in Cagliari province, Sardinia Island, Italy, hosts the world's largest power plant and the second largest European oil refinery and petrochemical park. This industrial estate produces a complex mixture of air pollutants, including benzene, heavy metals and polycyclic aromatic hydrocarbons. Thus, we conducted a cross-sectional study to evaluate the prevalence of malondialdehyde-deoxyguanosine adducts in the nasal epithelium of 75 representative children, aged 6-14 years, attending primary and secondary schools in Sarroch in comparison with 73 rural controls. Additionally, the levels of bulky DNA adducts were analysed in a subset of 62 study children. DNA damage was measured by (32)P-postlabelling methodologies. The air concentrations of benzene and ethyl benzene were measured in the school gardens of Sarroch and a rural village by diffusive samplers. Outdoor measurements were also performed in other Sarroch areas and in the proximity of the industrial estate. The outdoor levels of benzene and ethyl benzene were significantly higher in the school gardens of Sarroch than in the rural village. Higher concentrations were also found in other Sarroch areas and in the vicinity of the industrial park. The mean levels of malondialdehyde-deoxyguanosine adducts per 10(8) normal nucleotides ± standard error (SE) were 74.6±9.1 and 34.1±4.4 in the children from Sarroch and the rural village, respectively. The mean ratio was 2.53, 95% confidence interval (CI): 1.71-2.89, P < 0.001, versus rural controls. Similarly, the levels of bulky DNA adducts per 10(8) normal nucleotides ± SE were 2.9±0.4 and 1.6±0.2 in the schoolchildren from Sarroch and the rural village, respectively. The means ratio was 1.90, 95% CI: 1.25-2.89, P = 0.003 versus rural controls. Our study indicates that children residing near the industrial estate have a significant increment of DNA damage.

Maternal intake of flavonoids, known for their antioxidant properties, may affect the offspring's susceptibility to developing chronic diseases at adult age, especially those related to oxidative stress, via developmental programming. Therefore, we supplemented female mice with the flavonoids genistein and quercetin during gestation, to study their effect on the antioxidant capacity of lung and liver of adult offspring. Maternal intake of quercetin increased the expression of Nrf2 and Sod2 in fetal liver at gestational day 14.5. At adult age, in utero exposure to both flavonoids resulted in the increased expression of several enzymatic antioxidant genes, which was more pronounced in the liver than in the adult lung. Moreover, prenatal genistein exposure induced the nonenzymatic antioxidant capacity in the adult lung, partly by increasing glutathione levels. Prenatal exposure to both flavonoids resulted in significantly lower levels of oxidative stress-induced DNA damage in liver only. Our observations lead to the hypothesis that a preemptive trigger of the antioxidant defense system in utero had a persistent effect on antioxidant capacity and as a result decreased oxidative stress-induced DNA damage in the liver.

Background: Adverse biological effects from airborne pollutants are a primary environmental concern in highly industrialized areas. Recent studies linked air pollution exposures with altered blood Deoxyribo-nucleic acid (DNA) methylation, but effects from industrial sources and underlying biological mechanisms are still largely unexplored. Methods: The Ma Ta Phut industrial estate (MIE) in Rayong, Thailand hosts one of the largest steel, oil refinery and petrochemical complexes in south-eastern Asia. We measured a panel of blood DNA methylation markers previously associated with air pollution exposures, including repeated elements [long interspersed nuclear element-1 (LINE-1) and Alu] and genes [p53, hypermethylated-in-cancer-1 (HIC1), p16 and interleukin-6 (IL-6)], in 67 MIE workers, 65 Ma Ta Phut residents and 45 rural controls. To evaluate the role of DNA damage and oxidation, we correlated DNA methylation measures with bulky DNA and 3-(2-deoxy-ß-D-erythro-pentafuranosyl)pyrimido[1,2-a]purin-10(3H)-one deoxyguanosine (M1dG) adducts. Results: In covariate-adjusted models, MIE workers, compared with rural residents, showed lower LINE-1 (74.8% vs 78.0%; P < 0.001), p53 (8.0% vs 15.7%; P < 0.001) and IL-6 methylation (39.2% vs 45.0%; P = 0.027) and higher HIC1 methylation (22.2% vs 15.3%, P < 0.001). For all four markers, Ma Ta Phut residents exhibited methylation levels intermediate between MIE workers and rural controls (LINE-1, 75.7%, P < 0.001; p53, 9.0%, P < 0.001; IL-6, 39.8%, P = 0.041; HIC1, 17.8%, P = 0.05; all P-values vs rural controls). Bulky DNA adducts showed negative correlation with p53 methylation (P = 0.01). M1dG showed negative correlations with LINE-1 (P = 0.003) and IL-6 methylation (P = 0.05). Conclusions: Our findings indicate that industrial exposures may induce alterations of DNA methylation patterns detectable in blood leucocyte DNA. Correlation of DNA adducts with DNA hypomethylation suggests potential mediation by DNA damage. © The Author 2012; all rights reserved.

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.

Malondialdehyde (MDA), a biomarker of lipid peroxidation and oxidative stress, is a mutagenic and carcinogenic compound that can react with DNA to form several types of DNA adducts including the deoxyguanosine adduct (M1dG). The aim of this cross-sectional study was to evaluate the association between individual dietary and lifestyle habits and M1dG levels, measured in peripheral leukocytes in a large representative sample of the general population of Florence City (Italy). Selected anthropometric measurements, detailed information on dietary and lifestyle habits and blood samples were available for 313 adults of the Florence City Sample enrolled in the frame of European Prospective Investigation into Cancer and nutrition (EPIC) study. A multivariate regression analysis adjusted for selected individual characteristics possibly related to M1dG levels (sex, age, BMI, smoke, physical activity level, education level, total caloric intake and a Mediterranean dietary score) was performed to estimate the association between these parameters and M1dG levels. M1dG levels were significantly higher in women (P = 0.014) and lower in moderately active or active subjects (P = 0.037).We also found a significant inverse association with the Modified Mediterranean dietary score (P for trend = 0.049), particularly evident for the highest categories of adherence. Our results indicate that M1dG levels can be modulated by selected individual characteristics such as gender, physical activity and a Mediterranean dietary pattern.

Formaldehyde is a ubiquitous volatile organic compound widely used for various industrial purposes. Formaldehyde was reclassified by the International Agency for Research on Cancer as a human carcinogen, based on sufficient evidence for a casual role for nasopharyngeal cancer. However, the mechanisms by which this compound causes nasopharyngeal cancer are not completely understood. Therefore, we have examined the formaldehyde-induced toxicity in the nasal epithelia of the workers of a plastic laminate plant in Bra, Cuneo, Piedmont region, North-Western Italy, hence in the target site for formaldehyde-related nasal carcinogenesis. We have conducted a cross-sectional study aimed at comparing the frequency of 3-(2-deoxy-β-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) adducts, a biomarker of oxidative stress and lipid peroxidation, in 50 male exposed workers and 45 male controls using 32P-DNA post-labeling. The personal levels of formaldehyde exposure were analysed by gas-chromatography mass-spectrometry. The smoking status was estimated by measuring the concentrations of urinary cotinine by gas-chromatography mass-spectrometry. The air monitoring results showed that the exposure levels of formaldehyde were significantly greater for the plastic laminate plant workers, 211.4 ± 14.8 standard error (SE) μg m−3, than controls, 35.2 ± 3.4 (SE) μg m−3, P < 0.001. The levels of urinary cotinine were 1064 ± 118 ng ml−1 and 14.18 ± 2.5 ng ml−1 in smokers and non-smokers, respectively, P < 0.001. The M1dG adduct frequency per 108 normal nucleotides was significantly higher among the workers of the plastic laminate plant exposed to formaldehyde, 111.6 ± 14.3 (SE), compared to controls, 49.6 ± 3.4 (SE), P < 0.001. This significant association persisted also when personal dosimeters were used to measure the extent of indoor levels of formaldehyde exposure. No influences of smoking and age were observed across the study population. However, after categorization for occupational exposure, a significant effect was found in the controls, P = 0.018, where the levels of DNA damage were significantly correlated with the levels of urinary cotinine, regression coefficient (β) = 0.494 ± 0.000 (SE), P < 0.002. Our findings indicated that M1dG adducts constitute a potential mechanism of formaldehyde-induced toxicity. Persistent DNA damage contributes to the general decline of the physiological mechanisms designed to maintain cellular homeostasis.

The next-generation sequencing studies of breast cancer have reported that the tumour suppressor P53 (TP53) gene is mutated in more than 40% of the tumours. We studied the levels of oxidative lesions, including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), along the coding strand of the exon 5 in breast cancer patients as well as in a reactive oxygen species (ROS)-attacked breast cancer cell line using the ligation-mediated polymerase chain reaction technique. We detected a significant 'in vitro' generation of 8-oxodG between the codons 163 and 175, corresponding to a TP53 region with high mutation prevalence, after treatment with xanthine plus xanthine oxidase, a ROS-generating system. Then, we evaluated the occurrence of oxidative lesions in the DNA-binding domain of the TP53 in the core needle biopsies of 113 of women undergoing breast investigation for diagnostic purpose. An increment of oxidative damage at the -G- residues into the codons 163 and 175 was found in the cancer cases as compared to the controls. We found significant associations with the pathological stage and the histological grade of tumours. As the major news of this study, this largest analysis of genomic footprinting of oxidative lesions at the TP53 sequence level to date provided a first roadmap describing the signatures of oxidative lesions in human breast cancer. Our results provide evidence that the generation of oxidative lesions at single nucleotide resolution is not an event highly stochastic, but causes a characteristic pattern of DNA lesions at the site of mutations in the TP53, suggesting causal relationship between oxidative DNA adducts and breast cancer.

The Map-Ta-Phut Industrial Estate (MIE) in Rayong, Thailand, is the location of one of the largest industrial complexes in southeastern Asia. The MIE complex produces a mixture of air pollutants, including polycyclic aromatic hydrocarbons, compounds capable to induce the generation of DNA adducts. DNA adducts are considered to be a biomarker of carcinogen exposure; however, its production can be modulated by genetic susceptibility. Thus, we analysed the influence of EPHX1 His139Arg (A>G, rs2234922) and NQO1 Pro187Ser (C>T, rs1800566) involved in the metabolism of polycyclic aromatic hydrocarbons; MnSOD2 Val16Ala (C>T, rs1799725) a gene that acts against the free radical generation; APE1/Ref-1 Asp148Glu (T>G, rs3136820) a gene involved in the repair of DNA, and in the control of cell-cycle and apoptosis on leucocyte DNA adducts in 77 MIE workers, 69 Map-Ta-Phut residents, and 50 rural controls, Rayong, Thailand. We searched for associations with the 'sum of at-risk alleles' by combining the variant alleles of EPHX1, NQO1 and MnSOD2 together with the wild-type allele of APE1, since they appeared to influence lung cancer risk. Although our findings revealed significant associations between DNA adducts and the EPHX1 His139Arg and NQO1 Pro187Ser polymorphisms, the combination of at-risk alleles was found to affect DNA damage much stronger. DNA adducts were significantly increased in the individuals bearing 4 and =5 at-risk alleles [mean ratio (MR) = 1.55, 95% CI 1.10-2.18, P = 0.012, and MR = 2.11, 95% CI 1.27-3.51, P = 0.004, respectively)]. After correction for residence/employment categorisation, a significant increment was present in the MIE workers with =5 alleles (MR = 2.88, 95% CI 1.46-5.71, P = 0.003). Our data indicate relationships between the generation of DNA adducts and the enzymatic activities of EPHX and NQO1. The combination of unfavourable genetic variants seems to determine the individuals' susceptibility, rather than a single polymorphism. © The Author 2012. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved.

Diet has been shown to modulate M(1)dG adduct, a biomarker of oxidative stress and lipid peroxidation. Thus, we analysed the association between diet and M(1)dG in 120 controls and 67 Map Ta Phut industrial estate workers in Rayong, Thailand, to evaluate the influence of fruit and vegetables, and fried and charcoal-grilled/barbecued food consumption on M(1)dG. M(1)dG was decreased in controls reporting to consume 14-17 servings/week of fruit and vegetables (mean ratio [MR]= 0.35, CI 0.18-0.69, p< 0.05). Conversely, a non-statistically significant M(1)dG increment was detected in controls consuming 9-18 servings/week of fried food (MR = 1.33, CI 0.88-2.00, p = 0.168). No effect of charcoal-grilled/barbecued food was found. No effect of diet was observed in workers. An association with smoking was observed in controls (MR = 1.88, CI 1.14-3.10, p < 0.05), but not in workers. M(1)dG can induce mutations and/or methylation changes within the promoter regions of cancer-related genes, thus promotion of healthy eating practices should be recommended.

This study has analysed the generation of 3-(2-deoxy-β-D-erythro-pentafuranosyl) pyrimido [1,2-α] purin-10 (3H)-one deoxyguanosine adduct [M1dG], a biomarker of oxidative stress and lipid peroxidation, in breast fine-needle aspirate samples of 22 patients with breast cancer, at different clinical stages, in respect to 13 controls. The multivariate analysis show that M1dG adduct was higher in cases than in controls (Mean Ratio (MR) = 5.26, 95% CI = 3.16–8.77). Increased M1dG was observed in women with a tumour grade 3 and a pathological diameter 2 (MR = 7.61, 95% CI 3.91–14.80 and MR = 5.75, 95% CI = 3.13–10.59, respectively). A trend with increasing tumour grade and pathological diameter was present (MR = 1.98, 95% CI = 1.57–2.50 and MR = 2.44, 95% CI = 1.71–3.48, respectively). Not significant effects of age and smoking habit were found (MR = 1.58, 95% CI = 0.92–2.72 and MR = 1.68, 95% CI 0.88–3.20, respectively). An increment over the background frequency of M1dG can contribute to breast cancer development. Increasing severity of breast tumour can influence DNA damage level.

Formaldehyde is an ubiquitous pollutant to which humans are exposed. Pathologists can experience high formaldehyde exposure levels. Formaldehyde-among other properties-induce oxidative stress and free radicals, which react with DNA and lipids, leading to oxidative damage and lipid peroxidation, respectively. We measured the levels of air-formaldehyde exposure in a group of Italian pathologists and controls. We analyzed the effect of formaldehyde exposure on leukocyte malondialdehyde-deoxyguanosine adducts (M(1)-dG), a biomarker of oxidative stress and lipid peroxidation. We studied the relationship between air-formaldehyde and M(1)-dG adducts. Air-formaldehyde levels were measured by personal air samplers. M(1)-dG adducts were analyzed by a (32)P-postlabeling assay. Reduction room pathologists were significantly exposed to air-formaldehyde with respect to controls and to the pathologists working in other laboratory areas (p0.001). A significant difference for M(1)-dG adducts between exposed pathologists and controls was found (p = 0.045). The effect becomes stronger when the evaluation of air-formaldehyde exposure was based on personal samplers (p = 0.018). Increased M(1)dG adduct levels were only found in individuals exposed to air-formaldehyde concentrations higher than 66 microg/m(3). When the exposed workers and controls were subgrouped according to smoking, M(1)-dG tended to increase in all of the subjects, but a significant association between M(1)-dG and air-formaldehyde was only found in nonsmokers (p = 0.009). Air-formaldehyde played a role positive but not significant (r = 0.355, p = 0.075, Pearson correlation) in the formation of M(1)-dG, only in nonsmokers. Working in the reduction rooms and exposure to air-formaldehyde concentrations higher than 66 microg/m(3) are associated with increased levels of M(1)-dG adducts.

Background: Humans living near industrial point emissions can experience high levels of exposures to air pollutants. Map Ta Phut Industrial Estate in Thailand is the location of the largest steel, oil refinery, and petrochemical factory complexes in Southeast Asia. Air pollution is an important source of oxidative stress and reactive oxygen species, which interact with DNA and lipids, leading to oxidative damage and lipid peroxidation, respectively. Objective: We measured the levels of malondialdehyde-deoxyguanosine (dG) adducts, a biomarker of oxidative stress and lipid peroxidation, in petrochemical workers, nearby residents, and subjects living in a control district without proximity to industrial sources. Design: We conducted a cross-sectional study to compare the prevalence of malondialdehyde-dG adducts in groups of subjects experiencing various degrees of air pollution. Results: The multivariate regression analysis shows that the adduct levels were associated with occupational and environmental exposures to air pollution. The highest adduct level was observed in the steel factory workers. In addition, the formation of DNA damage tended to be associated with tobacco smoking, but without reaching statistical significance. A non significant increase in DNA adducts was observed after 4-6 years of employment among the petrochemical complexes. Conclusions: Air pollution emitted from the Map Ta Phut Industrial Estate complexes was associated with increased adduct levels in petrochemical workers and nearby residents. Considering the mutagenic potential of DNA lesions in the carcinogenic process, we recommend measures aimed at reducing the levels of air pollution.

Chronic inflammation has been recognized as a contributing factor in the pathogenesis of lung cancer. In this process, reactive oxygen species released by neutrophils may play an important role. The aim of the present study was to investigate the capacity of the major neutrophilic oxidant hypochlorous acid (HOCl), which is formed by myeloperoxidase (MPO), to induce DNA damage and mutagenicity in lung cells. HOCl was mutagenic in lung epithelial A549 cells in vitro, showing at physiological concentrations a significant induction of mutations in the HPRT gene. We studied three major types of DNA lesions that could be relevant for this HOCl-induced mutagenicity. Single strand DNA breakage and 8-oxo-7,8-dihydro-2'-deoxyguanosine were not found to be increased following HOCl treatment. On the other hand, HOCl caused a significant increase in the formation of 3-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M(1)dG), which can be formed by either malondialdehyde (MDA) or base propenals. We observed an increased MDA formation upon exposure of A549 cells to HOCl, but a role of base propenals cannot be excluded. In line with this, we observed 4-fold increased M(1)dG adduct levels in mice that were intratracheally instilled with lipopolysaccharide to induce a pulmonary inflammation with neutrophil influx. Depletion of circulating neutrophils significantly reduced pulmonary MPO activity as well as M(1)dG adducts levels, thereby providing a causal link between neutrophils/HOCl and pulmonary genotoxicity in vivo. Taken together, these data indicate that MPO catalysed formation of HOCl during lung inflammation should be considered as a significant source of neutrophil-induced genotoxicity.

The aim of this study was to assess the relationship between bulky DNA adducts in white blood cells (WBC) and lifestyle factors in a sample from the Spanish cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC). 296 subjects aged between 35 and 64 years, from five regions, were included. Food intake was estimated with a computerized version of dietary history questionnaire. Daily intake of polycyclic aromatic hydrocarbons (PAH) was estimated using a database with information on food content of potential carcinogens. Data on lifestyle and health factors were collected and DNA adducts measured using the nuclease P1 32P-postlabelling technique. Geometric means of adducts were similar for men and women (4.11/109 and 3.94/109 nucleotides, respectively). Highest levels of adduct were observed in non-smokers and non-occupationally exposed. Meat intake, oils and fats were associated with higher levels of adducts, but all non-statistically significant. Higher intakes of calcium, sodium and phosphorus were associated with lower adducts levels. Summarising, our study shows that bulky adducts measured by 32P-postlabelling in DNA from WBC do not correlate with the usual diet of healthy Spanish adults. Although it has been proposed that diet be the main source of PAH in nonsmokers without occupational exposure, DNA adducts do not seem to be suitable biomarkers of dietary PAH in general population.

Air pollution and particulate matter in urban areas have been associated with increased mortality from cardiovascular and respiratory diseases and increased cancer risk. Carcinogenic effects of particulate matter have been related to the contents of specific compounds, such as polycyclic aromatic hydrocarbons. The latter may form bulky DNA adducts, that may be considered as candidate markers of cancer risk. We have recently shown that traffic-exposed workers and the general population in Florence have divergent levels of DNA adducts, possibly related to different levels of exposure to genotoxic agents from vehicle emissions. In the current study, in a series of 214 Florence City healthy adults enrolled in a prospective study in the period 1993–1998 (152 residents / 62 traffic-exposed workers), we investigated the correlation between individual levels of DNA bulky adducts and PM 10 exposure scores based on daily environmental measures provided by the local Environmental Protection Agency for the whole study period, by two types of urban monitoring stations (high- and low-traffic). We found that PM 10 cumulative scores from high-traffic stations over the last 1–2 weeks prior to blood drawing significantly correlated ( r = 0.58, p = 0.02) with DNA adduct levels among non-smoking traffic-exposed workers (but not among residents with no occupational exposure to vehicle emissions). A multivariate regression analysis adjusted for possible confounders confirmed these findings. PM 10 scores from low-traffic stations did not show any correlation. These results show that DNA adduct levels in non-smoking workers reflect the average levels of exposure to PM 10 in high-traffic urban areas experienced over a time period of 1–2 weeks. Since DNA adduct levels have been found predictive of lung cancer risk, our findings provide clues relevant to the reduction of genotoxic damage and possibly cancer risk among traffic-exposed urban workers.

The (32)P-post-labelling assay has emerged as a major tool for detecting bulky DNA adducts in subjects exposed to carcinogens, especially aromatic compounds. However, the (32)P-post-labelling protocol still requires the use of high amounts of radioactivity, i.e. 25-50 muCi per sample, an obstacle that limits its use in large studies. The characterization of the DNA adducts measured is also limited. Methodological improvements and increased DNA adduct characterization are necessary to make this assay capable of achieving higher throughput. A new protocol was tested to ensure efficient hydrolysis to reduce the use of radioactive material and to obtain higher chromatography resolution. Different chromatography systems based on high-urea or ammonium hydroxide systems were also employed to characterize the adducts being measured. Improvements were tested by re-analysing DNA adducts in a group of police officers and urban residents in Genoa, Italy. The analysis of carcinogen-modified DNA standards was also included in the study for qualitative and quantitative comparison. An efficient DNA digestion was obtained using a method involving hydrolysis by micrococcal nuclease and a mixture of two spleen phosphodiesterases at fixed concentrations. A 72% reduction of the amount of radioactivity used for labelling was achieved in respect to the non-modified protocol without loss of DNA adduct sensitivity. An improved chromatography resolution was obtained by reducing the volume of sample to be spotted on the chromatogram. Lower volume of spotting sample can decrease sample diffusion and the formation of unresolved spots on the thin-layer chromatography plate. The amount of output produced using a single batch of carrier-free [gamma-(32)P]ATP was increased by about 3.5-fold. A complex pattern of DNA adducts was observed in leukocytes using both high-urea or isopropanol-ammonium hydroxide systems, two techniques effective in the detection of aromatic DNA adducts. The above observations indicate that DNA adducts being measured are likely to have been induced by aromatic compounds.

Tobacco smoking is a major risk factor for lung cancer causing, among other effects, oxidative stress and lipid peroxidation. Malondialdehyde (MDA)–DNA adducts can be induced by direct DNA oxidation and by lipid peroxidation. We measured the relationship between bronchial MDA–DNA adducts and tobacco smoking, cancer status, and selected polymorphisms in 43 subjects undergoing a bronchoscopic examination for diagnostic purposes. MDA–DNA adducts were higher in current smokers than in never smokers (frequency ratio (FR) = 1.51, 95% confidence interval (CI) 1.01–2.26). MDA–DNA adducts were also increased in lung cancer cases with respect to controls, but only in smokers (FR = 1.70, 95% CI 1.16–2.51). Subjects with GA and AA cyclin D1 (CCND1) genotypes showed higher levels of MDA–DNA adducts than those with the wild-type genotype (FR = 1.51 (1.04–2.20) and 1.45 (1.02–2.07)). Lung cancer cases with levels of MDA–DNA adducts over the median showed a worse, but not statistically significant, survival, after adjusting for age, gender, and packyears (hazard ratio = 2.48, 95% CI 0.65–9.44). Our findings reinforce the role of smoking in lung carcinogenesis through oxidative stress. Subjects who carry at least one variant allele of the CCND1 gene could accumulate DNA damage for altered cell-cycle control and reduced DNA repair proficiency.

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. (Cancer Res 2006; 66(13): 6871-6)