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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× 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 β-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.

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 Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

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., (© The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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(3 H )-one deoxyguanosine (M 1 dG) adducts, a biomarker of oxidative stress and lipid peroxidation, in 50 male exposed workers and 45 male controls using 32 P-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 M 1 dG adduct frequency per 10 8 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 M 1 dG 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.

Unlabelled: 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 (M1dG) 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 M1dG generation was measured by the (32)P-postlabelling assay. Significant higher levels of M1dG 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 M1dG 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 < 0.001, respectively. Null increments were observed in association with the pottery and the ceramic factories. After stratification for different exposures, silica-exposed workers that were co-exposed to organic solvents, and welding and exhaust fumes have significantly higher M1dG levels, 90.4±13.4 (SE), P = 0.014 vs., Control: Our data suggested that silica exposure might be associated with genotoxicity in the nasal epithelial cells of silica-exposed workers that handle of artificial marble conglomerates and quarry workers. Importantly, we observed that co-exposures to other respiratory carcinogens may have contributed to enhance the burden of M1dG adducts in the nasal epithelium of silica-exposed workers., (© The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 (β) = .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.

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.

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; MnSOD(2) 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 MnSOD(2) 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.

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-β-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M(1)dG) 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). M(1)dG 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.

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 (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.

We have conducted a randomized trial which investigated the ability of dietary changes (in particular diets rich in cruciferous vegetables and flavonoids), to increase urinary antimutagenicity and inhibit DNA damage in smokers. Ninety heavy smokers were recruited and randomly assigned to three groups, and were given three different diets. The first diet was based on flavonoid-rich foods, particularly cruciferous vegetables, but not based on supplementation; the second was a normal isocaloric diet (with an adequate administration of fruits and vegetables); and the third was based on supplementation of flavonoids in the form of green tea and soy products. DNA adducts were measured by (32)P-postlabelling in exfoliated bladder cells at different times since the start of the trial. In spite of randomization, subjects in the control group smoked more than those in the experimental groups, and this can explain the higher adduct levels at baseline. A slight decrease in bulky DNA adducts in exfoliated bladder cells was observed after 1 year since the end in the supplementation group and after 1 month in white blood cells. The only statistically significant association was found in a regression model that adjusted for smoking, in which the increase in flavonoid intake was associated with a decrease in adducts after 1 year (P = 0.02). These data suggest that adherence to a diet rich in cruciferous vegetables and flavonoids might reduce genotoxicity in the human urinary bladder of smokers, but they should be interpreted with caution owing to small numbers and the uneven distribution of smoking habits in the experimental groups. Smoking is the most important single preventable cause of cancer; at the present stage of knowledge it is totally unlikely that certain dietary habits can seriously counteract the effects of tobacco smoking.

Industrial and urban workers may be exposed to significant levels of air pollutants resulting from the incomplete combustion of organic matter. The authors performed a meta-analysis of 13 DNA-adduct studies ((32)P-DNA postlabeling technique) on occupational cohorts exposed to air pollution. The association between levels of DNA adducts and air pollution exposure was significant both in heavily exposed industrial workers and in less severely exposed urban workers. Moreover, in an analysis using the seven studies that reported measuring levels of benzo[a]pyrene (B(a)P), a typical marker of exposure, DNA adduct levels in exposed workers (versus those in referents) were significantly correlated with air levels of B(a)P. The relation between DNA adducts and B(a)P was found to be linear at low doses and sublinear at high doses, indicating that DNA adduct formation tends to reach some kind of saturation point at higher levels of exposure to the chemical mixtures present in fumes. When the authors examined the efficiency of DNA adduct production associated with increasing air pollution exposures, the production of DNA adducts per unit of exposure was significantly decreased at higher B(a)P exposure levels. These findings suggest that linear downward extrapolations based on DNA adduct levels associated with B(a)P concentrations of > or =20 ng/m(3) might be affected by underestimation bias.