Linking the generation of DNA adducts to lung cancer.

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.

Bulky DNA Adducts, Tobacco Smoking, Genetic Susceptibility, and Lung Cancer Risk.

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.

Magnetic Hyperthermia and Oxidative Damage to DNA of Human Hepatocarcinoma Cells.

Nanotechnology is addressing major urgent needs for cancer treatment. We conducted a study to compare the frequency of 3-(2-deoxy-ß-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.

3-(2-deoxy-ß-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine adducts of workers exposed to asbestos fibers.

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-ß-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 <0.001, respectively. A significant effect of smoking on heavy smokers was found (p=0.005). Our study gives additional support to the oxidative stress theory, where M1dG may reflect an additional potential mechanism of asbestos-induced toxicity.

8-Oxo-7,8-dihydro-2'-deoxyguanosine and other lesions along the coding strand of the exon 5 of the tumour suppressor gene P53 in a breast cancer case-control study.

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.

Formaldehyde-induced toxicity in the nasal epithelia of workers of a plastic laminate plant.

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-ß-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 (ß) = 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.

Oxidatively damaged DNA in the nasal epithelium of workers occupationally exposed to silica dust in Tuscany region, Italy.

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-ß-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.

Bisphenol-A exposures and behavioural aberrations: median and linear spline and meta-regression analyses of 12 toxicity studies in rodents.

Exposures to bisphenol-A, a weak estrogenic chemical, largely used for the production of plastic containers, can affect the rodent behaviour. Thus, we examined the relationships between bisphenol-A and the anxiety-like behaviour, spatial skills, and aggressiveness, in 12 toxicity studies of rodent offspring from females orally exposed to bisphenol-A, while pregnant and/or lactating, by median and linear splines analyses. Subsequently, the meta-regression analysis was applied to quantify the behavioural changes. U-shaped, inverted U-shaped and J-shaped dose-response curves were found to describe the relationships between bisphenol-A with the behavioural outcomes. The occurrence of anxiogenic-like effects and spatial skill changes displayed U-shaped and inverted U-shaped curves, respectively, providing examples of effects that are observed at low-doses. Conversely, a J-dose-response relationship was observed for aggressiveness. When the proportion of rodents expressing certain traits or the time that they employed to manifest an attitude was analysed, the meta-regression indicated that a borderline significant increment of anxiogenic-like effects was present at low-doses regardless of sexes (ß)=-0.8%, 95% C.I. -1.7/0.1, P=0.076, at ≤120 µg bisphenol-A. Whereas, only bisphenol-A-males exhibited a significant inhibition of spatial skills (ß)=0.7%, 95% C.I. 0.2/1.2, P=0.004, at ≤100 µg/day. A significant increment of aggressiveness was observed in both the sexes (ß)=67.9,C.I. 3.4, 172.5, P=0.038, at >4.0 µg. Then, bisphenol-A treatments significantly abrogated spatial learning and ability in males (P<0.001 vs. females). Overall, our study showed that developmental exposures to low-doses of bisphenol-A, e.g. ≤120 µg/day, were associated to behavioural aberrations in offspring.

Aromatic adducts and lung cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC) Spanish cohort.

In this case-cohort study, we examined the association between bulky DNA adducts and the risk of lung cancer within the European Prospective Investigation into Cancer and Nutrition (EPIC) Spanish cohort with an average 7-year follow-up, including 98 cases of primary lung cancer and 296 subjects randomly selected from the cohort. Aromatic adducts were measured using (32)P-postlabeling in leukocyte DNA from blood samples collected at enrollment. The association between DNA adducts and the risk of lung cancer was estimated using a Cox proportional hazards model with a modified partial likelihood. There was an overall significant increased risk for developing lung cancer when DNA adduct concentrations were doubled, with relative risk (RR) adjusting for all relevant confounders of 1.36 with 95% confidence interval (CI) 1.18-157. There was a significant increased risk for developing lung cancer when DNA adduct concentrations were doubled for current smokers and among subjects exposed to PAH at work; there was also a slightly higher increase among males than females. However, no statistically significant differences were observed for the effect of adduct levels across smoking status, sex or occupational exposure to PAH. A meta-analysis combined four prospective studies, including this study, resulting in a significant association among current smokers, with an overall estimate of 34% increase in the risk of lung cancer when doubling the level of aromatic DNA adducts in leukocytes.

Aberrant methylation of hypermethylated-in-cancer-1 and exocyclic DNA adducts in tobacco smokers.

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.

DNA adducts and combinations of multiple lung cancer at-risk alleles in environmentally exposed and smoking subjects.

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.

Exocycilic DNA Adducts in a Murine Model of Non-alcoholic Steatohepatitis.

INTRODUCTION: Non-alcoholic fatty liver disease is the most common hepatic disorder in Western countries. The transition from abnormal accumulation of lipids toward non-alcoholic steatohepatitis (NASH) represents a key step in the development of chronic liver pathologies. Oxidative stress and lipid peroxidation have often been proposed as mechanisms in the progression to steatohepatitis. METHODS: We have examined the hepatic levels of exocyclic DNA adducts, indicated from 3-(2-deoxy-ß-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) adduct, a biomarker of oxidative stress and lipid peroxidation, in a murine model of NASH using the 32P-DNA postlabeling assay. RESULTS: Our findings show that C57BL/6 mice fed with high-fat and cholesterol diet developed signs associated with NASH after eight weeks, whereas there was no evidence of steatosis in control mice. The score for steatohepatitis ranged from grade 2 to 3 for steatosis, inflammation, and fibrosis, showing that the experimental diet was able to induce pathologic alterations of the parenchyma in eight weeks. Higher levels of M1dG adducts were detected in the livers of C57BL/6 mice which developed experimental NASH after eight weeks of high-fat and cholesterol feed, 5.6 M 1dG ± 0.4 (SE) per 106 total nucleotides, as compared to control mice, 1.6 M1dG ± 0.4 (SE). The statistical analysis showed that the increment of oxidatively damaged DNA in mice with NASH raised on high-fat and cholesterol diet was statistically significant as compared to control mice, P=0.006. CONCLUSIONS: Our report suggests a link between NASH and M1dG in experimental animals fed with a diet rich in saturated fats and cholesterol. High-fat and cholesterol may act together in inducing a broader spectrum of oxidatively damaged DNA, including exocyclic DNA adducts, that may contribute to the decline of hepatocyte functions, from disturbance of critical pathways, such as transcription and replication, triggering transient or permanent cell-cycle arrest and cell-death, up to chromosomal instability.