Exposure to particulate matter (PM) has been associated with lung cancer risk in epidemiology investigations. Elemental components of PM have been suggested to have critical roles in PM toxicity, but the molecular mechanisms underlying their association with cancer risks remain poorly understood. DNA methylation has emerged as a promising biomarker for environmental-related diseases, including lung cancer. In this study, we evaluated the effects of PM elemental components on methylation of three tandem repeats in a highly-exposed population in Beijing, China. The Beijing Truck Driver Air Pollution Study was conducted shortly before the 2008 Beijing Olympic Games (June 15-July 27, 2008) and included 60 truck drivers and 60 office workers. On two days separated by 1-2 weeks, we measured blood DNA methylation of SATα, NBL2, D4Z4, and personal exposure to eight elemental components in PM2.5, including aluminum (Al), silicon (Si), sulfur (S), potassium (K), calcium (Ca) titanium (Ti), iron (Fe), and zinc (Zn). We estimated the associations of individual elemental component with each tandem repeat methylation in generalized estimating equations (GEE) models adjusted for PM2.5 mass and other covariates. Out of the eight examined elements, NBL2 methylation was positively associated with concentrations of Si (0.121, 95%CI: 0.030; 0.212, FDR=0.047) and Ca (0.065, 95%CI: 0.014; 0.115, FDR=0.047) in truck drivers. In office workers, SATα methylation was positively associated with concentrations of S (0.115, 95%CI: 0.034; 0.196, FDR=0.042). PM-associated differences in blood tandem-repeat methylation may help detect biological effects of the exposure and identify individuals who may eventually experience higher lung cancer risk.
Tandem repeats; DNA methylation; lung cancer
MicroRNAs (miRNAs) are environmentally sensitive inhibitors of gene expression that may mediate the effects of metal-rich particulate matter (PM) and toxic metals on human individuals. Previous environmental miRNA studies have investigated a limited number of candidate miRNAs and have not yet evaluated the functional effects on gene expression. In this study, we wanted to identify PM-sensitive miRNAs using microarray profiling on matched baseline and postexposure RNA from foundry workers with well-characterized exposure to metal-rich PM and to characterize miRNA relations with expression of candidate inflammatory genes. We applied microarray analysis of 847 human miRNAs and real-time PCR analysis of 18 candidate inflammatory genes on matched blood samples collected from foundry workers at baseline and after 3 days of work (postexposure). We identified differentially expressed miRNAs (fold change [FC] > 2 and p < 0.05) and correlated their expression with the inflammatory associated genes. We performed in silico network analysis in MetaCore v6.9 to characterize the biological pathways connecting miRNA-mRNA pairs. Microarray analysis identified four miRNAs that were differentially expressed in postexposure compared with baseline samples, including miR-421 (FC = 2.81, p < 0.001), miR-146a (FC = 2.62, p = 0.007), miR-29a (FC = 2.91, p < 0.001), and let-7g (FC = 2.73, p = 0.019). Using false discovery date adjustment for multiple comparisons, we found 11 miRNA-mRNA correlated pairs involving the 4 differentially expressed miRNAs and candidate inflammatory genes. In silico network analysis with MetaCore database identified biological interactions for all the 11 miRNA-mRNA pairs, which ranged from direct mRNA targeting to complex interactions with multiple intermediates. Acute PM exposure may affect gene regulation through PM-responsive miRNAs that directly or indirectly control inflammatory gene expression.
miRNA expression; integrative analysis; mRNA expression; inflammation; metal-rich particulate matter; microarray.
Worldwide lung cancer incidence is decreasing or leveling off among men, but rising among women. Sex differences in associations of tobacco carcinogens with lung cancer risk have been hypothesized, but the epidemiologic evidence is conflicting. We tested sex-smoking interaction in association with lung cancer risk within a population-based case-control study, the Environment and Genetics in Lung Cancer Etiology (EAGLE) Study (Lombardy, Italy, 2002–2005). Detailed lifetime smoking histories were collected by personal interview in 2,100 cases with incident lung cancer and 2,120 controls. Odds ratios and 95% confidence intervals for pack-years of cigarette smoking were estimated by logistic regression, adjusted for age, residence area, and time since quitting smoking. To assess sex-smoking interaction, we compared the slopes of odds ratios for logarithm of pack-years in a model for men and women combined. Overall, the slope for pack-years was steeper in men (odds ratio for female-smoking interaction = 0.39, 95% confidence interval: 0.24, 0.62; P < 0.0001); after restriction to ever smokers, the difference in slopes was much smaller (odds ratio for interaction = 0.63, 95% confidence interval: 0.29, 1.37; P = 0.24). Similar results were found by histological type. Results were unchanged when additional confounders were evaluated (e.g., tobacco type, inhalation depth, Fagerström-assessed nicotine dependence). These findings do not support a higher female susceptibility to tobacco-related lung cancer.
case-control studies; lung cancer; sex differences; smoking
Recent investigations have associated airborne Particulate Matter (PM) with increased coagulation and thrombosis, but underlying biological mechanisms are still incompletely characterized. DNA methylation is an environmentally-sensitive mechanism of gene regulation that could potentially contribute to PM-induced hypercoagulability. We aimed to test whether altered methylation mediates environmental effects on coagulation.
We investigated 63 steel workers exposed to a wide range of PM levels, as a work-related condition with well-characterized prothrombotic exposure. We measured personal PM10 (PM≤10 μm in aerodynamic diameter), PM1 (≤1 μm), and air metal components. We determined leukocyte DNA methylation of NOS3 (nitric-oxide-synthase-3) and EDN1 (endothelin-1) through bisulfite-pyrosequencing and we measured Endogenous Thrombin Potential (ETP), as a global coagulation-activation test after standardized triggers.
ETP increased in association with PM10 (β=20.0, 95%CI: 3.0, 37.0), PM1 (β=80.8 95%CI: 14.9, 146.7), and zinc (β=51.3, 95%CI: 0.01, 111.1) exposures. NOS3 methylation was negatively associated with PM10 (β=−0.2, 95%CI: −0.4, −0.03), PM1 (β=−0.8, 95%CI: −1.4, −0.1), zinc (β=−0.9, 95%CI: −1.4, −0.3) and iron (β=−0.7, 95%CI: −1.4, −0.01) exposures. Zinc exposure was negatively associated with EDN1 (β=−0.3, 95%CI: −0.8, −0.1) methylation. Lower NOS3 (β=−42.3; p<0.001) and EDN1 (β=−14.5; p=0.05) were associated with higher ETP. Statistical mediation analysis formally confirmed NOS3 and EDN1 hypomethylation as intermediate mechanisms for PM-related coagulation effects.
Our study showed for the first time, that gene hypomethylation contributes to environmentally-induced hypercoagulability.
Air pollution; DNA methylation; coagulation
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
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.
Air pollution; Ma Ta Phut; blood leucocytes; DNA methylation; DNA damage
Epidemiologic studies have reported that frequent consumption of quercetin-rich foods is inversely associated with lung cancer incidence. A quercetin-rich diet might modulate microRNA (miR) expression; however, this mechanism has not been fully examined.
miR expression data were measured by a custom-made array in formalin-fixed paraffin-embedded tissue samples from 264 lung cancer cases (144 adenocarcinomas and 120 squamous cell carcinomas). Intake of quercetin-rich foods was derived from a food-frequency questionnaire. In individual-miR-based analyses, we compared the expression of miRs (n=198) between lung cancer cases consuming high-versus-low quercetin-rich food intake using multivariate ANOVA tests. In family-miR-based analyses, we used Functional Class Scoring (FCS) to assess differential effect on biologically functional miRs families. We accounted for multiple testing using 10,000 global permutations (significance at p-valueglobal <0.10). All multivariate analyses were conducted separately by histology and by smoking status (former and current smokers).
Family-based analyses showed that a quercetin-rich diet differentiated miR expression profiles of the tumor suppressor let-7 family among adenocarcinomas (p-valueFCS<0.001). Other significantly differentiated miR families included carcinogenesis-related miR-146, miR-26, and miR-17 (p-valuesFCS<0.05). In individual-based analyses, we found that among former and current smokers with adenocarcinoma, 33 miRs were observed to be differentiated between highest-and-lowest quercetin-rich food consumers (23 expected by chance; p-valueglobal = 0.047).
We observed differential expression of key biologically functional miRNAs between high-versus-low consumers of quercetin-rich foods in adenocarcinoma cases.
Our findings provide preliminary evidence on the mechanism underlying quercetin-related lung carcinogenesis.
Ambient particular matter (PM) exposure has been associated with short- and long-term effects on cardiovascular disease (CVD). Telomere length (TL) is a biomarker of CVD risk that is modified by inflammation and oxidative stress, two key pathways for PM effects. Whether PM exposure modifies TL is largely unexplored.
To investigate effects of PM on blood TL in a highly-exposed population.
We measured blood TL in 120 blood samples from truck drivers and 120 blood samples from office workers in Beijing, China. We measured personal PM2.5 and Elemental Carbon (EC, a tracer of traffic particles) using light-weight monitors. Ambient PM10 was obtained from local monitoring stations. We used covariate-adjusted regression models to estimate percent changes in TL per an interquartile-range increase in exposure.
Covariate-adjusted TL was higher in drivers (mean=0.87, 95%CI: 0.74; 1.03) than in office workers (mean=0.79, 95%CI: 0.67; 0.93; p=0.001). In all participants combined, TL increased in association with personal PM2.5 (+5.2%, 95%CI: 1.5; 9.1; p=0.007), personal EC (+4.9%, 95%CI: 1.2; 8.8; p=0.01), and ambient PM10 (+7.7%, 95%CI: 3.7; 11.9; p<0.001) on examination days. In contrast, average ambient PM10 over the 14 days before the examinations was significantly associated with shorter TL (−9.9%, 95%CI: −17.6; −1.5; p=0.02).
Short-term exposure to ambient PM is associated with increased blood TL, consistent with TL roles during acute inflammatory responses. Longer exposures may shorten TL as expected after prolonged pro-oxidant exposures. The observed TL alterations may participate in the biological pathways of short- and long-term PM effects.
Particulate Matter; Personal Monitoring; Telomere length; Traffic pollution; China
Large fractions of the human population do not express GSTM1 and GSTT1 (GSTM1/T1) enzymes because of deletions in these genes. These variations affect xenobiotic metabolism and have been evaluated in relation to lung cancer risk, mostly based on null/present gene models. We measured GSTM1/T1 heterozygous deletions, not tested in genome-wide association studies, in 2120 controls and 2100 cases from the Environment And Genetics in Lung cancer Etiology (EAGLE) study. We evaluated their effect on mRNA expression on lung tissue and peripheral blood samples and their association with lung cancer risk overall and by histology types. We tested the null/present, dominant and additive models using logistic regression. Cigarette smoking and gender were studied as possible modifiers. Gene expression from blood and lung tissue cells was strongly down-regulated in subjects carrying GSTM1/T1 deletions by both trend and dominant models (p<0.001). In contrast to the null/present model, analyses distinguishing subjects with 0, 1 or 2 GSTM1/T1 deletions revealed several associations. There was a decreased lung cancer risk in never-smokers (OR=0.44;95%CI=0.23–0.82; p=0.01) and women (OR=0.50;95%CI=0.28–0.90; p=0.02) carrying 1 or 2 GSTM1 deletions. Analogously, male smokers had an increased risk (OR=1.13;95%CI=1.0–1.28; p=0.05) and women a decreased risk (OR=0.78;95%CI=0.63–0.97; p=0.02) for increasing GSTT1 deletions. The corresponding gene-smoking and gene-gender interactions were significant (p<0.05). Our results suggest that decreased activity of GSTM1/T1 enzymes elevates lung cancer risk in male smokers, likely due to impaired carcinogens’ detoxification. A protective effect of the same mutations may be operative in never-smokers and women, possibly because of reduced activity of other genotoxic chemicals.
GST; copy numbers; gene expression; lung cancer; smoking and gender differences
We examined occupational exposures among subjects with sinonasal cancer (SNC) recorded in a population-based registry in the Lombardy Region, the most populated and industrialized Italian region. The registry collects complete clinical information and exposure to carcinogens regarding all SNC cases occurring in the population of the region. In the period 2008–2011, we recorded 210 SNC cases (137 men, 73 women). The most frequent occupational exposures were to wood (44 cases, 21.0%) and leather dust (29 cases, 13.8%), especially among men: 39 cases (28.5%) to wood and 23 cases (16.8%) to leather dust. Exposure to other agents was infrequent (<2%). Among 62 subjects with adenocarcinoma, 50% had been exposed to wood dust and 30.7% to leather dust. The proportions were around 10% in subjects with squamous cell carcinoma and about 20% for tumors with another histology. The age-standardized rates (×100,000 person-years) were 0.7 in men and 0.3 in women. Complete collection of cases and their occupational history through a specialized cancer registry is fundamental to accurately monitor SNC occurrence in a population and to uncover exposure to carcinogens in different industrial sectors, even those not considered as posing a high risk of SNC, and also in extraoccupational settings.
Repetitive elements take up >40% of the human genome and can change distribution through transposition, thus generating subfamilies. Repetitive element DNA methylation has associated with several diseases and environmental exposures, including exposure to airborne pollutants. No systematic analysis has yet been conducted to examine the effects of exposures across different repetitive element subfamilies. The purpose of the study is to evaluate sensitivity of DNA methylation in differentially‒evolved LINE, Alu, and HERV subfamilies to different types of airborne pollutants.
We sampled a total of 120 male participants from three studies (20 high-, 20 low-exposure in each study) of steel workers exposed to metal-rich particulate matter (measured as PM10) (Study 1); gas-station attendants exposed to air benzene (Study 2); and truck drivers exposed to traffic-derived elemental carbon (Study 3). We measured methylation by bisulfite-PCR-pyrosequencing in 10 differentially‒evolved repetitive element subfamilies.
High-exposure groups exhibited subfamily-specific methylation differences compared to low-exposure groups: L1PA2 showed lower DNA methylation in steel workers (P=0.04) and gas station attendants (P=0.03); L1Ta showed lower DNA methylation in steel workers (P=0.02); AluYb8 showed higher DNA methylation in truck drivers (P=0.05). Within each study, dose–response analyses showed subfamily-specific correlations of methylation with exposure levels. Interaction models showed that the effects of the exposures on DNA methylation were dependent on the subfamily evolutionary age, with stronger effects on older LINEs from PM10 (p‒interaction=0.003) and benzene (p‒interaction=0.04), and on younger Alus from PM10 (p-interaction=0.02).
The evolutionary age of repetitive element subfamilies determines differential susceptibility of DNA methylation to airborne pollutants.
Environment; Exposures; DNA methylation; Repetitive elements; Subfamily
Background Exposure to occupational carcinogens is an important preventable cause of lung cancer. Most of the previous studies were in highly exposed industrial cohorts. Our aim was to quantify lung cancer burden attributable to occupational carcinogens in a general population.
Methods We applied a new job–exposure matrix (JEM) to translate lifetime work histories, collected by personal interview and coded into standard job titles, into never, low and high exposure levels for six known/suspected occupational lung carcinogens in the Environment and Genetics in Lung cancer Etiology (EAGLE) population-based case–control study, conducted in Lombardy region, Italy, in 2002–05. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated in men (1537 cases and 1617 controls), by logistic regression adjusted for potential confounders, including smoking and co-exposure to JEM carcinogens. The population attributable fraction (PAF) was estimated as impact measure.
Results Men showed an increased lung cancer risk even at low exposure to asbestos (OR: 1.76; 95% CI: 1.42–2.18), crystalline silica (OR: 1.31; 95% CI: 1.00–1.71) and nickel–chromium (OR: 1.18; 95% CI: 0.90–1.53); risk increased with exposure level. For polycyclic aromatic hydrocarbons, an increased risk (OR: 1.64; 95% CI: 0.99–2.70) was found only for high exposures. The PAFs for any exposure to asbestos, silica and nickel–chromium were 18.1, 5.7 and 7.0%, respectively, equivalent to an overall PAF of 22.5% (95% CI: 14.1–30.0). This corresponds to about 1016 (95% CI: 637–1355) male lung cancer cases/year in Lombardy.
Conclusions These findings support the substantial role of selected occupational carcinogens on lung cancer burden, even at low exposures, in a general population.
lung neoplasms; case–control study; carcinogens; occupational health
Short‐term exposures to fine (<2.5 μm aerodynamic diameter) ambient particulate‐matter (PM) have been related with increased blood pressure (BP) in controlled‐human exposure and community‐based studies. However, whether coarse (2.5 to 10 μm) PM exposure increases BP is uncertain. Recent observational studies have linked PM exposures with blood DNA hypomethylation, an epigenetic alteration that activates inflammatory and vascular responses. No experimental evidence is available to confirm those observational data and demonstrate the relations between PM, hypomethylation, and BP.
Methods and Results
We conducted a cross‐over trial of controlled‐human exposure to concentrated ambient particles (CAPs). Fifteen healthy adult participants were exposed for 130 minutes to fine CAPs, coarse CAPs, or HEPA‐filtered medical air (control) in randomized order with ≥2‐week washout. Repetitive‐element (Alu, long interspersed nuclear element‐1 [LINE‐1]) and candidate‐gene (TLR4, IL‐12, IL‐6, iNOS) blood methylation, systolic and diastolic BP were measured pre‐ and postexposure. After adjustment for multiple comparisons, fine CAPs exposure lowered Alu methylation (β‐standardized=−0.74, adjusted‐P=0.03); coarse CAPs exposure lowered TLR4 methylation (β‐standardized=−0.27, adjusted‐P=0.04). Both fine and coarse CAPs determined significantly increased systolic BP (β=2.53 mm Hg, P=0.001; β=1.56 mm Hg, P=0.03, respectively) and nonsignificantly increased diastolic BP (β=0.98 mm Hg, P=0.12; β=0.82 mm Hg, P=0.11, respectively). Decreased Alu and TLR4 methylation was associated with higher postexposure DBP (β‐standardized=0.41, P=0.04; and β‐standardized=0.84, P=0.02; respectively). Decreased TLR4 methylation was associated with higher postexposure SBP (β‐standardized=1.45, P=0.01).
Our findings provide novel evidence of effects of coarse PM on BP and confirm effects of fine PM. Our results provide the first experimental evidence of PM‐induced DNA hypomethylation and its correlation to BP.
air pollution; blood pressure; DNA methylation; epigenetics; mediation
Mitochondria have small mitochondrial DNA (mtDNA) molecules independent from the nuclear DNA, a separate epigenetic machinery that generates mtDNA methylation, and are primary sources of oxidative-stress generation in response to exogenous environments. However, no study has yet investigated whether mitochondrial DNA methylation is sensitive to pro-oxidant environmental exposures.
We sampled 40 male participants (20 high-, 20 low-exposure) from each of three studies on airborne pollutants, including investigations of steel workers exposed to metal-rich particulate matter (measured as PM1) in Brescia, Italy (Study 1); gas-station attendants exposed to air benzene in Milan, Italy (Study 2); and truck drivers exposed to traffic-derived Elemental Carbon (EC) in Beijing, China (Study 3). We have measured DNA methylation from buffy coats of the participants. We measured methylation by bisulfite-Pyrosequencing in three mtDNA regions, i.e., the transfer RNA phenylalanine (MT-TF), 12S ribosomal RNA (MT-RNR1) gene and “D-loop” control region. All analyses were adjusted for age and smoking.
In Study 1, participants with high metal-rich PM1 exposure showed higher MT-TF and MT-RNR1 methylation than low-exposed controls (difference = 1.41, P = 0.002); MT-TF and MT-RNR1 methylation was significantly associated with PM1 exposure (beta = 1.35, P = 0.025); and MT-RNR1 methylation was positively correlated with mtDNA copy number (r = 0.36; P = 0.02). D-loop methylation was not associated with PM1 exposure. We found no effects on mtDNA methylation from air benzene (Study 2) and traffic-derived EC exposure (Study 3).
Mitochondrial MT-TF and MT-RNR1 DNA methylation was associated with metal-rich PM1 exposure and mtDNA copy number. Our results suggest that locus-specific mtDNA methylation is correlated to selected exposures and mtDNA damage. Larger studies are needed to validate our observations.
Air pollutants; Mitochondria; DNA methylation
Background Estimates of global DNA methylation from repetitive DNA elements, such as Alu and LINE-1, have been increasingly used in epidemiological investigations because of their relative low-cost, high-throughput and quantitative results. Nevertheless, determinants of these methylation measures in healthy individuals are still largely unknown. The aim of this study was to examine whether age, gender, smoking habits, alcohol drinking and body mass index (BMI) are associated with Alu or LINE-1 methylation levels in blood leucocyte DNA of healthy individuals.
Methods Individual data from five studies including a total of 1465 healthy subjects were combined. DNA methylation was quantified by PCR-pyrosequencing.
Results Age [β = −0.011% of 5-methyl-cytosine (%5mC)/year, 95% confidence interval (CI) −0.020 to −0.001%5mC/year] and alcohol drinking (β = −0.214, 95% CI −0.415 to −0.013) were inversely associated with Alu methylation. Compared with females, males had lower Alu methylation (β = −0.385, 95% CI −0.665 to −0.104) and higher LINE-1 methylation (β = 0.796, 95% CI 0.261 to 1.330). No associations were found with smoking or BMI. Percent neutrophils and lymphocytes in blood counts exhibited a positive (β = 0.036, 95% CI 0.010 to 0.061) and negative (β = −0.038, 95% CI −0.065 to −0.012) association with LINE-1 methylation, respectively.
Conclusions Global methylation measures in blood DNA vary in relation with certain host and lifestyle characteristics, including age, gender, alcohol drinking and white blood cell counts. These findings need to be considered in designing epidemiological investigations aimed at identifying associations between DNA methylation and health outcomes.
Blood; DNA methylation; epigenetics; meta-analysis; repetitive elements
DNA methylation is increasingly proposed as a mechanism for underlying asthma-related inflammation. However, epigenetic studies are constrained by uncertainties on whether samples that can be easily collected in human individuals can provide informative results.
Two nasal cell DNA samples were collected on different days by nasal brushings from 35 asthmatic children aged between 8 and 11 years old. We correlated DNA methylation of IL-6, iNOS, Alu and LINE-1 with fractional exhaled nitric oxide, forced expiratory volume in 1 s and wheezing.
Fractional exhaled nitric oxide increased in association with lower promoter methylation of both IL-6 (+29.0%; p = 0.004) and iNOS (+41.0%; p = 0.002). Lower IL-6 methylation was nonsignificantly associated with wheezing during the week of the study (odds ratio = 2.3; p = 0.063).
Our findings support the use of nasal cell DNA for human epigenetic studies of asthma.
airway obstruction; asthma; children; DNA methylation; epigenetics; inflammation
Affordable early screening in subjects with high risk of lung cancer has great potential to improve survival from this deadly disease. We measured gene expression from lung tissue and peripheral whole blood (PWB) from adenocarcinoma cases and controls to identify dysregulated lung cancer genes that could be tested in blood to improve identification of at-risk patients in the future. Genome-wide mRNA expression analysis was conducted in 153 subjects (73 adenocarcinoma cases, 80 controls) from the Environment And Genetics in Lung cancer Etiology (EAGLE) study using PWB and paired snap-frozen tumor and non-involved lung tissue samples. Analyses were conducted using unpaired t-tests, linear mixed effects and ANOVA models. The area under the receiver operating characteristic curve (AUC) was computed to assess the predictive accuracy of the identified biomarkers. We identified 50 dysregulated genes in stage I adenocarcinoma versus control PWB samples (False Discovery Rate ≤0.1, fold change ≥1.5 or ≤0.66). Among them, eight (TGFBR3, RUNX3, TRGC2, TRGV9, TARP, ACP1, VCAN, and TSTA3) differentiated paired tumor versus non-involved lung tissue samples in stage I cases, suggesting a similar pattern of lung cancer-related changes in PWB and lung tissue. These results were confirmed in two independent gene expression analyses in a blood-based case-control study (n=212) and a tumor-non tumor paired tissue study (n=54). The eight genes discriminated patients with lung cancer from healthy controls with high accuracy (AUC=0.81, 95% CI=0.74–0.87). Our finding suggests the use of gene expression from PWB for the identification of early detection markers of lung cancer in the future.
microarray gene expression; peripheral blood; lung cancer; stage I
Alcohol abuse leads to earlier onset of aging-related diseases, including cancer at multiple sites. Shorter telomere length (TL) in peripheral blood leucocytes (PBLs), a marker of biological aging, has been associated with alcohol-related cancer risks. Whether alcohol abusers exhibit accelerated biological aging, as reflected in PBL-TL, has never been examined.
To investigated the effect of alcohol abuse on PBL-TL and its interaction with alcohol metabolic genotypes, we examined 200 drunk-driving traffic offenders diagnosed as alcohol abusers as per the Diagnostic and Statistical Manual of Mental Disorders [DSM-IV-TR] and enrolled in a probation program, and 257 social drinkers (controls). We assessed alcohol intake using self-reported drink-units/day and conventional alcohol abuse biomarkers (serum γ-glutamyltrasferase [GGT] and mean corpuscular volume of erythrocytes [MCV]). We used multivariable models to compute TL geometric means (GM) adjusted for age, smoking, BMI, diet, job at elevated risk of accident, genotoxic exposures.
TL was nearly halved in alcohol abusers compared to controls (GMs 0.42 vs. 0.87 relative T/S ratio; P<0.0001) and decreased in relation with increasing drink-units/day (P-trend=0.003). Individuals drinking >4 drink-units/day had substantially shorter TL than those drinking 4 drink-units/day (GMs 0.48 vs. 0.61 T/S, P=0.002). Carriers of the common ADH1B*1/*1 (rs1229984) genotype were more likely to be abusers (P=0.008), reported higher drink-units/day (P=0.0003), and exhibited shorter TL (P<0.0001). The rs698 ADH1C and rs671 ALDH2 polymorphisms were not associated with TL.
The decrease in PBL-TL modulated by the alcohol metabolic genotype ADH1B*1/*1 may represent a novel mechanism potentially related to alcohol carcinogenesis in alcohol abusers.
While lung cancer is largely caused by tobacco smoking, inherited genetic factors play a role in its etiology. Genome-wide association studies (GWAS) in Europeans have robustly demonstrated only three polymorphic variations influencing lung cancer risk. Tumor heterogeneity may have hampered the detection of association signal when all lung cancer subtypes were analyzed together. In a GWAS of 5,355 European smoking lung cancer cases and 4,344 smoking controls, we conducted a pathway-based analysis in lung cancer histologic subtypes with 19,082 SNPs mapping to 917 genes in the HuGE-defined “inflammation” pathway. We identified a susceptibility locus for squamous cell lung carcinoma (SQ) at 12p13.33 (RAD52, rs6489769), and replicated the association in three independent samples totaling 3,359 SQ cases and 9,100 controls (odds ratio=1.20, Pcombined=2.3×10−8).
The combination of pathway-based approaches and information on disease specific subtypes can improve the identification of cancer susceptibility loci in heterogeneous diseases.
Lung cancer; histology; squamous cell carcinoma; pathway analysis; RAD52
Background: Benzene is an established leukemogen at high exposure levels. Although low-level benzene exposure is widespread and may induce oxidative damage, no mechanistic biomarkers are available to detect biological dysfunction at low doses.
Objectives: Our goals were to determine in a large multicenter cross-sectional study whether low-level benzene is associated with increased blood mitochondrial DNA copy number (mtDNAcn, a biological oxidative response to mitochondrial DNA damage and dysfunction) and to explore potential links between mtDNAcn and leukemia-related epigenetic markers.
Methods: We measured blood relative mtDNAcn by real-time polymerase chain reaction in 341 individuals selected from various occupational groups with low-level benzene exposures (> 100 times lower than the Occupational Safety and Health Administration/European Union standards) and 178 referents from three Italian cities (Genoa, Milan, Cagliari).
Results: In each city, benzene-exposed participants showed higher mtDNAcn than referents: mtDNAcn was 0.90 relative units in Genoa bus drivers and 0.75 in referents (p = 0.019); 0.90 in Milan gas station attendants, 1.10 in police officers, and 0.75 in referents (p-trend = 0.008); 1.63 in Cagliari petrochemical plant workers, 1.25 in referents close to the plant, and 0.90 in referents farther from the plant (p-trend = 0.046). Using covariate-adjusted regression models, we estimated that an interquartile range increase in personal airborne benzene was associated with percent increases in mtDNAcn equal to 10.5% in Genoa (p = 0.014), 8.2% (p = 0.008) in Milan, 7.5% in Cagliari (p = 0.22), and 10.3% in all cities combined (p < 0.001). Using methylation data available for the Milan participants, we found that mtDNAcn was associated with LINE-1 hypomethylation (–2.41%; p = 0.007) and p15 hypermethylation (+15.95%, p = 0.008).
Conclusions: Blood MtDNAcn was increased in persons exposed to low benzene levels, potentially reflecting mitochondrial DNA damage and dysfunction.
benzene; biomarkers; low exposures; methylation; mitochondrial DNA copy number
Global DNA hypomethylation affecting repeat sequences has been reported in different cancer types. Herein, we investigated the methylation levels of repetitive DNA elements in chronic lymphocytic leukemia (CLL), their correlation with the major cytogenetic and molecular features, and clinical relevance in predicting therapy-free survival (TFS). A quantitative bisulfite-PCR Pyrosequencing method was used to evaluate methylation of Alu, long interspersed nuclear elements-1 (LINE-1) and satellite-α (SAT-α) sequences in 77 untreated early-stage (Binet A) CLL patients. Peripheral B-cells from 7 healthy donors were used as controls. Methylation levels (median %5mC) were lower in B-CLLs compared with controls (21.4 vs. 25.9; 66.8 vs. 85.7; 84.0, vs. 88.2 for Alu, LINE-1 and SAT-α, respectively) (p < 0.001). Among CLL patients, a significant association was observed with 17p13.1 deletion (16.8 vs. 22.4; 51.2 vs. 68.5; 52.6 vs. 85.0, for Alu, LINE-1 and SAT-α) but not with other major genetic lesions, IgVH mutation status, CD38 or ZAP-70 expression. Follow-up analyses showed that lower SAT-α methylation levels appeared to be an independent prognostic marker significantly associated with shorter TFS. Our study extended previous limited evidences in methylation of repetitive sequences in CLL suggesting an important biological and clinical relevance in the disease.
Alu; DNA methyltransferases; LINE-1; SAT-α; chronic lymphocytic leukemia
Particulate Matter (PM) exposure is critical in Beijing due to high population density and rapid increase in vehicular traffic. PM effects on blood pressure (BP) have been investigated as a mechanism mediating cardiovascular risks, but results are still inconsistent. The purpose of our study is to determine the effects of ambient and personal PM exposure on BP.
Before the 2008 Olympic Games (June 15-July 27), we examined 60 truck drivers and 60 office workers on two days, 1-2 weeks apart (n = 240). We obtained standardized measures of post-work BP. Exposure assessment included personal PM2.5 and Elemental Carbon (EC, a tracer of traffic particles) measured using portable monitors during work hours; and ambient PM10 averaged over 1-8 days pre-examination. We examined associations of exposures (exposure group, personal PM2.5/EC, ambient PM10) with BP controlling for multiple covariates.
Mean personal PM2.5 was 94.6 μg/m3 (SD = 64.9) in office workers and 126.8 (SD = 68.8) in truck drivers (p-value < 0.001). In all participants combined, a 10 μg/m3 increase in 8-day ambient PM10 was associated with BP increments of 0.98 (95%CI 0.34; 1.61; p-value = 0.003), 0.71 (95%CI 0.18; 1.24; p-value = 0.01), and 0.81 (95%CI 0.31; 1.30; p-value = 0.002) mmHg for systolic, diastolic, and mean BP, respectively. BP was not significantly different between the two groups (p-value > 0.14). Personal PM2.5 and EC during work hours were not associated with increased BP.
Our results indicate delayed effects of ambient PM10 on BP. Lack of associations with exposure groups and personal PM2.5/EC indicates that PM effects are related to background levels of pollution in Beijing, and not specifically to work-related exposure.
Particulate Matter; Personal Monitoring; Blood Pressure; Traffic Pollution; China
The molecular drivers that determine histology in lung cancer are largely unknown. We investigated whether microRNA (miR) expression profiles can differentiate histological subtypes and predict survival for non-small cell lung cancer.
We analyzed miR expression in 165 adenocarcinoma (AD) and 125 squamous cell carcinoma (SQ) tissue samples from the Environmental And Genetics in Lung cancer Etiology (EAGLE) study using a custom oligo array with 440 human mature antisense miRs. We compared miR expression profiles using t-tests and F-tests and accounted for multiple testing using global permutation tests. We assessed the association of miR expression with tobacco smoking using Spearman correlation coefficients and linear regression models, and with clinical outcome using log-rank tests, Cox proportional hazards and survival risk prediction models, accounting for demographic and tumor characteristics.
MiR expression profiles strongly differed between AD and SQ (global p<0.0001), particularly in the early stages, and included miRs located on chromosome loci most often altered in lung cancer (e.g., 3p21-22). Most miRs, including all members of the let-7 family, were down-regulated in SQ. Major findings were confirmed by QRT-PCR in EAGLE samples and in an independent set of lung cancer cases. In SQ, low expression of miRs down-regulated in the histology comparison was associated with 1.2 to 3.6-fold increased mortality risk. A 5-miR signature significantly predicted survival for SQ.
We identified a miR expression profile that strongly differentiated AD from SQ and had prognostic implications. These findings may lead to histology-based therapeutic approaches.
Particulate matter (PM) exposure has been linked to increased risk of cardiovascular disease, possibly resulting from hypercoagulability and thrombosis. Lung and systemic inflammation from PM inhalation may activate blood coagulation, but mechanisms for PM-related hypercoagulability are still largely unknown.
To identify coagulation mechanisms activated by PM in a population with well-characterized exposure.
We measured prothrombin time [PT], activated-partial-thromboplastin time [aPTT], Endogenous Thrombin Potentials [ETP] with/without exogenous triggers and with/without soluble thrombomodulin, tissue-plasminogen activator antigen [t-PA], D-dimer, and C-reactive protein [CRP] in 37 workers in a steel-production plant with well-characterized exposure to PM with aerodynamic diameter <1μm (PM1) and coarse PM (PM10-PM1). Blood samples were collected from each subject on the first (baseline) and last (post-exposure) day of a four-day workweek. We analysed differences between baseline and post-exposure levels using paired Student’s t-test. We fitted multivariate mixed-regression models to estimate the associations of inter-quartile range PM1 and coarse PM exposure with parameter levels.
None of the parameters showed any significant changes in post-exposure samples, compared to baseline. However, exposure levels were associated with shorter PT (β[PM1]=−0.33 sec, p=0.08; β[PMcoarse]=−0.33 sec, p=0.01), and higher ETP without exogenous triggers and with thrombomodulin (β[PM1]=+99 nM*min, p=0.02; β[PMcoarse]=+66 nM*min, p=0.05), t-PA (β[PM1]=+0.72 ng/mL, p=0.01; β[PMcoarse]=+0.88 ng/mL, p=0.04), and CRP (β[PM1]=+0.59 mg/L, p=0.03; β[PMcoarse]=+0.48 mg/L, p=0.01).
PM exposure did not show any short-term effect within the week of the study. The association of PM exposure with PT, ETP, CRP provides some evidence of long-term effects on inflammation and coagulation.
Coagulation; Endogenous Thrombin Potential; Environmental Risk Factors; Occupational Health; Particulate Matter