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1.  Susceptibility to particle health effects, miRNA and exosomes: rationale and study protocol of the SPHERE study 
BMC Public Health  2014;14(1):1137.
Despite epidemiological findings showing increased air pollution related cardiovascular diseases (CVD), the knowledge of the involved molecular mechanisms remains moderate or weak. Particulate matter (PM) produces a local strong inflammatory reaction in the pulmonary environment but there is no final evidence that PM physically enters and deposits in blood vessels. Extracellular vesicles (EVs) and their miRNA cargo might be the ideal candidate to mediate the effects of PM, since they could be potentially produced by the respiratory system, reach the systemic circulation and lead to the development of cardiovascular effects.
The SPHERE (“Susceptibility to Particle Health Effects, miRNAs and Exosomes”) project was granted by ERC-2011-StG 282413, to examine possible molecular mechanisms underlying the effects of PM exposure in relation to health outcomes.
The study population will include 2000 overweight (25 < BMI < 30 kg/cm2) or obese (BMI ≥ 30 kg/cm2) subjects presenting at the Center for Obesity and Work (Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy).
Each subject donates blood, urine and hair samples. Extensive epidemiological and clinical data are collected. Exposure to PM is assigned to each subject using both daily PM10 concentration series from air quality monitors and pollutant levels estimated by the FARM (Flexible air Quality Regional Model) modelling system and elaborated by the Regional Environmental Protection Agency.
The recruitment period started in September 2010 and will continue until 2015. At December 31, 2013 we recruited 1250 subjects, of whom 87% lived in the province of Milan.
Primary study outcomes include cardiometabolic and respiratory health effects. The main molecular mechanism we are investigating focuses on EV-associated microRNAs.
SPHERE is the first large study aimed to explore EVs as a novel potential mechanism of how air pollution exposure acts in a highly susceptible population. The rigorous study design, the availability of banked biological samples and the potential to integrate epidemiological, clinical and molecular data will also furnish a powerful base for investigating different complementary molecular mechanisms. Our findings, if confirmed, could lead to the identification of potentially reversible alterations that might be considered as possible targets for new diagnostic and therapeutic interventions.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2458-14-1137) contains supplementary material, which is available to authorized users.
PMCID: PMC4242553  PMID: 25371091
Particulate matter; Obesity; Cardiovascular effects; Extracellular vesicles; Exosomes; Microvesicles; miRNAs
Environment international  2012;48:10.1016/j.envint.2012.06.020.
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.
PMCID: PMC3821920  PMID: 22871507
Particulate Matter; Personal Monitoring; Telomere length; Traffic pollution; China
3.  Predictors of global methylation levels in blood DNA of healthy subjects: a combined analysis 
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.
PMCID: PMC3304518  PMID: 20846947
Blood; DNA methylation; epigenetics; meta-analysis; repetitive elements
4.  Ambient PM exposure and DNA methylation in tumor suppressor genes: a cross-sectional study 
Exposure to ambient air particles matter (PM) has been associated with increased risk of lung cancer. Aberrant tumor suppressor gene promoter methylation has emerged as a promising biomarker for cancers, including lung cancer. Whether exposure to PM is associated with peripheral blood leukocyte (PBL) DNA methylation in tumor suppressor genes has not been evaluated. In 63 male healthy steel workers with well-characterized exposure to metal-rich particles nearby Brescia, Italy, we evaluated whether exposure to PM and metal components was associated with PBL DNA methylation in 4 tumor suppressor genes (i.e., APC, p16, p53 and RASSF1A). Blood samples were obtained on the 1st (baseline) and 4th day (post-exposure) of the same work week and DNA methylation was measured using pyrosequencing. A linear mixed model was used to examine the correlations of the exposure with promoter methylation levels. Mean promoter DNA methylation levels of APC or p16 were significantly higher in post-exposure samples compared to that in baseline samples (p-values = 0.005 for APC, and p-value = 0.006 for p16). By contrast, the mean levels of p53 or RASSF1A promoter methylation was decreased in post-exposure samples compared to that in baseline samples (p-value = 0.015 for p53; and p-value < 0.001 for RASSF1A). In post-exposure samples, APC methylation was positively associated with PM10 (β = 0.27, 95% CI: 0.13-0.40), and PM1 (β = 0.23, 95% CI: 0.09-0.38). In summary, ambient PM exposure was associated with PBL DNA methylation levels of tumor suppressor genes of APC, p16, p53 and RASSF1A, suggesting that such methylation alterations may reflect processes related to PM-induced lung carcinogenesis.
PMCID: PMC3180673  PMID: 21878113
5.  Effects of Inhalable Particulate Matter on Blood Coagulation 
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.
PMCID: PMC3093960  PMID: 19922434
Coagulation; Endogenous Thrombin Potential; Environmental Risk Factors; Occupational Health; Particulate Matter
6.  Airborne particulate matter and mitochondrial damage: a cross-sectional study 
Environmental Health  2010;9:48.
Oxidative stress generation is a primary mechanism mediating the effects of Particulate Matter (PM) on human health. Although mitochondria are both the major intracellular source and target of oxidative stress, the effect of PM on mitochondria has never been evaluated in exposed individuals.
In 63 male healthy steel workers from Brescia, Italy, studied between April and May 2006, we evaluated whether exposure to PM was associated with increased mitochondrial DNA copy number (MtDNAcn), an established marker of mitochondria damage and malfunctioning. Relative MtDNAcn (RMtDNAcn) was determined by real-time PCR in blood DNA obtained on the 1st (time 1) and 4th day (time 2) of the same work week. Individual exposures to PM10, PM1, coarse particles (PM10-PM1) and airborne metal components of PM10 (chromium, lead, arsenic, nickel, manganese) were estimated based on measurements in the 11 work areas and time spent by the study subjects in each area.
RMtDNAcn was higher on the 4th day (mean = 1.31; 95%CI = 1.22 to 1.40) than on the 1st day of the work week (mean = 1.09; 95%CI = 1.00 to 1.17). PM exposure was positively associated with RMtDNAcn on either the 4th (PM10: β = 0.06, 95%CI = -0.06 to 0.17; PM1: β = 0.08, 95%CI = -0.08 to 0.23; coarse: β = 0.06, 95%CI = -0.06 to 0.17) or the 1st day (PM10: β = 0.18, 95%CI = 0.09 to 0.26; PM1: β = 0.23, 95%CI = 0.11 to 0.35; coarse: β = 0.17, 95%CI = 0.09 to 0.26). Metal concentrations were not associated with RMtDNAcn.
PM exposure is associated with damaged mitochondria, as reflected in increased MtDNAcn. Damaged mitochondria may intensify oxidative-stress production and effects.
PMCID: PMC2928195  PMID: 20696069
7.  Exposure to Metal-Rich Particulate Matter Modifies the Expression of Candidate MicroRNAs in Peripheral Blood Leukocytes 
Environmental Health Perspectives  2010;118(6):763-768.
Altered patterns of gene expression mediate the effects of particulate matter (PM) on human health, but mechanisms through which PM modifies gene expression are largely undetermined. MicroRNAs (miRNAs) are highly conserved, noncoding small RNAs that regulate the expression of broad gene networks at the posttranscriptional level.
We evaluated the effects of exposure to PM and PM metal components on candidate miRNAs (miR-222, miR-21, and miR-146a) related with oxidative stress and inflammatory processes in 63 workers at an electric-furnace steel plant.
We measured miR-222, miR-21, and miR-146a expression in blood leukocyte RNA on the first day of a workweek (baseline) and after 3 days of work (postexposure). Relative expression of miRNAs was measured by real-time polymerase chain reaction. We measured blood oxidative stress (8-hydroxyguanine) and estimated individual exposures to PM1 (< 1 μm in aerodynamic diameter), PM10 (< 10 μm in aerodynamic diameter), coarse PM (PM10 minus PM1), and PM metal components (chromium, lead, cadmium, arsenic, nickel, manganese) between the baseline and postexposure measurements.
Expression of miR-222 and miR-21 (using the 2−ΔΔCT method) was significantly increased in postexposure samples (miR-222: baseline = 0.68 ± 3.41, postexposure = 2.16 ± 2.25, p = 0.002; miR-21: baseline = 4.10 ± 3.04, postexposure = 4.66 ± 2.63, p = 0.05). In postexposure samples, miR-222 expression was positively correlated with lead exposure (β = 0.41, p = 0.02), whereas miR-21 expression was associated with blood 8-hydroxyguanine (β = 0.11, p = 0.03) but not with individual PM size fractions or metal components. Postexposure expression of miR-146a was not significantly different from baseline (baseline = 0.61 ± 2.42, postexposure = 1.90 ± 3.94, p = 0.19) but was negatively correlated with exposure to lead (β = −0.51, p = 0.011) and cadmium (β = −0.42, p = 0.04).
Changes in miRNA expression may represent a novel mechanism mediating responses to PM and its metal components.
PMCID: PMC2898851  PMID: 20061215
epigenetics; etiology; miRNA expression; particulate matter; peripheral blood leukocytes
8.  Association between leukocyte telomere shortening and exposure to traffic pollution: a cross-sectional study on traffic officers and indoor office workers 
Environmental Health  2009;8:41.
Telomere shortening in blood leukocytes has been associated with increased morbidity and death from cardiovascular disease and cancer, but determinants of shortened telomeres, a molecular feature of biological aging, are still largely unidentified. Traffic pollution has been linked with both cardiovascular and cancer risks, particularly in older subjects. Whether exposure to traffic pollution is associated with telomere shortening has never been evaluated.
We measured leukocyte telomere length (LTL) by real-time PCR in blood DNA from 77 traffic officers exposed to high levels of traffic pollutants and 57 office workers (referents). Airborne benzene and toluene, as tracers for traffic exposure, were measured using personal passive samplers and gas-chromatography/flame-ionization detector analysis. We used covariate-adjusted multivariable models to test the effects of the exposure on LTL and obtain adjusted LTL means and 95% Confidence Intervals (CIs).
Adjusted mean LTL was 1.10 (95%CI 1.04-1.16) in traffic officers and 1.27 in referents (95%CI 1.20-1.35) [p < 0.001]. LTL decreased in association with age in both traffic officers (p = 0.01) and referents (p = 0.001), but traffic officers had shorter LTL within each age category. Among traffic officers, adjusted mean relative LTL was shorter in individuals working in high (n = 45, LTL = 1.02, 95%CI 0.96-1.09) compared to low traffic intensity (n = 32, LTL = 1.22, 95%CI 1.13-1.31) [p < 0.001]. In the entire study population, LTL decreased with increasing levels of personal exposure to benzene (p = 0.004) and toluene (p = 0.008).
Our results indicate that leukocyte telomere length is shortened in subjects exposed to traffic pollution, suggesting evidence of early biological aging and disease risk.
PMCID: PMC2761867  PMID: 19772576
10.  Effects of Particulate Matter on Genomic DNA Methylation Content and iNOS Promoter Methylation 
Environmental Health Perspectives  2008;117(2):217-222.
Altered patterns of gene expression mediate the effects of particulate matter (PM) on human health, but mechanisms through which PM modifies gene expression are largely undetermined.
We aimed at identifying short- and long-term effects of PM exposure on DNA methylation, a major genomic mechanism of gene expression control, in workers in an electric furnace steel plant with well-characterized exposure to PM with aerodynamic diameters < 10 μm (PM10).
We measured global genomic DNA methylation content estimated in Alu and long interspersed nuclear element-1 (LINE-1) repeated elements, and promoter DNA methylation of iNOS (inducible nitric oxide synthase), a gene suppressed by DNA methylation and induced by PM exposure in blood leukocytes. Quantitative DNA methylation analysis was performed through bisulfite PCR pyrosequencing on blood DNA obtained from 63 workers on the first day of a work week (baseline, after 2 days off work) and after 3 days of work (postexposure). Individual PM10 exposure was between 73.4 and 1,220 μg/m3.
Global methylation content estimated in Alu and LINE-1 repeated elements did not show changes in postexposure measures compared with baseline. PM10 exposure levels were negatively associated with methylation in both Alu [β = −0.19 %5-methylcytosine (%5mC); p = 0.04] and LINE-1 [β = −0.34 %5mC; p = 0.04], likely reflecting long-term PM10 effects. iNOS promoter DNA methylation was significantly lower in postexposure blood samples compared with baseline (difference = −0.61 %5mC; p = 0.02).
We observed changes in global and gene specific methylation that should be further characterized in future investigations on the effects of PM.
PMCID: PMC2649223  PMID: 19270791
DNA methylation; epigenetics; etiology; interspersed repetitive sequences; nitric oxide synthase; particulate matter
11.  Environment And Genetics in Lung cancer Etiology (EAGLE) study: An integrative population-based case-control study of lung cancer 
BMC Public Health  2008;8:203.
Lung cancer is the leading cause of cancer mortality worldwide. Tobacco smoking is its primary cause, and yet the precise molecular alterations induced by smoking in lung tissue that lead to lung cancer and impact survival have remained obscure. A new framework of research is needed to address the challenges offered by this complex disease.
We designed a large population-based case-control study that combines a traditional molecular epidemiology design with a more integrative approach to investigate the dynamic process that begins with smoking initiation, proceeds through dependency/smoking persistence, continues with lung cancer development and ends with progression to disseminated disease or response to therapy and survival. The study allows the integration of data from multiple sources in the same subjects (risk factors, germline variation, genomic alterations in tumors, and clinical endpoints) to tackle the disease etiology from different angles. Before beginning the study, we conducted a phone survey and pilot investigations to identify the best approach to ensure an acceptable participation in the study from cases and controls. Between 2002 and 2005, we enrolled 2101 incident primary lung cancer cases and 2120 population controls, with 86.6% and 72.4% participation rate, respectively, from a catchment area including 216 municipalities in the Lombardy region of Italy. Lung cancer cases were enrolled in 13 hospitals and population controls were randomly sampled from the area to match the cases by age, gender and residence. Detailed epidemiological information and biospecimens were collected from each participant, and clinical data and tissue specimens from the cases. Collection of follow-up data on treatment and survival is ongoing.
EAGLE is a new population-based case-control study that explores the full spectrum of lung cancer etiology, from smoking addiction to lung cancer outcome, through examination of epidemiological, molecular, and clinical data. We have provided a detailed description of the study design, field activities, management, and opportunities for research following this integrative approach, which allows a sharper and more comprehensive vision of the complex nature of this disease. The study is poised to accelerate the emergence of new preventive and therapeutic strategies with potentially enormous impact on public health.
PMCID: PMC2464602  PMID: 18538025

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