Susceptibility to particle health effects, miRNA and exosomes: rationale and study protocol of the SPHERE study.

BACKGROUND: 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. METHODS/DESIGN: 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. DISCUSSION: 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.

Quantification of carcinogenic 4- to 6-ring polycyclic aromatic hydrocarbons in human urine by solid-phase microextraction gas chromatography-isotope dilution mass spectrometry.

Polycyclic aromatic hydrocarbons (PAHs) are pollutants found in living and working environments. The aim of this study was to develop a solid-phase microextraction (SPME) gas chromatography (GC)-isotope dilution mass spectrometry method for the quantification of 10 four- to six-ring PAHs in urine samples. Seven of the selected PAHs have been classified as carcinogenic. Under the final conditions, analytes were sampled with a 100-µm polydimethylsiloxane SPME fibre for 60 min at 80 °C and desorbed in the injection port of the GC at 270 °C. Fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, indeno[1,2,3-cd]pyrene and benzo[ghi]perylene were separated using a highly arylene-modified phase capillary column and quantified by MS using eight deuterated PAHs as surrogate internal standards. Limits of quantification (LOQ) were in the 0.5- to 2.2-ng/L range. Validation showed linear dynamic ranges up to 340 ng/L, inter- and intra-run precisions <20%, and accuracies within 20% of spiked concentrations. Matrix effect evaluation and the use of control charts to monitor process performances showed that the isotope dilution approach allowed for the control of bias sources. Urinary PAHs were above or equal to LOQ, depending on different compounds, in 58-100% (min-max), 40-100% and 5-39% of samples from coke oven workers (n = 12), asphalt workers (n = 10) and individuals not occupationally exposed to PAHs (n = 18), respectively. Chrysene was the most abundant PAH determined with median levels of 62.6, 6.9 and <0.6 ng/L, respectively. These results show that the method is suitable for quantifying carcinogenic PAHs in specimens from individuals with different levels of PAH exposure.

[Biological monitoring of PAH exposure among asphalt workers]. / Monitoraggio biologico dell'esposizione a IPA negli asfaltatori.

Aim of this work was the assessment of exposure to polycyclic aromatic hydrocarbons (PAHs) by urinary 1-hydroxypyrene (1-OHPyr) in asphalt workers. Median levels of 1-OHPyr resulted higher in asphalt workers than in controls (184 vs. < 20 ng/L, p < 0.001). The determinants of exposure of 1-OHPyr resulted smoking habit, the number of consecutive days at work and the job task; 1-OHPyr was also associated to urinary creatinine. End of work week 1-OHPyr is suggested as an useful indicator of occupational exposure to PAHs in bitumen fumes.

[Urinary benzene in biological monitoring of environmental exposure to low benzene concentrations]. / Il benzene urinario nel monitoraggio biologico dell'esposizione a bassi livelli ambientali.

INTRODUCTION: Conflicting opinions exist about urinary benzene (UB) as a reliable biomarker of exposure. Objective of our study is to evaluate the effect of low-level environmental exposure on UB levels. METHODS: We monitored UB excretion in 74 non-smoking non- occupationally exposed subjects; a questionnaire interview gathered information on relevant exposures during the day of monitoring. RESULTS: UB excretion was related (p < 0.05) to gender, sampling time, residence, and reported vehicular traffic, but not to passive smoking and body mass index. CONCLUSION: Our findings support the use of unmetabolized UB as a specific and sensitive biomarker of low-level exposure to benzene.

Shorter telomere length in peripheral blood lymphocytes of workers exposed to polycyclic aromatic hydrocarbons.

Shorter telomere length (TL) in peripheral blood lymphocytes (PBLs) is predictive of lung cancer risk. Polycyclic aromatic hydrocarbons (PAHs) are established lung carcinogens that cause chromosome instability. Whether PAH exposure and its molecular effects are linked with shorter TL has never been evaluated. In the present study, we investigated the effect of chronic exposure to PAHs on TL measured in PBLs of Polish male non-current smoking cokeoven workers and matched controls. PAH exposure and molecular effects were characterized using measures of internal dose (urinary 1-pyrenol), effective dose [anti-benzo[a]pyrene diolepoxide (anti-BPDE)-DNA adduct], genetic instability (micronuclei, MN) and DNA methylation [p53 promoter and Alu and long interspersed nuclear element-1 (LINE-1) repetitive elements, as surrogate measures of global methylation] in PBLs. TL was measured by real-time polymerase chain reaction. Cokeoven workers were heavily exposed to PAHs (79% exceeded the urinary 1-pyrenol biological exposure index) and exhibited lower TL (P = 0.038) than controls, as well as higher levels of genetic and chromosomal alterations [i.e. anti-BPDE-DNA adduct and MN (P < 0.0001)] and epigenetic changes [i.e. p53 gene-specific promoter and global methylation (P

Dermal exposure to polycyclic aromatic hydrocarbons in asphalt workers.

OBJECTIVES: To assess dermal exposure to 16 polycyclic aromatic hydrocarbons (PAHs) in asphalt workers by applying polypropylene pads to six body sites (neck, shoulder, upper arm, wrist, groin, ankle), to identify the compounds and exposure sites most representative, and to integrate dermal exposure results with environmental and biological data. METHODS: Twenty-four asphalt workers were recruited. Dermal exposure was assessed during a single work shift. Sixteen PAHs (from naphthalene to indeno[1,2,3-cd]pyrene) were quantified via gas chromatography-mass spectrometry. Airborne exposure, urinary PAHs and monohydroxy metabolites were also investigated. RESULTS: Phenanthrene (PHE), present in all samples, was the most abundant compound (median 0.805-1.825 ng/cm(2)). Benzo[a]pyrene (BaP) was present in 75% of the samples (0.046-0.101 ng/cm(2)). Wrist had the highest contamination, with median PHE, pyrene (PYR), and BaP concentrations of 1.825, 0.527, and 0.063 ng/cm(2). PHE and PYR on wrist correlated with almost all 3- to 4-ring PAHs (0.405< or =r< or =0.856), but not with BaP; BaP correlated with almost all 4- to 6- ring PAHs (0.584< or =r< or =0.633). Significant correlations were observed between PHE level, airborne exposure, and the corresponding urinary PHE and monohydroxy metabolites. For PYR, significant correlations existed only between urinary PYR and monohydroxy metabolites. Multiple linear regression analysis revealed that 42% of the end-of-shift monohydroxy metabolites were the result of airborne exposure, dermal exposure, and baseline levels of biomarkers. CONCLUSIONS: Dermal exposure to PAHs was in the low ng/cm(2) range. PHE or PYR and BaP were the most representative compounds and the wrist was the best location to perform dermal exposure assessments. Both dermal and airborne exposure contributed to the total body burden of PAHs, though the relative contribution was analyte-dependent.

Biomarkers of exposure and effect in Bulgarian petrochemical workers exposed to benzene.

Biomarkers of benzene exposure and effect were evaluated in 158 Bulgarian petrochemical workers and 50 matched controls. Air exposures to benzene averaged about 1.8 ppm, for workers and 0.02 ppm for controls. Urinary trans,trans-muconic acid, and S-phenylmuconic acid, showed dose response relationships with benzene air exposure. The dose response curve for DNA single strand breaks (SSB), but not for the metabolites, showed a saturation effect. NQO1 genotype had a significant effect on SSB. We conclude that the pathways for these metabolites may be distinct from those involved in some forms of genotoxic damage induced by benzene.

Urinary carcinogenic 4-6 ring polycyclic aromatic hydrocarbons in coke oven workers and in subjects belonging to the general population: role of occupational and environmental exposure.

AIM: A new solid phase microextraction-gas chromatography-mass spectrometry method (SPME-GC-MS) to detect urinary unmetabolized 3-, 6-ring polycyclic aromatic hydrocarbons (PAHs) was applied to coke oven workers and general population subjects with the aim to assess exposure to carcinogenic PAHs, to evaluate the role of occupational and environmental variables on PAHs levels, and to compare present results with those previously obtained with a less sensitive method. METHODS: A total of 104 coke oven workers (CW) from Poland [recruited in 2000 (CW-2000; n=55) and 2006 (CW-2006; n=49)], and 45 control subjects from the same area, provided urine spot samples for measurement of 10 PAHs (from phenanthrene to benzo[g,h,i]perylene). The comparison between the two methods was performed only on CW-2000 subjects. Information regarding personal characteristics and job variables was collected by a questionnaire. RESULTS: The new method enables the quantification of 5-, 6-ring PAHs; precision and accuracy were in the 7.3-20.8% and 89.4-110% range, respectively; in CW-2000 samples results obtained with the new and the old method were highly correlated (Lin's concordance correlation coefficients: from 0.790 to 0.965); the mean difference between measured PAHS increased with the molecular weight of the analytes (from +5 to +27%). Urinary PAHs were above or equal to the quantification limit, depending on the compound, in 67-100% (min-max), 26-100% and 6-100% of samples from CW-2000, CW-2006 and controls, respectively. Chrysene and benz[a]anthracene were the most abundant carcinogenic PAHs with median levels of 43.4, 13.4, and 2.3 ng/L and 45.9, 14.9, and 0.7 ng/L in CW-2000, CW-2006, and controls, respectively, while benzo[a]pyrene levels were 6.5, 0.7 and <0.5 ng/L. The multiple linear regression model showed that the determinants of exposure were the use of wood and/or coke for house heating for controls, and job title or the plant for CW-2006. CONCLUSIONS: Urinary benzo[a]pyrene and other carcinogenic PAHs were, for the first time, quantified in urine samples from both occupationally and environmentally exposed subjects. These results show that urinary PAHs can discriminate exposure at different levels. Moreover, the simultaneous determination of several PAHs allows for the development of excretion profiles to assess exposure to specific compounds.