Inflammation response and cytotoxic effects in human THP-1 cells of size-fractionated PM10 extracts in a polluted urban site.

To contribute to a greater characterization of the airborne particulate matter's toxicity, size-fractionated PM10 was sampled during different seasons in a polluted urban site in Torino, a northern Italian city. Three main size fractions (PM10 - 3 µm; PM3 - 0.95 µm; PM < 0.95 µm) extracts (organic and aqueous) were assayed with THP-1 cells to evaluate their effects on cell proliferation, LDH activity, TNFα, IL-8 and CYP1A1 expression. The mean PM10 concentrations were statistically different in summer and in winter and the finest fraction PM<0.95 was always higher than the others. Size-fractionated PM10 extracts, sampled in an urban traffic meteorological-chemical station produced size-related toxicological effects in relation to season and particles extraction. The PM summer extracts induced a significant release of LDH compared to winter and produced a size-related effect, with higher values measured with PM10-3. Exposure to size-fractionated PM10 extracts did not induce significant expression of TNFα. IL-8 expression was influenced by exposure to size-fractionated PM10 extracts and statistically significant differences were found between kind of extracts for both seasons. The mean fold increases in CYP1A1 expression were statistically different in summer and in winter; winter fraction extracts produced a size-related effect, in particular for organic samples with higher values measured with PM<0.95 extracts. Our results confirm that the only measure of PM can be misleading for the assessment of air quality moreover we support efforts toward identifying potential effect-based tools (e.g. in vitro test) that could be used in the context of the different monitoring programs.

PM10 in a background urban site: chemical characteristics and biological effects.

PM10 was sampled in a background urban site in Torino, a northern Italian city. PM10 extracts were tested with THP-1 and A-549 cells to evaluate their effects on cell proliferation, LDH activity, TNFα, IL8 and CYP1A1 expression, and genotoxic damage induction (Comet assay). Through Principal Component Analysis (PCA), it was observed that (1) the aqueous extracts induced the inhibition of cell proliferation in the warm season that clustered together to total ions, (2) organic extracts determined a winter cell viability reduction and (3) there was a genotoxic effect associated with PAH and metal concentrations. The analysed low PAH levels were unable to induce significant CYP1A1 expression. The results obtained confirmed that PM composition and seasonality play an important role in particle-induced toxicity. The presence of PM10-induced biological effects at a low polluted site suggested that a reduction of PM10 mass did not seem to be sufficient to reduce its toxicity.

Monitoring air pollution effects on children for supporting public health policy: the protocol of the prospective cohort MAPEC study.

INTRODUCTION: Genotoxic biomarkers have been studied largely in adult population, but few studies so far have investigated them in children exposed to air pollution. Children are a high-risk group as regards the health effects of air pollution and some studies suggest that early exposure during childhood can play an important role in the development of chronic diseases in adulthood. The objective of the project is to evaluate the associations between the concentration of urban air pollutants and biomarkers of early biological effect in children, and to propose a model for estimating the global risk of early biological effects due to air pollutants and other factors in children. METHODS AND ANALYSIS: Two biomarkers of early biological effects, DNA damage by the comet assay and the micronuclei (MN) test, will be investigated in oral mucosa cells of 6-8-year-old children. Concurrently, some toxic airborne pollutants (polycyclic aromatic hydrocarbon (PAH) and nitro-PAH) and in vitro air mutagenicity and toxicity in ultra-fine air particulates (PM0.5) will be evaluated. Furthermore, demographic and socioeconomic variables, other sources of exposures to air pollutants and lifestyle variables will be assessed by a structured questionnaire. The associations between sociodemographic, environmental and other exposure variables and biomarkers of early biological effect using univariate and multivariate models will be analysed. A tentative model for calculating the global absolute risk of having early biological effects caused by air pollution and other variables will be proposed. ETHICS AND DISSEMINATION: The project has been approved by the Ethics Committees of the local Health Authorities. The results will be communicated to local Public Health Agencies, for supporting educational programmes and health policy strategies. LIFE+2012 Environment Policy and Governance. LIFE12 ENV/IT/000614.

Involvement of nitro-compounds in the mutagenicity of urban Pm2.5 and Pm10 in Turin.

Fine particles can be active carriers of toxic compounds into the alveoli of the lungs. Among these compounds are numerous mutagens and carcinogens. The direct mutagenicity per unit mass of fine particulate matter (PM) is significantly higher than that of coarse particles, especially in urban areas. In this study, the mutagenic properties of urban PM2.5 and PM10 were evaluated, and the role of nitro-compounds was estimated. PM2.5 and PM10 samplings, and measurements of NOx and some PAHs were performed daily in 2007 in Turin, following a consolidated in vitro test - the Salmonella mutagenicity assay - conducted with organic extracts of PM2.5 and PM10. The mutagenic properties were assessed for each month of sampling with Salmonella typhimurium strain TA98 and TA98-derived strains: a nitroreductase-deficient mutant strain (TA98NR) and an additional nitroreductase-producing plasmid strain (YG1021). The annual measured mean levels of PM2.5 and PM10 were 34±20 and 48±18µg/m(3). The PM2.5/PM10 ratio ranged from 0.36 to 0.89. The Salmonella assay showed higher mutagenicity in autumn/winter (20±15 TA98NR; 54±39 TA98; 173±161 YG1021 net revertants/m(3)) compared with spring/summer (2±2 TA98NR; 7±8 TA98; 24±27 YG1021 net revertants/m(3)) (p<0.01). There are also statistically significant seasonal differences in the gravimetric analysis data. The number of TA98 net revertants per µg of PM2.5 is 6.5 times greater than per µg PM10. Moreover, the bioassay results showed an amplified response in the YG1021 strain and a reduced response in the TA98NR strain. The net revertant ratio TA98NR/YG1021 is 11±4 for organic extracts of PM2.5 and 13±6 for extracts of PM10 (p<0.01). There is a significant correlation between the NOx and PAH concentrations. These findings illustrate the relevant role of nitro compounds, and they underline the priority in improving preventive measures to reduce air pollution by nitrated molecules.

Endocrine disrupting activity in fruits and vegetables evaluated with the E-screen assay in relation to pesticide residues.

Food is likely to be one of the most important routes of human exposure to endocrine disrupting compounds (EDCs). In the present study, we evaluated the total estrogenic activity of fruits and vegetables, which was calculated using the human breast cancer cell line (MCF-7 BUS) proliferation assay (E-screen), in relation to pesticide residues. We analysed 44 food samples, 30 fruits and 14 vegetables. Of these samples, 10 did not contain any pesticide residues. The other 34 samples contained from 1 to 7 pesticide residues in concentrations ranging from 0.03 to 1.91 ppm. Estrogenic activity was detected in the 59% of samples tested. The positive controls used were 17-ß-estradiol (E2), the phytoestrogen genistein and the pesticide endosulfan. The average value of estradiol equivalency quantity (EEQ) for all positive samples was 0.15±0.32 µg/100g. A low correlation was found between the concentration of pesticide residues and the EEQ values (Spearman correlation r=0.376 and p=0.012). Using values obtained from the literature, we compared the estrogenic activity of food samples with the intrinsic content of phytoestrogens, but we found no correlations. Our results also suggested that the calculated intake of dietary EDCs might represent a concentration comparable to the normal endogenous estrogen concentration in human blood.

Meteo-climatic conditions influence the contribution of endotoxins to PM10 in an urban polluted environment.

A decrease in inhalable particulate matter (PM10) pollution is a top priority in urban areas of northern Italy. The sources of PM10 are both anthropogenic and natural. The former have been broadly investigated while the latter are less well known. Endotoxins are natural compounds of PM10 and are potentially toxic. Endotoxins are part of the outer membrane of Gram-negative bacteria. Their health effects are linked to environmental exposure. The effects mainly consist of respiratory symptoms, including pulmonary function decline. The occurrence of endotoxins has been proven in several occupational environments where organic materials supply an optimal substrate for bacteria growth. Knowledge about the presence of these contaminants in the environment is limited. The aim of this work is to evaluate the endotoxin levels of PM10 in the urban air of Turin, and to investigate the influence of seasonal and meteo-climatic factors. The sampling was conducted from January to December 2007. Endotoxin determination was performed by an LAL assay after extraction optimization. The PM10 levels ranged from 11.90 to 104.74 microg/m(3) (48.28 +/- 23.09) while the endotoxin levels ranged between 0.09 and 0.94 EU/m(3) (0.42 +/- 0.23). The seasonal trends of PM10 and endotoxin are inversely proportional. There is a statistically significant correlation between endotoxin and temperature (r = 0.532 p < 0.01), as well as between endotoxin and relative humidity (r = -0.457 p < 0.01). However, temperature has a predominant role. We observed that urban endotoxin concentrations are narrow in range and that the contribution of endotoxins to the total PM10 is only two millionths.

Analysis of environmental endocrine disrupting chemicals using the E-screen method and stir bar sorptive extraction in wastewater treatment plant effluents.

Endocrine disrupting chemicals (EDCs) have become a major issue in the field of environmental science due to their ability to interfere with the endocrine system. Recent studies show that surface water is contaminated with EDCs, many released from wastewater treatment plants (WWTP). This pilot study used biological (E-screen assay) and chemical (stir bar sorptive extraction-GC-MS) analyses to quantify estrogenic activity in effluent water samples from a municipal WWTP and in water samples of the recipient river, upstream and downstream of the plant. The E-screen assay was performed on samples after solid phase extraction (SPE) to determine total estrogenic activity; the presence of estrogenic substances can be evaluated by measuring the 17-beta-estradiol equivalency quantity (EEQ). Untreated samples were also assayed with an acute toxicity test (Vibrio fischeri) to study the correlation between toxicity and estrogenic disruption activity. Mean EEQs were 4.7 ng/L (+/-2.7 ng/L) upstream and 4.4 ng/L (+/-3.7 ng/L) downstream of the plant, and 11.1 ng/L (+/-11.7 ng/L) in the effluent. In general the WWTP effluent had little impact on estrogenicity nor on the concentration of EDCs in the river water. The samples upstream and downstream of the plant were non-toxic or weakly toxic (0

Chemical characteristics and mutagenic activity of PM10 in Torino, a northern Italian city.

Epidemiological studies of particulate matter (PM) have associated PM mass, as well as certain individual components of PM such as secondary particulate with adverse human health effects. For example genotoxic effects attributed to PM may relate to the content of organic compounds but also to the oxidative DNA damage generated by transition metals like iron. However the exact physiochemical mechanism by which PM produces adverse effects is not clear. The aims of this study were to evaluate (1) concentrations of PM10, (2) amounts of bioavailable iron associated with PM10, (3) amounts of secondary particulate expressed as SO4(=) and NO3(-) and (4) the mutagenic activities of PM10 organic extracts. Sampling was carried out in a meteochemical station located in Torino, a northern Italian city with high levels of PM10. The mean PM10 concentration in the considered period was 46.1+/-28.8 microg/m3, the iron mean concentration was 0.83 microg/m3 (+/-0.65 microg/m3) and the bioavailable Fe was 5.7% (+/-4.4%). The data showed that secondary particulate matter (as sum of sulfates and nitrates) constituted about 47% of PM10 total mass. Both iron and secondary species concentrations were positively associated with PM10 levels. Seasonal variations of PM10 concentration, iron level and secondary species amount were significant. Samples were tested for mutagenicity with Salmonella typhimurium strains TA98 and TA100, with and without metabolic activation and a positive response was observed especially for TA98. There were positive statistical associations between mutagenicity and PM10, bioavailable iron, sulfates and nitrates concentrations. Therefore, these results showed the usefulness of this biological approach for monitoring PM10.

The mutagenic hazards of environmental PM2.5 in Turin.

Owing to the large number of natural and anthropogenic sources, particulate matter (PM) may present several physical and chemical patterns in different areas. The finer PM2.5 fraction, which is now widely but not routinely measured in Europe, is considered to be the alveolar fraction of the ambient particles. Annual and winter mean concentrations of PM2.5 substantially vary in Europe, with higher concentrations in the South. The aims of this work were to (a) measure the PM2.5 levels in Turin over a long period, (b) evaluate mutagenic activities of organic extracts containing this collected complex mixture using the Ames test and (c) determine the level of polycyclic aromatic hydrocarbons (PAHs) in order to identify important mutagens in ambient air. Sampling was carried out from November 2001 to December 2004. The monthly mean of PM2.5 was 48.76+/-24.12 microg/m3. From the beginning to the end of the sample period there was a decrease in gravimetric levels, with annual means of 54.10+/-29.77 microg/m3 in 2002; 42.48+/-15.73 microg/m3 in 2003 and 45.89+/-24.92 microg/m3 in 2004. Samples were tested for mutagenicity using Salmonella typhimurium strains TA98 and TA100, with and without S9 mix metabolic activation. A positive genotoxic response was observed for TA98, with and without metabolic activation. The measured PAHs monthly mean level was 8.24+/-6.30 ng/m3, with values ranging from 0.20 to 21.38 ng/m3 Seasonal variation of gravimetric, mutagenic and PAH values was significant. The Salmonella assay results statistically correlated to PM2.5 and PAHs levels, but sometimes the mutagenic potencies were rather different despite an equal concentration of pollutant. The results confirm the usefulness of this biological approach to detect genotoxic properties of sampled PM2.5 and they show the variability of the mutagenic properties of the airborne mixture over time.

Airborne particulate matter: Ionic species role in different Italian sites.

Epidemiological studies have provided evidences for an association between exposure to elevated levels of ambient particulate matter (PM) and increased mortality and morbidity. However, the exact physiochemical nature of the responsible component is not clear. Secondary airborne PM formed from gas-phase pollutants contributes significantly to the most severe particulate air quality events. Although chemical formation for ionic species of aerosol have been observed, they have not been well reported for local variation. This work evaluates the amount of secondary particulate ionic species: sulfates (SO(4)(2-)) and nitrates (NO(3)(-)), chlorides (Cl(-)) and the mutagenic activities of PM10 extracts in different Italian sites (one Southern, one Central and three Northern; in one of the latter also PM2.5 has been evaluated). In general, mean secondary species concentration constitutes about 35-45% of PM10 mass in the North sites, 15% in the center site and 20% in the South site and it is positively associated with PM10 levels. There are significant local differences in the mean levels of PM10 ionic constituents: NO(3)(-) are predominant in northern cities, SO(4)(2-) are more equally distributed and coastal southern city is abundant in Cl(-). Samples were also tested for mutagenicity with Salmonella typhimurium strains TA98 and TA100, with and without metabolic activation; mutagenicity did not correlate with PM10 concentrations. The results showed the important roles and the geographical variability of PM secondary species in the total mass PM10 concentrations and the usefulness of this biological approach for monitoring PM to understand hazards from PM.

Application of semipermeable membrane device for assessing toxicity in drinking water.

Semipermeable membrane devices (SPMDs) mimic passive diffusive transport of bioavailable hydrophobic organic compounds through biological membranes and their partitioning between lipids and environmental levels. Our study was developed on a surface water treatment plant based in Turin, Northern Italy. The investigated plant treats Po River surface water and it supplies about 20% of the drinking water required by Turin city (about one million inhabitants). Surface water (input) and drinking water (output) were monitored with SPMDs from October 2001 to January 2004, over a period of 30 days. The contaminant residues, monthly extracted from SPMDs by dialysis in organic solvent, were tested with the Microtox acute toxic test and with the Ames mutagenicity test. Same extracts were also analyzed with gas chromatography--mass spectrometry technique in order to characterise the organic pollutants sampled, especially Polycyclic Aromatic Hydrocarbons (PAHs). Although the PAHs mean concentration is about one hundred times lower in the output samples, the mean toxic units are similar in drinking and surface water. Our data indicate that the SPMD is a suitable tool to assess the possible toxicity in drinking water.

Toxicity and estrogenic activity of a wastewater treatment plant in Northern Italy.

Many xenobiotics, widely diffused in the environment, have the potential to disrupt the delicate endocrine system balance of wildlife and humans. Substances showing hormone-like actions in living organisms are defined endocrine-disruptor chemicals and they may mimic, block, or interfere with the synthesis, release, transport, elimination, and binding of natural hormones. Different ecotoxicologic studies were applied to screen for the discharge of estrogenic and toxic substances into the aquatic environment from a small industrial wastewater treatment plant based in Northern Italy. Samples of wastewater (3 L) were taken at three different periods during the year from different points of the plant's process: three phases of treatment, and effluent. Untreated samples (1 L) were assayed with two toxicity tests: Microtox and Daphnia magna. A simplified proliferation test using human estrogen receptor-positive MCF-7 breast cancer cells (E-screen assay) was performed on 2-L samples after solid-phase extraction for the determination of total estrogenic activity; the presence of estrogenic substances was evaluated by measuring the proliferation rate in cells exposed to samples. The results of the Microtox assay employing the marine bacterium Vibrio fischeri were compared with data obtained from whole-effluent toxicity testing methods employing D. magna. Toxicity was found only for the influent point. The proliferative effect of the five points, relative to the positive control 17beta-estradiol (relative proliferation effect), was between 2.4% and 39%, and the 17beta-estradiol equivalent concentrations were between 0.06 and 3.94 ng/L. A first comparison between results from toxicity load and estrogenic activity was performed. At all process points, the effluent samples were considered acceptable (taking only toxicity into account), but we demonstrated that they had estrogenic activity in the river water indicating an input of estrogenic substances by way of the treatment plant.