Genotoxic and oxidative damage of PM0.5 organic extracts from different Italian towns: preliminary results of the MAPEC (Monitoring Air Pollution Effects on Children for supporting public health policy) study

Background – Air pollution is one of the most important worldwide health concern (WHO_Europe, 2013). In the last years, in both the US and Europe, new directives and regulations supporting more restrictive pollution limits were published (Krzyzanowski, 2008). However, the early effects of air pollution cannot be avoided, especially for the urban population (EEA, 2012). Several epidemiological and toxicological studies have documented the remarkable effect of particulate matter (PM) in increasing morbidity and mortality for cardiovascular disease, lung cancer and natural cause mortality. The finest fractions of PM (PM with aerodynamic diameter <2.5 µm and less) play a major role in causing chronic diseases (WHO_Europe, 2013; Cesaroni et al., 2014). The International Agency for Research on Cancer (IARC) has recently classified air pollution and fine PM as carcinogenic to human (1 Group) (Loomis et al. 2013). PM is of major toxicological interest due to its great heterogeneity and complexity. The structure and composition of PM influence the biological properties of particles. The chemical composition varies with season and region of sampling, photochemical-meteorological conditions and sources of emissions (Longhin et al, 2013). Different studies showed that PM induces several kinds of adverse cellular effects as cytotoxicity, mutagenicity, DNA damage and stimulation of cytokine production (Moller et al., 2015). Extracts of urban air particles can induce cancer in animals and are mutagenic in bacteria, plant and mammalian cells in vitro tests (Claxton et al, 2007; Traversi et al, 2015; Moller et al., 2015. The aim of the MAPEC (Monitoring Air Pollution Effects on Children for supporting public health policy) study is i) to evaluate the associations between air pollution and biomarkers of early biological effects in oral mucosa cells of 6-8 year old children recruited from first grade schools and ii) to propose a model for estimating the global risk of early biological effects due to air pollutants and other factors in school children. Methods and analysis – The study was performed in five Italian towns (Brescia, Torino, Lecce, Perugia and Pisa) characterized by different levels of airborne PM (PM10 annual average from 44 µg/m3 measured in Torino to 20 µg/m3 measured in Lecce) (Feretti et al., 2013). Two to five schools for each town were chosen to evaluate the variability of pollution within the same town. Child exposure to urban air pollution was evaluated by collecting ultrafine PM (PM0.5) in the school area, on the same day of biological sampling. PM samples were collected for 72h using a high-volume gravimetric air sampler and glass fiber filters in two different seasons (winter and spring). Gravimetric analysis of the collected filters (a total of 40 samples) was performed; then the filters were Soxhlet extracted with 200 mL of n-hexane-acetone (4:1). Each extract was separated into different aliquots destined to chemical analysis (polycyclic aromatic hydrocarbon (PAH) and Nitro-PAH) and biological tests (Comet assay, Ames test, Micronuclei test and cytotoxicity). The organic extracts were dried with rotavapor and suspended in dimethyl sulfoxide (DMSO) (1 m3/µL) for comet assay. The A549 (human alveolar carcinoma) cells were exposed (24h at 37°C) to serial dilutions of the PM organic extracts (10, 25, 50 m3 of air equivalent). The comet assay was performed under alkaline conditions (pH>13) with and without Fpg enzyme according to standard protocol. A medium-throughput approach (Shaposhnikov et al., 2010) was applied to the comet assay by using 12-gel units (Severn Biotech Ltd., UK). Results - The 12-gel slides were stained with ethidium bromide and examined under a fluorescence microscope; 100 cells per sample (two spots/sample dilution), random selected, were analysed using the Comet Assay IV (Perceptive Instruments, UK). The tail intensity (% DNA) was selected as the parameter to estimate DNA damage and oxidative stress. Results were expressed as genotoxic parameter per cubic meter of air equivalent. The analysis of PM0.5 samples collected in winter and spring seasons (2014-2015) in the 5 Italian towns is still ongoing. Preliminary data will be presented at the workshop. Conclusions - Results obtained with the comet assay in human cells (A549) will be used to evaluate the role of the PM0.5 on genotoxic and oxidative damage in oral mucosa cells of Italian children and will highlight the variability in genotoxic and oxidative stress of PM0.5 samples collected in urban site with different levels of airborne pollutants.