### Primary and oxidative DNA damage in salivary leokocytes as a tool for the evaluation of air pollution early biological effects in children: current status of the MAPEC (Monitoring Air Pollution Effects on Children for Supporting public health policy) study.

#### Abstract

Background - Air pollution is a global problem: airborne or deposited pollutants are present everywhere on the planet, from highly polluted to remote areas. Twenty per cent of the EU urban population lives in areas where the EU air quality 24-hour limit value for particulate matter (PM10) is exceeded. At present, PM is Europe's most problematic pollutant in terms of harm to health, as reported by European Environmental Agency (EEA) in the EEA Technical Report on Air quality in Europe, 2011. Among the top 30 most polluted cities in Europe, more than half are Italian; in particular, the Po Valley is the most polluted area in Europe, with the concentrations of PM10, PM2,5 and NOx being well above the EU reference values for many days of the year. Epidemiological studies have found a consistent association between exposure to air pollution, especially to PM, and the incidence and mortality for several chronic diseases such as lung cancer, cardiovascular diseases and diabetes (Lanki et al., 2015). Among the mechanisms responsible for these adverse effects, genotoxic damage is of particular concern. Children are a high risk group with respect to the short- and long-term effects of air pollution. Indeed recent data suggest that genetic damage occurring early in childhood can increase the risk of chronic diseases, including cancer, in adulthood. The aim of the MAPEC (Monitoring Air Pollution Effects on Children for supporting public health policy) study is to evaluate the associations between air pollution and biomarkers of early biological effects 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 (Feretti et al., 2013). Methods and analysis – Enrollment of children was performed in five Italian towns characterized by different concentrations of air polluntants: Brescia and Torino, in the Po Valley, in the Northern Italy, Pisa and Perugia located in Central Italy, and Lecce, in Southern Italy. The towns in Central and Southern Italy show lower PM values than towns in Northern Italy allowing us to make valuable comparisons between areas with higher and lower air pollution. About 1,000 children (200 children per town) aged 6-8 years were recruited in the study from first grade schools. The sample size of the study was determined in order to detect statistically significant differences in the studied parameters (i.e., primary DNA damage in lymphocytes from saliva and MN in buccal mucosa cells) among children living in towns with high air pollution and those living in less polluted towns. Biological samples were collected (1,000 × 2 = 2,000 samples) at two separate times, in winter and in late spring. In order to collect the cells for the comet assay, the children were asked to rinse their mouths twice with mineral water and the mouthwashes were then collected in tubes containing 25 ml of saline solution (NaCl 0.9%) to obtain leukocytes (Osswald et al., 2003). Leukocytes were processed in order to evaluate primary and oxidative (FPG) DNA damage caused by exposure to air pollutants. The comet assay in alkaline conditions (pH>13) was performed according to the standard protocol as well as to detect oxidative damage using FPG incubation (Azqueta et al., 2013). A medium-throughput approach (Shaposhnikov et al., 2010) was applied to the comet assay by using 12-gel units (Severn Biotech Ltd., UK). The slides (coded) were sent to the University of Parma for microscope analysis (blind). Results - The 12-gel slides were stained with ethidium bromide and examined under a ﬂuorescence microscope; 50 randomly selected comets per spot (two spots/subject) were analysed. DNA damage was measured using the tail intensity (% DNA) parameter (Comet Assay IV, Perceptive Instruments, UK. Microscope analysis of cells sampled on winter 2014 is still ongoing. Preliminary data will be presented at the conference. Conclusions - The main objective of the MAPEC study is to evaluate the associations in children between air pollutants and early biological effects, and to propose a model for estimating the global genotoxic risk.