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Air pollutants, especially PM, have been found to determine various effects on human health, including genotoxic effects. The aim of this study was to assess DNA damage with micronuclei (MN) and comet tests on buccal cells of 6–8 years old children living in an area with high air pollution. Both tests were repeated in the same children in two consecutive winters to compare the levels of DNA damage under different pollution conditions. A complete data set including lifestyle, air pollutants levels and biological sampling was available for 180 children in the two winters. A high mean MN frequency was found in both seasons, with higher value in the first (0.51 ± 0.59) than the second winter (0.40 ± 0.52), whereas DNA damage measured with comet test showed higher damage in the second versus the first winter (visual score 208.8 ± 67.1 vs. 173.2 ± 50.8). The associations between air pollutant levels (CO, NO2, SO2, benzene, O3, PM10, and PM2.5, PM0.5 and PAHs) and DNA damage were investigated at different lag times, and mainly, no significant association was found. This study on repeated measure of MN frequency and DNA damage in children’s buccal did not show an association with various air pollutants evaluated in an area with high levels of air pollution.

Additional file 1: Table S1. Levels of air pollutants monitored by Regional Agencies for Environmental Protection in the 3 weeks preceding the biological samplings in winter and spring. Mean (± standard deviation, SD), minimum and maximum levels of exposure are reported. Table S2. Analysis of the associations between MN frequency and children’s features. Incidence Rate Ratio (IRR), 95% Confidence Intervals (95% CIs) and p value are reported. Table S3. Analysis of the associations between MN frequency and air pollutant levels. For each compound, the complete set of pollutant measures included in the model are specified. The Incidence Rate Ratio (IRR), 95% Confidence Intervals (95% CIs) and p value are reported. Table S4. Analysis of the associations between MN frequency and dichotomized air pollutant variables. Number of samples (N) and mean MN frequency ± SD (MN/1000) are reported for children exposed to levels lower () than the EU Ambient Air Quality Directive limits. The associations are expressed as IRR and 95%CIs. Table S5. Analysis of the associations between MN frequency and dichotomized air pollutant variables. Number of samples (N) and mean MN frequency ± SD (MN/1000) are reported for children exposed to levels lower () than the WHO Air Quality Guidelines (AQG). The associations are expressed as IRR and 95%CIs.

Additional file 1: Table S1. Levels of air pollutants monitored by Regional Agencies for Environmental Protection in the 3 weeks preceding the biological samplings in winter and spring. Mean (± standard deviation, SD), minimum and maximum levels of exposure are reported. Table S2. Analysis of the associations between MN frequency and children’s features. Incidence Rate Ratio (IRR), 95% Confidence Intervals (95% CIs) and p value are reported. Table S3. Analysis of the associations between MN frequency and air pollutant levels. For each compound, the complete set of pollutant measures included in the model are specified. The Incidence Rate Ratio (IRR), 95% Confidence Intervals (95% CIs) and p value are reported. Table S4. Analysis of the associations between MN frequency and dichotomized air pollutant variables. Number of samples (N) and mean MN frequency ± SD (MN/1000) are reported for children exposed to levels lower () than the EU Ambient Air Quality Directive limits. The associations are expressed as IRR and 95%CIs. Table S5. Analysis of the associations between MN frequency and dichotomized air pollutant variables. Number of samples (N) and mean MN frequency ± SD (MN/1000) are reported for children exposed to levels lower () than the WHO Air Quality Guidelines (AQG). The associations are expressed as IRR and 95% CIs.

One of the objectives of the MAPEC-Life project is raising children's awareness on air quality and its health effects. To achieve this goal, we designed didactic tools for primary school students, including leaflets with more information for teachers, a cartoon, and three educational videogames. The tools were then tested with 266 children who attended six primary schools in four Italian cities. A control group of 51 children received only explanations from teachers. An improvement in knowledge after using the audiovisual package was demonstrated, with higher efficacy compared with the control group. In addition, the use of videogames was greatly appreciated.

Background Air pollution is a global problem: airborne or deposited pollutants can be found worldwide, from highly polluted to remote areas. Epidemiological studies attribute the most severe health effects from air pollution to particulate matter which has been associated with cardiovascular diseases, lung cancer and other chronic diseases. In 2013, the International Agency for Research on Cancer (IARC) classified air pollution and particulate matter as carcinogenic to human. Among the whole population, children are at a higher risk of suffering the health consequences of airborne chemicals, for various reasons. First, children have higher levels of physical activity, spend more time outside and have a higher air intake than adults [1]. Second, children are more vulnerable to the adverse effects of air pollution due to their small body size, fast growth rate and relatively immature organs (lungs in particular), body functions [2], immune system and cell repair mechanisms [3,4]. Lastly, some data suggest that genetic damage, caused by environmental pollutants, viruses or lifestyle factors, occurring early in life can increase the risk of carcinogenesis in adulthood [5]. Various studies have analysed the genotoxic effects of urban air pollution exposure in the general population and in highly exposed subjects [6-11]. In particular, a significant association was found between high levels of urban pollution (PM10 and ozone) and DNA damage detected by the comet assay in human blood lymphocytes and leukocytes [7,10] and nasal mucosa cells [9,10]. Significantly higher levels of DNA damage were found in outdoor workers (bus and taxi drivers and street vendors) compared to indoor office workers [11], and in airport personnel, who are more directly exposed to air pollution, compared to administrative office workers at the same airport, in both lymphocytes and exfoliated buccal cells [8]. As regard children, very few data are available on biomarkers of early effect of air pollution: some studies have shown a higher level of biomarkers of DNA damage in children living in more, compared to less, polluted areas [6]. The few works which investigated primary DNA damage using the comet assay showed a greater DNA damage in cells (buccal and nasal cells, lymphocytes, white blood cells or umbilical cord blood lymphocytes) of children exposed to high level of airborne pollutants [12-18]. Aim of the study The aim of this study was to investigate primary DNA damage in salivary leukocytes of pre-school children exposed to high levels of air pollution during the winter season, when the highest levels of particulate matter and other pollutants are usually found. Methods This study is part of the RESPIRA study (Italian acronym for Rischio ESPosizione Inquinamento aRia Atmosferica), a molecular epidemiology cross-sectional study, aiming to assess biomarkers of early effects in buccal cells of pre-school children living in Brescia, a highly polluted town in the Po Valley, Northern Italy. The children were recruited in 6 pre-schools located in different areas of the town. The study enrolled children aged 3–6 years, born in Italy to European parents, without malignant tumours, who had not undergone radiotherapy or chemotherapy in the previous 12 months or X-rays in the previous 3 months. Primary DNA damage was investigated using comet assay on leukocytes from children sputum collected during two consecutive winter seasons (2012 and 2013). The project was approved by the Ethics Committee of Local Unit Health of Brescia. The children’s parents provided their written informed consent to participate in this study. All the data collected were treated confidentially in accordance with current Italian legislation (privacy law). Before cell sampling, the children’s parents were interviewed using an ad hoc questionnaire designed to gather information on exposure to air pollutants from both indoor and outdoor sources, including some characteristics of the area of residence (e.g. traffic, factories), parents’ smoking habits, and children’s respiratory diseases and drug consumption. Moreover, chemical data regarding daily concentration of the most commonly measured air pollutants (CO, NO2, SO2, benzene, O3, PM10 and PM2.5) were retrieved from the freely available ARPA (Regional Agency for Environmental Protection) database to characterize urban air quality. For the collection of salivary leukocytes, the children rinsed their mouths twice with mineral water and the mouthwashes were collected in tubes containing 25 ml of saline solution (NaCl 0.9%) [19]. This simple and non-invasive method proved to be easily acceptable by both children and parents. Collected samples were immediately transferred to the laboratory and processed. Cell suspension in saline solution was centrifuged for 10 minutes at 1100g (at 4°C) and the pellet were re-suspended in 1 ml of PBS. Leukocyte viability was determined using the trypan blue exclusion technique; epithelial buccal cells also present in the mouthwash samples were not considered. The comet assay was performed on salivary leukocytes, according to the method of Singh et al. on white blood cells [20], with minor modifications. PBS cell suspension was centrifuged for 4 minutes at 8700g, and the pellet was re-suspended in 200 µl of LMA (0.7%) and layered onto pre-treated (NMA, 1%) slides. After overnight lysis (2.5 M NaCl, 100 mM Na2EDTA, 8 mM TrisHCl, 10% DMSO, 1% Tryton X-100, 4°C, pH 10), 20-minute unwinding and 20-minute electrophoresis (0.8 V/cm, 300mA) in alkaline buffer (10 mM Na2EDTA, 300 mM NaOH, 10% DMSO, pH>13) were performed. Lastly, the microgels were neutralized (0.4M TrisHCl, pH 7.5) and fixed with absolute ethanol (-20°C). For each electrophoresis, two slides prepared with human leukocytes from healthy donor peripheral blood treated with EMS (2 mM) for 1h at 37°C were added in the tank as internal control. To evaluate DNA damage, the slides were stained with ethidium bromide (10 µg/ml) and examined using a fluorescence microscope. DNA migration was measured by both percentage of DNA in the tail (Komet 5, Kinetic Imaging Ltd) and visual score, derived from visual classification of the comets into four different damage classes. Two hundred cells were analysed for each subject (100 cells/slide, 2 slides per subject). Results During the sampling months, PM10 and PM2.5 were very often over the EU limit values for daily means (50 and 25 mg/m3, respectively); similarly, daily concentration of NO2 exceeded the annual EU limit (40mg/m3) on all the days. On the contrary, the concentration of CO, ozone, SO2 and benzene remained always below the EU limit values during all the period. A total of 222 children (mean age ± SD: 4.35 ± 0.84 years; 56.9% males) were recruited for the study in 6 pre-schools, but only 215 cell samples were suitable for performing the comet assay. Microscope analysis of the slides is still ongoing. The preliminary data on 94 children showed a median TI value of 4.31% (range 0.59-30.80%, mean value: 5.46 ± 5.00%). Completed results will be presented during the Conference. Discussion. The value of percentage of DNA migrated in the tail reported above is a preliminary result regarding about half of the subjects recruited for the study. So, the analysis of the results cannot be done yet. However, some consideration can be made on the method used. In our project, we collected salivary leukocytes because they are directly exposed to air passage through the mouth and easy to retrieve especially in little children. However, we encountered some difficulties with this type of sample. First of all, not all children were able to spit a sufficient amount of saliva, especially the younger ones: we lost 7 subjects because they cannot split at all; in other cases, we retrieved very few cells and the reading of the slides became very difficult and time-consuming. Moreover, we found that the concentration of leukocytes in salivary samples was very variable from subject to subject and independent from the amount of collected material. Another problem that, in some cases, made slide analysis difficult was the presence of the epithelial buccal mucosa cells: even if they were easily discerned from salivary leukocytes because their membrane was not broken by the lysis solution, when they were too numerous, the leukocytes may be hidden and not available for the measure. For all these reasons, in some cases we could not count 100 nucleoids per slide. However, we decided to include the subjects in the final analysis of the results only if at least 60 nucleoids per subject (about 30 per slide, in 2 slides per subject) were found [21]. Besides primary DNA damage, the RESPIRA study investigated micronucleus frequency in epithelial buccal cells of the same children as another biomarkers of early effects. The results of this test [22] showed a weak, but significant, association between MN frequency and concentration of air pollutants, such as PM10, PM2.5 and NO2. Moreover, MN level in the children of Brescia was higher than usually found among children living in areas with low or medium-high levels of air pollution.

Background. Polychlorinated biphenyls (PCBs) have been recognized as human carcinogens and cause liver cancer in animal experimental studies. However, no study investigated their association with hepatocellular carcinoma (HCC) so far. This study aimed to evaluate the serum PCB concentration in HCC patients and in healthy subjects of the general population living in Brescia, North Italy, a highly industrialized area with heavy PCB environmental pollution due to the presence of a PCB producing factory. Methods. Lipid-adjusted PCB concentrations, computed as the sum of 24 congeners, were measured in the serum of 101 HCC patients and in 101 healthy subjects of the same age and gender.Results. Hepatitis B and C virus infection and history of heavy alcohol intake were found, alone and combined, in 87% of HCC patients. No difference was found in PCB serum concentration of HCC patients with and without, and according to, the major risk factors for liver disease. No significant difference was observed in serum total PCB concentration between HCC patients (median: 1081; range: 287.0-3182.0 ng/g lipid) and healthy subjects (median: 1199.3; range: 225.7-22825 ng/g lipid). PCB congeners 118, 138, 153, 156, 180 and 194 were the only ones found over the detection limit in at least 30% of HCC patients. The serum level of PCB 118, but not that of other congeners, was higher in HCC patients than in healthy subjects.Conclusion. These findings do not support the hypothesis that PCBs play an important role in HCC development, although a contribution by some specific congeners cannot be ruled out.