Comet assay in salivary leukocytes: evaluation of early effects of air pollution exposure in pre-school children.
air pollution exposure
alkalyne comet assay
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AbstractBackground 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 . 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 , 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 . 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 , 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 . 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 . 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%) . 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 , 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 . 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  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.