Correlation of PM10 oxidative potential with ecotoxicological and cytotoxicological potential measured at an urban background site in Italy

Atmospheric particulate matter is a concern in most of the European towns because it has potential negative effects on human health (Davidson et al., 2005; Lelieveld et al., 2015). Although the toxic effects of PM have been correlated with some of its chemical and physical properties, the toxicity mechanisms are not yet fully known. Different in vitro toxicological tests are often necessary to characterise potential health effects and, often, it is found significant correlation only among a few of the possible tests. In addition, contrasting results could be obtained comparing in vitro tests with acellular assays like those used to determine oxidative potential (Steenhof et al., 2011; Van Den Heuvel et al., 2018). The aim of the work was to study the oxidative potential (OP) of PM10, determined with the acellular DTT assay, in relationship with its ecotoxicological and cytotoxicological potential. The study was carried out on aqueous extracts of 10 samples of airborne PM10 randomly selected among the samples collected between 16/09/2017 and 25/12/2017. Samples were collected using a low-volume (2.3 m3 /h) sampler (SWAM, Fai Instruments srl) on 47 mm quartz fibre filters (Whatman) pre-fired at 700 °C for 2 hours in order to reduce contamination. Samples were exposed for 24 hours (starting at midnight) at the Environmental-Climate Observatory of ISAC-CNR in Lecce (Southern Italy), regional station of the Global Atmosphere Watch (GAW) network, characterised as an urban background site (Cesari et al., 2018). The aqueous extraction was performed in an ultrasonic bath for 80 min using 10 ml Milli-Q water. The ecotoxicological potential of PM10 was assessed by the bioluminescence inhibition assay based on the Gram-negative non-pathogenic bacterium Vibrio fischeri (Microtox® test), which physiologically emits light as a results of its metabolic activity. The natural bioluminescence of V. fisheri is inhibited by the exposure to a number of chemical pollutants, including organic and inorganic compounds (Abbass et al., 2018). Different exposure times (5, 15, and 30 mins) were used and inhibition results, obtained with five repetitions, are reported as a net effect corrected using field blanks. The cytotoxicological potential of PM10 was assessed on the same extracts by the MTT assay on the cell line A549. The MTT assay is based on a colorimetric reaction dependent on mitochondrial respiration of the cells and indirectly allows assessing the cellular energy capacity of a cell (Stockert et al., 2012). The MTT assay was applied to the A549 cell line, representative of the alveolar type II pneumocytes of the human lung (Foster et al., 1998). Cell mortality after 24h exposition is evaluated, in relative terms, considering the net effect of PM10 using field blanks for correction. Six repetitions were done. The water-soluble fraction of PM10 was also used for the analysis of the OP, performed with the dithiothreitol assay (DTT), a surrogate for cellular antioxidants, which analyses the rate of DTT depletion catalysed by chemical species present in the PM (Chirizzi et al., 2017). An aliquot of the extracts was diluted with deionised water (1:4 factor). Diluted samples were incubated at 37 °C with DTT (0.1 mM) in 0.1 M potassium phosphate buffer at pH 7.4 for times varying from 5 to 90 min. At designated times (specifically at 5, 10, 15, 20, 30, 45, 60, and 90 min) an aliquot of incubation mixture was picked up and 10% trichloroacetic acid was added to stop the reaction. Then, this reaction mixture was mixed with a solution containing 10 mM DTNB. The concentration of the formed 5-mercapto-2-nitrobenzoic acid was measured by its optical density absorption at 412 nm using a Eon BioTek Microplate Spectrophotometer. The consumption of DTT over time was determined through the linear fitting of the absorbance with the time in which it was made the withdrawal. The DTT depletion rate was used to determine OP values as DTT-activity 21 normalized in terms of sampled air volume (OPV) or in terms of mass of collected aerosols (OPM). The OPV and OPM values were comparable with previous measurements in this area or in other Italian towns (Chirizzi et al., 2017). In all the 10 samples analysed a significant inhibition of the Vibrio fisheri bioluminescence was observed as a results of the exposure of bacteria to the undiluted extracts for 5, 15 and 30 min, suggesting the presence in the PM10 of components able to induce an ecotoxic effect. Four samples (samples n. 2,3,4, and 7) showed a % of inhibition ranging from 30% to 50%, ascribable to a slight toxic effect, while six samples (samples 1,5,6, 8,9, and 10) showed a % of inhibition above 50% after 30 min exposure, suggesting the presence of a toxic effect. The correlation analysis between the sampled mass and the Vibrio fisheri bioluminescence inhibition showed a significant positive correlation (p<0.0462) for nine of the data pairs, while one sample (sample n. 6) was out of trend. As regards cytotoxicity, in all the 10 samples analysed a significant cell mortality was observed, ranging from 35% to 65% following exposure of the cells for 24h to the undiluted samples. 4 samples showed a slight cytotoxicity (mortality below 50%, samples n. 3,6,8, and 9), while the other showed a mortality higher than 50% (maximum mortality recorded 65%). The correlation analysis between the sampled mass and the A459 showed a highly significant positive correlation (p<0.0016) for eight of the data pairs, while two sample (sample n. 2 and 7) were out of trend. The OPV values were correlated with PM10 mass concentrations (Pearson 0.85). The specific oxidative potential (OPM) was well correlated with the results of the MTT assay (normalised for the mass collected). Instead, the correlation of OPM with Microtox test (normalised for the mass collected) was significantly lower. The correlation analysis between the Microtox test results and the MTT assay results on the same extracts was not significant, suggesting that each test, based on a specific biological system, can show its proper specificity and sensibility to different chemical components of PM. This highlight the need to improve the use of suites of biological assay in order to detect the multifaceted aspects associated to the toxicity of PM10 that can be the resulting aspect of multiple contaminants simultaneously present in the particulate. The financial support of the project PAPER (Paper Analyser for Particulate Exposure Risk, funded within POR Puglia FESR-FSE 2014-2020 – Asse prioritario 1 – Azione 1.6 – Bando Innonetwork – Aiuti a sostegno delle attività di R&S.). Abbas, M., M. Adil, S. Ehtisham-ul-Haque, B. Munir, M. Yameen, A. Ghaffar, G. Abba Sharc, M.A. Tahir, M. Iqbal. (2018) Sci. Tot. Environ. 626, 1295-1309. Cesari, D., De Benedetto, G. E., Bonasoni, P., Busetto, M., Dinoi, A., Merico, E., Chirizzi, D., Cristofanelli, P., Donateo, A., Grasso, F., Marinoni, A., Pennetta, A., Contini, D. (2017). Sci. Total Environ. 612, 202- 213. Chirizzi, D., Cesari D., Guascito, M.R., Dinoi, A., Giotta, L., Donateo, A., Contini, D., (2017) Atmos. Environ. 163, 1-8. Davidson, C.I., Phalen, R.F., Solomon, P.A., (2005). Aerosol Science and Technology 39, 737–749. Foster K.A., Oster C.G., Mayer M.M., Avery M.L., Audus K.L. (1998) Exp. Cell Res. 243, 359–366. 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