Testing particulate matter toxicity via in vitro methods: What should be tested?

Unlike other toxic substances, usually of a known chemical formula, air particulate matter (PM) is a mixture of solid and liquid particles. The most frequently used tests are in vitro in nature and examine ‘cell viability’ following 24-hour exposure to PM. In most cases, PM induces sub-toxic viability responses but other key cell functions are not detected. The aim of this study was to compare the toxicity profiles of engineered NPs: zinc oxide (ZnO), crystalline form of silicon oxide (SiO2), and nickel (Ni), which are frequently present in ambient air pollution. Three different assays (acellular and cellular) were chosen to test PM biological targets: (1) plasmid scission assay – detecting DNA damage (indicative of the ability to produce reactive oxygen species; ROS; Figure 1); (2) haemolysis assay – informing about red blood cells (RBCs) membranes integrity; (3) proliferation assay inspected on HUVEC (human umbilical vein endothelial cells) at 24, 48 and 72 hours post-exposure to NPs

Mimicking in vivo-like physiological properties of the human bronchial epithelium in in vitro 3-D cell cultures

The human respiratory muco-ciliary epithelium consists of different cell types, connected by tight junctions; forming a protective barrier against the external environment. The epithelium is attached to a basement membrane through which necessary nutrients are delivered. Apart from a thin layer of mucus, the apical part of the bronchial epithelium is exposed to the ambient air. Mimicking these conditions through the use of 3-dimensional (3-D) filter-well membrane technology, a functional and fully-differentiated muco-ciliary phenotype in vitro cell culture model can be obtained. By providing the in vivo-like pulmonary microenvironment primary bronchial cells are stimulated to differentiate into a pseudo-stratified epithelium containing, basal, ciliated, Clara, goblet, intermediate and serous cells. In addition to this in vivo-like morphology, the cultures attain physiological properties akin to the human bronchial epithelium. Cells form tight junctions, enabling the whole culture to function as a barrier, providing trans-epithelial electrical resistance (TEER). Day 15 of ALI culture conditions results in beating cilia that are fully-grown and spread the mucus produced by the goblet cells. Unlike ‘conventional’ cell cultures, where tested substances need to be suspended in submerged culture media, our 3-D model of the human bronchial epithelium enables in vitro toxicology testing of apically deposited aerosols and/or solubilised substances onto mucus protected cell surfaces. This mimics in vivo deposition through inhalation. Moreover, we observe the tissue (i.e. multi-cellular) response, rather than the effect of submerged cell monolayers (i.e. single cell type). The additional protective mechanisms, such as apical mucus, traps inhaled substances and the tight junctions (i.e. barrier function) provide a more in vivo-like response when compared to submerged monolayer cultures. Our 3-D model of the human bronchial epithelium maintains the in vivo complexity observed in the in-situ human lung and is a reliable in vitro tool that can be employed to obtain robust predictive in vivo responses for human endpoint data

An 11 year study of multipollutant correlations of urban aerosols in Krakow, Poland

Krakow is the most polluted, as far as particulate matter (PM) is concerned, city in Poland. This is an important public concern as Krakow is also the second largest populated Polish city. The specific geomorphological localization of Krakow influences the atmospheric dynamics over the city, resulting in still- or weak-winds promoting air pollution accumulation, especially during the heating season. Seasonal variability in concentrations and multipollutant correlations of gaseous pollutants (i.e. NO2, NO, NOx, SO2) and PM10, 2.5 measured over a period of January 2005 to December 2013 were investigated. Data for the study were obtained from reports published by the Voivodship Inspectorate for Environmental Protection in Krakow. A strong seasonal variation in PM10 concentration revealed that during warm months the European Union annual limit value of 40 µg/m3 was not exceeded, whereas during the heating season, it was exceeded more than twice (Figure 1). Normalized monthly concentration patterns of all investigated pollutants and temperatures revealed that NO2 had the most consistent concentration pattern over the year. Conversely, SO2, PM2.5 and PM10 levels varied greatly (e.g. SO2 concentrations in January were more than 100% greater and 54% lower than the monthly average in May). Moreover SO2 had the strongest negative correlation (r = -0.64) with temperature. Seasonal correlations between pairs of pollutants were the highest between NO and NOx (0.99) and between PM10 and PM2.5 in annual and seasonal terms. The non-heating season (May-August) was characterised by lower coefficients than the heating season (September-April), when coefficients were similar to the annual values. Additionally, the ratios between average concentrations of investigated pollutants were also higher in the heating season. Transmission electron microscopy (TEM) images confirmed that particles were consistent with the known morphology of fly-ash (Brown et al., 2011) and other combustion-derived PM (BéruBé et al., 1999; Figure 2). For example, individual carbonaceous spheres’ forming grape-like bunches of aggregates and agglomerates which are highly-respirable. Natural factors such as geomorphology, climate and weather conditions have been determined to be the perpetrators of air pollution accumulation over the city (Wlodarczyk et al., 2015). The main source of elevated pollution levels were traffic emissions (i.e. nitrogen compounds) during warm months and residential coal-burning during the heating season. In conclusion, high annual levels, especially for PM, are greatly affected by measurements from the heating season. This ‘seasonality’ in PM2.5 concentrations should be taken into account when treating PM2.5 as a proxy in epidemiological studies for Krakow; as people in colder months spend less time outdoors. Further analysis including in vitro toxicology of PM is required to assess its direct effects on human lung biology. Brown, P., Jones, T. and BéruBé, K. (2011). Environmental Pollution 159 (12):3324-3333. BéruBé, K., Williamson, B., Winters, C., et al., (1999). Atmospheric Environment, 33(10):1599-1614

Exposure and harm to combustion-derived wood particles

The human respiratory system is the gateway of entry for inhaled detritus from anthropogenic (e.g. combustion-derived (CD) particulate matter (PM; e.g. diesel exhaust and wood-burning PM). Adult humans inhale 20m3 of air and suspended debris (gases and particles) into the airways daily. Inhalation exposure to CDPM (Figure 1) is known to increase the risk of morbidity and mortality of lung and heart diseases in all exposed individuals. The physicochemical properties of size, surface area and presence of transition metals have been implicated as drivers of the oxidative capacity of CDPM. However, the precise role of reactive organic compounds (ROC) in ambient aerosols, present either in the gas or particle phase has not been fully-investigated for their relevance in the induction of the observed adverse health effects. When addressing the toxicity of inhalation hazards such as wood smoke CDPM, a model that resembles the human lung responding to toxic challenges is required. In our in vitro exposure studies, we utilised normal human bronchial epithelial (NHBE) cells grown at the air-liquid interface (ALI) using filter-well technology (Prytherch et al 2011), to create an in vivo-like 3-dimensional lung model. This model is a fully-differentiated, pseudo-stratified, muco-ciliary epithelium containing basal, serous, Clara, goblet and ciliated cells. NHBE cells were exposed to wood smoke derived from Spruce, Beech and Birch at a dose of 152µg/cm2: carbon black (CB; negative control; Monarch 120, Cabot UK; DQ12 quartz (positive control). Following exposure (24 hours), tissue integrity (i.e. transepithelial electrical resistance (TEER) was measured to reveal minor disruption to bronchial tissue integrity (Figure 2). However, changes in cellular energy levels (i.e. ATP) between the types of wood smokes (Figure 3), could infer the smoke acted as an irritant to the lung environment. Wood smoke exposure can depress the immune system and damage the layer of cells in the lungs that protect and cleanse the airways. Further work on the biological and histological impacts of wood smoke will allow us to reveal mechanisms behind the changes observed, as well identifying biomarkers of cell damage by specific CDPM ROCs. For vulnerable populations, such as people with asthma, chronic respiratory disease and those with cardiovascular disease, wood smoke is particularly harmful, even at short exposures it can prove dangerous. Wood smoke interferes with normal lung development in infants and children. It also increases children’s risk of lower respiratory infections such as bronchitis and pneumonia. Prytherch, Z., Job, C., Marshall, H., Oreffo, V., Foster, M. and BéruBé, K.A. (2011) Macro. Bios. 11, 1467–77. This work was supported by HICE (www.hice-vi.eu)

Exposure and harm to combustion-derived particles: Searching for biomarkers

The physicochemical properties of size, surface area and presence of transition metals have been implicated as drivers of the oxidative capacity of CDPM. However, the precise role of reactive organic compounds (ROC) in ambient aerosols, present either in the gas phase or the particle phase or in both phases, have not been fully-investigated for their relevance in the induction of the observed adverse health effects. Oxidation of fatty acids linked to the cell membrane phospholipids leads to many metabolites that have been used as markers of the process. Such metabolites have long been considered to be involved in two possibly inter-related processes: cell/tissue damage and signalling. As one approach to resolve the role played by ROCs, their effects on fatty acid and lipid metabolism in human lung tissues will be studied in detail by using the standard biochemical techniques and lipidomics