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

Do ferrous-minerals in Arctic circle coal fly-ash mediate respiratory toxicity in humans?

Svalbard is a Norwegian Archipelago located in the Arctic Circle that has a long history of coal-mining. The coal is used by the local power-station to provide electricity to the residential settlements, as well as being exported to countries, e.g. Norway and Germany. Bulk coal fly-ash (CFA) produced from the Longyearbyen Power-Station was collected by Dr Lisa Mol in 2014. Here we report the first geo-toxicological investigation of CFA derived from this Artic region. The bulk CFA (Figure 1a) from Svalbard was separated into inhalable particles (Figure 1b), as a means to test their oxidative capacity in the lung environment. Dry CFA was resuspended inside a rotating-drum fitted with a PM10-selective inlet head attached to an air-pump. The air-flow rates of 5, 10 and 20 litres per minute produced different sized (i.e. PM 1, 2.5, 10, respectively) particles for geo-toxicological analysis. Analysis of CFA physicochemical properties has been on-going (e.g. surface area, size, shape, number, metal chemistry) as they are known drivers of the oxidative capacity of particulate matter (PM). Knowledge of particle size informs on probable deposition sites within the lung, and thereby, estimation of toxic potential. For example, PM <0.1µm will have the greatest toxic effect(s) because nano-sized particles readily enter alveoli in the distal respiratory tract to provoke inflammation. The main cause of PM bioreactivity is believed to be through their induction of oxidative stress by generating reactive oxygen species (ROS). Ferrous minerals can be found in varying levels in CFA depending on the coal geochemistry, and have been indicated to cause the most damage through inducing ROS via Fenton reactions (Brown et al., 2011). Svalbard CFA was exposed to a neodymium magnet in order to extract magnetic components. All CFA fractions demonstrated a very high-presence of ferrous minerals. Accordingly, pure magnetite (Fe2O4) was selected to be a positive control particle for assessing CFA toxicity. Preliminary physicochemical characterisation involved field-emission scanning electron microscopy (FE-SEM) and associated energy dispersive X-ray microanalysis (EDX) to determine the inorganic elemental composition of bulk and inhalable-sized CFA fractions. The oxidative capacity of Svalbard CFA was determined by the Plasmid Scission Assay (PSA). PSA employs a ROS-sensitive plasmid that exhibits different DNA morphologies relative to specific levels of ‘damage’ (i.e. relaxed, linear and fragmented) versus DNA ‘un-damaged’ (i.e. super-coiled); that are captured via gel electrophoresis. Svalbard CFA caused DNA damage in a dose-responsive manner; thus inferring toxicity (Figure 2). Preliminary results have implied that a dose as low as 5µg/ml induced minor (i.e. relaxed) DNA damage, with the total amount of damage being increased gradually to approximately 40% as the CFA concentration increased. A TD40 (i.e. Toxic Dose 40%) refers to the dose required to kill 40% of the ‘test subject’ (e.g. plasmid DNA or lung cells) being challenged by a toxicant. Further analysis of this Artic CFA is required to determine the parameters causing bioreactivity. Conventional toxicology will include cellular and whole organism ROS-assays, including lysis of human red blood cells (haemolyses) and detection of bioluminescence in Vibrio fischeri, respectively, to confirm the acellular PSA results. The indirect effects that Svalbard CFA may have on human lungs will require investigation of soluble-components (via leaching in lung fluids) and bio-availability of other metals (using metal-chelators). *Brown, P., Jones, T. and BéruBé, K. (2011). Environmental Pollution 159 (12):3324-3333

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

Toxicological responses of normal human bronchial epithelium (NHBE) model exposed to settled dust samples from moisture damaged and reference schools

Exposure to indoor air in moisture damaged buildings is associated with deteriorating respiratory health, assumedly due to emissions from microbial growth and wet building materials. Previous studies of toxicological effects of mouldy house microbes have indicated that inflammation and cell death are important mechanisms. Aiming to gain further insight into function of respiratory epithelia, we studied the responses of normal human bronchial epithelium (NHBE) model after exposure to settled dust samples (n=9) collected from moisture damaged and reference schools in Spain, The Netherlands and Finland. The results were compared with immunotoxicological potential of the same samples in mouse RAW264.7 macrophage model. The effects of exposure on the NHBE model was assessed by measuring trans epithelial electric resistance (TEER) of the culture, changes in mucus production (Bradford assay), cell toxicity (ATP assay), cytokine levels in the culture media (ELISA) as well as morphological changes in cultured cells (light and scanning electron microscopy). The results showed that exposure to dust from moisture damaged schools was capable of inducing both increased TEER and mucus production and in some instances diminished TEER values indicating deterioration of tissue integrity and cell death. Similarly with the results from mouse macrophage model, the samples from The Netherlands and Spain were more toxic compared to samples from Finland. The findings suggest that the defence mechanisms present in respiratory epithelia are activated by dust from moisture damaged buildings, and exposure to high doses may damage the affected lung tissue

Toxicological responses of normal human bronchial epithelium (NHBE) model exposed to settled dust samples from moisture damaged and reference schools

Exposure to indoor air in moisture damaged buildings is associated with deteriorating respiratory health, assumedly due to emissions from microbial growth and wet building materials. Previous studies of toxicological effects of mouldy house microbes have indicated that inflammation and cell death are important mechanisms. Aiming to gain further insight into function of respiratory epithelia, we studied the responses of normal human bronchial epithelium (NHBE) model after exposure to settled dust samples (n=9) collected from moisture damaged and reference schools in Spain, The Netherlands and Finland. The results were compared with immunotoxicological potential of the same samples in mouse RAW264.7 macrophage model. The effects of exposure on the NHBE model was assessed by measuring trans epithelial electric resistance (TEER) of the culture, changes in mucus production (Bradford assay), cell toxicity (ATP assay), cytokine levels in the culture media (ELISA) as well as morphological changes in cultured cells (light and scanning electron microscopy). The results showed that exposure to dust from moisture damaged schools was capable of inducing both increased TEER and mucus production and in some instances diminished TEER values indicating deterioration of tissue integrity and cell death. Similarly with the results from mouse macrophage model, the samples from The Netherlands and Spain were more toxic compared to samples from Finland. The findings suggest that the defence mechanisms present in respiratory epithelia are activated by dust from moisture damaged buildings, and exposure to high doses may damage the affected lung tissue