In vitro NHBE model of the human bronchial epithelium for toxicological testing

The respiratory tract is the primary site of exposure to inhaled substances. A growing need exists for high throughput in vitro models of the respiratory epithelium, which can provide rapid, reliable safety and effective screening in preclinical drug development applications. Normal human bronchial epithelial (NHBE) cells were cultured at an air-liquid interface in order to produce an in vitro model of the respiratory epithelium for toxicological testing. Extensive biochemical and morphological characterisation during construct development revealed that the NHBE model formed a pseudo-stratified, fully-differentiated culture of muco-ciliary phenotype. Histochemical and immunohistochemical techniques allowed the identification of basal, Clara, goblet and ciliated cells. Developmental characterisation revealed a toxicological dosing window of 7 days, where the model was deemed to be fully-established. Fully-developed NHBE cultures were then exposed to classical pulmonary toxicants (CPT) Lipopolysaccharide, cadmium chloride, paraquat, Amiodarone and cigarette smoke. Conventional toxicology techniques (culture viability, trans-epithelial electrical resistance TEER and morphology) were utilised to monitor the NHBE response to each CPT. The NHBE model responded with both general and toxicant-specific defence/irritancy mechanisms, observed to take place in the human bronchial epithelium and as such, reflective of in vivo toxicity. The in vitro model was finally challenged with candidate respiratory drugs (AstraZeneca AZ ) to test the utility of the cell system as a drug pre-screening tool. Blind-exposure of AZ compounds were characterised (physicochemical/ biochemically/morphologically) in the in vitro model and compared to AZ in vivo (rat) parallel exposure, focusing on irritancy end-points. A comparison of in vitro to in vivo exposures resulted in a 76.9 - 85% correlation of irritancy responses.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

In vitro NHBE model of the human bronchial epithelium for toxicological testing

The respiratory tract is the primary site of exposure to inhaled substances. A growing need exists for high throughput in vitro models of the respiratory epithelium, which can provide rapid, reliable safety and effective screening in preclinical drug development applications. Normal human bronchial epithelial (NHBE) cells were cultured at an air-liquid interface in order to produce an in vitro model of the respiratory epithelium for toxicological testing. Extensive biochemical and morphological characterisation during construct development revealed that the NHBE model formed a pseudo-stratified, fully-differentiated culture of muco-ciliary phenotype. Histochemical and immunohistochemical techniques allowed the identification of basal, Clara, goblet and ciliated cells. Developmental characterisation revealed a toxicological dosing window of 7 days, where the model was deemed to be fully-established. Fully-developed NHBE cultures were then exposed to classical pulmonary toxicants (CPT) Lipopolysaccharide, cadmium chloride, paraquat, Amiodarone and cigarette smoke. Conventional toxicology techniques (culture viability, trans-epithelial electrical resistance TEER and morphology) were utilised to monitor the NHBE response to each CPT. The NHBE model responded with both general and toxicant-specific defence/irritancy mechanisms, observed to take place in the human bronchial epithelium and as such, reflective of in vivo toxicity. The in vitro model was finally challenged with candidate respiratory drugs (AstraZeneca AZ ) to test the utility of the cell system as a drug pre-screening tool. Blind-exposure of AZ compounds were characterised (physicochemical/ biochemically/morphologically) in the in vitro model and compared to AZ in vivo (rat) parallel exposure, focusing on irritancy end-points. A comparison of in vitro to in vivo exposures resulted in a 76.9 - 85% correlation of irritancy responses

An in vitro versus in vivo toxicogenomics investigation of prenatal exposures to tobacco smoke

Approximately 1 million women smoke during pregnancy despite evidence demonstrating serious juvenile and/or adult diseases being linked to early-life exposure to cigarette smoke. Susceptibility could be determined by factors in previous generations, i.e. pre-natal or ‘maternal’ exposures to toxins. Pre-natal exposure to airborne pollutants such as mainstream cigarette smoke has been shown to induce early-life insults (i.e. gene changes) in Offspring that serve as biomarkers for disease later in life. In this investigation, we have evaluated genome-wide changes in the lungs of mouse Dams and their juvenile Offspring exposed pre-natally to mainstream cigarette smoke. An additional lung model was tested alongside the murine model, as a means to find an alternative in vitro, human tissue-based replacement for the use of animals in medical research. Our toxicogenomic and bioinformatic results indicated that in utero exposure altered the genetic patterns of the foetus that could put them at greater risk for developing a range of chronic illnesses in later-life. The genes altered in the in vitro, cell culture model were reflected in the murine model of pre-natal exposure to MCS. The use of alternative in vitro models derived from human medical waste tissues could be viable options to achieve human end-point data and to conduct research that meets the remits for scientists to undertake the 3Rs practises

Iron-Rich Magnetic Coal Fly Ash Particles Induce Apoptosis in Human Bronchial Cells

Svalbard is an arctic archipelago where coal mining generates all electricity via the local coal-fired power station. Coal combustion produces a waste product in the form of particulate matter (PM) coal fly ash (CFA), derived from incombustible minerals present in the feed coal. PM ≤10 µm (diameter) may be “inhaled” into the human respiratory system, and particles ≤2.5 µm may enter the distal alveoli to disrupt normal pulmonary functions and trigger disease pathways. This study discovered that Svalbard CFA contained unusually high levels of iron-rich magnetic minerals that induced adverse effects upon human lungs cells. Iron is a well-characterised driver of reactive oxygen species (ROS) generation, a driving force for cell death and disease. CFA physicochemical characterisation showed non-uniform particle morphologies indicative of coal burnt at inefficient combustion temperatures. The bioreactivity (ROS generation) of PM2.5/10 fractions was measured using plasmid scission assay (PSA, DNA damage) and haemolysis assays (erythrocyte lysis), with PM2.5 CFA showing significant bioreactivity. CFA leached in mild acid caused a significant increase in toxicity, which could occur in CFA waste-stores. The CFA and leachates were exposed to a surrogate model of human bronchial epithelia that confirmed that CFA induced apoptosis in bronchial cells. This study shows that CFA containing magnetic iron-rich minerals mediated adverse reactions in the human lung, and thus CFA should be considered to be an environmental inhalation hazard

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