# Iron-Rich Magnetic Coal Fly Ash Particles Induce Apoptosis in Human Bronchial Cells
## 4 Discussion
### Lung Cell Exposure to the Svalbard CFA/Leachates  
Although two-dimensional (2D) cell systems, i.e., monocultures, have proven to be a valuable method for cell-based studies, their limitations have been increasingly recognized. Since almost all cells in the in vivo environment are surrounded by other cells and extracellular matrix in a three-dimensional (3D) fashion, 2D cell culture does not adequately consider the natural 3D environment of cells. As a result, 2D cell culture tests sometimes provide misleading and non-predictive data for in vivo responses. Consequently, our research group has been utilising 3D cell constructs, namely, the EpiAirway model, which provides physiologically relevant and reproducible results for our inhalation toxicology research. The most \"bioreactive\" PM/leachates were exposed to a 3D cell culture model of the human bronchial epithelium that was used as a proxy for testing inhalation exposure in humans that mimicked both \"occupational\" (e.g., coal miners) and \"environmental\" (e.g., indoor/outdoor environment) scenarios. The most bioreactive CFA PM and leachates were shown to elicit mild to moderate damage in the bronchial region of the lung, the first site of deposition for airborne detritus not captured and/or removed by the upper RT [14]. Inflammation of the respiratory tract by cytotoxic factors (e.g., exposure to bioreactive PM/leachates) may cause an increase in the permeability of the epithelium, leading to deterioration of the epithelial barrier junction, ultimately allowing luminal substances to access the underlying tissues [15]. Changes to the permeability of the epithelium caused by alterations of the tight junctions was a reliable indicator of cytotoxic effects upon the structured components of cells [16], such as membrane channels, pumps, and other junctions [17]. TEER measurements were a reliable indicator of tight junction function in cell cultures. The permeability of the junction may be increased or decreased by the phosphorylation or de-phosphorylation of regulatory proteins, such as claud [18]. Any decrease in TEER readings would be indicative of a disruption of the epithelial barrier. However, the TEER readings all indicated an increase in trans-epithelial resistance when compared to the controls. This was most likely caused by a defensive mechanism known as \"hormesis\", which has been suggested to be a beneficial biological action occurring in response to a low dose of an otherwise toxic substance [19]. In this context, it was a \"tightening\" of the tight junctions in the cells as a means to compensate for the exposure to the toxic compound. This phenomenon was most likely occurring in all of the samples tested, but was most apparent in the 1 h HCl leachate, which exhibited the highest TEER reading (138% of control). However, hormesis only takes place up until the point where the dose of the toxic compound becomes too much of an irritation for the cell and the process becomes reversed, whereby the cells prepare for cell death. This process was thought to be taking place with the 48 h HCl leachate, as this showed a significant decrease in TEER readings from the 1 h reading. The readings from the ATP assay can be equated to number of cells in a culture (once calibrated for each cell type), but as the EpiAirway had a multi cellular phenotype, this was not possible; however, it is a good indicator of cell number and culture viability according to metabolic activity of the cells. Increases in the levels of ATP would suggest increased metabolic activity, which could be caused by hormesis after exposure of the cells. A decrease in the ATP levels would show decreased metabolic activity most likely caused by the occurrence of cell death. Cells actively release ATP under increased stress, and this was observed to be occurring in the CFA and H2O leachates, which showed small increases in ATP versus the controls. This was an indication of the intracellular stress and the initiation of defence mechanisms.