Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Substance interaction with the cell membrane leading to lung fibrosis||KeyEvent|
Key Event Description
Loss of alveolar capillary membrane integrity
The alveolar-capillary membrane (ACM) is the gas exchange surface of the lungs that is only ~0.3µm thick and is the largest surface area within the lung that separates the interior of the body from the environment. It is comprised of the microvascular endothelium, interstitium, and alveolar epithelium. The cellular composition of ACM includes the type 1 alveolar epithelial cells, the capillary endothelial cells, and the endothelial and epithelial basement membranes. As a consequence of its anatomical position, and the large surface area, it is the first point of contact for any inhaled pathogens, particles or toxic substances. Thus, ACM is subjected to injury and rapidly repaired following the external insults without formation of fibrosis or scar tissue. The extent of ACM injury or how rapidly its integrity is restored is a pivotal determinant of whether the lung restores its normal functioning following an injury or is replaced by fibrotic lesion or scar tissue. Repeated exposure to or biopersistent toxic substances, pathogens or lung irritants leading to non-resolving inflammation contribute to the ACM injury. Chronic inflammation mediated by overexpression of cytokines such as IL-1, TGFb, T-helper type 2 cytokine IL-13 or exposure to specific proteinases initiate ACM injury leading to significant loss of the epithelium and endothelium of the ACM resulting in loss of the barrier. In patients diagnosed with idiopathic pulmonary fibrosis, both type 1 pneumocyte and endothelial cell injury with the ACM barrier loss is observed. In addition, in normal conditions, the epithelial barrier consisting of alveolar epithelial type 1 and type 2 cells establish close contacts with the neighbouring cells via cell-cell adhesions through the intercellular junction complexes such as tight junctions, adherens junction and desmosomes (Vareille M et al., 2011; Kulkarni T et al., 2016). The various proteins found at the epithelial cell surface play a role in maintaining the cellular adhesions and intercellular junction complexes, which are critical for maintaining the integrity of alveolar epithelium. Exposure to pro-fibrotic drug bleomycin destroys structural architecture of the tight junctions leading to increased permeability, epithelial death and loss of specialised proteins such as caludins required for repair and restoration of alveolar epithelial barrier. Thoracic radiation and bleomycin-induced lung injury result in decreased expression of E-cadherin and Aquaporin-5 expression – proteins involved in adherens and in transgenic mice lacking aquaporin-5, increased lung fibrosis is observed. Increased membrane permeability leading to efflux of protein-rich fluid into the peribronchovascular interstitium and the distal airspaces of the lung, disruption of normal fluid transport via downregulated Na channels or malfunctioning Na+/K+ATPase pumps, loss of surfactant production, increased expression of epithelial or endothelial cell markers such as intercellular adhesion molecule-1 (ICAM-1) or decreased expression of surfactant D are few of the markers of decreasing lung compliance arising from the lost integrity of ACM (Johnson ER et al., 2010).
Alveolar capillary membrane integrity loss is also associated with other events such as chronic inflammation and Reactive Oxygen Species (ROS) synthesis. Chronic inflammation and ROS synthesis are described below as associative events to ACM loss.
Chronic inflammation as an associative event in the loss of ACM
Chronic inflammation, by definition is associated with tissue injury (Wallace WA et al., 2007). In the presence of continuous stimulus (e.g., presence of biopersistent toxic fibres such as asbestos, multi walled carbon nanotubes) or following repeated stimulus (e.g., repeated exposure to silica or coal dust), the ensuing tissue injury fuels the inflammatory mechanisms leading to accumulation of immune cells, prolonged inflammation and aggravated tissue damage. This sustained and perpetuated immunological response is termed as chronic inflammation. The chronicity of the inflammatory process occurs after prolonged acute inflammation and is a crucial component of the lung fibrotic response. During the chronic inflammatory phase, active inflammation, tissue injury and destruction, and tissue repair processes proceed in tandem. Thus, the causative substance must contain unique physico-chemical properties that grant the material biopersistance in the pulmonary environment or the pulmonary system has to be repeatedly exposed to the same substance that perpetuates the tissue injury.
Although, increases in number of neutrophils are observed during chronic inflammation, mononuclear phagocytes (circulating monocytes, tissue macrophages) and lymphoid cells mark this phase. The macrophages, components of mononuclear phagocyte system, are the predominant cells in chronic inflammation. Macrophages are the key inflammatory cells linking inflammation with repair and fibrosis. Activated macrophages release a variety of cytokines, chemokines, growth factors, which when uncontrolled, lead to extensive tissue injury, cellular death and necrosis, the other characteristics of chronic inflammation. The other types of inflammatory cells found in chronic inflammation include eosinophils in allergen induced lung fibrosis, lymphocytes and epithelial cells.
Oxidative stress as an associative event in the ACM loss
Oxidative stress is an important event that influences the extent of lung injury (reviewed in MacNee W, 2001). Superoxide anion (O2-) and the hydroxyl radical (OH) are the common ROS found in the biological systems that are unstable due to unpaired electrons. ROS, when interacted with biomolecules, initiate their oxidation. As such, all tissues including lung have efficient antioxidant system to counteract the ROS induced oxidation. The antioxidant enzymes including superoxide dismutase, catalase and glutathione peroxidase act directly to inactivate ROS and associated reactions. In addition, phase II detoxifying enzymes, including glutathione-S-transferase, NADPH quinone oxidoreductase and glutamate-cysteine ligase catalytic act as indirect antioxidant enzymes. However, when the balance between oxidant and antioxidants is tipped towards oxidants, oxidative stress occurs. During the process, proteins, DNA and lipids are oxidized. Oxidative stress modulates the cellular signalling processes and contributes to oxidative stress-induced tissue injury. It also plays a role in the tissue injury caused by inflammatory processes in lungs. The infiltrating neutrophils and macrophages generate superoxide anion which is converted to hydrogen peroxide by superoxide dismutase enzyme. OH is formed by a secondary reaction in the presence of Fe2+. ROS can also be produced by NADPH oxidase present in phagocytes. The other enzyme that contributes to ROS synthesis during inflammatory processes is the myeloperoxidase from neutrophils. In a self-perpetuating loop, inflammatory cells generate oxidative stress leading to increased airspace epithelial permeability, increased cell death and increased expression of proinflammatory genes, all of which lead to secretion of inflammatory cytokines/chemokines leading to prolonged and chronic inflammation. Nrf2, a member of the cap’n’collar basic leucine zipper transcription factors is suggested to play an important role in orchestrating the antioxidant defence against ROS via antioxidant response element. Nrf2 is shown to be protective against pulmonary inflammation and fibrosis induced by oxidants (Kikuchi N et al., 2010).
Human idiopathic pulmonary fibrosis is suggested to result from increased ROS synthesis and imbalances in oxidant/antioxidant levels in distal alveolar space. Antioxidant treatment of mice attenuates bleomycin-induced oxidative stress and subsequent lung fibrosis (Wang HD, 2002). Several ENMs have been shown to induce oxidative stress (a state of redox disequilibrium), an event associated with their in vivo toxicity (Xia T, 2008). Bleomycin induced lung pathology involves oxidative stress. In mice treated with antioxidants, bleomycin-induced inflammation and pathology is attenuated (Kelly C, 2008).
How It Is Measured or Detected
Proteinosis, BAL fluid protein content
The compromised ACM integrity in vivo can be measured by measuring total protein or total albumin content in the BAL fluid derived from experimental animals exposed to lung toxicants or in human patients suffering from lung fibrosis. In addition to albumin, the total urea in BAL fluid is also a good indicator of the ACM integrity loss (Schmekel B et al., 1992).
The physical-chemical properties of chemicals, their intended applications and exposure levels must be considered as important factors influencing the extent of lung injury. Although not quantitative, in case of ENMs, the physical presence of ENMs (biopersistence) must be confirmed in lungs following exposure, using Transmission Electron Microscopy or Cytoviva Nanoscale Hyperspectral Microscope. High aspect ratio materials including ENMs injure the ACM in rodent models.
Cell type considerations
In vitro, assays with human cells are desired; however, the use of cells derived from experimental animals including alveolar macrophages, dendritic cells, epithelial cells, and neutrophils are routinely used. Primary cells are preferred over immortalised cell types that are in culture for a long period of time.
Upon interaction with toxic substances, phagocytic cells internalize toxic substances leading to respiratory burst and release of ROS. A fluorimetric assay that relies on the intracellular oxidation of 5- and 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate (carboxy H2DCFDA) (Molecular Probes) has been used to detect ROS release in cells in vitro (Decan et al., 2016); however, it is important to note that the results are not specific to the types of radicals detected. In addition, lipid peroxidation, protein oxidation, and protein carbonylation can be measured as indicative of oxidative stress using proteomics techniques (Riebeling et al., 2001). Measurement of intracellular glutathione levels using the ThiolTracker™ Violet assay (Decan et al., 2016) or glutathionylation of proteins is also used. Oxidative stress can also be measured by assessing the relevant genes and proteins associated with antioxidant pathways (Riebeling et al., 2001). Other biomolecule modifications such as nitrosylation, reflective of oxidative stress, can be measured by measuring nitrosylated tissue proteins, or increase in NO production, and nitrate/nitrite ratio in BAL. In addition to tissue analysis, acellular glutathione levels, antioxidants and NO production in BAL supernatant can be used to assess ROS synthesis. These methods have been routinely used to measure ROS release following exposures to several toxic substances including ENMs.
Cellular viability or cytotoxicity assays are the most commonly used endpoints to assess the leaky or compromised cell membrane. The most commonly employed method is the trypan blue exclusion assay – a dye exclusion assay where cells with intact membrane do not permit entry of the dye into cells and thus remain clear, whereas the dye diffuses into cells with damaged membrane turning them to blue colour. Other high throughput assays that use fluorescent DNA stains such as ethidium bromide or propidium iodide can also be used and cells that have incorporated the dye can be scored using flow cytometry.
Lactate dehydrogenase (LDH) release assay is a very sensitive cytotoxicity assay that measures the amount of LDH released in the media following membrane injury. The assay is based on measuring the reduction of NAD and conversion of a tetrazolium dye that is measured at a wavelength of 490 nm.
The Calcein AM assay depends on the hydrolysis of calcein AM ( a non-fluorescent hydrophobic compound that permeates live cells by simple diffusion) by non-specific intracellular esterases resulting in production of calcein, a hydrophilic and strongly fluorescent compound that is readily released into the cell culture media by the damaged cells.
Although the above mentioned assays work for almost all chemicals, insoluble substances such as ENMs can confound the assays by inhibiting the enzyme activity or interfering with the absorbance reading. Thus, care must be taken to include appropriate controls in the assay.
Transepithelial/transendothelial electrical resistance (TEER)
TEER is an accepted quantitative technique that measures the integrity of tight junctions in cell culture models of endothelial and epithelial cell monolayers. They are based on measuring ohmic resistance or measuring impedance across a wide range of frequencies.
The other methods include targeted qRT-PCR or ELISA assays for tight junction proteins, cell adhesion molecules and inflammatory mediators such as IFNg, IL-10, and IL-13.
Domain of Applicability
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- Tejaswini Kulkarni, Joao de Andrade, Yong Zhou, Tracy Luckhardt, and Victor J. Thannickal. Alveolar epithelial disintegrity in pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 311: L185–L191, 2016.
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- Norihiro Kikuchi, Yukio IshiiEmail, Yuko Morishima, Yuichi Yageta, Norihiro Haraguchi, Ken Itoh, Masayuki Yamamoto and Nobuyuki Hizawa. Nrf2 protects against pulmonary fibrosis by regulating the lung oxidant level and Th1/Th2 balance. Respiratory Research 2010 11:31
- Wang HD, Yamaya M, Okinaga S, Jia YX, Kamanaka M, Takahashi H, Guo LY, Ohrui T, Sasaki H. Bilirubin ameliorates bleomycin-induced pulmonary fibrosis in rats. Am J Respir Crit Care Med. 2002;165:406–411
- Xia T, Kovochich M, Liong M, et al. Comparison of the Mechanism of Toxicity of Zinc Oxide and Cerium Oxide Nanoparticles Based on Dissolution and Oxidative Stress Properties. ACS nano. 2008;2(10):2121-2134. doi:10.1021/nn800511k.
- Tian Xia, Michael Kovochich, Jonathan Brant, Matt Hotze, Joan Semp, Terry Oberley, Constantinos Sioutas, Joanne I. Yeh, Mark R. Wiesner, and Andre E. Nel. Comparison of the Abilities of Ambient and Manufactured Nanoparticles To Induce Cellular Toxicity According to an Oxidative Stress Paradigm. Nano Letters 2006 Vol. 6, No. 8 1794-1807.
- Kelly C. Teixeira, Fernanda S. Soares, Luís G.C. Rocha, Paulo C.L. Silveira, Luciano A. Silva, Samuel S. Valença, Felipe Dal Pizzol, Emilio L. Streck, Ricardo A. Pinho, Attenuation of bleomycin-induced lung injury and oxidative stress by N-acetylcysteine plus deferoxamine, In Pulmonary Pharmacology & Therapeutics, Volume 21, Issue 2, 2008, Pages 309-316.
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- Nathalie Decan, Dongmei Wu, Andrew Williams, Stéphane Bernatchez, Michael Johnston, Myriam Hill, Sabina Halappanavar, Characterization of in vitro genotoxic, cytotoxic and transcriptomic responses following exposures to amorphous silica of different sizes, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Volume 796, 2016, Pages 8-22.
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