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|
|Latent TGFbeta1 activation leads to pulmonary fibrosis||AdverseOutcome|
|Substance interaction with the lung cell membrane leading to lung fibrosis||AdverseOutcome|
|Carbon nanotubes, Multi-walled carbon nanotubes, single-walled carbon nanotubes, carbon nanofibres|
Key Event Description
Lung/pulmonary fibrosis – thickened alveolar septa
Fibrosis or scarring is a fixed end result of damage in a tissue not capable of regeneration with no possibility of restoring the original tissue architecture. In normal lung, an individual alveolus spans about 0.2mm in diameter and there are 300 million alveoli in an adult male lung. In between the two adjacent alveoli are two layers of alveolar epithelium resting on basement membrane, which consists of interstitial space, pulmonary capillaries, elastin and collagen fibres. Thus, the alveolar capillary membrane, where gas exchange takes place, is made up of the alveolar epithelium and alveolar endothelium (Gracey, 1968). In fibrotic disease however, a pronounced decrease in the number of capillaries within the alveolar septa is found with asymmetric deposition of collagen and cells between part of the surface of a capillary and the nearby alveolar lining. In areas where capillaries are not present, the alveolar capillary membrane is occupied with collagen and cells.
How It Is Measured or Detected
How it is measured
in vivo, histopathological analysis is used for assessing fibrotic lung disease. Morphometric analysis of the diseased area versus total lung area is used to quantitatively stage the fibrotic disease. Although, some inconsistencies can be introduced during the analysis due to the experience of the individual scoring the disease, the histological stain, etc., a numerical scale with grades from 0 to 8, originally developed by Ashcroft et al., 1988 is assigned to indicate the amount of fibrotic tissue in histological samples. This scale is applied to diagnose lung fibrosis in both human and animal samples. Modifications to this scoring system were proposed (Hubner, 2008), which enables morphological distinctions thus enabling a better grading of the disease. Using the modified scoring system, bleomycin induced lung fibrosis in rats was scored as follows: Grade 0 – normal lung, Grade 1 – isolated alveolar septa with gentle fibrotic changes, Grade 2 – knot like formation in fibrotic areas in alveolar septa, Grade 3 – contiguous fibrotic walls of alveolar septa, Grade 4 – single fibrotic masses, Grade 5 – confluent fibrotic masses, Grade 6 – large contiguous fibrotic masses, Grade 7 – air bubbles and Grade 8 – fibrotic obliteration. Further morphometric analysis can be conducted to quantify the total disease area (Nikota et al., 2017).
Lungs are formalin fixed and paraffin embedded such that an entire cross section of lung can be presented on a slide. The entire cross section is captured in a series of images using wide field light microscope. Areas of alveolar epithelium thickening and consolidated air space are identified. ImageJ software (freely available) is used to trace the total area (green line) and the diseased area (red line) imaged and quantified. The diseased area is equal to disease area/total area (Nikota et al., 2017).
In vitro, there is no single assay that can measure the alveolar thickness. However, a combination of assays spanning various KEs described above provide a measure of the extent of fibrogenesis potential of tested substances. qRT-PCR and ELISA assays measuring increased collagen, TGFβ1 and various pro-inflammatory mediators are used as sensitive markers of potential of substances to induce the adverse outcome of lung fibrosis.
Domain of Applicability
Evidence for Perturbation by Stressor
Bleomycin is a potent anti-tumour drug, routinely used for treating various types of human cancers (Umezawa H et al., 1967; Adamson IY, 1976). Lung injury and lung fibrosis are the major adverse effects of this drug in humans (Hay J et al., 1991). Bleomycin is shown to induce lung fibrosis in animals – such as dogs (Fleischman RW et al., 1971), mice (Adamson IY and Bowden DH, 1974), and hamsters (Snider GL et al., 1978) and is widely used as a model to study the mechanisms of fibrosis (reviewed in Moeller A et al., 2008; Gilhodes J-C et al., 2017).
- Umezawa H, Ishizuka M, Maeda K, Takeuchi T. Studies on bleomycin. Cancer. 1967 May;20(5):891-5.
- Adamson IY. Pulmonary toxicity of bleomycin. Environ Health Perspect. 1976 Aug;16:119-26.
- Hay J, Shahzeidi S, Laurent G Mechanisms of bleomycin induced lung damage. 1991 Arch Toxicol 65:81–94.
- Fleischman RW, Baker JR, Thompson GR, et al. Bleomycin-induced interstitial pneumonia in dogs. Thorax. 1971;26(6):675-682.
- Adamson IYR, Bowden DH. The Pathogenesis of Bleomycin-Induced Pulmonary Fibrosis in Mice. The American Journal of Pathology. 1974;77(2):185-198.
- Snider GL, Celli BR, Goldstein RH, O'Brien JJ, Lucey EC. Chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin. Lung volumes, volume-pressure relations, carbon monoxide uptake, and arterial blood gas studied. Am Rev Respir Dis. 1978 Feb; 117(2):289-97.
- Moeller A, Ask K, Warburton D, Gauldie J, Kolb M. The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis? The international journal of biochemistry & cell biology. 2008;40(3):362-382.
- Gilhodes J-C, Julé Y, Kreuz S, Stierstorfer B, Stiller D, Wollin L (2017) Quantification of Pulmonary Fibrosis in a Bleomycin Mouse Model Using Automated Histological Image Analysis. PLoS ONE 12(1): e0170561.
Carbon nanotubes, Multi-walled carbon nanotubes, single-walled carbon nanotubes, carbon nanofibres
Carbon nanotubes (CNTs) are allotropes of carbon, are made of rolled up sheet of graphene (single-walled carbon nanotubes) and are tubular in shape. A multi-walled carbon nanotube (MWCNT) is a multi-layered concentric cylinder of graphene sheets stacked one inside the other (N. Saifuddin et al., 2013). CNTs exhibit a combination of unique mechanical, thermal, and electronic properties and are highly desired commercially. They are light weight but their tensile strength is 50 times higher than that of steel, and they are stable chemically as well as in the environment. Consequently, they are produced in massive amounts and are increasingly incorporated in several industrial products.
CNTs are high aspect ratio materials and are shown to cause lung fibrosis in animals (Muller J et al., 2005; Porter DW et al., 2010). In an intelligence bulletin published by NIOSH on ‘Occupational exposure to carbon nanotubes and nanofibers’, NIOSH reviewed 54 individual animal studies investigating the pulmonary toxicity induced by CNTs and reported that half of those studies consistently showed lung fibrosis (NIOSH bulletin, 2013). However, the evidence is inconsistent and the occurrence of fibrotic pathology is influenced by the specific physical-chemical properties of CNTs (i.e. length, rigidity), their dispersion in exposure vehicle, and the mode of exposure.
- N. Saifuddin, A. Z. Raziah, and A. R. Junizah. Carbon Nanotubes: A Review on Structure and Their Interaction with Proteins. Journal of Chemistry, vol. 2013, Article ID 676815, 18 pages, 2013.
- Julie Muller, Franc¸ois Huaux, Nicolas Moreau, Pierre Misson, Jean-Francois Heilier,Monique Delos, Mohammed Arras, Antonio Fonseca, Janos B. Nagy, Dominique Lison Respiratory toxicity of multi-wall carbon nanotubes. Toxicology and Applied Pharmacology 207 (2005) 221–231.
- Dale W. Porter, Ann F. Hubbs, Robert R. Mercer, Nianqiang Wu, Michael G. Wolfarth, Krishnan Sriram, Stephen Leonard, Lori Battelli, Diane Schwegler-Berry, Sherry Friend, Michael Andrew, Bean T. Chen, Shuji Tsuruoka, Morinobu Endo, Vincent Castranova, Mouse pulmonary dose- and time course-responses induced by exposure to multi-walled carbon nanotubes. Toxicology, Volume 269, Issues 2–3, 2010, Pages 136-147.
- NIOSH: Occupational exposure to carbon nanotubes and nanofibers: current intelligence bulletin 65. 2013.
Regulatory Significance of the Adverse Outcome
- Gracey DR, Divertie MB and Brown Jr. AL. Alveolar-Capillary Membrane in Idiopathic Interstitial Pulmonary Fibrosis. 1968. American Review of Respiratory Disease, 98(1), pp. 16–21.
- Ashcroft, T., J.M. Simpson, and V. Timbrell. 1988. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J. Clin. Pathol. 41:467-470.
- Ralf-Harto Hübner, Wolfram Gitter, Nour Eddine El Mokhtari, Micaela Mathiak, Marcus Both,Hendrik Bolte, Sandra Freitag-Wolf, and Burkhard Bewig. 2008. BioTechniques 44:507-517.
- Nikota J, Banville A, Goodwin LR, Wu D, Williams A, Yauk CL, Wallin H, Vogel U, Halappanavar S. Stat-6 signaling pathway and not Interleukin-1 mediates multi-walled carbon nanotube-induced lung fibrosis in mice: insights from an adverse outcome pathway framework. Part Fibre Toxicol. 2017 Sep 13;14(1):37.