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Event: 923

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Increase, Proliferation of goblet cells

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Increased goblet cell proliferation

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Cellular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term
goblet cell

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Organ term
lung

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
cell proliferation goblet cell increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Decreased lung function KeyEvent Karsta Luettich (send email) Under development: Not open for comment. Do not cite Under Development

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
human Homo sapiens High NCBI
mouse Mus musculus High NCBI
rat Rattus norvegicus High NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help
Life stage Evidence
Adult High

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Term Evidence
Mixed High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

Cell proliferation is defined as the increase in cell number because of cell growth and division (Yada et al., 2014). It is a tightly regulated process. In adult organisms, cells are normally in a non-proliferative state, and cell proliferation primarily occurs to replace cells lost due to cell death. Because of tissue injury or exposure to chemicals, cell proliferation may take place at an accelerated pace, skewing the balance between cell growth and division and cell death.

Goblet cells proliferate under normal conditions (Chopra et al., 1981) and in response to a variety of stimulants including EGF, viruses, bacteria and allergens (Ichinose et al., 2006; Camateros et al., 2007; Shatos et al., 2003; Duh et al., 2000; Tamaoki et al., 2004) in colon, eye, nose and lung. EGFR ligands EGF, HB-EGF and TGFA induce goblet cell proliferation through MAPK, p38 and JNK in cultured conjunctival and bronchial goblet cells (Booth et al., 2001; Booth et al., 2007; Gu et al., 2008; Shatos et al., 2003).

How It Is Measured or Detected

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

Various methods for measuring cell proliferation exist. A number of recent reviews summarize the available methods, their principles, advantages and drawbacks (Riss et al., 2013; Cobb, 2013; Yadav et al., 2014; Adan et al., 2016; Romar et al., 2016). Broadly, cell proliferation assays can be categorized on the basis of their principle, and the 3 major categories are based on measurments of: 1) rate of DNA synthesis, 2) metabolic activity of cells, and 3) antigens associated with cell proliferation.

Assays that measure the rate of DNA synthesis

During cell proliferation, DNA replication occurs before cell division starts; therefore, the rate of DNA replication/synthesis is directly proportional to the rate of cell proliferation (Yadav et al., 2014). If cell proliferation is negatively affected by exposure, nascent DNA content would be lower in treated than in untreated cells. Conversely, if a chemical increases proliferation, the rate of DNA synthesis would increase.

Incubation of proliferating cells with labeled nuclotides such as 3H-thymidine and other thymidine analogs such as BrdU leads to their incorporation in newly synthesized DNA. Scintigraphy can then be used to detect the radiolabeled tracer and quantify the amount incorporated. BrdU can be detected by using anti-BrdU antibody-based staining and ELISA or flow cytometry or, if labeled with a fluorochrome, directly by reading fluorescent signals with a plate reader. Because directly labeled BrdU can also be detected microscopically, high-content imaging may prove to be another suitable alternative.

Because assays in this category are sensitive to cell cycle phase, synchronization of cells either by serum withdrawal (which accumulates cells in G1) or chemical inhibition of DNA synthesis (blocking cells in S phase) should be considered.

Assays that measure metabolic activity

Increased metabolic activity is typical for actively proliferating cells and is characterized by increasing ratios of NADPH/NADP, FADH/FAD, FMNH/FMN, and NADH/NAD. The oxidant capabilities of these metabolic intermediates cellular dehydrogenases or reductases can be exploited to convert e.g. tetrazolium salts to a formazan product, resulting in a colorimetric change which can be detected by measuring absorbance at an appropriate wavelength with a plate reader. There are several tetrazolium salts, including MTT, MTS/XTT, and WST, that can be used for this purpose. They are also included in various commercially available kits.

Other dyes that are sensitive to changes in metabolic activity, such as resazurin can also be used to determine the number of viable cells. Similar to the tetrazolium salts, resazurin is reduced in viable cells, giving rise to resorufin and dihydroresorufin, which have a different absorbance than resazurin and can be measured by using a plate reader. In addition, ATP concentration can serve as an indicator of cell number, because ATP levels are rapidly depleted and not restored through new synthesis when cell death occurs. Thus, ATP content directly correlates with the rate of proliferation of cells. Commonly, bio- or chemiluminescent assays, most of which are commercially available, are used for measuring ATP content.

Assays that assess antigens associated with cell proliferation

Proliferating cells express key proteins or antigens that are not present in non-proliferating cells, such as proliferating cell nuclear antigen (PCNA), Ki-67, topoisomerase IIB and phospho-histone H3. These can be detected with appropriate antibodies and imaging techniques. Immunocytochemistry/immunohistochemistry are commonly applied to verify proliferation in tissue sections, and here, tissue context may add insights into specific subpopulations of cells that stain positively for these markers. Semiquantitative image analysis may provide means to quantify a proliferation index. Flow cytometry and high-content imaging can be applied to cell cultures or ex vivo cell samples (e.g. PBMC). Although no standardized methods for the detection of these antigens exist, there are several well-described protocols for some of these markers for clinical application (Padmanabhan, 2015; Sun et al., 2016; Winther et al., 2016; Shen et al., 2017; Volynskaya et al., 2019; Nielsen et al., 2020)

It is worth noting here that at least one OECD Test Guideline (TG) refers to cell proliferation assays as essential components of the assessment of chemicals in vitro. Specifically, TG No. 487 (In Vitro Mammalian Cell Micronucleus Test) recommends the measurement of Relative Population Doubling (RPD) or Relative Increase in Cell Count (RICC) and Proliferation Index (PI) (OECD, 2016).

There are also a number of recent developments that changed the manner in which cell proliferation can be assessed today. For example, real-time cell analysis (RTCA) is based on electrical impedance measurements and allows for label-free, real-time, and continuous monitoring of cell adhesion, morphology, and rate of cell proliferation (Yan et al., 2018; Stefanowicz-Hajduk and Ochocka, 2020). 

Domain of Applicability

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

Proliferation of goblet cells was reported in human, mouse and rat (Tamaoki et al., 2004; Ichinose et al., 2006; Camateros et al., 2007; Shatos et al., 2003; Duh et al., 2000; Sydlik et al., 2006; Taniguchi et al., 2011).

Evidence for Perturbation by Stressor

Cigarette smoke

Treatment of primary human bronchial epithelial cells differentiated at the air-liquid interface with up to 20 µg/mL cigarette smoke total particulate matter induced a concentration dependent increase in the percentage of MUC5A-positive cells (Haswell et al., 2010). Similarly, repeated exposure of primary human bronchial epithelial cells differentiated at the air-liquid interface to smoke from 1R6F reference cigarettes (University of Kentucky) 3 times per week for up to 6 weeks significantly increased the MUC5AC-positive cell population starting from week 4 (Haswell et al., 2021).

Acrolein

Treatment of primary human bronchial epithelial cells differentiated at the air-liquid interface with up to 1 µM acrolein induced a concentration dependent increase in the percentage of MUC5A-positive cells (Haswell et al., 2010).

Exposure of Kunming mice to 4 ppm acrolein (nebulized) for 6 h per day, for up to 21 days caused a significant increase in the area of AB-PAS positive staining in the small airways (Liu et al., 2009).

Ozone

Exposure of ovalbumin-sensitized Balb/c mice to 100 or 250 ppb ozone for 3 h caused goblet cell metaplasia in bronchi and bronchioles and significantly increased the number of PAS-positive cells at 24 h post-exposure (Larsen et al., 2010).  

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help
Adan, A., Kiraz, Y., & Baran, Y. (2016). Cell proliferation and cytotoxicity assays. Curr. Pharm. Biotechnol. 17, 1213-1221.
 
Booth, B.W., Adler, K.B., Bonner, J.C., Tournier, F., and Martin, L.D. (2001). Interleukin-13 induces proliferation of human airway epithelial cells in vitro via a mechanism mediated by transforming growth factor-alpha. Am. J. Respir. Cell Mol. Biol. 25, 739–743.
 

Booth, B.W., Sandifer, T., Martin, E.L., and Martin, L.D. (2007). IL-13-induced proliferation of airway epithelial cells: mediation by intracellular growth factor mobilization and ADAM17. Respir. Res. 8, 51.

Camateros, P., Tamaoka, M., Hassan, M., Marino, R., Moisan, J., Marion, D., Guiot, M.-C., Martin, J.G., and Radzioch, D. (2007). Chronic asthma-induced airway remodeling is prevented by toll-like receptor-7/8 ligand S28463. Am. J. Respir. Crit. Care Med. 175, 1241–1249.

Chopra, D.P., Yeh, K., and Brockman, R.W. (1981). Isolation and characterization of epithelial cell types from the normal rat colon. Cancer Res. 41, 168–175.

Cobb, L. (2013). Cell Proliferation Assays and Cell Viability Assays. MATER METHODS 3, 2799.

Duh, G., Mouri, N., Warburton, D., and Thomas, D.W. (2000). EGF regulates early embryonic mouse gut development in chemically defined organ culture. Pediatr. Res. 48, 794–802.

Gu, J., Chen, L., Shatos, M.A., Rios, J.D., Gulati, A., Hodges, R.R., and Dartt, D.A. (2008). Presence of EGF growth factor ligands and their effects on cultured rat conjunctival goblet cell proliferation. Exp. Eye Res. 86, 322–334.

Haswell, L.E., Hewitt, K., Thorne, D., Richter, A., and Gaça, M.D. (2010). Cigarette smoke total particulate matter increases mucous secreting cell numbers in vitro: A potential model of goblet cell hyperplasia. Toxicol. in Vitro 24, 981-987. 

Haswell, L.E., Smart, D., Jaunky, T., Baxter, A., Santopietro, S., Meredith, S., et al. (2021). The development of an in vitro 3D model of goblet cell hyperplasia using MUC5AC expression and repeated whole aerosol exposures. Toxicol. Lett. 347, 45-57. 

Ichinose, T., Sadakane, K., Takano, H., Yanagisawa, R., Nishikawa, M., Mori, I., Kawazato, H., Yasuda, A., Hiyoshi, K., and Shibamoto, T. (2006). Enhancement of mite allergen-induced eosinophil infiltration in the murine airway and local cytokine/chemokine expression by Asian sand dust. J. Toxicol. Environ. Health A 69, 1571–1585.

Larsen, S.r.T., Matsubara, S., McConville, G., Poulsen, S.S., and Gelfand, E.W. (2010). Ozone increases airway hyperreactivity and mucus hyperproduction in mice previously exposed to allergen. J. Toxicol. Environm. Health A 73, 738-747.
 
Liu, D.-S., Wang, T., Han, S.-X., Dong, J.-J., Liao, Z.-L., He, G.-M., et al. (2009). p38 MAPK and MMP-9 cooperatively regulate mucus overproduction in mice exposed to acrolein fog. Int. Immunopharmacol. 9, 1228-1235.
 
Nielsen, T.O., Leung, S.C.Y., Rimm, D.L., Dodson, A., Acs, B., Badve, S., et al. (2020). Assessment of Ki67 in Breast Cancer: Updated Recommendations From the International Ki67 in Breast Cancer Working Group. JNCI 113, 808-819. 
 
OECD (2016). Test No. 487: In Vitro Mammalian Cell Micronucleus Test.
 
Padmanabhan, J. (2015). Immunostaining analysis of tissue cultured cells and tissue sections using phospho-Histone H3 (Serine 10) antibody. Methods Mol. Biol. 1288, 231-244. 
 
Riss, T.L., Moravec, R.A., Niles, A.L., Duellman, S., Benink, H.A., Worzella, T.J., et al. (2004-2013 [updated 2016 Jul 1] ). Cell viability assays. Bethesda, MD: Eli Lilly & Company and the National Center for Advancing Translational Sciences.
 
Romar, G. A., Kupper, T. S., & Divito, S. J. (2016). Research Techniques Made Simple: Techniques to Assess Cell Proliferation. J. Invest. Dermatol. 136, e1-e7.
 

Shatos, M.A., Ríos, J.D., Horikawa, Y., Hodges, R.R., Chang, E.L., Bernardino, C.R., Rubin, P.A.D., and Dartt, D.A. (2003). Isolation and characterization of cultured human conjunctival goblet cells. Invest. Ophthalmol. Vis. Sci. 44, 2477–2486.

Shen, Y., Vignali, P., and Wang, R. (2017). Rapid Profiling Cell Cycle by Flow Cytometry Using Concurrent Staining of DNA and Mitotic Markers. Bio Protoc. 7, e2517.

Sun, Y., Yang, K., Bridal, T., and Ehrhardt, A.G. (2016). Robust Ki67 detection in human blood by flow cytometry for clinical studies. Bioanalysis 8, 2399-2413. 

Sydlik, U., Bierhals, K., Soufi, M., Abel, J., Schins, R.P.F., and Unfried, K. (2006). Ultrafine carbon particles induce apoptosis and proliferation in rat lung epithelial cells via specific signaling pathways both using EGF-R. Am. J. Physiol. Lung Cell. Mol. Physiol. 291, L725–L733.

Tamaoki, J., Isono, K., Takeyama, K., Tagaya, E., Nakata, J., and Nagai, A. (2004). Ultrafine carbon black particles stimulate proliferation of human airway epithelium via EGF receptor-mediated signaling pathway. Am. J. Physiol. Lung Cell. Mol. Physiol. 287, L1127–L1133.

Taniguchi, K., Yamamoto, S., Aoki, S., Toda, S., Izuhara, K., and Hamasaki, Y. (2011). Epigen is induced during the interleukin-13-stimulated cell proliferation in murine primary airway epithelial cells. Exp. Lung Res. 37, 461–470.

Volynskaya, Z., Mete, O., Pakbaz, S., Al-Ghamdi, D., and Asa, S.L. (2019). Ki67 Quantitative Interpretation: Insights using Image Analysis. J. Pathol. Inform. 10, 8.

Winther, T.L., Arnli, M.B., Salvesen, Ø., and Torp, S.H. (2016). Phosphohistone-H3 Proliferation Index Is Superior to Mitotic Index and MIB-1 Expression as a Predictor of Recurrence in Human Meningiomas. Am. J. Clin. Pathol. 146, 510-520. 

Yadav K, Singhal N, Rishi V, Yadav H (2014). Cell Proliferation Assays. In: eLS. John Wiley & Sons, Ltd: Chichester. 

Yan, G., Du, Q., Wei, X., Miozzi, J., Kang, C., Wang, J., et al. (2018). Application of Real-Time Cell Electronic Analysis System in Modern Pharmaceutical Evaluation and Analysis. Molecules 23, 3280.