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Event: 870

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, Cell Proliferation

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

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

Organ term

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		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
pH Induced Nasal Tumors	KeyEvent	Cataia Ives (send email)	Open for citation & comment	EAGMST Under Review
Frustrated phagocytosis-induced lung cancer	KeyEvent	Arthur Author (send email)	Under development: Not open for comment. Do not cite	Under Development
Deposition of energy leading to lung cancer	KeyEvent	Brendan Ferreri-Hanberry (send email)	Under development: Not open for comment. Do not cite	EAGMST Approved
Frustrated phagocytosis leads to malignant mesothelioma	KeyEvent	Evgeniia Kazymova (send email)	Under development: Not open for comment. Do not cite
AHR leading to lung cancer via NRF2 tox path	KeyEvent	Allie Always (send email)	Under development: Not open for comment. Do not cite
Ionizing Radiation-Induced AML	KeyEvent	Allie Always (send email)	Under development: Not open for comment. Do not cite

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

Name
Ionizing Radiation

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
rat	Rattus norvegicus	High	NCBI
mouse	Mus musculus	High	NCBI
human	Homo sapiens	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
All life stages	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
Unspecific	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

In the context of cancer, one hallmark is the sustained and uncontrolled cell proliferation (Hanahan et al., 2011, Portt et al., 2011). When cells in the lung epithelium obtain a growth advantage due to mutations in critical genes that regulate cell cycle progression, they may begin to proliferate excessively, resulting in hyperplasia and potentially leading to the development of a tumour (Hanahan et al., 2011). It has been hypothesized that stressors such as radiation can result in cell inactivation and the replacement of these cells can initiate clonal expansion (Heidenreich adn Paretzke et al., 2008).

Sustained atrophy/degeneration olfactory epithelium under the influence of a cytotoxic agent leads to adaptive tissue remodeling. Cell types unique to olfactory epithelium, e.g. olfactory neurons, sustentacular cells and Bowmans glands, are replaced by cell types comprising respiratory epithelium or squamous epithelium.

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). ?

Two common methods of measuring cell proliferation in vivo are the use of Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) labeling (Pera, 1977), and Ki67 immunostaining (Grogan, 1988). BrdU is a synthetic analogue of the nucleoside Thymidine. BrDu is incorporated into DNA synthesized during the S1 phase of cell replication and is stable for long periods. Labeling of dividing cells by BrdU is accomplished by infusion, bolus injection, or implantation of osmotic pumps containing BrdU for a period of time sufficient to generate measureable numbers of labeled cells. Tissue sections are stained immunhistochemically with antibodies for BrdU and labeled cells are counted as dividing cells. Ki67 is a cellular marker of replication not found in quiescent cells (Roche, 2015). Direct immunohistochemical staining of cells for protein Ki67 using antibodies is an alternative to the use of BrdU, with the benefit of not requiring a separate treatment (injection for pulse-labeling). Cells positive for Ki67 are counted as replicating cells. Replicating cell number is reported per unit tissue area or per cell nuclei (Bogdanffy, 1997).

Assay Name	References	Description	OECD Approved Assay
CyQuant Cell Proliferation Assay	Jones et al., 2001	DNA-binding dye is added to cell cultures, and the dye signal is measured directly to provide a cell count and thus an indication of cellular proliferation	N/A
Nucleotide Analog Incorporation Assays (e.g. BrdU, EdU)	Romar et al., 2016, Roche; 2013	Nucleoside analogs are added to cells in culture or injected into animals and become incorporated into the DNA at different rates, depending on the level of cellular proliferation; Antibodies conjugated to a peroxidase or fluorescent tag are used for quantification of the incorporated nucleoside analogs using techniques such as ELISA, flow cytometry, or microscopy	Yes (No. 442B)
Cytoplasmic Proliferation Dye Assays	Quah & Parish, 2012	Cells are incubated with a cytoplasmic dye of a certain fluorescent intensity; Cell divisions decrease the intensity in such a way that the number of divisions can be calculated using flow cytometry measurements	N/A
Colourimetric Dye Assays	Vega-Avila & Pugsley, 2011; American Type Culture Collection	Cells are incubated with a dye that changes colour following metabolism; Colour change can be measured and extrapolated to cell number and thus provide an indication of cellular proliferation rates	N/A

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

Cell proliferation is a central process supporting development, tissue homeostasis and carcinogenesis, each of which occur in all vertebrates. This key event has been observed nasal tissues of rats exposed to the chemical initiator vinyl acetate. In general, cell proliferation is necessary in the biological development and reproduction of most organisms. This KE is thus relevant and applicable to all multicellular cell types, tissue types, and taxa.

Evidence for Perturbation by Stressor

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

Bogdanffy. et al. (1997). “FOUR-WEEK INHALATION CELL PROLIFERATION STUDY OF THE EFFECTS OF VINYL ACETATE ON RAT NASAL EPITHELIUM”, Inhalation Toxicology, Taylor & Francis. 9: 331-350.

Grogan. et al. (1988). “Independent prognostic significance of a nuclear proliferation antigen in diffuse large cell lymphomas as determined by the monoclonal antibody Ki-67”, Blood. 71: 1157-1160.

Hanahan, D. & R. A. Weinberg, (2011),” Hallmarks of cancer: the next generation”, Cell. 144(5):646-74. doi: 10.1016/j.cell.2011.02.013.

Heidenreich WF, Paretzke HG. (2008) Promotion of initiated cells by radiation-induced cell inactivation. Radiat Res. Nov;170(5):613-7. doi: 10.1667/RR0957.1. PMID: 18959457.

Jones, J. L. et al. (2001), Sensitive determination of cell number using the CyQUANT cell proliferation assay. Journal of Immunological Methods. 254(1-2), 85-98. Doi:10.1016/s0022-1759(01)00404-5.

Pera, Mattias and Detzer (1977). “Methods for determining the proliferation kinetics of cells by means of 5-bromodeoxyuridine”, Cell Tissue Kinet.10: 255-264. Doi: 10.1111/j.1365-2184.1977.tb00293.x.

Portt, L. et al. (2011), “Anti-apoptosis and cell survival: a review”, Biochim Biophys Acta. 21813(1):238-59. doi: 10.1016/j.bbamcr.2010.10.010.

Quah, J. C. B. & R. C. Parish (2012), “New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes”, Journal of Immunological Methods. 379(1-2), 1-14. doi: 10.1016/j.jim.2012.02.012.

Roche Applied Science, (2013), “Cell Proliferation Elisa, BrdU (Colourmetric) ». Version 16

Romar, A. G., S. T. Kupper & J. S. Divito (2015), “Research Techniques Made Simple: Techniques to Assess Cell Proliferation”, Journal of Investigative Dermatology. 136(1), e1-7. doi: 10.1016/j.jid.2015.11.020.

Vega-Avila, E. & K. M. Pugsley (2011), “An Overview of Colorimetric Assay Methods Used to Assess Survival or Proliferation of Mammalian Cells”, Proc. West. Pharmacol. Soc. 54, 10-4.