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  • <div id="title">
  • <h2>AOP ID and Title:</h2>
  • <div class="title">AOP 272: Deposition of energy leading to lung cancer</div>
  • <strong>Short Title: Deposition of energy leading to lung cancer</strong>
  • </div>
  • <h2>Graphical Representation</h2>
  • <img src="https://aopwiki.org/system/dragonfly/production/2019/09/04/15va66wk5z_aop_figure.png" height="500" width="700" alt=""/>
  • <img src="https://training.aopwiki.org/system/dragonfly/production/2019/09/04/15va66wk5z_aop_figure.png" height="500" width="700" alt=""/>
  • <div id="authors">
  • <h2>Authors</h2>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Samantha Sherman<sup>1</sup>, Zakara Said<sup>1</sup>, Baki Sadi<sup>1</sup>, Carole Yauk<sup>1,2</sup>, Danielle Beaton<sup>3</sup>, Ruth Wilkins<sup>1</sup> Robert Stainforth<sup>1</sup>, Nadine Adam<sup>1</sup>, &nbsp;Vinita Chauhan<sup>1,</sup>*</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><sup>1&nbsp;</sup>Consumer and Clinical Radiation Protection Bureau, Health Canada, Ottawa, ON, Canada</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><sup>2 </sup>Department of Biology, University of Ottawa, Ottawa, ON, Canada</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><sup>3&nbsp;</sup>Canadian Nuclear Laboratories, Chalk River, ON, Canada</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">*Corresponding author: Vinita Chauhan (vinita.chauhan@canada.ca)</span></span></p>
  • </div>
  • <div id="status">
  • <h2>Status</h2>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Author status</th>
  • <th scope="col">OECD status</th>
  • <th scope="col">OECD project</th>
  • <th scope="col">SAAOP status</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Open for citation &amp; comment</td>
  • <td>EAGMST Approved</td>
  • <td>WPHA/WNT Endorsed</td>
  • <td>1.56</td>
  • <td>Included in OECD Work Plan</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <div id="abstract">
  • <h2>Abstract</h2>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Despite its widespread recognition in chemical toxicology, the adverse outcome pathway (AOP) framework has not been fully explored in the radiation field to guide relevant research and subsequent risk assessment.&nbsp; Development of a radiation relevant AOP&nbsp;is described here using a case example of lung cancer.&nbsp; Lung cancer is a major public health problem world-wide, causing the deaths of an estimated 1.5 million people annually; it imposes a major health-care burden. Numerous environmental factors are known contributors including both chemical (e.g.. asbestos, air pollution and arsenic) and radiation stressors (e.g.. radon&nbsp; gas).&nbsp; Radon gas is the second leading cause of lung cancer in North America. Evidence suggests that environmental and indoor radon exposure constitutes a significant public health problem. The mechanism of lung cancer development from exposure to radon gas is unclear. Data suggest that cytogenetic damage from radon decay progeny may be an important contributor.&nbsp;&nbsp;This AOP defines a&nbsp;path to cancer&nbsp;using key events&nbsp; related to DNA damage response&nbsp;and repair. The molecular initiating event (MIE)&nbsp; which represents the first chemical interaction with the cell is identified as&nbsp;the&nbsp; deposition of ionizing energy.&nbsp; Energy deposited onto a cell can lead to multiple ionization events to targets such as DNA. This energy will break DNA double strands (KE1)&nbsp;and initiate&nbsp;double strand break (DSB) repair machinery. &nbsp;In higher eukaryotes, this occurs through non-homologous end joining (NHEJ) which is a quick and efficient, but error-prone process (KE2). If DSBs occur in regions of the DNA transcribing critical genes, then mutations (KE3) generated through faulty repair may alter the function of these genes or may cause chromosomal aberrations (KE4), resulting in genomic instability. These events will alter the functions of many gene products and impact cellular pathways such as cell growth, cell cycling, and apoptosis. With these alterations, cell proliferation (KE5) will be promoted by escaping the regulatory control and form hyperplasia in lung epithelial cells, leading eventually to lung cancer (AO) induction and metastasis . The overall&nbsp;weight of evidence for this AOP is strong.&nbsp;&nbsp; The uncertainties and inconsistencies surrounding this AOP are centred on dose-response relationships associated with dose, dose-rates and radiation quality. The proposed AOP will act as a case example to motivate more researchers in the radiation field to use the AOP framework to effectively exchange knowledge and identify research gaps in the area of low dose risk assessment. </span></span></p>
  • </div>
  • <div id="background">
  • <h3>Background</h3>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">According to the World Cancer Research Fund, lung cancer is a disease that poses a significant healthcare burden world-wide. (https://www.wcrf.org/dietandcancer/cancer-trends/worldwide-cancer-data (https://www.wcrf.org/dietandcancer/cancer-trends/worldwide-cancer-data)). It is the most commonly diagnosed cancer with the highest incidence of occurrence on a global scale (excluding non-melanoma skin cancers). It is a multi-faceted disease exhibiting various genetic lesions and involving the accumulation of multiple molecular abnormalities over time. It is responsible for 1.5 million deaths annually. There is convincing evidence to show that smoking is an important risk modulating factor to lung cancer development.&nbsp; This risk is increased by age at which one starts, the total number of years&nbsp; and number of cigarettes smoked/day.&nbsp; Studies highlight smoking leads to the largest (relative) increases for small cell carcinoma and squamous cell carcinoma and (Sobue et al., 1999 and Janssen-Heijnen et al., 2001). Other risk factors include lack of physical activity, genetic mutations, dietary factors, asbestos, air pollution (de Groot et al., 2012). Although the link between smoking and lung cancer has been well-established, environmental and indoor radiation exposure are also significant contributors. Risk assessment measures for defining acceptable exposure levels of radiation exposure still remain uncertain; including the scientific research to support the justifications. This is partially due to the assumption of a non-threshold and linear model at low doses with no consideration that cellular/tissue effects of low dose radiation exposure remain poorly understood. </span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Efforts were focused on developing a simple, unidirectional AOP to lung cancer using predominantly available data from radiation studies. Decades of research suggest that energy in the form of ionizing radiation can break DNA molecules. In vitro mutagenicity studies suggest that alterations in genes in the form of mutations, chromosomal aberrations and micronuclei formation may be important for cancer cell differentiation/proliferation and eventually neoplastic transformation (Harris, 1987). The MIE was selected to be &ldquo;deposition of energy&rdquo; as it is the initial measurable interaction at the macro-molecular level within an organism that can lead to a perturbation that initiates the AOP. The term accurately defines the initiating phenomena that manifest from any type of radiation insult (e.g. alpha- and beta-particles, photons, neutrons and heavy ions) and is distinguishable from chemical-based initiation events.&nbsp; Although the &ldquo;deposition of energy&rdquo; is itself a physical phenomenon (not biological) it is essential to describe the causal relationship between radiation insults and the stochastic onset of associated downstream biological damage. Historically, this relationship has been empirically observed and reported in the form of dose-response data. In addition, this MIE encapsulates the known varieties of radiation and their differing physical properties while still adhering to the stressor agnostic principles of the AOP framework. </span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">&nbsp;This AOP has brought together molecular and cellular based research in the radiation realm and defined a modular, simplistic path towards lung cancer. It has used data&ndash;rich key events to a classic targeted response onto a cell that is applicable to multiple radiation stressors (e.g. X-rays, gamma rays, alpha particles, beta particles, heavy ions, neutrons) and well supported thorough empirical evidence. The proposed&nbsp;AOP is not the only route to lung cancer it is likely to be one linear path in a network of multiple pathways that may include other critical events.&nbsp; This hypothetical AOP will be networked to AOP-296, AOP-322, AOP-293, AOP-294 and AOP-303 forming a larger network of KEs related inflammation, apoptosis, and oxidative stress, providing a more complete path to lung cancer. <span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">This AOP is also a case example of how existing evidence from radiation stressors can stregthen empirical evidence surrounding key events that may be non-radiation specific and vice versa. By using a radiation centric molecular initiating event (MIE), networks can be developed for multiple adverse outcomes distinct to a radiation response. As different radiation stressors can trigger the MIE, the AOP will have wide applicability. </span></span></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">It is our goal, with the development of this AOP to motivate radiation researchers to use this framework for bringing together research data, exchanging knowledge, identifying priority areas and better co-ordinating research in the low-dose ionizing radiation field.</span></span></p>
  • </div>
  • <div id="aop_summary">
  • <h2>Summary of the AOP</h2>
  • <h3>Events</h3>
  • <h3>Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)</h3>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Sequence</th>
  • <th scope="col">Type</th>
  • <th scope="col">Event ID</th>
  • <th scope="col">Title</th>
  • <th scope="col">Short name</th>
  • </tr>
  • </thead>
  • <tbody>
  • <tr>
  • <td>1</td>
  • <td>MIE</td>
  • <td>1686</td>
  • <td><a href="/events/1686">Deposition of Energy</a></td>
  • <td>Energy Deposition</td>
  • </tr>
  • <tr><td></td><td></td><td></td><td></td><td></td></tr>
  • <tr>
  • <td>2</td>
  • <td>KE</td>
  • <td>1635</td>
  • <td><a href="/events/1635">Increase, DNA strand breaks</a></td>
  • <td>Increase, DNA strand breaks</td>
  • </tr>
  • <tr>
  • <td>3</td>
  • <td>KE</td>
  • <td>155</td>
  • <td><a href="/events/155">Inadequate DNA repair</a></td>
  • <td>Inadequate DNA repair</td>
  • </tr>
  • <tr>
  • <td>4</td>
  • <td>KE</td>
  • <td>185</td>
  • <td><a href="/events/185">Increase, Mutations</a></td>
  • <td>Increase, Mutations</td>
  • </tr>
  • <tr>
  • <td>5</td>
  • <td>KE</td>
  • <td>1636</td>
  • <td><a href="/events/1636">Increase, Chromosomal aberrations</a></td>
  • <td>Increase, Chromosomal aberrations</td>
  • </tr>
  • <tr>
  • <td>6</td>
  • <td>KE</td>
  • <td>870</td>
  • <td><a href="/events/870">Increase, Cell Proliferation</a></td>
  • <td>Increase, Cell Proliferation</td>
  • </tr>
  • <tr><td></td><td></td><td></td><td></td><td></td></tr>
  • <tr>
  • <td></td>
  • <td>AO</td>
  • <td>1556</td>
  • <td><a href="/events/1556">Increase, lung cancer</a></td>
  • <td>Increase, lung cancer</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h3>Key Event Relationships</h3>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Upstream Event</th>
  • <th scope="col">Relationship Type</th>
  • <th scope="col">Downstream Event</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Quantitative Understanding</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/relationships/1977">Deposition of Energy</a></td>
  • <td>adjacent</td>
  • <td>Increase, DNA strand breaks</td>
  • <td>High</td>
  • <td>High</td>
  • </tr>
  • <tr>
  • <td><a href="/relationships/1911">Increase, DNA strand breaks</a></td>
  • <td>adjacent</td>
  • <td>Inadequate DNA repair</td>
  • <td>Moderate</td>
  • <td>Moderate</td>
  • </tr>
  • <tr>
  • <td><a href="/relationships/164">Inadequate DNA repair</a></td>
  • <td>adjacent</td>
  • <td>Increase, Mutations</td>
  • <td>Moderate</td>
  • <td>Moderate</td>
  • </tr>
  • <tr>
  • <td><a href="/relationships/1912">Inadequate DNA repair</a></td>
  • <td>adjacent</td>
  • <td>Increase, Chromosomal aberrations</td>
  • <td>High</td>
  • <td>Low</td>
  • </tr>
  • <tr>
  • <td><a href="/relationships/1978">Increase, Mutations</a></td>
  • <td>adjacent</td>
  • <td>Increase, Cell Proliferation</td>
  • <td>High</td>
  • <td>Low</td>
  • </tr>
  • <tr>
  • <td><a href="/relationships/1979">Increase, Chromosomal aberrations</a></td>
  • <td>adjacent</td>
  • <td>Increase, Cell Proliferation</td>
  • <td>Moderate</td>
  • <td>Low</td>
  • </tr>
  • <tr>
  • <td><a href="/relationships/1980">Increase, Cell Proliferation</a></td>
  • <td>adjacent</td>
  • <td>Increase, lung cancer</td>
  • <td>High</td>
  • <td>Low</td>
  • </tr>
  • <tr>
  • <td></td>
  • <td></td>
  • <td></td>
  • <td></td>
  • <td></td>
  • </tr>
  • <tr>
  • <td><a href="/relationships/1981">Deposition of Energy</a></td>
  • <td>non-adjacent</td>
  • <td>Increase, Mutations</td>
  • <td>High</td>
  • <td>High</td>
  • </tr>
  • <tr>
  • <td><a href="/relationships/1982">Deposition of Energy</a></td>
  • <td>non-adjacent</td>
  • <td>Increase, Chromosomal aberrations</td>
  • <td>High</td>
  • <td>High</td>
  • </tr>
  • <tr>
  • <td><a href="/relationships/1983">Deposition of Energy</a></td>
  • <td>non-adjacent</td>
  • <td>Increase, lung cancer</td>
  • <td>Moderate</td>
  • <td>Moderate</td>
  • </tr>
  • <tr>
  • <td><a href="/relationships/1931">Increase, DNA strand breaks</a></td>
  • <td>non-adjacent</td>
  • <td>Increase, Mutations</td>
  • <td>High</td>
  • <td>Low</td>
  • </tr>
  • <tr>
  • <td><a href="/relationships/1939">Increase, DNA strand breaks</a></td>
  • <td>non-adjacent</td>
  • <td>Increase, Chromosomal aberrations</td>
  • <td>High</td>
  • <td>Low</td>
  • </tr>
  • <tr>
  • <td><a href="/relationships/1984">Increase, Mutations</a></td>
  • <td>non-adjacent</td>
  • <td>Increase, lung cancer</td>
  • <td>High</td>
  • <td>Low</td>
  • </tr>
  • <tr>
  • <td><a href="/relationships/1985">Increase, Chromosomal aberrations</a></td>
  • <td>non-adjacent</td>
  • <td>Increase, lung cancer</td>
  • <td>Moderate</td>
  • <td>Moderate</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h3>Stressors</h3>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Name</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Ionizing Radiation</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <div id="overall_assessment">
  • <h2>Overall Assessment of the AOP</h2>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="background-color:white"><span style="color:black">The present AOP compiles data in the most simplified, modular path to lung cancer from an MIE of deposition of energy. An estimated 1.5 million people worldwide die of lung cancer annually (<a href="https://www.wcrf.org" style="color:blue; text-decoration:underline">https://www.wcrf.org</a>) with smoking being the leading cause globally, followed by radon gas. Multiple other environmental factors (e.g., asbestos, air pollution and arsenic) in combination with smoking can increase risk (Hubaux et al., 2012).&nbsp; Indeed, studies show that the histological lung profile of smokers is very different from non-smokers exposed to high radon levels (Kreuzer et al., 2000). This is in part due to the complexity of the interaction of each stressor with macromolecules within the cell. Therefore, at the different levels of biological organization, it is important to distinguish the mechanisms between lung cancer from smoking and that of radon exposure. Furthermore, studies show that residential radon gas can contribute to lung cancer, however, there remains uncertainty in risk estimates due to the dosimetry of the exposures from the lower doses (Samet et al., 2000 and 2006). The cellular/tissue effects of low dose radiation exposure are still not well understood, and the traditional linear no-threshold model cannot be assumed to accurately estimate risk (Ruhm et al., 2016; Shore et al., 2018).&nbsp; </span></span></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="background-color:white"><span style="color:black">Following AOP conventions, KEs were chosen based on the well-accepted understanding of lung cancer from radiation exposures (e.g., radon gas).&nbsp; Essential key events were identified that are routinely measured by modern and conventional assays. Hallmarks of cancer (i.e., evasion, angiogenesis, etc.) are not included in the AOP, but these could be developed separately and networked in the future. This AOP is the first to use an MIE that is radiation-specific, therefore, this pathway is envisioned to be networked to other health outcomes initiated from radiation exposures. Pathways could be created in parallel including additional KEs leading to non-targeted effects (i.e., immune response/suppression and adaptive response) or non-cancer outcomes.</span></span></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="background-color:white"><span style="color:black">Although decades of radiobiological and epidemiological research exist in the radiation field, it was a difficult to identify the required elements of the Bradford-Hill criteria (e.g., essentiality, incidence-concordance).&nbsp; This AOP is best supported by evidence from biological plausibility given the DNA damage response and repair is a well-established and reviewed pathway. &nbsp;It was noted that studies typically analyzed endpoints at a single time-point, which challenged fully understanding the timeframe of KEs initiation. A few studies used a broad dose-range but did not detail quantitative trends. Additionally, there was limited evidence supporting essentiality, particularly for the latter half of the pathway. This was evident in the KERs of inadequate DNA repair to mutations/chromosomal aberrations (CAs) and mutations/CAs to cellular proliferation, while the non-adjacent KERs (e.g., energy deposition to CAs or energy deposition to mutations) generally were well-supported. Another area of challenge was KERs that were linked directly to the MIE.&nbsp; For these KERs there were often inconsistencies in findings due to varied exposure parameters related to doses, dose-rates and radiation quality.&nbsp; Radiation attributes can modulate cancer progression by influencing the type and amount of damage. This aspect can complicate the quantitative understanding of the AOP, although qualitatively the outcomes were observed to be similar. Furthermore, no single study measured all the KEs in this AOP. The lack of studies showing essentiality formed the principal knowledge gap of the AOP. Data supporting dose- and temporal-effects could also be improved across various KERs through more well-conducted animal studies. Uncertainty surrounding modulating factors such as lifestyle, health status and individual radiosensitivity also reduced the strength of the KERs. Additional KEs addressing these factors could be incorporated in parallel as research on these KEs improves. </span></span></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="background-color:white"><span style="color:black">An assessment of the weight of evidence supporting this AOP determined a strong biological plausibility and a moderate level of empirical evidence. Mathematical simulations and cell-based studies have predominantly provided evidence for dose- and temporal-response relationships. Various factors can influence the initiation of the KEs, including cell type, radiation quality and dose-rate. Therefore, the amount of energy deposited (MIE) that is required to drive the KEs in a pathway leading to cancer remains undefined. This is especially relevant for conflicting concepts of hormesis and hypersensitivity at low doses and low dose-rates. Additionally, because of the nature of the MIE, absolute values of DSBs that are required to surpass the capability of DNA repair mechanisms, resulting in inadequate DNA repair, and the downstream events leading to cancer, remain unknown. The occurrence of tumorigenesis requires more than one 1 hit to the DNA molecule (Loeb et al., 2003), however, there is a low probability that a single ionization event to the DNA molecule will result in the pathway leading to lung cancer. In contrast, at higher doses, cancer formation may not occur, as apoptosis may be induced in damaged cells. Further research involving the development of quantitative and predictive models can strengthen the understanding of the AOP.</span></span></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="background-color:white"><span style="color:black">The present AOP can be expanded to include KEs related to oxidative stress, signaling pathways and inflammatory mediators. The uncertainties, inconsistencies and knowledge gaps identified in the AOP can inform areas for future research. The AOP demonstrates the framework as a means to compile data, exchange knowledge, and identify priority areas for research in the ionizing radiation field. The current version of this AOP was developed by a team of researchers with backgrounds primarily in AOP development, carcinogenesis, radiobiology, radiation physics and biomolecular epidemiology. However, due to the importance of radiation epidemiology in the international radiological protection system and its underlying assumptions, it seems essential to strengthen the epidemiological aspects of this AOP, a specific area of future improvement. With increased development of radiation relevant AOPs, the AOP framework will have a larger part in supporting the system of radiological protection.</span></span></span></span></p>
  • <h3>Domain of Applicability</h3>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>All life stages</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>human</td>
  • <td>Homo sapiens</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>rat</td>
  • <td>Rattus norvegicus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>mouse</td>
  • <td>Mus musculus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">This AOP is relevant to mammals (Eymin &amp; Gazzeri, 2009; Barron et al., 2014; Kurgan et al., 2017). The pathway leading to the development of lung cancer often occurs during adulthood but may be applicable at earlier life stages (Liu et al., 2015) and is independent of sex. In humans, however, genetic abnormalities/mutations suggestive of lung cancer risk seem to be influenced by ethnicity (Lloyd et al., 2013), smoking history (Lim et al., 2009; Sanders &amp; Albitar, 2010; Paik et al., 2012; Lloyd et al., 2013; Cortot et al., 2014; Minina et al., 2017, Cahoon et al., 2017), age (Lloyd et al., 2013), sex (Lim et al., 2009; Cortot et al., 2014) and genotype (Lim et al., 2009; Sanders &amp; Albitar, 2010; Kim et al., 2012; Paik et al., 2012; Leng et al. 2013; Cortot et al., 2014; Minina et al., 2017). Evidence supporting this AOP comes primarily from studies using bacterial DNA (Sutherland et al., 2000; Jorge et al., 2012), human fibroblast cells (Rothkamm &amp; Lo, 2003; Kuhne et al., 2005; Rydberg et al., 2005a), mice (Duan et al., 2008; Zhang &amp; Jasin, 2011), hamsters (Bracalente et al., 2013; Lin et al., 2014), lung cancer cell lines (Sato, Melville B. Vaughan, et al. 2006; Kurgan et al., 2017; Tu et al., 2018), and tissue samples (both with and without lung cancer) Sun et al., 2016; Tu et al., 2018 Warth et al., 2014.</span></span></p>
  • <h3>Essentiality of the Key Events</h3>
  • <table border="1" cellpadding="0" cellspacing="0" style="height:2510px; width:750px">
  • <tbody>
  • <tr>
  • <td rowspan="2" style="background-color:#dddddd; width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Support for Essentiality of KEs</strong></span></span></p>
  • </td>
  • <td style="background-color:#dddddd; width:140px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Defining Question</strong></span></span></p>
  • </td>
  • <td style="background-color:#dddddd; width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Strong</strong></span></span></p>
  • </td>
  • <td style="background-color:#dddddd; width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Moderate</strong></span></span></p>
  • </td>
  • <td style="background-color:#dddddd; width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Weak</strong></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:140px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Are downstream KEs and/or the AO prevented if an upstream KE is blocked?</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">No or contradictory experimental evidence of the essentiality of any of the KEs</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>MIE: </strong></span></span></p>
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Deposition of Energy</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:518px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Evidence for Essentiality of KE: Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">This event is difficult to test for essentiality as deposition of energy is a physical stressor and cannot be blocked/decreased using chemicals. &nbsp;However, there are a number of antioxidant studies demonstrating that treatment with various antioxidants prior to irradiation decreases the number of radiation-induced DSBs (results summarized in a review by Kuefner et al. 2015; Smith et al. 2017).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>KE1: </strong></span></span></p>
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Double-Strand Breaks, Increase</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:518px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Evidence for Essentiality of KE: Weak</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">A variety of different studies demonstrate that organisms with compromised DNA repair tend to have an increased incidence of DSBs. Inhibition studies have shown that addition of a DNA repair antagonist results in significant increases in DSBs at 6 and 12 hours post-irradiation (Dong et al. 2017). Similarly, knock-outs/knock-downs of DNA repair proteins also results in persisting DSBs post-irradiation (Rothkamm and Lo 2003; Bracalente et al. 2013; Mcmahon et al. 2016; Dong et al. 2017), with one DNA ligase IV-deficient human cell line showing DSBs 240 - 340 hours after radiation exposure (Mcmahon et al. 2016). Studies by Tatsumi-Miyajima et al., (1993) note the increased rate of supF mutation frequencies following the use of a restriction enzyme, <em>Aval, </em>which induces DSBs in different human fibroblast cell lines transfected with plasmids containing the <em>Aval </em>restriction site.&nbsp; Kurashige et al. (2017) have demonstrated a decrease in MN frequency following the reduction in DSBs by regulating NAC pre-treatment.</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>KE2: Inadequate DNA Repair, Increase</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:518px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Evidence for Essentiality of KE: Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is extensive evidence to demonstrate the essentiality of inadequate repair to downstream events. Studies show that inhibiting DNA repair results in a lack of DNA repair foci post-irradiation (Paull et al. 2000), while cells deficient in ATM (involved in DNA repair) show increased levels of incorrectly rejoined DSBs (Lobrich et al. 2000; Bucher et al. 2021). Similarly, chromosomal aberrations were more frequent after inhibition of various proteins involved in DNA repair (Chernikova et al. 1999; Heterodimer et al. 2002; Wilhelm et al. 2014). Furthermore, when knock-out cell lines (i.e., knock-out of genes involved in DNA repair to increase the incidence of &lsquo;inadequate&rsquo; repair) &nbsp;were examined for genomic abnormalities, increased incidence of chromosomal aberrations were clearly evident (Karanjawala et al. 1999; Cornforth and Bedford 1994; Patel et al. 1998; Simsek and Jasin 2010; Lin et al. 2014; Wilhelm et al. 2014; Mcmahon et al. 2016).&nbsp; Deficiencies in proteins involved in DNA repair also resulted in altered mutation frequencies relative to wild-type cases (Amundson and Chen 1996; Feldmann et al. 2000; Smith et al. 2003; Wessendorf et al. 2014; Perera et al. 2016). Mutation frequency increased following knocked-down BER-initiating glycosylases (OGG1, NEIL1, MYH, NTH1) in HEK293T human embryonic kidney cells transfected with plasmids that were either positive or negative for 8-oxodG (Suzuki et al., 2010). Moreover, G:C to T:A transversion frequency increased in all analyzed cells. Nallanthighal et al. (2017) demonstrated that inadequate DNA repair impacts MN induction in irradiated Ogg1-deficienct mice (compared to Oggff1+/+ mice).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>KE3: Mutations, Increase</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:518px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Evidence for Essentiality of KE: Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Numerous studies show a strong correlation between inadequate DNA repair and mutation incidence, as altered mutation frequencies were evident when there were deficiencies in the proteins involved in DNA repair (Amundson and Chen 1996; Feldmann et al. 2000; Smith et al. 2003; Wessendorf et al. 2014; Perera et al. 2016). Mutations in several different genes, including tumour suppressor gene TP53, have also been shown to increase cell proliferation rates (Hundley et al. 1997; Lang et al. 2004; Ventura et al. 2007; Welcker and Clurman 2008; Duan et al. 2008; Geng et al. 2017; Li and Xiong 2017); mutant or absent TP53 has likewise been implicated in carcinogenesis (Iwakuma and Lozano 2007; Muller et al. 2011; Kim and Lozano 2018). In terms of lung cancer specifically, there are many different studies showing that mutations in TP53, KRAS, and EGFR &nbsp;are associated with lung carcinogenesis. The conceptual &lsquo;removal&rsquo; or &lsquo;blocking&rsquo; of these mutations using conditional knock out models, inducible mutation models, and treatment with various antagonizing and agonizing compounds has been observed to reverse or prevent lung tumourigenesis in vivo (Roth et al. 1996; Fisher et al. 2001; Ventura et al. 2007; Iwakuma and Lozano 2007; Jia et al. 2016; Luo et al. 2019, Krasinski 2012). The lung tumourigenesis process was also observed to be expedited by exposure of Gprc5a knock-out mice to a known pulmonary carcinogen; this resulted in more somatic mutations and an increased tumour burden in a much shorter time frame relative to unexposed mice (Fujimoto et al. 2017).&nbsp; &nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>KE4: Chromosomal Aberrations, Increase</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:518px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Evidence for Essentiality of KE: Weak</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Many studies using a model with inadequate DNA repair (in the form of knock-out cell lines and DNA repair inhibitor studies) demonstrated that chromosomal aberrations were significantly increased when DNA repair was inadequate (Karanjawala et al.; Patel et al. 1998; Deniz Simsek and Jasin 2010; Lin et al. 2014; Wilhelm et al. 2014; Mcmahon et al. 2016, Cornforth 1994). The presence of chromosomal aberrations, particularly gene fusions and translocations, has also been associated with high rates of cellular proliferation (Li et al. 2007; Soda et al. 2007; Guarnerio et al. 2016).There also is support for the essentiality of CAs in the induction of cancer. There were significant increases in CAs (micronuclei, nucleoplasmic bridges and nuclear buds) in peripheral blood lymphocyte cultures after addition of a known pulmonary carcinogen to the cells (Lloyd et al. 2013). Furthermore, introduction of the BCR/ABL translocation in mice resulted in chronic myelogenous leukemia; this was accomplished by lethally irradiating the mice and performing a bone marrow transplant with cells that contained a retrovirus carrying the BCR/ABL translocation (Pear et al. 1998). Furthermore, tumour-inducing A549 cells, which are deficient in TSCL1 due to a loss of heterozygosity at chromosome 11, can induce detectable tumours within 3 weeks of injection; transfection of these A549 cells with genes to correct the TSCL1 deficiency and subsequent injection into mice results in fewer and slower-growing tumours (Kuramochi et al. 2001).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>KE5: </strong></span></span></p>
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Cell Proliferation, Increase</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:518px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Evidence for Essentiality of KE: Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rates of cellular proliferation have been shown to be increased when there are mutations in key genes associated with cell cycle control, including tumour suppressor gene TP53 (Hundley et al. 1997; Lang et al. 2004; Ventura et al. 2007; Welcker and Clurman 2008; Duan et al. 2008; Geng et al. 2017; Li and Xiong 2017). Cells transformed with various oncogenic mutations that suppressed tumour suppressor genes and enhanced activity of proto-oncogenes also showed increased cellular proliferation rates in the form of higher tumour volumes (Sato et al. 2017). Addition of inhibitors that blocked the pro-proliferative signaling pathway associated with KRAS and EGFR in these oncogenically-transformed cells resulted in lower rates of cellular proliferation (Sato et al. 2017). Similarly, several specific chromosomal gene fusions and translocations have also been associated with increasing the rate of cellular proliferation (Li et al. 2007; Soda et al. 2007; Guarnerio et al. 2016). In cancer cells known to harbor the Philadelphia chromosome (a translocation heavily implicated in the pathogenesis of acute lymphoblastic leukemia), addition of an ERB inhibitor resulted in decreased cellular proliferation rates in the cancer cells (Irwin et al. 2013). In another experiment where human ovarian cancer cells were treated with estrogen, there was an increase in the levels of micronuclei and a corresponding increase in the proliferation rates; addition of an antagonist maintained micronuclei frequencies and cell proliferation rates at control cell levels (Stopper et al. 2003). Cellular proliferation rates were decreased using both in vitro and in vivo carcinogenic models exposed to anti-cancer compounds, which highlights the importance of high cellular proliferation for carcinogenesis (Kassie et al. 2008; Lv et al. 2012; Wanitchakool et al. 2012; Pal et al. 2013; Warin et al. 2014; Tu et al. 2018). Genetic manipulations of genes involved in proliferation also resulted in modified cellular proliferation rates (Lv et al. 2012; Sun et al. 2016).</span></span></p>
  • <p>&nbsp;</p>
  • </td>
  • </tr>
  • </tbody>
  • </table>
  • <h3>Weight of Evidence Summary</h3>
  • <table border="1" cellpadding="0" cellspacing="0" style="height:5000px; width:750px">
  • <tbody>
  • <tr>
  • <td rowspan="2" style="background-color:#dddddd; width:141px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Support for Biological Plausibility of KERs</em></strong></span></span></p>
  • </td>
  • <td style="background-color:#dddddd; width:109px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Defining Question</em></strong></span></span></p>
  • </td>
  • <td style="background-color:#dddddd; width:137px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Strong</em></strong></span></span></p>
  • </td>
  • <td style="background-color:#dddddd; width:116px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Moderate</em></strong></span></span></p>
  • </td>
  • <td style="background-color:#dddddd; width:124px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Weak</em></strong></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:109px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><em>Is there a mechanistic relationship between KE<sub>up</sub> and KE<sub>down</sub> consistent with established biological knowledge?</em></span></span></p>
  • </td>
  • <td style="width:137px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><em>Extensive understanding of the KER based on extensive previous documentation and broad acceptance; Established mechanistic basis</em></span></span></p>
  • </td>
  • <td style="width:116px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><em>KER is plausible based on analogy to accepted biological relationships, but scientific understanding is &nbsp;not completely established</em></span></span></p>
  • </td>
  • <td style="width:124px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><em>There is empirical support for&nbsp; statistical association between KEs, but the structural or functional relationship between them is not understood</em></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:141px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Deposition of Energy (MIE)&nbsp;&nbsp;&nbsp; --&gt; Double-Strand Breaks, Increase (KE1)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:486px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Biological Plausibility of KER: </em>Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">It is well established that ionizing radiation can cause various types of DNA damage including single-strand and double-strand breaks (DSBs) (reviewed in Lomax et al. 2013). In particular, there is evidence for the deposition of energy and a resulting increase in DSBs (Ward 1988; Terato and Ide 2005; Goodhead 2006; Hada and Georgakilas 2008; Asaithamby and Chen 2011; Okayasu 2012; Lomax et al. 2013; Moore et al. 2014; Desouky et al. 2015; Sage and Shikazono 2017; Chadwick 2017; Franken et al., 2012; Frankenberg et al., 1999; Rydberg et al., 2002; Belli et al., 2000). Structural damage from the deposited energy can induce chemical modifications in the form of breaks to the phosphodiester backbone of both strands of the DNA. (Joiner 2009). DSBs are also often formed by indirect interactions with radiation through water molecules. Energy deposited on water molecules by radiation results in the production of reactive oxygen species that can then damage the DNA (Ward 1988; Desouky et al. 2015; Maier et al. 2016).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:141px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Deposition of Energy (MIE)&nbsp;&nbsp;&nbsp; --&gt; Mutations, Increase (KE3)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:486px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Biological Plausibility of KER: </em>Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Many studies across a variety of different models provide evidence that deposition of energy by ionizing radiation results in increased mutation frequencies (Russell et al. 1957; Winegar et al. 1994; Gossen et al. 1995; Suzuki and Hei 1996; Albertini et al. 1997; Dubrova et al. 1998; Dubrova et al. 2000; Canova et al. 2002; Dubrova et al. 2002; Dubrova and Plumb 2002; Masumura et al. 2002; Somers et al. 2004; Burr et al. 2007; Ali et al. 2012; Adewoye et al. 2015; Wilson et al. 2015; Bolsunovsky et al. 2016; Mcmahon et al. 2016; Matuo et al. 2018; Nagashima et al. 2018; Wu et al., 1999; Hei et al., 1997; Nagasawa and Little, 1999; Barnhart and Cox, 1979; Thacker at al., 1982; Zhu et al., 1982; Metting et al., 1992; Schwartz et al., 1991; Chen et al., 1984). Radiation-specific mutational signatures have been identified in a variety of radiation-induced tumours (Sherborne et al. 2015; Behjati et al. 2016), and there is extensive evidence that radiation increases germline mutations in both mice (Dubrova et al. 1998; Dubrova et al. 2000; Dubrova et al. 2002; Somers et al. 2004; Barber et al. 2009; Ali et al. 2012; Adewoye et al. 2015; Wilson et al. 2015) and humans (Dubrova et al. 2002; Dubrova and Plumb 2002).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:141px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Deposition of Energy (MIE)&nbsp;&nbsp;&nbsp; --&gt; Chromosomal Aberrations, Increase (KE4)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:486px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Biological Plausibility of KER: </em>Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Extensive and diverse data from human, animal and <em>in vitro</em>-based studies show ionizing radiation induces a rich variety of chromosomal aberrations (Bauchinger et al. 1994; Schmid et al. 2002; Thomas et al. 2003; Maffei et al. 2004; Tucker et al. 2005a; Tucker et al. 2005b; George et al. 2009; Meenakshi and Mohankumar 2013; Santovito et al. 2013; Arlt et al. 2014; Balajee et al. 2014; Han et al. 2014; Vellingiri et al. 2014; Suto et al. 2015; Adewoye et al. 2015; Cheki et al. 2016; Mcmahon et al. 2016; Morishita et al. 2016; Qian et al. 2016; Basheerudeen et al. 2017; Meenakshi et al. 2017; Abe et al. 2018; Jang et al. 2019; Puig et al., 2016; Barquinero et al., 2004; Curwen et al., 2012; Testa et al., 2018; Franken et al., 2012; Cornforth et al., 2002; Loucas et al., 2013; Nagasawa et al., 1990a; Nagasawa et al., 1990b; Edwards et al., 1980; Themis et al., 2013; Schmid et al., 1996; Mestres et al., 2004; Bilbao et al., 1989; Mill et al., 1996; Brooks, 1975; Tawn and Thierens, 2009; Durante et al., 1992; Hamza and Mohankumar, 2009; Takatsuji and Sasaki, 1984; Moquet et al., 2001; Purrott et al., 1980; duFrain et al., 1979).The mechanism leading from deposition of energy to chromosomal aberrations has been described in several reviews (Smith et al. 2003; Christensen 2014; Sage and Shikazono 2017). Other evidence derives from studies examining the mechanism of copy number variant formation (Arlt et al. 2014) and induction of radiation-induced chromothripsis (Morishita et al. 2016).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:141px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Double-Strand Breaks, Increase (KE1) --&gt; Inadequate DNA Repair, Increase (KE2)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:486px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Biological Plausibility of KER: </em>Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">&nbsp;It is well recognized that almost all types of DNA lesions will result in recruitment of repair enzymes and factors to the site of damage, and the pathway involved in the repair of DSBs has been well-documented in a number of reviews, many of which also discuss the error-prone nature of DNA repair (Van Gent et al. 2001; Hoeijmakers 2001a; Khanna and Jackson 2001; Lieber et al. 2003; San Filippo et al. 2008; Lieber et al. 2010; Polo and Jackson 2011; Schipler and Iliakis 2013; Vignard et al. 2013; Betermier et al. 2014; Mehta and Haber 2014; Moore et al. 2014; Rothkamm et al. 2015; Jeggo and Markus 2015; Chang et al. 2017; Lobrich and Jeggo 2017; Sage and Shikazono 2017) Error-prone repair processes are particularly important when DSBs are biologically induced and repaired during V(D)J recombination of developing lymphocytes(Jeggo et al. 1995; Malu et al. 2012) and during meiotic divisions to generate gametes (Murakami and Keeney 2008).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:141px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Inadequate DNA Repair, Increase (KE2) --&gt; Mutations, Increase (KE3)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:486px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Biological Plausibility of KER: </em>Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Decades of research have shown that DNA repair pathways are error prone and can cause mutations inherently (such as the error prone NHEJ) (Sishc and Davis 2017). This error-prone repair, however, may be due more to the structure of the DSB ends rather than the repair machinery; more complex breaks require more processing, increasing the likelihood that there will be errors in the DNA sequence upon completion of repair (Betermier et al. 2014; Waters et al. 2014). After being exposed to ionizing radiation, approximately 25 &ndash; 50% of double-strand breaks have been shown to be incorrectly repaired (L&ouml;brich et al. 1998; Kuhne et al. 2000; Lobrich et al. 2000).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:141px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Inadequate DNA Repair, Increase (KE2) --&gt; Chromosomal Aberrations, Increase (KE4)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:486px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Biological Plausibility of KER: </em>Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">DSBs are repaired by non-homologous end joining (NHEJ) and homologous recombination (HR). HR uses a template DNA strand to repair DNA damage, while the more error-prone NHEJ simply religates broken ends back together without the use of a template (van Gent et al. 2001; Hoeijmakers 2001; Jeggo and Markus 2015; Sishc and Davis 2017). Chromosomal aberrations may result if DNA repair is inadequate, meaning that the double-strand breaks are misrepaired or not repaired at all (Bignold, 2009; Danford, 2012; Schipler &amp; Iliakis, 2013). A multitude of different types of chromosomal aberrations can occur, depending on the timing and type of erroneous repair. Examples of chromosomal aberrations include copy number variants, deletions, translocations, inversions, dicentric chromosomes, nucleoplasmic bridges, nuclear buds, micronuclei, centric rings, and acentric fragments. A multitude of publications are available that provide details on how these various chromosomal aberrations are formed in the context of inadequate repair (Ferguson and Alt 2001; Venkitaraman 2002; Povirk 2006; Weinstock et al. 2006; Denis Simsek and Jasin 2010; Lieber et al. 2010; Fenech and Natarajan 2011; Danford 2012; Schipler and Iliakis 2013; Mizukami et al. 2014; Russo et al. 2015; Leibowitz et al. 2015; Rode et al. 2016; Vodicka et al. 2018). &nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:141px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Mutations, Increase (KE3) --&gt;&nbsp; Cell Proliferation, Increase (KE5)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:486px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Biological Plausibility of KER: </em>Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">It is clearly documented that when enough mutations accumulate in critical genes associated with cell cycling or proliferation, there is potential for uncontrollable cell proliferation to occur, which in some cases leads to carcinogenesis (Bertram 2001; Vogelstein and Kinzler 2004; Panov 2005, Lee and Muller 2010). In fact, one of the hallmarks of cancer is sustained proliferative signalling, and one of the enabling characteristics of this increased proliferation is genomic instability/mutations (Hanahan and Weinberg 2011). Thus mutations are particularly dangerous if they occur in proteins controlling the cell cycle checkpoint for entry into proliferation, such as RB and p53 (Lee and Muller 2010). Activating mutations in proto-oncogenes (Bertram 2001; Vogelstein and Kinzler 2004; Larsen and Minna 2011; Lee and Muller 2010) Lee and Muller 2010, inactivating mutations in tumour suppressor genes (Bertram 2001; Vogelstein and Kinzler 2004; Lee and Muller 2010; Fernandez-Antoran et al. 2019) and inactivating mutations in caretaker/stability genes (Vogelstein and Kinzler 2004; Hanahan and Weinberg 2011) are all associated with abnormal increases the rate of cellular proliferation.</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:141px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Chromosomal Aberrations, Increase (KE4) --&gt; Cell Proliferation, Increase (KE5)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:486px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Biological Plausibility of KER: </em>Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Chromosomal aberrations are formed when there is inadequate DNA repair (Bignold 2009; Danford 2012; Schipler and Iliakis 2013) or errors during mitosis (Levine and Holland 2018). Chromosomal aberrations have been shown to increase cell proliferation when the aberrations result in the activation of proto-oncogenes (Bertram 2001; Vogelstein and Kinzler 2004), the inactivation of tumour suppressor genes (Bertram 2001; Vogelstein and Kinzler 2004),, or the modification of caretaker/stability genes (Vogelstein and Kinzler 2004). Reviews documenting the contribution of CAs to cellular proliferation and/or cancer development (which implies high rates of cellular proliferation) are available (Mes-Masson and Witte 1987; Bertram 2001; Vogelstein and Kinzler 2004; Ghazavi et al. 2015; Kang et al. 2016). The link between chromosomal instability (CIN), which describes the rate of chromosome gains and losses, and cancer development has also been reviewed (Thompson et al. 2017; Gronroos 2018; Targa and Rancati 2018; Lepage et al. 2019).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:141px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Cell Proliferation, Increase (KE5) --&gt;&nbsp; Lung Cancer, Increase (AO)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:486px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Biological Plausibility of KER: </em>Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The&nbsp;means by &nbsp;which dysregulation of cell proliferation promotes the transformation of normal to carcinogenic&nbsp;cells has been heavily reviewed (Pucci et al. 2000; Bertram 2001; Panov 2005; Eymin and Gazzeri 2009; Hanahan and Weinberg 2011; Larsen and Minna 2011). The cell cycle is essential in controlling cellular proliferation rates, and requires a series of checkpoints to be passed before the cell can fully commit to the process of cell division (Pucci et al. 2000; Bertram 2001; Eymin and Gazzeri 2009; Hanahan and Weinberg 2011). One of the most important checkpoints requires the proper functioning of p53, RB, CDK4 and CDK6. The tumour suppressor p53 &nbsp;plays a particularly important role in stopping the cell cycle when there is DNA damage, and for triggering apoptosis when damage is too severe to be repaired (Bertram 2001; Hanahan and Weinberg 2011; Larsen and Minna 2011). Telomeres also play a role in controlling cell proliferation; when the telomeres become too short to protect the coding DNA, the cell enters into a state of replicative senescence (Bertram 2001; Hanahan and Weinberg 2011). All of these processes play a role in controlling the rate of cellular proliferation within a cell. Cancer may occur when these processes became dysregulated such that cells begin to proliferate at excessively high rates. High rates of proliferation are in fact one of the strongest hallmarks of cancer (Hanahan and Weinberg 2011), and uncontrolled proliferation can be accomplished through sustained proliferative signalling through activation of proto-oncogenes (Bertram 2001; Vogelstein and Kinzler 2004; Eymin and Gazzeri 2009; Hanahan and Weinberg 2011; Larsen and Minna 2011), evading growth suppressors and resisting cell death through suppression of tumour suppressor genes (Bertram 2001; Vogelstein and Kinzler 2004; Eymin and Gazzeri 2009; Hanahan and Weinberg 2011; Larsen and Minna 2011), and overcoming replicative senescence through expression of the telomere-lengthening enzyme telomerase (Bertram 2001; Panov 2005; Hanahan and Weinberg 2011; Larsen and Minna 2011). In lung cancer specifically, commonly activated proto-oncogenes include <em>EGFR, ERBB2, MYC, KRAS, MET, CCND1, CDK4 </em>and <em>BCL2</em>, while commonly inactivated tumour suppressor genes are <em>TP53, RB1, STK11, CDKN2A, FHIT, RASSF1A, </em>and <em>PTEN</em> (Larsen and Minna 2011). Telomerase is also activated in nearly all small cell lung cancer (SCLC) cases, and in over three-quarters of non-small cell lung cancer (NSCLC) cases (Panov 2005; Larsen and Minna 2011).</span></span></p>
  • <p>&nbsp;</p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; text-align:center; width:141px"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Double-Strand Breaks, Increase (KE1) --&gt; Mutations, Increase (KE3) </strong></span></span></td>
  • <td colspan="4" style="text-align:center; width:486px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Biological Plausibility of KER: </em>Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Mechanisms of DNA strand break repair have been extensively studied. It is accepted that non-homologous joining of broken ends can introduce deletions, insertions, or base substitution. In mamalian and yeast cells, both HR and NHEJ can lead to alteration in DNA sequence (Hicks &amp; Haber, 2010; Butning &amp; Nussenzweig, 2013; Byrne et al., 2014; Rodgers &amp; McVey, 2016; Dwivedi &amp; Haver, 2018).</span></span></p>
  • <p style="text-align:left">&nbsp;</p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; text-align:center; width:141px"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Double-Strand Breaks, Increase (KE1) --&gt; Chromosomal Aberrations, Increase (KE4)</strong></span></span></td>
  • <td colspan="4" style="text-align:center; width:486px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Biological Plausibility of KER: </em>Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">DNA strand breaks must occur for chromosomal aberrations to occur. Studies have shown DSBs leading to irreversible damage. The links between DSBs and the role DSB repairs has in preventing chromosomal aberrations is widely discussed, with several reviews available: (van Gent et al., 2001; Ferguson &amp; Alt, 2001; Hoeijmakers, 2001; Iliakis et al., 2004; Povirik, 2006; Weinstock et al., 2006; Natarajan &amp; Palitti, 2008; Lieber et al., 2010; Mehta &amp; Haber, 2014; Ceccaldi et al., 2016; Chang et al., 2017; Sishc &amp; Davis, 2017; Brunet &amp; Jasin, 2018).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:141px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Mutations, Increase (KE3) --&gt; Lung Cancer, Increase (AO)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:486px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Biological Plausibility of KER: </em>Moderate</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is strong biological plausibility for the relationship between mutations and lung cancer. Bioinformatics studies have identified unique mutation signature profiles associated with specific types of cancer, including lung adenocarcinoma, lung squamous cell carcinoma and lung small cell carcinoma (Alexandrov et al. 2013; Jia et al. 2014; George et al. 2015). Moreover, mutations/genome instability have been implicated as one of the &lsquo;enabling characteristics&rsquo; underlying the hallmarks of cancer (Hanahan and Weinberg 2011). Mutations are thought to promote tumourigenesis by modifying the expression of tumour suppressor genes, proto-oncogenes, and caretaker/stability genes in such a way that promotes cell proliferation and/or suppresses apoptosis (Vogelstein and Kinzler 2004; Panov 2005; Sanders and Albitar 2010; Hanahan and Weinberg 2011; Larsen and Minna 2011). &nbsp;Commonly mutated genes in lung cancer include <em>TP53, KRAS</em> and <em>EGFR</em>. Mutations in these genes, along with known lung cancer driver mutations, are thought to promote tumourigenesis by stimulating pro-proliferation signalling pathways such as the PI3K-AKT-mTOR pathway and RAS-REF-MEK pathway (Varella-garcia 2009; Sanders and Albitar 2010; Larsen and Minna 2011McCubrey 2006).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:141px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Chromosomal Aberrations, Increase (KE4) --&gt; Lung Cancer, Increase (AO)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:486px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Biological Plausibility of KER: </em>Moderate</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Chromosomal aberrations, encompassing chromosome-type aberrations, chromatid-type aberrations, micronuclei, and nucleoplasmic bridges, have all been found to be predictive of cancer risk in various human cohorts (Bonassi et al. 2000; Smerhovsky et al. 2002; Hagmar et al. 2004; Norppa et al. 2006; Boffetta et al. 2007; Bonassi et al. 2008; Lloyd et al. 2013; El-zein et al. 2014; Vodenkova et al. 2015; El-zein et al. 2017). Specific categories of CAs, including CNVs (Wrage et al. 2009; Shlien and Malkin 2009; Liu et al. 2013; Mukherjee et al. 2016; Zhang et al. 2016; Ohshima et al. 2017) and gene rearrangements (Bartova et al. 2000; Trask 2002; Sanders and Albitar 2010; Sasaki et al. 2010; Mao et al. 2011), have also been associated with cancer development. Chromosomal aberrations promote tumourigenesis through the alteration of pathways controlling cellular growth and apoptosis (Albertson et al. 2003; Sanders and Albitar 2010). The chromosomal aberration burden may be increased by factors such as aberrant centromeres, telomerase deficiencies paired with poor cell surveillance (Albertson et al. 2003), ionizing radiation (Hei et al. 1994; Weaver et al. 1997; Weaver et al. 2000), and the interplay between non-clonal and clonal CAs (Heng, Bremer, et al. 2006; Heng, Stevens, et al. 2006).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:141px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Deposition of Energy (MIE)&nbsp;&nbsp;&nbsp; --&gt; Lung Cancer, Increase (AO)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:486px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Biological Plausibility of KER: Strong</em></strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The deposition of energy, particularly by radon gas, has been associated heavily with lung cancer (Axelson 1995; Jostes 1996; Beir 1999; Kendall and Smith 2002a; Al-Zoughool and Krewski 2009; Robertson et al. 2013). Deposition of energy that triggers lung carcinogenesis in particular is thought to enter the body through inhalation (Beir 1999; Kendall and Smith 2002b). The inhaled particles are thought to deposit on lung tissue and decay, producing ionizing radiation (Axelson 1995; Beir 1999; Kendall and Smith 2002b; Al-Zoughool and Krewski 2009) that can direct the cell towards carcinogenesis (Axelson 1995; Beir 1999; Robertson et al. 2013). The process of radiation-induced carcinogenesis often follows three steps: initiation, promotion and progression. Initiation refers to the interaction between the radiation and the cell, and results in irreversible genetic changes. Promotion occurs when non-carcinogenic promoter is added to the initiated cells such that it synergistically increases oncogenesis, often through receptor-mediated epigenetic changes. Progression occurs at the point when the cells convert from benign to malignant, and is associated with rapid growth and further accumulation of genomic aberrations (NRC 1990; Pitot 1993).</span></span></p>
  • </td>
  • </tr>
  • </tbody>
  • </table>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">&nbsp;</span></span></p>
  • <table border="1" cellpadding="0" cellspacing="0" style="height:5359px; width:750px">
  • <tbody>
  • <tr>
  • <td rowspan="2" style="background-color:#dddddd; width:134px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Support for Empirical Evidence of KERs</em></strong></span></span></p>
  • </td>
  • <td style="background-color:#dddddd; width:133px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Defining Question </em></strong></span></span></p>
  • </td>
  • <td style="background-color:#dddddd; width:126px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Strong</em></strong></span></span></p>
  • </td>
  • <td style="background-color:#dddddd; width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Moderate</em></strong></span></span></p>
  • </td>
  • <td style="background-color:#dddddd; width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Weak</em></strong></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:133px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><em>Does empirical evidence support that a change in KE<sub>up</sub> leads to an appropriate change in KE<sub>down</sub>? Does KE<sub>up</sub> occur at lower doses and earlier time points than KE<sub>down</sub> and is the incidence of KE<sub>up</sub> &gt; than that for KE<sub>down</sub>?</em></span></span></p>
  • <p>&nbsp;</p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><em>Inconsistencies?</em></span></span></p>
  • </td>
  • <td style="width:126px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><em>Multiple studies showing dependent change in both events following exposure to a wide range of specific stressors (Extensive evidence for temporal, dose-response and incidence concordance); &nbsp;No or few critical data gaps or conflicting data</em></span></span></p>
  • </td>
  • <td style="width:123px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><em>Demonstrated dependent change in both events following exposure to a small number of specific stressors; Some evidence inconsistent with expected pattern that can be explained by factors such as the experimental design, technical considerations, differences between laboratories, etc.</em></span></span></p>
  • </td>
  • <td style="width:123px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><em>Limited or no studies reporting dependent change in both events following exposure to a specific stressor (i.e. endpoints never measured in the same study or not at all); And/or significant inconsistencies in empirical support across taxa and species that don&rsquo;t align with expected pattern for hypothesized AOP</em></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:134px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Deposition of Energy (MIE) --&gt; Double-Strand Breaks, Increase (KE1)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:504px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Empirical Support of KER: </em>Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Evidence exists for dose/incidence and temporal concordance between deposition of energy and the resultant formation of DNA double-strand breaks. With increasing ionizing radiation, there is an increase in the frequency of double-strand breaks (Aufderheide et al., 1987; Charlton et al. 1989; Sidjanin, 1993; Reddy et al., 1998; Frankenberg et al., 1999; Rogakou et al. 1999; Belli et al., 2000; Sutherland et al. 2000; Lara et al. 2001; Rydberg et al., 2002; Baumstark-Khan et al., 2003; Rothkamm and Lo 2003; Rogers et al., 2004; Kuhne et al. 2005; Sudprasert et al. 2006; Rube et al. 2008; Beels et al. 2009; Grudzenski et al. 2010; Liao et al., 2011; Franken et al., 2012; Bannik, 2013; Antonelli et al. 2015; Flegal et al., 2015; Markiewicz et al., 2015; Shelke and Das, 2015; Chadwick, 2017; Hamada, 2017; Allen et al., 2018; Cencer et al., 2018;&nbsp;Bains, 2019;&nbsp;Barnard, 2019; Ahmadi et al., 2021; Barnard, 2021). However, dose-rate and radiation quality play a crucial role in determining the degree of DNA damage. Temporally, DSBs have been evident at 3 - 30 minutes post-irradiation (Rogakou et al. 1999; Rothkamm and Lo 2003; Rube et al. 2008; Beels et al. 2009; Kuefner et al. 2009; Grudzenski et al. 2010; Antonelli et al. 2015; Cencer et al., 2018). A significant proportion of the DSBs are resolved within 5 hours of radiation (Kleiman, 1990; Sidjanin, 1993; Rogakou et al. 1999; Rube et al. 2008; Kuefner et al. 2009; Grudzenski et al. 2010; Bannik, 2013; Markiewicz et al., 2015; Shelke and Das 2015; Cencer et al., 2018), with a return to baseline levels by 24 hours in most cases (Aufderheide et al., 1987; Baumstark-Khan et al., 2003; Rothkamm and Lo 2003; Rube et al. 2008; Grudzenski et al. 2010; Bannik et al., 2013; Antonelli et al., 2015; Markiewicz et al., 2015; Russo et al., 2015; Dalke, 2018; Bains, 2019; Barnard, 2019; Ahmadi et al., 2021).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:134px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Deposition of Energy (MIE) --&gt; Mutations, Increase (KE3)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:504px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Empirical Support of KER: </em>Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Evidence exists for dose/incidence concordance between deposition of energy by ionizing radiation and a corresponding dose-dependent increase in mutation frequency (Suzuki and Hei 1996; Schmidt and Kiefer 1998; Kraemer et al. 2000;&nbsp;Canova et al. 2002; Bolsunovsky et al. 2016; Mcmahon et al. 2016; Matuo et al. 2018; Nagashima et al. 2018). The linear energy transfer of the radiation (Dubrova and Plumb 2002; Matuo et al. 2018), whether the radiation is chronic or acute (Russell 1958), the radiation type (Schmidt and Kiefer 1998; Masumura 2002), and the tissue being irradiated (Masumura 2002, Gossen 1995) all affect this dose-dependent increase. Temporally, it is well established that an increased incidence of mutations is reported after the deposition of energy by radiation (Winegar 1994, Gossen 1995, Albertini 1997, Dubrova 2002A, Matuo 2018, Canova 2002, Nagashima 2018, Masumura 2002, Russell 1958). Most of these studies, however, span over days and weeks, thus making it difficult to pinpoint exactly when mutations occur. Several studies report the manifestation of mutations within 2 - 3 days of irradiation (Winegar 1994, Masumura 2002, Gossen 1995), with an increased mutation frequency still elevated at 14 (Winegar 1994) and 21 days (Gossen 1995) after radiation exposure. At low doses (&lt;1 Gy) the induction of mutations in cells has been observed for high-LET radiation such as alpha particles (Wu et al., 1999; Hei et al., 1997; Nagasawa and Little, 1999; Barnhart and Cox, 1979; Thacker at al., 1982; Zhu et al., 1982; Metting et al., 1992; Schwartz et al., 1991; Chen et al., 1984; Albertini et al., 1997).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:134px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Deposition of Energy (MIE) --&gt;&nbsp; Chromosomal Aberrations, Increase (KE4)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:504px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Empirical Support of KER: </em>Strong</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Results from many studies indicate dose/incidence and temporal concordance between the deposition of energy and the increased frequency of chromosomal aberrations. There is strong evidence of a dose-dependent increase in a wide range of chromosomal aberrations in response to increasing radiation dose (Schmid 2002, Hande et al. 2003, Thomas 2003, Jang 2019, Abe 2018, Suto 2015, McMahon 2016, Tucker 2005A, Tucker 2005B, Arlt 2014, McMahon 2016, Balajee 2014,George 2009, Maffei 2004, Qian 2015; Puig et al., 2016; Barquinero et al., 2004; Curwen et al., 2012; Testa et al., 2018; Franken et al., 2012; Cornforth et al., 2002; Loucas et al., 2013; Nagasawa et al., 1990a; Nagasawa et al., 1990b; Edwards et al., 1980; Themis et al., 2013; Schmid et al., 1996; Mestres et al., 2004; Bilbao et al., 1989; Mill et al., 1996; Brooks, 1975; Tawn and Thierens, 2009; Durante et al., 1992; Hamza and Mohankumar, 2009; Takatsuji and Sasaki, 1984; Moquet et al., 2001; Purrott et al., 1980; duFrain et al., 1979). Temporally, it is well-established that chromosomal aberrations occur after exposure to radiation (Schmid 2002, Thomas 2003, Balajee 2014, Arlt 2014, George 2009, Suto 2015, Basheerudeen 2017, Tucker 2005A, Tucker 2005B, Abe 2018, Jang 2019), though the exact timing is difficult to pinpoint because most assays take place hours or days after the radiation exposure. One notable study did, however, document the presence of chromosomal aberrations within the first 20 minutes of irradiation, with the frequency increasing sharply until approximately 40 minutes, followed by a plateau (McMahon 2016). By 7 days post-irradiation, the frequencies of most chromosomal aberrations had declined (Tucker 2005A, Tucker 2005B). &nbsp;It should be noted that chromosomal aberrations induced by ionizing radiation are dependent on dose, dose-rate, and radiation type (Bender et al., 1988; Guerrero-Carbajal et al., 2003; Day et al., 2007, Suzuki 1996, Hande et al. 2003). &nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:134px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Double-Strand Breaks, Increase (KE1) --&gt; Inadequate DNA Repair, Increase (KE2)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:504px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Empirical Support of KER: </em>Moderate</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Results from many studies indicate dose/incidence and temporal concordance between the frequency of double-strand breaks and the rate of inadequate repair. As DNA damage accumulates in organisms, the incidence of in adequate DNA repair activity (in the form of non-repaired or misrepaired DSBs) also increases (Dikomey 2000, McMahon 2016, Kuhne 2005, Rydberg 2005, Kuhne 2000, Lobrich 2000). DNA damage and its ensuing repair also follow a very similar time course, with both events documented within minutes of a radiation stressor (Pinto 2005, Rothkamm 2003, Asaithambly 2009, Dong 2017, Paull 2000). Uncertainties in this KER include controversy surrounding how error-prone NHEJ truly is (Betemier 2014), differences in responses depending on the level of exposure of a genotoxic substance (Marples 2004), and confounding factors (such as smoking) that affect double-strand break repair fidelity (Scott 2006, Leng 2008).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:134px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Inadequate DNA Repair, Increase (KE2) --&gt; Mutations, Increase (KE3)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:504px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Empirical Support of KER: </em>Moderate</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There are several studies that indicate a dose/incidence concordance between inadequate DNA repair and an increased frequency of mutations. Inadequate DNA repair (Pt&aacute;cek et al. 2001; Mcmahon et al. 2016) and mutation frequencies (Mcmahon et al. 2016) have both been found to increase in a dose-dependent fashion with increasing doses of a radiation stressor. Moreover, specific genomic regions with inadequate DNA repair rates also were found to have increased mutation densities in cancer samples (Perera et al. 2016). Increased mutation frequencies have also been demonstrated in cases where more complex DNA repair is required (Smith et al. 2001). According to the results of this study, evidence of repaired DNA was present prior to the detection of mutations in cases of simple repair, whereas these two events occurred together at a later time point when more complex repair was required (Smith et al. 2001).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:134px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Inadequate DNA Repair, Increase (KE2) --&gt; Chromosomal Aberrations, Increase (KE4)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:504px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Empirical Support of KER: </em>&nbsp;Moderate</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is little empirical evidence available that directly examines the dose and incidence concordance between DNA repair and CAs within the same study. However, comparison of results from studies that measure either radiation-induced DNA repair or radiation-induced chromosomal aberrations demonstrate that the rate of double-strand break misrepair increases in a dose-dependent fashion with radiation doses between 0 - 80 Gy (Mcmahon et al. 2016), as does the incidence of chromosomal aberrations between doses of 0 - 10 Gy (Thomas et al. 2003; Tucker et al. 2005a; Tucker et al. 2005b; George et al. 2009; Arlt et al. 2014; Balajee et al. 2014; Han et al. 2014; Suto et al. 2015; Mcmahon et al. 2016). Similarly, there is not clear evidence of a temporal concordance between these two events. One study examining DNA repair and micronuclei in irradiated cells pre-treated with a DNA repair inhibitor found that both repair and micronuclei were present at 3 hours and 24 hours post-irradiation. This suggests that there may be temporal concordance (Chernikova et al. 1999). More research, however, is required to establish empirical evidence for this KER.</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:134px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Mutations, Increase (KE3)&nbsp;&nbsp;&nbsp; --&gt; Cell Proliferation, Increase (KE5)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:504px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Empirical Support of KER: </em>Moderate</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is little empirical evidence available that assesses the dose and incidence concordance between mutation frequency and cellular proliferation rates. The correlation between these two events is clear in human epidemiology studies examining the incidence between mutations in specific genes, such as <em>TP53</em> and <em>BRCA1</em>, and the proliferative status of human tumours (M Jarvis et al. 1998; Schabath et al. 2016). Another study introducing oncogenic mutations into mouse lung epithelial cells demonstrated that the addition of multiple oncogenic mutations to the cells resulted in increased tumour volumes over 40 days (suggestive of cell proliferation); in contrast, cells containing only one of these mutations did not show significant changes in tumour volumes (Sato et al. 2017). Unsurprisingly, there is also little empirical evidence available supporting a temporal concordance between these two events. One review explores the timing between these two events by comparing the somatic mutation theory of cancer and the stem cell division theory of cancer. In the somatic mutation theory, it is suggested that mutations accumulate and result in increased rates of cellular proliferation; the stem cell theory, however, states that high proliferation in stem cells allows the accumulation of mutations (L&oacute;pez-l&aacute;zaro 2018). More research is thus required to establish empirical evidence for this KER.</span></span></p>
  • <p>&nbsp;</p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:134px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Chromosomal Aberrations, Increase (KE4) &nbsp;&nbsp;&nbsp;--&gt; Cell Proliferation, Increase (KE5)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:504px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Empirical Support of KER: </em>Moderate</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is little empirical evidence available that assesses the dose and incidence concordance between chromosomal aberration frequency and cellular proliferation rates. There are several reviews available that discuss the structure and function of specific human cancer-associated chromosomal aberrations, including <em>BCR-ABL1</em>, <em>ALK</em> fusions, and <em>ETV6-RUNX1</em> (Mes-Masson and Witte 1987; Ghazavi et al. 2015; Kang et al. 2016). There was no identified evidence supporting dose and incidence concordance. Details from a study where estrogen-responsive cancer cells were treated with estrogen suggested the possibility of a temporal concordance, as both micronuclei levels and proliferation rates were higher in the estrogen-treated cells at 140 and 216 hours post-treatment (Stopper et al. 2003). Overall, however, more empirical evidence is required to support this KER.</span></span></p>
  • <p>&nbsp;</p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:134px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Cell Proliferation, Increase (KE5) &nbsp;&nbsp;&nbsp;--&gt; Lung Cancer, Increase (AO)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:504px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Empirical Support of KER: </em>Moderate</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is some empirical evidence of a dose and incidence concordance between cell proliferation and lung carcinogenesis. In a few experiments, rodent lungs exposed to various carcinogens showed increased levels of proliferation and developed squamous metaplasia (Zhong et al. 2005) or full-blown tumours (Kassie et al. 2008). Furthermore, nude mice injected with carcinogenic human NSCLC cells also developed tumours within a few weeks of the injection (Pal et al. 2013; Warin et al. 2014; Sun et al. 2016; Tu et al. 2018)(Sun 2016, Pal 2013, Tu 2018, Warin 2014). In terms of temporal concordance between these two events, studies are also limited. Multiple tumour xenograft experiments found that nude mice injected with NSCLC cells develop detectable tumours within two weeks of inoculation, which continued to increase in size over time (Sun 2016, Pal 2013, Tu 2018, Warin 2014). Examination of lung squamous metaplasia after 14 weeks of exposure to high levels of tobacco smoke showed increased cell proliferation markers in comparison to lungs from rats exposed to filtered air (Zhong et al. 2005). Similarly, lung tumours from mice that received carcinogens NNK and BaP orally over 4 weeks were also found to express proliferation markers when examined 27 weeks after the start of the experiment (Kassie et al. 2008).</span></span></p>
  • <p>&nbsp;</p>
  • <p>&nbsp;</p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; text-align:center; width:134px"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Double-Strand Breaks, Increase (KE1) --&gt; Mutations, Increase (KE3) </strong></span></span></td>
  • <td colspan="4" style="text-align:center; width:504px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Empirical Support of KER: </em>Moderate</strong></span></span></p>
  • <p style="text-align:left"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is some evidence demonstrating dose and temporal concordance between the two KEs, both in-viv and in-vitro. These studies used a variety of sources of ionizing radiation as stressors. The types of radiation testing this relationship include X-rays, gamma-rays, alpha particles and heavy ions. Example studies include: (in vitro) Rydberg et al., 2005; Kuhne et al., 2005, 2000; Dikomey et al., 2000; Lobrich et al., 2000, (in vivo) Ptacek et al., 2001. For a discussion of chemical stressors affecting this relationship, see AOP 296.</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; text-align:center; width:134px"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Double-Strand Breaks, Increase (KE1) --&gt; Chromosomal Aberrations, Increase (KE4) </strong></span></span></td>
  • <td colspan="4" style="text-align:center; width:504px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Empirical Support of KER: </em>Moderate</strong></span></span></p>
  • <p style="text-align:left"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Temporal concordance is clear in both in vitro and in vivo data. However, due to the differences in the methods used to measure strand breaks and chromosomal aberrations, the dose-response of these events often appear to be discordant. Examples of studies relating the links between DSBs and chromosomal aberrations include an in-vitro study of gamma-radiated lymphoblasted cell lines (Trenz et al.&nbsp;2003) isolated lymphocytes and whole blood samples (Sudpresert et al., 2006) and PL61 cells (Chernikova et al., 1999). Source of high linear energy transfer have also been probed, see Iliakis et al. (2019). </span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:134px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Mutations, Increase (KE3) &nbsp;&nbsp;&nbsp;--&gt; Lung Cancer, Increase (AO)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:504px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Empirical Support of KER: </em>Moderate</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Evidence for dose/incidence concordance comes from studies with similar radiological and biological conditions that assessed either the relationship between radiation exposure and mutations, or radiation exposure and cancer. Using various<em> in vitro </em>&nbsp;models, there was a dose-dependent relationship found for mutation induction and radiation dose (Suzuki and Hei 1996; Weaver et al. 1997; Canova et al. 2002), and for oncogenic transformations and radiation dose (Hei et al. 1994; Miller et al. 1995; Miller et al. 1999). Analyses of lung cancer incidences in radon-exposed rats and uranium miners echo these results (Monchaux et al. 1994; Lubin et al. 1995; Ramkissoon et al. 2018). Likewise, administration of a known pulmonary carcinogen to <em>Gprc5a</em> knock-out mice resulted in an increased rate of tumourigenesis and increased mutation accumulation relative to saline-treated mice (Fujimoto et al. 2017). Increasing the number of mutations<em> in vitro </em>&nbsp;and <em>&nbsp;in vivo</em> resulted in cells becoming increasingly oncogenic (Sato, Melville B Vaughan, et al. 2006; Sasai et al. 2011) and mice sporting a faster rate of lung tumourigenesis (Fisher et al. 2001; Kasinski and Slack 2012), respectively. In terms of temporal concordance, there is some evidence from separate studies indicating&nbsp;that mutations precede tumourigenesis (Hei et al. 1994; Lubin et al. 1995; Hei et al. 1997; Miller et al. 1999; Fujimoto et al. 2017) , particulary in Cre-inducible models where Cre expression must be induced for the mutations to be expressed (Fisher et al. 2001; Kasinski and Slack 2012).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:134px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Chromosomal Aberrations, Increase (KE4)&nbsp;&nbsp; &nbsp;--&gt; Lung Cancer, Increase (AO)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:504px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Empirical Support of KER: </em>Moderate</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Evidence for dose/incidence concordance comes from epidemiological studies of radon-exposed uranium miners that found there was an increased CA load with increasing radon exposure (Smerhovsky et al. 2002), and an increased risk of lung cancer with increased cumulative radon exposure (Tirmarchel et al. 1993; Smerhovsky et al. 2002; Vacquier et al. 2008; Walsh et al. 2010). <em>In vivo</em> and <em>in vitro</em> studies have also shown a dose-dependent increase in CAs in lung and non-lung cell lines (Nagasawa et al. 1990; Deshpande et al. 1996; Yamada et al. 2002; Stevens et al. 2014) and lung cells of rodents with increasing radiation dose (A.L. Brooks et al. 1995; Khan et al. 1995; Werner et al. 2017), and a dose-dependent increase in oncogenic transformation in non-lung cells lines (Robertson et al. 1983; Miller et al. 1996) &nbsp;and in rodent lung tumours with increasing radiation dose (Monchaux et al. 1994; Yamada et al. 2017) Furthermore, there are several published reviews that provide evidence for associations between radon exposure and the appearance of CAs, and radon exposure and the incidence of lung cancer (Jostes 1996; Al-Zoughool and Krewski 2009; Robertson et al. 2013). Likewise, more CAs were found to accumulate in larger tumours (To et al. 2011) and in increasingly more oncogenic lung tissue lesions (Thibervile et al. 1995; Wistuba et al. 1999). There is also evidence for temporal concordance as, the time gap between radiation exposure and the increased incidence of CAs is hours to days (Nagasawa et al. 1990; A.A.L. Brooks et al. 1995; Deshpande et al. 1996; Yamada et al. 2002; Stevens et al. 2014; Werner et al. 2017), while the time gap between radiation exposure and the development of oncogenic transformations or lung tumours is weeks, months or years (Robertson et al. 1983; Tirmarchel et al. 1993; Miller et al. 1996; Pear et al. 1998; Kuramochi et al. 2001; Yamada et al. 2017).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:134px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Deposition of Energy (MIE)&nbsp; --&gt; Lung Cancer, Increase (AO)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:504px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Empirical Support of KER: </em>Moderate</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is strong evidence of the relationship between radiation exposure and lung carcinogenesis in human epidemiological studies that assess radon exposure and the risk of lung cancer. Results from numerous studies assessing indoor residential radon exposure and outdoor radon exposure in miners suggest that there is a positive association between cumulative radon exposure and lung cancer risk (Lubin et al. 1995; Hazelton et al. 2001; Darby et al. 2005; Krewski et al. 2005; Krewski et al. 2006; TAl-Zoughool and Krewski 2009; Torres-Dur&aacute;n et al. 2014; Kreuzer et al. 2015; Sheen et al. 2016; Rodr&iacute;guez-Mart&iacute;nez et al. 2018; Ramkissoon et al. 2018; Rage et al. 2020). Several<em> in vitro</em> studies showed that cells could be induced to obtain oncogenic characteristics through radiation exposure (Hei et al. 1994; Miller et al. 1995). Likewise, irradiation of rats at radon levels comparable to those experienced by uranium miners resulted in a dose-dependent increase in lung carcinoma incidence (Monchaux et al. 1994). There is also evidence of temporal concordance, as the oncogenic characteristics of the radon-exposed cells were not evident until weeks after the irradiation (Hei et al. 1994; Miller et al. 1995), while tumours took months to years to grow (Hei et al. 1994; Monchaux et al. 1994). In humans, the risk of lung cancer was also found to increase with increasing time since exposure (Hazelton et al. 2001) at a mean time of 15 years (A&szlig;enmacher et al. 2019) and with longer periods of exposure (Lubin et al.1995).</span></span></p>
  • </td>
  • </tr>
  • </tbody>
  • </table>
  • <h3>Quantitative Consideration</h3>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is strong biological plausibility and empirical evidence to suggest a qualitative link between the deposition of energy on DNA to the final adverse outcome of lung cancer. This evidence has been derived predominately from laboratory studies and through mathematical simulations using cell-based models. The studies show both dose and temporal-response relationships for a select KEs. The quantitative thresholds to initiate each of the KEs are not definitive and have been shown to vary with factors such as the cell type, dose-rate of exposure and radiation quality. Thus, an absolute amount of deposited energy (MIE) to drive a key event forward to a path of cancer is not yet definable. This is particularly relevant to low doses and low dose-rates of radiation exposure where the biology is interplayed with conflicting concepts of hormesis, hypersensitivity and the linear no threshold theory. Furthermore due to the stochastic nature of the MIE, it remains difficult to identify specific threshold values of DSBs needed to overwhelm the DNA repair machinery to cause &ldquo;inadequate&rdquo; DNA repair leading to downstream genetic abnormalities and eventually cancer. With a radiation stressor, a single hit to the DNA molecule could drive a path forward to lung cancer; however this is with low probability. &nbsp;Empirical modeling of cancer probability vs. mean radiation dose and time to lethality, does provide a good visualization of the effective thresholds (Raabe 2011). However, in general there is considerable uncertainty surrounding the connection of biologically contingent observations and stochastic energy deposition. </span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Raabe OG. Toward improved ionizing radiation safety standards. Health Phys 101: 84&ndash;93; 2011.</span></span></p>
  • <table border="1" cellpadding="0" cellspacing="0" style="height:4386px; width:750px">
  • <tbody>
  • <tr>
  • <td rowspan="2" style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Support for Quantitative Understanding of KERs</em></strong></span></span></p>
  • </td>
  • <td style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Defining Question</em></strong></span></span></p>
  • </td>
  • <td style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Strong</em></strong></span></span></p>
  • </td>
  • <td style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Moderate</em></strong></span></span></p>
  • </td>
  • <td style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Weak</em></strong></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:128px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><em>What is the extent to which a change in KE<sub>down </sub>can be predicted from KE<sub>up</sub>? What is the precision with which uncertainty in the prediction of KE<sub>down</sub> can be quantified? What is the extent to which known modulating factors or feedback mechanisms can be accounted for? What is the extent to which the relationships can be reliably generalized across the applicability domain of the KER?</em></span></span></p>
  • </td>
  • <td style="width:128px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><em>Change in KE<sub>down</sub> can be precisely predicted based on a relevant measure of KE<sub>up</sub>; Uncertainty in the quantitative prediction can be precisely estimated from the variability in the relevant KE<sub>up</sub> measure; Known modulating factors and feedback/ feedforward mechanisms are accounted for in the quantitative description; Evidence that the quantitative relationship between the KEs generalizes across the relevant applicability domain of the KER</em></span></span></p>
  • </td>
  • <td style="width:128px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><em>Change in KE<sub>down</sub> can be precisely predicted based on relevant measure of KE<sub>up</sub>; Uncertainty in the quantitative prediction is influenced by factors other than the variability in the relevant KE<sub>up</sub> measure; Quantitative description does not account for all known modulating factors and/or known feedback/ feedforward mechanisms; Quantitative relationship has only been demonstrated for a subset of the overall applicability domain of the KER </em></span></span></p>
  • </td>
  • <td style="width:128px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><em>Only a qualitative or semi-quantitative prediction of the change in KE<sub>down</sub> can be determined from a measure of KE<sub>up</sub>; Known modulating factors and feedback/ feedforward mechanisms are not accounted for; Quantitative relationship has only been demonstrated for a narrow subset of the overall applicability domain of the KER </em></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Deposition of Energy (MIE) --&gt; Double-Strand Breaks, Increase (KE1)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:511px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Quantitative Understanding of KER: Strong</em></strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The vast majority of studies examining energy deposition and incidence of DSBs suggest a positive, linear relationship between these two events (Aufderheide et al., 1987; Sidjanin, 1993; Frankenberg et al., 1999; Sutherland et al. 2000; Lara et al. 2001; Baumstark-Khan et al., 2003; Rothkamm and Lo 2003; Kuhne et al. 2005; Rube et al. 2008; Grudzenski et al. 2010; Bannik et al., 2013; Shelke and Das 2015; Antonelli et al. 2015; Dalke, 2018). Predicting the exact number of DSBs from the deposition of energy, however, appears to be highly dependent on the biological model, the type of radiation and the radiation dose range, as evidenced by the differing calculated DSB rates across studies (Charlton et al. 1989; Rogakou et al. 1999; Sutherland et al. 2000; Lara et al. 2001; Rothkamm and Lo 2003; Kuhne et al. 2005; Rube et al. 2008; Grudzenski et al. 2010; Antonelli et al. 2015) .</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Deposition of Energy (MIE) --&gt; Mutations, Increase (KE3)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:511px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Quantitative Understanding of KER: Strong</em></strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Most studies indicate a positive, linear relationship between the radiation dose and the mutation frequency (Russell et al. 1957; Albertini et al. 1997; Canova et al. 2002; Dubrova et al. 2002; Nagashima et al. 2018). In order to predict the number of mutations induced by a particular dose of radiation, parameters such as the type of radiation, the radiation&rsquo;s LET, and the type of model system being used should be taken into account (Albertini et al. 1997; Dubrova et al. 2002; Matuo et al. 2018; Nagashima et al. 2018). Predicting the mutation frequency at particular time-points, however, would be very difficult owing to our limited time scale knowledge.</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Deposition of Energy (MIE) --&gt;&nbsp; Chromosomal Aberrations, Increase (KE4)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:511px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Quantitative Understanding of KER: Strong</em></strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Most studies indicate a positive, linear-quadratic relationship between the deposition of energy by ionizing radiation and the frequency of chromosomal aberrations (Schmid et al. 2002; Suto et al. 2015; Abe et al. 2018; Jang et al. 2019). Equations describing this relationship were given in a number of studies (Schmid et al. 2002; George et al. 2009; Suto et al. 2015; Abe et al. 2018; Jang et al. 2019), with validation of the dose-response curve performed in one particular case (Suto et al. 2015). In terms of time scale predictions, this may still be difficult owing to the often-lengthy cell cultures required to assess chromosomal aberrations post-irradiation. For translocations in particular, however, one study defined a linear relationship between time and translocation frequency at lower radiation doses (0 - 0.5 Gy) and a linear quadratic relationship at higher doses (0.5 - 4 Gy) (Tucker et al. 2005b).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Double-Strand Breaks, Increase (KE1) &nbsp;--&gt; Inadequate DNA Repair, Increase (KE2)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:511px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Quantitative Understanding of KER: </em>Moderate</strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">According to studies examining DSBs and DNA repair after exposure to a radiation stressor, there was a positive linear relationship between DSBs and radiation dose (Lobrich et al. 2000; Rothkamm and Lo 2003; Kuhne et al. 2005; Asaithamby and Chen 2009), and a linear-quadratic relationship between the number of misrejoined DSBs and radiation dose (Kuhne et al. 2005) which varied according to LET (Rydberg et al. 2005b) and dose-rate (Dikomey and Brammer 2000) of the radiation. Overall, 1 Gy of radiation may induce between 35 and 70 DSBs (Dubrova et al. 2002; Rothkamm and Lo 2003), with 10 - 15% being misrepaired at 10 Gy (Mcmahon et al. 2016) and 50 - 60% being misrepaired at 80 Gy (Lobrich et al. 2000; Mcmahon et al. 2016). Twenty-four hours after radiation exposure the frequency of misrepair appeared to remain relatively constant around 80%, a rate that was maintained for the next ten days of monitoring (Kuhne et al. 2000).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Inadequate DNA Repair, Increase (KE2) --&gt; Mutations, Increase (KE3)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:511px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Quantitative Understanding of KER: Moderate</em></strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Positive relationships have been reported between radiation stressor and inadequate DNA repair, radiation stressor and mutation frequency (Mcmahon et al. 2016), and inadequate DNA repair and mutation frequency (Perera et al. 2016). It has been found that 10 - 15% of DSBs are misrepaired at 10 Gy (Mcmahon et al. 2016) and 50 - 60% at 80 Gy (Lobrich et al. 2000; Mcmahon et al. 2016), with mutation rates varying from 0.1 - 0.2 mutation per 10<sup>4</sup> cells at 1 Gy and 0.4 - 1.5 mutation per 10<sup>4</sup> cells at 6 Gy (Mcmahon et al. 2016).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Inadequate DNA Repair, Increase (KE2) --&gt; Chromosomal Aberrations, Increase (KE4)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:511px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Quantitative Understanding of KER: Weak</em></strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">A direct quantitative understanding of the relationship between inadequate DNA repair and chromosomal aberrations has not been established. However, some data has been generated using studies from radiation stressor studies. At a radiation dose of 10 Gy, the rate of DSB misrepair was found to be approximately 10 - 15% (Lobrich et al. 2000); this rate increased to 50 - 60% at a radiation exposure of 80 Gy (Kuhne et al. 2000; Lobrich et al. 2000; Mcmahon et al. 2016). It is not known, however, how this rate of misrepair relates to chromosomal aberration frequency. Results from one study using a DNA repair inhibitor suggested that as adequate DNA repair declines, the chromosomal aberration frequency increases (Chernikova et al. 1999). &nbsp;The time scale between inadequate repair and chromosomal aberration frequency has also not been well established.</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Mutations, Increase (KE3)&nbsp;&nbsp;&nbsp; --&gt; Cell Proliferation, Increase (KE5)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:511px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Quantitative Understanding of KER: Weak</em></strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">&nbsp;Quantitative understanding of the relationship between these two events has not been well established. There are, however, some studies that have examined how cellular proliferation changes over time in the presence of mutations. In cells harbouring mutations in critical genes, higher proliferation rates were evident by the fourth day in culture (Lang et al. 2004; Li and Xiong 2017) and higher rates of population doublings were evident by passage 7 (Li and Xiong 2017) relative to wild-type cells. DNA synthesis (which could be indicative of cellular proliferation) was higher in p53-/- cells than in wild-type cells for the first 6 days of culture, and increased to drastically higher levels in the knock-out cells until the end of the experiment at day 10 (Lang et al. 2004). <em>In vivo</em>, mice injected with oncogenically-transformed cells containing multiple mutations had detectable tumour growth by 10 - 12 days post-inoculation. These volumes continued increasing over the 40-day experiment (Sato et al. 2017).&nbsp;&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Chromosomal Aberrations, Increase (KE4)&nbsp;&nbsp;&nbsp; --&gt; Cell Proliferation, Increase (KE5)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:511px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Quantitative Understanding of KER: Weak</em></strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Quantitative understanding of the relationship between these two events has not been well established. . Although studies that directly assessed the time scale between chromosomal aberrations and cell proliferation rates were not identified, differences in cellular proliferation rates for cells with different CA-related manipulations or treatments were evident within the first 3 days of culture (Stopper et al. 2003; Li et al. 2007; Soda et al. 2007; Irwin et al. 2013; Guarnerio et al. 2016).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Cell Proliferation, Increase (KE5)&nbsp;&nbsp;&nbsp; --&gt; Lung Cancer, Increase (AO)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:511px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Quantitative Understanding of KER: Weak</em></strong> </span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Quantitative understanding of the relationship between these two events has not been well established. Human non-carcinogenic cells are thought to undergo 50 &ndash; 70 cell divisions before the telomeres can no longer support cell division (Panov 2005); this number would presumably be higher in cancer cells, but&nbsp; quantitative data was not able to be identified. There are some studies available, however, that provide some details regarding the timing between these two events. <em>In vitro</em> experiments using lung cancer cell lines demonstrated that expression levels of key proteins involved in the regulation of the cell cycle and/or proliferation were modified by chemical inhibitors within the first 48 hours of treatment (Lv et al. 2012; Wanitchakool et al. 2012; Pal et al. 2013; Sun et al. 2016). <em>In vivo</em> studies using xenograft nude mice found that tumours were detected within two weeks of NSCLC-cell inoculation, and continued to grow over the experimental period (Pal et al. 2013; Warin et al. 2014; Sun et al. 2016; Tu et al. 2018). Differences in tumour growth rates between mice treated with an anti-cancer drug and those left untreated were also evident within 13 - 27 days (Pal et al. 2013; Sun et al. 2016; Tu et al. 2018), with significant differences in cell proliferation markers and tumour numbers or sizes at time of harvest (22 days - 27 weeks) (Kassie et al. 2008; Pal et al. 2013; Warin et al. 2014; Sun et al. 2016; Tu et al. 2018).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; text-align:center; width:128px"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Double-Strand Breaks, Increase (KE1) --&gt; Mutations, Increase (KE3)<span style="background-color:#ffa07a"> </span></strong></span></span></td>
  • <td colspan="4" style="text-align:center; width:511px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Quantitative Understanding of KER: Weak</em></strong></span></span></p>
  • <p style="text-align:left"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is overall limited quantitiative understanding of the relationship between DSBs and increased mutation rates. McMahon et al., 2016 compiled data from multiple studies spanning different human and mouse cell lines to model the IR dose-dependent increase in mutation rate. However, further quantitiative studies into this relationship are required to provide a better quantitiative understanding.</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; text-align:center; width:128px"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Double-Strand Breaks, Increase (KE1) --&gt; Chromosomal Aberrations, Increase (KE4) </strong></span></span></td>
  • <td colspan="4" style="text-align:center; width:511px">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Quantitative Understanding of KER: Weak</em></strong></span></span></p>
  • <p style="text-align:left"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Similarly to the non-adjacent relationship above (KE1 -&gt; KE4), there is overall limited quantitiative understanding of the relationship between DSBs and increased rates of chromosomal aberrations. McMahon et al., 2016 compiled data from multiple studies spanning different human and mouse cell lines to model the IR dose-dependent increase in the rate of chromosomal aberrations. However, further quantitiative studies into this relationship are required to provide a better quantitiative understanding.</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Mutations, Increase (KE3)&nbsp;&nbsp;&nbsp; --&gt; Lung Cancer, Increase (AO)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:511px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Quantitative Understanding of KER: Weak</em></strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Finding studies addressing the quantitative relationship between mutations and cancer directly was particularly challenging. However, many studies indicated that there was a positive, dose-dependent increase in mutations with increasing radiation dose (Suzuki and Hei 1996; Canova et al. 2002). A similar positive, dose-dependent relationship was found for the oncogenic transformations in cell and the radiation dose (Miller et al. 1995), and the incidence of lung cancer in rats and their cumulative radon exposure (Monchaux et al. 1994). Epidemiological studies examining lung cancer in radon-exposed uranium miners found a positive, linear relationship between lung cancer and cumulative radon exposure (Lubin et al. 1995; Ramkissoon et al. 2018). In terms of time-scale, mutations were evident in 2 weeks following irradiation (Hei et al. 1997), whereas oncogenic transformations took 7 weeks to develop following radiation exposure (Miller et al. 1999). <em>In vivo</em> models with injected tumour cells, inherent mutations, exposure to carcinogens, or Cre-induced mutations showed tumour growth months after exposure to the tumour-inducing insult (Hei et al. 1994; Fisher et al. 2001; Kasinski and Slack 2012; Fujimoto et al. 2017).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Chromosomal Aberrations, Increase (KE4)&nbsp;&nbsp;&nbsp; --&gt; Lung Cancer, Increase (AO)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:511px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Quantitative Understanding of KER: Moderate</em></strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is evidence of a positive, linear relationship between radiation dose and CAs (Nagasawa et al. 1990; A.L. Brooks et al. 1995; Khan et al. 1995; Yamada et al. 2002; Stevens et al. 2014), radiation dose and oncogenic transformations (Miller et al. 1996), as well as radon exposure and the risk of lung cancer mortality (Tirmarchel et al. 1993; Walsh et al. 2010). The latter relationship was found to be exponentially modified, however, by factors such as the age at median exposure, the time since median exposure, and the radon exposure rate (Walsh et al. 2010). Equations defining these relationships were derived in a number of different studies (Tirmarchel et al. 1993; A.L. Brooks et al. 1995; Khan et al. 1995; Miller et al. 1996; Girard et al. 2000; Yamada et al. 2002; Walsh et al. 2010; Stevens et al. 2014). In terms of time scale, micronuclei were documented in cells of the rodent lung as early as 0.2 days (Khan et al. 1995), and were found to persist for days to weeks (Khan et al. 1995; Deshpande et al. 1996; Werner et al. 2017). Oncogenic transformations, on the other hand, took weeks to develop (Robertson et al. 1983; Miller et al. 1996), while lung tumours took months or years to develop following radiation exposure (Tirmarchel et al. 1993; Yamada et al. 2017). Delivery of an agent carrying a cancer-related CA resulted in tumour growth within 21 - 31 days of its injection into mice (Pear et al. 1998; Kuramochi et al. 2001).</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:#dddddd; width:128px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Deposition of Energy (MIE) --&gt; Lung Cancer, Increase (AO)</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:511px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em>Evidence for Quantitative Understanding of KER: Moderate</em></strong></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Quantitative understanding has been well-established for this KER. According to current Canadian guidelines developed by Health Canada, annual residential radon levels should not exceed 200 Bq/m<sup>3</sup>. Similarly, the WHO recommends that the national annual residential radon levels not exceed 100 Bq/m<sup>3</sup> where possible; if there are geographic or national constraints that make this target unachievable, the national standard should not be higher than 300 Bq/m<sup>3 </sup>(World Health Organization - Radon Guide 2009). Positive relationships between radon exposure and lung cancer have been established using <em>in vitro </em>models (Miller 1995), <em>in vivo</em> models(Monchaux et al. 1994) and results from human epidemiological studies (Lubin et al. 1995; Hazelton et al. 2001; Darby et al. 2005; Krewski et al. 2005; Krewski et al. 2006; Rodr&iacute;guez-Mart&iacute;nez et al. 2018; Ramkissoon et al. 2018). Unsurprisingly, oncogenic transformation in cells were found weeks after radiation exposure (Miller et al. 1995), sizable tumours developed months after irradiation in mice (Hei et al. 1994) and lung cancer was found years after exposure in humans (Lubin et al. 1995; Darby et al. 2005; Torres-Dur&aacute;n et al. 2014; Rodr&iacute;guez-Mart&iacute;nez et al. 2018; Ramkissoon et al. 2018).</span></span></p>
  • </td>
  • </tr>
  • </tbody>
  • </table>
  • <h2><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Quantification of AOP KERs</strong></span></span></h2>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The development of quantitative AOPs (qAOPs) has been demonstrated in other fields such as chemical toxicology (Zgheib et al., 2019) and similar objectives are warranted for AOPs with ionizing radiation stressors. The quantification of an AOP can help expedite the development of an AOP by reducing the original long-form and qualitative nature of an AOP to tables and graphs that summarize particular features e.g. dose ranges considered, radiation types included etc. Quantification is achieved by extracting numerical information from the underlying supporting evidence of KERs. The quantification of four key event relationships (KERs) from this AOP has been completed. The KERs which have been quantified are as follows:</span></span></p>
  • <ol>
  • <li>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Energy deposition leads to Increase, DNA strand breaks (Ad-KER1)</span></span></p>
  • </li>
  • <li>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Energy deposition leads to Increase, mutations (NAd-KER1)</span></span></p>
  • </li>
  • <li>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Energy deposition leads to Increase, Chromosomal aberrations (NAd-KER2)</span></span></p>
  • </li>
  • <li>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Energy deposition leads to Increase, lung cancer (NAd-KER7)</span></span></p>
  • </li>
  • </ol>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">For each of the KERs listed above, all relevant publications from those used to support the AOP were considered for quantification. In some cases, the measure of dose-response featured in one publication could not be reconciled with the measure adopted by another. For example, in the study of energy deposition leading to an increase in DNA strand breaks, Sudprasert et al. (2006) use a measure of <em>olive moment </em>from the Comet assay technique, whereas Sutherland et al. (2000) measure the relative site frequency compared to a benchmark instance of DNA damage. Due to variations such as these, not all studies that contribute qualitatively to supporting the weight of evidence of a given KER is eligible for quantification. In the case of the four KERs considered above, the most common measure of response across studies was adopted ensure the largest data sample possible. These response measured were as follows (in same order for each KER listed above):</span></span></p>
  • <ol>
  • <li>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ad-KER1 - DNA DSBs / cell</span></span></p>
  • </li>
  • <li>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER1 - Mutations / 10<sup>6</sup> cells</span></span></p>
  • </li>
  • <li>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER2 - Chromosomal aberrations / 100 cells</span></span></p>
  • </li>
  • <li>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER7 - Relative risk (RR) of lung cancer</span></span></p>
  • </li>
  • </ol>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The quantification of these four key event relationships (KERs) from this AOP has been completed as detailed in Stainforth et al., 2021. The process of quantification first involves digitizing data from publications. Results provided from tables were used directly. For figures (e.g. scatter or bar-charts) information was obtained by using the WebPlotDigitizer-4.2 authored by Rohatgi (2019). Full information of all quantified studies and respective references can be found in Tables 1-7, <a href="https://docs.google.com/document/d/1eZ26ePaTENgdKMgT_Dp0n_CF8xCWPOekwOwdylSUxNo/edit?usp=sharing">here</a>.</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The two dominant radiation types featured in the AOP are from photon and alpha-particle sources, see Table 1 below. Upstream KERs describing Ad-KER1, NAd-KER1 and NAd-KER2 are respectively composed of datasets with 298, 176 and 629 data points with 59%, 39% and 57% from photon sources and 35%, 52% and 42% from alpha-particle sources. The AO (NAd-KER7) is 100% characterized by radon (alpha-particle emitter) with a total of 33 data points. </span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">A graphical representation of the four quantified KERs is shown in Figure 1. This AOP is best documented for alpha-particles but could potentially support further data relevant to lung cancer incidence from photon radiation sources. The scope of the AOP could be extended with additional data from proton and heavy ion sources. This would encapsulate research areas such as space-travel where galactic radiation is predominantly composed of protons, and to a lesser extent, heavy ions (Chancellor et al., 2014). Overall, Figure 1 and Table 1 demonstrate how reviewing supporting empirical evidence through a quantitative <em>lens</em> reduces the description of an AOP to tables and graphs that can be used to identify inconsistencies and potential missing information across KERs and radiation types. </span></span><br />
  • &nbsp;</p>
  • <table align="center" border="1" cellpadding="0" cellspacing="0" style="height:1558px; width:765px">
  • <tbody>
  • <tr>
  • <td rowspan="2" style="width:76px">
  • <p style="text-align:center">&nbsp;</p>
  • </td>
  • <td colspan="4" style="width:491px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Radiation quality</strong></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Photons</strong></span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Protons</strong></span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Alpha-particles</strong></span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Heavy ions</strong></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>KER</strong></span></span></p>
  • </td>
  • <td colspan="4" style="width:491px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Values of dose, response, time and dose rate quoted as [minimum, maximum, average]</strong></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center">&nbsp;</p>
  • </td>
  • <td colspan="4" style="width:491px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Dose [Gy]</strong></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ad-KER1</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[1.2x10<sup>3</sup>, 80, 7.9]</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.5, 0.5, 0.5]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.1, 713, 203]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.5, -, -]</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER1</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[1.7x10<sup>-5</sup>, 14, 2.4]</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[1.24, 3.74, 2.5]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[3.4x10<sup>-5</sup>, 2.4, 0.6]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[10, 20, 11.8]</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER2</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[6.3x10<sup>-4</sup>, 10, 1.8]</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[4.3x10<sup>-4</sup>, 6.9, 0.7]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.15, 1.5, 0.7]</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER7</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[4.8x10<sup>-2</sup>, 2.63, 0.9]</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[7.89x10<sup>-3</sup>, 10.1, 0.63]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center">&nbsp;</p>
  • </td>
  • <td colspan="4" style="width:491px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Response measures [DNA DSBs / cell (Ad-KER1), Mutant frequency / 10<sup>6 </sup>cells (NAd-KER1), CAs / 100 cells (NAd-KER2), Increase in lung cancer RR [%] (NAd-KER7)]</strong></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ad-KER1</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[5x10<sup>-3</sup>, 2.8x10<sup>3</sup>, 244]</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.34, 10.1, 5.3]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[1.3, 3x10<sup>4</sup>, 9.31x10<sup>3</sup>]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.4, 8.8, 4.3]</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER1</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.3, 1.9x10<sup>3</sup>, 148]</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[1.7, 3.8x10<sup>3</sup>, 279]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.4, 19.4, 4]</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER2</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.01, 584, 44.8]</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.08, 314, 34.9]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[13.2, 138, 5.7]</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER7</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[2.7, 166, 64.4]</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[-17.9, 942, 84.4]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center">&nbsp;</p>
  • </td>
  • <td colspan="4" style="width:491px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Time [hours (Ad-KER1), days (Ad-KER1, NAd-KER2), years (NAd-KER7)]</strong></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ad-KER1</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.02, 72, 10.6]</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.03, 24, 6.5]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.02, 24, 0.5]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.25, 24, 6.5]</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER1</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[6.9x10<sup>-4</sup>, 67, 5.3]</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[6.9x10<sup>-4</sup>, -, -]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[6.94x10<sup>-4</sup>, 6, 1.4]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[6.94x10<sup>-4</sup>, 2, 0.1]</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER2</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[6.9x10<sup>-4</sup>, 56, 1.2]</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[6.94x10<sup>-4</sup>, 362, 23.6]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[6.94x10<sup>-4</sup>, -, -]</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER7</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[40, -, -]</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[5.7, 39.0, 18.5]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center">&nbsp;</p>
  • </td>
  • <td colspan="4" style="width:491px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Dose rate [Gy/min]</strong></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ad-KER1</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.03, 2, 0.9]</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.08, 100, 51.5]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER1</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[1.1x10<sup>-6</sup>, 1.2, 0.5]</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[2x10<sup>-3</sup>, 3.6, 1.3]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[1, 5, 4.8]</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER2</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[1.7x10<sup>-3</sup>, 5.9, 0.9]</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[5.3x10<sup>-6</sup>, 2.3, 0.4]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[0.5, -, -]</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER7</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[2.27x10<sup>-9</sup>, 1.25x10<sup>-7</sup>, 4.15x10<sup>-8</sup>]</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">[7.7x10<sup>-10</sup>, 3.4x10<sup>-6</sup>, 1.8x10<sup>-7</sup>]</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N/A</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center">&nbsp;</p>
  • </td>
  • <td colspan="4" style="width:491px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>% data points for KER dataset with valid dose and response values (number of data points)</strong></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ad-KER1</span></span></p>
  • </td>
  • <td style="width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">59 (177)</span></span></p>
  • </td>
  • <td style="width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">3 (8)</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">35 (105)</span></span></p>
  • </td>
  • <td style="width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">3 (8)</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="height:6px; width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER1</span></span></p>
  • </td>
  • <td style="height:6px; width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">40 (75)</span></span></p>
  • </td>
  • <td style="height:6px; width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">3 (6)</span></span></p>
  • </td>
  • <td style="height:6px; width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">48 (91)</span></span></p>
  • </td>
  • <td style="height:6px; width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">9 (17)</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="height:6px; width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER2</span></span></p>
  • </td>
  • <td style="height:6px; width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">56 (344)</span></span></p>
  • </td>
  • <td style="height:6px; width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">0 (0)</span></span></p>
  • </td>
  • <td style="height:6px; width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">43 (262)</span></span></p>
  • </td>
  • <td style="height:6px; width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">1 (10)</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="height:9px; width:76px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">NAd-KER7</span></span></p>
  • </td>
  • <td style="height:9px; width:132px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">12 (6)</span></span></p>
  • </td>
  • <td style="height:9px; width:113px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">0 (0)</span></span></p>
  • </td>
  • <td style="height:9px; width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">88 (44)</span></span></p>
  • </td>
  • <td style="height:9px; width:123px">
  • <p style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">0 (0)</span></span></p>
  • </td>
  • </tr>
  • </tbody>
  • </table>
  • <div style="text-align:center">&nbsp;</div>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Table 1: Summary&nbsp; of the quantified datasets from four KERs of the AOP. Data is categorized by both KER and radiation type. Values of dose, response measure, time since irradiation and dose rate are quoted in terms of &lsquo;[minimum, maximum, average]&rsquo; values. &lsquo;N/A&rsquo; denotes fields where there was no data. The final set of rows denote the percentages of dose-response data of a given KER associated with a given radiation type.</span></span></p>
  • <p>&nbsp;</p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><img alt="" src="https://aopwiki.org/system/dragonfly/production/2021/06/22/48rfkw4nca_Figure_3_21_June_2021.jpg" style="height:805px; width:800px" /></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Figure 1: Quantified datasets of the four KERs in graphical form. Each row of plots represents a KER in the following order from top to bottom: Ad-KER1, NAd-KER1, NAd-KER2 and NAd-KER7. The response measure for each KER is shown along the y-axis of each plot, and from left to right the dose, time and dose rate along the x-axes respectively.</span></span></p>
  • <p>&nbsp;</p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Shown in Figure 2 below is a comparison between the two dominat radiation sources; alpha-particles (green) and photon radiation (back). For each of the response measures shown in Figure 2, different symbols denote different end-points or variants of the response as measured for each KER. In the case of chromsomal aberrations (bottom-left) there is a distinct difference in the response of different chromsomal aberration types among a given radiation type e.g. for alpha-particles PCC rings (solid stars) can be 10-100 times less abundant than dicentric chromsomal types (solid circles).</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">While these differences and variations are embraced by the standard AOP construction, it should be questioned if the quantitative form of these variations is of use for constructing predictive models, and whether such an application is limited only to those of direct response-response relationships where the level of variation may be reduced. Even then, such response-response relationships would need to account for radiation type effects between each KE e.g. differing cell survival rates and the fraction of total DNA damage attributable to single strand breaks (SSBs), DSBs and complex/clustered damage. These are both very different between photon and alpha-particle sources (Franken et al., 2012; Nikjoo et al., 2001). This ultimately constrains any quantitative formalism of an AOP to be radiation type specific.</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><img alt="" src="https://aopwiki.org/system/dragonfly/production/2021/06/22/4y67nh2bpi_Figure_4_2021_06_21.jpg" style="height:707px; width:690px" /></span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Figure 2: Quantified dose-response of the four KERs in graphical form. Data is focussed on the comparison between photon and alpha-particle radiation types, in addition to the response variants for each type of response. Data is evaluated for the low-dose range of 0-2 Gy for time periods following exposure &lt; 60 minutes for Ad-KER1 (top-left), NAd-KER1 (top-right), and NAd-KER2 (bottom-left). No restriction on the time value for data points plotted for NAd-KER7 (bottom-right) has been made.</span></span></p>
  • <p><span style="font-size:9px">Stainforth, R. et al., (2021), Challenges in the quantificaton approach to a radiation relevant&nbsp;adverse outcome patway for lung cancer. Int J Radiat Biol. 97(1):85-101.</span></p>
  • </div>
  • <div id="considerations_for_potential_applicaitons">
  • <h2>Considerations for Potential Applications of the AOP (optional)</h2>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">At present the AOP framework is not readily used to support regulatory decision-making in radiation protection practices. The goal of developing this AOP is to bring attention to the framework to the radiation community as an effective means to organize knowledge, &nbsp;identify gaps &nbsp;and co-ordinate research.&nbsp; We have used lung cancer as the case example due to its relevance to radon risk assessment and broadly because it can be represented as a simplified targeted path with a molecular initiating event that is specific to a radiation insult. &nbsp;From this AOP, more complex networks can form which are relevant to both radiation and chemical exposure scenarios. Furthermore, as &nbsp;mechanistic knowledge surrounding low dose radiation exposures becomes clear, this information can be incorporated into the AOP. &nbsp;By developing this AOP, we have supported the necessary efforts highlighted by the international and national radiation protection agencies such as, the United Nations Scientific Committee on the Effects of Atomic Radiation, International Commission of Radiological Protection, International Dose Effect Alliance and the Electric Power Research Institute Radiation Program to consolidate and enhance the knowledge in understanding of low dose radiation exposures from the cellular to organelle levels within the biological system.</span></span></p>
  • </div>
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  • </div>
  • <div id="appendicies">
  • <h2>Appendix 1</h2>
  • <h3>List of MIEs in this AOP</h3>
  • <h4><a href="/events/1686">Event: 1686: Deposition of Energy</a></h4>
  • <h5>Short Name: Energy Deposition</h5>
  • <h4>AOPs Including This Key Event</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP ID and Name</th>
  • <th scope="col">Event Type</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/272">Aop:272 - Deposition of energy leading to lung cancer</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/432">Aop:432 - Deposition of Energy by Ionizing Radiation leading to Acute Myeloid Leukemia</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/386">Aop:386 - Deposition of ionizing energy leading to population decline via inhibition of photosynthesis</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/387">Aop:387 - Deposition of ionising energy leading to population decline via mitochondrial dysfunction</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/388">Aop:388 - Deposition of ionising energy leading to population decline via programmed cell death</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/435">Aop:435 - Deposition of ionising energy leads to population decline via pollen abnormal</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/216">Aop:216 - Deposition of energy leading to population decline via DNA strand breaks and follicular atresia</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/238">Aop:238 - Deposition of energy leading to population decline via DNA strand breaks and oocyte apoptosis</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/311">Aop:311 - Deposition of energy leading to population decline via DNA oxidation and oocyte apoptosis</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/299">Aop:299 - Deposition of energy leading to population decline via DNA oxidation and follicular atresia</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/441">Aop:441 - Ionizing radiation-induced DNA damage leads to microcephaly via apoptosis and premature cell differentiation</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/444">Aop:444 - Ionizing radiation leads to reduced reproduction in Eisenia fetida via reduced spermatogenesis and cocoon hatchability</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/470">Aop:470 - Deposition of energy leads to vascular remodeling</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/473">Aop:473 - Energy deposition from internalized Ra-226 decay lower oxygen binding capacity of hemocyanin</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/478">Aop:478 - Deposition of energy leading to occurrence of cataracts</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/482">Aop:482 - Deposition of energy leading to occurrence of bone loss</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/483">Aop:483 - Deposition of Energy Leading to Learning and Memory Impairment</a></td>
  • <td>MolecularInitiatingEvent</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Stressors</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Name</th></tr>
  • </thead>
  • <tbody>
  • <tr><td>Ionizing Radiation</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Biological Context</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Level of Biological Organization</th></tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr><td>Molecular</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <h3>Evidence for Perturbation by Stressor</h3>
  • <h4>Overview for Molecular Initiating Event</h4>
  • <p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">It is well documented that ionizing radiation( (eg.&nbsp;X-rays, gamma, photons, alpha, beta, neutrons, heavy ions)&nbsp;leads to energy deposition on the atoms and molecules of the substrate. Many studies, have demonstrated that the type of radiation and distance from source has an impact on the pattern of energy deposition (Alloni, et al. 2014). High linear energy transfer (LET) radiation has been associated with higher-energy deposits (Liamsuwan et al., 2014) that are more densely-packed and cause more complex effects within the particle track (Hada and Georgakilas, 2008; Okayasu, 2012ab; Lorat et al., 2015; Nikitaki et al., 2016) in comparison to low LET radiation. Parameters such as mean lineal energy, dose mean lineal energy, frequency mean specific energy and dose mean specific energy can impact track structure of the traversed energy into a medium (Friedland et al., 2017)</span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">. The detection of energy deposition by ionizing radiation can be demonstrated with the use of fluorescent nuclear track detectors (FNTDs). FNTDs used in conjunction with fluorescent microscopy, are able to visualize radiation tracks produced by ionizing radiation (Niklas et al., 2013; Kodaira et al., 2015; Sawakuchi and Akselrod, 2016). In addition, these FNTD chips can quantify the LET of primary and secondary radiation tracks up to 0.47 keV/um (Sawakuchi and Akselrod, 2016). This co-visualization of the radiation tracks and the cell markers enable the mapping of the radiation trajectory to specific cellular compartments, and the identification of accrued damage (Niklas et al., 2013; Kodaira et al., 2015). There are no known chemical initiators or prototypes that can mimic the MIE.</span></span></p>
  • <h4>Domain of Applicability</h4>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>human</td>
  • <td>Homo sapiens</td>
  • <td>Moderate</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>rat</td>
  • <td>Rattus norvegicus</td>
  • <td>Moderate</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>mouse</td>
  • <td>Mus musculus</td>
  • <td>Moderate</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>nematode</td>
  • <td>Caenorhabditis elegans</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=6239" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>zebrafish</td>
  • <td>Danio rerio</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>thale-cress</td>
  • <td>Arabidopsis thaliana</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=3702" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Scotch pine</td>
  • <td>Pinus sylvestris</td>
  • <td>Moderate</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=3349" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Daphnia magna</td>
  • <td>Daphnia magna</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=35525" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Chlamydomonas reinhardtii</td>
  • <td>Chlamydomonas reinhardtii</td>
  • <td>Moderate</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=3055" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>common brandling worm</td>
  • <td>eisenia fetida</td>
  • <td>Moderate</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=6396" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Lemna minor</td>
  • <td>Lemna minor</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=4472" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Salmo salar</td>
  • <td>Salmo salar</td>
  • <td>Low</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8030" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>All life stages</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>Low</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Energy can be deposited into any substrate, both living and non-living; it is independent of age, taxa, sex, or life-stage. </span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Taxonomic applicability: </strong>This MIE is not taxonomically specific. &nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Life stage applicability: </strong>This MIE is not life stage specific.&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Sex applicability: </strong>This MIE is not sex specific.&nbsp;</span></span></p>
  • <h4>Key Event Description</h4>
  • <p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:&quot;Times New Roman&quot;,serif">Deposition of energy refers to events where energetic subatomic particles, nuclei, or electromagnetic radiation deposit energy in the media through which they transverse. The energy may either be sufficient (</span></span></span><span style="font-size:12.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">e.g. ionizing radiation) or insufficient (e.g. non-ionizing radiation) to ionize atoms or molecules<span style="background-color:white"> (Beir et al.,1999</span>). &nbsp;</span></span></span></span></p>
  • <p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">Ionizing radiation can cause the ejection of electrons from atoms and molecules, thereby</span><span style="background-color:white"><span style="font-family:&quot;Times New Roman&quot;,serif"> resulting in their ioniz</span></span><span style="font-family:&quot;Times New Roman&quot;,serif">ation</span></span> <span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:&quot;Times New Roman&quot;,serif">and the </span></span></span><span style="font-size:12.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">breakage of</span></span> <span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:&quot;Times New Roman&quot;,serif">chemical bonds. The energy of these subatomic particles or electromagnetic waves mostly range from 124 KeV to 5.4 MeV and is dependent on the source and type of radiation (Zyla et al., 2020). To be ionizing the incident radiation must have sufficient energy to free electrons from atomic or molecular electron orbitals. The energy deposited can induce direct and indirect ionization events and this</span></span></span><span style="font-size:12.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif"> can be via internal (injections, inhalation, or absorption of radionuclides) or external exposure from radiation fields -- this also applies to non-ionizing radiation. </span></span></span></span></p>
  • <p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">Ionizing radiation can cause the ejection of electrons from atoms and molecules, thereby</span><span style="background-color:white"><span style="font-family:&quot;Times New Roman&quot;,serif"> resulting in their ioniz</span></span><span style="font-family:&quot;Times New Roman&quot;,serif">ation</span></span> <span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:&quot;Times New Roman&quot;,serif">and the </span></span></span><span style="font-size:12.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">breakage of</span></span> <span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:&quot;Times New Roman&quot;,serif">chemical<span style="font-size:16px"> <span style="font-family:Times New Roman,Times,serif">bonds. </span></span></span></span></span></span></span><span style="font-size:14px"><span style="font-family:Times New Roman,Times,serif"><span style="font-size:16px">The excitation of molecules can also occur without ionization.&nbsp;</span>T</span></span><span style="font-size:16px"><span style="font-family:Times New Roman,Times,serif">hese events are stochastic and unpredictable.&nbsp;</span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:&quot;Times New Roman&quot;,serif"><span style="font-size:16px">Th</span>e energy of these subatomic particles or electromagnetic waves mostly range from 124 KeV to 5.4 MeV and is dependent on the source and type of radiation (Zyla et al., 2020). To be ionizing the incident radiation must have sufficient energy to free electrons from atomic or molecular electron orbitals. The energy deposited can induce direct and indirect ionization events and this</span></span></span><span style="font-size:12.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif"> can be via internal (injections, inhalation, or absorption of radionuclides) or external exposure from radiation fields -- this also applies to non-ionizing radiation. </span></span></span></span></p>
  • <p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:&quot;Times New Roman&quot;,serif">Direct ionization is the principal path where charged particles interact with biological structures such as DNA, proteins or membranes to cause biological damage. Photons, which are electromagnetic waves can also deposit energy to cause direct ionization. Ionization of water, which is a major constituent of tissues and organs, produces free radical and molecular species, which themselves can indirectly damage critical targets such as DNA (Beir et al., 1999; </span></span></span><span style="font-size:12.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">Balagamwala et al., 2013) or alter cellular processes. Given the fundamental nature of energy deposition by radioactive/unstable nuclei, nucleons or elementary particles in material, this process is universal to all biological contexts. </span></span></span></span></p>
  • <p style="text-align:justify"><span style="font-size:16px"><span style="font-family:Times New Roman,Times,serif">Not all electromagnetic radiation is ionizing; as the incident radiation must have sufficient energy to free electrons from the electron orbitals of the atom or molecule. The energy deposited can induce direct and indirect ionization events and can result from internal (injections, inhalation, ingestion) or external exposure.&nbsp;</span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:&quot;Times New Roman&quot;,serif">Direct ionization is the principal path where charged particles interact with biological structures such as DNA, proteins or membranes to cause biological damage. Photons, which are electromagnetic waves can also create electrons that&nbsp;can&nbsp;cause direct ionization.&nbsp;</span></span></span></span></span><span style="font-size:16px"><span style="font-family:Times New Roman,Times,serif">Indirect ionization&nbsp;produces free radicals from&nbsp;other molecules, specifically water, which can then transform to cause damage to critical targets such as DNA.</span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="font-family:&quot;Times New Roman&quot;,serif"><span style="font-size:16px"><span style="font-family:Times New Roman,Times,serif"> </span></span>Ionization of water, which is a major constituent of tissues and organs, produces free radical and molecular species, which themselves can indirectly damage critical targets such as DNA (Beir et al., 1999; </span></span></span><span style="font-size:12.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">Balagamwala et al., 2013) or alter cellular processes. Given the fundamental nature of energy deposition by radioactive/unstable nuclei, nucleons or elementary particles in material, this process is universal to all biological contexts. </span></span></span></span></p>
  • <p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">The spatial structure of ionizing energy deposition along the resulting particle track is represented as linear energy transfer (LET) (Hall and Giaccia, 2018 UNSCEAR, 2020). High LET refers to energy mostly above 10 keV &mu;m<sup>-1</sup> which produces more complex, dense structural damage than low LET radiation (below 10 keV &mu;m<sup>-1</sup>). Low-LET particles produce sparse ionization events such as photons (X- and gamma </span></span><span style="font-size:12.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">rays), as well as high-energy protons. Low LET radiation </span></span><span style="font-size:12.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">travels farther into tissue but deposits smaller amounts of energy, whereas high LET radiation, which includes heavy ions, alpha particles and high-energy neutrons, does not travel as far but deposits larger amounts of energy into tissue at the same absorbed dose. The biological effect of the deposition of energy can be modulated by varying dose and dose rate of exposure, such as acute, chronic, or fractionated exposures (Hall and Giaccia, 2018).</span></span></span></span></p>
  • <p style="text-align:justify"><span style="font-size:16px"><span style="font-family:Times New Roman,Times,serif">Energy&nbsp;deposition differs with&nbsp;the&nbsp;linear energy transfer (LET) defined as deposition of energy per unit distance (Hall and Giaccia, 2018 UNSCEAR, 2020). High LET radiation refers to energy mostly above 10 keV &mu;m-1 which often produces more complex, dense structural damage than low LET radiation (below 10 keV &mu;m-1). High LET radiation includes heavy ions, alpha particles and high-energy neutrons. Low-LET radiation&nbsp;such as photons (X- and gamma rays), electrons as well as high-energy protons produces sparse ionization events. Low LET radiation travels farther into tissue but deposits smaller amounts of energy, whereas typically high LET particles,&nbsp;do not travel as far but deposits larger amounts of energy into tissue at the same absorbed dose. The biological effect of the deposition of energy can be modulated by varying dose and dose rate of exposure, such as in acute, chronic, or fractionated exposures (Hall and Giaccia, 2018).&nbsp;</span></span></p>
  • <p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">Non-ionizing radiation is electromagnetic waves that does not have enough energy to break bonds and induce ion formation but it can cause molecules to excite and vibrate faster resulting in biological effects. </span></span><span style="font-size:12.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">Examples of non-ionizing radiation include radio waves (wavelength: 100 km-1m), microwaves (wavelength: 1m-1mm), infrared radiation (wavelength: 1mm- 1 um), visible light (wavelengths: 400-700 nm), and ultraviolet radiation of longer wavelengths such as UVB (wavelengths: 315-400nm) and UVA (wavelengths: 280-315 nm). UVC radiation (X-X nm) is, in contrast to UVB and UVA, considered to be a type of ionizing radiation. </span></span></span></span></p>
  • <p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">Non-ionizing radiation is electromagnetic waves that does not have enough energy to break bonds and induce ion formation but it can cause molecules to excite and vibrate faster resulting in ensuing biological effects. </span></span><span style="font-size:12.0pt"><span style="font-family:&quot;Times New Roman&quot;,serif">Examples of non-ionizing radiation include radio waves (wavelength: 100 km-1m), microwaves (wavelength: 1m-1mm), infrared radiation (wavelength: 1mm- 1 um), visible light (wavelengths: 400-700 nm), and ultraviolet radiation of longer wavelengths such as UVB (wavelengths: 315-400nm) and UVA (wavelengths: 280-315 nm). UVC radiation (200-289&nbsp;nm) is, in contrast to UVB and UVA, considered to be a type of ionizing radiation<span style="font-size:16px"><span style="font-family:Times New Roman,Times,serif">.&nbsp;</span></span></span></span></span></span><span style="font-size:16px"><span style="font-family:Times New Roman,Times,serif">Exposure to non-ionizing radiation occurs either from natural or anthropogenic sources, and include radio waves used for communication (broadcasting and cell phones), microwaves used in cooking food and in radar systems, infrared radiation emitted by warm objects or used in remote controls, thermal imaging and medical treatments. Visible light is the range of electromagnetic radiation and is commonly used in photosynthesis in primary producers. UV radiation has key functions in melanisation (tanning) of a number of species and exhibits key signalling roles in navigation and communication (e.g insects, aquatic invertebrates and fish), locomotory and predatory behavior (e.g. reptiles, birds and crustaceans) and growth and development (e.g. plants). UV radiation is also used in some medical treatments such as skin diseases (e.g. psoriasis, eczema, vitiligo and skin cancers).&nbsp;&nbsp;</span></span></p>
  • <h4>How it is Measured or Detected</h4>
  • <table border="1" bordercolor="#ccc" cellpadding="5" cellspacing="0" style="border-collapse:collapse">
  • <tbody>
  • <tr>
  • <td style="background-color:#eeeeee; text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Radiation type</strong></span></span></td>
  • <td style="background-color:#eeeeee; text-align:center">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Assay Name</strong></span></span></p>
  • </td>
  • <td style="background-color:#eeeeee; text-align:center">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>References</strong></span></span></p>
  • </td>
  • <td style="background-color:#eeeeee; text-align:center">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Description</strong></span></span></p>
  • </td>
  • <td style="background-color:#eeeeee; text-align:center">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>OECD Approved Assay</strong></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ionizing radiation</span></span></td>
  • <td style="text-align:center">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Monte Carlo Simulations (Geant4)</span></span></p>
  • </td>
  • <td style="text-align:center">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Douglass et al., 2013; Douglass et al. 2012; Zyla et al., 2020</span></span></p>
  • </td>
  • <td style="text-align:center">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Monte Carlo simulations are based on a computational algorithm that mathematically models the deposition of energy into materials.</span></span></p>
  • </td>
  • <td style="text-align:center">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">No</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ionizing radiation</span></span></td>
  • <td style="text-align:center">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Fluorescent Nuclear Track Detector (FNTD)</span></span></p>
  • </td>
  • <td style="text-align:center">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sawakuchi, 2016; Niklas, 2013; Koaira &amp; Konishi,&nbsp;2015</span></span></p>
  • </td>
  • <td style="text-align:center">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">FNTDs are biocompatible chips with crystals of aluminium oxide doped with carbon and magnesium; used in conjuction with fluorescent microscopy, these FNTDs allow for the visualization and the linear energy transfer (LET) quantification of tracks produced by the deposition of energy into a material.</span></span></p>
  • </td>
  • <td style="text-align:center">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">No</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ionizing radiation</span></span></td>
  • <td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Tissue equivalent proportional counter (TEPC)</span></span></td>
  • <td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Straume et al, 2015</span></span></td>
  • <td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Measure the LET spectrum and calculate the dose equivalent.</span></span></td>
  • <td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">No</span></span></td>
  • </tr>
  • <tr>
  • <td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ionizing radiation</span></span></td>
  • <td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">alanine dosimeters/NanoDots</span></span></td>
  • <td style="text-align:center">
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Lind et al. 2019;&nbsp;Xie et al., 2022</span></span></p>
  • </td>
  • <td style="text-align:center">&nbsp;</td>
  • <td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">No</span></span></td>
  • </tr>
  • <tr>
  • <td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Non-ionizing radiation</span></span></td>
  • <td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">UV meters or radiameters</span></span></td>
  • <td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Xie et at., 2020</span></span></td>
  • <td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">UVA/UVB (irradiance intensity), UV dosimeters (accumulated irradiance over time), Spectrophoto meter (absorption of UV by a substance or material)</span></span></td>
  • <td style="text-align:center"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">No</span></span></td>
  • </tr>
  • </tbody>
  • </table>
  • <p>&nbsp;</p>
  • <h4>References</h4>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Balagamwala, E. H. et al. (2013), &ldquo;Introduction to radiotherapy and standard teletherapy techniques&rdquo;,<em> Dev Ophthalmol,</em> Vol. 52, Karger, Basel, https://doi.org/10.1159/000351045&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Beir, V. et al. (1999), &ldquo;The Mechanistic Basis of Radon-Induced Lung Cancer&rdquo;, in <em>Health Risks of Exposure to Radon: BEIR V</em>I, National Academy Press, Washington, D.C., https://doi.org/10.17226/5499&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Douglass, M. et al. (2013), &ldquo;Monte Carlo investigation of the increased radiation deposition due to gold nanoparticles using kilovoltage and megavoltage photons in a 3D randomized cell model&rdquo;<em>, Medical Physics</em>, Vol. 40/7, American Institute of Physics, College Park, https://doi.org/10.1118/1.4808150&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Douglass, M. et al. (2012), &ldquo;Development of a randomized 3D cell model for Monte Carlo microdosimetry simulations.&rdquo;, <em>Medical Physics</em>, Vol. 39/6, American Institute of Physics, College Park, https://doi.org/10.1118/1.4719963&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Hall, E. J. and Giaccia, A.J. (2018), <em>Radiobiology for the Radiologist</em>, 8th edition, Wolters Kluwer, Philadelphia. &nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kodaira, S. and Konishi, T. (2015), &ldquo;Co-visualization of DNA damage and ion traversals in live mammalian cells using a fluorescent nuclear track detector.&rdquo;, <em>Journal of Radiation Research</em>, Vol. 56/2, Oxford University Press, Oxford, https://doi.org/10.1093/jrr/rru091&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Lind, O.C.,&nbsp;D.H. Oughton and&nbsp;Salbu B. (2019), &quot;The NMBU FIGARO low dose irradiation facility&quot;,&nbsp;<em>International Journal of Radiation Biology</em>, Vol. 95/1, Taylor &amp; Francis, London,&nbsp;https://doi.org/10.1080/09553002.2018.1516906.</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sawakuchi, G.O. and Akselrod, M.S. (2016), &ldquo;Nanoscale measurements of proton tracks using fluorescent nuclear track detectors.&rdquo;,<em> Medical Physics</em>, Vol. 43/5, American Institute of Physics, College Park, https://doi.org/10.1118/1.4947128&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Straume, T. et al. (2015), &ldquo;Compact Tissue-equivalent Proportional Counter for Deep Space Human Missions.&rdquo;,<em> Health physics,</em> Vol. 109/4, Lippincott Williams &amp; Wilkins, Philadelphia, https://doi.org/10.1097/HP.0000000000000334&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Niklas, M. et al. (2013), &ldquo;Engineering cell-fluorescent ion track hybrid detectors.&rdquo;, <em>Radiation Oncology</em>, Vol. 8/104, BioMed Central, London, https://doi.org/10.1186/1748-717X-8-141&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">UNSCEAR (2020), <em>Sources, effects and risks of ionizing radiation</em>, United Nations.&nbsp;</span></span></p>
  • <p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Xie, Li. et al. (2022), &quot;Ultraviolet B Modulates Gamma Radiation-Induced Stress Responses in Lemna Minor at Multiple Levels of Biological Organisation&quot;, <em>SSRN</em>, Elsevier, Amsterdam,&nbsp;http://dx.doi.org/10.2139/ssrn.4081705&nbsp;.</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Zyla, P.A. et al. (2020)<em>, Review of particle physics: Progress of Theoretical and Experimental Physics,</em> 2020 Edition, Oxford University Press, Oxford.&nbsp;</span></span></p>
  • <p>&nbsp;</p>
  • <p>&nbsp;</p>
  • <h3>List of Key Events in the AOP</h3>
  • <h4><a href="/events/1635">Event: 1635: Increase, DNA strand breaks</a></h4>
  • <h5>Short Name: Increase, DNA strand breaks</h5>
  • <h4>AOPs Including This Key Event</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP ID and Name</th>
  • <th scope="col">Event Type</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/296">Aop:296 - Oxidative DNA damage leading to chromosomal aberrations and mutations</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/272">Aop:272 - Deposition of energy leading to lung cancer</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/322">Aop:322 - Alkylation of DNA leading to reduced sperm count</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/216">Aop:216 - Deposition of energy leading to population decline via DNA strand breaks and follicular atresia</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/238">Aop:238 - Deposition of energy leading to population decline via DNA strand breaks and oocyte apoptosis</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/478">Aop:478 - Deposition of energy leading to occurrence of cataracts</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/483">Aop:483 - Deposition of Energy Leading to Learning and Memory Impairment</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/470">Aop:470 - Deposition of energy leads to vascular remodeling</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Stressors</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Name</th></tr>
  • </thead>
  • <tbody>
  • <tr><td>Ionizing Radiation</td></tr>
  • <tr><td>Topoisomerase inhibitors</td></tr>
  • <tr><td>Radiomimetic compounds</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Biological Context</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Level of Biological Organization</th></tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr><td>Molecular</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Domain of Applicability</h4>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>human and other cells in culture</td>
  • <td>human and other cells in culture</td>
  • <td></td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>All life stages</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <p><span style="font-size:11px"><strong>Taxonomic applicability: </strong>DNA strand breaks are relevant to all species, including vertebrates such as humans, that contain DNA (Cannan &amp; Pederson, 2016). &nbsp;</span></p>
  • <p><span style="font-size:11px"><strong>Life stage applicability: </strong>This key event is not life stage specific as all life stages display strand breaks. However, there is an increase in baseline levels of DNA strand breaks seen in older individuals though it is unknown whether this change due to increased break induction or a greater retention of breaks due to poor repair (White &amp; Vijg, 2016).&nbsp;</span></p>
  • <p><span style="font-size:11px"><strong>Sex applicability:</strong> This key event is not sex specific as both sexes display evidence of strand breaks. In some cell types, such as peripheral blood mononuclear cells, males show higher levels of single strand breaks than females (Garm et al., 2012).&nbsp;</span></p>
  • <p><span style="font-size:11px"><strong>Evidence for perturbation by a stressor: </strong>There are studies demonstrating that increased DNA strand breaks can result from exposure to multiple stressor types including ionizing &amp; non-ionizing radiation, chemical agents, and oxidizing agents (EPRI, 2014; Hamada, 2014; Cencer et al., 2018; Cannan &amp; Pederson, 2016; Yang et al., 1998). &nbsp;</span></p>
  • <h4>Key Event Description</h4>
  • <p>DNA strand breaks can occur on a single strand (SSB) or both strands (double strand breaks; DSB). SSBs arise when the phosphate backbone connecting adjacent nucleotides in DNA is broken on one strand. DSBs are generated when both strands are simultaneously broken at sites that are sufficiently close to one another that base-pairing and chromatin structure are insufficient to keep the two DNA ends juxtaposed. As a consequence, the two DNA ends generated by a DSB can physically dissociate from one another, becoming difficult to repair and increasing the chance of inappropriate recombination with other sites in the genome (Jackson, 2002).&nbsp;SSB can turn into DSB if the replication fork stalls at the lesion leading to fork collapse.</p>
  • <p>DNA strand breaks are a type of damage resulting from the hydrolysis of phosphodiester groups in the backbone of DNA molecules (Gates, 2009) and can occur on a single strand (single strand breaks; SSBs) or both strands (double strand breaks; DSBs) (Cannan and Pederson, 2016; Tamanoi and Yoshikawa, 2022; Tripathy et al., 2021). SSBs arise when the phosphate backbone connecting adjacent nucleotides in DNA is broken on one strand. DSBs are generated when both strands are simultaneously broken at sites that are sufficiently close to one another that base-pairing and chromatin structure are insufficient to keep the two DNA ends juxtaposed. As a consequence, the two DNA ends generated by a DSB can physically dissociate from one another, becoming difficult to repair and increasing the chance of inappropriate recombination with other sites in the genome (Jackson, 2002).&nbsp;SSBs can also turn into DSBs if the replication fork stalls at the lesion leading to fork collapse. It is also worth noting that there are error-prone and error-free forms of DSB repair (Jackson, 2002), and that the SSB repair pathway are distinct form the DSB repair pathways.</p>
  • <p>Strand breaks are intermediates in various biological events, including DNA repair (e.g., excision repair), V(D)J recombination in developing lymphoid cells and chromatin remodeling in both somatic cells and germ cells. The spectrum of damage&nbsp; can be complex, particularily if the stressor is from large amounts of deposited energy which can result in complex lesions and clustered damage defined as two or more oxidized bases, abasic sites or starnd breaks on opposing DNA strands within a few helical turns. These lesions are more difficult to repair and have been studied in many types of models&nbsp; (Barbieri et al., 2019 and Asaithamby et al., 2011)<span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">. DSBs and complex&nbsp;lesions &nbsp;are of particular concern, as they are considered the most lethal and deleterious type of DNA lesion. If misrepaired or left unrepaired, DSBs may drive the cell towards genomic instability, apoptosis or tumorigenesis (Beir, 1999).</span></span></p>
  • <p>Strand breaks are intermediates in various biological events, including DNA repair (e.g., base excision repair), V(D)J recombination in developing lymphoid cells and chromatin remodeling in both somatic cells and germ cells. The spectrum of damagecan be intricate,&nbsp; resulting in complex lesions, leading to mutations, and clustered damage defined as two or more oxidized bases, abasic sites or strand breaks on opposing DNA strands within a few helical turns. These lesions are more difficult to repair and have been studied in many types of models&nbsp; (Barbieri et al., 2019 and Asaithamby et al., 2011)<span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">. </span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">DSBs and complex&nbsp;lesions &nbsp;are of particular concern, as they are considered the most lethal and deleterious type of DNA lesion. If misrepaired or left unrepaired, DSBs may drive the cell towards genomic instability, apoptosis or tumorigenesis (Beir, 1999).</span></span></p>
  • <p>&nbsp;</p>
  • <h4>How it is Measured or Detected</h4>
  • <p style="text-align:center"><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">Please refer to the table below for details regarding these and other methodologies for detecting DNA DSBs.</span></span></p>
  • <p style="text-align:center">&nbsp;</p>
  • <table cellspacing="0" class="Table" style="border-collapse:collapse; border:none; margin-left:15px">
  • <tbody>
  • <tr>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:1px double #2b2b2b; height:37px; vertical-align:top; width:85px">
  • <p style="text-align:center"><strong><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Assay Name</span></span></strong></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:1px double #2b2b2b; height:37px; vertical-align:top; width:89px">
  • <p style="text-align:center"><strong><span style="font-size:11px"><span style="font-family:Arial,sans-serif">References</span></span></strong></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:1px double #2b2b2b; height:37px; vertical-align:top; width:228px">
  • <p style="text-align:center"><strong><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Description</span></span></strong></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #7f7f7f; border-top:1px double #2b2b2b; height:37px; vertical-align:top; width:46px">
  • <p style="margin-left:1px; margin-right:1px; text-align:center"><strong><span style="font-size:11px"><span style="font-family:Arial,sans-serif">OECD&nbsp;</span></span></strong><strong><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Approved Assay</span></span></strong></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:61px; vertical-align:top; width:85px">
  • <p style="margin-left:10px; margin-right:9px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Comet Assay (Single Cell Gel Eletrophoresis - Alkaline)</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:61px; vertical-align:top; width:89px">
  • <p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Collins, 2004; Olive&nbsp;</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">and Banath, 2006; Platel et al., 2011; Nikolova et al., 2017</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:61px; vertical-align:top; width:228px">
  • <p style="margin-left:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">To detect SSBs or DSBs, single cells are encapsulated in agarose on a slide, lysed, and subjected to gel electrophoresis at an alkaline pH (pH &gt;13); DNA fragments are forced to move, forming a &quot;comet&quot;-like&nbsp;</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">appearance</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:61px; vertical-align:top; width:46px">
  • <p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">Yes (No. 489)</span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:61px; vertical-align:top; width:85px">
  • <p style="margin-left:11px; margin-right:10px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Comet Assay (Single Cell Gel Eltrophoresis - Neutral)</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:61px; vertical-align:top; width:89px">
  • <p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Collins, 2014; Olive&nbsp;</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">and Banath, 2006; Anderson and Laubenthal, 2013; Nikolova et al., 2017</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:61px; vertical-align:top; width:228px">
  • <p style="margin-left:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">To detect DSBs, single cells are encapsulated in agarose on a slide, lysed, and subjected to gel electrophoresis at a neutral pH; DNA fragments, which are not denatured at the neutral pH, are forced to move, forming a &quot;comet&quot;-</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">like appearance</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:61px; vertical-align:top; width:46px">
  • <p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:37px; vertical-align:top; width:85px">
  • <p style="margin-left:1px; margin-right:-1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif"><span style="font-family:&quot;MS UI Gothic&quot;,sans-serif">&gamma;</span>-H2AX Foci Quantification - Flow&nbsp;</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Cytometry</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:37px; vertical-align:top; width:89px">
  • <p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Rothkamm and Horn, 2009; Bryce et al.,&nbsp;</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">2016</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:37px; vertical-align:top; width:228px">
  • <p style="margin-left:26px; margin-right:-1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Measurement of <span style="font-family:&quot;MS UI Gothic&quot;,sans-serif">&gamma;</span>-H2AX immunostaining in cells by flow cytometry, normalized to total levels of H2AX</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:37px; vertical-align:top; width:46px">
  • <p style="text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">N/A</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:37px; vertical-align:top; width:85px">
  • <p style="margin-left:12px; margin-right:11px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif"><span style="font-family:&quot;MS UI Gothic&quot;,sans-serif">&gamma;</span>-H2AX Foci Quantification -&nbsp;</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Western Blot</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:37px; vertical-align:top; width:89px">
  • <p style="margin-left:9px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Burma et al., 2001; Revet et al., 2011</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:37px; vertical-align:top; width:228px">
  • <p style="margin-left:14px; margin-right:9px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Measurement of <span style="font-family:&quot;MS UI Gothic&quot;,sans-serif">&gamma;</span>-H2AX immunostaining in cells by Western blotting, normalized to total levels of H2AX</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:37px; vertical-align:top; width:46px">
  • <p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:49px; vertical-align:top; width:85px">
  • <p style="margin-left:12px; margin-right:11px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif"><span style="font-family:&quot;MS UI Gothic&quot;,sans-serif">&gamma;</span>-H2AX Foci Quantification - Microscopy</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; vertical-align:top; width:89px">
  • <p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Redon et al., 2010; Mah et al., 2010; Garcia-Canton et al.,&nbsp;</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">2013</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; vertical-align:top; width:228px">
  • <p style="text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Quantification of <span style="font-family:&quot;MS UI Gothic&quot;,sans-serif">&gamma;</span>-H2AX immunostaining by counting <span style="font-family:&quot;MS UI Gothic&quot;,sans-serif">&gamma;</span>- H2AX foci visualized with a microscope</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:49px; vertical-align:top; width:46px">
  • <p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:37px; vertical-align:top; width:85px">
  • <p style="margin-left:12px; margin-right:11px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif"><span style="font-family:&quot;MS UI Gothic&quot;,sans-serif">&gamma;</span>-H2AX Foci Detection&nbsp;-</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">&nbsp;</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">ELISA and flow cytometry</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:37px; vertical-align:top; width:89px">
  • <p style="text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Ji et al., 2017;&nbsp;</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Bryce et al., 2016</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:37px; vertical-align:top; width:228px">
  • <p style="margin-right:-1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Detection of <span style="font-family:&quot;MS UI Gothic&quot;,sans-serif">&gamma;</span>-H2AX in cells by ELISA, normalized to total levels of H2AX; &gamma;H2AX foci detection&nbsp;can be high-throughput and automated using flow cytometry-based immunodetection.</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:37px; vertical-align:top; width:46px">
  • <p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:61px; vertical-align:top; width:85px">
  • <p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">Pulsed Field Gel Electrophoresis (PFGE)</span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:61px; vertical-align:top; width:89px">
  • <p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Ager et al., 1990; Gardiner et al., 1985; Herschleb et al., 2007; Kawashima et&nbsp;</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">al., 2017</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:61px; vertical-align:top; width:228px">
  • <p style="margin-left:9px; margin-right:8px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">To detect DSBs, cells are embedded and lysed in agarose, and the released DNA undergoes gel electrophoresis in which the direction of the voltage is periodically alternated; Large DNA fragments are thus&nbsp;</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">able to be separated by size</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:61px; vertical-align:top; width:46px">
  • <p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:73px; vertical-align:top; width:85px">
  • <p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">The TUNEL&nbsp;</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">(Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling) Assay</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:73px; vertical-align:top; width:89px">
  • <p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">Loo, 2011</span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:73px; vertical-align:top; width:228px">
  • <p style="margin-left:5px; margin-right:4px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">To detect strand breaks, dUTPs added to the 3&rsquo;OH end of a strand break by the DNA polymerase terminal deoxynucleotidyl transferase (TdT) are tagged with a fluorescent dye or a reporter enzyme to allow visualization (We note that this method is typically used to measure apoptosis)</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #2b2b2b; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:73px; vertical-align:top; width:46px">
  • <p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:49px; vertical-align:top; width:85px">
  • <p style="margin-left:7px; margin-right:6px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif"><em>In Vitro&nbsp;</em>DNA Cleavage Assays using&nbsp;</span></span><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Topoisomerase</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; vertical-align:top; width:89px">
  • <p style="text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Nitiss, 2012</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; vertical-align:top; width:228px">
  • <p style="margin-left:15px; margin-right:15px; text-align:center"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">Cleavage of DNA can be achieved using purified topoisomerase; DNA strand breaks can then be separated and quantified using gel electrophoresis</span></span></p>
  • </td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:49px; vertical-align:top; width:46px">
  • <p style="text-align:center"><span style="font-family:Arial,sans-serif; font-size:11px">N/A</span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:85px"><strong><span style="font-size:11px">PCR assay&nbsp;</span></strong></td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:89px"><strong><span style="font-size:11px">Figueroa‑Gonz&aacute;lez &amp; P&eacute;rez‑Plasencia, 2017&nbsp;</span></strong></td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:228px"><strong><span style="font-size:11px">Assay of strand breaks through the observation of DNA amplification prevention. Breaks block Taq polymerase, reducing the number of DNA templates, preventing amplification&nbsp;</span></strong></td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:49px; text-align:center; vertical-align:top; width:46px"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">N/A</span></span></td>
  • </tr>
  • <tr>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:85px"><strong><span style="font-size:11px">Sucrose density gradient centrifuge&nbsp;</span></strong></td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:89px"><strong><span style="font-size:11px">Raschke et al. 2009&nbsp;</span></strong></td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:228px"><strong><span style="font-size:11px">Division of DNA pieces by density, increased fractionation leads to lower density pieces, with the use of a sucrose cushion&nbsp;</span></strong></td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:49px; text-align:center; vertical-align:top; width:46px"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">N/A</span></span></td>
  • </tr>
  • <tr>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:85px"><strong><span style="font-size:11px">Alkaline Elution Assay&nbsp;</span></strong></td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:89px"><strong><span style="font-size:11px">Kohn, 1991&nbsp;</span></strong></td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:228px"><strong><span style="font-size:11px">Cells lysed with detergent-solution, filtered through membrane to remove all but intact DNA&nbsp;</span></strong></td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:49px; text-align:center; vertical-align:top; width:46px"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">N/A</span></span></td>
  • </tr>
  • <tr>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:1px double #2b2b2b; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:85px"><strong><span style="font-size:11px">Unwinding Assay&nbsp;</span></strong></td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:89px"><strong><span style="font-size:11px">Nacci et al. 1992&nbsp;</span></strong></td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #2b2b2b; border-top:none; height:49px; text-align:center; vertical-align:top; width:228px"><strong><span style="font-size:11px">DNA is stored in alkaline solutions with DNA-specific dye and allowed to unwind following removal from tissue, increased strand damage associated with increased unwinding&nbsp;</span></strong></td>
  • <td style="background-color:whitesmoke; border-bottom:1px double #7f7f7f; border-left:none; border-right:1px double #7f7f7f; border-top:none; height:49px; text-align:center; vertical-align:top; width:46px"><span style="font-size:11px"><span style="font-family:Arial,sans-serif">N/A</span></span></td>
  • </tr>
  • </tbody>
  • </table>
  • <h4>References</h4>
  • <p>Ager, D. D. et al. (1990). &ldquo;Measurement of Radiation- Induced DNA Double-Strand Breaks by Pulsed-Field Gel Electrophoresis.&rdquo; Radiat Res. 122(2), 181-7.</p>
  • <p>Anderson, D. &amp; Laubenthal J. (2013), &ldquo;Analysis of DNA Damage via Single-Cell Electrophoresis. In: Makovets S, editor. DNA Electrophoresis. Totowa.&rdquo;, NJ: Humana Press. p 209-218.</p>
  • <p>Asaithamby, A., B. Hu and D.J. Chen. (2011) Unrepaired clustered DNA lesions induce chromosome breakage in human cells. Proc Natl Acad Sci U S A 108(20): 8293-8298 .</p>
  • <p>Barbieri, S., G. Babini, J. Morini et a l (2019). . Predicting DNA damage foci and their experimental readout with 2D microscopy: a unified approach applied to photon and neutron exposures. Scientific Reports 9(1): 14019</p>
  • <p>Beir, V. et al. (1999), &ldquo;The Mechanistic Basis of Radon-Induced Lung Cancer&rdquo;, in Health Risks of Exposure to Radon: BEIR VI, National Academy Press, Washington, D.C., https://doi.org/10.17226/5499&nbsp;</p>
  • <p>Bryce, S. et al. (2016), &ldquo;Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach.&rdquo;, Environ Mol Mutagen. 57:171-189. Doi: 10.1002/em.21996.</p>
  • <p>Burma, S. et al. (2001), &ldquo;ATM phosphorylates histone H2AX in response to DNA double-strand breaks.&rdquo;, J Biol Chem, 276(45): 42462-42467. doi:10.1074/jbc.C100466200</p>
  • <p>Cannan, W.J. and D.S. Pederson (2016), &quot;Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin.&quot;, Journal of Cellular Physiology, Vol.231/1, Wiley, New York, https://doi.org/10.1002/jcp.25048. &nbsp;</p>
  • <p>Cannan, W.J. and D.S. Pederson (2016), &quot;Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin.&quot;, Journal of Cellular Physiology, Vol.231/1, Wiley, New York, https://doi.org/10.1002/jcp.25048.&nbsp;&nbsp;&nbsp;</p>
  • <p>Cencer, C. et al. (2018), &ldquo;PARP-1/PAR Activity in Cultured Human Lens Epithelial Cells Exposed to Two Levels of UVB Light&rdquo;, Photochemistry and Photobiology, Vol.94/1, Wiley-Blackwell, Hoboken, https://doi.org/10.1111/php.12814. &nbsp;</p>
  • <p>Charlton, E. D. et al. (1989), &ldquo;Calculation of Initial Yields of Single and Double Stranded Breaks in Cell Nuclei from Electrons, Protons, and Alpha Particles.&rdquo;, &nbsp;Int. J. Radiat. Biol. 56(1): 1-19. doi: 10.1080/09553008914551141.</p>
  • <p>Collins, R. A. (2004), &ldquo;The Comet Assay for DNA Damage and Repair. Molecular Biotechnology.&rdquo;, Mol Biotechnol. 26(3): 249-61. doi:10.1385/MB:26:3:249</p>
  • <p>Durdik, M et al. (2015), &ldquo;Imaging flow cytometry as a sensitive tool to detect low-dose-induced DNA damage by analyzing 53BP1 and &gamma;H2AX foci in human lymphocytes.&rdquo; Cytometry. Part A. 87(12): 1070-8. Doi:10.1002/cyto.a.22731&nbsp;</p>
  • <p>EPRI (2014), Epidemiology and mechanistic effects of radiation on the lens of the eye: Review and scientific appraisal of the literature, EPRI, California.&nbsp;</p>
  • <p>Figueroa‑Gonz&aacute;lez, G. and C. P&eacute;rez‑Plasencia. (2017), &ldquo;Strategies for the evaluation of DNA damage and repair mechanisms in cancer&rdquo;, Oncology Letters, Vol.13/6, Spandidos Publications, Athens, https://doi.org/10.3892/ol.2017.6002.&nbsp;</p>
  • <p>Garcia-Canton, C. et al. (2013), &ldquo;Assessment of the in vitro p-H2AX assay by High Content Screening asa novel genotoxicity test.&rdquo;, Mutat Res. 757:158-166.&nbsp; Doi: &nbsp;10.1016/j.mrgentox.2013.08.002</p>
  • <p>Gardiner, K. et al. (1986), &ldquo;Fractionation of Large Mammalian DNA Restriction Fragments Using Vertical Pulsed-Field Gradient Gel Electrophoresis.&rdquo;, &nbsp;Somatic Cell and Molecular Genetics. 12(2): 185-95.Doi: 10.1007/bf01560665.</p>
  • <p>Garm, C. et al. (2012), &ldquo;Age and gender effects on DNA strand break repair in peripheral blood mononuclear cells&rdquo;, Aging Cell, Vol.12/1, Blackwell Publishing Ltd, Oxford, https://doi.org/10.1111/acel.12019.&nbsp;</p>
  • <p>Guo, X. et al. (2018), &ldquo;Acetylation of 53BP1 dictates the DNA double strand break repair pathway.&rdquo; Nucleic acids research. 46(2): 689-703. doi:10.1093/nar/gkx1208&nbsp;</p>
  • <p>Hamada, N. (2014), &ldquo;What are the intracellular targets and intratissue target cells for radiation effects?&rdquo;, Radiation research, Vol. 181/1, The Radiation Research Society, Indianapolis, https://doi.org/10.1667/RR13505.1.&nbsp;</p>
  • <p>Herschleb, J. et al. (2007), &ldquo;Pulsed-field gel electrophoresis.&rdquo;, &nbsp;Nat Protoc. 2(3): 677-684. doi:10.1038/nprot.2007.94</p>
  • <p>Iliakis, G. et al. (2015), &ldquo;Alternative End-Joining Repair Pathways Are the Ultimate Backup for Abrogated Classical Non-Homologous End-Joining and Homologous Recombination Repair: Implications for the Formation of Chromosome Translocations.&rdquo;, &nbsp;Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 2(3): 677-84. doi: 10.1038/nprot.2007.94</p>
  • <p>Jackson, S. (2002). &ldquo;Sensing and repairing DNA double-strand breaks.&rdquo;, &nbsp;Carcinogenesis. 23:687-696. Doi:10.1093/carcin/23.5.687.</p>
  • <p>Ji, J. et al. (2017), &ldquo;Phosphorylated fraction of H2AX as a measurement for DNA damage in cancer cells and potential applications of a novel assay.&rdquo;, &nbsp;PLoS One. 12(2): e0171582. doi:10.1371/journal.pone.0171582</p>
  • <p>Kawashima, Y.(2017), &ldquo;Detection of DNA double-strand breaks by pulsed-field gel electrophoresis.&rdquo;, &nbsp;Genes Cells 22:84-93. Doi: 10.1111/gtc.12457.</p>
  • <p>Khoury, L. et al. (2013), &ldquo;Validation of high-throughput genotoxicity assay screening using cH2AX in-cell Western assay on HepG2 cells.&rdquo;, Environ Mol Mutagen, 54:737-746. Doi: &nbsp;10.1002/em.21817.</p>
  • <p>Khoury, L. et al. (2016), &ldquo;Evaluation of four human cell lines with distinct biotransformation properties for genotoxic screening.&rdquo;, Mutagenesis, 31:83-96. Doi: <a href="https://doi.org/10.1093/mutage/gev058" target="_blank">10.1093/mutage/gev058</a>.</p>
  • <p>Kohn, K.W. (1991), &ldquo;Principles and practice of DNA filter elution&rdquo;, Pharmacology &amp; Therapeutics, Vol.49/1, Elsevier, Amsterdam, https://doi.org/10.1016/0163-7258(91)90022-E.&nbsp;</p>
  • <p>Loo, DT. (2011), &ldquo;In Situ Detection of Apoptosis by the TUNEL Assay: An Overview of Techniques. In: Didenko V, editor. DNA Damage Detection In Situ, Ex Vivo, and In Vivo. Totowa.&rdquo;, NJ: Humana Press. p 3-13.doi: <a href="https://doi.org/10.1007/978-1-60327-409-8_1" target="_blank">10.1007/978-1-60327-409-8_1</a>.</p>
  • <p>Mah, L. J. et al. (2010), &ldquo;Quantification of gammaH2AX foci in response to ionising radiation.&rdquo;, &nbsp;J Vis Exp(38). doi:10.3791/1957.</p>
  • <p>Nacci, D. et al. (1992), &ldquo;Application of the DNA alkaline unwinding assay to detect DNA strand breaks in marine bivalves&rdquo;, Marine Environmental Research, Vol.33/2, Elsevier BV, Amsterdam, https://doi.org/10.1016/0141-1136(92)90134-8.&nbsp;</p>
  • <p>Nikolova, T., F. et al. (2017), &ldquo;Genotoxicity testing: Comparison of the &gamma;H2AX focus assay with the alkaline and neutral comet assays.&rdquo;, &nbsp;Mutat Res&nbsp;822:10-18. Doi: <a href="https://doi.org/10.1016/j.mrgentox.2017.07.004" target="_blank">10.1016/j.mrgentox.2017.07.004</a>.</p>
  • <p>Nitiss, J. L. et al. (2012), &ldquo;Topoisomerase assays. &rdquo;, Curr Protoc Pharmacol. Chapter 3: Unit 3 3.</p>
  • <p>OECD. (2014). Test No. 489: &ldquo;In vivo mammalian alkaline comet assay.&rdquo; &nbsp;OECD Guideline for the Testing of Chemicals, Section 4 .</p>
  • <p>Olive, P. L., &amp; Ban&aacute;th, J. P. (2006), &ldquo;The comet assay: a method to measure DNA damage in individual cells.&rdquo;, &nbsp;Nature Protocols. 1(1): 23-29. doi:10.1038/nprot.2006.5.</p>
  • <p>Platel A. et al. (2011), &ldquo;Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the thymidine kinase gene-mutation assay and the&nbsp;<em>in vitro&nbsp;</em>modified comet assay: Determination of No-Observed-Genotoxic-Effect-Levels.&rdquo;, &nbsp;Mutat Res 726:151-159. Doi: 10.1016/j.mrgentox.2011.09.003.</p>
  • <p>Popp, H. D. et al. (2017), &ldquo;Immunofluorescence Microscopy of &gamma;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks&rdquo;, Journal of visualized experiments, 129: 56617, doi:10.3791/56617&nbsp;</p>
  • <p>Raschke, S., J. Guan and G. Iliakis. (2009), &ldquo;Application of alkaline sucrose gradient centrifugation in the analysis of DNA replication after DNA damage&rdquo;, Methods in Molecular Biology, Vol.521, Humana Press, Totowa, https://doi.org/10.1007/978-1-60327-815-7_18.&nbsp;</p>
  • <p>Redon, C. et al. (2010), &ldquo;The use of gamma-H2AX as a biodosimeter for total-body radiation exposure in non-human primates.&rdquo;, &nbsp;PLoS One. 5(11): e15544. doi:10.1371/journal.pone.0015544</p>
  • <p>Revet, I. et al. (2011), &ldquo;Functional relevance of the histone &gamma;H2Ax in the response to DNA damaging agents.&rdquo; Proc Natl Acad Sci USA.108:8663-8667. Doi: 10.1073/pnas.1105866108</p>
  • <p>Rogakou, E.P. et al. (1998), &ldquo;DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139.&rdquo; , J Biol Chem, 273:5858-5868. Doi: 10.1074/jbc.273.10.5858</p>
  • <p>Rothkamm, K. &amp; Horn, S. (2009), &ldquo;&gamma;-H2AX as protein biomarker for radiation exposure.&rdquo;, &nbsp;Ann Ist Super Sanit&agrave;, 45(3): 265-71.</p>
  • <p>Tamanoi, F., &amp; Yoshikawa, K. (2022), &ldquo;Overview of DNA damage and double-strand breaks&rdquo;,&nbsp; The Enzymes, Vol.51, 1&ndash;5. https://doi.org/10.1016/bs.enz.2022.08.001&nbsp;&nbsp;</p>
  • <p>Tripathy, B. K., Pal, K., Shabrish, S., &amp; Mittra, I. (2021), &ldquo;A New Perspective on the Origin of DNA Double-Strand Breaks and Its Implications for Ageing&rdquo; Genes, Vol.12/2, 163. <a href="https://doi.org/10.3390/genes12020163" rel="noreferrer noopener" target="_blank">https://doi.org/10.3390/genes12020163</a>&nbsp;&nbsp;</p>
  • <p>White, R.R. and J. Vijg. (2016), &ldquo;Do DNA Double-Strand Breaks Drive Aging?&rdquo;, Molecular Cell, Vol.63, Elsevier, Amsterdam, http://doi.org/10.1016/j.molcel.2016.08.004.&nbsp;</p>
  • <p>Yang, Y. et al. (1998), &ldquo;The effect of catalase amplification on immortal lens epithelial cell lines&rdquo;, Experimental Eye Research, Vol.67/6, Academic Press Inc, Cambridge, https://doi.org/10.1006/exer.1998.0560. &nbsp;</p>
  • <h4><a href="/events/155">Event: 155: Inadequate DNA repair</a></h4>
  • <h5>Short Name: Inadequate DNA repair</h5>
  • <h4>Key Event Component</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Process</th>
  • <th scope="col">Object</th>
  • <th scope="col">Action</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>DNA repair</td>
  • <td>deoxyribonucleic acid</td>
  • <td>functional change</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>AOPs Including This Key Event</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP ID and Name</th>
  • <th scope="col">Event Type</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/15">Aop:15 - Alkylation of DNA in male pre-meiotic germ cells leading to heritable mutations</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/141">Aop:141 - Alkylation of DNA leading to cancer 2</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/139">Aop:139 - Alkylation of DNA leading to cancer 1</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/296">Aop:296 - Oxidative DNA damage leading to chromosomal aberrations and mutations</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/272">Aop:272 - Deposition of energy leading to lung cancer</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/322">Aop:322 - Alkylation of DNA leading to reduced sperm count</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/397">Aop:397 - Bulky DNA adducts leading to mutations</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/432">Aop:432 - Deposition of Energy by Ionizing Radiation leading to Acute Myeloid Leukemia</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/443">Aop:443 - Alcohol Induced DNA damage and mutations leading to Metastatic Breast Cancer</a></td>
  • <td><a href="/aops/443">Aop:443 - DNA damage and mutations leading to Metastatic Breast Cancer</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/478">Aop:478 - Deposition of energy leading to occurrence of cataracts</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Stressors</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Name</th></tr>
  • </thead>
  • <tbody>
  • <tr><td>Ionizing Radiation</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Biological Context</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Level of Biological Organization</th></tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr><td>Cellular</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Domain of Applicability</h4>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>mouse</td>
  • <td>Mus musculus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>rat</td>
  • <td>Rattus norvegicus</td>
  • <td>Moderate</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Syrian golden hamster</td>
  • <td>Mesocricetus auratus</td>
  • <td>Moderate</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10036" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Homo sapiens</td>
  • <td>Homo sapiens</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>cow</td>
  • <td>Bos taurus</td>
  • <td>Low</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9913" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>All life stages</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <p>The retention of adducts has been directly measured in many different types of eukaryotic somatic cells (in vitro and in vivo). In male germ cells, work has been done on hamsters, rats and mice. The accumulation of mutation and changes in mutation spectrum has been measured in mice and human cells in culture. Theoretically, saturation of DNA repair occurs in every species (prokaryotic and eukaryotic). The principles of this work were established in prokaryotic models. Nagel et al. (2014) have produced an assay that directly measures DNA repair in human cells in culture.</p>
  • <p>NHEJ is primarily used by vertebrate multicellular eukaryotes, but it also been observed in plants. Furthermore, it has recently been discovered that some bacteria (Matthews et al., 2014) and yeast (Emerson et al., 2016) also use NHEJ. In terms of invertebrates, most lack the core DNA-PK<sub>cs</sub> and Artemis proteins; they accomplish end joining by using the RA50:MRE11:NBS1 complex (Chen et al., 2001).&nbsp; HR occurs naturally in eukaryotes, bacteria, and some viruses (Bhatti et al., 2016).</p>
  • <p><strong>Taxonomic applicability:</strong> Inadequate DNA repair is applicable to all species, as they all contain DNA (White &amp; Vijg, 2016). &nbsp;</p>
  • <p><strong>Life stage applicability:</strong> This key event is not life stage specific as any life stage can have poor repair, though as individuals age their repair process become less effective (Gorbunova &amp; Seluanov, 2016).&nbsp;</p>
  • <p><strong>Sex applicability: </strong>There is no evidence of sex-specificity for this key event, with initial rate of DNA repair not significantly different between sexes (Trzeciak et al., 2008).&nbsp;</p>
  • <p><strong>Evidence for perturbation by a stressor: </strong>Multiple studies demonstrate that inadequate DNA repair can occur as a result of stressors such as ionizing and non-ionizing radiation, as well as chemical agents (Kuhne et al., 2005; Rydberg et al., 2005; Dahle et al., 2008; Seager et al., 2012; Wilhelm, 2014; O&rsquo;Brien et al., 2015). &nbsp;</p>
  • <h4>Key Event Description</h4>
  • <p>DNA lesions may result from the formation of DNA adducts (i.e., covalent modification of DNA by chemicals), or by the action of agents such as radiation that may produce strand breaks or modified nucleotides within the DNA molecule. These DNA lesions are repaired through several mechanistically distinct pathways that can be categorized as follows:</p>
  • <ol>
  • <li><strong>Damage reversal</strong> acts to reverse the damage without breaking any bonds within the sugar phosphate backbone of the DNA. The most prominent enzymes associated with damage reversal are photolyases (Sancar, 2003) that can repair UV dimers in some organisms, and O6-alkylguanine-DNA alkyltransferase (AGT) (Pegg 2011) and oxidative demethylases (Sundheim et al., 2008), which can repair some types of alkylated bases.</li>
  • <li><strong>Excision repair</strong> involves the removal of a damaged nucleotide(s) through cleavage of the sugar phosphate backbone followed by re-synthesis of DNA within the resultant gap. Excision repair of DNA lesions can be mechanistically divided into:&nbsp;
  • <p style="margin-left:40px"><strong>a) Base excision repair (BER)</strong><span style="font-size:1rem"> (Dianov and H&uuml;bscher, 2013), in which the damaged base is removed by a damage-specific glycosylase prior to incision of the phosphodiester backbone at the resulting abasic site.</span></p>
  • <p style="margin-left:40px"><strong>a) Base excision repair (BER)</strong><span style="font-size:1rem"> (Dianov and H&uuml;bscher, 2013), in which the damaged base is removed by a damage-specific glycosylase prior to incision of the phosphodiester backbone at the resulting abasic site.&nbsp;</span>This leads to an intermediate that contains a DNA strand break, whereby DNA ligase is then recruited to seal the ends of the DNA.</p>
  • <p style="margin-left:40px"><strong>b) Nucleotide excision repair (NER)</strong> (Sch&auml;rer, 2013), in which the DNA strand containing the damaged nucleotide is incised at sites several nucleotides 5&rsquo; and 3&rsquo; to the site of damage, and a polynucleotide containing the damaged nucleotide is removed prior to DNA resynthesis within the resultant gap.&nbsp;</p>
  • <p style="margin-left:40px"><strong>b) Nucleotide excision repair (NER)</strong> (Sch&auml;rer, 2013), in which the DNA strand containing the damaged nucleotide is incised at sites several nucleotides 5&rsquo; and 3&rsquo; to the site of damage, and a polynucleotide containing the damaged nucleotide is removed prior to DNA resynthesis within the resultant gap&nbsp;and sealing of the ends by DNA ligase.&nbsp;&nbsp;</p>
  • <p style="margin-left:40px"><strong>c) Mismatch repair (MMR)</strong> (Li et al., 2016)&nbsp;&nbsp;which does not act on DNA lesions but does recognize mispaired bases resulting from replication errors. In MMR the strand containing the misincorporated base is removed prior to DNA resynthesis.</p>
  • <p style="margin-left:40px">The major pathway that removes oxidative DNA damage is base excision repair (BER), which can be either monofunctional or bifunctional; in mammals, a specific DNA glycosylase (OGG1: 8-Oxoguanine glycosylase) is responsible for excision of 8-oxoguanine (8-oxoG) and other oxidative lesions (Hu et al., 2005; Scott et al., 2014; Whitaker et al., 2017). We note that long-patch BER is used for the repair of clustered oxidative lesions, which uses several enzymes from DNA replication pathways (Klungland and Lindahl, 1997). These pathways are described in detail in various reviews e.g., (Whitaker et al., 2017).&nbsp;</p>
  • </li>
  • <li><strong>Single strand break repair (SSBR)&nbsp;</strong>involves different proteins and enzymes depending on the origin of the SSB (e.g., produced as an intermediate in excision repair or due to direct chemical insult) but the same general steps of repair&nbsp;are taken for all SSBs: detection, DNA end processing, synthesis, and ligation&nbsp;(Caldecott, 2014). Poly-ADP-ribose polymerase1 (PARP1)&nbsp;detects and binds&nbsp;unscheduled SSBs (i.e., not deliberately induced during excision repair) and synthesizes&nbsp;PAR as a signal to the downstream factors in repair.&nbsp;PARP1 is not required to initiate SSBR of BER intermediates. The XRCC1 protein complex is then recruited to the site of damage and&nbsp;acts as a scaffold for proteins and enzymes&nbsp;required for repair.&nbsp;Depending on the nature of the damaged termini of the DNA strand, different enzymes are required for end processing to generate the substrates that&nbsp;DNA polymerase &beta; (Pol&beta;;&nbsp;short patch repair) or Pol&nbsp;&delta;/&epsilon; (long patch repair)&nbsp;can bind to synthesize&nbsp;over the gap. Synthesis&nbsp;in long-patch repair&nbsp;displaces a single stranded flap which is excised by flap endonuclease 1 (FEN1). In short-patch repair, the XRCC1/Lig3&alpha; complex joins the two ends after synthesis. In&nbsp;long-patch repair, the PCNA/Lig1 complex ligates the ends. (Caldecott, 2014).&nbsp;</li>
  • <li><strong>Double strand break repair (DSBR)</strong> is necessary to preserve genomic integrity when breaks occur in both strands of a DNA molecule. There are two major pathways for DSBR: homologous recombination (HR), which operates primarily during S phase in dividing cells, and nonhomologous end joining (NHEJ), which can function in both dividing and non-dividing cells (Teruaki Iyama and David M. Wilson III, 2013).&nbsp;</li>
  • <li><strong>Single strand break repair (SSBR)&nbsp;</strong>involves different proteins and enzymes depending on the origin of the SSB (e.g., produced as an intermediate in excision repair or due to direct chemical insult) but the same general steps of repair&nbsp;are taken for all SSBs: detection, DNA end processing, synthesis, and ligation&nbsp;(Caldecott, 2014). Poly-ADP-ribose polymerase1 (PARP1)&nbsp;detects and binds&nbsp;unscheduled SSBs (i.e., not deliberately induced during excision repair) and synthesizes&nbsp;PAR as a signal to the downstream factors in repair.&nbsp;PARP1 is not required to initiate SSBR of BER intermediates. The XRCC1 protein complex is then recruited to the site of damage&nbsp;where a common DNA intermediate as BER was generated, and&nbsp;acts as a scaffold for proteins and enzymes&nbsp;required for repair.&nbsp;Depending on the nature of the damaged termini of the DNA strand, different enzymes are required for end processing to generate the substrates that&nbsp;DNA polymerase &beta; (Pol&beta;;&nbsp;short patch repair) or Pol&nbsp;&delta;/&epsilon; (long patch repair)&nbsp;can bind to synthesize&nbsp;over the gap, although end processing is generally done by polynucleotide kinase. Synthesis&nbsp;in long-patch repair&nbsp;displaces a single stranded flap which is excised by flap endonuclease 1 (FEN1). In short-patch repair, the XRCC1/Lig3&alpha; complex joins the two ends after synthesis. In&nbsp;long-patch repair, the PCNA/Lig1 complex ligates the ends. (Caldecott, 2014).&nbsp;</li>
  • <li><strong>Double strand break repair (DSBR)</strong> is necessary to preserve genomic integrity when breaks occur in both strands of a DNA molecule. There are two major pathways for DSBR: homologous recombination (HR), which operates primarily during the S phase of&nbsp;dividing cell types, and nonhomologous end joining (NHEJ), which can function in both dividing and non-dividing cell types. No repair occurs in the M phase&nbsp;(Teruaki Iyama and David M. Wilson III, 2013).&nbsp;DNA repair in mitosis is controversial (Mladenov et al., 2023).</li>
  • </ol>
  • <p style="margin-left:40px">In higher eukaryotes such as mammals, NHEJ is usually the preferred pathway for DNA DSBR. Its use, however, is dependent on the cell type, the gene locus, and the nuclease platform (Miyaoka et al., 2016). The use of NHEJ is also dependent on the cell cycle; NHEJ is generally not the pathway of choice when the cell is in the late S or G2 phase of the cell cycle, or in mitotic cells when the sister chromatid is directly adjacent to the double-strand break (DSB) (Lieber et al., 2003). In these cases, the HR pathway is commonly used for repair of DSBs. Despite this, NHEJ is still used more commonly than HR in human cells. Classical NHEJ (C-NHEJ) is the most common NHEJ repair mechanism, but alternative NHEJ (alt-NHEJ) can also occur, especially in the absence of C-NHEJ and HR.</p>
  • <p style="margin-left:40px">Complex lesions can be created by a single mutagen and can be more difficult to repair, as the mechanism behind how different repair pathways cooperate to address this is still unclear (Aleksandrov et al., 2018). In higher eukaryotes such as mammals, NHEJ is usually the preferred pathway for DNA DSBR. Its use, however, is dependent on the cell type, the gene locus, and the nuclease platform (Miyaoka et al., 2016). The use of NHEJ is also dependent on the cell cycle; NHEJ is generally not the pathway of choice when the cell is in the late S or G2 phase of the cell cycle, or in mitotic cells when the sister chromatid is directly adjacent to the double-strand break (DSB) (Lieber et al., 2003). In these cases, the HR pathway is commonly used for repair of DSBs. Despite this, NHEJ is still used more commonly than HR in human cells. Classical NHEJ (C-NHEJ) is the most common NHEJ repair mechanism, but alternative NHEJ (alt-NHEJ) can also occur, especially in the absence of C-NHEJ and HR.</p>
  • <p style="margin-left:40px">The process of C-NHEJ in humans requires at least seven core proteins: Ku70, Ku86, DNA-dependent protein kinase complex (DNA-PK<sub>cs&nbsp;</sub>), Artemis, X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and DNA ligase IV (Boboila et al., 2012). When DSBs occur, the Ku proteins, which have a high affinity for DNA ends, will bind to the break site and form a heterodimer. This protects the DNA from exonucleolytic attack and acts to recruit DNA-PK<sub>cs</sub>, thus forming a trimeric complex on the ends of the DNA strands. The kinase activity of DNA-PK<sub>cs&nbsp;</sub>is then triggered, causing DNA-PK<sub>cs&nbsp;</sub>to auto-phosphorylate and thereby lose its kinase activity; the now phosphorylated DNA-PK<sub>cs</sub>&nbsp;dissociates from the DNA-bound Ku proteins. The free DNA-PK<sub>cs</sub>&nbsp;phosphorylates Artemis, an enzyme that possesses 5&rsquo;-3&rsquo; exonuclease and endonuclease activity in the presence of DNA-PK<sub>cs</sub>&nbsp;and ATP. Artemis is responsible for &lsquo;cleaning up&rsquo; the ends of the DNA. For 5&rsquo; overhangs, Artemis nicks the overhang, generally leaving a blunt duplex end. For 3&rsquo; overhangs, Artemis will often leave a four- or five-nucleotide single stranded overhang (Pardo et al., 2009; Fattah et al., 2010; Lieber et al., 2010). Next, the XLF and XRCC4 proteins form a complex which makes a channel to bind DNA and aligns the ends for efficient ligation via DNA ligase IV (Hammel et al., 2011).</p>
  • <p style="margin-left:40px">The process of C-NHEJ in humans requires at least seven core proteins: Ku70, Ku86, DNA-dependent protein kinase complex (DNA-PK<sub>cs&nbsp;</sub>), Artemis, X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and DNA ligase IV (Boboila et al., 2012). When DSBs occur, the Ku proteins, which have a high affinity for DNA ends, will bind to the break site and form a heterodimer. This protects the DNA from exonucleolytic attack and acts to recruit DNA-PK<sub>cs</sub>, the catalytic subunit,&nbsp;thus forming a trimeric complex on the ends of the DNA strands. Alternative NHEJ, or alt NHEJ, uses small similar sequences in two broken DNA ends to join them together. Unlike the usual repair method (cNHEJ), aNHEJ doesn&#39;t need specific proteins like LIG4 and KU. Instead, it relies on the MRN complex to process the breaks. However, alt NHEJ tends to cause mutations by adding or removing bits of DNA during the repair (Chaudhuri and Nussenzweig, 2017). The kinase activity of DNA-PK<sub>cs&nbsp;</sub>is then triggered, causing DNA-PK<sub>cs&nbsp;</sub>to auto-phosphorylate and thereby lose its kinase activity; the now phosphorylated DNA-PK<sub>cs</sub>&nbsp;dissociates from the DNA-bound Ku proteins. The free DNA-PK<sub>cs</sub>&nbsp;phosphorylates Artemis, an enzyme that possesses 5&rsquo;-3&rsquo; exonuclease and endonuclease activity in the presence of DNA-PK<sub>cs</sub>&nbsp;and ATP. Artemis is responsible for &lsquo;cleaning up&rsquo; the ends of the DNA. For 5&rsquo; overhangs, Artemis nicks the overhang, generally leaving a blunt duplex end. For 3&rsquo; overhangs, Artemis will often leave a four- or five-nucleotide single stranded overhang (Pardo et al., 2009; Fattah et al., 2010; Lieber et al., 2010). Next, the XLF and XRCC4 proteins form a complex which makes a channel to bind DNA and aligns the ends for efficient ligation via DNA ligase IV (Hammel et al., 2011).</p>
  • <p style="margin-left:40px">The process of alt-NHEJ is less well understood than C-NHEJ. &nbsp;Alt-NHEJ is known to involve slightly different core proteins than C-NHEJ, but the steps of the pathway are essentially the same between the two processes (reviewed in Chiruvella et al., 2013). It is established, however, that alt-NHEJ is more error-prone in nature than C-NHEJ, which contributes to incorrect DNA repair. Alt-NHEJ is thus considered primarily to be a backup repair mechanism (reviewed in Chiruvella et al., 2013).&nbsp;</p>
  • <p style="margin-left:40px">The process of alt-NHEJ is less well understood than C-NHEJ and is a lower fidelity mechanism. &nbsp;Alt-NHEJ is known to involve slightly different core proteins than C-NHEJ and required microhomology repeats, but the steps of the pathway are essentially the same between the two processes (reviewed in Chiruvella et al., 2013). It is established, however, that alt-NHEJ is more error-prone in nature than C-NHEJ, which contributes to incorrect DNA repair. Alt-NHEJ is thus considered primarily to be a backup repair mechanism (reviewed in Chiruvella et al., 2013).&nbsp;</p>
  • <p style="margin-left:40px">In contrast to NHEJ, HR takes advantage of similar or identical DNA sequences to repair DSBs (Sung and Klein, 2006). The initiating step of HR is the creation of a 3&rsquo; single strand DNA (ss-DNA) overhang. Combinases such as RecA and Rad51 then bind to the ss-DNA overhang, and other accessory factors, including Rad54, help recognize and invade the homologous region on another DNA strand. From there, DNA polymerases are able to elongate the 3&rsquo; invading single strand and resynthesize the broken DNA strand using the corresponding sequence on the homologous strand.</p>
  • <p style="margin-left:40px">In contrast to NHEJ, HR takes advantage of similar or identical DNA sequences to repair DSBs and is not error-prone (Sung and Klein, 2006). The initiating step of HR is the creation of a 3&rsquo; single strand DNA (ss-DNA) overhang. Combinases such as RecA and Rad51 then bind to the ss-DNA overhang, and other accessory factors, including Rad54, help recognize and invade the homologous region on another DNA strand. From there, DNA polymerases are able to elongate the 3&rsquo; invading single strand and resynthesize the broken DNA strand using the corresponding sequence on the homologous strand.</p>
  • <p>&nbsp;</p>
  • <p><strong><u>Fidelity of DNA Repair</u></strong></p>
  • <p><br />
  • Most DNA repair pathways are extremely efficient. However, in principal, all DNA repair pathways can be overwhelmed when the DNA lesion burden exceeds the capacity of a given DNA repair pathway to recognize and remove the lesion. Exceeded repair capacity may lead to toxicity or mutagenesis following DNA damage. Apart from extremely high DNA lesion burden,&nbsp;inadequate repair&nbsp;may arise through several different specific mechanisms. For example, during repair of DNA containing O6-alkylguanine adducts, AGT irreversibly binds a single O6-alkylguanine lesion and as a result is inactivated (this is termed suicide inactivation, as its own action causes it to become inactivated). Thus, the capacity of AGT to carry out alkylation repair can become rapidly saturated when the DNA repair rate exceeds the de novo synthesis of AGT (Pegg, 2011).</p>
  • <p>A second mechanism relates to cell specific differences in the cellular levels or activity of some DNA repair proteins. For example, XPA is an essential component of the NER complex. The level of XPA that is active in NER is low in the testes, which may reduce the efficiency of NER in testes as compared to other tissues (K&ouml;berle et al., 1999). Likewise, both NER and BER have been reported to be deficient in cells lacking functional p53 (Adimoolam and Ford, 2003; Hanawalt et al., 2003; Seo and Jung, 2004). A third mechanism relates to the importance of the DNA sequence context of a lesion in its recognition by DNA repair enzymes. For example, 8-oxoguanine (8-oxoG) is repaired primarily by BER; the lesion is initially acted upon by a bifunctional glycosylase, OGG1, which carries out the initial damage recognition and excision steps of 8-oxoG repair. However, the rate of excision of 8-oxoG is modulated strongly by both chromatin components (Menoni et al., 2012) and DNA sequence context (Allgayer et al., 2013) leading to significant differences in the repair of lesions situated in different chromosomal locations.</p>
  • <p>DNA repair is also remarkably error-free. However, misrepair can arise during repair under some circumstances. DSBR is notably error prone, particularly when breaks are processed through NHEJ, during which partial loss of genome information is common at the site of the double strand break (Iyama and Wilson, 2013).&nbsp;This is because NHEJ rejoins broken DNA ends without the use of extensive homology; instead, it uses the microhomology present between the two ends of the DNA strand break to ligate the strand back into one. When the overhangs are not compatible, however, indels (insertion or deletion events),&nbsp;duplications, translocations, and inversions in the DNA can occur. These changes in the DNA may lead to significant issues within the cell, including alterations in the gene determinants for cellular fatality (Moore et al., 1996).</p>
  • <p>Activation of mutagenic DNA repair pathways to withstand cellular or replication stress either from endogenous or exogenous sources can promote cellular viability, albeit at a cost of increased genome instability and mutagenesis (Fitzgerald et al., 2017). These salvage DNA repair pathways including, Break-induced Replication (BIR) and Microhomology-mediated Break-induced Replication (MMBIR). BIR repairs one-ended DSBs and has been extensively studied in yeast as well as in mammalian systems. BIR and MMBIR are linked with heightened levels of mutagenesis, chromosomal rearrangements and ensuing genome instability (Deem et al., 2011; Sakofsky et al., 2015; Saini et al., 2017; Kramara et al., 2018). In mammalian genomes BIR-like synthesis has been proposed to be involved in late stage Mitotic DNA Synthesis (MiDAS) that predominantly occurs at so-called Common Fragile Sites (CFSs) and maintains telomere length under s conditions of replication stress that serve to promote cell viability (Minocherhomji et al., 2015; Bhowmick et al., 2016; Dilley et al., 2016).&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</p>
  • <p>Misrepair may also occur through other repair pathways.&nbsp;Excision repair pathways require the resynthesis of DNA and rare DNA polymerase errors during gap resynthesis will result in mutations (Brown et al., 2011). Errors may also arise during gap resynthesis when the strand that is being used as a template for DNA synthesis contains DNA lesions (Kozmin and Jinks-Robertson, 2013). In addition, it has been shown that sequences that contain tandemly repeated sequences, such as CAG triplet repeats, are subject to expansion during gap resynthesis that occurs during BER of 8-oxoG damage (Liu et al., 2009).</p>
  • <h4>How it is Measured or Detected</h4>
  • <p>There is no test guideline for this event. The event is usually inferred from measuring the retention of DNA adducts or the creation of mutations as a measure of lack of repair or incorrect repair. These &lsquo;indirect&rsquo; measures of its occurrence are crucial to determining the mechanisms of genotoxic chemicals and for regulatory applications (i.e., determining the best approach for deriving a point of departure). More recently, a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) has been developed to directly measure&nbsp;the ability of human cells to repair plasmid reporters (Nagel et al., 2014).</p>
  • <p><u><strong>Indirect Measurement</strong></u></p>
  • <p>In somatic and spermatogenic cells, measurement of DNA repair is usually inferred by measuring DNA adduct formation/removal. Insufficient repair is inferred from the retention of adducts and from increasing adduct formation with dose. Insufficient DNA repair is also measured by the formation of increased numbers of mutations and alterations in mutation spectrum. The methods will be specific to the type of DNA adduct that is under study.</p>
  • <p>Some EXAMPLES are given below for alkylated DNA.</p>
  • <p>DOSE-RESPONSE CURVE FOR ALKYL ADDUCTS/MUTATIONS: It is important to consider that some adducts are not mutagenic at all because they are very effectively repaired. Others are effectively repaired, but if these repair processes become overwhelmed mutations begin to occur. The relationship between exposure to mutagenic agents and the presence of adducts (determined as adducts per nucleotide) provide an indication of whether the removal of adducts occurs, and whether it is more efficient at low doses. A sub-linear DNA adduct curve suggests that less effective repair occurs at higher doses (i.e., repair processes are becoming saturated). A sub-linear shape for the dose-response curves for mutation induction is also suggestive of repair of adducts at low doses, followed by saturation of repair at higher doses. Measurement of a clear point of inflection in the dose-response curve for mutations suggests that repair does occur, at least to some extent, but reduced repair efficiency arises above the breakpoint. A lack of increase in mutation frequencies (i.e., flat line for dose-response) for a compound showing a dose-dependent increase in adducts would imply that the adducts formed are either not mutagenic or are effectively repaired.</p>
  • <p>DOSE-RESPONSE CURVE FOR ALKYL ADDUCTS/MUTATIONS: It is important to consider that some adducts are not mutagenic at all because they are very effectively repaired. Others are effectively repaired, but if these repair processes become overwhelmed mutations begin to occur. The relationship (shape of dose-response curve) between exposure to mutagenic agents and mutations&nbsp;provide an indication of whether the removal of adducts occurs, and whether it is more efficient at low doses. Sub-linear dose-response curves (hockey stick or j-shape curves) for mutation induction indicates that adducts are not converted to mutations at low doses. This suggests the effective repair of adducts at low doses, followed by saturation of repair at higher doses (Clewell et al., 2019). Thus, measurement of a clear point of inflection in the dose-response curve for mutations suggests that repair does occur, at least to some extent, at low dosees but that reduced repair efficiency arises above the inflection point. A lack of increase in mutation frequencies (i.e., flat line for dose-response) for a compound showing a dose-dependent increase in adducts would imply that the adducts formed are either not mutagenic or are effectively repaired.</p>
  • <p>RETENTION OF ALKYL ADDUCTS: Alkylated DNA can be found in cells long after exposure has occurred. This indicates that repair has not effectively removed the adducts. For example, DNA adducts have been measured in hamster and rat spermatogonia several days following exposure to alkylating agents, indicating lack of repair (Seiler et al., 1997; Scherer et al., 1987).</p>
  • <p>MUTATION SPECTRUM: Shifts in mutation spectrum (i.e., the specific changes in the DNA sequence) following a chemical exposure (relative to non-exposed mutation spectrum) indicates that repair was not operating effectively to remove specific types of lesions. The shift in mutation spectrum is indicative of the types of DNA lesions (target nucleotides and DNA sequence context) that were not repaired. For example, if a greater proportion of mutations occur at guanine nucleotides in exposed cells, it can be assumed that the chemical causes DNA adducts on guanine that are not effectively repaired.</p>
  • <p><br />
  • <u><strong>Direct Measurement</strong></u></p>
  • <p>Nagel et al. (2014) we developed a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) to measures the ability of human cells to repair plasmid reporters. These reporters contain different types and amounts of DNA damage and can be used to measure repair through by NER, MMR, BER, NHEJ, HR and MGMT.</p>
  • <p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">Please refer to the table below for additional details and methodologies for detecting DNA damage and repair.</span></span></p>
  • <table border="1" cellpadding="1" cellspacing="1" style="height:2082px; width:629px">
  • <tbody>
  • <tr>
  • <td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong>Assay Name</strong></span></td>
  • <td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong>References</strong></span></td>
  • <td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong>Description</strong></span></td>
  • <td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong>DNA Damage/Repair Being Measured</strong></span></td>
  • <td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong>OECD Approved Assay</strong></span></td>
  • </tr>
  • <tr>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Dose-Response Curve for Alkyl Adducts/ Mutations</span></span></td>
  • <td>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Lutz 1991</span></span></p>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">&nbsp;</span></span></p>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Clewell 2016</span></span></p>
  • </td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Creation of a curve plotting the stressor dose and the abundance of adducts/mutations; Characteristics of the resulting curve can provide information on the efficiency of DNA repair</span></span></td>
  • <td>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Alkylation,</span></span></p>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">oxidative damage, or DSBs</span></span></p>
  • </td>
  • <td style="text-align:center"><span style="font-size:14px">N/A</span></td>
  • </tr>
  • <tr>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Retention of Alkyl Adducts</span></span></td>
  • <td>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Seiler 1997</span></span></p>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">&nbsp;</span></span></p>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Scherer 1987</span></span></p>
  • </td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Examination of DNA for alkylation after exposure to an alkylating agent; Presence of alkylation suggests a lack of repair</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Alkylation</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px">N/A</span></td>
  • </tr>
  • <tr>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Mutation Spectrum</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Wyrick 2015</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Shifts in the mutation spectrum after exposure to a chemical/mutagen relative to an unexposed subject can provide an indication of DNA repair efficiency, and can inform as to the type of DNA lesions present</span></span></td>
  • <td>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Alkylation,</span></span></p>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">oxidative damage, or DSBs</span></span></p>
  • </td>
  • <td style="text-align:center"><span style="font-size:14px">N/A</span></td>
  • </tr>
  • <tr>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">DSB Repair Assay (Reporter constructs)</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:times new roman,serif"><span style="font-family:arial,sans-serif">Mao</span></span><span style="font-family:arial,sans-serif"> et al., 2011</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px">Transfection of a GFP reporter construct (and DsRed control) where the GFP signal is only detected if the DSB is repaired; GFP signal&nbsp; is quantified using fluorescence microscopy or flow cytometry</span></td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">DSBs</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px">N/A</span></td>
  • </tr>
  • <tr>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Primary Rat Hepatocyte DNA Repair Assay</span></span></td>
  • <td>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Jeffrey and Williams, 2000</span></span></p>
  • <p style="text-align:center"><span style="font-size:14px"><u><span style="font-family:arial,sans-serif">&nbsp;</span></u></span></p>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Butterworth et al., 1987</span></span></p>
  • </td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Rat primary hepatocytes are cultured with a <sup>3</sup>H-thymidine solution in order to measure DNA synthesis in response to a stressor in non-replicating cells; Autoradiography is used to measure the amount of <sup>3</sup>H incorporated in the DNA post-repair</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Unscheduled DNA synthesis in response to DNA damage</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px">N/A</span></td>
  • </tr>
  • <tr>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Repair synthesis measurement by </span><sup><span style="font-family:arial,sans-serif">3</span></sup><span style="font-family:arial,sans-serif">H-thymine incorporation</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Iyama and Wilson, 2013</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Measure DNA synthesis in non-dividing cells as indication of gap filling during excision repair</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Excision repair</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px">N/A</span></td>
  • </tr>
  • <tr>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Comet Assay with Time-Course</span></span></td>
  • <td>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Olive et al., 1990</span></span></p>
  • <p style="text-align:center"><span style="font-size:14px"><u><span style="font-family:arial,sans-serif">&nbsp;</span></u></span></p>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Trucco et al., 1998</span></span></p>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">-</span></span></p>
  • <p style="text-align:center">Dunkenberger et al., 2022&nbsp;</p>
  • </td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Comet assay is performed with a time-course; Quantity of DNA in the tail should decrease as DNA repair progresses</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Comet assay is performed with a time-course </span></span>under alkaline conditions to detect SSBs and DSBs.<span style="font-size:14px"><span style="font-family:arial,sans-serif">&nbsp;Quantity of DNA in the tail should decrease as DNA repair progresses</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">DSBs</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">&nbsp;</span><span style="font-family:times new roman,serif"><a href="https://read.oecd-ilibrary.org/environment/test-no-489-in-vivo-mammalian-alkaline-comet-assay_9789264264885-en"><span style="font-family:arial,sans-serif">Yes</span></a></span><u><span style="font-family:arial,sans-serif"> (No. 489)</span></u></span></td>
  • </tr>
  • <tr>
  • <td style="text-align:center">Flow Cytometry&nbsp;&nbsp;&nbsp;</td>
  • <td>Corneo et al., 2007&nbsp;&nbsp;&nbsp;</td>
  • <td style="text-align:center">The alt-NHEJ flow cytometer method involves utilizing an extrachromosomal substrate. Green fluorescent protein (GFP) expression is indicative of successful alt-NHEJ activity, contingent on the removal of 10 nucleotides from each end of the DNA and subsequent rejoining within a 9-nucleotide microhomology region. This approach provides a quantitative and visual means to measure the efficiency of alternative non-homologous end joining in cellular processes.&nbsp;&nbsp;&nbsp;</td>
  • <td style="text-align:center">Alt NHEJ</td>
  • <td style="text-align:center">No</td>
  • </tr>
  • <tr>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Pulsed Field Gel Electro-phoresis (PFGE) with Time-Course</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:times new roman,serif"><span style="font-family:arial,sans-serif">Biedermann</span></span><u><span style="font-family:arial,sans-serif"> </span></u><span style="font-family:arial,sans-serif">et al., 1991</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px">PFGE assay with a time-course; Quantity of small DNA fragments should decrease as DNA repair&nbsp; progresses</span></td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">DSBs</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px">N/A</span></td>
  • </tr>
  • <tr>
  • <td>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Fluorescence -Based Multiplex Flow-Cytometric Host Reactivation Assay </span></span></p>
  • <p style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">(FM-HCR)</span></span></p>
  • </td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Nagel et al., 2014</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">Measures the ability of human cells to repair plasma reporters, which contain different types and amounts of DNA damage; Used to measure repair processes including HR, NHEJ, BER, NER, MMR, and MGMT</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif">HR, NHEJ, BER, NER, MMR, or MGMT</span></span></td>
  • <td style="text-align:center"><span style="font-size:14px">N/A</span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:14px">Alkaline Unwinding Assay with Time Course&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">Nacci et al. 1991&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">DNA is stored in alkaline solutions with DNA-specific dye and allowed to unwind following removal from tissue, increased strand damage associated with increased unwinding. Samples analyzed at different time points to compare remaining damage following repair opportunities&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">DSBs&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">Yes (<u><span style="font-family:arial,sans-serif">No. 489)</span></u>&nbsp;</span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:14px">Sucrose Density Gradient Centrifugation with Time Course&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">Larsen et al. 1982&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">Strand breaks alter the molecular weight of the DNA piece. DNA in alkaline solution centrifuged into sugar density gradient, repeated set time apart. The less DNA breaks identified in the assay repeats, the more repair occurred&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">SSBs&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">N/A</span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:14px">y-H2AX Foci Staining with Time Course&nbsp;</span></td>
  • <td style="text-align:center">
  • <p><span style="font-size:14px">Mariotti et al. 2013&nbsp;</span></p>
  • <p><span style="font-size:14px">Penninckx et al. 2021&nbsp;</span></p>
  • </td>
  • <td style="text-align:center"><span style="font-size:14px">Histone H2AX is phosphorylated in the presence of DNA strand breaks, the rate of its disappearance over time is used as a measure of DNA repair&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">DSBs&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">N/A</span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:14px">Alkaline Elution Assay with Time Course&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">Larsen et al. 1982&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">DNA with strand breaks elute faster than DNA without, plotted against time intervals to determine the rate at which strand breaks repair&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">SSBs&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">N/A</span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:14px">53BP1 foci Detection with Time Course&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">Penninckx et al. 2021&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">53BP1 is recruited to the site of DNA damage, the rate at which its level decreases over time is used to measure DNA repair&nbsp;</span></td>
  • <td style="text-align:center"><span style="font-size:14px">DSBs</span></td>
  • <td style="text-align:center"><span style="font-size:14px">N/A&nbsp;</span></td>
  • </tr>
  • </tbody>
  • </table>
  • <p>&nbsp;</p>
  • <h4>References</h4>
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  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Bronstein, S.M. et al. (1992), &quot;Efficient repair of O6-ethylguanine, but not O4-ethylthymine or O2-ethylthymine, is dependent upon O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair activities in human cells&quot;, <em>Cancer Research</em>, 52(7): 2008-2011.&nbsp;</span></span></p>
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  • <h4><a href="/events/185">Event: 185: Increase, Mutations</a></h4>
  • <h5>Short Name: Increase, Mutations</h5>
  • <h4>Key Event Component</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Process</th>
  • <th scope="col">Object</th>
  • <th scope="col">Action</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>mutation</td>
  • <td>deoxyribonucleic acid</td>
  • <td>increased</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>AOPs Including This Key Event</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP ID and Name</th>
  • <th scope="col">Event Type</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/15">Aop:15 - Alkylation of DNA in male pre-meiotic germ cells leading to heritable mutations</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/141">Aop:141 - Alkylation of DNA leading to cancer 2</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/139">Aop:139 - Alkylation of DNA leading to cancer 1</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/294">Aop:294 - Increased reactive oxygen and nitrogen species (RONS) leading to increased risk of breast cancer</a></td>
  • <td>AdverseOutcome</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/293">Aop:293 - Increased DNA damage leading to increased risk of breast cancer</a></td>
  • <td>AdverseOutcome</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/296">Aop:296 - Oxidative DNA damage leading to chromosomal aberrations and mutations</a></td>
  • <td>AdverseOutcome</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/272">Aop:272 - Deposition of energy leading to lung cancer</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/397">Aop:397 - Bulky DNA adducts leading to mutations</a></td>
  • <td>AdverseOutcome</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/443">Aop:443 - Alcohol Induced DNA damage and mutations leading to Metastatic Breast Cancer</a></td>
  • <td><a href="/aops/443">Aop:443 - DNA damage and mutations leading to Metastatic Breast Cancer</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/478">Aop:478 - Deposition of energy leading to occurrence of cataracts</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Stressors</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Name</th></tr>
  • </thead>
  • <tbody>
  • <tr><td>Ionizing Radiation</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Biological Context</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Level of Biological Organization</th></tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr><td>Molecular</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Domain of Applicability</h4>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Mus musculus</td>
  • <td>Mus musculus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>medaka</td>
  • <td>Oryzias latipes</td>
  • <td>Moderate</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8090" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>rat</td>
  • <td>Rattus norvegicus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Homo sapiens</td>
  • <td>Homo sapiens</td>
  • <td>Moderate</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>All life stages</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <p><strong>Taxonomic applicability:</strong> Mutations can occur in any organism and in any cell type, and are the fundamental material of evolution. The test guidelines described above range from analysis from prokaryotes, to rodents, to human cells in vitro. Mutations have been measured in virtually every human tissue sampled in vivo.</p>
  • <p><strong>Life stage applicability:</strong> This key event is not life stage specific as all stages of life have DNA that can be mutated; however, baseline levels of mutations are seen to increase with age (Slebos et al., 2004; Kirkwood, 1989).&nbsp;</p>
  • <p><strong>Sex applicability:</strong> This key event is not sex specific as both sexes undergo mutations. Males have a higher mutation rate than females (Hedrick, 2007).&nbsp;</p>
  • <p><strong>Evidence for perturbation by a stressor:</strong> Many studies demonstrate that increased mutations can occur as a result of ionizing radiation (Sankaranarayanan &amp; Nikjoo, 2015; Russell et al., 1957; Winegar et al., 1994; Gossen et al., 1995). &nbsp;</p>
  • <h4>Key Event Description</h4>
  • <p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">A mutation is a change in DNA sequence. Mutations can thus alter the coding sequence of genes, potentially leading to malformed or truncated proteins. Mutations can also occur in promoter regions, splice junctions, non-coding RNA, DNA segments, and other functional locations in the genome. These mutations can lead to various downstream consequences, including alterations in gene expression. There are several different types of mutations including missense, nonsense, insertion, deletion, duplication, and frameshift mutations, all of which can impact the genome and its expression in unique ways. </span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Missense mutations are the substitution of one base in the codon with another. This change is significant because the three bases in a codon code for a specific amino acid and the new combination may signal for a different amino acid to be formed. Nonsense mutations also result from changes to the codon bases, but in this case, they cause the generation of a stop codon in the DNA strand where there previously was not one. This stop codon takes the place of a normal coding triplet, preventing its translation into an amino acid. This will cause the translation of the strand to prematurely stop. Both missense and nonsense mutations can result from substitutions, insertions, or deletions of bases (Chakarov et al. 2014). &nbsp;</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Insertion and deletion mutations are the addition and removal of bases from the strand, respectively. These often accompany a frameshift mutation, as the alteration in the number of bases in the strand causes the frame of the base reader to shift by the added or reduced number, altering the amino acids that are produced if that number is not devisable by three. Codons come in specific orders, sectioned into groups of three. When the boundaries of which three bases are included in one group are changed, this can change the whole transcriptional output of the strand (Chakaroy et al. 2014).&nbsp;</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Mutations can be propagated to daughter cells upon cellular replication. Mutations in stem cells (versus terminally differentiated non-replicating cells) are the most concerning, as these will persist in the organism. The consequence of the mutation, and thus the fate of the cell, depends on the location (e.g., coding versus non-coding) and the type (e.g., nonsense versus silent) of mutation.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Mutations can occur in somatic cells or germ cells (sperm or egg).</span></span></p>
  • <h4>How it is Measured or Detected</h4>
  • <p>Mutations can be measured using a variety of both OECD and non-OECD mutagenicity tests. Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed.</p>
  • <p><strong>Somatic cells:</strong> The Salmonella mutagenicity test (Ames Test) is generally used as part of a first tier screen to determine if a chemical can cause gene mutations. This well-established test has an OECD test guideline (OECD TG 471, 2020). A variety of bacterial strains are used, in the presence and absence of a metabolic activation system (e.g., rat liver microsomal S9 fraction), to determine the mutagenic potency of chemicals by dose-response analysis. A full description is found in Test No. 471: Bacterial Reverse Mutation Test (OECD, 2016).</p>
  • <p>A variety of in vitro mammalian cell gene mutation tests are described in OECD&rsquo;s Test Guidelines 476 (2016) and 490 (2015). TG 476 (2016) is used to identify substances that induce gene mutations at the hprt (hypoxanthine-guanine phosphoribosyl transferase) gene, or the transgenic xprt (xanthine-guanine phosphoribosyl transferase) reporter locus. The most commonly used cells for the HPRT test include the CHO, CHL and V79 lines of Chinese hamster cells, L5178Y mouse lymphoma cells, and TK6 human lymphoblastoid cells. The only cells suitable for the XPRT test are AS52 cells containing the bacterial xprt (or gpt) transgene (from which the hprt gene was deleted).</p>
  • <p>The new OECD TG 490 (2015) describes two distinct in vitro mammalian gene mutation assays using the thymidine kinase (tk) locus and requiring two specific tk heterozygous cells lines: L5178Y tk+/-3.7.2C cells for the mouse lymphoma assay (MLA) and TK6 tk+/- cells for the TK6 assay. The autosomal and heterozygous nature of the thymidine kinase gene in the two cell lines enables the detection of cells deficient in the enzyme thymidine kinase following mutation from tk+/- to tk-/-.</p>
  • <p>It is important to consider that different mutation spectra are detected by the different mutation endpoints assessed. The non-autosomal location of the hprt gene (X-chromosome) means that the types of mutations detected in this assay are point mutations, including base pair substitutions and frameshift mutations resulting from small insertions and deletions. Whereas, the autosomal location of the transgenic xprt, tk, or gpt locus allows the detection of large deletions not readily detected at the hemizygous hprt locus on X-chromosomes. Genetic events detected using the tk locus include both gene mutations (point mutations, frameshift mutations, small deletions) and large deletions.</p>
  • <p>The transgenic rodent mutation assay (OECD TG 488, 2020) is the only assay capable of measuring gene mutation in virtually all tissues in vivo. Specific details on the rodent transgenic mutation reporter assays are reviewed in Lambert et al. (2005, 2009). The transgenic reporter genes are used for detection of gene mutations and/or chromosomal deletions and rearrangements resulting in DNA size changes (the latter specifically in the lacZ plasmid and Spi- test models) induced in vivo by test substances (OECD, 2009, OECD, 2011; Lambert et al., 2005). Briefly, transgenic rodents (mouse or rat) are exposed to the chemical agent sub-chronically. Following a manifestation period, genomic DNA is extracted from tissues, transgenes are rescued from genomic DNA, and transfected into bacteria where the mutant frequency is measured using specific selection systems.</p>
  • <p>The Pig-a (phosphatidylinositol glycan, Class A) gene on the X chromosome codes for a catalytic subunit of the N-acetylglucosamine transferase complex that is involved in glycosylphosphatidyl inositol (GPI) cell surface anchor synthesis. Cells lacking GPI anchors, or GPI-anchored cell surface proteins are predominantly due to mutations in the Pig-a gene. Thus, flow cytometry of red blood cells expressing or not expressing the Pig-a gene has been developed for mutation analysis in blood cells from humans, rats, mice, and monkeys. The assay is described in detail in Dobrovolsky et al. (2010). Development of an OECD guideline for the Pig-a assay is underway. In addition, experiments determining precisely what proportion of cells expressing the Pig-a mutant phenotype have mutations in the Pig-a gene are in progress (e.g., Nicklas et al., 2015, Drobovolsky et al., 2015). A recent paper indicates that the majority of CD48 deficient cells from 7,12-dimethylbenz[a]anthracene-treated rats (78%) are indeed due to mutation in Pig-a (Drobovolsky et al., 2015).</p>
  • <p><br />
  • <strong>Germ cells:</strong> Tandem repeat mutations can be measured in bone marrow, sperm, and other tissues using single-molecule PCR. This approach has been applied most frequently to measure repeat mutations occurring in sperm DNA. Isolation of sperm DNA is as described above for the transgenic rodent mutation assay, and analysis of tandem repeats is done using electrophoresis for size analysis of allele length using single-molecule PCR. For expanded simple tandem repeat this involved agarose gel electrophoresis and Southern blotting, whereas for microsatellites sizing is done by capillary electrophoresis. Detailed methodologies for this approach are found in Yauk et al. (2002) and Beal et al. (2015).</p>
  • <p>Mutations in rodent sperm can also be measured using the transgenic reporter model (OECD TG 488, 2020). A description of the approach is found within this published TG. Further modifications to this protocol have been made as of 2022 for the analysis of germ cells. Detailed methodology for detecting mutant frequency arising in spermatogonia is described in Douglas et al. (1995), O&#39;Brien et al. (2013); and O&#39;Brien et al. (2014). Briefly, male mice are exposed to the mutagen and killed at varying times post-exposure to evaluate effects on different phases of spermatogenesis. Sperm are collected from the vas deferens or caudal epididymis (the latter preferred). Modified protocols have been developed for extraction of DNA from sperm.</p>
  • <p>A similar transgenic assay can be used in transgenic medaka (Norris and Winn, 2010).</p>
  • <p><br />
  • Please note, gene mutations that occur in somatic cells in vivo (OECD Test. No. 488, 2020) or in vitro (OECD Test No. 476: In vitro Mammalian Cell Gene Mutation Test, 2016), or in bacterial cells (i.e., OECD Test No. 471, 2020) can be used as an indicator that mutations in male pre-meiotic germ cells may occur for a particular agent (sensitivity and specificity of other assays for male germ cell effects is given in Waters et al., 1994). However, given the very unique biological features of spermatogenesis relative to other cell types, known exceptions to this rule, and the small database on which this is based, inferring results from somatic cell or bacterial tests to male pre-meiotic germ cells must be done with caution. That mutational assays in somatic cells may predict mutations in germ cells has not been rigorously tested empirically (Singer and Yauk, 2010). The IWGT working group on germ cells specifically addressed this gap in knowledge in their report (Yauk et al., 2015) and recommended that additional research address this issue. Mutations can be directly measured in humans (and other species) through the application of next-generation sequencing. Although single-molecule approaches are growing in prevalence, the most robust approach to measure mutation using next-generation sequencing today requires clonal expansion of the mutation to a sizable proportion (e.g., sequencing tumours; Shen et al., 2015), or analysis of families to identify germline derived mutations (reviewed in Campbell and Eichler, 2013; Adewoye et al., 2015).</p>
  • <p><span style="font-size:14px"><span style="font-family:arial,sans-serif">Please refer to the table below for additional details and methodologies for measuring mutations. </span></span></p>
  • <table border="1" cellpadding="1" cellspacing="1" style="height:2351px; width:633px">
  • <tbody>
  • <tr>
  • <td style="background-color:#eeeeee; text-align:center">A<strong>ssay Name</strong></td>
  • <td style="background-color:#eeeeee; text-align:center"><strong>References </strong></td>
  • <td style="background-color:#eeeeee; text-align:center"><strong>Description </strong></td>
  • <td style="background-color:#eeeeee; text-align:center"><strong>OECD Approved Assay</strong></td>
  • </tr>
  • <tr>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">Assorted Gene Loci Mutation Assays</span></td>
  • <td>
  • <p style="text-align:center"><span style="font-family:arial,sans-serif; font-size:11pt">Tindall et al., 1989; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:times new roman,serif; font-size:12pt"><span style="font-family:arial,sans-serif; font-size:11pt">Kruger</span></span><span style="font-family:arial,sans-serif; font-size:11pt"> et al., 2015</span></span></span></p>
  • </td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">After exposure to a chemical/mutagen, mutations can be measured by the ability of exposed cells to form colonies in the presence of specific compounds that would normally inhibit colony growth; Usually only cells -/- for the gene of interest are able to form colonies</span></td>
  • <td>N/A</td>
  • </tr>
  • <tr>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">TK Mutation Assay</span></td>
  • <td>
  • <p style="text-align:center"><span style="font-family:arial,sans-serif; font-size:11pt">Yamamoto et al., 2017; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Liber et al., 1982; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Lloyd and Kidd, 2012</span></span></span></span></span></p>
  • </td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">After exposure to a chemical/mutagen, mutations are detected at the thymidine kinase (TK) loci&nbsp;of L5178Y wild-type mouse lymphoma TK (+/-) cells by measuring resistance to lethaltriflurothymidine (TFT); Only TK-/- cells are able to form colonies</span></td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">Yes&nbsp;(No. 490)</span></td>
  • </tr>
  • <tr>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">HPRT Mutation Assay</span></td>
  • <td>
  • <p style="text-align:center"><span style="font-family:arial,sans-serif; font-size:11pt">Ayres et al., 2006; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Parry and Parry, 2012</span></span></span></p>
  • </td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">Similar to TK Mutation Assay above, X-linked HPRT mutations produced in response to chemical/mutagen exposure can be measured through colony formation in the presence of 6-TG or 8-azoguanine; Only HPRT-/- cells are able to form colonies</span></td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">Yes&nbsp;(No. 476)</span></td>
  • </tr>
  • <tr>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">Salmonella Mutagenicity Test (Ames Test)</span></td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">OECD, 1997</span></td>
  • <td>After exposure to a chemical/mutagen, point mutations are detected by analyzing the growth capacity of different bacterial strains in the presence and absence of various metabolic activation systems&nbsp;</td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">Yes (No. 471)</span></td>
  • </tr>
  • <tr>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">PIG-A / PIG-O Assay</span></td>
  • <td>
  • <p style="text-align:center"><span style="font-family:arial,sans-serif; font-size:11pt">Kruger et al., 2015; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Nakamura, 2012; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Chikura, 2019</span></span></span></span></span></p>
  • </td>
  • <td>After exposure to a chemical/mutagen, mutations&nbsp; in PIG-A or PIG-O (which decrease the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor protein) are assessed by the colony-forming capabilities of cells after <em>in vitro</em> exposure, or by flow cytometry of blood samples after <em>in vivo </em>exposure</td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">N/A</span></td>
  • </tr>
  • <tr>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">Single Molecule PCR</span></td>
  • <td>
  • <p style="text-align:center"><span style="font-family:arial,sans-serif; font-size:11pt">Kraytsberg &amp; Khrapko, 2005; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Yauk, 2002</span></span></span></p>
  • </td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">This PCR technique uses a single DNA template, and is often employed for detection of mutations in microsatellites, recombination studies, and generation of polonies</span></td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">N/A</span></td>
  • </tr>
  • <tr>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">ACB-PCR</span></td>
  • <td>
  • <p>Myers et al., 2014 (Textbook, pg 345-363); Banda et al.,&nbsp; 2013; Banda et al.,&nbsp; 2015; Parsons et al., 2017</p>
  • </td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">Using this PCR technique, single base pair substitution mutations within oncogenes or tumour suppressor genes can be detected by selectively amplifying specific point mutations within an allele and selectively blocking amplification of the wild-type allele </span></td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">N/A</span></td>
  • </tr>
  • <tr>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">Transgenic Rodent Mutation Assay </span></td>
  • <td>
  • <p style="text-align:center"><span style="font-family:arial,sans-serif; font-size:11pt">OECD 2013; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Lambert 2005; <span style="font-family:arial,sans-serif; font-size:11pt"><span style="font-family:arial,sans-serif; font-size:11pt">Lambert 2009</span></span></span></span></span></p>
  • </td>
  • <td>This <em>in vivo</em> test detects gene mutations using transgenic rodents that possess transgenes and reporter genes; After<em> in vivo</em> exposure to a chemical/mutagen, the transgenes are analyzed by transfecting bacteria with the reporter gene and examining the resulting phenotype</td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">Yes (No. 488)</span></td>
  • </tr>
  • <tr>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">Conditionally inducible transgenic mouse models</span></td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">Parsons 2018 (Review)</span></td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">Inducible mutations linked to fluorescent tags are introduced into transgenic mice; Upon exposure of the transgenic mice to an inducing agent, the presence and functional assessment of the mutations can be easily ascertained due to expression of the linked fluorescent tags </span></td>
  • <td>N/A</td>
  • </tr>
  • <tr>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">Error</span><span style="font-family:arial,sans-serif; font-size:12pt">-</span><span style="font-family:arial,sans-serif; font-size:11pt">Corrected Next Generation Sequencing (NGS)</span></td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">Salk 2018 (Review)</span></td>
  • <td><span style="font-family:arial,sans-serif; font-size:11pt">This technique detects rare subclonal mutations within a pool of heterogeneous DNA samples through the application of new error-correction strategies to NGS; At present, few laboratories in the world are capable of doing this, but commercial services are becoming available (e.g., Duplex sequencing at TwinStrand BioSciences) </span></td>
  • <td>N/A&nbsp;</td>
  • </tr>
  • </tbody>
  • </table>
  • <p>&nbsp;</p>
  • <h4>References</h4>
  • <p>Adewoye, A.B. et al. (2015), &quot;The genome-wide effects of ionizing radiation on mutation induction in the mammalian germline&quot;, <em>Nat. Commu.</em>, 6:6684. Doi: 10.1038/ncomms7684.</p>
  • <p>Ayres, M. F. et al. (2006), &nbsp;&ldquo;Low doses of gamma ionizing radiation increase hprt mutant frequencies of TK6 cells without triggering the mutator phenotype pathway&rdquo;, &nbsp;<em>Genetics and Molecular Biology</em>. 2(3): 558-561. Doi:10.1590/S1415-4757200600030002.</p>
  • <p>Banda M, Recio L, and Parsons BL. (2013), &ldquo;ACB-PCR measurement of spontaneous and furan-induced H-ras codon 61 CAA to CTA and CAA to AAA mutation in B6C3F1 mouse liver&rdquo;, <em>Environ Mol Mutagen</em>. 54(8):659-67. Doi:10.1002/em.21808.</p>
  • <p>Banda, &nbsp;M. et al. (2015), &ldquo;Quantification of Kras mutant fraction in the lung DNA of mice exposed to aerosolized particulate vanadium pentoxide by inhalation&rdquo;, &nbsp;<em>Mutat Res Genet Toxicol Environ Mutagen</em>. 789-790:53-60. Doi: 10.1016/j.mrgentox.2015.07.003</p>
  • <p>Campbell, C.D. &amp; E.E. Eichler (2013), &quot;Properties and rates of germline mutations in humans&quot;, <em>Trends Genet</em>., 29(10): 575-84. Doi: &nbsp;10.1016/j.tig.2013.04.005</p>
  • <p>Chakarov, S. et al. (2014), &ldquo;DNA damage and mutation. Types of DNA damage&rdquo;, BioDiscovery, Vol.11, Pensoft Publishers, Sofia, https://doi.org/10.7750/BIODISCOVERY.2014.11.1.</p>
  • <p>Chikura, S. et al. (2019), &ldquo;Standard protocol for the total red blood cell Pig-a assay used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society&rdquo;, &nbsp;<em>Genes Environ</em>.&nbsp; 27:41-5. Doi: 10.1186/s41021-019-0121-z.</p>
  • <p>Dobrovolsky, V.N. et al. (2015), &quot;CD48-deficient T-lymphocytes from DMBA-treated rats have de novo mutations in the endogenous Pig-a gene. CD48-Deficient T-Lymphocytes from DMBA-Treated Rats Have De Novo Mutations in the Endogenous Pig-a Gene&quot;, Environ. Mol. Mutagen., (6): 674-683. Doi: 10.1002/em.21959.</p>
  • <p>Douglas, G.R. et al. (1995), &quot;Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice&quot;, <em>Proceedings of the National Academy of Sciences of the United States of America</em>, 92(16): 7485-7489. Doi: 10.1073/pnas.92.16.7485.</p>
  • <p>Gossen, J.A. et al. (1995), &quot;Spontaneous and X-ray-induced deletion mutations in a LacZ plasmid-based transgenic mouse model&quot;, Mutation Research, 331/1, Elsevier, Amsterdam, https://doi.org/10.1016/0027-5107(95)00055-N.&nbsp;</p>
  • <p>Hedrick, P.W. (2007), &ldquo;Sex: Differences In Mutation, Recombination, Selection, Gene Flow, And Genetic Drift&rdquo;, Evolution, Vol.61/12, Wiley, Hoboken, https://doi.org/10.1111/j.1558-5646.2007.00250.x.&nbsp;</p>
  • <p>Kirkwood, T.B.L. (1989), &ldquo;DNA, mutations and aging&rdquo;, Mutation Research, Vol.219/1, Elsevier B.V., Amsterdam, https://doi.org/10.1016/0921-8734(89)90035-0</p>
  • <p>Kraytsberg,Y. &amp; &nbsp;Khrapko, K. (2005), &nbsp;&ldquo;Single-molecule PCR: an artifact-free PCR approach for the analysis of somatic mutations&rdquo;, &nbsp;<em>Expert Rev Mol Diagn</em>. 5(5):809-15. Doi: 10.1586/14737159.5.5.809.</p>
  • <p>Kr&uuml;ger, T. C., Hofmann, M., &amp; Hartwig, A. (2015), &ldquo;The in vitro PIG-A gene mutation assay: mutagenicity testing via flow cytometry based on the glycosylphosphatidylinositol (GPI) status of TK6 cells&rdquo;, <em>Arch Toxicol</em>. 89(12), 2429-43. Doi: 10.1007/s00204-014-1413-5.</p>
  • <p>Lambert, I.B. et al. (2005), &quot;Detailed review of transgenic rodent mutation assays&quot;, <em>Mutat Res.</em>, 590(1-3):1-280. Doi: 10.1016/j.mrrev.2005.04.002.</p>
  • <p>Liber, L. H., &amp; Thilly, G. W. (1982), &nbsp;&ldquo;Mutation assay at the thymidine kinase locus in diploid human lymphoblasts&rdquo;, &nbsp;<em>Mutation Research</em>. 94: 467-485. Doi:10.1016/0027-5107(82)90308-6.</p>
  • <p>Lloyd, M., &amp; Kidd, D. (2012), &ldquo;The Mouse Lymphoma Assay. In: Parry J., Parry E. (eds) Genetic Toxicology, Methods in Molecular Biology (Methods and Protocols), 817. Springer, New York, NY.</p>
  • <p>Myers, M. B. et al., (2014), &ldquo;ACB-PCR Quantification of Somatic Oncomutation&rdquo;, &nbsp;<em>Molecular Toxicology Protocols, Methods in Molecular Biology</em>. DOI: 10.1007/978-1-62703-739-6_27</p>
  • <p>Nakamura, J. et al., (2012), &ldquo;Detection of PIGO-deficient cells using proaerolysin: a valuable tool to investigate mechanisms of mutagenesis in the DT40 cell system&rdquo;, <em>PLoS One</em>.7(3): e33563. Doi:10.1371/journal.pone.0033563.</p>
  • <p>Nicklas, J.A., E.W. Carter and R.J. Albertini (2015), &quot;Both PIGA and PIGL mutations cause GPI-a deficient isolates in the Tk6 cell line&quot;, Environ. Mol. Mutagen., 6(8):663-73. Doi: 10.1002/em.21953.</p>
  • <p>Norris, M.B. and R.N. Winn (2010), &quot;Isolated spermatozoa as indicators of mutations transmitted to progeny&quot;, Mutat Res., 688(1-2): 36&ndash;40. Doi: 10.1016/j.mrfmmm.2010.02.008.</p>
  • <p>O&#39;Brien, J.M. et al.(2013), &quot;No evidence for transgenerational genomic instability in the F1 or F2 descendants of Muta&trade;Mouse males exposed to N-ethyl-N-nitrosourea&quot;, <em>Mutat. Res</em>., 741-742:11-7. Doi: 10.1016/j.mrfmmm.2013.02.004.</p>
  • <p>O&#39;Brien, J.M. et al. (2014), &quot;Transgenic rodent assay for quanitifying male germ cell mutation frequency&quot;, <em>Journal of Visual Experimentation</em>, Aug 6;(90). Doi: 10.3791/51576.</p>
  • <p>O&rsquo;Brien, J.M. et al. (2015), &quot;Sublinear response in lacZ mutant frequency of Muta&trade; Mouse spermatogonial stem cells after low dose subchronic exposure to N-ethyl-N-nitrosourea&quot;, <em>Environ. Mol. Mutagen.</em>, 6(4): 347-355. Doi: 10.1002/em.21932.</p>
  • <p>OECD (2020), Test No. 471: Bacterial Reverse Mutation Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.</p>
  • <p>OECD (2016), Test No. 476: In vitro Mammalian Cell Gene Mutation Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.</p>
  • <p>OECD (2009), Detailed Review Paper on Transgenic Rodent Mutation Assays, Series on Testing and Assessment, N&deg; 103, ENV/JM/MONO 7, OECD, Paris.</p>
  • <p>OECD (2020), Test No. 488: Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.</p>
  • <p>OECD (2016), Test. No. 490: In vitro mammalian cell gene mutation mutation tests using the thymidine kinase gene, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.</p>
  • <p>OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.</p>
  • <p>Parry MJ, &amp; Parry ME. 2012. Genetic Toxicology Principles and Methods. Humana Press. Springer Protocols.</p>
  • <p>Parsons BL, McKim KL, Myers MB. 2017. Variation in organ-specific PIK3CA and KRAS mutant levels in normal human tissues correlates with mutation prevalence in corresponding carcinomas. Environ Mol Mutagen. 58(7):466-476. Doi: 10.1002/em.22110.</p>
  • <p>Parsons BL. Multiclonal tumor origin: Evidence and implications<em>. Mutat Res</em>. 2018. 777:1-18. doi: 10.1016/j.mrrev.2018.05.001.</p>
  • <p>Russell, W.L. et al. (1957), &quot;Radiation Dose Rate and Mutation Frequency.&quot;, Science, Vol.128/3338, American Association for the Advancement of Science, Washington, https://doi.org/10.1126/science.128.3338.1546.</p>
  • <p>Salk JJ, Schmitt MW, &amp;Loeb LA. (2018), &ldquo;Enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations&rdquo;, <em>Nat Rev Genet</em>. 19(5):269-285. Doi: 10.1038/nrg.2017.117.</p>
  • <p>Sankaranarayanan, K. &amp; H. Nikjoo (2015), &quot;Genome-based, mechanism-driven computational modeling of risks of ionizing radiation: The next frontier in genetic risk estimation?&quot;, Mutation Research, Vol.764, Elsevier, Amsterdam, https://doi.org/10.1016/j.mrrev.2014.12.003.&nbsp;</p>
  • <p>Shen, T., S.H. Pajaro-Van de Stadt, N.C. Yeat and J.C. Lin (2015), &quot;Clinical applications of next generation sequencing in cancer: from panels, to exomes, to genomes&quot; <em>Front. Genet.</em>, 6: 215. Doi: 10.3389/fgene.2015.00215.</p>
  • <p>Singer, T.M. and C.L. Yauk CL (2010), &quot;Germ cell mutagens: risk assessment challenges in the 21st century&quot;, <em>Environ. Mol. Mutagen.</em>, 51(8-9): 919-928. Doi: 10.1002/em.20613.</p>
  • <p>Slebos, R.J.C. et al. (2004), &ldquo;Mini-and microsatellite mutations in children from Chernobyl accident cleanup workers&rdquo;, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Vol.559/1-2, Elsevier, Amsterdam, https://doi.org/10.1016/j.mrgentox.2004.01.003.&nbsp;</p>
  • <p>Tindall, R. K., &amp; Stankowski Jr., F. L. (1989), &nbsp;&ldquo;Molecular analysis of spontaneous mutations at the GPT locus in Chinese hamster ovary (AS52) cells&rdquo;, <em>Mutation Research</em>, 220, 241-53. Doi: 10.1016/0165-1110(89)90028-6.</p>
  • <p>Waters, M.D. et al. (1994), &quot;The performance of short-term tests in identifying potential germ cell mutagens: a qualitative and quantitative analysis&quot;, <em>Mutat. Res.</em>, 341(2): 109-31. Doi: 10.1016/0165-1218(94)90093-0.</p>
  • <p>Winegar, R.A. et al. (1994), &quot;Radiation-induced point mutations, deletions and micronuclei in lacI transgenic mice&quot;, Mutation Research, Vol.307/2, Elsevier, Amsterdam, https://doi.org/10.1016/0027-5107(94)90258-5.&nbsp;</p>
  • <p>Yamamoto, A. et al. (2017), &ldquo;Radioprotective activity of blackcurrant extract evaluated by in vitro micronucleus and gene mutation assays in TK6 human lymphoblastoid cells&rdquo;,<em> Genes and Environment. </em>39: 22. Doi: 10.1186/s41021-017-0082-z.</p>
  • <p>Yauk, C.L. et al. (2002), &quot;A novel single molecule analysis of spontaneous and radiation-induced mutation at a mouse tandem repeat locus&quot;, Mutat. Res., 500(1-2): 147-56. Doi: 10.1016/s0027-5107(02)00005-2.</p>
  • <p>Yauk, C.L. et al. (2015), &quot;Approaches for Identifying Germ Cell Mutagens: Report of the 2013 IWGT Workshop on Germ Cell Assays&quot;, <em>Mutat. Res. Genet. Toxicol. Environ. Mutagen.</em>, 783: 36-54. Doi: 10.1016/j.mrgentox.2015.01.008.</p>
  • <p>Yeat and J.C. Lin. 2015. Clinical applications of next generation sequencing in cancer: from panels, to exomes, to genomes. <em>Front. Genet</em>., 6: 215. Doi: 10.3389/fgene.2015.00215.</p>
  • <h4><a href="/events/1636">Event: 1636: Increase, Chromosomal aberrations</a></h4>
  • <h5>Short Name: Increase, Chromosomal aberrations</h5>
  • <h4>AOPs Including This Key Event</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP ID and Name</th>
  • <th scope="col">Event Type</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/296">Aop:296 - Oxidative DNA damage leading to chromosomal aberrations and mutations</a></td>
  • <td>AdverseOutcome</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/272">Aop:272 - Deposition of energy leading to lung cancer</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/478">Aop:478 - Deposition of energy leading to occurrence of cataracts</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Stressors</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Name</th></tr>
  • </thead>
  • <tbody>
  • <tr><td>Ionizing Radiation</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Biological Context</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Level of Biological Organization</th></tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr><td>Cellular</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Domain of Applicability</h4>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>human</td>
  • <td>Homo sapiens</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>rat</td>
  • <td>Rattus norvegicus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>mouse</td>
  • <td>Mus musculus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>All life stages</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <p><strong>Taxonomic applicability:</strong> CAs are possible in nucleated cells of any species (Ferguson-Smith, 2015).&nbsp;&nbsp;</p>
  • <p><strong>Life stage applicability:</strong> This key event is not life stage specific as subjects of all ages have chromosomes that can be improperly structured. However, older individuals have naturally higher baseline levels of CAs (Vick et al., 2017). Individuals born with stable type aberrations will retain them throughout their lifetime (Gardner et al., 2011).&nbsp;</p>
  • <p><strong>Sex applicability: </strong>This key event is not sex specific, with both sexes experiencing CAs at comparable rates (Ka&scaron;uba et al., 1995).&nbsp;</p>
  • <p><strong>Evidence for perturbation by a stressor:</strong> Many studies have provided evidence to support increased CAs occurring as a result of exposure to ionizing radiation (Franken et al., 2012; Cornforth et al., 2002; Loucas et al., 2013).&nbsp;&nbsp;</p>
  • <h4>Key Event Description</h4>
  • <p>Structural chromosomal aberrations describe&nbsp;the damage to chromosomes&nbsp;that results from breaks along the DNA and may lead to deletion, addition, or rearrangement of sections in the chromosome. Chromosomal aberrations can be divided in two major categories: chromatid-type or chromosome-type depending on whether one or both chromatids are involved, respectively. They can be further classified as rejoined or non-rejoined aberrations. Rejoined aberrations include translocations, insertions, dicentrics and rings, while unrejoined aberrations include acentric fragments and breaks (Savage, 1976). Some of these aberrations are stable (i.e., reciprocal translocations) and can persist for many years (Tucker and Preston, 1996). Others are unstable (i.e., dicentrics, acentric fragments) and decline at each cell division because of clonogenic inactivation&nbsp;(Boei et al., 1996). These events may be detectable after cell division and such damage to DNA is irreversible. Chromosomal aberrations are&nbsp;associated with&nbsp; clonogenic inactivation&nbsp;and carcinogenicity (Mitelman, 1982).</p>
  • <p>Chromosomal aberrations (CA) refer to a missing, extra or irregular portion of chromosomal DNA. These DNA changes in the chromosome structure may be produced by different double strand break (DSB) repair mechanisms (Obe et al., 2002).</p>
  • <p>There are 4 main types of CAs: deletions, duplications, translocations, and inversions. Deletions happen when a portion of the genetic material from a chromosome is lost. Terminal deletions occur when an end piece of the chromosome is cleaved. Interstitial deletions arise when a chromosome breaks in two separate locations and rejoins incorrectly, with the center piece being omitted. Duplications transpire when there is any addition or rearrangement of excess genetic material; types of duplications include transpositions, tandem duplications, reverse duplications, and displaced duplications (Griffiths et al., 2000). Translocations result from a section of one chromosome being transferred to a non-homologous chromosome (Bunting and Nussenzweig, 2013). When there is an exchange of segments on two non-homologous chromosomes, it is called a reciprocal translocation. Inversions occur in a single chromosome and involve both of the ends breaking and being ligated on the opposite ends, effectively inverting the DNA sequence.&nbsp;&nbsp;&nbsp;&nbsp;<br />
  • &nbsp;</p>
  • <p>A fifth type of CA that can occur in the genome is the copy number variant (CNV). CNVs, which may comprise greater than 10% of the human genome (Shlien et al., 2009; Zhang et al., 2016; Hastings et al., 2009),&nbsp; are deletions or duplications that can vary in size from 50 base pairs (Arlt et al., 2012; Arlt et al., 2014; Liu et al., 2013) up into the megabase pair range (Arlt et al., 2012; Wilson et al., 2015; Arlt et al., 2014; Zhang et al., 2016). CNV regions are especially enriched in large genes and large active transcription units (Wilson et al., 2015), and are of particular concern when they cause deletions in tumour suppressor genes or duplications in oncogenes (Liu et al., 2013; Curtis et al., 2012)<em>. </em>There are two types of CNVs: recurrent and non-recurrent. Recurrent CNVs are thought to be produced through a recombination process during meiosis known as non-allelic homologous recombination (NAHR) (Arlt et al., 2012; Hastings et al., 2009). These recurrent CNVs, also called germline CNVs, could be inherited and are thus common across different individuals (Shlien et al., 2009; Liu et al., 2013). Non-recurrent CNVs are believed to be produced in mitotic cells during the process of replication. Although the mechanism is not well studied, it has been suggested that stress during replication, in particular stalling replication forks, prompt microhomology-mediated mechanisms to overcome the replication stall, which often results in duplications or deletions. Two models that have been proposed to explain this mechanism include the Fork Stalling and Template Switching (FoSTeS) model, and the Microhomology-Mediated Break-Induced Replication (MMBIR) model (Arlt et al., 2012; Wilson et al., 2015; Lee et al., 2007; Hastings et al., 2009).</p>
  • <p>&nbsp;</p>
  • <p>CAs can be classified according to whether the chromosome or chromatid is affected by the aberration. Chromosome-type aberrations (CSAs) include chromosome-type breaks, ring chromosomes, marker chromosomes, and dicentric chromosomes; chromatid-type aberrations (CTAs) refer to chromatid breaks and chromatid exchanges (Bonassi et al., 2008; Hagmar et al., 2004). When cells are blocked at the cytokinesis step, CAs are evident in binucleated cells as micronuclei (MN; small nucleus-like structures that contain a chromosome or a piece of a chromosome that was lost during mitosis) and nucleoplasmic bridges (NPBs; physical connections that exist between the two nuclei) (El-Zein et al., 2014). Other CAs can be assessed by examining the DNA sequence, as is the case when detecting copy number variants (CNVs) (Liu et al., 2013)<em>.</em></p>
  • <p>CAs can be classified according to whether the chromosome or chromatid is affected by the aberration. Chromosome-type aberrations (CSAs) include chromosome-type breaks, ring chromosomes, marker chromosomes, and dicentric chromosomes; chromatid-type aberrations (CTAs) refer to chromatid breaks and chromatid exchanges (Bonassi et al., 2008; Hagmar et al., 2004). When cells are blocked at the cytokinesis step, When cells are blocked at the cytokinesis step, micronuclei (MN; small nucleus-like structures that contain a chromosome or a piece of a chromosome that was lost during mitosis) can appear in the cytoplasm of binucleated cells. These micronuclei are an indication of CAs and are often related to dicentric chromosomes. Dicentric chromosomes can also cause nucleoplasmic bridges (NPBs; physical connections that exist between the two nuclei) (El-Zein et al., 2014). Other CAs can be assessed by examining the DNA sequence, as is the case when detecting copy number variants (CNVs) (Liu et al., 2013)<em>.</em></p>
  • <p>OECD defines clastogens as &lsquo;any substance that causes structural chromosomal aberrations in populations of cells or organisms&rsquo;.</p>
  • <h4>How it is Measured or Detected</h4>
  • <p>CAs can be detected before and after cell division. Widely used assays are described in the table below,&nbsp;however there may be other comparable methods that are not listed.&nbsp;</p>
  • <table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none; width:0px">
  • <tbody>
  • <tr>
  • <td style="background-color:#f2f2f2; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:37px; width:130px">
  • <p style="margin-left:4px; margin-right:7px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><strong>Assay</strong></span></span></p>
  • </td>
  • <td style="background-color:#f2f2f2; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:37px; width:113px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-family:&quot;Arial&quot;,sans-serif">References</span></strong></span></span></p>
  • </td>
  • <td style="background-color:#f2f2f2; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:37px; width:274px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-family:&quot;Arial&quot;,sans-serif">Description</span></strong></span></span></p>
  • </td>
  • <td style="background-color:#f2f2f2; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:37px; width:142px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="font-family:&quot;Arial&quot;,sans-serif">OECD-approved assay</span></strong></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:95px; text-align:center; width:130px">Premature Chromosome Condensation (PCC)&nbsp;</td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:95px; text-align:center; width:113px">Prasanna et al., 2000; Okayasu et al., 2019&nbsp;</td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:95px; text-align:center; width:274px">&nbsp;Cells are exposed to mitosis-promoting factors (MPF) following cell fusion, causing the chromosomes to condense prematurely. In another approach, cells are exposed to protein phosphatase inhibitors, such as type 1 and 2A protein phosphatases, also causing premature chromosome condensation.&nbsp;</td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:95px; text-align:center; width:142px">N/A&nbsp;</td>
  • </tr>
  • <tr>
  • <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:95px; text-align:center; width:130px">Chromosomal G-banding&nbsp;</td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:95px; text-align:center; width:113px">Schwatz, 1990&nbsp;</td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:95px; text-align:center; width:274px">Use of Giesma dye to stain chromosomal bands, abnormalities determined by the presence of altered morphology &nbsp;</td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:95px; text-align:center; width:142px">N/A&nbsp;</td>
  • </tr>
  • <tr>
  • <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:95px; width:130px">
  • <p style="margin-left:4px; margin-right:7px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Fluorescent In Situ&nbsp; Hybridization (FISH)</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:95px; width:113px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Beaton et al., 2013; Pathak</span></span></span></p>
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">et al., 2017</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:95px; width:274px">
  • <p style="margin-left:5px; margin-right:5px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Fluorescent assay of metaphase chromosomes that can detect CAs through chromosome painting and microscopic analysis</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:95px; width:142px">
  • <p style="margin-left:3px; margin-right:5px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">N/A</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:95px; text-align:center; width:130px">Micronuclei (MN) Assay via Microscopy<em> in vitro &nbsp;</em></td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:95px; text-align:center; width:113px">OECD, 2016a</td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:95px; text-align:center; width:274px">Micronuclei are scored in vitro using microscopy &nbsp;</td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:95px; text-align:center; width:142px">Yes (No. 487)&nbsp;</td>
  • </tr>
  • <tr>
  • <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:97px; width:130px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Cytokinesis Block Micronucleus (CBMN)</span></span></span></p>
  • <p style="margin-right:3px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Assay with Microscopy in vitro</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:97px; width:113px">
  • <p style="margin-right:1px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Fenech, 2000; OECD, 2016a</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:97px; width:274px">
  • <p style="margin-left:5px; margin-right:5px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Cells are cultured with cytokinesis blocking agent, fixed to slides, and undergo MN quantification using microscopy.</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:97px; width:142px">
  • <p style="text-align:center">&nbsp;</p>
  • <p style="margin-right:-1px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Yes (No.487)</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:97px; width:130px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Micronucleus (MN)</span></span></span></p>
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Assay by Microscopy in vivo</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:97px; width:113px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">OECD, 2016b</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:97px; width:274px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Cells are fixed on slides and MN are scored using microscopy. Red blood cells can also be scored for MN using flow cytometry (see below)</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:97px; width:142px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Yes</span></span></span></p>
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">(No. 474)</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:79px; width:130px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">CBMN with Imaging Flow Cytometry</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:79px; width:113px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Rodrigues et al., 2015</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:79px; width:274px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Cells are cultured with cytokinesis blocking agent, fixed in solution, and imaged with flow cytometry to quantify MN</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:79px; width:142px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">N/A</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:58px; width:130px">
  • <p style="margin-left:3px; margin-right:3px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Flow cytometry detection of MN</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:58px; width:113px">
  • <p style="margin-right:9px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Dertinger et al., 2004; Bryce et al., 2007; OECD 2016a, 2016b</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:58px; width:274px">
  • <p style="margin-left:5px; margin-right:5px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">In vivo and in vitro flow cytometry-based, automated micronuclei measurements are also done without cytokinesis block. MN analysis in vivo is performed in peripheral blood cells to detect MN in erythrocytes and reticulocytes.</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:58px; width:142px">
  • <p style="text-align:center">&nbsp;</p>
  • <p style="text-align:center">&nbsp;</p>
  • <p style="margin-right:-1px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Yes (No.487; No. 474)</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:56px; width:130px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">High-throughput biomarker assays (indirect measures to confirm clastogenicity)</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:56px; width:113px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Bryce et al. 2014, 2016, 2018</span></span></span></p>
  • <p style="text-align:center">&nbsp;</p>
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Khoury et al., 2013, Khoury et al., 2016)</span></span></span></p>
  • <p style="text-align:center">&nbsp;</p>
  • <p style="text-align:center">&nbsp;</p>
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Hendriks et al., 2012, 2016; Wink et al., 2014</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:56px; width:274px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Multiplexed biomarkers can be measured by flow cytometry are used to discern clastogenic and aneugenic mechanisms for MN induction.</span><span style="font-family:&quot;Arial&quot;,sans-serif"> Flow cytometry-based quantification of &gamma;H2AX foci and p53 protein expression (Bryce et al., 2016).</span></span></span></p>
  • <p style="text-align:center">&nbsp;</p>
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Prediscreen Assay&ndash; In-Cell Western -based quantification of &gamma;H2AX</span></span></span></p>
  • <p style="text-align:center">&nbsp;</p>
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Green fluorescent protein reporter assay to detect the activation of stress signaling pathways, including DNA damage signaling including a reporter porter that is associated with DNA double strand breaks.</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:56px; width:142px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">N/A</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:193px; width:130px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Dicentric Chromosome Assay (DCA)</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:193px; width:113px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Abe et al., 2018</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:193px; width:274px">
  • <p style="margin-right:25px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Cells are fixed on microscope slides, chromosomes are stained, and the number of dicentric chromosomes are quantified</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:193px; width:142px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">N/A</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:65px; width:130px">
  • <p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">High content imaging</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:65px; width:113px">
  • <p style="margin-left:2px; margin-right:2px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">Shahane et al., 2016</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:65px; width:274px">
  • <p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">DNA can be stained using fluorescent dyes and micronuclei can be scored high-throughput microscopy image analysis.</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:65px; width:142px">
  • <p style="text-align:center">&nbsp;</p>
  • <p style="margin-right:-3px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">N/A</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:193px; width:130px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Chromosomal aberration test</span></span></span></p>
  • <p style="margin-left:1px; margin-right:1px; text-align:center">&nbsp;</p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:193px; width:113px">
  • <p style="margin-left:2px; margin-right:2px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">OECD, 2016c; 2016d; 20l16e</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:193px; width:274px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">In vitro, the cell cycle is arrested at metaphase after 1.5 cell cycle following 3-6 hour exposure</span></span></span></p>
  • <p style="text-align:center">&nbsp;</p>
  • <p style="margin-left:1px; margin-right:1px; text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">In vivo, the test chemical is administered as a single treatment and bone marrow is collected 18-24 hrs later (TG 475), while testis is collected 24-48 hrs later (TG 483). The cell cycle is arrested with a metaphase-arresting chemical (e.g., colchicine) 2-5 hours before cell collection. Once cells are fixed and stained on microscope slides, chromosomal aberrations are scored</span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:193px; width:142px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Yes.</span></span></span></p>
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">In vitro (No. 473)</span></span></span></p>
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Arial,sans-serif">In vivo (No. 475 and No. 483)</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:193px; width:130px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Array Comparative Genomic Hybridization (aCGH) or SNP</span></span></span></p>
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Microarray</span></span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:193px; width:113px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Adewoye et al., 2015;&nbsp;</span></span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Wilson et al., 2015; Arlt et&nbsp;</span></span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">al., 2014;&nbsp;</span></span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Redon et al., 2006; Keren,&nbsp;</span></span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">2014;&nbsp;</span></span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Mukherjee, </span><span style="font-family:&quot;Arial&quot;,sans-serif">2017</span></span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:193px; width:274px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">CNVs are most commonly detected using global DNA microarray technologies; This method, however, is unable to detect balanced CAs, such as inversions</span></span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:193px; width:142px">
  • <p style="text-align:center">&nbsp;</p>
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">N/A</span></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:193px; width:130px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Next Generation Sequencing (NGS): Whole Genome Sequencing (WGS) or</span></span></span></p>
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Whole Exome Sequencing (WES)</span></span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:193px; width:113px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">Liu, 2013;</span><span style="font-family:&quot;Arial&quot;,sans-serif"> Shen, 2016; Mukherjee, 2017</span></span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:193px; width:274px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">CNVs are detected by fragmenting the genome and using NGS to sequence either the entire genome (WGS), or only the exome (WES); Challenges with this methodology include only being able to detect CNVs in exon-rich areas if using WES, the computational investment required for the storage and analysis of these large datasets, and the lack of computational algorithms available for effectively detecting somatic CNVs</span></span></span></p>
  • </td>
  • <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:193px; width:142px">
  • <p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:&quot;Arial&quot;,sans-serif">N/A</span></span></span></p>
  • </td>
  • </tr>
  • </tbody>
  • </table>
  • <p>&nbsp;</p>
  • <h4>References</h4>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Abe, Y et al. (2018), &ldquo;Dose-response curves for analyzing of dicentric chromosomes and chromosome translocations following doses of 1000 mGy or less, based on irradiated peripheral blood samples from five healthy individuals&rdquo;, &nbsp;<em>J Radiat Res</em>. 59(1), 35-42. doi:10.1093/jrr/rrx052</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Adewoye, A.B.et al. (2015), &ldquo;The genome-wide effects of ionizing radiation on mutation induction in the mammalian germline&rdquo;, <em>Nat. Commun</em>. 6:66-84. doi: 10.1038/ncomms7684.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Arlt MF, Wilson TE, Glover TW. (2012), &ldquo;Replication stress and mechanisms of CNV formation&rdquo;, <em>Curr Opin Genet Dev</em>. 22(3):204-10. doi: 10.1016/j.gde.2012.01.009.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Arlt, MF. Et al. (2014), &ldquo;Copy number variants are produced in response to low-dose ionizing radiation in cultured cells&rdquo;, <em>Environ Mol Mutagen</em>. 55(2):103-13. doi: 10.1002/em.21840.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Beaton, L. A. et al. (2013), &ldquo;Investigating chromosome damage using fluorescent in situ hybridization to identify biomarkers of radiosensitivity in prostate cancer patients&rdquo;, Int J Radiat Biol. 89(12): 1087-1093. doi:10.3109/09553002.2013.825060</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Boei, J.J., Vermeulen, S., Natarajan, A.T. (1996), &ldquo;Detection of chromosomal aberrations by fluorescence in situ hybridization in the first three postirradiation divisions of human lymphocytes&rdquo;, Mutat Res, 349:127-135. Doi: 10.1016/0027-5107(95)00171-9.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Bonassi, S. &nbsp;(2008),&rdquo;Chromosomal aberration&nbsp;frequency&nbsp;in&nbsp;lymphocytes&nbsp;predicts&nbsp;the&nbsp;risk of&nbsp;cancer: results from a pooled cohort study of 22 358 subjects in 11 countries&rdquo;, <em>Carcinogenesis.</em> 29(6):1178-83. doi: 10.1093/carcin/bgn075.</span></span></p>
  • <p><span style="font-size:14px">Bryce SM, Bemis JC, Avlasevich SL, Dertinger SD. In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity. Mutat Res. 2007 Jun 15;630(1-2):78-91. doi: 10.1016/j.mrgentox.2007.03.002. Epub 2007 Mar 19. PMID: 17434794; PMCID: PMC1950716.</span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Bryce, S. et al. (2014), &ldquo;Interpreting In VitroMicronucleus Positive Results: Simple Biomarker Matrix Discriminates Clastogens, Aneugens, and Misleading Positive Agents&rdquo;, Environ Mol Mutagen, 55:542-555. Doi:10.1002/em.21868.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Bryce, S. et al.(2016), &ldquo;Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach&rdquo;, Environ Mol Mutagen, 57:171-189. Doi: 10.1002/em.21996.</span></span></p>
  • <p><span style="font-size:14px">Bryce SM, Bernacki DT, Smith-Roe SL, Witt KL, Bemis JC, Dertinger SD. Investigating the Generalizability of the MultiFlow &reg; DNA Damage Assay and Several Companion Machine Learning Models With a Set of 103 Diverse Test Chemicals. Toxicol Sci. 2018 Mar 1;162(1):146-166. doi: 10.1093/toxsci/kfx235. PMID: 29106658; PMCID: PMC6059150.</span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Bunting, S. F., &amp; Nussenzweig, A. (2013), &ldquo;End-joining, translocations and cancer&rdquo;, Nature Reviews Cancer.13 (7): 443-454. doi:10.1038/nrc3537</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Cornforth, M.N., S.M. Bailey, and E.H. Goodwin. (2002), &ldquo;Dose Responses for Chromosome Aberrations Produced in Noncycling Primary Human Fibroblasts by Alpha Particles, and by Gamma Rays Delivered at Sublimating Low Dose Rates&rdquo;, Radiation Research, Vol.158, Radiation Research Society, Indianapolis, https://doi.org/10.1667/0033-7587(2002)158[0043:DRFCAP]2.0.CO;2. &nbsp;</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Curtis, C. et al. (2012), &ldquo;The&nbsp;genomic&nbsp;and&nbsp;transcriptomic&nbsp;architecture&nbsp;of 2,000&nbsp;breast tumours&nbsp;reveals novel subgroups&rdquo;, Nature. 486(7403):346-52. doi: 10.1038/nature10983.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Dertinger, S.D. et al.(2004), &ldquo;Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood&rdquo;, Environ Mol Mutagen, 44:427-435. Doi:&nbsp;<a href="https://doi.org/10.1002/em.20075" target="_blank">10.1002/em.20075</a>.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">El-Zein, RA. Et al. (2014), &ldquo;The&nbsp;cytokinesis-blocked&nbsp;micronucleus assay&nbsp;as a&nbsp;strong&nbsp;predictor&nbsp;of&nbsp;lung cancer: extension of a&nbsp;lung cancer&nbsp;risk prediction model&rdquo;, &nbsp;Cancer&nbsp;Epidemiol Biomarkers Prev. 23(11):2462-70. doi: 10.1158/1055-9965.EPI-14-0462.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Fenech, M. (2000), &ldquo;The in vitro micronucleus technique&rdquo;, Mutation Research. 455(1-2), 81-95. Doi: 10.1016/s0027-5107(00)00065-8</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Ferguson-Smith, M.A. (2015), &ldquo;History and evolution of cytogenetics&rdquo;, Molecular Cytogenetics, Vol.8/19, Biomed Central, London, https://doi.org/10.1186/s13039-015-0125-8.&nbsp;</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Franken, N.A.P. et al. (2012), &ldquo;Relative biological effectiveness of high linear energy transfer alpha-particles for the induction of DNA-double-strand breaks, chromosome aberrations and reproductive cell death in SW-1573 lung tumour cells&rdquo;, Oncology Reports, Vol.27, Spandidos Publications, Athens, https://doi.org/10.3892/or.2011.1604.&nbsp;</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Gardner, R.M., G.R. Sutherland, and L.G. Shaffer. (2011), &ldquo;Chapter 1: Elements in Medical Cytogenetics&rdquo; in Chromosome abnormalities and genetic counseling (No. 61), Oxford University Press, USA, pp.7-15.&nbsp;</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Griffiths, A. J. F., Miller, J. H., &amp; Suzuki, D. T. (2000), &ldquo;An Introduction to Genetic Analysis&rdquo;, 7th edition. New York: W. H. Freeman. Available from: https://www.ncbi.nlm.nih.gov/books/NBK21766/</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Hagmar, L. et al. (2004), &ldquo;Impact&nbsp;of&nbsp;types&nbsp;of&nbsp;lymphocyte&nbsp;chromosomal aberrations&nbsp;on&nbsp;human&nbsp;cancer risk: results from Nordic and Italian cohorts&rdquo;, Cancer Res. 64(6):2258-63.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Hastings PJ, Ira G &amp; Lupski JR. (2009), &ldquo;A microhomology-mediated&nbsp;break-induced&nbsp;replication&nbsp;model&nbsp;for the&nbsp;origin&nbsp;of human copy number variation&rdquo;. PLoS Genet. 2009 Jan;5(1): e1000327. doi: 10.1371/journal.pgen.1000327.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Hendriks, G. et al. (2012), &ldquo;The ToxTracker assay: novel GFP reporter systems that provide mechanistic insight into the genotoxic properties of chemicals&rdquo;, Toxicol Sci, 125:285-298. Doi: 10.1093/toxsci/kfr281.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Hendriks, G. et al. (2016), &ldquo;The Extended ToxTracker Assay Discriminates Between Induction of DNA Damage, Oxidative Stress, and Protein Misfolding&rdquo;, Toxicol Sci, 150:190-203. Doi: 10.1093/toxsci/kfv323.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Ka&scaron;uba, V., et al. (1995), &ldquo;Chromosome aberrations in peripheral blood lymphocytes from control individuals&rdquo;, Mutation Research Letters, Vol.346/4, Elsevier, Amsterdam, https://doi.org/10.1016/0165-7992(95)90034-9.&nbsp;</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Keren, B. (2014),&rdquo;The advantages of SNP arrays over CGH arrays&rdquo;, Molecular Cytogenetics.7( 1):I31. Doi: 10.1186/1755-8166-7-S1-I31.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Khoury, L., Zalko, D., Audebert, M. (2016), &ldquo;Evaluation of four human cell lines with distinct biotransformation properties for genotoxic screening&rdquo;, Mutagenesis. 31:83-96. Doi: 10.1093/mutage/gev058.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Khoury, L., Zalko, D., Audebert, M. (2013), &ldquo;Validation of high-throughput genotoxicity assay screening using cH2AX in-cell Western assay on HepG2 cells&rdquo;, Environ Mol Mutagen, 54:737-746. Doi: 10.1002/em.21817.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Lee JA, Carvalho CM, Lupski JR. (2007). &ldquo;Replication mechanism for generating nonrecurrent rearrangements&nbsp;associated&nbsp;with&nbsp;genomic&nbsp;disorders&rdquo;, Cell. 131(7):1235-47. Doi: 10.1016/j.cell.2007.11.037.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Liu B. et al. (2013). &ldquo;Computational methods for detecting copy number variations in cancer genome using next generation sequencing: principles and challenges&rdquo;, Oncotarget. 4(11):1868-81. Doi: 10.18632/oncotarget.1537.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Loucas, B.D., et al. (2013), &ldquo;Chromosome Damage in Human Cells by Gamma Rays, Alpha Particles and Heavy Ions: Track Interactions in Basic Dose-Response Relationships&rdquo;, Radiation Research, Vol.179/1, Radiation Research Society, Indianapolis, https://doi.org/10.1667/RR3089.1.&nbsp;</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Mitelman, F. (1982), &ldquo;Application of cytogenetic methods to analysis of etiologic factors in carcinogenesis&rdquo;, IARC Sci Publ, 39:481-496.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Mukherjee. S. et al. (2017),&nbsp;&ldquo;Addition&nbsp;of&nbsp;chromosomal&nbsp;microarray&nbsp;and&nbsp;next generation sequencing&nbsp;to&nbsp;FISH&nbsp;and classical cytogenetics enhances genomic profiling of myeloid malignancies, Cancer Genet. 216-217:128-141. doi: 10.1016/j.cancergen.2017.07.010.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Obe, G. et al. (2002), &ldquo;Chromosomal Aberrations: formation, Identification, and Distribution&rdquo;, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 504(1-2), 17-36. Doi: 10.1016/s0027-5107(02)00076-3.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Savage, J.R. (1976), &ldquo;Classification and relationships of induced chromosomal structual changes&rdquo;, J Med Genet, 13:103-122. Doi: 10.1136/jmg.13.2.103.</span></span></p>
  • <p><span style="font-size:14px">Shahane SA, Nishihara K, Xia M. High-Throughput and High-Content Micronucleus Assay in CHO-K1 Cells. Methods Mol Biol. 2016;1473:77-85. doi: 10.1007/978-1-4939-6346-1_9. PMID: 27518626.</span></p>
  • <p>OECD (2016a),&nbsp;<em>Test No. 487: In Vitro Mammalian Cell Micronucleus Test</em>, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris,&nbsp;<a href="https://doi.org/10.1787/9789264264861-en" title="">https://doi.org/10.1787/9789264264861-en</a>.</p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">OECD (2016a), Test No. 487: In Vitro Mammalian Cell Micronucleus Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264264861-en.&nbsp;</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">OECD. (2016b), &ldquo;Test No. 474: Mammalian Erythrocyte Micronucleus Test. OECD Guideline for the Testing of Chemicals, Section 4.&rdquo;Paris: OECD Publishing.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">OECD. (2016c), &ldquo;In Vitro Mammalian Chromosomal Aberration Test 473.&rdquo;</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">OECD. (2016d). Test No. 475: Mammalian Bone Marrow Chromosomal Aberration Test. OECD Guideline for the Testing of Chemicals, Section 4.&nbsp;Paris: OECD Publishing.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">OECD. (2016e). Test No. 483: Mammalian Spermatogonial Chromosomal Aberration Test. Paris: OECD Publishing.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Okayasu, R. and C. Liu. (2019), &ldquo;G1 premature chromosome condensation (PCC) assay&rdquo;, Methods in molecular biology, Humana Press, Totowa, https://doi.org/10.1007/978-1-4939-9432-8_4. &nbsp;</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Pathak, R., Koturbash, I., &amp; Hauer-Jensen, M. (2017), &ldquo;Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice&rdquo;, J Vis Exp(119). doi:10.3791/55162.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Prasanna, P. G. S., N. D. Escalada and W. F. Blakely (2000), &ldquo;Induction of premature chromosome condensation by a phosphatase inhibitor and a protein kinase in unstimulated human peripheral blood lymphocytes: a simple and rapid technique to study chromosome aberrations using specific whole-chromosome DNA hybridization probes for biological dosimetry&rdquo;, Mutation Research, Vol. 466/2, Elsevier B.V., Amsterdam, https://doi/org/10.1016/S1383-5718(00)00011-5&nbsp;</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Redon, R. et al. (2006), &ldquo;Global&nbsp;variation&nbsp;in&nbsp;copy&nbsp;number&nbsp;in the&nbsp;human genome&rdquo;, Nature.&nbsp;444(7118):444-54. 10.1038/nature05329.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Rodrigues, M. A., Beaton-Green, L. A., &amp; Wilkins, R. C. (2016), &ldquo;Validation of the Cytokinesis-block Micronucleus Assay Using Imaging Flow Cytometry for High Throughput Radiation Biodosimetry&rdquo;, Health Phys. 110(1): 29-36. doi:10.1097/HP.0000000000000371</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Schwartz, G. G. (1990), &ldquo;Chromosome aberrations. Biological Markers in Epidemiology (BS Hulka, TC Wlwosky, and JD Griffith, Eds.)&rdquo;, Oxford University Press, Oxford, pp.147-172. &nbsp;</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Shahane S, Nishihara K, Xia M. (2016), &ldquo;High-Throughput and High-Content Micronucleus Assay in CHO-K1 Cells&rdquo;, In: Zhu H, Xia M, editors. High-Throughput Screening Assays in Toxicology. New York, NY: Humana Press. p 77-85.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Shen.TW, &nbsp;(2016),&rdquo;Concurrent&nbsp;detection&nbsp;of&nbsp;targeted&nbsp;copy&nbsp;number&nbsp;variants&nbsp;and&nbsp;mutations&nbsp;using a myeloid malignancy next generation sequencing panel allows comprehensive genetic analysis using a single testing strategy&rdquo;, Br J Haematol. 173(1):49-58. doi: 10.1111/bjh.13921.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Shlien A, Malkin D. (2009), &ldquo;Copy&nbsp;number&nbsp;variations&nbsp;and&nbsp;cancer&rdquo;, Genome Med.&nbsp;1(6):62. doi: 10.1186/gm62.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Tucker, J.D., Preston, R.J. (1996), &ldquo;Chromosome aberrations, micronuclei, aneuploidy, sister chromatid exchanges, and cancer risk assessment&rdquo;, Mutat Res, 365:147-159.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Vick, E. et al. (2017), Age-related chromosomal aberrations in patients with diffuse large B-cell lymphoma, American Society of Hematology, https://doi.org/10.1182/blood.V130.Suppl_1.1571.1571&nbsp;</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Wilson, TE. et al.&nbsp; (2015), &ldquo;Large transcription units unify copy number variants and common fragile sites arising under replication stress&rdquo;, Genome Res. 25(2):189-200. doi: 10.1101/gr.177121.114.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Wink, S. et al. (2014), &ldquo;Quantitative high content imaging of cellular adaptive stress response pathways in toxicity for chemical safety assessment&rdquo;, Chem Res Toxicol, 27:338-355.</span></span></p>
  • <p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Zhang N, Wang M, Zhang P, Huang T. 2016. Classification&nbsp;of&nbsp;cancers&nbsp;based&nbsp;on&nbsp;copy number variation&nbsp;landscapes. Biochim Biophys Acta.&nbsp; 1860(11 Pt B):2750-5. doi: 10.1016/j.bbagen.2016.06.003.</span></span></p>
  • <h4><a href="/events/870">Event: 870: Increase, Cell Proliferation</a></h4>
  • <h5>Short Name: Increase, Cell Proliferation</h5>
  • <h4>Key Event Component</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Process</th>
  • <th scope="col">Object</th>
  • <th scope="col">Action</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>cell proliferation</td>
  • <td>epithelial cell</td>
  • <td>increased</td>
  • </tr>
  • <tr>
  • <td>cell proliferation</td>
  • <td>mesothelial cell</td>
  • <td>increased</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>AOPs Including This Key Event</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP ID and Name</th>
  • <th scope="col">Event Type</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/136">Aop:136 - Intracellular Acidification Induced Olfactory Epithelial Injury Leading to Site of Contact Nasal Tumors</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/303">Aop:303 - Frustrated phagocytosis-induced lung cancer</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/272">Aop:272 - Deposition of energy leading to lung cancer</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/409">Aop:409 - Frustrated phagocytosis leads to malignant mesothelioma</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/420">Aop:420 - Aryl hydrocarbon receptor activation leading to lung cancer through sustained NRF2 toxicity pathway</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/432">Aop:432 - Deposition of Energy by Ionizing Radiation leading to Acute Myeloid Leukemia</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/451">Aop:451 - Interaction with lung resident cell membrane components leads to lung cancer</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/478">Aop:478 - Deposition of energy leading to occurrence of cataracts</a></td>
  • <td>KeyEvent</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Stressors</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Name</th></tr>
  • </thead>
  • <tbody>
  • <tr><td>Ionizing Radiation</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Biological Context</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Level of Biological Organization</th></tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr><td>Cellular</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Domain of Applicability</h4>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>rat</td>
  • <td>Rattus norvegicus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>mouse</td>
  • <td>Mus musculus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>human</td>
  • <td>Homo sapiens</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>All life stages</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <p><span style="color:#27ae60"><strong>&nbsp;</strong></span>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. <span style="font-family:arial,helvetica,sans-serif">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.</span></p>
  • <p><span style="font-size:16px"><strong>Life stage applicability: </strong>This key event is not life stage specific (Fujimichi and Hamada, 2014; Barnard et al., 2022). </span></p>
  • <p><span style="font-size:16px"><strong>Sex applicability:</strong> This key event is not sex specific (Markiewicz et al., 2015).</span></p>
  • <p><strong>Evidence for perturbation by a stressor:</strong> There is a large body of evidence supporting the effectiveness of ionizing radiation, UV, and mechanical wounding as stressors for increased cell proliferation. These stressors can be subdivided into X-rays (van Sallmann, 1951; Ramsell and Berry, 1966; Richards, 1966; Riley et al., 1988; Riley et al., 1989; Kleiman et al., 2007; Pendergrass et al., 2010; Fujimichi and Hamada, 2014, Markiewicz et al., 2015; Bahia et al., 2018), 60Co &gamma;-rays (Hanna and O&rsquo;Brien, 1963; Barnard et al., 2022; McCarron et al., 2021), 137Cs &gamma;-rays (Andley and Spector, 2005), neutrons (Richards, 1966; Riley et al., 1988; Riley et al., 1989), 40Ar (Worgul et al., 1986), 56Fe (Riley et al., 1989), UVB (S&ouml;derberg et al., 1986; Andley et al., 1994; Cheng et al., 2019), UVC (Trenton and Courtois, 1981), and mechanical wounding (Riley et al., 1989).</p>
  • <h4>Key Event Description</h4>
  • <p>Throughout their life, cells replicate their organelles and genetic information before dividing to form two new daughter cells, in a process known as cellular proliferation. This replicative process is known as the cell cycle and is subdivided into various stages notably, G1, S, G2, and M in mammals. G1 and G2 are gap phases, separating mitosis and DNA synthesis. Differentiated cells typically remain in G1; however,&nbsp;quiescent cells reside in an optional phase just before G1, known as G0.&nbsp;&nbsp;</p>
  • <p>Progression through the cycle is dependent on sufficient nutrient availability to provide optimal nucleic acid, protein, and lipid levels, as well as sufficient cell mass. To this end, the cell cycle is mediated by three major checkpoints: the restriction (R) point, or G1/S checkpoint, controlling entry into S phase, the G2/M checkpoint, controlling entry into mitosis, and one more controlling entry into cytokinesis. If conditions are ideal for division, cells will pass the restriction point (G1/S) and begin the activation and expression of genes used for duplicating centrosomes and DNA, eventually leading to proliferation (Cuy&agrave;s et al., 2014).&nbsp;&nbsp;</p>
  • <p>Various protein complexes, known as cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs) regulate passage through each phase by activating and inhibiting specific processes (Lovicu et al., 2014). The CDKs are responsible for controlling progression through the cell cycle. They promote DNA synthesis and mitosis, and therefore cell division (Barnum &amp; O&rsquo;Connell, 2014). Furthermore, growth factors are required to stimulate cell division, but after passing through the restriction point at G1 they are no longer necessary (Lovicu et al., 2014).&nbsp;&nbsp;</p>
  • <p>In the context of cancer, one hallmark is the sustained and uncontrolled cell proliferation (Hanahan et al., 2011, Portt et al., 2011). When cells 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 tumor. This is often achieved through oncogene activation and inactivation of tumor suppressor genes (Hanahan et al., 2011). Cell inactivation and the replacement of these cells can initiate clonal expansion (Heidenreich adn Paretzke et al., 2008).&nbsp;</p>
  • <p>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.</p>
  • <h4>How it is Measured or Detected</h4>
  • <p>Two common methods of measuring cell proliferation in vivo are the use of Bromodeoxyuridine (5-bromo-2&#39;-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).&nbsp;Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed.</p>
  • <p>Two common methods of measuring cell proliferation in vivo are the use of Bromodeoxyuridine (5-bromo-2&#39;-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.&nbsp;Similarly, 5-iodo-2&#39;-deoxyuridine (IdU) is another analogue of thymidine used to measure cell proliferation as it is also incorporated into DNA during its synthesis (Devine &amp; Behbehani, 2021). 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).&nbsp;Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed.</p>
  • <table border="1" cellpadding="1" cellspacing="1" style="height:298px; width:595px">
  • <tbody>
  • <tr>
  • <td style="background-color:#dddddd; text-align:center"><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif"><strong>Assay Name</strong></span></span></td>
  • <td style="background-color:#dddddd; text-align:center"><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px"><strong>References</strong></span></span></td>
  • <td style="background-color:#dddddd; text-align:center"><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif"><strong>Description</strong></span></span></td>
  • <td style="background-color:#dddddd; text-align:center"><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px"><strong>OECD Approved Assay</strong></span></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">CyQuant Cell Proliferation Assay</span></span></td>
  • <td><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">Jones et al., 2001</span></span></td>
  • <td><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">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</span></span></td>
  • <td>N/A</td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Nucleotide Analog Incorporation Assays (e.g. BrdU, EdU)</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Romar et al., 2016, Roche; 2013</span></span></td>
  • <td><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">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</span></span></td>
  • <td><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">Yes (No. 442B)</span></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Cytoplasmic Proliferation Dye Assays</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Quah &amp; Parish, 2012</span></span></td>
  • <td><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">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</span></span></td>
  • <td><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">N/A</span></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Colourimetric Dye Assays</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Vega-Avila &amp; Pugsley, 2011; American Type Culture Collection</span></span></td>
  • <td><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">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</span></span></td>
  • <td><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">N/A</span></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px">BrdU, Ki67, IdU Quantification - Flow Cytometry&nbsp;</span></td>
  • <td><span style="font-size:12px">Ligasov&aacute; et al., 2017; Devine &amp; Behehani, 2021; Kim &amp; Sederstrom, 2015</span></td>
  • <td><span style="font-size:12px">Measurement of cell proliferation biomarkers by flow cytometry, normalized to total levels of BrdU, Ki67 or IdU.&nbsp;&nbsp;&nbsp;</span></td>
  • <td><span style="font-size:12px">No</span></td>
  • </tr>
  • </tbody>
  • </table>
  • <p>&nbsp;</p>
  • <h4>References</h4>
  • <p>Andley, U. P. et al. (1994), &ldquo;Modulation of lens epithelial cell proliferation by enhanced prostaglandin synthesis after UVB exposure&rdquo;, Investigative Ophthalmology &amp; Visual Science, Vol. 35/2, Rockville, pp<span style="font-size:16px">. 374-381&nbsp;&nbsp;</span></p>
  • <p>Andley, U. and A. Spector (2005), &ldquo;Peroxide resistance in human and mouse lens epithelial cell lines is related to long-term changes in cell biology and architecture&rdquo;, Free Radical Biology &amp; Medicine, Vol. 39/6, Elsevier B.V, United States, https://doi.org/10.1016/j.freeradbiomed.2005.04.028&nbsp;</p>
  • <p>Bahia, S. et al. (2018), &ldquo;Oxidative and nitrative stress-related changes in human lens epithelial cells following exposure to X-rays&rdquo;, International journal of radiation biology, Vol. 94/4, England, <a href="https://doi.org/10.1080/09553002.2018.1439194" rel="noreferrer noopener" target="_blank">https://doi.org/10.1080/09553002.2018.1439194</a>&nbsp;</p>
  • <p>Barnard, S. et al. (2022), &ldquo;Lens Epithelial Cell Proliferation in Response to Ionizing Radiation.&rdquo;, Radiation Research, Vol. 197/1, Radiation Research Society, United States, <a href="https://doi.org/10.1667/RADE-20-00294.1" rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/RADE-20-00294.1</a>&nbsp;</p>
  • <p>Barnum, K. and M. O&rsquo;Connell (2014), &ldquo;Cell cycle regulation by checkpoints&rdquo;, in Cell cycle control, Springer, New York, http://doi.org/ 10.1007/978-1-4939-0888-2&nbsp;</p>
  • <p><span style="font-size:16px"><span style="font-family:arial,sans-serif">Bogdanffy. et al. (1997). &ldquo;FOUR-WEEK INHALATION CELL PROLIFERATION STUDY OF THE EFFECTS OF VINYL ACETATE ON RAT NASAL EPITHELIUM&rdquo;, Inhalation Toxicology, Taylor &amp; Francis. 9: 331-350.</span></span></p>
  • <p>Cheng, T. et al. (2019), &ldquo;lncRNA H19 contributes to oxidative damage repair in the early age-related cataract by regulating miR-29a/TDG axis&rdquo;, Journal of cellular and molecular medicine, Vol. 23/9, Wiley Subscription Services, Inc. England, https://doi.org/10.1111/jcmm.14489&nbsp;</p>
  • <p>Cuy&agrave;s, E. et al. (2014), &ldquo;Cell cycle regulation by the nutrient-sensing mammalian target of rapamycin (mTOR) pathway&rdquo;, in Cell cycle control, Springer, New York, http://dx.doi.org/ 10.1007/978-1-4939-0888-2&nbsp;</p>
  • <p>Devine,R. D, and G. K. Behbehani (2021), &ldquo;Use of the Pyrimidine Analog, 5-Iodo-2&#39;-Deoxyuridine (IdU) with Cell Cycle Markers to establish Cell Cycle Phases in a Mass Cytometry Platform&rdquo;, Journal of visualized experiments. (176). doi:10.3791/60556&nbsp;&nbsp;</p>
  • <p>Fujimichi, Y. and N. Hamada (2014), &ldquo;Ionizing irradiation not only inactivates clonogenic potential in primary normal human diploid lens epithelial cells but also stimulates cell proliferation in a subset of this population&rdquo;, PloS one, Vol. 9/5, e98154, Public Library of Science, United States, <a href="https://doi.org/10.1371/journal.pone.0098154" rel="noreferrer noopener" target="_blank">https://doi.org/10.1371/journal.pone.0098154</a>&nbsp;</p>
  • <p><span style="font-size:16px"><span style="font-family:arial,sans-serif">Grogan. et al. (1988). &ldquo;Independent prognostic significance of a nuclear proliferation antigen in diffuse large cell lymphomas as determined by the monoclonal antibody Ki-67&rdquo;, Blood. 71: 1157-1160.</span></span></p>
  • <p><span style="font-size:16px">Hanna, C. and J. E. O&rsquo;Brien (1963), &ldquo;Lens epithelial cell proliferation and migration in radiation cataracts&rdquo;, Radiation research, Academic Press, Inc, United States, <a href="https://doi.org/10.2307/3571405" rel="noreferrer noopener" target="_blank">https://doi.org/10.2307/3571405</a>&nbsp;</span></p>
  • <p><span style="font-size:16px"><span style="font-family:arial,sans-serif">H</span><span style="font-family:arial,sans-serif">anahan, D. &amp; R. A. Weinberg, (2011),&rdquo; Hallmarks&nbsp;of&nbsp;cancer: the&nbsp;next&nbsp;generation&rdquo;, Cell.&nbsp;144(5):646-74. doi: 10.1016/j.cell.2011.02.013.</span></span></p>
  • <p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">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. </span></span></p>
  • <p><span style="font-size:16px"><span style="font-family:arial,sans-serif">Jones, J. L. et al. </span><span style="font-family:arial,sans-serif">(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.</span></span></p>
  • <p>Kim, K. H. and&nbsp;Sederstrom J. M. (2015), &ldquo;Assaying Cell Cycle Status Using Flow Cytometry.&rdquo; Current protocols in molecular biology, 111:28.6.1-28.6.11., doi:10.1002/0471142727.mb2806s111&nbsp;&nbsp;</p>
  • <p>Kleiman, N. J. et al. (2007), &ldquo;Mrad9 and Atm haplinsufficiency enhance spontaneous and X-ray-induced cataractogenesis in mice&rdquo;, Radiation research, Vol. 168/5, Radiation Research Society, United States, <a href="https://doi.org/10.1667/rr1122.1" rel="noreferrer noopener" target="_blank">https://doi.org/10.1667/rr1122.1</a>&nbsp;</p>
  • <p>Ligasov&aacute;, A. et al. (2017), &ldquo;Cell cycle profiling by image and flow cytometry: The optimised protocol for the detection of replicational activity using 5-Bromo-2&#39;-deoxyuridine, low concentration of hydrochloric acid and exonuclease III.&rdquo; PloS one, 12(4): e0175880, doi:10.1371/journal.pone.0175880&nbsp;&nbsp;</p>
  • <p>Lovicu, J. et al (2014), &ldquo;Lens epithelial cell proliferation&rdquo;, in Lens epithelium and posterior capsular opacification, Springer, Tokyo, http://dx.doi.org/ 10.1007/978-4-431-54300-8_4&nbsp;</p>
  • <p>Markiewicz, E. et al. (2015), &ldquo;Nonlinear ionizing radiation-induced changes in eye lens cell proliferation, cyclin K1 expression and lens shape&rdquo;, Open biology, Vol. 5/4, The Royal Society, England, <a href="https://doi.org/10.1098/rsob.150011" rel="noreferrer noopener" target="_blank">https://doi.org/10.1098/rsob.150011</a>&nbsp;</p>
  • <p>McCarron, R. A. et al. (2021), &ldquo;Radiation-induced lens opacity and cataractogenesis: a lifetime study using mice of varying genetic backgrounds&rdquo;, Radiation research, Vol. 197/1, Radiation Research Society, United States, https://doi.org/10.1667/RADE-20-00266.1&nbsp;</p>
  • <p>Pendergrass, W. et al. (2010), &ldquo;X-ray induced cataract is preceded by LEC loss, and coincident with accumulation of cortical DNA, and ROS; similarities with age-related cataracts&rdquo;, Molecular vision, Vol. 16, Molecular Vision, United States, pp. 1496-1513&nbsp;</p>
  • <p><span style="font-size:16px"><span style="font-family:arial,sans-serif">Pera, Mattias and Detzer (1977). &ldquo;Methods for determining the proliferation kinetics of cells by means of 5-bromodeoxyuridine&rdquo;, Cell Tissue Kinet.10: 255-264. Doi: 10.1111/j.1365-2184.1977.tb00293.x.</span></span></p>
  • <p><span style="font-size:16px"><span style="font-family:arial,sans-serif">Portt, L. et al. (2011), &ldquo;Anti-apoptosis&nbsp;and&nbsp;cell survival: a&nbsp;review&rdquo;, Biochim Biophys Acta. 21813(1):238-59. doi: 10.1016/j.bbamcr.2010.10.010.</span></span></p>
  • <p><span style="font-size:16px"><span style="font-family:arial,sans-serif">Quah, J. C. B. &amp; R. C. Parish (2012), &ldquo;New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes&rdquo;, Journal of Immunological Methods. 379(1-2), 1-14. doi: 10.1016/j.jim.2012.02.012.</span></span></p>
  • <p>Ramsell, T. G. and R. J. Berry (1966), &ldquo;Recovery from X-ray damage to the lens. The effects of fractionated X-ray doses observed in rabbit lens epithelium irradiated in vivo&rdquo;, British Journal of Radiology, Vol. 39/467, England, pp. 853-858&nbsp;</p>
  • <p>Riley, E. F. et al. (1988), &ldquo;Recovery of murine lens epithelial cells from single and fractionated doses of X rays and neutrons&rdquo;, Radiation Research, Vol. 114/3, Academic Press Inc, Oak Brook, https://doi.org/10.2307/3577127&nbsp;</p>
  • <p>Riley, E. F. et al. (1989), &ldquo;Comparison of recovery from potential mitotic abnormality in mitotically quiescent lens cells after X, neutron, and 56Fe irradiations&rdquo;, Radiation Research, Vol. 119/2, United States, pp. 232-254&nbsp;</p>
  • <p>Richards, R. D. (1966), &ldquo;Changes in lens epithelium after X-ray or neutron irradiation (mouse and rabbit)&rdquo;, Transactions of the American Ophthalmological Society, Vol. 64, United States, pp. 700-734&nbsp;</p>
  • <p><span style="font-family:arial,sans-serif">Roche Applied Science, (2013), &ldquo;Cell Proliferation Elisa, BrdU (Colourmetric)&nbsp;&raquo;. Version 16</span></p>
  • <p><span style="font-family:arial,sans-serif">Romar, A. G., S. T. Kupper &amp; J. S. Divito (2015), &ldquo;Research Techniques Made Simple: Techniques to Assess Cell Proliferation&rdquo;,&nbsp; Journal of Investigative Dermatology. 136(1), e1-7. doi: 10.1016/j.jid.2015.11.020.</span></p>
  • <p>S&ouml;derberg, P. G. et al. (1986), &ldquo;Unscheduled DNA synthesis in lens epithelium after in vivo exposure to UV radiation in the 300 nm wavelength region&rdquo;, Acta Ophthalmologica, Vol. 64/2, Blackwell Publishing Ltd, Oxford, UK, https://doi.org/10.1111/j.175<span style="font-size:16px">5-3768.1986.tb06894.x&nbsp;</span></p>
  • <p>Trenton, J. A. and Y. Courtois (1981), &ldquo;Evolution of the distribution, proliferation and ultraviolet repair capacity of rat lens epithelial cells as a function of maturation and aging&rdquo;, Mechanisms of Ageing and Development, Vol. 15/3, Elsevier, Ireland, https://doi.org/1016/0047-6374(81)90134-2&nbsp;</p>
  • <p><span style="font-size:16px"><span style="font-family:arial,sans-serif">Vega-Avila, E. &amp; K. M. Pugsley (2011), &ldquo;An Overview of Colorimetric Assay Methods Used to Assess Survival or Proliferation of Mammalian Cells&rdquo;, Proc. West. Pharmacol. Soc. 54, 10-4.</span></span></p>
  • <p>von Sallmann, L. (1951), &ldquo;Experimental studies on early lens changes after x-ray irradiation III. Effect of X-radiation on mitotic activity and nuclear fragmentation of lens epithelium in normal and cysteine-treated rabbits&rdquo;, Transactions of the American Ophthalmological Society, Vol. 48, United States, pp. 228-242&nbsp;</p>
  • <p>Worgul, B. V. et al. (1986), &ldquo;Accelerated heavy particles and the lens II. Cytopathological changes&rdquo;, Investigative Ophthalmology and Visual Science, Vol 27/1, pp. 108-114&nbsp;</p>
  • <h3>List of Adverse Outcomes in this AOP</h3>
  • <h4><a href="/events/1556">Event: 1556: Increase, lung cancer</a></h4>
  • <h5>Short Name: Increase, lung cancer</h5>
  • <h4>AOPs Including This Key Event</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP ID and Name</th>
  • <th scope="col">Event Type</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/272">Aop:272 - Deposition of energy leading to lung cancer</a></td>
  • <td>AdverseOutcome</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Stressors</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Name</th></tr>
  • </thead>
  • <tbody>
  • <tr><td>Ionizing Radiation</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Biological Context</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr><th scope="col">Level of Biological Organization</th></tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr><td>Organ</td></tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Domain of Applicability</h4>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>human</td>
  • <td>Homo sapiens</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>rat</td>
  • <td>Rattus norvegicus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>mouse</td>
  • <td>Mus musculus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>All life stages</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">Lung cancer and subsequent metastasis occurs in multicellular eukaryotic vertebrate organisms that have lungs.</span></span></p>
  • <h4>Key Event Description</h4>
  • <p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">Abnormally high levels of cell proliferation in the lungs may eventually culminate in the formation of malignant tumours and thus lung cancer. The term lung cancer refers to all malignant neoplasms arising from the bronchial, bronchiolar, and alveolar epithelium (Keshamouni et al., 2009). The cellular origin(s) of lung cancer remains largely unknown. It has been speculated that different tumour histopathological subtypes arise from distinct cells of origin localized in defined microenvironments. Histological characteristics of lung cancers, as defined by light microscopy, have led to the categorization of lung cancers into four main subtypes: small cell carcinoma, adenocarcinoma, squamous cell carcinoma, and large cell carcinoma (Beasly et al., 2005). These histological subtypes are grouped under one of the two umbrella terms used to describe lung cancers:&nbsp; small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The term SCLC refers to small cell carcinoma. The term NSCLC, which represents approximately 85% of all lung cancers (Molina et al., 2008), encompasses squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. These three tumour types are grouped together due to similarities in their prognosis and management (Keshamouni et al., 2009); patients with NSCLC often have poor prognoses and low 5-year survival rates due to the high metastatic potential of the tumours (Spira and Ettinger, 2004; Herbst et al., 2008). Some of the most common sites for lung cancer metastasis are the other lobe of the lungs, skeleton, adrenal glands, liver, and brain (Simon et al., 2015). </span></span></p>
  • <h4>How it is Measured or Detected</h4>
  • <table border="1" cellpadding="1" cellspacing="1" style="height:1664px; width:645px">
  • <tbody>
  • <tr>
  • <td style="background-color:#dddddd; text-align:center"><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif"><strong>Assay Name</strong></span></span></td>
  • <td style="background-color:#dddddd"><span style="font-size:12px"><strong><span style="font-family:arial,helvetica,sans-serif">Reference</span></strong></span></td>
  • <td style="background-color:#dddddd; text-align:center"><span style="font-size:12px"><strong><span style="font-family:arial,helvetica,sans-serif">Description</span></strong></span></td>
  • <td style="background-color:#dddddd; text-align:center"><span style="font-size:12px"><strong><span style="font-family:arial,helvetica,sans-serif">OECD Approved Assay</span></strong></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Computed Tomography (CT) Scans: CT, High-Resolution CT (HRCT), and&nbsp;&nbsp; Positron Emission Tomography-CT (PET-CT)</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Bach et al., 2012; Ollier et al., 2014</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">CT scans are described as a 3D X-ray; They provide cross-sections of organs/tissues/bones, and can thus be used to detect tumours</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">N/A</span></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Magnetic Resonance Imaging (MRI)</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Khalil et al., 2016; Wu et al., 2011 </span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">This technique uses magnetic fields and radio waves (NOT ionizing radiation) to generate a picture of organs, and can thus be used to detect tumours</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">N/A</span></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Sputum Analysis</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Hubers et al., 2013</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Sputum is collected and analyzed for a variety of markers, including mutations in <em>KRAS</em> and <em>TP53</em>, specific RNA/protein biomarkers, and chromosomal aberrations</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">N/A</span></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Bronchoscopy: Conventional White Light Bronchoscopy, Autofluorescence Bronchoscopy (AFB), and Endobronchial Ultrasonography (EBUS)</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Ikeda et al., 2007</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Bronchoscope (usually with a camera) is passed down through the throat to the lungs to provide a visual of the respiratory tract; Traditionally, visualization has been performed using conventional white light, but new technologies have also allowed for visualization using fluorescence and ultrasound technologies </span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">N/A</span></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Transbronchial Needle Aspiration</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Navani et al., 2015; Aziz, 2012</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">A needle is used to aspirate a tissue sample from a lesion of suspected lung cancer&nbsp; for analysis</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">N/A</span></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Analysis of Volatile Organic Compounds in the Breath</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Zhou et al., 2017</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Volatile organic compounds, which may act as lung cancer biomarkers, are&nbsp; collected from the breath and quantified (mostly using mass spectrometry)</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">N/A</span></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Cell Transformation Assays</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Redpath et al., 1987</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Measurement of the tumourigenicity of a tumour/biopsy sample by analyzing changes in cell physiology and morphology in response to tumour-inducing radiation or chemicals </span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Yes (No. 231)</span></span></td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Rodent Two-Year Cancer Bioassays</span></span></p>
  • <p>&nbsp;</p>
  • <p><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">(Carcinogenicity Studies)</span></span></p>
  • </td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Matsumo, 2012; Nambiar, 2014; Maronpot, 2015</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Animals are exposed to a possible carcinogen for a long period of time (often two years), allowing for long-term cancer-related studies</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Yes (No. 451)</span></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Window Chamber Models</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Moeller, 2004; Schafer, 2014; Chen, 2016</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Window chambers are implanted into the animal to observe tumour progression in living animals using imaging techniques such as <em>in vivo</em> microscopy, MRI or nuclear imaging</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">N/A</span></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Xenograft Assays</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Wang, 2018; Shi, 2017; Jin, 2018; Wang, 2017; Zhou, 2012</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Tumour cells (usually human) are grown <em>in vitro</em> and injected into animals to induce tumour growth and/or to test the tumourigenicity of the injected cells</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">N/A</span></span></td>
  • </tr>
  • </tbody>
  • </table>
  • <p>&nbsp;</p>
  • <h4>Regulatory Significance of the AO</h4>
  • <p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">At present the AOP framework is not readily used to support regulatory decision-making in radiation protection practices.The goal of developing this AOP is to bring attention to the framework as an effective means to organize knowledge and identify gaps associated with the mechanistic understanding of low dose radiation exposures. We have used lung cancer as the case example due to its relevance to both radiation and chemical risk assessment. This AOP will help build the concept of an &ldquo;all hazards&rdquo; approach to risk assessment, as it will be the first &nbsp;with a molecular initiating event that is specific to a radiation insult. This in turn could serve to identify networks that are critical to both radiation and chemical exposure scenarios and contribute to prioritizing co-exposures of relevance to risk assessment. By developing this AOP, we will support the necessary efforts highlighted by the international and national radiation protection agencies such as, the United Nations Scientific Committee on the Effects of Atomic Radiation, International Commission of Radiological Protection, International Dose Effect Alliance and the Electric Power Research Institute Radiation Program to consolidate and enhance the knowledge in understanding the mechanisms of low dose radiation exposures from the cellular to organelle levels within the system. </span></span></p>
  • <h4>References</h4>
  • <p><span style="font-size:12px">Aziz, F. (2012), &ldquo;Endobronchial ultrasound-guided transbronchial needle aspiration for staging of lung cancer: a concise review&rdquo;, Transl Lung Cancer Res, 1(3), 208-213. doi:10.3978/j.issn.2218-6751.2012.09.08.</span></p>
  • <p><span style="font-size:12px">Bach, P. B. et al. (2012), &ldquo;Benefits and harms of CT screening for lung cancer: a systematic review&rdquo;, JAMA, 307(22), 2418-2429. doi:10.1001/jama.2012.5521</span></p>
  • <p><span style="font-size:12px">Beasley, M. B., Brambilla, E., &amp; Travis, W. D. (2005), &ldquo;The 2004 World Health Organization classification of lung tumors&rdquo;, Seminars in Roentgenology, 40(2), 90-97. doi:10.1053/j.ro.2005.01.001</span></p>
  • <p><span style="font-size:12px">Chen Y, Maeda A, Bu J, DaCosta R. (2016), &ldquo;Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow&rdquo;, J Vis Exp. (113). doi: 10.3791/54205.</span></p>
  • <p><span style="font-size:12px">Herbst, R. S., Heymach, J. V., &amp; Lippman, S. M. (2008), &ldquo;Lung cancer&rdquo;, N Engl J Med. 359, 1367&ndash; 80.</span></p>
  • <p><span style="font-size:12px">Hubers, A. J. et al. (2013), &ldquo;Molecular sputum analysis for the diagnosis of lung cancer&rdquo;, Br J Cancer. 109(3), 530-537. doi:10.1038/bjc.2013.393</span></p>
  • <p><span style="font-size:12px">Ikeda, N. et al. (2007), &ldquo;Comprehensive diagnostic bronchoscopy of central type early stage lung cancer&rdquo;, Lung Cancer, 56(3), 295-302. doi:10.1016/j.lungcan.2007.01.009</span></p>
  • <p><span style="font-size:12px">Jin, Y. et al. (2018), &ldquo;Simvastatin inhibits the development of radioresistant esophageal cancer cells by increasing the radiosensitivity and reversing EMT process via the PTEN-PI3K/AKT pathway&rdquo;, Exp Cell Res.362(2):362-369. Doi: 10.1016/j.yexcr.2017.11.037.</span></p>
  • <p><span style="font-size:12px">Keshamouni, V., Arenberg, D., &amp; Kalemkerian, G. (2009), &ldquo;Lung Cancer Metastasis: Novel Biological Mechanisms and Impact on Clinical Practice&rdquo;, Springer Science + Business Media. Doi: 10.1007/978-1-4419-0772-1.</span></p>
  • <p><span style="font-size:12px">Khalil, A.et al. (2016), &ldquo;Contribution of magnetic resonance imaging in lung cancer imaging&rdquo;, Diagnostic and Interventional Imaging, 97(10), 991-1002. doi:10.1016/j.diii.2016.08.015</span></p>
  • <p><span style="font-size:12px">Maronpot RR, Thoolen RJ, Hansen B. (2015), &ldquo;Two-year carcinogenicity study of acrylamide in Wistar Han rats with in utero exposure&rdquo;,Exp Toxicol Pathol. 67(2):189-95. doi: 10.1016/j.etp.2014.11.009.</span></p>
  • <p><span style="font-size:12px">Matsumoto, M. et al. (2012), &ldquo;Carcinogenicity of ortho-phenylenediamine dihydrochloride in rats and mice by two-year drinking water treatment&rdquo;, &nbsp;Arch Toxicol. 86(5):791-804. doi: 10.1007/s00204-012-0800-z.</span></p>
  • <p><span style="font-size:12px">Moeller, BJ. et al.(2004), &ldquo;Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: role of reoxygenation, free radicals, and stress granules&rdquo;, Cancer Cell. 5(5):429-41.</span></p>
  • <p><span style="font-size:12px">Molina JR. et al. (2008), &ldquo;Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship&rdquo;, Mayo Clin Proc. 83(5):584-94. doi: 10.4065/83.5.584.</span></p>
  • <p><span style="font-size:12px">Nambiar PR. et al. (2015), &ldquo;Two-year carcinogenicity study in rats with a nonnucleoside reverse transcriptase inhibitor&rdquo;, Toxicol Pathol. 43(3):354-65. doi: 10.1177/0192623314544381.</span></p>
  • <p><span style="font-size:12px">Navani, N. et al. (2015), &ldquo;Lung cancer diagnosis and staging with endobronchial ultrasound-guided transbronchial needle aspiration compared with conventional approaches: an open-label, pragmatic, randomised controlled trial&rdquo;, Lancet Respir Med. 3(4), 282-9. doi: 10.1016/S2213-2600(15)00029-6</span></p>
  • <p><span style="font-size:12px">Ollier, M. et al. (2014), &ldquo;Chest CT scan screening for lung cancer in asbestos occupational exposure: a systematic review and meta-analysis&rdquo;, Chest. 145(6), 1339-1346. doi:10.1378/chest.13-2181</span></p>
  • <p><span style="font-size:12px">Redpath, J. L. et al. (1987), &ldquo;Neoplastic Transformation of Human Hybrid Cells by y Radiation: A Quantitative Assay&rdquo;, Radiat.Res. 110, 468-472.</span></p>
  • <p><span style="font-size:12px">Schafer R, Leung HM, Gmitro AF. (2014), &ldquo;Multi-modality imaging of a murine mammary window chamber for breast cancer research&rdquo;, Biotechniques. 57(1):45-50. Doi: 10.2144/000114191.</span></p>
  • <p><span style="font-size:12px">Sher, T., Dy, G. K., &amp; Adjei, A. A. (2008), &ldquo;Small cell lung cancer&rdquo;, MayoClin Proc. 83(3), 335-367. doi: 10.4065/83.3.355</span></p>
  • <p><span style="font-size:12px">Shi ZM. Et al.(2017), &ldquo;Downregulation of miR-218 contributes to epithelial-mesenchymal transition and tumor metastasis in lung cancer by targeting Slug/ZEB2 signaling&rdquo;, Oncogene. 36(18):2577-2588. Doi: 0.1038/onc.2016.414.</span></p>
  • <p><span style="font-size:12px">Simon,&nbsp; G.R., &amp; Brustugun, O.T. (2015), &ldquo;Metastatic Patterns of Lung Cancer&rdquo;, Oncolex Oncology Encyclopedia.&nbsp;<a href="http://oncolex.org/Lung-cancer/Background/MetastaticPatterns">http://oncolex.org/Lung-cancer/Background/MetastaticPatterns</a>.</span></p>
  • <p><span style="font-size:12px">Spira, A., &amp; Ettinger, D. S. (2004), &ldquo;Multidisciplinary management of lung cancer&rdquo;,Engl J Med. 350(4), 379&ndash;92. doi: 10.1056/NEJMra035536</span></p>
  • <p><span style="font-size:12px">Wang T. et al. (2017), &ldquo;Role of Nrf2 signaling pathway in the radiation tolerance of patients with head and neck squamous cell carcinoma: an in vivo and in vitro study&rdquo;, Onco Targets Ther. 2017 Mar 23;10:1809-1819.</span></p>
  • <p><span style="font-size:12px">Wang L. et al. (2018), &ldquo;K-ras mutation promotes ionizing radiation-induced invasion and migration of lung cancer in part via the Cathepsin L/CUX1 pathway&rdquo;, Exp Cell Res. 362(2):424-435. Doi: 10.1016/j.yexcr.2017.12.006.</span></p>
  • <p><span style="font-size:12px">Wu, N. Y. et al. (2011), &ldquo;Magnetic resonance imaging for lung cancer detection: experience in a population of more than 10,000 healthy individuals&rdquo;, BMC Cancer, 11, 242. doi:10.1186/1471-2407-11-242.</span></p>
  • <p><span style="font-size:12px">Zhou, J. et al. (2012), &ldquo;Antitumor activity of Endostar combined with radiation against human nasopharyngeal carcinoma in mouse xenograft models&rdquo;, Oncol Lett. 4(5):976-980. Doi: 10.3892/ol.2012.856.</span></p>
  • <p><span style="font-size:12px">Zhou, J. et al. (2017), &ldquo;Review of recent developments in determining volatile organic compounds in exhaled breath as biomarkers for lung cancer diagnosis&rdquo;, Anal Chim Acta, 996, 1-9. doi:10.1016/j.aca.2017.09.021</span></p>
  • <h2>Appendix 2</h2>
  • <h2>List of Key Event Relationships in the AOP</h2>
  • <div id="evidence_supporting_links">
  • <h3>List of Adjacent Key Event Relationships</h3>
  • <div>
  • <h4><a href="/relationships/1977">Relationship: 1977: Energy Deposition leads to Increase, DNA strand breaks</a></h4>
  • <h4>AOPs Referencing Relationship</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP Name</th>
  • <th scope="col">Adjacency</th>
  • <th scope="col">Weight of Evidence</th>
  • <th scope="col">Quantitative Understanding</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/272">Deposition of energy leading to lung cancer</a></td>
  • <td>adjacent</td>
  • <td>High</td>
  • <td>High</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/216">Deposition of energy leading to population decline via DNA strand breaks and follicular atresia</a></td>
  • <td>adjacent</td>
  • <td>High</td>
  • <td></td>
  • </tr>
  • <tr>
  • <td><a href="/aops/238">Deposition of energy leading to population decline via DNA strand breaks and oocyte apoptosis</a></td>
  • <td>adjacent</td>
  • <td></td>
  • <td></td>
  • </tr>
  • <tr>
  • <td><a href="/aops/478">Deposition of energy leading to occurrence of cataracts</a></td>
  • <td>adjacent</td>
  • <td>High</td>
  • <td>High</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/470">Deposition of energy leads to vascular remodeling</a></td>
  • <td>adjacent</td>
  • <td>High</td>
  • <td>High</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/483">Deposition of Energy Leading to Learning and Memory Impairment</a></td>
  • <td>adjacent</td>
  • <td>High</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Evidence Supporting Applicability of this Relationship</h4>
  • <div>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>mouse</td>
  • <td>Mus musculus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>human</td>
  • <td>Homo sapiens</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>rat</td>
  • <td>Rattus norvegicus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>bovine</td>
  • <td>Bos taurus</td>
  • <td>Low</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9913" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>rabbit</td>
  • <td>Oryctolagus cuniculus</td>
  • <td>Low</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9986" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>Pig</td>
  • <td>Pig</td>
  • <td>Low</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <div>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>All life stages</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <div>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">This KER is plausible in all life stages, sexes, and organisms with DNA. The majority of the evidence is from In vivo adult mice and human In vitro models that do not specify the sex.&nbsp;</span></span></p>
  • <h4>Key Event Relationship Description</h4>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Direct deposition of ionizing energy refers to imparted energy interacting directly with the DNA double helix and producing randomized damage. This can be in the form of double strand breaks (DSBs), single-strand breaks, base damage, or the crosslinking of DNA to other molecules (Smith et al., 2003; Joiner, 2009; Christensen, 2014; Sage and Shikazono, 2017). Among these, the most detrimental type of DNA damage to a cell is DSBs. They are caused by the breaking of the sugar-phosphate backbone on both strands of the DNA double helix molecule, either directly across from each other or several nucleotides apart (Ward, 1988; Iliakis et al., 2015). This occurs when high-energy subatomic particles interact with the orbital electrons of the DNA causing ionization (where electrons are ejected from atoms) and excitation (where electrons are raised to higher energy levels) (Joiner, 2009). The number of DSBs produced and the complexity of the breaks is highly dependent on the amount of energy deposited on and absorbed by the cell. This can vary as a function of the dose-rate (Brooks et al., 2016) and the radiation quality which is a function of its linear energy transfer (LET) (Sutherland et al., 2000; Nikjoo et al., 2001; Jorge et al., 2012). LET describes the amount of energy that an ionizing particle transfers to media per unit distance (Smith et al., 2003; Okayasu, 2012a; Christensen et al., 2014).&nbsp;High LET radiation, such as alpha particles, heavy ion particles, and neutrons&nbsp;can deposit larger quantities of energy within a single track than low LET radiation, such as &gamma;-rays,&nbsp;X-rays, electrons, and protons&nbsp;(Kadhim et al., 2006; Franken et al., 2012; Frankenberg et al., 1999; Rydberg et al., 2002; Belli et al., 2000; Antonelli et al., 2015). As such, radiation with higher LETs tends to produce more complex, dense structural damage, particularly in the form of clustered damage, in comparison to lower LET radiation (Nikjoo et al., 2001; Terato and Ide, 2005; Hada and Georgakilas, 2008; Okayasu, 2012a; Lorat et al., 2015; Nikitaki et al., 2016). Thus, the complexity and yield of clustered DNA damage increases with ionizing density (Ward, 1988; Goodhead, 2006). However, clustered damage can also be induced even by a single radiation track through a cell.</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Direct deposition of ionizing energy refers to imparted energy interacting directly with the DNA double helix and producing randomized damage. This can be in the form of double strand breaks (DSBs), single-strand breaks, base damage, or the crosslinking of DNA to other molecules (Smith et al., 2003; Joiner, 2009; Christensen, 2014; Sage and Shikazono, 2017). Among these, the most detrimental type of DNA damage to a cell is DSBs. They are caused by the breaking of the sugar-phosphate backbone on both strands of the DNA double helix molecule, either directly across from each other or several nucleotides apart (Ward, 1988; Iliakis et al., 2015). This occurs when high-energy subatomic particles interact with the orbital electrons of the DNA causing ionization (where electrons are ejected from atoms) and excitation (where electrons are raised to higher energy levels) (Joiner, 2009). The number of DSBs produced and the complexity of the breaks is highly dependent on the amount of energy deposited on and absorbed by the cell. This can vary as a function of the dose-rate (Brooks et al., 2016) and the radiation quality which is a function of its linear energy transfer (LET) (Sutherland et al., 2000; Nikjoo et al., 2001; Jorge et al., 2012). LET describes the amount of energy that an ionizing particle transfers to media per unit distance (Smith et al., 2003; Okayasu, 2012a; Christensen et al., 2014).&nbsp;High LET radiation, such as alpha particles, heavy ion particles, and neutrons&nbsp;can deposit larger quantities of energy within a single track than low LET radiation, such as &gamma;-rays,&nbsp;X-rays, electrons, and protons&nbsp;(Kadhim et al., 2006; Franken et al., 2012; Frankenberg et al., 1999; Rydberg et al., 2002; Belli et al., 2000; Antonelli et al., 2015). As such, radiation with higher LETs tends to produce more complex, dense structural damage, particularly in the form of clustered damage, in comparison to lower LET radiation (Nikjoo et al., 2001; Terato and Ide, 2005; Hada and Georgakilas, 2008; Okayasu, 2012a; Lorat et al., 2015; Nikitaki et al., 2016). Some data reports that low dose and low LER radiation can lead to complex lesions, which can cause unrepairable DNA damage. However, determining the actual frequency of the complexity of these lesions has proven challenging (Wilkinson et al., 2023). The complexity and yield of clustered DNA damage increases with ionizing density (Ward, 1988; Goodhead, 2006). However, clustered damage can also be induced even by a single radiation track through a cell.</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">While the amount of DSBs produced depends on the radiation dose (see dose concordance), it also depends on several other factors. As the LET&nbsp;increases, the complexity of DNA damage increases, decreasing the repair rate, and increasing toxicity (Franken et al., 2012; Antonelli et al., 2015).</span></span></p>
  • <h4>Evidence Supporting this KER</h4>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Overall Weight of Evidence for this KER: High</span></span></p>
  • <strong>Biological Plausibility</strong>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The biological rationale linking the direct deposition of energy on DNA with an increase in DSB formation is strongly supported by numerous literature reviews that are available on this topic (J .F. Ward, 1988; Lipman, 1988; Hightower, 1995; Terato &amp; Ide, 2005; Goodhead, 2006; Kim &amp; Lee, 2007; Asaithamby et al., 2008; Hada &amp; Georgakilas, 2008; Jeggo, 2009; Clement, 2012; Okayasu, 2012b; Stewart, 2012; M. E. Lomax et al., 2013; EPRI, 2014; Hamada, 2014; Moore et al., 2014; Desouky et al., 2015; Ainsbury, 2016; Foray et al., 2016; Hamada &amp; Sato, 2016; Hamada, 2017a; Sage &amp; Shikazono, 2017; Chadwick, 2017; Wang et al., 2021; Nagane et al., 2021; Sylvester et al., 2018; Baselet et al., 2019). Ionizing radiation can be in the form of high energy particles (such as alpha particles, beta particles, or charged ions) or high energy photons (such as gamma-rays or X-rays). Ionizing radiation can break the DNA within chromosomes both directly and indirectly, as shown through using velocity sedimentation of DNA through neutral and alkaline sucrose gradients. The most direct path entails a collision between a high-energy particle or photon and a strand of DNA.</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Additionally, excitation of secondary electrons in the DNA allows for a cascade of ionization events to occur, which can lead to the formation of multiple damage sites (Joiner, 2009). As an example, high-energy electrons will traverse a DNA molecule in a mammalian cell within 10<sup>-18</sup>&nbsp;s and 10<sup>-14</sup>&nbsp;s, resulting in 100,000 ionizing events per 1 Gy dose in a 10 &mu;m cell (Joiner, 2009). The amount of damage can be influenced by factors such as the cell cycle stage and chromatin structure. It has been shown that in more condensed, packed chromatin structures such as those present in intact cells and heterochromatin, it is more difficult for the DNA to be damaged (Radulescu et al., 2006; Agrawala et al., 2008; Falk et al., 2008; Venkatesh et al., 2016). In contrast, DNA damage is more easily induced in lightly-packed chromatin such as euchromatin and&nbsp;nucleoids, (Radulescu et al., 2006; Falk et al., 2008; Venkatesh et al., 2016).</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Of the possible radiation-induced DNA damage types, DSB is considered to be the most harmful to the cell, as there may be severe consequences if this damage is not adequately repaired (Khanna &amp; Jackson, 2001; Smith et al., 2003; Okayasu, 2012a; M. E. Lomax et al., 2013; Rothkamm et al., 2015).</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">A considerable fraction of DSBs can also be formed in cells through indirect mechanisms.&nbsp; In this case, deposited energy can split water molecules near DNA, which can generate a significant quantity of reactive oxygen species in the form of hydroxyl free radicals (Ward, 1988; Wolf, 2008; Desouky et al., 2015; Maier et al., 2016, Cencer et al., 2018; Bains, 2019; Ahmadi et al., 2021). Estimates using models and experimental results suggest that hydroxyl radicals may be present within nanoseconds of energy deposition by radiation (Yamaguchi et al., 2005). These short-lived but highly reactive hydroxyl radicals may react with nearby DNA. This will produce DNA damage, including single-strand breaks and DSBs (Ward, 1988; Sasaki, 1998; Desouky et al., 2015; Maier et al., 2016). DNA breaks are especially likely to be produced if the sugar moiety is damaged, and DSBs occur when two single-strand breaks are in close proximity to each other&nbsp;(Ward, 1988).</span></span></p>
  • <strong>Empirical Evidence</strong>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Empirical data strongly supports this KER. The evidence presented below is summarized in table 1. The types of DNA damage produced by ionizing radiation and the associated mechanisms, including the induction of DSBs, are reviewed by Lomax et al. (2013) and documents produced by international radiation governing frameworks (Valentin, 1998; UNSCEAR, 2000). Other reviews also highlight the relationship between the deposition of energy by radiation and DSB induction, and discuss the various methods available to detect these DSBs (Terato &amp; Ide, 2005; Rothkamm et al., 2015; Sage &amp; Shikazono, 2017). A visual representation of the time frames and dose ranges probed by the dedicated studies discussed here is shown in Figures 1 &amp; 2 below.</span></span></p>
  • <p>&nbsp;</p>
  • <p>&nbsp;</p>
  • <p><img alt="" src="https://aopwiki.org/system/dragonfly/production/2022/10/12/4zw4nw353c_ke1_mie_dsb_dose_v2.png" style="height:734px; width:1000px" /></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Figure 1: Plot of example studies (y-axis) against equivalent dose (Sv) used to determine the empirical link between direct deposition of energy and DSBs. The z-axis denotes the equivalent dose rate used in each study. The y-axis is ordered from low LET to high LET from top to bottom.&nbsp;</span></span></p>
  • <p>&nbsp;</p>
  • <p>&nbsp;</p>
  • <p><img alt="" src="https://aopwiki.org/system/dragonfly/production/2022/10/12/a85jspx5_ke1_mie_dsb_time_v2.png" style="height:706px; width:1000px" /></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Figure 2: Plot of example studies (y-axis) against time scales used to determine the empirical link between direct deposition of energy and DSBs. The z-axis denotes the equivalent dose rate used in each study. The y-axis is ordered from low LET to high LET from top to bottom.&nbsp;</span></span></p>
  • <p>&nbsp;</p>
  • <p>&nbsp;</p>
  • <p><u><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dose Concordance</span></span></u></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is evidence in the literature suggesting a dose concordance between the direct deposition of energy by ionizing radiation and the incidence (Grudzenski et al., 2010) of DNA DSBs. Results from&nbsp;in vitro&nbsp;(Aufderheide et al., 1987; Sidjanin, 1993; Bucolo, 1994; Frankenberg et al., 1999; Rogakou et al., 1999; Belli et al., 2000; Sutherland et al., 2000; Lara et al., 2001; Rydberg et al., 2002; Baumstark-Kham et al., 2003; Rothkamm and Lo, 2003; Long, 2004; Kuhne et al., 2005; Sudprasert et al., 2006; Beels et al., 2009; Grudzenski et al., 2010; Liao, 2011; Franken et al., 2012; Bannik et al., 2013; Shelke &amp; Das, 2015; Antonelli et al., 2015;&nbsp;Markiewicz et al., 2015; Allen, 2018; Dalke, 2018; Bains, 2019; Ahmadi et al., 2021;&nbsp;Sabirzhanov et al., 2020; Ungvari et al., 2013; Rombouts et al., 2013; Baselet et al., 2017</span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">), in vivo&nbsp;(Reddy, 1998; Sutherland et al., 2000; Rube et al., 2008; Beels et al., 2009; Grudzenski et al., 2010; Markiewicz et al., 2015; Barnard, 2018; Barnard, 2019; Barnard, 2022;&nbsp;Schmal et al., 2019; Barazzuol et al., 2017; Geisel et al., 2012</span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">),&nbsp;ex vivo&nbsp;(Rube et al., 2008; Flegal et al., 2015) &nbsp;and simulation studies (Charlton et al., 1989) suggest that there is a positive, linear, dose-dependent increase in DSBs with increasing deposition of energy across a wide range of radiation types (iron ions, X-rays, ultrasoft X-rays, gamma-rays, photons, UV light, and alpha particles)&nbsp;and radiation doses (1 mGy - 100 Gy) (Aufderheide et al., 1987; Sidjanin, 1993; Frankenberg et al., 1999; Sutherland et al., 2000; de Lara et al., 2001; Baumstark-Khan et al., 2003; Rothkamm &amp; Lo, 2003; Kuhne et al., 2005; Rube et al., 2008; Grudzenski et al., 2010; Bannik et al., 2013; Shelke &amp; Das, 2015; Antonelli et al., 2015;&nbsp;Dalke, 2018;&nbsp;Barazzuol et al., 2017; Ungvari et al., 2013; Rombouts et al., 2013; Baselet et al., 2017; Geisel et al., 2012</span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">). DSBs have been predicted to occur at energy deposition levels as low as 75 eV (Charlton et al., 1989). </span></span></p>
  • <p>&nbsp;</p>
  • <p>&nbsp;</p>
  • <p><u><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Time Concordance</span></span></u></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is evidence suggesting a time concordance between the direct deposition of energy and the incidence of DSBs. A number of different models and experiments have provided evidence of ionizing radiation-induced foci (IRIF), which can be used to infer DSB formation seconds (Mosconi et al., 2011) or minutes after radiation exposure (Rogakou et al., 1999; Rothkamm and Lo, 2003; Rube et al., 2008; Beels et al., 2009; Kuefner et al., 2009; Grudzenski et al., 2010; Antonelli et al., 2015; Acharya et al., 2010; Sabirzhanov et al., 2020; Rombouts et al., 2013; N&uuml;bel et al., 2006; Baselet et al., 2017; Zhang et al., 2017</span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">).&nbsp;&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is evidence suggesting a time concordance between the direct deposition of energy and the incidence of DSBs. A number of different models and experiments have provided evidence of ionizing radiation-induced foci (IRIF), which can be used to infer DSB formation seconds (Mosconi et al., 2011) or minutes after radiation exposure (Rogakou et al., 1999; Rothkamm and</span></span><span style="font-size:11px"><span style="font-family:Arial,Helvetica,sans-serif"> </span>L&ouml;brich</span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">, 2003; Rube et al., 2008; Beels et al., 2009; Kuefner et al., 2009; Grudzenski et al., 2010; Antonelli et al., 2015; Acharya et al., 2010; Sabirzhanov et al., 2020; Rombouts et al., 2013; N&uuml;bel et al., 2006; Baselet et al., 2017; Zhang et al., 2017).&nbsp;&nbsp;</span></span></p>
  • <p>&nbsp;</p>
  • <p>&nbsp;</p>
  • <p><u><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Essentiality</span></span></u></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Deposition of energy is essential for DNA strand breaks. They can also be caused through other routes, such as oxidative stress (Cadet et al., 2012), but under normal physiological conditions deposition of energy is necessary. This was tested through many studies using&nbsp; various indicators such as 53BP1 foci/cell, &gamma;H2AX foci/cell, DNA migration, and the amount of DNA in tails for the comet assay. Various organisms such as humans, mice, rabbits, guinea pigs, and cattle were used. They showed that without the deposition of energy, there was only a negligible amount of DNA strand breaks (Aufderheide et al., 1987; Sidjanin, 1993; Bucolo, 1994; Reddy, 1998; Rogers, 2004; Bannik et al., 2013; Dalke, 2018; Bains, 2019; Barnard, 2019; Barnard, 2021). </span></span></p>
  • <strong>Uncertainties and Inconsistencies</strong>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Uncertainties and inconsistencies in this KER are as follows:</span></span></p>
  • <ul>
  • <li><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Studies have shown that dose-rates (Brooks et al., 2016) and radiation quality (Sutherland et al., 2000; Nikjoo et al., 2001; Jorge et al., 2012) are factors that can influence the dose-response relationship.&nbsp;&nbsp;</span></span></li>
  • <li><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Low-dose radiation has been observed to have beneficial effects and may even invoke protection against spontaneous genomic damage (Feinendegen, 2005; Day et al., 2007; Feinendegen et al., 2007; Shah et al., 2012; Nenoi et al., 2015; Dalke, 2018). This protective effect has been documented in&nbsp;in vivo&nbsp;and&nbsp;in vitro,&nbsp;as reviewed by ICRP (2007) and UNSCEAR (2008) and can vary depending on the cell type, the tissue, the organ, or the entire organism (Brooks et al., 2016).</span></span></li>
  • <li><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Depositing ionizing energy is a stochastic event; as such this can influence the location, degree and type of DNA damage imparted on a cell. As an example, studies have shown that mitochondrial DNA may also be an important target for genotoxic effects of ionizing radiation (Wu et al., 1999).</span></span></li>
  • </ul>
  • <h4>Quantitative Understanding of the Linkage</h4>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Quantitative understanding of this linkage suggests that DSBs can be predicted upon exposure to ionizing radiation. This is dependent on the biological model, the type of radiation and the radiation dose. In general, 1 Gy of radiation is thought to result in 3000 damaged bases (Maier et al., 2016), 1000 single-strand breaks, and 40 DSBs (Ward, 1988; Foray et al., 2016; Maier et al., 2016) . The table below provides representative examples of the calculated DNA damage rates across different model systems, most of which are examining DNA DSBs.</span></span></p>
  • <p>&nbsp;</p>
  • <p>&nbsp;</p>
  • <p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dose Concordance&nbsp;</span></span></strong></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The following tables provide representative examples of the relationship, unless otherwise indicated, all data is significantly significant.&nbsp;</span></span></p>
  • <table border="1">
  • <tbody>
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  • <td>
  • <p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Reference&nbsp;</span></span></strong></p>
  • </td>
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  • <p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Experiment Description&nbsp;</span></span></strong></p>
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  • <p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Result&nbsp;</span></span></strong></p>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ward, 1988&nbsp;</span></span></p>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Cells containing approximately 6 pg of DNA were exposed to 1 Gy.&nbsp;</span></span></p>
  • </td>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Under the assumption of 6 pg of DNA per cell. 60 eV of energy deposited per event over a total of 1 Gy. Deoxyribose (2.3 pg/cell): 14,000 eV deposited, 235 events. Bases (2.4 pg/cell): 14.7 keV deposited, 245 events. Phosphate (1.2 pg/cell): 7,300 eV deposited, 120 events. Bound water (3.1 pg/cell): 19 keV deposited, 315 events. Inner hydration shell (4.2 pg/cell): 25,000 eV deposited 415 events.&nbsp;</span></span></p>
  • </td>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Charlton, 1989&nbsp;</span></span></p>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In-silico. A computer simulation/model was used to test various types of radiation with doses from 0 to 400 eV (energy deposited) on the amount of DNA damage produced.&nbsp;</span></span></p>
  • </td>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Simulated dose-concordance prediction of increase in number of DSBs/54 nucleotide pairs as direct deposition of energy increases in the range 75-400 eV. In the range 100 - 150 eV: 0.38 DSBs/54 nucleotide pairs and at 400 eV: ~0.80 DSBs per 64 nucleotide pairs.&nbsp;</span></span></p>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sutherland, 2000&nbsp;</span></span></p>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human cells were exposed to <sup>137</sup>Cs &gamma;-rays (0 &ndash; 100 Gy, 0.16 &ndash; 1.6 Gy/min). The frequency of DSBs was determined using gel electrophoresis.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Using isolated bacteriophage T7 DNA and 0-100 Gy of &gamma; radiations, observed a response of 2.4 DSBs per megabase pair per Gy.&nbsp;</span></span></p>
  • </td>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rogakou et al., 1999&nbsp;</span></span></p>
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  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Normal human fibroblasts (IMR90) and human breast cancer cells (MCF7 were exposed to 0.6 and 2 Gy <sup>137</sup>Cs &gamma;-rays delivered at 15.7 Gy/min. The number of DSBs were determined by immunoblotting for &gamma;-H2AX.&nbsp;</span></span></p>
  • <p>&nbsp;</p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Radiation doses of 0.6 Gy &amp; 2 Gy to normal human fibroblasts (IMR90) and MCF7 cells resulted in 10.1 &amp; 12.2 DSBs per nucleus on average (0.6 Gy), respectively; increasing to 24 &amp; 27.1 DSBs per nucleus (2 Gy).&nbsp;</span></span></p>
  • </td>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kuhne et al., 2005&nbsp;</span></span></p>
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  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Primary human skin fibroblasts (HSF2) were exposed to 0 &ndash; 70 Gy <sup>60</sup>Co &gamma;-rays (0.33 Gy/min), X-rays (29 kVp, 1.13 Gy/min), and CKX-rays (0.14 Gy/min). The number of DSBs were determined with pulsed-field gel electrophoresis.&nbsp;</span></span></p>
  • </td>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">&gamma;-ray and X-ray irradiation of primary human skin fibroblasts (HSF2) at 0 - 70 Gy. &gamma;-rays: (6.1 &plusmn; 0.2) x 10-9 DSBs per base pair per Gy, X-rays: (7.0 &plusmn; 0.2) x 10-9 DSBs per base pair per Gy. CKX -rays: (12.1 &plusmn; 1.9) x 10-9 DSBs per base pair per Gy.&nbsp;</span></span></p>
  • </td>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rothkamm, 2003&nbsp;</span></span></p>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Primary human fibroblast cell lines MRC-5 (lung), HSF1 and HSF2 (skin), and180BR (deficient in DNA ligase IV) were exposed to 1 mGy &ndash; 100 Gy X-rays (90 kV). Low doses were delivered at 6 &ndash; 60 mGy/min and high doses were delivered at 2 Gy/min. The number of DSBs were determined with pulsed-field gel electrophoresis.&nbsp;</span></span></p>
  • </td>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">X-ray irradiation of primary human fibroblasts (MRC-5) in the range 1 mGy - 100 Gy, 35 DSBs per cell per Gy.&nbsp;</span></span></p>
  • </td>
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  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Grudzenski et al, 2010&nbsp;</span></span></p>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Primary human fibroblasts (HSF1) and C57BL/6NCrl adult mice were exposed to X-rays (2.5 &ndash; 200 mGy, 70 mGy/min), and photons (10 mGy &ndash; 1 Gy, 2 Gy/min (100 mGy and 1 Gy), and 0.35 Gy/min (10 mGy)). &gamma;-H2AX immunofluorescence was observed to determine DSBs.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">X-rays irradiating primary human fibroblasts (HSF1) in the range 2.5 - 100 mGy yielded a response of 21 foci per Gy. When irradiating adult C57BL/6NCrl mice with photons a response of 0.07 foci per cell at 10 mGy was found. At 100 mGy the response was 0.6 foci per cell and finally, at 1 Gy; 8 foci per cell.&nbsp;</span></span></p>
  • </td>
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  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">de Lara, 2001&nbsp;</span></span></p>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Chinese hamster cells (V79-4) were exposed to 0 &ndash; 20 Gy of<sup>&nbsp; 60</sup>Co &gamma;-rays (2 Gy/min), and ultrasoft X-rays (0.7 &ndash; 35 Gy/min): carbon-K shell (0.28 keV), copper L-shell (0.96 keV), aluminum K-shell (1.49 keV), and titanium K-shell (4.55 keV). The number of DSBs were determined with pulsed-field gel electrophoresis.&nbsp;</span></span></p>
  • </td>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">V79-4 cells irradiated with &gamma;-rays and ultrasoft X-rays (carbon K-shell, copper L-shell, aluminium K-shell and titanum K-shell) in the range 0 - 20 Gy. Response (DSBs per Gy per cell): &gamma;-rays: 41, carbon K-shell: 112, copper L-shell: 94, aluminum K-shell: 77, titanium K-shell: 56.&nbsp;</span></span></p>
  • </td>
  • </tr>
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  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">R&uuml;be et al., 2008&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo. Brain, lung, heart and small intestine tissue from adult SCID, A-T, BALB/c and C57BL/6NCrl mice; Whole blood and isolated lymphocytes from BALB/c and C57BL/6NCrl mice were exposed to 0.1 &ndash; 2 Gy of photons (whole body irradiation, 6 MV, 2 Gy/min) and X-rays (whole body irradiation, 90 kV, 2 Gy/min). &gamma;-H2AX foci were determined with immunochemistry to measure DSBs.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Linear dose-dependent increase in DSBs in the brain, small intestine, lung and heart of C57BL/6CNrl mice after whole-body irradiation with 0.1 - 1.0 Gy of radiation. 0.8 foci per cell (0.1 Gy) and 8 foci per cell (1 Gy).&nbsp;</span></span></p>
  • </td>
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  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Antonelli et al., 2015&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Primary human foreskin fibroblasts (AG01522) were exposed to 0 &ndash; 1 Gy of <sup>136</sup>Cs &gamma;-rays (1 Gy/min), protons (0.84 MeV, 28.5 keV/um), carbon ions (58 MeV/u, 39.4 keV/um), and alpha particles (americium-241, 0.75 MeV/u, 0.08 Gy/min, 125.2 keV/um). &gamma;-H2AX foci were determined with immunochemistry to measure DSBs.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Linear dose-dependent increase in the number of DSBs from 0 - 1 Gy for &gamma;-rays and alpha particles as follows: &gamma;-rays: 24.1 foci per Gy per cell nucleus, alpha particles: 8.8 foci per Gy per cell nucleus.&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Barnard et al., 2019&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo. 10-week-old female C57BL/6 mice were whole-body exposed to 0.5, 1, and 2 Gy of 60Co &gamma;-rays at 0.3, 0.063, and 0.014 Gy/min. p53 binding protein 1 (53BP1) foci were determined via immunofluorescence.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Central LECs showed a linear increase in mean 53BP1 foci/cell with the maximum dose and dose-rate displaying a 78x increase compared to control. Peripheral LECs and lower dose rates displayed similar results, with slightly fewer foci.&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Central LECs showed a linear increase in mean 53BP1 foci/cell with the maximum dose and dose-rate displaying a 78x increase compared to control. Peripheral LECs and lower dose rates displayed similar results, with slightly fewer foci.&nbsp;</span></span><span style="font-size:11px">Although an increase in dose-response was observed, an inverse-dose rate response was reported, with higher 53BP1 foci persisting at lower dose rates.</span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ahmadi et al., 2021&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human LEC cells were exposed to 137Cs &gamma;-rays at doses of 0, 0.1, 0.25, and 0.5 Gy and dose rates of 0.065 and 0.3 Gy/min. DNA strand breaks were measured using the comet assay.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Human LECs showed a gradual increase in the tail from the comet assay with the maximum dose and dose-rate displaying a 3.7x increase compared to control. Lower dose-rates followed a similar pattern with a lower amount of strand breaks.&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Hamada et al., 2006&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Primary normal human diploid fibroblast (HE49) cells were exposed to 0.1, 0.5, and 4 Gy X-rays at 240 kV with a dose rate of 0.5 Gy/min. The number of H2AX foci/cell, which represented DNA strand breaks, was determined 6 &ndash; 7 minutes after irradiation through fluorescence microscopy.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cells displayed a linear increase in the number of H2AX foci/cell, with the maximum dose displaying a 125x increase compared to control.&nbsp;</span></span></p>
  • <p>&nbsp;</p>
  • </td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dubrova &amp; Plumb, 2002</span></span></td>
  • <td>&nbsp;</td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">At 1 Gy observe 70 DSBs, 1000 single-strange breaks and 2000 damaged DNA bases per cell per Gy.</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sabirzhanov et al., 2020</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Rat cortical neurons were exposed to 2, 8 or 32 Gy of X rays (320 kV) at a dose rate of 1.25 Gy/min. Western blot was used to measure &gamma;-H2AX, p-ataxia telangiectasia mutated (ATM) and p- ATM/RAD3-related (ATR) levels.&nbsp;&nbsp;</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In rat cortical neurons, p-ATM increased at 2, 8, and 32 Gy, with a 15-fold increase at 8 and 32 Gy. &gamma;-H2AX levels increased at 8 and 32 Gy.&nbsp;</span></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Geisel et al., 2012&nbsp;</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo. Patients with suspected coronary artery disease receiving X-rays from computed tomography or conventional coronary angiography had levels of DSBs assessed in blood lymphocytes by &gamma;-H2AX fluorescence.&nbsp;</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There was a correlation between effective dose (in mSv) and DSBs. For both conventional coronary angiography and computed tomography, a dose of 10 mSv produced about 2-fold more DNA DSBs than a dose of 5 mSv.&nbsp;</span></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ungvari et al., 2013&nbsp;</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Rat cerebromicrovascular endothelial cells and hippocampal neurons were irradiated with 2-10 Gy of <sup>137</sup>Cs gamma rays. DNA strand breaks were assessed with the comet assay.&nbsp;</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">DNA damage increased at all doses (2-10 Gy). In the control, less than 5% of DNA was in the tail, while by 6 Gy, 35% of the DNA was in the tail in cerebromicrovascular endothelial cells and 25% was in the tail in neurons.&nbsp;</span></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rombouts et al., 2013&nbsp;</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. EA.hy926 cells and human umbilical vein endothelial cells were irradiated with various doses of X-rays (0.25 Gy/min). &gamma;-H2AX foci were assessed with immunofluorescence.&nbsp;</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">More &gamma;-H2AX foci were observed at higher doses in both cell types. In human umbilical vein endothelial cells, few foci/nucleus were observed at 0.05 Gy, with about 23 at 2 Gy. In EA.hy926 cells, few foci/nucleus were observed at 0.05 Gy, with about 37 at 2 Gy.&nbsp;</span></span></td>
  • </tr>
  • <tr>
  • <td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Baselet et al., 2017&nbsp;</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human telomerase-immortalized coronary artery endothelial cells were irradiated with various doses of X-rays (0.5 Gy/min). Immunocytochemical staining was performed for &gamma;-H2AX and 53BP1 foci.&nbsp;</span></span></td>
  • <td><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Doses of 0.05 and 0.1 Gy did not increase the number of &gamma;-H2AX foci, but 0.5 Gy increased foci number by 5-fold and 2 Gy by 15-fold. A dose of 0.05 Gy did not increase the number of 53BP1 foci, but 0.1 Gy, 0.5 Gy and 2 Gy increased levels by 3-fold, 7-fold and 8-fold, respectively.</span></span>&nbsp;</td>
  • </tr>
  • </tbody>
  • </table>
  • <p>&nbsp;</p>
  • <p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Time Concordance&nbsp;</span></span></strong></p>
  • <table border="1">
  • <tbody>
  • <tr>
  • <td>
  • <p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Reference&nbsp;</span></span></strong></p>
  • </td>
  • <td>
  • <p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Experiment Description&nbsp;</span></span></strong></p>
  • </td>
  • <td>
  • <p><strong><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Result&nbsp;</span></span></strong></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rogakou et al., 1999&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Normal human fibroblasts (IMR90), human breast cancer cells (MCF7), human astrocytoma cells (SF268), Indian muntjac Muntiacus muntjak normal skin fibroblasts, Xenopus laevisA6 normal kidney cells, Drosophila melanogaster epithelial cells, and Saccharomyces cerevisiae were exposed to 0.6, 2, 20, 22, 100, and 200 Gy 137Cs &gamma;-rays. Doses below 20 Gy were delivered at 15.7 Gy/min and other doses were delivered in 1 minute. DNA breaks were visualized using &gamma;-H2AX antibodies and microscopy.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">DSBs were present at 3 min and persisted from 15 - 60 min.&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Hamada &amp; Woloschak, 2017&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. human LECs were exposed to 0.025 Gy X-rays at 0.42 &ndash; 0.45 Gy/min. 53BP1 foci were measured via indirect immunofluorescence.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In cells immediately exposed to 0.025 Gy, the level of 53BP1 foci/cell increased to 3.3x relative to control 0.5 h post-irradiation.&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Hamada et al., 2006&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Primary normal human diploid fibroblast (HE49) cells were exposed to 0.1, 0.5, and 4 Gy (deposition of energy) at 240 kV with a dose rate of 0.5 Gy/min. The number of H2AX foci/cell, which represented DNA strand breaks, was determined through fluorescence microscopy.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In cells immediately exposed to 0.5 Gy, 11% of cells had 18 foci six min post-irradiation, compared to 90% of controls having 0 foci.&nbsp;&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Acharya et al., 2010&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human neural stem cells were exposed to 1, 2 and 5 Gy of &gamma;-rays at a dose rate of 2.2 Gy/min. The levels of &gamma;-H2AX phosphorylation post irradiation were assessed by immunocytochemistry, fluorescence-activated cell sorting (FACS) analysis and &gamma;-H2AX foci enumeration.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The number of cells positive for nuclear &gamma;-H2AX foci peaked at 20 min post-irradiation. After 1h, this level quickly declined.&nbsp;&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Schmal et al., 2019&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo. Juvenile and adult C57BL/6 mice were exposed to whole body 6-MV photons at 2 Gy/min. Irradiations were done in 5x, 10x, 15x and 20x fractions of 0.1 Gy. Double staining for NeuN and 53BP1 was used to quantify DNA damage foci and the possible accumulation in the hippocampal dentate gyrus.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">To assess possible accumulation of persisting 53BP1-foci during fractionated radiation, juvenile and adult mice were examined 72 h after exposure to 5&times;, 10&times;, 15&times;, or 20&times; fractions of 0.1 Gy, compared to controls. The number of persisting 53BP1-foci increased significantly in both juvenile and adult mice during fractionated irradiation (maximum at 1 m post-IR).&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dong et al., 2015&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo. C57BL/6J mice were exposed to 2 Gy of X-rays at 2 Gy/min using a 6 MV source. &gamma;-H2AX foci were assessed with immunofluorescence in the brain.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">At 0.5 h, about 14 &gamma;-H2AX foci/cell were present. This decreased linearly to about 2 foci/cell at 24 h, with no foci/cell from 48 h to 6 weeks.&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Barazzuol et al., 2017&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo. C57BL/6 mice were exposed to 0.1 or 2 Gy of X-rays (250 kV) at a rate of 0.5 Gy/min. 53BP1 foci were quantified with immunofluorescence in neural stem cells and neuron progenitors in the lateral ventricle.&nbsp;&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">At both 0.5 and 6 h post-irradiation, increased 53BP1 foci were observed, with the highest level at 0.5 h.&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sabirzhanov et al., 2020&nbsp;</span></span></p>
  • <p>&nbsp;</p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Rat cortical neurons were exposed to 2, 8 or 32 Gy of X rays (320 kV) at a dose rate of 1.25 Gy/min. Western blot was used to measure &gamma;-H2AX, p-ATM and p-ATR levels.&nbsp;&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In rat cortical neurons, &gamma;-H2AX, p-ATM and p-ATR all increased at 30 minutes post-irradiation, with a sustained increase until 6 h.&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Zhang et al., 2017&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. HT22 hippocampal neuronal cellsT were irradiated with X-rays (320 kVp) at 8 or 12 Gy at a dose rate of 4 Gy/min. The comet assay was preformed to assess the DNA double strand breaks in HT22 cells. Western blot was used to measure &gamma;-H2AX and p-ATM.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">At 8 Gy, the comet assay showed an increased tail moment at both 30 minutes and 24 h post-irradiation. At 12 Gy, p-ATM was increased over 4-fold at both 30 minutes and 1 h post-irradiation. &gamma;-H2AX was increased over 3-fold at 30 minutes post-irradiation and almost 2-fold at 1 and 24 h.&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Geisel et al., 2012&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo. Patients with suspected coronary artery disease receiving X-rays from computed tomography or conventional coronary angiography had levels of DSBs assessed in blood lymphocytes by &gamma;-H2AX fluorescence.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">DSBs were increased at 1 h post-irradiation and returned to pre-irradiation levels by 24 h.&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Park et al., 2022&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human aortic endothelial cells were irradiated with 137Cs gamma rays at 4 Gy (3.5 Gy/min). &gamma;-H2AX was measured with western blot. p-ATM and 53BP1 were determined with immunofluorescence.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">&gamma;-H2AX, p-ATM, and 53BP1 were shown increased at 1 h post-irradiation and slightly decreased for the rest of the 6 h but remained elevated above the control.&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kim et al., 2014&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human umbilical vein endothelial cells were irradiated with 4 Gy of 137Cs gamma rays. &gamma;-H2AX levels were determined with immunofluorescence.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">&gamma;-H2AX foci greatly increased at 1 and 6 h post-irradiation, with the greatest increase at 1 h.&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dong et al., 2014&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human umbilical vein endothelial cells were irradiated with 2 Gy of 137Cs gamma rays. &gamma;-H2AX levels were determined with immunofluorescence.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">&gamma;-H2AX foci increased 8-fold at 3 h, 7-fold at 6 h, and 2-fold at 12 and 24 h post-irradiation.&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Rombouts et al., 2013&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. EA.hy926 cells and human umbilical vein endothelial cells were irradiated with X-rays (0.25 Gy/min). &gamma;-H2AX foci were assessed with immunofluorescence.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The greatest increase in &gamma;-H2AX foci was observed 30 minutes post-irradiation, while levels were still slightly elevated at 24 h.&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">N&uuml;bel et al., 2006&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human umbilical vein endothelial cells were irradiated with gamma rays at 20 Gy. DNA strand breaks were assessed with the comet assay and western blot for &gamma;-H2AX.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The olive tail moment increased 5-fold immediately after irradiation and returned to control levels by 4 h. A large increase in &gamma;-H2AX was observed at 0.5 h post-irradiation, with lower levels at 4 h but still above the control.&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Baselet et al., 2017&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vitro. Human telomerase-immortalized coronary artery endothelial cells were irradiated with various doses of X-rays (0.5 Gy/min). Immunocytochemical staining was performed for &gamma;-H2AX and 53BP1 foci.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Increased &gamma;-H2AX and 53BP1 foci were observed at 0.5 h post-irradiation, remaining elevated at 4 h but returning to control levels at 24 h.&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Gionchiglia et al., 2021&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In vivo. Male CD1 and B6/129 mice were irradiated with X-rays at 10 Gy. Brain sections were single or double-stained with antibodies against &gamma;-H2AX and p53BP1.&nbsp;&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">In the forebrain, cerebral cortex, hippocampus and subventricular zone (SVZ)/ rostral migratory stream (RMS)/ olfactory bulb (OB), &gamma;H2AX and p53BP1 positive cells increased at both 15 and 30 minutes post-irradiation, with the greatest increase at 30 minutes.&nbsp;</span></span></p>
  • </td>
  • </tr>
  • </tbody>
  • </table>
  • <p>&nbsp;</p>
  • <p>&nbsp;</p>
  • <strong>Response-response relationship</strong>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">There is evidence of a response-response relationship between the deposition of energy and the frequency of DSBs. In studies encompassing a variety of biological models, radiation types and radiation doses, a positive, linear relationship was found between the radiation dose and the number of DSBs (Aufderheide et al., 1987; Sidjanin, 1993; Frankenberg et al., 1999; Sutherland et al., 2000; de Lara et al., 2001; Baumstark-Khan et al., 2003; Rothkamm &amp; Lo, 2003; Kuhne et al., 2005; Rube et al., 2008; Grudzenski et al., 2010; Bannik et al., 2013; Shelke &amp; Das, 2015; Antonelli et al., 2015;&nbsp;Hamada, 2017b;&nbsp;Dalke, 2018; Barazzuol et al., 2017; Geisel et al., 2012; Ungvari et al., 2013; Rombouts et al., 2013; Baselet et al., 2017</span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">). There were, however, at least four&nbsp;exceptions reported. When human blood lymphocytes were irradiated with X-rays&nbsp;in vitro, a linear relationship was only found for doses ranging from 6 - 500 mGy; at low doses from 0 - 6 mGy, there was a quadratic relationship reported (Beels et al., 2009). Secondly, simulation studies predicted that there would be a non-linear increase in DSBs as energy deposition increased, with a saturation point at higher LETs (Charlton et al., 1989).&nbsp;Furthermore, primary normal human fibroblasts exposed to 1.2 &ndash; 5 mGy X-rays at 5.67 mGy/min showed a supralinear relationship, indicating at low doses, the DSBs are mostly due to radiation-induced bystander effects. Doses above 10 mGy showed a positive linear relationship (Ojima et al., 2008). Finally, in the human lens epithelial cell line SRA01/04, DNA strand breaks appeared immediately after exposure to UVB (0.14 J/cm2) and were repaired after 30 minutes. They then reappeared after 60 and 90 minutes. Both were once again repaired within 30 minutes. However, the two subsequent stages of DNA strand breaks did not occur when exposed to a lower dose of UVB (0.014 J/cm2) (Cencer et al., 2018). &nbsp;</span></span></p>
  • <strong>Time-scale</strong>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Data from temporal response studies suggests that DSBs likely occur within seconds to minutes of energy deposition by ionizing radiation. In a variety of biological models, the presence of DSBs has been well documented within 10 - 30 minutes of radiation exposure (Rogakou et al., 1999; Rube et al., 2008; Beels et al., 2009; Kuefner et al., 2009; Grudzenski et al., 2010; Antonelli et al., 2015; Acharya et al., 2010; Dong et al., 2015; Barazzuol et al., 2017; Sabirzhanov et al., 2020; Rombouts et al., 2013; N&uuml;bel et al., 2006; Baselet et al., 2017; Zhang et al., 2017; Gionchiglia et al., 2021</span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">); there is also evidence that DSBs may actually be present within 3 - 5 minutes of irradiation (Kleiman, 1990; Rogakou et al., 1999; Rothkamm &amp; Lo, 2003; Rube et al., 2008; Grudzenski et al., 2010; Cencer et al., 2018). Interestingly, one study that focussed on monitoring the cells before, during and after irradiation by taking photos every 5, 10 or 15 seconds found that foci indicative of DSBs were present 25 and 40 seconds after collision of the alpha particles and protons with the cell, respectively. The number of foci were found to increase over time until plateauing at approximately 200 seconds after alpha particle exposure and 800 seconds after proton exposure (Mosconi et al., 2011).</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">After the 30 minute mark, DSBs have been shown to rapidly decline in number. By 24 hours post-irradiation, DSB numbers had declined substantially in systems exposed to radiation doses between 40 mGy and 80 Gy (Aufderheide et al., 1987; Baumstark-Khan et al., 2003; Rothkamm &amp; Lo, 2003; Rube et al., 2008; Grudzenski et al., 2010; Bannik et al., 2013; Markiewicz et al., 2015; Russo et al., 2015; Antonelli et al., 2015; Dalke, 2018; Bains, 2019; Barnard, 2019; Ahmadi et al., 2021; Dong et al., 2015; Dong et al., 2014; Sabirzhanov et al., 2020; Rombouts et al., 2013; Baselet et al., 2017; Gionchiglia et al., 2021</span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">), with the sharpest decrease documented within the first 5 h (Kleiman, 1990; Sidjanin, 1993; Rogakou et al., 1999; Rube et al., 2008; Kuefner et al., 2009; Grudzenski et al., 2010; Bannik, 2013; Markiewicz et al., 2015; Shelke and Das, 2015; Cencer et al., 2018; Acharya et al., 2010; Park et al., 2022; Kim et al., 2014; N&uuml;bel et al., 2006</span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">). Interestingly, DSBs were found to be more persistent when they were induced by higher LET radiation (Aufderheide et al., 1987, Baumstark-Khan et al., 2003; Antonelli et al., 2015). </span></span></p>
  • <strong>Known modulating factors</strong>
  • <p>&nbsp;</p>
  • <table border="1">
  • <tbody>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Modulating Factor&nbsp;</strong></span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Details &nbsp;</strong></span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Effects on the KER &nbsp;</strong></span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>References &nbsp;</strong></span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Nitroxides&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Increased concentration&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Decreased DNA strand breaks.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">DeGraff et al., 1992; Citrin &amp; Mitchel, 2014&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">5-fluorouracil&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Increased concentration&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Increased DNA strand breaks.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">De Angelis et al., 2006; Citrin &amp; Mitchel, 2014&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Thiols&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Increased concentration&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Decreased DNA strand breaks.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Milligan et al., 1995; Citrin &amp; Mitchel, 2014&nbsp;</span></span></p>
  • </td>
  • </tr>
  • <tr>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cisplatin&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Increased concentration&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Decreased DNA break repair.&nbsp;</span></span></p>
  • </td>
  • <td>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sears &amp; Turchi; Citrin &amp; Mitchel, 2014&nbsp;</span></span></p>
  • </td>
  • </tr>
  • </tbody>
  • </table>
  • <strong>Known Feedforward/Feedback loops influencing this KER</strong>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Not identified.</span></span></p>
  • <h4>References</h4>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Agrawala, P.K. et al. (2008), &quot;Induction and repairability of DNA damage caused by ultrasoft X-rays: Role of core events.&quot;, Int. J. Radiat. Biol., 84(12):1093&ndash;1103. doi:10.1080/09553000802478083.</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ahmadi, M. et al. (2021), &ldquo;Early responses to low-dose ionizing radiation in cellular lens epithelial models&rdquo;, Radiation research, Vol.197/1, <em>Radiation Research Society</em>, United States, https://doi.org/10.1667/RADE-20-00284.1&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ainsbury, E. A. et al. (2016), &ldquo;Ionizing radiation induced cataracts: Recent biological and mechanistic developments and perspectives for future research&rdquo;, <em>Mutation research. Reviews in mutation research</em>, Vol. 770, Elsevier B.V., Amsterdam, https://doi.org/10.1016/j.mrrev.2016.07.010&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Alexander, J. L. and Orr-Weaver, T. L. (2016), &ldquo;Replication fork instability and the consequences of fork collisions from replication&rdquo;, <em>Genes &amp; Development</em>, Vol. 30/20, Cold Spring Harbor Laboratory Press, https://doi.org/ 10.1101/gad.288142.116&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Allen, C. H. et al. (2018), &ldquo;Raman micro-spectroscopy analysis of human lens epithelial cells exposed to a low-dose-range of ionizing radiation&rdquo;, <em>Physics in medicine &amp; biology</em>, Vol. 63/2, IOP Publishing, Bristol, https://doi.org/10.1088/1361-6560/aaa176&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Antonelli, A.F. et al. (2015), &quot;Induction and Repair of DNA DSB as Revealed by H2AX Phosphorylation Foci in Human Fibroblasts Exposed to Low- and High-LET Radiation: Relationship with Early and Delayed Reproductive Cell Death&quot;, Radiat. Res.&nbsp;183(4):417-31,&nbsp;doi:10.1667/RR13855.1.</span></span></p>
  • <p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Acharya, M. et al. (2010), &ldquo;Consequences of ionizing radiation-induced damage in human neural stem cells&rdquo;, Free Radical Biology and Medicine. 49(12):1846-1855, doi:10.1016/j.freeradbiomed.2010.08.021.&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Asaithamby, A. et al. (2008), &quot;Repair of HZE-Particle-Induced DNA Double-Strand Breaks in Normal Human Fibroblasts.&quot;, Radiat Res. 169(4):437&ndash;446. doi:10.1667/RR1165.1.</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Aufderheide, E. et al. (1987), &ldquo;Heavy ion effects on cellular DNA: Strand break induction and repair in cultured diploid lens epithelial cells&rdquo;, <em>International journal of radiation biology and related studies in physics, chemistry and medicin</em>e, Vol. 51/5, Taylor &amp; Francis, London, https://doi.org/10.1080/09553008714551071&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Bannik, K. et al. (2013), &ldquo;Are mouse lens epithelial cells more sensitive to &gamma;-irradiation than lymphocytes?&rdquo;, <em>Radiation and environmental biophysics</em>, Vol. 52/2, Springer-Verlag, Berlin/Heidelberg, https://doi.org/10.1007/s00411-012-0451-8&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Bains, S. K. et al. (2019), &ldquo;Effects of ionizing radiation on telomere length and telomerase activity in cultured human lens epithelium cells&rdquo;, I<em>nternational journal of radiation biology</em>, Vol. 95/1, Taylor &amp; Francis, Abingdon, https://doi.org/10.1080/09553002.2018.1466066&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Barazzuol, L et al. (2017), &ldquo;A coordinated DNA damage response promotes adult quiescent neural stem cell activation. PLOS Biology, 15(5). <a href="https://doi.org/10.1371/journal.pbio.2001264" rel="noreferrer noopener" target="_blank">https://doi.org/10.1371/journal.pbio.2001264</a>&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Barnard, S. G. R. et al. (2018), &ldquo;Dotting the eyes: mouse strain dependency of the lens epithelium to low dose radiation-induced DNA damage&rdquo;, <em>International journal of radiation biology</em>, Vol. 94/12, Taylor &amp; Francis, Abingdon, https://doi.org/10.1080/09553002.2018.1532609&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Barnard, S. G. R. et al. (2019), &ldquo;Inverse dose-rate effect of ionising radiation on residual 53BP1 foci in the eye lens&rdquo;, <em>Scientific Reports</em>, Vol. 9/1, Nature Publishing Group, England, https://doi.org/10.1038/s41598-019-46893-3&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Barnard, S. G. R. et al. (2022), &ldquo;Radiation-induced DNA damage and repair in lens epithelial cells of both Ptch1 (+/-) and Ercc2 (+/-) mutated mice&rdquo;, <em>Radiation Research</em>, Vol. 197/1, Radiation Research Society, United States, https://doi.org/10.1667/RADE-20-00264.1&nbsp;</span></span></p>
  • <p>&nbsp;</p>
  • <p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Baselet, B. et al. (2019), &ldquo;Pathological effects of ionizing radiation: endothelial activation and dysfunction&rdquo;, Cellular and molecular life sciences, Vol. 76/4, Springer Nature, https://doi.org/10.1007/s00018-018-2956-z&nbsp;</span></span></p>
  • <p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Baselet, B. et al. (2017), &ldquo;Functional Gene Analysis Reveals Cell Cycle Changes and Inflammation in Endothelial Cells Irradiated with a Single X-ray Dose&rdquo;, Frontiers in pharmacology, Vol. 8, Frontiers, https://doi.org/10.3389/fphar.2017.00213&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Baumstark-Khan, C., J. Heilmann, and H. Rink (2003), &lsquo;Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation&rdquo;, <em>Advances in space research</em>, Vol. 31/6, Elsevier Ltd, England, https://doi.org/10.1016/S0273-1177(03)00095-4&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Beels, L. et al. (2009), &quot;g-H2AX Foci as a Biomarker for Patient X-Ray Exposure in Pediatric Cardiac Catheterization&quot;, Are We Underestimating Radiation Risks?&quot;:1903&ndash;1909. doi:10.1161/CIRCULATIONAHA.109.880385.</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Belli M, Cherunbini R, Vecchia MD, Dini V, Moschini G, Signoretti C, Simon G, Tabocchini MA, Tiveron P. 2000. DNA DSB induction and rejoining in V79 cells irradiated with light ions: a constant field gel electrophoresis study. Int J Radiat Biol. 76(8):1095-1104.</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Brooks, A.L., D.G. Hoel &amp; R.J. Preston (2016), &quot;The role of dose rate in radiation cancer risk: evaluating the effect of dose rate at the molecular, cellular and tissue levels using key events in critical pathways following exposure to low LET radiation.&quot;, Int. J. Radiat. Biol. 92(8):405&ndash;426. doi:10.1080/09553002.2016.1186301.</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Bucolo, C. et al. (1994), &ldquo;The effect of ganglioside on oxidation-induced permeability changes in lens and in epithelial cells of lens and retina&rdquo;, <em>Experimental eye research,</em> Vol. 58/6, Elsevier Ltd, London, https://doi.org/10.1006/exer.1994.1067&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cabrera et al. (2011), &ldquo;Antioxidants and the integrity of ocular tissues&rdquo;, in Veterinary medicine international, SAGE-Hindawi Access to Research, United States. DOI: 10.4061/2011/905153&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cadet, J. et al. (2012), &ldquo;Oxidatively generated complex DNA damage: tandem and clustered lesions&rdquo;, Cancer letters, Vol. 327/1, Elsevier Ireland Ltd, Ireland. https://doi.org/10.1016/j.canlet.2012.04.005&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cannan, W.J. &amp; D.S. Pederson (2016), &quot;Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin.&quot;, J. Cell Physiol. 231(1):3&ndash;14. doi:10.1002/jcp.25048.&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Cencer, C. S. et al. (2018), &ldquo;PARP-1/PAR activity in cultured human lens epithelial cells exposed to two levels of UVB light&rdquo;, Photochemistry and photobiology, Vol. 94/1, Wiley, Hoboken, https://doi.org/10.1111/php.12814&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Chadwick, K.H., (2017), Towards a new dose and dose-rate effectiveness factor (DDREF)? Some comments., J Radiol Prot., 37:422-433. doi: 10.1088/1361-6498/aa6722.&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Charlton, D.E., H. Nikjoo &amp; J.L. Humm (1989), &quot;Calculation of initial yields of single- and double-strand breaks in cell nuclei from electrons, protons and alpha particles.&quot;, Int. J. Rad. Biol., 53(3):353-365, DOI: 10.1080/09553008814552501&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Christensen, D.M. (2014), &quot;Management of Ionizing Radiation Injuries and Illnesses, Part 3: Radiobiology and Health Effects of Ionizing Radiation.&quot;, 114(7):556&ndash;565. doi:10.7556/jaoa.2014.109.&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Citrin, D.E. &amp; J.B. Mitchel (2014), &quot;Public Access NIH Public Access.&quot;, 71(2):233&ndash;236. doi:10.1038/mp.2011.182.doi.&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dalke, C. et al. (2018), &ldquo;Lifetime study in mice after acute low-dose ionizing radiation: a multifactorial study with special focus on cataract risk&rdquo;, Radiation and environmental biophysics, Vol. 57/2, Springer Berlin Heidelberg, Berling/Heidelberg, https://doi.org/10.1007/s00411-017-0728-z&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Day, T.K. et al. (2007), &quot;Adaptive Response for Chromosomal Inversions in pKZ1 Mouse Prostate Induced by Low Doses of X Radiation Delivered after a High Dose.&quot;, Radiat Res. 167(6):682&ndash;692. doi:10.1667/rr0764.1.&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">De Angelis, P. M. et al. (2006), &ldquo;Cellular response to 5-fluorouracil (5-FU) in 5-FU-resistant colon cancer cell lines during treatment and recovery&rdquo;, Molecular Cancer, Vol. 5/20, BioMed Central, https://doi.org/10.1186/1476-4598-5-20&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">DeGraff, W. G. et al. (1992), &ldquo;Nitroxide-mediated protection against X-ray- and neocarzinostatin-induced DNA damage&rdquo;, Free Radical Biology and Medicine, Vol. 13/5, Elsevier, https://doi.org/10.1016/0891-5849(92)90142-4&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Desouky, O., N. Ding &amp; G. Zhou (2015), &quot;ScienceDirect Targeted and non-targeted effects of ionizing radiation.&quot;, J. Radiat. Res. Appl. Sci. 8(2):247&ndash;254. doi:10.1016/j.jrras.2015.03.003.&nbsp;</span></span></p>
  • <p>&nbsp;</p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dong et al. (2015), &ldquo;Relationship between irradiation-induced neuro-inflammatory environments and impaired cognitive function in the developing brain of Mice. International Journal of Radiation Biology, 91(3):224&ndash;239. <a href="https://doi.org/10.3109/09553002.2014.988895" rel="noreferrer noopener" target="_blank">https://doi.org/10.3109/09553002.2014.988895</a>&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dong, X. et al. (2014), &ldquo;NEMO modulates radiation-induced endothelial senescence of human umbilical veins through NF-&kappa;B signal pathway&rdquo;, Radiation Research, Vol. 183/1, BioOne, https://doi.org/10.1667/RR13682.1&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Dubrova, Y.E. &amp; M.A. Plumb (2002), &quot;Ionising radiation and mutation induction at mouse minisatellite loci The story of the two generations&quot;, Mutat. Res. 499(2):143&ndash;150.&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">EPRI (2014), Epidemiology and mechanistic effects of radiation on the lens of the eye: Review and scientific appraisal of the literature, EPRI, California&nbsp;</span></span></p>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sears, C. R. and J. J. Turchi (2012), &ldquo;Complex cisplatin-double strand break (DSB) lesions directly impair cellular non-homologous end-joining (NHEJ) independent of downstream damage response (DDR) pathways&rdquo;, Journal of biological chemistry, Vol 287/29, The American Society for Biochemistry and Molecular Biology, Inc, USA, https://doi.org/ 10.1074/jbc.M112.344911&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Shah, D.J., R.K. Sachs &amp; D.J. Wilson (2012), &quot;Radiation-induced cancer: A modern view.&quot; Br. J. Radiol. 85(1020):1166&ndash;1173. doi:10.1259/bjr/25026140.&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Shelke, S. &amp; B. Das (2015), &quot;Dose response and adaptive response of non- homologous end joining repair genes and proteins in resting human peripheral blood mononuclear cells exposed to &gamma; radiation.&quot;, (December 2014):365&ndash;379. doi:10.1093/mutage/geu081.&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sidjanin, D., S. Zigman and J. Reddan (1993), &ldquo;DNA damage and repair in rabbitlens epithelial cells following UVA radiation&rdquo;, Current eye research, Vol. 12/9, Informa UK Ltd, Oxford, https://doi.org/10.3109/02713689309020382&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Smith, J. et al. (2003), &quot;Impact of DNA ligase IV on the delity of end joining in human cells.&quot;, Nucleic Acids Research. 31(8):2157-2167.doi:10.1093/nar/gkg317.&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Smith, T.A. et al. (2017), &quot;Radioprotective agents to prevent cellular damage due to ionizing radiation.&quot; Journal of Translational Medicine.15(1).doi:10.1186/s12967-017-1338-x.&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Stewart, F. A. et al. (2012), &ldquo;ICRP publication 118: ICRP statement on tissue reactions and early and late effects of radiation in normal tissues and organs &ndash; threshold doses for tissue reactions in a radiation protection context&rdquo;, Annals of the ICRP, Vol, 41/1-2, Elsevier Ltd, London, https://doi.org/10.1016/j.icrp.2012.02.001&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sudprasert, W., P. Navasumrit &amp; M. Ruchirawat (2006), &quot;Effects of low-dose &gamma; radiation on DNA damage, chromosomal aberration and expression of repair genes in human blood cells.&quot;, Int. J. Hyg. Envrion. Health, 209:503&ndash;511. doi:10.1016/j.ijheh.2006.06.004.&nbsp;</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sutherland, B.M. et al. (2000), &quot;Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation.&quot;, J. of Rad. Res. 43 Suppl(S):S149-52. doi: 10.1269/jrr.43.S149</span></span></p>
  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sylvester, C. B. et al. (2018), &ldquo;Radiation-Induced Cardiovascular Disease: Mechanisms and Importance of Linear Energy Transfer&rdquo;, Frontiers in cardiovascular medicine, Vol. 5, Frontiers, https://doi.org/10.3389/fcvm.2018.00005&nbsp;</span></span></p>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Ward, J. F. (1988), &quot;DNA Damage Produced by Ionizing Radiation in Mammalian Cells: Identities, Mechanisms of Formation, and Reparability.&quot;, Prog. Nucleic Acid Res. Mol. Biol. 35(C):95&ndash;125. doi:10.1016/S0079-6603(08)60611-X.&nbsp;</span></span></p>
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  • <p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Wolf, N. et al. (2008), &ldquo;Radiation cataracts: Mechanisms involved in their long delayed occurrence but then rapid progression&rdquo;, Molecular vision, Vol. 14/34-35, Molecular Vision, Atlanta, pp. 274-285&nbsp;</span></span></p>
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  • <p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Zhang, L. et al. (2017), &ldquo;The inhibitory effect of minocycline on radiation-induced neuronal apoptosis via AMPK&alpha;1 signaling-mediated autophagy.&rdquo;, Sci Rep.7(1):16373. doi: 10.1038/s41598-017-16693-8.&nbsp;</span></span></p>
  • </div>
  • <div>
  • <h4><a href="/relationships/1911">Relationship: 1911: Increase, DNA strand breaks leads to Inadequate DNA repair</a></h4>
  • <h4>AOPs Referencing Relationship</h4>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">AOP Name</th>
  • <th scope="col">Adjacency</th>
  • <th scope="col">Weight of Evidence</th>
  • <th scope="col">Quantitative Understanding</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td><a href="/aops/296">Oxidative DNA damage leading to chromosomal aberrations and mutations</a></td>
  • <td>adjacent</td>
  • <td>High</td>
  • <td>Low</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/272">Deposition of energy leading to lung cancer</a></td>
  • <td>adjacent</td>
  • <td>Moderate</td>
  • <td>Moderate</td>
  • </tr>
  • <tr>
  • <td><a href="/aops/478">Deposition of energy leading to occurrence of cataracts</a></td>
  • <td>adjacent</td>
  • <td>Moderate</td>
  • <td>Moderate</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • <h4>Evidence Supporting Applicability of this Relationship</h4>
  • <div>
  • <strong>Taxonomic Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Term</th>
  • <th scope="col">Scientific Term</th>
  • <th scope="col">Evidence</th>
  • <th scope="col">Links</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>human</td>
  • <td>Homo sapiens</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>mouse</td>
  • <td>Mus musculus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090" target="_blank">NCBI</a></td>
  • </tr>
  • <tr>
  • <td>rat</td>
  • <td>Rattus norvegicus</td>
  • <td>High</td>
  • <td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116" target="_blank">NCBI</a></td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <div>
  • <strong>Life Stage Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Life Stage</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>All life stages</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <div>
  • <strong>Sex Applicability</strong>
  • <div class="table-responsive">
  • <table class="table table-bordered table-fullwidth">
  • <thead class="thead-light">
  • <tr>
  • <th scope="col">Sex</th>
  • <th scope="col">Evidence</th>
  • </tr>
  • </thead>
  • <tbody class="tbody-striped">
  • <tr>
  • <td>Unspecific</td>
  • <td>High</td>
  • </tr>
  • </tbody>
  • </table>
  • </div>
  • </div>
  • <p><span style="font-size:12px">This KER is plausible in all life stages, sexes, and organisms with DNA. The majority of the evidence is from in vivo adult mice with no specification on sex, and in vitro human models that do not specify sex.&nbsp;</span></p>
  • <h4>Key Event Relationship Description</h4>
  • <p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">The maintenance of DNA integrity is essential for genomic stability; for this reason cells have multiple response mechanisms that enable the repair of damaged DNA. Thus when DNA double strand breaks (DSBs) occur, the most detrimental type of lesion, the cell will initiate repair machinery. These mechanisms are not foolproof, and emerging evidence suggests that closely spaced lesions can compromise the repair machinery. The two most common DSB repair mechanisms are non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is initiated in G1 and early S phases of the cell cycle (Lieber et al., 2003) and is preferentially used to repair DSB damage (Godwint et al., 1994), as it is rapid and more efficient than HR (Lliakis, 1991; Jeggo, 1998; Mao et al., 2008). In higher-order eukaryotes such as humans, NHEJ is the favoured DNA repair mechanism because of the large non-coding regions within the genome. NHEJ can occur through one of two subtypes: canonical NHEJ (C‐NHEJ) or alternative non-homologous end joining (alt-NHEJ). C-NHEJ, as the name suggests, simply ligates the broken ends back together. In contrast, alt‐NHEJ occurs when one strand of the DNA on either side of the break is resected to repair the lesion (B&eacute;termier et al., 2014). Both repair mechanisms are error‐prone, meaning insertions and deletions are sometimes formed due to the DSBs being repaired imperfectly (Thurtle-Schmidt and Lo, 2018). However, alt-NHEJ is considered more error-prone than C-NHEJ, as studies have shown that it more often leads to chromosomal aberrations (Zhu et al., 2002; Guirouilh-Barbat et al., 2007; Simsek &amp; Jasin, 2010).&nbsp;HR is mostly operative during S and G2 phases because of the presence of the sister chromatid that can be used as template for repair (Van Gent et al 2001). Because of the reliance on the undamaged sister chromatid to repair the DSB, HR is less error-prone than NEHJ. Nevertheless, defects in HR are known to contribute to genomic instability and the formation of chromosomal aberrations (Deans et al 2000) </span></span></p>
  • <p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">The maintenance of DNA integrity is essential for genomic stability; for this reason cells have multiple response mechanisms that enable the repair of damaged DNA. Thus when DNA double strand breaks (DSBs) occur, the most detrimental type of lesion, the cell will initiate repair machinery. These mechanisms are not foolproof, and emerging evidence suggests that closely spaced lesions can compromise the repair machinery. The two most common DSB repair mechanisms are non-homologous end joining (NHEJ) and homologous recombination (HR). </span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">The latter predominates in stem cells as they are frequently in the replicative phase of the cell cycle (Choi et al., 2020).</span></span>&nbsp;<span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">NHEJ is initiated in G1 and early S phases of the cell cycle (Lieber et al., 2003) and is preferentially used to repair DSB damage (Godwint et al., 1994), as it is rapid and more efficient than HR (Lliakis, 1991; Jeggo, 1998; Mao et al., 2008). In higher-order eukaryotes such as humans, NHEJ is the favoured DNA repair mechanism because of the large non-coding regions within the genome. </span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">However, when other repair mechanisms (e.g., NHEJ, HR) are compromised, single strand annealing, which is a low fidelity mechanism may be involved (Chang et al., 2017).&nbsp;</span></span><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">NHEJ can occur through one of two subtypes: canonical NHEJ (C‐NHEJ) or alternative non-homologous end joining (alt-NHEJ). C-NHEJ, as the name suggests, simply ligates the broken ends back together. In contrast, alt‐NHEJ occurs when one strand of the DNA on either side of the break is resected to repair the lesion (B&eacute;termier et al., 2014). All&nbsp;repair mechanisms are error‐prone, meaning that insertions and deletions are sometimes formed due to the DSBs being repaired imperfectly (Thurtle-Schmidt and Lo, 2018). However, alt-NHEJ is considered more error-prone than C-NHEJ, as studies have shown that it more often leads to chromosomal aberrations (Zhu et al., 2002; Guirouilh-Barbat et al., 2007; Simsek &amp; Jasin, 2010).&nbsp;HR is&nbsp;operative during late S and G2 phases where the sister chromatid&nbsp;can be used as template for error-free repair (Van Gent et al 2001). Because of the reliance on the undamaged sister chromatid to repair the DSB, HR is less error-prone than NEHJ. Nevertheless, defects in HR are known to contribute to genomic instability and the formation of chromosomal aberrations (Deans et al 2000) </span></span></p>
  • <p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">There is extensive evidence that DNA repair capacity can be overwhelmed or saturated in the presence of high numbers of strand breaks. This is demonstrated by decades of studies showing dose-related increases in chromosomal exchanges, chromosomal breaks and micronuclei following exposure to double-strand break inducers. Inadequate repair not only refers to overwhelming of DNA repair machinery, but also the use of repair mechanisms that are error-prone (i.e., misrepair is considered inadequate repair).</span></span></p>
  • <p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">There is extensive evidence that DNA repair capacity can be overwhelmed or saturated in the presence of high numbers of strand breaks. </span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">For example, with multiple single strand breaks (SSBs) in close proximity that can lead to DSBs (Caldecott, 2024).&nbsp;</span></span><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px">This is demonstrated by decades of studies showing dose-related increases in chromosomal exchanges, chromosomal breaks and micronuclei following exposure to double-strand break inducers. </span></span><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Additionally, the loss of heterozygosity (LOH)&nbsp; is an example of how during the repair of incorrect DNA that uses HR, t