CHEBI:16991deoxyribonucleic acidCL:0000066epithelial cellCL:0000077mesothelial cellUBERON:0002405immune systemGO:0002526acute inflammatory responseD009154mutationGO:0006281DNA repairGO:0008283cell proliferationGO:0044414suppression of host defenses7functional change1increasedIonizing Radiation<p>Ionizing radiation can vary in energy, dose, charge, and in the spatial distributions of energy transferred to other matter (linear energy transfer per unit length or LET) (ICRU 1970). At the same dose, low and high LET both generate energy deposition events, including many higher energy events (Goodhead and Nikjoo 1989). However, they differ in the spatial distribution and upper range of intensity of energy deposited. Lower LET such as gamma rays sparsely deposit many individual excitations or small clusters of excitations of low energy (Goodhead 1988). In contrast, high LET such as alpha particles have fewer tracks but readily transfer their energy to matter and therefore deposit their energy over a much smaller area (Goodhead 1994). Consequently, alpha and other high LET particles penetrate less deeply into tissue, interactions are densely focused on a narrow track, and individual energy depositions can be large (Goodhead 1988). These different energy deposition patterns can lead to differences in radiation effects including the pattern of DNA damage.</p>
<p>Exposure to ionizing radiation can come from natural and industrial sources. Space and terrestrial radiation includes a range of LET particles, while diagnostic radiation methods such as X-ray imaging, mammography and CT scans use low LET X-rays. Radiation therapy can use an external beam to direct radiation on a focused tissue area, or deposit solid or liquid radioactive materials in the body that release (mostly gamma) radiation internally. External radiotherapy typically uses X-rays but is moving towards higher LET charged particles such as protons and heavy ions (Durante, Orecchia et al. 2017).</p>
2019-05-03T12:36:362019-05-07T12:12:13Estrogen2019-05-08T11:40:272019-05-08T11:40:27WCS_9606human10116rat10090mouse6239nematodeWCS_7955zebrafish3702thale-cress3349Scotch pineWCS_35525Daphnia magna3055Chlamydomonas reinhardtiiWCS_6396common brandling wormWCS_4472Lemna minor8030Salmo salarWikiUser_28Vertebrates10036Syrian golden hamster9606Homo sapiens9913cow10090Mus musculusDeposition of EnergyEnergy DepositionMolecular<p><span style="color:#0000ff"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Deposition of energy refers to events where subatomic particles or electromagnetic radiation deposit energy in the media through which they transverse. The energy may either be sufficient (e.g. ionizing radiation) or insufficient (e.g. non-ionizing radiation) to ionize atoms or molecules (Beir, et al. 1999). </span></span></span></p>
<p><span style="color:#0000ff"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Ionizing radiation can cause the ejection of electrons from atoms and molecules, thereby </span></span></span><span style="color:#e74c3c"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">resulting in their ionization and the breakage of chemical bonds</span></span></span><span style="color:#0000ff"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">. The energy of these subatomic particles or electromagnetic waves ranges from 124 KeV to 5.4 MeV, and is dependent on the source and type of radiation (Zyla et al., 2020). Not all electromagnetic radiation is ionizing; as the incident radiation must have sufficient energy to free electrons from the atom or molecule’s electron orbitals. The energy can induce direct and indirect ionization events and </span></span></span><span style="color:#e74c3c"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">can be via internal (injections, inhalation, injection) or external exposure</span></span></span><span style="color:#0000ff"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">. Direct ionization is the principal path where charged particles interact with DNA to cause a biological damage. Photons, which are electromagnetic waves can also cause direct ionization. Indirect ionization produces free radicals of other molecules, specifically water, which can transform to damage critical targets such as DNA (Beir, et al. 1999; </span></span></span><span style="color:#e74c3c"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Balagamwala et al., 2013</span></span></span><span style="color:#0000ff"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">). Given the fundamental nature of energy deposition by nuclei, nucleons or elementary particles in material, this process is universal to all biological contexts.</span></span></span></p>
<p><span style="color:#e74c3c"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Energy deposition is influenced by the linear energy transfer (LET) (Hall and Giaccia, 2018 UNSCEAR, 2020). High LET refers to energy above 10 keV μm-1 which produces more complex, dense structural damage than low LET radiation (below 10 keV μm-1). Low-LET particles produce sparse ionization events such as photons (X- and gamma rays), as well as high-energy protons. Low LET radiation 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></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px"><span style="color:#e74c3c">Non-ionizing radiation is a type of electromagnetic radiation that lacks the energy to ionize atoms or molecules. 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. 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 that we can see and that is commonly used in photosynthesis in primary producers. UV radiation has key functions in melanisation (tanning) of a number of species, and exhibit 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). </span></span></span></p>
<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; <span style="color:#e74c3c">Zyla et al., 2020</span></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 & Konishi, 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; Xie et al., 2022</span></span></p>
</td>
<td style="text-align:center"> </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> </p>
<p><span style="color:#0000ff"><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></span></p>
<p><span style="color:#e74c3c"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Taxonomic applicability: </strong>This MIE is not taxonomically specific. </span></span></span></p>
<p><span style="color:#e74c3c"><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. </span></span></span></p>
<p><span style="color:#e74c3c"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Sex applicability: </strong>This MIE is not sex specific. </span></span></span></p>
LowUnspecificHighAll life stagesModerateModerateModerateHighHighHighModerateHighModerateModerateHighLow<p><span style="color:#e74c3c"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Balagamwala, E. H. et al. (2013), “Introduction to radiotherapy and standard teletherapy techniques”,<em> Dev Ophthalmol,</em> Vol. 52, Karger, Basel, https://doi.org/10.1159/000351045 </span></span></span></p>
<p><span style="color:#0000ff"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Beir, V. et al. (1999), “The Mechanistic Basis of Radon-Induced Lung Cancer”, in <em>Health Risks of Exposure to Radon: BEIR V</em>I, National Academy Press, Washington, D.C., https://doi.org/10.17226/5499 </span></span></span></p>
<p><span style="color:#0000ff"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Douglass, M. et al. (2013), “Monte Carlo investigation of the increased radiation deposition due to gold nanoparticles using kilovoltage and megavoltage photons in a 3D randomized cell model”<em>, Medical Physics</em>, Vol. 40/7, American Institute of Physics, College Park, https://doi.org/10.1118/1.4808150 </span></span></span></p>
<p><span style="color:#0000ff"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Douglass, M. et al. (2012), “Development of a randomized 3D cell model for Monte Carlo microdosimetry simulations.”, <em>Medical Physics</em>, Vol. 39/6, American Institute of Physics, College Park, https://doi.org/10.1118/1.4719963 </span></span></span></p>
<p><span style="color:#e74c3c"><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. </span></span></span></p>
<p><span style="color:#0000ff"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Kodaira, S. and Konishi, T. (2015), “Co-visualization of DNA damage and ion traversals in live mammalian cells using a fluorescent nuclear track detector.”, <em>Journal of Radiation Research</em>, Vol. 56/2, Oxford University Press, Oxford, https://doi.org/10.1093/jrr/rru091 </span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Lind, O.C., D.H. Oughton and Salbu B. (2019), "The NMBU FIGARO low dose irradiation facility", <em>International Journal of Radiation Biology</em>, Vol. 95/1, Taylor & Francis, London, https://doi.org/10.1080/09553002.2018.1516906.</span></span></p>
<p><span style="color:#0000ff"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Sawakuchi, G.O. and Akselrod, M.S. (2016), “Nanoscale measurements of proton tracks using fluorescent nuclear track detectors.”,<em> Medical Physics</em>, Vol. 43/5, American Institute of Physics, College Park, https://doi.org/10.1118/1.4947128 </span></span></span></p>
<p><span style="color:#0000ff"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Straume, T. et al. (2015), “Compact Tissue-equivalent Proportional Counter for Deep Space Human Missions.”,<em> Health physics,</em> Vol. 109/4, Lippincott Williams & Wilkins, Philadelphia, https://doi.org/10.1097/HP.0000000000000334 </span></span></span></p>
<p><span style="color:#0000ff"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Niklas, M. et al. (2013), “Engineering cell-fluorescent ion track hybrid detectors.”, <em>Radiation Oncology</em>, Vol. 8/104, BioMed Central, London, https://doi.org/10.1186/1748-717X-8-141 </span></span></span></p>
<p><span style="color:#e74c3c"><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. </span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">Xie, Li. et al. (2022), "Ultraviolet B Modulates Gamma Radiation-Induced Stress Responses in Lemna Minor at Multiple Levels of Biological Organisation", <em>SSRN</em>, Elsevier, Amsterdam, http://dx.doi.org/10.2139/ssrn.4081705 .</span></span></p>
<p><span style="color:#e74c3c"><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. </span></span></span></p>
<p> </p>
<p> </p>
2019-08-22T09:44:232023-04-28T08:40:04Increase in reactive oxygen and nitrogen species (RONS)Increase in RONSMolecular<p>Reactive oxygen and nitrogen species (RONS) are highly reactive oxygen- and nitrogen-based molecules that often contain or generate free radicals. Key molecules include superoxide ([O2]•−), hydrogen peroxide (H2O2), hydroxyl radical ([OH]•), lipid peroxide (ROOH)<em>, </em>nitric oxide ([NO]•, and peroxynitrite ([ONOO-]) (Dickinson and Chang 2011; Egea, Fabregat et al. 2017)</p>
<p>RONS are generated in the course of cellular respiration, metabolism, cell signaling, and inflammation (Dickinson and Chang 2011; Egea, Fabregat et al. 2017). Superoxide and hydrogen peroxide are commonly produced by the mitochondrial electron transport chain and cytochrome c and by membrane bound NADPH oxidases and related molecules. Hydrogen peroxide is also made by the endoplasmic reticulum in the course of protein folding. Nitric oxide is produced at the highest levels by nitric oxide synthase in endothelial cells and phagocytes. The other species are produced by reactions with superoxide or peroxide, or by other free radicals or enzymes.</p>
<p>RONS activity is principally local. Most reactive oxygen species (ROS) have short half-lives, ranging from nano- to milliseconds, so diffusion is limited, while reactive nitrogen species (RNS) nitric oxide or peroxynitrate can survive long enough to diffuse across membranes (Calcerrada, Peluffo et al. 2011). Consequently, local concentrations of ROS are much higher than average cellular concentrations and signaling is typically controlled by colocalization with redox buffers (Dickinson and Chang 2011; Egea, Fabregat et al. 2017). The effects of ROS and RNS are countered by cellular antioxidants, with glutathione and peroxiredoxins playing a major role (Dickinson and Chang 2011). Glutathione is slower but broad acting, while peroxiredoxins act quickly and are specific to peroxides. Peroxiredoxins are effective at low peroxide concentrations but can be deactivated at higher concentrations, suggesting the cellular response to peroxides may sometimes be non-linear.</p>
<p>Although their existence is limited temporally and spatially, reactive oxygen species (ROS) interact with other RONS or with other nearby molecules to produce more ROS and participate in a feedback loop to amplify the ROS signal, which can increase Reactive Nitrogen Species (RNS). Both ROS and RNS also move into neighboring cells and ROS can increase intracellular RONS signaling in neighboring cells (Egea, Fabregat et al. 2017).</p>
<p>RONS can modify a range of targets including amino acids, lipids, and nucleic acids to inactivate or alter target functionality (Calcerrada, Peluffo et al. 2011; Dickinson and Chang 2011; Go and Jones 2013; Ravanat, Breton et al. 2014; Egea, Fabregat et al. 2017). For example, phosphatases including the tumor suppressor PTEN can be reversibly deactivated by oxidation, and the movement of HDAC4 is peroxide dependent. Elevated ROS are implicated in proliferation and maintenance of stem cell population size (Dickinson and Chang 2011) and conversely in differentiation of stem cells and oncogene-induced senescence (Egea, Fabregat et al. 2017).</p>
<p>RONS is typically measured using fluorescent or other probes that react with RONS to change state, or by measuring the redox state of proteins or DNA (Dickinson and Chang 2011; Wang, Fang et al. 2013; Griendling, Touyz et al. 2016). Optimal methods for RONS detection have high sensitivity, selectivity, and spatiotemporal resolution to distinguish transient and localized activity, but most methods lack one or more of these parameters.</p>
<p>Molecular probes that indicate the presence of RONS species vary in specificity and kinetics (Dickinson and Chang 2011; Wang, Fang et al. 2013; Griendling, Touyz et al. 2016). Small molecule fluorescent probes can be applied to any tissue in vitro, but cannot be finely targeted to different cellular compartments. The non-selective probe DCHF was widely used in the past, but can produce false positive signals and is no longer recommended. Newer more selective small molecule probes such as boronate-based molecules are being developed but are not yet widely used. Alternatively, fluorescent protein-based probes can be genetically engineered, expressed in vivo, and targeted to cellular compartments and specific cells. However, these probes are very sensitive to pH in the physiological range and must be carefully controlled. EPR (electron paramagnetic resonance spectroscopy) provide the most direct and specific detection of free radicals, but requires specialized equipment.</p>
<p>Alternative methods involve the detection of redox-dependent changes to cellular constituents such as proteins, DNA, lipids, or glutathione (Dickinson and Chang 2011; Wang, Fang et al. 2013; Griendling, Touyz et al. 2016). However, these methods cannot generally distinguish between the oxidative species behind the changes, and cannot provide good resolution for kinetics of oxidative activity.</p>
<p>Table 1. Common methods for detecting oxidative activity</p>
<table border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr>
<td style="height:22px; width:133px">
<p><strong>Target</strong></p>
</td>
<td style="height:22px; width:126px">
<p><strong>Name</strong></p>
</td>
<td style="height:22px; width:144px">
<p><strong>Method</strong></p>
</td>
<td style="height:22px; width:235px">
<p><strong>Strengths/Weaknesses</strong></p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Hydrogen peroxide- extracellular</strong></p>
</td>
<td style="height:22px; width:126px">
<p>AmplexRed</p>
</td>
<td style="height:22px; width:144px">
<p>Small molecule fluorescent probes</p>
</td>
<td style="height:22px; width:235px">
<p>Can be applied to any tissue in vitro.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Hydrogen peroxide- mitochondrial</strong></p>
</td>
<td style="height:22px; width:126px">
<p>MitoPy1</p>
</td>
<td style="height:22px; width:144px">
<p>Small molecule fluorescent probes</p>
</td>
<td style="height:22px; width:235px">
<p>Can be applied to any tissue in vitro.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Hydrogen peroxide</strong></p>
</td>
<td style="height:22px; width:126px">
<p>HyPer</p>
</td>
<td style="height:22px; width:144px">
<p>Protein-based fluorescent probes</p>
</td>
<td style="height:22px; width:235px">
<p>Sensitive, can be targeted to specific cells and compartments. Slower and pH sensitive.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Hydrogen peroxide</strong></p>
</td>
<td style="height:22px; width:126px">
<p>HyPer3</p>
</td>
<td style="height:22px; width:144px">
<p>Protein-based fluorescent probes</p>
</td>
<td style="height:22px; width:235px">
<p>Rapid kinetics and larger dynamic range, can be targeted to specific cells and compartments. Sensitive to pH, less sensitive to H2O2.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Hydrogen peroxide</strong></p>
</td>
<td style="height:22px; width:126px">
<p>Boronate-based indicators</p>
</td>
<td style="height:22px; width:144px">
<p>Small molecule fluorescent probe</p>
</td>
<td style="height:22px; width:235px">
<p>Selective for H2O2 but can interact with peroxynitrite.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Superoxide- intracellular</strong></p>
</td>
<td style="height:22px; width:126px">
<p>DHE (dihydroethidium)</p>
</td>
<td style="height:22px; width:144px">
<p>Small molecule fluorescent probe</p>
</td>
<td style="height:22px; width:235px">
<p>Can be applied to any tissue in vitro, but not targeted to different compartments.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Superoxide- intracellular</strong></p>
</td>
<td style="height:22px; width:126px">
<p>cpYFP</p>
</td>
<td style="height:22px; width:144px">
<p>Protein-based fluorescent probes</p>
</td>
<td style="height:22px; width:235px">
<p>Reversible. Can be targeted to specific cells and compartments.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Superoxide- mitochondrial</strong></p>
</td>
<td style="height:22px; width:126px">
<p>MitoSox</p>
</td>
<td style="height:22px; width:144px">
<p>Small molecule fluorescent probe</p>
</td>
<td style="height:22px; width:235px">
<p>Can be applied to any tissue in vitro.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Superoxide- mitochondrial</strong></p>
</td>
<td style="height:22px; width:126px">
<p>mt-cpYFP</p>
</td>
<td style="height:22px; width:144px">
<p>Protein-based fluorescent probes</p>
</td>
<td style="height:22px; width:235px">
<p>Reversible. Can be targeted to specific cells and compartments.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Superoxide- extracellular</strong></p>
</td>
<td style="height:22px; width:126px">
<p>nitroblue tetrazolium</p>
</td>
<td style="height:22px; width:144px">
<p>Small molecule fluorescent probe</p>
</td>
<td style="height:22px; width:235px">
<p>Can be applied to any tissue in vitro.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Superoxide- intracellular or extracelluar</strong></p>
</td>
<td style="height:22px; width:126px">
<p>various trityl probes</p>
</td>
<td style="height:22px; width:144px">
<p>EPR</p>
</td>
<td style="height:22px; width:235px">
<p>Very specific, but requires specialized equipment, not as sensitive in tissue.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Nitric oxide</strong></p>
</td>
<td style="height:22px; width:126px">
<p>Fe[DETC]2 and</p>
<p>Fe[MGD]2,</p>
</td>
<td style="height:22px; width:144px">
<p>EPR</p>
</td>
<td style="height:22px; width:235px">
<p>Very specific, but requires specialized equipment, not as sensitive in tissue.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Nitric oxide</strong></p>
</td>
<td style="height:22px; width:126px">
<p>DAF-FM</p>
</td>
<td style="height:22px; width:144px">
<p>Small molecule fluorescent probe</p>
</td>
<td style="height:22px; width:235px">
<p>Can be applied to any tissue in vitro, but not targeted to different compartments</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Peroxynitrite</strong></p>
</td>
<td style="height:22px; width:126px">
<p>EMPO</p>
</td>
<td style="height:22px; width:144px">
<p>EPR</p>
</td>
<td style="height:22px; width:235px">
<p>Very specific, but requires specialized equipment, not as sensitive in tissue.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Peroxynitrite</strong></p>
</td>
<td style="height:22px; width:126px">
<p>Boronate-based indicators</p>
</td>
<td style="height:22px; width:144px">
<p>Small molecule fluorescent probe</p>
</td>
<td style="height:22px; width:235px">
<p>Selective for H2O2 but can interact with (is inhibited by) peroxynitrite.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Peroxynitrite</strong></p>
</td>
<td style="height:22px; width:126px">
<p>8-nitroguanine (DNA) content</p>
</td>
<td style="height:22px; width:144px">
<p>HPLC-MS/MS</p>
</td>
<td style="height:22px; width:235px">
<p>Destruction of sample required for measurement.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Non-specific oxidation</strong></p>
</td>
<td style="height:22px; width:126px">
<p>DCHF</p>
</td>
<td style="height:22px; width:144px">
<p>Small molecule fluorescent probe</p>
</td>
<td style="height:22px; width:235px">
<p>Very non selective, and can produce false positive signals.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Non-specific oxidation</strong></p>
</td>
<td style="height:22px; width:126px">
<p>roGFP or FRET</p>
</td>
<td style="height:22px; width:144px">
<p>Protein-based fluorescent probes</p>
</td>
<td style="height:22px; width:235px">
<p>Slow acting. Good to look at steady state activity.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Non-specific oxidation</strong></p>
</td>
<td style="height:22px; width:126px">
<p>ratio of reduced to oxidized glutathione or cysteine</p>
</td>
<td style="height:22px; width:144px">
<p>Redox state detectors</p>
</td>
<td style="height:22px; width:235px">
<p>Slow acting. Good to look at steady state activity. Destruction of sample required for measurement.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Non-specific oxidation</strong></p>
</td>
<td style="height:22px; width:126px">
<p>8-oxoguanine (DNA) or protein carbonyl content</p>
</td>
<td style="height:22px; width:144px">
<p>HPLC-MS/MS</p>
</td>
<td style="height:22px; width:235px">
<p>Destruction of sample required for measurement.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:133px">
<p><strong>Non-specific oxidation</strong></p>
</td>
<td style="height:22px; width:126px">
<p>TBARS (thiobarbituric acid reactive substance)</p>
</td>
<td style="height:22px; width:144px">
<p>Lipid peroxidation</p>
</td>
<td style="height:22px; width:235px">
<p>Destruction of sample required for measurement.</p>
</td>
</tr>
</tbody>
</table>
<p>This KE is broadly applicable across species.</p>
<p style="margin-left:.5in"><a name="_ENREF_1">Calcerrada, P., G. Peluffo, et al. (2011). "Nitric oxide-derived oxidants with a focus on peroxynitrite: molecular targets, cellular responses and therapeutic implications." <u>Curr Pharm Des</u> <strong>17</strong>(35): 3905-3932.</a></p>
<p style="margin-left:.5in"><a name="_ENREF_2">Dickinson, B. C. and C. J. Chang (2011). "Chemistry and biology of reactive oxygen species in signaling or stress responses." <u>Nature chemical biology</u> <strong>7</strong>(8): 504-511.</a></p>
<p style="margin-left:.5in"><a name="_ENREF_3">Egea, J., I. Fabregat, et al. (2017). "European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS)." <u>Redox biology</u> <strong>13</strong>: 94-162.</a></p>
<p style="margin-left:.5in"><a name="_ENREF_4">Go, Y. M. and D. P. Jones (2013). "The redox proteome." <u>J Biol Chem</u> <strong>288</strong>(37): 26512-26520.</a></p>
<p style="margin-left:.5in"><a name="_ENREF_5">Griendling, K. K., R. M. Touyz, et al. (2016). "Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System: A Scientific Statement From the American Heart Association." <u>Circulation research</u> <strong>119</strong>(5): e39-75.</a></p>
<p style="margin-left:.5in"><a name="_ENREF_6">Ravanat, J. L., J. Breton, et al. (2014). "Radiation-mediated formation of complex damage to DNA: a chemical aspect overview." <u>Br J Radiol</u> <strong>87</strong>(1035): 20130715.</a></p>
<p style="margin-left:.5in"><a name="_ENREF_7">Wang, X., H. Fang, et al. (2013). "Imaging ROS signaling in cells and animals." <u>Journal of molecular medicine</u> <strong>91</strong>(8): 917-927.</a></p>
2019-05-03T14:00:042019-05-08T12:30:20Increase, DNA damageIncrease, DNA DamageMolecular<p>DNA nucleotide damage, single, and double strand breaks occur in the course of cellular operations such as DNA repair and replication and can be induced directly and in neighboring “bystander” cells by internal or external stressors like reactive oxygen species, chemicals, and radiation. Ionizing radiation and RONS such as hydroxyl radicals or peroxide can create a range of lesions (a change in molecular structure) in the base of the nucleotide, with guanine particularly vulnerable because of its low redox potential (David, O'Shea et al. 2007). The same stressors can also break the sugar (deoxyribose)-phosphate backbone creating a single strand break. Simultaneous proximal breaks in both strands of DNA form double strand breaks, which are considered to be more destructive and mutagenic than lesions or single strand breaks. Double strand breaks can generate chromosomal abnormalities including changes in chromosomal number, breaks and gaps, translocations, inversions, and deletions (Yang, Craise et al. 1992; Haag, Hsu et al. 1996; Ponnaiya, Cornforth et al. 1997; Yang, Georgy et al. 1997; Unger, Wienberg et al. 2010; Behjati, Gundem et al. 2016; Morishita, Muramatsu et al. 2016).</p>
<p>However, DNA lesions and single strand breaks can also be destructive and mutagenic. Lesions can lead to point mutations (David, O'Shea et al. 2007) or single strand breaks (Regulus, Duroux et al. 2007). Lesions and single strand breaks can also promote the formation of double strand breaks: replication fork collapse and double strand breaks sometimes occur during mitosis when the replisome encounters an unrepaired single strand break (Kuzminov 2001), and clustered lesions and closely opposed single strand breaks can also form double strand breaks (Chaudhry and Weinfeld 1997; Vispe and Satoh 2000; Shiraishi, Shikazono et al. 2017). Complex damage consists of any combination of closely opposed DNA lesions, abasic sites, crosslinks, single, or double strand breaks in proximity. While classically induced by ionizing radiation, there is also evidence that it can be induced by oxidative activity (Sharma, Collins et al. 2016) or even by a single oxidizing particle (Ravanat, Breton et al. 2014). Complex damage is more difficult to repair (Kuhne, Rothkamm et al. 2000; Stenerlow, Hoglund et al. 2000; Pinto, Prise et al. 2005; Rydberg, Cooper et al. 2005).</p>
<p>DNA damage and resulting repair activity can trigger a halt in the cell cycle, cell death (apoptosis), and cause permanent changes to DNA including deletions, translocations, and sequence changes. DNA damage is also associated with an increase in genomic instability - the new appearance of DNA damage including double strand breaks, mutations, and chromosomal damage following repair of initial damage in affected cells or in clonal descendants or neighbors of DNA damaged cells. The mechanism behind this long term DNA damage is not clear, but telomere erosion appears to play a major role (Murnane 2012; Sishc, Nelson et al. 2015). Genomic instability is more common and longer lasting following complex damage (Ponnaiya, Cornforth et al. 1997), and is influenced by multiple factors including variants in DNA repair genes (Ponnaiya, Cornforth et al. 1997; Yu, Okayasu et al. 2001; Yin, Menendez et al. 2012), RONS (Dayal, Martin et al. 2008), estrogen (Kutanzi and Kovalchuk 2013), caspases (Liu, He et al. 2015), and telomeres (Sishc, Nelson et al. 2015).</p>
<p>DNA damage can be studied in isolated DNA, fixed cells, or living cells. Types of damage that can be detected include single and double strand breaks, nucleotide damage, complex damage, and chromosomal or telomere damage. The OECD test guideline for DNA synthesis Test No. 486 (OECD 1997) detects nucleotide excision repair, so it will reflect the formation of bulky DNA adducts but not the majority of oxidative damage to nucleotides, which is typically repaired via the Base Excision Repair pathway. The OECD test guideline alkaline comet assay Test No. 489 (OECD 2016) detects single and double strand breaks, including those arising from repair as well as some (alkali sensitive) nucleotide lesions including some lesions from oxidative damage. OECD tests for chromosomal damage and micronuclei Test No. 473, 475, 483, and 487 measure longer term effects of DNA damage but these tests require the damaged cell to subsequently undergo replication (OECD 2016; OECD 2016; OECD 2016; OECD 2016). They can therefore reflect a wider range of sources of DNA damage including changes in mitosis. Finally, tests for mutations reveal past DNA damage that resulted in a heritable change, and these are described in the key event ‘Increase in Mutation’.</p>
<p>Many other (non-test guideline) techniques have been used to examine specific forms of DNA damage (Table 1). Double strand breaks are commonly reported because of the significant risk attributed to breaks and the relative ease of detecting and quantifying them. Historically, single and double strand breaks were measured using gel electrophoresis, but are now commonly visualized microscopically using fluorescent or other labeled probes for double and single strand break repair such as H2AX and XRCC2. Base lesions can also be detected using labeled probes for base excision repair enzymes, or by chemical methods such as mass spectroscopy. Refinements on these methods can be used to characterize complex or clustered damage, in which various forms of damage occur in close proximity on a DNA molecule (Lorat, Timm et al. 2016; Nikitaki, Nikolov et al. 2016).</p>
<p>Certain challenges are common to all methods of detecting DNA damage. In the time required to initiate the detection method, some DNA may already be repaired, leading to undercounting of damage. On the other hand, apoptotic DSBs may be incorrectly included in a measurement of direct (non-apoptotic) induction of DSB damage unless controlled. All methods have difficulty distinguishing individual components of clustered lesions, and microscopic methods may undercount disparate breaks that are processed together in repair centers (Barnard, Bouffler et al. 2013). Methods that use isolated DNA (gel electrophoresis, analytical chemistry) are vulnerable to artifacts and must ensure that the DNA sample is protected from oxidative damage during extraction (Pernot, Hall et al. 2012; Barnard, Bouffler et al. 2013; Ravanat, Breton et al. 2014).</p>
<p>Table 1. Common methods of detecting DNA damage</p>
<table border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr>
<td style="height:22px; width:127px">
<p><strong>Target</strong></p>
</td>
<td style="height:22px; width:167px">
<p><strong>Name</strong></p>
</td>
<td style="height:22px; width:133px">
<p><strong>Method</strong></p>
</td>
<td style="height:22px; width:211px">
<p><strong>Strengths/Weaknesses</strong></p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Nucleotide damage</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Single cell gel electrophoresis (comet assay) with restriction enzymes (Collins 2004)</p>
</td>
<td style="height:22px; width:133px">
<p>Gel electrophoresis</p>
<p> </p>
</td>
<td style="height:22px; width:211px">
<p>A variant of the comet assay in which restriction enzymes allow the identification of different types of nucleotide damage.</p>
<p>The comet assay is more sensitive than PFGE, detecting damage from 0.1 Gy ionizing radiation (Pernot, Hall et al. 2012). A reproducible high-throughput application of the assay is available (Ge, Prasongtanakij et al. 2014; Sykora, Witt et al. 2018), and the test requires only a small (single cell) sample. Requires destruction of the cell.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Nucleotide damage</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Labeled probes including Biotrin OxyDNA and anti- 8-oxoguanine-DNA glycosylase (OGG1) for oxidative damage and AP</p>
<p>endonuclease (APE1) for Base Excision Repair of less bulky lesions such as oxidative damage.</p>
</td>
<td style="height:22px; width:133px">
<p>Microscopy, FACS</p>
</td>
<td style="height:22px; width:211px">
<p>Most useful with FACS or other measures of average or relative intensity, as locations and numbers of damaged nucleotides can be difficult to distinguish using fluorescence microscopy. (Ogawa, Kobayashi et al. 2003; Nikitaki, Nikolov et al. 2016).</p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Nucleotide damage</strong></p>
</td>
<td style="height:22px; width:167px">
<p>High performance liquid chromatography (HPLC), tandem mass spectrometry (MS/MS)</p>
</td>
<td style="height:22px; width:133px">
<p>Analytical chemistry</p>
</td>
<td style="height:22px; width:211px">
<p>Capable of quantifying low levels of specific nucleotide lesions (Madugundu, Cadet et al. 2014; Ravanat, Breton et al. 2014). Requires destruction of the cell.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Nucleotide damage</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Unscheduled DNA synthesis test OECD Test Guideline 486 (OECD 1997)</p>
</td>
<td style="height:22px; width:133px">
<p>Autoradiography</p>
</td>
<td style="height:22px; width:211px">
<p>Measures DNA damage that is repaired using Nucleotide Excision Repair - mostly bulky adducts (OECD (Organisation for Economic Co-operation and Development) 2016).</p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Non-specific DNA strand breaks</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Single cell gel electrophoresis (comet assay), alkali conditions</p>
<p>OECD Test Guideline 489 (OECD 2016)</p>
</td>
<td style="height:22px; width:133px">
<p>Gel electrophoresis</p>
</td>
<td style="height:22px; width:211px">
<p>When used in alkali conditions, the comet assay reveals single and double strand breaks and alkali-sensitive nucleotide lesions. See single cell gel electrophoresis (comet assay) with restriction enzymes above for further comments. </p>
<p> </p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Single strand breaks</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Labeled probe pXRCC1 (Lorat, Brunner et al. 2015)</p>
</td>
<td style="height:22px; width:133px">
<p>Microscopy</p>
</td>
<td style="height:22px; width:211px">
<p>Fluorescent probes can label single strand breaks in cells, while immunogold labeling is able to distinguish multiple single strand breaks in clusters (Lorat, Timm et al. 2016; Nikitaki, Nikolov et al. 2016).</p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Double strand breaks</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Single cell gel electrophoresis (comet assay), neutral conditions</p>
</td>
<td style="height:22px; width:133px">
<p>Gel electrophoresis</p>
</td>
<td style="height:22px; width:211px">
<p>Neutral conditions help minimize the release of single strand breaks coiled DNA and alkali lesions, allowing the measurement of double strand breaks. Since single strand breaks can still appear, assay is not very sensitive or specific to double strand breaks (Pernot, Hall et al. 2012). See single cell gel electrophoresis (comet assay) with restriction enzymes above for further comments.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Double strand breaks</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Pulsed field gel electrophoresis (PFGE)</p>
</td>
<td style="height:22px; width:133px">
<p>Gel electrophoresis</p>
</td>
<td style="height:22px; width:211px">
<p>Permits the quantitative measurement of double strand breaks, and can be combined with immunoblotting to detect DNA-associated proteins (Lobrich, Rydberg et al. 1995; Kawashima, Yamaguchi et al. 2017). Considered less sensitive than comet assay, but detected damage from 0.25 Gy ionizing radiation (Gradzka and Iwanenko 2005). Requires destruction of the cell.</p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Double strand breaks</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Labeled probes including phosphorylated H2AX, 53BP1, Ku70, ATM (Lorat, Brunner et al. 2015)</p>
</td>
<td style="height:22px; width:133px">
<p>Microscopy</p>
</td>
<td style="height:22px; width:211px">
<p>Fluorescent probes can label individual double breaks in cells allowing for quantification, with immunogold labeling resolving breaks in clusters (Lorat, Timm et al. 2016; Nikitaki, Nikolov et al. 2016). Sensitive: detects damage from 0.001 Gy ionizing radiation (Rothkamm and Lobrich 2003; Ojima, Ban et al. 2008).</p>
</td>
</tr>
<tr>
<td style="height:22px; width:127px">
<p><strong>Chromosomal damage</strong></p>
</td>
<td style="height:22px; width:167px">
<p>Chromosomal aberrations and micronuclei</p>
<p>OECD Test Guidelines 473, 475, 483, and 487 (OECD 2016; OECD 2016; OECD 2016; OECD 2016)</p>
</td>
<td style="height:22px; width:133px">
<p>Microscopy</p>
</td>
<td style="height:22px; width:211px">
<p>Detects major DNA damage resulting from large breaks and rearrangements, or mitotic failures. Damage does not appear until DNA undergoes mitosis, so slower and limited to damage in replicating cells. Insensitive tosmall deletions and substitutions.</p>
</td>
</tr>
</tbody>
</table>
CL:0000255eukaryotic cell<p><a name="_ENREF_1">Barnard, S., S. Bouffler, et al. (2013). "The shape of the radiation dose response for DNA double-strand break induction and repair." Genome integrity 4(1): 1.</a></p>
<p><a name="_ENREF_2">Behjati, S., G. Gundem, et al. (2016). "Mutational signatures of ionizing radiation in second malignancies." Nat Commun 7: 12605.</a></p>
<p><a name="_ENREF_3">Chaudhry, M. A. and M. Weinfeld (1997). "Reactivity of human apurinic/apyrimidinic endonuclease and Escherichia coli exonuclease III with bistranded abasic sites in DNA." The Journal of biological chemistry 272(25): 15650-15655.</a></p>
<p><a name="_ENREF_4">Collins, A. R. (2004). "The comet assay for DNA damage and repair: principles, applications, and limitations." Molecular biotechnology 26(3): 249-261.</a></p>
<p><a name="_ENREF_5">David, S. S., V. L. O'Shea, et al. (2007). "Base-excision repair of oxidative DNA damage." Nature 447(7147): 941-950.</a></p>
<p><a name="_ENREF_6">Dayal, D., S. M. Martin, et al. (2008). "Hydrogen peroxide mediates the radiation-induced mutator phenotype in mammalian cells." Biochem J 413(1): 185-191.</a></p>
<p><a name="_ENREF_7">Ge, J., S. Prasongtanakij, et al. (2014). "CometChip: a high-throughput 96-well platform for measuring DNA damage in microarrayed human cells." Journal of visualized experiments : JoVE(92): e50607.</a></p>
<p><a name="_ENREF_8">Gradzka, I. and T. Iwanenko (2005). "A non-radioactive, PFGE-based assay for low levels of DNA double-strand breaks in mammalian cells." DNA repair 4(10): 1129-1139.</a></p>
<p><a name="_ENREF_9">Haag, J. D., L. C. Hsu, et al. (1996). "Allelic imbalance in mammary carcinomas induced by either 7,12-dimethylbenz[a]anthracene or ionizing radiation in rats carrying genes conferring differential susceptibilities to mammary carcinogenesis." Mol Carcinog 17(3): 134-143.</a></p>
<p><a name="_ENREF_10">Kawashima, Y., N. Yamaguchi, et al. (2017). "Detection of DNA double-strand breaks by pulsed-field gel electrophoresis." Genes to cells : devoted to molecular & cellular mechanisms 22(1): 84-93.</a></p>
<p><a name="_ENREF_11">Kuhne, M., K. Rothkamm, et al. (2000). "No dose-dependence of DNA double-strand break misrejoining following alpha-particle irradiation." International journal of radiation biology 76(7): 891-900.</a></p>
<p><a name="_ENREF_12">Kutanzi, K. and O. Kovalchuk (2013). "Exposure to estrogen and ionizing radiation causes epigenetic dysregulation, activation of mitogen-activated protein kinase pathways, and genome instability in the mammary gland of ACI rats." Cancer Biol Ther 14(7): 564-573.</a></p>
<p><a name="_ENREF_13">Kuzminov, A. (2001). "Single-strand interruptions in replicating chromosomes cause double-strand breaks." Proceedings of the National Academy of Sciences of the United States of America 98(15): 8241-8246.</a></p>
<p><a name="_ENREF_14">Liu, X., Y. He, et al. (2015). "Caspase-3 promotes genetic instability and carcinogenesis." Mol Cell 58(2): 284-296.</a></p>
<p><a name="_ENREF_15">Lobrich, M., B. Rydberg, et al. (1995). "Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends." Proceedings of the National Academy of Sciences of the United States of America 92(26): 12050-12054.</a></p>
<p><a name="_ENREF_16">Lorat, Y., C. U. Brunner, et al. (2015). "Nanoscale analysis of clustered DNA damage after high-LET irradiation by quantitative electron microscopy--the heavy burden to repair." DNA repair 28: 93-106.</a></p>
<p><a name="_ENREF_17">Lorat, Y., S. Timm, et al. (2016). "Clustered double-strand breaks in heterochromatin perturb DNA repair after high linear energy transfer irradiation." Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology 121(1): 154-161.</a></p>
<p><a name="_ENREF_18">Madugundu, G. S., J. Cadet, et al. (2014). "Hydroxyl-radical-induced oxidation of 5-methylcytosine in isolated and cellular DNA." Nucleic acids research 42(11): 7450-7460.</a></p>
<p><a name="_ENREF_19">Morishita, M., T. Muramatsu, et al. (2016). "Chromothripsis-like chromosomal rearrangements induced by ionizing radiation using proton microbeam irradiation system." Oncotarget 7(9): 10182-10192.</a></p>
<p><a name="_ENREF_20">Murnane, J. P. (2012). "Telomere dysfunction and chromosome instability." Mutation research 730(1-2): 28-36.</a></p>
<p><a name="_ENREF_21">Nikitaki, Z., V. Nikolov, et al. (2016). "Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)." Free radical research 50(sup1): S64-S78.</a></p>
<p><a name="_ENREF_22">OECD (1997). Test No. 486: Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo.</a></p>
<p><a name="_ENREF_23">OECD (2016). Test No. 473: In Vitro Mammalian Chromosomal Aberration Test.</a></p>
<p><a name="_ENREF_24">OECD (2016). Test No. 475: Mammalian Bone Marrow Chromosomal Aberration Test.</a></p>
<p><a name="_ENREF_25">OECD (2016). Test No. 483: Mammalian Spermatogonial Chromosomal Aberration Test.</a></p>
<p><a name="_ENREF_26">OECD (2016). Test No. 487: In Vitro Mammalian Cell Micronucleus Test.</a></p>
<p><a name="_ENREF_27">OECD (2016). Test No. 489: In Vivo Mammalian Alkaline Comet Assay.</a></p>
<p><a name="_ENREF_28">OECD (Organisation for Economic Co-operation and Development) (2016). Overview of the set of OECD Genetic Toxicology Test Guidelines and updates performed in 2014–2015. No. 238.</a></p>
<p><a name="_ENREF_29">Ogawa, Y., T. Kobayashi, et al. (2003). "Radiation-induced oxidative DNA damage, 8-oxoguanine, in human peripheral T cells." International journal of molecular medicine 11(1): 27-32.</a></p>
<p><a name="_ENREF_30">Ojima, M., N. Ban, et al. (2008). "DNA double-strand breaks induced by very low X-ray doses are largely due to bystander effects." Radiation research 170(3): 365-371.</a></p>
<p><a name="_ENREF_31">Pernot, E., J. Hall, et al. (2012). "Ionizing radiation biomarkers for potential use in epidemiological studies." Mutation research 751(2): 258-286.</a></p>
<p><a name="_ENREF_32">Pinto, M., K. M. Prise, et al. (2005). "Evidence for complexity at the nanometer scale of radiation-induced DNA DSBs as a determinant of rejoining kinetics." Radiation research 164(1): 73-85.</a></p>
<p><a name="_ENREF_33">Ponnaiya, B., M. N. Cornforth, et al. (1997). "Induction of chromosomal instability in human mammary cells by neutrons and gamma rays." Radiation research 147(3): 288-294.</a></p>
<p><a name="_ENREF_34">Ponnaiya, B., M. N. Cornforth, et al. (1997). "Radiation-induced chromosomal instability in BALB/c and C57BL/6 mice: the difference is as clear as black and white." Radiation research 147(2): 121-125.</a></p>
<p><a name="_ENREF_35">Ravanat, J. L., J. Breton, et al. (2014). "Radiation-mediated formation of complex damage to DNA: a chemical aspect overview." Br J Radiol 87(1035): 20130715.</a></p>
<p><a name="_ENREF_36">Regulus, P., B. Duroux, et al. (2007). "Oxidation of the sugar moiety of DNA by ionizing radiation or bleomycin could induce the formation of a cluster DNA lesion." Proceedings of the National Academy of Sciences of the United States of America 104(35): 14032-14037.</a></p>
<p><a name="_ENREF_37">Rothkamm, K. and M. Lobrich (2003). "Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses." Proceedings of the National Academy of Sciences of the United States of America 100(9): 5057-5062.</a></p>
<p><a name="_ENREF_38">Rydberg, B., B. Cooper, et al. (2005). "Dose-dependent misrejoining of radiation-induced DNA double-strand breaks in human fibroblasts: experimental and theoretical study for high- and low-LET radiation." Radiation research 163(5): 526-534.</a></p>
<p><a name="_ENREF_39">Sharma, V., L. B. Collins, et al. (2016). "Oxidative stress at low levels can induce clustered DNA lesions leading to NHEJ mediated mutations." Oncotarget 7(18): 25377-25390.</a></p>
<p><a name="_ENREF_40">Shiraishi, I., N. Shikazono, et al. (2017). "Efficiency of radiation-induced base lesion excision and the order of enzymatic treatment." International journal of radiation biology 93(3): 295-302.</a></p>
<p><a name="_ENREF_41">Sishc, B. J., C. B. Nelson, et al. (2015). "Telomeres and Telomerase in the Radiation Response: Implications for Instability, Reprograming, and Carcinogenesis." Front Oncol 5: 257.</a></p>
<p><a name="_ENREF_42">Stenerlow, B., E. Hoglund, et al. (2000). "Rejoining of DNA fragments produced by radiations of different linear energy transfer." International journal of radiation biology 76(4): 549-557.</a></p>
<p><a name="_ENREF_43">Sykora, P., K. L. Witt, et al. (2018). "Next generation high throughput DNA damage detection platform for genotoxic compound screening." Sci Rep 8(1): 2771.</a></p>
<p><a name="_ENREF_44">Unger, K., J. Wienberg, et al. (2010). "Novel gene rearrangements in transformed breast cells identified by high-resolution breakpoint analysis of chromosomal aberrations." Endocrine-related cancer 17(1): 87-98.</a></p>
<p><a name="_ENREF_45">Vispe, S. and M. S. Satoh (2000). "DNA repair patch-mediated double strand DNA break formation in human cells." The Journal of biological chemistry 275(35): 27386-27392.</a></p>
<p><a name="_ENREF_46">Yang, T.-H., L. M. Craise, et al. (1992). "Chromosomal changes in cultured human epithelial cells transformed by low- and high-LET radiation." Adv Space Res 12(2-3): 127-136.</a></p>
<p><a name="_ENREF_47">Yang, T. C., K. A. Georgy, et al. (1997). "Initiation of oncogenic transformation in human mammary epithelial cells by charged particles." Radiat Oncol Investig 5(3): 134-138.</a></p>
<p><a name="_ENREF_48">Yin, Z., D. Menendez, et al. (2012). "RAP80 is critical in maintaining genomic stability and suppressing tumor development." Cancer research 72(19): 5080-5090.</a></p>
<p><a name="_ENREF_49">Yu, Y., R. Okayasu, et al. (2001). "Elevated breast cancer risk in irradiated BALB/c mice associates with unique functional polymorphism of the Prkdc (DNA-dependent protein kinase catalytic subunit) gene." Cancer Res 61(5): 1820-1824.</a></p>
2016-11-29T18:41:302019-05-08T12:28:46Increased Pro-inflammatory mediatorsIncreased pro-inflammatory mediatorsTissue<p>Inflammatory mediators are soluble, diffusible molecules that act locally at the site of tissue damage and infection, and at more distant sites. They can be divided into exogenous and endogenous mediators.</p>
<p>Exogenous mediators of inflammation are bacterial products or toxins like endotoxin or LPS. Endogenous mediators of inflammation are produced from within the (innate and adaptive) immune system itself, as well as other systems. They can be derived from molecules that are normally present in the plasma in an inactive form, such as peptide fragments of some components of complement, coagulation, and kinin systems. Or they can be released at the site of injury by a number of cell types that either contain them as preformed molecules within storage granules, e.g. histamine, or which can rapidly switch on the machinery required to synthesize the mediators.</p>
<p>Table1: a non-exhaustive list of examples for pro-inflammatory mediators</p>
<table border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr>
<td style="width:253px">
<p><strong>Classes of inflammatory mediators</strong></p>
</td>
<td style="width:361px">
<p><strong>Examples</strong></p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Pro-inflammatory cytokines</p>
</td>
<td style="width:361px">
<p>TNF-a, Interleukins (IL-1, IL-6, IL-8), Interferons (IFN-g), chemokines (CXCL, CCL, GRO-α, MCP-1), GM-CSF</p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Prostaglandins</p>
</td>
<td style="width:361px">
<p>PGE2</p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Bradykinin</p>
</td>
<td style="width:361px">
<p> </p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Vasoactive amines</p>
</td>
<td style="width:361px">
<p>histamine, serotonin</p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Reactive oxygen species (ROS)</p>
</td>
<td style="width:361px">
<p>O<sup>2-</sup>, H<sub>2</sub>O<sub>2</sub></p>
</td>
</tr>
<tr>
<td style="width:253px">
<p>Reactive nitrogen species (RNS)</p>
</td>
<td style="width:361px">
<p>NO, iNOS</p>
</td>
</tr>
</tbody>
</table>
<p>The increased production of pro-inflammatory mediators can have negative consequences on the parenchymal cells leading even to cell death, as described for TNF-a or peroxynitrite on neurons (Chao et al., 1995; Brown and Bal-Price, 2003). <span style="color:#2980b9">Along with TNF-α, IL-1β and IL-6 have been shown to exhibit negative consequences on neurogenesis and neuronal precursor cell proliferation when overexpressed. IFN-γ is also associated with neuronal damage, although it is not as extensively studied compared to TNF-α, IL-1β and IL-6.</span> In addition, via a feedback loop, they can act on the reactive resident cells thus maintaining or exacerbating their reactive state; and by modifying elements of their signalling pathways, they can favour the M1 phenotypic polarization and the chronicity of the inflammatory process (Taetzsch et al., 2015).</p>
<p>Basically, this event occurs equally in various tissues and does not require tissue-specific descriptions. Nevertheless, there are some specificities such as the release of glutamate by brain reactive glial cells (Brown and Bal-Price, 2003; Vesce et al., 2007).The differences may rather reside in the type of insult favouring the increased expression and/or release of a specific class of inflammatory mediators, as well the time after the insult reflecting different stages of the inflammatory process. For these reasons, the analyses of the changes of a battery of inflammatory mediators rather than of a single one is a more adequate measurement of this KE.</p>
<p style="margin-right:13px; text-align:justify"><span style="font-family:Arial,Helvetica,sans-serif"><strong><span style="font-size:7pt"><span style="font-size:11.0pt">Regulatory examples using the KE</span></span></strong></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:7pt"><span style="font-size:11.0pt">CD54 and CD 86 as well as IL-8 expression is used to assess skin sensitization potential (OECD TG 442E). IL-2 expression is used to assess immunotoxicity (and will become an OECD test guideline); for the latter see also doi: 10.1007/s00204-018-2199-7. </span></span></span></p>
<p> </p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>When activated, resident macrophages (Kupffer cells) release inflammatory mediators including cytokines, chemokines, lysosomal, and proteolytic enzymes and are a main source of TGF-β1 - the most potent pro-fibrogenic cytokine. Following the role of TGF-β is described in more detail.</p>
<p>Transforming growth factor β (TGF-β) is a pleiotropic cytokine with potent regulatory and</p>
<p>inflammatory activity [Sanjabi et al., 2009; Li and Flavell, 2008a;2008b]. The multi-faceted effects of TGF-β on numerous immune functions are cellular and environmental context dependent [Li et al., 2006]. TGF-β binds to TGF-β receptor II (TGF-βRII) triggering the kinase activity of the cytoplasmic domain that in turn activates TGF-βRI. The activated receptor complex leads to nuclear translocation of Smad molecules,</p>
<p>and transcription of target genes [Li et al., 2006a]. The role of TGF-β as an immune modulator of T cell activity is best exemplified by the similarities between TGF-β1 knockout and T cell specific</p>
<p>TGF-β receptor II knockout mice [Li et al., 2006b; Marie et al., 2006;Shull et al., 1992]. The animals in both of these models develop severe multi-organ autoimmunity and succumb to death within a few weeks after birth [Li et al., 2006b; Marie et al., 2006; Shull et al., 1992]. In addition, in mice where TGF-β signaling is blocked specifically in T cells, the development of natural killer T (NKT) cells, natural regulatory T (nTreg) cells, and CD8+ T cells was shown to be dependent on TGF-β signaling in the thymus [Li et al., 2006b; Marie et al., 2006].</p>
<p>TGF-β plays a major role under inflammatory conditions. TGF-β in the presence of IL-6 drives the differentiation of T helper 17 (Th17) cells, which can promote further inflammation and augment autoimmune conditions [Korn et al., 2009]. TGF-β orchestrates the differentiation of both Treg and Th17 cells in a concentration-dependent manner [Korn et al., 2008]. In addition, TGF-β in combination with IL-4, promotes the differentiation of IL-9- and IL-10-producing T cells, which lack</p>
<p>suppressive function and also promote tissue inflammation [Dardalhon et al., 2008; Veldhoen et al., 2008]. The biological effects of TGF-β under inflammatory conditions on effector and memory CD8+ T cells are much less understood. In a recent study, it was shown that TGF-β has a drastically opposing role on naïve compared to antigen-experienced/memory CD8+ T cells [Filippi et al., 2008]. When cultured <em>in vitro</em>, TGF-β suppressed naïve CD8+ T cell activation and IFN-γ production, whereas TGF-β enhanced survival of memory CD8+ T cells and increased the production of IL-17 and IFN-γ [Filippi et al., 2008]. TGF-β also plays an important role in suppressing the cells of the innate immune system.</p>
<p>The transforming growth factor beta (TGF-β) family of cytokines are ubiquitous, multifunctional, and essential to survival. They play important roles in growth and development, inflammation and repair, and host immunity. The mammalian TGF-β isoforms (TGF-β1, β2 and β3) are secreted as latent precursors and have multiple cell surface receptors of which at least two mediate signal transduction. Autocrine and paracrine effects of TGF-βs can be modified by extracellular matrix, neighbouring cells and other cytokines. The vital role of the TGF-β family is illustrated by the fact that approximately 50% of TGF-1 gene knockout mice die in utero and the remainder succumb to uncontrolled inflammation after birth. The role of TGF-β in homeostatic and pathogenic processes suggests numerous applications in the diagnosis and treatment of various diseases characterised by inflammation and fibrosis. [Clark and Coker, 1998; Santibañez et al., 2011; Pohlers et al., 2009] Abnormal TGF-β regulation and function are implicated in a growing number of fibrotic and inflammatory pathologies, including pulmonary fibrosis, liver cirrhosis, glomerulonephritis and diabetic nephropathy, congestive heart failure, rheumatoid arthritis, Marfan syndrome, hypertrophic scars, systemic sclerosis, myocarditis, and Crohn’s disease. [Gordon and Globe,2008] TGF-β1 is a polypeptide member of the TGF-β superfamily of cytokines. TGF-β is synthesized as a non-active pro-form, forms a complex with two latent associated proteins latency-associated protein (LAP) and latent TGF- β binding protein (LTBP) and undergoes protolithic cleavage by the endopeptidase furin to generate the mature TGF-β dimer. Among the TGF-βs, six distinct isoforms have been discovered although only the TGF-β1, TGF-β2 and TGF-β3 isoforms are expressed in mammals, and their human genes are located on chromosomes 19q13, 1q41 and 14q24, respectively. Out of the three TGF-β isoforms (β1, β2 and β3) only TGF-β1 was linked to fibrogenesis and is the most potent fibrogenic factor for hepatic stellate cells. [Roberts, 1998; Govinden and Bhoola, 2003]. During fibrogenesis, tissue and blood levels of active TGF-β are elevated and overexpression of TGF-β1 in transgenic mice can induce fibrosis. Additionally, experimental fibrosis can be inhibited by anti-TGF-β treatments with neutralizing antibodies or soluble TGF-β receptors [Qi et al.; 1999; Shek and Benyon , 2004; De Gouville et al., 2005; Chen et al., 2009]. TGF-β1 induces its own mRNA to sustain high levels in local sites of injury. The effects of TGF-β1 are classically mediated by intracellular signalling via Smad proteins. Smads 2 and 3 are stimulatory whereas Smad 7 is inhibitory. [Parsons et al., 2013; Friedman, 2008; Kubiczkova et al., 2012] Smad1/5/8, MAP kinase (mitogen-activated protein) and PI3 kinase are further signalling pathways in different cell types for TGF-β1 effects.</p>
<p>TGF-β is found in all tissues, but is particularly abundant in bone, lung, kidney and placental tissue. TGF-β is produced by many, but not all parenchymal cell types, and is also produced or released by infiltrating cells such as lymphocytes, monocytes/macrophages, and platelets. Following wounding or inflammation, all these cells are potential sources of TGF-β. In general, the release and activation of TGF-β stimulates the production of various extracellular matrix proteins and inhibits the degradation of these matrix proteins. [Branton and Kopp, 1999]</p>
<p>TGF-β 1 is produced by every leukocyte lineage, including lymphocytes, macrophages, and dendritic cells, and its expression serves in both autocrine and paracrine modes to control the differentiation, proliferation, and state of activation of these immune cells. [Letterio and Roberts; 1998]</p>
<p>In the liver TGF-β1 is released by activated Kupffer cells, liver sinusoidal endothelial cells, and platelets; in the further course of events also activated hepatic stellate cells express TGF-β1. Hepatocytes do not produce TGF-β1 but are implicated in intracellular activation of latent TGF-β1. [Roth et al., 1998; Kisseleva and Brenner, 2007; Kisseleva and Brenner, 2008; Poli, 2000; Liu et al., 2006]</p>
<p>TGF-β1 is the most established mediator and regulator of epithelial-mesenchymal-transition (EMT) which further contributes to the production of extracellular matrix. It has been shown that TGF-β1 mediates EMT by inducing snail-1 transcription factor and tyrosine phosphorylation of Smad2/3 with subsequent recruitment of Smad4. [Kolios et al., 2006; Bataller and Brenner, 2005; Guo and Friedman,2007; Brenner,2009; Kaimori et al., 2007; Gressner et al., 2002; Kershenobich Stalnikowitz and Weisssbrod, 2003; Li et al., 2008; Matsuoka and Tsukamoto, 1990; Kisseleva and Brenner, 2008; Poli, 200; Parsons et al., 2007; Friedman 2008; Liu et al., 2006]</p>
<p>TGF-β1 induces apoptosis and angiogenesis in vitro and in vivo through the activation of vascular endothelial growth factor (VEGF) High levels of VEGF and TGF-β1 are present in many tumors. Crosstalk between the signalling pathways activated by these growth factors controls endothelial cell apoptosis and angiogenesis. [Clark and Coker; 1998]</p>
<p> </p>
<p><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:11pt">T<span style="font-size:14px">he specific type of measurement(s) might vary with tissue, environment and context and will need to be described for different tissue contexts as used within different AOP descriptions</span></span><span style="font-size:14px">.</span></span></p>
<p><span style="font-size:14px">In general, quantification of inflammatory markers can be done by:</span></p>
<ul>
<li><span style="font-size:14px">qRT-PCR (mRNA expression)</span></li>
<li><span style="font-size:14px">ELISA</span></li>
<li><span style="font-size:14px">Immunocytochemistry</span></li>
<li><span style="font-size:14px">Immunoblotting</span></li>
</ul>
<p><span style="font-size:14px">For descriptions of techniques, see Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al., 2014</span><br />
</p>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>There are several assays for TGB-β1 measurement available.</p>
<p>e.g. Human TGF-β1 ELISA Kit. The Human TGF-β 1 ELISA (Enzyme –Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human TGF-β1 in serum, plasma, cell culture supernatants, and urine. This assay employs an antibody specific for human TGF-β1 coated on a 96-well plate. Standards and samples are pipetted into the wells and TGF-β1 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human TGF-β1 antibody is added. After washing away unbound biotinylated antibody, HRP- conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and colour develops in proportion to the amount of TGF-β1 bound. The StopSolution changes the colour from blue to yellow, and the intensity of the colour is measured at 450 nm [Mazzieri et al., 2000]</p>
<p><span style="color:#2980b9">Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed.</span></p>
<table border="1">
<tbody>
<tr>
<td>
<p><span style="color:#2980b9">Assay </span></p>
</td>
<td>
<p><span style="color:#2980b9">Reference </span></p>
</td>
<td>
<p><span style="color:#2980b9">Description </span></p>
</td>
<td>
<p><span style="color:#2980b9">OECD Approved Assay </span></p>
</td>
</tr>
<tr>
<td>
<ul>
<li>
<p><span style="color:#2980b9">RT-qPCR </span></p>
</li>
<li>
<p><span style="color:#2980b9">Q-PCR </span></p>
</li>
</ul>
</td>
<td>
<p><span style="color:#2980b9">(Veremeyko et al., 2012; Alwine et al, 1977; Forlenza et al., 2012) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Measures mRNA expression of cytokines, chemokines and inflammatory markers </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Immunoblotting (western blotting) </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Lee et al., 2008) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Uses antibodies specific to proteins of interest, can used to detect presence of pro-inflammatory mediators in samples of cell or tissue lysate </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Whole blood stimulation assay </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Thurm & Halsey, 2005) </span></p>
</td>
<td>
<p><span style="color:#2980b9"> Detects inflammatory cytokines in blood </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Imaging tests </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Rollins & Miskolci, 2014) </span></p>
</td>
<td>
<p><span style="color:#2980b9">A qualitative technique using a cytokine specific antibodies and fluorophores can be used to visualize expression patterns, subcellular location of the target and protein-protein interactions. </span></p>
<p><span style="color:#2980b9">Common examples include double immunofluorescence confocal microscopy or other molecular imaging modalities. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Flow-cytometry </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Karanikas et al., 2000) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Detects the intracellular cytokines with stimulation. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Immunoassays (ex. enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISpot), radioimmunoassay) </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Amsen et al., 2009; Engvall & Perlmann, 1972; Ji & Forsthuber, 2016; Goldsmith, 1975) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Plate based assay technique using antibodies to detect presence of a protein in a liquid sample. </span></p>
<p><span style="color:#2980b9">Can be used to identify presence of an inflammatory cytokine of interest especially when in low concentrations. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Inflammatory cytokine arrays </span></p>
<p> </p>
</td>
<td>
<p><span style="color:#2980b9">(Amsen et al., 2009) </span></p>
<p> </p>
</td>
<td>
<p><span style="color:#2980b9">Similar to the ELISA, except using a membrane-based rather than plate-based approach. Can be used to measure multiple cytokine targets concurrently. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="color:#2980b9">Immunohistochemistry (IHC) </span></p>
</td>
<td>
<p><span style="color:#2980b9">(Amsen et al., 2009; Coons et al., 1942) </span></p>
</td>
<td>
<p><span style="color:#2980b9">Immobilized tissue or cell cultures are stained using antibodies for specificity of ligands of interest. Versions of the assays can be used to visualize localization of inflammatory cytokines. </span></p>
</td>
<td>
<p><span style="color:#2980b9">No </span></p>
</td>
</tr>
</tbody>
</table>
<p><span style="font-size:14px"><strong>LIVER:</strong></span></p>
<p>Human [Santibañez et al., 2011]</p>
<p>Rat [Luckey and Petersen, 2001]</p>
<p>Mouse [Nan et al., 2013]</p>
<p><strong>BRAIN:</strong></p>
<p><span style="font-size:14px">Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al., 2014</span></p>
<p> </p>
<p><span style="color:#2980b9"><strong>Taxonomic applicability</strong>: The inflammatory response and increase of the pro-inflammatory mediators has been observed across species from simple invertebrates such as Daphnia to higher order vertebrates (Weavers & Martin, 2020).</span></p>
<p><span style="color:#2980b9"><strong>Life stage applicability</strong>: This key event is not life stage specific (Kalm et al., 2013; Veeraraghan et al., 2011; Hladik & Tapio, 2016). </span></p>
<p><span style="color:#2980b9"><strong>Sex applicability</strong>: Most studies conducted were on male models, although sex-dependent differences in pro-inflammatory markers have been previously reported (Cekanaviciute et al., 2018; Parihar et al., 2020). </span></p>
<p><span style="color:#2980b9"><strong>Evidence for perturbation by a prototypic stressor</strong>: There is evidence of the increase of pro-inflammatory mediators following perturbation from a variety of stressors including exposure to ionizing radiation. (Abdel-Magied et al., 2019; Cho et al., 2017; Gaber et al., 2003; Ismail et al., 2016; Kim et al. 2002; Lee et al., 2010; Parihar et al., 2018).</span></p>
Not SpecifiedUnspecificNot SpecifiedAll life stagesNot SpecifiedNot Specified<p> <span style="color:windowtext">Brown GC, Bal-Price A (2003) Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol 27: 325-355</span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Dong Y, Benveniste EN (2001) Immune Function of Astrocytes. Glia 36: 180-190</span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Falsig J, Latta M, Leist M. Defined inflammatory states in astrocyte cultures correlation with susceptibility towards CD95-driven apoptosis. J Neurochem. 2004 Jan;88(1):181-93. </span></span></p>
<p><span style="font-size:14px"><span style="color:windowtext">Falsig J, Pörzgen P, Lund S, Schrattenholz A, Leist M. The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function. J Neurochem. 2006 Feb;96(3):893-907. </span></span></p>
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<p><span style="font-size:14px"><span style="color:windowtext"> <strong>LIVER:</strong></span></span></p>
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2017-11-28T09:00:542023-03-21T15:50:49Increase, Mutations in Critical GenesIncrease, Mutations in Critical GenesMolecular<p>Respiratory metaplasia requires increases in cell division of local stem cells, which replace olfactory specific cell types with respiratory tissue cell types. The same process occurs during squamous metaplasia. Cell division during respiratory metaplasia occurs under conditions of cellular stress and cytotoxicity, both of which can increase the probability of mutation, as cells exert less effective control over fidelity of the genome<sup id="cite_ref-1" class="reference"><a href="#cite_note-1">[1]</a></sup>.
</p><p>Although the presence of tumors implies accumulation of DNA mutations<sup id="cite_ref-2" class="reference"><a href="#cite_note-2">[2]</a></sup>, and increased cell division is known to increase mutations<sup id="cite_ref-3" class="reference"><a href="#cite_note-3">[3]</a></sup>, direct measurement in vivo is challenging. Traditional methods of assessing the mutagenic potential of acetate ester metabolites<sup id="cite_ref-4" class="reference"><a href="#cite_note-4">[4]</a></sup>, for example in vitro systems such as the AMES assay, are appropriate for assessing the direct mutagenic potential<sup id="cite_ref-5" class="reference"><a href="#cite_note-5">[5]</a></sup>, but not for the indirect mutagenic potential of cell proliferation induced by respiratory metaplasia. Transgenic models, for example Big Blue <sup id="cite_ref-6" class="reference"><a href="#cite_note-6">[6]</a></sup> are capable of measuring specific types of DNA damage in vivo.
</p><p>DNA mutation is an obligate step in carcinogenesis.
</p>CL:0000255eukaryotic cellHighHighHigh<ol class="references">
<li id="cite_note-1"><span class="mw-cite-backlink"><a href="#cite_ref-1">↑</a></span> <span class="reference-text">Cohen and Ellwein (1991). Genetic errors, cell proliferation, and carcinogenesis. Cancer Res. 51: 6493-6505, Cohen, Purtilo and Ellwein (1991). Ideas in pathology. Pivotal role of increased cell proliferation in human carcinogenesis. Mod Pathol. 4: 371-382, Cohen (1995). Role of cell proliferation in regenerative and neoplastic disease. Toxicol Lett. 82-83: 15-21, Counts and Goodman (1995). Principles underlying dose selection for, and extrapolation from, the carcinogen bioassay: dose influences mechanism. Regul Toxicol Pharmacol. 21: 418-421</span>
</li>
<li id="cite_note-2"><span class="mw-cite-backlink"><a href="#cite_ref-2">↑</a></span> <span class="reference-text">Bertram (2000). The molecular biology of cancer. Mol Aspects Med. 21: 167-223, Hanahan and Weinberg (2000). The hallmarks of cancer. Cell. 100: 57-70</span>
</li>
<li id="cite_note-3"><span class="mw-cite-backlink"><a href="#cite_ref-3">↑</a></span> <span class="reference-text">Preston-Martin, Pike, Ross, Jones and Henderson (1990). Increased cell division as a cause of human cancer. Cancer Res. 50: 7415-7421, Cohen, Purtilo and Ellwein (1991). Ideas in pathology. Pivotal role of increased cell proliferation in human carcinogenesis. Mod Pathol. 4: 371-382</span>
</li>
<li id="cite_note-4"><span class="mw-cite-backlink"><a href="#cite_ref-4">↑</a></span> <span class="reference-text">Albertini (2013). Vinyl acetate monomer (VAM) genotoxicity profile: relevance for carcinogenicity. Crit Rev Toxicol. 43: 671-706</span>
</li>
<li id="cite_note-5"><span class="mw-cite-backlink"><a href="#cite_ref-5">↑</a></span> <span class="reference-text">Albertini (2013). Vinyl acetate monomer (VAM) genotoxicity profile: relevance for carcinogenicity. Crit Rev Toxicol. 43: 671-706</span>
</li>
<li id="cite_note-6"><span class="mw-cite-backlink"><a href="#cite_ref-6">↑</a></span> <span class="reference-text">Manjanatha, Shelton, Aidoo, Lyn-Cook and Casciano (1998). Comparison of in vivo mutagenesis in the endogenous Hprt gene and the lacI transgene of Big Blue(R) rats treated with 7, 12-dimethylbenz[a]anthracene. Mutat Res. 401: 165-178</span>
</li>
</ol>2016-11-29T18:41:272017-09-16T10:16:38Acute Myeloid LeukemiaAMLIndividual2021-12-03T07:45:422021-12-03T07:45:42Inadequate DNA repairInadequate DNA repairCellular<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:
<p style="margin-left:40px"><strong>a) Base excision repair (BER)</strong><span style="font-size:1rem"> (Dianov and Hübscher, 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>b) Nucleotide excision repair (NER)</strong> (Schärer, 2013), in which the DNA strand containing the damaged nucleotide is incised at sites several nucleotides 5’ and 3’ to the site of damage, and a polynucleotide containing the damaged nucleotide is removed prior to DNA resynthesis within the resultant gap. </p>
<p style="margin-left:40px"><strong>c) Mismatch repair (MMR)</strong> (Li et al., 2016) 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). </p>
</li>
<li><strong>Single strand break repair (SSBR) </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 are taken for all SSBs: detection, DNA end processing, synthesis, and ligation (Caldecott, 2014). Poly-ADP-ribose polymerase1 (PARP1) detects and binds unscheduled SSBs (i.e., not deliberately induced during excision repair) and synthesizes PAR as a signal to the downstream factors in repair. PARP1 is not required to initiate SSBR of BER intermediates. The XRCC1 protein complex is then recruited to the site of damage and acts as a scaffold for proteins and enzymes required for repair. Depending on the nature of the damaged termini of the DNA strand, different enzymes are required for end processing to generate the substrates that DNA polymerase β (Polβ; short patch repair) or Pol δ/ε (long patch repair) can bind to synthesize over the gap. Synthesis in long-patch repair displaces a single stranded flap which is excised by flap endonuclease 1 (FEN1). In short-patch repair, the XRCC1/Lig3α complex joins the two ends after synthesis. In long-patch repair, the PCNA/Lig1 complex ligates the ends. (Caldecott, 2014). </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). </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">The process of C-NHEJ in humans requires at least seven core proteins: Ku70, Ku86, DNA-dependent protein kinase complex (DNA-PK<sub>cs </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 </sub>is then triggered, causing DNA-PK<sub>cs </sub>to auto-phosphorylate and thereby lose its kinase activity; the now phosphorylated DNA-PK<sub>cs</sub> dissociates from the DNA-bound Ku proteins. The free DNA-PK<sub>cs</sub> phosphorylates Artemis, an enzyme that possesses 5’-3’ exonuclease and endonuclease activity in the presence of DNA-PK<sub>cs</sub> and ATP. Artemis is responsible for ‘cleaning up’ the ends of the DNA. For 5’ overhangs, Artemis nicks the overhang, generally leaving a blunt duplex end. For 3’ 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. 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). </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’ 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’ invading single strand and resynthesize the broken DNA strand using the corresponding sequence on the homologous strand.</p>
<p> </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, inadequate repair 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 (Köberle 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). 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), 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). </p>
<p>Misrepair may also occur through other repair pathways. 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>
<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 ‘indirect’ 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 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>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><span style="color:#0000cd">Assay Name</span></strong></span></td>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong><span style="color:#0000cd">References</span></strong></span></td>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong><span style="color:#0000cd">Description</span></strong></span></td>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><strong><span style="color:#0000cd">DNA Damage/Repair Being Measured</span></strong></span></td>
<td style="background-color:#eeeeee; text-align:center"><span style="font-size:14px"><span style="color:#0000cd"><strong>OECD Approved Assay</strong></span></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"> </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"> </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 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"> </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"> </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>
</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">DSBs</span></span></td>
<td style="text-align:center"><span style="font-size:14px"><span style="font-family:arial,sans-serif"> </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"><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 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 </span></td>
<td style="text-align:center"><span style="font-size:14px">Nacci et al. 1991 </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 </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">Yes (<u><span style="font-family:arial,sans-serif">No. 489)</span></u> </span></td>
</tr>
<tr>
<td><span style="font-size:14px">Sucrose Density Gradient Centrifugation with Time Course </span></td>
<td style="text-align:center"><span style="font-size:14px">Larsen et al. 1982 </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 </span></td>
<td style="text-align:center"><span style="font-size:14px">SSBs </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 </span></td>
<td style="text-align:center">
<p><span style="font-size:14px">Mariotti et al. 2013 </span></p>
<p><span style="font-size:14px">Penninckx et al. 2021 </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 </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</span></td>
</tr>
<tr>
<td><span style="font-size:14px">Alkaline Elution Assay with Time Course </span></td>
<td style="text-align:center"><span style="font-size:14px">Larsen et al. 1982 </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 </span></td>
<td style="text-align:center"><span style="font-size:14px">SSBs </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 </span></td>
<td style="text-align:center"><span style="font-size:14px">Penninckx et al. 2021 </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 </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 </span></td>
</tr>
</tbody>
</table>
<p> </p>
<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). 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 & Vijg, 2016). </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 & Seluanov, 2016). </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). </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’Brien et al., 2015). </p>
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<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Seo, Y.R. and H.J. Jung (2004), "The potential roles of p53 tumor suppressor in nucleotide excision repair (NER) and base excision repair (BER)", <em>Exp. Mol. Med.</em>, 36(6): 505-509. Doi: 10.1038/emm.2004.64.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Sundheim, O. et al. (2008), "AlkB demethylases flip out in different ways",<em> DNA Repair (Amst)</em>., 7(11): 1916-1923. Doi: <a href="https://doi.org/10.1016/j.dnarep.2008.07.015" target="_blank">10.1016/j.dnarep.2008.07.015</a>.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Sung, P., & H. Klein, (2006), “Mechanism of homologous recombination: mediators and helicases take on regulatory functions”, <em>Nat Rev Mol Cell Biol</em>, 7(10), 739-750. Doi:10. 1038/nrm2008.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Trucco, C., et al., (1998), “DNA repair defect i poly(ADP-ribose) polymerase-deficient cell lines”, Nucleic Acids Research. 26(11): 2644–2649. Doi: 10.1093/nar/26.11.2644.</span></span></p>
<p><span style="font-size:14px">Trzeciak, A.R. et al. (2008), “Age, sex, and race influence single-strand break repair capacity in a human population”, Free Radical Biology & Medicine, Vol. 45, Elsevier, Amsterdam, https://doi.org/10.1016/j.freeradbiomed.2008.08.031. </span></p>
<p><span style="font-size:14px">White, R.R. and J. Vijg. (2016), “Do DNA Double-Strand Breaks Drive Aging?”, Molecular Cell, Vol.63, Elsevier, Amsterdam, http://doi.org/10.1016/j.molcel.2016.08.004. </span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">Wyrick, J.J. & S. A. Roberts, (2015), “Genomic approaches to DNA repair and mutagenesis”, DNA Repair (Amst). 36:146-155. doi: 10.1016/j.dnarep.2015.09.018.</span></span></p>
<p><span style="font-size:14px"><span style="font-family:arial,helvetica,sans-serif">van Zeeland, A.A., A. de Groot and A. Neuhäuser-Klaus (1990), "DNA adduct formation in mouse testis by ethylating agents: a comparison with germ-cell mutagenesis", <em>Mutat. Res.</em>, 231(1): 55-62.</span></span></p>
2016-11-29T18:41:232023-04-24T11:01:39Increase, Cell ProliferationIncrease, Cell ProliferationCellular<p><span style="color:#e74c3c">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, quiescent cells reside in an optional phase just before G1, known as G0. </span></p>
<p><span style="color:#e74c3c">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 (Cuyàs et al., 2014). </span></p>
<p><span style="color:#e74c3c">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 & O’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).</span> </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. <span style="color:#e74c3c">This is often achieved through oncogene activation and inactivation of tumor suppressor genes</span> (Hanahan et al., 2011). Cell inactivation and the replacement of these cells can initiate clonal expansion (Heidenreich adn Paretzke et al., 2008). </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>
<p>Two common methods of measuring cell proliferation in vivo are the use of Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) labeling (Pera, 1977), and Ki67 immunostaining (Grogan, 1988). BrdU is a synthetic analogue of the nucleoside Thymidine. BrDu is incorporated into DNA synthesized during the S1 phase of cell replication and is stable for long periods. Labeling of dividing cells by BrdU is accomplished by infusion, bolus injection, or implantation of osmotic pumps containing BrdU for a period of time sufficient to generate measureable numbers of labeled cells. Tissue sections are stained immunhistochemically with antibodies for BrdU and labeled cells are counted as dividing cells. Ki67 is a cellular marker of replication not found in quiescent cells (Roche, 2015). Direct immunohistochemical staining of cells for protein Ki67 using antibodies is an alternative to the use of BrdU, with the benefit of not requiring a separate treatment (injection for pulse-labeling). Cells positive for Ki67 are counted as replicating cells. Replicating cell number is reported per unit tissue area or per cell nuclei (Bogdanffy, 1997). <span style="color:#e74c3c">Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed.</span></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="color:#0000cd"><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif"><strong>Assay Name</strong></span></span></span></td>
<td style="background-color:#dddddd; text-align:center"><span style="font-family:arial,helvetica,sans-serif"><span style="color:#0000cd"><span style="font-size:12px"><strong>References</strong></span></span></span></td>
<td style="background-color:#dddddd; text-align:center"><span style="color:#0000cd"><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif"><strong>Description</strong></span></span></span></td>
<td style="background-color:#dddddd; text-align:center"><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px"><span style="color:#0000cd"><strong>OECD Approved Assay</strong></span></span></span></td>
</tr>
<tr>
<td><span style="color:#0000cd"><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">CyQuant Cell Proliferation Assay</span></span></span></td>
<td><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px"><span style="color:#0000cd">Jones et al., 2001</span></span></span></td>
<td><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px"><span style="color:#0000cd">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></span></td>
<td><span style="color:#0000cd">N/A</span></td>
</tr>
<tr>
<td><span style="color:#0000cd"><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Nucleotide Analog Incorporation Assays (e.g. BrdU, EdU)</span></span></span></td>
<td><span style="color:#0000cd"><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Romar et al., 2016, Roche; 2013</span></span></span></td>
<td><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px"><span style="color:#0000cd">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></span></td>
<td><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px"><span style="color:#0000cd">Yes (No. 442B)</span></span></span></td>
</tr>
<tr>
<td><span style="color:#0000cd"><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Cytoplasmic Proliferation Dye Assays</span></span></span></td>
<td><span style="color:#0000cd"><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Quah & Parish, 2012</span></span></span></td>
<td><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px"><span style="color:#0000cd">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></span></td>
<td><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px"><span style="color:#0000cd">N/A</span></span></span></td>
</tr>
<tr>
<td><span style="color:#0000cd"><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Colourimetric Dye Assays</span></span></span></td>
<td><span style="color:#0000cd"><span style="font-size:12px"><span style="font-family:arial,helvetica,sans-serif">Vega-Avila & Pugsley, 2011; American Type Culture Collection</span></span></span></td>
<td><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px"><span style="color:#0000cd">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></span></td>
<td><span style="font-family:arial,helvetica,sans-serif"><span style="font-size:12px"><span style="color:#0000cd">N/A</span></span></span></td>
</tr>
</tbody>
</table>
<p> </p>
<p><span style="color:#27ae60"><strong> </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"><span style="color:#0000cd">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></span></p>
<p><span style="color:#e74c3c"><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="color:#e74c3c"><strong>Sex applicability:</strong> This key event is not sex specific (Markiewicz et al., 2015). </span></p>
<p><span style="color:#e74c3c"><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 γ-rays (Hanna and O’Brien, 1963; Barnard et al., 2022; McCarron et al., 2021), 137Cs γ-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 (Söderberg et al., 1986; Andley et al., 1994; Cheng et al., 2019), UVC (Trenton and Courtois, 1981), and mechanical wounding (Riley et al., 1989).</span></p>
HighUnspecificHighAll life stagesHighHighHigh<p><span style="color:#e74c3c">Andley, U. P. et al. (1994), “Modulation of lens epithelial cell proliferation by enhanced prostaglandin synthesis after UVB exposure”, Investigative Ophthalmology & Visual Science, Vol. 35/2, Rockville, pp. 374-381 </span></p>
<p><span style="color:#e74c3c">Andley, U. and A. Spector (2005), “Peroxide resistance in human and mouse lens epithelial cell lines is related to long-term changes in cell biology and architecture”, Free Radical Biology & Medicine, Vol. 39/6, Elsevier B.V, United States, https://doi.org/10.1016/j.freeradbiomed.2005.04.028 </span></p>
<p><span style="color:#e74c3c">Bahia, S. et al. (2018), “Oxidative and nitrative stress-related changes in human lens epithelial cells following exposure to X-rays”, International journal of radiation biology, Vol. 94/4, England, </span><a href="https://doi.org/10.1080/09553002.2018.1439194" rel="noreferrer noopener" target="_blank"><span style="color:#e74c3c">https://doi.org/10.1080/09553002.2018.1439194</span></a><span style="color:#e74c3c"> </span></p>
<p><span style="color:#e74c3c">Barnard, S. et al. (2022), “Lens Epithelial Cell Proliferation in Response to Ionizing Radiation.”, Radiation Research, Vol. 197/1, Radiation Research Society, United States, </span><a href="https://doi.org/10.1667/RADE-20-00294.1" rel="noreferrer noopener" target="_blank"><span style="color:#e74c3c">https://doi.org/10.1667/RADE-20-00294.1</span></a><span style="color:#e74c3c"> </span></p>
<p><span style="color:#e74c3c">Barnum, K. and M. O’Connell (2014), “Cell cycle regulation by checkpoints”, in Cell cycle control, Springer, New York, http://doi.org/ 10.1007/978-1-4939-0888-2 </span></p>
<p><span style="color:mediumblue; font-family:arial,sans-serif; font-size:9pt">Bogdanffy. et al. (1997). “FOUR-WEEK INHALATION CELL PROLIFERATION STUDY OF THE EFFECTS OF VINYL ACETATE ON RAT NASAL EPITHELIUM”, Inhalation Toxicology, Taylor & Francis. 9: 331-350.</span></p>
<p> </p>
<p><span style="color:#e74c3c">Cheng, T. et al. (2019), “lncRNA H19 contributes to oxidative damage repair in the early age-related cataract by regulating miR-29a/TDG axis”, Journal of cellular and molecular medicine, Vol. 23/9, Wiley Subscription Services, Inc. England, https://doi.org/10.1111/jcmm.14489 </span></p>
<p><span style="color:#e74c3c">Cuyàs, E. et al. (2014), “Cell cycle regulation by the nutrient-sensing mammalian target of rapamycin (mTOR) pathway”, in Cell cycle control, Springer, New York, http://dx.doi.org/ 10.1007/978-1-4939-0888-2 </span></p>
<p><span style="color:#e74c3c">Fujimichi, Y. and N. Hamada (2014), “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”, PloS one, Vol. 9/5, e98154, Public Library of Science, United States, </span><a href="https://doi.org/10.1371/journal.pone.0098154" rel="noreferrer noopener" target="_blank"><span style="color:#e74c3c">https://doi.org/10.1371/journal.pone.0098154</span></a><span style="color:#e74c3c"> </span></p>
<p><span style="color:mediumblue; font-family:arial,sans-serif; font-size:9pt">Grogan. et al. (1988). “Independent prognostic significance of a nuclear proliferation antigen in diffuse large cell lymphomas as determined by the monoclonal antibody Ki-67”, Blood. 71: 1157-1160.</span></p>
<p><span style="color:#e74c3c">Hanna, C. and J. E. O’Brien (1963), “Lens epithelial cell proliferation and migration in radiation cataracts”, Radiation research, Academic Press, Inc, United States, </span><a href="https://doi.org/10.2307/3571405" rel="noreferrer noopener" target="_blank"><span style="color:#e74c3c">https://doi.org/10.2307/3571405</span></a><span style="color:#e74c3c"> </span></p>
<p><span style="color:mediumblue; font-family:arial,sans-serif; font-size:9pt">H</span><span style="color:mediumblue; font-family:arial,sans-serif; font-size:9pt">anahan, D. & R. A. Weinberg, (2011),” Hallmarks of cancer: the next generation”, Cell. 144(5):646-74. doi: 10.1016/j.cell.2011.02.013.</span></p>
<p><span style="color:#0000cc"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">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></span></p>
<p><span style="color:mediumblue; font-family:arial,sans-serif; font-size:9pt">Jones, J. L. et al. </span><span style="color:mediumblue; font-family:arial,sans-serif; font-size:9pt">(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></p>
<p> </p>
<p><span style="color:#e74c3c">Kleiman, N. J. et al. (2007), “Mrad9 and Atm haplinsufficiency enhance spontaneous and X-ray-induced cataractogenesis in mice”, Radiation research, Vol. 168/5, Radiation Research Society, United States, </span><a href="https://doi.org/10.1667/rr1122.1" rel="noreferrer noopener" target="_blank"><span style="color:#e74c3c">https://doi.org/10.1667/rr1122.1</span></a><span style="color:#e74c3c"> </span></p>
<p><span style="color:#e74c3c">Lovicu, J. et al (2014), “Lens epithelial cell proliferation”, in Lens epithelium and posterior capsular opacification, Springer, Tokyo, http://dx.doi.org/ 10.1007/978-4-431-54300-8_4 </span></p>
<p><span style="color:#e74c3c">Markiewicz, E. et al. (2015), “Nonlinear ionizing radiation-induced changes in eye lens cell proliferation, cyclin K1 expression and lens shape”, Open biology, Vol. 5/4, The Royal Society, England, </span><a href="https://doi.org/10.1098/rsob.150011" rel="noreferrer noopener" target="_blank"><span style="color:#e74c3c">https://doi.org/10.1098/rsob.150011</span></a><span style="color:#e74c3c"> </span></p>
<p><span style="color:#e74c3c">McCarron, R. A. et al. (2021), “Radiation-induced lens opacity and cataractogenesis: a lifetime study using mice of varying genetic backgrounds”, Radiation research, Vol. 197/1, Radiation Research Society, United States, https://doi.org/10.1667/RADE-20-00266.1 </span></p>
<p><span style="color:#e74c3c">Pendergrass, W. et al. (2010), “X-ray induced cataract is preceded by LEC loss, and coincident with accumulation of cortical DNA, and ROS; similarities with age-related cataracts”, Molecular vision, Vol. 16, Molecular Vision, United States, pp. 1496-1513 </span></p>
<p><span style="color:mediumblue; font-family:arial,sans-serif; font-size:9pt">Pera, Mattias and Detzer (1977). “Methods for determining the proliferation kinetics of cells by means of 5-bromodeoxyuridine”, Cell Tissue Kinet.10: 255-264. Doi: 10.1111/j.1365-2184.1977.tb00293.x.</span></p>
<p><span style="color:mediumblue; font-family:arial,sans-serif; font-size:9pt">Portt, L. et al. (2011), “Anti-apoptosis and cell survival: a review”, Biochim Biophys Acta. 21813(1):238-59. doi: 10.1016/j.bbamcr.2010.10.010.</span></p>
<p><span style="color:mediumblue; font-family:arial,sans-serif; font-size:9pt">Quah, J. C. B. & R. C. Parish (2012), “New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes”, Journal of Immunological Methods. 379(1-2), 1-14. doi: 10.1016/j.jim.2012.02.012.</span></p>
<p> </p>
<p><span style="color:#e74c3c">Ramsell, T. G. and R. J. Berry (1966), “Recovery from X-ray damage to the lens. The effects of fractionated X-ray doses observed in rabbit lens epithelium irradiated in vivo”, British Journal of Radiology, Vol. 39/467, England, pp. 853-858 </span></p>
<p><span style="color:#e74c3c">Riley, E. F. et al. (1988), “Recovery of murine lens epithelial cells from single and fractionated doses of X rays and neutrons”, Radiation Research, Vol. 114/3, Academic Press Inc, Oak Brook, https://doi.org/10.2307/3577127 </span></p>
<p><span style="color:#e74c3c">Riley, E. F. et al. (1989), “Comparison of recovery from potential mitotic abnormality in mitotically quiescent lens cells after X, neutron, and 56Fe irradiations”, Radiation Research, Vol. 119/2, United States, pp. 232-254 </span></p>
<p><span style="color:#e74c3c">Richards, R. D. (1966), “Changes in lens epithelium after X-ray or neutron irradiation (mouse and rabbit)”, Transactions of the American Ophthalmological Society, Vol. 64, United States, pp. 700-734 </span></p>
<p><span style="color:mediumblue; font-family:arial,sans-serif; font-size:9pt">Roche Applied Science, (2013), “Cell Proliferation Elisa, BrdU (Colourmetric) ». </span><span style="color:mediumblue; font-family:arial,sans-serif; font-size:9pt">Version 16</span></p>
<p><span style="color:mediumblue; font-family:arial,sans-serif; font-size:9pt">Romar, A. G., S. T. Kupper & J. S. Divito (2015), “Research Techniques Made Simple: Techniques to Assess Cell Proliferation”, Journal of Investigative Dermatology. 136(1), e1-7. doi: 10.1016/j.jid.2015.11.020.</span></p>
<p> </p>
<p><span style="color:#e74c3c">Söderberg, P. G. et al. (1986), “Unscheduled DNA synthesis in lens epithelium after in vivo exposure to UV radiation in the 300 nm wavelength region”, Acta Ophthalmologica, Vol. 64/2, Blackwell Publishing Ltd, Oxford, UK, https://doi.org/10.1111/j.1755-3768.1986.tb06894.x </span></p>
<p><span style="color:#e74c3c">Trenton, J. A. and Y. Courtois (1981), “Evolution of the distribution, proliferation and ultraviolet repair capacity of rat lens epithelial cells as a function of maturation and aging”, Mechanisms of Ageing and Development, Vol. 15/3, Elsevier, Ireland, https://doi.org/1016/0047-6374(81)90134-2 </span></p>
<p><span style="color:mediumblue; font-family:arial,sans-serif; font-size:9pt">Vega-Avila, E. & K. M. Pugsley (2011), “An Overview of Colorimetric Assay Methods Used to Assess Survival or Proliferation of Mammalian Cells”, Proc. West. Pharmacol. Soc. 54, 10-4.</span></p>
<p> </p>
<p><span style="color:#e74c3c">von Sallmann, L. (1951), “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”, Transactions of the American Ophthalmological Society, Vol. 48, United States, pp. 228-242 </span></p>
<p><span style="color:#e74c3c">Worgul, B. V. et al. (1986), “Accelerated heavy particles and the lens II. Cytopathological changes”, Investigative Ophthalmology and Visual Science, Vol 27/1, pp. 108-114 </span></p>
<p> </p>
2016-11-29T18:41:272023-04-24T11:08:07Suppression, Immune systemSuppression, Immune systemIndividual2016-11-29T18:41:242016-12-03T16:37:49Deposition of Energy by Ionizing Radiation leading to Acute Myeloid LeukemiaIonizing Radiation-Induced AML<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Calibri",sans-serif">Kimberly Appelgate<sup>1</sup>, Christophe Badie<sup>2</sup>, Dag Anders Brede<sup>3</sup>, Fieke Dekkers<sup>4,5,</sup>, Maria Gomolka<sup>6</sup>, Dmitry Klokov<sup>7,8</sup>,Yevgeniya Le<sup>9,10</sup>, Katalin Lumniczky<sup>11</sup></span></span></span></p>
<p> </p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><sup><span style="background-color:white"><span style="font-family:"Calibri",sans-serif"><span style="color:#212121">1</span></span></span></sup><span style="background-color:white"><span style="font-family:"Calibri",sans-serif"><span style="color:#212121">Radiology, University of Kentucky College of Medicine, Lexington, 40506-9983, UNITED STATES.</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><sup><span style="font-family:"Calibri",sans-serif">2</span></sup><span style="font-family:"Calibri",sans-serif">Christophe Badie/Cancer Mechanisms and Biomarkers group/Radiation Effects Department/Radiation, Chemical & Environmental Hazards/Harwell Campus</span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Calibri",sans-serif">Chilton, Didcot, Oxfordshire OX11 ORQ United Kingdom/ UK Health Security Agency </span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><sup><span style="font-family:"Calibri",sans-serif">3</span></sup><span style="background-color:white"><span style="font-family:"Calibri",sans-serif"><span style="color:#212121">Centre for Environmental Radioactivity (CERAD), Faculty of Environmental Sciences and Natural Resource Management (MINA), Norwegian University of Life Sciences (NMBU), 1432 Ås, Norway.</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><sup><span style="background-color:white"><span style="font-family:"Calibri",sans-serif"><span style="color:#212121">4</span></span></span></sup><span style="background-color:white"><span style="font-family:"Calibri",sans-serif"><span style="color:#212121">Mathematical Institute, Utrecht University, Utrecht, 3508 TA, The Netherlands.</span></span></span> </span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><sup><span style="font-family:"Calibri",sans-serif">5</span></sup><span style="background-color:white"><span style="font-family:"Calibri",sans-serif"><span style="color:#212121">Netherlands National Institute for Public Health and the Environment, Bilthoven, The Netherlands.</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><sup><span style="font-size:12.0pt">6</span></sup><span style="font-size:12.0pt">Federal Office for Radiation Protection│ Radiationbiology │ WR1 Ingolstädter Landstr.1</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">85764 Oberschleissheim</span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><sup><span style="font-family:"Calibri",sans-serif">7</span></sup><span style="background-color:white"><span style="font-family:"Calibri",sans-serif"><span style="color:#212121">Experimental Radiotoxicology and Radiobiology Laboratory, Institute for Radiological Protection and Nuclear Safety, 92262 Fontenay-aux-Roses, France.</span></span></span> </span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><sup><span style="font-family:"Calibri",sans-serif">8</span></sup><span style="background-color:white"><span style="font-family:"Calibri",sans-serif"><span style="color:#212121">Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON K1N 6N5, Canada.</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><sup><span style="font-family:"Calibri",sans-serif">9</span></sup><span style="background-color:white"><span style="font-family:"Calibri",sans-serif"><span style="color:#212121">Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada.</span></span></span></span></span></p>
<p><br />
<span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Calibri",sans-serif"><sup>10</sup></span><span style="background-color:white"><span style="font-family:"Calibri",sans-serif"><span style="color:#212121">Radiobiology and Health, Canadian Nuclear Laboratories, Chalk River, ON, Canada.</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><sup><span style="font-family:"Calibri",sans-serif">11</span></sup><span style="background-color:white"><span style="font-family:"Calibri",sans-serif"><span style="color:#212121">Unit of Radiation Medicine, Department of Radiobiology and Radiohygiene, National Public Health Centre, 1221 Budapest, Hungary.</span></span></span></span></span></p>
<p> </p>
Under development: Not open for comment. Do not cite<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">It is well documented that ionizing radiation( (eg. X-rays, gamma, photons, alpha, beta, neutrons, heavy ions) 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>
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