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Key Event Title
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Cyp2E1 Activation Leading to Liver Cancer||KeyEvent||Francina Webster (send email)||Open for citation & comment||EAGMST Under Review|
|Oxidative stress and Developmental impairment in learning and memory||KeyEvent||Marie-Gabrielle Zurich (send email)||Under development: Not open for comment. Do not cite||EAGMST Under Review|
|Oxidative stress in chronic kidney disease||KeyEvent||Frederic Y. Bois (send email)||Under development: Not open for comment. Do not cite|
|TLR9 activation leading to ARDS and MOD||KeyEvent||Gillina Bezemer (send email)||Under development: Not open for comment. Do not cite|
|Oxidative stress [MIE] Leading to Decreased Lung Function [AO]||MolecularInitiatingEvent||Karsta Luettich (send email)||Open for comment. Do not cite|
|All life stages||High|
Key Event Description
Oxidative stress is defined as an imbalance in the production of reactive oxygen species (ROS) and antioxidant defenses. High levels of oxidizing free radicals can be very damaging to cells and molecules within the cell. As a result, the cell has important defense mechanisms to protect itself from ROS. For example, Nrf2 is a transcription factor and master regulator of the oxidative stress response. During periods of oxidative stress, Nrf2-dependent changes in gene expression are important in regaining cellular homeostasis (Nguyen, et al. 2009) and can be used as indicators of the presence of oxidative stress in the cell.
In addition to the directly damaging actions of ROS, cellular oxidative stress also changes cellular activities on a molecular level. Redox sensitive proteins have altered physiology in the presence and absence of ROS, which is caused by the oxidation of sulfhydryls to disulfides (2SH àSS) on neighboring amino acids (Antelmann and Helmann 2011). Importantly Keap1, the negative regulator of Nrf2, is regulated in this manner (Itoh, et al. 2010).
Protection against oxidative stress is relevant for all tissues and organs, although some tissues may be more susceptible. For example, the brain possesses several key physiological features, such as high O2 utilization, high polyunsaturated fatty acids content, presence of autooxidable neurotransmitters, and low antioxidant defenses as compared to other organs, that make it highly susceptible to oxidative stress (Halliwell, 2006; Emerit and al., 2004; Frauenberger et al., 2016).
How It Is Measured or Detected
Oxidative Stress. Direct measurement of ROS is difficult because ROS are unstable. The presence of ROS can be assayed indirectly by measurement of cellular antioxidants, or by ROS-dependent cellular damage:
- Detection of ROS by chemiluminescence (https://www.sciencedirect.com/science/article/abs/pii/S0165993606001683)
- Detection of ROS by chemiluminescence is also described in OECD TG 495 to assess phototoxic potential.
- Glutathione (GSH) depletion. GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html).
- TBARS. Oxidative damage to lipids can be measured by assaying for lipid peroxidation using TBARS (thiobarbituric acid reactive substances) using a commercially available kit.
- 8-oxo-dG. Oxidative damage to nucleic acids can be assayed by measuring 8-oxo-dG adducts (for which there are a number of ELISA based commercially available kits),or HPLC, described in Chepelev et al. (Chepelev, et al. 2015).
Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assay for Nrf2 activity include: - Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus;
- Western blot for increased Nrf2 protein levels;
- Western blot of cytoplasmic and nuclear fractions to observe translocation of Nrf2 protein from the cytoplasm to the nucleus;
- qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences);
- Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway (e.g., Jackson et al. 2014);
- OECD TG422D describes an ARE-Nrf2 Luciferase test method;
- In general, there are a variety of commercially available colorimetric or fluorescent kits for detecting Nrf2 activation.
Domain of Applicability
Oxidative stress is produced in, and can occur in, any species from bacteria through to humans.
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
Antelmann, H., Helmann, J.D., 2011. Thiol-based redox switches and gene regulation. Antioxid. Redox Signal. 14, 1049-1063.
Chepelev, N.L., Kennedy, D.A., Gagne, R., White, T., Long, A.S., Yauk, C.L., White, P.A., 2015. HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, in Cultured Cells and Animal Tissues. J. Vis. Exp. (102):e52697. doi, e52697.
Emerit, J., Edeas, M., Bricaire, F., 2004. Neurodegenerative diseases and oxidative stress. Biomed. Pharmacotherapy. 58(1): 39-46.
Frauenberger, E.A., Scola, G., Laliberté, V.L.M., Duong, A., Andreazza, A.C., 2015. Redox modulations, Antioxidants, and Neuropsychitrica Disorders. Ox. Med. Cell. Longevity. Vol. 2016, Article ID 4729192.
Halliwell, B., 2006. Oxidative stress and neurodegeneration: where are we now? J. Neurochem. 97(6):1634-1658.
Itoh, K., Mimura, J., Yamamoto, M., 2010. Discovery of the negative regulator of Nrf2, Keap1: a historical overview. Antioxid. Redox Signal. 13, 1665-1678.
Jackson, A.F., Williams, A., Recio, L., Waters, M.D., Lambert, I.B., Yauk, C.L., 2014. Case study on the utility of hepatic global gene expression profiling in the risk assessment of the carcinogen furan. Toxicol. Applied Pharmacol.274, 63-77.
Nguyen, T., Nioi, P., Pickett, C.B., 2009. The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. J. Biol. Chem. 284, 13291-13295.
OECD (2018), Test No. 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264229822-en.
OECD (2019), Test No. 495: Ros (Reactive Oxygen Species) Assay for Photoreactivity, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/915e00ac-en.