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Event: 1392

Key Event Title

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Oxidative Stress

Short name

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Oxidative Stress

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Biological Context

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Level of Biological Organization
Molecular

Cell term

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

Organ term

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signaling by that receptor). Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help

AOP Name	Role of event in AOP	Point of Contact	Author Status	OECD Status
Cyp2E1 Activation Leading to Liver Cancer	KeyEvent	Agnes Aggy (send email)	Open for citation & comment	WPHA/WNT Endorsed
Oxidative stress and Developmental impairment in learning and memory	KeyEvent	Brendan Ferreri-Hanberry (send email)	Under development: Not open for comment. Do not cite	EAGMST Approved
Oxidative stress in chronic kidney disease	KeyEvent	Brendan Ferreri-Hanberry (send email)	Under development: Not open for comment. Do not cite
TLR9 activation leading to Multi Organ Failure and ARDS	KeyEvent	Cataia Ives (send email)	Under development: Not open for comment. Do not cite
Oxidative stress Leading to Decreased Lung Function	MolecularInitiatingEvent	Brendan Ferreri-Hanberry (send email)	Open for comment. Do not cite
Ox stress-mediated CFTR/ASL/CBF/MCC impairment	MolecularInitiatingEvent	Arthur Author (send email)	Open for comment. Do not cite
ox stress-mediated FOXJ1/cilia/CBF/MCC impairment	MolecularInitiatingEvent	Agnes Aggy (send email)	Open for comment. Do not cite
tau-AOP	KeyEvent	Brendan Ferreri-Hanberry (send email)	Under development: Not open for comment. Do not cite
Inhibition of Mt-ETC complexes leading to kidney toxicity	KeyEvent	Agnes Aggy (send email)	Under development: Not open for comment. Do not cite
PM-induced respiratory toxicity	KeyEvent	Cataia Ives (send email)	Under development: Not open for comment. Do not cite
AhR activation leads to increased diabetes risk	KeyEvent	Arthur Author (send email)	Under development: Not open for comment. Do not cite

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help

Term	Scientific Term	Evidence	Link
rodents	rodents	High	NCBI
Homo sapiens	Homo sapiens	High	NCBI

Life Stages

An indication of the the relevant life stage(s) for this KE. More help

Life stage	Evidence
All life stages	High

Sex Applicability

An indication of the the relevant sex for this KE. More help

Term	Evidence
Mixed	High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. More help

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

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA). Do not provide detailed protocols. More help

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

Assay Type & Measured Content	Description	Dose Range Studied	Assay Characteristics (Length / Ease of use/Accuracy)
ROS Formation in the Mitochondria assay (Shaki et al., 2012)	“The mitochondrial ROS measurement was performed flow cytometry using DCFH-DA. Briefly, isolated kidney mitochondria were incubated with UA (0, 50, 100 and 200 μM) in respiration buffer containing (0.32 mM sucrose, 10 mM Tris, 20 mM Mops, 50 μM EGTA, 0.5 mM MgCl2, 0.1 mM KH2PO4 and 5 mM sodium succinate) [32]. In the interval times of 5, 30 and 60 min following the UA addition, a sample was taken and DCFH-DA was added (final concentration, 10 μM) to mitochondria and was then incubated for 10 min. Uranyl acetate-induced ROS generation in isolated kidney mitochondria were determined through the flow cytometry (Partec, Deutschland) equipped with a 488-nm argon ion laser and supplied with the Flomax software and the signals were obtained using a 530-nm bandpass filter (FL-1 channel). Each determination is based on the mean fluorescence intensity of 15,000 counts.”	0, 50, 100 and 200 μM of Uranyl Acetate	Long/ Easy High accuracy
Mitochondrial Antioxidant Content Assay Measuring GSH content (Shaki et al., 2012)	“GSH content was determined using DTNB as the indicator and spectrophotometer method for the isolated mitochondria. The mitochondrial fractions (0.5 mg protein/ml) were incubated with various concentrations of uranyl acetate for 1 h at 30 °C and then 0.1 ml of mitochondrial fractions was added into 0.1 mol/l of phosphate buffers and 0.04% DTNB in a total volume of 3.0 ml (pH 7.4). The developed yellow color was read at 412 nm on a spectrophotometer (UV-1601 PC, Shimadzu, Japan). GSH content was expressed as μg/mg protein.”	0, 50, 100, or 200 μM Uranyl Acetate
H₂O₂ Production Assay Measuring H₂O₂ Production in isolated mitochondria (Heyno et al., 2008)	“Effect of CdCl₂ and antimycin A (AA) on H₂O₂ production in isolated mitochondria from potato. H₂O₂ production was measured as scopoletin oxidation. Mitochondria were incubated for 30 min in the measuring buffer (see the Materials and Methods) containing 0.5 mM succinate as an electron donor and 0.2 µM mesoxalonitrile 3‐chlorophenylhydrazone (CCCP) as an uncoupler, 10 U horseradish peroxidase and 5 µM scopoletin.” (	0, 10, 30 μM Cd²⁺ 2 μM antimycin A
Flow Cytometry ROS & Cell Viability (Kruiderig et al., 1997)	“For determination of ROS, samples taken at the indicated time points were directly transferred to FACScan tubes. Dih123 (10 mM, final concentration) was added and cells were incubated at 37°C in a humidified atmosphere (95% air/5% CO2) for 10 min. At t 5 9, propidium iodide (10 mM, final concentration) was added, and cells were analyzed by flow cytometry at 60 ml/min. Nonfluorescent Dih123 is cleaved by ROS to fluorescent R123 and detected by the FL1 detector as described above for Dc (Van de Water 1995)”		Strong/easy medium
DCFH-DA Assay Detection of hydrogen peroxide production (Yuan et al., 2016)	Intracellular ROS production was measured using DCFH-DA as a probe. Hydrogen peroxide oxidizes DCFH to DCF. The probe is hydrolyzed intracellularly to DCFH carboxylate anion. No direct reaction with H₂O₂to form fluorescent production.	0-400 µM	Long/ Easy High accuracy
H2-DCF-DA Assay Detection of superoxide production (Thiebault et al., 2007)	This dye is a stable nonpolar compound which diffuses readily into the cells and yields H2-DCF. Intracellular OH or ONOO- react with H2-DCF when cells contain peroxides, to form the highly fluorescent compound DCF, which effluxes the cell. Fluorescence intensity of DCF is measured using a fluorescence spectrophotometer.	0–600 µM	Long/ Easy High accuracy
CM-H2DCFDA Assay	Come back and explain the flow cytometry determination of oxidative stress from Pan et al. (2009)

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided). More help

Oxidative stress is produced in, and can occur in, any species from bacteria through to humans.

References

List of the literature that was cited for this KE description. More help

Al Dera, H. S. (2016). Protective effect of resveratrol against aluminum chloride induced nephrotoxicity in rats. Saudi Med J, 37(4), 369-378. doi:10.15537/smj.2016.4.13611

Andjelkovic, M., Djordjevic, A. B., Antonijevic, E., Antonijevic, B., Stanic, M., Kotur-Stevuljevic, J., . . . Bulat, Z. (2019). Toxic effect of acute cadmium and lead exposure in rat blood, liver, and kidney. International Journal of Environmental Research and Public Health, 16, 247. doi:10.3390/ijerph16020274

Antelmann, H., Helmann, J.D., 2011. Thiol-based redox switches and gene regulation. Antioxid. Redox Signal. 14, 1049-1063.

Belyaeva, E. A., Sokolova, T. V., Emelyanova, L. V., & Zakharova, I. O. (2012). Mitochondrial electron transport chain in heavy metal-induced neurotoxicity : Effects of cadmium , mercury , and copper. Thescientificworld, 2012, 1-14. doi:10.1100/2012/136063

Bhadauria, S., & Flora, S. J. S. (2007). Response of arsenic-induced oxidative stress, DNA damage, and metal imbalance to combined administration of DMSA and monoisoamyl-DMSA during chronic arsenic poisoning in rats. Cell Biol Toxicol, 23, 91-104. doi:10.1007/s10565-006-0135-8

Buelna-Chontal, M., Franco, M., Hernandez-Esquivel, L., Pavon, N., Rodriguez-Zalvala, J. S., Correa, F., . . . Chavez, E. (2017). CDP-choline circumvents mercury-induced mitochondrial damage and renal dysfunction. Cell Biology International, 41, 1356-1366. doi:10.1002/cbin.10871

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.

Chtourou, Y., Garoui, E. m., Boudawara, T., & Zeghal, N. (2012). Protective role of silymarin against manganese-induced nephrotoxicity and oxidative stress in rat. Environ Toxicol, 29, 1147-1154. doi:10.1002/tox.21845

Emerit, J., Edeas, M., Bricaire, F., 2004. Neurodegenerative diseases and oxidative stress. Biomed. Pharmacotherapy. 58(1): 39-46.

Ferreira, G. K., Cardoso, E., Vuolo, F. S., Michels, M., Zanoni, E. T., Carvalho-Silva, M., . . . Paula, M. M. S. (2015). Gold nanoparticles alter parameters of oxidative stress and energy metabolism in organs of adult rats. Biochem. Cell Biol., 93, 548-557. doi:10.1139/bcb-2015-0030

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.

Heyno, E., Klose, C., & Krieger-Liszkay, A. (2008). Origin of cadmium-induced reactive oxygen species production: Mitochondrial electron transfer versus plasma membrane NADPH oxidase. New Phytologist, 179, 687-699. doi:10.1111/j.1469-8137.2008.02512.x

Hao Y, Ren J, Liu C, Li H, Liu J, Yang Z, Li R, Su Y. (2014). Zinc Protects Human Kidney Cells from Depleted Uraniuminduced Apoptosis. Basic Clin Pharmacol Toxicol. 114(3):271-80. doi: 10.1111/bcpt.12167.

Huerta-García, E., Perez-Arizti, J. A., Marquez-Ramirez, S. G., Delgado-Buenrostro, N. L., Chirino, Y. I., Iglesias, G. G., & Lopez-Marure, R. (2014). Titanium dioxide nanoparticles induce strong oxidative stress and mitochondrial damage in glial cells. Free Radical Biology and Medicine, 73, 84-94. doi:10.1016/j.freeradbiomed.2014.04.026

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.

Jozefczak, M., Bohler, S., Schat, H., Horemans, N., Guisez, Y., Remans, T., . . . Cuypers, A. (2015). Both the concentration and redox state of glutathione and ascorbate influence the sensitivity of arabidopsis to cadmium. Annals of Botany, 116(4), 601-612. doi:10.1093/aob/mcv075

Kharroubi, W., Dhibi, M., Mekni, M., Haouas, Z., Chreif, I., Neffati, F., . . . Sakly, R. (2014). Sodium arsenate induce changes in fatty acids profiles and oxidative damage in kidney of rats. Environ Sci Pollut Res, 21, 12040-12049. doi:10.1007/s11356-014-3142-y

Kruidering, M., Van De Water, B., De Heer, E., Mulder, G. J., & Nagelkerke, J. F. (1997). Cisplatin-induced nephrotoxicity in porcine proximal tubular cells: Mitochondrial dysfunction by inhibition of complexes I to IV of the respiratory chain. The Journal of Pharmacology and Experimental Therapeutics, 280(2), 638-649.

Liu, S., Xu, L., Zhang, T., Ren, G., & Yang, Z. (2010). Oxidative stress and apoptosis induced by nanosized titanium dioxide in PC12 cells. Toxicology, 267, 172-177. doi:10.1016/j.tox.2009.11.012

Miyayama, T., Arai, Y., Suzuki, N., & Hirano, S. (2013). Mitochondrial electron transport is inhibited by disappearance of metallothionein in human bronchial epithelial cells follwoing exposure to silver nitrate. Toxicology, 305, 20-29. doi:10.1016/j.tox.2013.01.004

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.

Pan, Y., Leifer, A., Ruau, D., Neuss, S., Bonrnemann, J., Schmid, G., . . . Jahnen-Dechent, W. (2009). Gold nanoparticles of diameter 1.4 nm trigger necrosis by oxidative stress and mitochondrial damage. Small, 5(8), 2067-2076. doi:10.1002/smll.200900466

Shaki, F., Hosseini, M. J., Ghazi-Khansari, M., & Pourahmad, J. (2012). Toxicity of depleted uranium on isolated rat kidney mitochondria. Biochimica Et Biophysica Acta - General Subjects, 1820(12), 1940-1950. doi:10.1016/j.bbagen.2012.08.015

Soussi A, Gargouri M, El Feki A. (2018). Effects of co-exposure to lead and zinc on redox status, kidney variables, and histopathology in adult albino rats. Toxicol Ind Health. 34(7):469-480. doi: 10.1177/0748233718770293.

Thiébault, C., Carrière, M., Milgram, S., Simon, A., Avoscan, L., & Gouget, B. (2007). Uranium induces apoptosis and is genotoxic to normal rat kidney (NRK-52E) proximal cells. Toxicological Sciences : An Official Journal of the Society of Toxicology, 98(2), 479-487. doi:kfm130 [pii]

Turk, E., Kandemir, F. M., Yildirim, S., Caglayan, C., Kucukler, S., & Kuzu, M. (2019). Protective effect of hesperidin on sodium arsenite-induced nephrotoxicity and hepatotoxicity in rats. Biological Trace Element Research, 189, 95-108. doi:10.1007/s12011-018-1443-6

Tyagi, R., Rana, P., Gupta, M., Khan, A. R., Bhatnagar, D., Bhalla, P. J. S., . . . Kushu, S. (2011). Differntial biochemical response of rat kidney towards low and high doses of NiCl2 as revealed by NMR spectroscopy. Journal of Applied Toxicology, 33, 134-141. doi:10.1002/jat.1730

Wang, L., Li, J., Li, J., & Liu, Z. (2009). Effects of lead and/or cadmium on the oxidative damage of rat kidney cortex mitochondria. Biol.Trace Elem.Res., 137, 69-78. doi:10.1007/s12011-009-8560-1

Yeh, Y., Lee, Y., Hsieh, Y., & Hwang, D. (2011). Dietary taurine reduces zinc-induced toxicity in male wistar rats. Journal of Food Science, 76(4), 90-98. doi:10.1111/j.1750-3841.2011.02110.x

Yuan, Y., Zheng, J., Zhao, T., Tang, X., & Hu, N. (2016). Uranium-induced rat kidney cell cytotoxicity is mediated by decreased endogenous hydrogen sulfide (H2S) generation involved in reduced Nrf2 levels. Toxicology Research, 5(2), 660-673. doi:10.1039/C5TX00432B