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Key Event Title
|Level of Biological Organization|
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|
|Cholestatic Liver Injury induced by Inhibition of the Bile Salt Export Pump (ABCB11)||KeyEvent||Mathieu Vinken (send email)||Under development: Not open for comment. Do not cite||Under Development|
|PDK inhibition- HCC||KeyEvent||Charles Wood (send email)||Under Development: Contributions and Comments Welcome|
|Hypertension||MolecularInitiatingEvent||Frazer Lowe (send email)||Not under active development||Under Development|
|JNK, FOXO and WNT alteration leading to reproductive failure: Multi-OMICS approach||KeyEvent||Jinhee Choi (send email)||Under development: Not open for comment. Do not cite|
|All life stages||High|
Key Event Description
Oxidative stress corresponds to an imbalance between the rate of oxidant production and that of their degradation. The term oxidative stress indicates the outcome of oxidative damage to biologically relevant macromolecules such as nucleic acids, proteins, lipids and carbohydrates. This occurs when oxidative stress-related molecules, generated in the extracellular environment or within the cell, exceed cellular antioxidant defenses. Major reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and superoxide anion, as well as 4-hydroxy- 2,3-nonenal (HNE) and related 4-hydroxy-2,3-alkenals (HAKs), major aldehydic end-products of lipid peroxidation, can act as potential mediators able to affect signal transduction pathways as well as the proliferative and functional response of target cells. H2O2 and superoxide anion may be also generated as molecular messengers within the cell as part of the cellular response to defined growth factors, cytokines and other mediators. The final consequence at tissue, cellular and molecular level is primarily affected by the steady state concentration of oxidative stress-related molecules. The main biological targets of free radicals are proteins, lipids and DNA.
Major consequences of reaction of ROS, HAKs and NO with biologically relevant macromolecules that can mediate pathophysiological effects:
ROS: DNA: oxidation, strand breaks, genotoxicity Proteins: oxidation, fragmentation, formation of carbonyls Lipids: lipid peroxidation and degradation
HAKs: DNA: adducts (low doses), strand breaks, genotoxicity (high doses) Proteins: adducts (Michael type reactions on Lys, Cys and His residues)
NO: DNA: oxidation, strand breaks Proteins: oxidation, nitrosation, nitration (nytrosylation of tyrosine) Lipids: lipid peroxidation and degradation
Continued oxidative stress can lead to chronic inflammation. Oxidative stress can activate a variety of transcription factors including NF-κB, AP-1, p53, HIF-1α, PPAR-γ, β-catenin/Wnt, and Nrf2. Activation of these transcription factors can lead to the expression of over 500 different genes, including those for growth factors, inflammatory cytokines and chemokines, which can activate inflammatory pathways.   
Glutathione (GSH) oxidation refers to the conversion of reduced glutathione to its oxidized form glutathione disulfide (GSSG) in the presence of oxidative species. GSH plays an important role as an anti-oxidant in regulating cellular redox homeostasis, and is mainly present in the cell as the reduced form (98%). Deficiency in GSH or a decrease in GSH/GSSG ratio results in decreased anti-oxidant function and increased susceptibility to oxidative stress, thus making it a marker of cellular redox status. An imbalance in GSH/GSSG ratio has been implicated in the onset and progression of human diseases, such as neurodegenerative diseases, cancers, pulmonary diseases and cardiovascular diseases (Ballatori et al., 2009; Kalinina et al., 2014)
How It Is Measured or Detected
measuring oxidative stress
Agents for ROS detection are primarily fluorescence based, but recently luminescent based detections have been introduced. The biggest difficulty reported with much of the cellular ROS research has been with the lack of reporter agents specific for discrete molecules. ROS moieties by their nature are reactive with a number of different molecules; as such designing reporter agents has been difficult. With more specific chemistries, particularly for hydrogen peroxide, the specific mechanisms for regulation will be elucidated.
Reduced glutathione (GSH) is regenerated from its oxidized form (GSSH) by the action of an NADPH dependent reductase GSSH + NADPH + H+ à 2 GSH + NADP+ Due to the rapid nature of the reduction of GSSH relative to its synthesis or secretion, the ratio of GSH to GSSH is a good indicator of oxidative stress within cells. GSH and GSSH levels can be determined by HPLC, capillary electrophoresis, or biochemically in microplates. Several different assays have been designed to measure glutathione in samples. By using a luciferin derivative in conjunction with glutathione S-transferase enzyme the amount of GSH would be proportional to the luminescent signal generated when luciferase is added in a subsequent step. Total glutathione can be determined colorimetrically by reacting GSH with DTNB (Ellman’s reagent) in the presence of glutathione reductase. Glutathione reductase reduces GSSH to GSH, which then reacts with DTNB to produce a yellow colored 5-thio-2-nitrobenzoic acid (TNB), which absorbs at 412 nm.
Lipid peroxidation is one of the most widely used indicators of free radical formation, a key indicator of oxidative stress. Measurement of lipid peroxidation has historically relied on the detection of thiobarbituric acid (TBA) reactive compounds such as malondialdehyde generated from the decomposition of lipid peroxidation products. While this method is controversial in that it is quite sensitive, but not necessarily specific to MDA, it remains the most widely used means to determine lipid peroxidation. This reaction, which takes place under acidic conditions at 90-100ºC, results in an adduct that can be measured colorimetrically at 532 nm or by fluorescence using a 530 nm excitation wavelength and a 550 nm emission wavelength. A number of commercial assay kits are available for this assay using absorbance or fluorescence detection technologies. The formation of F2-like prostanoid derivatives of arachidonic acid, termed F2-isoprostanes (IsoP) has been shown to be specific for lipid peroxidation. Unlike the TBA assay, measurement of IsoP appears to be specific to lipid peroxides, they are stable and are not produced by any enzymatic pathway making interpretation easier. There have been a number of commercial ELISA kits developed for IsoPs, but interfering agents in samples requires partial purification of samples prior to running the assay. The only reliable means for detection is through the use of GC/MS, which makes it expensive and limits throughput.
Superoxide detection is based on the interaction of superoxide with some other compound to create a measurable result. The reduction of ferricytochrome c to ferrocytochrome c has been used in a number of situations to assess the rate of superoxide formation. While not completely specific for superoxide this reaction can be monitored colorimetrically at 550 nm. Chemiluminescent reactions have been used for their potential increase in sensitivity over absorbance-based detection methods. The most widely used chemiluminescent substrate is Lucigenin, but this compound has a propensity for redox cycling, which has raised doubts about its use in determining quantitative rates of superoxide production. Coelenterazine has also been used as a chemiluminescent substrate. Hydrocyanine dyes are fluorogenic sensors for superoxide and hydroxyl radical. These dyes are synthesized by reducing the iminium cation of the cyanine (Cy) dyes with sodium borohydride. While weakly fluorescent, upon oxidation their fluorescence intensity increases 100 fold. In addition to being fluorescent, oxidation also converts the molecule from being membrane permeable to an ionic impermeable moiety. The most characterized of these probes are Hydro-Cy3 and Hydro-Cy5.
Hydrogen peroxide (H2O2) is the most important ROS in regards to mitogenic stimulation or cell cycle regulation. There are a number of fluorogenic substrates, which serve as hydrogen donors that have been used in conjunction with horseradish peroxidase (HRP) enzyme to produce intensely fluorescent products. The more commonly used substrates include diacetyldichloro-fluorescein, homovanillic acid, and Amplex® Red. In these examples, increasing amounts of H2O2 form increasing amounts of fluorescent product.
Nitric Oxide The free radical nitric oxide (•NO) is produced by a number of different cell types with a variety of biological functions. Regardless of the source or role, the free radical •NO has a very short half life (t½= 4 seconds), reacting with several different molecules normally present to form either nitrate (NO3-) or nitrite (NO2-) A commonly used method for the indirect determination of •NO is the determination of its composition products nitrate and nitrite colorimetrically. This reaction requires that nitrate (NO3) first be reduced to nitrite (NO2), typically by the action of nitrate reductase. Subsequent determination of nitrite by a two step process provides information on the “total” of nitrate and nitrite. In the presence of hydrogen ions nitrite forms nitrous acid, which reacts with sulfanilamide to produce a diazonium ion. This then coupled to N-(1-napthyl) ethylenediamine to form the chromophore which absorbs at 543 nm. Nitrite only determinations can then be made in a parallel assay where the samples were not reduced prior to the colorimetric assay. Actual nitrate levels are then calculated by the subtraction of nitrite levels from the total. 
Domain of Applicability
The concentrations of GSH and GSSG have been shown in tissues of human and laboratory animals, including rats, mice and cows (Chen et al., 2010; Giustarini et al., 2013).
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
Ballatori, N., Krance, S.M., Notenboom, S., Shi, S., Tieu, K., and Hammond, C.L. (2009). Glutathione dysregulation and the etiology and progression of human diseases. Biol. Chem. 390, 191–214.
Chen, C.-A., Wang, T.-Y., Varadharaj, S., Reyes, L.A., Hemann, C., Talukder, M.A.H., Chen, Y.-R., Druhan, L.J., and Zweier, J.L. (2010). S-glutathionylation uncouples eNOS and regulates its cellular and vascular function. Nature 468, 1115–1118.
Giustarini, D., Dalle-Donne, I., Milzani, A., Fanti, P., and Rossi, R. (2013). Analysis of GSH and GSSG after derivatization with N-ethylmaleimide. Nat. Protoc. 8, 1660–1669.
Held P., 2010 Biotek, Measurement of ROS in Cells, http://www.biotek.com/assets/tech_resources/ROS%20Application%20Guide.pdf
Kalinina, E.V., Chernov, N.N., and Novichkova, M.D. (2014). Role of glutathione, glutathione transferase, and glutaredoxin in regulation of redox-dependent processes. Biochem. Biokhimii︠a︡ 79, 1562–1583.
Kamencic, H., Lyon, A., Paterson, P.G., and Juurlink, B.H. (2000). Monochlorobimane fluorometric method to measure tissue glutathione. Anal. Biochem. 286, 35–37.
Parola, M. and Robino, G. (2001). Oxidative stress-related molecules and liver fibrosis. J Hepatol. 35, 297-306
Reuter S. et al., (2010) Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med.49, 1603-1616
Sánchez-Valle V. et al., (2012) Role of oxidative stress and molecular changes in liver fibrosis: a review. Curr Med Chem. 19, 4850-4860
Tipple, T.E., and Rogers, L.K. (2012). Methods for the Determination of Plasma or Tissue Glutathione Levels. Methods Mol. Biol. Clifton NJ 889, 315–324