Suppression of Keap1 cysteine oxidation

7722-84-1

MHAJPDPJQMAIIY-UHFFFAOYSA-N

Hydrogen peroxide

Hydrogen peroxide, Adeka Super EL Albone DS Anti-Keim 50 Asepticper Baquashock Clarigel Gold Crest whitestrips Crestal Whitestrips Crystacide Dentasept Deslime LP Dihydrogen dioxide Hybrite Hydrogen dioxide Hydrogen peroxide aq. solns. Hydroperoxide Inhibine Interox ST 50 Lase Peroxide Lensan A Magic Bleaching Metrokur Microcyn 60 Mirasept Nite White Excel 2 NSC 19892 Odosat D Opalescence Xtra Opalescence Xtra Boost OxiDate Oxigenal Oxyfull Oxysept Oxysept I Pegasyl PERHYDROL Peroxaan Peroxclean peroxido de hidrogeno peroxyde d'hydrogene Quasar Brite Select Bleach Superoxol T-Stuff UN 2014 UN 2015 UN 2984 Wasserstoffperoxid WASSERSTOFFPEROXID <5% WASSERSTOFFPEROXID >20-35% WASSERSTOFFPEROXID >35% WASSERSTOFFPEROXID 5-20% Whiteness HP Whitespeed Xtra White Zerosil

DTXSID2020715

7440-43-9

BDOSMKKIYDKNTQ-UHFFFAOYSA-N

Cadmium

Cadimium CADMIUM BLUE CADMIUM, IN PLATTEN, STANGEN, BROCKEN,KOERNER

DTXSID1023940

7440-66-6

HCHKCACWOHOZIP-UHFFFAOYSA-N

Zinc

Zn Asarco L 15 C.I. Pigment Black 16 Merrillite NC-Zinc Rheinzink Stapa TE Zinc AT UF (metal) UN 1436 Zinc dust Zinc Dust 3 Zinc Dust 500 mesh Zinc Dust LS 2 Zinc Dust MCS Zinc Flakes GTT ZINC METAL ZINC MOSSY ZINC STRIP ZINC, MOSSY Zincsalt GTT

DTXSID7035012

CHEBI:26208

CHEBI polyunsaturated fatty acid

MP:0001860

MP liver inflammation

GO:0034440

GO lipid oxidation

WIKI occurrence

WIKI increased

All-trans retinoic acid

2022-02-15T10:43:46

Brusatol

2025-11-04T10:27:06

ML385

2025-11-04T10:27:28

Hydrogen peroxide

2019-05-19T17:21:21

Cadmium

2017-10-25T08:33:12

Zinc

2022-02-04T15:05:00

10090

NCBI mouse

WCS_9606

common toxicological species human

10116

NCBI rat

WikiUser_6

ApacheUser fish

WikiUser_17

mammals

WikiUser_25

Wikiuser: Cyauk human and other cells in culture

69158

NCBI Rodentia sp.

Suppression of Keap1 cysteine oxidation

Molecular

AOPWiki 2025-11-04T10:12:12

2025-11-04T10:12:12

NFE2/Nrf2 repression

Molecular

AOPWiki 2017-06-02T16:27:39

2017-06-02T16:27:39

Increase, Ferroptosis

Ferroptosis

Cellular

AOPWiki 2022-04-07T09:20:22

2022-04-07T09:20:22

Inflammation, Liver

Organ

Approximately 29 million people in the European Union suffer from a chronic liver condition <a href="#cite_note-Blachier2013-1">[1]</a>. Inflammation is a crucial link that is related to many of these conditions, with the potential for the development of cirrhosis or primary liver cancer which represent the end-stage of liver pathology and are often associated with mortality: chronic hepatitis (A-E), non-alcoholic steatohepatitis (NASH) which is the progressive form of non-alcoholic fatty liver disease (NAFLD), primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC) <a href="#cite_note-Blachier2013-1">[1]</a>. Drug-induced liver injury (DILI) still is a major problem in drug development as its early detection is problematic, and acute liver inflammation is the most common symptom. DILI is the main cause for withdrawal of drugs from the pharmaceutical market <a href="#cite_note-2">[2]</a>. Liver inflammation is marked by an increased influx of neutrophils, following the secretion of signaling factors such as CXC chemokines and macrophage inflammatory protein 2 (MIP-2) from damaged cells <a href="#cite_note-3">[3]</a>. Kupffer cells (KCs), the resident macrophages of the liver and accounting for about 15-20% of total cell numbers in a healthy liver. They are the gatekeepers in the liver, as they monitor the blood that enters this organ <a href="#cite_note-Kermanizadeh2012-4">[4]</a><a href="#cite_note-Arrese2016-5">[5]</a>. Activation of KCs by activation of toll like receptors, for example, leads to the recruitment of further inflammatory cells as well as amplified KC activation. This, in turn, activates Hepatic stellate cells (HSCs) <a href="#cite_note-Arrese2016-5">[5]</a> which can link liver inflammation to further severe outcomes such as development of fibrosis A list of drugs generally known to induce DILI can be found here <a href="#cite_note-6">[6]</a>.

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible? Liver inflammation is usually confirmed by analysis of histological features, marked by influx of inflammatory cells (mainly neutrophils) which can be stained by using Haematoxylin and eosin <a href="#cite_note-Huebsch2006-7">[7]</a>. In mice, neutrophil influx can be analysed using a mouse MPO ELISA kit for lysed tissue <a href="#cite_note-Kermanizadeh2012-4">[4]</a>. mRNA expression levels of inflammatory cytokines in tissue samples can be determined by using real-time PCR as described in <a href="#cite_note-Cui2011-8">[8]</a>. Plasma levels of pro-inflammatory cytokines can be analysed by enzyme linked immunosorbent assay) ELISA using commercial kits <a href="#cite_note-Ma2009-9">[9]</a>.

<a href="#cite_note-Huebsch2006-7">[7]</a>: human (representative for general application in patients, as liver inflammation is commonly found in patients with DILI) <a href="#cite_note-Cui2011-8">[8]</a><a href="#cite_note-Kermanizadeh2012-4">[4]</a><a href="#cite_note-Ma2009-9">[9]</a>: mouse (nanomaterial-induced) <a href="#cite_note-10">[10]</a>: rat (nanomaterial-induced)

UBERON:0002107

UBERON liver

High High Moderate

<ol class="references"> <li id="cite_note-Blachier2013-1">↑ <a href="#cite_ref-Blachier2013_1-0">1.0</a> <a href="#cite_ref-Blachier2013_1-1">1.1</a> Blachier M, Leleu H, Peck-Radosavljevic M, Valla DC, Roudot-Thoraval F. The burden of liver disease in Europe: a review of available epidemiological data. J Hepatol. 2013 Mar;58(3):593-608 </li> <li id="cite_note-2"><a href="#cite_ref-2">↑</a> Larrey D. Epidemiology and individual susceptibility to adverse drug reactions affecting the liver. Semin Liver Dis. 2002;22(2):145-55 </li> <li id="cite_note-3"><a href="#cite_ref-3">↑</a> Jaeschke H. Inflammation in response to hepatocellular apoptosis. Hepatology. 2002 Apr;35(4):964-6 </li> <li id="cite_note-Kermanizadeh2012-4">↑ <a href="#cite_ref-Kermanizadeh2012_4-0">4.0</a> <a href="#cite_ref-Kermanizadeh2012_4-1">4.1</a> <a href="#cite_ref-Kermanizadeh2012_4-2">4.2</a> Kermanizadeh A, Brown DM, Hutchison GR, Stone V. Engineered Nanomaterial Impact in the Liver following Exposure via an Intravenous Route–The Role of Polymorphonuclear Leukocytes and Gene Expression in the Organ. Journal of Nanomed & Nanotechnol 2012;04(01):1–7 </li> <li id="cite_note-Arrese2016-5">↑ <a href="#cite_ref-Arrese2016_5-0">5.0</a> <a href="#cite_ref-Arrese2016_5-1">5.1</a> Arrese M, Cabrera D, Kalergis AM, Feldstein AE. Innate Immunity and Inflammation in NAFLD/NASH. Dig Dis Sci. 2016 May;61(5):1294-303 </li> <li id="cite_note-6"><a href="#cite_ref-6">↑</a> Ortega-Alonso A, Stephens C, Lucena MI, Andrade RJ. Case Characterization, Clinical Features and Risk Factors in Drug-Induced Liver Injury. Int J Mol Sci. 2016 May 12;17(5) </li> <li id="cite_note-Huebsch2006-7">↑ <a href="#cite_ref-Huebsch2006_7-0">7.0</a> <a href="#cite_ref-Huebsch2006_7-1">7.1</a> Huebscher SG. Histological assessment of non-alcoholic fatty liver disease. Histopathol. 2006;49:450–465 </li> <li id="cite_note-Cui2011-8">↑ <a href="#cite_ref-Cui2011_8-0">8.0</a> <a href="#cite_ref-Cui2011_8-1">8.1</a> Cui Y, Liu H, Zhou M, Duan Y, Li N, Gong X, Hu R, Hong M, Hong F. Signaling pathway of inflammatory responses in the mouse liver caused by TiO2 nanoparticles. 2011; J. Biomed. Mater. Res. - Part A 96 A:221–229 </li> <li id="cite_note-Ma2009-9">↑ <a href="#cite_ref-Ma2009_9-0">9.0</a> <a href="#cite_ref-Ma2009_9-1">9.1</a> Ma L, Zhao J, Wang J, Liu J, Duan Y, Liu H, Li N, Yan J, Ruan J, Wang H, Hong F. The Acute Liver Injury in Mice Caused by Nano-Anatase TiO2. Nanoscale Res Lett. 2009 Aug 1;4(11):1275-85 </li> <li id="cite_note-10"><a href="#cite_ref-10">↑</a> Alarifi S, Ali ., Al-Doaiss AA, Ali BA, Ahmed M, Al-Khedhairy AA. Histologic and apoptotic changes induced by titanium dioxide nanoparticles in the livers of rats. Intern J Nanomed. 2013;8:3937–3943 </li> </ol> AOPWiki 2016-11-29T18:41:27

2017-09-16T10:16:41

Increase, Lipid peroxidation

Increase, LPO

Molecular

Lipid peroxidation is an oxidative degradation process affecting lipids, particularly polyunsaturated fatty acids in cellular and organelle membranes. The process is initiated when oxidants, including free radicals and reactive oxygen species, abstract hydrogen atoms from susceptible lipid chains. This generates lipid radicals that react with molecular oxygen to form lipid peroxyl radicals and lipid hydroperoxides. These products can propagate chain reactions, producing additional oxidized lipids and secondary reactive aldehydes such as malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE), and related hydroxyalkenals (Esterbauer et al., 1991; Yin et al., 2011; Ayala et al., 2014).     As a key event, increased lipid peroxidation represents a measurable increase in oxidized lipid products relative to an appropriate control state. The event may reflect direct oxidative damage to membrane lipids, increased formation of lipid hydroperoxides, increased accumulation of MDA or 4-HNE, or increased abundance of specific oxidized phospholipid or fatty acid species. Because lipid peroxidation products can alter membrane fluidity, permeability and signaling, the event is relevant both as a marker of oxidative damage and as a potential contributor to downstream mitochondrial dysfunction, loss of membrane integrity, cytotoxicity and impaired growth (Esterbauer et al., 1991; Uchida, 2003; Ayala et al., 2014). This KE should be described independently of any specific upstream or downstream event. In an AOP context, lipid peroxidation is commonly downstream of oxidative stress and upstream of events related to decreased mitochondrial coupling, cellular injury, or altered membrane-dependent biological processes. However, the KE itself is defined only by the increased lipid oxidation state and its measurable biochemical products.

No OECD Test Guideline is currently dedicated specifically to measurement of lipid peroxidation as a standalone endpoint. Nevertheless, the KE can be measured using several well-established biochemical and analytical methods. Scientific confidence is highest when methods quantify specific lipid peroxidation products or oxidized lipid species directly, and lower when nonspecific colorimetric assays are used without appropriate controls or confirmatory methods.   <table align="center" cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none"> <thead> <tr> <td style="background-color:#d9eaf7; border-bottom:1px solid #bfbfbf; border-left:1px solid #bfbfbf; border-right:1px solid #bfbfbf; border-top:1px solid #bfbfbf; vertical-align:top; width:130px"> Measurement approach </td> <td style="background-color:#d9eaf7; border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:1px solid #bfbfbf; vertical-align:top; width:139px"> Endpoint measured </td> <td style="background-color:#d9eaf7; border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:1px solid #bfbfbf; vertical-align:top; width:158px"> Representative method names </td> <td style="background-color:#d9eaf7; border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:1px solid #bfbfbf; vertical-align:top; width:302px"> Scientific confidence and limitations </td> </tr> </thead> <tbody> <tr> <td style="border-bottom:1px solid #bfbfbf; border-left:1px solid #bfbfbf; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:130px"> TBARS / MDA assays </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:139px"> Thiobarbituric acid reactive substances, often interpreted as MDA or MDA-like products </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:158px"> TBARS assay; spectrophotometric or fluorometric MDA assays </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:302px"> Widely used and sensitive, but not fully specific because TBA can react with compounds other than MDA. Best used as a screening or comparative indicator of lipid peroxidation, particularly when supported by extraction, HPLC separation or additional markers (Buege and Aust, 1978; Ohkawa et al., 1979; Janero, 1990; Draper and Hadley, 1990). </td> </tr> <tr> <td style="border-bottom:1px solid #bfbfbf; border-left:1px solid #bfbfbf; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:130px"> 4-HNE and hydroxyalkenal assays </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:139px"> 4-hydroxy-2-nonenal and related reactive aldehydes </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:158px"> ELISA, immunoblotting of HNE-protein adducts, HPLC or LC-MS quantification </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:302px"> Mechanistically informative because 4-HNE is a major bioactive lipid peroxidation product. Antibody-based methods can detect protein adducts, whereas chromatographic or mass spectrometric methods improve specificity (Esterbauer et al., 1991; Uchida, 2003; Ayala et al., 2014). </td> </tr> <tr> <td style="border-bottom:1px solid #bfbfbf; border-left:1px solid #bfbfbf; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:130px"> Lipid hydroperoxide assays </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:139px"> Primary lipid hydroperoxides </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:158px"> FOX assay; iodometric assays; commercial lipid hydroperoxide kits </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:302px"> Useful for detecting relatively early lipid peroxidation products. Hydroperoxides can be unstable and sample handling is critical. FOX-based methods provide a simple approach for lipid hydroperoxide detection (Jiang et al., 1992). </td> </tr> <tr> <td style="border-bottom:1px solid #bfbfbf; border-left:1px solid #bfbfbf; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:130px"> Chromatography and mass spectrometry </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:139px"> Specific oxidized fatty acids, oxidized phospholipids, oxylipins or oxidized lipid classes </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:158px"> HPLC, GC, LC-MS/MS, lipidomics </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:302px"> High specificity and quantitative power when standards and validated workflows are available. These methods can distinguish individual oxidized lipid species and are preferred for detailed mechanistic studies (Yin et al., 2011; Li et al., 2019). </td> </tr> <tr> <td style="border-bottom:1px solid #bfbfbf; border-left:1px solid #bfbfbf; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:130px"> Fluorescent probes and imaging </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:139px"> Oxidation-sensitive fluorescent signal in cellular lipids </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:158px"> BODIPY 581/591 C11 and related lipid oxidation probes </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; vertical-align:top; width:302px"> Useful for cell-based or imaging applications and spatial localization, but probe specificity, photoxidation and calibration must be considered. Best used with complementary biochemical or analytical endpoints. </td> </tr> </tbody> </table>

The biological domain of applicability for this KE is broad because lipid membranes and oxidizable fatty acids are widely conserved biological features. The event is applicable wherever lipid substrates susceptible to oxidation are present and where oxidants can access those substrates. The KE is therefore relevant across many biological systems, including unicellular algae, invertebrates, fish, mammals and human-derived cells. The current evidence base is strongest in mammalian systems because lipid peroxidation chemistry and analytical methods have been extensively studied there, but ecotoxicological evidence supports relevance in algae, crustaceans, mollusks and fish.     The KE is not intrinsically limited by sex or life stage. However, the magnitude of lipid peroxidation and its downstream consequences may be modified by lipid composition, antioxidant capacity, oxygen availability, temperature, metabolic rate, nutritional status, metal availability, and exposure duration. Organisms or tissues enriched in polyunsaturated fatty acids, exposed to high oxygen flux, or experiencing antioxidant depletion may be particularly susceptible. In photosynthetic organisms, lipid peroxidation may also occur in chloroplast and thylakoid membranes; in animals, mitochondria and plasma membranes are common sites of interest.     Within the ROS-growth AOP network, this KE is especially relevant as a molecular damage event linking oxidative stress to impaired mitochondrial membrane function, decreased coupling of oxidative phosphorylation, reduced ATP production, cell injury, and decreased growth. Nevertheless, this KE should remain modular: it may be reused in other AOPs whenever increased lipid oxidation products are measured as a consequence of oxidative stress or other lipid-damaging perturbations.

UBERON:0000062

UBERON organ

CL:0000000

CL cell

High Unspecific

High

All life stages

Moderate High

AOP-Wiki. 2026. Key Event 1445: Increase, Lipid peroxidation. AOP-Wiki. Available at: https://aopwiki.org/events/1445. Accessed 14 May 2026. Alam MR, Ehiguese FO, Vitale D, Martín-Díaz ML. 2022. Oxidative stress response to hydrogen peroxide exposure of Mytilus galloprovincialis and Ruditapes philippinarum: reduced embryogenesis success and altered biochemical response of sentinel marine bivalve species. Environmental Chemistry and Ecotoxicology 4:97-105. Ayala A, Munoz MF, Arguelles S. 2014. Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Medicine and Cellular Longevity 2014:360438. https://doi.org/10.1155/2014/360438. Belaid C, Sbartai I. 2021. Assessing the effects of Thiram to oxidative stress responses in a freshwater bioindicator cladoceran (Daphnia magna). Chemosphere 268:128808. https://doi.org/10.1016/j.chemosphere.2020.128808. Buege JA, Aust SD. 1978. Microsomal lipid peroxidation. Methods in Enzymology 52:302-310. https://doi.org/10.1016/S0076-6879(78)52032-6. Cong B, Liu C, Wang L, Chai Y. 2020. The impact on antioxidant enzyme activity and related gene expression following adult zebrafish (Danio rerio) exposure to dimethyl phthalate. Animals 10(4):717. https://doi.org/10.3390/ani10040717. Draper HH, Hadley M. 1990. Malondialdehyde determination as index of lipid peroxidation. Methods in Enzymology 186:421-431. https://doi.org/10.1016/0076-6879(90)86135-I. Esperanza M, Cid A, Herrero C, Rioboo C. 2015. Acute effects of a prooxidant herbicide on the microalga Chlamydomonas reinhardtii: screening cytotoxicity and genotoxicity endpoints. Aquatic Toxicology 165:210-221. https://doi.org/10.1016/j.aquatox.2015.06.004. Esterbauer H, Schaur RJ, Zollner H. 1991. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radical Biology and Medicine 11(1):81-128. https://doi.org/10.1016/0891-5849(91)90192-6. Janero DR. 1990. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radical Biology and Medicine 9(6):515-540. https://doi.org/10.1016/0891-5849(90)90131-2. Jiang ZY, Hunt JV, Wolff SP. 1992. Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein. Analytical Biochemistry 202(2):384-389. https://doi.org/10.1016/0003-2697(92)90122-N. Knauert S, Knauer K. 2008. The role of reactive oxygen species in copper toxicity to two freshwater green algae. Journal of Phycology 44(2):311-319. https://doi.org/10.1111/j.1529-8817.2008.00471.x. Li L, Zhong S, Shen X, Li Q, Xu W, Tao Y, Yin H. 2019. Recent development on liquid chromatography-mass spectrometry analysis of oxidized lipids. Free Radical Biology and Medicine 144:16-34. https://doi.org/10.1016/j.freeradbiomed.2019.06.006. Moore TD, Martin-Creuzburg D, Yampolsky LY. 2023. Diet effects on longevity, heat tolerance, lipid peroxidation and mitochondrial membrane potential in Daphnia. Oecologia 202(1):151-163. https://doi.org/10.1007/s00442-023-05382-1. Ohkawa H, Ohishi N, Yagi K. 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95(2):351-358. https://doi.org/10.1016/0003-2697(79)90738-3. Ouillon N, Sokolov EP, Otto S, Rehder G, Sokolova IM. 2021. Effects of variable oxygen regimes on mitochondrial bioenergetics and reactive oxygen species production in a marine bivalve, Mya arenaria. Journal of Experimental Biology 224(4):jeb237156. https://doi.org/10.1242/jeb.237156. Tseng YC, Chen RD, Lucassen M, Schmidt MM, Dringen R, Abele D, Hwang PP. 2011. Exploring uncoupling proteins and antioxidant mechanisms under acute cold exposure in brains of fish. PLoS ONE 6(3):e18180. https://doi.org/10.1371/journal.pone.0018180. Uchida K. 2003. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Progress in Lipid Research 42(4):318-343. https://doi.org/10.1016/S0163-7827(03)00014-6. Yin H, Xu L, Porter NA. 2011. Free radical lipid peroxidation: mechanisms and analysis. Chemical Reviews 111(10):5944-5972. https://doi.org/10.1021/cr200084z. AOPWiki 2017-06-29T08:04:27

2026-06-23T06:46:14

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AOPWiki 2025-11-04T10:18:10

2025-11-04T10:18:10

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AOPWiki 2025-11-04T10:19:07

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AOPWiki 2026-01-19T09:15:22

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AOPWiki 2026-01-19T09:15:47

2026-01-19T09:15:47

Suppression of Keap1 cysteine oxidation leading to liver inflammation

Keap1 cysteine oxidation,liver failure

Cataia Ives

Young Jun Kim1 and Bongsuk Choi2 1 KIST Europe, Saarbruecken 66123, Germany 2. Hanpoong Pharm & Foods Co., Ltd.11 Guretdeul 3-gil, Deokjin-gu, Jeonju-si, Jeollabuk-do, 54843, Republic of Korea. email : bongsuk333@hanpoong.co.kr

2.7

The Adverse Outcome Pathway (AOP) for Suppression of Keap1 cysteine oxidation Leading to Liver Failure describes a mechanistic sequence linking a redox-regulatory Molecular Initiating Event (MIE)—suppression of oxidation (or adduction) of reactive Keap1 cysteines—to the adverse outcome of liver failure. When Keap1 cysteines remain reduced, the Keap1–Cul3–RBX1 E3 ligase complex persistently ubiquitinates Nrf2, resulting in Nrf2 pathway inhibition. Because Nrf2 transactivates key antioxidant and thiol-metabolism genes (e.g., SLC7A11, GPX4, GCLC/GCLM, HO-1, NQO1), its inhibition diminishes glutathione/NADPH regeneration and cystine import, lowering the hepatocyte’s capacity to detoxify reactive species. This deficit predisposes membranes to lipid peroxidation overload (phospholipid hydroperoxides, PL-OOH), particularly under high labile iron and PUFA-rich conditions, and culminates in ferroptosis-biased hepatocellular death. Progression of cell death precipitates organ-level dysfunction—hyperbilirubinemia, coagulopathy (INR ≥1.5), encephalopathy, and marked transaminase elevation—defining liver failure. The pathway is supported by strong biological plausibility (Keap1–Nrf2 control of antioxidant/ferroptosis-resistance programs) and robust empirical evidence at early KEs (ARE-target suppression, redox capacity loss, lipid peroxidation). Rescue with Nrf2 activators (e.g., sulforaphane, tBHQ), thiol donors (GSH-EE, NAC), and ferroptosis antagonists (ferrostatin-1, liproxstatin-1, deferoxamine) provides functional support for KERs. Prototypical stressors include conditions or agents that maintain Keap1 in a reduction-competent, Nrf2-repressive state (excess thiol buffering; Keap1–Cul3 hyperactivity) or that indirectly depress Nrf2 tone. This AOP aids chemical hazard identification, supports read-across for redox-active materials, and guides discovery of liver-safe formulations by prioritizing protection of the Nrf2–SLC7A11–GPX4 axis. This AOP provides a mechanistic framework for how suppression of Keap1 cysteine oxidation keeps Nrf2 repressed, diminishing hepatocellular antioxidant capacity and promoting iron-dependent lipid peroxidation and ferroptosis, ultimately leading to liver failure. The Keap1–Nrf2 node integrates xenobiotic, oxidative, and metabolic stress in hepatocytes; its dysfunction has broad consequences for glutathione synthesis, cystine uptake (SLC7A11), NADPH homeostasis, detoxifying enzymes (NQO1, HO-1), and lipid peroxide reduction via GPX4. This pathway is relevant to pharmaceuticals, botanicals, industrial chemicals, and mixture contexts that dampen Nrf2 signaling or elevate iron/PUFA susceptibility. Applications span regulatory toxicology, drug-induced liver injury (DILI) risk assessment, and formulation design.

1. Identify and Characterize Key Events (KEs) 1.1 Molecular Initiating Event (MIE) Focus: Establish suppression of Keap1 cysteine oxidation as the MIE that keeps Keap1–Cul3 active and inhibits Nrf2. Approach: <ul> <li> Biotin-switch/LC–MS to quantify Keap1 cysteine oxidation/adducting status. </li> <li> Nrf2 nuclear translocation (IF/Western) and ARE-luciferase assays ± redox modulators. Outcome: Define conditions that maintain reduced Keap1 and thresholds for Nrf2 suppression. </li> </ul> 1.2 Downstream KEs Focus: Characterize the cascade from Nrf2 inhibition → loss of thiol/antioxidant capacity → lipid peroxidation overload → ferroptosis → liver failure. Approach: <ul> <li> qPCR/Western for SLC7A11, GPX4, GCLC/GCLM, HO-1, NQO1. </li> <li> Redox metrics: GSH/GSSG, NADPH/NADP+, labile iron pool (calcein quench). </li> <li> Lipid peroxidation: BODIPY-C11, 4-HNE/MDA, GPX4 activity. </li> <li> Cell death mode: ferroptosis rescue (ferrostatin-1, liproxstatin-1, DFO) vs apoptosis markers. </li> <li> Liver function readouts in vivo (ALT/AST, bilirubin, INR, ICG clearance). Outcome: Temporal and mechanistic links among KEs and AO. </li> </ul> 2. Define Key Event Relationships (KERs) 2.1 Biological Plausibility <ul> <li> Map Keap1–Cul3–Nrf2 control to ARE gene networks that guard against lipid peroxidation/ferroptosis. 2.2 Empirical Support </li> <li> Dose–response and time-course linking MIE/KE1 suppression to KE2 lipid peroxidation and KE4 cell death; rescue with Nrf2 activators/thiol donors/ferroptosis inhibitors. 2.3 Quantitative Understanding </li> <li> Build response–response models: ARE-target suppression (KE1) vs BODIPY-C11 & GPX4 loss (KE2); KE composites vs ALT/AST/INR. </li> </ul> 3. Address Modulating Factors <ul> <li> Age, baseline Nrf2 tone, iron overload, membrane PUFA/ACSL4, pentose-phosphate/NADPH capacity, co-medications that alter redox/iron. </li> </ul> 4. Expand Domain of Applicability 4.1 Taxonomic: Human hepatocytes (primary, HepaRG), rodents; supportive evidence in other vertebrates. 4.2 Life Stage & Sex: Adult/aged livers (higher iron, comorbidities) often more sensitive; sex hormones may modulate iron handling and Nrf2 tone.

adjacent

Moderate

High adjacent

Moderate

Moderate adjacent

Moderate

High adjacent

Moderate

High

High Male Moderate Female

Moderate

Adult, reproductively mature

High High

This AOP links a well-defined redox control point—Keap1–Cul3-mediated repression of Nrf2 maintained by suppression of Keap1 cysteine oxidation—to liver failure through coherent biochemical and cellular mechanisms. Biological plausibility is high; empirical support is strong for early KEs (Nrf2 inhibition; redox capacity loss; lipid peroxidation) and moderate–strong for hepatocellular ferroptosis contributing to organ-level failure. Further work should refine quantitative thresholds (e.g., composite KE2 scores that predict INR elevation) and characterize population variability (iron burden, baseline Nrf2 tone). <table> <thead> <tr> <th>Domain</th> <th>Relevance</th> <th>Evidence</th> </tr> </thead> <tbody> <tr> <td>Taxonomic Relevance</td> <td>Humans, rodents</td> <td>Conservation of Keap1–Nrf2; ferroptosis machinery conserved.</td> </tr> <tr> <td>Life Stage</td> <td>Adults, elderly</td> <td>Higher iron burden and comorbidities increase sensitivity.</td> </tr> <tr> <td>Sex</td> <td>Both</td> <td>Minor modulation possible via iron handling/hormones.</td> </tr> <tr> <td>Molecular/Cellular</td> <td>Hepatocytes (Keap1–Nrf2–SLC7A11–GPX4)</td> <td>Central to redox and lipid-peroxide detox.</td> </tr> <tr> <td>Stressors</td> <td>Redox-active environments inhibiting Keap1 Cys oxidation</td> <td>Map to chemicals/conditions that depress Nrf2 tone.</td> </tr> </tbody> </table>

<table cellspacing="0" style="border-collapse:collapse; width:459px"> <tbody> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:58px; text-align:center; vertical-align:middle; white-space:normal; width:113px">Key Event (KE)</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; text-align:center; vertical-align:middle; white-space:normal; width:78px">Essentiality</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; text-align:center; vertical-align:middle; white-space:normal; width:266px">Rationale and Evidence</td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:76px; vertical-align:middle; white-space:normal; width:113px">MIE: Suppression of Keap1 cysteine oxidation</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:78px">Strong</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:266px">Maintains Keap1–Cul3 repression of Nrf2; reversing with Nrf2 activators restores KE1.</td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:76px; vertical-align:middle; white-space:normal; width:113px">KE1: Nrf2 pathway inhibition</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:78px">Strong</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:266px">Necessary for reductions in SLC7A11/GPX4; genetic/chemical rescue prevents downstream KEs.</td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:76px; vertical-align:middle; white-space:normal; width:113px">KE2: Lipid-peroxidation overload</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:78px">Strong</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:266px">Required for ferroptosis; lipid peroxide scavengers/ferroptosis inhibitors block KE4.</td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:76px; vertical-align:middle; white-space:normal; width:113px">KE3: Hepatocyte ferroptosis</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:78px">Strong</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:266px">Blocking ferroptosis improves biochemical function and prevents AO progression.</td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:76px; vertical-align:middle; white-space:normal; width:113px">AO: Liver failure</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:78px">Outcome</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:middle; white-space:normal; width:266px">Resultant organ-level dysfunction from cumulative hepatocellular loss.</td> </tr> </tbody> </table>

1. MIE: Suppression of Keap1 cysteine oxidation → Nrf2 inhibition Biological Plausibility: Strong (Keap1–Cul3 represses Nrf2 when Keap1 Cys remain reduced). Empirical Support: Moderate–Strong (ARE-reporter/Nrf2 nuclear assays respond to redox manipulation; reversal by Nrf2 activators). 2. KE1: Nrf2 pathway inhibition (ARE targets down) Biological Plausibility: Strong (Nrf2 drives SLC7A11/GPX4/GCLC). Empirical Support: Strong (loss of these targets lowers GSH/NADPH and raises susceptibility to lipid peroxidation). 3. KE2: Lipid-peroxidation overload Biological Plausibility: Strong (PL-OOH accumulation with GPX4 shortfall). Empirical Support: Strong (BODIPY-C11↑; 4-HNE/MDA↑; rescued by ferrostatin-1/liproxstatin-1/DFO). 4. KE3: Hepatocyte ferroptosis Biological Plausibility: Strong (iron-dependent, GPX4-limited death). Empirical Support: Moderate–Strong (mode-specific rescue; histology/biochemistry improvements). 5. AO: Liver failure Biological Plausibility: Strong (massive hepatocellular loss impairs function). Empirical Support: Moderate (preclinical models; translational biomarkers align).

<div> </div> <table> <thead> <tr> <th>Modulating Factor (MF)</th> <th>Influence or Outcome</th> <th>KER(s) involved</th> </tr> </thead> <tbody> <tr> <td>Labile iron pool (↑)</td> <td>Lowers threshold for KE2/KE3</td> <td>KE2→KE3/KE4</td> </tr> <tr> <td>Membrane PUFA / ACSL4 (↑)</td> <td>Amplifies lipid-peroxidation</td> <td>KE1→KE2</td> </tr> <tr> <td>Baseline Nrf2 tone (low)</td> <td>Sensitizes to KE1 suppression</td> <td>MIE→KE1</td> </tr> <tr> <td>NADPH supply (low, PPP defects)</td> <td>Worsens GSH regeneration</td> <td>KE1→KE2</td> </tr> <tr> <td>Co-medications (DDI)</td> <td>Can depress Nrf2/thiol metabolism</td> <td>MIE→KE1; KE1→KE2</td> </tr> <tr> <td>Age/comorbidities (NAFLD, hemochromatosis)</td> <td>Increase iron/oxidative load</td> <td>KE2→KE4; AO</td> </tr> </tbody> </table>

<table cellspacing="0" style="border-collapse:collapse; width:469px"> <tbody> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:39px; text-align:center; vertical-align:middle; white-space:normal; width:117px">Key Event / Relationship</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; text-align:center; vertical-align:middle; white-space:normal; width:117px">Quantitative Evidence</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; text-align:center; vertical-align:middle; white-space:normal; width:117px">Thresholds</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; text-align:center; vertical-align:middle; white-space:normal; width:117px">Temporal Concordance</td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:76px; text-align:center; vertical-align:middle; white-space:normal; width:117px">MIE (Keap1 Cys oxidation suppressed)</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; text-align:center; vertical-align:middle; white-space:normal; width:117px">% decrease in Keap1 Cys oxidation vs Nrf2 nuclear levels</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; text-align:center; vertical-align:middle; white-space:normal; width:117px">≥30–50% Nrf2 nuclear reduction impacts ARE output</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; text-align:center; vertical-align:middle; white-space:normal; width:117px">0.5–2 h</td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:76px; text-align:center; vertical-align:middle; white-space:normal; width:117px">KE1 (Nrf2 inhibited)</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; text-align:center; vertical-align:middle; white-space:normal; width:117px">Composite ARE score vs GSH/NADPH</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; text-align:center; vertical-align:middle; white-space:normal; width:117px">≥30% drop in ARE score lowers GSH/NADPH</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; text-align:center; vertical-align:middle; white-space:normal; width:117px">2–6 h</td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:76px; text-align:center; vertical-align:middle; white-space:normal; width:117px">KE1→KE2</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; text-align:center; vertical-align:middle; white-space:normal; width:117px">ARE score vs BODIPY-C11 & GPX4 activity</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; text-align:center; vertical-align:middle; white-space:normal; width:117px">GPX4 activity ≤70% + BODIPY-C11 ≥150% baseline predicts KE3</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; text-align:center; vertical-align:middle; white-space:normal; width:117px">4–12 h</td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:76px; text-align:center; vertical-align:middle; white-space:normal; width:117px">KE2→KE3</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; text-align:center; vertical-align:middle; white-space:normal; width:117px">Lipid-ROS vs ferroptosis markers</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; text-align:center; vertical-align:middle; white-space:normal; width:117px">Lipid-ROS ≥200% baseline with iron-rescue sensitivity</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; text-align:center; vertical-align:middle; white-space:normal; width:117px">6–24 h</td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:76px; text-align:center; vertical-align:middle; white-space:normal; width:117px">KE3→AO</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; text-align:center; vertical-align:middle; white-space:normal; width:117px">Ferroptosis burden vs ALT/AST, INR</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; text-align:center; vertical-align:middle; white-space:normal; width:117px">Model-dependent; sustained KE3 correlates with INR ≥1.5</td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; text-align:center; vertical-align:middle; white-space:normal; width:117px">≥24–72 h</td> </tr> </tbody> </table>

This AOP supports screening and prioritization of redox-active chemicals, read-across for materials impacting the Keap1–Nrf2 axis, and risk mitigation in drug/formulation design by monitoring Nrf2–SLC7A11–GPX4 integrity and iron/lipid peroxidation loads. In DILI programs, composite KE2 metrics (BODIPY-C11↑ + GPX4↓ + LIP↑) can flag ferroptosis risk before overt injury, while Nrf2 activation or ferroptosis inhibitors serve as mechanistic countermeasures in exploratory studies.

Not Specified

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2026-06-26T16:12:16