Suppression of Keap1 cysteine oxidation leading to liver inflammation

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

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:

• Biotin-switch/LC–MS to quantify Keap1 cysteine oxidation/adducting status.

• Nrf2 nuclear translocation (IF/Western) and ARE-luciferase assays ± redox modulators. Outcome: Define conditions that maintain reduced Keap1 and thresholds for Nrf2 suppression.

1.2 Downstream KEs Focus: Characterize the cascade from Nrf2 inhibition → loss of thiol/antioxidant capacity → lipid peroxidation overload → ferroptosis → liver failure. Approach:

• qPCR/Western for SLC7A11, GPX4, GCLC/GCLM, HO-1, NQO1.

• Redox metrics: GSH/GSSG, NADPH/NADP+, labile iron pool (calcein quench).

• Lipid peroxidation: BODIPY-C11, 4-HNE/MDA, GPX4 activity.

• Cell death mode: ferroptosis rescue (ferrostatin-1, liproxstatin-1, DFO) vs apoptosis markers.

• Liver function readouts in vivo (ALT/AST, bilirubin, INR, ICG clearance). Outcome: Temporal and mechanistic links among KEs and AO.

2. Define Key Event Relationships (KERs) 2.1 Biological Plausibility

• Map Keap1–Cul3–Nrf2 control to ARE gene networks that guard against lipid peroxidation/ferroptosis. 2.2 Empirical Support

• 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

• Build response–response models: ARE-target suppression (KE1) vs BODIPY-C11 & GPX4 loss (KE2); KE composites vs ALT/AST/INR.

3. Address Modulating Factors

• Age, baseline Nrf2 tone, iron overload, membrane PUFA/ACSL4, pentose-phosphate/NADPH capacity, co-medications that alter redox/iron.

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.

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).

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.

1. Cai et al., “A novel KEAP1 inhibitor, tiliroside, activates NRF2 to protect against acetaminophen-induced liver injury” (2025, PMC11868432)​

2. Cai et al., “USP25 regulates KEAP1-NRF2 anti-oxidation axis and its inactivation protects acetaminophen-induced liver injury in male mice” (2023, Nature Comm)​

3. Sato et al., “Sensor systems of KEAP1 uniquely detecting oxidative and electrophilic stresses in vivo” (2024, Science Direct)​

4. Zhou et al., “The Nrf2 Pathway in Liver Diseases” (2022, Front Cell Dev Biol)​

5. Bardallo et al., “Nrf2 and oxidative stress in liver ischemia/reperfusion injury” (2022, FEBS J)​

6. Yang et al., “ELANE enhances KEAP1 protein stability and reduces Nrf2 activity, promoting MAFLD” (2025, Cell Death Disease)​

7. Seedorf et al., “Selective disruption of NRF2-KEAP1 interaction leads to hepatoprotection” (2022, PMC9971056)​

8. “KEAP1 retention in phase-separated p62 bodies drives pathological NRF2 activation and liver injury when autophagy is impaired” (2025, PMC12238652)​

9. Zhou et al., “The role of Keap1-Nrf2 signaling pathway in the treatment of liver diseases” (2024, ScienceDirect)​

10. “CH7450924, a KEAP1-NRF2 interaction inhibitor, suppresses inflammatory cytokine expression in the kidney and liver” (2025, Nature Sci Rep)​

11. “Roles of the Keap1/Nrf2 pathway and mitophagy in liver diseases” (2025, PubMed 41116207)​

12. Mohs et al., “Nrf2 target gene expression supplementation after Keap1 knockout in stress response” (2021, referenced in Front Cell Dev Biol)​

13. Shin et al., “CDDO-Im prevents hepatic lipid accumulation via Nrf2 activation” (2009, referenced in Front Cell Dev Biol)​

14. Sano et al., “Nrf2 activation in fatty liver disease model” (2021, referenced in Front Cell Dev Biol)​

15. Yan et al., “Natural Nrf2 activators alleviate NAFLD by Keap1 regulation” (2018, referenced in Front Cell Dev Biol)​

16. Shen et al., “Natural Nrf2 activators in NAFLD prevention” (2019, referenced in Front Cell Dev Biol)​

17. Yang et al., “Ginkgolide B as Nrf2 activator protects liver” (2020, referenced in Front Cell Dev Biol)​

18. Cuadrado et al., “Nrf2 activation as oncology preventive mechanism” (2019, referenced in Front Cell Dev Biol)​

19. Villanueva et al., “Nrf2 role in viral hepatitis and NAFLD” (2019, referenced in Front Cell Dev Biol)​

20. Taguchi et al., “Nrf2 activation protects from aflatoxin B1 hepatotoxicity” (2016, referenced in Front Cell Dev Bio)

21. Takafumi Suzuki et al., "Molecular Mechanism of Cellular Oxidative Stress Sensing by Keap1"(2019, Cell reports)