| Step Number | Molecular Event | Key Molecules/Proteins Involved | Cellular Location | Timing (in mouse model) | Ontology Terms (GO/CHEBI) |
|---|---|---|---|---|---|
| 1 | CYP2E1-mediated bioactivation of acetaminophen to the reactive metabolite NAPQI | Acetaminophen (APAP), CYP2E1, CYP1A2, CYP3A4, NAPQI | Smooth ER / microsomes of centrilobular hepatocytes | Earliest initiating event; minutes after overdose | GO: xenobiotic metabolic process; GO: monooxygenase activity; CHEBI: acetaminophen; CHEBI: N-acetyl-p-benzoquinone imine (pqac-00000001, pqac-00000005) |
| 2 | Rapid depletion of hepatic glutathione (GSH), reducing detoxification capacity | Glutathione, NAPQI, GCLC | Cytosol and mitochondria | ~30 min | GO: glutathione metabolic process; GO: cellular detoxification; CHEBI: glutathione (pqac-00000002, pqac-00000012) |
| 3 | Covalent protein adduct formation, especially on mitochondrial proteins | NAPQI-protein adducts, cysteine residues on mitochondrial proteins | Mitochondria, especially pericentral hepatocytes | Begins early; adducts detectable by ~2 h | GO: protein alkylation; GO: mitochondrial protein-containing complex; CHEBI: protein adduct (pqac-00000001, pqac-00000003, pqac-00000012) |
| 4 | Mitochondrial oxidant stress initiated by superoxide release from respiratory complex III | Respiratory complex III, superoxide, mitochondrial adducted proteins | Mitochondrial inner membrane / intermembrane space | Early after adduct formation; within first few hours | GO: mitochondrial electron transport, ubiquinol to cytochrome c; GO: superoxide metabolic process; CHEBI: superoxide (pqac-00000001, pqac-00000004) |
| 5 | Redox-sensitive MAPK signaling cascade activates JNK | ASK1, MKK4, JNK, oxidant stress | Cytosol | JNK phosphorylation by ~1–2 h | GO: MAPK cascade; GO: response to oxidative stress; GO: protein phosphorylation (pqac-00000002, pqac-00000004, pqac-00000012) |
| 6 | Phospho-JNK translocates to mitochondria and binds Sab, amplifying dysfunction | p-JNK, Sab (SH3BP5), p-Src, Bax, 14-3-3 | Outer mitochondrial membrane | Peaks around ~6 h; sustained in severe injury | GO: protein targeting to mitochondrion; GO: regulation of mitochondrial membrane permeability; GO: intrinsic apoptotic signaling pathway in response to oxidative stress (pqac-00000002, pqac-00000004, pqac-00000006) |
| 7 | Amplified ROS from complex I and formation of peroxynitrite | Complex I, superoxide, nitric oxide, peroxynitrite, nitrotyrosine | Mitochondrial matrix / inner membrane | First several hours; downstream of JNK mitochondrial signaling | GO: reactive oxygen species metabolic process; GO: nitric oxide metabolic process; GO: protein nitration; CHEBI: nitric oxide; CHEBI: peroxynitrite (pqac-00000001, pqac-00000002, pqac-00000008) |
| 8 | Mitochondrial permeability transition (MPT) and complete membrane depolarization | Cyclophilin D, MPT pore, loss of membrane potential | Mitochondria | After sustained oxidant/peroxynitrite stress; several hours | GO: mitochondrial permeability transition pore complex; GO: regulation of mitochondrial membrane potential; GO: mitochondrial depolarization (pqac-00000001, pqac-00000006) |
| 9 | Release and nuclear translocation of endonucleases causing DNA fragmentation, the “point of no return” | Endonuclease G, AIF, nuclear DNA | Mitochondria to nucleus | Downstream of MPT; several hours, preceding terminal cell death | GO: DNA fragmentation; GO: nuclear DNA catabolic process; GO: protein localization to nucleus (pqac-00000001, pqac-00000005, pqac-00000006) |
| 10 | Programmed oncotic necrosis of hepatocytes | Necrotic hepatocytes, ATP depletion, mitochondrial failure | Centrilobular/pericentral hepatocytes | Major injury phase within ~6–24 h | GO: necrotic cell death; GO: programmed necrotic cell death; GO: loss of plasma membrane integrity (pqac-00000005, pqac-00000006) |
| 11 | Release of DAMPs from necrotic cells | HMGB1, ATP, mitochondrial DNA, nuclear DNA, histones, uric acid | Extracellular space / hepatic sinusoids | Follows necrosis; prominent by ~6–24 h | GO: release of sequestered calcium ion into cytosol; GO: inflammatory response; GO: pattern recognition receptor signaling pathway; CHEBI: ATP; CHEBI: uric acid (pqac-00000038, pqac-00000040) |
| 12 | Sterile inflammation and regenerative response | Kupffer cells, neutrophils, monocyte-derived macrophages, CCL2/MCP-1, CXCL2/MIP-2, IL-10, complement, hepatocyte proliferative programs | Liver sinusoids, necrotic interface, peri-necrotic zones | Neutrophils peak ~24 h; macrophages increase later during repair; regeneration over ~24–96 h | GO: sterile inflammatory response; GO: neutrophil chemotaxis; GO: monocyte chemotaxis; GO: phagocytosis; GO: liver regeneration (pqac-00000038, pqac-00000039, pqac-00000040) |


*Table: This table summarizes the accepted mechanistic sequence of acetaminophen hepatotoxicity from metabolic activation through mitochondrial failure, necrotic death, DAMP release, and inflammatory repair. It is useful for mapping disease biology to ontology terms and timing relationships in the standard mouse model.*