Oxidation-driven mtDNA B-Z transition activates ZBP1 to mediate acetaminophen hepatotoxicity

Acetaminophen (APAP) overdose induces mitochondrial damage in hepatocytes, leading to secondary toxicity that resists N-acetylcysteine (NAC) treatment and culminates in hepatocyte death and acute liver failure (ALF)1. The underlying mechanisms remain poorly understood, often necessitating liver transplantation2. Here, we identify oxidative modification-driven B-to-Z transitions in mitochondrial DNA (mtDNA) as the central pathological driver of APAP-induced ALF. Upon APAP exposure, oxidized mtDNA fragments leak into the cytosol, activating ZBP1 signaling via its Zα domain. This recognition triggers ZBP1-dependent apoptotic signaling, and genetic inhibition of either Zbp1, Mavs or Casp8 mitigates liver damage and improves survival. Using synthetic dGdC duplexes, we demonstrate that 8-oxoG substitution, even under physiological salt concentrations, is sufficient to induce Z-DNA formation, enabling specific ZBP1 binding through its Zα domain. The 8-oxoG repair enzyme OGG13, activated by TH107854, removes 8-oxoG modifications and reverses the Z-DNA conformation back to B-DNA. In mice with lethal APAP toxicity, delayed NAC treatment results in 50% mortality. In contrast, TH10785 monotherapy increases survival rate to 90%, while its combination with NAC achieves 100% survival. These results define the oxidized mtZ-DNA-ZBP1 axis as a critical driver of APAP hepatotoxicity, providing fundamental insights into DNA conformational dynamics and therapeutic opportunities in drug-induced liver failure.