Acetaminophen (APAP) overdose can cause liver injury and is the leading cause of acute liver failure in Western countries. Hepatocellular necrosis induced by APAP involves the formation of a reactive metabolite, triggering mitochondrial oxidant stress and peroxynitrite formation. Iron-catalyzed protein nitration is critical for mitochondrial dysfunction and cell death in the absence of lipid peroxidation (LPO). However, co-treatment of APAP and ferrous sulfate aggravated protein nitration and liver injury but also triggered extensive LPO (measured as malondialdehyde and hydroxy eicosatetraenoic acid (HETE) species). The objective of this study was to evaluate whether the aggravated injury under these conditions is caused by a combination of protein nitration and LPO or if LPO is now the dominant injury mechanism. To test this, C57BL/6J mice were co-treated with APAP (300mg/kg) and a moderate dose of ferrous sulfate (0.15mmol/kg) for 6h. Some animals also received a dose of Mito-TEMPO, the mitochondria-targeted SOD mimetic, or minocycline, an inhibitor of mitochondrial iron uptake. Although Mito-TEMPO and minocycline eliminated protein nitration and liver injury after APAP alone, these interventions did not affect LPO and only had a moderate effect on protein nitration and liver injury in the APAP+Fe²⁺ group, suggesting LPO as the main mechanism of cell death. Consistent with these findings, delayed treatment with clinically relevant antidotes N-acetylcysteine and fomepizole did not reduce LPO or liver injury. Thus, liver injury after APAP+Fe²⁺ is no longer primarily driven by mitochondrial oxidant stress and peroxynitrite-mediated necrosis but by lipid peroxidation and a ferroptosis-like cell death.

Keywords: 4-methylpyrazole; N-acetylcysteine; drug hepatotoxicity; ferroptosis; lipid peroxidation; peroxynitrite.

Copyright © 2025 The Author(s). Published by Elsevier B.V. All rights reserved.

对乙酰氨基酚（APAP）过量可导致肝脏损伤，并且是西方国家急性肝衰竭的主要原因。APAP诱导的肝细胞坏死涉及活性代谢物的形成，触发线粒体氧化应激和过氧亚硝酸盐的生成。在没有脂质过氧化（LPO）的情况下，铁催化的蛋白硝化对于线粒体功能障碍和细胞死亡至关重要。然而，在给予APAP和硫酸亚铁的同时治疗会加剧蛋白硝化和肝脏损伤，但也触发了广泛的脂质过氧化（通过丙二醛和羟基二十碳四烯酸 (HETE) 物种测量）。本研究的目的是评估在这种条件下加重的损伤是否是由蛋白硝化和LPO的组合引起的，还是LPO已成为主要的损伤机制。为了测试这一点，将C57BL/6J小鼠与APAP（300 mg/kg）和中剂量硫酸亚铁（0.15 mmol/kg）共处理了6小时。一些动物还接受了线粒体靶向SOD模拟物Mito-TEMPO或抑制线粒体铁摄取的米诺环素治疗。尽管Mito-TEMPO和米诺环素消除了单独使用APAP后的蛋白硝化和肝脏损伤，但在APAP+Fe²⁺组中这些干预措施对LPO没有影响，并且仅对蛋白硝化和肝损伤有轻微的影响，这表明LPO是细胞死亡的主要机制。与这一发现一致的是，在临床相关的解毒剂N-乙酰半胱氨酸和甲吡唑延迟治疗后，LPO或肝脏损伤并未减少。因此，在APAP+Fe²⁺后的肝脏损伤不再主要由线粒体氧化应激和过氧亚硝酸盐介导的坏死驱动，而是由脂质过氧化以及类似铁死亡的细胞死亡所驱动。

关键词：4-甲基吡唑；N-乙酰半胱氨酸；药物肝毒性；铁死亡；脂质过氧化；过氧亚硝酸盐。

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