Hu Jiangting, Kholmukhamedov Andaleb, Lindsey Christopher C, Beeson Craig C, Jaeschke Hartmut, Lemasters John J
Center for Cell Death, Injury & Regeneration, Medical University of South Carolina, Charleston, SC 29425, United States; Department of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425, United States.
Department of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425, United States.
Free Radic Biol Med. 2016 Aug;97:418-426. doi: 10.1016/j.freeradbiomed.2016.06.024. Epub 2016 Jun 23.
Acetaminophen (APAP) overdose causes hepatotoxicity involving mitochondrial dysfunction and the mitochondrial permeability transition (MPT). Iron is a critical catalyst for ROS formation, and reactive oxygen species (ROS) play an important role in APAP-induced hepatotoxicity. Previous studies show that APAP disrupts lysosomes, which release ferrous iron (Fe(2+)) into the cytosol to trigger the MPT and cell killing. Here, our aim was to investigate whether iron released from lysosomes after APAP is then taken up into mitochondria via the mitochondrial electrogenic Ca(2+), Fe(2+) uniporter (MCFU) to cause mitochondrial dysfunction and cell death. Hepatocytes were isolated from fasted male C57BL/6 mice. Necrotic cell killing was assessed by propidium iodide fluorimetry. Mitochondrial membrane potential (ΔΨ) was visualized by confocal microscopy of rhodamine 123 (Rh123) and tetramethylrhodamine methylester (TMRM). Chelatable Fe(2+) was monitored by quenching of calcein (cytosol) and mitoferrofluor (MFF, mitochondria). ROS generation was monitored by confocal microscopy of MitoSox Red and plate reader fluorimetry of chloromethyldihydrodichlorofluorescein diacetate (cmH2DCF-DA). Administered 1h before APAP (10mM), the lysosomally targeted iron chelator, starch-desferal (1mM), and the MCFU inhibitors, Ru360 (100nM) and minocycline (4µM), decreased cell killing from 83% to 41%, 57% and 53%, respectively, after 10h. Progressive quenching of calcein and MFF began after ~4h, signifying increased cytosolic and mitochondrial chelatable Fe(2+). Mitochondria then depolarized after ~10h. Dipyridyl, a membrane-permeable iron chelator, dequenched calcein and MFF fluorescence after APAP. Starch-desferal, but not Ru360 and minocycline, suppressed cytosolic calcein quenching, whereas starch-desferal, Ru360 and minocycline all suppressed mitochondrial MFF quenching and mitochondrial depolarization. Starch-desferal, Ru360 and minocycline also each decreased ROS formation. Moreover, minocycline 1h after APAP decreased cell killing by half. In conclusion, release of Fe(2+) from lysosomes followed by uptake into mitochondria via MCFU occurs during APAP hepatotoxicity. Mitochondrial iron then catalyzes toxic hydroxyl radical formation, which triggers the MPT and cell killing. The efficacy of minocycline post-treatment shows minocycline as a possible therapeutic agent against APAP hepatotoxicity.
对乙酰氨基酚（APAP）过量会导致肝毒性，涉及线粒体功能障碍和线粒体通透性转换（MPT）。铁是活性氧（ROS）形成的关键催化剂，而ROS在APAP诱导的肝毒性中起重要作用。先前的研究表明，APAP会破坏溶酶体，溶酶体将亚铁离子（Fe(2+)）释放到细胞质中，从而引发MPT和细胞死亡。在这里，我们的目的是研究APAP后从溶酶体释放的铁是否通过线粒体电致钙（2+）、铁（2+）单向转运体（MCFU）进入线粒体，从而导致线粒体功能障碍和细胞死亡。从禁食的雄性C57BL/6小鼠中分离肝细胞。通过碘化丙啶荧光法评估坏死细胞的杀伤情况。通过罗丹明123（Rh123）和四甲基罗丹明甲酯（TMRM）的共聚焦显微镜观察线粒体膜电位（ΔΨ）。通过钙黄绿素（细胞质）和线粒体铁荧光探针（MFF，线粒体）的淬灭来监测可螯合的Fe(2+)。通过MitoSox Red的共聚焦显微镜观察和二氯二氢荧光素二乙酸甲酯（cmH2DCF-DA）的酶标仪荧光法监测ROS的产生。在APAP（10mM）给药前1小时给予溶酶体靶向铁螯合剂淀粉去铁胺（1mM）以及MCFU抑制剂Ru360（100nM）和米诺环素（4µM），10小时后细胞杀伤率分别从83%降至41%、57%和53%。钙黄绿素和MFF在约4小时后开始逐渐淬灭，表明细胞质和线粒体中可螯合的Fe(2+)增加。线粒体在约10小时后去极化。联吡啶是一种可透过膜的铁螯合剂，在APAP后使钙黄绿素和MFF荧光恢复。淀粉去铁胺可抑制细胞质中钙黄绿素的淬灭，但Ru360和米诺环素则不能，而淀粉去铁胺、Ru360和米诺环素均能抑制线粒体MFF的淬灭和线粒体去极化。淀粉去铁胺、Ru360和米诺环素也均能减少ROS的形成。此外，APAP给药1小时后给予米诺环素可使细胞杀伤率减半。总之，在APAP肝毒性过程中，溶酶体释放Fe(2+)，随后通过MCFU进入线粒体。线粒体铁随后催化有毒羟基自由基的形成，从而引发MPT和细胞死亡。米诺环素治疗后的疗效表明米诺环素可能是一种治疗APAP肝毒性的药物。
