Translocation of iron from lysosomes to mitochondria during acetaminophen-induced hepatocellular injury: Protection by starch-desferal and minocycline

Abstract

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 (Fe2+) 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 Ca2+, Fe2+ 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 Fe2+ 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 1 h before APAP (10 mM), the lysosomally targeted iron chelator, starch-desferal (1 mM), and the MCFU inhibitors, Ru360 (100 nM) and minocycline (4 μM), decreased cell killing from 83% to 41%, 57% and 53%, respectively, after 10 h. Progressive quenching of calcein and MFF began after ~4 h, signifying increased cytosolic and mitochondrial chelatable Fe2+. Mitochondria then depolarized after ~10 h. 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 1 h after APAP decreased cell killing by half. In conclusion, release of Fe2+ 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.

Acetaminophen Hepatotoxicity

“…76 While lysosomal iron release may be due, in part, to lysosomal instability seen after APAP overdose, 77 iron uptake into mitochondria after APAP overdose is mediated by the calcium uniporter. 78…”

Acetaminophen Toxicity: Novel Insights Into Mechanisms and Future Perspectives

“…Another trigger for induction of the MPTP in the context of APAP-induced liver injury is lysosomal iron, whose translocation to the mitochondria has been shown to occur in mouse hepatocytes treated with APAP, where it then induced opening of the MPTP 75 . This iron release could be due to lysosomal instability which has been documented after APAP treatment 76 , and lysosomal iron translocation seems to occur through the calcium uniporter, since treatment with either an iron chelator or inhibitor of the uniporter prevented mitochondrial free radical generation and membrane depolarization 77 . While the induction of the MPTP has been considered to be a catastrophic feature of APAP-induced cell signaling, recent research suggests that this too could be adjusted depending on the dose of APAP, where treatment of animals with a low 150mg/kg overdose of APAP resulted in transient JNK activation and reversible induction of the MPTP 20 , from which cells may be able to recover.…”

Toxicological Property of Acetaminophen: The Dark Side of a Safe Antipyretic/Analgesic Drug?

“…For instance, preventive effects of inhibition of mitochondrial permeability transition pore (MPTP) opening by cyclosporin A against APAP liver injury in mouse model 100) and in cellular model 103,104) have been reported. MPTP opening seems activated by phosphorylated JNK translocation into mitochondria 105) and some factors, such as translocated Bcl-2-associated X (Bax), 106) cyclophilin D, 107,108) and iron ion, 109,110) seem also involved. Dysfunction of mitochondrial respiratory chain and induction of mitochondrial oxidative stress, which accompany superoxide and subsequent reactive radical peroxynitrite production, are also critical factors in APAP hepatotoxicity.…”