Date of Award

2015

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Drug Discovery and Biomedical Sciences

College

College of Graduate Studies

First Advisor

John J. Lemasters

Second Advisor

Lorne J. Hofseth

Third Advisor

Anna-Liisa Nieminen

Fourth Advisor

Don C. Rockey

Fifth Advisor

Zhi Zhong

Abstract

Acetaminophen (APAP) is a threshold hepatotoxicant whose overdose produces a fulminant hepatic necrosis. Therapeutic doses are non-hepatotoxic but high therapeutic dosing may induce hepatotoxicity in some vulnerable patients. The underlying mechanism of APAP hepatotoxicity involves mitochondrial dysfunction, including respiratory inhibition, mitochondrial oxidant stress, onset of the mitochondrial permeability transition (MPT) and loss of the mitochondrial membrane potential (ΔΨ). Iron-mediated reactive oxygen species (ROS) formation is essential in oxidative stress. Previous studies show that iron released from lysosomes is taken up into mitochondria during APAP hepatotoxicity, which triggers the MPT and cell killing. Here, my aim was to investigate mitochondrial mechanisms and the role of iron in hepatotoxicity in vitro and in vivo after various doses of APAP. Mouse hepatocytes and C57BL/6 mice were administered APAP in the presence and absence of NIM811 (MPT inhibitor), starch-desferal (lysosomally targeted iron chelator), Ru360 and minocycline (inhibitors of the mitochondrial Ca,Fe uniporter [MCFU], or Nacetylcysteine (NAC, an antioxidant). Necrotic cell killing was determined by propidium iodide (PI) fluorometry. Chelatable Fe2+, mitochondrial membrane potential and ROS were monitored by confocal/ multiphoton microscopy of different fluorophores. Liver injury was assessed by ALT release and liver necrosis. In vivo studies showed that high dose APAP (300 mg/kg) caused ALT release, liver necrosis, irreversible mitochondrial dysfunction and cell death. By contrast, low dose APAP causes reversible mitochondrial dysfunction associated with transient JNK activation and translocation to mitochondria of hepatocytes without ALT release or necrosis in vivo. NIM811 attenuated these changes at both low and high APAP, indicating that the MPT is the likely principal mechanism of mitochondrial dysfunction. In searching for the upstream inducer of MPT that may participate in APAP hepatotoxicity, we examined the role of iron. After exposure of hepatocytes to APAP, confocal/multiphoton microscopy showed that iron translocates from lysosomes into mitochondria both in vitro and in vivo. Mitochondria take up this Fe2+ via the mitochondrial MCFU to trigger formation of reactive oxygen species (ROS) and the MPT. The iron chelator, starch-desferal, and the MCFU inhibitors, Ru360 and minocycline, protected against APAP-induced liver injury. In addition, minocycline post-treatment at 4 h after APAP showed protection in vivo, whereas NAC was ineffective at this late time point. Taken together, the data suggest that release of chelatable Fe2+ from lysosomes followed by uptake into mitochondria via MCFU occurs during APAP hepatotoxicity, which in turn catalyzes ROS formation and triggers iron-dependent MPT and cell killing. The efficacy of minocycline posttreatment compared to NAC shows minocycline as a new therapeutic agent against APAP hepatotoxicity.

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