Lysosomal iron mobilization and induction of the mitochondrial permeability transition in acetaminophen-induced toxicity to mouse hepatocytes

Abstract

Acetaminophen induces the mitochondrial permeability transition (MPT) in hepatocytes. Reactive oxygen species (ROS) trigger the MPT and play an important role in AAP-induced hepatocellular injury. Because iron is a catalyst for ROS formation, our aim was to investigate the role of chelatable iron in MPT-dependent acetaminophen toxicity to mouse hepatocytes. Hepatocytes were isolated from fasted male C3Heb/FeJ mice. Necrotic cell killing was determined by propidium iodide fluorometry. Mitochondrial membrane potential was visualized by confocal microscopy of tetramethylrhodamine methylester. Chelatable ferrous ion was monitored by calcein quenching, and 70 kDa rhodamine-dextran was used to visualize lysosomes. Cell killing after acetaminophen (10mM) was delayed and decreased by more than half after 6 h by 1mM desferal or 1mM starch-desferal. In a cell-free system, ferrous but not ferric iron quenched calcein fluorescence, an effect reversed by dipyridyl, a membrane-permeable iron chelator. In hepatocytes loaded with calcein, intracellular calcein fluorescence decreased progressively beginning about 4 h after acetaminophen. Mitochondria then depolarized after about 6 h. Dipyridyl (20mM) dequenched calcein fluorescence. Desferal and starch-desferal conjugate prevented acetaminophen-induced calcein quenching and mitochondrial depolarization. As calcein fluorescence became quenched, lysosomes disappeared, consistent with release of iron from ruptured lysosomes. In conclusion, an increase of cytosolic chelatable ferrous iron occurs during acetaminophen hepatotoxicity, which triggers the MPT and cell killing. Disrupted lysosomes are the likely source of iron, and chelation of this iron decreases acetaminophen toxicity to hepatocytes.

FIG. 1.

Acetaminophen-induced killing in mouse hepatocytes: protection by desferal and starch-desferal. Mouse hepatocytes were treated with desferal (1mM), starch-desferal (sDesferal, 1mM), or no addition 30 min before exposure to acetaminophen (AAP, 10mM). Cell viability was determined by propidium iodide fluorometry. Control represents hepatocytes not exposed to acetaminophen. Values are means ± SE from three or more hepatocyte isolations.

FIG. 2.

Quenching of calcein fluorescence by ferrous iron but not ferric iron. Calcein-free acid (2μM) was added to HDM, and ferric chloride (FeCl₃, 10μM) or ferrous ammonium sulfate ((Fe(NH₄)₂(SO4)₂, 10μM) was subsequently added, followed by dipyridyl (DPD, 20mM) after 10 min. Fluorescence was measured with a plate reader, as described in “Materials and Methods” section.

FIG. 3.

Calcein quenching and mitochondrial depolarization after acetaminophen. Hepatocytes were exposed to acetaminophen (AAP, 10mM) in the presence of fructose (20mM) plus the glycine (5mM) to prevent plasma membrane failure and cell death after mitochondrial failure. After 2.5 h, hepatocytes were loaded with TMRM (100nM), PI (3μM), and calcein-AM (1μM), and calcein-free acid (300μM) was then added to the extracellular medium, as described in “Materials and Methods” section. After 6 h, dipyridyl (DPD, 20mM) was added. Lastly, digitonin (Dig, 375μM) was added to permeabilize the plasma membrane.

FIG. 4.

Inhibition of acetaminophen-induced calcein quenching, mitochondrial depolarization, and inner membrane permeabilization desferal and starch-desferal. Hepatocytes were incubated with desferal (1mM) (A) or starch-desferal (sDesferal, 1mM) (B) for 30 min before exposure to acetaminophen, as described in Figure 3.

FIG. 5.

Release of chelatable iron after treatment with glycylphenylalanine 2-napthylamide. Mouse hepatocytes were isolated from mice injected with rhodamine-dextran (Rho-Dex) and then loaded with calcein-AM, as described in “Materials and Methods” section. Hepatocytes were then incubated with the lysomotropic detergent, GPN (100μM), and the red fluorescence of rhodamine-dextran and the green fluorescence of calcein were imaged by laser scanning confocal microscopy.

FIG. 6.

Acetaminophen-dependent lysosomal degradation in parallel with calcein quenching: prevention by desferal. Mouse hepatocytes were isolated from mice injected with rhodamine-dextran and then loaded with calcein-AM, as described in “Materials and Methods” section. In the presence of fructose plus glycine, hepatocytes were then exposed to acetaminophen (AAP, 10mM) (A), acetaminophen after 30-min treatment with desferal (Desf, 1mM) (B), or no treatment (C). The red fluorescence of rhodamine-dextran and the green fluorescence of calcein were imaged by laser scanning confocal microscopy.

FIG. 7.

Lack of protection by cyclosporin A against acetaminophen-induced lysosomal breakdown and calcein quenching. Mouse hepatocytes were loaded with rhodamine-dextran and calcein, as described in Figure 6 and treated with acetaminophen (AAP) in the presence of fructose plus glycine and cyclosporin A (CsA, 1μM).