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. Author manuscript; available in PMC: 2009 Nov 1.
Published in final edited form as: Hepatology. 2008 Nov;48(5):1644–1654. doi: 10.1002/hep.22498

TRANSLOCATION OF IRON FROM LYSOSOMES INTO MITOCHONDRIA IS A KEY EVENT DURING OXIDATIVE STRESS-INDUCED HEPATOCELLULAR INJURY*

Akira Uchiyama 1,2, Jae-Sung Kim 3, Kazuyoshi Kon 2, Hartmut Jaeschke 4, Kenichi Ikejima 2, Sumio Watanabe 2, John J Lemasters 1,5
PMCID: PMC2579320  NIHMSID: NIHMS57410  PMID: 18846543

Abstract

Iron overload exacerbates various liver diseases. In hepatocytes, a portion of non-heme iron is sequestered in lysosomes and endosomes. The precise mechanisms by which lysosomal iron participates in hepatocellular injury remain uncertain. Here, our aim was to determine the role of intracellular movement of chelatable iron in oxidative stress-induced killing to cultured hepatocytes from C3Heb mice and Sprague-Dawley rats. Mitochondrial polarization and chelatable iron were visualized by confocal microscopy of tetramethylrhodamine methylester (TMRM) and quenching of calcein, respectively. Cell viability and hydroperoxide formation (a measure of lipid peroxidation) were measured fluorometrically using propidium iodide and chloromethyl dihydrodichlorofluorescein, respectively. After collapse of lysosomal/endosomal acidic pH gradients with bafilomycin (50 nM), an inhibitor of the vacuolar proton-pumping ATPase, cytosolic calcein fluorescence became quenched. Desferal and starch-desferal (1 mM) prevented bafilomycin-induced calcein quenching, indicating that bafilomycin induced release of chelatable iron from lysosomes/endosomes. Bafilomycin also quenched calcein fluorescence in mitochondria, which was blocked by 20 μM Ru360, an inhibitor of the mitochondrial calcium uniporter, consistent with mitochondrial iron uptake by the uniporter. Bafilomycin alone was not sufficient to induce mitochondrial depolarization and cell killing, but in the presence of low dose tert-butylhydroperoxide (25 μM), bafilomycin enhanced hydroperoxide generation leading to mitochondrial depolarization and subsequent cell death. Taken together, the results are consistent with the conclusion that bafilomycin induces release of chelatable iron from lysosomes/endosomes, which is taken up by mitochondria. Oxidative stress and chelatable iron thus act as two “hits” synergistically promoting toxic radical formation, mitochondrial dysfunction and cell death. This pathway of intracellular iron translocation is a potential therapeutic target against oxidative stress-mediated hepatotoxicity.

Keywords: bafilomycin, calcein, hepatocyte, iron, lysosome, mitochondrial permeability transition, oxidative stress

INTRODUCTION

Chelatable iron and other transition metals such as copper catalyze formation of highly reactive hydroxyl radical (OH•) from H2O2 and superoxide (O2•-), which damages DNA, proteins and membranes (1). Cytoprotection by desferal in various models of oxidative stress and hypoxia/ischemia suggests a role for iron in the pathogenesis of injury (2-6). In hepatocytes, generation of reactive oxygen species (ROS), including H2O2, O2•-, and OH•, contributes to lethal cell injury from oxidative stress, various hepatotoxicants and ischemia/reperfusion (7-9). In particular, ROS formation initiates onset of the mitochondrial permeability transition (MPT) caused by opening of non-specific permeability transition (PT) pores in the mitochondrial inner membrane (5, 10). Open PT pores conduct all solutes up to 1500 Da to cause mitochondrial depolarization, uncoupling of oxidative phosphorylation and large amplitude mitochondrial swelling. These events lead either to necrosis from ATP depletion or apoptosis from release of proapoptotic proteins such as cytochrome c.

Although an essential nutrient, iron in excess is a human toxicant causing acute hepatocellular necrosis after accidental overdose and chronic hepatic injury in hereditary hemochromatosis (11). Excess iron also may aggravate diabetes, cancer, cardiovascular disease and alcoholic and nonalcoholic steatohepatitis (12, 13, 13-16). In cells and tissues, two pools of iron exist. The first pool is “non-chelatable” iron that is sequestered in ferritin or in structural components of proteins (e.g., heme, iron-sulfur complexes) and which cannot be removed by conventional iron chelators like desferal. The second pool is “chelatable” iron that represents free iron and iron bound less strongly to a wide variety of anionic intracellular molecules. Estimated to be 5 μM in hepatocytes (17), chelatable iron is accessible to desferal and other iron chelators. Increases of intracellular chelatable iron contribute to death of sinusoidal endothelial cells and hepatocytes after cold I/R (18, 19). Moreover, direct addition to hepatocytes of a membrane permeable Fe3+ complex causes the MPT and consequent necrotic and apoptotic cell killing (20), whereas the iron chelator, TPEN, attenuates caspase activation and apoptosis in postischemic livers (21).

The precise mechanisms by which iron is mobilized to contribute to hepatocellular injury after oxidative and other stresses remain poorly understood. Here using laser scanning confocal microscopy, we show that lysosomes are a source of rapidly mobilized chelatable iron and that chelatable iron released by lysosomes is rapidly taken up into mitochondria by the calcium uniporter. Inside mitochondria, this iron is available to catalyze toxic ROS cascades.

MATERIALS AND METHODS

Hepatocyte isolation and culture

All experiments were conducted using protocols approved by the Institutional Animal Care and Use Committee. Hepatocytes were isolated from 25 to 30 g overnight-fasted male C3Heb/FeJ mice (Jackson Laboratory, Bar Harbor, ME) and 200-250 g overnight-fasted male Sprague-Dawley rats (Charles River, Wilmington, MA) by collagenase perfusion, as described previously (22). Hepatocytes were resuspended in Waymouth’s medium MB-752/1 (GIBCO, Grand Island, NY) containing 2 mM L-glutamine, 10% fetal bovine serum, 100 nM insulin (Squibb-Novo, Princeton, NJ), 100 nM dexamethasone (LyphoMed, Rosemont, IL), 100 units/mL penicillin and 100 μg/mL streptomycin. Cell viability was greater than 92%, as determined by trypan blue exclusion. Hepatocytes were plated in 24-well microtiter plates (1.5 × 105 cells per well) and 35-mm Petri dishes (6.0 × 105 cells) with 14-mm glass coverslips for imaging (MatTek, Ashland, MA). Plates and coverslips were coated with 0.1% Type-1 rat-tail collagen (Sigma, St. Louis, MO). Hepatocytes were allowed to attach for 4 h in humidified 5% CO2, 95% air at 37°C. Subsequently, hepatocytes were washed once and incubated in Waymouth’s medium 752/1 containing 20 mM N-2-hydroxyethyl-piperazine-N’-2’-ethanesulfonic acid (HEPES, Sigma) buffer supplemented with 10% fetal bovine serum, 100 nM insulin, 100 nM dexamethasone, 100 units/mL penicillin and 100 μg/mL streptomycin at pH 7.4 (Waymouth/HEPES).

Fluorometric assay of cell viability and reactive oxygen species

After attachment to 24-well plates, mouse hepatocytes were washed once and replaced with Waymouth/HEPES containing 30 μM propidium iodide (PI, Molecular Probes/Invitrogen, Eugene, OR) or 10 μM chloromethyldihydrodichlorofluorescein diacetate (cmH2DCF-DA, Molecular Probes/Invitrogen). Cell killing (PI) and ROS production (cmH2DCF-DA) were assessed using a multiwell fluorescence plate reader (BMG Lab Technologies, Germany), as previously described (23, 24). Increased PI fluorescence correlates closely with trypan blue uptake, whereas as conversion of de-esterified non-fluorescent cmH2DCF-DA to green fluorescing chloromethyldichlorofluorescein (cmDCF) signifies production of organic hydroperoxides that are formed after lipid peroxidation (25). Fluorescence does not arise from a direct reaction with H2O2. Rather, cmDCF fluorescence is a general indicator of ROS production and not an indicator for a specific species of oxygen radical.

In some experiments, hepatocytes were preincubated 1 h with 1 mM desferal (Sigma), 1 mM desferal conjugated to hydroxyethystarch (starch-desferal, >10 kDa, Biomedical Frontiers, Minneapolis, MN) or 20 μM Ru360 (Calbiochem, San Diego, CA) at 37°C. After pretreatment, hepatocytes were then incubated for 1 h in the presence and absence of 50 nM bafilomycin A1 (Calbiochem) before addition of tert-butylhydroperoxide (t-BuOOH, 25 μM, Sigma).

Loading of fluorophores

Hepatocytes plated on glass coverslips in Waymouth/HEPES were loaded for 20 min with calcein-AM (1 μM, Molecular Probes/Invitrogen) or 10 μM cmH2DCF-DA and/or tetramethylrhodamine methylester (TMRM, 100 nM, Molecular Probes/Invitrogen). The hepatocytes were then incubated in Waymouth/HEPES containing 300 μM calcein free acid, 50 nM TMRM and/or 3 μM PI, as indicated.

In some experiments, 70 kDa rhodamine-dextran (5 mg/100g body weight, i.p., Sigma) was injected into rats 12 h prior to hepatocyte isolation to label lysosomes (26). After isolation, 6 h-cultured hepatocytes loaded with rhodamine-dextran were cold-loaded with 1 μM calcein-AM for 1 h at 4 °C and washed. The cells were then incubated overnight in Waymouth/HEPES at 37°C. This procedure produced selective labeling of lysosomes with rhodamine-dextran and both mitochondria and lysosomes with calcein, as shown previously (27).

Laser scanning confocal microscopy

Hepatocytes loaded with the various combinations of fluorophores were placed in environmental chambers at 37°C on the stages of Zeiss LSM 410 and LSM 510 laser scanning confocal microscopes (Zeiss, Germany). Red fluorescence (TMRM, PI, Rho-Dex) was excited at 543 or 568-nm, and emission was imaged at >590-nm. Green fluorescence (calcein, cmDCF) was excited at 488-nm, and emission was collected through a 515 to 560-nm band pass filter.

Statistics

Data are presented as means ± S.E. Images shown are representative of 3 or more experiments. Statistical analysis was performed by Student t test or ANOVA, using p < 0.05 as the criterion of significance.

RESULTS

Bafilomycin-induced increase of chelatable iron in the cytosol

Cultured mouse hepatocytes were loaded with calcein, a fluorophore whose fluorescence is rapidly and stoichiometrically quenched by transition metal ions, such as iron, copper, cobalt and nickel (28). Calcein-loaded hepatocytes were treated with bafilomycin (50 nM), an inhibitor of the vacuolar proton-pumping ATPase that collapses acidic lysosomal/endosomal pH gradients (29). Bafilomycin caused quenching of calcein loaded into the cytosol that was evident within 60 min and even more marked after 2 h (Fig. 1B). Loss of calcein fluorescence was not due to passive leak into the extracellular medium, since calcein fluorescence decreased to substantially below the fluorescence of exogenous calcein free acid (300 μM) placed in the extracellular medium. Quenching of calcein fluorescence was a specific effect of bafilomycin, since intracellular calcein fluorescence declined only slightly in hepatocytes not exposed to bafilomycin (Fig. 1A).

Figure 1. Inhibition of bafilomycin-induced intracellular calcein quenching by desferal and starch-desferal in mouse hepatocytes.

Figure 1

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Mouse hepatocytes were co-loaded with TMRM (100 nM) and calcein-AM (1 μM) and incubated with PI (3 μM), calcein free acid (300 μM) and TMRM (50 nM) in the extracellular medium with and without desferal (1 mM) or starch-desferal (1 mM desferal equivalency), as described in MATERIALS AND METHODS. Green fluorescence of calcein and red fluorescence of TMRM and PI were then imaged by laser scanning confocal microscopy before (0 min) and at 60 and 120 min after no further addition (A, Control), 50 nM bafilomycin (B, Baf), bafilomycin in the presence of desferal (C, Baf+DFO) and bafilomycin in the presence of starch-desferal (D, sBaf+DFO). Note the marked decrease of green calcein fluorescence in the cytosol in B after bafilomycin addition, which did not occur during the control incubation (A) and which was suppressed in the presence of desferal (C) and starch-desferal (D). TMRM fluorescence was maintained under all conditions, and PI did not label nuclei. Each experiment is typical of 3 or more replicates.

To determine whether quenching of calcein in the cytosol was a consequence of an increase of chelatable iron, hepatocytes were treated with desferal or starch-desferal prior to exposure to bafilomycin. Desferal and starch-desferal are specific iron chelators, but starch-desferal is membrane-impermeable and can enter cells only via endocytosis. Desferal and starch-desferal (1 mM) both largely blocked calcein quenching after bafilomycin (Fig. 1C and D), which supported the conclusion that calcein quenching was iron-mediated.

After background subtraction, average calcein fluorescence was quantified for individual hepatocytes in the various groups. In untreated hepatocytes, calcein fluorescence decreased 17% after 2 h (Fig. 2). By contrast after bafilomycin, calcein fluorescence decreased 54% (p<0.01 vs. untreated). Desferal and starch-desferal blocked bafilomycin-induced calcein quenching almost completely, and calcein fluorescence decreased by only 16% and 13%, respectively, after bafilomycin plus desferal and bafilomycin plus starch-desferal (p<0.01 vs. bafilomycin alone) (Fig. 2).

Figure 2. Quantitation of calcein quenching after bafilomycin treatment.

Figure 2

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Mouse hepatocytes were loaded with calcein and treated as described in Fig. 1. Average calcein fluorescence of individual hepatocytes after background subtraction was determined at 60 and 120 min of incubation as the percentage of fluorescence prior to additions (0 min). Baf, bafilomycin; DFO, desferal; sDFO, starch-desferal; *, p < 0.01 compared to other groups (n = 2 to 5 hepatocytes per group).

After the treatment with bafilomycin, cytosolic calcein was quenched but TMRM fluorescence remained essentially unchanged both with and without treatment with desferal and starch-desferal (Fig. 1). Thus, mitochondria remained polarized during up 2 h of incubation with bafilomycin, which indicated lack of onset of the MPT.

Contribution of chelatable iron to cytotoxicity after oxidative stress

Mitochondrial glutathione peroxidase reduces t-BuOOH to t-butanol, which promotes mitochondrial oxidative stress by depleting NADPH and glutathione (30). When hepatocytes were exposed to a relatively low dose of t-BuOOH (25 μM), little cell killing occurred as evaluated by PI fluorometry (Fig. 3A). Similarly, bafilomycin alone caused no cell killing over untreated cells. However, the combination of t-BuOOH plus bafilomycin caused substantial cytotoxicity over either t-BuOOH or bafilomycin alone (Fig. 3A). Treatment with desferal and starch-desferal prevented cytotoxicity by t-BuOOH plus bafilomycin and restored cell killing to nearly the same as untreated cells (Fig. 3B).

Figure 3. Synergistic cell killing after bafilomycin plus t-BuOOH: protection by desferal and starch-desferal.

Figure 3

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Viability of mouse hepatocytes was assessed by PI fluorometry, as described in MATERIALS AND METHODS. In A, hepatocytes were exposed to t-BuOOH (25 μM) with and without 60 min of pretreatment with 50 nM bafilomycin (Baf). In B, hepatocytes were treated with desferal (1 mM) or starch-desferal (1 mM desferal equivalency) or no addition prior to bafilomycin plus t-BuOOH treatment. In both panels, “None” represents hepatocytes incubated without any additions. Values are means ± SE from 3 or more hepatocyte isolations.

The MPT after t-BuOOH plus bafilomycin

To further characterize cellular responses after t-BuOOH with and without bafilomycin, confocal microscopy was performed of mouse hepatocytes that were loaded with calcein and TMRM and then incubated in the presence of PI and calcein free acid in the extracellular medium. After exposure to low-dose t-BuOOH (25 μM) alone, virtually no loss of TMRM or quenching of calcein fluorescence occurred after 1 h, although a small decrease of both calcein and TMRM fluorescence became evident after 2 h (Fig. 4A). By contrast after exposure to t-BuOOH plus bafilomycin together, all TMRM fluorescence was lost within 1 h (Fig. 4B). In 1 of the 3 cells in the field, loss of viability had already occurred, as shown by nuclear staining with PI. In the remaining cells, calcein fluorescence was decreased, especially in the middle cell showing large surface blebs as a sign of cellular stress. After 90 min, all cells in the field had lost viability, as shown by PI labeling and the equilibration of intra- and extracellular calcein fluorescence. Nonetheless, calcein fluorescence inside non-viable cells was somewhat less than outside due to space filling structures (e.g., endoplasmic reticulum and nuclei) within the dead cells (Fig. 4B). Complete loss of TMRM fluorescence followed by cell death was consistent with onset of the MPT, as shown previously for hepatocytes exposed to higher concentrations of t-BuOOH (30).

Figure 4. Mitochondrial depolarization and cell death after bafilomycin plus t-BuOOH: protection by desferal and starch-desferal.

Figure 4

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Mouse hepatocytes were loaded, as described in Fig. 1, and exposed to 25 μM t-BuOOH alone (A), t-BuOOH plus 50 nM bafilomycin (Baf) (B), t-BuOOH plus bafilomycin after pretreatment with desferal (DFO, 1 mM) (C) and t-BuOOH plus bafilomycin after pretreatment with starch-desferal (DFO, 1 mM desferal equivalency) (D). After t-BuOOH alone (A), note that red mitochondrial TMRM fluorescence was retained and green calcein quenching did not occur. When t-BuOOH was combined with bafilomycin, calcein quenching, loss of TMRM and cellular blebbing occurred within 60 min followed by nuclear PI labeling with 2 h (B). Desferal and starch-desferal prevented calcein quenching, loss of TMRM fluorescence and nuclear labeling with PI (C and D). Each experiment is typical of 3 or more replicates.

Protection against mitochondrial depolarization by iron chelators

Calcein- and TMRM-loaded hepatocytes were pretreated with desferal (1 mM) before exposure to t-BuOOH plus bafilomycin. In the presence of desferal, mitochondria did not release TMRM fluorescence after up to 2 h of incubation (Fig. 4C). Similarly, quenching of calcein fluorescence was much decreased in comparison to hepatocytes not treated with desferal (Fig. 4C, compare to 4B). Moreover, the hepatocytes did not lose viability as shown by lack of nuclear staining with PI. Similarly, starch-desferal blocked mitochondrial depolarization and calcein quenching (Fig. 4D). In this last experiment, TMRM had not yet fully equilibrated into mitochondria at the time of baseline imaging (Fig. 4D, 0 min), and TMRM fluorescence actually increased during incubation to reach stable steady state mitochondrial loading. Thus, both iron chelators prevented the MPT and cell death induced by low-dose t-BuOOH plus bafilomycin.

Formation of reactive oxygen species after t-BuOOH plus bafilomycin

To assess ROS formation after various treatments, we incubated mouse hepatocytes with cmH2DCF-DA and measured development of cmDCF fluorescence after exposure to bafilomycin, t-BuOOH, and bafilomycin plus t-BuOOH. Bafilomycin alone did not increase hydroperoxide formation at all in comparison to untreated hepatocytes (Fig. 5A). t-BuOOH alone caused an approximate doubling of the cmDCF signal in comparison to untreated cells. By contrast, the combination of bafilomycin plus t-BuOOH led to a 4-fold increased signal (Fig. 5A). Iron chelation with either desferal or starch-desferal completely blocked hydroperoxide formation after bafilomycin plus t-BuOOH to levels observed in untreated cells (Fig. 5B).

Figure 5. ROS formation after bafilomycin plus t-BuOOH: protection by desferal and starch-desferal.

Figure 5

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Mouse hepatocytes were incubated with cmH2DCF-DA (10 μM, and fluorescence was measured using a fluorescence plate reader. In A, hepatocytes were exposed to t-BuOOH (25 μM) with and without 60 min pretreatment with 50 nM bafilomycin (Baf) in comparison to bafilomycin alone. In B, hepatocytes were treated with desferal (DFO, 1 mM), starch-desferal (sDFO, 1 mM desferal equivalency) or no addition prior to bafilomycin plus t-BuOOH. In both panels, “Control” represents hepatocytes incubated without any additions. Values are means ± SE from 3 or more hepatocyte isolations.

Lysosomal integrity after bafilomycin

To this point, our findings strongly implicated the lysosomal/endosomal compartment as a source of mobilized chelatable iron, because bafilomycin increased cytosolic chelatable iron, as shown by calcein quenching. Moreover, starch desferal, an iron chelator that gains entrance by endocytosis into the endosomal/lysosomal compartment but not the cytosol, blocked the bafilomycin-induced increase of chelatable iron in the cytosol. Bafilomycin-induced release of chelatable iron suggested that iron was retained in lysosomes due to transport coupled to the pH gradient. Alternatively, bafilomycin may induce lysosomal swelling and rupture to cause iron release. To discriminate between these possibilities, lysosomes of rat hepatocytes were loaded with 70 kDa rhodamine-dextran. The mitochondria of the hepatocytes were also loaded with calcein, as described below. In untreated hepatocytes, rhodamine-dextran fluorescence persisted essentially unchanged for at least 2 h (Fig. 6A, bottom panels). Although individual lysosomes moved in and out of the plane of section during the incubation, rhodamine-dextran fluorescence remained punctuate, and no release of red rhodamine-dextran fluorescence into the cytosol was observed. These findings confirmed the expectation that lysosomes remained intact during the normal incubation. Similarly after exposure to bafilomycin, rhodamine-dextran fluorescence remained punctate and did not move into the cytosol after as long as 2 h (Fig. 6B, bottom panels). Thus, bafilomycin was not causing lysosomal fragility and rupture for at least 2 h of observation. These results indicated that iron release was likely linked to bafilomycin-induced collapse of lysosomal/endosomal pH gradients rather than to disintegration of individual lysosomes.

Figure 6. Calcein quenching in mitochondria and unquenching in lysosomes after bafilomycin.

Figure 6

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Rat hepatocytes were loaded with 70 kDa rhodamine-dextran (Rhod-Dex) and co-loaded with calcein by cold ester loading/warm incubation, as described in MATERIALS AND METHODS. The hepatocytes were then exposed to no addition (Control) (A), bafilomycin (Baf, 50 nM) (B), bafilomycin after pretreatment with desferal (DFO, 1 mM) (C), and bafilomycin after pretreatment with starch-desferal (sDFO, 1 mM desferal equivalency) (D). Red fluorescence of rhodamine-dextran and green fluorescence of calcein were imaged by laser scanning confocal microscopy. Bafilomycin was added after collection of a baseline image (0 min) and then after 60 and 120 min. Note that mitochondrial calcein fluorescence and lysosomal rhodamine-dextran fluorescence did not change during the control incubation (A). By contrast, mitochondrial calcein was quenched markedly after bafilomycin, whereas lysosomal calcein fluorescence co-localizing with rhodamine-dextran appeared to increase (B). In the presence of desferal (C) and starch-desferal (D), mitochondrial calcein quenching was suppressed, whereas the increase of lysosomal calcein fluorescence appeared to be more marked. Rhodamine-dextran did not leak from lysosomes under any condition. Arrows identify representative lysosomal structures. Each experiment is typical of 3 or more replicates.

Increased chelatable iron in mitochondria after bafilomycin

To assess whether chelatable iron released from lysosomes moves into mitochondria, we used a cold ester loading/warm incubation protocol to load calcein selectively into the mitochondria of rhodamine-dextran-loaded rat hepatocytes. When calcein-AM is loaded at colder temperatures into rat hepatocytes, mitochondrial esterases de-esterify a portion of the calcein-AM to trap calcein free acid in the mitochondrial matrix as well as the cytosol (27, 30). Similarly, lysosomal esterases lead to lysosomal loading of calcein. Subsequent warm incubation overnight then causes loss of cytosolic calcein through an anion transporter in the plasma membrane, but mitochondrial and lysosomal calcein is retained. However, attempts to cold ester load calcein into the mitochondria of mouse hepatocytes were unsuccessful, possibly because of low activity of the mitochondrial esterase (data not shown). Accordingly, we continued these experiments using rat hepatocytes.

After calcein labeling of mitochondria and lysosomes by cold loading/warm incubation, compartmentation of calcein was quite stable during a normal incubation, and no redistribution of calcein from either organelle into the cytosol was observed even after 2 h (Fig. 6A, top panels). By contrast, when hepatocytes were exposed to bafilomycin, mitochondrial calcein fluorescence progressively and substantially decreased over 2 h (Fig. 6B, top panels). Calcein fluorescence in rhodamine-dextran-labeled lysosomes after bafilomycin did not decrease but instead appeared to increase (Fig. 6B, arrows). Thus as inferred from calcein quenching and unquenching, bafilomycin caused lysosomal chelatable iron to decrease and mitochondrial chelatable iron to increase.

In hepatocytes co-loaded with rhodamine-dextran in lysosomes and calcein in both mitochondria and lysosomes, desferal and starch-desferal each suppressed bafilomycin-induced quenching of mitochondrial calcein fluorescence (Fig. 6C and D). Desferal suppressed quenching of mitochondrial calcein fluorescence somewhat more strongly than starch-desferal. Desferal and starch-desferal also promoted unquenching of lysosomal calcein fluorescence after bafilomycin over the course of 2 h of incubation (Fig. 6C and D, arrows). Taken together, these findings indicated that quenching of calcein fluorescence in mitochondria after bafilomycin was due specifically to an increase of mitochondrial chelatable iron in association with a decrease of lysosomal chelatable iron.

Iron uptake into mitochondria by the calcium uniporter

Ru360 is a highly specific inhibitor of the mitochondrial electrogenic calcium uniporter (31). When hepatocytes were co-loaded with rhodamine-dextran in lysosomes and calcein in both mitochondria and lysosomes, exposure to bafilomycin in the presence of Ru360 (20 μM) decreased mitochondrial calcein quenching (Fig. 7). However in contrast to desferal and starch-desferal, Ru360 did not induce an increase of calcein fluorescence in lysosomes (Fig. 7, arrows).

Figure 7. Suppression of mitochondrial calcein quenching after bafilomycin by Ru360.

Figure 7

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Rat hepatocytes were loaded with rhodamine-dextran and calcein, as described in Fig. 6, and exposed to 50 nM bafilomycin (Baf) in the presence of 20 μM Ru360. Note that in comparison to bafilomycin alone (Fig. 6B), mitochondrial calcein quenching was suppressed by Ru360. Arrows identify representative lysosomal structures. One experiment is typical of 3 or more replicates.

DISCUSSION

Release of lysosomal iron into the cytosol by bafilomycin

The major finding of the present work was that the lysosomal/endosomal compartment of cultured hepatocytes is a reservoir of chelatable iron, which is released upon inhibition of the proton-pumping vacuolar ATPase with bafilomycin (Fig. 1). Chelatable iron released into the cytosol was in part taken up into mitochondria via the electrogenic calcium uniporter residing in the mitochondrial inner membrane (Fig. 6 and 7). Bafilomycin-induced release of chelatable iron acted synergistically with the oxidant, t-BuOOH, to augment hydroperoxide formation, onset of the MPT and cell death (Fig. 3, 4 and 5). Suppression of these effects by desferal and starch-desferal confirmed the specific role of chelatable iron in augmenting cytotoxicity.

Quenching of calcein fluorescence by ferrous iron

Transition metals rapidly and stoichiometrically quench calcein fluorescence with the relative potency: Cu > Ni > Co > Fe2+ >> Mn > Zn > Pb > Fe3+ > Ca, Mg, Hg (32). Although copper and cobalt quench calcein fluorescence, quenching after bafilomycin treatment was prevented by desferal and starch-desferal. These chelators are highly specific for iron and do not chelate copper, cobalt or nickel (33). Aluminum is the only other biologically relevant metal that is chelated by desferal, but aluminum does not quench calcein fluorescence. Thus, calcein quenching most likely represents an increase of chelatable iron, specifically ferrous iron (Fe2+). The magnitude of increase of chelatable iron was substantial, since the decrease of intracellular calcein fluorescence was roughly the magnitude of the fluorescence of calcein (300 μM) placed to the extracellular medium. Assuming a 1 to 1 stoichiometry of chelatable iron to quenched calcein, bafilomycin led to an increase of chelatable iron in the range of 300 μM.

Two hit hypothesis of iron-catalyzed hydroxyl radical formation

In the presence of H2O2 (and O2•- dismutating to H2O2), Fe2+ catalyzes OH• formation and lipid peroxidation (1). After bafilomycin, increased chelatable iron by itself was not sufficient to enhance ROS production (increased cmDCF formation) (Fig. 5), the MPT (mitochondrial depolarization) (Fig. 4) or cell killing (PI uptake) (Fig. 3). Rather, two “hits” of oxidant stress and increased chelatable iron were needed to promote ROS formation, the MPT and cell killing. Lack of cytotoxicity after bafilomycin alone was not due to iron chelation by calcein, since cell killing and mitochondrial depolarization after bafilomycin did not occur in the absence of calcein loading (data not shown), which is consistent with the observation that most Fe-chelates are redox active, unlike desferal (34). Moreover, previous experiments with calcein did not show cytoprotection by the fluorophore (30).

Mitochondrial iron overloading causing mitochondrial ROS formation, the MPT and cell death may contribute to a variety of hepatic diseases, such as hepatotoxicity from iron overdose, hemochromatosis, and alcoholic and nonalcoholic steatohepatitis (12-14, 16, 35). Direct addition of membrane permeable iron complexes induces the MPT and killing of hepatocytes (20). Moreover, in isolated mitochondria, Fe2+ induces of the MPT at concentrations comparable to the ~300 μM increase of chelatable iron observed here (36). In conditions associated with lysosomal fragility and breakdown, such as lipotoxicity and high cytokine exposure (37-39), release of iron from lysosomes and uptake into mitochondria may also contribute to oxidative stress, MPT induction and activation of death pathways. Similar mechanisms also play a role in Wilson’s diseases with copper replacing iron as the transition metal promoting oxidative stress (40). These possibilities will need to be explored in future studies.

Cytoprotection by desferal and starch-desferal via chelation of lysosomal iron

Desferal is highly polar and poorly permeant through membranes, and high doses of desferal (0.5-1 mM) are required to block hepatocyte killing after I/R and oxidative stress, which may signify its poor penetration into the cytoplasm (41). Alternatively, desferal may enter through endocytosis into the acidic endosomal/lysosomal compartment, as has been suggested (42). To assess the latter possibility, hepatocytes were exposed to bafilomycin in the presence of desferal conjugated to hydroxyethyl starch (starch-desferal, 1 mM desferal equivalency). The >10 kDa starch-desferal only enters cells by endocytosis. Strikingly, starch-desferal prevented bafilomycin-induced calcein quenching as effectively as desferal (Fig. 1 and 2). These findings are consistent with the conclusion that lysosomes/endosomes release chelatable iron after bafilomycin and that desferal and starch-desferal prevent this release by chelating the intralumenal iron store of these organelles. Indeed after calcein loading into lysosomes, bafilomycin treatment especially in combination with desferal or starch-desferal caused unquenching of calcein fluorescence, signifying a decrease of intralumenal chelatable iron within the lysosomes (Fig. 6). Other approaches to measuring changes of intralumenal lysosomal chelatable iron, such as homogenizing hepatocytes and isolating lysosomes by density gradient centrifugation, were considered but not pursued, since chelatable iron contained in lysosomes would most likely be released before the lysosomes could be purified.

Fe2+/H+ exchange as a mechanism of lysosomal iron release

Changes of chelatable iron may also contribute to normal physiological processes. In Kupffer cells, chelatable iron increases transiently after LPS stimulation, and iron chelators block NFκB activation and cytokine formation (43-45). Iron chelation also produces hypoxia inducible factor-1α transactivation and expression of transferrin receptors (46, 47). An important question is how intracellular iron is mobilized. Proteolysis in lysosomes and proteosomes recycles iron for biosynthetic reactions(48). Similarly, heme oxygenase releases iron as heme is degraded. Ferritin and hemosiderin store excess iron in a non-reactive highly chelated form. In plasma, transferrin binds almost all non-heme iron at 2 iron binding sites. Plasma transferrin is 5-10 μM and transferrin iron occupancy is ~30%, which translates to plasma iron of 3-6 μM (49). The endosomal/lysosomal compartment continuously receives iron by transferrin receptor-mediated endocytosis (50, 51), but how iron is released into the cytosol for cellular needs, such as synthesis of iron-containing proteins, is not known (52). A membrane iron transporter, divalent metal transporter-1 (DMT1), mediates H+/Fe2+ symport by enterocytes across the plasma membrane and early endosomes into the cytosol but does not mediate iron release from lysosomes and late endosomes (52, 53). In our experiments, alkalinization of lysosomes/endosomes with bafilomycin caused release of chelatable iron into the cytosol without disrupting the integrity of individual lysosomes. This finding suggests that a Fe2+/H+ exchange mechanism may be important in lysosomal iron retention at low lysosomal pH and release at high lysosomal pH.

During oxidative stress and hypoxia/ischemia, lysosomes rupture in hepatocytes and other cells (26, 48, 54, 55). In cell lines, lysosomal rupture is a source of iron release and consequent pro-oxidant cell damage (56-58). Lysosomal degradation also occurs in models of apoptosis to hepatocytes (59). Overall, chelatable iron may act both as a dynamic regulator of cellular function and as a mediator of cell injury. Like the better characterized calcium ion, chelatable iron may represent both a signal regulating normal cellular responses and an intracellular mediator of toxicity when iron homeostasis is dysregulated.

Mitochondrial iron uptake after lysosomal iron release

Using a technique of cold-loading followed by warm incubation, we were able to load mitochondria selectively with calcein (Fig. 6). After bafilomycin treatment, mitochondrial calcein fluorescence decreased, signifying an increase of chelatable iron within the mitochondria. Both desferal and starch desferal suppressed mitochondrial calcein quenching after bafilomycin (Fig. 6). Thus, chelatable iron released into the cytosol by bafilomycin from lysosomes and endosomes was being taken up into mitochondria.

Previous studies show that isolated mitochondria accumulate Fe2+ electrogenically via the mitochondrial Ca2+ uniporter (60). Fe3+ is not transported. We confirmed this observation in intact hepatocytes by showing that Ru360, a highly specific inhibitor of the calcium uniporter (31), inhibited bafilomycin-induced quenching of mitochondrial calcein fluorescence (Fig. 7). Thus, mitochondria took up at least a portion of chelatable iron released by lysosomes after bafilomycin via the calcium uniporter. In previous studies of ischemia/reperfusion injury and oxidative stress with t-BuOOH, ROS formation assessed with DCF occurred primarily within mitochondria (5, 24). Such ROS formation promoted onset of the MPT and subsequent cell death, since mitochondrial depolarization, inner membrane permeabilization and loss of cell viability were blocked by desferal and antioxidants (5, 24). Thus, the two hits of ROS generation and increased chelatable iron may be occurring within mitochondria to promote the MPT and cell death.

Conclusion

Iron potentiates injury in a variety of diseases of the liver and other organs. Storage of chelatable iron in the lysosomal compartment and the mobilization of lysosomal iron by various stressors may be important events exacerbating hepatocellular injury. In particular, injury from oxidant stress may involve the two “hits” to mitochondria of increased O2•-/H2O2 and increased chelatable iron to promote formation of highly reactive and toxic OH•. Understanding of these mechanisms may lead to new strategies to minimize iron-dependent hepatocellular injury in various liver diseases.

Abbreviations used are

calcein-AM

acetoxymethyl ester of calcein

cmH2DCF-DA

chloromethyldihydrodichlorofluorescein diacetate

MPT

mitochondrial permeability transition

PI

propidium iodide

ROS

reactive oxygen species

t-BuOOH

tert-butylhydroperoxide

TMRM

tetramethylrhodamine methylester

Footnotes

*

This work was supported, in part, by Grants DK070195, DK073336, DK37034, and C06 RR015455 from the National Institutes of Health.

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