Tài liệu Báo cáo khoa học: Intralysosomal iron chelation protects against oxidative stress-induced cellular damage doc

Intralysosomal iron chelation protects against oxidative stress-induced cellular damage Tino Kurz1, Bertil Gustafsson2 and Ulf T Brunk1 Division of Pharmacology, Faculty of Health Sciences, Linkoping University, Sweden ă Department of Pathology and Cytology, University Hospital, Linkoping, Sweden ă Keywords cell death; lysosomes; mitochondria; redoxactive iron; salicylaldehyde isonicotinoyl hydrazone Correspondence U T Brunk, Department of Pharmacology, University Hospital, SE-581 85 Linkoping, ă Sweden Fax: +46 13 149106 Tel: +46 13 221515 E-mail: Ulf.Brunk@imv.liu.se (Received March 2006, revised 28 April 2006, accepted 15 May 2006) doi:10.1111/j.1742-4658.2006.05321.x Oxidant-induced cell damage may be initiated by peroxidative injury to lysosomal membranes, catalyzed by intralysosomal low mass iron that appears to comprise a major part of cellular redox-active iron Resulting relocation of lytic enzymes and low mass iron would result in secondary harm to various cellular constituents In an effort to further clarify this still controversial issue, we tested the protective effects of two potent iron chelators – the hydrophilic desferrioxamine (dfo) and the lipophilic salicylaldehyde isonicotinoyl hydrazone (sih), using cultured lysosome-rich macrophage-like J774 cells as targets dfo slowly enters cells via endocytosis, while the lipophilic sih rapidly distributes throughout the cell Following dfo treatment, long-term survival of cells cannot be investigated because dfo by itself, by remaining inside the lysosomal compartment, induces apoptosis that probably is due to iron starvation, while sih has no lasting toxic effects if the exposure time is limited Following preincubation with mm dfo for h or 10 lm sih for a few minutes, both agents provided strong protection against an ensuing $LD50 oxidant challenge by preventing lysosomal rupture, ensuing loss of mitochondrial membrane potential, and apoptotic ⁄ necrotic cell death It appears that once significant lysosomal rupture has occurred, the cell is irreversibly committed to death The results lend strength to the concept that lysosomal membranes, normally exposed to redox-active iron in high concentrations, are initial targets of oxidant damage and support the idea that chelators selectively targeted to the lysosomal compartment may have therapeutic utility in diminishing oxidant-mediated cell injury Exposing cells in culture to increasing oxidative stress triggers a range of cellular events Depending on the cell type, these may include enhanced proliferation or growth arrest, DNA damage, protein and lipid oxidation, apoptosis, and finally necrosis [1] This points to an important physiological role of redox regulation in cellular homeostasis [2,3] While most current studies suggest a direct effect of oxidative stress on DNA and mitochondria followed by apoptosis or necrosis, recent research has established a critical role for lysosomes in the initiating phase of impairment [4–13] Because hydrogen peroxide, added in moderate concentrations as a bolus, may be consumed within minutes [14] and most cellular alterations, including apoptosis, not occur until hours later, any satisfactory hypothesis on the mechanisms behind oxidative stress-induced cellular damage must provide firm and distinct links between the triggering events and the ultimate cellular injuries There are indications that hydrogen peroxide per se has little harmful effects and that an interaction Abbreviations AO, acridine orange base; dfo, desferrioxamine; pHPA, p-hydroxy-phenylacetic acid; PI, propidium iodide; sih, salicylaldehyde isonicotinoyl hydrazone (N¢-[(1Z)-(2-hydroxyphenyl)methylene]isonicotinohydrazide); SSM, sulfide-silver method; TMRE, tetramethylrhodamine ethyl ester; Ym, mitochondrial membrane potential 3106 FEBS Journal 273 (2006) 3106–3117 ª 2006 The Authors Journal compilation ª 2006 FEBS T Kurz et al between redox-active iron and hydrogen peroxide is required in order to initiate damage [10,12,15] The acidic vacuolar compartment is rich in low mass iron that would put these organelles in focus as initial targets for oxidative damage [16–19] Lysosomes, which together with late endosomes constitute the acidic vacuolar compartment, are the main cellular structures for normal autophagic turnover of organelles and longlived proteins [20–22] Autophagic degradation of ferruginous material, such as ferritin and cytochromes, is responsible for the intralysosomal occurrence of redoxactive low molecular weight iron [23] before it is transported out of the lysosomal compartment for use in a variety of anabolic processes, e.g synthesis of ironcontaining macromolecules, while excess iron is stored in ferritin This, along with the participation of iron in Fenton-type reactions producing hydroxyl radicals (HO•), or similarly reactive iron-centered [oxidoiron(IV)] radicals, would account for the sensitivity of lysosomes to oxidative stress that, if intense enough, may result in lysosomal rupture and release to the cytosol of harmful contents with ensuing cellular damage, including apoptosis and necrosis [4–6,24,25] Although Christian de Duve, the discoverer of lysosomes, envisaged such a possibility by nicknaming lysosomes ‘suicide bags’ [26], lysosomes are today often – although wrongly, we believe – considered to be sturdy organelles that usually not rupture until cells are already dead and necrotic We have previously shown that cells exposed for 1–3 h to high (‡ mm) concentrations of desferrioxamine, dfo (either in free form or as a high molecular weight conjugate to starch, HMW-dfo), are substantially, although not fully, protected against oxidative stress [10] dfo (or HMW-dfo), being a strong hydrophilic iron chelator that firmly binds all six coordinates of iron, preventing its iron(II) ⁄ iron(III) redox-cycling, does not pass membranes but is fluid phase-endocytosed by cells in culture, passes through late endosomes and, because of the extensive fusion and fission activities of the lysosomal compartment, is transferred to most lysosomes [13,27–30] As dfo remains intralysosomal, it will act as a sink for cellular iron in transit through the lysosomal compartment and, thus, within hours, cells will start to become affected by iron starvation and finally die [5,12] Interestingly, dfo-induced apoptosis, and a variety of other apoptogenic stimuli, involves lysosomal destabilization, suggesting this phenomenon to be related to apoptosis in general and not only to oxidative stress [5,6,31,32] Recently, it was demonstrated that the lipophilic iron chelator sih (salicylaldehyde isonicotinoyl hydra- Oxidative stress and intralysosomal iron zone, systematic name: N¢-[(1Z)-(2-hydroxyphenyl) methylene]isonicotinohydrazide) fully protects against oxidative stress-induced mitochondrial and cellular damage when present in low concentrations during the oxidative stress period [15,33] sih was first synthesized in 1953 [34] and its iron binding ability was demonstrated 20 years ago [35] Its binding constant for Fe(III) is 1050 at pH 7.4 [36] In the present study, by exposing lysosome-rich macrophage-like J774 cells to oxidative stress, either in the presence of sih or following pretreatment with dfo, we find strong evidence for a primary role of lysosomal redox-active iron in oxidative stress-induced cell damage and a close correlation between initial lysosomal rupture and later development of cellular damage and death Results Hydrogen peroxide-induced mitochondrial injury and cell death are downstream effects of lysosomal rupture In order to confirm and add to earlier findings on the sequence of events with respect to lysosomal rupture, mitochondrial injury and apoptosis ⁄ necrosis following oxidative stress [37], we first assessed lysosomal stability by cytofluorometric evaluation of alterations in green and red fluorescence, respectively, of cells vitally stained with acridine orange (AO) before (AO-relocation test [6,10,13,38–40]) and at h after the oxidative stress period (AO-uptake test [6,10,13,32,40,41]) AO is a weak base (pKa $10) that, due to proton trapping, preferentially distributes within the acidic vacuolar (lysosomal) cellular compartment [4–6,24,25,42–44] Due to its metachromatic properties, this probe fluoresces red inside lysosomes, where it is highly concentrated, and weakly green in the cytosol and the nucleus, where it is much less concentrated When used as a vital stain at low concentrations, the intercalation of AO into RNA and DNA is very low and does not disturb the evaluation of lysosomal stability Ordinary photomultipliers are about 10-fold more sensitive to green than to red photons, making the AO-relocation test useful for the early detection of a limited number of ruptured lysosomes As shown in Fig 1, unprotected cells showed lysosomal destabilization that was detectable by the AO-relocation method as early as 15 following the end of the oxidative stress period (peroxidative lysosomal membrane damage develops by time) Using the AO-uptake technique, unprotected cells showed a substantial increase in ‘pale’ cells (cells with a reduced FEBS Journal 273 (2006) 3106–3117 ª 2006 The Authors Journal compilation ª 2006 FEBS 3107 Oxidative stress and intralysosomal iron T Kurz et al Fig Lysosomal rupture is an early event after oxidative stress Acridine orange (AO)relocation assay Cells (106) were preloaded with the metachromatic fluorophore and lysosomotropic base AO Following two washing steps in culture medium, they were then exposed for 30 to 100 lM H2O2 in mL NaCl ⁄ Pi with ⁄ without 10 lM sih and returned to standard culture conditions for 15 Lysosomal stability was assayed by the AO-relocation technique using flow cytofluorometry in the green FL1 channel Due to release of AO from ruptured lysosomes into the cytoplasm, oxidative stress resulted in an early (15 min) increase of the mean value for the green cytoplasmic fluorescence that was significantly prevented by sih-protection (mean ± SD; ***P < 0.001; n ¼ 8) Examples of green fluorescence histograms are shown above each bar Fig Iron chelation protects against lysosomal rupture by oxidative stress Acridine orange (AO)-uptake assay Cells, either protected by sih or not, were exposed to oxidative stress as described for Fig Other cells were initially pretreated for h with mM dfo under otherwise standard culture conditions before exposure to the same oxidative stress Following end of the stress period, cells were returned to standard culture conditions for an additional period of h when lysosomal stability was assessed using the AO-uptake method Ruptured lysosomes not take up AO resulting in a population of cells with reduced red fluorescence (‘pale’ cells) The number of ‘pale’ cells was reduced highly significantly in cells protected by sih or dfo (mean ± SD; ***P < 0.001; n > 6) For each bar a representative histogram of red fluorescence, with ‘pale cells’ gated, is given number of intact lysosomes) after h (Fig 2), when these cells started to show apoptotic alterations as described previously [22] Cells protected by the lipophilic iron chelator sih, or the hydrophilic iron chelator dfo 3108 were highly protected against both early and late lysosomal rupture [13] dfo is known to induce iron starvation and related cell death and cannot be used in long-term experiments FEBS Journal 273 (2006) 3106–3117 ª 2006 The Authors Journal compilation ª 2006 FEBS T Kurz et al Oxidative stress and intralysosomal iron Fig Disruption of mitochondrial membrane potential is a down-stream effect of lysosomal rupture Cells, protected against oxidative stress by sih or not, were exposed to 100 nM tetramethylrhodamine ethyl ester (TMRE) for 15 under standard culture conditions 1–8 h following the oxidative stress period Red fluorescence was analyzed in the FL3 channel by flow cytofluorometry Damaged mitochondria with depolarized membranes show reduced TMRE uptake In unprotected cultures, significant mitochondrial damage was observed only and h after end of the oxidative stress period, while sih-protected cells showed almost no increase in damaged mitochondria (mean ± SD; ***P < 0.001; **P < 0.01; n ¼ 4) At top of the panel, examples of histograms are given showing red fluorescence h following end of oxidative stress Cells with reduced red fluorescence were gated [5] dfo is taken up by endocytosis [27,28], although this is not generally recognized (often it is just considered to pass membranes very slowly), remains intralysosomal and causes iron starvation [5,12] Thus, only the lipophilic sih, which quickly redistributes, was used in the remaining experiments As is evident from Fig 3, mitochondrial membrane potential was preserved by sih protection, indicating (as suggested before [6,22,37,40,45]) that mitochondrial damage is secondary to lysosomal rupture and related to iron-catalyzed intralysosomal peroxidation Similarly, sih-protection prevented the oxidative stress-induced decline in cell numbers (Fig 4) and postapoptotic necrosis (Fig 5) Actually, sih-protected cells multiplied similarly to control cells if exposed to sih only during the oxidative stress period (Fig 4), while cells exposed to >5 lm sih for long periods of time finally all died by iron-starvation (results not shown) Cells degraded hydrogen peroxide as shown before [22], and pretreatment with dfo or sih did not influence the rate (results not shown) In all experiments, cells exposed to sih without oxidative stress behaved like unexposed controls Fig sih-protected cells retain normal proliferation capacity following oxidative stress Cells were seeded (500 000 ⁄ well) and 24 h later exposed to oxidative stress, with or without sih-protection as described before Directly after end of the oxidative stress period (0 h) and again after return to standard culture conditions for another 12 and 24 h, cells were fixed in 4% formaldehyde in NaCl ⁄ Pi and counted in five predefined areas per dish (mean ± SD; ***P < 0.001; **P < 0.01; *P < 0.05; n.s., nonsignificant; n ¼ 4) After a short lag-phase, sih-protected cells continued to proliferate normally, while about half of the unprotected cells underwent apoptotic ⁄ necrotic cell death FEBS Journal 273 (2006) 3106–3117 ª 2006 The Authors Journal compilation ª 2006 FEBS 3109 Oxidative stress and intralysosomal iron T Kurz et al Fig sih-protected cells show no decrease in viability following oxidative stress Cells were seeded (500 000 ⁄ well) and 24 h later exposed protected or unprotected to oxidative stress as described before and were then returned to standard culture conditions for another 24 h The cells were then scraped, exposed to 40 lgỈmL)1 propidium iodide for 90 min, centrifuged and washed in NaCl ⁄ Pi ⁄ centrifuged twice Red fluorescence of PI-stained nuclei was measured by flow cytofluorometry in the FL3 channel (mean ± SD; ***P < 0.001; n ¼ 3) sih strongly protected cells against oxidative stress-induced postapoptotic necrosis Examples of red fluorescence histograms are given PI-positive cells were gated Cellular labile iron is located predominantly inside lysosomes To prove that cellular labile iron is located predominantly inside lysosomes, we utilized the sulfide-silver method (SSM), which is a very sensitive cytochemical technique to demonstrate heavy metals [18,41,46] As iron is the dominating heavy metal in most normal cells (the exceptions being some zinc-containing neurons and endocrine cells), it can be considered specific for this transition metal As shown in Fig (and our previous publications, reviewed in [45]), the outcome of the reaction is a mainly granular staining with a distinct lysosomal pattern when the SSM is applied to normal J774 cells in culture The SSM is an auto-catalytic procedure and a short development time results in few stained lysosomes, while a longer development results in a more general staining of lysosomes (Fig 6, compare parts A, B and C) This shows that the amount of redox-active iron varies between lysosomes, probably depending on the extent of recent engagement in autophagic degradation of ferruginous material [22,47], explaining why there is a pronounced heterogeneity between lysosomes with respect to sensitivity to oxidative stress [47] The fact that some cells and individual lysosomes resist oxidative stress better than others is considered an important and not well understood phenomenon [48] We have previously suggested that the difference in cellular and lysosomal amount of redox-active iron could be a major cause [47] 3110 In order to show that the SSM does indeed demonstrate iron, we exposed some cell cultures to 30 lm FeCl3 for h before the SSM In the culture medium, iron ions complex with phosphate groups under the formation of a hydrated iron phosphate complex, which is endocytosed by cells and transported to the lysosomal compartment [22,41] Following such iron uptake, a lysosomal staining pattern was obvious already after 30 development time, while the control cells showed no labeling (Fig 6A and D) Discussion Using the cytochemical SSM and the calcein technique in combination with induction of lysosomal rupture, we have here and previously demonstrated that lysosomes contain a major part of the cellular redox-active low mass iron, making the lysosomal compartment notably vulnerable to oxidative stress [12,18,22,41] The lysosomal concentration of low mass iron differs between the lysosomes of individual cells as well as between different cells, probably reflecting the participation of individual lysosomes in the autophagic degradation of ferruginous material These variations may explain the obvious differences in the sensitivity to oxidative stress of individual lysosomes of the same cell and of different cells of the same population [47,48] Lysosomes are a heterogeneous group of vesicular structures and, at a given point in time, some lysosomes are performing degradation, while others are ‘resting’ [20–22,47] Those engaged in the degradation FEBS Journal 273 (2006) 3106–3117 ª 2006 The Authors Journal compilation ª 2006 FEBS T Kurz et al Oxidative stress and intralysosomal iron Fig Cytochemical demonstration of iron by the sulfide-silver method (SSM) With increasing development time (30–60 min) control cells (A–C) show an increasingly intense lysosomal pattern of black granular silver precipitates, indicating the presence of lysosomal low molecular weight iron While there was no granular staining after a 30-min period of development (A), occasional granules were evident in some cells (examples are indicated by arrow heads) after 40 (B) and a distinctly granular staining with a lysosomal pattern was found in all cells after 60 of development (C) Cells exposed for h to a hydrated iron phosphate complex (obtained by adding FeCl3 to the culture medium to a final concentration of 30 lM) showed many stained lysosomes after development for 30 (D) when the control cells (A) were still empty This finding reflects the fluid phase endocytosis of the iron phosphate complex, as well as the capacity of the method to demonstrate iron of iron-containing macromolecules may contain a high concentration of low-mass iron, while a ‘resting’ lysosome would contain almost none [21,49,50], explaining our finding (Fig 6) that some lysosomes contain much more iron than others Although iron is an essential transition metal required for many vital functions, including electron transport, it is potentially dangerous because of its capacity to participate in Fenton-type reactions (Eqn 1) Fe2ỵ ỵ H2 O2 ! Fe3ỵ ỵ HO ỵ OH 1ị Hydroxyl radicals (HO•) are short lived ($10)9 s), extremely reactive, and able to bring about oxidative injury to a variety of biomolecules Consequently, cells and organisms handle iron with great care and usually hide it within stable metallo-organic complexes, thereby preventing hydrogen peroxide from encountering redox-active iron; an exception being low mass iron in late endosomes and lysosomes as well as, perhaps, a small amount of low mass iron in transit from these structures for storage in ferritin or use in the synthesis of iron-containing macromolecules [10–12] As lysosomes break under oxidative stress, as proven here and earlier with the AO-relocation and -uptake tests [6,10,13,32,38–41], we may assume that a certain fraction of lysosomal low mass iron exists in iron(II) form Most probably this is related to the low lysosomal pH and presence of reducing equivalents, such as cysteine [51–53] At pH 5, cysteine reduces iron(III) to iron(II), as has been shown before [22] This reduction of iron (Eqn 2) is driven by the removal of Cys-S• (Eqn 3) and the subsequent reduction of dioxygen (Eqn 4) [54,55] Cys SH ỵ Fe3ỵ ! Cys S ỵ Fe2ỵ ỵ Hỵ 2ị Cys S ỵ Cys SH ! Cys S S Cysị ỵ Hỵ 3ị  Cys S S Cysị ỵ O2 ! Cys S S Cys ỵ O2 4ị Autophagy is a normal and continuously ongoing process that allows a fine-tuned turnover of organelles and most long-lived macromolecules being of major importance for intracellular iron turnover [21,49,50,56] Due to intralysosomal degradation of a large variety of biomolecules, including many ferruginous materials, FEBS Journal 273 (2006) 3106–3117 ª 2006 The Authors Journal compilation ª 2006 FEBS 3111 Oxidative stress and intralysosomal iron T Kurz et al such as ferritin, mitochondrial complexes and various other metalloproteins, low-mass iron is set free intralysosomally As an effect, a substantial part of such iron seems to be temporarily harbored within these organelles before being transported, by not yet well characterized carrier systems, to the cytosol and used for anabolic purposes or stored in ferritin [11,12,22,23,57] Differences between cells of different origin with respect to lysosomal stability to oxidative stress may reflect a divergence in their capacity to degrade hydrogen peroxide, while differences in amounts of intralysosomal redox-active iron may explain intra- and intercellular variation [47,58] Here we exposed 106 cells in mL NaCl ⁄ Pi to a bolus dose of 100 lm hydrogen peroxide that is rapidly and exponentially degraded under these conditions Due to efficient intracellular degradation of hydrogen peroxide, a steep gradient across the plasma membrane is established and the actual concentration of hydrogen peroxide sensed by the lysosomes would probably not exceed $15 lm soon after the start of the oxidative stress [14] As lysosomes not contain any catalase or glutathione peroxidase, this initial magnitude of oxidative stress proved sufficient to induce lysosomal rupture that initially was of limited scale Lysosomal rupture accelerated by time through a lipid peroxidation chain reaction until it initiated apoptosis, probably by a direct or indirect effect on mitochondrial stability [37,59,60] This lysosomal rupture results in release to the cytosol of powerful hydrolytic enzymes and redox-active iron that, depending on the magnitude of the rupture, may initiate a variety of cellular injuries, including reparative autophagocytosis, apoptosis and necrosis [45] Clearly, lysosomes are not the sturdy organelles they once were believed to be, breaking only late during necrotic (accidental) cell death Oxidative stress-induced lysosomal rupture, secondary mitochondrial injury and final cell death were almost fully prevented by a pre-exposure to the potent iron chelators sih and dfo, as was also shown previously [37,45], suggesting that the damaging effect of hydrogen peroxide per se is of minor importance in comparison to its iron-mediated effect on lysosomal stability Even if the importance of lysosomal rupture for oxidative stress-induced damage, including apoptosis and DNA damage, has been shown before [5,10– 13], this view is still controversial A major reason for the present lack of general acceptance of an initiating role of lysosomal break in oxidative stress-induced injury may be that dfo, which often has been used to chelate intralysosomal iron, works like a double-edged sword It does prevent early lysosomal damage under 3112 oxidative stress, but after some time dfo itself induces apoptosis [5] The reason for this phenomenon seems to be that dfo following endocytotic uptake remains inside the lysosomal compartment where it scavenges and acts as a sink for iron that is in transit through the compartment as a result of autophagy [11,12,21,49,50] The lipophilic iron chelator sih does not, however, have this disadvantage sih swiftly penetrates membranes and binds iron very strongly throughout the cell in a non redox-active form, also at lysosomal pH [36] In a recent study, it was shown that in the presence of sih, cells show no mitochondrial damage following oxidative stress, indicating the need of free iron for such damage [15] Here we add the information that the defense is mediated by lysosomal stabilization and that cells protected by sih during oxidative stress and then brought back to normal culture conditions continue to proliferate normally Because of its lipophilicity, sih enters and leaves cells rapidly and, in contrast to dfo, it can be used to chelate intracellular redox-active iron for a limited period of time without causing long-lasting effects A key aspect of oxidative regulation of physiological processes is the disparity of the time-scales involved The apoptotic ⁄ necrotic process takes several hours to fully develop, although cells need to be exposed to hydrogen peroxide for only a short period of time to be committed to apoptosis ⁄ necrosis The very sensitive AO-relocation technique to detect early lysosomal rupture allowed us to observe a strong correlation between a hydrogen peroxideinduced cellular modification that occurred rapidly, by induction of partial lysosomal rupture, and the signs of apoptosis ⁄ necrosis hours later Both processes showed the same dose-dependent response to hydrogen peroxide and were inhibited by dfo- and sih-mediated iron chelation during the period of oxidative stress Release of lysosomal contents initiates a process that results in mitochondrial destabilization as well as further lysosomal rupture [37] In this context, it is worth mentioning that the change in mitochondrial membrane potential that was demonstrated, as well as release of cytochrome c from mitochondria under similar conditions [37,61] does not occur until 1–3 h after the end of exposure to hydrogen peroxide and long after the lysosomal rupture is observed Interestingly, we observed a progressive decrease in the number of intact lysosomes over time when the cells were no longer under oxidative stress, which is in accordance with a self-amplifying loop and cross-talk between lysosomes and mitochondria (Fig 7), as previously pointed FEBS Journal 273 (2006) 3106–3117 ª 2006 The Authors Journal compilation ª 2006 FEBS T Kurz et al Oxidative stress and intralysosomal iron Fig The lysosomal-mitochondrial pathway of cell death; a tentative scheme Slightly modified from Zhao et al [37], the scheme shows intralysosomal Fenton-type reactions resulting from oxidative stress Lysosomal contents are released to the cytosol following lysosomal destabilization and may activate pro-apoptotic proteins, such as Bid [59,60], and ⁄ or attack mitochondria with release of cytochrome c and enhanced production of superoxide Released lysosomal enzymes (LE) may also activate cytosolic phospholipases, which in turn may attack mitochondria and lysosomes, inducing a self-amplifying loop Released redox-active iron may bind to nuclear and mitochondrial DNA and induce site-specific damage under continuous oxidative stress [10,12] out as the foundation of the lysosomal-mitochondrial axis theory of apoptosis [37] Taken together, the findings of this study suggest that the major effect of harmful oxidative stress, rather than being a direct effect on targets such as DNA and mitochondria, is a result of lysosomal rupture due to intralysosomal peroxidative events, with ensuing relocalization of lysosomal hydrolytic enzymes and lowmass iron Experimental procedures Chemicals Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, penicillin and streptomycin were from Gibco (Paisley, UK) Acridine orange base was from Gurr (Poole, UK), while silver lactate was from Fluka AG (Buchs, Switzerland) Glutaraldehyde was from Bio-Rad (Cambridge, MA, USA), and ammonium sulfide and hydroquinone were from BDH Ltd (Poole, UK) dfo was from Ciba-Geigy (Basel, Switzerland) sih was a kind gift from D Richardson (University of New South Wales, Sydney, Australia) All other chemicals were from Sigma (St Louis, MO, USA) Cell culture and exposure to hydrogen peroxide with ⁄ without iron chelator protection Murine macrophage-like J774 cells (ATCC, Manassas, VA, USA) were grown in DMEM supplemented with 10% fetal bovine serum, mm l-glutamine, 100 ImL)1 penicillin and 100 lgỈmL)1 streptomycin, at 37 °C in humidified air with 5% CO2 The cells were subcultivated twice a week, plated at a concentration of · 106 cells per well in six-well plates, with or without cover-slips, and typically subjected to oxidative stress after another 24 h Concentrations of hydrogen peroxide and exposure times (in relation to cell density) and exposure to sih and dfo were established in preliminary experiments In final experiments, control and chelator-protected cells were oxidatively stressed (or not) for 30 by exposure to a bolus dose of 100 lm H2O2 in mL NaCl ⁄ Pi at 37 °C Note that under these conditions the H2O2 concentration declines quickly (t1 ⁄ $15 min) to < 20 lm after 30 (see below) dfo (1 mm) was added to the culture medium under otherwise standard conditions h before oxidative stress sih was prepared as a 10 mm solution in dimethyl sulfoxide and then diluted to a mm stock solution in absolute ethanol To produce a final concentration of 10 lm sih during the oxidative stress exposure, some of the stock was added to the NaCl ⁄ Pi immediately prior to the addition of hydrogen peroxide No sih pretreatment was found necessary After the oxidative stress period, cells were directly analyzed or returned to standard culture conditions and assayed at indicated periods of time In some experiments, cells were incubated for h in complete medium with FeCl3 added to a concentration of 30 lm (resulting in the formation of a nonsoluble iron phosphate complex that is endocytosed and transported into the lysosomal compartment) FEBS Journal 273 (2006) 3106–3117 ª 2006 The Authors Journal compilation ª 2006 FEBS 3113 Oxidative stress and intralysosomal iron T Kurz et al Degradation of hydrogen peroxide To ensure that the observed resistance to oxidative stress was not an effect of enhanced H2O2 catabolism induced by the iron-chelating drugs, the rate of H2O2 clearance was determined Control cells and cells protected by the iron chelators (in concentrations described above) were exposed to a bolus dose of 100 lm H2O2 in mL NaCl ⁄ Pi at 37 °C During a 60-min period, aliquots (50 lL) were sampled for H2O2 analysis by the horseradish peroxidase-mediated H2O2-dependent p-hydroxy-phenylacetic acid (pHPA) oxidation technique [62] Fluorescence intensity was read (kex315 nm; kem410 nm) using a RF-540 spectrofluorometer (Shimadzu, Kyoto, Japan) connected to a DR-3 data recorder Lysosomal membrane stability assay Six hours after the oxidative stress period (see above), cells were exposed to 10 lgỈmL)1 acridine orange (AO) in complete medium at 37 °C for 15 min, detached by scraping and collected for flow cytofluorometric assessment of lysosomal AO-uptake AO is a metachromatic fluorophore and a lysosomotropic base (pKa ¼ 10.3), which becomes charged (AOH+) and retained by proton trapping within acidic compartments, mainly secondary lysosomes (pH 4.5–5.5) When normal cells are excited by blue light, highly concentrated lysosomal AO emits an intense red fluorescence, while nuclei and cytosol show weak diffuse green fluorescence In AOuptake experiments, red fluorescence was measured (FL3 channel) using a Becton-Dickinson FACScan (Becton-Dickinson, Mountain View, CA, USA) equipped with a 488 nm argon laser Cells with a reduced number of intact, AO-accumulating lysosomes (here termed ‘pale’ cells) were detected as described earlier [6,10,13,32,40,41] The AO-relocation technique [6,10,13,38–40] was used to measure early lysosomal damage For this assay, cells were preloaded with AO (10 lgỈmL)1) for 15 in complete culture medium, rinsed with culture medium and kept under standard culture conditions for a further 15 before being exposed to oxidative stress After the oxidative stress period cells were returned to standard culture conditions for 15 and then scraped and assayed by flow cytofluorometry The increase in green cytoplasmic fluorescence, due to the release of AO from ruptured lysosomes, was measured in the FL1 channel cellquest software (BD Biosciences, Franklin Lakes, NJ, USA) was used for acquisition and analyses Mitochondrial membrane potential assay Mitochondrial membrane potential (Ym) was measured by flow cytofluorometry, using the cationic and lipophilic dye tetramethylrhodamine ethyl ester (TMRE), which 3114 accumulates in the mitochondrial matrix Decreased Ym is indicated by a reduction of the TMRE-induced red fluorescence At different points of time (1–8 h) following the end of oxidative stress (see above), cells were incubated with TMRE in complete culture medium (100 nm; 15 min; 37 °C) and assayed by flow cytofluorometry Red (FL3 channel) fluorescence was recorded in a log scale and analyzed using the cellquest software Cells with reduced red fluorescence were gated Assessment of cell proliferation Five hundred thousand cells were seeded per well and exposed for 30 to oxidative stress (or not) 24 h later with 10 lm sih present (or not) Directly after oxidative stress, and after another 12 and 24 h under standard culture conditions, cells were washed in NaCl ⁄ Pi and fixed in 4% formaldehyde in NaCl ⁄ Pi For each condition cells were counted in five predefined areas of two separate dishes The cell proliferation experiments were done twice Assessment of postapoptotic necrotic cells Cells were seeded and treated as described above for the cell proliferation assay The magnitude of oxidative stress applied is known to induce apoptosis but little direct necrosis [22] After 24 h under standard conditions following the oxidative stress, cells were scraped and exposed in the dark for 90 to 40 lgỈmL)1 propidium iodide in complete culture medium at 22 °C Cells were then centrifuged and washed in NaCl ⁄ Pi twice before red fluorescence was analyzed by flow cytofluorometry using the FL3 channel Propidium iodide does not cross the plasma membrane of normal or early apoptotic cells, while it penetrates into postapoptotic necrotic cells and binds to nuclear DNA Cytochemical assay of lysosomal reactive iron For evaluation of cellular low-mass iron, we used the autometallographic sulfide-silver method as previously described [18], modified (high pH; high S2–) from Timm [46] Cells were grown on cover-slips and exposed, or not, for h to an insoluble hydrated iron phosphate complex, obtained by addition of FeCl3 to complete culture medium to a final concentration of 30 lm Cells were rinsed briefly in NaCl ⁄ Pi (22 °C) prior to fixation with 2% glutaraldehyde in 0.1 m sodium cacodylate buffer with 0.1 m sucrose (pH 7.2) for h at 22 °C The fixation was followed by five short rinses in glass-distilled water at 22 °C Cells were then sulfidated at pH $9 with 1% (w ⁄ v) ammonium sulfide in 70% (v ⁄ v) ethanol for 15 Following careful rinsing in glassdistilled water for 10 at 22 °C, development was performed using a physical, colloid-protected developer containing silver-lactate and hydroquinon (the method is an FEBS Journal 273 (2006) 3106–3117 ª 2006 The Authors Journal compilation ª 2006 FEBS T Kurz et al autocatalytic one and the precipitation of metallic silver on the FeS core is dependent on time and the amount of initiating FeS) The reaction was performed in the dark at 26 °C for various periods of time (30–60 min) Following dehydration in a graded series of ethanol solutions and mounting in Canada balsam, the cells were examined and photographed, using transmitted light, under an Axioscope microscope (Zeiss, Oberkochen, Germany) connected to a Zeiss ZVS-47E digital camera easy image measurement 2000 software (version 2.3, Bergstrom Instruments AB, ă Solna, Sweden) was used for image acquisition Oxidative stress and intralysosomal iron 10 Statistical analysis Results are given as mean ± SD Statistical comparisons were made using analysis of variance (anova), whereby pair-wise multiple comparisons were made using Tukey’s adjustment For comparison of two means, Student’s t-test was used P