H2O2-Mediated Damage to Lysosomal Membranes of J-774 Cells

The effects of hydrogen peroxide on cell viability and, in particular, on lysosomal integrity were investigated in a model system of cultured, established, macrophage-like J-774 cells. The cells were found to rapidly degrade added hydrogen peroxide, withstanding concentrations 250μM without cell death; however, all tested concentrations (100-500/μM) substantially decreased cellular ATP to approximately the same degree. Concentrations of hydrogen peroxide 500/μM resulted in a pronounced and rapid decrease in cell viability preceded by the loss of lysosomal integrity, as judged by the relocalization of acridine orange, a lysosomotropic weak base, in pre-labelled cells. Hydrogen peroxide-induced relocalization of acridine orange and cell death were either enhanced or much prevented, according to if the cells were initially allowed to endocytose ferric iron or the specific iron-chelator deferoxamine, respectively. Depletion of ATP, however, was not associated with the loss of lysosomal integrity and viability regardless of iron or deferoxamine pretreatment. Pre-exposure to E-64, an inhibitor of lysosomal thiol proteases, resulted in the reduction of both lysosomal membrane damage and cell death. The results are interpreted as indicating (i) generation of hydroxyl radicals within the secondary lysosomal compartment due to the occurrence of reactive ferrous iron, leading to (ii) peroxidative alterations of the lysosomal membrane resulting in (iii) loss of lysosomal membrane integrity with dissipation of the proton gradient and leakage of lysosomal contents, including hydrolytic enzymes, into the cell sap. The partial protection by E-64 may result from hydroxyl radical scavening by accumulated non-degraded autophagocytosed lysosomal material, and/or decreased availability of reactive redox-cycling iron due to decreased enzymatic digestion of autophagocytosed iron-containing metalloproteins. Moreover, our results show that the normal lysosomal content of iron, capable of redox cycling, of the cell line under study is enough to induce oxidative damage leading to loss of lysosomal integrity. It is suggested that lysosomal damage may be an important cause of cell degeneration under conditions of increased intra- or extracellular hydrogen peroxide-formation.     

Exposure of cells to nonlethal concentrations of hydrogen peroxide induces degeneration-repair mechanisms involving lysosomal destabilization

The cytotoxicity of hydrogen peroxide is, at least partly, mediated by the induction of intralysosomal iron-catalyzed oxidative reactions with damage to lysosomal membranes and leakage of destructive contents. We hypothesize that minor such leakage may be nonlethal, and the ensuing cellular degeneration repairable. Consequently, we investigated, using a model system of cultured J-774 cells, the effects of hydrogen peroxide in moderate concentrations on cellular viability, lysosomal membrane integrity, morphology, and ATP and reduced glutathione concentrations. These parameters were initially estimated directly after a 30 min exposure to a bolus dose of hydrogen peroxide in phosphate buffered saline at 37°C, and then again following subsequent recovery periods of different lengths under ordinary culture conditions. All cells survived an exposure to 250 μM hydrogen peroxide for 30 min, whereas 350 and 500 μM exposure was lethal to a small fraction of cells. The oxidative stress caused early, time- and dose-dependent, partial relocalization of the lysosomotropic weak base acridine orange from the lysosomal compartment to the cytosol. This phenomenon is known to parallel leakage of damaging lysosomal contents such as hydrolytic enzymes. There were also signs of cellular damage in the form of surface blebbing and increased autophagocytosis, more marked with the higher doses of hydrogen peroxide. Also found was a rapid depletion of ATP and GSH. These alterations were all reversible, as long as cells were exposed to nonlethal amounts of hydrogen peroxide. Based on these and previous findings, we suggest that lysosomes are less stable organelles than has hitherto been assumed. Restricted lysosomal leakage might be a common event, for example, during sublethal oxidative stress, causing reversible, degenerative alterations, which are repaired by autophagocytosis.     

Cathepsin D-Bax death pathway in oxidative stressed neuroblastoma cells

Hydrogen peroxide, the major oxidoradical species in the central nervous system, has been involved in neuronal cell death and associated neurodegenerative diseases. In this study, we have investigated the involvement of the lysosomal pathway in the cytotoxic mechanism of hydrogen peroxide in human neuroblastoma cells. Alteration of lysosomal and mitochondrial membrane integrity was shown to be an early event in the lethal cascade triggered by oxidative stress. Desferrioxamine (DFO), an iron chelator that abolishes the formation of reactive oxygen species within lysosomes, prevented lysosome leakage, mitochondrial permeabilization and caspase-dependent apoptosis in hydrogen peroxide-treated cells. Inhibition of cathepsin D, not of cathepsin B, as well as small-interference RNA-mediated silencing of the cathepsin D gene prevented hydrogen peroxide-induced injury of mitochondria, caspase activation, and TUNEL-positive cell death. Cathepsin D activity was shown indispensable for translocation of Bax onto mitochondrial membrane associated with oxidative stress. DFO abolished both the cytosolic relocation of Cathepsin D and the mitochondrial relocation of Bax in hydrogen peroxide-treated cells. siRNA-mediated down-regulation of Bax expression protected the cells from oxidoradical injury. The present study identifies the lysosome as the primary target and the axis cathepsin D-Bax as the effective pathway of hydrogen peroxide lethal activity in neuroblastoma cells.     

Starvation-induced autophagocytosis paradoxically decreases the susceptibility to oxidative stress of the extremely oxidative stress-sensitive NIT insulinoma cells

Summary

Glucose and amino acid starvation of cells in culture generally enhances their sensitivity to oxidative stress. This is explained by compensatory autophagocytosis, which results in increased amounts of lysosomal low-molecular-weight, redox-active iron, due to the degradation of metallo-proteins, with a potential increase in iron-catalyzed, intralysosomal oxidative reactions. Such reactions diminish the stability of lysosomal membranes, with resultant leakage of hydrolytic enzymes into the cytosol and ensuing cellular degeneration, often of apoptotic type. However, starvation of NIT insulinoma cells, which are normally remarkably sensitive to oxidative stress, actually attenuated the sensitivity to such stress. We found that starved NIT cells rapidly synthesized ferritin. Moreover, ferritin was found to be autophagocytosed, and the lysosomes were stabilized, as assayed by the acridine orange relocation test. We hypothesize that compensatory autophagocytosis during starvation increases the cytosolic pool of redox-active iron, as a reflection of enhanced transportation of low-molecular-weight iron from autophagic lysosomes to the cytosol, resulting in ferritin induction. The newly formed ferritin would, in turn, become autophagocytosed and bind redox-active lysosomal iron in a non-redox-active form. We also suggest that the proposed mechanism may be a way for oxidative stress-sensitive cells to compensate partly for their failing capacity to degrade hydrogen peroxide before it leaks into the acidic vacuolar apparatus and induces intralysosomal oxidative stress. The insulin-producing beta cell may belong to this type of cells.     

PDCD4基因在过氧化氢诱导喉癌细胞凋亡中的作用

目的探讨过氧化氢诱导喉癌细胞Hep-2凋亡过程中PDCD4基因表达的变化。方法以体外培养的喉癌细胞Hep-2为实验材料,不同浓度的过氧化氢作用于Hep-2细胞,噻唑蓝(MTT)比色法测定细胞生存率,采用吖啶橙染色、Ho33342/PI荧光双染进行形态学观察,RT-PCR及Western blot检测PDCD4 mRNA水平及蛋白表达的变化,评价在过氧化氢诱导喉癌细胞Hep-2凋亡过程中PDCD4基因的作用。结果过氧化氢(200μmol/L)作用Hep-2细胞24h,能够显著抑制细胞增殖,并诱导细胞凋亡,同时引起pdcd4 mRNA水平显著上调,PDCD4蛋白表达显著增加。结论本研究首次报道PDCD4基因可能在氧化胁迫诱导喉癌细胞凋亡中起关键作用。     

Visualization of exocytotic secretory processes of mast cells by fluorescence techniques

Secretory processes via exocytosis in rat peritoneal mast cells were visualized by two complementary fluorescence techniques; one staining pre-exocytotic granules with a basic probe and the other staining post-exocytotic granules with acidic probes. Granules within mast cells were selectively stained with acridine orange and emitted orange yellow fluorescence. Upon stimulation with compound 48/80, release of acridine orange from granules was observed both in population and single cell measurements. This release was seen in some localized area of mast cells. Opening of pores between plasma membranes and granule membranes was monitored using acidic fluorescence probes such as 6-carboxyfluorescein or lucifer yellow CH. Not only granules located at peripheral region, but also granules near the core region participated in exocytosis. The existence of junctions between these granules was suggested. TMA-DPH, a lipophilic membrane probe, which was localized at plasma membrane before stimulation, diffused into granule membranes after stimulation. This shows that after stimulation, some constituents of plasma and granule membranes were mixed. Even after extensive degranulation, mast cells extruded acidic probes, indicating the plasma membranes still play a role of barrier. Activation of lateral motion of granules preceding to exocytosis was not observed. It was concluded that the visualization of secretory processes by fluorescence and image processing techniques will be useful for the study of molecular mechanisms underlying exocytosis.     

Rapid assessment of islet viability with acridine orange and propidium iodide

Summary A simple, rapid method for estimating the viability of isolated islets of Langerhans with fluorescent dyes is described. Low concentrations of acridine orange and propidium iodide (AO/PI) were used to visualize living and dead islet cells simultaneously. AO/PI-stained islets can be divided into three distinct groups. Group A islets fluoresce green, contain insulin, and have normal ultrastructure; group C islets fluoresce primarily red, contain little or no insulin, and have cells with disrupted cellular membranes. Group B islets fluoresce red, green, and yellow. The yellow color is due to the addition of two primary colors from the superimposed red and green fluorescing cells. In this assay, the interpretation that red islet cells are dead and green islet cells are alive was confirmed by sequentially staining single islet cells with AO/PI and trypan blue. The observation that red islets are dead was confirmed by heat-killing, enzymatically damaging, treating with ethanol, or depriving islets of nutrients and observing the red fluorescence. This assay should be useful in studies where the assessment of islet viability is essential. Preliminary reports of this work were presented at two meetings and were published in abstract form (24,25). This research was supported in part by the National Institutes of Health, Bethesda, MD, grant DK 18115.     

Lipofuscin Accumulation in Cultured Retinal Pigment Epithelial Cells Causes Enhanced Sensitivity to Blue Light Irradiation

Lipofuscin accumulates with age within secondary lysosomes of retinal pigment epithelial (RPE) cells of humans and many animals. The autofluorescent lipofuscin pigment has an excitation maximum within the range of visible blue light, while it is emitting in the yellow-orange area. This physico-chemical property of the pigment indicates that it may have a photo-oxidative capacity and, consequently, then should destabilize lysosomal membranes of blue-light exposed RPE. To test this hypothesis, being of relevance to the understanding of age-related macular degeneration, cultures of heavily lipofuscin-loaded RPE cells were blue-light–irradiated and compared with respect to lysosomal stability and cell viability to relevant controls. To rapidly convert primary cultures of RPE, obtained from neonatal rabbits, into aged, lipofuscin-loaded cells, they were allowed to phagocytize artificial lipofuscin that was prepared from outer segments of bovine rods and cones. Following blue-light irradiation, lysosomal membrane stability was measured by vital staining with the lysosomotropic weak base, and metachromatic fluorochrome, acridine orange (AO). Quantifying red (high AO concentration within intact lysosomes with preserved proton gradient over their membranes) and green fluorescence (low AO concentration in nuclei, damaged lysosomes with decreased or lost proton gradients, and in the cytosol) allowed an estimation of the lysosomal membrane stability after blue-light irradiation. Cellular viability was estimated with the delayed trypan blue dye exclusion test. Lipofuscin-loaded blue-light–exposed RPE cells showed a considerably enhanced loss of both lysosomal stability and viability when compared to control cells. It is concluded that the accumulation of lipofuscin within secondary lysosomes of RPE sensitizes these cells to blue light by inducing photo-oxidative alterations of their lysosomal membranes resulting in a presumed leakage of lysosomal contents to the cytosol with ensuing cellular degeneration of apoptotic type. The suggested mechanism may have bearings on the development of age-related macular degeneration. © 1997 Elsevier Science Inc.     

Role of oxidative stress in lysosomal membrane permeabilization and apoptosis induced by gentamicin, an aminoglycoside antibiotic

Gentamicin, an aminoglycoside antibiotic used to treat severe bacterial infections, may cause acute renal failure. At therapeutic concentrations, gentamicin accumulates in lysosomes and induces apoptosis in kidney proximal tubular cells. In gentamicin-treated renal LLC-PK1 cells, acridine orange release from lysosomes, previously interpreted as lysosomal membrane permeabilization, precedes the apoptotic cascade that develops during incubation with gentamicin. However, the link between gentamicin lysosomal accumulation and apoptosis remains unclear. We here examined if reactive oxygen species (ROS) production could account for gentamicin-induced acridine orange release and apoptosis, and the implication of iron in these events. We found that gentamicin induced ROS production prior to, and at lower drug concentrations than required for, acridine orange release and apoptosis. ROS antioxidant or scavenger, catalase, and N-acetylcysteine largely prevented these events. Vital confocal imaging revealed that gentamicin-induced ROS production occurs in lysosomes. Deferoxamine, an iron chelator, which is endocytosed and accumulates in lysosomes, largely prevented gentamicin-induced ROS production as well as apoptosis. Direct evidence for gentamicin-induced permeabilization of lysosomal membrane was provided by showing the release into the cytosol of Lucifer yellow, a membrane-impermeant endocytic tracer with a comparable molecular weight as gentamicin. Altogether, our data demonstrate a key role of lysosomal iron and early ROS production in gentamicin-induced lysosomal membrane permeabilization and apoptosis.     

Mechanism of apoptosis induced by a lysosomotropic agent, L-Leucyl-L-Leucine methyl ester

Lysosomes are fundamental for cell growth, and thus inhibition of the lysosomal function often leads to cell death. L-Leucyl-L-leucine methyl ester (LeuLeuOMe) is a lysosomotropic agent that induces apoptosis of certain immune cells. LeuLeuOMe is taken up through receptor-mediated endocytosis, and then converted into (LeuLeu)_n-OMe (n>3) by dipeptidyl peptidase I (DPPI) in lysosomes, which reportedly causes rupture of the lysosomes and DNA fragmentation. In this study we examined how lysosomal damage causes DNA fragmentation in LeuLeuOMe-treated HL-60 cells. When acridine orange was employed to monitor lysosomal membrane integrity, orange or red granular fluorescence was seen in normal cells. In contrast, LeuLeuOMe-treated cells showed orange, yellow or green cellular fluorescence all over the cytoplasm, suggesting that LeuLeuOMe induced a loss of lysosomal membrane integrity. The loss was inhibited by a DPPI inhibitor, GlyPheCHN₂ (GFCHN₂), but not by a caspase-3 inhibitor, Ac-DEVD-CHO, indicating that a condensation product was responsible for the loss. LeuLeuOMe also induced the activation of caspase-3-like protease and DNA fragmentation, both of which were inhibited by either GFCHN₂ or Ac-DEVD-CHO. Therefore, the activation of caspase-3-like protease links the loss of lysosomal membrane integrity to DNA fragmentation during apoptosis induced by LeuLeuOMe.     

Oxidative stress,growth factor starvation and Fas activation may all cause apoptosis through lysosomal leak

Abstract

Oxidative stress, growth factor starvation, and activation of the Fas/APO-1/CD95 receptor all induce apoptosis in a variety of cell-types, including the established human Jurkat T-cell line. Oxidative stress, in the form of exposure of the cells to a bolus dose of hydrogen peroxide, results in intra-lysosomal, iron-catalyzed oxidative reactions. This is accompanied by a time- and dose-dependent lysosomal destabilization — as evaluated by a decreased lysosomal uptake of the metachromatic fluorochrome, and weak base, acridine orange —in combination with leakage to the cytosol of lysosomal contents, including hydrolytic enzymes. Moderate lysosomal rupture is followed by apoptosis within initially intact plasma membranes, while necrosis and cell lysis are associated with a more complete lysosomal breach. Prior endocytosis of the potent iron-chelator desferrioxamine,resulting in binding of intralysosomal low molecular weight iron in a non-redox active form, largely prevents not only oxidative stress-induced lysosomal labilization, but apoptosis as well. When apoptosis is induced by the use of a monoclonal IgM anti-human Fas/APO-1/CD95 receptor antibody, the apoptotic process is again found to be accompanied by lysosomal leak. It is, however, not prevented by a preceding endocytosis of desferrioxamine and, consequently, could not be a function of intralysosomal iron-catalyzed oxidative reactions,but must be due to other mechanisms. Growth factor starvation of Jurkat cultures for a few days results in a high proportion of apoptotic cells, which contain lysosomes many of which have lost their proton gradient and appear to have released their contents. Overall, our results indicate that lysosomal leakage/rupture precedes apoptosis in Jurkat cells regardless of the initiating agent, but that such rupture may occur through multiple mechanisms. Lysosomal enzymes, leaking out of their normal vacuolar compartment, may then induce apoptosis, perhaps by proteolytic activation of the caspase-family of enzymes. Regardless of the precise mechanism, these observations suggest that partial rupture of the acidic vacuolar compartment may be one of the finalpathways in apoptosis.     

Lysosomal heterogeneity between and within cells with respect to resistance against oxidative stress

The prevailing opinion on lysosomal endurance is that, as long as the cells are still alive, these organelles are generally quite stable and, thus, do not induce cell damage by leaking their numerous powerful hydrolytic enzymes to the cytosol. We suggest that this opinion is basically wrong and consider that many lysosomes are quite vulnerable, especially to oxidative stress. Moreover, we suggest that cellular degeneration, including apoptosis as well as necrosis, follows upon lysosomal disruption. We have found differing stability of lysosomal membranes to oxidative stress, not only among different cell types, but also between cells of the same type and between lysosomes of individual cells. We suggest that cellular resistance to oxidative stress is mainly a function of three parameters: (i) the capacity to degrade hydrogen peroxide before it reaches, and may diffuse into, the acidic vacuolar compartment; (ii) the resistance to reactive oxygen species of lysosomal membranes; and (iii) the intralysosomal amounts of redox-active, low molecular weight iron. Iron-catalysed intralysosomal reactions, if pronounced enough, result in peroxidation and destabilization of the lysosomal membrane. Owing to differences in the cellular synthesis of hydrogen peroxide-degrading enzymes, degree of autophagocytotic degradation of iron-containing metalloproteins, lysosomal localization within the cytoplasm and intralysosomal iron chelation, the above three parameters may vary between both different and similar cells and between lysosomes of individual cells as well, explaining their observed variability with respect to resistance against oxidative stress     

Lysosomal heterogeneity between and within cells with respect to resistance against oxidative stress

The prevailing opinion on lysosomal endurance is that, as long as the cells are still alive, these organelles are generally quite stable and, thus, do not induce cell damage by leaking their numerous powerful hydrolytic enzymes to the cytosol. We suggest that this opinion is basically wrong and consider that many lysosomes are quite vulnerable, especially to oxidative stress. Moreover, we suggest that cellular degeneration, including apoptosis as well as necrosis, follows upon lysosomal disruption. We have found differing stability of lysosomal membranes to oxidative stress, not only among different cell types, but also between cells of the same type and between lysosomes of individual cells. We suggest that cellular resistance to oxidative stress is mainly a function of three parameters: (i) the capacity to degrade hydrogen peroxide before it reaches, and may diffuse into, the acidic vacuolar compartment; (ii) the resistance to reactive oxygen species of lysosomal membranes; and (iii) the intralysosomal amounts of redox-active, low molecular weight iron. Iron-catalysed intralysosomal reactions, if pronounced enough, result in peroxidation and destabilization of the lysosomal membrane. Owing to differences in the cellular synthesis of hydrogen peroxide-degrading enzymes, degree of autophagocytotic degradation of iron-containing metalloproteins, lysosomal localization within the cytoplasm and intralysosomal iron chelation, the above three parameters may vary between both different and similar cells and between lysosomes of individual cells as well, explaining their observed variability with respect to resistance against oxidative stress This revised version was published online in November 2006 with corrections to the Cover Date.     

Lysosomal heterogeneity between and within cells with respect to resistance against oxidative stress

The prevailing opinion on lysosomal endurance is that, as long as the cells are still alive, these organelles are generally quite stable and, thus, do not induce cell damage by leaking their numerous powerful hydrolytic enzymes to the cytosol. We suggest that this opinion is basically wrong and consider that many lysosomes are quite vulnerable, especially to oxidative stress. Moreover, we suggest that cellular degeneration, including apoptosis as well as necrosis, follows upon lysosomal disruption. We have found differing stability of lysosomal membranes to oxidative stress, not only among different cell types, but also between cells of the same type and between lysosomes of individual cells. We suggest that cellular resistance to oxidative stress is mainly a function of three parameters: (i) the capacity to degrade hydrogen peroxide before it reaches, and may diffuse into, the acidic vacuolar compartment; (ii) the resistance to reactive oxygen species of lysosomal membranes; and (iii) the intralysosomal amounts of redox-active, low molecular weight iron. Iron-catalysed intralysosomal reactions, if pronounced enough, result in peroxidation and destabilization of the lysosomal membrane. Owing to differences in the cellular synthesis of hydrogen peroxide-degrading enzymes, degree of autophagocytotic degradation of iron-containing metalloproteins, lysosomal localization within the cytoplasm and intralysosomal iron chelation, the above three parameters may vary between both different and similar cells and between lysosomes of individual cells as well, explaining their observed variability with respect to resistance against oxidative stress This revised version was published online in November 2006 with corrections to the Cover Date.     

Influence of oxidized low-density lipoproteins (LDL) on the viability of osteoblastic cells

Cardiovascular diseases have recently been noted as potential risk factors for osteoporosis development. Although it is poorly understood how these two pathologies are related, it is a known fact that oxidized low-density lipoproteins (OxLDL) constitute potential determinants for both of them. The current study investigated the metabolism of OxLDL by osteoblasts and its effect on osteoblastic viability. The results obtained show that OxLDL are internalized but not degraded by osteoblasts while they can selectively transfer their CE to these cells. It is also demonstrated that OxLDL induce proliferation at low concentrations but cell death at high concentrations. This reduction of osteoblast viability was associated with lysosomal membrane damage caused by OxLDL as demonstrated by acridine orange relocalization. Accordingly, chloroquine, an inhibitor of lysosomal activity, accentuated cell death induced by OxLDL. Finally, we demonstrate that osteoblasts have the capacity to oxidize LDL and thereby potentially increase the local concentration of OxLDL. Overall, the current study confirms the potential role of OxLDL in the development of osteoporosis given its influence on osteoblastic viability.     

Glucose-6-phosphate dehydrogenase deficiency: a new hypothesis for an old disease

Summary

We have previously shown insulinoma (HIT-T15 and RINm5F) cells in culture to be very sensitive, in comparison with a reference cell line (J-774), to the oxidative stress that is created when alloxan reacts extracellularly with reducing agents, forming superoxide and hydrogen peroxide. The toxic effects are prevented by catalase added to the medium, suggesting that alloxan does not need to be taken up in order to affect cells. Rather, alloxan seems to exert its action through extracellular formation of hydrogen peroxide that influences the stability of the cells' lysosomes following diffusion into them. To further analyse the mechanisms in operation, we studied the influence of induced autophagocytosis on the sensitivity to ensuing oxidative stress. Starvation for 60–120 min in PBS at 37°C markedly enhanced autophagocytosis and, in parallel, increased the cytotoxic effect and lysosomal vulnerability of ensuing exposure to hydrogen peroxide, while not significantly changing the antioxidative status or the energy balance. Autophagocytosis increased the size of the intralysosomal pool of reactive, low-molecular-weight, iron, probably by degradation of metallo-proteins, as shown by autometallography and HPLC demonstration of desferrioxamine-reactive intracellular iron. Moreover, exposure to the iron-chelator desferrioxamine before treatment with hydrogen peroxide prevented lysosomal destabilization and cellular death of both starved and control cells, further proving the importance of intralysosomal iron for the response to oxidative stress. We hypothesize that β-cells which, like insulinoma cells, have a weak antioxidative defence system under conditions of enhanced general autophagocytosis, or crinophagy, might become vulnerable to even low, or moderate, oxidative stress.     

Imidazoline drugs stabilize lysosomes and inhibit oxidative cytotoxicity in astrocytes

Oxidative stress is a primary pathogenesis in the brain, which is particularly vulnerable to oxidative stress. Maintenance of astrocyte functions under oxidative stress is essential to prevent neuronal injuries and to recover neuronal functions in various pathologic conditions. Imidazoline drugs have affinities for imidazoline receptors, which are highly distributed in the brain, and have been shown to be neuroprotective. This study presented the protective effects of several imidazoline drugs against oxidative cytotoxicity, in primary cultures of astrocytes. Imidazoline drugs, such as idazoxan, guanabenz, guanfacine, BU224, and RS-45041-190, showed protective effects against naphthazarin-induced oxidative cytotoxicity, as evidenced by LDH release and Hoechst 33342/propidium iodide staining. The imidazoline drugs stabilized lysosomes and inhibited naphthazarin-induced lysosomal destabilization, as evidenced by acridine orange relocation. Guanabenz inhibited, the leakage of lysosomal cathepsin D to cytosol, the decreased mitochondrial potential, and the release of mitochondrial cytochrome c, which were induced by naphthazarin. The lysosomal destabilization by oxidative stress and other apoptotic signals and subsequent cathepsin D leakage to the cytosol can induce apoptotic changes of mitochondria and eventually cell death. Therefore, lysosomal stabilization by imidazoline drugs may be ascribed to their protective effects against oxidative cytotoxicity.     

Oxidative stress, growth factor starvation and Fas activation may all cause apoptosis through lysosomal leak

Oxidative stress, growth factor starvation, and activation of the Fas/APO-1/CD95 receptor all induce apoptosis in a variety of cell-types, including the established human Jurkat T-cell line. Oxidative stress, in the form of exposure of the cells to a bolus dose of hydrogen peroxide, results in intralysosomal, iron-catalyzed oxidative reactions. This is accompanied by a time- and dose-dependent lysosomal destabilization--as evaluated by a decreased lysosomal uptake of the metachromatic fluorochrome, and weak base, acridine orange--in combination with leakage to the cytosol of lysosomal contents, including hydrolytic enzymes. Moderate lysosomal rupture is followed by apoptosis within initially intact plasma membranes, while necrosis and cell lysis are associated with a more complete lysosomal breach. Prior endocytosis of the potent iron-chelator desferrioxamine, resulting in binding of intralysosomal low molecular weight iron in a non-redox active form, largely prevents not only oxidative stress-induced lysosomal labilization, but apoptosis as well. When apoptosis is induced by the use of a monoclonal IgM anti-human Fas/APO-1/CD95 receptor antibody, the apoptotic process is again found to be accompanied by lysosomal leak. It is, however, not prevented by a preceding endocytosis of desferrioxamine and, consequently, could not be a function of intralysosomal iron-catalyzed oxidative reactions, but must be due to other mechanisms. Growth factor starvation of Jurkat cultures for a few days results in a high proportion of apoptotic cells, which contain lysosomes many of which have lost their proton gradient and appear to have released their contents. Overall, our results indicate that lysosomal leakage/rupture precedes apoptosis in Jurkat cells regardless of the initiating agent, but that such rupture may occur through multiple mechanisms. Lysosomal enzymes, leaking out of their normal vacuolar compartment, may then induce apoptosis, perhaps by proteolytic activation of the caspase-family of enzymes. Regardless of the precise mechanism, these observations suggest that partial rupture of the acidic vacuolar compartment may be one of the final pathways in apoptosis.     

Lysosomal origin of the ferric iron required for cell killing by hydrogen peroxide

The lysosomotropic amines methylamine (40 mM) and chloroquine (125 mM) prevented the killing of cultured hepatocytes by hydrogen peroxide generated in the medium by glucose oxidase. Maximum protection required several hours preincubation with either amine. Sensitivity of the hepatocytes to H2O2 was restored either by the addition of ferrous or ferric iron to the culture medium, or by incubating the cells for 4 hours in the absence of either amine prior to treatment with H2O2. Neither methylamine nor chloroquine had any effect on the cell killing by t-butyl hydroperoxide, a hepatotoxin that does not require iron. The protective effect of the lysosomotropic amines was distinguished from that of the ferric iron chelator deferoxamine in two ways: 1) deferoxamine protected hepatocytes from H2O2 toxicity but did not require a pretreatment period; and 2) in contrast to methylamine or chloroquine, deferoxamine had no effect on lysosomal pH as assessed by the fluorescent probe acridine orange. The data suggest that a lysosomal pool is the source of the ferric iron necessary for the killing of hepatocytes by H2O2.     

Cell sensitivity to oxidative stress is influenced by ferritin autophagy

To test the consequences of lysosomal degradation of differently iron-loaded ferritin molecules and to mimic ferritin autophagy under iron-overload and normal conditions, J774 cells were allowed to endocytose heavily iron loaded ferritin, probably with some adventitious iron (Fe-Ft), or iron-free apo-ferritin (apo-Ft). When cells subsequently were exposed to a bolus dose of hydrogen peroxide, apo-Ft prevented lysosomal membrane permeabilization (LMP), whereas Fe-Ft enhanced LMP. A 4-h pulse of Fe-Ft initially increased oxidative stress-mediated LMP that was reversed after another 3h under standard culture conditions, suggesting that lysosomal iron is rapidly exported from lysosomes, with resulting upregulation of apo-ferritin that supposedly is autophagocytosed, thereby preventing LMP by binding intralysosomal redox-active iron. The obtained data suggest that upregulation of the stress protein ferritin is a rapid adaptive mechanism that counteracts LMP and ensuing apoptosis during oxidative stress. In addition, prolonged iron starvation was found to induce apoptotic cell death that, interestingly, was preceded by LMP, suggesting that LMP is a more general phenomenon in apoptosis than so far recognized. The findings provide new insights into aging and neurodegenerative diseases that are associated with enhanced amounts of cellular iron and show that lysosomal iron loading sensitizes to oxidative stress.