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.     

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.     

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.     

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.     

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.     

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.     

The lysosomal-mitochondrial axis theory of postmitotic aging and cell death

Aging (senescence) is characterized by a progressive accumulation of macromolecular damage, supposedly due to a continuous minor oxidative stress associated with mitochondrial respiration. Aging mainly affects long-lived postmitotic cells, such as neurons and cardiac myocytes, which neither divide and dilute damaged structures, nor are replaced by newly differentiated cells. Because of inherent imperfect lysosomal degradation (autophagy) and other self-repair mechanisms, damaged structures (biological "garbage") progressively accumulate within such cells, both extra- and intralysosomally. Defective mitochondria and aggregated proteins are the most typical forms of extralysosomal "garbage", while lipofuscin that forms due to iron-catalyzed oxidation of autophagocytosed or heterophagocytosed material, represents intralysosomal "garbage". Based on findings that autophagy is diminished in lipofuscin-loaded cells and that cellular lipofuscin content positively correlates with oxidative stress and mitochondrial damage, we have proposed the mitochondrial-lysosomal axis theory of aging, according to which mitochondrial turnover progressively declines with age, resulting in decreased ATP production and increased oxidative damage. Due to autophagy of ferruginous material, lysosomes contain a pool of redox-active iron, which makes these organelles particularly susceptible to oxidative damage. Oxidant-mediated destabilization of lysosomal membranes releases hydrolytic enzymes to the cytosol, eventuating in cell death (either apoptotic or necrotic depending on the magnitude of the insult), while chelation of the intralysosomal pool of redox-active iron prevents these effects. In relation to the onset of oxidant-induced apoptosis, but after the initiating lysosomal rupture, cytochrome c is released from mitochondria and caspases are activated. Mitochondrial damage follows the release of lysosomal hydrolases, which may act either directly or indirectly, through activation of phospholipases or pro-apoptotic proteins such as Bid. Additional lysosomal rupture seems to be a consequence of a transient oxidative stress of mitochondrial origin that follows the attack by lysosomal hydrolases and/or phospholipases, creating an amplifying loop system.     

Novel cellular defenses against iron and oxidation: ferritin and autophagocytosis preserve lysosomal stability in airway epithelium

Adsorbed to a variety of particles, iron may be carried to the lungs by inhalation thereby contributing to a number of inflammatory lung disorders. Redox-active iron is a potent catalyst of oxidative processes, but intracellularly it is bound primarily to ferritin in a non-reactive form and probably is catalytically active largely within the lysosomal compartment. Damage to the membranes of these organelles causes the release to the cytosol of a host of powerful hydrolytic enzymes, inducing apoptotic or necrotic cell death. The results of this study, using cultured BEAS-2B cells, which are adenovirus transformed human bronchial epithelial cells, and A549 cells, which have characteristics similar to type II alveolar epithelial cells, suggest that the varying abilities of different types of lung cells to resist oxidative stress may be due to differences in intralysosomal iron chelation. Cellular ferritin and iron were assayed by ELISA and atomic absorption, while plasma and lysosomal membrane stability were evaluated by the acridine orange uptake and trypan blue dye exclusion tests, respectively. Normally, and also after exposure to an iron complex, A549 cells contained significantly more ferritin (2.26 +/- 0.60 versus 0.63 +/- 0.33 ng/microg protein, P <0.001) and less iron (0.96 +/- 0.14 versus 1.48 +/- 0.21 ng/microg protein, P <0.05) than did BEAS-2B cells. Probably as a consequence, iron-exposed A549 cells displayed more stable lysosomes (P <0.05) and better survival (P <0.05) following oxidative stress. Following starvation-induced autophagocytosis, which also enhances resistance to oxidant stress, the A549 cells showed a significant reduction in ferritin, and the BEAS-2B cells did not. These results suggest that intralysosomal ferritin enhances lysosomal stability by iron-chelation, preventing Fenton-type chemistry. This notion was further supported by the finding that endocytosis of apoferritin, added to the medium, stabilized lysosomes (P <0.001 versus P <0.01) and increased survival (P <0.01 versus P <0.05) of iron-loaded A549 and BEAS-2B cells. Assuming that primary cell lines of the alveolar and bronchial epithelium behave in a similar manner as these respiratory cell lines, intrabronchial instillation of apoferritin-containing liposomes may in the future be a treatment for iron-dependent airway inflammatory processes.     

-Lipoic acid and -lipoamide prevent oxidant-induced lysosomal rupture and apoptosis

Abstract

-Lipoic acid (LA) and its corresponding derivative, -lipoamide (LM), have been described as antioxidants, but the mechanisms of their putative antioxidant effects remain largely uncharacterised. The vicinal thiols present in the reduced forms of these compounds suggest that they might possess metal chelating properties. We have shown previously that cell death caused by oxidants may be initiated by lysosomal rupture and that this latter event may involve intralysosomal iron which catalyzes Fenton-type chemistry and resultant peroxidative damage to lysosomal membranes. Here, using cultured J774 cells as a model, we show that both LA and LM stabilize lysosomes against oxidative stress, probably by chelating intralysosomal iron and, consequently, preventing intralysosomal Fenton reactions. In preventing oxidant-mediated apoptosis, LM is significantly more effective than LA, as would be expected from their differing capacities to enter cells and concentrate within the acidic lysosomal compartment. As previously reported, the powerful iron-chelator, desferrioxamine (Des) (which also locates within the lysosomal compartment), also provides protection against oxidant-mediated cell death. Interestingly, although Des enhances the partial protection afforded by LA, it confers no additional protection when added with LM. Therefore, the antioxidant actions of LA and LM may arise from intralysosomal iron chelation, with LM being more effective in this regard.     

Intralysosomal iron: a major determinant of oxidant-induced cell death

As a result of continuous digestion of iron-containing metalloproteins, the lysosomes within normal cells contain a pool of labile, redox-active, low-molecular-weight iron, which may make these organelles particularly susceptible to oxidative damage. Oxidant-mediated destabilization of lysosomal membranes with release of hydrolytic enzymes into the cell cytoplasm can lead to a cascade of events eventuating in cell death (either apoptotic or necrotic depending on the magnitude of the insult). To assess the importance of the intralysosomal pool of redox-active iron, we have temporarily blocked lysosomal digestion by exposing cells to the lysosomotropic alkalinizing agent, ammonium chloride (NH(4)Cl). The consequent increase in lysosomal pH (from ca. 4.5 to > 6) inhibits intralysosomal proteolysis and, hence, the continuous flow of reactive iron into this pool. Preincubation of J774 cells with 10 mM NH(4)Cl for 4 h dramatically decreased apoptotic death caused by subsequent exposure to H(2)O(2), and the protection was as great as that afforded by the powerful iron chelator, desferrioxamine (which probably localizes predominantly in the lysosomal compartment). Sulfide-silver cytochemical detection of iron revealed a pronounced decrease in lysosomal content of redox-active iron after NH(4)Cl exposure, probably due to diminished intralysosomal digestion of iron-containing material coupled with continuing iron export from this organelle. Electron paramagnetic resonance experiments revealed that hydroxyl radical formation, readily detectable in control cells following H(2)O(2) addition, was absent in cells preexposed to 10 mM NH(4)Cl. Thus, the major pool of redox-active, low-molecular-weight iron may be located within the lysosomes. In a number of clinical situations, pharmacologic strategies that minimize the amount or reactivity of intralysosomal iron should be effective in preventing oxidant-induced cell death.     

Lipofuscin: mechanisms of age-related accumulation and influence on cell function

The accumulation of lipofuscin within postmitotic cells is a recognized hallmark of aging occurring with a rate inversely related to longevity. Lipofuscin is an intralysosomal, polymeric substance, primarily composed of cross-linked protein residues, formed due to iron-catalyzed oxidative processes. Because it is undegradable and cannot be removed via exocytosis, lipofuscin accumulation in postmitotic cells is inevitable, whereas proliferative cells efficiently dilute it during division. The rate of lipofuscin formation can be experimentally manipulated. In cell culture models, oxidative stress (e.g., exposure to 40% ambient oxygen or low molecular weight iron) promotes lipofuscin accumulation, whereas growth at 8% oxygen and treatment with antioxidants or iron-chelators diminish it. Lipofuscin is a fluorochrome and may sensitize lysosomes to visible light, a process potentially important for the pathogenesis of age-related macular degeneration. Lipofuscin-associated iron sensitizes lysosomes to oxidative stress, jeopardizing lysosomal stability and causing apoptosis due to release of lysosomal contents. Lipofuscin accumulation may also diminish autophagocytotic capacity by acting as a sink for newly produced lysosomal enzymes and, therefore, interfere with recycling of cellular components. Lipofuscin, thus, may be much more directly related to cellular degeneration at old age than was hitherto believed.     

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.     

Autophagy, ageing and apoptosis: the role of oxidative stress and lysosomal iron

As an outcome of normal autophagic degradation of ferruginous materials, such as ferritin and mitochondrial metalloproteins, the lysosomal compartment is rich in labile iron and, therefore, sensitive to the mild oxidative stress that cells naturally experience because of their constant production of hydrogen peroxide. Diffusion of hydrogen peroxide into the lysosomes results in Fenton-type reactions with the formation of hydroxyl radicals and ensuing peroxidation of lysosomal contents with formation of lipofuscin that amasses in long-lived postmitotic cells. Lipofuscin is a non-degradable polymeric substance that forms at a rate that is inversely related to the average lifespan across species and is built up of aldehyde-linked protein residues. The normal accumulation of lipofuscin in lysosomes seems to reduce autophagic capacity of senescent postmitotic cells--probably because lipofuscin-loaded lysosomes continue to receive newly formed lysosomal enzymes, which results in lack of such enzymes for autophagy. The result is an insufficient and declining rate of autophagic turnover of worn-out and damaged cellular components that consequently accumulate in a way that upsets normal metabolism. In the event of a more substantial oxidative stress, enhanced formation of hydroxyl radicals within lysosomes jeopardizes the membrane stability of particularly iron-rich lysosomes, specifically of autophagolysosomes that have recently participated in the degradation of iron-rich materials. For some time, the rupture of a limited number of lysosomes has been recognized as an early upstream event in many cases of apoptosis, particularly oxidative stress-induced apoptosis, while necrosis results from a major lysosomal break. Consequently, the regulation of the lysosomal content of redox-active iron seems to be essential for the survival of cells both in the short- and the long-term.     

Endogenous ferritin protects cells with iron-laden lysosomes against oxidative stress

Previous studies have shown that a variety of mammalian cell types, including macrophages, contain small amounts of redox-active iron in their lysosomes. Increases in the level of this iron pool predispose the cell to oxidative stress. Limiting the availability of intralysosomal redox-active iron could therefore represent potential cytoprotection for cells under oxidative stress.

In the present study we have shown that an initial 6 h exposure of J774 macrophages to 30 μM iron, added to the culture medium as FeCl₃, increased the lysosomal iron content and their sensitivity to H₂O₂-induced (0.25 mM for 30 min) oxidative stress. Over time (24-72 h), however, the cells were desensitized to the cytotoxic effects of H₂O₂; most likely as a consequence of both lysosomal iron exocytosis and of ferritin synthesis (demonstrated by atomic absorption spectrophotometry, autometallography, and immunohistochemistry). When the cells were exposed to a second dose of iron, their lysosomal content of iron increased again but the cells became no further sensitized to the cytotoxic effects of H₂O₂. Using the lysosomotropic weak base, acridine orange, we demonstrated that after the second exposure to iron and H₂O₂, lysosomes remained intact and were no different from control cells which were exposed to H₂O₂ but not iron.

These data suggest that the initial induction of ferritin synthesis leads to enrichment of lysosomes with ferritin via autophagocytosis. This limits the redox-availability of intralysosomal iron and, in turn, decreases the cells' sensitivity to oxidative stress. These in vitro observations could also explain why cells under pathological conditions, such as haemochromatosis, are apparently able to withstand high iron concentrations for some time in vivo.     

On the Cytoprotective Role of Ferritin in Macrophages and its Ability to Enhance Lysosomal Stability

Macrophages have a great capacity to take up (eg. by endocytosis and phagocytosis) exogenous sources of iron which could potentially become cytotoxic, particularly following the intralysosomal formation of low-molecular weight, redox active iron, and under conditions of oxidative stress. Following autophago-cytosis of endogenous ferritin/apoferritin, these compounds may serve as chelators of such lysosomal iron and counteract the occurrence of iron-mediated intralysosomal oxidative reactions. Such redox-reactions have been shown to lead to destabilisation of lysosomal membranes and result in leakage of damaging lysosomal contents to the cytosol. In this study we have shown: (i) human monocyte-derived macrophages to accumulate ferritin in response to iron exposure; (ii) iron to destabilise macrophage secondary lysosomes when the cells are exposed to H₂O₂; and (iii) endocytosed apoferritin to act as a stabiliser of the acidic vacuolar compartment of iron-loaded macrophages. While the endogenous ferritin accumulation which was induced by iron exposure was not sufficient to protect cells from the damaging effects of H₂O₂, exogenously added apoferritin, as well as the potent iron chelator desferrioxamine, afforded significant protection. It is suggested that intralysosomal formation of haemosiderin, from partially degraded ferritin, is a protective strategy to suppress intralysosomal iron-catalysed redox reactions. However, under conditions of severe macrophage lysosomal iron-overload, induction of ferritin synthesis is not enough to completely prevent the enhanced cytotoxic effects of H₂O₂.     

Oxidative stress induces macroautophagy of amyloid β-protein and ensuing apoptosis

There is increasing evidence for the toxicity of intracellular amyloid β-protein (Aβ) to neurons and the involvement of lysosomes in this process in Alzheimer disease (AD). We have recently shown that oxidative stress, a recognized determinant of AD, enhances macroautophagy and leads to intralysosomal accumulation of Aβ in cultured neuroblastoma cells. We hypothesized that oxidative stress promotes AD by stimulating macroautophagy of Aβ that further may induce cell death by destabilizing lysosomal membranes. To investigate such possibility, we compared the effects of hyperoxia (40% ambient oxygen) in cultured HEK293 cells that were transfected with an empty vector (Vector), wild-type APP (APPwt), or Swedish mutant APP (APPswe). Exposure to hyperoxia for 5 days increased the number of cells with Aβ-containing lysosomes, as well as the number of apoptotic cells, compared to normoxic conditions. The rate of apoptosis in all three cell lines demonstrated dependence on intralysosomal Aβ content (Vector < APPwt < APPswe). Furthermore, the degree of apoptosis was positively correlated with lysosomal membrane permeabilization, whereas inhibitors of macroautophagy and lysosomal function decreased oxidant-induced apoptosis and diminished the differences in apoptotic response between different cell lines. These results suggest that oxidative stress can induce neuronal death through macroautophagy of Aβ and consequent lysosomal membrane permeabilization, which may help explain the mechanisms behind neuronal loss in AD.     

Lethal hydrogen peroxide toxicity involves lysosomal iron-catalyzed reactions with membrane damage

Secondary lysosomes contain low-molecular weight iron-complexes as a consequence of normal autophagocytotic degradation of various metallo-proteins. Thus, entry of hydrogen peroxide into these organelles may induce ironcatalyzed oxidative reactions with ensuing damage to lysosomal membranes and leakage of destructive contents. The amount of lysosomal reactive iron and the cellular capacity to degrade hydrogen peroxide would then be important determining factors in cellular resistance to oxidative stress. The effects of hydrogen peroxide on cell viability and, in particular, on lysosomal membrane integrity, evaluated by acridine orange, lucifer yellow, neutral red, and cathepsin D relocalization, were investigated in a model system of cultured J-774 cells. The protective effect of the iron-chelator desferal was studied after exposure to the drug under ordinary culture conditions and after inhibition of cellular endocytosis. Hydrogen peroxide-exposure (500 μM in PBS, 37°C, 5–90 min) was manifested as a time-dependent decrease in cell viability. This was preceded by a rapid reduction of the proton gradient across the lysosomal membranes, as judged by relocalization of acridine orange. Another early sign of damage was plasma membrane blebbing, found on many cells within minutes after the initiation of hydrogen peroxide-exposure. The cells also showed a partial redistribution of the lysosomal markers lucifer yellow, neutral red, and cathepsin D, indicating lysosomal destabilization. The pre-exposure of cells to desferal in culture prevented all these phenomena, unless endocytotic uptake of the drug was prevented.     

Lysosomal labilization

The lysosomal compartment is the place for cellular degradation of endocytosed and autophagocytosed material and a center for normal turnover of organelles as well as most long-lived proteins. Lysosomes were long considered stable structures that broke and released their many hydrolytic enzymes only following necrotic cell death. It is now realized that lysosomes instead are quite vulnerable, although in a heterogeneous way. Their exposure to a number of events, such as oxidative stress, lysosomotropic detergents and aldhydes, as well as overexpression of the p53 protein, causes time-and-dose-dependent lysosomal rupture that is followed by apoptosis or necrosis. Partial lysosomal rupture has often been found to be an early upstream event in apoptosis, while necrosis results from fulminant lysosomal rupture. Consequently, factors influencing the stability of lysosomes, for instance their content of labile and redox-active iron, seem to be essential for the survival of cells.     

Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells

Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other iron-binding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX.