ARPE-19 retinal pigment epitheliaL CELLS are highly resistant to oxidative stress and exercise strict control over their lysosomal redox-active iron

Normal retinal pigment epithelial (RPE) cells are postmitotic, long-lived and basically not replaced. Daily, they phagocytose substantial amounts of lipid-rich material (photoreceptor outer segment discs), and they do so in the most oxygenated part of the body – the retina. One would imagine that this state of affairs should be associated with a rapid formation of the age pigment lipofuscin (LF). However, LF accumulation is slow and reaches significant amounts only late in life when, if substantial, it often coincides with or causes age-related macular degeneration. LF formation occurs inside the lysosomal compartment as a result of iron-catalyzed peroxidation and polymerization. This process requires phagocytosed or autophagocytosed material under degradation, but also the presence of redox-active low mass iron and hydrogen peroxide. To gain some information on how RPE cells are able to evade LF formation, we investigated the response of immortalized human RPE cells (ARPE-19) to oxidative stress with/without the protection of a strong iron-chelator. The cells were found to be extremely resistant to hydrogen peroxide-induced lysosomal rupture and ensuing cell death. This marked resistance to oxidative stress was not explained by enhanced degradation of hydrogen peroxide, but to a certain extent further increased by the potent lipophilic iron chelator SIH. The cells were also able to survive, and even replicate, at high concentrations of SIH and showed a high degree of basal autophagic flux. We hypothesize that RPE cells have a highly developed capacity to keep lysosomal iron in a non-redox-active form, perhaps by pronounced autophagy of iron-binding proteins in combination with an ability to rapidly relocate low mass iron from the lysosomal compartment.     

Endosomal and lysosomal effects of desferrioxamine: protection of HeLa cells from hydrogen peroxide-induced DNA damage and induction of cell-cycle arrest

The role of endosomal/lysosomal redox-active iron in H2O2-induced nuclear DNA damage as well as in cell proliferation was examined using the iron chelator desferrioxamine (DFO). Transient transfections of HeLa cells with vectors encoding dominant proteins involved in the regulation of various routes of endocytosis (dynamin and Rab5) were used to show that DFO (a potent and rather specific iron chelator) enters cells by fluid-phase endocytosis and exerts its effects by chelating redox-active iron present in the endosomal/lysosomal compartment. Endocytosed DFO effectively protected cells against H2O2-induced DNA damage, indicating the importance of endosomal/lysosomal redox-active iron in these processes. Moreover, exposure of cells to DFO in a range of concentrations (0.1 to 100 microM) inhibited cell proliferation in a fluid-phase endocytosis-dependent manner. Flow cytometric analysis of cells exposed to 100 microM DFO for 24 h showed that the cell cycle was transiently interrupted at the G2/M phase, while treatment for 48 h led to permanent cell arrest. Collectively, the above results clearly indicate that DFO has to be endocytosed by the fluid-phase pathway to protect cells against H2O2-induced DNA damage. Moreover, chelation of iron in the endosomal/lysosomal cell compartment leads to cell cycle interruption, indicating that all cellular labile iron is propagated through this compartment before its anabolic use is possible.     

OxLDL-induced macrophage cytotoxicity is mediated by lysosomal rupture and modified by intralysosomal redox-active iron

Oxidized low density lipoprotein (oxLDL) is believed to play a central role in atherogenesis. LDL is oxidized in the arterial intima by mechanisms that are still only partially understood. OxLDL is then taken up by macrophages through scavenger receptor-mediated endocytosis, which then leads to cellular damage, including apoptosis. The complex mechanisms by which oxLDL induces cell injury are mostly unknown. This study has demonstrated that oxLDL-induced damage of macrophages is associated with iron-mediated intralysosomal oxidative reactions, which cause partial lysosomal rupture and ensuing apoptosis. This series of events can be prevented by pre-exposing cells to the iron-chelator, desferrioxamine (DFO), whereas it is augmented by pretreating the cells with a low molecular weight iron complex. Since both DFO and the iron complex would be taken up by endocytosis, and thus directed to the lysosomal compartment, the results suggest that the normal contents of lysosomal low molecular weight iron may play an important role in oxLDL-induced cell damage, presumably by catalyzing intralysosomal fragmentation of lipid peroxides and the formation of toxic aldehydes and oxygen-centered radicals.     

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.     

The role of lysosomes in iron metabolism and recycling

Iron is the most abundant transition metal in the earth's crust. It cycles easily between ferric (oxidized; Fe(III)) and ferrous (reduced; Fe(II)) and readily forms complexes with oxygen, making this metal a central player in respiration and related redox processes. However, 'loose' iron, not within heme or iron-sulfur cluster proteins, can be destructively redox-active, causing damage to almost all cellular components, killing both cells and organisms. This may explain why iron is so carefully handled by aerobic organisms. Iron uptake from the environment is carefully limited and carried out by specialized iron transport mechanisms. One reason that iron uptake is tightly controlled is that most organisms and cells cannot efficiently excrete excess iron. When even small amounts of intracellular free iron occur, most of it is safely stored in a non-redox-active form in ferritins. Within nucleated cells, iron is constantly being recycled from aged iron-rich organelles such as mitochondria and used for construction of new organelles. Much of this recycling occurs within the lysosome, an acidic digestive organelle. Because of this, most lysosomes contain relatively large amounts of redox-active iron and are therefore unusually susceptible to oxidant-mediated destabilization or rupture. In many cell types, iron transit through the lysosomal compartment can be remarkably brisk. However, conditions adversely affecting lysosomal iron handling (or oxidant stress) can contribute to a variety of acute and chronic diseases. These considerations make normal and abnormal lysosomal handling of iron central to the understanding and, perhaps, therapy of a wide range of diseases.     

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.     

OxLDL-induced macrophage cytotoxicity is mediated by lysosomal rupture and modified by intralysosomal redox-active iron

Oxidized low density lipoprotein (oxLDL) is believed to play a central role in atherogenesis. LDL is oxidized in the arterial intima by mechanisms that are still only partially understood. OxLDL is then taken up by macrophages through scavenger receptor-mediated endocytosis, which then leads to cellular damage, including apoptosis. The complex mechanisms by which oxLDL induces cell injury are mostly unknown. This study has demonstrated that oxLDL-induced damage of macrophages is associated with iron-mediated intralysosomal oxidative reactions, which cause partial lysosomal rupture and ensuing apoptosis. This series of events can be prevented by pre-exposing cells to the iron-chelator, desferrioxamine (DFO), whereas it is augmented by pretreating the cells with a low molecular weight iron complex. Since both DFO and the iron complex would be taken up by endocytosis, and thus directed to the lysosomal compartment, the results suggest that the normal contents of lysosomal low molecular weight iron may play an important role in oxLDL-induced cell damage, presumably by catalyzing intralysosomal fragmentation of lipid peroxides and the formation of toxic aldehydes and oxygen-centered radicals.     

The radioprotective agent, amifostine, suppresses the reactivity of intralysosomal iron

Amifostine (2-[(3-aminopropyl)amino]ethane-thiol dihydrogen phosphate ester; WR-2721) is a radioprotective agent used clinically to minimize damage from radiation therapy to adjacent normal tissues. This inorganic thiophosphate requires dephosphorylation to produce the active, cell-permeant thiol metabolite, WR-1065. The activation step is presumably catalyzed by membrane-bound alkaline phosphatase, activity of which is substantially higher in the endothelium of normal tissues. This site-specific delivery may explain the preferential protection of normal versus neoplastic tissues. Although it was developed several decades ago, the mechanisms through which this agent exerts its protective effects remain unknown. Because WR-1065 is a weak base (pKa = 9.2), we hypothesized that the drug should preferentially accumulate (via proton trapping) within the acidic environment of intracellular lysosomes. These organelles contain abundant 'loose' iron and represent a likely initial target for oxidant- and radiation-mediated damage. We further hypothesized that, within the lysosomal compartment, the thiol groups of WR-1065 would interact with this iron, thereby minimizing iron-catalyzed lysosomal damage and ensuing cell death. A similar mechanism of protection via intralysosomal iron chelation has been invoked for the hexadentate iron chelator, desferrioxamine (DFO; although DFO enters the lysosomal compartment by endocytosis, not proton trapping). Using cultured J774 cells as a model system, we found substantial accumulation of WR-1065 within intracellular granules as revealed by reaction with the thiol-binding fluorochrome, BODIPY FL L-cystine. These granules are lysosomes as indicated by co-localization of BODIPY staining with LysoTracker Red. Compared to 1 mM DFO, cells pre-treated with 0.4 microM WR-1065 are protected from hydrogen peroxide-mediated lysosomal rupture and ensuing cell death. On a molar basis in this experimental system, WR-1065 is approximately 2500 times more effective than DFO in preventing oxidant-induced lysosomal rupture and cell death. This increased effectiveness is most likely due to the preferential concentration of this weak base within the acidic lysosomal apparatus. By electron spin resonance, we found that the generation of hydroxyl radical, which normally occurs following addition of hydrogen peroxide to J774 cells, is totally blocked by pretreatment with either WR-1065 or DFO. These findings suggest a single and plausible explanation for the radioprotective effects of amifostine and may provide a basis for the design of even more effective radio- and chemoprotective drugs.     

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.     

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.     

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     

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.     

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.     

Does the calcein-AM method assay the total cellular 'labile iron pool' or only a fraction of it?

The calcein-AM (calcein-acetoxymethyl ester) method is a widely used technique that is supposed to assay the intracellular 'labile iron pool' (LIP). When cells in culture are exposed to this ester, it passes the plasma membrane and reacts with cytosolic unspecific esterases. One of the reaction products, calcein, is a fluorochrome and a hydrophilic alcohol to which membranes are non-permeable and which, consequently, is retained within the cytosol of cells. Calcein fluorescence is quenched following chelation of low-mass labile iron, and the degree of quenching gives an estimate of the amounts of chelatable iron. However, a requirement for the assay to be able to demonstrate cellular LIP in total is that such iron be localized in the cytosol and not in a membrane-limited compartment. For some time it has been known that a major part of cellular, redox-active, labile, low-mass iron is temporarily localized in the lysosomal compartment as a result of the autophagic degradation of ferruginous materials, such as mitochondrial complexes and ferritin. Even if some calcein-AM may escape cytosolic esterases and enter lysosomes to be cleaved by lysosomal acidic esterases, the resulting calcein does not significantly chelate iron at 相似文献   

The radioprotective agent,amifostine, suppresses the reactivity of intralysosomal iron

Abstract

Amifostine (2-[(3-aminopropyl)amino]ethane-thiol dihydrogen phosphate ester; WR-2721) is a radioprotective agent used clinically to minimize damage from radiation therapy to adjacent normal tissues. This inorganic thiophosphate requires dephosphorylation to produce the active, cell-permeant thiol metabolite, WR-1065. The activation step is presumably catalyzed by membrane-bound alkaline phosphatase, activity of which is substantially higher in the endothelium of normal tissues. This site-specific delivery may explain the preferential protection of normal versus neoplastic tissues. Although it was developed several decades ago, the mechanisms through which this agent exerts its protective effects remain unknown. Because WR-1065 is a weak base (pKa = 9.2), we hypothesized that the drug should preferentially accumulate (via proton trapping) within the acidic environment of intracellular lysosomes. These organelles contain abundant 'loose' iron and represent a likely initial target for oxidant- and radiation-mediated damage. We further hypothesized that, within the lysosomal compartment, the thiol groups of WR-1065 would interact with this iron, thereby minimizing iron-catalyzed lysosomal damage and ensuing cell death. A similar mechanism of protection via intralysosomal iron chelation has been invoked for the hexadentate iron chelator, desferrioxamine (DFO; although DFO enters the lysosomal compartment by endocytosis, not proton trapping). Using cultured J774 cells as a model system, we found substantial accumulation of WR-1065 within intracellular granules as revealed by reaction with the thiol-binding fluorochrome, BODIPY FL L-cystine. These granules are lysosomes as indicated by co-localization of BODIPY staining with LysoTracker Red. Compared to 1 mM DFO, cells pre-treated with 0.4 μM WR-1065 are protected from hydrogen peroxide-mediated lysosomal rupture and ensuing cell death. On a molar basis in this experimental system, WR-1065 is approximately 2500 times more effective than DFO in preventing oxidant-induced lysosomal rupture and cell death. This increased effectiveness is most likely due to the preferential concentration of this weak base within the acidic lysosomal apparatus. By electron spin resonance, we found that the generation of hydroxyl radical, which normally occurs following addition of hydrogen peroxide to J774 cells, is totally blocked by pretreatment with either WR-1065 or DFO. These findings suggest a single and plausible explanation for the radioprotective effects of amifostine and may provide a basis for the design of even more effective radio- and chemoprotective drugs.     

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.     

-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.     

Lysosomal membrane permeabilization causes oxidative stress and ferritin induction in macrophages

Moderate lysosomal membrane permeabilization (LMP) is an important inducer of apoptosis. Macrophages are professional scavengers and are rich in hydrolytic enzymes and iron. In the present study, we found that LMP by lysosomotropic detergent MSDH resulted in early up-regulation of lysosomal cathepsins, oxidative stress and ferritin up-regulation, and cell death. Lysosomotropic base NH₄Cl reduced the ferritin induction and oxidative stress in apoptotic cells induced by MSDH. Cysteine cathepsin inhibitors significantly protected cell death and oxidative stress, but had less effect on ferritin induction. We conclude that oxidative stress induced by lysosomal rupture causes ferritin induction with concomitant mitochondrial damage, which are the potential target for prevention of cellular oxidative stress and cell death induced by typical lysosomotropic substances in different disorders.     

Radiation-induced lysosomal iron reactivity: implications for radioprotective therapy

A novel mechanism of radiosensitization involves radiation-enhanced autophagy of damaged mitochondria and various metalloproteins, by which iron accumulates within lysosomes. Hydrogen peroxide, formed by the radiolytic cleavage of water, generates in the presence of lysosomal redox-active iron extremely reactive hydroxyl radicals by Fenton-type chemistry. Subsequent peroxidative damage of lysosomal membranes initiates release of harmful content from ruptured lysosomes that triggers a cascade of events eventuating in DNA damage and apoptotic or necrotic cell death. This article reviews the role of lysosomal destabilization in radiation-induced cell damage and death. The potential effects of iron chelation therapy targeted to the lysosomes for protection of normal tissues against unwanted effects by radiation is also discussed.     

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.