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

doi:10.1016/S0891-5849(03)00109-6

Free Radical Biology and Medicine

Volume 34, Issue 10, 15 May 2003, Pages 1243-1252

https://doi.org/10.1016/S0891-5849(03)00109-6 Get rights and content

Abstract

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 (NH₄Cl). 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 NH₄Cl for 4 h dramatically decreased apoptotic death caused by subsequent exposure to H₂O₂, 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 NH₄Cl 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 H₂O₂ addition, was absent in cells preexposed to 10 mM NH₄Cl. 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.

Introduction

Lysosomes are responsible for the normal turnover of organelles and long-lived proteins by autophagocytotic degradation 1, 2, 3. The ongoing decomposition of iron-containing metalloproteins within these acidic organelles is accompanied by the release of redox-active iron which, upon export from the lysosome, may be a major intracellular source of “free” iron for the continued synthesis of new iron-containing proteins 4, 5, 6. This system of iron recycling may also be important in the turnover of ferritin, the intralysosomal digestion of which could permit release of metabolically useable iron. However, the details of this iron export system are still unclear 6, 7, 8, 9, 10, 11, 12, 13, 14.

These considerations raise the possibility that intralysosomal redox-active iron could represent a clear and present danger in the event that cells are exposed to oxidant stress. The resultant formation of hydroxyl radicals (HO^•) or, more likely, iron-centered radicals (ferryl or perferryl) could then damage and destabilize lysosomal membranes 4, 5, 6. The release into the cytosol of moderate amounts of lysosomal hydrolytic enzymes is known to lead to apoptosis secondary to activation of the caspase cascade, while necrosis will result if lysosomal breach is pronounced 14, 15, 16.

In order to assess the importance of this intralysosomal pool of redox-active iron in cellular oxidant sensitivity, we exposed macrophage-like J774 cells to the lysosomotropic base ammonia (NH₃) by adding ammonium chloride (NH₄Cl) to the medium. The entry of NH₃ into the acidic lysosomal compartment causes alkalinization (with pH increasing from ca. 4.5 to > 6), thereby preventing intralysosomal degradation of iron-containing metalloproteins by the specialized lysosomal proteases, which have pH optima of 4 to 5. We hypothesized that such treatment should prevent intralysosomal release of reactive iron, but allow the continued transport of preexisting iron into the cytosol. The diminution of redox-active lysosomal iron, we reasoned, should decrease cellular sensitivity to oxidant stress. Overall, our results support the idea that the majority of redox-active iron is normally located within the lysosomal compartment and that depletion of this pool does, indeed, provide powerful protection against oxidant-induced cell death.

Section snippets

Chemicals

Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were from GIBCO (Paisley, UK); hydrogen peroxide and 5,5′-dimethyl-1-pyrroline N-oxide (DMPO) from Sigma-Aldrich (Steinheim, Germany); acridine orange base (AO) from Gurr (Poole, UK); and NH₄Cl and silver-lactate were from Fluka AG (Buchs, Switzerland). Glutaraldehyde was from Bio-Rad (Cambridge, MA, USA), ammonium sulfide and hydroquinone from BDH Ltd (Poole, UK), and propidium iodide (PI) from

NH₄Cl, DFO, or iron complex treatments do not influence hydrogen peroxide degradation

Cells exposed to NH₄Cl (for 15 min or 4 h), to added iron or to DFO (in previously given concentrations) degraded H₂O₂ (starting concentration = 50 μM) at rates similar to that of control cells (t_1/2 ≈ 10 min) (n = 3; results not shown). This indicates that the antioxidant effects described below do not derive simply from accelerated H₂O₂ clearance.

Inhibition of lysosomal degradation decreases intralysosomal redox-active iron

The cytochemical sulfide-silver method is an extremely sensitive technique that can be used to demonstrate the presence of iron and several other

Discussion

A number of earlier reports support the likelihood that iron-driven oxidation reactions are an important mediator of oxidant-induced cell death. In support of the importance of iron in these reactions, the marked protective effects of the iron-chelator DFO are often cited. Interestingly, it appears that DFO localizes predominantly (or perhaps even exclusively) intralysosomally 4, 17, 18. If this is correct, insofar as iron-driven reactions are important in sensitizing cells to oxidant killing,

Abbreviations

AO—acridine orange
DFO—desferrioxamine
DMEM—Dulbecco’s modified Eagle’s medium
DMPO—5,5′-dimethyl-1-pyrroline N-oxide
EPR—electron paramagnetic resonance
FBS—fetal bovine serum
NH₄Cl—ammonium chloride
PBS—phosphate-buffered saline
PI—propidium iodide

Acknowledgements

We thank Dr. Robert Bjorklund for skillful technical assistance and Dr. Des Richardson for helpful discussions. Supported by the Swedish Medical Research Council and the Swedish Cancer society (grants no. 4481 and no. 4296 to U.T.B.), and by the ÖLL Research Foundation, the Lions Foundation, and the Research Funds of the Linköping University Hospital, Sweden (grants to H.L.P.). J.W.E. was the recipient of a Visiting Professorship from the Linköping University Hospital and of support from the

References (36)

K. Öllinger et al.
Cellular injury induced by oxidative stress is mediated through lysosomal damage
Free Radic. Biol. Med.
(1995)
U.T. Brunk et al.
Exposure of cells to non-lethal concentrations of hydrogen peroxide induces degeneration-repair mechanisms involving lysosomal destabilization
Free Radic. Biol. Med.
(1995)
A.M. Konijn et al.
The cellular labile iron pool and intracellular ferritin in K562 cells
Blood
(1999)
M. Tabuchi et al.
Human NRAMP2/DMT1, which mediates iron transport across endosomal membranes, is localized to late endosomes and lysosomes in HEp-2 cells
J. Biol. Chem.
(2000)
P.E. Starke et al.
Lysosomal origin of the ferric iron required for cell killing by hydrogen peroxide
Biochem. Biophys. Res. Commun.
(1985)
W. Li et al.
Induction of cell death by the lysosomotropic detergent MSDH
FEBS Lett
(2000)
J.B. Lloyd et al.
Evidence that desferrioxamine cannot enter cells by passive diffusion
Biochem. Pharmacol.
(1991)
P.C. Panus et al.
Detection of H₂O₂ release from vascular endothelial cells
Free Radic. Biol. Med.
(1993)
J.M. Zdolsek et al.
Visualization of iron in cultured macrophagesa cytochemical light and electron microscopic study using autometallography
Free Radic. Biol. Med.
(1993)
G.R. Buettner
Spin trappingESR parameters of spin adducts
Free Radic. Biol. Med.
(1987)

H.A. Franch et al.

A mechanism regulating proteolysis of specific proteins during renal tubular cell growth

J. Biol. Chem.

(2001)

C. Brander et al.

Inhibition of human NK cell-mediated cytotoxicity by exposure to ammonium chloride

J. Immunol. Methods

(2001)

S. Nyholm et al.

Role of ribonucleotide reductase in inhibition of mammalian cell growth by potent iron chelators

J. Biol. Chem.

(1993)

K.L. Schalinske et al.

Phosphorylation and activation of both iron regulatory proteins 1 and 2 in HL-60 cells

J. Biol. Chem.

(1996)

B. Halliwell et al.

Role of free radicals and catalytic metal ions in human diseasean overview

Methods Enzymol

(1990)

W. Breuer et al.

Iron acquired from transferrin by K562 cells is delivered into a cytoplasmic pool of chelatable iron(II)

J. Biol. Chem.

(1995)

S. Epsztejn et al.

Fluorescence analysis of the labile iron pool of mammalian cells

Anal. Biochem.

(1997)

W. Breuer et al.

Newly delivered transferrin iron and oxidative injury

FEBS Lett

(1997)

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Original contributionIntralysosomal iron: a major determinant of oxidant-induced cell death

Abstract

Introduction

Section snippets

Chemicals

NH4Cl, DFO, or iron complex treatments do not influence hydrogen peroxide degradation

Inhibition of lysosomal degradation decreases intralysosomal redox-active iron

Discussion

Abbreviations

Acknowledgements

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Blood

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

FEBS Lett

Biochem. Pharmacol.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

J. Biol. Chem.

J. Immunol. Methods

J. Biol. Chem.

J. Biol. Chem.

Methods Enzymol

J. Biol. Chem.

Anal. Biochem.

FEBS Lett

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

NH₄Cl, DFO, or iron complex treatments do not influence hydrogen peroxide degradation