Original contributionCold-induced apoptosis of hepatocytes: mitochondrial permeability transition triggered by nonmitochondrial chelatable iron
Introduction
Hypothermia is widely used as a protective principle during the transport and storage of organs and tissues. Although hypothermia has strong beneficial effects, it can also trigger cellular injury: in previous studies we and others have shown that hypothermia is a strong inducer of apoptosis in diverse cell types relevant for transplantation medicine, such as hepatocytes, liver endothelial cells, and renal proximal tubular cells 1, 2, 3, 4. This cold-induced apoptosis is triggered by an exposure to low temperatures (e.g., 4°C) for several hours and develops its morphological and biochemical features—such as membrane blebbing, nuclear condensation, chromatin condensation, and DNA fragmentation—as well as a major part of the final cell injury during rewarming of the cells to 37°C.
In hepatocytes, liver endothelial cells, and kidney cells, cold-induced apoptosis is mediated by reactive oxygen species (ROS; 1, 2, 3, 4, 5, 6, 7), or, more precisely, by an iron-dependent formation of reactive oxygen species 1, 2, 4, 5, 6, 7. For cultured hepatocytes we have previously shown that an alteration of the cellular chelatable iron pool plays a crucial role in the development of this apoptosis: hypothermia induces a rapid increase in the cellular chelatable iron pool that seems to cause oxidative injury in the absence of an increased release of the primary reactive oxygen species formed by cells, superoxide anion radicals (O2•−) and hydrogen peroxide (H2O2). This iron-dependent pathway constitutes the largely predominant pathway in cold-induced apoptosis of hepatocytes after cold incubation in cell culture medium 1, 5 and virtually the only pathway in cold-induced apoptosis occurring after cold incubation in the clinically used preservation solutions, University of Wisconsin (UW) solution [6] and histidine-tryptophan-ketoglutarate (HTK) solution [7].
In a recent study on cultured hepatocytes, in which the cellular chelatable iron pool was experimentally increased by incubation with the membrane-permeable iron complex Fe (III)/8-hydroxyquinoline, we could confirm this unusual mechanism of an ROS-mediated injury: an increase in the cellular chelatable iron pool was indeed sufficient to trigger apoptosis in cultured rat hepatocytes (manuscript submitted for publication). This apoptosis triggered by an experimentally induced increase in the cellular chelatable iron pool caused a mitochondrial permeability transition (MPT) that was decisively involved in the apoptotic pathway. Therefore, we here set out to study whether in cold-induced apoptosis the increase in the cellular chelatable iron pool also causes a mitochondrial permeability transition and, if so, whether this permeability transition is involved in the pathway leading to cold-induced apoptosis. Furthermore, we tried to assess the role of mitochondrial chelatable iron—i.e., of the pool with the highest concentration of chelatable iron in hepatocytes—in cold-induced apoptosis using mitochondrion-selective iron indicators and chelators.
Section snippets
Animals
Male Wistar rats (250–310 g) were obtained from the Zentrales Tierlaboratorium (Universitätsklinikum Essen). Animals were kept under standard conditions with free access to food and water. All animals received humane care in compliance with the institutional guidelines.
Chemicals
Leibovitz L-15 medium was obtained from Gibco (Eggenstein, Germany), deferoxamine mesylate (Desferal) from Novartis Pharma (Nuremberg, Germany) and 1,10-phenanthroline from Sigma-Aldrich (Taufkirchen, Germany). Propidium iodide,
Effects of hypothermia and rewarming on mitochondrial membrane potential and MPT induction
When hepatocytes were loaded with 500 nM of the cationic fluorophore tetramethylrhodamine methyl ester (TMRM) for 20 min, they showed a selectively mitochondrial staining with this mitochondrial, membrane potential-sensitive dye, and, in the presence of a maintenance dose of 100 nM TMRM [9], the signal remained stable. When loaded cells were cooled down to 4°C, an immediate, slight increase in fluorescence intensity, most likely due to physical effects [20], and some mitochondrial shortening
Discussion
The data presented here show that a mitochondrial permeability transition plays a crucial role in the signaling pathway of cold-induced apoptosis and that the occurrence of this MPT is iron-dependent. Furthermore, they suggest that cytosolic, not mitochondrial chelatable iron is involved in the induction of this MPT.
Mitochondria seem to be a prime target of cold-induced cell injury/apoptosis: the sequence hypothermia/rewarming led to a rapid loss of the mitochondrial membrane potential and an
Abbreviations
a.u.—arbitrary units
BHT—butylated hydroxytoluene
DMSO—dimethyl sulfoxide
2,2′-DPD—2,2′-dipyridyl
HBSS—Hanks′ balanced salt solution
HTK solution—Histidine-Tryptophan-Ketoglutarate solution
KH buffer—Krebs-Henseleit buffer
LDH—lactate dehydrogenase
MPT—mitochondrial permeability transition
NBT—nitroblue tetrazolium
PIH—pyridoxal isonicotinoyl hydrazone
RDA—rhodamine B-[(2,2′-bipyridin-4-yl)aminocarbonyl]benzyl ester
ROS—reactive oxygen species
RPA—rhodamine B-[(1,10-phenanthrolin-5-yl)amino-carbonyl]benzyl
Acknowledgements
This study was supported by the Deutsche Forschungsgemeinschaft (RA 960/1-1). We would like to thank Mrs. E. Hillen and Ms. B. Lammers for their excellent technical assistance.
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