Biomedical subjects

Ursula Rauen

Publications and source records attributed to Ursula Rauen.

At least 19 recordsLinked to original sources

PubMed2007

Assessment of chelatable mitochondrial iron by using mitochondrion-selective fluorescent iron indicators with different iron-binding affinities.

Chelatable cellular iron, and chelatable mitochondrial iron in particular, has yet to be well characterized, so the overall strength with which these "loosely bound" iron ions (presumably mainly Fe(II)) are intracellularly/intramitochondrially bound is unclear. We have previously reported the first selective mitochondrial iron indicator: rhodamine B 4-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester (RPA). With this compound as a model, we have now developed two additional mitochondrial iron indicators with very different iron-binding affinities and have applied these to the study of the chelatable iron pool in the mitochondria of isolated rat liver cells. With the new indicator rhodamine B 4-[(2,2'-bipyridin-4-yl)aminocarbonyl]benzyl ester (RDA), with 2,2'-bipyridine as chelating unit (log beta(3)=17.5), essentially the same iron concentration (16.0+/-1.9 microM) was determined as with RPA (log beta(3)=21.1), despite the four orders of magnitude difference in Fe(II)-binding affinity. This not only demonstrates the reliability of the procedure, but also confirms that iron complexation by these indicators does not induce any significant release of iron from the iron-storage proteins on the timescale of the experiment. In contrast, the indicator rhodamine B 4-[bis(pyridin-2-ylmethyl)aminomethyl]benzyl ester (PIRO), with an N,N-bis(pyridin-2-ylmethyl)amine group as chelating component (log beta(2)=12.2), could not compete against the array of endogenous ligands. The intramitochondrial concentrations of the three indicators were determined to be in the range of 100 microM: that is, about three orders of magnitude lower than the total concentration of endogenous compounds that might chelate iron ions. It is therefore estimated that chelatable mitochondrial iron ions are bound by endogenous ligands with apparent stability constants (log K(app)) of between 9 and 14.

Animals↗

PubMed2006

Nitric oxide increases toxicity of hydrogen peroxide against rat liver endothelial cells and hepatocytes by inhibition of hydrogen peroxide degradation.

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) show cooperativity in their cytotoxic action. The present study was performed to decipher the mechanisms underlying this phenomenon. In cultured liver endothelial cells and in cultured, glutathione-depleted hepatocytes, the combined exposure to NO (released by spermine NONOate, 1 mM) and H(2)O(2) (released by glucose oxidase) induced cell injury that was far higher than the injury elicited by NO or H(2)O(2) alone. In both cell types, the addition of the NO donor increased H(2)O(2) steady-state levels, although with different kinetics: in hepatocytes, the increase in H(2)O(2) levels was already evident at early time points while in liver endothelial cells it became evident after > or =2 h of incubation. NO exposure inhibited H(2)O(2) degradation, assessed after addition of 50 microM, 200 microM, or 4 mM authentic H(2)O(2), significantly in both cell types. However, again, early and delayed inhibition was observed. The late inhibition of H(2)O(2) degradation in endothelial cells was paralleled by a decrease in glutathione peroxidase activity. Glutathione peroxidase inactivation was prevented by hypoxia or by ascorbate, suggesting inactivation by reactive nitrogen oxide species (NO(x)). Early inhibition of H(2)O(2) degradation by NO, in contrast, could be mimicked by the catalase inhibitor azide. Together, these results suggest that the cooperative effect of NO and H(2)O(2) is due to inhibition of H(2)O(2) degradation by NO, namely to inhibition of catalase by NO itself (predominant in hepatocytes) and/or to inhibition of glutathione peroxidase by NO(x) (prevailing in endothelial cells).

Animals↗

PubMed2006

Sodium as the major mediator of NO-induced cell death in cultured hepatocytes.

NO has been shown to induce cellular injury via inhibition of the mitochondrial respiratory chain and/or oxidative/nitrosative stress. Here, we studied which mechanism and downstream mediator is responsible for NO toxicity to hepatocytes. When cultured rat hepatocytes were incubated with spermineNONOate (0.01-2 mM) at 2, 5, 21 and 95% O(2) in Krebs-Henseleit buffer (37 degrees C), spermineNONOate caused concentration-dependent hepatocyte death (lactate dehydrogenase release, propidium iodide uptake) with morphological features of both apoptosis and necrosis. Increasing O(2) concentrations protected hepatocytes from NO-induced injury. Steady-state NO concentrations were lower at higher O(2) concentrations, suggesting formation of reactive nitrogen oxide species. Despite this, the scavenger ascorbic acid was hardly protective. In contrast, at equal NO concentrations loss of viability was higher at lower O(2) concentrations and inhibitors of hypoxic injury, fructose and glycine (10 mM), strongly decreased NO-induced injury. Upon addition of spermineNONOate, the cytosolic Na(+) concentration rapidly increased. The increase in sodium depended on the NO/O(2) ratio and was paralleled by hepatocyte death. Sodium-free Krebs-Henseleit buffer strongly protected from NO-induced injury. SpermineNONOate also increased cytosolic calcium levels but the Ca(2+) chelator quin-2-AM did not diminish cell injury. These results show that - in analogy to hypoxic injury - a sodium influx largely mediates the NO-induced death of cultured hepatocytes. Oxidative stress and disturbances in calcium homeostasis appear to be of minor importance for NO toxicity to hepatocytes.

Animals↗

PubMed2006

Cold-induced injury to porcine corneal endothelial cells and its mediation by chelatable iron: implications for corneal preservation.

PURPOSE: During hypothermic storage of the cornea, corneal endothelial damage restricts storage times. We previously reported a new, iron-dependent mechanism of cold-induced injury to cultured liver cells. In this study, we sought to evaluate whether corneal endothelial cells incur a similar kind of injury. METHODS: Cultured porcine corneal endothelial cells were exposed to 4 degrees C in either cell culture medium, Krebs-Henseleit buffer, Optisol-GS solution, or McCarey-Kaufman medium for 5 hours to 14 days and then rewarmed under cell culture conditions (3 hours). The cultures were assessed for lethal cell injury (LDH release); cellular, nuclear, and mitochondrial morphologic alterations; lipid peroxidation; and mitochondrial membrane potential. RESULTS: Corneal endothelial cells sustained substantial injury following cold storage and rewarming in cell culture medium (47% +/- 8% and 64% +/- 20% cell death after 2 and 5 days cold storage, respectively). The injury displayed some apoptotic features, and cells lost mitochondrial membrane potential before cell death occurred. The iron chelators deferoxamine, 1,10-phenanthroline, and 2,2'-dipyridyl and the antioxidant butylated hydroxytoluene completely inhibited this cell injury. Marked iron-dependent cell injury and lipid peroxidation also occurred during and after cold incubation in Krebs-Henseleit buffer and, most importantly, iron-dependent cell injury was also observed after cold incubation in Optisol solution and in McCarey-Kaufman medium. CONCLUSIONS: Cultured porcine corneal endothelial cells incur a strong iron-dependent injury elicited by hypothermia. This cold-induced injury might provide an explanation for the known corneal endothelial susceptibility to hypothermic preservation injury, which thus might be amenable to therapeutic interventions (ie, by iron chelators).

2,2'-Dipyridyl↗

PubMed2005

ROS-mediated genotoxicity of asbestos-cement in mammalian lung cells in vitro.

Asbestos is a known carcinogen and co-carcinogen. It is a persisting risk in our daily life due to its use in building material as asbestos-cement powder. The present study done on V79-cells (Chinese hamster lung cells) demonstrates the cytotoxic and genotoxic potential of asbestos-cement powder (ACP) in comparison with chrysotile asbestos. A co-exposure of chrysotile and ACP was tested using the cell viability test and the micronucleus assay. The kinetochore analysis had been used to analyse the pathway causing such genotoxic effects. Thiobarbituric acid-reactive substances were determined as evidence for the production of reactive oxygen species. Both, asbestos cement as well as chrysotile formed micronuclei and induced loss of cell viability in a concentration- and time-dependent way. Results of TBARS analysis and iron chelator experiments showed induction of free radicals in ACP- and chrysotile exposed cultures. CaSO4 appeared to be a negligible entity in enhancing the toxic potential of ACP. The co-exposure of both, ACP and chrysotile, showed an additive effect in enhancing the toxicity. The overall study suggests that asbestos-cement is cytotoxic as well as genotoxic in vitro. In comparison to chrysotile the magnitude of the toxicity was less, but co-exposure increased the toxicity of both.

Journal Article↗

PubMed2004

Friedreich's ataxia, no changes in mitochondrial labile iron in human lymphoblasts and fibroblasts: a decrease in antioxidative capacity?

Friedreich's ataxia (FRDA) is caused by low expression of frataxin, a small mitochondrial protein. Studies with both yeast and mammals have suggested that decreased frataxin levels lead to elevated intramitochondrial concentrations of labile (chelatable) iron, and consequently to oxidative mitochondrial damage. Here, we used the mitochondrion-selective fluorescent iron indicator/chelator rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzylester (RPA) to determine the mitochondrial chelatable iron of FRDA patient lymphoblast and fibroblast cell lines, in comparison with age- and sex-matched control cells. No alteration in the concentration of mitochondrial chelatable iron could be observed in patient cells, despite strongly decreased frataxin levels. Uptake studies with (55)Fe-transferrin and iron loading with ferric ammonium citrate revealed no significant differences in transferrin receptor density and iron responsive protein/iron regulatory element binding activity between patients and controls. However, sensitivity to H(2)O(2) was significantly increased in patient cells, and H(2)O(2) toxicity could be completely inhibited by the ubiquitously distributing iron chelator 2,2'-dipyridyl, but not by the mitochondrion-selective chelator RPA. Our data strongly suggest that frataxin deficiency does not affect the mitochondrial labile iron pool or other parameters of cellular iron metabolism and suggest a decreased antioxidative defense against extramitochondrial iron-derived radicals in patient cells. These results challenge current concepts favoring the use of mitochondrion-specific iron chelators and antioxidants to treat FRDA.

Adolescent↗

PubMed2004

Protection against iron- and hydrogen peroxide-dependent cell injuries by a novel synthetic iron catalase mimic and its precursor, the iron-free ligand.

Hydrogen peroxide is involved in many types of cell injury and exerts most of its injurious effects in conjunction with chelatable iron. We previously described a synthetic nonporphyrin iron-containing catalase mimic, TAA-1/Fe. Its ligand TAA-1 was designed for application in biological systems in which it is supposed to fulfill a dual task: it should chelate cellular labile iron and thus form the active catalase mimic, thereby decreasing levels of redox-active iron and enhancing the degradation of hydrogen peroxide. Here, we tested these novel compounds in cellular systems, i.e., in cultured hepatocytes and liver endothelial cells. Both the iron complex, i.e., the complete mimic, and the ligand, i.e., the putative precursor of this mimic, provided protection against endothelial cell injury induced by exogenous hydrogen peroxide. Furthermore, the ligand--but not (or less so) the complex--strongly protected both cell types against iron-dependent hypothermic injury and hepatocytes against iron-induced cell injury and against iron-dependent, histidine-induced injury. Together, these results demonstrate that the putative catalase mimic precursor TAA-1 is able to protect cells against iron- and/or hydrogen peroxide-dependent cell injuries and that--in line with our initial concept--it is likely to exert its protection by both iron chelation and hydrogen peroxide degradation.

Animals↗

PubMed2004

Iron-induced mitochondrial permeability transition in cultured hepatocytes.

BACKGROUND/AIMS: We previously described that the cold-induced apoptosis of cultured hepatocytes and liver endothelial cells is mediated by an increase in the cellular chelatable iron pool-in the absence of any increase in O(2)(.-)/H(2)O(2) formation. As this is an unusual mechanism, we here set out to assess whether an increase in cellular chelatable iron per se is sufficient to trigger cell injury/apoptosis. METHODS: Cultured rat hepatocytes were acutely loaded with iron using the membrane-permeable complex Fe(III)/8-hydroxyquinoline and incubated under otherwise 'physiological' conditions. RESULTS: Incubation with Fe(III)/8-hydroxyquinoline (15 microM/30 microM) increased the cellular chelatable iron and induced strong hepatocellular injury with morphological features of apoptosis, but also of necrosis. The iron-induced cell injury was oxygen-dependent, and although it was not inhibitable by extracellular catalase, it was strongly inhibited by the novel membrane-permeable catalase mimic TAA-1/Fe. The experimentally induced increase in cellular chelatable iron triggered a mitochondrial permeability transition (MPT) as assessed using double-staining with calcein and tetramethylrhodamine methyl ester. The MPT inhibitor cyclosporine A partially and the well-known inhibitor combination trifluoperazine+fructose completely inhibited the iron-induced cell injury/apoptosis. CONCLUSIONS: These results show that iron per se can induce cell injury/apoptosis and that this injury is mediated via an MPT.

Animals↗

PubMed2004

New insights into the cellular and molecular mechanisms of cold storage injury.

Solid organ grafts, but also other biologic materials requiring storage for a few hours to a few days, are usually stored under hypothermic conditions. To decrease graft injury during cold storage, organ preservation solutions were developed many years ago. However, since then, modern biochemical and cell biologic methods have allowed further insights into the molecular and cellular mechanisms of cold storage injury, including further insights into alterations of the cellular ion homeostasis, the occurrence of a mitochondrial permeability transition, and the occurrence of free-radical-mediated hypothermic injury and cold-induced apoptosis. These new aspects of cold storage injury, which are not covered by preservation solutions in current clinical use and offer the potential for improvement of organ and tissue preservation, are presented here.

Animals↗

PubMed2003

Cold-induced apoptosis of hepatocytes: mitochondrial permeability transition triggered by nonmitochondrial chelatable iron.

We previously described that the cold-induced apoptosis of cultured hepatocytes is mediated by an increase in the cellular chelatable iron pool. We here set out to assess whether a mitochondrial permeability transition (MPT) is involved in cold-induced apoptosis. When cultured hepatocytes were rewarmed after 18 h of cold (4 degrees C) incubation in cell culture medium or University of Wisconsin solution, the vast majority of cells rapidly lost mitochondrial membrane potential. This loss was due to MPT as assessed by confocal laser scanning microscopy and as evidenced by the inhibitory effect of the MPT inhibitors trifluoperazine plus fructose. The occurrence of the MPT was iron-dependent: it was strongly inhibited by the iron chelators 2,2'-dipyridyl and deferoxamine. Addition of trifluoperazine plus fructose also strongly inhibited cold-induced apoptosis, suggesting that the MPT constitutes a decisive intermediate event in the pathway leading to cold-induced apoptosis. Further experiments employing the non-site-specific iron indicator Phen Green SK and specifically mitochondrial iron indicators and chelators (rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester, RPA, and rhodamine B-[(2,2'-bipyridin-4-yl)aminocarbonyl]benzyl ester, RDA) suggest that it is the cold-induced increase in cytosolic chelatable iron that triggers the MPT and that mitochondrial chelatable iron is not involved in this process.

Animals↗

PubMed2003

Cold-induced apoptosis of rat liver endothelial cells: contribution of mitochondrial alterations.

BACKGROUND: Maintenance of the integrity of the vascular endothelium is a critical issue in liver preservation, but hypothermia, applied for cellular protection, induces apoptotic cell death in liver endothelial cells. This cold-induced apoptosis is mediated by an iron-dependent formation of reactive oxygen species. Here, we study the involvement of mitochondria in this process. METHODS: Cultured rat liver endothelial cells were incubated in cold University of Wisconsin solution for 18 hr and subsequently rewarmed in cell culture medium. Mitochondrial morphology and membrane potential were evaluated using laser scanning microscopy. RESULTS: During cold incubation in University of Wisconsin solution, a marked, progressive mitochondrial shortening and a reduction in mitochondrial membrane potential occurred. Rewarming of the cells led to mitochondrial ultracondensation, complete loss of the mitochondrial membrane potential, and subsequent apoptotic cell death. The inhibitors of mitochondrial permeability transition, trifluoperazine and fructose, or iron chelation with deferoxamine did not affect mitochondrial shortening during cold incubation but inhibited ultracondensation, loss of mitochondrial membrane potential, and loss of viability during rewarming. Moreover, in these protected cells, an almost complete reestablishment of the mitochondrial membrane potential and morphology could be observed; the few mitochondria that were irreversibly damaged were incorporated into autophagosomes during cellular recovery. CONCLUSION: Two apparently independent mitochondrial alterations take place during cold incubation and subsequent rewarming of liver endothelial cells. Cold-induced mitochondrial shortening represents a reversible process, whereas iron-mediated mitochondrial permeability transition and ultracondensation during rewarming are irreversible and constitute an important mediator of cold-induced apoptosis.

Animals↗

PubMed2003

Stress kinase phosphorylation is increased in pacing-induced heart failure in rabbits.

In hearts with chronic left ventricular (LV) systolic dysfunction secondary to hypertension or myocardial infarction, MAPK phosphorylation and/or activity are increased. Whether other settings of LV dysfunction not associated with ischemia-reperfusion are also characterized by increased MAPK phosphorylation or activity is unknown. After 3 wk of rapid LV pacing (400 beats/min), eight rabbits displayed clinical signs of heart failure (HF), and echocardiography revealed an increase in LV end-diastolic diameter from 15.6 +/- 0.7 (means +/- SE) to 18.8 +/- 0.7 mm and a reduced shortening fraction from 31 +/- 1to10 +/- 2% (both P < 0.05). Morphological alterations in HF included increased numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cardiomyocytes, extent of fibrosis, and cross-sectional cardiomyocyte area. Total p38 MAPK did not differ between failing and normal hearts (n = 8). However, p38 MAPK phosphorylation [164,488 +/- 29,323 vs. 43,565 +/- 14,817 arbitrary units (AU), P < 0.05, densitometry] and the activities of p38 MAPK-alpha and -beta were increased in failing compared with normal hearts (149,441 +/- 38,381 and 170,430 +/- 32,952 vs. 68,815 +/- 28,984 and 81,788 +/- 22,774 AU, respectively, both P < 0.05). In failing compared with normal hearts, total and phosphorylated JNK46 and JNK54 MAPK were increased, whereas total and phosphorylated ERK MAPK remained unchanged. In pacing-induced HF, p38 and JNK MAPK phosphorylation as well as p38 MAPK activity was increased. Further studies will have to define whether or not chronic specific blockade of MAPK activity can interfere with apoptosis/fibrosis and thereby attenuate the progression of HF.

Animals↗

PubMed2003

Cold-induced apoptosis of rat liver endothelial cells: involvement of the proteasome.

BACKGROUND: We have previously shown that in cultured liver endothelial cells the mere sequence of hypothermia and rewarming induces a pronounced, iron-dependent apoptosis that is likely to contribute to hepatic preservation injury. Here we study the involvement of proteases in this cold-induced apoptosis. METHODS: Cultured liver endothelial cells were incubated in preservation solutions at 4 degrees C in the absence or presence of protease inhibitors. Cell injury and different protease activities were assessed. RESULTS: Cold incubation of liver endothelial cells in University of Wisconsin or histidine-tryptophan-ketoglutarate (HTK) solution led to marked cell injury, which was strongly inhibited by the protease inhibitor 3,4-dichloroisocoumarin (DCI) (lactate dehydrogenase release: 86.0+/-2.6% in HTK and 4.0+/-0.4% in HTK + DCI after 24 hr of cold incubation). Determination of protease activity showed a doubling in the activity of a Suc-Leu-Leu-Val-Tyr-AMC-cleaving protease and some increase in a relatively low Suc-Ala-Ala-Ala-AMC-cleaving activity after 8 hr of cold incubation in HTK solution or 16 hr in University of Wisconsin solution. Further characterization of the protease activities showed that they belonged to two different proteases, with the major activity being calcium independent, inhibited by DCI, MG-132, and lactacystin, and strongly decreased by immunoprecipitation with an antiproteasome antibody. Preincubation of the cells with the iron chelator deferoxamine prevented the cold-induced increase of both activities. CONCLUSION: These results show that (i) the proteasome and possibly, in addition, a serine protease are involved in cold-induced apoptosis of liver endothelial cells and (ii) the protease activation is downstream from the increase in intracellular chelatable iron.

Adenosine Triphosphatases↗

PubMed2003

Hypothermia causes a marked injury to rat proximal tubular cells that is aggravated by all currently used preservation solutions.

Cold preservation results in cell death via iron-dependent formation of reactive oxygen species, leading to apoptosis during rewarming. We aimed to study cold-induced damage (i.e., injury as a consequence of hypothermia itself and not cold ischemia) in proximal tubular cells (PTC) in various preservation solutions presently applied and to clarify the role of mitochondria in this injury. Primary cultures of rat PTC were incubated at 4 degrees C for 24 h in culture medium, UW, Euro-Collins or HTK solution with and without the iron chelator desferal and rewarmed at 37 degrees C in culture medium. Cell damage, morphology, and apoptosis were studied and mitochondrial membrane potential was assessed by fluorescence microscopy. Cold incubation of PTC in culture medium followed by rewarming caused marked cell damage compared to warm incubation alone (LDH release 39+/-10% vs. 1.6+/-0.3%). Cold-induced damage was aggravated in all preservation solutions (LDH release 85+/-2% for UW; similar in Euro-Collins and HTK). After rewarming, cells showed features suggestive for apoptosis. Desferal prevented cell injury in all solutions (e.g., 8+/-2% for UW). Mitochondrial membrane potential was lost during rewarming and this loss could also be inhibited by desferal. Trifluoperazine, which is known to inhibit mitochondrial permeability transition (MPT), was able to prevent cold-induced injury (LDH 85+/-5% vs. 12+/-2%). We conclude that cold-induced injury occurs in PTC and is aggravated by UW, Euro-Collins, and HTK solution. Iron-dependent MPT is suggested to play a role in this damage. Strategies to prevent cold-induced injury should aim at reducing the availability of "free" iron.

Adenosine↗

PubMed2003

Effect of quercetin on hypoxic injury in freshly isolated rat proximal tubules.

The bioflavonoid quercetin, which has antioxidant properties, protects renal tubular epithelial cells from oxidant-induced injury by inhibiting lipid peroxidation. We examined the effect of quercetin on hypoxia-induced injury in freshly isolated rat renal proximal tubules. Hypoxia induced rapid loss of cellular ATP, followed by functional and structural alterations measured as a decrease in tubular potassium content and sequentially by an increase in lactate dehydrogenase release. Furthermore, hypoxia increased lipid peroxidation, measured as thiobarbituric acid-reactive substances. Quercetin significantly inhibited hypoxia-induced functional and structural tubular injury in addition to lipid peroxidation but did not alter hypoxia-induced ATP depletion. These results demonstrate the potency of the bioflavonoid quercetin in protecting proximal tubules from hypoxic injury, which is independent of tubular energy metabolism and may be related to the inhibition of lipid peroxidation.

Acute Kidney Injury↗

PubMed2002

Enhancement of iron toxicity in L929 cells by D-glucose: accelerated(re-)reduction.

It has recently been shown that an increase in the cellular chelatable iron pool is sufficient to cause cell damage. To further characterize this kind of injury, we artificially enhanced the chelatable iron pool in L929 mouse fibroblasts using the highly membrane-permeable complex Fe(III)/8-hydroxyquinoline. This iron complex induced a significant oxygen-dependent loss of viability during an incubation period of 5 h. Surprisingly, the addition of L-glucose strongly enhanced this toxicity whereas no such effect was exerted by L-glucose and 2-deoxyglucose. The assumption that this increase in toxicity might be due to an enhanced availability of reducing equivalents formed during the metabolism of L-glucose was supported by NAD(P)H measurements which showed a 1.5-2-fold increase in the cellular NAD(P)H content upon addition of L-glucose. To assess the influence of this enhanced cellular reducing capacity on iron valence we established a new method to measure the reduction rate of iron based on the fluorescent iron(II) indicator PhenGreen SK. We could show that the rate of intracellular iron reduction was more than doubled in the presence of L-glucose. A similar acceleration was achieved by adding the reducing agents ascorbate and glutathione (the latter as membrane-permeable ethyl ester). Glutathione ethyl ester, as well as the thiol reagent N -acetylcysteine, also caused a toxicity increase comparable with L-glucose. These results suggest an enhancement of iron toxicity by L-glucose via an accelerated (re-)reduction of iron with NAD(P)H serving as central electron provider and ascorbate, glutathione or possibly NAD(P)H itself as final reducing agent.

Animals↗

PubMed2002

Selective determination of mitochondrial chelatable iron in viable cells with a new fluorescent sensor.

Mitochondrial chelatable ("redox-active") iron is considered to contribute to several human diseases, but has not yet been characterized in viable cells. In order to determine this iron pool, we synthesized a new fluorescent indicator, rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester (RPA). In a cell-free system, RPA fluorescence was strongly and stoichiometrically quenched by Fe(2+). RPA selectively accumulated in the mitochondria of cultured rat hepatocytes. The intramitochondrial RPA fluorescence was quenched when iron was added to the cells in a membrane-permeant form. It increased when the mitochondrial chelatable iron available to the probe was experimentally decreased by the membrane-permeant transition metal chelators pyridoxal isonicotinoyl hydrazone and 1,10-phenanthroline. The concentration of mitochondrial chelatable iron in cultured rat hepatocytes, quantified from the increase in RPA fluorescence after addition of pyridoxal isonicotinoyl hydrazone, was found to be 12.2 +/- 4.9 microM. Inhibition of haem synthesis with succinylacetone did not alter the signal obtained in hepatocytes, but a rapid increase in the concentration of mitochondrial chelatable iron was observed in human erythroleukaemia K562 cells. In conclusion, RPA enables the selective determination of the highly physiologically and pathophysiologically interesting mitochondrial pool of chelatable iron in intact cells and to record the time course of alterations of this pool.

Animals↗

PubMed2002

Cold-induced apoptosis of rat liver cells in University of Wisconsin solution: the central role of chelatable iron.

Although University of Wisconsin (UW) solution aims at the prevention of cold-induced cell injury, it failed to protect against cold-induced apoptosis of hepatocytes and liver endothelial cells: when incubated in UW solution at 4 degrees C for 24 hours and subsequently rewarmed at 37 degrees C, 72% +/- 8% of rat hepatocytes and 81% +/- 5% of liver endothelial cells lost viability. In both cell types, the observed cell damage occurred under an apoptotic morphology; it appeared to be mediated by a rapid increase in the cellular chelatable iron pool by a factor > or =2 (as determined in hepatocytes) and subsequent formation of reactive oxygen species (ROS). Consequently, this cell injury was decreased by iron chelators to 6 to 25% (hepatocytes) and 4% +/- 2% (liver endothelial cells). Deferoxamine nearly completely inhibited the occurrence of apoptotic morphology in both cell types. In liver endothelial cells, cold-induced apoptosis occurring during rewarming after 24 hours of cold incubation in UW solution was far more pronounced than in cell culture medium (loss of viability: 81% +/- 5% vs. 28% +/- 13%), but viability could even be maintained for 2 weeks of cold incubation by use of deferoxamine. In conclusion, this pathological mechanism might be an explanation for the strong endothelial cell injury known to occur after cold preservation. With regard to the extent of this iron-mediated injury, addition of a suitable iron chelator to UW solution might markedly improve the outcome of liver preservation.

Adenosine↗