The labile iron pool in hepatocytes: prooxidant-induced increase in free iron precedes oxidative cell injury

Anna Stäubli and Urs A. Boelsterli

Institute of Toxicology, Swiss Federal Institute of Technology and University of Zurich, CH-8603 Schwerzenbach, Switzerland

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The labile iron pool (LIP) represents the nonferritin-bound, redox-active iron that has been implicated in oxidative stress and cell injury. Here we examined whether alterations in LIP can be detected in cultured murine hepatocytes and whether increases in LIP are related to the oxidative damage inflicted by the redox cycling drug nitrofurantoin (NFT). Early changes in LIP were monitored with the metal-sensitive fluorescent probe calcein (CA), the fluorescence of which is quenched on binding to iron. Short-term exposure (<1 h) to NFT reduced the CA fluorescence signal by 30%, indicating that the amount of LIP-associated iron had increased. Prolonged exposure (>= 2 h) to NFT caused oxidative cell injury. The addition of the cell-permeable ferrous iron chelator 2,2'-bipyridyl not only prevented the quenching of CA fluorescence but also partially protected from NFT toxicity. It is concluded that reductive stress-induced increase in LIP is an essential event that precedes oxidative cell damage in intact hepatocytes.

cultured murine hepatocytes; nitrofurantoin; calcein; reductive stress; iron chelators

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

IRON-DEPENDENT PROCESSES play a pivotal role in the development of prooxidant-induced cell injury. Specifically, the generation of hydroxyl radicals from hydrogen peroxide and the formation of aldehydes and lipid peroxy radicals from lipid hydroperoxides are catalyzed by redox-active metals, including iron (20, 30, 35). The bulk of the intracellular iron is protein bound and therefore not available for these redox reactions. However, there is evidence for the existence of a cytosolic "free" (i.e., non-protein bound) labile iron pool (LIP), distinct from ferritin, which is catalytically active and participates in those reactions involved in the production of injurious oxygen species (34, 36). The LIP represents that portion of the intracellular iron that is chelatable by deferoxamine (DFO) and other commonly used chelators (2, 6, 7). LIP-associated iron is in a dynamic equilibrium with other sequestered iron forms in the cells (10) and is bound to cytosolic low-affinity ligands that have not yet been identified.

A widely used method to indirectly quantify the LIP involves the measurement of bleomycin/iron-dependent DNA degradation or the direct assay of oxidized 2-deoxyribose in homogenized tissue samples (19, 36). More recently, the LIP has been quantitated by several other techniques, including the use of DFO-chelatable iron-59 (34) and electron paramagnetic resonance spectroscopy (26). It is, however, difficult to measure free iron concentration without disrupting the cells. As a consequence of tissue homogenization, the existing equilibrium between free and bound iron, as well as its oxidation state, may be altered (16). With the advent of the use of calcein (CA) as an iron-sensitive fluorescent probe, it has become possible to quantitate the basal content, as well as to assess relative changes, of LIP-associated iron in intact cells. With this method, a series of elegant studies has characterized the dynamics of the LIP in human cell lines (6-8, 10, 12, 44). As yet this technique has not been applied to primary cultures of hepatocytes, although hepatic parenchymal cells play an important role both in the uptake and storage of iron (1, 3), as well as in the metabolic activation of prooxidant chemicals.

In hepatocytes, LIP-associated iron plays a pivotal role in catalyzing reactive oxygen species-induced cell injury provoked both by prooxidant chemicals (13, 23, 25) and posthypoxic reoxygenation (11, 24). Therefore, alterations in LIP-associated iron concentrations could have important repercussions on the extent of hepatocellular damage. In addition, these changes could indirectly be involved in the regulation of gene expression, because a number of oxygen radicals play a major role in the activation of transcription factors and subsequent induction of other regulatory proteins (28, 38). Transient increases in LIP are not only caused by excessive iron uptake from extracellular sources but can also be driven by prooxidant-mediated intracellular reductive stress, which results in reductive iron release from intracellular stores, including ferritin (15, 33, 35, 40, 41). Because most of the data on intracellular iron release were generated in cell-free systems, it has proven difficult to establish a causal link between reductive iron release precipitated by a prooxidant chemical and the ensuing cellular consequences, including oxidative cell injury.

The objective of this study was to adapt the CA technique for cultured hepatocytes and to monitor in situ the possible alterations of LIP-associated iron in intact cells. In particular, we investigated the mechanistic role of increases in LIP in the early pathogenesis of cell injury induced by the prooxidant drug nitrofurantoin (NFT).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals. 2,2'-Bipyridyl (BIP) was obtained from Fluka (Buchs, Switzerland). NFT, probenecid, DFO mesylate, dexamethasone, BSA, insulin, and Williams' medium E (WME) without phenol red were obtained from Sigma Chemical (St. Louis, MO). Cyclosporin A was from Novartis (Basel, Switzerland), and CA-acetoxymethyl ester (CA-AM) was from Molecular Probes Europe (Leiden, The Netherlands). FCS and penicillin/streptomycin were obtained from BioConcept (Basel, Switzerland), and collagenase (type A) from Clostridium histolyticum was from Boehringer (Mannheim, Germany).

Animals. All experiments were approved by the institutional supervisory board and performed according to federal regulations. Male C57BL/6J/Zur mice (8-10 wk of age), obtained from Biological Research Laboratories (Füllinsdorf, Switzerland), were kept in Macrolone cages with wood shavings as bedding at 24 ± 2°C and 55 ± 5% relative humidity in a 12:12-h light-dark cycle (7 AM to 7 PM light) with free access to water and Nafag 857 mouse pellets (Gossau, Switzerland).

Isolation and culture of hepatocytes. Fed mice were anesthetized with pentobarbital sodium, and hepatocytes were isolated by a two-step liver perfusion method (4), as described previously for rats (17), except that the perfusion was run in a retrograde manner. Briefly, after an incision of the exposed right ventricle, a small gavage needle attached to the tubing was inserted via the ventricle into the vena cava caudalis. After ligation of the vena cava caudalis distal to the vena iliaca communis, the portal vein was cut and the two-step liver perfusion started. Perfusate flow was adjusted to 7 ml/min. After mechanical disruption of the liver capsule, the liver cells were collected in WME and serially filtered (30-, 50-, and 80-mesh) through an 85-ml Cellector (Bellco Biotechnology, Vineland, NJ) tissue sieve. Typically, 10 to 25 × 106 cells were obtained from one mouse liver. Viability, assessed by trypan blue exclusion after two sedimentation steps (15 min at 4°C), ranged between 80% and 95%. The cells were seeded in 120 µg/ml collagen (type IV; Pentapharm, Basel, Switzerland)-coated six-well Primaria dishes (Becton-Dickinson, Oxnard, CA) at a density of 8.3 × 104 viable cells/cm2 in 2 ml/well WME, which was supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (0.1 mg/ml), insulin (100 nM), and dexamethasone (100 nM). After an attachment period of 3 h at 37°C in 5% CO2-95% air, the medium was replaced by serum-free WME supplemented with the antibiotics and hormones. For all experiments, cells were precultured overnight before use.

Determination of LIP in hepatocytes. The intracellular LIP was measured with a fluorimetric assay using the metal-sensitive probe CA (7). In cell-free systems, CA binds to both iron(II) and iron(III) with stability constants of 1014 and 1024 M-1, respectively, which results in quenching the fluorescence (8). In the cytosol, however, CA-chelatable LIP is primarily composed of iron(II) (8, 9). Because CA is insensitive to calcium and magnesium ions up to 1 mM at physiological pH and the intracellular concentration of other CA-binding metals is very low, it is generally accepted that the decrease in CA fluorescence represents iron binding (7, 8). We have modified the assay, originally developed for human erythroleukemia K-562 cells in suspension (7), for murine hepatocytes cultured in six-well plates. Hepatocytes, precultured in WME for 18-20 h, were loaded with 0.5 µM CA-AM in calcium- and bicarbonate-free modified Krebs-Henseleit buffer (KHB), consisting of 20 mM HEPES, pH 7.4, 119 mM NaCl, 4.9 mM KCl, 0.96 mM KH2PO4, and 5 mM glucose, for 0-30 min at 37°C. AM rapidly penetrates across the hepatocyte plasma membrane and is hydrolyzed intracellularly to release free CA. After loading, the cultures were washed of excess CA-AM three times with KHB. Cellular CA fluorescence was recorded in a CytoFluor 2300 fluorescence plate reader (Millipore) using a filter combination with an excitation wavelength of 485 nm (22-nm bandwidth) and an emission wavelength of 530 nm (25-nm bandwidth). Cell cultures without CA-AM were used as blank to correct for nonspecific autofluorescence. The initial baseline level of fluorescence represented the total amount of both free and metal-bound CA loaded into the hepatocytes. To characterize the responsiveness of CA fluorescence toward different concentrations of intracellular free iron, cells were preloaded with ferrous ammonium sulfate (FAS) or pretreated with the cell-permeable ferrous iron chelator BIP.

Exposure to prooxidant drugs. NFT, probenecid, cyclosporin A, and BIP were added from stock solutions in DMSO. The final DMSO concentration did not exceed 0.1%, and an equal concentration of DMSO was present in all cell cultures, including controls. NFT-induced cell injury was assessed by measuring leakage of lactate dehydrogenase (LDH) into the culture medium (32). LDH activity was determined spectrophotometrically with a test kit (Boehringer) by means of a Cobas/Fara autoanalyzer. Enzyme activity in the medium was determined by serial sampling and was expressed as a percentage of the total of intracellular and extracellular LDH activity.

Fluorescence microscopy imaging. Flurorescence microscopy was performed with a Nikon Diaphot inverted fluorescence microscope. Fluorescence images were acquired with a Kodak DCS digital system. A 440- to 490-nm excitation filter, a 510-nm dichroic mirror, and a 520-nm emission filter were used. Cultured cells were analyzed immediately after loading with CA-AM and subsequent washing off the extracellular medium containing excess CA-AM.

Other biochemical assays. Total protein was determined according to the method of Bradford (5), using BSA as standard protein.

Statistical analysis. Each incubation was done in duplicate or triplicate and repeated at least three times with different cell preparations. Differences among groups were analyzed by ANOVA/Scheffé's F-test. All statistical analyses were performed with the StatView software package (Microsoft). P <=  0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In situ determination of LIP in cultured murine hepatocytes. When cultured murine hepatocytes were incubated with CA-AM, an intracellular fluorescence signal became readily apparent, reflecting uptake of the compound into the cells, where the ester was readily hydrolyzed to the fluorescent CA (Fig. 1). Cell-associated fluorescence, measured after replacing the extracellular medium with fresh KHB that did not contain CA-AM, reached a steady-state level after 20 min and remained stable thereafter. Hence, for all subsequent experiments, hepatocyte cultures were routinely loaded with CA-AM for 30 min. In contrast, the total intracellular plus extracellular fluorescence still increased after 30 min incubation. This indicates that CA-AM was hydrolyzed in the extracellular compartment and/or that free CA was not completely trapped inside cells but either leaked out or was actively transported across the hepatocellular plasma membrane.


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Fig. 1.   Time course of calcein (CA) accumulation in cultured murine hepatocytes. Overnight-precultured cells were incubated with 0.5 µM CA-AM in Krebs-Henseleit buffer (KHB). At the indicated time points, fluorescence in the culture dishes was measured with a CytoFluor 2300 plate reader at 485/530 nm. Both the total of intracellular + extracellular CA fluorescence (square ), with the CA-AM-containing medium still present, and the intracellular CA fluorescence (bullet ), after the medium was replaced with fresh CA-AM-free medium, are shown. Data are means ± SD from 3 independent cell preparations.

Because the free anionic form of CA is a substrate for the multidrug resistance-associated protein (MRP) in human tumor cell lines (14, 22), we next addressed the question of whether CA might be exported from hepatocytes by the murine homolog of MRP protein. The presence of probenecid (100 µM), an inhibitor of MRP-mediated transport (14), during loading of cells with CA-AM did not, however, alter the steady-state levels of cell-associated CA compared with cells loaded with CA-AM in the absence of probenecid [732 ± 311 vs. 739 ± 206 relative fluorescence units (RFU), probenecid-treated vs. untreated cells, respectively]. Similarly, probenecid did not alter the extracellular fluorescence (not shown). This indicates that CA loss from the hepatocytes via the probenecid-sensitive multidrug carrier was negligible under the conditions used and did not represent a critical factor that would influence the fluorescence readouts. To ascertain that CA was not lost from cells by mechanisms other than MRP-mediated transport, cells loaded with CA for 30 min were washed, and the fluorescence was repeatedly measured over 10 min. No significant decrease in fluorescence was recorded.

On the other hand, the bulky CA-AM ester has been shown to be a substrate for the multidrug transporter MDR1 in MDR1-expressing human cell lines (14, 21, 27). Therefore, we determined in murine hepatocytes loaded with CA-AM whether the ester might be expelled into the extracellular medium via the murine analog of this carrier. Accordingly, we loaded cells with CA-AM in the presence of cyclosporin A, an inhibitor of MDR-mediated transport. Cyclosporin A, added at a concentration (10 µM) that is nontoxic in hepatocytes and does not induce oxidative stress (42), significantly decreased the cell-associated fluorescence by 60% compared with cells without cyclosporin A (245 ± 105 vs. 617 ± 209 RFU, cyclosporin A-treated vs. untreated cells, respectively), whereas the total of intracellular and extracellular fluorescence remained unchanged (not shown). These data suggest that cyclosporin A may inhibit the uptake of CA-AM rather than prevent its egress from cells. Consequently, for all subsequent experiments, cells were loaded with CA-AM without the inclusion of transport inhibitors, because the fluorophore was well retained within the hepatocytes.

The degree of cell-associated fluorescence is, however, not necessarily proportional to the total amount of intracellular CA, because the presence of cytoplasmic free iron quenches the fluorescence of CA (6, 8, 10). To ascertain whether CA fluorimetry could be used to monitor relative changes in LIP in primary cultures of hepatocytes, we first exposed cells to an extracellular source of iron. Hepatocytes were incubated with various concentrations of FAS for 60 min before the cells were loaded with CA-AM. Because no transferrin was added, cellular uptake of iron most likely occurred via nontransferrin-mediated transport mechanisms, including endocytosis or metal-specific carriers (18). Exposure to extracellular iron resulted in concentration-dependent quenching of the intracellular CA fluorescence that became significant at 10 µM FAS and decreased the signal by 70% at 50 µM FAS (Fig. 2). This indicates that iron was taken up into the cultured hepatocytes and transiently incorporated into the LIP. To further substantiate the specificity for iron of the changes in fluorescence, the effect of the cell-permeable iron chelator BIP was investigated. The addition of BIP (100 µM) to FAS-pretreated hepatocytes, before the cells were loaded with CA, dequenched in part CA fluorescence (Fig. 2). Full recovery of the signal to untreated control values or higher was not observed at this concentration, because BIP chelates iron(II) only and had to compete for binding with CA. Collectively, the data indicate that CA fluorescence in hepatocytes readily reflected the changes in the amount of available LIP-associated iron.


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Fig. 2.   Quenching of the hepatocellular CA fluorescence signal after iron uptake from the extracellular medium as a function of added extracellular iron. Hepatocytes were incubated with KHB alone (untreated controls) or KHB in the presence of various concentrations of ferrous ammonium sulfate (FAS) (bullet ) for 60 min at 37°C. The cells were washed and loaded with CA-AM for 30 min, and intracellular CA fluorescence was determined. To some cultures exposed to 50 µM FAS, 100 µM 2,2'-bipyridyl (BIP) was added before cells were loaded with CA-AM (square ). Data are means ± SD of 3 independent cell preparations. * P < 0.05, significantly different from untreated control.

Increase in hepatocellular LIP by NFT and its prevention by BIP. To further explore the role of LIP during oxidative cell injury, we monitored changes in CA fluorescence after exposing hepatocytes to the prooxidant drug NFT. We have chosen NFT as a model compound, because NFT is a well-known redox cycling drug that produces massive amounts of superoxide anion in cultured murine hepatocytes (32). For the present study, it was important to record fluorescence before the onset of overt cell injury, as subtle changes in plasma membrane permeability may have confounded the results. Therefore, we chose NFT concentrations not exceeding 300 µM and limited the time of exposure to 1 h. Figure 3 shows that CA fluorescence in cells exposed to 200 µM NFT, a nontoxic concentration, was decreased by ~30% after 1 h, whereas no significant quenching of fluorescence was observed after 20 min of exposure. In contrast, exposure of cells to 300 µM NFT resulted in a significant decrease in CA fluorescence (by ~20%) after 20 min. This indicates that intracellular LIP increased in cells exposed to NFT as a function of time and concentration and that this increase in free iron was clearly detectable before the onset of cell injury.


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Fig. 3.   Time-dependent decrease in intracellular fluorescence after exposure of cultured hepatocytes to nitrofurantoin (NFT). Hepatocytes were incubated in KHB alone (square ) or in KHB containing 200 µM NFT (bullet ) or 300 µM NFT (black-triangle). After the NFT-containing medium was washed off, the cells were loaded with CA-AM, and intracellular fluorescence was recorded. Control (100%) = normalized data of KHB alone for each time point. Data are means ± SD of 3 independent cell preparations. *P < 0.05, significantly different from untreated concurrent control at the given time point.

Fluorescence microscopy imaging of cultured murine hepatocytes exposed to NFT confirmed the quantitative measurements (Fig. 4). Untreated hepatocytes loaded with CA-AM for 30 min revealed that the fluorescent probe had accumulated in the cytosol and that the signal was intense and evenly distributed across the cell (Fig. 4A). In contrast, cells preexposed to 300 µM NFT for 1 h and subsequently loaded with CA-AM under identical conditions exhibited greatly reduced intracellular fluorescence, and the negative staining of nuclei and mitochondria (43) became apparent (Fig. 4B).


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Fig. 4.   Fluorescence microscopy analysis of CA fluorescence in cultured murine hepatocytes. Untreated cells (A) or cells pretreated with NFT (300 µM; B) for 60 min were loaded with CA-AM for 30 min, washed of excess CA-AM, and examined under a fluorescence microscope using an excitation filter of 440-490 nm, a dichroic mirror of 510 nm, and an emission filter of 520 nm. Magnification, ×750.

If the quenching of CA fluorescence was due to reductive release of ferrous iron from intracellular stores, then it should be possible to dequench the signal by trapping the released iron with a ferrous iron chelator, thus preventing the interaction of free iron with CA. To test this hypothesis, NFT-pretreated cells were incubated with BIP immediately before the addition of CA-AM. Figure 5 demonstrates that BIP (500 µM) treatment abrogated the NFT effects and effectively restored the fluorescence to the values recorded in untreated control cells.


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Fig. 5.   Reversing effects of BIP on NFT-induced decrease in CA fluorescence. Hepatocytes were incubated for 60 min in KHB alone or in KHB containing 300 µM NFT. Where indicated, BIP (500 µM) was added immediately before cells were loaded with CA-AM for 30 min, and intracellular fluorescence was measured. Control (100%) = CA fluorescence of untreated cells in the absence of BIP. Data are means ± SD of 3 independent cell preparations, each done in duplicate wells. * P < 0.05, significantly different from KHB control. ** P < 0.05, significantly different from NFT without BIP.

Protection by iron chelators from NFT-induced oxidative cell injury. We have previously shown that NFT depletes hepatocellular glutathione within 1 h and subsequently produces oxidative cell injury to cultured murine hepatocytes, manifested as increases in protein carbonyl formation, lipid peroxidation, and increased LDH release (32). To explore whether there is a causal relationship between oxidative cell damage and increases in LIP, we next examined the effects of the iron chelators BIP and DFO on the early phases of NFT-induced cell injury. Commensurate with our previous results, 300 µM NFT alone caused massive increases in LDH release (>40% leakage after 2 h incubation), whereas extracellular LDH activity in untreated controls was low (~5%) (Fig. 6). The presence of BIP delayed the onset and significantly reduced the extent of LDH release by >60% to a level that was not different from untreated controls (Fig. 6). However, BIP was not able to prevent toxicity at a later time point, i.e., after 4 h incubation (data not shown), suggesting that the intracellular BIP-iron(II) complex was not stable for prolonged periods of time and/or that a ferric iron chelator was necessary for protection. The addition of DFO, featuring a high-affinity constant for ferric iron, similarly prevented NFT-mediated cell injury. The high concentration of DFO (5 mM) was necessary, because DFO, in contrast to the more lipophilic BIP, penetrates into cells more slowly (31). In contrast to BIP, DFO afforded full protection over the entire culture period of 4 h (data not shown). Collectively, these data provide further evidence for a direct role of LIP-associated intracellular iron in the precipitation of NFT injury to hepatocytes.


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Fig. 6.   Cytoprotective effect of iron chelators BIP and deferoxamine (DFO) against NFT-induced acute lethal cell injury. Hepatocytes in KHB were exposed to 300 µM NFT alone, NFT + 100 µM BIP, or NFT + 5 mM DFO. Lactate dehydrogenase (LDH) was serially determined at the indicated time points. BIP alone or DFO alone (not shown) did not increase LDH over untreated control (UC). Data are means ± SD of 3 independent cell preparations. * P < 0.05, significantly different from untreated control. ** P < 0.05, significantly different from NFT alone.

    DISCUSSION
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Materials & Methods
Results
Discussion
References

Release of intracellular iron from iron-sequestering proteins and increases in redox-active LIP are generally believed to facilitate molecular damage catalyzed by iron-dependent processes. Alterations in the concentrations of free iron are, however, difficult to monitor in intact cells. This study was designed to evaluate the CA fluorescence method as a tool to assess such changes driven by prooxidants in cultured hepatocytes. The results demonstrate that the increase in LIP, triggered by the reductive metabolism of the redox cycling drug NFT, can be readily detected in intact cells. Furthermore, this study provides evidence that the increase in LIP-associated iron not only precedes but is also causally related to the development of prooxidant-induced lethal cell injury in hepatocytes.

These conclusions were derived from a number of observations. First, the alterations in CA fluorescence were selectively induced by iron, which is complexed by CA and quenches the signal intensity of the fluorophore (7, 16). This selectivity was reflected by the fact that the CA signal was decreased by iron taken up from the culture medium and, inversely, increased by a chelator that sequestered LIP-associated iron. It has to be taken into account that CA is not specific for iron but can also form complexes with other bivalent metals including copper, nickel, and cobalt. The intracellular concentrations of these latter metals are, however, negligible under physiological conditions. In addition, calcium that may be released from intracellular compartments during oxidative damage may also constitute a potential source of interference. However, recent data suggest that calcium, even at a 1,000-fold excess concentration over iron, binds weakly to CA, if at all (8). Furthermore, our observation that the prooxidant-induced decrease in CA fluorescence was dequenched by the addition of the iron chelator BIP makes it highly unlikely that alterations in cell volume (water space) or in net CA uptake may have caused the shift in CA fluorescence intensity. Possible effects of the iron-binding indicator CA itself on mobilization and redistribution of storage iron cannot be excluded but appear to be minimal at early time points, because in untreated CA-loaded cells the fluorescence remained stable for at least 10 min.

Second, after exposure of cells to NFT, the LIP significantly increased (after 20 min) before any LDH leakage became apparent. On the other hand, these changes in free iron occurred concomitantly with superoxide production, which was readily apparent immediately after the addition of NFT (32). This sequence of events suggests that iron is primarily released by reducing equivalents rather than as a consequence of oxidative cell damage. It also suggests that the reductive stress posed by NFT bioactivation is a crucial event in the pathogenesis of NFT toxicity. The present study did not answer the question of whether superoxide was responsible for releasing iron or whether the NFT anion radical itself was also involved in iron mobilization. It is well known that reductants other than superoxide anion can mediate iron release from ferritin (15, 33), provided that the redox potential of these agents is below that of ferritin (16). The fact that the redox potential of NFT (V = -0.26) is slightly lower than that of ferritin (V -0.23) (29) offers a plausible explanation for the iron-releasing capacity of NFT. In fact, NFT can release iron from ferritin in solution at a rate two- to threefold over the basal control rate (29, 39). A similar mechanism of iron release has been previously demonstrated for paraquat (40), as well as for hyperthermia-induced oxidative damage to the liver (37). In contrast to these earlier studies, which were all performed in cell-free systems, the present data provide evidence that the prooxidant NFT is able to increase the LIP in intact hepatic parenchymal cells.

Finally, our observation that the addition of BIP not only reversed the alterations in CA fluorescence but also greatly reduced cell injury underlines the pivotal role of LIP in oxidative tissue damage. BIP is known as a strong lipophilic ferrous iron chelator that inhibits the Fenton reaction, because the corresponding iron chelate prevents single-electron exchange with hydrogen peroxide (16). Similarly, the full cytoprotective effect provided by DFO emphasizes the crucial role of LIP-associated iron in NFT toxicity to hepatocytes.

This study confirms and extends a series of elegant reports in which LIP levels were quantitated by CA fluorescence in cell lines (6, 8, 12, 44) and in which iron was delivered to cells by the physiological route, i.e., via transferrin (7, 9, 10). In one of these studies, a positive correlation was found between transferrin-stimulated increased iron levels and the extent of cytotoxicity induced by the mitochondrial prooxidant tert-butyl hydroperoxide (9). Here, increases in LIP were induced without the external addition of transferrin-bound iron. Instead, we concluded that the free iron was recruited from reductive release of protein-bound intracellular iron stores.

Collectively, the data demonstrate that the CA fluorescence technique can be adapted for cultured hepatocytes to monitor alterations in LIP. The results demonstrate that exposure of hepatocytes to NFT rapidly augmented the LIP and that this preceded the manifestations of oxidative cell injury. It is concluded that the iron chelator-sensitive increase in LIP is essential and causally related to prooxidant-induced cell injury triggered by NFT. Targeting of LIP iron, therefore, continues to offer a potential tool to prevent chemically induced oxidative cell injury.

    ACKNOWLEDGEMENTS

These studies were supported in part by the Hartmann Müller Foundation for Medical Research (Zurich, Switzerland) and by the Foundation for Scientific Research (University of Zurich, Schwerzenbach, Switzerland).

    FOOTNOTES

A portion of this work was presented at the 4th Annual Meeting of the Oxygen Society, in San Francisco, CA, November 20-24, 1997.

Address for reprint requests: U. A. Boelsterli, F. Hoffmann-LaRoche, Pharma Nonclinical R&D, Toxicology 73/216A, CH-4070 Basel, Switzerland.

Received 30 October 1997; accepted in final form 24 February 1998.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 274(6):G1031-G1037
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