Transient increase of the labile iron pool in HepG2 cells by intravenous iron preparations Brigitte Sturm, Hans Goldenberg and Barbara Scheiber-Mojdehkar Department of Medical Chemistry, University of Vienna, Austria Intravenous iron, used for the treatment of anemia in chronic renal failure and other diseases, represents a possible source of free iron in tissue cells, particularly in the liver. In this study we examined the effect of different sources of intravenous iron (IVI) on the labile iron pool (LIP) which represents the nonferritin-bound, redox-active iron that is implicated in oxidative stress and cell injury. Furthermore, we examined the role of the LIP for the synthesis of ferritin. We used HepG2 cells as a well known model for hepatoma cells and monitored the LIP with the metal-sensitive fluor- escent probe, calcein-AM, the fluorescence of which is quenched on binding to iron. We showed that steady state LIP levels in HepG2 cells were increased transiently, up to three-fold compared to control cells, as an adaptive response to long-term IVI exposure. In relation to the amount of iron in the LIP, the ferritin levels increased and the iron content of ferritin decreased. As any fluctuation in the LIP, even when it is only transient (e.g. after exposure to intravenous iron in this study), may result either in impairment of synthesis of iron containing proteins or in cell injury by pro-oxidants. Such findings in nonreticuloendothelial cells may have important implications in the generation of the adverse effects of chronic iron exposure reported in dialysis patients. Keywords: intravenous iron; labile iron pool; ferritin; liver; protein synthesis. Parenteral iron preparations are used widely for the treatment of iron deficiency anemia in patients under chronic hemodialysis. The iron supplementation is neces- sary to support erythropoiesis initiated by exogenous erythropoietin [1–3]. As intestinal absorption seems to be insufficient to meet the iron demand in recombinant human erythropoietin (r-HuEPO) treated dialysis patients [4], most of them require intravenous iron to sustain adequate erythropoiesis. Multiple parenteral iron formulations exist for adminis- tration to patients with end-stage renal disease [5]. The preparations are complexes of ferric iron with polymeric carbohydrates like dextran or sugars like sucrose or gluconate that form polynuclear complexes with the metal [6]. Recently, ferric pyrophosphate (Fe-PP) has also been used as a direct dialysis supplement [7]. These iron complexes are thought to be taken up by macrophages, degraded in the cells from where the iron is delivered to transferrin and further to the erythroblastic cells of the bone marrow. However, in a recent study [8] we showed, that parenteral iron preparations add iron to epithelial cells, like the human hepatoma cells HepG2 as well, and influence their iron metabolism accordingly: by stimulation of nontransferrin bound iron uptake, by deactivation of the iron regulatory protein IRP1, which results in increased ferritin synthesis, and by increased expression of the divalent metal transporter, DMT-1. These findings may have important implications on the possible toxicity of parenteral iron preparations for nonreticulo- endothelial cells. This is particularly true for liver hepato- cytes, as the liver is also the main sink for excess iron either from transferrin or from nontransferrin sources. As the half-life of intravenous iron is several hours, depending on the molecular properties of the individual preparations [6,9], the tissues of the body are confronted with this form of iron at relatively high concentrations (in the range between 10 and 500 l M ) depending on the dose used and the rate of its infusion. Further, a recent study suggesting that the life expectancy of dialysis patients may be dependent on the dosage regimen of intravenous iron (IVI) underscores the need of investi- gation of the biochemical and pathobiochemical conse- quences of its accumulation [10]. The administration of large doses of parenteral iron may therefore be associated with morbidity and mortality, in particular from infections. These concerns arise, in part, from the known role of iron as a growth factor for bacteria [11,12], its suspected inhibition of neutrophil and endothelial function [13–18], the induc- tion of protein oxidation [19], the ability to initiate oxidative reactions [5] and clinical studies relating iron overload to infectious morbidity [20–23]. The primary source of danger stems from the potential release of iron into the plasma as Ôlabile plasma ironÕ [24], as well as from the so-called cellular labile iron pool (LIP), Correspondence to B. Scheiber-Mojdehkar, Department of Medical Chemistry, Waehringerstr. 10, A-1090 Vienna, Austria. Fax: + 43 1 4277 60881, Tel.: + 43 1 4277 60827, E-mail: barbara.scheiber-mojdehkar@univie.ac.at Abbreviations: LIP, labile iron pool; IVI, intravenous iron; EPO, erythropoietin; Tf, transferrin; Fe, ferrum; Fe-PP, ferric-pyrophos- phate; IRP, iron regulatory protein; IRE, iron responsive element; ROS, reactive oxygen species; SIH, isonicotinoyl salicylaldehyde hydrazone; DMEM, Dulbecco’s minimal essential medium; DTPA, diethylene-triamine-pentaacetate; calcein-AM, calcein-acetoxy- methylester; Ca-Fe, calcein-iron complex; AAS, atomic absorption spectrophotometry. (Received 29 May 2003, revised 9 July 2003, accepted 18 July 2003) Eur. J. Biochem. 270, 3731–3738 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03759.x whose size mirrors all aspects of intracellular iron homeo- stasis. The chemical composition of the LIP has remained essentially elusive, but it may be implicated in generation of oxidative cell damage [25–27]. In this study, we demonstrate the quantitative relation- ship between concentration of iron from the preparation and rate of increase of the labile iron pool, using HepG2 human hepatoma cells as a cell culture model. The initiation of translation of ferritin by an increase in the labile iron pool (LIP) and the subsequent incorporation of labile iron into newly synthesized ferritin, followed by a decrease in the LIP needs several hours. ÔFreeÕ iron or labile iron is the part of intracellular iron not bound to enzymes or other proteins binding it firmly and thus available for binding to low-affinity sites, but also able to initiate toxic radical reactions. Thus, the cells have to resist an increased intracellular labile iron pool for a time window between iron challenge by the preparations, incorporation into the LIP, synthesis of ferritin and subsequent decrease of the LIP by incorporation into ferritin. These effects of parenteral iron preparations in nonreticuloendothelial cells should not be neglected when judging the applied dosage of intravenous iron. Materials and methods Materials Calcein and its acetoxymethylester (calcein-AM) were obtained from Molecular Probes. The iron chelator, salicylaldehyde isonicotinoyl hydrazone (SIH), was a gen- erous gift from P. Ponka (Lady Davis Institute for Medical Research, Montreal, Canada) and was prepared as 5 m M stock solution in dimethylsulfoxide. Diethylene triamine pentaacetate (DTPA), Fe-PP, cycloheximide and Hepes were from Sigma. Iron preparations (intravenous iron, IVI) The preparations for testing were Venofer (ferric saccharate) from Vifor (St. Gallen, Switzerland); Ferrlecit (ferric gluconate) from Rhone-Poulenc Rorer (A. Nattermann and Cie) and INFeD (ferric dextran) from Schein Pharma- ceuticals. The preparations were dissolved in phosphate buffered saline [NaCl/P i (m M ):137,NaCl;2.7,KCl;1.45, Na 2 HPO 4 ;8.45,Na 2 HPO 4 Æ12 H 2 O, pH 7.3] and freshly prepared for each experiment. Cell culture Human hepatoma HepG2 cells were cultured in DMEM containing 10% (v/v) fetal bovine serum, 2 m ML -glutamine and gentamicin (50 lgÆmL )1 ). Cells were treated with tryp- sin (1.25 ·) and resuspended in DMEM and seeded on 48-well tissue culture plates at a density of 1 · 10 6 cellsÆmL )1 . After 2 days, the cells were in the log-phase and were used for the experiments. Iron loading Cells were incubated with IVI at the indicated concen- trations at 37 °C for the indicated times. Then any surface-bound iron was removed by washing the cells with DMEM containing 50 l M DTPA and two more washings with DMEM alone. IVI induced cell injury was assessed by measuring leakage of lactate dehydrogenase (LDH) into the culture medium [28]. LDH activity was determined spectrophotometrically with a test kit (Boehringer) by means of Cobra Integra 700 autoanalyzer (Roche, Swit- zerland). Enzyme activity in the medium was calculated as percentage of the total intracellular and extracellular LDH activity. Toxicity of the iron preparations to HepG2 cells was tested by a neutral red cytotoxicity assay [29]. After preincubation of the cells with parenteral iron, cells were washed and incubated with neutral red for 3 h. Then the cells were washed with NaCl/P i and incubated with 200 lL of 50% ethanol, 1% acetic acid (v/v) in distilled water for 20 min and absorbance at 540 nm was measured in a fluorescence plate reader (Victor II) from Perkin Elmer. Iron uptake into the LIP In order to show that parenteral iron preparations increase the cellular LIP, HepG2-cells were first incubated with the fluorescent metal sensor, calcein-AM (0.25 l M ) at 37 °C in DMEM, buffered with 20 m M Hepes for 15 min. After calcein-loading, the cells were washed three times and reincubated in DMEM, containing 20 m M Hepes and anti-calcein Igs [made by M. Hermann, Department of Medical Biochemistry, University of Vienna, Austria (method by Breuer et al. Hebrew Uni- versity, Jerusalem, Israel [30])] were added for quenching extracellular probe fluorescence. Baseline fluorescence was measured in a fluorescence plate reader (Victor II) from Perkin Elmer (excitation 485 nm, emission 535 nm) at 37 °C. Then various amounts of the iron preparations were added and quenching of calcein fluorescence by incorporated iron into the LIP was assayed continuously for 15 min. Measurement of the cellular LIP after iron loading with IVI Iron loaded cells (see above) were incubated with 0.25 l M calcein-AM for 15 min at 37 °C in DMEM, buffered with 20 m M Hepes. The cell monolayer was then washed free of excess calcein-AM and reincubated with DMEM containing 20 m M Hepes and a fluorescence-quenching anti-calcein Ig that was added to eliminate all extracellular fluorescence. Calcein fluorescence was measured in a fluorescence plate reader (Victor II) from Perkin Elmer (excitation 485 nm, emission 535 nm) at 37 °C. After stabilization of the signal, the amount of intracellular iron, bound to calcein (Ca-Fe), was assessed by addition of 100 l M of the fast permeating chelator isonicotinoyl salicylaldehyde hydrazone (SIH). Inhibition of protein synthesis Cells were preincubated with IVI (75 l M ) and cyclohexi- mide (15 lgÆmL )1 ) for the indicated times. The cell mono- layer was then washed free of any surface-bound iron with DMEM containing 50 l M DTPA and two more washings 3732 B. Sturm et al. (Eur. J. Biochem. 270) Ó FEBS 2003 with DMEM alone. Finally, the cellular LIP was measured as described above. Ferritin quantification by ELISA Cells were incubated with 75 l M of IVI for the indicated times. The cell monolayer was then washed free of any surface-bound iron with DMEM containing 50 l M DTPA and two more washings with DMEM alone. The cells were lysed on ice in NP-40 lysis buffer containing 1% NP-40 and 1m M phenylmethanesulfonyl fluoride in 150 m M NaCl, 50 m M Tris, pH 8.0. The lysates were centrifuged at 7500 g for 10 min at 4 °C and the supernatants were collected and stored at )80 °C until used. Lysates were analyzed for cellular ferritin content by using a human ferritin ELISA (BioCheckInc.,Burlingame,CA,USA).Theassaysystem utilizes a rabbit anti-ferritin Ig for solid phase immobiliza- tion and a mouse monoclonal anti-ferritin Ig in the Ig-enzyme (horseradish peroxidase) conjugate solution. Protein concentrations were determined using the Bradford method (Bio-Rad). Iron content of ferritin During the last step of the ferritin-ELISA (see above) the ferritin detaches from the surface of the wells and the iron content in the supernatant was quantified by atomic absorption spectrophotometry (AAS) (Hitachi). The iron content of ferritin was calculated from the iron concentra- tion in the supernatant and the amount of ferritin within the same well. Statistical analysis Results are presented as mean ± SEM from three inde- pendent experiments. Each experiment was carried out in triplicate. Ferritin content was measured in duplicate. Differences were examined for statistical significance using the paired t-test. All experiments showed P <0.03 or smaller. Data were analyzed with GRAPH PAD PRISM software. Results Effect of IVI on the LIP IVI taken up by HepG2 cells entered the labile iron pool (Fig. 1). The LIP was assessed by the calcein-based method. Cells were incubated with calcein-AM and baseline fluor- escence was registered. Then various concentrations of IVI were added and changes in calcein-fluorescence were measured. Within the first 15 min of incubation with IVI, the LIP increased (i.e. baseline fluorescence decreased) between 8 and 25% depending on the iron source and the concentration of iron calculated from the stoichiometric composition. (Table 1). Exact concentrations could not be obtained reliably because the cell-free calibration and the assessment in the cellular system were apparently not exactly equal. Ferric pyrophospate nominally represents ÔfreeÕ iron and was most effective, followed by Ferrlecit, Venofer and INFeD. This order corresponds to the known physico-chemical stability of the iron complexes [6]. Adaptive response of the LIP to extracellular IVI Exposure to extracellular IVI resulted in concentration dependent quenching of the intracellular calcein fluores- cence (Fig. 1, Table 1). This indicates that iron from extracellular IVI was taken up into the cultured hepatocytes and transiently incorporated into the LIP. To further substantiate the adaptive response of the cells to the iron challenge by the intravenous iron preparations, LIP meas- urements at different time points after iron addition to the culture medium were performed. Within the time frame between 0 and 24 h of incubation with IVI, the LIP changed in different ways depending on the source of iron (Fig. 2). With all preparations the increase of the LIP was dependent on the concentration of extracellular iron. The highest increase in the LIP was found with Fe-PP (up to threefold compared to control) after 2 h followed by a subsequent decrease to the control value after 8 h. With the other iron Fig. 1. Effect of IVI (Venofer) on the LIP in HepG2 cells. Cells were loaded with calcein-AM (0.25 l M ), washed and incubated with DMEM, containing 20 m M Hepes and anti-calcein Ig. After registra- tion of the baseline fluorescence 25, 75 or 150 l M iron from the IVI preparation Venofer were added. Control cells were incubated with cell culture medium alone. Iron taken up into the LIP was assessed by measuring the decrease in calcein fluorescence within 15 min at 37 °C. Shown are the mean ± SEM from triplicates of three independent experiments. Table 1. Effect of IVI on the LIP (% decrease of basic calcein fluor- escence). Cells were loaded with calcein-AM (0.25 l M ), washed and incubated with DMEM containing 20 m M Hepes and anti-calcein Ig. After registration of the baseline fluorescence, 25, 75 or 150 l M iron from different IVI preparations (Venofer, Ferrlecit, INFeD, Fe-PP) were added. Control cells were incubated with cell culture medium alone. Calcein fluorescence was determined within 15 min at 37 °C. Quenching of fluorescence was referred to percentage of control. Shown are the mean ± SEM from triplicates of three independent experiments. Preparation IVI concentration (l M iron) 25 75 150 Venofer 8.3 ± 2.5 16.4 ± 2.2 17.8 ± 4.5 Ferrlecit 10.8 ± 4.0 16.9 ± 2.2 18.6 ± 0.8 INFeD 7.8 ± 2.7 10.1 ± 0.7 13.4 ± 1.3 Fe-PP 19.3 ± 0.3 21.4 ± 0.9 25.2 ± 1.5 Ó FEBS 2003 Intravenous iron and the labile iron pool (Eur. J. Biochem. 270) 3733 preparations, the maxima and the time course were quantitatively different, i.e. the maxima were reached later (after 4 h with Ferrlecit, and after 6 h with Venofer and INFeD), were smaller and the decrease to the baseline was slower, but in principle all IVI sources showed a similar behaviour. The transient increase in LIP after exposure to extracel- lular IVI was not caused by cell damage as assessed by means of lactate dehydrogenase release (LDH-release to the medium was less than 5% of total LDH with 150 l M IVI) and neutral red cytotoxicity test (neutral red incorporation was not changed compared to untreated cells after exposure to 150 l M IVI for 24 h) (not shown). Effect of protein synthesis inhibitors on the adaptive response of the LIP to extracellular IVI In order to confirm that the observed decrease of the LIP upon prolonged exposure to IVI (Fig. 2) was due to the synthesis of protein, presumably ferritin (see below), the cells were incubated with IVI and cycloheximide, to block cytosolic protein synthesis and the LIP was assessed following different incubation times (0–8 h). The conse- quence was a further strong increase in the LIP in cycloheximide and IVI-treated cells (Fig. 3) compared to the time phase corresponding to the decline in cells with normal protein synthesis (exposed to IVI alone) (Fig. 2A– D). When cycloheximide was present during IVI exposure, all iron sources behaved similarly and the increase in the LIP did not appear to be limited. After 8 h with all IVI preparations the LIP was increased up to sevenfold compared to control. This means that high amounts of iron can enter the LIP. In comparison, inhibition of prokaryotic protein synthesis did not have any effect to the LIP (data not shown). Changes in ferritin content In order to confirm that the observed decrease of the LIP upon prolonged exposure to IVI (Fig. 2) was due to newly synthezised ferritin, HepG2 cells were first exposed to 75 l M iron from IVI and then ferritin content was assessed. The Fig. 2. Adaptive response of the LIP to extracellular IVI. Cells were preincubated with extracellular IVI (25–75 l M iron) for up to 24 h (A) Venofer; (B) Ferrlecit; (C) INFeD; (D) Fe-PP. Control cells were incubated with the cell culture medium alone. Then cells were loaded with calcein-AM (0.25 l M ), washed and incubated with DMEM, containing 20 m M Hepes and anti-calcein Ig. After registration of the baseline fluorescence, the amount of intracellular metal bound to calcein (Ca-Fe) was assessed by addition of 100 l M of the fast permeating chelator SIH. Calcein fluorescence was measured when the signal reached full fluorescence and remained stable (after 2 min). Shown are the mean ± SEM from triplicates of three independent experiments. 3734 B. Sturm et al. (Eur. J. Biochem. 270) Ó FEBS 2003 cellular ferritin content increased with time and the rate of the increase paralleled the increase in the LIP in the first few hours of incubation, but was steeper in the time between 4 and 24 h for the iron sources with apparently slower iron release, namely Venofer and INFeD (Fig. 4). Whereas with Fe-PP and Ferrlecit, a cellular ferritin, content of 15 ng ferritin per mg protein was already reached after 8 h of incubation, it needed 24 h of incubation with Venofer and INFeDtoreachthesameferritincontent.Thetimecourse of ferritin increase corresponded to the decrease in LIP back to the steady-state level: whereas with Fe-PP and Ferrlecit the LIP was back to control level after 8 h, this took more time with the two other iron preparations (Fig. 2A–D). Apparently, the higher the initial increase in the LIP, the faster ferritin synthesis is turned on, leading to quicker disappearance of labile iron. Molar ratio of iron and ferritin Iron from all iron preparations tested increased the labile iron pool and as a consequence, ferritin biosynthesis was up-regulated and at the same time the LIP decreased. Therefore we assessed the time course of the molar ratio of iron and ferritin in HepG2 cells following IVI exposure for 0–24 h. The decrease in the iron content of ferritin paralleled the increase in ferritin content itself (Fig. 5). The faster the initial increase in ferritin, the faster the decrease of its iron content from 4000 iron atoms in untreated control cells down to a common end-value of approximately 800 iron atoms per molecule of ferritin following exposure to IVI. Discussion Parenteral iron preparations are used widely for the treatment of iron deficiency anemia in patients undergoing chronic hemodialysis. The iron supplementation is neces- sary to support erythropoiesis initiated by exogenous erythropoietin [1]. The safety and efficacy of the intravenous iron prepara- tions in use is generally accepted. However, in a retrospec- tive analysis of data from Medicar dialysis patients, Collins et al. [31] found a significant relationship between the frequency of IVI dosing and increased risk of death from infection. There is also some debate about whether frequent low-dosage IVI administration is safer than less frequent high dosage [32–34]. Therefore, much concern has been raised recently about the potential toxicity of chronic iron exposure in dialysis patients. These concerns relate to the following concepts: (a) parenchymal cell iron overload with Fig. 3. Effect of protein synthesis on the adaptive response of the LIP to IVI. HepG2 cells were incubated for 0–8 h with IVI (75 l M iron) and cycloheximide (15 lgÆmL )1 ). The control was incubated with cyclo- heximide without IVI. Then cells were loaded with calcein-AM, washed and incubated with DMEM, containing 20 m M Hepes and anti-calcein Ig. After registration of the baseline fluorescence, the amount of intracellular metal, bound to calcein (Ca-Fe), was assessed by addition of 100 l M of the fast permeating chelator SIH. Calcein fluorescence was measured when the signal reached full fluorescence and remained stable (after 2 min). Shown are the mean ± SEM from triplicates of three independent experiments. Fig. 4. Synthesis of ferritin during long-time exposure to 75 l M iron from IVI. HepG2cellswereexposedtoIVIbetween0and24h, washed to remove surface bound iron, lysed, sonicated and stored at )80 °C until used. The ferritin content of the lysate was determined by ELISA as described in the Materials and methods section and corre- lated to a standard curve. Shown are the mean ± SEM from dupli- cates of three independent experiments. Fig. 5. Molar ratio of iron and ferritin. Cells were incubated with 75 l M iron from IVI between 0 and 24 h. Then cells were washed to remove surface bound iron, lysed, and the ferritin content of the lysate was determined by ELISA. The iron content of ferritin was measured by AAS in the supernatant of the ELISA which included the total determined ferritin. Shown are the mean ± SEM from duplicates of three independent experiments. Ó FEBS 2003 Intravenous iron and the labile iron pool (Eur. J. Biochem. 270) 3735 possible permanent organ damage (e.g. liver cirrhosis or pancreatic fibrosis, cancer or myocardial infarction); (b) increased incidence of infections and (c) increased free radical generation from free iron causing increased oxidant- mediated tissue injury. The iron complexes are thought to be taken up by macrophages, degraded in the cells from where the iron is delivered to transferrin and further to the erythroblastic cells of the bone marrow. However, in a recent study, we showed the ability of parenteral iron preparations to deliver iron to cells others than the reticuloendothelial cells, their effect on intracellular iron metabolism and indirectly on the labile iron pool of the human hepatoma cells HepG2 [8]. The polymers increase the uptake rate for nontransferrin bound iron, inactivate the IRE-binding activity of the iron regulatory protein IRP1 [35,36] and stimulate ferritin synthesis in these cells, which is characteristic for the effects seen with labile iron. Effects of these iron complexes on the labile iron pool in this cell culture model may have important implications on the possible toxicity of parenteral iron preparations for nonreticuloendothelial cells, as initiation of iron-mediated oxidative cell injury is generally ascribed to the labile iron pool, formally also called Ôchelatable iron poolÕ because of its accessibility to iron chelators [30,37]. This LIP is a normal part of the total cellular iron, but it is kept small and tightly regulated by the control mechanisms of cellular iron homeostasis. When this balance gets out of control, free iron can accumulate and cause oxidative damage, mainly by reaction with ever-present reactive oxygen species (ROS) like superoxide, hydrogen peroxide or organic peroxides [38–40]. When the cellular LIP rises, the iron regulatory proteins (IRPs) lose their ability to bind to iron responsive elements (IRE) in several mRNAs. This, among other effects, leads to an increase in the synthesis of ferritin, the major iron storage protein. Iron bound to ferritin is harmless; thus ferritin is the major defense against iron toxicity. Oxidative stress appar- ently inactivates binding of IRP to IRE too and this initiates cellular protection [41]. In hepatocytes, incubation with 100 l M low molecular weight iron for 18 h doubled the LIP [42] and significantly increased their ferritin content. We also show that iron from the parenteral preparations enter the LIP in a time- and concentration dependent manner. We chose the concentra- tions between 25 and 75 l M iron because the fluorescence- based method is limited with respect to the amount of iron in the LIP. Higher concentrations of IVI lead to statistically invalid and rather erratic results. Moreover, this concentra- tion range corresponds to what can be expected in the plasma of recipient patients. The uptake of IVI is rather fast: within the first 15 min of incubation with IVI, the LIP increases between 8 and 25% depending on the iron source tested. Due to the fact that the uptake was performed in medium without any supplemen- tation it shows that IVI can be taken up directly by the cells without preceding release to mediating chelators. After long-time exposure of HepG2 cells to IVI, we could show that an adaptive response of the LIP took place. The time response and the maximal changes in the LIP differed with the iron complex used: Fe-PP achieved its maximal LIP already after 2 h of incubation while Ferrlecit had its maxima after 4 h. In both cases, the LIP decreased to the control value after 8 h. In comparison, Venofer and INFeD needed about 6 h of incubation to have maximal LIP and the decrease to the control value took longer than 8 h. There was not only a time- and concentration-dependent signifi- cant difference but also the level of the increase of the LIP varied tremendously. While Fe-PP increased the LIP up to threefold compared to control, INFeD could increase the LIP only up to 1.5-fold. In general, with all iron preparations, inhibition of cytosolic protein synthesis by cycloheximide resulted in a significant increase of the LIP that did not seem to be limited. This would mean that most of the iron from the LIP is incorporated into ferritin. In comparison, inhibition of prokaryotic (and thus also mitochondrial) protein synthesis (data not shown) did not have any effect to the LIP. The increase in ferritin by the iron preparations showed a pattern of behavior similar to the increase of the LIP. The more iron appeared in the LIP the faster the synthesis of ferritin took place. But in general, at the endpoint (24 h) of our IVI uptake experiments, the ferritin content was almost the same in all cases. HepG2 cells cultivated under normal tissue culture conditions (DMEM-medium supplemented with 10% fetal calf serum) are relatively iron poor. Accordingly, they have a very low ferritin content. In this study, we show that the ferritin of these cells is almost iron-saturated (4000 iron atoms per molecule ferritin) and after uptake of iron from the iron complexes into the LIP, the cells change their metabolism according to the amount of incorporated iron into the LIP. Control cells have highly iron loaded ferritins: under these conditions iron from the preparations taken up by the cells is not immediately scavenged by existing ferritin and therefore can increase the labile iron pool. As the LIP is suspected to regulate cellular iron metabolism (and possibly also other known/or yet unknown enzymes or proteins with/or without iron responsive elements) according to its size, it is necessary that the size of the LIP is really sensitive to incoming iron. With iron-poor ferritin, this sensitivity to incoming iron would be much weaker: it could immediately scavenge all new iron from the LIP and almost no increase in the LIP could result. The consequence of this scenario would be that the size of the LIP is less dependent on nontransferrin- bound iron uptake and therefore the cells need much more time and higher amounts of incoming (and possible toxic) iron to accommodate their metabolism according to the iron challenge. We show that the content of iron stored in ferritin paralleled the synthesis of ferritin and that in turn paralleled the size of the LIP. That means that there is a relationship between the size of the LIP, ferritin synthesis and the iron content of ferritin. Further we conclude that the iron from the LIP is not stored in existing ferritin but is incorporated into newly synthesized ferritin. Compared to the increase in ferritin expression, the total amount of iron added to the cells in the form of polymeric complexes is comparatively small. Thus, the increase in total iron-containing ferritin is also neglectably small compared to the total ferritin content of the cells. This is not unreasonable, as the biosynthesis of ferritin is a means of protection from possible iron toxicity, which the cells turn on after iron signalling and which then 3736 B. Sturm et al. (Eur. J. Biochem. 270) Ó FEBS 2003 precedes any further iron loading. Moreover, though we show in this study that parenteral iron preparations enter the cells and add iron to the LIP, it does not mean that all incorporated parenteral iron can enter the LIP and has to be taken up by newly synthesized ferritin. Parenteral iron preparations mimic ferritin-like molecules and it is therefore quite possible that they can exist in this form beside ferritin into the cell. In which form parenteral iron is stored in the cells is not known and is subject of further investigations. Altogether, our results show that parenteral iron prepa- rations enter HepG2-cells, add iron to the labile iron pool and that the cells adapt their iron metabolism according to the size of incoming iron by highly increasing ferritin biosynthesis as a means of protection from further iron loading. LIP levels return to the constitutive level of normal tissue culture due to incorporation of labile iron into ferritin. As any fluctuation in the LIP, even when it is only transient (such as that following exposure to intravenous iron) may result either in impairment of synthesis of iron containing proteins or in cell injury by pro-oxidants [43], such findings in nonreticuloendothelial cells may have important impli- cations in the generation of the adverse effects of chronic iron exposure reported in dialysis patients. Acknowledgements This work was supported by the Austrian Research Found (# FWF P147842-PAT) and Hochschuljubilaeumsstiftung der Stadt Wien (# H-83/2000). References 1. Sunder-Plassmann, G. & Ho ¨ rl, W.H. (1995) Importance of iron supply for erythropoietin therapy. Nephrol. Dial. Transplant. 10, 2070–2076. 2. Besarab, A., Kaiser, J.W. & Frinak, S. (1999) A study of par- enteral iron regimens in hemodialysis patients. Am.J.KidneyDis. 34, 21–28. 3. MacDougall, I.C. (2001) Present and future strategies in the treatment of renal anemia. Nephrol. Dial. Trans. 16, 50–55. 4. Kooistra, M.P., Niemantsverdreit, E.C., van Es, A., Mol-Beer- man,N.M.,Strycenberg,A.&Marx,J.J.M.(1998)Ironabsorp- tion in erythropoietin-treated hemodialysis patients: Effects of iron availability, inflammation and aluminium. Nephrol. Dial. Trans. 13, 82–88. 5. Zager, R.A., Johnson, A.C., Hanson, S.Y. & Wasse, H. (2002) Parenteral iron formulations: a comparative toxicologic analysis and mechanisms of cell injury. Am.J.KidneyDis.40, 90–103. 6. Geisser, P., Baer, M. & Schaub, E. (1992) Structure/histotoxicity relationship of parenteral iron. Drug Res. 42, 1439–1452. 7. Gupta, A. & Crumbliss, A.L. (2000) Treatment of iron deficiency anemia: Are monomeric iron compounds suitable for parenteral administration? J. Laboratory Clin. Med. 136, 371–378. 8. Scheiber-Mojdehkar, B., Sturm, B., Plank, L., Kyzer, I. & Gold- enberg, H. (2003) Influence of parenteral iron preparations on non-transferrin bound iron uptake, the iron regulatory protein and the expression of ferritin and the divalent metal transporter DMT-1 in HepG2 human hepatoma cells. Biochem. Pharm. 65, 1973–1978. 9. Danielson, B.G., Salmonson, T., Derendorf, H. & Geisser, P. (1996) Pharmacokinetics of iron (III)-sucrose complex after a single intravenous dose in healthy volunteers. Drug. Res. 46, 615–621. 10. Feldman,H.I.,Santanna,J.,Guo,W.,Furst,H.,Franklin,E., Joffe, M., Marcus, S. & Faich, G. (2002) Iron administration and clinical outcomes in hemodialysis patients. J. Am. Soc. Nephrol. 13, 734–744. 11. Williams, P. & Griffiths, E. (1992) Bacteria transferrin receptors- structure, function and contribution to virulence. Med. Microbiol. Immunol. 181, 301–322. 12. Payne, S.M. (1988) Iron and virulence in the family Enterobac- teriaceae. Crit.Rec.Microbiol.16, 81–111. 13. Flament, J., Goldman, M., Waterlot, Y., Dupont, E., Wybran, J. & Vanherweghem, J.L. (1986) Impairment of phagocyte oxidative metabolism in hemodialyzed patients with iron overload. Clin. Nephrol. 25, 27–30. 14. Cantinieaux, B., Boelaert, J., Hariga, C. & Fondu, P. (1988) Impaired neutrophil defense against Cersinis enterocolitica in patients with iron overload who are undergoing dialysis. J. Laboratory Clin. Med. 111, 524–528. 15. Boelaert, J.R., Cantinieaux, B., Hariga, C. & Fondu, P. (1990) Recombinant erythropoietin reverses polymorphonuclear granu- locyte dysfunction in iron-overloaded dialysis patients. Nephrol. Dial. Transplant. 5, 504–417. 16. Veys, N., Vanholder, R. & Ringoir, S. (1992) Correction of defi- cient phagocytosis during erythropoietin treatment in main- tenance hemodialysis patients. Am. J. Kidney Dis. 19, 58–62. 17. Patruta, S.I., Edlinger, R., Sunder-Plassmann, G. & Hoerl, W.H. (1998) Neutrophil impairment associated with iron therapy in hemodialysis patients with functional iron deficiency. J. Am. Soc. Nephrol. 9, 655–663. 18. Rooyakkers, T.M., Stroes, E.S.G., Kooistra, M.P., van Faassen, E.E., Hider, R.C., Rabelink, T.J. & Marx, J.J.M. (2002) Ferric saccharate induces oxygen radical stress and endothelial dys- function in vivo. Eur. J. Clin. Invest. 32, 9–16. 19. Tovbin, D., Mazor, D., Vorobiov, M., Chaimovitz, C. & Meyerstein, N. (2002) Induction of protein oxidation by intra- venous iron in hemodialysis patients: Role of inflammation. Am. J. Kidney Dis. 40, 1005–1012. 20. Mossey, R.T. & Sondheimer, J. (1985) Listeriosis in patients with long-term hemodialysis and transfusional iron overload. Am. J. Med. 7, 397–400. 21. Seifert,A.,vonHerrath,D.&Schaefer,K.(1987)Ironoverload, but not treatment with desferrioxamine favours the development of septemia in patients on maintenance hemodialysis. Q. J. Med. 65, 1025–1024. 22. Boelaert, J.R., Daneels, R.F., Schurgers, M.L., Matthys, E.G., Grodts, B.Z. & Van Landuyt, H.W. (1990) Iron overload in hemodialysis patients inreases the risk of bacteraemia: a pro- spective study. Nephrol. Dial. Transplant. 5, 130–134. 23. Hoen, B., Kessler, M., Hestin, D. & Mayeux, D. (1995) Risk factors for bacterial infections in chronic hemodialysis adult patients: a multicenter prospective survey. Nephrol. Dial. Trans- plant. 10, 377–381. 24. Esposito, B.P., Breuer, W., Slotki, I. & Cabantchik, Z.I. (2002) Labile iron in parenteral iron formulations and its potential for generating plasma nontransferrin-bound iron in dialysis patients. Eur. J. Clin. Invest. 43, 42–49. 25. Breuer, W., Greenberg, E. & Cabantchik, Z.I. (1997) Newly delivered transferrin iron and oxidative cell injury. FEBS Lett. 403, 213–219. 26. Picard, V., Renaudie, F., Porcher, C., Hentze, M.W., Grand- champ, B. & Beaumont, C. (1996) Overexpression of the ferritin H subunit in cultured erythroid cells changes the intracellular iron distribution. Blood 87, 2057–2064. 27. Lipinski, P., Drapier, J C., Oliveira, L., Retmanska, H., Soc- hanowicz, B. & Kruszewski, M. (2000) Intracellular iron status as a hallmark of mammalian cell susceptibility to oxidative stress: Ó FEBS 2003 Intravenous iron and the labile iron pool (Eur. J. Biochem. 270) 3737 a study of L5178Y mouse lymphoma cell lines differentially sensitive to H 2 O 2 . Blood 95, 2960–2966. 28. Amador, E., Dorfmann, L.E. & Wacker, W.E.C. (1963) Serum lactic dehydrogenase: an analytical assessment of current assays. Clin. Chem. 9, 391–395. 29. Babich, H., Sardana, M.K. & Borenfreund, E. (1988) Acute cytotoxicities of polynuclear aromatic hydrocarbons determined in vitro with the human liver tumour line, HepG2. Cell Biol. Toxicol. 4, 295–309. 30. Breuer, W., Epsztejn, S. & Cabantchik, Z.I. (1995) Iron acquired from transferrin by K562 cells is delivered into a cytoplasmic pool of chelatable iron. J. Biol. Chem. 270, 24209–24215. 31. Collins, A., Ebben, J. & Ma, J. (1997) Frequent IV iron dosing is associated with higher infectious deaths. J. Am. Soc. Nephrol. 8, 190 A. 32. MacDougall, I.C. (2000) Intravenous administration of iron in epoetin-treated hemodialysis patients-which drugs, which regi- men? Nephrol. Dial. Trans. 15, 1743–1745. 33. Besarab, A. (1999) Parenteral Iron Therapy: Safety and Efficacy. Seminars Dialysis. 12, 237–242. 34. Besarab, A., Frinak, S. & Yee, J. (1999) An indistinct balance: The safety and efficacy of parenteral iron therapy. J. Am. Soc. Nephrol. 10, 2029–2043. 35. Kuehn, L.C. & Hentze, M.W. (1992) Coordination of cellular iron metabolism by post-transcriptional gene regulation. J. Inorg. Biochem. 47, 183–195. 36. Klausner, R.D., Rouault, T.A. & Harford, J.B. (1993) Regulating the fate of messenger RNA:the control of cellular iron metabo- lism. Cell 72, 19–28. 37. Jacobs, A. (1977) Low molecular weight intracellular iron trans- port compounds. Blood 5, 4331–4336. 38. Halliwell, B. & Gutteridge, J.M.C. (1990) Role of free radicals and catalytic metal ions in human disease: An overview. Methods Enzymol. 186, 1–85. 39. Rothman,R.J.,Serroni,A.&Farber,J.L.(1992)Cellularpoolof transient ferric iron, chelatable by desferrioxamine and distinct from ferritin that is involved in oxidative cell injury. Mol. Phar- macol. 42, 703–710. 40. Staeubli, A. & Boelsterli, U.A. (1998) The labile iron pool in hepatocytes: prooxidant-induced increase in free iron precedes oxidative cell injury. Am.J.Physiol.274, G1031–G1037. 41. Cairo, G., Castrusini, E., Minotti, G. & Bernelli-Zazzera, A. (1996) Superoxide and hydrogen peroxide-dependent inhibition of iron regulatory protein activity: a protective stratagem against oxidative injury. FASEB. J. 10, 1326–1335. 42. Zanninelli, G., Loreal, O., Brissot, P., Konijn, A.M., Slotki, I.N., Hider, R.C. & Cabantchik, Z.I. (2001) The labile iron pool of hepatocytes in chronic and acute iron overload and chelator- inducedirondeprivation.J. Hepatol. 36, 39–46. 43. Kakhlon, O. & Cabantchik, Z.I. (2002) The labile iron pool: Characterization, measurement, and participation in cellular processes. Free Radic. Biol. Med. 33, 1037–1046. 3738 B. Sturm et al. (Eur. J. Biochem. 270) Ó FEBS 2003 . Transient increase of the labile iron pool in HepG2 cells by intravenous iron preparations Brigitte Sturm, Hans Goldenberg and Barbara Scheiber-Mojdehkar Department of Medical Chemistry,. The initiation of translation of ferritin by an increase in the labile iron pool (LIP) and the subsequent incorporation of labile iron into newly synthesized ferritin, followed by a decrease in the. ferritin content increased with time and the rate of the increase paralleled the increase in the LIP in the first few hours of incubation, but was steeper in the time between 4 and 24 h for the iron