Báo cáo khoa học: Transient increase of the labile iron pool in HepG2 cells by intravenous iron preparations Brigitte Sturm, Hans Goldenberg and Barbara Scheiber-Mojdehkar doc

Transient increase of the labile iron pool in HepG2 cellsby intravenous iron preparations Brigitte Sturm, Hans Goldenberg and Barbara Scheiber-Mojdehkar Department of Medical Chemistry,

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 lM) 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 oxidainduc-tion [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
labile plasma iron [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)

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
Free 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 mM

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/Pi(mM): 137, NaCl; 2.7, KCl; 1.45,

Na2HPO4; 8.45, Na2HPO4Æ12 H2O, 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 mM L-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· 106cellsÆmL)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 37C for the indicated times Then any

surface-bound iron was removed by washing the cells with DMEM containing 50 lMDTPA 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/Piand 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 lM)

at 37C in DMEM, buffered with 20 mM Hepes for

15 min After calcein-loading, the cells were washed three times and reincubated in DMEM, containing 20 mM

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

37C 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 lM

calcein-AM for 15 min at 37C in DMEM, buffered with

20 mM Hepes The cell monolayer was then washed free

of excess calcein-AM and reincubated with DMEM containing 20 mM 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 37C After stabilization of the signal, the amount of intracellular iron, bound to calcein (Ca-Fe), was assessed by addition of

100 lM of the fast permeating chelator isonicotinoyl salicylaldehyde hydrazone (SIH)

Inhibition of protein synthesis Cells were preincubated with IVI (75 lM) 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 DTPA and two more washings

with DMEM alone Finally, the cellular LIP was measured

as described above

Ferritin quantification by ELISA

Cells were incubated with 75 lM of IVI for the indicated

times The cell monolayer was then washed free of any

surface-bound iron with DMEM containing 50 lMDTPA

and two more washings with DMEM alone The cells were

lysed on ice in NP-40 lysis buffer containing 1% NP-40 and

1 mM phenylmethanesulfonyl fluoride in 150 mM NaCl,

50 mMTris, pH 8.0 The lysates were centrifuged at 7500 g

for 10 min at 4C 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

(BioCheck Inc., Burlingame, CA, USA) The assay system

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

free 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)

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

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 lMIVI)

and neutral red cytotoxicity test (neutral red incorporation

was not changed compared to untreated cells after exposure

to 150 lMIVI 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 lM

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.

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

INFeD to reach the same ferritin content The time course

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 HepG2 cells were exposed to IVI between 0 and 24 h,

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.

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
chelatable iron pool 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 lM 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 lMiron 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

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).

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