The intracellular distribution of iron taken up as NTBI changed in the presence of chelators suggesting that the chelators may act intracellularly as well as at the cell membrane.. In co
Trang 1Multidentate pyridinones inhibit the metabolism
of nontransferrin-bound iron by hepatocytes and hepatoma cells
Anita C G Chua1, Helen A Ingram1, Kenneth N Raymond2and Erica Baker1
1
Physiology, School of Biomedical and Chemical Sciences, University of Western Australia, Crawley, Western Australia, Australia, 2
Department of Chemistry, University of California, Berkeley, California, USA
The therapeutic effect of iron (Fe) chelators on the
poten-tially toxic plasma pool of nontransferrin-bound iron
(NTBI), often present in Fe overload diseases and in some
cancer patients during chemotherapy, is of considerable
interest In the present investigation, several multidentate
pyridinones were synthesized and compared with their
bidentate analogue, deferiprone (DFP; L1, orally active) and
desferrioxamine (DFO; hexadentate; orally inactive) for
their effect on the metabolism of NTBI in the rat
hepato-cyte and a hepatoma cell line (McArdle 7777, Q7)
Hepa-toma cells took up much less NTBI than the hepatocytes
(< 10%) All the chelators inhibited NTBI uptake
(80–98%) much more than they increased mobilization of Fe
from cells prelabelled with NTBI (5–20%) The hexadentate
pyridinone,
N,N,N-tris(3-hydroxy-1-methyl-2(1H)-pyridi-none-4-carboxaminoethyl)amine showed comparable
acti-vity to DFO and DFP There was no apparent correlation
between Fe status, Fe uptake and chelator activity in hepatocytes, suggesting that NTBI transport is not regulated
by cellular Fe levels The intracellular distribution of iron taken up as NTBI changed in the presence of chelators suggesting that the chelators may act intracellularly as well as
at the cell membrane In conclusion (a) rat hepatocytes have
a much greater capacity to take up NTBI than the rat hep-atoma cell line (Q7), (b) all chelators bind NTBI much more effectively during the uptake phase than in the mobilization
of Fe which has been stored from NTBI and (c) while DFP is the most active chelator, other multidentate pyridinones have potential in the treatment of Fe overload, particularly at lower, more readily clinically available concentrations, and during cancer chemotherapy, by removing plasma NTBI Keywords: non-transferrin bound iron; liver cells; iron che-lation therapy; chemotherapy
Iron (Fe) is transported in blood plasma bound tightly in a
nontoxic form to the plasma iron-binding protein,
trans-ferrin (Tf) Under normal conditions, Tf is 20–50%
saturated with Fe Howev er, in some cases, particularly
when the concentration of Fe in the plasma exceeds the
Fe-binding capacity of Tf, there is additional Fe circulating
in non-Tf bound forms (NTBI) This is of particular
concern in diseases of Fe overload such as the genetic
disorder hemochromatosis [1–3], in which there is an
abnormally high absorption of Fe leading to saturation of
the plasma Tf Patients with the hereditary anemia
thalassemia [4,5] also have increased plasma Fe, primarily
due to the obligatory treatment of the anemia with blood
transfusions The contribution of plasma NTBI to the
toxicity associated with Fe overload in these disorders is
uncertain, as is the form of NTBI Significant levels of NTBI in plasma also occur in cancer as a result of some chemotherapeutic regimes [6–8] The source of this Fe, its toxicity, and whether it can be cleared by the liver or taken
up by cancer cells and used in Fe-dependent reactions essential for growth and proliferation, is uncertain Hence, it
is of interest to investigate the uptake and metabolism of NTBI in normal and cancer cells, and the effect of Fe chelators on these processes
In the present study we have characterized these processes
in the rat hepatocyte and its neoplastic counterpart, the rat hepatoma cell line (Q7) The form of NTBI used was ferric citrate, as several studies indicate that citrate (normal plasma concentration, 70–150 lM) may be a major NTBI transport molecule in the plasma under Fe overload conditions, and is also implicated in intracellular Fe metabolism [3,9,10] An important aspect of this work was the assessment of the effect of novel Fe chelators on the uptake and fate of NTBI and to investigate the potential of these chelators for therapeutic use in Fe overload diseases and cancer chemotherapy Desferrioxamine (DFO), the only chelator in widespread clinical use, is expensive and not active when given orally [11,12] Deferiprone (DFP, L1; 1,2-dimethyl,3-OH pyridin-4-one; CP 20), the most pro-mising alternative, is in extensive clinical trials and is orally active However, there is some evidence of toxicity [13,14] which may be related to its bidentate nature, due to the formation of transient intermediate Fe complexes with chelator/Fe ratios of 1 : 1 and 2 : 1 before formation of the stable hexadentate 3 : 1 complex The present study
Correspondence to E Baker, Physiology, School of Biomedical and
Chemical Sciences, Faculty of Life and Physical Sciences,
University of Western Australia, 35 Stirling Highway,
Crawley 6009, Western Australia.
Fax: + 61 8 93801025, Tel.: + 61 8 93803932,
E-mail: baker@cyllene.uwa.edu.au
Abbreviations: DFO, desferrioxamine; DFP, deferiprone (L1);
MEM, minimum essential medium; NTBI, nontransferrin-bound
iron; PIH, pyridoxal isonicotinoyl hydrazone; Tf, transferrin;
Tren-N-Me,3,2-HOPO,
N,N,N-tris(3-hydroxy-1-methyl-2(1H)-pyridinone-4-carboxaminoethyl)amine.
(Received 16 October 2002, revised 7 February 2003,
accepted 14 February 2003)
Trang 2documents the effects of DFP and novel tetradentate and
hexadentate analogues on NTBI uptake and mobilization
from rat hepatocytes and hepatoma cells DFO was
included as a reference chelator
Materials and methods
Animals
Hepatocytes were obtained from 7- to 10-week-old-male
Wistar rats The procedure was approved by the Animal
Experimentation Ethics Committee of the University of
Western Australia and is in accordance with the Australian
Code of Practice for the care and use of animals for scientific
purposes as well as the guidelines published by the National
Institutes of Health, USA Animals were fed on the normal
chow diet (control) or diet supplemented with carbonyl Fe
and had access to water ad libitum
Reagents
59Fe as FeCl3 was obtained from Dupont (North Ryde,
Australia) Collagenase H and pronase were from
Boehrin-ger Mannheim (Mannheim, Germany) Eagle’s Minimum
Essential Medium (MEM) was purchased from Flow
Laboratories (Irvine, Scotland) Foetal bovine serum and
insulin were both supplied by Commonwealth Serum
Laboratories (Melbourne, Australia), Fungizone was from
Trace Bioscience (Sydney, Australia), penicillin and
gluta-mine from Gibco BRL (Auckland, New Zealand) and
streptomycin sulphate from Calbiochem (La Jolla, USA)
The synthetic medium, Ultroser G was purchased from
Sepracor (la Garenne, France) Hepes and bovine serum
albumin (BSA) were from Sigma (St Louis, USA) Tris
(hydroxymethyl) methylamine was from BDH Chemicals,
Australia All other chemicals were of analytical reagent
quality and purchased from Sigma or Ajax (Sydney,
Australia)
Chelators
DFO was purchased from Sigma (St Louis, USA) The
multidentate pyridinones were provided by the research
group of K N Raymond (University of California,
Berkeley, USA) and prepared as described previously in
US patents no 5,624,901, April 29, 1997
3-Hydroxy-2-(1H)-Pyridinone Chelating Agents and no 5,892,029,
April 6, 1999 3-Hydroxy-2(1H)-Pyridinone Chelating
Agents
The structures of the chelators used are presented in
Fig 1, and include the hexadentate
N,N,N-tris(3-hyd-roxy-1-methyl-2(1H)-pyridinone-4-carboxaminoethyl)amine
(Tren-N-Me-3,2-HOPO), Tren-Bis-3,2-HOPO-Bis-acetic
acid and DFO, tridentate pyridoxal isonicotinoyl hydrazone
(PIH), tetradentate 4LI-Me-3,2-HOPO (4L1) and
5LIO-Me-3,2-HOPO (5L1O) and the bidentate deferiprone
(DFP)
Protein purification
Ferritin was isolated using the method of Huebers and
colleagues [15] and used to raise an antiserum in rabbits [16]
Radiolabelling of ferric citrate Ferric citrate solution was prepared by adding 56FeSO4/
59FeCl3 (both in 0.1M HCl; pH 1.3) at a molar ratio of
10 : 1 Trisodium citrate (pH 8.0) was then added to the mixture to yield a Fe/citrate ratio of 1 : 100 (pH 5.5) The mixture was incubated for 10 min at 37C and then added
to an isotonic solution of MEM containing 20 mMHepes/ Tris and 10 mg BSAÆmL)1to give a final concentration of
1 lMFe and 100 lMcitrate at pH 7.4 It has been shown that all the Fe is converted to the ferric form over the 10 min incubation [10]
Isolation and culture of rat hepatocytes Adult rat hepatocytes were isolated and cultured after liver perfusion with collagenase (0.05%) as described previously [10,16]
Culture of rat hepatoma cells The rat hepatoma cell line, McA-RH 7777 (McArdle Laboratory for Cancer Research, Wisconsin) was grown in MEM containing 100 lgÆmL)1streptomycin, 3.75 lgÆmL)1 Fungizone, 100 UÆmL)1 penicillin and 10% fetal bovine serum, and were seeded on tissue culture plates The plates were used when cells had reached 90–100% confluency
Experimental procedures Uptake studies Hepatocytes and hepatoma cells were washed with Hank’s balanced salt solution and the medium replaced with the incubation medium (MEM/Hepes/Tris/ BSA, pH 7.4) containing the radiolabelled ferric citrate with
or without chelators (0.1 and 1 mM) The cells were incubated for 0–3 h at 37C, after which the amount of radioactivity internalized by the cells was estimated by incubation with pronase (1 mgÆmL)1balanced salt solution) for 30 min at 4C to release external membrane-bound Fe [10,17] Cells were then centrifuged and separated into supernatant (membrane-bound Fe), and cell pellet (inter-nalized Fe) An aliquot of the cell suspension was taken for DNA estimation, by the method of Hinegardner [18,19] All data were calculated as nmol FeÆg)1 DNA to correct for variation in cell density The efficacy of chelators was calculated from changes in internalized Fe levels expressed
as a percentage of the control in each experiment Some uptake data were also expressed as molecules per cell using our measured values of 21 ± 2 pg DNA per hepatocyte (mean ± SEM; n ¼ 5) and 31 ± 1 pg DNA per hepatoma cell (mean ± SEM; n ¼ 5)
Mobilization studies Cells were preincubated with the radiolabelled ferric citrate in MEM containing Hepes/Tris/ BSA (pH 7.4) at 37C for 2 h The cell monolayer was then washed before reincubation with the control medium (no chelator) or the test media (with chelators) for 2 h The efflux medium was then collected and the cells treated as in the uptake experiments
Subcellular fractionation In several experiments the incorporation of radioactive Fe into stroma-mitochondrial
1690 A C G Chua et al (Eur J Biochem 270) FEBS 2003
Trang 3membrane, ferritin and ferritin-free cytosol was also
measured as previously described [16]
Fe-loading in vivo Hepatocytes were Fe-loaded in vivo by
feeding 3-week-old male Wistar rats 2% (20 gÆkg)1diet)
carbonyl Fe (pure form of elemental Fe) for 8 weeks prior
to cell isolation [20,21] The nonheme Fe levels in the
livers of the Fe-loaded animals were 10-fold greater than
the controls In the isolated Fe-loaded hepatocytes,
nonheme Fe was sixfold greater, 0.89 ± 0.08 and
5.56 ± 1.3 nmolÆlg)1 DNA for control and Fe-loaded
rats, respectively (n¼ 6) Uptake and mobilization studies
were performed on these Fe-loaded hepatocytes for comparison with normal hepatocytes, using a wider range
of chelators
Toxicity studies Aspartate aminotransferase (AST) release from cells was measured using the optimized AST kit purchased from Sigma This was carried out at every step
of the experiments to assess chelator toxicity The morpho-logy of the cells was also monitored
Results are expressed as mean ± SD unless stated otherwise Student’s unpaired t-test was used to determine any significant difference at the 95% confidence level
Fig 1 Structures of chelators DFP (bidentate); PIH (tridentate); 4L1, 5L1, 5L1O (tetradentate); DFO, Tren-Me-3,2-HOPO, Tren-bis-3,2-HOPO (hexadentate).
Trang 4Kinetics of NTBI uptake
Hepatocytes took up and rapidly internalized NTBI over
time, up to at least 3 h incubation (Fig 2A) Fe uptake by
hepatoma cells was linear over the same 3 h time period but
the rate of binding and internalization was very much
slower (Fig 2B) The membrane-bound Fe uptake was
approximately 10% of the total Fe uptake at 3 h in both the
hepatocytes and hepatoma cells There was a marked
difference in NTBI uptake between the two cell types, with
hepatocytes internalizing 19624 ± 4068 (n¼ 11) nmol Fe
per g DNA per 2 h, over 10-fold greater than hepatoma
cells, which took up 1457 ± 124 (n¼ 9) nmol Fe per g
DNA over a 2-h incubation When uptake was expressed as
atoms Fe per cell, the values for Fe internalization were
(239 ± 50)· 106and (26 ± 2)· 106for hepatocytes and
hepatoma cells, respectively
Mobilization of NTBI The release of Fe from membrane-bound and intracellular pools was measured during reincubation after a 2-h preincubation with59Fe-citrate in hepatocytes and hepa-toma cells There was little release of Fe from either Fe pool
in both cell types over 2 h reincubation in the absence of added chelators (not shown) Internalized Fe in hepatoma cells decreased slightly ( 5%) The membrane bound
Fe also fell slightly The hepatocytes exhibited similar characteristics
Effect of chelators on uptake of NTBI The effects of the chelators on NTBI uptake by hepatocytes and Q7 hepatoma cells were marked and similar All chelators decreased uptake to 20% or less of the controls DFP and DFO almost completely abolished NTBI uptake
at both 0.1 and 1 mM chelator concentrations (Fig 3) The other pyridinones tested in this series were also effective (80–90% inhibition) On the whole, the chelators were slightly more effective at reducing NTBI uptake in hepato-cytes than in hepatoma cells The hexadentate molecule Tren-N-Me-3,2-HOPO decreased Fe uptake by 90% in hepatocytes but only 80% in hepatoma cells at the same concentration The tetradentate molecule, 5L1O, was also active, decreasing uptake to about 10% of the control in both cell types
Kinetics of NTBI uptake in the presence of chelators (Fig 4) showed that DFP and DFO were the most active chelators, almost blocking membrane and internalized Fe uptake at all time points in hepatocytes (Fig 4), and in hepatoma cells (not shown) The tridentate chelator, PIH, was almost as effective Tren-N-Me-3,2-HOPO, a hexaden-tate chelator like DFO, with a similar Fe-binding affinity
Fig 2 Kinetics of NTBI uptake from ferric citrate by (A) hepatocytes
and (B) hepatoma cells Cells were incubated for 0–3 h with
radio-labelled ferric citrate (1 l M Fe:100 l M citrate) at 37 C Internalized
Fe (s) and membrane bound Fe (h) are shown.
Fig 3 Effect of chelators on NTBI uptake from citrate (1 l M
Fe/100 l M citrate) by hepatocytes (h) and hepatoma cells (j) Cells were incubated for 2 h with or without chelators at 0.1 and 1 m M at
37 C Tren HOPO, Tren-N-Me-3,2-HOPO.
1692 A C G Chua et al (Eur J Biochem 270) FEBS 2003
Trang 5but orally active, was not as effective as DFO in inhibiting
NTBI uptake but still reduced uptake to less than 10% of
the control
Effect of chelators on Fe mobilization after
preincubation with NTBI
All the chelators studied were less effective in mobilizing
cellular59Fe than blocking NTB-59Fe uptake (Fig 3) and
were similar in both hepatocytes and hepatoma cells
(Fig 5) DFP was the most active chelator in both cell
types, releasing 20% of intracellular59Fe taken up ov er
2 h in hepatoma cells and in hepatocytes (Fig 5)
Tren-N-Me-3,2-HOPO, DFO and 5L1O were less effective,
releasing approximately 10% at 1 mM Both DFP and
Tren-N-Me-3,2-HOPO at 0.1 mM reduced the amount of
internalized Fe in hepatoma cells Interestingly, an increase
in chelator concentration by 10-fold had no significant effect
on cellular Fe, suggesting Fe mobilization is limited by the
size of an intracellular chelatable Fe pool or permeability of
the Fe–chelator complex
In view of the similar efficacy of the multidentate
pyridinones to DFO, further experiments were conducted
using a wider range of pyridinones on normal and
Fe-loaded hepatocytes
Comparison of normal and Fe-loaded hepatocytes Effect of hepatocyte Fe loading on NTBI uptake There was no apparent difference in the uptake and internalization
of NTBI by normal hepatocytes and Fe-loaded hepatocytes
at any time point (Fig 6A) The mean rates of Fe internalization were 25349 ± 4022 and 24061 ± 635 nmol
Fe per g DNA per 2 h for normal and Fe-loaded hepatocytes, respectively The proportion of NTBI incor-porated into ferritin increased with time to 60% after 2 h (Fig 6B) The difference between normal and Fe-loaded cells was not statistically significant
Effect of hepatocyte Fe-loading on chelator activity in Fe uptake studies The activities of DFO and five pyridinone chelators were compared in normal and Fe-loaded hepato-cytes (Table 1) The bidentate DFP and the hexadentate DFO almost totally inhibited NTBI uptake in both cell types The hexadentate Tren-Bis-3,2-HOPO-Bis-acetic acid was as effectiv e as DFP and DFO, reducing Fe uptake to approximately 1% of the control, while the other chelators 5L1O, 4L1 and Tren-N-Me-3,2-HOPO were less active, although inhibition was still 90–95% Apart from Tren-N-Me-3,2-HOPO, Fe-loading in vivo did not affect the efficacy of the chelators at the concentration used
Effect of hepatocyte Fe-loading on chelator activity in Fe mobilization studies All chelators decreased internalized
Fe levels slightly compared to the controls (Table 1) There was, however, little difference in the activity of the chelators
in Fe-loaded hepatocytes compared to normal hepatocytes DFP, 5L1O and Tren-N-Me-3,2-HOPO reduced internal-ized Fe significantly to 75–80% control (P < 0.05), in the normal hepatocytes The decrease in internalized Fe caused
by chelators in the Fe-loaded hepatocytes was similar
Fig 5 Effect of chelators on Fe internalized from NTBI over a 2-h reincubation at 37 °C in hepatocytes (h) and hepatoma cells (j) Cells were prelabelled for 2 h with radioactive ferric citrate (1 l M Fe/100 l M
citrate) and then reincubated with chelators Tren HOPO, Tren-N-Me-3,2-HOPO.
Fig 4 Kinetics of NTBI uptake in the presence or absence of chelators
by hepatocytes Cells were incubated with ferric citrate (1 l M Fe/
100 l M citrate) ± 0.1 mMTren HOPO (s), DFP (n), DFO (e) and
PIH (h), for up to 1 h at 37 C Kinetics of NTBI uptake in the
presence of DFO and DFP were very similar, hence symbols overlap.
Tren HOPO, Tren-N-Me-3,2-HOPO.
Trang 6Effect of chelators and Fe status on subcellular
distribu-tion of NTBI in hepatocytes The effect of the chelators on
the intracellular distribution of Fe in normal and Fe-loaded
hepatocytes in the Fe uptake and mobilization studies are
shown in Tables 2 and 3, respectively In the Fe uptake
studies in normal hepatocytes, all chelators caused a major
shift of Fe from the ferritin fraction to the cytosolic
compartment, and a slight shift to the membrane-bound
fraction, particularly by 4L1 (Table 2) This change in
intracellular distribution with all chelators suggests they act
intracellularly as well as extracellularly In contrast, only
DFP, DFO and Tren-N-Me-3,2-HOPO caused a major shift
in Fe distribution to the cytosolic compartment in Fe-loaded hepatocytes There was little effect on intracellular distribu-tion of Fe by 5L1O, while 4L1 increased Fe accumuladistribu-tion in the membrane-bound compartment, as seen in normal hepatocytes In the Fe mobilization studies, there appeared
to be a shift from the membrane-bound fraction to the cytosolic compartments, while the proportion of Fe incor-porated into ferritin remained within the range obtained for the control (Table 3) The changes in the cellular distribution
of Fe in the Fe uptake studies were much more marked compared to those observed in the Fe mobilization studies
Toxicity studies Chelators showed no apparent cytotoxicity Table 4 shows AST release values observed for hepatocytes and a typical hepatoma cell line (Q7) There was no obvious morpho-logical change detected by phase contrast microscopy and only 1–6% of cellular AST was released under all conditions
by all chelators
Discussion The main aims of this study were: (a) To examine NTBI uptake and metabolism in normal and Fe-loaded hepato-cytes, and neoplastic liver cells, using rat hepatocytes and the rat hepatoma cell line (Q7); (b) to determine the effect of chelators on uptake, intracellular distribution and mobiliza-tion of NTBI; (c) to assess and compare the activity and toxicity of novel tetradentate and hexadentate pyridinones with bidentate DFP, whose toxicity may be due to the transient formation of reactive ligand-Fe species before forming the stable Fe(L)3species (see Richardson, 2001 [41]) Both hepatocytes and hepatoma cells took up NTBI in the form of Fe-citrate, however, the hepatocytes accumu-lated NTBI at a far greater rate than the rat hepatoma cell line in confluent culture (over 10-fold faster) In addition, hepatocyte uptake of NTBI was not regulated by intracel-lular Fe levels, as judged by the lack of effect of a sixfold increase in hepatocyte nonheme Fe This strongly suggests a role for the liver in binding, storing and detoxifying excess body Fe in the form of plasma NTBI While these hepatoma cells took up much less NTBI than hepatocytes, they did take up a significant amount Indeed, another study on other hepatoma cell lines has shown a much higher uptake
of NTBI [22] but the uptake times and concentration employed were different to that used in the current study This is of concern as several studies have shown the presence
of NTBI in the plasma of patients undergoing chemother-apy for cancer [6] This may be Fe derived from reticulo-endothelial cells, accumulating in the plasma while the marrow is ablated Thus it appears possible that cancer cells could take up NTBI and utilize it in cell proliferation
In contrast to NTBI uptake by Q7 cells, the uptake of Tf-bound Fe (TBI) by receptor-mediated endocytosis is much lower in hepatocytes in comparison with hepatoma cells [23] and regulated by intracellular nonheme Fe levels [24] However, the intracellular distribution of Fe from these two sources are similar (Table 2 cf 16, 25) and competition studies indicate that NTBI and TBI have at least one common step in uptake by hepatocytes [25–27]
Fig 6 Kinetics of NTBI uptake and incorporation into ferritin in
nor-mal and iron-loaded cells (A) Kinetics of NTBI uptake from ferric
citrate by normal (h) and Fe-loaded (s) hepatocytes Cells were
incubated for 0–2 h with radiolabelled ferric citrate (1 l M Fe/100 l M
citrate) at 37 C (B) Fe incorporation into ferritin by normal (h) and
Fe-loaded (s) hepatocytes as a percentage of the total, over 0–2 h
uptake.
1694 A C G Chua et al (Eur J Biochem 270) FEBS 2003
Trang 7Chelator uptake studies revealed that DFO and DFP
were the most potent compounds DFO, the only widely
used chelator in clinical therapy for Fe overload diseases,
almost completely abolished NTBI uptake in both the
hepatocytes and hepatoma cells DFO could possibly
chelate Fe directly from citrate as it has a much higher
affinity for Fe The ferrioxamine complex (Fe-chelator
complex) does not donate Fe to cells [16] DFP, like DFO, almost completely blocked NTBI uptake Its mechanism of inhibition may be similar to that of DFO DFO is a relatively slow-permeating chelator compared to DFP, as it
is a much more hydrophilic molecule Hence its ability to rapidly (within 2 min) and almost totally block Fe uptake from citrate, with the same kinetics as DFP suggests that
Table 1 Effect of chelators on internalization of NTBI by normal and loaded hepatocytes, and on Fe internalized from NTBI by normal and iron-loaded hepatocytes For uptake assays, cells were incubated for 2 h at 37 C in the presence or absence of 1 m M chelator, after which the cells were treated with pronase as described in Materials and methods For mobilization results, cells were incubated for 2 h with radiolabelled ferric citrate at
37 C and then reincubated with medium with or without 1 m M chelator for 2 h at 37 C The cells were then treated with pronase as described in Materials and methods Results are the mean and standard deviation of iron internalized by normal and iron-loaded hepatocytes from three separate experiments, and are expressed as a percentage of the control.
Chelator
Uptake (% control cells) Mobilization (% control cells) Normal Fe-loaded Normal Fe-loaded DFP 0.18 ± 0.09 0.10 ± 0.05 77.7 ± 6.4 81.0 ± 10.8 4L1 5.16 ± 0.94 4.80 ± 0.59 82.9 ± 7.3 85.7 ± 16.0 5L1O 9.31 ± 0.78 10.39 ± 1.42 76.3 ± 5.8 85.7 ± 14.0 Tren-N-Me-3,2-HOPO 9.41 ± 0.45 4.25 ± 0.31 80.4 ± 7.5 80.6 ± 12.6 Tren-Bis-3,2-HOPO 1.36 ± 0.48 0.72 ± 0.02 91.1 ± 7.6 96.1 ± 24.7 DFO 0.11 ± 0.05 0.10 ± 0.03 89.7 ± 9.1 87.6 ± 13.2
Table 2 The effect of chelators on the cellular distribution of iron taken up from NTBI by normal and Fe-loaded hepatocytes Cells were incubated with radiolabelled ferric citrate for 2 h in the presence or absence of 1 m M chelator at 37 C The cells were then fractionated into the subcellular compartments; membrane-bound, cytosol and ferritin, as described in Materials and methods Results are presented as mean ± SD, from three separate experiments, and are expressed as a percentage of total iron taken up from NTBI.
Chelator
Normal hepatocytes Fe-loaded hepatocytes Membrane Cytosol Ferritin Membrane Cytosol Ferritin Control 15.8 ± 4.5 7.2 ± 1.7 77.0 ± 2.8 26.0 ± 1.1 5.8 ± 0.3 68.2 ± 0.8 DFP 19.2 ± 0.5 34.7 ± 10.5 46.2 ± 10.9 27.1 ± 6.4 30.6 ± 3.2 42.3 ± 9.6 DFO 20.2 ± 16.0 46.0 ± 3.0 33.8 ± 13.0 25.6 ± 2.5 47.4 ± 27.4 27.1 ± 24.9 Tren-N-Me-3,2-HOPO 23.6 ± 5.4 40.1 ± 22.5 36.4 ± 17.0 23.6 ± 6.4 18.3 ± 6.1 58.0 ± 0.4 Tren-Bis-3,2-HOPO 24.8 ± 5.0 19.3 ± 10.3 56.0 ± 5.3 30.8 ± 17.8 9.6 ± 3.5 48.6 ± 3.2 5L1O 27.8 ± 10.8 26.3 ± 19.4 45.8 ± 8.6 23.0 ± 8.8 8.8 ± 4.5 67.4 ± 5.2 4L1 31.8 ± 0.9 20.6 ± 14.2 47.7 ± 13.3 38.2 ± 2.8 15.8 ± 4.0 53.0 ± 1.3
Table 3 The effect of chelators on the cellular distribution of iron taken up from NTBI by normal and Fe-loaded hepatocytes following a 2 h reincubation with chelators Cells were incubated with radiolabelled ferric citrate for 2 h at 37 C, followed by reincubation with medium containing
no chelator (control) or chelators at 1 m M for 2 h at 37 C Cells were then fractionated into the subcellular compartments; membrane bound, cytosol and ferritin, as described in Materials and methods Results are presented as mean ± SD, from three separate experiments, and are expressed as a percentage of total iron taken up from NTBI.
Chelator
Normal hepatocytes (% total) Fe-loaded hepatocytes (% total) Membrane Cytosol Ferritin Membrane Cytosol Ferritin Control 15.1 ± 1.9 5.4 ± 2.0 79.5 ± 3.8 19.6 ± 5.2 3.9 ± 1.8 76.5 ± 4.1 DFP 9.9 ± 1.4 10.4 ± 1.3 79.7 ± 2.1 10.8 ± 1.9 9.9 ± 0.6 79.3 ± 2.0 DFO 7.9 ± 0.8 8.6 ± 2.1 82.3 ± 1.6 11.9 ± 3.9 8.8 ± 3.4 80.2 ± 1.4 Tren-N-Me-3,2-HOPO 8.9 ± 0.4 10.4 ± 0.6 80.4 ± 1.5 12.7 ± 4.4 7.0 ± 1.4 80.2 ± 3.2 Tren-Bis-3,2-HOPO 11.8 ± 1.5 8.6 ± 1.2 79.6 ± 1.0 14.7 ± 2.9 5.1 ± 1.1 81.1 ± 2.2 5L1O 13.2 ± 2.9 8.9 ± 2.5 78.0 ± 1.4 15.2 ± 2.5 5.8 ± 2.1 79.0 ± 3.5 4L1 12.9 ± 1.9 7.0 ± 0.8 80.0 ± 2.4 15.7 ± 4.5 4.2 ± 0.9 81.6 ± 6.1
Trang 8locus of action of both chelators is extracellular However, changes in the intracellular distribution of Fe (Table 2), with a big increase in the proportion of Fe in the cytosol suggest that all the chelators including DFO and DFP, act
in part intracellularly, mobilizing Fe from ferritin but forming Fe-chelator complexes too hydrophilic to permeate cell membranes, thus accumulating in the cytosol
Tren-N-Me-3,2-HOPO was only slightly less effective than DFP, and showed no toxicity even at 1 mM, as assessed from the lack of change in cell morphology and AST release (Table 4) This is most encouraging as higher ligand denticity (the number of Fe binding sites per molecule) leads to a smaller chelator concentration require-ment for complexation (for full details, see [28]) Thus, the hexadentate molecule with six Fe binding ligands, e.g Tren-N-Me-3,2-HOPO (Fig 1) will retain its high affinity for Fe
at low, therapeutically relevant concentrations, contrasting with a sharp decrease in affinity for the bidentate molecule, DFP, which requires three molecules to provide the six Fe binding sites necessary for stable complexation [28] In addition, Tren-N-Me-3,2-HOPO was almost as active as DFO in vitro and there is some evidence suggesting that it may be orally active in the promotion of Fe excretion in Fe-loaded rats [29] Tetradentate 5L1O, like Tren-N-Me-3,2-HOPO, was almost as effective as DFP and DFO It is possible that 5L1O binds Fe in a less stable intermediate form than Tren-N-Me-3,2-HOPO and DFO, which may be less permeable to cells
The chelators markedly altered the intracellular distribu-tion of Fe in the uptake studies (Table 2), with a much greater proportion of Fe in the ferritin-free cytosolic compartment This suggests that the chelators may also be acting intracellularly, inhibiting Fe incorporation into ferritin In comparison, the proportion of59Fe in ferritin did not change significantly in the Fe mobilization studies, although there was a slight increase in59Fe in the cytosol, derived from the membrane-bound fraction (Table 3) There was also a relatively low amount of Fe mobilized,
at least in the 2 h reincubation period used in this study These results indicate that the chelators may be acting on Fe present in a small transient or labile intracellular Fe pool, with limited access to Fe already incorporated into ferritin DFP has been postulated to exert an effect on the intracellular labile Fe pool and ferritin in cancer cells (melanoma), by diffusing into cells and chelating Fe from these Fe pools, thus causing significant Fe mobilization [30] Our results do suggest the presence of an intracellular Fe pool but do not suggest DFP has the ability to mobilize
Fe directly from ferritin in hepatocytes It has been proposed that there are at least two distinct intracellular
Fe subpools in rat hepatocytes, one of which is affiliated with Fe from endosomes, the other from lysosomal release
of Fe [31,32]
Even though the nonheme Fe level in hepatocytes Fe-loaded in vivo was 6-fold greater than that of the control hepatocytes, there was no significant effect of Fe-loading on NTBI uptake, in agreement with our previous observations [10] There was also no apparent effect of Fe-loading on intracellular distribution and chelator activity DFP, 5L1O and Tren-N-Me-3,2-HOPO were the most effective chela-tors in reducing internalized Fe following reincubation after prelabelling both normal and Fe-loaded hepatocytes
1696 A C G Chua et al (Eur J Biochem 270) FEBS 2003
Trang 9Fe-loading in vitro with ferric ammonium citrate, however,
can lead to an up-regulation of NTBI uptake mechanisms
[33–36], although this may be accompanied by a risk of cell
membrane damage due to lipid peroxidation [37] There was
no cytotoxicity apparent in the hepatocytes or hepatoma
cells in the present work in the presence or absence of any
chelator (even at 1 mM) However, the exposure times were
relatively short
The use of DFP as a therapeutic drug in the treatment of
Fe overload diseases is controversial DFP administration
has been found to cause toxic effects in some studies [13,
38–40] while not in others [41–44] Despite this controversy,
DFP is still considered to have potential as a chelator,
particularly for the treatment of Fe overload [45] Also,
Olivieri et al [13] only reported toxicity in patients
admini-stered with DFP for about 4.5 years In 1998, Wonke and
colleagues [46] administered DFP and DFO as a combined
form of therapy and found no toxicity from either drug,
with a promising drop in serum ferritin levels in patients
accompanied by an increased urinary Fe excretion While
DFO and DFP are used to treat Fe overload, their potential
as antineoplastic agents is also being assessed DFP inhibits
Fe uptake and cellular proliferation in liver cells [47,48]
However, further investigations are required to assess
DFP’s potential as an anticancer drug Our preliminary
assessment suggests multidentate pyridinones such as
Tren-N-Me-3,2-HOPO are also potential candidates for the
treatment of Fe overload, particularly as they may be orally
active [29] Further studies with appropriate detailed dose–
response curves and varying exposure times to these
chelators are warranted
Acknowledgements
The current work was supported by the National Health and Medical
Research Council of Australia and NIH grant DK57814 (KNR) The
authors would also like to thank Sharyn Baker, Anthony Kicic, Jide Xu
and Kristy Clarke Jurchen for skillful technical assistance.
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