Tài liệu Báo cáo khoa học: Multidentate pyridinones inhibit the metabolism of nontransferrin-bound iron by hepatocytes and hepatoma cells docx

Multidentate pyridinones inhibit the metabolism
of nontransferrin-bound iron by hepatocytes and hepatoma cells
Anita C. G. Chua
1
, Helen A. Ingram
1
, Kenneth N. Raymond
2
and Erica Baker
1
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%
saturatedwithFe.However,insomecases,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 l
M
) 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:
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)
Eur. J. Biochem. 270, 1689–1698 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03525.x
documents 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
59
Fe as FeCl
3
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 Ô3-Hydroxy-2-
(1H)-Pyridinone Chelating AgentsÕ and no. 5,892,029,

April6,1999Ô3-Hydroxy-2(1H)-Pyridinone Chelating
AgentsÕ.
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
56
FeSO
4
/
59
FeCl
3
(both in 0.1
M
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 37 °C and then added
to an isotonic solution of MEM containing 20 m
M

Hepes/
Tris and 10 mg BSAÆmL
)1
to give a final concentration of
1 l
M
Fe and 100 l
M
citrate 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
)1
streptomycin, 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 m
M
). The cells were
incubated for 0–3 h at 37 °C, after which the amount of
radioactivity internalized by the cells was estimated by
incubation with pronase (1 mgÆmL
)1
balanced salt solution)
for 30 min at 4 °C 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 37 °C 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) Ó FEBS 2003
membrane, 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
)1
diet)
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 (n ¼ 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).
Ó FEBS 2003 Pyridinones inhibit iron uptake by liver cells (Eur. J. Biochem. 270) 1691
Results
Kinetics 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 (n ¼ 11) nmol Fe
per g DNA per 2 h, over 10-fold greater than hepatoma
cells, which took up 1457 ± 124 (n ¼ 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) · 10
6
and (26 ± 2) · 10
6
for 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 with
59

Fe-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 m
M
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) Ó FEBS 2003
but 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
cellular
59
Fe than blocking NTB-
59
Fe 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 intracellular
59
Fetakenupover
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 m
M
.BothDFPand
Tren-N-Me-3,2-HOPO at 0.1 m
M
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
wasaseffectiveasDFPandDFO,reducingFeuptaketo
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),forupto1hat37°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.
Ó FEBS 2003 Pyridinones inhibit iron uptake by liver cells (Eur. J. Biochem. 270) 1693
Effect 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 accumulation 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)
3
species (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) Ó FEBS 2003
Chelator 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 iron-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
Control 100 100 100 100
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
Ó FEBS 2003 Pyridinones inhibit iron uptake by liver cells (Eur. J. Biochem. 270) 1695
locus 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 m
M
,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 of
59
Fe in ferritin
did not change significantly in the Fe mobilization studies,
although there was a slight increase in
59
Fe 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.
Table 4. The effect of chelators on aspartate aminotransferase (AST) release from hepatocytes and Q7 hepatoma cells. Cells were incubated with ferric citrate for 2 h at 37 °C in the presence or absence of
chelators (uptake studies) or were incubated with 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 (mobilization
studies). AST release was measured at every step of the experiments as described in Materials and methods. Results are the mean and standard deviation from a typical experiment (n ¼ 3), and are expressed
as a percentage of total AST present in the cells.
Chelator
Uptake (% total) Mobilization (% total)
Hepatocytes Q7 Hepatocytes Q7

Incubation
medium
Membrane
fraction
Incubation
medium
Membrane
fraction
Incubation
medium
Reincubation
medium
Membrane
fraction
Incubation
medium
Reincubation
medium
Membrane
fraction
Control 3.63 ± 0.21 0.83 ± 0.23 0.89 ± 0.17 0.32 ± 0.36 2.47 ± 0.43 2.45 ± 0.17 0.76 ± 0.10 2.59 ± 0.61 1.29 ± 0.30 0.58 ± 0.47
DFP 0.1 m
M
3.03 ± 0.19 0.61 ± 0.05 1.04 ± 0.13 0.38 ± 0.18 2.78 ± 0.08 2.21 ± 0.09 0.57 ± 0.12 1.79 ± 0.14 0.70 ± 0.51 0.16 ± 0.24
DFP 1 m
M
5.33 ± 1.59 0.87 ± 0.43 1.05 ± 0.16 0.42 ± 0.16 2.74 ± 0.32 2.43 ± 0.22 0.58 ± 0.17 2.16 ± 0.46 1.21 ± 0.18 0.05 ± 0.00
DFO 0.1 m
M
3.10 ± 0.88 0.46 ± 0.11 0.76 ± 0.42 0.20 ± 0.16 2.43 ± 0.41 3.49 ± 0.55 0.56 ± 0.26 1.73 ± 0.91 1.20 ± 0.87 0.39 ± 0.03

DFO 1 m
M
2.54 ± 0.50 0.69 ± 0.05 0.78 ± 0.20 0.43 ± 0.12 2.61 ± 0.19 3.27 ± 0.36 0.54 ± 0.06 2.04 ± 0.32 1.15 ± 0.16 0.14 ± 0.21
Tren-N-Me-3,2-HOPO 0.1 m
M
3.03 ± 0.03 0.25 ± 0.36 0.84 ± 0.24 0.32 ± 0.28 2.56 ± 0.80 2.31 ± 0.71 0.49 ± 0.28 1.36 ± 0.92 1.88 ± 0.06 0.27 ± 0.20
Tren-N-Me-3,2-HOPO 1 m
M
0.56 ± 0.12 0.56 ± 0.12 2.04 ± 1.40 0.32 ± 0.36 2.50 ± 0.28 3.52 ± 0.50 0.32 ± 0.15 1.93 ± 0.41 2.07 ± 0.28 0.04 ± 0.01
Tren-Bis-3,2-HOPO 1 m
M
4.47 ± 4.65 1.22 ± 0.72 0.84 ± 0.73 0.66 ± 0.69 2.56 ± 0.31 3.64 ± 1.10 0.54 ± 0.04 1.90 ± 0.25 2.44 ± 0.68 0.04 ± 0.03
5L1O 1 m
M
1.36 ± 0.63 0.45 ± 0.21 3.36 ± 0.79 0.18 ± 0.15 2.31 ± 0.15 3.91 ± 0.82 0.50 ± 0.03 2.05 ± 0.36 1.78 ± 0.51 0.56 ± 0.28
1696 A. C. G. Chua et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Fe-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 m
M
). 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.
References
1. Batey, R.G., Fong, P.L.C., Shamir, S. & Sherlock, S. (1980) A
non-transferrin-bound serum iron in idiopathic hemochromatosis.
Dig. Dis. Sci. 25, 340–346.
2. Gutteridge, J.M.C., Rowley, D.A., Griffiths, E. & Halliwell, B.
(1985) Low-molecular-weight iron complexes and oxygen

radical reactions in idiopathic hemochromatosis. Clin. Sci. 68,
463–467.
3. Grootveld, M., Bell, J.D., Halliwell, B., Aruoma, O.I., Bomford,
A. & Sadler, P.J. (1989) Non-transferrin-bound iron in plasma or
serum from patients with idiopathic hemochromatosis. Char-
acterization by high performance liquid chromatography and
nuclear magnetic resonance spectroscopy. J. Biol. Chem. 264,
4417–4422.
4. Hershko,C.,Graham,G.,Bates,G.&Rachmilewitz,E.A.(1978)
Non-specific serum iron in thalassaemia: an abnormal serum iron
fraction of potential toxicity. Br.J.Haematol.40, 235–263.
5. Graham, G., Bates, G.W., Rachmilewitz, E.A. & Hershko, C.
(1979) Nonspecific serum iron in thalassaemia: quantitation and
chemical reactivity. Am. J. Hematol. 6, 207–217.
6. Bradley, S.J., Gosriwitana, I., Srichairatanakool, S., Hider, R.C.
& Porter, J.B. (1997) Non-transferrin-bound iron induced by
myeloablative chemotherapy. Br.J.Haematol.99, 337–343.
7. Carmine, T.C., Evans, P., Bruchelt, G., Evans, R., Handgretinger,
R., Niethammer, D. & Halliwell, B. (1995) Presence of iron
catalytic for free radical reactions in patients undergoing chemo-
therapy: implications for therapeutic management. Cancer Lett.
94, 219–226.
8. Breuer, W., Hershko, C. & Cabantchik, Z.I. (2000) The
importance of non-transferrin bound iron in disorders of iron
metabolism. Transfus. Sci. 23, 185–192.
9. Sarkar, B. (1970) State of Fe (III) in normal human serum: low
molecular weight and protein ligands besides transferrin. Can. J.
Biochem. 48, 1339–1350.
10. Baker, E., Baker, S.M. & Morgan, E.H. (1998) Characterisation
of non-transferrin-bound iron (ferric citrate) uptake by rat hepa-

tocytes in culture. Biochim. Biophys. Acta 1380, 21–30.
11. Hershko, C. (1994) Control of disease by selective iron depletion: a
novel therapeutic strategy utilizing iron chelators. Baillieres Clin.
Haematol. 7, 965–1000.
12. Hider, R.C., Choudhury, R., Rai, B.L., Dehkordi, L.S. & Singh, S.
(1996) Design of orally active iron chelators. Acta Haematol. 95,
6–12.
13. Olivieri, N.F., Brittenham, G.M., McLaren, C.E., Templeton,
D.M.,Cameron,R.G.,McClelland,R.A.,Burt,A.D.&Fleming,
K.A. (1998) Long-term safety and effectiveness of iron-chelation
therapy with deferiprone for thalassaemia major. N.Engl.J.Med.
339, 417–423.
14. Wong, A., Alder, V., Robertson, D., Papadimitriou, J., Maserei,
J., Berdoukas, V., Kontoghiorghes, G., Taylor, E. & Baker, E.
(1997) Liver iron depletion and toxicity of the iron chelator
Deferiprone (L1, CP20) in the guinea pig. Biometals 10, 247–256.
15. Huebers, H., Huebers, E., Rummel, W. & Crichton, R.R. (1976)
Isolation and characterization of iron-binding proteins from rat
intestinal mucosa. Eur. J. Biochem. 66, 447–455.
16. Baker, E., Page, M. & Morgan, E.H. (1985) Transferrin and iron
release from rat hepatocytes in culture. Am.J.Physiol.248,G93–
G97.
17. Richardson, D.R. & Baker, E. (1992) Two mechanisms of iron
uptake from transferrin by melanoma cells: The effect of desfer-
rioxamine and ferric ammonium citrate. J. Biol. Chem. 267,
13972–13979.
18. Hinegardner, R.T. (1971) An improved fluorometric assay for
DNA. Anal. Biochem. 39, 197–201.
19. Yeoh, G.C.T., Bennett, F.A. & Oliver, I.T. (1979) Hepatocte
differentiation in culture. Appearance of tyrosine aminotrans-

ferase. Biochem. J. 180, 153–160.
20. Bacon, B.R., Tavill, A.S., Brittenham, G.M., Park, C.H. &
Recknagel, R.O. (1983) Hepatic lipid peroxidation in vivo in rats
with chronic iron overload. J. Clin. Invest. 71, 429–439.
21. Nakamura, T., Mori, M. & Awai, M. (1988) Process of carbonyl
iron absorption from the rat duodenal mucosa. Acta Physiol. Jpn.
38, 1377–1390.
22. Hirsh, M., Konijn, A.M. & Iancu, T.C. (2002) Acquisition, sto-
rage and release of iron by cultured hepatoma cells. J. Hepatol. 36,
30–38.
23. Baker, E., Trinder, D., Torrance, J., Page, M. & Morgan, E.H.
(1988) Characteristics of iron exchange between transferrin and
hepatocytes in culture. In Thalassaemia, Pathophysiology and
Management (Fucharoen,S.,Rawley,P.T.&Paul,N.W.,eds),pp.
41–61. Alan R. Liss, New York, USA.
24. Trinder, D., Batey, R., Morgan, E.H. & Baker, E. (1990) Effect of
cellular iron concentration on iron uptake by hepatocytes. Am. J.
Physiol. 259, G611–G617.
25. Baker, E., Richardson, D.R., Gross, S. & Ponka, P. (1992) Eva-
luation of the iron chelation potential of hydrazones of pyridoxal,
salicylaldehyde and 2-hydroxy-1-naphthylaldehyde using the
hepatocyte in culture. Hepatol. 15, 492–501.
Ó FEBS 2003 Pyridinones inhibit iron uptake by liver cells (Eur. J. Biochem. 270) 1697
26. Graham, R.M., Morgan, E.H. & Baker, E. (1998) Characterisa-
tion of citrate and iron citrate uptake by cultured rat hepatocytes.
J. Hepatol. 29, 603–613.
27. Trinder, D. & Morgan, E.H. (1997) Inhibition of uptake of
transferrin-bound iron by hepatoma cells by non-transferrin
bound iron. Hepatol. 26, 691–698.
28. O’Sullivan, B., Xu, J. & Raymond, K.N. (2000) New multidentate

chelators for iron. In Iron Chelators: New Development Strategies,
pp. 177–208. Saratoga Publishing Group, Ponte Vedra, Florida,
USA.
29. Yokel, R.A., Fredenburg, A.M., Durbin, P.W., Xu, J., Rayens,
M.K. & Raymond, K.N. (2000) The hydroxypyridononate Tren-
(Me-3,2-HOPO) is a more orally active iron chelator than its
bidentate analaogue. J. Pharm. Sci. 89, 545–555.
30. Richardson, D.R., Ponka, P. & Baker, E. (1994) The effect of
the iron (III) chelator, desferrioxamine, on iron and transferrin
uptake by the human malignant melanoma cell. Cancer Res. 54,
685–689.
31. Pippard, M.J., Johnson, D.K. & Finch, C.A. Hepatocytes iron
kinetics in the rat explored with an iron chelator. Br.J.Haematol.
52, 221–224.
32. Morgan, E.H. & Baker, E. (1986) Iron uptake and metabolism by
hepatocytes. Fed. Proc. 45, 2801–2816.
33. Parkes, J.G., Hussain, R.A., Olivieri, N.F. & Templeton, D.M.
(1993) Effects of iron loading on uptake, speciation, and chelation
of iron in cultured myocardial cells. J. Laboratory Clin. Med. 122,
36–47.
34. Parkes, J.G., Randell, E.W., Olivieri, N.F. & Templeton, D.M.
(1995) Modulation of iron loading and chelation of the uptake
of non-transferrin-bound iron by human liver cells. Biochim.
Biophys. Acta 1243, 373–380.
35. Parkes, J.G., Olivieri, N.F. & Templeton, D.M. (1997) Char-
acterisation of Fe
2+
and Fe
3+
transport by iron-loaded cardiac

myocytes. Toxicol. 117, 141–151.
36. Randell, E.W., Parkes, J.G., Olivieri, N.F. & Templeton, D.M.
(1994) Uptake of non-transferrin-bound iron by both reductive
and nonreductive processes is modulated by intracellular iron.
J. Biol. Chem. 269, 16046–16053.
37. Richardson, D.R. & Ponka, P. (1995) Identification of a
mechanism of iron uptake by cells which is stimulated by hydroxyl
radicals generated via the iron-catalysed Haber-Weiss reaction.
Biochim. Biophys. Acta 1269, 105–114.
38. Agarwal, M.B., Gupte, S.S., Viswanathan, C., Vasandani, D.,
Ramanathan, J., Desai, N., Puniyani, R.R. & Chablani, A.T.
(1992) Long-term assessment of efficacy and safety of L1, an oral
iron chelator, in transfusion dependent thalassaemia: Indian trial.
Br.J.Haematol.82, 460–466.
39. Al-Refaie, F.N., Hershko, C., Hoffbrand, A.V., Kosaryan,
M., Olivieri, N.F., Tondury, P. & Wonke, B. (1995) Results
of long-term deferiprone (L1) therapy: a report by the Inter-
national Study Group on oral iron chelators. Br.J.Haematol.91,
224–229.
40. Pati, H.P. & Choudhury, V.P. (1999) Deferiprone (L1) associated
neutropenia in beta thalassaemia major: an Indian experience.
Eur. J. Haematol. 63, 267–268.
41. Tondury, P., Kontoghiorghes, G.J., Ridolfi-Luthy, A., Hirt, A.,
Hoffbrand, A.V., Lottenbach, A.M., Sonderegger, T. & Wagner,
P. (1990) L1 (1,2-dimethyl-3-hydroxypyrid-4-one) for oral iron
chelation in patients with beta-thalassaemia major. Br.J.Hae-
matol. 76, 550–553.
42. Richardson, D.R. (2001) The controversial role of deferiprone
in the treatment of thalassemia. J. Laboratory Clin. Med. 137,
324–329.

43. Aydinok,Y.,Nisli,G.,Kavakli,K.,Coker,C.,Kantar,M.&
Cetingul, N. (1999) Sequential use of deferiprone and desferriox-
amine in primary school children with thalassaemia major in
Turkey. Acta Haematol. 102, 17–21.
44. Diav-Citrin, O., Anatackovic, G. & Koren, G. (1999) An
investigation into variability in the therapeutic response to defer-
iprone in patients with thalassaemia major. Ther. Drug Monit. 21,
74–81.
45. Taher, A., Chamoun, F.M., Koussa, S., Saad, M.A., Khoriaty,
A.I., Neeman, R. & Mourad, F.H. (1999) Efficacy and side effects
of deferiprone (L1) in thalassaemia patients not compliant with
deferoxamine. Acta Haematol. 101, 173–177.
46. Wonke, B., Wright, C. & Hoffbrand, A.V. (1998) Combined
therapy with deferiprone and desferrioxamine. Br.J.Haematol.
361–364.
47. Chenoufi, N., Drenou, B., Loreal, O., Pigeon, C., Brissot, P. &
Lescoat, G. Antiproliferative effect of deferiprone on the Hep G2
cell line. Biochem. Pharmacol. 56, 431–437.
48. Cragg,L.,Hebbel,R.P.,Miller,W.,Solovery,A.,Selby,S.&
Enright, H. (1998) The iron chelator L1 potentiates oxidative
DNA damage in iron-loaded liver cells. Blood 92, 632–638.
1698 A. C. G. Chua et al.(Eur. J. Biochem. 270) Ó FEBS 2003