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Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 632-638
The Iron Chelator L1 Potentiates Oxidative DNA Damage in
Iron-Loaded Liver Cells
By
Louise Cragg,
Robert P. Hebbel,
Wesley Miller,
Alex Solovey,
Scott Selby, and
Helen Enright
From the Department of Medicine-Hematology, University of Minnesota
Medical School, Minneapolis, MN.
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ABSTRACT |
Iron-mediated carcinogenesis is thought to occur through the
generation of oxygen radicals. Iron chelators are used in attempts to
prevent the long term consequences of iron overload. In particular, 1,2-dimethyl-3-hydroxypyrid-4-one (L1), has shown promise as an effective chelator. Using an established hepatocellular model of iron
overload, we studied the generation of iron-catalyzed oxidative DNA
damage and the influence of iron chelators, including L1, on such
damage. Iron loading of HepG2 cells was found to greatly exacerbate
hydrogen peroxide-mediated DNA damage. Desferrithiocin was protective
against iron/hydrogen peroxide-induced DNA damage; deferoxamine had no
effect. In contrast, L1 exposure markedly potentiated hydrogen
peroxide-mediated oxidative DNA damage in iron-loaded liver cells.
However, when exposure to L1 was maintained during incubation with
hydrogen peroxide, L1 exerted a protective effect. We interpret this as
indicating that L1's potential toxicity is highly dependent on the
L1:iron ratio. In vitro studies examining iron-mediated ascorbate
oxidation in the presence of L1 showed that an L1:iron ratio must be at
least 3 to 1 for L1 to inhibit the generation of free radicals; at
lower concentrations of L1 increased oxygen radical generation occurs.
In the clinical setting, such potentiation of iron-catalyzed oxidative
DNA damage at low L1:iron ratios may lead to long-term toxicities that
might preclude administration of L1 as an iron chelator. Whether this
implication in fact extends to the in vivo situation will have to be
verified in animal studies.
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INTRODUCTION |
IRON IS THOUGHT to play a role in
carcinogenesis through the generation of oxygen free
radicals.1,2 In the hereditary iron overload condition of
increased iron absorption, hemochromatosis, hepatic iron accumulation
causes fibrosis, cirrhosis, and ultimately hepatocellular carcinoma in
25% to 35% of untreated homozygotes.3-6 The prevalence of
neoplastic disease may be higher in  -thalassemia heterozygotes than
in the general population.7 In addition, epidemiological
studies have implicated increased body iron stores in the pathogenesis
of lung and intestinal carcinomas.8-10 Iron, mainly in its
non-protein-bound, low molecular weight form, causes cellular damage by
participating in the generation of the hydroxyl radical,11
thought to be the principal effector of oxidative DNA damage and
mutagenesis.12
Iron chelators are presently used in attempts to prevent the long-term
consequences of iron overload. Deferoxamine is the most widely used
agent, but has significant disadvantages, including its high cost,
parenteral method of administration, toxicity, and frequent
noncompliance, leading to a search for a safe, orally active
alternative.11 Recently, encouraging results have been reported using the orally active iron chelator
1,2-dimethyl-3-hydroxypyrid-4-one (L1). This agent is presently being
studied in clinical trials and appears to be effective with an
acceptable toxicity profile.13-16 Both deferoxamine and L1
have been shown to inhibit oxygen radical formation in vitro, but their
use as therapeutic agents for this purpose has not yet been fully
examined.17,18
Using an established hepatocellular model of iron overload, we studied
the generation of iron-catalyzed oxidative DNA damage in the form of
DNA single-strand breaks. In addition, we examined the influence of
iron chelators, including L1, on such iron-mediated DNA damage.
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MATERIALS AND METHODS |
Cell culture.
The human hepatocellular line HepG2 (American Type Culture Collection,
Rockville, MD) was grown in monolayer in Dulbecco's Modified Eagle
Media supplemented with 10% fetal bovine serum (FBS), 100 U/mL
penicillin G, 100 µg/mL streptomycin, and 25 ng/mL amphotericin B, at
37°C in 5% CO2. As indicated below, in some experiments, cells were maintained for short periods in media without
serum and rendered iron-free by the addition of a chelating resin,
iminodiacetic acid (iron-free media). Except for the deliberate addition of iron, all buffers were similarly made iron-free, the resin
being removed by filtration immediately before use.
Iron loading of HepG2 cells.
Nontransferrin-bound iron loading of HepG2 cells using ferric citrate
was performed as previously described.19,20 Ferric citrate
solution (final concentration of ferric iron 10 mmol/L; molar
iron:citrate ratio 1:10) was freshly prepared for each experiment. Cells were exposed to 1 mmol/L ferric citrate in media for 20 hours.
Cell viability was maintained under these conditions for at least this
length of time. Both HepG2 cells in media alone and in media with
iron-free citrate served as controls.
Exposure of iron-loaded HepG2 cells to hydrogen peroxide.
After exposure to ferric citrate, cells were washed twice with
iron-free phosphate buffered saline (PBS) and exposed to 100 µM
hydrogen peroxide in iron-free media for 30 minutes at 37°C. After
hydrogen peroxide exposure, cells were washed twice and harvested by
trypsinization. Viable cells were isolated by Nycoprep gradient
centrifugation. Cell viability was assessed by trypan blue exclusion
and was consistently greater than 90%. DNA single-strand breaks were
then assessed as described below.
Exposure of iron-loaded HepG2 cells to iron chelators.
For some experiments, iron-loaded HepG2 cells were incubated with
various iron chelators before hydrogen peroxide exposure. For this,
cells were washed with iron-free PBS and then incubated for 1 hour with
one of the following iron chelators in iron-free media: deferoxamine
mesylate, desferrithiocin, or L1. Concentrations used are indicated in
the result section and figure legends. After exposure to a chelator,
cells were again washed with iron-free PBS before exposure to hydrogen
peroxide. In experiments in which L1 exposure was maintained,
incubation with hydrogen peroxide occurred in iron-free media
containing 1 mmol/L L1.
Measurement of DNA single-strand breaks by alkaline elution.
Alkaline elution was performed as described,21,22 with some
modifications.23 Briefly, HepG2 cells in exponential growth phase were labeled with [3H]thymidine (35 Ci/mmol,
1µCi/mL, 1 µmol/L in tissue culture medium) for 24 hours and then
exposed to oxidant stress with or without iron chelators as described
above. Ice-cold suspensions of 2 × 105 radiolabeled
cells were then lysed on 2.0 µm polycarbonate filters using lysis
solution (25 mmol/L Na2 EDTA, 2% sodium dodecyl
sulfate (SDS), 40 mmol/L glycine, pH 9.6, 0.85 mg/mL proteinase K).
Single-strand breaks were assessed by alkaline elution of DNA from the
filters using elution buffer (20 mmol/L Na2EDTA, 54 mmol/L tetraethylammonium hydroxide, pH 12.3). The elution rate was
0.03 mL/minute with fractions collected at 30 minute intervals.
Radioactivity in each fraction and that remaining on the filter was
determined by liquid scintillation counting (Beckman LS 5000TD, Palo
Alto, CA). All studies were performed in dim light to
prevent ultraviolet light-induced DNA damage. To ensure that measured
DNA damage was not caused by available intracellular iron during cell
lysis or DNA elution, additional control experiments were performed.
The addition of 10 µmol/L ferrous iron to the cell lysis and DNA
elution buffers did not increase DNA strand breaks, indicating little
oxygen radical generation under the conditions of cell lysis and DNA
elution (4°C; pH 12.3). Furthermore, the addition of deferoxamine
during cell lysis and DNA elution did not result in decreased DNA
damage. Results are reported as the mean ± standard error of the mean of three experiments performed in duplicate unless otherwise stated.
Ascorbate oxidation by iron: Effect of iron chelators.
Fresh solutions of 5 mmol/L phosphate buffer (pH 7.4) containing 100 µmol/L sodium ascorbate, 30 µmol/L ferric iron, and increasing concentrations of L1 (0 to 180 µmol/L) or deferoxamine (0 to 90 µmol/L) were incubated for 40 minutes at room temperature. The rate
of ascorbate consumption was measured by absorbance spectroscopy (Beckman DU 70 spectrophotometer) at 265 nm at 10 and 40 minutes. A 0.1 decrease in absorbance has been determined to be equivalent to 70 µmol/L of oxidized ascorbate.24
Statistics.
For alkaline elution experiments, unless otherwise stated, all values
are presented as the mean ± standard error of the mean of a given
number of independent experiments. The data were evaluated using the
unpaired Student's t-test: P values less than .05 were considered to be statistically significant. For ascorbate oxidation experiments, a single representative experiment is shown, with points
indicating the mean ± standard deviation of 9 replicates.
Materials.
The following materials were purchased from Sigma (St Louis, MO):
deferoxamine mesylate, sodium ascorbate, ferric iron, hypoxanthine, 2-deoxy-D-ribose, xanthine oxidase, thiobarbituric acid, and
trichloroacetic acid. Hydrogen peroxide and proteinase K were obtained
from Fischer Biotech (Pittsburgh, PA). Nycoprep was
purchased from GIBCO BRL (Gaithersburg, MD),
3H-thymidine from ICN Corp (Costa Mesa, CA),
and the 2 µmol/L polycarbonate filters from Costar (Cambridge,
MA). Desferrithiocin was a gift from I. Schraufstätter (Research Institute of Scripps Clinic, La Jolla,
CA) and L1 was kindly provided by Drs N. Olivieri (Hospital for Sick
Children, Toronto, Canada) and R. McClelland (University of Toronto,
Toronto, Canada). The L1 was synthesized as described by
Kontoghiorghes and Sheppard25 and was greater than 98%
pure.26
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RESULTS |
DNA damage in iron-loaded HepG2 cells.
Oxidative DNA damage after iron loading of HepG2 cells with subsequent
hydrogen peroxide exposure is shown in Fig
1. Iron loading alone did not lead to increased DNA single-strand
breaks compared with control cells exposed to media. Even in the
absence of iron loading, hydrogen peroxide caused a significant degree of strand cleavage, with approximately 50% of the total cellular DNA
eluting through the filter. When iron-loaded cells were exposed to
hydrogen peroxide, oxidative DNA damage was greatly exacerbated (85%
DNA eluting through the filter). No strand cleavage was observed in
control cells exposed to citrate buffer alone (data not shown).
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| Fig 1.
Generation of DNA single-strand breaks in iron-loaded
HepG2 cells exposed to hydrogen peroxide. Iron-loaded cells were
exposed to 100 µmol/L hydrogen peroxide for 30 minutes. Cells were
harvested and DNA single-strand breaks were quantified by alkaline
elution. Shown are DNA strand breaks in control cells exposed to media alone ( ), iron-loaded cells ( ), hydrogen peroxide-exposed cells, and iron-loaded cells exposed to hydrogen peroxide. The results are the
mean ± SEM of three experiments performed in duplicate (*P < .03 compared with hydrogen peroxide exposed cells).
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Influence of iron chelators on oxidative DNA damage in iron-loaded
HepG2 cells.
The influence of the iron chelators deferoxamine (which penetrates
cells poorly), and of desferrithiocin and L1 (both of which are readily
cell permeable) on hydrogen peroxide-mediated DNA damage in
iron-loaded HepG2 cells was examined. Deferoxamine, desferrithiocin,
and L1 form hexadentate, tridentate, and bidentate ligands with iron,
respectively.27 Hence, to compare these chelators at
equipotent iron-chelating concentrations, the following concentrations were used: deferoxamine, 0.33 mmol/L; desferrithiocin, 0.67 mmol/L; and
L1, 1 mmol/L. Iron-loaded HepG2 cells were incubated with each chelator
for 1 hour, then washed in iron-free PBS before exposure to hydrogen
peroxide. Determination of DNA single-strand breaks by alkaline elution
was then performed. Although deferoxamine failed to protect cells from
iron/hydrogen peroxide-mediated oxidative DNA damage, desferrithiocin
did have a protective effect, with a dramatic decrease in the amount of
DNA single strand breaks generated (Fig 2).
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| Fig 2.
Effect of deferoxamine and desferrithiocin on
iron/hydrogen peroxide-mediated DNA single-strand breaks. (A)
Iron-loaded HepG2 cells were exposed to 0.33 mmol/L deferoxamine for 1 hour before exposure to 100 µmol/L hydrogen peroxide for 30 minutes.
(B) Iron-loaded HepG2 cells were incubated with 0.67 mmol/L
desferrithiocin before exposure to hydrogen peroxide. Shown are DNA
strand breaks in control cells exposed to media alone, iron-loaded
cells exposed to hydrogen peroxide, iron-loaded cells exposed to
deferoxamine before exposure to hydrogen peroxide, and iron-loaded
cells exposed to desferrithiocin before exposure to hydrogen peroxide.
The results are the mean ± SEM of three experiments performed in
duplicate (*P < .03 compared with iron-loaded cells exposed
to hydrogen peroxide).
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In contrast to desferrithiocin and deferoxamine, L1 markedly
potentiated hydrogen peroxide-mediated oxidative DNA damage in iron-loaded liver cells (Fig 3). Exposure
of control cells to L1 alone yielded no damage, and exposure to L1
followed by hydrogen peroxide caused slightly increased damage compared
with exposure to hydrogen peroxide alone (data not shown). The effects
of 1 mmol/L and 10 mmol/L L1 on hydrogen peroxide-induced DNA damage were then compared. The alkaline elution profiles obtained showed no
differences in the DNA-damage potentiating effect at either concentration, with 94% of the total DNA eluting through the filter at
1 mmol/L L1 and 95% at 10 mmol/L L1. In contrast to this potentiation of damage after sequential exposure of iron-loaded cells to L1 and
hydrogen peroxide (with cell washing in between), when L1 exposure was
maintained during incubation with hydrogen peroxide, L1 did protect
against oxidative DNA damage (Fig 4). This
suggests that the ability of L1 to influence iron-catalyzed oxygen
radical generation is highly dependent on the effective intracellular L1:iron ratio. Furthermore, inclusion of desferrithiocin during incubation with L1 partially reversed its DNA-damage potentiating effect (Fig 5).
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| Fig 3.
Effect of L1 on iron/hydrogen peroxide-induced DNA
single-strand breaks. Iron-loaded HepG2 cells were incubated in 1 mmol/L L1 for 1 hour, followed by exposure to 100 µmol/L hydrogen
peroxide for 30 minutes. Shown are DNA strand breaks in control cells
exposed to media alone, iron-loaded cells exposed to hydrogen peroxide, and iron-loaded cells exposed to L1 before exposure to hydrogen peroxide. The results are the mean ± SEM of four experiments
performed in duplicate (*P < .03 compared with iron-loaded
cells exposed to hydrogen peroxide).
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| Fig 4.
Effect of sustained L1 exposure on iron/hydrogen
peroxide-mediated DNA single-strand breaks. Iron-loaded liver cells
were incubated in 1 mmol/L L1 for 1 hour, followed by exposure to 100 µmol/L hydrogen peroxide for 30 minutes during which exposure to 1 mmol/L L1 was maintained. Illustrated is a representative experiment
performed in duplicate and the values obtained were averaged. Shown are
DNA single-strand breaks in control cells exposed to media alone,
iron-loaded cells exposed to hydrogen peroxide, and iron-loaded cells
exposed to hydrogen peroxide in the presence of L1.
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| Fig 5.
Combined effect of desferrithiocin and L1 on
iron/hydrogen peroxide-mediated DNA single-strand breaks. Iron-loaded
liver cells were simultaneously exposed to 1 mmol/L L1 and 0.67 mmol/L
desferrithiocin for 1 hour, followed by incubation with 100 µmol/L
hydrogen peroxide for 30 minutes in the absence of either chelator.
Illustrated is a representative experiment performed in duplicate and
the values obtained were averaged. Shown are DNA single-strand breaks in control cells exposed to media alone, iron-loaded cells exposed to
hydrogen peroxide, iron-loaded cells exposed to desferrithiocin followed by hydrogen peroxide, iron-loaded cells exposed to L1 followed
by hydrogen peroxide, and iron-loaded cells exposed to both
desferrithiocin and L1 followed by hydrogen peroxide.
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Iron-mediated ascorbate oxidation in the presence of increasing
concentrations of iron chelator.
The measurement of ascorbate oxidation to compare the influence of
different iron chelators on iron-dependent free radical damage has
previously been described.24 We used this method to examine
the influence of the L1:iron ratio on oxygen radical generation in
vitro and compared this to the effect of deferoxamine. Even at low
concentrations, addition of deferoxamine to the reaction solution
caused a decrease in ascorbate consumption
(Fig 6A). Ascorbate consumption continued
to decrease in a dose-dependent manner until a plateau was reached at
30 µmol/L deferoxamine (equimolar concentration with iron). In
contrast, L1 showed a bimodal effect on ascorbate oxidation (Fig 6B).
Initially, at low L1 concentration, augmentation of ascorbate oxidation
was observed. The initial rate of oxidation increased from 42 µmol/L/0.5 hour to 70 µmol/L/0.5 hour at 70 µmol/L L1. A further
increase in L1 concentration resulted in decreased ascorbate
consumption, dropping below the baseline oxidation level only at 90 µmol/L L1 (L1:iron ratio 3:1).
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| Fig 6.
Ascorbate oxidation by iron: effect of deferoxamine and
L1. Increasing concentrations of chelator were added to fresh solutions of phosphate buffer containing 100 µmol/L sodium ascorbate and 30 µmol/L ferric ion. The rate of ascorbate consumption was measured by
absorbance spectroscopy at 265 nm at 10 and 40 minutes. (A) Ascorbate
oxidation in response to increasing concentrations of deferoxamine (0 to 90 µmol/L). (B) Ascorbate oxidation in response to increasing
concentrations of L1 (0 to 180 µmol/L). Shown is a single
representative experiment with points indicating the mean ± the
standard deviation of nine replicates.
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DISCUSSION |
Current evidence suggests that transition metals, in particular iron,
react with hydrogen peroxide in cell nuclei leading to oxygen radical
generation and consequent DNA damage.28-30 The iron-catalyzed generation of oxygen radicals is believed to mediate its
mutagenic and carcinogenic effects.31 In situations of iron overload, as occurs in thalassemia and hemochromatosis, iron chelators have been shown to prevent and reverse iron-mediated tissue
damage.32 In addition, in vitro data suggest that iron
chelators can inhibit iron-catalyzed oxygen radical
formation.17,18 This has led us to postulate that
administration of iron chelators may decrease the risk of
carcinogenesis in hemochromatosis. However, our results suggest that
the iron chelator L1 may, under some circumstances, sensitize cells to
iron-induced DNA damage.
Iron-mediated oxidative DNA damage has previously been studied in
iron-loaded hepatic nuclei33 and in murine hybridoma
cells.34 The use of the human hepatocellular line HepG2 for
the study of iron-catalyzed DNA damage may be more physiologically
relevant.35 Effective iron-loading of HepG2 cells using
55Fe complexed with citrate has been previously
shown.19 Iron citrate was used because citrate is one of
the main physiologic chelators comprising nontransferrin-bound iron. In
iron overload states, elevated plasma levels of nontransferrin-bound
iron are thought to be an important cause of hepatic iron
loading.36 Furthermore, studies using this methodology have
shown that cellular iron accumulation occurred at concentrations (400 µg/100 mg protein) similar to those found in hepatic tissue from
patients with hemochromatosis.3,37 For the present studies,
we did not quantitate bulk iron uptake by HepG2 cells, nor potential
chelator-induced bulk iron removal, because such measurements would
show nothing about the iron compartment that is relevant to DNA damage,
ie, nuclear iron, which cannot be accurately quantitated. The marked
sensitization of iron-loaded cells to hydrogen peroxide-mediated
oxidative DNA damage that we observed however, implies effective iron
loading resulting in an increased amount of available iron for
participation in the Fenton reaction. It is likely that iron-catalyzed
oxidative DNA damage fueled by lower levels of endogenous hydrogen
peroxide occurs over time in vivo, as shown by animal data showing
evidence of increasing oxidative DNA damage with aging.38
Iron chelators have been shown to inhibit the production of oxygen
radical species resulting in decreased lipid peroxidation and oxidative
DNA damage both in vitro and in vivo.11,28,39,40 The iron chelators used in the current study included deferoxamine, desferrithiocin, and L1. All three chelators have high iron affinity and specificity. Deferoxamine's failure to affect oxidative DNA damage
in this model probably relates to its poor cellular permeability. Indeed, cellular uptake of deferoxamine and subsequent release of
intracellular iron has been shown to occur more slowly than for
hydroxypyridione chelators.41,42 In contrast,
desferrithiocin, which readily penetrates cells,27 does
inhibit iron-mediated DNA strand cleavage, as expected.
The paradoxical potentiation of iron-mediated DNA damage by L1 cannot
be explained by a direct toxic effect of the chelator, because exposure
of HepG2 cells to L1 alone yielded no damage. Rather, L1's effect
might be explained by the stoichiometry of its interaction with iron.
L1 forms a bidentate ligand with iron as follows:
The
log cumulative association constants for these reactions are 15, 27, and 36 respectively.43 Low concentrations of L1 may in fact
facilitate iron-mediated oxygen radical generation.11,44 We
hypothesized that this is due to a greater facility of
[FeL]2+ or [FeL2]+ as compared
with free or protein-bound iron to participate in the Fenton reaction,
thereby leading to increased hydroxyl radical production.27
Thus, after exposure to 1 mmol/L L1, the resultant intracellular L1
concentration during hydrogen peroxide exposure may not be high enough
to fully chelate iron as [FeL3], thereby allowing
increased DNA damage. We postulated that increasing the extracellular
L1 concentration to 10 mmol/L would result in an increased
intracellular L1:iron ratio, with increased [FeL3]
formation and decreased iron-catalyzed oxidative DNA damage. Instead,
we found that exposure to 10 mmol/L L1 caused similar potentiation of
iron-mediated DNA damage as 1 mmol/L L1. This failure of increased L1
concentration to reduce iron-mediated oxidative DNA damage may be due
to L1's relatively high cellular permeability.45,46 After
exposure to both L1 concentrations, the iron-loaded HepG2 cells had
been washed in iron-free PBS before exposure to hydrogen peroxide.
During washing and hydrogen peroxide exposure, noniron-bound L1 could
rapidly diffuse down its concentration gradient out of cells, resulting
in lower intracellular levels of L1 that would lead to potentiation of
iron-catalyzed oxidative DNA damage. Thus, increasing the initial
concentration of L1 from 1 to 10 mmol/L would not necessarily result in
an increased intracellular concentration of L1 during hydrogen peroxide
exposure when oxidative DNA damage is produced.
When experimental conditions were modified to maintain L1 exposure
during incubation with hydrogen peroxide, thereby maintaining the
intracellular L1 concentration, a dramatic reduction in iron-catalyzed DNA damage ensued. This supports our hypothesis that potentiation of
iron-mediated DNA damage by L1 is dependent on the intracellular L1:iron ratio. The protective effect against DNA damage of continuous incubation with L1 also argues against the possibility that
potentiation of oxidative DNA damage is due to an inhibitory effect on
DNA repair. Furthermore, addition of a second chelator,
desferrithiocin, partially reversed L1's DNA damaging effect. This is
probably mediated by competitive inhibition between the two chelators
and supports our hypothesis that L1's toxic effect is due to its iron chelating properties.
The in vitro observation of a bimodal pattern of ascorbate oxidation by
iron in the presence of increasing concentrations of L1 supports the
concept that L1's ability to potentiate or to inhibit iron-catalyzed
oxygen free radical formation in vivo is highly dependent on the
L1:iron ratio. It appears that this ratio must be at least 3:1 for L1
to inhibit the generation of free radicals; at lower concentrations of
L1 increased oxygen radical generation occurs.
L1 has been developed as a potential alternative to deferoxamine in the
treatment of iron overload, to be used on a widespread clinical scale
in patients with iron overload conditions such as thalassemia and
hemochromatosis. Initial clinical experience with L1 has shown it to be
an effective chelator, inducing increased urinary iron excretion,
decreased ferritin levels and decreased hepatic iron.15,16
However, the potentiation of iron-catalyzed oxidative DNA damage at low
L1:iron ratios that we observed in these in vitro studies of HepG2
cells has important potential clinical implications. L1 is given orally
on a daily basis with an elimination half-life of approximately 90 minutes.47 The maximum plasma concentration of L1 after a
single oral dose (50 mg/kg) in patients with iron overload is
approximately 300 µmol/L.47 This plasma concentration is
lower than that used experimentally (1 mmol/L). As iron-catalyzed
oxidative DNA damage in vitro occurs at low L1:iron ratios, one might
expect that at pharmacologically achieved levels of L1 in vivo,
iron-mediated DNA damage would be even more likely to occur. The main
toxicities of L1 recognized at this time include severe neutropenia,
agranulocytosis, arthropathy, gastrointestinal intolerance, and liver
enzyme abnormalities.11,48-50 The mechanism of these
toxicities is not known, though acceleration of hydroxyl radical
formation by incompletely complexed L1:iron has been
proposed.11,49,51 Controlled animal studies have shown that
iron-overloaded gerbils administered the hydroxypyridione CP94
(1,2-diethyl-3-hydroxypyrid-4-one) developed cardiac and hepatic
fibrosis.52 There have been no reports of an increased incidence of malignancy in patients receiving L1. However, the long
latent period required for the development of human malignancy does not
preclude such an effect. Notwithstanding these implications, our
findings in this limited in vitro study will have to be corroborated in
vivo by appropriate animal studies to determine if they are clinically
relevant.
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FOOTNOTES |
Submitted June 24, 1997;
accepted March 6, 1998.
Supported in part by National Institute of Health grants CA65021
(H.E.), HL30160, and HL37528 (R.P.H.).
Address Correspondence to Helen Enright, MD, The Adelaide and Meath
Hospital, Iallaght, Dublin 24, Ireland.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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Abstract
Introduction
Abstract
![Fe<SUP>3+</SUP> + L<SUP>−</SUP> ↔ [FeL]<SUP>2+</SUP>](/content/vol92/issue2/fulltext/632/img001.gif)
![Fe<SUP>3+</SUP> + 2L<SUP>−</SUP> ↔ [FeL<SUB>2</SUB>]<SUP>+</SUP>](/content/vol92/issue2/fulltext/632/img002.gif)
![Fe<SUP>3+</SUP> + 3L<SUP>−</SUP> ↔ [FeL<SUB>3</SUB>]](/content/vol92/issue2/fulltext/632/img003.gif)