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Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada
Received February 23, 2003; accepted May 20, 2003
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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;
Hasinoff et al., 1998). There
is now considerable evidence to indicate that this toxicity may be due to
iron-dependent oxygen free radical formation
(Meyers, 1998) on the
relatively unprotected cardiac muscle. Dexrazoxane likely acts by diffusing
into the cell and hydrolyzing (Hasinoff,
1994a,1994b,
1998;
Hasinoff et al., 1998) to its
ring-opened metal ion-binding form, ADR-925
(Fig. 1), which has a structure
similar to that of EDTA. ADR-925 may then either remove iron from the
iron-doxorubicin complex (Buss and
Hasinoff, 1993) or bind free or loosely bound iron, thus
preventing oxygen radical formation. ADR-925 binds Fe2+
and Fe3+ with formation constants of 1010 and
1018.2 M–1, respectively, compared with
formation constants of 1014.3 and 1025.1
M–1 for EDTA
(Huang et al., 1982;
Diop et al., 2000). Thus,
dexrazoxane, which is easily permeable to cells
(Dawson, 1975), can be
considered a neutral prodrug analog of EDTA.
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We previously showed in isolated hepatocytes
(Hasinoff et al., 1994), in a
rat model (Schroeder and Hasinoff,
2002), and in humans
(Schroeder et al., 2003) that
dexrazoxane was rapidly metabolized to the one-ring open compounds B
and C, and ADR-925 (Fig.
1). This metabolism may be due, in part, to the ability of
dihydropyrimidinase, which is present in the liver and kidney, but not the
heart, to enzymatically hydrolyze dexrazoxane to B and C, but
not to ADR-925 (Hasinoff et al.,
1998). We also recently showed that dihydroorotase, which is
present in all three of these organs, enzymatically hydrolyzes B and
C to ADR-925 but does not act on dexrazoxane
(Schroeder et al., 2002). We
also previously used a neonatal cardiac myocyte model to compare the ability
of dexrazoxane and deferiprone to protect against doxorubicin-induced
cytotoxicity (Barnabé et al.,
2002). Given the rapid appearance of these metabolites in plasma,
we decided to examine whether these metabolites could prevent
doxorubicin-induced damage to isolated neonatal rat myocytes and, thus,
whether they might be useful as drugs. To accompany these studies, we also
investigated the uptake of dexrazoxane and its metabolites in myocytes by
following the fluorescence dequenching of an intracellular iron-calcein
complex (Barnabé et al.,
2002; Esposito et al.,
2002; Hasinoff,
2002; Kakhlon and Cabantchik,
2002).
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). As determined by high-performance liquid
chromatography, B and C contained less than 1.0 and 1.6 mol%
ADR-925, respectively. The ApoAlert DNA fragmentation assay TUNEL kit was from
BD Biosciences Clontech (Palo Alto, CA). Nonlinear least-squares curve fitting
was done with SigmaPlot (SPSS Science, Chicago, IL). The errors shown were
least-squares calculated S.E. values. Where significance is indicated, an
unpaired t test was used with p < 0.05 considered as
significant.
Myocyte Isolation and Culture. Ventricular myocytes were isolated
from 1- to 3-day-old Sprague-Dawley rats as described
(Barnabé et al., 2002;
Hasinoff, 2002). Briefly,
minced ventricles were serially digested with collagenase and trypsin in
Dulbecco's phosphate-buffered saline /1% (w/v) glucose at 37°C in the
presence of DNase and preplated in large Petri dishes to remove fibroblasts.
The preparation, which was typically greater than 90% viable by trypan blue
exclusion, yielded an almost confluent layer of uniformly beating heart
myocytes by day 2. For the LDH release experiments, the myocyte-rich
supernatant was plated on day 0 in 24-well plastic culture dishes (5 x
105 myocytes/well, 750 µl/well) in DF-15 to yield an almost
confluent layer of beating heart myocytes by day 2. On days 2 and 3, the
medium was replaced with 750 µl of fresh DF-10 containing 10% (v/v) fetal
bovine serum. To lower the background LDH levels, on day 4, 24 h before the
drug treatments, the medium was changed to DF-2 and again on day 5 just before
the addition of drugs. The animal protocol was approved by the University of
Manitoba Animal Care Committee.
LDH Determination and Drug Treatments. LDH released into the myocyte
growth medium was determined as previously described
(Barnabé et al., 2002;
Hasinoff, 2002). In all
experiments at the end of the 3-h doxorubicin treatment, the myocytes were
washed (two 20-min incubations followed by replacement with fresh medium) with
fresh DF-2 medium containing 0, 20, or 100 µM dexrazoxane, B, or
ADR-925, respectively, and maintained in this medium for a further 72 h.
Starting on day 6 after plating, samples (80 µl) of the myocyte supernatant
were collected every 24 h for 3 days after treatment with doxorubicin and/or
dexrazoxane, B, or ADR-925. The samples were frozen at –80°C
and analyzed within 1 week. After the last supernatant sample was taken, the
myocytes were lysed with 250 µl of 1% (v/v) Triton X-100/2 mM EDTA/1 mM
dithiothreitol/0.1 M phosphate buffer (pH 7.8) for 20 min at room temperature.
The total cellular LDH activity, from which the percentage of LDH was
calculated, was determined from the activity of the lysate plus the activity
of three 80-µl samples previously taken. The LDH activity was determined in
quadruplicate, in a kinetic assay in a 96-well plate in a Molecular Devices
Corp. (Sunnyvale, CA) plate reader. The initial velocity of the LDH-catalyzed
reaction of NAD+ with lactate to produce NADH and pyruvate was
determined by measuring the rate of increase in absorbance at 340 nm at
25°C. The assay buffer contained 2.4 mM NAD+ and 290 mM sodium
lactate in 28 mM Tris buffer (pH 8.8).
Calcein Loading of Myocytes and Displacement of Iron from the
Fluorescence-Quenched Intracellular Calcein-Iron Complex by Dexrazoxane, B, C,
and ADR-925. Calcein was loaded into attached myocytes 6 to 10 days after
plating in 96-well plates (125,000 myocytes/well, 200 µl of medium/well),
essentially as described (Cabantchik et
al., 1996; Zanninelli et al.,
1997; Barnabé et al.,
2002; Hasinoff,
2002). Briefly, myocytes were incubated with 125 nM calcein-AM
(Molecular Probes), the cell-permeant acetoxymethyl ester of calcein, for 5
min at 37°C in serum-free DF-0 medium, followed by three changes of medium
at room temperature to remove extracellular ester. The kinetic fluorescence
measurements were conducted on a BMG Labtechnologies Inc. (Durham, NC)
Fluostar Galaxy fluorescence plate reader (
ex of 485 nm,
em of 520 nm, 30°C) equipped with excitation and
emission probes directed to the bottom of the plate. To reduce background
fluorescence from DF-0 medium, the medium was changed to HBS buffer (100
µl) a couple of minutes before the addition of the drug. After initial
baseline fluorescence intensity data were collected, dexrazoxane or its
metabolites were added to the attached myocytes by pipetting stock drug
solution into the well and gently mixing the solution with a pipette. The
increase in fluorescence as the drug displaced iron from the
fluorescence-quenched trapped intracellular calcein-iron complex was recorded
relative to untreated controls as a function of time. The initial velocities
(v) for the fluorescence change occurring upon the addition of
chelators were calculated by linear least-squares fits of the
fluorescence-time data over the first 20 min for dexrazoxane and 50 min for
the metabolites. The neutral iron chelator deferiprone
(Fig. 1), which rapidly enters
cells and displaces iron from its complex with calcein, was used a positive
control (Zanninelli et al.,
1997; Barnabé et al.,
2002).
Solution Kinetics of the Reaction of ADR-925 and B with
Fe2+-Calcein. The displacement of
Fe2+ from its complex with calcein by ADR-925 and
B was followed in the fluorescence plate reader at 30°C in HBS
buffer containing 1 mM ascorbic acid and 0.1 mg/ml catalase. Because the
Fe2+-calcein complex is rapidly oxidized to
Fe3+-calcein under aerobic conditions
(Breuer et al., 1995), ascorbic
acid was used to maintain the iron in its ferrous state. The catalase was used
to decompose H2O2 formed from oxidation of
Fe2+-calcein and prevent the oxidation of calcein to a
nonfluorescent product (Petrat et al.,
2002). The Fe2+-calcein complex (1 µM)
was formed by adding FeSO4 to calcein in the well of the 96-well
plate, mixing, and waiting 10 s for the reaction to complete. The chelator was
then added, and the fluorescence was followed with time. The initial
velocities were calculated from the first 0.7 to 1.5 min of the reaction.
TUNEL Assay of Doxorubicin- and Dexrazoxane-Treated Myocytes, Epifluorescence Microscopy, and Image Analysis. To determine whether doxorubicin treatment of myocytes induced apoptosis, a TUNEL assay was carried out along with a nuclear propidium iodide counterstain to determine the percentage of apoptotic cells as per the manufacturer's directions. The terminal deoxynucleotidyl transferase catalyzes incorporation of fluorescein-dUTP at the free 3'-hydroxy ends of fragmented DNA. During late-stage apoptosis, cellular endonucleases cleave DNA between nucleosomes. The wet-mounted fixed RNase and anti-fade-treated cells were imaged on a Zeiss Axioscop 2 MOT epifluorescence microscope with fluorescein and propidium iodide filter sets, respectively. The percentage of TUNEL-positive cells in 20 randomly chosen fields (approximately 125 cells/field) per treatment was determined from counting the number of TUNEL-positive cells in the green plane relative to the total number of cells in the red plane.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; Barnabé et al.,
2002). The data of Fig.
2a also show that preincubation of the myocytes with either 20 or
100 µM dexrazoxane for 3 h before doxorubicin treatment resulted in a
significant reduction in doxorubicin-induced LDH release similar to what we
observed before (Barnabé et al.,
2002). The data of Fig. 2, b
and c, show that preincubation of the myocytes with either 20 or
100 µM B or ADR-925 for 3 h before doxorubicin treatment did not
result in any significant decrease in doxorubicin-induced LDH release. In
similar experiments carried out using 0.6 µM doxorubicin, preincubation
with either 20 or 100 µM B also did not reduce doxorubicin-induced
LDH release (data not shown).
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Kinetics of Displacement of Intracellular Iron from Its Complex with
Calcein by ADR-925, Metabolites B and C, and Dexrazoxane in Myocytes. As
shown in Fig. 3a, the addition
of various concentrations of ADR-925 to attached calcein-loaded myocytes in
HBS resulted in increases in fluorescence intensity consistent with the
removal of iron from the trapped intracellular iron-calcein complex. Upon the
addition of 50 to 1000 µM ADR-925, the initial rate of the fluorescence
change increased with an increase in the ADR-925 concentration as seen
previously (Hasinoff, 2002). A
plot of the initial velocities (v) as a function of ADR-925
concentration (Fig. 3b)
suggested that ADR-925 displacement of iron from its complex with calcein was
a saturable process. A nonlinear least-squares fit of the initial velocity
data v to the Michaelis-Menten equation, v =
Vmax · [ADR-925]/(1 + Km
· [ADR-925]) gave a Km of 103 ± 26
µM.
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Because ADR-925 binds Ca2+ and
Mg2+ with formation constants of 106.9 and
105.1 M–1
(Huang et al., 1982),
respectively, in vivo, in plasma the ADR-925 is likely complexed to
Ca2+ and to Mg2+, to a lesser
extent. Thus, to determine the effect of Ca2+ and
Mg2+ in the medium on the ADR-925-induced fluorescence
dequenching of the intracellular iron-calcein complex in attached myocytes,
the experiments were repeated in the presence of Ca2+
and Mg2+ at concentrations typically found in plasma
(Sheppard and Kontoghiorghes,
1993). As can be seen from Fig.
4a, the initial rate of calcein fluorescence dequenching by 500
µM ADR-925 was greatly decreased (6.6-fold) by the addition of 2.5 mM
Ca2+ to the HBS medium, but not by the addition of 1.0
mM Mg2+. A control experiment
(Fig. 4b) was carried out with
deferiprone, which has a low affinity for Ca2+ and
Mg2+ (Sheppard and
Kontoghiorghes, 1993). As previously seen, deferiprone in HBS
caused a rapid fluorescence dequenching of calcein
(Barnabé et al., 2002).
However, in contrast to the ADR-925 results, in the presence of a mixture of
Ca2+ and Mg2+, the
deferiprone-induced fluorescence dequenching was only slightly affected.
Similar results with both ADR-925 and deferiprone were seen with
calcein-loaded myocytes in Hanks' buffer containing 1.25 mM
Ca2+, 0.81 mM Mg2+, 20 mM HEPES
(pH 7.4). Probenecid is well known to inhibit the uptake of organic anions
into cells (Tsuji et al.,
1990). To determine whether uptake of dianionic ADR-925
(Fig. 1) was inhibited by
probenecid, ADR-925 fluorescence dequenching of calcein-loaded myocytes was
determined in the absence and presence of 1 mM probenecid. The prior addition
of probenecid to HBS had no effect on ADR-925-induced fluorescence dequenching
of myocytes.
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Because significant levels of the intermediate metabolites B and
C are found in plasma after the administration of dexrazoxane to both
rats (Schroeder and Hasinoff,
2002) and humans (Schroeder et
al., 2003), the ability of B and C to displace iron
from the trapped intracellular iron-calcein complex were also examined. As
seen from Fig. 5, b and c, both
B and C were able to dequench calcein fluorescence in myocytes
in a concentration-dependent manner, although at rates that were,
respectively, 5.1- and 3.9-fold lower than ADR-925 at a 500 µM drug
concentration. The rate at which dexrazoxane dequenched calcein fluorescence
(Fig. 5a) was much lower than
either B, C, or ADR-925 (maximally 7.4-fold less) and, as
previously seen, did not measurably vary with the dexrazoxane concentration
(Hasinoff, 2002).
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Solution Kinetics of the Displacement of Fe2+
from Its Complex with Calcein by ADR-925 and Metabolite B. The kinetics
of the displacement of Fe2+ from its complex with
calcein by ADR-925 and B were studied in solution under reducing
conditions, as would exist in the cell, to see whether these reactions might
be rate-limiting and, thus, whether they could affect the observed rate of
fluorescence dequenching observed in myocytes. As shown in
Fig. 6a, upon the addition of
ADR-925 to Fe2+-calcein, a rapid
(t1/2 of 2.5 min at 100 µM chelator) increase in
fluorescence resulted, due to the displacement of Fe2+
from its complex with calcein. Under conditions where ADR-925 was present in
large excess, the fluorescence increased exponentially with time
(Fig. 6a). To obtain
kobs, the observed pseudofirst-order rate constant for the
reaction, the observed fluorescence changes were nonlinear least-squares
fitted to a three-parameter exponential equation:
 | (1) |
F
is the total change in fluorescence, and F0 is the
fluorescence at time 0. The plots of kobs as a function of
the ADR-925 concentrations are shown in
Fig. 6b and increase with an
increase in chelator concentration. However, the reaction of B with
Fe2+-calcein was much slower (t1/2
of 10 min at 100 µM B) and could not be accurately fit to eq. 1
because it displayed both a fast initial phase and a slower second phase. To
obtain a kobs for the first initial reaction,
kobs was calculated from v/F,
which assumed that the fast initial reaction was a first-order process. The
values of kobs initially increased and then leveled off at
about 100 µM B (Fig.
6b). In the presence of 2.5 mM Ca2+
in the medium, the kobs for the reaction of ADR-925 and
B with Fe2+-calcein decreased from 1.4- to
3.4-fold, between 100 and 1000 µM ADR-925. However, the presence of
Ca2+ in the medium increased the rate at which B
displaced Fe2+ from its complex with calcein. The
kobs increased from 3.0- to 4.5-fold, between 50 and 1000
µM B. A kobs was also determined at 200 µM
EDTA in the absence of any added Ca2+. The
kobs for EDTA under these conditions was 0.53
min–1 compared with 0.43
min–1 for ADR-925.
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TUNEL Assay of Doxorubicin- and Dexrazoxane-Treated Myocytes. To
determine whether doxorubicin induced apoptosis in myocytes and whether
dexrazoxane was able to prevent doxorubicin-induced apoptosis, a TUNEL assay
was carried out on myocytes. As shown in
Fig. 7, myocytes that were
treated with doxorubicin for 3 h, washed, and TUNEL-assayed 72 h later showed
a significant (p < 0.001) increase in the proportion of
TUNEL-positive myocytes compared with untreated controls. Myocytes that were
pretreated with 100 µM dexrazoxane for 3 h, then treated with doxorubicin
for a further 3 h, and washed with medium containing dexrazoxane showed a
significant (p < 0.001) reduction in the proportion of
TUNEL-positive myocytes compared with doxorubicin-treated myocytes. Thus,
dexrazoxane acted as an antiapoptotic agent. Dexrazoxane was not able to
reduce the proportion of TUNEL-positive myocytes to control values (p
< 0.001). This result is consistent with the LDH results of
Fig. 2a in which LDH levels of
dexrazoxane-treated myocytes did not reach levels of control myocytes.
Dexrazoxane-treated myocytes also showed a significant (p < 0.001)
increase in the proportion of TUNEL-positive myocytes compared with untreated
controls. This result might be due to the fact that dexrazoxane is also a
strong inhibitor of DNA topoisomerase II
(Hasinoff et al., 1995) that
can induce apoptosis in K562 cells
(Hasinoff et al., 2001). The
high percentage of TUNEL-positive control apoptotic myocytes is likely a
result of the collagenase/trypsin tissue dissociation treatment used to obtain
the primary cell culture.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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;
Schroeder et al., 2003). The
peak plasma concentrations of dexrazoxane, ADR-925, B, and C in
humans dosed at 1500 mg/m2 are 211, 30, 19, and 9 µM,
respectively (Schroeder et al.,
2003). The concentrations of doxorubicin used in these studies
were even smaller (1.5 compared with 12 µM) than the plasma concentrations
seen clinically at the end of a 60 mg/m2 doxorubicin infusion
period (t1/2
of 1.8 h)
(Hochster et al., 1992). Thus,
the doxorubicin concentrations and the time over which the myocytes were
exposed to doxorubicin were also in a pharmacologically relevant range.
The rapid rate of displacement of Fe2+ from its
complex by calcein by ADR-925 in solution
(Fig. 6b) in either the
presence or absence of Ca2+ suggests that this reaction
was not likely to be rate-limiting when ADR-925 was added to calcein-loaded
myocytes. However, the slower rate at which B displaced
Fe2+ (Fig.
6b) suggests that this reaction partly limited the kinetics of
fluorescence dequenching in calcein-loaded myocytes in the absence of added
Ca2+ (Fig.
5b). We previously showed that B and C are also
chelators that are capable of displacing Fe3+ from its
complex with doxorubicin (Buss and
Hasinoff, 1993). The similarity in the kobs
values for the ADR-925 and EDTA displacement reactions in solution suggests
that there was a common rate-determining step that was most likely the
breaking of an Fe2+-calcein bond. The
Ca2+-induced decrease in rate of displacement of
Fe2+ from its complex with calcein in solution is likely
due to the requirement that the Ca2+-ADR-925 complex
dissociate before it can complex Fe2+ released from
calcein. We previously showed that Ca2+ binds to
B (Buss and Hasinoff,
1997), although likely much less strongly than to ADR-925. The
reason for the Ca2+-induced increase in rate of reaction
of B with Fe2+-calcein is not known but may be
due to a faster dissociation of the weaker Ca2+-B
complex.
Our recent pharmacokinetic studies showed that dexrazoxane was rapidly
metabolized in rats (Schroeder and
Hasinoff, 2002) and in humans
(Schroeder et al., 2003) to
the one-ring open compounds B and C and ADR-925
(Fig. 1). Given the rapid
metabolism of dexrazoxane in vivo, we tested the hypothesis that circulating
B or ADR-925 in plasma might be the active form of dexrazoxane that
protects myocytes from doxorubicin-induced LDH release. However, as shown in
Fig. 2, neither B nor
ADR-925 protected myocytes against doxorubicin. The inability of ADR-925 to
protect myocytes from doxorubicin-induced damage is in accord with the
inability of ICRF-198 (the racemic form of ADR-925) to protect hamsters from
acute toxic effects of the doxorubicin analog daunorubicin
(Herman et al., 1985). The
reason that both B and ADR-925 do not protect may be due to their being
anionic species that enter cells more slowly than neutral dexrazoxane does
(Dawson, 1975).
To determine whether the lack of uptake of these anionic metabolites
prevented them from protecting myocytes, B-, C-, and
ADR-925-induced fluorescence dequenching of the iron-calcein complex as they
were taken up in myocytes was determined. However, the results of
Fig. 3 show that there was a
saturable uptake of ADR-925 into myocytes. The fact that ADR-925 uptake was
saturable suggests that dianionic ADR-925 may have been taken up by an anion
transport system that was, however, not inhibited by probenecid. However, in
the presence of Ca2+ concentrations similar to those in
plasma, the rate of entry of ADR-925 was greatly reduced. This probably
occurred because ADR-925 can form a complex with Ca2+
(K of 106.9 M–1;
Huang et al., 1982). Although
the presence of 2.5 mM Ca2+ did decrease the rate of the
reaction of ADR-925 with Fe2+-calcein in solution, this
decrease was not wholly consistent with the large decrease in rate of
fluorescence dequenching of calcein in myocytes that was caused by the
addition of Ca2+ to the medium
(Fig. 4a). However, the reduced
rate of uptake of the Ca2+-ADR-925 complex may, in part,
be responsible for the inability of ADR-925 to protect myocytes from
doxorubicin.
The results of Fig. 5 show
that B and C were taken up by myocytes more rapidly than
ADR-925. However, the uptake of B did not result in its protecting
myocytes from doxorubicin (Fig.
2). Together, these results suggested that these anionic
metabolites did not have the same access that dexrazoxane had to iron pools in
critical cellular compartments. Doxorubicin has been shown by fluorescence
microscopy to localize in the mitochondria of myocytes
(Swift and Sarvazyan, 2000)
with doxorubicin-induced dichlorofluorescin oxidation occurring close to the
mitochondria (Sarvazyan,
1996). Cardiac mitochondria are also a prominent site of injury by
doxorubicin (Gianni et al.,
1983; Sokolove and Shinaberry,
1988). We have shown that dexrazoxane reduces doxorubicin-induced
oxidation of intracellular dichlorofluorescin and prevents mitochondrial
damage (Hasinoff et al.,
2003). Thus, dexrazoxane may be protecting mitochondria because it
is neutral and permeable to mitochondria, whereas the anionic metabolites
B and ADR-925 are not. The intracellular Ca2+
concentration is so low that the ADR-925 would not be present as its neutral
Ca2+-ADR-925 complex. The calcein in myocytes is located
mainly in the cytoplasm and, thus, our experiments do not give us any
information on the displacement of iron from the mitochondria. The results of
Fig. 7 showing that dexrazoxane
reduced doxorubicin-induced apoptosis are consistent with its ability to
prevent daunorubicin-induced apoptosis of myocytes
(Sawyer et al., 1999).
An alternative, although less likely possibility, is that dexrazoxane did
not act by preventing iron-based oxidative damage, but through its ability to
catalytically inhibit DNA topoisomerase II (Ki 13 µM)
(Hasinoff et al., 1995). It
is, however, unclear how preventing doxorubicin from targeting topoisomerase
II could protect myocytes. Dexrazoxane is clearly able to prevent oxygen free
radical damage inasmuch as it has been shown that dexrazoxane can protect
myocytes from hypoxia-reoxygenation and other drug-induced free radical damage
(Hasinoff, 2002;
Hasinoff et al., 1998).
The fact that dexrazoxane only very slowly removed iron from its complex
with calcein in myocytes (Fig.
5a) is explained by the fact that at the pH and temperature of
this study, it is only slowly hydrolyzed to its one-ring open intermediates
B and C (Fig. 2)
(t1/2 of 14 and 29 h, respectively) and then to ADR-925
(t1/2 23 h) (Hasinoff,
1994b). We previously showed that ferrous ion strongly promoted
the ring opening of B and C to ADR-925
(Buss and Hasinoff, 1995).
Given that the intracellular environment of the cell is highly reducing, free
or loosely bound intracellular iron would be expected to be largely present in
the ferrous state. Thus, by reacting with ferrous ion, the strongly chelating
ADR-925 could be formed intracellularly more quickly. From the kinetics of the
formation of B and C
(Hasinoff, 1994b), and
assuming rapid ferrous ion-promoted ring opening, it can be estimated that a
3-h preincubation with 100 µM dexrazoxane could yield an intracellular
ADR-925 concentration of 20 µM. The free or loosely bound iron
concentration in cells is not known with certainty but has been estimated
using calcein to be 1.3 µM in mouse leukemia cells
(Picard et al., 1998). Thus,
assuming that similar concentrations of free iron were present in myocytes, a
3-h preincubation with dexrazoxane would produce more than enough ADR-925 to
chelate all of the free iron in the myocytes.
In conclusion, this study showed that whereas dexrazoxane was able to protect cardiac myocytes from doxorubicin-induced damage, its B and ADR-925 metabolites were not. Thus, these metabolites are unlikely to be useful on their own as antioxidant drugs. The ability of B, ADR-925, and dexrazoxane to displace iron from intracellular trapped iron-calcein complex suggests that these metabolites were taken up into the myocyte and bound iron. The fact that the these anionic metabolites did not protect myocytes from doxorubicin suggests either that the metabolites do not have access to the same cellular compartments that neutral dexrazoxane has, or that they are taken up too slowly to protect, or, alternatively, that dexrazoxane protects myocytes through its ability to inhibit topoisomerase II through some unknown mechanism.
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Footnotes |
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ABBREVIATIONS: DF-x, Dulbecco's modified Eagle's
medium/Ham's F-12 medium with 50 mM HEPES (pH 7.4) (x is v/v %
serum); HBS, HEPES/NaCl buffer (20/150 mM, pH 7.4); kobs,
pseudofirst-order rate constant; TUNEL, terminal deoxynucleotidyl
transferase-mediated dUTP nick-end-labeling; LDH, lactate dehydrogenase;
t1/2, half-time;
t1/2, beta phase pharmacokinetic
elimination half-time; v, initial velocity; ex,
em, fluorescence excitation and emission wavelengths,
respectively.
Address correspondence to: Dr. Brian Hasinoff, Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba R3T 2N2 Canada. E-mail: B_Hasinoff{at}UManitoba.ca
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Breuer W, Epsztejn S, and Cabantchik ZI (1995) Iron
acquired from transferrin by K562 cells is delivered into a cytoplasmic pool
of chelatable iron(II). J Biol Chem
270:
24209–24215.
Buss JL and Hasinoff BB (1993) The one-ring open hydrolysis product intermediates of the cardioprotective agent ICRF-187 (dexrazoxane) displace iron from iron-anthracycline complexes. Agents Actions 40: 86–95.[CrossRef][Medline]
Buss JL and Hasinoff BB (1995) Ferrous ion strongly promotes the ring opening of the hydrolysis intermediates of the antioxidant doxorubicin cardioprotective agent ICRF-187 (dexrazoxane). Arch Biochem Biophys 317: 121–127.[CrossRef][Medline]
Buss JL and Hasinoff BB (1997) Metal ion-promoted hydrolysis of the antioxidant cardioprotective agent dexrazoxane (ICRF-187) and its one-ring open hydrolysis products to its metal-chelating active form. J Inorg Biochem 68: 101–108.[CrossRef]
Cabantchik ZI, Glickstein H, Milgram P, and Breuer W (1996) A fluorescence assay for assessing chelation of intracellular iron in a membrane model system and in mammalian cells. Anal Biochem 233: 221–227.[CrossRef][Medline]
Dawson KM (1975) Studies on the stability and cellular distribution of dioxopiperazines in cultured BHK-21S cells. Biochem Pharmacol 24: 2249–2253.[CrossRef][Medline]
Diop NK, Vitellaro LK, Arnold P, Shang M, and Marusak RA (2000) Iron complexes of the cardioprotective agent dexrazoxane (ICRF-187) and its desmethyl derivative, ICRF-154: solid state structure, solution thermodynamics and DNA cleavage activity. J Inorg Biochem 78: 209–216.[CrossRef][Medline]
Esposito BP, Epsztejn S, Breuer W, and Cabantchik ZI (2002) A review of fluorescence methods for assessing labile iron in cells and biological fluids. Anal Biochem 304: 1–18.[CrossRef][Medline]
Gianni L, Corden BJ, and Myers CE (1983) The biochemical basis of anthracycline toxicity and anti-tumor activity. Rev Biochem Toxicol 5: 1–82.
Hasinoff BB (1994a) An HPLC and spectrophotometric study of the hydrolysis of ICRF-187 (dexrazoxane, (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane) and its one-ring opened intermediates. Int J Pharm 107: 67–76.[CrossRef]
Hasinoff BB (1994b) Pharmacodynamics of the hydrolysis-activation of the cardioprotective agent (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane. J Pharm Sci 83: 64–67.[CrossRef][Medline]
Hasinoff BB (1998) Chemistry of dexrazoxane and analogues. Semin Oncol 25 (Suppl 10): 3–9.[Medline]
Hasinoff BB (2002) Dexrazoxane (ICRF-187) protects cardiac myocytes against hypoxia-reoxygenation damage. Cardiovasc Toxiciol 2: 111–118.
Hasinoff BB, Abram ME, Barnabé N, Khelifa T, Allan WP, and
Yalowich JC (2001) The catalytic DNA topoisomerase II inhibitor
dexrazoxane (ICRF-187) induces differentiation and apoptosis in human leukemia
K562 cells. Mol Pharmacol
59:
453–461.
Hasinoff BB, Hellmann K, Herman EH, and Ferrans VJ (1998) Chemical, biological and clinical aspects of dexrazoxane and other bisdioxopiperazines. Curr Med Chem 5: 1–28.[Medline]
Hasinoff BB, Kuschak TI, Yalowich JC, and Creighton AM (1995) A QSAR study comparing the cytotoxicity and DNA topoisomerase II inhibitory effects of bisdioxopiperazine analogs of ICRF-187 (dexrazoxane). Biochem Pharmacol 50: 953–958.[CrossRef][Medline]
Hasinoff BB, Schnabl KL, Marusak RA, Patel D, and Huebner E (2003) Dexrazoxane (ICRF-187) protects cardiac myocytes against doxorubicin by preventing damage to mitochondria. Cardiovasc Toxiciol, in press.
Hasinoff BB, Venkataram S, Singh M, and Kuschak TI (1994) Metabolism of the cardioprotective agents dexrazoxane (ICRF-187) and levrazoxane (ICRF-186) by the isolated hepatocyte. Xenobiotica 24: 977–987.[Medline]
Herman EH, El-Hage AN, Creighton AM, Witiak DT, and Ferrans VJ (1985) Comparison of the protective effect of ICRF-187 and structurally related analogues against acute daunorubicin toxicity in Syrian golden hamsters. Res Commun Chem Pathol Pharmacol 48: 39–55.[Medline]
Hershko C, Link G, Tzahor M, Kaltwasser JP, Athias P, Grynberg A, and Pinson A (1993) Anthracycline cytoxicity is potentiated by iron and inhibited by deferoxamine: studies in rat heart cells in culture. J Lab Clin Med 122: 245–251.[Medline]
Hochster H, Liebes L, Wadler S, Oratz R, Wernz JC, Meyers M, Green
M, Blum RH, and Speyer JL (1992) Pharmacokinetics of the
cardioprotector ADR-529 (ICRF-187) in escalating doses combined with
fixed-dose doxorubicin. J Natl Cancer Inst
84:
1725–1730.
Huang Z-X, May PM, Quinlan KM, Williams DR, and Creighton AM (1982) Metal binding by pharmaceuticals. Part 2. Interactions of Ca(II), Cu(II), Fe(II), Mg(II), Mn(II) and Zn(II) with the intracellular hydrolysis products of the antitumor agent ICRF-159 and its inactive homologue ICRF-192. Agents Actions 12: 536–542.[CrossRef][Medline]
Kakhlon O and Cabantchik ZI (2002) The labile iron pool: characterization, measurement and participation in cellular processes. Free Radic Biol Med 33: 1037–1046.[CrossRef][Medline]
Meyers CE (1998) The role of iron in doxorubicin-induced cardiomyopathy. Semin Oncol 25 (Suppl 10): 10–14.
Petrat F, de Groot H, Sustmann R, and Rauen U (2002) The chelatable iron pool in living cells: a methodically defined quantity. Biol Chem 383: 489–502.[CrossRef][Medline]
Picard V, Epsztejn S, Santambrogio P, Cabantchik ZI, and Beaumont C
(1998) Role of ferritin in the control of the labile iron pool in
murine erythroleukemia cells. J Biol Chem
273:
15382–15386.
Sarvazyan N (1996) Visualization of doxorubicin-induced oxidative stress in isolated cardiac myocytes. Am J Physiol 271: H2079–H2085.
Sawyer DB, Fukazawa R, Arstall MA, and Kelly RA (1999)
Daunorubicin-induced apoptosis in rat cardiac myocytes is inhibited by
dexrazoxane. Circ Res
84:
257–265.
Schroeder PE, Davidson JN, and Hasinoff BB (2002)
Dihydroorotase catalyzes the ring-opening of the hydrolysis intermediates of
the cardioprotective drug dexrazoxane (ICRF-187). Drug Metab
Dispos 30:
1431–1435.
Schroeder PE and Hasinoff BB (2002) The doxorubicin-cardioprotective drug dexrazoxane undergoes metabolism in the rat to its metal ion-chelating form ADR-925. Cancer Chemother Pharmacol 50: 509–513.[CrossRef][Medline]
Schroeder PE, Jensen PB, Sehested M, Hofland KF, Langer SW, and Hasinoff BB (2003) Metabolism of dexrazoxane (ICRF-187) used as a rescue agent in cancer patients treated with high-dose etoposide. Cancer Chemother Pharmacol, in press.
Sheppard LN and Kontoghiorghes GJ (1993) Competition between deferiprone, desferrioxamine and other chelators for iron and the effect of other metals. Arzneim-Forsch 43: 659–663.[Medline]
Sokolove PM and Shinaberry RG (1988) Na+-independent release of Ca2+ from rat heart mitochondria. Biochem Pharmacol 37: 803–812.[CrossRef][Medline]
Swift LM and Sarvazyan N (2000) Localization of dichlorofluorescin in cardiac myocytes: implications for assessment of oxidative stress. Am J Physiol 278: H982–H990.
Tsuji A, Terasaki T, Tamai I, and Takeda K (1990) In
vivo evidence for carrier-mediated uptake of beta-lactam antibiotics through
organic anion transport systems in rat kidney and liver. J
Pharmacol Exp Ther 253:
315–320.
Zanninelli G, Glickstein H, Breuer W, Milgram P, Brissot P, Hider
RC, Konijn AM, Libman J, Shanzer A, and Cabantchik ZI (1997)
Chelation and mobilization of cellular iron by different classes of chelators.
Mol Pharmacol 51:
842–852.
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) or treated with 1.5 µM doxorubicin (
), 100 µM dexrazoxane
and 1.5 µM doxorubicin (
), 20 µM dexrazoxane and 1.5 µM
doxorubicin (
), or 100 µM dexrazoxane (
). The data were from a
typical single experiment in which the percentage of LDH release was measured
in four separate wells. Where error bars are not seen, they were smaller than
the size of the symbol. b, plot of cumulative percentage of LDH release from
cardiac myocytes that were untreated (
) of 2.5 mM Ca2+. The reaction was
followed in HBS buffer at 30°C containing 1 mM ascorbic acid and 0.1 mg/ml
catalase. At time 0, the chelator was added to the preformed
Fe2+-calcein complex. The
Fe2+-calcein complex was formed by adding
Fe2+ to calcein (each 1 µM final concentration). The
fluorescence shown before time 0 was that of calcein before the addition of
Fe2+. Upon the addition of Fe2+ to
calcein, there was a rapid drop in fluorescence corresponding to binding of
Fe2+ to calcein (bottom trace).