© 1999 American Heart Association, Inc.
Original Contribution |
Daunorubicin-Induced Apoptosis in Rat Cardiac Myocytes Is Inhibited by Dexrazoxane
From the Cardiovascular Division, Brigham and Women's Hospital and Harvard Medical School, Boston, Mass. The current affiliation for Dr Sawyer is Cardiology Division, Boston University Medical Center, Boston, Mass. The current affiliation for Dr Arstall is Cardiology Unit, The Queen Elizabeth Hospital, Woodville, South Australia.
Correspondence to Ralph A. Kelly, MD, Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115. E-mail rakelly{at}rics.bwh.harvard.edu
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Abstract |
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Abstract—The clinical efficacy of anthracycline antineoplastic agents is limited by a high incidence of severe and usually irreversible cardiac toxicity, the cause of which remains controversial. In primary cultures of neonatal and adult rat ventricular myocytes, we found that daunorubicin, at concentrations
1 µmol/L, induced myocyte programmed cell death
within 24 hours, as defined by several complementary techniques. In
contrast, daunorubicin concentrations 
10 µmol/L induced
necrotic cell death within 24 hours, with no changes characteristic of
apoptosis. To determine whether reactive oxygen species play a
role in daunorubicin-mediated apoptosis, we monitored the
generation of hydrogen peroxide with dichlorofluorescein
(DCF). However, daunorubicin (1 µmol/L) did not increase DCF
fluorescence, nor were the antioxidants
N-acetylcysteine or the combination of
-tocopherol and ascorbic acid able to prevent
apoptosis. In contrast, dexrazoxane (10 µmol/L), known
clinically to limit anthracycline cardiac toxicity, prevented
daunorubicin-induced myocyte apoptosis, but not necrosis
induced by higher anthracycline concentrations (10 µmol/L).
The antiapoptotic action of dexrazoxane was mimicked by the
superoxide-dismutase mimetic porphyrin
manganese(II/III)tetrakis(1-methyl-4-peridyl)porphyrin (50
µmol/L). The recognition that anthracycline-induced cardiac myocyte
apoptosis, perhaps mediated by superoxide anion generation,
occurs at concentrations well below those that result in myocyte
necrosis, may aid in the design of new therapeutic strategies to limit
the toxicity of these drugs.
Key Words: cardiotoxicity • dexrazoxane • superoxide anion • antioxidant
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The anthracycline antibiotics have been a mainstay of cancer chemotherapy for more than 20 years, although the development of a dilated cardiomyopathy often limits the cumulative dose that can be delivered.1 2 There appear to be 2 types of clinical cardiotoxicity, which are not necessarily mechanistically related. One type occurs rapidly; is characterized by electrocardiographic changes, arrhythmias, and a reversible decrease in ventricular contractile function; and can occur after a single dose of anthracycline.3 The more common delayed form is characterized by a largely irreversible decrease in ventricular contractile function, the incidence of which is in direct proportion to the cumulative anthracycline dose.1 At cumulative doses of 430 to 600 mg/m2 of doxorubicin, the prevalence of ventricular contractile dysfunction rises to 60% of patients.4
However, the mechanism(s) by which anthracyclines causes irreversible myocardial injury remains unclear. Data have been presented that implicate free-radical formation,5 6 lipid peroxidation,7 mitochondrial impairment,8 9 alterations in calcium handling,10 and direct suppression of muscle-specific gene expression.11 12 13 The quinone moiety of the anthracyclines is known to act as a catalyst for the formation of reactive oxygen species, including superoxide anion and hydrogen peroxide.8 The unique sensitivity of the myocardium to anthracyclines may be due to the low levels of catalase and superoxide dismutase (SOD) found in cardiac myocytes.14 Therefore, approaches to prevention of anthracycline-induced cardiac injury have centered on the use of antioxidants to minimize the generation of free radicals, with mixed results.15 16 17 Recently, simultaneous administration of the iron chelator dexrazoxane with anthracyclines has been shown to markedly reduce the risk of cardiomyopathy in patients.18 Despite its documented clinical utility, the mechanisms by which dexrazoxane prevents cardiac damage remain unknown.
The chemotherapeutic benefits of anthracyclines appear to be derived
from induction of programmed cell death (apoptosis) in
malignant tissues.19 20 Therefore, we examined whether the
anthracycline daunorubicin could induce programmed cell death in
isolated ventricular myocytes. At concentrations relevant
to those achieved clinically (1 µmol/L), daunorubicin was found
to induce myocyte apoptosis. Only at higher concentrations
(10 µmol/L) did daunorubicin induce necrosis in isolated
cardiac myocytes. Both dexrazoxane and the SOD mimetic
manganese(II/III)tetrakis(1-methyl-4-peridyl)porphyrin (MnTMPyP) had no
effect on daunorubicin-induced necrosis, yet did inhibit
apoptotic cell death in cardiac myocytes.
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Chemicals
Daunorubicin,
-tocopherol, ascorbic acid, SOD,
fumonisin B1, N-acetylcysteine (NAC),
and pyrrolidinedithiocarbamate (PDTC) were from Sigma. MnTMPyP was from
Calbiochem. Dexrazoxane was from Pharmacia.
Isolation and Culture of Cardiac Myocytes
Neonatal rat ventricular myocytes were isolated from
1-day-old Sprague-Dawley pups as previously
described,21 22 using serial digestion with trypsin and
collagenase in HBSS (Gibco BRL). After preplating to
minimize nonmyocyte contamination, cells were plated at a
density of 1000/mm2. Neonatal myocytes were
cultured at 37°C, 5% CO2 in DMEM supplemented
with 7% FCS (Gibco BRL) for 48 to 72 hours, with addition of fresh
medium before treatment with daunorubicin. Using this method, we
routinely obtained cultures with <5% nonmyocytes.
Calcium-tolerant adult rat ventricular myocytes were
isolated from male Sprague-Dawley rats (225 to 275 g) as
previously described.21 Primary adult myocyte isolates
were cultured in defined medium consisting of DMEM with 25
mmol/L HEPES and 44 mmol/L NaHCO3 with
4.2 mmol/L L-glutamine (Gibco BRL), supplemented with
100 IU/mL penicillin, 100 µg/mL streptomycin, 2 mg/mL BSA, 2
mmol/L L-carnitine, 5 mmol/L creatine, 5 mmol/L
taurine, 0.1 nmol/L triiodothyronine, and 100 nmol/L insulin from
bovine pancreas (Sigma). All culture dishes used for adult myocytes
were pretreated with laminin (Gibco BRL) as previously
described.21 Adult myocytes were cultured for 18 to 24
hours before treatment with daunorubicin. After incubation with
daunorubicin for 24 hours, cells were fixed in 4% formaldehyde in PBS
before staining for quantification of apoptotic cell death.
Detection of Apoptosis and Necrosis
Quantification of DNA Fragmentation by Gel Electrophoresis
Myocyte-membrane integrity was measured using the ability of
cells to exclude trypan blue. For detection of DNA fragmentation using
gel electrophoresis, DNA was isolated from neonatal myocytes. Culture
medium was removed and centrifuged at 3000g for 5
minutes to collect any detached myocytes. Adherent cells were lysed
with a hypotonic lysis buffer (10 mmol/L Tris-HCl [pH 7.4],
10 mmol/L EDTA, 0.5% Triton X-100) and then pooled with the
pellet made up of detached cells. After incubation at 4°C for 10
minutes, lysates were centrifuged at 10 000g for 15
minutes. Supernatants were incubated with RNase A for 1 hour at 37°C,
followed by proteinase K for 1 hour at 37°C. DNA was precipitated
overnight at 4°C with 0.1 volume of 5 mol/L NaCl and 1 volume of
isopropanol and then electrophoresed on a 2.5% agarose gel and
visualized under UV light after staining with ethidium bromide.
Comet Assay
The comet assay was also used to detect DNA fragmentation in
individual cells.23 Briefly, cells were suspended by
treatment with trypsin and pooled with suspended cells in PBS. Cells
were then embedded in 1% low–melting-temperature agarose in PBS and
layered onto frosted slides in a sandwich between layers of 1%
agarose/PBS. Cells were lysed by incubating slides in 2.5 mol/L NaCl,
100 mmol/L EDTA (pH 8), 10 mmol/L Tris (pH 8), 1% SDS, and
1% Triton X-100 for 1 hour. Electrophoresis was carried out at 22 V
for 20 minutes at neutral pH so as to only assay for double-stranded
DNA cleavage. DNA was stained with 5 µmol/L YOYO-1 (Molecular
Probes). The validity of this method for the detection of
double-stranded DNA breaks was assessed by treatment of cells with
H2O2. After
H2O2 treatment, there was
evidence of DNA fragmentation and comet formation under alkaline
conditions, but no comets were seen under neutral conditions, as would
be expected for single-stranded DNA breaks.
Terminal Deoxynucleotidyl Transferase–Mediated
Nick-End Labeling (TUNEL) Assay
Apoptosis was detected in adult myocytes using the TUNEL
assay. Labeling (3' end) of DNA with digoxigenin was done and stained
with peroxidase substrate via a horseradish peroxidase–conjugated
anti-digoxigenin antibody using a commercially available kit
(Apoptag, Oncor) following the manufacturer's directions
with the modification of diluting the terminal
deoxynucleotidyl transferase enzyme 8-fold. Cells
were counterstained with methyl green (Sigma) and magnified at x400.
In these experiments, many cell fragments were seen on the coverslips
that stained positively. Cells were counted as apoptotic as
long as they retained some cytoplasm surrounding a positively stained
nucleus or nuclear fragment. For each experimental condition,
contiguous visual fields were counted to accumulate data on at least
200 total myocytes per condition per experiment.
Detection of Chromatin Condensation With Hoescht 33258
Neonatal myocytes were also stained with Hoescht 33258 for
detection of chromatin condensation characteristic of
apoptosis. Fixed cells were stained for 30 minutes in PBS
containing 10 µmol/L Hoescht 33258 as well as 2 U/mL Texas
Red-conjugated phalloidin (Molecular Probes) to stain filamentous
actin.
Flow Cytometric Analysis of Propidium Iodide
(PI)–Stained Cells
Flow cytometric analysis of cells fixed in 70%
ethanol/PBS and stained with PI (Sigma) was performed to quantify the
percentage of cells undergoing apoptosis. This method is based
on the observation that cells undergoing apoptosis when fixed
in ethanol and stained with PI have a hypodiploid quantity of DNA and
localize in a broad area below the
G0/G1 peak on a PI
histogram.24 Briefly, cells were suspended as described
above, with care to include any cells that had already detached and
fixed in 70% ethanol. This fixation is necessary to allow small
fragments of DNA to leave the cell. If flow cytometry could not be
performed on the same day, then samples were stored at –20°C in
ethanol for no longer than 1 week. Cells were rinsed once with PBS and
then suspended in PBS with PI (20 µg/mL) and RNase A (5 Kunitz
units/mL, Sigma). Analyses were performed after a
minimum 30-minute incubation at room temperature. Ten thousand cells
from each sample were counted using a Becton Dickinson FACScan
(excitation, 488; emission, 590). Gating was performed on the cells to
exclude very small debris with >2 logs weaker staining for PI than
G0 cells. The hypodiploid population of cells
remaining was considered apoptotic. The cutoff at the lower
limit of the G0 peak was determined by eye. There
was some variability in the absolute number of apoptotic
myocytes under control and treated conditions, and for this reason,
each set of experiments is compared with its own internal positive and
negative controls for statistical comparison.
ELISA for Histone-Bound DNA Fragments
Apoptosis was also quantified in neonatal
ventricular myocytes using a commercially available ELISA
for histone-bound DNA fragments (Boehringer). Cells were plated
in a 24-well culture dish and grown to confluence. Lysis of cells was
done according to the manufacturer's directions after 2 washes in PBS.
Samples were run in triplicate, with optical density (OD) measured at
405 nm. The apoptosis enhancement factor was calculated for
each group of experiments as (OD treatment/OD control)x100, after
subtraction of background OD405.
Myocyte Oxidative Stress
2',7'-Dichlorofluorescein (DCF) was used as a
measure of the oxidative stress induced by daunorubicin.25
Flow cytometry was also used to measure levels of reactive oxygen
species in myocytes.26 Myocytes were incubated in the
presence or absence of daunorubicin in the culture medium for 3 hours,
with or without 1 hour of preincubation with antioxidants. DCF
diacetate (DCFDA) was added for the last hour. DCFDA was prepared in
ethanol and diluted into myocyte culture medium to a final
concentration of 5 mmol/L. DCFDA is taken up by cells and on
deacetylation forms a nonfluorescent DCF. On
oxidation, this becomes DCF.27 Cells were trypsinized but
not fixed, and the mean fluorescence was measured for 10 000
cells using a Becton Dickinson FACScan with excitation at 488 nm and
emission at 530 nm (±21 nm). Background fluorescence increased
following addition of daunorubicin because of the fluorescence
of the anthracycline, with some overlap in the emission spectra for the
2 fluorochromes.25 Thus, specific background for each
concentration of daunorubicin was subtracted to obtain a corrected DCF
fluorescence.
Statistical Analysis
All data are presented as mean+SEM. Comparison of groups
was done using ANOVA, followed by Dunnett multiple-comparison test.
Statistical significance was achieved when P<0.05.
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Daunorubicin Induces Concentration-Dependent Apoptosis and Necrosis in Neonatal and Adult Ventricular Myocytes
Isolated neonatal ventricular myocytes undergo morphological changes consistent with apoptosis when exposed to 1 µmol/L daunorubicin for 24 hours (Figure 1A
and 1B). A significant fraction
of the daunorubicin-treated myocytes shrank, although most remained
attached to coverslips (Figure 1B). Staining with Hoescht 33258
does not clearly show chromatin condensation because of the intrinsic
fluorescence of daunorubicin bound to the DNA in these cells.
However, the single-cell electrophoresis ("comet") assay for
apoptosis demonstrates double-stranded DNA
fragmentation,23 as evidenced by the "tail" of nucleic
acid, indicative of apoptosis in myocytes treated with 1
µmol/L daunorubicin (Figure 1C and 1D). DNA
fragmentation on gel electrophoresis indicative of apoptosis
was also observed (Figure 2A). Untreated
neonatal myocytes, or those exposed to 0.1 µmol/L daunorubicin
for 18 hours, exhibited little DNA fragmentation, whereas 1
µmol/L daunorubicin produced clear DNA fragmentation. These and
subsequent experiments were done with 18-hour treatments of
daunorubicin to look for evidence of DNA fragmentation before onset of
morphological changes.
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Myocytes exposed to 1 µmol/L daunorubicin for 18 hours excluded
trypan blue, similarly to untreated myocytes (Figure 2B
), as
expected for cells early in apoptosis. However, myocytes
exposed to 10 µmol/L daunorubicin had increased uptake of trypan
blue. Importantly, and in contrast to myocytes exposed to lower
concentrations of the anthracycline, there was no detectable increase
in DNA laddering either at 6 or 18 hours with 10 µmol/L
daunorubicin (6 hour data not shown). This loss of membrane integrity
with 10 µmol/L daunorubicin, in the absence of DNA fragmentation
at these higher concentrations of drug, is consistent with
necrosis.
When apoptotic cell death in neonatal myocytes exposed to
1 µmol/L daunorubicin was quantified by flow cytometry (Figure 3), the percentage of apoptotic
cells increased from 5% to 15% (n=11 independent experiments,
P<0.001; Figure 3C). The baseline rate of
apoptosis is variable, from <1% in some experiments to as
much as 9%. In all experiments, there was a higher percentage of
apoptosis in the daunorubicin-treated cells. These data were
supported by less quantitative assays as well. Using the comet assay,
the percentage of apoptotic cells increased from 
1% in
untreated myocytes to 13% in myocytes exposed to 1 µmol/L
daunorubicin. This concentration of drug, when apoptosis was
quantified by ELISA assay for histone-bound DNA fragments, resulted in
an "apoptosis enhancement factor" of 18 (range 2.4 to 55.6,
n=3 independent experiments). While each of these methods gave a
similar magnitude of induction of apoptosis by 1 µmol/L
daunorubicin, flow cytometry showed the least variability, likely
because of the large number of cells that can be analyzed in
each experiment.
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We also verified that a similar phenomenon occurred in the adult
ventricular myocyte phenotype. Adult myocytes in
primary culture in defined medium were exposed to 1 µmol/L
daunorubicin for 18 hours and examined for evidence of DNA
fragmentation with the TUNEL technique (Figure 4). Exposure to 1 µmol/L
daunorubicin increased the fraction of TUNEL-positive myocytes from 6%
to 22% (n=3, P<0.005; Figure 4B). Exposure to
10 µmol/L daunorubicin also increased the number of
apoptotic myocytes in adult ventricular myocyte
primary isolates. However, as in neonatal myocytes, the majority of
cells became rounded up and failed to exclude trypan blue, indicating
that there was also necrotic cell death at this higher concentration of
drug (data not shown).
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Daunorubicin, Oxidative Stress, and Apoptosis
Apoptosis induced in neoplastic cells by anthracyclines
had been shown to be mediated by the activation of ceramide
synthase, at least in some cell lines,19 and is
inhibitable by the fungal toxin fumonisin B1. However, pretreatment of
ventricular myocytes for 3 hours with fumonisin B1 (25
µmol/L) did not protect against daunorubicin-induced
apoptosis, as assessed by the comet, ELISA, or flow cytometric
assays (data not shown; n=3 independent experiments), indicating that
activation of ceramide synthase does not appear to be required for
anthracycline-induced apoptosis in cardiac myocytes.
Anthracyclines are also known to catalyze the formation of superoxide
anion, hydrogen peroxide, and hydroxyl radical,8 and these
reactive oxygen species are thought to mediate, at least in part, the
cardiac toxicity of anthracyclines. We examined whether the
antioxidants NAC (100 µmol/L), or -tocopherol
(1 µmol/L) in combination with ascorbic acid (1 mmol/L),
could affect daunorubicin-induced apoptosis when administered 3
hours before the anthracycline. We loaded myocytes with DCFDA to
confirm that these antioxidants were decreasing the level of reactive
oxygen species in both the absence and presence of daunorubicin.
Exposure of myocytes to antioxidants in the absence of daunorubicin
caused a clear decrease in DCF fluorescence, consistent
with a decline in the level of reactive oxygen species (Figure 5A). Even in the presence of
daunorubicin, both antioxidant treatments maintained a level of DCF
fluorescence that was lower than that observed in control
myocytes not exposed to antioxidants. However, neither the antioxidant
NAC nor the combination of -tocopherol and ascorbic acid
ameliorated the increase in apoptotic-cell death induced by
1 µmol/L daunorubicin (Figure 5B). Thus, it would appear
that daunorubicin-induced apoptosis in myocytes does not occur
through increases in DCF-detectable reactive oxygen species. Moreover,
as shown in Figure 5B, treatment of cells with either NAC or the
antioxidant PDTC (100 µmol/L), which also decreased the level of
DCF fluorescence, caused a significant increase in myocyte
apoptosis over baseline, control levels, even in the absence of
daunorubicin.
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Daunorubicin-Induced Apoptosis and Superoxide
Anion
The ability of both PDTC and daunorubicin to induce
apoptosis in cardiac myocytes suggested a potential mechanism
of action. It is known that PDTC chelates copper and thereby
inactivates the copper-dependent cytosolic and
extracellular forms of SOD.28 We examined whether
superoxide anion could be a mediator of daunorubicin-induced
apoptosis by determining whether the superoxide anion
dismutase-mimetic porphyrin MnTMPyP29 or exogenous SOD
could inhibit anthracycline-induced programmed cell death. While
exogenous SOD had no effect on apoptosis (data not shown),
MnTMPyP (50 µmol/L) markedly diminished
daunorubicin-induced apoptosis, as determined by flow
cytometric analysis of PI-labeled myocytes (Figure 6A).
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We next examined whether dexrazoxane, used clinically to prevent
anthracycline-induced cardiomyopathy in
humans,18 could inhibit daunorubicin-induced
apoptosis in vitro. Dexrazoxane is used typically at 10:1 molar
ratios with anthracylines to prevent cardiac toxicity in both
experimental animal preparations and in humans.18 30 As
shown in Figure 6A, myocyte apoptosis induced by
1 µmol/L daunorubicin, quantified by measuring the hypodiploid
fraction or flow cytometry of PI-labeled cells, was prevented when
10 µmol/L dexrazoxane added to the medium of neonatal myocytes
at the same time as anthracycline. This was confirmed by DNA
electrophoretic analysis for DNA fragmentation (data not
shown). In contrast, 100 µmol/L dexrazoxane had no effect on
necrosis induced by 10 µmol/L daunorubicin, measured by trypan
blue exclusion (Figure 6B). Dexrazoxane alone, at either 10
µmol/L or 100 µmol/L, had no effect on myocyte
apoptosis or necrosis in the absence of the anthracycline.
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Anthracyclines and Apoptosis
The mechanism(s) by which the anthracyclines cause cardiotoxicity has been the subject of much investigation and debate for more than 2 decades. The data presented here suggest that this largely permanent loss of contractile function may occur, in part, through initiation of programmed cell death of ventricular myocytes. Early in vitro work with cultured cardiac myocytes had shown direct cardiotoxicity of anthracyclines at concentrations that approximated those relevant clinically. In retrospect, the results of these investigators appear to suggest that these drugs initiated programmed cell death of myocytes. For example, Tobin and Abbott31 found that 1.7 µmol/L doxorubicin produced death of some myocytes in primary culture, and ultrastructural analysis showed nuclear fragmentation, segregation, and chromatin clumping, characteristics now considered typical for cells undergoing programmed cell death. However, Lampidis et al32 found effects of doxorubicin on cultured neonatal rat ventricular myocytes over a broad concentration range (0.1 to 100 µmol/L). They described rapid myocyte degeneration at the upper end of this concentration range, but at concentrations of 1.8 µmol/L and below, myocytes continued to beat spontaneously for >24 hours. This is similar to our finding that there is a switch from overt myocyte necrosis to initiation of programmed cell death that occurred at daunorubicin concentrations between 1 and 10 µmol/L. Newman et al33 also found that a 24-hour exposure to 1.8 µmol/L daunorubicin caused no decrease in viability of neonatal myocytes, as assessed by trypan blue exclusion, although there was a significant accumulation of cardiac muscle–specific enzymes in cell culture media, indicative of some myocyte damage.
Many apparently distinct effects of anthracyclines on cardiac myocytes have been found, and it is unclear what role each of these play in the cumulative toxicity of these drugs. Anthracyclines at concentrations at or below 1 µmol/L induce increases in myocyte intracellular calcium, probably mediated by a cAMP-dependent protein kinase10 and by activation of L-type calcium channels.34 Importantly, doxorubicin at similar, clinically relevant concentrations has been shown by Kurabayashi et al11 12 35 in a series of reports to inhibit the transcription of a number of striated muscle–specific genes, as well as to activate specific genetic responses that are independent of stress-related activator protein-1, protein kinase A, or protein kinase C activation. Recently, using an mRNA differential-display strategy, these investigators demonstrated a rapid decline in mRNA of nuclear genes coding for proteins involved in myocyte energy production, as well as of a previously unrecognized negative regulator of cardiac-specific gene expression termed CARP (for cardiac Adriamycin-responsive proteins).36 These authors speculate that this sequence of events could have been initiated by a signal-transduction cascade triggered by oxygen radicals.37 It is possible that myocyte apoptosis is a consequence of 1 or more of these electrophysiological or transcriptional events triggered by the anthracyclines.
Although programmed cell death has been clearly documented now to participate in the tumoricidal action of anthracyclines, apoptosis has not been recognized in the heart with these drugs. Zhang et al38 have reported evidence of apoptosis in the kidney and intestines, but not the heart, of spontaneously hypertensive rats after treatment with doxorubicin. Our results suggest that programmed myocyte cell death should occur relatively early after anthracycline administration. When Unverferth et al39 obtained myocardial biopsies within 24 hours of doxorubicin administration, the most striking features they identified were mitochondrial swelling and nucleolar contraction. It is tempting to speculate that the chromatin condensation occurring in these myocytes represented a transient wave of programmed cell death in the myocardium. Incremental small decreases in ventricular systolic function might not be readily apparent clinically, and the delay in the clinical appearance of cardiomyopathy in these patients is likely a function of the imperfect correlation between left-ventricular function and symptomatic heart failure. Indeed, when sensitive provocative testing has been performed, such as exercise echocardiography, decrements in contractile function have been evident even at low cumulative anthracycline doses.40 Finally, it has been noted recently that a rise in serum levels of cardiac-specific troponin can be detected following a single dose of anthracycline.41 This observation is consistent with the concept that actual myocyte death, and not only changes in myofibrillar organization, myocyte-specific gene expression, or cellular electrophysiology, may occur at low cumulative drug concentrations.
Anthracyclines and Reactive Oxygen Species
The inhibition of daunorubicin-induced apoptosis in
myocytes by the iron chelator dexrazoxane and the SOD mimetic MnTMPyP
indicates that reactive oxygen species play a role in the triggering of
apoptosis in myocytes. Early investigations into the mechanisms
contributing to anthracycline-induced cardiotoxicity suggested that
drug-induced oxygen-radical cascades played a role in the cardiotoxic
effects of these drugs. Biochemical and biophysical measurements in
cardiac mitochondrial preparations showed that the quinone moiety of
the anthracyclines was reduced by 1 electron at complex I of the
mitochondrial electron transport chain to form superoxide
anion.42 43 At anthracycline concentrations from 20 to
200 µmol/L, there was a rapid increase in production of
superoxide anion, with subsequent increases in
H2O2 and
OH
by submitochondrial
particles.8 44 45 In the intact heart, formation of
OH peaks at Adriamycin concentrations
of 1 µmol/L and is inhibited by treatment with exogenous SOD,
catalase, and dexrazoxane.46 Iron acts as a cofactor in
the formation of reactive oxygen species catalyzed by the quinone group
of anthracyclines, and thus dexrazoxane may bind iron and displace it
from iron-anthracycline complexes.47 Iron also acts as a
cofactor in the formation of OH by the Fenton
reaction from H2O2, and
dexrazoxane may be working at this level as well.
H2O2 alone appears less
important for the induction of myocyte apoptosis. Although
Sarvazyan25 was able to show rapid increases in DCF
fluorescence indicative of significant oxidative stress in
single ventricular myocytes treated with Adriamycin
doses at or above 80 µmol/L, we were unable to detect any
increase in DCF fluorescence in myocytes treated with 1
µmol/L daunorubicin at 3 hours. This suggests that at these lower,
clinically relevant concentrations, the increase in formation of
H2O2 in the myocyte may be
relatively small, although we cannot exclude the possibility that a
longer exposure to daunorubicin at these concentrations might result in
a measurable increase in DCF fluorescence. Moreover, treatment
of myocytes with NAC or the combination of -tocopherol
and ascorbic acid caused an easily detectable decrease in DCF
fluorescence, consistent with their "antioxidant"
effects, yet neither of these antioxidant treatments was able to
prevent daunorubicin-induced apoptosis. Although interpretation
of the NAC experiments is complicated by independent effects of NAC on
myocyte apoptosis, together, these antioxidant experiments
suggest that H2O2 is not an
essential mediator of the apoptotic signal induced by
daunorubicin.
In contrast to H2O2,
superoxide anion is poorly quenched by NAC.48 In addition,
at neutral pH in the presence of metal ions, cysteine and other
sulfhydryl compounds exhibit autoxidation behavior and, at
concentrations such as those used here for NAC, can produce superoxide
anion at quantities measurable by SOD-inhibitable reduction of
cytochrome c.49 Thus, the increase in apoptosis
observed in the presence of NAC may also have been due to generation of
superoxide anion or other reactive oxygen species. Likewise, superoxide
may also be a mediator of the increased apoptosis caused by
PDTC, since PDTC is known to increase the level of superoxide anion
both by the inhibition of Cu/Zn-SOD and by autoxidation.28
Interestingly, both NAC and PDTC have been shown to cause
apoptosis in vascular smooth muscle cells through a mechanism
that was attributed to the antioxidant properties of these
compounds.50 Given the multiple effects of these
compounds, including inhibition of nuclear factor 
B,51
further investigation is necessary before firm conclusions can be drawn
regarding the mechanisms of NAC- and PDTC-induced
apoptosis.
The ability of the SOD-mimetic MnTMPyP to inhibit daunorubicin-induced
apoptosis is the strongest evidence that superoxide anion
mediates a selective apoptotic signal. The narrow window of
daunorubicin concentrations that result in induction of
apoptosis without necrosis may be due to a limited range of
drug concentrations in which superoxide anion is selectively increased
without an appreciable rise in the formation of additional cytotoxic
oxygen species, such as
H2O2 and
OH. However, the fact that both an iron
chelator and an SOD mimetic gave the same result suggests that
daunorubicin-induced apoptosis may involve downstream
products of superoxide. Interestingly, overexpression of
manganese-SOD in mice protected against some of the early mitochondrial
damage after Adriamycin treatment, as well as the level of
serum markers of myocyte death, although myocyte apoptosis was
not examined in this study.52
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Acknowledgments |
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This article is dedicated to Thomas W. Smith, who died during its preparation. This work was supported by a grant (HL 36141) to R.A.K. and a National Research Service Award and grant HL03878-01 to D.B.S. from the National Institutes of Health. M.A.A. received funding from the Thomas W. Smith Research Fellowship at Harvard Medical School and from the National Heart Foundation of Australia Ralph Reader Overseas Research Fellowship. We thank Jing Wang for technical assistance.
Received July 30, 1998; accepted November 19, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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