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NEOPLASIA
Oxidative DNA base modifications in peripheral blood mononuclear cells of
patients treated with high-dose infusional doxorubicin
James H. Doroshow, Timothy W. Synold, George Somlo, Steven A. Akman, and Ewa Gajewski
In prior studies, it was demonstrated that
the redox metabolism of doxorubicin
leads to the formation of promutagenic
oxidized DNA bases in human chromatin,
suggesting a potential mechanism for
doxorubicin-related second malignancies. To determine whether a similar type
of DNA damage is produced in the clinic,
peripheral blood mononuclear cell DNA
from 15 women treated with infusional
doxorubicin (165 mg/m2) as a single agent
was examined for 14 modified bases by
gas chromatography/mass spectrometry
with selected ion monitoring. Prior to the
96-hour doxorubicin infusion, 13 different
oxidized bases were present in all DNA
samples examined. Chemotherapy, producing a steady-state level of 0.1 ␮M
doxorubicin, increased DNA base oxidation up to 4-fold compared to baseline
values for 9 of the 13 bases studied.
Maximal base oxidation was observed 72
to 96 hours after doxorubicin treatment
was begun; the greatest significant increases were found for Thy Gly (4.2-fold),
5-OH-Hyd (2.5-fold), FapyAde (2.4-fold),
and 5-OH-MeUra (2.4-fold). The level of
the promutagenic base FapyGua increased 1.6-fold (P < .02), whereas no
change in 8-OH-Gua levels was observed
in peripheral blood mononuclear cell DNA
during the doxorubicin infusion. These
results suggest that DNA base damage
similar to that produced by ionizing radiation occurs under clinical conditions in
hematopoietic cells after doxorubicin exposure. If doxorubicin-induced DNA base
oxidation occurs in primitive hematopoietic precursors, these lesions could
contribute to the mutagenic or toxic effects of the anthracyclines on the bone
marrow. (Blood. 2001;97:2839-2845)
© 2001 by The American Society of Hematology
Introduction
The anthracycline antibiotic doxorubicin plays an important role in
the treatment of a wide variety of hematologic malignancies as well
as breast cancer and osteogenic sarcoma.1 Although many competing hypotheses exist to explain the antineoplastic mechanism(s) of
action of doxorubicin, there is little doubt that this drug interacts
pleiotropically with DNA.2 In addition to DNA interactions that
may be important for the therapeutic effects of the drug, doxorubicin is a well-characterized mutagen.3,4 When used clinically in
combination with cyclophosphamide, doxorubicin is associated
with a dramatically increased risk of second malignancy, particularly acute myelomonocytic leukemia.5,6 However, the specific
DNA lesions underlying the carcinogenic effect of doxorubicin
remain to be elucidated.7
Early investigations of the doxorubicin-DNA interaction characterized the ability of the planar anthracycline ring to intercalate into
DNA8 with more recent studies demonstrating a special affinity of
the drug for dGdC-rich regions flanked by A:T base pairs.9
Unfortunately, little evidence has been developed to demonstrate
that intercalation of DNA by doxorubicin, per se, could explain the
varied biochemical alterations (or mutagenicity) produced by this
drug.10 Furthermore, because of the intercalative function of the
anthracycline ring, preclinical studies of the doxorubicin-DNA
interaction initially focused on the ability of the anthracycline to
inhibit DNA and RNA synthesis as well as specific DNA poly-
merases.11-13 However, the doxorubicin concentrations required for
inhibition of these enzymes were found to be in excess of those
achievable clinically, which may explain the lack of correlation
between doxorubicin-related inhibition of bulk DNA or RNA
synthesis and tumor cell killing.14,15
More recent studies of the doxorubicin-DNA interaction have
focused on the effect of the anthracycline on the nuclear matrixassociated enzyme topoisomerase II.16 Doxorubicin inhibits topoisomerase II through the formation of DNA strand passage
intermediates that can be detected as protein-associated DNA
single- and double-strand breaks linked to the enzyme; intercalation of DNA by anthracyclines is not required for inhibition of
topoisomerase II.17 However, in certain cell lines the formation and
disappearance of doxorubicin-related DNA breaks has not correlated with tumor cell killing; in others, DNA single-strand cleavage
is modest and double-strand breaks are essentially undetectable at
supralethal drug concentrations.18,19 Because doxorubicin also
exerts more cytotoxicity than expected per DNA break,20 it is likely
that mechanisms of DNA damage other than those related to
topoisomerase II operate in parallel with the formation of the
doxorubicin–DNA–topoisomerase II ternary complex.
Continued interest in the nature of the interaction of doxorubicin with DNA has recently provided new insights into potential
mechanisms of anthracycline-induced mutagenicity. It now seems
From the Department of Medical Oncology and Therapeutics Research, City
of Hope Comprehensive Center, Duarte, CA, and Department of Cancer
Biology, Comprehensive Cancer Center of Wake Forest University, WinstonSalem, NC.
Association for Cancer Research. 1998;39:489.
Submitted March 27, 2000; accepted January 10, 2001.
Reprints: James H. Doroshow, Department of Medical Oncology, City of Hope
National Medical Center, 1500 E Duarte Rd, Duarte, CA 91010; e-mail:
[email protected].
Supported by grants CA 33572, CA 63265, and CA 62505 from the National
Cancer Institute.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
These results were reported, in part, in the Proceedings of the American
© 2001 by The American Society of Hematology
BLOOD, 1 MAY 2001 䡠 VOLUME 97, NUMBER 9
2839
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2840
BLOOD, 1 MAY 2001 䡠 VOLUME 97, NUMBER 9
DOROSHOW et al
clear that redox activation of doxorubicin by H2O2, dithiothreitol
and Fe3⫹, or formaldehyde produces a unique alkylating species
capable of covalently binding DNA.21-23 The discovery of this
novel drug adduct, although not yet demonstrated in clinical
material, provides the first evidence for a long sought after,
potentially lethal, covalently bound doxorubicin species that could
provide a critical link in our understanding of the doxorubicinDNA interaction. Because previous studies have demonstrated that
flavin dehydrogenase–mediated redox cycling of doxorubicin leads
to reactive oxygen metabolism, including the formation of H2O2, in
mammalian nuclei as well as intact cells,24,25 it is reasonable to
suggest that doxorubicin-induced oxidative modifications in DNA
might also play an important role in either the therapeutic or
carcinogenic properties of this agent.
In a previous study, we examined whether the flavoenzymerelated, one-electron reduction of doxorubicin induced DNA base
modifications in isolated human chromatin.26 Redox cycling of
doxorubicin catalyzed by NADH dehydrogenase generated a wide
range of promutagenic oxidized DNA bases, including Thy Gly3
and 8-OH-Gua, typical of base modifications produced by a
chemical species with the characteristics of the hydroxyl radical
(䡠 OH).27-29 Furthermore, base oxidation was significantly increased
by Fe3⫹, and significantly decreased by substitution of the non–
redox cycling anthracycline 5-iminodaunorubicin for doxorubicin.
Our in vitro experiments, as well as recent interest in the
mechanism of anthracycline-related second malignancies, stimulated a further examination of free radical metabolism in patients
treated with doxorubicin. In the study presented here, we evaluated
the potential of high-dose infusional doxorubicin to produce base
oxidation in peripheral blood mononuclear cell (PBMC) DNA. We
found that doxorubicin administered for 96 hours as the first agent
of a multiagent conditioning regimen for women undergoing
high-dose chemotherapy with bone marrow stem cell support30
significantly increased levels of 9 different oxidized bases detectable in PBMC DNA by gas chromatography/mass spectrometry
(GC/MS) with selected ion monitoring. The pattern of base
modification was similar, in part, to our previous in vitro study with
human chromatin and suggests the involvement of 䡠 OH in the
oxidation of PBMC DNA after doxorubicin exposure in the clinic.
Patients, materials, and methods
Materials
Doxorubicin hydrochloride was purchased from Pharmacia & Upjohn
(Kalamazoo, MI); Tris base, proteinase K, and sodium dodecyl sulfate
(SDS) were obtained from Boehringer Mannheim (Indianapolis, IN).
EDTA and sodium chloride were from Mallinckrodt (Paris, KY). Tergitol
type NP-40, RNAse A, 2-amino-6,8-dihydroxypurine (8-OH-Gua), azaT,
formic acid, and Histopaque-1077 were purchased from Sigma Chemical
(St Louis, MO). Ethyl alcohol [200 proof] was from Quantum Chemical
(Tuscola, IL). Spectra/Por dialysis membranes with a molecular weight
cutoff of 3500 were purchased from Spectrum Companies (Gardena, CA).
2-OH-Ade was from ACROS Organic (Fairlawn, NJ). BSTFA and acetonitrile were purchased from Pierce (Rockford, IL). Pure samples of 5-OH-5MeHyd, 5-OH-Ura, 5-OH-6H-Thy, 5-OH-Cyt, 5-OH-MeUra, Thy Gly,
5,6diOH-Ura, FapyAde, 8-OH-Ade, Xan, and FapyGua were kindly
provided by Dr Miral Dizdaroglu of the National Institute of Standards and
Technology, Gaithersburg, MD. Dulbecco phosphate-buffered saline solution (PBS) without calcium and magnesium was purchased from Irvine
Scientific (Santa Ana, CA). Only deionized, glass-distilled water was used
in this study.
Clinical treatment program, pharmacokinetic determinations,
and PBMC sampling
The PBMCs for determination of DNA base modification and plasma for
doxorubicin pharmacokinetics were obtained from patients entered in a clinical
trial of high-dose infusional doxorubicin, cyclophosphamide, and paclitaxel with
autologous peripheral blood progenitor cell support for women with high-risk
primary and chemotherapeutically responsive metastatic breast cancer. The
clinical trial and blood sampling studies were approved by the Institutional
Review Board of the City of Hope National Medical Center. All patients gave
their voluntary, written informed consent for participation in the investigational
treatment program. Blood from 15 patients of median age 47 years (range, 33-63
years) treated between December 1996 and February 1998 was examined for
modified DNA bases; an evaluation of doxorubicin pharmacokinetics was
performed for 6 patients.
The preliminary clinical results of the high-dose chemotherapy regimen
used for this study have recently been reported.30 The initial therapeutic
component involved a 96-hour, continuous intravenous infusion of doxorubicin at a dose of 165 mg/m2, followed by high-dose cyclophosphamide 100
mg/kg, and escalating doses of paclitaxel delivered over 24 hours.
Peripheral blood progenitor cells (at least 8 ⫻ 108 mononuclear cells/kg)
were administered after the completion of high-dose chemotherapy. To
examine the pharmacokinetics of doxorubicin administered at this dose and
schedule, as well as oxidized DNA base levels, 20-mL blood samples were
collected in EDTA-coated tubes from a peripheral intravenous line contralateral to the site of treatment immediately prior to the start of the infusion and
48, 72, and 96 hours after the initiation of doxorubicin. Plasma was
separated, and doxorubicin concentrations were determined by highperformance liquid chromatography (HPLC) with fluorescence detection
using daunorubicin as the internal standard.31 PBMCs were isolated from
whole blood obtained at these time points by Ficoll-Hypaque gradient.
Briefly, each 20-mL sample of anticoagulated whole blood was diluted 1:3
in PBS and layered onto Histopaque-1077. Following centrifugation at
1500g for 20 minutes, the PBMC-containing interface was transferred to a
15-mL conical centrifuge tube and washed once in ice-cold PBS. The
viability of the PBMC was always more than 90% as assessed by exclusion
of trypan blue dye. After the wash, the cell pellet was resuspended in 0.5 mL
TEN buffer (10 mM Tris HCl, pH 7.8, 1 mM EDTA, 0.15 M NaCl) and
stored at ⫺20°C until all samples from each patient were collected. Prior to
further study, cells were defrosted and all samples from each patient were
simultaneously processed further for DNA isolation, hydrolysis, derivatization, and GC/MS analysis.
DNA preparation
DNA from PBMCs was prepared with no exposure to phenol according to the
modified procedure of Nicolaides and Stoeckert.32 Ten percent Tergitol NP-40
was added to a final concentration 0.6%. Samples were gently shaken for 2
minutes on ice; nuclei were obtained by centrifugation at 1400g for 10 minutes at
4°C. Collected nuclei were resuspended in nuclear lysis buffer (10 mM Tris HCl,
pH 8.0, 2 mM EDTA, 0.4 M NaCl) and digested overnight at 37°C with gentle
shaking after the additions of 10% SDS (final concentration 0.6%) and proteinase
K solution (2 mg/mL in 1% SDS and 2 mM EDTA). The final proteinase K
concentration was 0.27 mg/mL. The next day solubilized nuclei were incubated
with RNAse (final concentration 0.10 mg/mL) at 50°C for 2 hours. Proteins were
precipitated from nuclei by adding 6 M NaCl (saturated solution) to a final
concentration of 1.5 M, shaking vigorously for 30 seconds; samples were then
centrifuged at 10 000g for 10 minutes at room temperature. Supernatants were
removed, and DNA was precipitated with 2 volumes of ethanol kept at ⫺20°C
for at least 1 hour and then centrifuged at 10 000g. DNA precipitate was rinsed
with 70% ethanol and centrifuged again at 10 000g. Dry DNA was dissolved in
water at room temperature. After centrifuging again to remove undissolved
debris, DNA samples were dialyzed overnight at room temperature against water.
DNA concentration was estimated by measuring the optical density at 260 nm
using a Milton Roy Spectronic 1201 Spectrophotometer.
Measurement of oxidative DNA base modifications by GC/MS
Each set of patient DNA samples obtained before or at specific times during
the doxorubicin infusion contained the same amount of DNA by estimation
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BLOOD, 1 MAY 2001 䡠 VOLUME 97, NUMBER 9
DOXORUBICIN-INDUCED DNA BASE MODIFICATION
Table 1. Experimentally derived relative molar response factors and oxidized
DNA base content in peripheral blood mononuclear cells
of patients prior to doxorubicin
Oxidized DNA
base level
(nmol/mg DNA)*
Oxidized DNA
base level
(oxidized bases/
106 bases)†
Ion
used
RMRF*
Thy Gly
259
0.49 ⫾ 0.13
5.98 ⫾ 6.99
1914 ⫾ 2237
5-OH-Hyd
317
—
0.81 ⫾ 1.25
259 ⫾ 400
FapyAde
354
1.66 ⫾ 0.55
0.64 ⫾ 0.64
205 ⫾ 205
5-OHMe-Ura
358
0.31 ⫾ 0.07
0.60 ⫾ 0.85
192 ⫾ 272
5,6diOH-Ura
417
2.35 ⫾ 0.87
1.20 ⫾ 0.83
384 ⫾ 266
5-OH-5-MeHyd
331
0.40 ⫾ 0.10
1.64 ⫾ 2.66
525 ⫾ 851
5-OH-Ura
329
0.72 ⫾ 0.51
0.60 ⫾ 0.54
192 ⫾ 173
DNA base
FapyGua
442
8.20 ⫾ 6.1
6.62 ⫾ 6.24
2118 ⫾ 1997
Xan
353
0.45 ⫾ 0.30
4.71 ⫾ 3.97
1507 ⫾ 1270
2-OH-Ade
352
0.25 ⫾ 0.13
0.33 ⫾ 0.77
106 ⫾ 246
5-OH-Cyt
328
0.46 ⫾ 0.11
0.73 ⫾ 0.72
234 ⫾ 230
8-OH-Ade
352
0.32 ⫾ 0.12
1.52 ⫾ 0.73
486 ⫾ 237
8-OH-Gua
440
4.66 ⫾ 2.76
13.30 ⫾ 9.76
4256 ⫾ 3123
RMRF indicates relative molar response factor.
*Mean ⫾ SD.
†1 nmol oxidized base/mg DNA is 32 molecules of the modified base/105 DNA
bases.
2841
RMRF, because 5-OH-Hyd was detected in the PBMC DNA of the patients
treated with doxorubicin, we assumed the RMRF for this compound to be
the same as that calculated for 5-OH-5-Me-Hyd due to the similarity of their
MS spectra. Having established the RMRFs for these bases, the amount of
each modified base in our DNA samples, with azaT as the internal standard,
was calculated using the following equation:
nmol modified base/mg DNA
(nmol azaT)(peak area of modified base ion)
⫻ (RMRF for the modified base)
⫽
(peak area of the aza T ion)(mg DNA in sample)
The amount of each modified base in each extracted DNA sample was
calculated per milligram of DNA sample. Our calibration samples did not
contain additional DNA or any other “dummy mass” to offset destruction or
alteration of bases during formic acid hydrolysis and derivatization. It has
been assumed that the destructive effect of hydrolysis on the modified DNA
bases may, in part, be quenched by the bulk of the DNA in our patient
samples. Furthermore, additional DNA was not added to the calibration
samples because there is no unambiguous method to ensure that the effects
of hydrolysis are identical on modified and unmodified bases. The DNA
amount was determined by measuring the optical density of each DNA
sample at 260 nm (OD260 ⫽ 1 for a DNA concentration of 50 ␮g/mL).
Statistical methods
of OD260. A known amount of azaT was added to each sample as an internal
standard for quantitative determination of modified DNA bases. For
GC/MS measurements, all samples in a series were lyophilized, hydrolyzed
with 66% formic acid at 140°C for 45 minutes, lyophilized again, and
derivatized with a mixture of BSTFA/acetonitrile (4:1) at 130°C for 45
minutes. For measurements of the relative molar response factors (RMRFs)
needed for quantitative calculations of the modified bases in DNA samples
with azaT as an internal standard, samples containing known amounts of 13
DNA modified bases monitored in these studies and the known amount of
azaT were prepared and kept frozen. 5-OH-Hyd was not included because it
was not available. Periodically, during the course of this work, we
lyophilized and derivatized these samples in the same manner as the patient
DNA samples, and performed GC/MS analysis to calculate RMRFs for all
modified bases. These averages of the RMRFs were used for calculations of
the modified bases in the tested DNA samples from PBMCs. Each set of
patient samples was examined by GC/MS in 1 day with the same instrument
tuning. A Hewlett-Packard (Palo Alto, CA) model 5890A gas chromatograph with a model 5970B mass spectral detector was used for these
studies. The injector port was kept at 250°C, interface at 280°C, and oven
temperature was kept at 150°C for 2 minutes followed by a gradient of
8°C/min up to 260°C; then the oven was kept at 260°C for an additional 2
minutes. A Hewlett-Packard Ultra 2 (cross-linked methyl-siloxane) column
(12 meters, 0.20 mm ID, film thickness 0.33 m) was used. The column head
pressure was 50 kPa with a 20:1 split. Using these conditions, 14 oxidative
products of DNA bases and azaT were detected simultaneously in one
chromatogram.
To compare changes in the content of modified bases in our DNA
samples quantitatively, azaT was used as an internal standard; the areas of
the peaks of the ions of interest were estimated using the integration
software package of the Hewlett-Packard GC/MS. The RMRF for each
compound with respect to the internal standard is described by the
following equation33:
RMRF ⫽
(amount of oxidized base standard)
(amount of internal standard)
⫻
(peak area of internal standard ion)
(peak area of oxidized base standard ion)
The average value for the RMRF of each modified DNA base measured in
this trial is shown in Table 1. These values were obtained from GC/MS
measurements performed during the same time period in which modified
DNA bases were quantitated in the patient samples for this study. Although
an authentic sample of 5-OH-Hyd was not available for determination of its
All samples obtained from a single patient were analyzed by GC/MS on the
same day to minimize day-to-day assay variability. The levels of modified
bases were normalized to pretreatment values determined in each individual. A 2-tailed, paired t test was used, after consultation with Dr Jeff
Longmate of the City of Hope Department of Biostatistics, to compare the
fold change in each modified base after doxorubicin treatment to its
pretreatment value using the entire (n ⫽ 15) patient sample; NS is not
significant, P ⬎ .05.
Results
Identification of modified DNA bases by GC/MS
The chromatogram shown in Figure 1A using known standards is
representative of 12 modified DNA bases monitored in this study.
These compounds are: 5-OH-5MeHyd, 5-OH-Ura, 5-OH-Cyt,
5-OHMe-Ura, cis and trans Thy Gly, 5,6diOH-Ura, FapyAde,
8-OH-Ade, 2-OH-Ade, Xan, FapyGua, and 8-OH-Gua. Not included in this chromatogram are 5-OH-6-H-Thy, which was also
monitored but was not detected in our DNA samples, and
5-OH-Hyd, a pure sample of which was not available. However,
5-OH-Hyd (monitored ion 317) was identified in all of the DNA
samples we studied and was found to elute after 5-OH-5-MeHyd.
The internal standard azaT (monitored ion 256) elutes before
5-OH-5-MeHyd. Panels B and C of Figure 1 demonstrate typical
chromatograms from PBMCs of a patient immediately before she
received high-dose, infusional doxorubicin.
Quantitative measurement of modified bases in DNA from
PBMCs of doxorubicin-treated patients
The content (mean ⫾ SD) of 13 modified bases found in PBMC
DNA prior to anthracycline chemotherapy is shown in Table 1. The
relative amounts of the individual modified bases varied by over
20-fold prior to doxorubicin treatment. Following initiation of the
doxorubicin infusion, an increase in oxidized base content was
detected in PBMC DNA in both a time-dependent and base-specific
fashion. As shown in Table 2, significant increases in modified
DNA base content were observed primarily at the completion of the
96-hour anthracycline infusion; however, a significant rise in Thy
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BLOOD, 1 MAY 2001 䡠 VOLUME 97, NUMBER 9
DOROSHOW et al
Figure 1. Selected ion chromatograms obtained during the GC/MS analysis of PBMC DNA. (A) Represents a
mixture of trimethylsilylated standards. (B) The chromatogram demonstrates the higher abundance ions and the
chromatogram in panel C shows the lower abundance ions
of a trimethylsilylated hydrolysate of DNA isolated from the
PBMCs of a representative patient entered into this trial
before initiation of the doxorubicin infusion. Experimental
details are given in “Patients, materials, and methods.”
Peak indicates DNA base (ion monitored): 1, 6-azathymine
(m/z 256) (internal standard); 2, 5-OH-5-MeHyd (m/z 331);
3, 5-OH-Hyd (m/z 317); 4, 5-OH-Ura (m/z 329); 5, 5-OHMeUra (m/z 358); 6, 5-OH-Cyt (m/z 328); 7, 7a, cis and
trans Thy glycol (m/z 259); 8, 5,6-dihydroxyuracil (m/z
417); 9, FapyAde (m/z 354); 10, 8-OH-Ade (m/z 352); 11,
Xan (m/z 353); 12, 2-OH-Ade (m/z 352); 13, FapyGua (m/z
442); 14, 8-OH-Gua (m/z 440).
Gly, 5-OH-Hyd, and 5-OH-Ura levels was also found 72 hours
after drug treatment was begun. The average fold increase over
baseline values was highest for Thy Gly followed by 5-OH-Hyd,
FapyAde, 5-OHMe-Ura, 5,6-diOH-Ura, and 5-OH-5-MeHyd.
Smaller, but significant increases were also observed for 5-OH-Ura
and FapyGua (Table 2). No change in the levels of 3 modified
bases, 5-OH-Cyt, 8-OH-Ade, and 8-OH-Gua, was observed during
the 96 hours of doxorubicin treatment. Although oxidized DNA
base levels varied between patients (as can be appreciated by
review of the SDs for pretreatment values seen in Table 1), the
baseline for individual patients was quite stable. As shown in Table
2, for the most part no significant alterations in oxidized DNA base
levels were observed for the first 48 hours after the doxorubicin
infusion had begun.
Doxorubicin levels
The concentration of doxorubicin in plasma after initiation of a 96-hour
continuous intravenous infusion rises quickly to a steady-state value of
60 ng/mL (0.1 ␮M) that is maintained for the duration of treatment.
Table 2. Oxidized DNA base content in peripheral blood mononuclear cells
of patients receiving a 96-hour infusion of 165 mg/m2 doxorubicin
0h
48 h
72 h
96 h
Thy Gly
DNA base
1
1.5 ⫾ 1.2
1.9 ⫾ 1.1*
4.2 ⫾ 4.6*
5-OH-Hyd
1
1.3 ⫾ 0.7
1.9 ⫾ 1.1*
2.5 ⫾ 1.3*
FapyAde
1
1.9 ⫾ 2.2
1.6 ⫾ 1.5
2.4 ⫾ 1.9*
5-OHMe-Ura
1
0.9 ⫾ 0.4
1.4 ⫾ 1.1
2.4 ⫾ 2.1†
5,6diOH-Ura
1
1.2 ⫾ 0.7
1.4 ⫾ 1.0
2.0 ⫾ 1.8†
5-OH-5-MeHyd
1
1.4 ⫾ 1.3
2.2 ⫾ 2.6
1.8 ⫾ 1.0*
5-OH-Ura
1
1.7 ⫾ 1.7
1.5 ⫾ 0.8†
1.8 ⫾ 1.3*
FapyGua
1
1.9 ⫾ 1.5
1.6 ⫾ 1.2
1.6 ⫾ 0.8*
Xan
1
1.4 ⫾ 0.9
1.4 ⫾ 0.9
1.4 ⫾ 0.7
2-OH-Ade
1
1.6 ⫾ 0.8†
2.1 ⫾ 2.6
1.4 ⫾ 1.0
5-OH-Cyt
1
1.0 ⫾ 0.5
0.9 ⫾ 0.4
1.1 ⫾ 0.9
8-OH-Ade
1
1.0 ⫾ 0.7
1.0 ⫾ 0.7
1.0 ⫾ 0.8
8-OH-Gua
1
1.1 ⫾ 0.9
1.0 ⫾ 0.9
0.7 ⫾ 0.5
N ⫽ 15. Data represent the fold increase (⫾ SD) in oxidized DNA base levels
normalized to pretreatment values for the entire patient population 48, 72, and 96 h
after the initiation of infusional doxorubicin.
PBMC indicates peripheral blood mononuclear cell.
*P ⬍ 0.02 versus PBMC DNA from patients before doxorubicin infusion began.
†P ⬍ 0.05 versus PBMC DNA from patients before doxorubicin infusion began.
Discussion
The oxidative metabolism of doxorubicin, which generates reactive
oxygen species during cycles of reduction and oxidation of the
anthracycline quinone, has generally been accepted to play an
important role in the cardiac toxicity of this drug.34 However, the
importance of drug-induced free radical formation for doxorubicin
toxicity in other tissues has been questioned. To determine whether
by-products of the reductive activation of doxorubicin could be
observed under clinical conditions in patients receiving anthracycline therapy, and to provide potential insights into the mechanism
by which doxorubicin increases the rate of second malignancies of
the hematopoietic system, we evaluated PBMC DNA from 15
women receiving doxorubicin as a 96-hour intravenous infusion for
the presence of oxidized base lesions known to be promutagenic.
Methodologic issues related to oxidized DNA base quantitation
The methodology used in this study for the calculation of doxorubicin-induced oxidative DNA base damage has been described
previously.35,36 The calculated number of nanomoles of each
modified base per milligram DNA is a function of (1) the RMRF for
this base, (2) how the amount of DNA in the sample was estimated,
and (3) whether a constant fraction of DNA-bound modified bases
was released during hydrolysis. However, RMRFs can be determined in several different ways, including the method described in
this study, with values that may vary substantially.37 Furthermore,
the ion peak response of most modified DNA bases can change
with the tuning of the GC/MS. This means that RMRFs may differ
significantly if they are calculated from multiple injections of
mixtures of known amounts of modified DNA bases and an internal
standard performed at different times before and after the GC/MS
has been tuned. However, this last possibility produced only a small
degree of error in the studies presented here. We examined the same
set of standards with different instrument tuning during a period of
5 months and found the calculated RMRFs to vary by less than
10%. Finally, for the purposes of this study, the DNA content in
each tested sample was estimated by OD260, and a constant fraction
of modified bases was assumed to have been released from the
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BLOOD, 1 MAY 2001 䡠 VOLUME 97, NUMBER 9
DNA backbone during hydrolysis in all samples processed and
hydrolyzed in identical fashion.36
Oxidative base modifications in PBMC DNA during infusional
doxorubicin therapy
The objective of this work was to determine whether doxorubicin
produced oxidative damage to PBMC DNA of patients undergoing
a 96-hour doxorubicin infusion. In our study, doxorubicin was
delivered as a prolonged intravenous infusion to diminish its
cardiotoxic potential,38 while retaining therapeutic activity, in the
setting of high-dose chemotherapy for women with responsive
metastatic and high-risk primary breast cancer.30 Under these
conditions, the steady-state doxorubicin plasma level was 0.1 ␮M
when the drug was infused for 96 hours at a total dose of 165
mg/m2; this level can be compared to the typical peak doxorubicin
concentration of 0.6 to 0.9 ␮M, which rapidly decays below 0.05
␮M after bolus administration of a 60 mg/m2 dose.39 In the major
clinical studies of doxorubicin- and epirubicin-related leukemias,
the anthracycline antibiotics were delivered as an intravenous bolus
at doses that ranged from 60 to 70 mg/m2 per administration.5
We monitored the content of 14 modified bases in DNA isolated
from PBMCs of 15 patients before and during high-dose infusional
doxorubicin treatment and found 13 modified bases present in all
DNA samples examined. Infusional doxorubicin significantly increased DNA base oxidation (up to 4-fold) over baseline levels for
9 of the 13 species monitored beginning, for the most part, 72 hours
after anthracycline treatment was initiated. Maximal base oxidation
was observed at the end of the 96-hour infusion. Unfortunately,
because of the design of our clinical trial, repair of doxorubicininduced DNA base oxidation could not usefully be examined. This
was due to the possibility that alkylation-related DNA damage
from high-dose cyclophosphamide (100 mg/kg) administered immediately following completion of the doxorubicin infusion could
have substantially altered interpretation of DNA base oxidation
results subsequent to the 96-hour time point.
Two previous studies have examined the production of oxidized
DNA base lesions in patients treated with an anthracycline.40,41 In
the work of Faure and colleagues, levels of 5-(hydroxymethyl)uracil (HMUra) and 8-oxo-deoxyguanine (8-oxo-dGuo) were determined in 24-hour urine collections following doxorubicincontaining combination chemotherapy in patients treated for a wide
variety of malignancies. A significant, 25% increase in urinary
HMUra was found in the 24 hours following chemotherapy; no
change in 8-oxo-dGuo concentration was observed. Olinski and
associates examined oxidative DNA base damage by GC/MS in
lymphocytes of cancer patients receiving single-agent chemotherapy with the doxorubicin analogue epirubicin.41 Treatment was
administered as a short intravenous bolus of epirubicin at a dose of
70 mg/m2; DNA base damage was evaluated 1 and 24 hours after
therapy. In concert with the results presented in the current report,
the greatest increase (2- to 2.5-fold) for any single oxidized base
after treatment with epirubicin was shown for Thy Gly. Significant
increases in 5-OH-Ura and 2-OH-Ade, as well as significant
interpatient variability in overall DNA base oxidation, were also
found in both series. However, comparison of Table 2 in the present
study with the data of Olinski and associates also demonstrates
substantive differences. Other than for Thy Gly, DNA base
oxidation observed following bolus epirubicin was substantially
smaller (⬍ 1.5-fold) compared to that following infusional doxorubicin. Furthermore, we observed increases in other modified bases,
namely, 5-OH-Hyd, FapyAde, 5-OHMe-Ura, 5,6diOH-Ura, 5-OH5-MeHyd, FapyGua, and Xan during the 96-hour infusion of
DOXORUBICIN-INDUCED DNA BASE MODIFICATION
2843
doxorubicin not reported in the other study. On the other hand, we
did not observe changes in 5-OH-Cyt, 8-OH-Ade, or 8-OH-Gua
levels following infusional doxorubicin. However, it must be
recognized that because of substantial variations in the pretreatment levels of these 3 bases, as well as the potential for artifactual
alterations in 8-OH-Gua levels due to our hydrolysis conditions,42 a
definitive conclusion regarding the effect of doxorubicin on the
production of these 3 oxidized bases in PBMC may not be possible
in the context of our clinical trial.36
The explanation for the greater degree and wider array of
oxidized DNA base damage following infusional doxorubicin
remains to be determined but could be related to the substantially
greater systemic exposure provided by the 96-hour dosing schedule,43 leading to the consumption of intracellular antioxidants, as
well as the possibility that longer-term drug treatment may
transiently overwhelm the DNA repair capacity of the hematopoietic compartment. In fact, recent in vitro studies from our
laboratory suggest that DNA repair enzyme messenger RNAs may
be down-regulated following continuous doxorubicin exposure at
precisely the time when increased DNA base oxidation begins to be
quantifiable (data not shown). In any case, the importance of these
observations is related to the increased frequency of infusional
doxorubicin usage in the clinic, especially during the treatment of
both hematologic malignancies and solid tumors in the pediatric
age group, as part of a strategy to decrease the potentially
devastating long-term side effects of doxorubicin cardiac toxicity
in the young.44
Mechanisms of doxorubicin-induced PBMC DNA
base oxidation
The spectrum of oxidized DNA base damage observed in this study
is similar to that produced by ionizing radiation (and thus,
presumably, is due to a species with the chemical reactivity of
䡠 OH).33,45 It is also similar to our previous study in which
doxorubicin-related 䡠 OH formation oxidized DNA bases in vitro in
human chromatin.26 Only Xan has not routinely been observed in
DNA after exposure to systems generating 䡠 OH; however, Xan has
been detected in DNA extracted from human tumors at levels
greater than that in surrounding normal tissues.46 It has been
suggested that Xan could be formed by attack of 䡠 OH at the C-2 of
guanine, or by deamination of guanine by other species.47 5-OH-6H-Thy was not detected in any of our DNA samples. This
compound is formed from thymine where, in the first step, 䡠 OH
reacts with the thymine molecule to form the 5-hydroxy-6-yl
radical. Further oxidation forms Thy Gly, and reduction forms
5-OH-6H-Thy.27 Apparently, in PBMC DNA exposed to doxorubicin, the formation of Thy Gly is favored over 5-OH-6-H-Thy.
Potential role of DNA base oxidation in the mechanism
of action and mutagenicity of doxorubicin
In addition to supporting the possibility that the oxidative metabolism of doxorubicin occurs under clinically relevant conditions, this
study provides insight into potential mechanisms of doxorubicin
toxicity and mutagenicity for hematopoietic cells. Reactive oxygen
species are known to produce a wide range of DNA lesions
including apurinic sites, strand breaks, and DNA-protein crosslinks, in addition to base oxidation. However, the promutagenic
effects of several oxidized bases have been very well characterized.48 Thy Gly, FapyGua, 5-OH-Ura, and 5-OHMe-Ura, which
were increased 1.6- to 4.2-fold after doxorubicin treatment in this
study, have all been shown to be potentially mutagenic under
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2844
BLOOD, 1 MAY 2001 䡠 VOLUME 97, NUMBER 9
DOROSHOW et al
specific experimental conditions.28,49 In a previous study,50 we
demonstrated that 1.5- to 4-fold increases in the levels of FapyGua,
Thy Gly, FapyAde, and 5-OH-Hyd observed following exposure of
a reporter plasmid to an 䡠 OH-generating system in vitro (similar to
the changes found in the current trial) were associated with a
significantly increased mutation frequency when the oxidized
plasmid was transfected into monkey kidney CV-1 cells. The
predominant lesions observed in that report occurred at C-G base
pairs. It is also known that FapyGua may lead to GC3CG
transversions,51 2-OH-Ade pairs with adenine and guanine,52
5-OH-Ura leads to GC3AT transitions and GC3CG transversions,53 and 5-OH-5-Me-Ura is known to be mutagenic.54,55
Furthermore, ring-fragmented DNA bases (such as FapyGua), as
well as Thy Gly, have also been shown to block DNA replication or
increase reading error frequency by DNA polymerase, leading
either to potentially mutagenic or cytotoxic DNA lesions.48,56,57
Similar inefficiencies in DNA polymerase function may be produced by conformational changes in DNA that occur as a consequence of DNA base oxidation. Thus, if doxorubicin-related DNA
base oxidation occurs clinically either in primitive hematopoietic
precursors or in tumor cells (in addition to PBMC), there is
substantial experimental precedent for the contribution of these
lesions to the toxic effects of the anthracycline.
In conclusion, we observed a significant increase in 9 oxidatively modified DNA bases in the PBMC of cancer patients
undergoing a 96-hour doxorubicin infusion. The largest increases
found were for Thy Gly, 5-OH-Hyd, FapyAde, 5-OHMe-Ura, as
well as FapyGua. These results strongly suggest that doxorubicinenhanced reactive oxygen metabolism occurs in the clinic and
produces potentially mutagenic DNA base lesions that can be
detected in PBMCs. However, further studies will be required to
define the precise mechanisms of oxidatively modified DNA base
mutagenesis or toxicity, and the kinetics of doxorubicin-induced
oxidized DNA base formation and repair, in both normal and
malignant hematopoietic precursors.
Acknowledgment
We wish to thank Lynn Baltazar for her expert secretarial assistance
in the preparation of this manuscript.
Appendix
Abbreviations: thymine glycol, Thy Gly; 8-hydroxyguanine, 8-OH-Gua;
6-azathymine, azaT; 2-hydroxy-6-aminopurine, 2-OH-Ade; N, O-bis(trimethylsilyl) trifluoroacetamide, BSTFA; 5-hydroxy-5-methylhydantoin,
5-OH-5-MeHyd; 5-hydroxyuracil, 5-OH-Ura; 5-hydroxy-6-hydrothymine, 5-OH-6H-Thy; 5-hydroxycytosine, 5-OH-Cyt; 5-(hydroxymethyl)uracil, 5-OH-MeUra; 5,6-dihydroxyuracil, 5,6diOH-Ura; 4,6-diamino-5formamidopyrimidine, FapyAde; 8-hydroxyadenine, 8-OH-Ade; Xanthine,
Xan; 2,6-diamino-4-hydroxy-5-formamidopyrimidine, FapyGua; 5-hydroxyhydantoin, 5-OH-Hyd.
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2001 97: 2839-2845
doi:10.1182/blood.V97.9.2839
Oxidative DNA base modifications in peripheral blood mononuclear cells of
patients treated with high-dose infusional doxorubicin
James H. Doroshow, Timothy W. Synold, George Somlo, Steven A. Akman and Ewa Gajewski
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