|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
NEOPLASIA
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, Winston-Salem, NC.
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 µM 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) 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
polymerases.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 matrix-associated 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 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
doxorubicin-DNA 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 flavoenzyme-related,
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
(·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
·OH in the oxidation of PBMC DNA after
doxorubicin exposure in the clinic.
Materials
Clinical treatment program, pharmacokinetic determinations, and
PBMC sampling
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 high-performance 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 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 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:
 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 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:
Statistical methods 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.
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 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 µM) that is maintained for the duration of treatment.
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 DNA backbone during hydrolysis in all samples processed and hydrolyzed in identical fashion.36Oxidative 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 µM 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 µM, which rapidly decays below 0.05 µM 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.5We 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 doxorubicin-induced 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 doxorubicin-containing 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-OH-5-MeHyd, FapyGua, and Xan during the 96-hour infusion of 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 ·OH).33,45 It is also similar to our previous study in which doxorubicin-related ·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 ·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 ·OH at the C-2 of guanine, or by deamination of guanine by other species.47 5-OH-6-H-Thy was not detected in any of our DNA samples. This compound is formed from thymine where, in the first step, ·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 cross-links, 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 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 ·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 GC CG
transversions,51 2-OH-Ade pairs with adenine and
guanine,52 5-OH-Ura leads to GCAT transitions and GCCG 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 doxorubicin-enhanced 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.
We wish to thank Lynn Baltazar for her expert secretarial assistance in the preparation of this manuscript.
Submitted March 27, 2000; accepted January 10, 2001.
Supported by grants CA 33572, CA 63265, and CA 62505 from the National Cancer Institute.
These results were reported, in part, in the Proceedings of the American Association for Cancer Research. 1998;39:489.
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.
Reprints: James H. Doroshow, Department of Medical Oncology, City of Hope National Medical Center, 1500 E Duarte Rd, Duarte, CA 91010; e-mail: jdoroshow{at}coh.org.
1. Doroshow JH. Anthracyclines and anthracenediones. In: Chabner BA,Longo DL, eds. Cancer Chemotherapy and Biotherapy. Philadelphia, PA: Lippincott-Raven; 1996:409-434. 2. Gewirtz DA. A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem Pharmacol. 1999;57:727-741[CrossRef][Medline] [Order article via Infotrieve].
3.
Westendorf J, Marquardt H, Marquardt H.
Structure-activity relationship of anthracycline-induced genotoxicity in vitro.
Cancer Res.
1984;44:5599-5604
4.
Au WW, Butler MA, Matney TS, Loo TL.
Comparative structure-genotoxicity study of three aminoanthraquinone drugs and doxorubicin.
Cancer Res.
1981;41:376-379
5.
Pedersen-Bjergaard J, Sigsgaard TC, Nielsen D, et al.
Acute monocytic or myelomonocytic leukemia with balanced chromosome translocations to band 11q23 after therapy with 4-epi-doxorubicin and cisplatin or cyclophosphamide for breast cancer.
J Clin Oncol.
1992;10:1444-1451
6.
Hoffmann L, Moller P, Pedersen-Bjergaard J, Waage A, Pedersen M, Hirsch FR.
Therapy-related acute promyelocytic leukemia with t(15;17) (q22;q12) following chemotherapy with drugs targeting DNA topoisomerase II: a report of two cases and a review of the literature.
Ann Oncol.
1995;6:781-788 7. Anderson RD, Berger NA. International Commission for Protection Against Environmental Mutagens and Carcinogens. Mutagenicity and carcinogenicity of topoisomerase-interactive agents. Mutat Res. 1994;309:109-142[CrossRef][Medline] [Order article via Infotrieve]. 8. Calendi E, Marco A, Reggiani M, et al. On physicochemical interactions between daunomycin and nucleic acids. Biochem Biophys Acta. 1965;103:25-54. 9. Chaires JB, Fox KR, Herrera JE, Britt M, Waring MJ. Site and sequence specificity of the daunomycin-DNA interaction. Biochemistry. 1987;26:8227-8236[CrossRef][Medline] [Order article via Infotrieve]. 10. Sen D, Crothers DM. Influence of DNA-binding drugs on chromatin condensation. Biochemistry. 1986;25:1503-1509[CrossRef][Medline] [Order article via Infotrieve].
11.
Meriwether WD, Bachur NR.
Inhibition of DNA and RNA metabolism by daunorubicin and adriamycin in L1210 mouse leukemia.
Cancer Res.
1972;32:1137-1142
12.
Goodman MF, Bessman MJ, Bachur NR.
Adriamycin and daunorubicin inhibition of mutant T4 DNA polymerases.
Proc Natl Acad Sci U S A.
1974;71:1193-1196
13.
Zunino F, Ganbetta R, DiMarco A, et al.
A comparison of the effects of daunomycin and adriamycin on various DNA polymerases.
Cancer Res.
1975;35:754-760 14. Siegfried JM, Sartorelli AC, Tritton TR. Evidence for the lack of relationship between inhibition of nucleic acid synthesis and cytotoxicity of adriamycin. Cancer Biochem Biophys. 1983;6:137-142[Medline] [Order article via Infotrieve]. 15. Schott B, Robert J. Comparative cytotoxicity, DNA synthesis inhibition and drug incorporation of eight anthracyclines in a model of doxorubicin-sensitive and resistant rat glioblastoma cells. Biochem Pharmacol. 1989;38:167-172[CrossRef][Medline] [Order article via Infotrieve].
16.
Kohn KW.
Beyond DNA cross-linking: history and prospects of DNA-targeted cancer treatment 17. Pommier Y. DNA topoisomerase I and II in cancer chemotherapy: update and perspectives. Cancer Chemother Pharmacol. 1993;32:103-108[CrossRef][Medline] [Order article via Infotrieve].
18.
Zwelling LA, Kerrigan D, Michaels S.
Cytotoxicity and DNA strand breaks by 5-iminodaunorubicin in mouse leukemia L1210 cells: comparison with adriamycin and 4'-(9-acridinylamino)methanesulfon-m-anisidide.
Cancer Res.
1982;42:2687-2691 19. Fornari FA, Randolph JK, Yalowich JC, Ritke MK, Gewirtz DA. Interference by doxorubicin with DNA unwinding in MCF-7 breast tumor cells. Mol Pharmacol. 1994;45:649-656[Abstract]. 20. Zwelling LA, Bales E, Altschuler E, Mayes J. Circumvention of resistance by doxorubicin, but not by idarubicin, in a human leukemia cell line containing an intercalator-resistant form of topoisomerase II: evidence for a non-topoisomerase II-mediated mechanism of doxorubicin cytotoxicity. Biochem Pharmacol. 1993;45:516-520[CrossRef][Medline] [Order article via Infotrieve]. 21. Taatjes DJ, Gaudiano G, Resing K, Koch TH. Alkylation of DNA by the anthracycline, antitumor drugs adriamycin and daunomycin. J Med Chem. 1996;39:4135-4138[CrossRef][Medline] [Order article via Infotrieve].
22.
Zeman SM, Phillips DR, Crothers DM.
Characterization of covalent adriamycin-DNA adducts.
Proc Natl Acad Sci U S A.
1998;95:11561-11565 23. Luce RA, Sigurdsson ST, Hopkins PB. Quantification of formaldehyde-mediated covalent adducts of adriamycin with DNA. Biochemistry. 1999;38:8682-8690[CrossRef][Medline] [Order article via Infotrieve].
24.
Doroshow JH.
Role of hydrogen peroxide and hydroxyl radical formation in the killing of Ehrlich tumor cells by anticancer quinones.
Proc Natl Acad Sci U S A.
1986;83:4514-4518
25.
Bachur NR, Gee MV, Friedman RD.
Nuclear catalyzed antibiotic free radical formation.
Cancer Res.
1982;42:1078-1081 26. Akman SA, Doroshow JH, Burke TG, Dizdaroglu M. DNA base modifications induced in isolated human chromatin by NADH dehydrogenase-catalyzed reduction of doxorubicin. Biochemistry. 1992;31:3500-3506[CrossRef][Medline] [Order article via Infotrieve]. 27. Dizdaroglu M. Chemistry of free radical damage to DNA and nucleoprotein. In: Halliwell B,Aruoma OI, eds. DNA and Free Radicals. New York, NY: Ellis Horwood; 1993:19-39.
28.
Basu AK, Loechler EL, Leadon SA, Essigmann JM.
Genetic effects of thymine glycol: site-specific mutagenesis and molecular modeling studies.
Proc Natl Acad Sci U S A.
1989;86:7677-7681 29. Shibutani S, Takeshita M, Grollman AP. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature. 1991;349:431-434[CrossRef][Medline] [Order article via Infotrieve]. 30. Somlo G, Doroshow JH, Synold T, et al. High-dose adriamycin (A), cyclophosphamide (C) and paclitaxel (T) for patients (PTS) with high-risk and responsive stage IV breast cancer (BC) [abstract]. Proc Am Soc Clin Oncol. 1997;16:110a. 31. Riley CA, Crom WR, Evans WE. Loop-column extraction and liquid chromatographic analysis of doxorubicin and three metabolites in plasma. Ther Drug Monit. 1985;7:455-460[Medline] [Order article via Infotrieve]. 32. Nicolaides NC, Stoeckert CJ Jr. A simple, efficient method for the separate isolation of RNA and DNA from the same cells. Biotechniques. 1990;8:154-156[Medline] [Order article via Infotrieve]. 33. Nackerdien Z, Olinski R, Dizdaroglu M. DNA base damage in chromatin of gamma-irradiated cultured human cells. Free Radic Res Commun. 1992;16:259-273[Medline] [Order article via Infotrieve].
34.
Singal PK, Iliskovic N.
Doxorubicin-induced cardiomyopathy.
N Engl J Med.
1998;339:900-905 35. Dizdaroglu M. Chemical determination of oxidative DNA damage by gas chromatography-mass spectrometry. Methods Enzymol. 1994;234:3-16[CrossRef][Medline] [Order article via Infotrieve]. 36. Senturker S, Dizdaroglu M. The effect of experimental conditions on the levels of oxidatively modified bases in DNA as measured by gas chromatography-mass spectrometry: How many modified bases are involved? Prepurification or not? Free Radic Biol Med. 1999;27:370-380[CrossRef][Medline] [Order article via Infotrieve]. 37. Swarts SG, Smith GS, Miao L, Wheeler KT. Effects of formic acid hydrolysis on the quantitative analysis of radiation-induced DNA base damage products assayed by gas chromatography/mass spectrometry. Radiat Environ Biophys. 1996;35:41-53[CrossRef][Medline] [Order article via Infotrieve]. 38. Legha SS, Benjamin RS, Mackay B, et al. Reduction of doxorubicin cardiotoxicity by prolonged continuous intravenous infusion. Ann Intern Med. 1982;96:133-139.
39.
Benjamin RS, Riggs CE Jr, Bachur NR.
Plasma pharmacokinetics of adriamycin and its metabolites in humans with normal hepatic and renal function.
Cancer Res.
1977;37:1416-1420 40. Faure H, Mousseau M, Cadet J, et al. Urine 8-oxo-7,8-dihydro-2-deoxyguanosine vs. 5-(hydroxymethyl) uracil as DNA oxidation marker in adriamycin-treated patients. Free Radic Res. 1998;28:377-382[CrossRef][Medline] [Order article via Infotrieve].
41.
Olinski R, Jaruga P, Foksinski M, Bialkowski K, Tujakowski J.
Epirubicin-induced oxidative DNA damage and evidence for its repair in lymphocytes of cancer patients who are undergoing chemotherapy.
Mol Pharmacol.
1997;52:882-885
42.
Collins A, Cadet J, Epe B, Gedik C.
Problems in the measurement of 8-oxoguanine in human DNA: report of a workshop, DNA oxidation, held in Aberdeen, UK, 19-21 January, 1997.
Carcinogenesis.
1997;18:1833-1836 43. Synold T, Doroshow JH. Anthracycline dose intensity: clinical pharmacology and pharmacokinetics of high-dose doxorubicin administered as a 96-hour continuous intravenous infusion. J Infusion Chemother. 1996;6:69-73. 44. Bukowski R. Cytoprotection in the treatment of pediatric cancer: review of current strategies in adults and their application to children. Med Pediatr Oncol. 1999;32:124-134[CrossRef][Medline] [Order article via Infotrieve]. 45. Gajewski E, Rao G, Nackerdien Z, Dizdaroglu M. Modification of DNA bases in mammalian chromatin by radiation-generated free radicals. Biochemistry. 1990;29:7876-7882[CrossRef][Medline] [Order article via Infotrieve]. 46. Olinski R, Zastawny T, Budzbon J, Skokowski J, Zegarski W, Dizdaroglu M. DNA base modifications in chromatin of human cancerous tissues. FEBS Lett. 1992;309:193-198[CrossRef][Medline] [Order article via Infotrieve]. 47. Spencer JP, Jenner A, Chimel K, et al. DNA damage in human respiratory tract epithelial cells: damage by gas phase cigarette smoke apparently involves attack by reactive nitrogen species in addition to oxygen radicals. FEBS Lett. 1995;375:179-182[CrossRef][Medline] [Order article via Infotrieve].
48.
Feig DI, Reid TM, Loeb LA.
Reactive oxygen species in tumorigenesis.
Cancer Res.
1994;54:1890s-1894s
49.
Jaruga P, Dizdaroglu M.
Repair of products of oxidative DNA base damage in human cells.
Nucleic Acids Res.
1996;24:1389-1394 50. Akman SA, Forrest GP, Doroshow JH, Dizdaroglu M. Mutation of potassium permanganate and hydrogen peroxide-treated plasmid pZ189 replicating in CV-1 monkey kidney cells. Mutat Res. 1991;261:123-130[CrossRef][Medline] [Order article via Infotrieve]. 51. Ono T, Negishi K, Hayatsu H. Spectra of superoxide-induced mutations in the lacI gene of a wild-type and a mutM strain of Escherichia coli K-12. Mutat Res. 1995;326:175-183[Medline] [Order article via Infotrieve].
52.
Kamiya H, Ueda T, Ohgi T, Matsukage A, Kasai H.
Misincorporation of dAMP opposite 2-hydroxyadenine, an oxidative form of adenine.
Nucleic Acids Res.
1995;23:761-766
53.
Purmal AA, Kow YW, Wallace SS.
Major oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil, exhibit sequence context-dependent mispairing in vitro.
Nucleic Acids Res.
1994;22:72-78
54.
Boorstein RJ, Teebor GW.
Mutagenicity of 5-hydroxymethyl-2'-deoxyuridine to Chinese hamster cells.
Cancer Res.
1988;48:5466-5470
55.
Levy DD, Teebor GW.
Site directed substitution of 5-hydroxymethyluracil for thymine in replicating phi X-174am3 DNA via synthesis of 5-hydroxymethyl-2'-deoxyuridine-5'-triphosphate.
Nucleic Acids Res.
1991;19:3337-3343 56. Clark JM, Beardsley GP. Functional effects of cis-thymine glycol lesions on DNA synthesis in vitro. Biochemistry. 1987;26:5398-5403[CrossRef][Medline] [Order article via Infotrieve]. 57. Evans J, Maccabee M, Hatahet Z, et al. Thymine ring saturation and fragmentation products: lesion bypass, misinsertion and implications for mutagenesis. Mutat Res. 1993;299:147-156[CrossRef][Medline] [Order article via Infotrieve].
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-5-formamidopyrimidine, FapyAde; 8-hydroxyadenine, 8-OH-Ade; Xanthine, Xan; 2,6-diamino-4-hydroxy-5-formamidopyrimidine, FapyGua; 5-hydroxyhydantoin, 5-OH-Hyd.
© 2001 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  J. H. Doroshow Redox modulation of chemotherapy-induced tumor cell killing and normal tissue toxicity. J Natl Cancer Inst, February 15, 2006; 98(4): 223 - 225. [Full Text] [PDF]
|
|
|
|
 |
|
 D. D. Kennedy, R. M. Santella, Q. Wang, E. J. Ladas, and K. M. Kelly 8-oxo-dG Elevated in Children During Leukemia Treatment Integr Cancer Ther, December 1, 2004; 3(4): 301 - 309. [Abstract] [PDF]
|
|
|
|
 |
|
 G. Minotti, P. Menna, E. Salvatorelli, G. Cairo, and L. Gianni Anthracyclines: Molecular Advances and Pharmacologic Developments in Antitumor Activity and Cardiotoxicity Pharmacol. Rev., June 1, 2004; 56(2): 185 - 229. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
fifteenth Bruce F. Cain Memorial Award Lecture.
Cancer Res.
1996;56:5533-5546