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King Saud University
College of Pharmacy
Department of Pharmacology
Influence of Dexrazoxane on Etoposide-Induced
Chromosomal Instability in Mice: A Mechanistic Study
‫تأثير الديكسرازوكسان على عدم االستقرار الكروموسومي‬
‫ دراسة ألية‬:‫المحدث بواسطة االتوبوسيد فى الفئران‬
Master Research Proposal Submitted to the Department of
Pharmacology
By
Al-Anteet A.A.
‫أالء أحمد العنتيت‬
B. Pharm (2004)
1429 H
2008 G
1. INTRODUCTION
1.1. General Overview:
Over the last 3-5 decades, treatment of cancer has relied primarily on the use
of various cytotoxic chemotherapeutic agents. The use of topoisomerase II-interactive
agents in the treatment of cancer has opened new possibilities for improving quality of
life of cancer patients and for the cure of disease. Despite the therapeutic benefits of
topoisomerase II-targeted anticancer drugs, there is strong circumstantial evidence that
the topoisomerase II-targeted drugs can increase the frequency of cells bearing
mutations [reviewed by 1]. These cells can develop resistance to the therapeutic
agents or may lead to the development of secondary tumours and abnormal
reproductive outcomes [1, 2]. Hence, it is vital to determine the frequency and
severity of mutagenicity following treatment with topoisomerase II-interactive agents
and develop strategies to reduce its occurrence in normal cells.
1.2. Topoisomerases inhibitors:
Topoisomerase II is an essential enzyme which alters DNA topology by
transiently creating and resealing DNA double strand breaks to enable the passage of
one DNA strand through another [3]. It is important in the relaxation of DNA
supercoils generated by cellular processes, such as transcription, recombination and
replication. It is also essential for the condensation of chromosomes and their
segregation during mitosis [3]. To date, there are two general classes of topoisomerase
II inhibitors that interfere with enzyme catalysis at distinct points of the enzyme
reaction. DNA topoisomerase II inhibitors, such as etoposide and doxorubicin,
stabilize cleaved DNA-topoisomerase II complexes, generating high levels of
enzyme-mediated DNA breaks. These drugs can convert this natural essential enzyme
into a potential cellular toxin. Hence, to distinguish their unique mechanism of action,
they are referred to as topoisomerase II "poisons" [Figures. 1 and 2].
In contrast to the complex-stabilizing topoisomerase II inhibitors, catalytic
inhibitors block topoisomerase II at specific stages in its catalytic cycle. Such
blockade can occur either prior to formation of DNA double-strand breaks (e.g.,
aclarubicin, merbarone) or subsequent to religation of these breaks (e.g., dexrazoxane)
[Fig. 1] [4]. Inhibition of topoisomerase II catalytic activity impairs critical DNA
processes such as replication, transcription, and recombination and mitotic failure
[Fig. 2]. Topoisomerase II poisons are, by far, the most frequently used class of
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topoisomerase II inhibitors in clinical chemotherapy. Whereas topoisomerase II
poisons are used for their antitumor activities, catalytic inhibitors could be utilized as
antineoplastic agents (e.g., aclarubicin and merbarone), cardioprotectors (e.g.,
dexrazoxane), or modulators that can increase the efficacy of other agents (e.g.,
suramin and novobiocin) [5].
1.3. Topoisomerase II inhibitors induce chromosomal instability
Since topoisomerase II is involved in DNA replication, chromosome
condensation and de-condensation, chromosome segregation and transcription during
mitosis and meiosis, the possibility exists that topoisomerase II inhibitors may have
potential mutagenic effects on mitotic and meiotic cells. In fact, after application of
topoisomerase II poisons, damage to DNA may result as DNA fragmentation,
chromosomal breaks, and micronucleus formation causing chromosomal instability,
and may lead to mutagenesis, carcinogenesis, or finally to apoptotic cell death [Fig.
2]. Follow-up studies of patients who received etoposide therapy revealed an
increased incidence of acute myeloid leukemia characterized by site-specific
rearrangements in the mixed multiple leukemia gene on chromosome 11q23 [6]. In
addition, a significant increase in the frequency of aneuploid sperm during the first 18
months following initiation of etoposide-including regimen was reported [7]. In
animals, etoposide is a somatic and germ-cell mutagen capable of inducing both
numerical and structural chromosome aberrations [8-14]. Such events may have
important consequences in cancer chemotherapy. Firstly, mutations induced in
somatic cells may lead to the development of secondary tumours from cells that were
not originally neoplastic. Secondly, induced somatic mutations may lead to drug
resistance, limiting further therapeutic response. Thirdly, mutations induced in germ
cells may be transmitted to the progency and pose a genetic hazard to future
generations.
The majority of the literature has described catalytic inhibitors which produce
low levels of topoisomerase II-mediated DNA cleavage as having only modest or
even no clastogenic activity [15, 16]. In contrast, in a few studies measuring
chromosomal alterations merbarone has been reported to produce significant
genotoxic effects both in vivo and in vitro [17, 18]. Additionally, in a few studies
measuring chromosomal damage dexrazoxane has been reported to produce
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significant genotoxic effects in vitro [18, 19]. However, to our knowledge, the in vivo
genotoxic effects of dexrazoxane have never been reported.
1.4. Dexrazoxane
The bisdioxopiperazine dexrazoxane (also known as ICRF-187, Cardioxane
and Zinecard) was originally developed as an antitumour agent. However,
dexrazoxane now is clinically used to reduce doxorubicin-induced cardiotoxicity [20].
Under physiological conditions dexrazoxane undergoes a slow ring-opening
hydrolysis to ADR-925, an analogue of EDTA. Dexrazoxane likely exerts its
cardioprotective effects through ADR-925 by virtue of its ability to strongly chelate
free iron, or to quickly and efficiently remove iron from its complex with doxorubicin
[20], thus reducing doxorubicin-induced iron-based oxygen free radical damage.
Since dexrazoxane is effective in inhibiting doxorubicin’s ability to damage
cardiac cells, there are concerns that the drug may, as a protective agent, diminish the
effectiveness of various chemotherapeutics. There is some clinical and in vitro data
supporting this concern. Hasinoff et al. [21] demonstrated that if Chinese hamster
ovary cells are exposed to dexrazoxane in vitro prior to the administration of
doxorubicin or daunorubicin, a significant antagonism of the antitumor activity
occurs. Alternatively, they showed that if dexrazoxane is administered simultaneously
with or after doxorubicin or daunorubicin, significant additive growth inhibitory
effects occur [21]. Additionally, Holm et al. [22] reported that dexrazoxane rescued
healthy mice from lethal doses of etoposide. Using an L1210 intracranial inoculation
model in mice, Holm and his colleagues have shown that the LD10 of etoposide in
mice increased 3.6-fold when used together with nontoxic dexrazoxane doses. Also,
there was a significant increase in lifespan of mice treated with etoposide and
dexrazoxane as compared to etoposide alone. They concluded that tumour cells in the
brain were reached by cytotoxic levels of etoposide, whereas normal tissues in the
periphery were protected by dexrazoxane. This is because the lipophilic drug
etoposide passes the blood-brain barrier to a much greater extent than the hydrophilic
drug dexrazoxane.
Moreover, combining etoposide and dexrazoxane synergizes with radiotherapy
and improves survival in mice with central nervous system tumours [23]. The
improved survival from radiotherapy following dexrazoxane and etoposide is difficult
to be explained, however, a pharmacokinetics based explanation is attractive. The
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prolonged co-exposure of the cerebral tumour to etoposide and low concentrations of
dexrazoxane enhance the outcome from radiotherapy whereas, extracerebrally, the
much higher dexrazoxane concentration counteracts the toxic myelosuppression
effects [24].
In one clinical trial, interim analysis appeared to indicate that dexrazoxane
might interfere with the therapeutic efficacy of 5-fluorouracil, adriamycin,
cyclophosphamide (FAC) regimen. But upon mature analysis time to progression,
which in the interim analysis was significantly shorter in the dexrazoxane-treated
group, the efficacy had become essentially identical for the two groups [25]. In
another clinical study, the median survival of patients with advanced breast cancer
had increased in patients treated with dexrazoxane plus FAC regimen compared to
those treated with FAC alone [26]. Therefore, the original concern that dexrazoxane
may interfere with a FAC regimen was not confirmed by these clinical trials.
In addition, in vitro studies where tumour cells were exposed to doxorubicin in
the absence or presence of dexrazoxane, demonstrate that the presence of dexrazoxane
significantly delays the appearance of P-glycoprotein-mediated multidrug resistance.
Therefore, the increased survival observed in the clinical studies, mentioned above, is
most likely due to a delay in the appearance of P-glycoprotein-mediated multidrug
resistance, which allows the regimen to remain active for longer periods of times [27].
In contrast, it was reported that dexrazoxane was not able to prevent the induction of
P-glycoprotein following exposure to the tubulin-interacting agent vincristine [28].
This suggests that dexrazoxane may only be useful in the prevention of multidrug
resistance induced by topoisomerase II inhibitors. This could be due to a decrease in
the
number
of
anthracycline-mediated
cleavable
complexes.
Alternatively,
dexrazoxane might be able to modify the transcription pattern induced by
anthracyclines and/or their associated reactive oxygen species, thereby inhibiting the
transcriptional activation of P-glycoprotein.
1.5. Mechanisms of antimutagens.
Antimutagens are compounds capable of lowering the frequency of mutations.
They have diverse mechanisms of action, such as activating cellular systems which
intercept and detoxify mutagens, decreasing genotoxic agent uptake and transport,
stimulating DNA damage repair, and/or elimination of heavily damaged cells via
apoptosis [29]. Apoptosis can be perceived as a repair process in genotoxically
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damaged tissues, capable of reducing the level of mutation by the selective
elimination of heavily damaged cells [29]. The mechanism of apoptosis is linked
through several proto-oncogenes (e.g., c-myc) and tumour suppressor genes (e.g.,
p53) to other biological processes essential for the maintenance of cellular
homeostasis, such as proliferation and the repair of DNA damage. Cells bearing DNA
damage activate genes, the products of which function in DNA repair and/or cell
cycle checkpoint activation [30]. The best studied process is the activation of the
p53 gene in response to DNA damage, which triggers a transcription of genes
involved in the cell cycle arrest (providing the cell with additional time for DNA
repair) and/or apoptosis [30].
Dexrazoxane
Figure 1. The catalytic cycle of DNA topoisomerase II.
The topoisomerase II poisons act by stabilizing stage 4, where the DNA strand is
cleaved. The catalytic inhibitors act either at stage 1 or at stage 2 or at stage 3 or at
stage 6 [4].
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Catalytic Inhibitors
Topoisomerase Poisons
Normal Cell Growth
Dexrazoxane
Low
High
Etoposide
Abnormal Growth
Genomic instability
Cell Death
Mitotic failure & apoptosis
Figure 2. DNA topoisomerase II: essential enzymes and cellular toxins [1].
1.6. Hypothesis
Considering the widespread use of etoposide in clinical oncology and the
ability of dexrazoxane to improve the therapeutic outcome from etoposide prompted
us to investigate whether dexrazoxane in combination with etoposide can ameliorate
etoposide-induced chromosomal instability in mice normal tissues.
The concept of providing protection against chromosomal instability in nontumor tissues will represent a promising approach of attacking the unwanted toxicity
from conventional cytotoxic chemotherapy, and, if successful, will allow the safe use
of increased drug doses for the benefit of future cancer patients.
2. SPECIFIC AIM
The objective of the current investigation is to determine whether dexrazoxane
can protect against chromosomal instability induced by etoposide in mice
genotoxically-damaged cells, and, if so, the possible mechanisms underlying this
amelioration will be assessed.
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To fulfil this aim, various cytogenetic and mechanistic techniques will be applied such
as:
1. Mitotic chromosomal aberrations
2. Micronuclei formations
3. Mitotic activity at both metaphase and interphase stages
4. Oxidative damage
5. Apoptosis
3. MATERIALS AND METHODS
1. Animals. Experiments will be performed with male Swiss albino mice (SWR)
aged 6-10 weeks and weighing 25-30 g. Animals will be obtained from the
Experimental Animal Care Center, King Saud University and will be
maintained on a 12 h light/dark cycle with mouse standard pellet food and
water ad libitum. All experiments on animals will be carried out according to
the Guidelines of the Animal Care and Use Committee, King Saud University,
Saudia Arabia.
2. Chemicals. Etoposide and dexrazoxane will be obtained from the
Developmental Therapeutics Program, National Cancer Institute, Bethesda,
MD, USA. Etoposide will be dissolved in 10% DMSO and dexrazoxane will
be dissolved in 0.9% NaCl. All other chemicals and reagents will be of highest
analytical grade.
3. Experimental protocol. Animals will be randomly assigned into seven groups
of ten mice each, as follows:
 Group 1: mice will serve as a control group and will be intraperitoneally
injected with 10% DMSO in 0.9% NaCl.
 Group 2: mice will be intraperitoneally injected with 40 mg/kg
cyclophosphamide as a positive control mutagen.
 Group 3: mice will be intraperitoneally injected with 1 mg/kg etoposide.
 Group 4: mice will be intraperitoneally injected with 20 mg/kg
etoposide.
 Group 5: mice will be intraperitoneally injected with 125 mg/kg
dexrazoxane.
 Group 6: mice will be intraperitoneally injected with 125 mg/kg
dexrazoxane thirty min before etoposide 1 mg/kg treatment.
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 Group 7: mice will be intraperitoneally injected with 125 mg/kg
dexrazoxane thirty min before etoposide 20 mg/kg treatment.
10% DMSO have previously been shown to be non-mutagen in mice [8, 10, 11].
The doses of etoposide were selected on the basis of its effectiveness in inducing
mutations and are within the dose range used for human chemotherapy [8-14]. A
dose of 125 mg/kg dexrazoxane has previously been shown to be the optimal
protective dose against etoposide-induced myelosuppression and weight loss
toxicities in mice and is corresponding to a clinically relevant dose in humans of
375 mg/m2 [24]. All drugs will be administered within 1 h following preparation.
The animals will be sacrificed by cervical dislocation 24 h after administration of
etoposide to estimate the following parameters.
1. Bone marrow mitotic chromosomal aberrations, micronuclei formations
and mitotic activity: will be performed according to the modified
techniques of Adler [31].
2. Oxidative damage: will be assessed by measuring the reduced
glutathione level according to the protocol described by Tietze [32].
3. Apoptosis; it will be measured by using annexin V detection kit
according to the methods of Vermes et al. [33].
4. STATISTICAL ANALYSIS
The results will be expressed as mean ± standard deviation. An unpaired
Student's t test will be used to assess the significant difference between two groups.
One-way ANOVA will be performed when more than two groups are compared with
a single control, and then significance of differences between individual groups within
the set will be assessed by a multiple comparison test (Dunnett) using a computer
software program (GRAPHPAD INSTAT). The 0.05 level of probability will be used
as the criterion for significance.
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