‫المملكة العربية السعودية‬
‫وزارة التعليم العالي‬
‫جامعة الملك سعود‬
‫عمادة البحث العلمي‬
‫مركز بحوث‪...........‬‬
‫نموذج طلب دعم مشروع بحثي للدراسات العليا‬
‫عنوان البحث ‪:‬‬
‫تأثير الديكسرازوكسان على عدم االستقرار الكروموسومي المستحث بواسطة االتوبوسيد فى الفئران‬
‫اسم الطالب أو الطالبة والقسم‪ :‬أالء أحمد العنتيت‬
‫كلية الصيدلة‬
‫قسم علم األدوية‬
‫اسم المشرف ‪ :‬الدكتور صبري محمد عطية‬
‫الميزانية المقترحة ‪ :‬سبعين ألف لاير‬
‫ملخص البحث‬
‫‪ ‬مشكلة البحث ‪ :‬ان معالجة السرطان في السنوات األخيرة اصبحت تعتمد بشكل أساسي على السمية الخلوية‬
‫للعالج الكيمياوي‪ .‬وبما أن استخدام األدوية المتفاعلة مع انزيمات توبوأيزومرازمن النوع الثاني أثبتت فعاليتها‬
‫في تحسين حياة مرضى السرطان فقد شاع استخدامها كثيرا بالرغم من وجود دراسات عديدة أثبتت بأن هذه‬
‫األدوية تسبب طفرة في الخاليا والتي تتطور الى سرطان من نوع ثاني أو ممكن ان تصبح مقاومة لعالج‬
‫الدواء او ظهور نسل غير طبيعي‬
‫‪ ‬أهمية البحث ‪ :‬لذلك من المهم ان نحدد تطور وخطورة تلك الطفرات في الخاليا بسبب استخدام هذه االدوية‬
‫وبأعتبارنا لالستخدام الشائع لدواء االتوبوسيد في عالج السرطان وقدرة دواء الديكسرازوكسان على تحسين‬
‫فعالية العالج مع االتوبوسيد ‪ .‬هذا قد حفزنا لنبحث بأمكانية الديكسرازوكسان في تحسين عدم االستقرار‬
‫الكرومسوي المحدث باالتوبوسيد في الخاليا الطبيعية للفئران‪ .‬وذلك ممكن أن يسمح لنا بزيادة الجرعات‬
‫لمرضى السرطان بشكل امن وقتل أكثر للخاليا السرطانية مع حماية الخاليا الغير خبثة من االثار الجانبية‬
‫لعقار االتوبوسيد‪.‬‬
‫‪ ‬أهداف البحث ‪ :‬تحديد امكانية الديكسرزوكسان بحماية الخاليا الطبيعية من تأثير األتوبوسيد على عدم‬
‫االستقرار الجيني فيها وذلك بقياس ‪ -1‬طفرة الكروموسونات ‪ -2‬تكون النوية في خاليا نخاع العظام ‪ -3‬حيوية‬
‫االنقسام في طور الميتا والنترفاس ‪ -4‬عامل األكسدة ‪ – 5‬موت الخاليا المنظم (‪)apoptosis‬‬
‫‪ ‬منهج البحث تحديد الطفرة على شكل و عدد الكروموسومات باستخدام المجهر الضوئي‪ ,‬تكون النوية في‬
‫خاليا نخاع العظام باستخدام المجهر الضوئي‪ ,‬تحديد عامل االكسدة باستخدام جهاز سبكتروفوتوميتر‪ ,‬قياس‬
‫موت الخاليا باستخدام جهاز الفلوسيتوميتر‬
King Saud University
College of Pharmacy
Department of Pharmacology
INFLUENCE OF DEXRAZOXANE ON ETOPOSIDEINDUCED CHROMOSOMAL INSTABILITY IN MICE
‫تأثير الديكسرازوكسان على عدم االستقرار الكروموسومي المستحث بواسطة االتوبوسيد فى الفئران‬
Master Research Proposal Submitted to the Department of Pharmacology
By
Alaa A. Al-Anteet
‫أالء أحمد العنتيت‬
B. Pharm (2004)
1429 (H)
2008 (G)
1
1. Introduction
1.1. 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 [1]. DNA topoisomerase II is a target for a number of
clinically useful anti-tumour agents, in part because it is essential for cell survival. 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, amsacrine and doxorubicin stabilize cleaved DNAtopoisomerase II complexes. In contrast to the complex-stabilizing topoisomerase II
inhibitors, merbarone and the bisdioxopiperazines (such as dexrazoxane) block the
catalytic activity of the enzyme [1, 2, 3]. Specifically, the bisdioxopiperazines have
been reported to stabilize topoisomerase II in a closed-clamp configuration around the
DNA, whereas agents such as merbarone have been implicated recently in blocking the
topoisomerase II-mediated DNA cleavage reaction. Because these drugs do not stabilize
DNA-topoisomerase II complexes (i.e., they do not induce DNA strand breaks), they
are termed “catalytic inhibitors” of topoisomerase II [2].
1.2. Topoisomerase II inhibitors induce chromosomal instability
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 [4, 5] [Fig. 1]. Follow-up studies of patients who received
topoisomerase II inhibitors therapy revealed an increased incidence of acute myeloid
leukaemia characterized by site-specific rearrangements in the mixed multiple
leukaemia gene on chromosome 11q23 [6]. In addition, a significant increase in the
frequency of aneuploid sperm during the first 18 months following initiation of
topoisomerase II poisons-including regimen was reported [7]. In animals, topoisomerase
II poisons are somatic and germ-cell mutagens 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
2
further therapeutic response. Thirdly, mutations induced in germ cells may be
transmitted to the progeny 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 significant genotoxic effects in vitro
[18, 19]. However, to our knowledge, the in vivo genotoxic effects of dexrazoxane have
never been reported.
Catalytic Inhibitors
Topoisomerase Poisons
Normal Cell Growth
Dexrazoxane
High
Low
Etoposide
Abnormal Growth
Chromosomal instability
Cell Death
Mitotic failure and apoptosis
Figure 2. DNA topoisomerase II: an essential enzyme and cellular toxins [5].
1.3. Dexrazoxane
Dexrazoxane was originally developed as an anti-tumour agent. However,
dexrazoxane now is clinically used to reduce doxorubicin-induced cardiotoxicity [20].
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, a
significant antagonism of the anti-tumour activity occurs. Alternatively, they showed
that if dexrazoxane is administered simultaneously with or after doxorubicin, 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
3
LD10 of etoposide in mice increased 3.6-fold when used together with non-toxic
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 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].
1.4. 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 [25].
2. Hypothesis:
The hypothesis of providing protection against chromosomal instability in nontumour tissues will represent a promising approach of attacking the unwanted toxicity
from conventional cytotoxic chemotherapy, this will allow the safe use of increased
drug doses for the benefit of future cancer patients.
3. Rational of the Investigation:
Considering the widespread use of etoposide in clinical oncology and the ability
of dexrazoxane to improve the therapeutic outcome from etoposide prompt the
investigation of whether dexrazoxane in combination with etoposide can ameliorate
etoposide-induced chromosomal instability in mice normal tissues.
4
3. Research Objectives:
The objectives of the current investigation are:
3.1. To determine whether dexrazoxane can protect against chromosomal instability
induced by etoposide in mice genotoxically-damaged cells by mitotic
chromosomal aberrations, micronuclei formations and mitotic activity at both
metaphase and interphase stages.
3.2. The possible mechanisms underlying this amelioration will be assessed by
oxidative damage and apoptosis.
4. Materials and Methods:
4.1. 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, Kingdom of Saudi
Arabia.
4.2. 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 analytical grade.
4.3. Experimental protocol: Animals will be randomly assigned into 16 groups of 10
mice each, as follows:
 Group 1: mice will serve as a control group and will be intraperitoneally
(i.p.) injected with 10% DMSO in 0.9% NaCl.
 Group 2: mice will be i.p. injected with 40 mg/kg cyclophosphamide as a
positive control mutagen.
 Groups 3-6: mice will be i.p. injected with etoposide in 4 doses (0.5, 1, 10 or
20 mg/kg).
 Groups 7-8: mice will be i.p. injected with dexrazoxane in 2 doses (125 or
250 mg/kg).
5
 Groups 9-12: mice will be i.p. injected with 125 mg/kg dexrazoxane 30 min
before etoposide 0.5, 1, 10 or 20 mg/kg treatment.
 Group 13-16: mice will be i.p. injected with 250 mg/kg dexrazoxane 30 min
before etoposide 0.5, 1, 10 or 20 mg/kg treatment.
10% DMSO have previously been shown to be non-mutagen in mice [9, 10]. The doses
of etoposide were selected on the basis of its effectiveness in inducing mutations in
mice [8-14] and the selected doses are within the dose range used for human
chemotherapy [26]. Doses of 125 and 250 mg/kg dexrazoxane have previously been
shown to be the optimal protective doses 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 [27].
2. Oxidative damage: will be assessed by measuring the reduced glutathione
level according to the protocol described by Tietze [28].
3. Apoptosis: it will be measured by using annexin V detection kit according
to the methods of Vermes et al. [29].
4.4. Statistical Analysis: Significant differences of mean percentage between
individual treatment groups and solvent control or between the two treatment
groups (etoposide alone or etoposide plus dexrazoxane) will be determined on
an animal to animal basis by the non-parametric Mann-Whitney U-test and ChiSquare test [30]. Differences between groups will assessed by one way analysis
of variance using the GraphPad InStat software package for Windows. Results
will be considered significantly different if the p value is  0.05.
6
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7
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8
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