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\begin{document}

\title{Protective Effect of Dimethyl Fumarate on Doxorubicin-induced
Genotoxicity in Rats}



\author[1]{Sara Al_kenany }%
\author[1]{nada al-shawi}%
\affil[1]{University of Baghdad Bab Al-Moadham Campus College of Pharmacy}%


\vspace{-1em}



  
  \date{May 22, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Background and Purpose Doxorubicin is a broad-spectrum antineoplastic
agent; however, however, its genotoxic/cytotoxic effects limit its
clinical application. Dimethyl fumarate (DMF) is an FDA-approved oral
drug shown to have antioxidant, anti-inflammatory and antimutagenic
effects via activating Nrf2 antioxidant pathway. The present study aimed
to investigate the possible protective effect of DMF against
doxorubicin-induced chromosomal and DNA damage in rat bone marrow cells.
Experimental Approach Wistar Albino rats of both sexes were administered
DMF orally (15mg/kg once daily for 14 days) alone or with doxorubicin
which was injected as a single dose (90 mg/kg at day 14) to induce
toxicity. The blood samples were collected 24 hours after doxorubicin's
injection from all groups to measure the serum levels of MDA, GSH, SOD,
and GPx1 and bone marrow was harvested to assess chromosomal aberration,
micronucleus, and comet assays. Key Results The rats in the
doxorubicin-only group exhibited a significant decrease in mitotic index
and depleted GSH and antioxidants enzymes serum levels with a
significant elevation in MDA serum level, \% DNA in Tail, micronucleus
appearance and chromosomal aberrations compared to the control group;
DMF pretreatment prior to doxorubicin exposure, significantly-reduced \%
DNA in Tail, micronucleus appearance, and chromosomal aberrations,
improved mitotic index, restored GSH level and antioxidant enzymes
activity compared doxorubicin-only group. Conclusion and Implication
This study revealed that DMF alone has no DNA-damaging or clastogenic
activities; DMF has protective effects against the genotoxicity induced
by doxorubicin; thus, DMF might be a potential chemoprotective agent
against doxorubicin-induced toxicity in cancer chemotherapy%
\end{abstract}%



\sloppy


\textbf{Protective Effect of Dimethyl Fumarate on Doxorubicin-induced
Genotoxicity in Rats}

Running title ``DMF Alleviate DOX-Related Genotoxicity''

Sara Al-Kenany*\textsuperscript{1}, Nada Al-Shawi\textsuperscript{1}

\textsuperscript{1}Department of Pharmacology and Toxicology, College of
Pharmacy, University of Baghdad, Baghdad Iraq

\textbf{*} Corresponding author

E-Mail: s90dokja@gmail.com

ORCID: 0000-0003-2934-5208

\textbf{Words count} 3026.

\textbf{Acknowledgements}

The authors are thankful to the College of Pharmacy, University of
Baghdad, for providing the experimental animals.

\textbf{Author contribution statement}

All authors contributed to the study's concept and design; SA
contributed to the experimental design, conducted the experiments,
analysed the data, and wrote the original manuscript. NA contributed to
the experimental design, confirmed the authenticity of all raw data, and
participated in revising the manuscript. All authors read and approved
the final manuscript.

\textbf{Conflicts of interest}

The authors report no conflicts of interest.

\textbf{Ethics statements}

The animal study was reviewed and approved by The Graduate Studies and
Ethics Committees of the College of Pharmacy, University of Baghdad.

\textbf{Data availability statement}

The data that support the findings of this study are available from the
corresponding author upon reasonable request. Some data may not be made
available because of privacy or ethical restrictions.

\textbf{Funding}

Self-funding.

\textbf{Background and Purpose}

Doxorubicin is a broad-spectrum antineoplastic agent; however, its
genotoxic/cytotoxic effects limit its clinical application. Dimethyl
Fumarate is an FDA-approved to treat multiple sclerosis shown to have
antioxidant, anti-inflammatory and antimutagenic effects via activating
Nrf2 pathway. The present study aimed to investigate the possible
protective effect of DMF against doxorubicin-induced chromosomal and DNA
damage in rat bone marrow cells.

\textbf{Experimental Approach}

Wistar Albino rats of both sexes were administered DMF orally (15mg/kg
once daily for 14 days) alone or with doxorubicin which was injected as
a single dose (90 mg/kg at day 14) to induce toxicity. The blood samples
were collected 24 hours after doxorubicin's injection from all groups to
measure the serum levels of MDA, GSH, SOD, and GPx1 and bone marrow was
harvested to assess chromosomal aberration, micronucleus, and comet
assays.

\textbf{Key Results}

The rats in the doxorubicin-only group exhibited a significant decrease
in mitotic index and depleted GSH and antioxidants enzymes serum levels
with a significant elevation in MDA serum level, \% DNA in Tail,
micronucleus appearance and chromosomal aberrations compared to the
control group; DMF pretreatment prior to doxorubicin exposure,
significantly-reduced \% DNA in Tail, micronucleus appearance, and
chromosomal aberrations, improved mitotic index, restored GSH level and
antioxidant enzymes activity compared doxorubicin-only group.

\textbf{Conclusion and Implication}

This study revealed that DMF alone has no DNA-damaging or clastogenic
activities; DMF has protective effects against the genotoxicity induced
by doxorubicin; thus, DMF might be a potential chemoprotective agent
against doxorubicin-induced toxicity in cancer chemotherapy.

\textbf{Keywords:} Doxorubicin; Dimethyl Fumarate; Genotoxicity;
Chromosomal Aberration; Micronucleus assay; Comet assay.

\textbf{What is already known}

DOX therapy is associated with myelosuppression and~genotoxicity, which
may lead to secondary malignancy.

DOX-induced genotoxicity/cytotoxicity due to the increased level of
oxidative stress and DNA damage in normal cells.

\textbf{What does this study add}

Oxidative stress inhibition by dimethyl fumarate pretreatment
ameliorates DOX-related genotoxic adverse effects in rats' bone marrow
cells.

\textbf{What is the clinical significance}

DMF has promising chemoprotective effects against DOX-related oxidative
stress-induced genotoxic/cytotoxic effects, which might allow the use of
DMF as an adjuvant in chemotherapy

\textbf{1. Introduction}

Doxorubicin (DOX), also known as adriamycin, is an important member of
the anthracyclines group of chemotherapeutic drugs; and it has a broad
anti-tumour spectrum; where it is used alone or in combination with
other chemotherapeutic agents worldwide in the treatment of
haematological malignancies, solid tumours, soft tissue sarcomas,
small-cell lung, and breast carcinoma; moreover, doxorubicin is also the
principal component in the management of Hodgkin's disease and lymphomas
(1). However, the dose-dependent response relation of doxorubicin in
many anticancer regimens has been well-defined; an increase in its dose
restricts its use due to the development of severe cardiotoxicity, in
addition to other cytotoxic effects on normal cells and a substantial
negative impact on patient's health, which poses a significant hurdle in
doxorubicin clinical application (2)(3).

The anti-tumour activity of DOX is mediated through its direct
intercalating with deoxyribonucleic acid (DNA) and by interfering with
the function of many enzymes that are necessary for DNA replication,
including topoisomerase-II; where it stabilises the DNA-topoisomerase-II
intermediate complex and this, in turn, leads to the distortion of DNA
repairing, which consequently results in DNA double-stranded breakage
and nuclei fragmentation with condensed chromatin (4)(5).

In addition, oxidative stress (OS) and overproduction of free radicals
is an essential part of the doxorubicin mechanism of action, where the
metabolism of DOX in the body is mediated by NADPH-dependent cytochrome
P-450 that generates free radicals such as semiquinone, quinone,
hydrogen peroxide (H\textsubscript{2}O\textsubscript{2}), superoxide
anion (O\textsubscript{2}\textsuperscript{*-}), and hydroxyl radical
(OH\textsuperscript{\textbf{@}} \selectlanguage{english}) which can deplete glutathione and
exhaust antioxidants enzymes, increase lipid, protein and nucleic acid
peroxidation (6). Furthermore, the lipid peroxidation product
malondialdehyde (MDA) can interact with the DNA; this consequently can
cause inhibition of DNA replication and chromosomal damage through the
formation of DNA adducts; these cytotoxic actions not only affect cancer
cells but also can affect normal cells triggering mutation and
chromosomal abnormalities including chromosomal aberrations and DNA
damage (7)(8). Therefore, enhancing the cellular antioxidant response
could reduce doxorubicin-induced oxidative damage.

Dimethyl fumarate (DMF) is a fumaric acid-derived small molecule that
exhibits potent antioxidant and anti-inflammatory properties; DMF is a
disease-modifying agent under the brand name ``Tecfidera'' that has been
FDA-approved to be used to treat patients with severe psoriasis and
relapsing-remitting multiple sclerosis (RRMS) (9). DMF's antioxidant and
anti-inflammatory mechanism of action is thought to involve the
activation of the nuclear factor erythroid 2-related factor 2 (Nrf2),
which controls the expression of various genes that regulates
antioxidant and detoxification processes (10). The activation of the
Nrf2 pathway by DMF has been demonstrated in several studies; DMF
treatment showed increased Nrf2 protein levels and gene expression,
which was accompanied by increased expression of downstream target
genes, including heme oxygenase 1 (HO-1) and NAD(P)H: quinone
oxidoreductase 1 (NQO1), reduced Nf-kB, TGF-\selectlanguage{greek}β \selectlanguage{english}signalling and cell
senescence (11)(12)(13)(14). The present study was designed to test the
possible protective effect of DMF against doxorubicin-induced
genotoxicity in rats through the assessment of the extent of chromosomal
aberrations (CAs), micronucleus appearance (MN) and mitotic index (MI)
in addition to the utilisation of the comet assay a sensitive technique
to measure the extent of oxidative DNA damage.

\textbf{2. Materials and methods}

\textbf{2.1. Chemicals}

DMF (Purity \textgreater{}97\%, CAS no.624-49-7) and Colchicine (Purity
\textgreater{}95\%, CAS no.64-86-8) were acquired from Sigma-Aldrich
(St. Louis, MO 63103, USA). Doxorubicin (Doxorubicin HCl 50mg powder for
injection, Khandelwal Labs, India) was purchased from local pharmacies.
Fetal calf serum (FCS) was obtained from Capricon Scientific GmbH, South
America, Giemsa stain (Sigma Chemicals, USA). Polysorbate 20 (Tween-20)
was from Sinopharm chemical reagent Co., Ltd, China. All solvents and
chemicals used were of analytical grade.

\textbf{2.2. Dose selection, preparation, and mode of administration}

Doxorubicin was dissolved in 0.9\% normal saline, and a single dose of
90mg/kg body weight (BW) was intraperitoneally (IP) injected based on
its success in inducing chromosomal damage in Wister rats (15).

In addition, DMF was prepared as a suspension using 5\% tween-20 in
double distilled water (DDW); where it was freshly prepared each day
just before treatment and orally administered to rats by the utilisation
of oral gavage as a single dose of 15mg/kg/day which was selected based
on the previously-shown protective effect (16).

\textbf{2.3. Animals and experimental design}

The Graduate Studies and the Ethical Committees of the College of
Pharmacy, University of Baghdad, approved the study protocol.

Wistar Albino experimental rats of both sexes aged six weeks with an
average weight of 150gm were utilized in this study; since the animals
were acquired and maintained in the College of Pharmacy Experimental
Animal House, University of Baghdad, Iraq; in addition, the experimental
rats were acclimatized for one week prior to starting the experiment;
the rats were housed under controlled conditions of a light/dark cycle
(12hours), temperature at (23\selectlanguage{ngerman}±2°C) and humidity (50±5\%); and had free
access to a standard commercial diet, which was purchased from the local
market, and tap water \emph{ad libitum} .

The experimental animals (32 Rats) were randomly assigned into four
groups (n=8) as follows:

\textbf{Group I} : Each rat was orally administered vehicle only (5\%
tween in DDW) \emph{via} oral gavage for 14 consecutive days. Then a
single dose of normal saline (NaCl) (0.9\%) was IP injected 1 hour after
the last vehicle administration on day 14. This group served as the
control group.

\textbf{Group II} : Each rat was orally administered DMF only
(15mg/kg/day) for 14 consecutive days.

\textbf{Group III} : Each rat was orally-administered vehicle (5\% tween
in DDW) only \emph{via} oral gavage for 14 consecutive days. Then a
single dose of DOX (90mg/kg) was IP injected 1hour after the last
vehicle administration on day 14. This group served as the model group.

\textbf{Group IV} : Each rat was orally administered DMF (15mg/kg/day)
for 14 consecutive days, then a single dose of DOX (90mg/kg) was IP
-injected 1 hour after the last DMF treatment on day 14.

Twenty-four hours after DOX injection (i.e., at day 15), rats were
anaesthetised using diethyl ether, blood samples were collected from the
Jugular vein in non-heparinized tubes and were left to clot at room
temperature, then centrifuged for 20 min at 4000 rpm to obtain serum and
stored at -20°C for biochemical analysis; the animals were sacrificed by
cervical dislocation, and the rats' femoral bone marrow (BM) was
harvested and processed for genotoxicity evaluations (17).

\textbf{2.4. Assessment of Oxidative stress parameters}

The obtained serum was used to assess the oxidative/antioxidant status
by measuring malondialdehyde (MDA), Glutathione (GSH), superoxide
dismutase (SOD) and glutathione peroxidase (GPx) levels using
commercially available ELISA kits obtained from MyBioSource (San Diego,
US).

\textbf{2.5. Preparation of bone marrow (BM) cells for the genotoxic
evaluations}

\textbf{2.5.1. Evaluation of the Chromosomal aberrations (CAs) and the
Mitotic Index (MI)}

BM cells were prepared according to the colchicine-hypotonic citrate
technique for CAs. Briefly, 2 hours before sacrifice, rats were
IP-injected with colchicine (2mg/kg BW); the femur bone of each animal
was taken and cleaned from tissues and muscles, and then the femoral
marrow was flushed out using (0.075M) potassium chloride into a
centrifuge tube and centrifuged at 2000rpm for 10minutes. Additionally,
the cells pellets were fixed in (1:3) acetic acid/methanol (v/v), which
was repeated three times; then the cell suspension was dropped on coded
and sterile cleaned frosted slides and then dried followed by staining
with 10\% Giemsa stain; and finally, the slides were examined under a
light microscope (Japan, Meiji). The frequency of CAs was scored in at
least 100 meta-phase plates per animal; moreover, the chromatid -gaps
and -break chromosome gaps and breaks, ring, deletion and exchanges were
recorded; and the MI was obtained by counting at least 1000 cells per
animal for dividing cells (18) (19).

\textbf{2.5.2. Evaluation of the Micronuclei (MN) Appearance}

The bone marrow cells were prepared following the method of Schmid (20)
with certain adjustments described by Bhilwade et al. (2004) (21). Each
animal's femur was taken, and the excess tissues and muscles were
removed. The femoral marrow was flushed out using fetal bovine serum
(FBS) into a centrifuge tube and centrifuged at 1500 rpm for 10 min to
obtain cells pellet, which was thoroughly mixed, then smeared on coded
and cleaned frosted slides, air-dried and fixed with absolute methanol.
The slides were stained in May--Gruenwald for 5min, then with 10\%
Giemsa for 10 min, followed by thorough washing with DW. The slides were
dried and examined under a light microscope. At least 1000 cells/animal
were screened for scoring the frequency of micronucleated polychromatic
erythrocytes (MNPCEs) (22).

\textbf{2.6. Single-cell gel electrophoresis} \textbf{(SCGE) /Comet
assay}

The comet assay was performed following the method described by
Dhawan\emph{et al.} (23) based on the original procedure developed by
Singh et al. (24). Concisely, the BM was flushed out with the chilled
Hanks'~Balanced Salt Solution~(HBSS) buffer into a microcentrifuge tube;
then 5\selectlanguage{greek}μ\selectlanguage{english}l was mixed with 75\selectlanguage{greek}μ\selectlanguage{english}l of 0.5\% low melting agarose solution
prepared in 0.9\% normal saline and transferred onto frosted slides,
which were kept in lysis buffer (20 mM EDTA, 10\% DMSO and 0.1\% Triton
X-100) for 2 hours at 4 \selectlanguage{ngerman}°C. Then the slides were removed from the lysis
buffer and placed on a horizontal electrophoresis gel box; then the
slides were kept in freshly prepared alkaline buffer (Electrophoresis
Buffer) with pH\textgreater{}13 for 20 min to unwind the DNA strands.
Electrophoresis was carried out for 30 min at 24 volts
(\textasciitilde{}0.74 V/cm), 300 milliamperes. The slides were gently
washed in a neutralising buffer for 5min, which was repeated twice to
remove the alkaline buffer and then dried. The slides were stained with
80\selectlanguage{greek}μ\selectlanguage{english}L 1X Ethidium Bromide, and a minimum of 50 cells/slide was captured
using a 40x objective on a fluorescent microscope. The comet images were
analysed using ``Open Comet'' digital imaging software. The percent (\%)
DNA in Tail, which is the fraction of DNA in the comet tail divided by
the total amount of DNA associated with a cell multiplied by 100, was
measured to assess the extent of oxidative DNA damage.

\textbf{2.7. Statistical Analysis}

The data are demonstrated as Mean \selectlanguage{ngerman}\textbf{±} Standard deviation (SD),
and the statistical significance among groups was determined using
one-way analysis of variance (ANOVA) test followed by Tukey's post-hoc
test for multiple comparisons using GraphPad Prism version 9.5.0.
The\emph{P} values\textless{}0.05 were regarded as statistically
significant.

\textbf{3. Results}

\textbf{3.1. Effect of DMF on oxidative stress parameters}

The antioxidant activity of DMF was assessed in rats following acute DOX
exposure, as oxidative stress plays a vital role in DOX-induced
cytotoxicity. Administration of DOX alone \textbf{(Group III)} caused a
highly significant increase (p \textless{} 0.0001) in the MDA level in
comparison to the control group \textbf{(Group I)} ; However,
pre-treatment with DMF \textbf{(Group IV)} significantly ameliorated the
increase in MDA serum level \textbf{Fig. 1A} . Additionally, DOX
alone\textbf{(Group III)} led to significant GSH depletion, as well as
SOD and GPx-1 exhaustion, compared to the control group \textbf{(Group
I).}In contrast, pre-treatment with DMF prior to DOX exposure
\textbf{(Group IV)} significantly increased the levels of GSH and
restored SOD and GPx-1 activity levels in comparison to the DOX group
\textbf{(Group III),} as shown in \textbf{Fig. 1B-D.} Furthermore, DMF
alone\textbf{(Group II)} caused no significant difference in MDA level
(p \textgreater{} 0.05) \textbf{Fig. 1A} and significantly increased SOD
serum level (p \textless{} 0.001) when compared to the control group

\textbf{(Group I) Fig. 1C.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1. Effect of DMF on Oxidative Stress markers in Wister
rats. DMF, Dimethyl fumarate; DOX, doxorubicin.} A) serum level of MDA,
B) serum level of GSH, C) serum level of SOD, D) serum level of GPx1;
Data are expressed as (mean\selectlanguage{ngerman}±SD), (n=8); *** (p \textless{} 0.001), ****
(p \textless{} 0.0001) vs DOX-only group; ns (P\textgreater{}0.05), no
significant difference.

\textbf{3.2. Effect of DMF on Mitotic index (MI) and Chromosomal
Aberrations (CAs)}

Table 1 showed that in \textbf{Group II} {[}rats orally administered DMF
alone (15mg/kg/day){]}, there was a non-significant difference in the
frequency of TCAs (\emph{P} \textgreater{}0.05) in compassion
to\textbf{Group I} (control).

Furthermore, rats IP injected with a single dose of DOX (90mg/kg)
(\textbf{Group III} ) exhibited a significant increase\emph{(P}
\textless{}0.05) in the frequency of all structural CAs types, total
chromosome aberrations, and abnormal metaphases compared to the control
(\textbf{Group I)} rats.

However, in \textbf{Group IV} (DMF pre-treatment prior to DOX (90mg/kg)
exposure), there were significant reductions \emph{(P} \textless{}0.05)
in the frequency of TCA, chromatid break, ring formation, and
chromosomal break compared to such frequency values in \textbf{Group
III} , with no significant change in the chromatid and chromosomal gap
appearance, chromosomal fragment deletion, acentric and dicentric
chromosome when compared to (\textbf{Group III} ) rats.

Concerning the mitotic index (MI), table 1 showed that there was a
non-significant difference in the percentage of MI (\emph{P}
\textgreater{}0.05) in \textbf{Group II} rats (DMF alone) when compared
to the corresponding index in control (\textbf{Group I} ). Moreover, in
rats IP-injected with a single dose of DOX (\textbf{Group III} ) there
was a significant decrease \emph{(P} \textless{}0.05) in the MI value
compared to the corresponding index in control (\textbf{Group I} ).

However, in \textbf{Group IV} (DMF pre-treatment prior to DOX exposure),
there was a significant increase \emph{(P} \textless{}0.05) in MI
compared to the corresponding index in \textbf{Group III (} DOX only).

\textbf{Table 1. Effect of DMF on the mitotic index (MI) and structural
chromosomal aberrations (CAs) in Wister rats' bone marrow cells}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllll@{}}
\toprule
\textbf{Groups} & \textbf{MI \%} & \textbf{Structural type of
aberration} & \textbf{Structural type of aberration} &
\textbf{Structural type of aberration} & \textbf{Structural type of
aberration} & \textbf{Structural type of aberration} &
\textbf{Structural type of aberration} & \textbf{Structural type of
aberration} & \textbf{Structural type of aberration} & \textbf{TCA
\%}\tabularnewline
& & Chromatid Break & Chromatid gap & Acentric & Dicentric & Ring &
Deletion & Chromosome break & Chromosome gap &\tabularnewline
\textbf{Control} & 8.5 \selectlanguage{ngerman}± 0.58 & 0.06 \selectlanguage{ngerman}± 0.008 & 0.07 \selectlanguage{ngerman}± 0.008 & 0.25 \selectlanguage{ngerman}±
0.032 & 0.21 \selectlanguage{ngerman}± 0.041 & 0.21 \selectlanguage{ngerman}± 0.01 & 0.04 \selectlanguage{ngerman}± 0.01 & 0.07 \selectlanguage{ngerman}± 0.01 & 0.06 \selectlanguage{ngerman}±
0.01 & 0.125 \selectlanguage{ngerman}± 0.008\tabularnewline
\textbf{DMF} & 8.6 \selectlanguage{ngerman}± 0.45 & 0.06 \selectlanguage{ngerman}± 0.008 & 0.06 \selectlanguage{ngerman}± 0.01 & 0.22 \selectlanguage{ngerman}± 0.06 &
0.19 \selectlanguage{ngerman}± 0.01 & 0.21 \selectlanguage{ngerman}± 0.01 & 0.03 \selectlanguage{ngerman}± 0.01 & 0.06 \selectlanguage{ngerman}± 0.01 & 0.06 \selectlanguage{ngerman}± 0.01 &
0.114 \selectlanguage{ngerman}±0.007\tabularnewline
\textbf{Dox} & 5.4 \selectlanguage{ngerman}± 0.98 \textsuperscript{*} & 0.06 \selectlanguage{ngerman}± 0.09
\textsuperscript{*} & 0.12 \selectlanguage{ngerman}± 0.04 \textsuperscript{*} & 0.31 \selectlanguage{ngerman}± 0.07 &
0.23 \selectlanguage{ngerman}± 0.04 & 0.28 \selectlanguage{ngerman}± 0.05 \textsuperscript{*} & 0.07 \selectlanguage{ngerman}±
0.01\textsuperscript{*} & 0.10 \selectlanguage{ngerman}± 0.03 \textsuperscript{*} & 0.11 \selectlanguage{ngerman}± 0.03
\textsuperscript{*} & 0.166 \selectlanguage{ngerman}± 0.021\textsuperscript{*}\tabularnewline
\textbf{DMF + Dox} & 6.7 \selectlanguage{ngerman}± 1.03 \textsuperscript{\#} & 0.07 \selectlanguage{ngerman}±
0.01\textsuperscript{\#} & 0.08 \selectlanguage{ngerman}± 0.02 & 0.26 \selectlanguage{ngerman}± 0.03 & 0.20 \selectlanguage{ngerman}± 0.01 &
0.23 \selectlanguage{ngerman}± 0.03 \textsuperscript{\#} & 0.06 \selectlanguage{ngerman}± 0.01 & 0.08 \selectlanguage{ngerman}± 0.01
\textsuperscript{\#} & 0.09 \selectlanguage{ngerman}± 0.04 & 0.136 \selectlanguage{ngerman}± 0.012
\textsuperscript{\#}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Data are expressed as (mean±SD), (n=8). TCA, total chromosomal
aberration. DMF, Dimethyl Fumarate; DOX, Doxorubicin

* \emph{p} \textless{} 0.05 vs vehicle-only (control/Group I)

\# \emph{p} \textless{} 0.05 vs. doxorubicin-only (Group III)

\textbf{3.3. Effect of DMF on Micronucleus (MN) appearance}

Table 2 showed no significant difference in the appearance of
micronucleated polychromatic erythrocytes (Mn-PCEs\%) in Group II rats
compared to the control group.

Furthermore, table 2 also showed that acute exposure to DOX (90mg/kg)
(\textbf{Group III} ) caused a significant increase\emph{(P}
\textless{}0.05) in the frequency of MN appearance in comparison to such
appearance in control (\textbf{Group I} ). in contrast, DMF pretreatment
prior to DOX Exposure (\textbf{Group IV} ) caused a significant decrease
(\emph{P} \textless{}0.05) in the appearance of Mn-PCEs when compared to
\textbf{Group III} .

\textbf{Table 2. Effect of Dimethyl Fumarate (DMF) on the frequency of
micronucleated polychromatic erythrocytes (\%Mn-PCEs) appearance in
Wister rats' bone marrow cells}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
& \textbf{Groups} & \textbf{\%} \textbf{Mn-PCEs}\tabularnewline
\textbf{I} & \textbf{Control (vehicle-only)} & 6.91 \selectlanguage{ngerman}±
0.64\tabularnewline
\textbf{II} & \textbf{DMF-only} & 6.58 \selectlanguage{ngerman}± 0.36\tabularnewline
\textbf{III} & \textbf{Dox-only} & 10.21 \selectlanguage{ngerman}± 0.66
\textsuperscript{*}\tabularnewline
\textbf{IV} & \textbf{DMF + Dox} & 9.45 \selectlanguage{ngerman}± 0.39
\textsuperscript{\#}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Data are expressed as (mean±SD), (n=8); \% Mn-Es: numbers of
micronucleated cells/total erythrocytes scored.

* \emph{p} \textless{} 0.05 vs the vehicle-only (control)

\# \emph{p} \textless{} 0.05 vs the doxorubicin-only/model (Group III)

\textbf{3.4. Effect of DMF on oxidative DNA damage/comet assay}

There was a non-significant difference in the appearance of the comet,
which can be seen in the form of intact nuclei with supercoiled
undamaged DNA without comet tail in \textbf{Group II} (DMF only)
compared to that in \textbf{Group I} (control) \textbf{Fig. 2A} ; in
addition, DMF alone produced non-statistically significant differences
(\emph{P} \textgreater{}0.05) in the DNA damage (\% DNA in tail)
compared to the control \textbf{fig. 2B} .

While acute exposure to DOX (\textbf{Group III)} showed damaged abnormal
nuclei with DNA strand breaks in the form of comet tail emerging as a
hollow area; moreover, there was a significant increase (\emph{P}
\textless{}0.0001) in \% DNA in tail value in \textbf{Group III}
compared to the control group \textbf{fig 2B} . However, the BM cells
from the rats pretreated with DMF prior to DOX (\textbf{Group
IV)}exhibited an improvement in the comet appearance; in addition, to a
significant inhibition (\emph{P} \textless{}0.0001) of DNA damage (i.e.,
a reduction in the value of the \% DNA in tail) compared to\textbf{Group
III} .\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. Effect of DMF on DNA damage in Wister rats. DMF,
Dimethyl fumarate; DOX, doxorubicin.}

A: Photomicrographs showing comet of rats' bone marrow stained with
Ethidium Bromide.

B: DNA damage measured as \% DNA in tail; Data are expressed as
(mean\selectlanguage{ngerman}±SD), (n=8); **** (\emph{p} \textless{} 0.0001) vs DOX-only group;
ns (\emph{P} \textgreater{}0.05), no significant difference.

\textbf{Discussion}

Although DOX is used for treating a broad range of solid tumours,
nevertheless, its use is associated with severe adverse effects such as
cardiotoxicity and myelosuppression (2)(3). The reduced efficacy of DOX
in treating cancer and the significant genotoxicity was reported to be
related to the direct DNA damage caused by such chemotherapeutic drug
and its ability to interact with the DNA molecule and interfering with
the activity of topoisomerase-II enzyme, thus, interfering with DNA
replication and repair; furthermore, DOX may have an indirect effect
through the generation of free radicals, and this consequently leads to
the depletion of the antioxidants, increasing lipid, protein and nucleic
acid peroxidation, DNA double-strand breakage and chromosomal aberration
in normal cells (4)(7). Therefore, reducing the DOX induced unwanted
effects on normal cells would enable broader use in chemotherapeutic
regimens and improve outcomes in cancer patients.

In the current study, the data showed that acute exposure to DOX caused
a significant increase in the serum level of MDA, depleted GSH and
antioxidant enzymes, induced structural chromosomal aberration and
increased micronuclei appearance in addition to stimulated DNA damage
compared to those in control (\textbf{Group I} ) as shown in Fig 1A,
tables 1 and 2, and figure 2 A\&B, respectively. These results are
agreeable with previous studies stating that DOX caused oxidative
stress, clastogenic changes and DNA damage (25)(26)(27). In addition,
DOX has a noticeable inhibitory effect on the cell division and the
mitotic index value, which is agreeable with the earlier findings
reported in previous studies (15)(28)(29). Furthermore, the results
revealed that DMF (15mg/kg/day) alone caused a non-significant
difference in TCAs, MN appearance and the \% DNA in Tail of the comet;
this might indicate that DMF may have no clastogenic or DNA damaging
effects on rats' BM \emph{in vivo} .

However, DMF (15mg/kg/day) orally administered for 14 days to rats prior
to doxorubicin (90mg/kg) exposure (\textbf{Group IV} ) significantly
reduced MDA level, restored GSH level and antioxidant enzymes activity
compared to \textbf{Group III} which supports its antioxidant role.
Moreover, in rats of \textbf{Group IV} , DMF significantly reduced
(\emph{P} \textless{}0.05) TCAs, MN appearance and \% DNA in Tail
compared to those in the control group; thus, it efficiently protected
against DOX-induced genotoxic effects in rats' BM {[}Fig. 1B-D, Tables 1
and 2 and Fig. 2B{]}.

There are no previous studies regarding the protective effect of DMF
against genotoxicity induced by DOX \emph{in vivo} . Thus, the present
study possibly is the first that demonstrates the modulatory effect of
DMF pre-treatment on DOX-induced chromosomal and DNA damage effects in
rats' BM \emph{in vivo} .

The mechanism underlying DMF anticlastogenic effect against DOX could be
related to its antioxidant activity, as it might reduce the DOX-mediated
free radicals' generation, inhibited the formation of DNA adduct, and
reduced DNA and chromosomal damage since DOX is well-renowned for
inducing cellular oxidative stress (OS) and subsequent DNA damage;
what's more, DMF showed to have an anticarcinogenic/antimutagenic
activity in animal models by modulating NrF2 axis (30)(31); furthermore,
DMF was previously-reported to show a protective activity against
myocardial Ischemic/Reperfusion which is related to DMF mediated
improved cellular viability, reduced oxidative stress and enhanced the
expression of Nrf2\selectlanguage{english}-regulated antioxidative genes; in addition, DMF was
reported to prevent apoptosis, increase the survival rate and
proliferation of human adipose-derived mesenchymal stem cells (hASCs)
against oxidative stress which is mediated by upregulation of HO-1 and
NQO-1 expression (32). Similarly, DMF has been shown to induce apoptosis
in cancer and damaged cells, which may facilitate the reduction in the
appearance of chromosomal damage (33).

\textbf{Conclusion}

According to results obtained from this study, it can be concluded that
oral administration of DMF (15mg/kg/day) alone has no DNA damaging
effect or clastogenic activity. Its administration prior to IP injection
of DOX improved the mitotic index and reduced the extent of DNA damage
and chromosomal damage, and MN appearance in Wistar rats BM cells under
the present experimental conditions; thus, DMF might be a potential
chemoprotective agent against doxorubicin-induced adverse effect in
cancer chemotherapy; consequently, DMF might help prevent reproductive
abnormalities, secondary malignancy development and myelosuppression in
cancer patients.

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\textbf{Figures legend}

\textbf{Figure 1. Effect of DMF on Oxidative Stress markers in Wister
rats. DMF, Dimethyl fumarate; DOX, doxorubicin.}

serum level of MDA, B) serum level of GSH, C) serum level of SOD, D)
serum level of GPx1.

Data are expressed as (mean\selectlanguage{ngerman}±SD), (n=8); *** (p \textless{} 0.001), ****
(p \textless{} 0.0001) vs DOX-only group; ns (P\textgreater{}0.05), no
significant difference.

\textbf{Figure 2. Effect of DMF on DNA damage in Wister rats. DMF,
Dimethyl fumarate; DOX, doxorubicin.}

A: Photomicrographs showing comet of rats' bone marrow stained with
Ethidium Bromide.

B: DNA damage measured as \% DNA in tail.

Data are expressed as (mean±SD), (n=8); **** (\emph{p} \textless{}
0.0001) vs DOX-only group; ns (\emph{P} \textgreater{}0.05), no
significant difference.

\selectlanguage{english}
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