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El-Sayed et al. World Journal of Pharmacy and Pharmaceutical Sciences
EFFECT OF CIPROFLOXACIN, VITAMIN E AND THEIR
COMBINATION ON DNA FRAGMENTATION AND BONE MARROW
CYTOGENICITY
El-Sayed M. G. A.*1, A. A. A. El-Komy
1, Hanan M. Sobhy
2 and Shimaa G. Saied
2
1Department of Pharmacology, Faculty of Veterinary Medicine, Benha Univerisity.
2Department of Biochemistry, Animal Health Research Institute, Dokki, Giza.
ABSTRACT
The antibacterial activities of the fluorinated 4-quinolone,
ciprofloxacin has been ascribed to a marked inhibition of bacterial
DNA gyrase. However, evidence is accumulating that ciprofloxacin
may cause liver DNA damage. Vitamin E is a free radical scavenger
and antioxidant. This study aimed to investigate the influence of
ciprofloxacin in recommended dose on liver and spleen deoxy-
ribonucleic acid as well as on micronuclei formation in bone marrow
erythrocytes and the effects of vitamin E on these changes.Sixty adult
male Sprague Dawley rats were divided into four equal groups:
control, ciprofloxacin treated, vitamin E treated and ciprofloxacin and
vitamin E treated. DNA fragmentation was evidenced in liver and
spleen cells of ciprofloxacin treated rats.Micronuclei formation significantly increased in
ciprofloxacin treated rats compared to control group. Vitamin E administration reduced liver
DNA fragmentation caused by ciprofloxacin in rats. It is suggested that.
KEYWORDS: Ciprofloxacin, Vitamin E, DNA, Cytogenicity.
INTRODUCTION
Ciprofloxacin is a flourinated quinolone carboxylic acid derivative which was developed for
use exclusively in animals. It exhibits a wide spectrum of antimicrobial activity, like other
fluoroquinolones including some Gram positive, Gram negative bacteria and Mycoplasma.[1]
Ciprofloxacin acts by the inhibition of the A subunit of the bacterial DNA gyrase which is an
essential enzyme for DNA replication and synthesis. This synthetic drug acts as bactericidal
Article Received on
21 Jan. 2018,
Revised on 11 Feb. 2018,
Accepted on 02 March 2018
DOI: 10.20959/wjpps20183-11080
*Corresponding Author
El-Sayed M. G. A.
Department of
Pharmacology, Faculty of
Veterinary Medicine, Benha
Univerisity.
drshimaa2016@yahoo.com
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 7.421
Volume 7, Issue 3, 1045-1060 Research Article ISSN 2278 – 4357
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El-Sayed et al. World Journal of Pharmacy and Pharmaceutical Sciences
at relatively low concentration[2]
The type II topoisomerases DNA gyrase and topoisomerase
IV is the target of quinolones.[3]
Vitamin E is the major lipid soluble antioxidant responsible for protecting membranes against
lipid peroxidation and is the term for a group of tocopherals and tocotrienols, of which -
tocopherots, has the highest biological activity.[4]
Most alpha-tocopheral is located in the
mitochondrial fractions and in the endoplasmic reticulum of the cells.[5]
The possible protective effect of vitamin E to the genotoxicity induced by fipronil, a
phenylpyrazol insecticide classified as a carcinogen by United States Environmental
Protection Agency was studied. The structural chromosome aberrations in bone marrow cells
and deoxy-ribonucleic acid damage in the lymphocytes was found to be significantly higher
in the male and female rats exposed to fipronil as compared to their respective controls. The
average degree of protection (male and female animals combined together) shown by
pretreatment of vitamin E against fipronil-induced genotoxicity was 63.28%: chromosome
aberrations; 47.91%: micronuclei formation; and 74.70%: deoxy-ribonucleic acid damage.[6]
The aim of the present work was undertaken to study the cytotoxic effect of ciprofloxacin on
cellular deoxy-ribonucleic acid in vivo and to declare the role of vitamin E as an antioxidant
to protect cellular deoxy-ribonucleic acid. Also to study Effect of ciprofloxacin, vitamin E
and their combination on bone marrow cytogenicity.
MATERIALS AND METHODS
Materials
1-Drugs
Ciprofloxacin is a flouroquinolone available as white to yellowish white powder under trade
name CIPROVAN®
20% (Turkey)-it is easily soluble in water. The drug used directly after
reconstitution in water for oral administration. Vitamin E is available as oral gelatinous
capsules contain yellowish oily substance. It was purchased from PHARCO Company,
Egypt. It is chemically α-Tochopherol, 5,7,8- trimethyltocol. Ciprofloxacin and vitamin E
were administrated in a dose of 12.5mg/ Kg b.wt and 100mg/kg b.wt by stomach tube once
daily for one week.[7,8]
Agrose gel (1%), TAE buffer (0.5x Tris Acetate EDTA buffer),
ethydium/bromide and Marker (M) 100-bp ladder size marker were obtained from Sigma
company. Kits for DNA extraction obtained from Intron Company, Korea.
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2-Rats
Sixty mature male rats of an average body weight 150-180 grams of Sprague Dawley strain
were obtained from Animal Health Research Institute Giza, Egypt. Basal diet supplying the
essential vitamins and trace elements was prepared.[9]
METHODS
Experimental design
The experiment was done in the pharmacology unit, Biochemistry, Toxicology and Food
Deficiency Department, Animal Health Research Institute. Animals were acclimatized to
laboratory conditions for one week. The animals were kept under hygienic conditions, housed
in metal cages containing wood shaving as bedding material. Rats kept under controlled
temperature at 23°C ± 2°C and maintained on a 12/12 h light and dark cycle during the
experiment with nonstop access to rat chow and tap water. The use of animals in the study
was in accordance to the guidelines of National Research Council for the care and use of
Laboratory Animals. Rats fed on basal diet and water supply was given ad-libtium all over
the period of the experiment. Rats were divided into four groups which were group (1) that
act as negative control, group (2) that given ciprofloxacin, group (3) that given vitamin E and
group (4) that given vitamin E as pretreatment followed by ciprofloxacin for one week.Liver
and spleen from each sacrificed rat were collected in sterilized eppindrof tubes and kept in
deep freeze (-80oC) for DNA fragmentation study. Also, bone marrow samples extruded from
femur of control and treated rats were collected separately and fixed immediately with
ethanol for cytogenic assay.
DNA fragmentation [DNA ladder Assay]: DNA Damage Inhibition Analysis was done in
Genetic Research Center El-Sadat University. The extent of DNA fragmentation (DNA
ladder) has been assayed by electrophoresing genomic DNA samples, isolated from normal
as well as treated rat livers and spleens; on agrose/ethydium bromide gel.[10]
High quality
genomic DNA was extracted from (-80oC preserved liver and spleen samples of all treated
and control groups) by precipitation of protein and other contents and further precipitation of
high molecular weight genomic DNA by absolute ethanol[11]
The amplified products of
different exons were verified in 1% (W/V) agrose gel. DNA molecules are separated by
applying an electric field to move the negatively charged molecules through an agrose matrix.
Shorter molecules move faster and migrate farther than longer ones because shorter
molecules migrate more easily through the pores of the gel.
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Bone marrow cytogenetic assay: In order to investigate the ameliorating effect of vitamin E
on the possible mutagenic effects of ciprofloxacin, the micronucleus test was performed to
detect chromosomal damage associated with the treatment. Micromuclei were identified as
dark blue staining bodies in the cytoplasm of the poly chromatic erythrocytes (PCEs). Using
standard laboratory technique[12]
,bone marrow cells of control and treated rats were extruded
with a pin into a clean dry glass slide and homogenized with two drops of fetal calf serum on
the slide, air dried, fixed in absolute methanol and stained with Giemsa 5% in phosphate
buffer pH 6.8.The pollychromatic erythrocytes (PCEs, 1000 per animal) were screened for
micronuclei, and the changes in the mitotic activity[13,14]
were assessed on the basis of the
ratio of polychromatic to normochromatic erythrocytes (PCE/NCE ratio).
STATISTICAL ANALYSIS
Statistical analysis was performed using one-way analysis of variance (ANOVA) for multiple
comparisons of all groups. Means and there standard errors (SE) were calculated using the
SPSS 16 software, followed by Least Significant Difference test (LSD) to calculate the
statistical difference between various groups. A value of P<0.05 was considered statistically
significant.[15]
RESULTS
Effect of ciprofloxacin, vitamin E and its combination on DNA fragmentation
Liver DNA fragmentation on agrose/ethydium bromide gel, DNA isolated was loaded onto
1% (W/V) agrose gels compared to Marker (M) 100-bp ladder size marker at one day post
last treatment as in figure (1). Lane (1) represented DNA isolated from liver tissues of
negative control rats revealed a single band corresponding to intact nuclear DNA. Lane (2)
represented DNA isolated from ciprofloxacin treated liver samples revealed smear formation
that indicated DNA damage and necrotic cell death to hepatic cells. Lane (3) represented
DNA extracted from liver samples treated with vitamin E revealed intact DNA as one nuclear
band. Lane (4) represented DNA extracted from liver samples of rats treated with both
ciprofloxacin and vitamin E at the same time which revealed incomplete protective action of
vitamin E to nuclear DNA fragmentation.
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Figure. (1): DNA isolated from experimental liver tissues 1 day post last treatment on
agarose/ethydium bromide gel.
Liver DNA extracted from liver samples at 7 days post last treatment. The protective effect of
vitamin E to the necrotic cell death is clearly visible in lane (4), as the gels revealed no smear
formation while only one migrating band of low molecular weight in each lane in comparison
with smear and band formation in lane (2) that represent DNA extracted from liver cells of
ciprofloxacin treated rats as represented in figure (2).
Figure. (2): DNA isolated from experimental liver tissues 7 days after stopping of
treatment on agarose/ethydium bromide gel.
DNA extracted from liver samples at 14 days post last treatment showed that both lane (2)
and lane (4) revealed weak staining quality of extracted genomic DNA from liver samples
suggesting DNA diminution in hepatocytes. Lane (4) showed no smear formation in
comparison with lane (2) because of a consequence of low DNA degradation as represented
in figure (3).
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Figure. (3): DNA fragmentation on agarose/ethydium bromide gel. DNA isolated from
experimental liver tissues 14 days after stop of treatment.
DNA fragmentation on agarose/ethydium bromide gel isolated from spleen of rats at one day
after stop of treatment was represented in figure (4). Lane (1): represents DNA extracted and
electrophoresed from spleen of negative control samples revealed a single nuclear band. Lane
(2) represents DNA extracted from spleen samples of ciprofloxacin treated rats exhibited
multiple fragments evidenced by a ladder – like separation of DNA fragments on the gel.
Lane (3) represented DNA extracted from spleen samples of vitamin E treated rats, exhibited
intact nuclear DNA. Lane (4) represents DNA extracted from spleen samples ciprofloxacin
and vitamin E treated rats revealed that vitamin E is effective to some extent in protection of
the cells and DNA fragmentation.
Figure. (4): DNA isolated from experimental spleen tissues 1 day after stop of treatment
agrose / ethydium bromide gel.
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DNA fragmentation on agarose/ethydium bromide gels, isolated from spleen samples of rats
at 7 days after stop of treatment. Lane (2) showed ladder like separation of genomic DNA
fragments to some extent due to stopping of treatment on week later, while in lane (4), no
ladder like fragmentation as found in figure (5).
Figure. (5): DNA isolated from experimental spleen tissues 7 days after stopping of
treatment on agarose/ethydium bromide gel.
DNA extracted from spleen samples at 14 days post last treatment was represented in figure
(6). Lane (2) represents DNA extracted from spleen samples of ciprofloxacin treated rats. It
revealed smear formation but no clear ladder – like separation of genomic DNA as a result of
decrease oxidative stress of ciprofloxacin on spleen cells by time. Lane (4) revealed intact
migrating bands and slight smear formation, due to antioxidant role of vitamin E on DNA
damage induced by ciprofloxacin.
Figure. (6): DNA fragmentation on agarose/ethydium bromide gel. DNA isolated from
experimental spleen tissues 14 days after stop of treatment.
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Effect of ciprofloxacin, vitamin E and its combination on bone marrow cytogenicity
The obtained results showed that treatment with ciprofloxacin produced significant decrease in normochromatic erythrocytes (NCE) at one and
seven days post last treatment and increase in multinucleated polychromatic erythrocytes (MPCE) as well as the ratio of polychromatic
erythrocytes to normochromatic erythrocytes (PCE/NCE) compared to control group. Vitamin E treatment produced no change in
normochromatic erythrocyts (NCE) 1, 7and 14 days post last treatment compared with control group. Rats treated with both ciprofloxacin and
vitamin E (group 4) indicated significant increase in normochromatic erythrocytes (NCE) compared to ciprofloxacin treated group 1and 7 days
post last treatment as well as decrease in multinucleated polychromatic erythrocytes(MPCE) and the ratio of polychromatic erythrocytes to
normochromatic erythrocytes(PCE/NCE) compared to control group (group1), 1and 7 days post last treatment as illustrated in table 1 and figure
7,8 and 9.
Table. (1): Effect
of ciprofloxacin, vitamin E and their combination on the incidence of multinucleated polychromatic
erythrocytes(MPCE), normochromatic erythrocytes(NCE) and polychromatic erythrocytes / normochromatic erythrocytes
ratio(PCE/NCE), (n=5).
Groups PCE
NCE MPCE PCE/NCE
Time post treatment Time post treatment Time post treatment
1 day 7days 14days 1 day 7days 14days 1 day 7days 14days
Group (1) 5000 2156.67±
9.63a
2159.4±
12.46a
2148.98±
23.82a
5.32±
0.20a
5.25±
0.15a
5.15±
0.13a
2.22±
0.30a
2.39±
0.17a
2.55±
0.11a
Group (2) 5000 740.39±
11.49 b
824.82±
29.77b
2130.21±
5.00b
14.58±
0.43 b
9.22±
0.072b
5.63±
0.11b
6.77±
0.22 b
5.81±
0.24 b
2.83±
0.26b
Group (3) 5000 2153.86±
20.19c
2183.9±
31.58c
2156.92±
5.59c
5.86±
0.12c
5.67±
0.27c
5.26±
0.14c
2.10±
0.10a
2.32±
0.09a
2.03±
0.14a
Group (4) 5000 944.63±
32.18 b
961.79±
18.02 b
2149.81±
41.09b
9.14±
0.24 d
7.04±
0.13 d
5.75±
0.15d
3.78±
0.12 c
3.70±
0.10 c
2.76±
0.18b
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Figure. (7): Effect
of ciprofloxacin, vitamin E and their combination on
Normochromatic erythrocytes (NCE).
Figure (8): Effect
of ciprofloxacin, vitamin E and their combination on multinucleated
polychromatic erythrocytes.
Figure (9): Effect
of ciprofloxacin, vitamin E and their combination on polychromatic
erythrocytes/ Normochromatic erythrocytes.
G(1):Untreated control rats.
G((2):Rats treated with ciprofloxacin(12.5 mg/kg b.wt) for 7 days.
G(3):Rats treated with vitamin E (100mg/kg b.wt.) for 7 days.
G(4):Rats treated with ciprofloxacin (12.5 mg/kg b.wt) and vitamin E(100mg/kg b.wt.) for 7
days.
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DISCUSSION
It is known that ciprofloxacin could inhibit of gyrase, a specific prokaryot enzyme that
catalyses the conversion of relaxed DNA into negatively supercoiled DNA. Acombination of
gyrase and topoisomerase-I is required to correct DNA topology during replication and
transcription. The inhibition of gyrase by quinolones triggers replication arrest and cell
death.[16]
The obtained results showed that the cellular DNA isolated from splenic and hepatic
cells, one day post last treatment in ciprofloxacin treated group evidenced clear fragmentation
and distinct ladder-like appearance especially in splenic DNA. Explaining the results,
Bredberg et al., (1991)[17]
reported that ciprofloxacin-induced marked induction of double-
strand DNA breaks in human lymphoblastoid cells. Moreover, Albertini et al., (1995)[18]
declared that, ciprofloxacin inhibit resolution of the normally transient topoisomerase II-
DNA cleavage complex resulting in chromosome stickness. This was supported later by
Gürbay et al., (2007)[19]
, who reported that ciprofloxacin generated reactive oxygen species
(Hydrogen peroxide) which considered one of most free radical causing cellular protein
damage and apoptosis in primary cultures of rat astrocytes and normal human fibroblast cells.
On the other hand, the obtained results of DNA fragmentation in ciprofloxacin treated rats
seven days post last treatment showed DNA damage in hepatic cells, suggesting the
continuation of impact action of ciprofloxacin through generation of reactive oxygen species
resulted in liver cellular damage.[20,21]
Results of splenic DNA fragmentation, seven days
following last treatment, are in harmony with that obtained by Klaudia and Alina, (2015)[22]
who reported that enrofloxacin, florfenical and ceftiofur administrated in therapeutic doses
may decrease the percentage of B and T cells subset in lymphatic organs and might influence
on the proportion of B and T cells in chicken spleen and thymus. The results of DNA
fragmentation are parallel fomer studies.[23-26]
The obtained results, 14 days post treatment for
hepatic and splenic genomic DNA indicated decreased smear formation isolated form
ciprofloxacin treated rats and low DNA degradation, this could be explained by that the
ciprofloxacin concentration, decrease in the cellular fluid by time, so it is evidenced from the
study that the effect of ciprofloxacin on DNA might be reversible. In contrary with our results
at one and 7 days post treatment, Herbold et al., (2001)[27]
mentioned that ciprofloxacin tested
for in vivo genotoxicity revealed no genotoxic effect using the following test systems:
micronucleus test in bone marrow of mice, cytogenetic chromosome analysis, in Chinese
hamster, dominant lethal assay in male mice and UDS tests in primary rat and mouse
hepatocytes in vivo. In addition, ciprofloxacin not found to be carcinogenic in two rodent
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long-term bioassays. On the other hand Smart et al., (2008)[28]
suggested that DNA-douple
strand breaks are not induced by 0-100 g/ml ciprofloxacin (0-10 hours exposure) in human
derived cells, therefore ciprofloxacin –induced clastogenicity in such cells arised from DNA
damage is different to direct DNA double strand breaks, moreover, the study didn't rule out
the possibility of other types of ciprofloxacin-mediated DNA damage as single strand DNA
lesions.
Regarding DNA fragmentation in hepatic cells, in ciprofloxacin and vitamin E group(G1),
one and seven days post treatment, Lane (4) in figure (1) and (2) revealed partial protection
by vitamin E, as decreased, smear formation with migration of low molecular weight nucleic
acid bands. Furthermore, vitamin E has no protective action on splenic DNA degradation by
ciprofloxacin in group(4),one day post treatment as evidenced by ladder-like separation of
splenic DNA, showed in lane (4), figure (4). One the other hand, vitamin E, showed a clear
protection in lane (4) in both figure (5) and (6) that representing DNA isolated from splenic
cells of ciprofloxacin treated rats, seven and 14 days respectively following last treatment, as
that genomic DNA has a likely an intact appearance with high molecular weight and low
degradation model.
Explaining the obtained results, about role of vitamin E in monitoring cytotoxic effect of
ciprofloxacin on hepatic and splenic cells, we can say that, vitamin E is mainly a chain-
breaking antioxidant against membranal damage, but probably not as effective against the
damage at mitochondrial level[19],
so vitamin E couldn't reverse the induction of oxidative
DNA damage by ciprofloxacin in hepatic and splenic cells as evidenced by the obtained
results one day post treatment. Moreover, vitamin E should be used as a pretreatment to this
drug, this supported by Mozdarani and Salimi, (2006)[29]
who declared that administration of
vitamin E before irradiation effectively reduced the frequency of chromosomal abnormalities.
The partial protective effect of vitamin E, seven and 14 days post treatment, declared its role
as radical scavenger. Similar results of vitamin E were also observed.[6,30-33]
According to the results of ciprofloxacin effect on micronuclei formation in bone marrow
erythrocytes, The administration of ciprofloxacin at therapeutic dose for 7 days causing
increase in multinucleated polychromatic erythrocytes statistically following stopping of
administration by one and seven days in comparison with control group. The micronecleus
assay is devised primarily for evaluating the ability of test agent to induce structural and/or
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numerical chromosomal damages. This results come in accordance with Curry et
al.,(1996)[23]
who found that structural chromosomal aberrations detected in metaphase cells
was attributed to ciprofloxacin-inhibition of topoisomerase II prior to mitosis while
micronuclei arised in binucleated cells as a result of this effect which interfers with
chromosomal separation. In the same manner, Olajuyigbe et al.,(2011)[34]
declared that
apossible mechanism for the induction of micronuclei in the mouse bone marrow cells is that
ciprofloxacin inhibited topoisomerase II in mice exposed to different concentrations of
ciprofloxacin (16,32,80,160, 320 µg/ml). In-vitro study by Ṥekeroğlu et al.,(2017).[26]
investigated the cytotoxic and genotoxic potentials of ofloxacin in cultured human peripheral
lymphocytes. Cultures were treated with 30, 60 and 120 μg/ml of ofloxacin for 48 hours.
Ofloxacin significantly induced chromosomal aberrations at all concentrations and sister
chromatid exchanges at higher concentrations (60 and 120 μg/ml) compared with solvent
control. El-Habit, Ola, et al (2001)[35]
suggested that the direct assessment of genotoxicity of
ofloxacin was achieved by scoring the incidence of multinucleated polychromatic
erythrocytes and multinucleated normochromatic erythrocytes. The results demonstrated a
positive dose response relationship, which suggested that ofloxacin is mutagenic at high
doses. The equivalent therapeutic dose, however, did not induce marked increase in the
incidence of micronucleated erythrocytes. The discrepancies noted between different reports
can be attributed to differences in dose, rout of administration, duration of treatment or test
system. In contrary with the present study, Herbold et al., (2001)[27]
mentioned that
ciprofloxacin tested for in vivo genotoxicity revealed no significant increase in the number of
micronucleated erythrocytes in bone marrow of mice.
However,vitamin E in combination with ciprofloxacin decreased the level of micronuclei
formation and polychromatic erythrocytes to normochromatic erythrocytes ratio. This
supported by Chorvatovičová.,et al., (1991)[36]
who found that the micronucleus test in bone
marrow showed that vitamin C caused antimutagenic effect against bichromate, in both rats
and guinea pigs,while the effect of vitamin E was demonstrated only in an increase of the
ratio of normochromatic erythrocytes to polychromatic erythrocytes, i.e., in a decrease of the
cytotoxic but not the mutagenic effects of hexavalent chromium. Moreover, Konopacka and
Rzeszowska-Wolny (2001)[37]
decleared that in-vitro study, human lymphocytes treated with
vitamin E at a concentration above 2 mg/ml showed a decrease at the level of radiation-
induced micronuclei when compared to the cells irradiated without vitamin treatment. Singh
et al., (2008)[8]
demonstrated that vitamin E along with atrazine, decrease the percentage of
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El-Sayed et al. World Journal of Pharmacy and Pharmaceutical Sciences
micronuclei as compared to atrazine treated rats. Furthermore, Badgujar et al.,(2017)[6]
suggested that adult male and female animals gavaged with various doses of fipronil (2.5,
12.5, and 25 mg/kg body weight to evaluate micronucleus test (mice), chromosome
aberration, and comet assay (rats), respectively and were pretreated with vitamin E orally
(400 mg/kg body weight) for 5 days prior to administration of fipronil (12.5 mg/kg).
Pretreatment of vitamin E induced a decrease in fipronil-induced micronuclei formation by
47.91%.
CONCLUSION
The results of the present study suggested that ciprofloxacin induce genotoxicity on basis of
cellular deoxy-ribonucleic acid. Moreover,micronuclei formation and increase in immature
erythrocytes were evidenced. Treatment with a known antioxidant, vitamin E has shown
protective effect to DNA fragmentation.
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