الفهرس 
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Abstract
Methotrexate (MTX) is an antineoplastic, antipsoriatic and
antirheumatic agent belongs to the group of antimetabolites as it
inhibits folic acid metabolism. The present work is mainly
concerned with the study of the genotoxic effect of it on bone
marrow chromosomes and DNA of male albino mice Mus
musculus.
Sixty CD-1 male mice of nearly the same age (16-18 weeks
old) were individually weighed 24 ± 2 g and randomly divided
into four groups, one control group and three treated groups with
different doses of methotrexate. Each group consisted of fifteen
mice. The control group was injected intraperitonealy with
1ml/kg b.wt. distilled water, while treated group (1) was
intraperitoneally injected with 2.5 mg / kg b. wt., single dose at
the first day of the experiment and sacrificed by cervical
dislocation after 24, 48 and 72 hour of treatment, treated group
(2) was intraperitoneally injected with methotrexate 5 mg / kg
b.wt., single dose at the first day of the experiment and
sacrificed by cervical dislocation after 24, 48 and 72 hour of
treatment and treated group (3) was intraperitoneally injected
with methotrexate 10 mg / kg b.wt., single dose at the first day
of experiment and sacrificed by cervical dislocation after 24, 48
and 72 hour of treatment.
The present study showed that the normal mouse
chromosomes are 40 telocentric chromosomes. They are divided
into five distinct groups; each contains the chromosomes of
nearly equal lengths.
The current results showed that the tested doses of
methotrexate induced structural and numerical chromosomal
aberrations in male albino mice bone marrow cells which were
highly significant increased (P< 0.001) by dose and time.
Structural aberrations were chromosomal and chromatid gaps,
fragments, centromeric attenuation, deletion, centric fusion, ring
formation, end to end association and beaded chromosomes.
Also, methotrexate treatment caused numerical aberration in the
form of polyploidy.
The present results indicated also that the highest mean of
total aberrations was recorded in treated group (3) which treatedwith methotrexate (10 mg / kg b.wt. at 72 hour post treatment),
while the lower rate of total aberrations was recorded in treated
group (1) was treated with methotrexate (2.5 mg / kg b.wt. at 24
hour post treatment). In addition, the present results showed that
gaps and fragments had the highest rate of appearance among all
types of aberrations.
In the present study, methotrexate treatment inhibited the
mitotic rate or cellular proliferation of bone marrow cells in the
three groups treated with 2.5, 5 and 10 mg / kg b.wt. of
methotrexate as compared to control group. Also, the present
results declared that the reduction in mitotic indices in
methotrexate treated groups when compared to the control one
and it was dose and time dependent. A small different average
of percentages of mitotic indices (18 % & 14 %) was observed
between the control and group 1 after 24 hr of treatment,
respectively) and a highly elevated average of percentages of
mitotic indices (18 % & 1.2 %) was observed between the
control and group 3 after 72 hr of treatment, respectively.
Results of micronucleus assay in the present study showed
that methotrexate treatment induced genotoxicity in bone
marrow cells and the number of micronucleated polychromatic
erythrocytes (MNPCEs) was gradually increased significantly (P
< 0.001) and it was dose and time in the treated groups with
methotrexate when compared to control one. Also, cytotoxicity
test showed that the ratio of polychromatic
erythrocytes/normochromatic erythrocytes was gradually
increased significantly (P < 0.001) by dose and time in the
treated groups with methotrexate when compared to the control
one.
The current results of comet assay showed that treatment
with methotrexate significantly increased the mean of DNA
damage in peripheral blood leukocytes of the treated mice in
dose and time dependent manner. Statistical analysis showed
that the mean of DNA damage scored by comet assay was
highly significant increased (P< 0.001) in group 3 (10 mg / kg b.
wt. of methotrexate) after 24, 48 and 72 hour of treatment and in
group 1 (2.5 mg / kg b. wt. of methotrexate) and group 2 (5 mg /
kg b. wt. of methotrexate) after 72 hour of treatment.
However, the randomly amplified polymorphism DNAPolymerase
Chain Reaction (RAPD-PCR) analysis showed
different range of DNA modifications in the DNA content of the
treated mice with methotrexate in comparison with untreated
control. The changes occurring in RAPD profiles included
variation in band density (Bd) as well as gain of new bands (+)
and/or loss of normal bands (-) following methotrexate
treatment.
In conclusion, results of the present study indicated that
methotrexate was highly clastogenic and cytotoxic as it induced
very harmful genetic damage on bone marrow chromosomes
and DNA content of male albino mice even after a low dose and
single treatment. Therefore, the therapeutic uses of methotrexate
should be restricted to a very narrow range border owing to its
harmful genotoxic effect.