Evaluation of the Cytogenetic Status and DNA Integrity of Human... Rhazya Stricta

Journal of Applied Sciences Research, 5(8): 986-994, 2009
© 2009, INSInet Publication
Evaluation of the Cytogenetic Status and DNA Integrity of Human Lymphocytes after
Exposure to an Aqueous Extract of Rhazya Stricta Leaves in Vitro
1
1
Nabih A. Baeshen, 1Jamal S.M. Sabir, 1Salah E. M. Abo-Aba and 2Sameer H. Qari
Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah,
Department of Biological Sciences, Teachers Collage, Umm Al Qura University, Makkah,
Saudia Arabia
2
Abstract: Human lymphocyte culture cells were treated with three different concentrations (6, 12 and 24
g/liter) of R. stricta aqueous leaf extract. Samples were collected at three different intervals (24, 48 and
72h) for each concentration. Following each treatment, cell samples were subjected to cytogenetic as well
as comet test assays. The cytogenetic assay revealed a significant decrease in mitotic index that was
inversely proportional with concentration and exposure time. Many aberrations, including high percentage
of interphase, increased micronuclei, viscosity, colchicines metaphase and two nuclei-cells were detected.
Microscopic observation showed necrosis of most of the treated cells at all used concentrations, an
indicative possibility of anticancer activity. Comet test detected DNA lesions that were proportionally
concentration- and exposure time-dependent too. The present data strongly suggest that the aqueous extract
of the R. stricta leaves has mutagenic, clastogenic and possibly anticancer activities on human
lymphocytes in vitro.
Key words: Rhazya stricta, cytogenetic assay, comet assay (SCGE), micronuclei test, DNA lesion,
clastogenesis, mutagenesis.
tissues and/or special cell types, its sensitivity for
detecting low levels of DNA damage, and its
requirement for small numbers of cells per sample,
[2 4 ,1 7 ,1 3 ,1 5 ,2 5 ,2 0 ,2 1 ,2 9 ,2 7 ,2 6 ,1 4 ]
. As a continuation for the battery
of tests of genotoxicity, we utilized cytogenetic and
comet test assays to evaluate the effects of the aqueous
extract of the Rhazya stricta leaves on the genetic
material of human lymphocyte culture.
INTRODUCTION
Rhazya stricta Decne belongs to Apocynaceae
family and is widely distributed in Saudi Arabia as
well as throughout the world. Its leaf extracts were
prescribed in folkloric medicine for the treatment of
various disorders such as diabetes, sore throat,
helminthiasis, inflammatory conditions and rheumatism
[1 ,4 ,3 ]
. The plant extract contains mainly alkaloids,
glycoside, flavonoides, tannins and triterpenes [2 2 ,6 ,5 ].
Several studies on rats and mice reported that the leaf
extract causes sedation, analgesia, a decrease in motor
activity; furthermore, it has anti-depressant and antioxidant activities, as well as complex effects on brain
endogenous monoamine oxidase activity and centrallymediated hypotension [3 ,2 3 ,2 ]. In addition, genotoxic effect
of the R. stricta leaf aqueous extract was demonstrated,
for the first time on S. cerevicae auxotrophic mutant by
the genuine study of Baeshin et al.[1 0 ] and, more
recently, by Baeshin et al.[8 ,9 ] in the course of
undertaken battery of tests of genotoxicity. Anticancer
activities were ascribed to the indole alkaloids of R.
stricta [1 6 ].
The comet assay or single-cell gel (SCG) test is a
micro-gel electrophoretic technique that measures DNA
damage at a single cell-level. It is increasingly being
used in genotoxicity testing. The advantages of the in
vivo comet assay include its applicability to various
M ATERIALS AND M ETHODS
M aterials: Plant identification was carried out
according to Batanouny and Baeshin [1 1 ]. Rhazya stricta
Decne was collected on April (2006) from naturally
growing plants in their natural habitats located along
the roadsides of Jeddah-Makkah Highway (Fig. 1).
Leaves were collected in plastic bags in the field,
transferred in their bags to a fridge, kept overnight and
then subjected to extraction in the following day.
Plant leaves were washed several times under
running water followed by distilled water. The leaves
were cut into small pieces and the following
concentrations were made: 6gm/L (this concentration
is widely used in a traditional folkloric use), 12gm/L
and 24gm/L. Before blending process to get the
homogenized leaves extract, leaves were soaked for
24hr. for each concentration. After blending, leaves
Corresponding Autuor: Nabih A. Baeshen, Department of Biological Sciences, Faculty of Science, King Abdulaziz
University, Jeddah, Saudi Arabia
986
J. Appl. Sci. Res., 5(8): 986-994, 2009
extract of each concentration was separately filtered
with filter papers; the filtrates were either used directly
in the experiment or kept in the fridge for no longer
than three days for future use [8 ].
Peripheral blood samples were collected from adult
healthy individuals, who were free of any drug
treatment, viral infection, hepatitis at any time, or
recently exposed to ionizing radiation and having no
history of smoking.
Comet Assay (SCGE): Agarose gel was prepared by
dissolving 65mg of agarose solution in 10ml of PBS
solution in microwave. Low-melting agarose solution
was prepared by dissolving 50mg of low-melting
agarose powder in 10ml of PBS solution. 300 µl of the
prepared agarose was dropped on a surface of comet
slide; slides were then covered with slide covers, air
bubbles were removed, slides were heated for
distribution of agarose and cooled for an hour in a
fridge. Covers were then carefully removed; 10µl of
the cell suspension were added to 90µl of low-melting
agarose solution, mixed completely and one drop was
added to each slide. Then each slide was covered.
M ethods:
Obtaining and Culturing Human Lymphocytes: 0.5
ml of blood was added to 4.5 ml of complete RPM I
medium [2 9 ] in a sterile centrifuge tube. The different
concentrations of prepared Rhazia stricta leaf extracts
were added to the test groups; meanwhile, distilled
water was added to the control culture. Caps tightly
screwed onto tubes and placed at 37 o C in an incubator
to initiate the culture for 48 hr.[2 6 ,1 4 ].
Preparation of Lysing Solution: Lysing solution was
prepared by dissolving 164.1gm sodium choride,
37.2gm EDTA 1.2gm Tris and 12gm sodium hydroxide
in 700 ml deionized water in a clean flask. The flask
was then shacked to complete dissolving; then 10gm
SD S were added to the flask. Slides soaked in a
solution composed of 1% of Triton X-100 and 10%
DEMSO and cooled in the fridge for 1 h before use.
Harvesting, Fixation and Slide Preparation: The
treated lymphocyte cultures were collected after 48
hours and centrifuged at 1000 rpm for 10 mins.
Supernatant was discarded and cell pellets were
resuspended in 8 ml warmed 0.075 M K CL (hypotonic
solution). Tubes were then incubated in water bath at
37 o C for 20 min. 0.5 ml of cold Carnoy fixative
solution was added to each tube and pipetted gently to
break up cell clumps. The tubes were then centrifuged
and sup ernatants were discarded. Cells were
resuspended by a drop-wise addition of cold fixative
solution. Centrifugation procedure was then repeated
twice, wherein each time the old fixative was discarded
and a new 0.5 ml fixative solution was added to each
tube; after which tubes were kept at 4 o C over night.
D N A U nw inding and Electrophoresis: D N A
unwinding and electrophoresis solution was prepared by
dissolving 12gm Sodium hydroxide, 0.372gm EDTA in
a liter of deionized water at pH 1.3 and cooled for an
hour before use. Slides were removed from lysing
solution and washed with deionized water, then
transferred to electrophoresis tank, left for 20 min. to
complete DNA Unwinding. DNA electrophoresis was
run at 25V and 300 mA for 30 min. in a cold room.
After 30 min, slides were removed and washed with
deionized water, transferred to neutralizing solution
(24.2gm Tris dissolved in 500ml deionized water, pH
7.5) for 15 min, then slides were removed and stained.
Each slide was stained with 10% Gimsa stain (about
300 slides were examined from each treatment), then
chromosomes was visualized by light microscope.
Slide Preparation and Staining: The preserved tubes
were centrifuged and old fixative was discarded, cell
pellets were resuspended in 0.2 ml of a new fixative
solution. Cells were dropped on cleaned slides, then
slides were placed on a hot plate at 37 o C to dry. Slides
were kept in a dry place until staining and microscopic
examination. Slides were stained with 10% Gimsa stain
(10 gm Gimsa stain powder dissolved in 100 ml
phosphate buffer solution, Ph 6.8, filtered and stored in
a dark bottle) for 20 min. Then slides were briefly
washed in distilled water three times and kept in dry
place.
Statistical Analysis: Data of the survival cells
percentage and frequency of comet cells were subjected
to linear regression analysis for the detection of linear
relationship between concentration of the extract or
time of exposure and chromosomal aberrations using
Microsoft Excel 2003 for MS-W indows.
RESULTS AND DISCUSSION
Preparing Sample for Comet Assay: Fixed cells were
transferred to sterile eppendorf tubes containing 1ml of
PBS (8gm sodium hydroxide, 0.2gm Potassium
chloride, 1.1gm sodium phosphate and 0.2 gm
potassium dihydrogen phosphate dissolved in one liter
deionized water). Then, tubes were stored at -20 o C
until use.
Aqueous Extract of Rhazya Stricta Leaves Inhibits
Proliferation of Human Lymphocytes: To assess
growth-promoting or -inhibiting ability of the aqueous
extract of R. stricta leaves on human lymphocytes, the
cells were incubated with increasing doses of the
extract for different time intervals and their mitotic
index were monitored. The data in Table (1) and Fig.
987
J. Appl. Sci. Res., 5(8): 986-994, 2009
aqueous extract of R. stricta leaves has cytogenetic
aberrations abilities.
(2) show that the extract has a significant (p>0.05)
growth-inhibiting ability, in a dose- and exposure timedependent manner. This because, although incubating
the cells with the lowest concentration (6gm/L) of the
extract for 24hr decreased mitotic index to 85.57%,
longer time incubation (72hr.) resulted in a very
noticeable decrease, giving 15.094% versus to 90.09%
(control group). Furthermore, the inhibiting effect of
the extract was very obvious after doubling its
concentration, since exposing the cells to 12gm/L of
the extract for 24, 48 and 72hrs brought about
reduction down to 24.76%, 16.6% and 12.5%,
respectively. Finally, further escalation of the extract
concentration (24gm/L) at the above mentioned time
frame was able to diminish the mitotic index as low as
to 23.42%, 9.96% and 4.975%, respectively. Taken
together, these data illustrate that the aqueous extract
of R. stricta leaves is able to diminish lymphocytic
mitotic ability.
Fig. 1: Rhazya stricta Decne plant in Saudi Arabia as
found in Mecca Jeddah high way road from
harmal valley.
Aqueous Extract of Rhazya Stricta Leaves Induces
Chromosomal Aberrations on Human Lymphocytes:
Next, we decided to test whether the extract is able to
induce any cytogenetic aberrations on the lymphocytes.
In our efforts to perfume this task, we exposed the
cells to escalated doses, and for different periods, to
the extract, then their karyotypic (chromosomal)
pictures were investigated. Table (2) and Fig. (3) show
the percentages of the chromosomal aberrations
emerged after such type of test. Once more, the
shortest exposure time (24hr.) and the lowest
concentration (6gm/L) of the extract elicited a
noticeable aberration, 5.88% versus 0.67% (control
group). M oreover, the effects of the higher
concentrations (12gm/L and 24 gm/L) were much more
pronounced, giving chromosomal aberrations of 11.7%
and 13.1%, respectively. By token, doubling exposure
time (48hr.) for the same escalated doses (6gm/L,
12gm/L and 24gm/L) brought about a more or less
more chromosomal aberrations, 10.5%, 11.0% and
13.0% , respectively. Finally, coupling the longest
exposure time (72hr.) with the escalated concentrations
(6gm/L, 12gm/L and 24 gm/L) of the extract did not
bring noticeable further aberrations, 10.3%, 10.7% and
13.0%, respectively, indicating that exposing the cells
to the extract for only 48hr was enough (saturating) to
trigger maximal chromosomal aberration.
There were several aberrations, Table (2) and Figs.
(3-7) records the appearance of binucleat and trinucleat
cells, in addition to some chromosomal aberrations,
such as chromosomal gaps, fragments, stickiness,
chromatin bridges, cholchicin and C-metaphase period.
Other aberrations included cell death reflected by
appearance of darkly stained nuclei, fragmented DNA
materials, in addition to appearance of ghost cells
(nuclei-free cells). Therefore, we concluded that the
Fig. 2: The Effect of different concentrations of
aqueous extract of Rhazia stricta leaves with
different exposing period on mitotic index of
human blood lymphocytes comparing with
control experiment.
Fig. 3: T he effect of treatments with different
concentrations of aqueous extract of Rhazia
stricta leaves at different exposing times on
p e rc e n t c hro m o s om a l a b e rra tio ns and
micronuclei produced in human blood
lymphocyte cells comparing with control
experiment.
988
J. Appl. Sci. Res., 5(8): 986-994, 2009
Percentage of norm al m itotic cell division in m itotic hum an blood lym phocytes (H PBL) after treatm ent with different concentrations
(6gm ,12gm , and 24gm /L) of aqueous extract of Rhazia stricta for different exposing tim es (24hr., 84hr.and 72hr.).
Treatm ents
Percent of norm al m itotic cell division
Total D ividing cells Total cells M itotic index
--------------------------------------------------------------------------------------------------------------Conc. Gm /L
Tim eh. Interphase
prophase
M etaphase
Anaphase Telophase
0
100
55.4
19.8
4.95
9.90
910
1010
90.09
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------6
24
145
60.6
14.9
4.97
4.97
860
1005
85.57
-------------------------------------------------------------------------------------------------------------------------------------------------------------48
700
13.8
1.98
4.95
9.9
310
1010
30.69*
-------------------------------------------------------------------------------------------------------------------------------------------------------------72
900
3.77
1.88
4.71
4.7
160
1060
15.094*
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------12
24
790
19.04
2.85
1.904
0.95
260
1050
24.76*
-------------------------------------------------------------------------------------------------------------------------------------------------------------48
850
12.7
1.96
0.980
0.982
170
1020
16.6*
-------------------------------------------------------------------------------------------------------------------------------------------------------------72
880
10.4
1.29
0.298
0.497
126
1006
12.5*
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------24
24
850
12.6
1.80
0
9.0
260
1110
23.42*
-------------------------------------------------------------------------------------------------------------------------------------------------------------48
904
8.96
0.99
0
0
100
1004
9.96*
-------------------------------------------------------------------------------------------------------------------------------------------------------------72
955
4.97
0
0
0
50
1005
4.975*
*significant difference (com pare with control experim ent) at 95% (p>0.05)
Table 1:
Table 2: Percentage of m icronuclei and chrom osom al aberration on hum an blood lym phocytes at different exposing tim es after treatm ents with
different concentrations (6-12-24gm /L) of aqueous extract of Rhazia stricta leaves.
Treatm ents
Chrom osom al aberrations
M icronuclei
Two nucleus Three nucleus D isturbance Total aberration
-----------------------------------------------------------------------------------------------cells
cells
cells
percents
Tim e
Conc. 1
2
3
4
5
6
7
8
0
0
0.00
0.00 0.50
0.00 0.00
1.9
0.0
0.00
2.97
0.00
1.98
0.67
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------24
6
3.98
0.00 2.99
1.00 3.98
5.9
2.9
5.97
11.9
5.97
19.9
5.88*
-----------------------------------------------------------------------------------------------------------------------------------------------------------------12
2.97
0.99 7.92
2.28 6.63
6.9
4.9
6.93
14.8
9.90
64.3
11.7*
-----------------------------------------------------------------------------------------------------------------------------------------------------------------24
1.89
0.94 13.2
7.17 10.9
6.6
6.6
7.55
9.43
9.43
70.7
13.1*
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------48
6
0.95
1.90 8.10
1.19 4.48
3.8
3.8
4.76
9.52
0.95
76.1
10.5*
-----------------------------------------------------------------------------------------------------------------------------------------------------------------12
0.98
0.98 8.82
2.97 7.23
2.9
1.9
3.92
5.88
0.98
85.2
11.0*
-----------------------------------------------------------------------------------------------------------------------------------------------------------------24
0.50
0.20 9.94
8.49 9.43
3.9
1.9
3.98
7.95
7.95
89.4
13.0*
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------72
6
0.45
0.18 3.60
0.80 2.39
3.6
2.7
3.60
9.91
9.91
81.0
10.3*
-----------------------------------------------------------------------------------------------------------------------------------------------------------------12
0.00
0.00 1.99
2.18 3.17
4.8
2.9
2.99
11.9
7.97
94.6
10.7*
-----------------------------------------------------------------------------------------------------------------------------------------------------------------24
0.00
0.00 0.30
7.36 7.26
1.9
1.0
1.00
4.98
0.50
89.5
13.0*
*Significant difference (com pare with control experim ent)at 95% (p>0.05)
8
7
6
M n-D isturbance M n-Binuclatecell M n-M onocells
5
4
3
2
1
Sticknees Chrom atin B ridges Chrom osom al Fragm ents C-M etaphase Chrom osom al Gap
Table (3) and Figs. (8-9) illustrate that all
treatments induced statistically significant increase in
mean comet tail length indicating the extract induced
DNA damage in treated cells. The table displays that
most cells had a tail length between 1-5 microns.
However, maximum increase in mean comet tail length
(longer than 7 microns) was observed at 72hr for all
tested concentrations of the extract. In addition, the
mean comet tail length showed a clear time-dependent
increase from 24hr to 72hr for all treatments.
Aqueous Extract of Rhazya Stricta Leaves Induces
Lesions in the Dna of Human Lymphocytes: Next,
we conducted our efforts to assess DNA damage at the
level of single cells by comet test (single-cell gel test).
Comet tail length is an important parameter in
evaluating the DNA damage and applications of this
assay for detecting DNA damage in individual cells are
briefly reviewed in T ice [2 4 ]. Rojas et al.[1 7 ]. Cotelle and
Férard [1 3 ]. Kassie et al.[1 5 ]. Tice et al.[2 5 ] . Singh and
Stephens [2 1 ] . Zijno et al.[2 9 ]. W aters et al.[2 7 ] and
Battershill et al.[8 ].
989
J. Appl. Sci. Res., 5(8): 986-994, 2009
Fig. 4: Normal mitotic cell division of normal human blood lymphocyte cells; (1) divided cells; (2) interphase;
(3) cell at prophase; (4) cell at metaphase; (5) cell at anaphase period and (6) cells at final period.
Fig. 5: Chromosomal aberrations and micronuclei produced in human blood lymphocyte cells (HPBL) after
exposing to different concentrations of aqueous extract of Rhazia stricta leaves: (1) three nucleus cell; (2)
two nucleus cell; (3and 4) micronuclei in two and one nucleolus cells; (5) colchicines metaphase period;
(6) chromosomal vacuoles; (7) Chromosomal Fragments; (8 and 9) central cells; (10) normal cells (left)
with dead cell (right) and (11,12 and13) death cells.
990
J. Appl. Sci. Res., 5(8): 986-994, 2009
Fig. 6: Different micronuclei produced in human blood lymphocytes (HPBL) after exposing to aqueous extract
of Rhazia stricta leaves at different exposure times.
Fig. 7: Two and three micronuclei produced human blood lymphocytes (HPBL) after exposing to different
concentrations of aqueous extract of Rhazia stricta leaves at different exposure times.
Table 3: Illustrate DN A D am age by using Com et assay test on hum an blood lym phocytes (H PB L) after exposing it to different concentrations
of aqueous extract of Rhazia stricta leaves at different exposing tim es.
Treatm ents
N o. of cells with tail M ean no. of cell with tail in period
----------------------------------------------------------------------------------------------Conc. (gm /L) Tim e (hr.)
1
2
3
4
5
0
98
1
1
0
0
2
2
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------6
24
74
9
8
6
3
26
32.7*
---------------------------------------------------------------------------------------48
66
9
11
8
6
34
---------------------------------------------------------------------------------------72
62
12
9
9
8
38
991
J. Appl. Sci. Res., 5(8): 986-994, 2009
Table 3: Continue
12
24
70
10
8
6
6
30
35.5*
---------------------------------------------------------------------------------------48
65
13
8
7
7
35
---------------------------------------------------------------------------------------72
60
14
9
8
9
40
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------24
24
73
8
8
6
5
27
29.7*
----------------------------------------------------------------------------------------48
69
11
7
7
6
31
----------------------------------------------------------------------------------------72
69
9
6
9
7
31
*Significant difference (com pared with control experim ent) at 95% (p>0.05)
Class (1)
N o tail
N orm al D N A
Class (2)
Tail lenth1-3*
Light D N A dam age
D N A D am age classification by tail size
Class (3)
Tail lenth3-5*
M ed. DN A dam age
Class (4)
Tail lenth5-7*
D ep D N A dam age
Class (5)
Tail lenth $7*
Full D N A D am aged
Stephens [2 1 ]. Zijno et al. [2 9 ]. W aters et al. [2 7 ] and
Battershill et al.[8 ].
Table (3) and Figs. (8-9) illustrate that all
treatments induced statistically significant increase in
mean comet tail length indicating the extract induced
DNA damage in treated cells. The table displays that
most cells had a tail length between 1-5 microns.
However, maximum increase in mean comet tail length
(longer than 7 microns) was observed at 72hr for all
tested concentrations of the extract. In addition, the
mean comet tail length showed a clear time-dependent
increase from 24hr to 72hr for all treatments.
Figs. (5, 6, and 7) and shapes (8 and 9) showed
that exposing period of lymphatic blood cells with
Rhazia stricta extracts had significant effect on
prodution comet cells produced, it proved as shown in
Table (3) increasing of the mean number of comet cells
at all treatment periods (24,48 and 72 hr.) comparing
with control experiment at p>0.05 also Table (3)
showed comet cells found and different DNA damage
phases which, from 2 and 3 and less common at phase
5.
Fig. 8: Percentage of comet lymphocyte cells (HPBL)
after exposing it to different concentrations of
aqueous extract of Rhazia stricta leaves
comparing with control experiment.
Discussion: In earlier studies we proved that the
aqueous extract of the R. stricta had a genotoxic
effects on S. cereviscae [1 0 ], on Asperillus terreus [8 ] and
on root tip meristems of Allium cepa [9 ]. In this study
we conducted our efforts to test the genotoxic effects
of the R. stricta extract on mammalian cells, namely
human lymphocytes. To achieve this goal we measured
the mitotic index of the human lymphocytes treated
with different concentrations of and for varied intervals
to the R. stricta extract. W e found that the extract
decreased the lymphocytic mitotic index in a manner
that was steadily dependent on the concentration and
exposure time for the applied treatment. Furthermore,
the decrease of mitotic index was highly significant
(p>0.05) for all tested treatments. These findings are
comparable to other previous studies [9 ,7 ,2 8 ,1 9 ].
Fig. 9: Percentage of comet lymphocyte cells (HPBL)
after exposing it to different periods of time to
aqueous extract of Rhazia stricta leaves
comparing with control experiment.
Aqueous Extract of Rhazya Stricta Leaves Induces
Lesions in the DNA of Human Lymphocytes: Next,
we conducted our efforts to assess DNA damage at the
level of single cells by comet test (single-cell gel test).
Comet tail length is an important parameter in
evaluating the DNA damage and applications of this
assay for detecting DNA damage in individual cells are
briefly reviewed in Tice [2 4 ]. Rojas et al. [1 7 ]. Cotelle and
Férard [1 3 ]. Kassie et al.[1 5 ]. Tice et al.[2 5 ]. Singh and
992
J. Appl. Sci. Res., 5(8): 986-994, 2009
Fig. 10: DNA Damage classification by tail size, (1) Illustrate how measuring comet, classified to head and tail,
(2-7) normal cells without tail, (3-8) class two cells (4-9) class three cells, (5-10)and (6-11) class four
and fifth cells respectively.
Mechanistically, diminishing mitotic index could be
due to preventing cells from proceeding into prophase
or from diminishing the mitotic phases following the
prophase. In either scenario, this diminishment indicates
on an alteration in the lymphocytic genetic materials,
such as DNA lesions, or interference with the cell
cycle [1 8 ]. This could be due to formation a complex
between one, or more, component(s) of the extract and
the DNA replication machinery or the DNA per se.
Other findings deserve attention are the induction
of the chromosomal aberrations that clearly went worse
parallel with the escalated concentrations and the
exposure time of the extract. The most frequent
chromosomal aberrations were stickiness, disturbance of
c h r o m o s o m e s , C - m e t a p h a s e , f r a g m e n ts , a n d
chromosomal gap and binucleate telophase. These
aberrations, once more, reflect the ability of candidate
component(s) of the extract to interfere with the
formation of the mitotic fibers; these findings are quite
consistent with the cytogenetic aberrations shown
earlier by and Sarbhoy et al.[1 9 ]. This mostly indicates
that the R. stricta extract has an anti-cancerous ability.
The microscopic investigation displayed too an
extensive cell death, which could be due necrosis,
rather than apoptosis. This is reflected by appearance
of darkly stained nuclei, fragmented DNA materials, in
addition to appearance of ghost cells (nuclei-free cells).
These cytogenetic aberrations may underlie necrotic
behavior of lymphocytes. A fact supporting the notion
that the R stricta extract mostly has an anti-cancerous
ability.
The picture emerged from the comet test further
extends and complements the findings of the
cytogenetic data. The frequency of single-strand breaks
showed a clear dose-related increase in human
lymphocytes. Maximum DNA damage was observed at
72hr post-treatment for all tested concentrations of the
extract, when compared with the controls. These
findings indicate that a particular ingredient in the
extract triggered multiform DNA damages e.g. strand
breakage, DNA-protein cross-links, DNA-DNA crosslinks, a candidate extract-DNA adducts and base
modifications in cells.
In summary, the current study demonstrates that
incubating human lymphocytes with the aqueous extract
of R. stricta leaves induced cytotoxicity, cell death and
DNA damage, which imply that the Razia extract has
mutagenic, clastogenic and anti-cancerous activities.
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