Full Journal - Journal of Cell and Molecular Biology - Haliç Üniversitesi

42 Teodora A. Staykova et al.
bioaccumulation rate and when fallen into the
organism, they are slowly released, causing a number
of damages. According to Nikolov (1987) the studying
of the mutagenic effect of heavy metals on different
test-objects is of current interest, since increasing
environmental pollution with chemical mutagens
possess a real threat to the presence and future of
mankind. At the same time, the number of studies on
cytotoxic and mutagenic effects of heavy-metal and
cyanide contaminated waters in Southwest Bulgaria is
rather limited. This motivated the present study.
Material and methods
Samples of spring water, used by the local population
for drinking and irrigation, were taken from the region
of Panagjurishte and subjected to chemical analysis.
The contents of copper, arsenic, cadmium, lead, and
cyanides (mg/dm 3 ) were determined using the method
of automatic photometry (apparatus SQ 118 - Merk).
The results for the contents of the heavy metals and
cyanides tested were compared with the hygienic
norms adopted for the country (maximum permissible
concentrations – MPC) according to the Ordinance No
7/1986.
Fiskesjö (1985, 1988, 1995), Sabti (1989) have
been shown that the Allium test to be a useful tool for
the detection of potentially genotoxic substances in air
and water screening programmes. Chang et al. (1997)
have used Allium cepa for testing of genotoxic effect
of a lead-contaminated soil. Moraes and Jordao (2001)
evaluated the cytogenotoxic effects of the waste water
in Allium cepa root meristems, too.
To conduct the cytogenetic analysis, temporary
squash preparations were made from Allium cepa root
meristem. The sprouted roots were fixed in a Clarke’s
fixator (acetic acid glaciale : distilled water = 3:1) for
4 hrs, washed in 96% and 70% ethanol and stored in
70% ethanol in a cold chamber at 4 0 C until making the
microscope slides. Hydrolyzed roots in 3N HCl at
temperature of 24 0 C for 10 min, were treated with
45% CH 3COOH for 30 min and stained for 2 hrs in
acetocarmine. After staining, the root meristems were
separated and squashed in 45% CH 3COOH (Ivanova et
al., 2002). A control sample No1 (rootlets sprouted in
distilled water) and a test sample No2 (rootlets
sprouted in the water tested for heavy-metal and
cyanide amounts) were examined. The microscopic
preparations were analysed to determine the cell
division intensity calculating the mitotic index (MI).
The latter was determined as a ratio between the
number of dividing cells (N') and the total number of
cells analysed (N) in promiles:
N'
MI = 1000 ‰
N
The indices of each phase of actual cell division (I
phase) were also calculated, i.e.: the prophase index (I
prophase), metaphase (I metaphase), anaphase (I
anaphase) and telophase (I telophase) as ratios
between the number of cells in the respective phase
(N'') and the number of dividing cells (N') in per cents:
N''
I phase = 100 %
N'
The mutation frequency, analysed through the
anaphase/telophase and micronucleus test for
mutagenicity, was calculated as percentage of the total
number of analysed cells (N). The microscope
examination was made at magnification 400x.
Results
Chemical water analysis
The lead (Pb) content of the test sample was 0.3
mg/dm 3 , exceeding six times the MPC (0.05). In the
same sample, the contents of heavy metals copper
(Cu), zinc (Zn), cadmium (Cd) and arsenic (As) were
below the PMC values accepted for the country.
The presence of cyanides (CN) in the tested water
sample was established. Their amount was 0.018
mg/dm 3 . According to the hygienic norms for
Bulgarian waters (Ordinance No 7/1986), no cyanide
amounts were permitted in them.
Cytogenetic analysis
To compare cell division rate in determining the
mitotic index, we examined a total of 2876 cells for
the control (No1) and 12334 cells for the test sample
(No2). A lower mitotic index in the test sample was