Journal of Environmental Protection, 2013, 4, 38-42
http://dx.doi.org/10.4236/jep.2013.46A005 Published Online June 2013 (http://www.scirp.org/journal/jep)
Analysis of Sheep Lymphocyte Chromosomal Aberrations
after Exposition to Chlortetracycline
Irena Šutiaková1, Václav Šutiak2, Mária Tulenková1
1Department of Biology, Faculty of Humanities and Natural Sciences, University of Prešov, Prešov, Slovak Republic; 2St. Elizabeth
University College of Health and Social Work, Bratislava, Slovak Republic.
Received April 19th, 2013; revised May 17th, 2013; accepted June 7th, 2013
Copyright © 2013 Irena Šutiaková et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Tetracyclines are broad spectrum antibiotics and one type of various their compounds—chlortetracycline has been suc-
cessfully used for our cytogenetic studies. We have selected for these studies the chromosomal assay because it is well-
known cytogenetic biomarker, which has been used to assess DNA damage at the chromosomal level. We analyzed the
chromosomal aberrations and mitotic index in peripheral lymphocytes of sheep exposed by chlortetracycline chloride in
pure tap water. To the food of the experimental group of animals (à n = 6) were added also 0.35 g of preparation
Aureovit 12 C 80 plv. a. u. v. per kg b. w. and day (i.e. 168 mg of chlortetracycline hydrochloride/kg b. w.) daily in
food. The frequencies of aberrant cells (ABC) in the experimental and control groups of sheep were stated to differ sig-
nificantly (P = < 0.001). In experimental group, chromatid breaks were the dominant type of chromosomal aberrations.
No statistical differences in mitotic index values were found in the both groups (P > 0.05). Increased frequencies of
chromosome aberrations in peripheral lymphocytes of sheep exposed by chlortetracycline in food, suggested a potential
hazard which needs the attention from the viewpoint of human and animal health. This knowledge is useful for all, for
the human and veterinary medicine, as well as for the aquaculture. Many of these chlortetracycline substances for their
environmental persistence and chemical unstability are present in the environment for various periods, and as a result,
they are responsible for several effects on human and animal health through food chain.
Keywords: Antibiotics; Ovine Lymphocytes; Chromosomal Assay; Genotoxicity; Cytotoxicity; Health; Environment
1. Introduction
Tetracyclines (TCs) are broad spectrum antibiotics that
have been successfully used worldwide in human and ve-
terinary medicine (mainly for medical therapy of dis-
eases) and in aquaculture [1-4]. However their effect may
be significantly limited [5]. Many of these substances for
their environmental persistence and chemical unstability,
are present in the environment for long period, and as a
result they are responsible for several effects on human
and animal health through food chain [6]. TCs are known
to possess limited stability on aqueous media and abiotic
degradation products or reversible epimers may be formed
through hydrolysis or photolysis, including epi-TCs and
anhydro-TCs, as well as iso-TCs for chlortetracycline
(CTC), [7,8]. Capleton et al. [9] proposed a method for
veterinary medicine active ingredients according to esti-
mates of their potential for indirect human exposure via
the environment and their toxicity profile. Greater em-
phasis was given to more serious health endpoints, such
as carcinogenity and mutagenicity. Genotoxicity is the
genetic damage, due to DNA damaging agents including
drugs by different mechanisms. Cytotoxic and genotoxic
properties of tetracycline. HCl (TC) have been studied in
human fibroblast experiments and human peripheral lym-
phocytes [10-12].
Cytogenetic endpoints (chromosomal aberrations, mi-
cronuclei and sister chromatid exchanges) are most fre-
quently used in hazard identification assays as the crite-
rion in the risk assessment process [13-16]. It is neces-
sary to investigate how TCs may affect target organ-
isms-microorganisms and also non-target organisms. Ac-
cording to van der Schalie et al. [17], the use of non-
human organisms, as early warning systems for human
health risk is not new. Sentinel animal models could in-
volve mammalian or non-mammalian species, domestic
animals, or wildlife species. Sentinel animal populations
would be exposed to a single chemical or a complex

Analysis of Sheep Lymphocyte Chromosomal Aberrations after Exposition to Chlortetracycline 39
mixture, or to different media (e.g. air, water, soil, and
sediment) in various locations.
CTC is usually administered in the feed or water for
prophylactic and therapeutic purposes. To provide addi-
tional genotoxicity data, we studied the effect of chlor-
tetracycline chloride (preparation Aureovit 12 C 80 plv.
a.u.v.) in peripheral blood lymphocytes of sheep after its
subchronical administration on the induction chromoso-
mal aberrations, as cytogenetic endpoint.
2. Materials and Methods
2.1. Experimental Animals and Nutrition
In the experiment two-months old Slovak merino ewes
weighing 15.2 ± 1.05 kg were used. The animals were
purchased from a private breeder. Prior to their inclusion
in the experiments, they were nursed by mothers in such
a way that they had limited access to drinking water and
received no additional feed. Two weeks before the expe-
riment the lambs were adapted to experimental housing
conditions.
The animals were fed with the ČOJ-2 feeding mixture
ration for animals and with meadow hay at doses of 250 -
300 g of feeding mixture and 350 g dose of hay per each
animal, respectively. Water was obtained at the “Výcho-
doslovenské vodárne a kanalizácie” enterprise in Košice,
where chlorine levels and water pH were determined ac-
cording to the valid standard STN 75 7111 [18]. For 30
days the lambs in the control group were given the same
amounts of feed and water, they had been receiving dur-
ing the adaptation period.
2.2. Scheme of the Experiment
The animals of the experimental group (n = 6) received
the ČOJ-2 feeding mixture for animals and it was sup-
plemented in this group only with chlortetracycline hy-
drochloride (preparation Aureovit 12 C 80 plv. a.u.v.,
containing 8% of the ingredient, 0.5 kg packaging) in
such a way that each lamb consumed 0.35 g of the pre-
paration per kg live weight (i.e. 168 mg of chlortetra-
cycline hydrochloride/kg b.w./day, added to the feeding
mixture. The total quantity of the feeding mixture and
meadow hay supplied was the same also in the control
group (also n = 6 animals), however without CTC.
2.3. Lymphocyte Cultures
After 30 days, blood samples were taken from jugular
vein (v. jugularis) from each animal individually into
glass tubes containing heparin (100 IU per ml blood).
Heparinized blood (0.4 ml) was cultured at 37.5˚C for 48
h with 7.0 ml chromosome medium S-chromo-cell sup-
plemented with FCS, PHA, and L-glutamine (PAN Sys-
tems GmbH, Biotechnologische Produkte, Germany).
Antibiotics were added to the culturing medium as fol-
lows: penicillin G 100 IU/ml, streptomycin 100 µg/ml
and 7.5% NaHCO3. Two hours prior to harvest the cul-
tures were treated with the spindle inhibitor colchicin
(Fluka Biochemika) at the concentration of 10 µg/ml to
arrest cells in metaphase. The squashes were stained with
a 10% solution of Giemsa-Romanovski stain in phosphate
buffer (pH = 7.0).
2.4. Cytogenetic Assay
For each donor, 100 well spread metaphases were scored
for chromosomal aberrations studies (magnification
1000×) using microscope (Nikon). Images were taken by
imaging microscopy (Nikon) using CCD-100 camera
system (Mitsubishi). The images were processed using
the Animal and Photostyler software system. The chro-
mosomal aberrations were scored according to the classi-
fication criteria suggested by Savage [19]. The identifi-
cation of the individual chromosome pairs was carried
out according to the standardized karyotyping of the do-
mestic sheep [20]. Chromatid breaks were distinguished
from gaps when the centric piece was displaced with re-
spect to the chromosome axis, or the size of the discon-
tinuity exceeded the width of the chromatid. Gap aberra-
tions were excluded from the total number of aberrations
and considered separately. Chromosome exchanges were
classified as stable (translocations, when detection was
possible) and unstable (dicentric, ring and fragments).
2.5. Mitotic Index
In the chromosomal aberration study the mitotic index
(MI) was evaluated by counting at least 1000 cells per
treatment and dividing the number of splitting cells (me-
taphases) by the total number of cells.
2.6. Statistical Evaluation of the Results
The Sigma Stat program (Statistical software™, Jandel
Scientific) was employed for statistical evaluation of the
results. The statistically significant differences between
experimental and control groups were determined using
analysis of variance (ANOVA) followed by Tukey test at
the 95% confidence level.
3. Results and Discussion
The effect of CTC on the frequency of chromosome ab-
errations in peripheral lymphocytes of ewe lambs is
summarized in Table 1. The experimental group of ani-
mals exhibited a significantly higher frequency of genetic
damage (20.50% ± 5.24% aberrant cells) (ABC) com-
pared to control group animals (4.00% ± 1.095% ABC; P
=< 0.001, ANOVA followed by Tukey test). In the ex-
perimental group, chromatid breaks were the dominant

Analysis of Sheep Lymphocyte Chromosomal Aberrations after Exposition to Chlortetracycline 40
Table 1. Chromosomal aberrations in ewe lamb peripheral lymphocytes after treatment with high doses of chlortetracycline.
Chromosomal aberrations
Group No. of metap.
cell analyzed B1 B2 G1 G2
ExchangesABC % B/C G/C MI[29]
Cont. 1 100 3 2 1 0 0 5 1.103
2 100 3 0 3 2 0 3 0.98
3 100 1 3 0 0 0 3 1.203
4 100 4 1 1 0 0 5 1.05
5 100 4 1 1 0 0 5 0.932
6 100 3 0 4 0 0 3 1.148
Mean 3.17 0.83 2.17 0.33 0.00 4.00 0.04 0.022 1.07
± SD 0.75 0.75 1.33 0.82 1.095 0.10
± SEM 0.31 0.32 0.54 0.33 0.45 0.42
Exper. 1 100 21 2 32 6 3 24 0.89
2 100 18 1 26 4 0 18 1.06
3 100 24 1 39 7 2 26 1.21
4 100 15 1 23 7 2 17 0.96
5 100 23 1 31 10 3 25 1.28
6 100 13 1 29 5 2 13 1.13
Mean 19.00 1.17 30.00 6.50 2.00 20.50 0.24 0.36 1.09
± SD 4.43 0.41 5.51 2.07 1.09 5.24 0.15
± SEM 1.81 0.17 2.55 0.85 0.45 2.14 0.06
=<0.001*! =0.363ns =<0.001*! =<0.001*! =0.001* =<0.001*! = 0.791ns
NS—no significant difference; *statistical significance in ANOVA; !statistical significance in Tukey test (P < 0.05); SD—standard deviation; SEM—standard
error mean; ABC—aberrant cells; B1, B2, G1, G2 = chromatid and chromosome breaks and gaps; B/C= number of breaks per cell; G/C = number of gaps per
cell; Exper. = experimental group; Control = cont. group; No. of metap. = Number of methaphases; [29]Preston et al., 1987.
type of aberrations (19.00 ± 4.43; P =< 0.001, ANOVA
followed by Tukey test). Most chemicals are S-depend-
ent clastogens supporting the rise of chromatid-type ab-
errations. S-dependent mutagens exert the indirect effects
on chromosomes in peripheral lymphocytes in vivo be-
cause replication occurrence only in stimulated cell cul-
tures [21,22]. As shown in Table 1, in experimental
group the frequencies of chromatid gaps (P =< 0.001,
ANOVA followed Tukey test), chromosome gaps (P =<
0.001, ANOVA followed Tukey test) and exchanges (P =
0.001, ANOVA) were significantly higher than those in
the control group. According to Brøgger [23] gaps are
sensitive indicators of exposure to genotoxic drugs, they
serve mainly at low doses as a “guard” parameter. On the
other hand cells with chromosome aberrations may go on
to become cancerous, and chromosome deletions and
translocations are observed in most cancer cells [22,24].
Other aberrations were represented mainly by associa-
tions of acrocentric chromosomes.
The classic cytogenetic technique, without differential
staining, permit a rapid overall analysis of tested cells and
can detect most cells carrying the so-called unstable ab-
errations (chromosome and chromatid breaks, deletions,
fragments, rings, dicentric and chromatid exchanges). Al-
though these aberrations tend to have a lethal effect on
the cells, it must be pointed out that other structural ab-
errations, the so-called stable aberrations, do not interfere
with the division of chromosomes and allow the cell to
survive [25,26]. The induction of DNA strand breaks and
DNA repair were determined in human fibroblasts after
treatment with tetracycline in the presence and absence
of light. In all experiments human fibroblasts were more
sensitive to incubations of TC in the light than in the dark.
CTC induced single-strand breaks in isolated PM 2 DNA
in the dark, however to a lower extent than in the pres-
ence of light [10]. Çelik and Eke [12] studied the geno-
toxic and cytotoxic effect of tetracycline on peripheral
blood lymphocytes using micronucleus assay, SCE test

Analysis of Sheep Lymphocyte Chromosomal Aberrations after Exposition to Chlortetracycline 41
and measuring mitotic activity and proliferation index
(PRI) or/and nuclear division index (NDI) respectively in
vitro. Their results indicated that tetracycline is able to
induce both cytotoxic and moderate genotoxic effects in
cultured human blood lymphocytes in vitro. No statistical
differences were found in MI values in the groups (Table
1; P > 0.05). Cellular proliferation is a phenomenon that
may be causally associated with the induction of chromo-
somal damage for a number of compounds which are
non-DNA reactive and thus are threshold in vitro clasto-
gens [27]. Changes in biochemical biomarkers (antioxi-
dant enzymes, catalase and superoxide dismutase) and
DNA damage in soil on the earthworm Eisenia fetida ex-
posed to tetracycline and chlortetracycline were investi-
gated. Compared to enzyme activities, DNA damage as a
biomarker was more sensitive and more suitable for de-
tection low concentration exposure the genotoxicity of
contaminants in terestrial environment [28]. The both an-
tibiotics induced significant genotoxicity on earthworm
Eisenia fetida in a dose—dependent manner (P < 0.01).
4. Conclusion
On the whole, when we consider our results together
with other published data our results demonstrate that
chlortetracycline chloride (preparation Aureovit 12 C 80
plv. a.u.v.) induced a significant increase in chromoso-
mal aberrations. These findings suggest a potential geno-
toxic hazard of this antibacterial drug as it may become
capable of attacking the animal genetic material. In our
experimental group of animals, chromatid breaks were
the dominant type of chromosomal aberrations. Our ex-
periences demonstrated that chromosomal damage stud-
ies may be used as the sensitive and effective biomarker
procedure for detecting the genotoxicity effect of hazar-
dous agents from the viewpoint of animal health. This is
the argument also for the further studies and other exa-
mination especially in connection with the human health
state because residues of hazardous agents in animal food-
stuffs may be also their hazard factor.
5. Acknowledgements
This work was supported by the Grant Vega ME SR No.
1/0545/08.
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