Mutation Research 534 (2003) 9399

Evaluation of the genotoxic potential of lambda-cyhalothrin using

nuclear and nucleolar biomarkers on fish cells
Tolga avas , Serap Ergene-Gzkara
Department of Biology, Faculty of Sciences and Letters, Mersin University, 33342, Mersin, Turkey
Received 28 May 2002; received in revised form 27 August 2002; accepted 22 September 2002

Abstract
Micronucleus formation in fish erythrocytes, as an indicator of chromosomal damage, has been increasingly used to detect
the genotoxic potential of environmental contaminants. Nucleolar organizer regions (NORs) stained with colloidal silver
techniques indicate sites of active RNA transcription. The number and size of NORs in interphase nuclei reflect cellular
activities such as proliferation and differentiation of cells. In this study, nuclear (micronucleus frequency) and nucelolar
(changes in quantitative characteristics of nucleoli) biomarkers were used to evaluate the functional and structural genotoxic
effects of the pyrethroid insecticide lambda-cyhalothrin on Garra rufa (Pisces: Cyprinidae). The frequency of micronuclei was
examined in blood smears obtained from fishes exposed to three different concentrations (0.005, 0.01, 0.05 g/l) for a period
of 36 h. Nucleolar parameters (the average number of nucleoli per cell; the volume of a single nucleolus; and the percentage
of cells with heteromorphic paired nucleoli) were examined in epithelial cells obtained from the edge of caudal fins at the 90th
and 180th minutes of exposure. Results of both tests demonstrated the genotoxic potential of pyrethroid lambda-cyhalothrin
on G. rufa. The frequency of micronucleated erythrocytes was significantly increased while the nucleolar parameters were
repressed by lambda-cyhalothrin treatment. Our results confirmed that the use of nucleolar biomarkers on fish fin cells, in
addition to micronucleus test, could provide valuable information in aquatic genotoxicity studies.
2002 Elsevier Science B.V. All rights reserved.
Keywords: Genotoxicity; Lambda-cyhalothrin; Nucleolar biomarker; Micronucleus test; Garra rufa

1. Introduction immediate and persistent activity against a large vari-

ety of arthropods harmful both to human and animal
Synthetic pyrethroids are a diverse class of more health and to vegetal production. These types of
than 1000 powerful broad spectrum insecticides that halogenated and lipophylic compounds are generally
are environmentally compatible by virtue of their recognized as potent neurotoxicants, characterized by
moderate persistence, low volatility and poor aque- high insecticidal properties and low mammalian tox-
ous mobility in soil [1]. In recent years, the use of icity [2]. In many countries lambda-cyhalothrin has
synthetic pyrethroids has increased due to their ob- been successfully used for the control of infectious
vious advantages. Lambda-cyhalothrin is one of the disease vectors, such as mosquitoes, triatomine bugs
newer synthetic pyrethroid insecticides with effective, and other arthropods [3,4]. On the other hand, it is
also known that synthetic pyrethroids are extremely
Corresponding author. Tel.: +90-324-3610001; toxic to fish and aquatic invertebrates [5].
fax: +90-324-3610047. Several cytogenetic methods using different end-
E-mail address: tcavas@mersin.edu.tr (T. avas). points, such as chromosomal aberrations, sister

1383-5718/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S 1 3 8 3 - 5 7 1 8 ( 0 2 ) 0 0 2 4 6 - 2
94 T. avas, S. Ergene-Gzkara / Mutation Research 534 (2003) 9399

chromatid exchange and micronucleus formation,

have been developed on aquatic organisms to assess
the genotoxicity of chemicals [6,7]. However, the mi-
cronucleus (MN) test has been considered as the most
suitable and effective method to use in fishes, because
of its simplicity and ease of scoring [8]. MN is com-
posed of small chromatin fragments which arise as a
result of chromosome breaks after clastogenic action Fig. 1. Structure of lambda-cyhalothrin (C23 H19 ClF3 NO3 ).
or whole chromosomes that do not migrate during
anaphase as a result of aneugenic affects [9]. The
efficacy of this test system as an indicator of struc- no: 71-43-2) was used as a positive control. All test
tural genomic damage has already been proven and solutions were freshly prepared before each experi-
the MN test has been successfully used as a measure ment.
of genotoxic stress in fish, under both laboratory and
field conditions [1012]. 2.2. Fish
Nucleolar organiser regions (NOR) are segments
of DNA coding ribosomal genes, which can be his- G. rufa is a common species with good availability
tologically detected by silver staining technique [13]. in most rivers in the province of Mersin. It is very easy
Morphological NOR parameters in interphase nuclei to handle and acclimate to laboratory conditions. All
are currently under investigation as markers of cellu- fish were collected from the Muftu river. The weight
lar proliferation in relation to RNA synthesis [14,15]. and length of the specimens were 2.50.5 g and 4.5
In recent years, the specificity of changes in nucleolar 0.5 cm, respectively. Before the experiments, fish were
characteristics in plant and animal cells and the po- acclimated to the laboratory conditions for 3 weeks
tential use of nucleolar parameters in assessment of and were not fed during the experiments.
cytogenetic toxicity have been shown [1619].
In this study, a set of nucleolar biomarkers in in- 2.3. Experimental design
terphase nuclei (the average number of nucleoli per
cell; the volume of a single nucleolus; and the per- Three specimens were analyzed for each treatment.
centage of cells with heteromorphic paired nucleoli), Fish were placed in aquariums containing three dif-
together with the nuclear biomarker (MN test) were ferent concentrations (0.005, 0.01, and 0.05 g/l) of
used to evaluate the functional and structural geno- lambda-cyhalothrin. These concentrations were se-
toxic effects of lambda-cyhalothrin on a cyprinid lected on the basis of literature data and our previous
fish Garra rufa (Heckel, 1843), a common repre- investigations. As a positive control, benzene at a
sentative of fresh-water ecosystems in the Mersin concentration of 10 mg/l, was used. For nucleolar
region. analysis, epithelial cells were sampled from the edge
of caudal fins at the 90th and 180th minutes of fish
exposure. The 36-h exposure was selected to eval-
2. Materials and methods uate the MN frequency, because in most studies on
fish peak values for MN in erythrocytes have been
2.1. Chemicals obtained after 2448 h [2022]. After 36 h the fish
were sacrificed and blood samples were obtained for
Lambda-cyhalothrin (-cyano-3-phenoxybenzyl-3- analysis of micronuclei.
(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethyl cy-
clopropanecarboxylate; CAS no: 91465-08-6) is the 2.4. Nucleolar analysis
most commonly and profusely used pyrethroid pes-
ticide in the Mersin region. A commercial formula- Nucleolar analyses were carried out on small
tion of lambda-cyhalothrin, named Karate (Zeneca), pieces of the fin edges collected and fixed in a 3:1
was used in the experiments (Fig. 1). Benzene (CAS alcoholacetic acid, at the 90th and 180th minutes
 T. avas, S. Ergene-Gzkara / Mutation Research 534 (2003) 9399 95

of exposure. Each sample was analyzed as a mixture Table 1

of fin cells of three fishes. Air-dried slides were pre- Frequency of micronucleated erythrocytes in specimens of G. rufa
exposed to different treatments
pared and then stained with 50% AgNO3 solution
for 56 min at 60 C according to the technique of Treatment Concentration MN frequency
Howell and Black [23]. (mean S.E.)
The number of nucleoli was counted in 1500 in- Negative control 3.0 0.58
Positive control 10 ppm (mg/l) 9.7 0.34
terphase cells in every sample under light-microscope
(Olympus) at 1000 magnification. Sizes of spherical Lambda-cyhalothrin 0.005 g/l 3.3 0.66
and intact nucleoli were measured in 200 cells in ev- 0.01 g/l 5.7 0.34
0.05 g/l 8.3 0.66
ery case at the same magnification using a micrometer
P < 0.05.
eyepiece. Paired nucleoli were also visually classified
P < 0.01.
into homomorphic (having nearly equal sizes) and het-
eromorphic (having different sizes) at the maximum
magnification [24].

2.5. Micronucleus test

Blood samples for the smears were obtained from

the caudal vein of the specimens investigated. After
fixation in pure ethanol for 20 min, the prepared slides
were left for air drying, than the smears were stained
with 10% Giemsa solution for 25 min.
From each fish, three slides were prepared and from
each slide 1500 erythrocytes were scored under 1000
magnification to determine the frequency of micronu-
cleated cells, which was calculated per 1000 cells ().

2.6. Statistical analysis Fig. 2. Micronucleated erythrocytes (arrows) in Garra rufa exposed
to lambda-cyhalothrin. Giemsa stained blood smear (magnification,
The statistical significance of the differences in 1000).
mean values, between treatment and control groups,
were determined with the Students t-test at 0.05 level.
are shown in Table 2 and Fig. 3, respectively. Changes
of nucleolar characteristics during the experiment
3. Results are also shown in Fig. 4. The insecticide treatment
reduced the proportion of cells with heteromorphic
paired nucleoli (PNhet) (Fig. 4a). This decrease was
The results of the micronucleus test are summarized
in Table 1. After 36 h, the frequencies of micronu-
cleated erythrocytes were increased in all treatment
groups. This increase was significantly different at
the two highest doses 0.05 (P < 0.01) and 0.01 g/l
(P < 0.05), compared with the negative control. The
benzene treatment also caused a significant increase
(P < 0.01) in the frequency of micronucleated ery-
throcytes. Examples of micronucleated erythrocytes
Fig. 3. Classification of nucleolar characteristics in the nuclei of
are shown in Fig. 2. fish fin cells. (a) Single nucleolus; (b) homomorphic paired nu-
Average values of nucleolar parameters and classi- cleoli; (c) heteromorphic paired nucleoli; (d) tri-nucleoli (magni-
fication of silver stained nucleoli in fin cells of G. rufa fication, 1500).
96 T. avas, S. Ergene-Gzkara / Mutation Research 534 (2003) 9399

Table 2
Average values of nucleolar characteristics in fin cells of G. rufa under control and experimental conditions
Nucleolar Duration Negative control Positive control Concentrations of lambda-cyhalothrin
characteristics (min)
0.005 g/l 0.01 g/l 0.05 g/l

PNhet (%) 90 61.3 39.1 57.2 48.3 43.1

180 63.2 36 50.2 42.4 35.3
nN 90 1.50 0.70 1.35 0.60 1.45 0.69 1.42 0.68 1.37 0.64
180 1.47 0.70 1.29 0.56 1.43 0.68 1.40 0.66 1.31 0.56
VSn 90 4.56 0.17 3.42 0.11 4.38 0.16 3.95 0.15 3.62 0.10
180 4.54 0.18 3.21 0.10 4.15 0.10 4.11 0.11 3.13 0.08
PNhet: cells with heteromorphic paired nucleoli; nN : number of nucleoli per cell; VSn : volume of a single nucleolus.
P < 0.05.
P < 0.01.
P < 0.001.

significant in all dose and time treatment groups, ex- human lymphocytes and reported weak or no geno-
cept for the 0.005 g/l at 90 min group (P > 0.05). toxic potential.
The number of nucleoli (nN ) in fin cells was also de- Information on the genotoxic effects of pyrethroid
creased in the positive control (P < 0.001) and treat- insecticides on fish species is also limited. In our
ment groups (Fig. 4b). This decrease was insignificant previous experiments, we observed that cypermethrin
at the lowest concentration 0.005 g/l (P > 0.05). treatment caused a significant increase in MN fre-
Similarly, lambda-cyhalothrin treatment caused a sta- quency in gill cells of Oreochromis niloticus [29].
tistically significant decrease in the volume of single On the other hand, genotoxic evaluation of lambda-
nucleolus (VSn ) in all groups (Fig. 4c) with one ex- cyhalothrin on fish was first performed by Campana
ception, the lowest concentration 0.005 g/l at 90 min et al. [21], who reported an increase in the MN fre-
(P > 0.05). quency in erythrocytes of Cheirodon. i. interruptus
exposed to different doses of this insecticide. In our
study, lambda-cyhalothrin treatment also caused an
4. Discussion increase in the frequency of micronucleated erythro-
cytes in G. rufa, with the exception of the lowest
The data on the genotoxic effects of synthetic concentration.
pyrethroids are rather controversial and different stud- The set of NOR characteristics used in this study re-
ies reported different results depending on the test flect different mechanisms of the regulation of nucleoli
system or organism used in the experiments. Accord- [30]. For example, the number of nucleoli per cell cor-
ing to Bhunya and Pati [25] cypermethrin caused responds to the number of active centers of ribosomal
micronuclei in mouse erythrocytes. Miadokova et al. RNA synthesis in interphase nuclei [31]; the size of
[26] also reported that supercypermethrin treatments a single nucleolus is representative of transcriptional
gave positive results for gene conversion in Saccha- activity of rDNA clusters [32]; and the percentage of
romyces cerevisiae and for frequency of aberrant cells with PNhet characterizes the specific mechanism
anaphasetelophases in root tips of Hordeum vulgare of regulation of paired nucleolar organizer regions,
and Vicia faba. However, allethrin was tested in the which are the most widespread type of the nucleolar
Drosophila wing spot test, and reported to be un- composition among plant and animal cells [24].
able to induce genotoxic effects [27]. Surrales et al. In our study, all analyzed nucleolar parameters
[28] also investigated the genotoxic activity of five responded in a dose-dependent way to treatments
pyrethroid insecticides (cypermethrin, deltamethrin, with lambda-cyhalothrin and benzene for 90 and
fenpropathrin, fenvalerate and permethrin) in cultured 180 min. In general, lambda-cyhalothrin repressed
 T. avas, S. Ergene-Gzkara / Mutation Research 534 (2003) 9399 97

Fig. 4. (a) Changes in the percent of heteromorphic paired nucleoli (PNhet, %); (b) the average number of nucleoli per cell (nN ), and (c)
the volume of a single nucleolus (VSn ) in fin cells of Garra rufa, after different treatments ( P < 0.05; P < 0.01; P < 0.001;
LCT, lambda-cyhalothrin).

all analyzed nucleolar parameters in fin cells, it respond to toxic impact very quickly, were also con-
reduced the number of active nucleolar organizer firmed by other studies. Arkhipchuk [17] showed
regions, lowered transcriptional activity of rRNA that the size of single nucleoli in cells of Allium
genes, and inhibited the specific activity of paired fistulosum decreased in only 15 min after irradia-
nucleoli in fin cells of G. rufa. These data, indi- tion of plant roots. Calin and Dragomir [19] showed
cating that morphologic nucleolar parameters can that the number and size of nucleoli in the liver
98 T. avas, S. Ergene-Gzkara / Mutation Research 534 (2003) 9399

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