ISSN 1022-7954, Russian Journal of Genetics, 2008, Vol. 44, No. 3, pp. 273–278. © Pleiades Publishing, Inc., 2008.

GENERAL
GENETICS

Genotoxic Action of Fungicide Conan 5FL (Hexaconazole)

on Mammalian Cells In Vivo and In Vitro*
S. Yilmaza, H. Aksoya, F. Ünala, M. Çelikb, and D. Yüzba şio ğ lua
1 Gazi University, Science Faculty, Department of Biology, Teknikokullar, Ankara, 06500 Türkiye;
e-mail: funal@gazi.edu.tr
·
2 Sütçü Imam University, Science Faculty, Department of Biology, Kahramanmara ş , Türkiye
Received October 28, 2006

Abstract—The genotoxic effects of fungicide Conan 5FL (containing 50 g/L hexaconazole) in mouse bone-
marrow cells and human lymphocytes have been evaluated. Three different concentrations of Conan 5FL
(17.50, 35.0, and 70.0 µg/mL for human lymphocytes and 17.50, 35.0, and 70.0 mg/kg for mouse bone marrow
cells) were studied. Conan 5FL induced significant increases (except 17.5 mg/kg for mouse bone marrow) in
the frequency of chromosomal aberrations (CAs) in both test systems. This fungicide caused structural and
numerical abnormalities in both mammalian cells. These are sister chromatid union, chromatid and chromo-
some breaks, fragments, dicentric and ring chromosomes, and polyploidy. Significant increase was found in
induction and in minimum-maximum numbers of sister chromatid exchanges (SCEs) at all treatments compared
with the negative control. Conan 5FL did not affect the replication index (RI) in human lymphocyte cultures, how-
ever, it significantly decreased the mitotic index (MI) in all treatment concentrations in both test systems. Using
of Conan 5FL should be reconsidered due to its possible cytotoxic, clastogenic and mutagenic effects.
DOI: 10.1134/S1022795408030058

INTRODUCTION of DNA damage following exposure to genotoxic

Great amount of pesticides are used for agricultural
applications worldwide each year. Pesticides are highly
biologically active chemicals. They may interact with
DNA and damage its structure. Such interaction may be
critical for the manifestation of carcinogenic properties
of different chemicals [1]. Epidemiological data
showed that there is an increase in the number of cancer
cases in persons involved in agricultural production
using pesticides [1–3]. Due to increasing evidence of
mutagenic, carcinogenic and teratogenic effects in
experimental test systems, interest on the pesticide tox-
icity has especially increased over the past years. The
toxicity of a pesticide can be measured by several ways;
one of them is chromosomal aberration (CA) test that
can be applied either in vivo or in vitro. CA is micro-
scopically visible changes in the single DNA molecule
of chromosomes and chromatids [4]. Test measuring
chromosomal aberrations in nucleated bone marrow
cells in rodents or in human lymphocytes in culture can
detect a wide spectrum of changes in chromosomal
integrity. Therefore assays detecting chromosomal
aberrations are acceptable for detecting clastogens [5].
Another test method is in vitro sister chromatid
exchanges (SCEs). The readily quantifiable nature of
SCEs with a high sensitivity for revealing toxicant-
DNA interactions and the demonstrated ability of geno-
toxic chemicals to induce significant increases in SCEs
has resulted in this end-point being used as an indicator
* The text was submitted by the authors in English.
agents [6–10].
In this study, we investigated in vivo and in vitro
genotoxic effects of Conan 5FL (containing 50 g/L
hexaconazole). Conan 5FL is a broad spectrum triazole
fungicide which exhibits its antifungal activity by
inhibiting fungal ergosterol biosynthesis. It is widely
used on crops such as wheat, barley, and orchard fruits
[11]. The genotoxic effects of hexaconazole has been
reported as negative by Ames test in S. typhimurium
and E. coli [12, 13], cytogenetic assay in human lym-
phocytes [14], unscheduled DNA synthesis test in rat
hepatocytes [15], micronucleus test in C57/BL/6J mice
(male + female) bone marrow cells [16]. In contrary to
these results, hexaconazole is reported to be toxic at
200 µg/mL but no clear information is available for the
concentrations under 200 µg/mL, exposure times and
assay conducted [14]. Hexaconazole also slightly
increased the incidence of benign Leydig cell tumors in
rats [17, 18]. On the other hand, there is no information
on the effects of Conan 5FL (commercial form) in
mouse bone marrow cells and in human lymphocytes.
The purpose of this study is to determine whether
Conan 5FL has any effect in chromosome aberrations
and mitotic division in in vivo mouse bone marrow
cells and, in chromosome aberrations, sister chromatid
exchanges and mitotic division in in vitro human lym-
phocytes and to compare the results. This study was
planned due to following reasons: (1) some pesticides
have negative effects in some organisms as indicated
above but show positive effects in the others; (2) both in
bacterial and mammalian cells, positive and negative

273
274 YILMAZ et al.
effects were reported in the same test system; (3) there
is no detailed information about the concentrations of
hexaconazole and assay used in human lymphocytes in
study given as a report; (4) in agriculture, only commer-
cial form is used; (5) there is no study using and com-
paring both human lymphocytes in culture and in vivo
mouse bone marrow cells for the effects of Conan 5FL.
In vitro and in vivo methods should be considered to be
complementary tools for addressing particular issues of
genotoxicity.
MATERIALS AND METHODS
Chemicals. The test substance Conan 5FL is obtained
from Agrochemicals Research Institute in Ankara. The
other chemicals mitomycin C (CAS no. 50-07-7) and bro-
modeoxyuridine (CAS no. 59-14-3) were obtained from
Sigma. All chemicals were dissolved in distilled water.
The active ingredient of Conan 5FL is hexaconazole
(IUPAC Name: (RS)-22-(2,4-dichlorophenyl)-1-(1H-
1,2,4-triazol-1-yl)hexan-2-ol; chemical abstract name:
(±)-α-butyl-α-(2,4-dichlorophenyl)-1H-1,2,4-triazole-1-
ethanol; molecular formula: C14H17Cl2N3O; company
name: MRK Universal LTD; purity: >85% pure; stability:
Stable for at least 6 years at ambient temperatures [19]).
Chromosome aberration analysis in mouse bone
marrow cells. In the present investigation, Swiss Albino
mice (8–10 weeks old) with an average body weight of
25–28 g were used as test animals. Four male animals
were used for each treatment and control groups. The
animals were maintained under conventional labora-
tory conditions at room temperature of 25 ± 2°C on a
12 h dark/12 h light cycle. Three different doses, 17.5,
35.0, and 70.0 mg/kg, were selected and doses were
administrated intraperitoneally as a single acute dose
(24 h). An untreated control and a positive control mito-
mycin-C (2 mg/kg) were also used for testing the validity
of assay. To arrest mitosis, colchicine (5 mg/kg) was
injected intraperitoneally 2 h before the animals were
sacrificed by cervical dislocation. The hind femurs
were stripped proximally, and the bone marrow was
aspirated in 0.075 M KCl (37°C) and kept in 37°C for
30 min. At the end of the treatment, the suspension was
centrifuged for 10 min at 1000 rpm and then cells were
centrifuged in 3 : 1 cold methanol:acetic acid. Cells were
fixed with three changes of fixative, and spread on glass
slides and air-dried. One-day-old slides were stained
with 5% Giemsa prepared in Sorensen buffer solution.
One hundred well spread metaphases were analyzed
for the CAs per animal (totally 400 metaphases per con-
centration). The numbers of abnormal cells were deter-
mined for each animal. Mitotic index (MI) was also
examined by scoring a total of 1000 cells from each
animal.
Chromosome aberration and sister chromatid
exchange analyses in human lymphocytes. Peripheral
blood samples were obtained from two healthy non-
smoking male and female donors aged 26–27 years.
RUSSIAN JOURNAL OF GENETICS
Heparinized blood (1/10 : 2 mL venous blood contain-
ing 0.2 mL heparin) was added to 2.5 mL Chromosome
Medium B (Biochrome) supplemented with 10 µg/mL
bromodeoxyuridine. The cultures were incubated for
72 h at 37°C and colchicine (final concentration
0.06 µg/mL) was added to each culture 2 h before har-
vesting. The cells were then harvested by centrifugation
(1200 rpm, 10 min), and the pellet was resuspended in
0.075 M KCl for 30 min at 37°C. Cells were centri-
fuged again and fixed in cold methanol:acetic acid
(3 : 1). Fixation process repeated for three times. Slides
were prepared by the conventional air-drying technique
and stained with Giemsa or fluorescence plus Giemsa
technique according to Speit and Houpter [20] method
with some modifications.
A hundred well spread metaphases per donor
(totally 200 metaphases per concentration) were ana-
lyzed for the CA assays, 50 s mitosis for the SCE assays
for each experimental concentrations. The mitotic index
was also determined by scoring 1000 cells from each
donor. In the SCE study, a total of 200 cells (100 cells
from each donor) were scored to determine the replica-
tion index (RI). Each metaphase was classified as being
in the first (M1), second (M2), and third (M3) division.
The replication index was calculated as follows: RI =
M1 + 2M2 + 3M3/N. Here, N is the total number of
metaphase scored [21].
Statistical analysis. For the statistical analysis of the
results, z-test was used for the percentage of abnormal
cell, CA/cell, RI, and MI and Student’s t-test was used
for SCEs. Dose-response relationships were deter-
mined from the correlation and regression coefficients
for the percentage of abnormal cell, CA/cell, MI, and
mean SCE.
RESULTS
Table 1 shows the types and numbers of chromo-
somal aberrations for the mouse bone marrow cells.
Conan 5FL increased abnormal cell frequency and
CA/cell ratio in a dose dependent manner (r = 0.96 in
both). This increase was significant in 35 and 70 mg/kg
concentrations. Both structural and numerical aberra-
tions were observed. Structural aberrations are sister
chromatid union, chromatid and chromosome breaks
and fragments. Sister chromatid union (51.72%) was
the most common abnormality. Numerical aberration is
polyploidy. On the other hand, Conan 5FL decreased
mitotic index significantly in a dose dependent manner
(r = –0.90) in all treatments compared with negative
control in mouse bone marrow cells (Table 1).
In vitro results were summarized in Tables 2 and 3.
Conan 5FL significantly increased the frequency of
structural aberrations and CA/cell ratio in a dose-
dependent manner (r = 0.99 in both). It induced six
types of structural aberrations in human lymphocytes:
chromatid and chromosome breaks, sister chromatid
union, fragment, ring and dicentric chromosomes. Chro-
Vol. 44 No. 3 2008
 GENOTOXIC ACTION OF FUNGICIDE CONAN 5FL (HEXACONAZOLE) 275

Table 1. The chromosomal aberrations in mouse bone marrow cells treated with Conan 5FL+

Numerical
aberration
Treatment Structural aberrations
Abnormal
Test substance CA/cell ± SE MI ± SE (%)
cell ± SE (%)
doses
period (h) ctb csb scu ace p
(µg/mL)
Negative control 0.00 1 – 3 – – 1.00 ± 0.50 0.010 ± 0.005 4.55 ± 0.33
Positive control 24 2.00 13 – 3 1 3 5.00 ± 1.09 0.050 ± 0.011 2.85 ± 0.26
Conan 5FL 24 17.50 6 1 4 – – 2.75 ± 0.82 0.028 ± 0.008 3.38 ± 0.29*
35.00 4 2 11 – 1 4.50 ± 1.04* 0.045 ± 0.010* 2.78 ± 0.26**
70.00 7 1 12 1 4 6.25 ± 1.21** 0.063 ± 0.012** 2.45 ± 0.24**
Frequency of ab- 31.03 6.89 51.72 1.72
normalities (%)
+ 400 metaphases were scored for each treatment in CA. 4000 metaphases were scored for each concentration in MI. ctb: chromatid break,
csb: chromosome break, scu: sister chromatid union, ace: acentric fragment, p: polyploidy.
* Significant from the control p < 0.01 (z-test).
** Significant from the control p < 0.001 (z-test).

Table 2. Chromosomal aberrations in human lymphocytes treated with Conan 5FL+

Numerical
aberration
Treatment Structural aberrations
Abnormal
Test substance CA/cell ± SE
cell ± SE (%)
doses
period (h) ctb cte csb scu dic ace r p
(µg/mL)
Negative control 0.00 2 – – – – 1 – – 1.50 ± 0.86 0.015 ± 0.008
Positive control 24 0.10 22 10 6 2 3 8 – – 24.50 ± 3.04 0.255 ± 0.030
Conan 5FL 24 17.50 10 – 3 – – 1 – 1 7.00 ± 1.80* 0.075 ± 0.019*
35.00 12 – 1 2 1 1 – – 8.50 ± 1.97* 0.085 ± 0.020*
70.00 17 – 2 1 1 4 1 – 12.00 ± 2.30** 0.130 ± 0.070**
Frequency of 68.33 10.00 5.00 3.33 11.66 1.66
abnormalities (%)
+200 metaphases were scored for each treatment. ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, scu: sister chromatid
union, dic: dicentric, ace: fragment, r: ring chromosome, p: polyploidy.
* Significant from the control p < 0.01 (z-test).
** Significant from the control p < 0.001 (z-test).

matid exchange was observed in MMC treatment only.
Chromatid break (68.33%) was the most common
abnormality. Conan 5FL has also induced polyploidy, a
numerical aberration, at only 17.5 µg/mL concentra-
tion.
Conan 5FL significantly increased the number of
SCE/cell in all treatments compared with negative con-
trol in human lymphocytes (Table 3). Significant dose-
response correlation was observed in SCE analyses (r =
0.98). Minimum–maximum numbers of SCEs were
also increased with increasing of the concentration of
Conan 5FL. The number of SCE/cell and maximum
numbers of SCEs were higher than those of positive
control at 35 and 70 µg/mL concentrations, minimum
SCE amount was also higher at 70 µg/mL than those of
the positive control. Conan 5FL did not effect the RI,
however, it significantly decreased mitotic index at all
treatment concentrations in a dose dependent manner (r =
–0.99) in human lymphocytes (Table 3).
DISCUSSION
Chromosomal aberrations in mouse bone marrow
cells and in human lymphocytes in culture are very
important assays for the detection of genotoxic poten-
tial of physical and chemical agents. The results
obtained in these in vivo and in vitro studies demon-

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276 YILMAZ et al.

Table 3. Sister chromatid exchange, replication index and mitotic index frequency in human lymphocytes treated with Conan
5FL+
Treatment
min–max
Test substance dose SCE/cell ± SE M1 M2 M3 RI ± SE MI ± SE (%)
period (h) SCE
(µg/mL)
Negative control 0.00 2–11 4.76 ± 0.34 36 32 132 2.48 ± 0.06 9.46 ± 0.65
Positive control 24 0.10 5–19 10.04 ± 0.44 35 44 121 2.43 ± 0.04 2.90 ± 0.38
Conan 5FL 24 17.50 2–16 7.38 ± 0.45a 39 72 89 2.25 ± 0.04 7.70 ± 0.60*
35.00 3–26 12.18 ± 0.69a 51 102 47 1.98 ± 0.03 6.30 ± 0.54**
70.00 7–30 15.46 ± 0.80a 43 106 51 2.04 ± 0.03 2.90 ± 0.38**
+ 50 metaphases were scored for each concentration in SCE. 200 metaphases were scored for each concentration in RI. 2000 metaphases
were scored for each concentration in MI.
a Significant from the control p < 0.05 (t-test).
* Significant from the control p < 0.05 (z-test).
** Significant from the control p < 0.001 (z-test).

strate that Conan 5FL induced chromosomal aberra-
tions in a dose-dependent manner. Sister chromatid
union, chromatid and chromosome breaks, fragment
and polyploidy were observed in both test systems. Two
highest doses of Conan 5FL induced a significant
amount of abnormal cells (%) and CA/cell a both test
systems. In addition, in human lymphocytes, the lowest
dose induced significant increase in both abnormal
cells (%) and CA/cells. Abnormal cell frequency and
CA/cell ratio was higher at 70 mg/kg in mouse bone
marrow cells than dose of the positive control, MMC.
The frequency of structural chromosome aberrations
and CA/cell ratio were higher in human lymphocyte
than those of mouse bone marrow cells, after the treat-
ment of Conan 5FL. In contrary, induction of polyp-
loidy by Conan 5FL was higher in mouse bone marrow
cells than those of human lymphocytes in culture.
Aberrations induced by Conan 5FL are the results of
clastogenic action of chemical [22, 23]. Chromatid
breaks suggest that the chemical acts mostly in the late
S or G2 phase of the cell cycle [24]. Chromatid
exchange was observed in only MMC treatment in this
study. Several other pesticides have been studied to
evaluate their genotoxic effects. Adikari and Grover
[25] used 2,4 D, dimecron and vitavax and they
observed chromatid breaks, chromatid and chromo-
some fragments, ring chromosomes and dicentric chro-
mosomes in rat bone marrow cells. Due to their effects,
these pesticides are reported as clastogenic. Carbosul-
fan induced significant dose dependent increase in the
frequency of CAs in mice. It induced chromatid and
isochromatid gaps and breaks, chromatid exchanges
and sister chromatid union [10]. Rencüzog° ullari et al.
[26] reported that etoxazole, an acaricide, induced CAs
in human lymphocytes and, has a potential genotoxic
effect. They observed chromatid and chromosome
breaks, sister chromatid union, dicentric chromosomes,
ring chromosome, chromatid exchanges and polyp-
loidy. Karathane LC, a systemic fungicide and a non-
systemic acaricide, increased CAs in human peripheral
lymphocytes in a dose-dependent manner. This pesti-
cide induced eight types of aberrations. These are chro-
matid break, fragment, sister chromatid union, dicen-
tric chromosomes, gap, ring chromosomes, chromatid
exchanges and polyploidy [27]. All these studies show
that pesticides may have genotoxic potential in mam-
malian cells.
In this investigation, Conan 5FL significantly
decreased mitotic indices in a dose-dependent manner
in vivo and in vitro studies. However, this fungicide did
not affect the RI value. Its mitotic inhibition was more
effective in human lymphocytes in culture than those of
mouse bone marrow cells. Inhibition was 70% in
human and 47% in mouse. A decrease in mitotic index
has also been reported to many other pesticides such as
etoxazole, karathane LC and sodium arsenite [26-28].
Decreasing of the MI could be due to blocking of G2
preventing the cell from entering mitosis [29] or
decreasing of the ATP level and the pressure from the
energy production centre [30, 31]. Inhibition of certain
cell cycle specific enzymes such as DNA polymerase,
which is necessary for the synthesis of DNA precursors
as well as other enzymes more directly involved with
spindle production, assembly or orientation, may also
explain the reported antimitotic effect [32].
Conan 5FL significantly increased the frequency of
SCEs in all treatment in a dose dependent manner in
human lymphocytes. Not only the frequence of SCEs,
but also minimum-maximum numbers of SCEs
increased with increasing of Conan 5FL. SCE analysis
is one of the most sensitive genotoxic assays and has
been widely used to detect mutagenic and carcinogenic
potential of chemicals [22, 33]. The frequency of SCEs
in eukaryotic cells is increased by exposure to geno-
toxic agents that induce DNA damage [33]. Fungicide
Karathane LC induced the formation of SCEs in
20 µg/mL concentrations at 24 h and all concentrations
at 48 h treatments compared to the control [27]. Acari-
cide etoxazole induced SCEs at all concentrations and

RUSSIAN JOURNAL OF GENETICS Vol. 44 No. 3 2008

 GENOTOXIC ACTION OF FUNGICIDE CONAN 5FL (HEXACONAZOLE)
treatment periods in a dose-dependent manner [26].
Soloneski et al. [34] observed that the concentrations of
50 and 100 µg/mL Zineb and its commercial formula-
tion Azzurro induced a significant increase in SCE fre-
quency. All these studies indicate that pesticides may be
mutagenic and induce the formation of SCEs.
Despite negative results reported for the genotoxic-
ity of hexaconazole, in this study, the findings of a sig-
nificant increase of CAs and SCEs in human peripheral
lymphocytes and a significant increase of CAs in
mouse bone marrow cells indicate a potential cytoge-
netic hazard due to Conan 5FL treatment. Both chro-
mosomal aberrations and sister chromatid exchanges
are very important biomarkers for the detection of DNA
damage induced by chemicals indicating that the chem-
ical has a clastogenic and mutagenic effects. The high-
est concentration of Conan 5FL used by fungicide appli-
cators/sprayers, in the field or greenhouses is 500 mg/L.
This concentration is almost 7–28 times higher that
those of our test concentrations according to active
ingredient hexaconazole. It is therefore advisable to be
careful in using of this fungicide as it may lead to alter-
ations in the genetic material.
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