Mutation Research 649 (2008) 155–160

Available online at www.sciencedirect.com

Genotoxicity testing of fluconazole in vivo and in vitro

Deniz Yuzbasıoglu a, Fatma Unal a,∗, Serkan Yılmaz a,Huseyin Aksoy b, Mustafa Celik c

a Gazi University, Science Faculty, Department of Biology, Ankara, Turkiyeb Sakarya University, Science Faculty, Department of Biology, Sakarya, Turkiye

c Kahramanmaras Sutcu Imam University, Science Faculty, Department of Biology, Kahramanmaras, Turkiye

Received 12 February 2007; received in revised form 8 August 2007; accepted 30 August 2007Available online 18 October 2007

Abstract

The genotoxic effects of the antifungal drug fluconazole (trade name triflucan) were assessed in the chromosome aberration (CA)test in mouse bone-marrow cells in vivo and in the chromosome aberration, sister chromatid exchange (SCE) and micronucleus (MN)tests in human lymphocytes. Fluconazole was used at concentrations of 12.5, 25.0 and 50.0 mg/kg for the in vivo assay and 12.5, 25.0and 50.0 �g/ml were used for the in vitro assay. In both test systems, a negative and a positive control (MMC) were also included.Six types of structural aberration were observed: chromatid and chromosome breaks, sister chromatid union, chromatid exchange,fragments and dicentric chromosomes. Polyploidy was observed in both the in vivo and in vitro systems. In the in vivo test, fluconazoledid not significantly increase the frequency of CA. In the in vitro assays, CA, SCE and MN frequencies were significantly increasedin a dose-dependent manner compared with the negative control. The mitotic, replication and cytokinesis-block proliferation indices

(CBPI) were not affected by treatments with fluconazole. According to these results, fluconazole is clastogenic and aneugenic inhuman lymphocytes, but these effects could not be observed in mice. Further studies should be conducted in other test systems toevaluate the full genotoxic potential of fluconazole.© 2007 Elsevier B.V. All rights reserved.

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Keywords: Fluconazole; Antifungal; Chromosomal aberrations (CA);

1. Introduction

Fluconazole (trade name triflucan) is a member ofthe bis-triazole class of antifungal agents, and a highlyselective inhibitor of fungal cytochrome P-450 sterolC-14 alpha demethylation [1]. Fluconazole is an impor-

tant drug in obstetrics and gynecology for treatment ofvaginal candidiasis. It is also used for the treatment oforopharyngeal, esophageal and urinary tract infections,

∗ Corresponding author. Tel.: +90 312 202 1181;fax: +90 312 212 2279.

E-mail address: funal@gazi.edu.tr (F. Unal).

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1383-5718/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.mrgentox.2007.08.012

hromatid exchange (SCE); Micronucleus (MN) assay

eritonitis and cryptococcal meningitis. In addition, flu-onazole is used to treat fungal infections in people withsuppressed immune system, such as cancer chemother-py or organ-transplant patients, and AIDS patients [2].igh doses of fluconazole have been shown to be ter-

togenic in rodents in vivo [3] and in vitro [4]. Apecific teratogenic effect on the branchial arch appara-us has been described in cultured whole mouse [4] andat embryos [5]. Fluconazole also induced teratogenicffects in tunicate Phallusia mammillata [2], and it was

ecently identified as a possible human teratogen [6]. Inontrast, fluconazole was not mutagenic in four strainsf Salmonella typhimurium and in the mouse lymphoma5178Y system. Cytogenetic studies in vivo (murine

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one-marrow cells) and in vitro (human lymphocytes)howed no evidence of chromosomal mutations [7].owever, there are chemicals that give negative results

n bacteria, but are mutagenic when tested in other organ-sms and in other test systems [8]. In both bacterial andammalian cells, positive and negative effects have been

eported in the same test system [9–12]. Furthermore, its generally necessary to use more than one test system tobtain a full evaluation of the genotoxicity of a drug or itsetabolites [13]. For these reasons, we decided to pro-

ide additional genotoxicity data for the antifungal druguconazole, investigating the induction of chromosomalberrations (CA) in mouse bone-marrow cells and chro-osomal aberrations, sister chromatid exchange (SCE)

nd micronuclei (MN) in cultured human lymphocytes.

. Material and methods

.1. Chemicals

The test substance fluconazole (CAS no. 86386-73-4) wasbtained from Pfizer (Turkey). Mitomycin C (CAS no. 50-07-), bromodeoxyuridine (CAS no. 59-14-3) and cytochalasin

(CAS no. 14930-96-2) were obtained from Sigma. Thehemical structure of fluconazole (2-(2,4-difluorophenyl)-1,3-is(1H-1,2,4-triazol-1-yl)-2-propanol) is as shown in Fig. 1.

.2. Animals and their treatment for measurement ofhromosome aberrations

Male Swiss albino mice (8–10 weeks old) weighing 25–28 gere used for the experiment. The mice were maintained in sep-

rate cages at room temperature (20 ± 1 ◦C) and 12-h light:12-hark cycle. The animals were divided into five groups contain-ng four mice each. Three dose levels of fluconazole (12.5,5.0, 50.0 mg/kg) were given intraperitoneally for 24 h. An

ntreated control and a positive control (mitomycin C, 2 mg/kg)ere also used to test the validity of the assay. In order to arrestitosis, colchicine (5 mg/kg) was injected intraperitoneally 2 h

efore the animals were sacrificed by cervical dislocation. For

Fig. 1. The chemical structure of fluconazole.

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search 649 (2008) 155–160

one-marrow preparations, both hind femora were isolated andhe adherent muscle removed. The marrow was flushed outn 0.075 M KCl and kept at 37 ◦C for 30 min. At the end ofhe treatment, the suspension was centrifuged for 10 min at000× rpm and the supernatant was discharged. The cells werexed with three 10-min changes of fixative, methanol:aceticcid (3:1). Cells were then spread on pre-cleaned slides andir-dried. One day old slides were stained with 5% Giemsarepared in Sorensen buffer.

.3. Human lymphocyte culture for chromosomeberration and sister chromatid exchange tests

Peripheral blood was taken with heparinized syringes fromwo healthy individuals, one male and one female. Wholelood (0.2 ml) was added to 2.5 ml Chromosome Medium(Biochrome) supplemented with 10 �g/ml bromodeoxyuri-

ine. Human lymphocytes were incubated at 37 ◦C for 72 hnd treated with fluconazole at 12.5, 25.0 and 50.0 �g/ml for4 h. Colchicine (0.06 �g/ml) was added to the cultures duringhe last 2 h. The cultured cells were treated with a hypotonicolution of 0.075 M KCI for 30 min at 37 ◦C and then fixedith cold methanol:acetic acid (3:1). The cells were fixed with

hree changes of fixative. Slides were prepared by dropping andir-drying. For chromosome aberrations, slides were stainedith 5% Giemsa (pH 6.8) prepared in Sorensen buffer, for0–25 min, washed in distilled water, dried at room tempera-ure and mounted with depex. For the SCE study, the slides weretained by use of the FPG technique according to the methodescribed by Speit and Haupter [14], with some modifications.

.4. Micronucleus test in cultured human lymphocytes

Whole blood was added to 2.5 ml Chromosome Medium BBiochrome). Human lymphocytes were incubated at 37 ◦C for2 h and treated with fluconazole at 12.5, 25.0 and 50.0 �g/mluring the last 48 h. Cytocalasin-B (5.2 �g/ml) was added torrest cytokinesis at 44 h after the start of the culture. Then,he cells were harvested by centrifugation (1000 rpm, 10 min),nd the pellet was re-suspended in a hypotonic solution of.075 M KCI for 5 min at 4 ◦C. Cells were re-centrifuged andxed three times in cold methanol:acetic acid (3:1). In the lastxative, 1% formaldehyde was added to preserve the cyto-lasm. Slides were prepared by dropping and air-drying. Slidesere stained with 5% Giemsa (pH 6.8) in Sorensen buffer for0–25 min, washed in distilled water, dried at room tempera-ure and mounted with depex.

.5. Slide evaluation

In mice, 100 well-spread metaphases per animal were ana-

yzed for the CAs (total: 400 metaphases per concentration).he number of abnormal cells per animal was determined. Theitotic index (MI, number of cells undergoing mitosis/1000

ells) was also examined. In human lymphocytes, a hundredell-spread metaphases were analyzed for the CA assay per

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donor (total: 200 metaphases per dose level), and 25 s mitosesper donor (total: 50 s mitoses per dose level) were analyzed forthe SCE assay for each experimental dose level. In addition,1000 cells were analyzed to obtain the mitotic index. In theSCE study, a total of 200 cells (100 cells from each donor)were scored for the replication index (RI), calculated accord-ing to the following formula: RI = M1 + 2M2 + 3M3/N, whereM1, M2 and M3 represent the number of cells undergoing first,second and third mitotic divisions, respectively, and N the totalnumber of metaphases scored [15]. Micronuclei were scoredfrom 1000 binucleated cells per donor (total: 2000 binucleatedcells per dose level). Cell proliferation was evaluated using thecytokinesis-block proliferation index (CBPI), which indicatesthe average number of cell cycles. Five hundred lymphocytes(total: 1000 lymphocytes per dose level) were scored to eval-uate the percentage of cells with 1, 2, 3 and 4 nuclei. CBPIwas calculated according to Surrales et al. [16] as follows:[1 × N1] + [2 × N2] + [3 × (N3 + N4)]/N where N1–N4 representthe number of cells with 1–4 nuclei, respectively, and N is thetotal number of cells scored.

2.6. Statistical analysis

For the statistical analysis of the results, the z-test was usedfor the percentage of abnormal cells, CA/cell, RI and MI, andthe t-test was used for SCE [17,18]. For MN analysis, differ-ences between treated samples and controls were tested withthe z-test.

Dose–response relationships were determined from thecorrelation and regression coefficients for the percentage ofabnormal cells, CA/cell, SCE and mean MN.

3. Results and discussion

Fluconazole induced four types of structural chromo-some aberration in mouse bone-marrow cells (Table 1).The dominant type of aberration was chromatid breaks.Chromosome breaks, sister chromatid union and frag-

ments were also induced. Fluconazole increased thenumber of CA in a dose-dependent manner (r = 0.88) butthis increase was not statistically significant comparedwith the negative control in the in vivo test. In addition,

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Table 1Chromosomal aberrations in mouse bone-marrow cells treated with fluconazo

Test substance Treatment Structural aberrations

Period (h) Doses (mg/kg) ctb scu csb f

Control 0.0 1 – – –MMC 24 2.0 13 1 – 1FC 24 12.5 – – – –

25.0 2 1 – –50.0 1 – 1 1

ctb, chromatid break; scu, sister chromatid union; csb, chromosome break; f,were scored for each treatment.

earch 649 (2008) 155–160 157

uconazole induced polyploidy. These kinds of aberra-ion were also observed with other antifungal drugs. Forxample, miconazole induced structural aberrations suchs gaps, centric fusion, chromosome-chromatid breaks,eletions and polyploidy in mouse bone-marrow cells19].

In human lymphocyte cultures, fluconazole inducedix types of structural aberration: chromatid andhromosome breaks, sister chromatid union, dicen-ric chromosomes, chromatid exchange and fragments.luconazole also induced numerical aberrations (poly-loidy). The frequency of abnormal cells and the numberf CA per cell were increased significantly and in aose-dependent manner (r = 0.97 and 0.98, respectively)Table 2). Note that chromatid breaks were the most com-on abnormality, like in mice in vivo. Biswas et al. [20]

eported that the occurrence of chromosome aberrations,specially breaks, would indicate that the chemical pos-ibly acted after chromosome duplication at the G2 phasef the cell cycle.

Fluconazole significantly increased the frequencyf SCE in all treatments in a dose-dependent mannerr = 0.90) (Table 3). Although the molecular mecha-isms of SCE formation and their biological significanceemain unclear, there is strong support for the likelihoodhat reciprocal exchanges between two sister chromatidsrise in cells exposed to genotoxic agents that are capa-le of inducing DNA damage that interferes with DNAeplication [21]. Many studies have shown the inductionf SCE by different drugs, including antifungal agents22–24]. On the other hand, the mitotic index decreasedith increasing dose levels. However, this reduction wasot statistically significant. Changes in the RI were alsoot significant.

In order to evaluate possible clastogenic and/or aneu-enic effects, the cytokinesis-block micronucleus assay

as conducted. Fluconazole induced micronuclei at a

tatistically significant level in a dose-dependent mannerr = 0.76) (Table 4). Chromosomal breaks or interfer-nce with the mitotic process, resulting in lagging

le

Numerical aberrations Abnormal cell ± S.E.(%)

CA/Cell ± S.E.

p

– 0.25 ± 0.50 0.003 ± 0.0023 4.50 ± 1.04 0.045 ± 0.0112 0.50 ± 0.35 0.005 ± 0.0041 1.00 ± 0.50 0.010 ± 0.0051 1.00 ± 0.50 0.010 ± 0.005

fragment; p, polyploidy; FC, fluconazole. Four hundred metaphases

158 D. Yuzbasıoglu et al. / Mutation Research 649 (2008) 155–160

Table 2Total chromosomal aberrations in human lymphocytes treated with fluconazole

Test substance Treatment Aberrations Abnormal cell ± S.E.(%)

CA/Cell ± S.E.

Period (h) Doses (�g/ml) ctb csb f dc scu cte p

Control 0.0 – – – – – – – 0.00 ± 0.00 0.000 ± 0.00MMC 24 0.1 13 3 2 4 3 8 2 17.50 ± 2.69 0.175 ± 0.027FC 24 12.5 4 – 1 2 1 – – 4.00 ± 1.38* 0.040 ± 0.014*

25.0 6 – 3 – 1 – – 4.50 ± 1.47* 0.050 ± 0.070*

50.0 12 2 2 1 – 1 1 8.50 ± 1.97** 0.095 ± 0.020**

ctb, chromatid break; scu, sister chromatid union; dc, dicentric; csb, chromosome break; cte, chromatid exchange; f, fragment; p, polyploidy; FC,fluconazole. Two hundred metaphases were scored for each treatment.

* Significantly different from the negative control P < 0.05 (z-test).** Significantly different from the negative control P < 0.001 (z-test).

Table 3Sister chromatid exchange, replicative and mitotic indices in human lymphocytes treated with fluconazole

Test substance Treatment Min–max SCE SCE/cell ± S.E. M1 M2 M3 RI ± S.E. MI ± S.E.

Period (h) Dose (�g/ml)

Control 2–10 5.54 ± 0.26 53 53 94 2.21 ± 0.034 5.70 ± 0.51MMC 24 0.10 12–67 38.10 ± 2.14 63 52 85 1.81 ± 0.064 5.10 ± 0.49FC 24 12.5 4–15 8.48 ± 0.39* 58 50 92 2.17 ± 0.088 5.60 ± 0.51

25.0 3–17 8.60 ± 0.44* 52 46 102 2.25 ± 0.089 5.35 ± 0.5050.0 3–19 10.04 ± 0.66* 30 48 122 2.46 ± 0.084 5.00 ± 0.49

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ifty metaphases were scored for each dose level in the SCE test. Twetaphases were scored for each dose level for the MI; FC, fluconazo* Significantly different from the negative control P < 0.05 (t-test).

f the chromosomal material during cell division, leadso the formation of this type of damage [25]. However,he CBPI was not affected by fluconazole treatment.riseofulvin, another antifungal drug, increased theicronucleus frequency; it is a strong aneuploidy-

nducing agent in peripheral human lymphocytes [26].bou-Eisha et al. [24] investigated the genotoxicity

f the antimicrobial drug sulfamethoxazole in cultureduman lymphocytes: it induced a slight increase in SCEnd MN frequencies. Induction of MN by other drugsas also reported in several studies [13,20,23,27].

pe4(

able 4he micronucleus frequency and cytokinesis-block proliferation index in hum

est substance Treatment BN cells scored Distraccor

Period (h) Doses (�g/ml) (1)

ontrol 2000 6MC 48 0.10 2000 100

C 48 12.5 2000 3225.0 2000 4050.0 2000 35

total of 2000 binucleate cells were scored for each dose level in the miuconazole.* Significantly different from the negative control P < 0.05 (z-test).

red metaphases were scored for each dose level for the RI, and 2000

Fluconazole inhibited the cytochrome P450-mediatedonversion of lanosterol to ergosterol, a main compo-ent in the fungal cell wall, like other azole antifungalhemicals [28]. Depletion of ergosterol makes the cellembrane more fluid, reduces the activity of fungal

nzymes and inhibits cell growth. Teratological studiesn vitro and in vivo in rats, as well as in patients with acute

romyelocytic leukaemia have shown that the inhibitoryffect of azole derivatives is targeted to CYP26, a P-50 enzyme that mediates the catabolism of retinoic acidRA) [29–31]. RA is an important vitamin A derivative

an lymphocytes treated with fluconazole

ibution of BN cellsding to the no. of MN

MN (%) CBPI

(2) (3)

0 0 0.30 ± 0.12 1.697 ± 0.4111 0 6.10 ± 0.54 1.439 ± 0.381 0 1.70 ± 0.29* 2.315 ± 0.480 0 2.00 ± 0.31* 2.077 ± 0.451 1 2.00 ± 0.31* 1.979 ± 0.44

cronucleus test, and 1000 lymphocytes were scored for CBPI; FC,

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that has a wide range of biological activities during dif-ferentiation and morphogenesis [32]. In mammals, flu-conazole increased endogenous RA levels by inhibitingthe cytochrome P-450 (CYP26)-mediated catabolism ofRA. While retinol and retinoic acid show cytotoxicityat concentrations above 50 �M, their lower doses sig-nificantly inhibited both cytotoxicity and mutation rateinduced by chemical mutagens in CHO cells [33]. In thepresent study, the non-significant increase in aberrationsin fluconazole-treated mice may result from inhibitionof CYP26, which causes a small increase in retinoicacid level and inhibits genotoxicity in vivo. It may alsoresult from involvement of a detoxification process in thewhole animal. A number of factors may also influencethe time of appearance of chemically induced aberrationsin in vivo studies, such as compound solubility, rate anddistribution of biotransport, availability at the target siteas influenced by time, and cell permeability [34].

On the other hand, the genotoxic activity of flucona-zole in human lymphocytes in vitro may be due tobio-activation by CYP2E1, which is found in humanlymphocytes as well as in other tissues. Chemical inter-actions with this enzyme produce free oxygen radicals[35–37], which can cause various aberrant chromosomesin human lymphocytes. Aneugenic effects of fluconazolemay be due to inhibition of Op18/stathmin, which playsa crucial role in the regulation of microtubule dynam-ics during cell cycle progression [38]. If Op18/stathminactivity is inhibited by fluconazole, some errors mayoccur such as de-stabilization of microtubules, abnor-mal organization of the mitotic spindle and micronucleusformation.

In summary, fluconazole induces clastogenesis, DNAeffects and aneugenesis in human lymphocytes. How-ever, it should be investigated in other mammalian testsystem(s) for its genotoxic effects in vivo. Furthermore,biomonitoring studies should also be conducted withpatients receiving therapy with this drug.

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