Genotoxicity testing of fluconazole in vivo and in vitro

Available online at www.sciencedirect.com

Mutation Research 649 (2008) 155–160

Genotoxicity testing of fluconazole in vivo and in vitro a,∗ , Serkan Yılmaz a , ¨ Deniz Y¨uzbas¸ıo˘glu a , Fatma Unal H¨useyin Aksoy b , Mustafa C ¸ elik c a

Gazi University, Science Faculty, Department of Biology, Ankara, T¨urkiye Sakarya University, Science Faculty, Department of Biology, Sakarya, T¨urkiye ˙ Kahramanmara¸s S¨ut¸cu¨ Imam University, Science Faculty, Department of Biology, Kahramanmara¸s, T¨urkiye b

c

Received 12 February 2007; received in revised form 8 August 2007; accepted 30 August 2007 Available 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, 25.0 and 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, fluconazole did not significantly increase the frequency of CA. In the in vitro assays, CA, SCE and MN frequencies were significantly increased in 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 in human lymphocytes, but these effects could not be observed in mice. Further studies should be conducted in other test systems to evaluate the full genotoxic potential of fluconazole. © 2007 Elsevier B.V. All rights reserved. Keywords: Fluconazole; Antifungal; Chromosomal aberrations (CA); Sister chromatid exchange (SCE); Micronucleus (MN) assay

1. Introduction Fluconazole (trade name triflucan) is a member of the bis-triazole class of antifungal agents, and a highly selective inhibitor of fungal cytochrome P-450 sterol C-14 alpha demethylation [1]. Fluconazole is an important drug in obstetrics and gynecology for treatment of vaginal candidiasis. It is also used for the treatment of oropharyngeal, esophageal and urinary tract infections,

∗ Corresponding author. Tel.: +90 312 202 1181; fax: +90 312 212 2279. ¨ E-mail address: [email protected] (F. Unal).

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

peritonitis and cryptococcal meningitis. In addition, fluconazole is used to treat fungal infections in people with a suppressed immune system, such as cancer chemotherapy or organ-transplant patients, and AIDS patients [2]. High doses of fluconazole have been shown to be teratogenic in rodents in vivo [3] and in vitro [4]. A specific teratogenic effect on the branchial arch apparatus has been described in cultured whole mouse [4] and rat embryos [5]. Fluconazole also induced teratogenic effects in tunicate Phallusia mammillata [2], and it was recently identified as a possible human teratogen [6]. In contrast, fluconazole was not mutagenic in four strains of Salmonella typhimurium and in the mouse lymphoma L5178Y system. Cytogenetic studies in vivo (murine

156

D. Y¨uzba¸sıo˘glu et al. / Mutation Research 649 (2008) 155–160

bone-marrow cells) and in vitro (human lymphocytes) showed no evidence of chromosomal mutations [7]. However, there are chemicals that give negative results in bacteria, but are mutagenic when tested in other organisms and in other test systems [8]. In both bacterial and mammalian cells, positive and negative effects have been reported in the same test system [9–12]. Furthermore, it is generally necessary to use more than one test system to obtain a full evaluation of the genotoxicity of a drug or its metabolites [13]. For these reasons, we decided to provide additional genotoxicity data for the antifungal drug fluconazole, investigating the induction of chromosomal aberrations (CA) in mouse bone-marrow cells and chromosomal aberrations, sister chromatid exchange (SCE) and micronuclei (MN) in cultured human lymphocytes. 2. Material and methods 2.1. Chemicals The test substance fluconazole (CAS no. 86386-73-4) was obtained from Pfizer (Turkey). Mitomycin C (CAS no. 50-077), bromodeoxyuridine (CAS no. 59-14-3) and cytochalasin B (CAS no. 14930-96-2) were obtained from Sigma. The chemical structure of fluconazole (2-(2,4-difluorophenyl)-1,3bis(1H-1,2,4-triazol-1-yl)-2-propanol) is as shown in Fig. 1. 2.2. Animals and their treatment for measurement of chromosome aberrations Male Swiss albino mice (8–10 weeks old) weighing 25–28 g were used for the experiment. The mice were maintained in separate cages at room temperature (20 ± 1 ◦ C) and 12-h light:12-h dark cycle. The animals were divided into five groups containing four mice each. Three dose levels of fluconazole (12.5, 25.0, 50.0 mg/kg) were given intraperitoneally for 24 h. An untreated control and a positive control (mitomycin C, 2 mg/kg) were also used to test the validity of the assay. In order to arrest mitosis, colchicine (5 mg/kg) was injected intraperitoneally 2 h before the animals were sacrificed by cervical dislocation. For

bone-marrow preparations, both hind femora were isolated and the adherent muscle removed. The marrow was flushed out in 0.075 M KCl and kept at 37 ◦ C for 30 min. At the end of the treatment, the suspension was centrifuged for 10 min at 1000× rpm and the supernatant was discharged. The cells were fixed with three 10-min changes of fixative, methanol:acetic acid (3:1). Cells were then spread on pre-cleaned slides and air-dried. One day old slides were stained with 5% Giemsa prepared in Sorensen buffer. 2.3. Human lymphocyte culture for chromosome aberration and sister chromatid exchange tests Peripheral blood was taken with heparinized syringes from two healthy individuals, one male and one female. Whole blood (0.2 ml) was added to 2.5 ml Chromosome Medium B (Biochrome) supplemented with 10 ␮g/ml bromodeoxyuridine. Human lymphocytes were incubated at 37 ◦ C for 72 h and treated with fluconazole at 12.5, 25.0 and 50.0 ␮g/ml for 24 h. Colchicine (0.06 ␮g/ml) was added to the cultures during the last 2 h. The cultured cells were treated with a hypotonic solution of 0.075 M KCI for 30 min at 37 ◦ C and then fixed with cold methanol:acetic acid (3:1). The cells were fixed with three changes of fixative. Slides were prepared by dropping and air-drying. For chromosome aberrations, slides were stained with 5% Giemsa (pH 6.8) prepared in Sorensen buffer, for 20–25 min, washed in distilled water, dried at room temperature and mounted with depex. For the SCE study, the slides were stained by use of the FPG technique according to the method described by Speit and Haupter [14], with some modifications. 2.4. Micronucleus test in cultured human lymphocytes Whole blood was added to 2.5 ml Chromosome Medium B (Biochrome). Human lymphocytes were incubated at 37 ◦ C for 72 h and treated with fluconazole at 12.5, 25.0 and 50.0 ␮g/ml during the last 48 h. Cytocalasin-B (5.2 ␮g/ml) was added to arrest cytokinesis at 44 h after the start of the culture. Then, the cells were harvested by centrifugation (1000 rpm, 10 min), and the pellet was re-suspended in a hypotonic solution of 0.075 M KCI for 5 min at 4 ◦ C. Cells were re-centrifuged and fixed three times in cold methanol:acetic acid (3:1). In the last fixative, 1% formaldehyde was added to preserve the cytoplasm. Slides were prepared by dropping and air-drying. Slides were stained with 5% Giemsa (pH 6.8) in Sorensen buffer for 20–25 min, washed in distilled water, dried at room temperature and mounted with depex. 2.5. Slide evaluation

Fig. 1. The chemical structure of fluconazole.

In mice, 100 well-spread metaphases per animal were analyzed for the CAs (total: 400 metaphases per concentration). The number of abnormal cells per animal was determined. The mitotic index (MI, number of cells undergoing mitosis/1000 cells) was also examined. In human lymphocytes, a hundred well-spread metaphases were analyzed for the CA assay per

D. Y¨uzba¸sıo˘glu et al. / Mutation Research 649 (2008) 155–160

donor (total: 200 metaphases per dose level), and 25 s mitoses per donor (total: 50 s mitoses per dose level) were analyzed for the SCE assay for each experimental dose level. In addition, 1000 cells were analyzed to obtain the mitotic index. In the SCE study, a total of 200 cells (100 cells from each donor) were scored for the replication index (RI), calculated according to the following formula: RI = M1 + 2M2 + 3M3 /N, where M1 , M2 and M3 represent the number of cells undergoing first, second and third mitotic divisions, respectively, and N the total number of metaphases scored [15]. Micronuclei were scored from 1000 binucleated cells per donor (total: 2000 binucleated cells per dose level). Cell proliferation was evaluated using the cytokinesis-block proliferation index (CBPI), which indicates the average number of cell cycles. Five hundred lymphocytes (total: 1000 lymphocytes per dose level) were scored to evaluate the percentage of cells with 1, 2, 3 and 4 nuclei. CBPI was calculated according to Surrales et al. [16] as follows: [1 × N1 ] + [2 × N2 ] + [3 × (N3 + N4 )]/N where N1 –N4 represent the number of cells with 1–4 nuclei, respectively, and N is the total number of cells scored. 2.6. Statistical analysis For the statistical analysis of the results, the z-test was used for the percentage of abnormal cells, CA/cell, RI and MI, and the t-test was used for SCE [17,18]. For MN analysis, differences between treated samples and controls were tested with the z-test. Dose–response relationships were determined from the correlation and regression coefficients for the percentage of abnormal cells, CA/cell, SCE and mean MN.

3. Results and discussion Fluconazole induced four types of structural chromosome aberration in mouse bone-marrow cells (Table 1). The dominant type of aberration was chromatid breaks. Chromosome breaks, sister chromatid union and fragments were also induced. Fluconazole increased the number of CA in a dose-dependent manner (r = 0.88) but this increase was not statistically significant compared with the negative control in the in vivo test. In addition,

157

fluconazole induced polyploidy. These kinds of aberration were also observed with other antifungal drugs. For example, miconazole induced structural aberrations such as gaps, centric fusion, chromosome-chromatid breaks, deletions and polyploidy in mouse bone-marrow cells [19]. In human lymphocyte cultures, fluconazole induced six types of structural aberration: chromatid and chromosome breaks, sister chromatid union, dicentric chromosomes, chromatid exchange and fragments. Fluconazole also induced numerical aberrations (polyploidy). The frequency of abnormal cells and the number of CA per cell were increased significantly and in a dose-dependent manner (r = 0.97 and 0.98, respectively) (Table 2). Note that chromatid breaks were the most common abnormality, like in mice in vivo. Biswas et al. [20] reported that the occurrence of chromosome aberrations, especially breaks, would indicate that the chemical possibly acted after chromosome duplication at the G2 phase of the cell cycle. Fluconazole significantly increased the frequency of SCE in all treatments in a dose-dependent manner (r = 0.90) (Table 3). Although the molecular mechanisms of SCE formation and their biological significance remain unclear, there is strong support for the likelihood that reciprocal exchanges between two sister chromatids arise in cells exposed to genotoxic agents that are capable of inducing DNA damage that interferes with DNA replication [21]. Many studies have shown the induction of SCE by different drugs, including antifungal agents [22–24]. On the other hand, the mitotic index decreased with increasing dose levels. However, this reduction was not statistically significant. Changes in the RI were also not significant. In order to evaluate possible clastogenic and/or aneugenic effects, the cytokinesis-block micronucleus assay was conducted. Fluconazole induced micronuclei at a statistically significant level in a dose-dependent manner (r = 0.76) (Table 4). Chromosomal breaks or interference with the mitotic process, resulting in lagging

Table 1 Chromosomal aberrations in mouse bone-marrow cells treated with fluconazole Test substance

Treatment Period (h)

Control MMC FC

24 24

Structural aberrations

Numerical aberrations

Doses (mg/kg)

ctb

scu

csb

f

p

0.0 2.0 12.5 25.0 50.0

1 13 – 2 1

– 1 – 1 –

– – – – 1

– 1 – – 1

– 3 2 1 1

Abnormal cell ± S.E. (%) 0.25 4.50 0.50 1.00 1.00

± ± ± ± ±

0.50 1.04 0.35 0.50 0.50

CA/Cell ± S.E.

0.003 0.045 0.005 0.010 0.010

± ± ± ± ±

0.002 0.011 0.004 0.005 0.005

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

158

D. Y¨uzba¸sıo˘glu et al. / Mutation Research 649 (2008) 155–160

Table 2 Total chromosomal aberrations in human lymphocytes treated with fluconazole Test substance

Treatment Period (h)

Control MMC FC

24 24

Abnormal cell ± S.E. (%)

Aberrations Doses (␮g/ml)

ctb

csb

f

dc

scu

cte

p

0.0 0.1 12.5 25.0 50.0

– 13 4 6 12

– 3 – – 2

– 2 1 3 2

– 4 2 – 1

– 3 1 1 –

– 8 – – 1

– 2 – – 1

0.00 17.50 4.00 4.50 8.50

± ± ± ± ±

0.00 2.69 1.38* 1.47* 1.97**

CA/Cell ± S.E.

0.000 0.175 0.040 0.050 0.095

± ± ± ± ±

0.00 0.027 0.014* 0.070* 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 3 Sister chromatid exchange, replicative and mitotic indices in human lymphocytes treated with fluconazole Test substance

Control MMC FC

Treatment Period (h)

Dose (␮g/ml)

24 24

0.10 12.5 25.0 50.0

Min–max SCE

SCE/cell ± S.E.

M1

M2

M3

RI ± S.E.

2–10 12–67 4–15 3–17 3–19

5.54 38.10 8.48 8.60 10.04

± ± ± ± ±

53 63 58 52 30

53 52 50 46 48

94 85 92 102 122

2.21 1.81 2.17 2.25 2.46

0.26 2.14 0.39* 0.44* 0.66*

± ± ± ± ±

0.034 0.064 0.088 0.089 0.084

MI ± S.E.

5.70 5.10 5.60 5.35 5.00

± ± ± ± ±

0.51 0.49 0.51 0.50 0.49

Fifty metaphases were scored for each dose level in the SCE test. Two hundred metaphases were scored for each dose level for the RI, and 2000 metaphases were scored for each dose level for the MI; FC, fluconazole. * Significantly different from the negative control P < 0.05 (t-test).

of the chromosomal material during cell division, leads to the formation of this type of damage [25]. However, the CBPI was not affected by fluconazole treatment. Griseofulvin, another antifungal drug, increased the micronucleus frequency; it is a strong aneuploidyinducing agent in peripheral human lymphocytes [26]. Abou-Eisha et al. [24] investigated the genotoxicity of the antimicrobial drug sulfamethoxazole in cultured human lymphocytes: it induced a slight increase in SCE and MN frequencies. Induction of MN by other drugs was also reported in several studies [13,20,23,27].

Fluconazole inhibited the cytochrome P450-mediated conversion of lanosterol to ergosterol, a main component in the fungal cell wall, like other azole antifungal chemicals [28]. Depletion of ergosterol makes the cell membrane more fluid, reduces the activity of fungal enzymes and inhibits cell growth. Teratological studies in vitro and in vivo in rats, as well as in patients with acute promyelocytic leukaemia have shown that the inhibitory effect of azole derivatives is targeted to CYP26, a P450 enzyme that mediates the catabolism of retinoic acid (RA) [29–31]. RA is an important vitamin A derivative

Table 4 The micronucleus frequency and cytokinesis-block proliferation index in human lymphocytes treated with fluconazole Test substance

Control MMC FC

Treatment

BN cells scored

Period (h)

Doses (␮g/ml)

48 48

0.10 12.5 25.0 50.0

2000 2000 2000 2000 2000

Distribution of BN cells according to the no. of MN (1)

(2)

(3)

6 100 32 40 35

0 11 1 0 1

0 0 0 0 1

MN (%)

0.30 6.10 1.70 2.00 2.00

± ± ± ± ±

0.12 0.54 0.29* 0.31* 0.31*

CBPI

1.697 1.439 2.315 2.077 1.979

± ± ± ± ±

0.41 0.38 0.48 0.45 0.44

A total of 2000 binucleate cells were scored for each dose level in the micronucleus test, and 1000 lymphocytes were scored for CBPI; FC, fluconazole. * Significantly different from the negative control P < 0.05 (z-test).

D. Y¨uzba¸sıo˘glu et al. / Mutation Research 649 (2008) 155–160

that has a wide range of biological activities during differentiation and morphogenesis [32]. In mammals, fluconazole increased endogenous RA levels by inhibiting the cytochrome P-450 (CYP26)-mediated catabolism of RA. While retinol and retinoic acid show cytotoxicity at concentrations above 50 ␮M, their lower doses significantly inhibited both cytotoxicity and mutation rate induced by chemical mutagens in CHO cells [33]. In the present study, the non-significant increase in aberrations in fluconazole-treated mice may result from inhibition of CYP26, which causes a small increase in retinoic acid level and inhibits genotoxicity in vivo. It may also result from involvement of a detoxification process in the whole animal. A number of factors may also influence the time of appearance of chemically induced aberrations in in vivo studies, such as compound solubility, rate and distribution of biotransport, availability at the target site as influenced by time, and cell permeability [34]. On the other hand, the genotoxic activity of fluconazole in human lymphocytes in vitro may be due to bio-activation by CYP2E1, which is found in human lymphocytes as well as in other tissues. Chemical interactions with this enzyme produce free oxygen radicals [35–37], which can cause various aberrant chromosomes in human lymphocytes. Aneugenic effects of fluconazole may be due to inhibition of Op18/stathmin, which plays a crucial role in the regulation of microtubule dynamics during cell cycle progression [38]. If Op18/stathmin activity is inhibited by fluconazole, some errors may occur such as de-stabilization of microtubules, abnormal organization of the mitotic spindle and micronucleus formation. In summary, fluconazole induces clastogenesis, DNA effects and aneugenesis in human lymphocytes. However, it should be investigated in other mammalian test system(s) for its genotoxic effects in vivo. Furthermore, biomonitoring studies should also be conducted with patients receiving therapy with this drug. References [1] M.A. Pfaller, S.A. Messer, R.J. Hollis, R.N. Jones, G.V. Doern, M.E. Brandt, R.A. Hajjeh, Trends in species distribution and susceptibility to fluconazole among blood stream isolates of Candida species in the United States, Diagn. Microbiol. Infect. Dis. 33 (1999) 217–222. [2] S. Groppelli, G. Zega, M. Biggiogero, F. De Bernardi, C. Sotgia, R. Pennati, Fluconazole induces teratogenic effects in the tunicate Phallusia mammillata, Environ. Toxicol. Pharmacol. 23 (2007) 265–271. [3] M. Tachibana, Y. Noguchi, A.M. Monro, Toxicology of fluconazole in experimental animals, in: R.A. Fromtling (Ed.), Recent Trends in the Discovery, Development and Evaluation of Antifungal Agents, Barcelona, 1987, pp. 93–102.

159

[4] G.M. Tiboni, Second branchial arch anomalies induced by fluconazole, a bistriazole antifungal agent, in cultured mouse embryos, Res. Commun. Chem. Pathol. Pharmacol. 79 (1993) 381–384. [5] E. Menegola, M.L. Broccia, F. Di Renzo, E. Giavini, Pathogenic pathways in fluconazole-induced branchial arch malformations, Birth Defects Res. A Clin. Mol. Teratol. 67 (2003) 16–124. [6] J.E. Polifka, J.M. Friedman, Medical genetics: 1. Clinical teratology in the age of genomics, CMAJ 167 (2002) 265–273. [7] http://www.pfizer.com/pfizer/download/uspi diflucan.pdf Diflucan (Fluconazole), Description, Pfizer (2004). [8] M.C. Cimino, New OECD genetic toxicology guidelines and interpretation of results, in: W.N. Chog (Ed.), Genetic Toxicology and Cancer Risk Assessment, Marcel Dekker, Inc., New York, 2001, pp. 223–248. [9] K.P.S. Rao, M.A. Rahiman, Cytogenetic effects of ribavirin on mouse bone marrow, Mutat. Res. 224 (1989) 213–218. [10] M.D. Phillips, B. Nascimbeni, R.R. Tice, M.D. Shelby, Induction of micronuclei in mouse bone marrow cells: an evaluation of nucleoside analogues used in the treatment of AIDS, Environ. Mol. Mutagen. 18 (3) (1991) 168–183. [11] K. Mortelmans, S. Haworth, T. Lawlor, W. Speck, B. Tainer, E. Zeiger, Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals, Environ. Mol. Mutagen. 8 (Suppl. 7) (1986) 1–119. [12] S.A. Rasool, M.A. Khan, A.Z. Alvi, M.N. Umer, Genetic activity of trimethoprim in the Salmonella/microsomal screening system, Mutat. Res. 188 (1987) 197–200. [13] L.M. Formigli, I. Ferrari, C.K. Grisolia, Evaluation of genotoxic and cytotoxic potential of Thiola (N-2Mercaptopropionylglycine), a medicine used in the treatment of humans contaminated with mercury, Environ. Mol. Mutagen. 39 (2002) 18–21. [14] G. Speit, S. Houpter, On the mechanism of differential giemsa staining of bromodeoxyuridine substitute chromosomes. II. Differences between the demonstration of sister chromatid differantiation and replication patterns, Hum. Genet. 70 (1985) 126–129. [15] E.L. Schneider, J. Lewis, Aging and sister chromatid exchange: VIII. Effect of the aging environment on sister chromatid exchange induction and cell cycle kinetics in Ehrlich ascites tumor cells, Mech. Ageing Dev. 17 (1981) 327–330. [16] J. Surrales, N. Xamena, A. Creus, J. Catalan, H. Norppa, R. Marcos, Induction of micronuclei by five pyrethroid insecticides in whole blood and isolated human lymphocytes cultures, Mutat. Res. 341 (1995) 169–184. [17] E. Renc¨uzogulları, B.A. T¨uyl¨u, M. Topaktas¸, H.B. Ila, A. Karyaldız, M. Arslan, S. Budak Diler, Genotoxicity of aspartame, Drug Chem. Toxicol. 27 (3) (2004) 257–268. ¨ [18] D. Y¨uzbas¸ıo˘glu, M. C ¸ elik, S. Yılmaz, F. Unal, H. Aksoy, Clastogenicity of the fungicide afugan in cultured human lymphocytes, Mutat. Res. 604 (1–2) (2006) 53–59. [19] N.H.A. Hassan, Miconazole genotoxicity in mice, J Appl. Toxicol. 17 (1997) 313–319. [20] S.J. Biswas, S. Pathak, A.R. Khuda-Bukhsh, Assesment of the genotoxic and cytotoxic potential of an anti-epileptic drug, phenobarbital, in mice: a time course study, Mutat. Res. 563 (2004) 1–11. [21] J.D. Tucker, A. Auletta, M.C. Cimino, K.L. Dearfield, D. Jacobson-Kram, R.R. Tice, A.V. Carrano, Sister-chromatid exchange: second report of the Gene-Tox program, Mutat. Res. 297 (1993) 101–180.

160

D. Y¨uzba¸sıo˘glu et al. / Mutation Research 649 (2008) 155–160

[22] J.L. Wilmer, S.E. Bloom, A cytogenetic approach for detecting the selective toxicity of drugs in avian embrionic B and T lymphocytes, Mutat. Res. 253 (1991) 161–172. [23] A. Abou-Eisha, A. Creus, R. Marcos, Genotoxic evaluation of the antimicrobial drug trimethoprim in cultured human lymphocytes, Mutat. Res. 440 (1999) 157–162. [24] A. Abou-Eisha, R. Marcos, A. Creus, Genotoxicity studies on the antimicrobial drug sulfamethoxazole in cultured human lymphocytes, Mutat. Res. 564 (2004) 51–56. [25] R.D. Gudi, S.S. Sandhu, S.R. Athwal, Kinetochore identification in micronuclei in mouse bone marrow erythrocytes: an assay for the detection of aneuploidy-inducing agents, Mutat. Res. 234 (1990) 263–268. [26] P. Kolachana, M.T. Smith, Induction of kinetochore-positive micronuclei in human lymphocytes by the anti-fungal drug griseofulvin, Mutat. Res. 322 (1994) 151–159. [27] K. Narayana, U.J.A. D’souza, K.P. Seetharama Rao, The genotoxic and cytotoxic effects of ribavirin in rat bone marrow, Mutat. Res. 521 (2002) 179–185. [28] H. Van Den Bossche, P. Marichal, J. Gorrens, M.C. Coene, G. Willemsens, D. Bellens, I. Roels, H. Moereels, P.A. Janssen, Biochemical approaches to selective antifungal activity. Focus on azole antifungals, Mycoses 32 (Suppl. 1) (1989) 35–52. [29] J.P. Van Wauwe, M.C. Coene, J. Goossens, W. Cools, J. Monbaliu, Effects of cytochrome P450 inhibitors on the in vivo metabolism of all-trans-retinoic acid in rats, J. Pharmacol. Exp. Ther. 252 (1990) 365–369. [30] E.L. Schwartz, S. Hallam, R.E. Gallagher, P.H. Wiernik, Inhibition of all-trans–retinoic acid metabolism by fluconazole in

[31]

[32] [33]

[34]

[35]

[36]

[37]

[38]

vitro and in patients with acute promyelocytic leukemia, Biochem. Pharmacol. 50 (1995) 923–928. K. Vanier, A. Mattiussi, D. Johnston, Interaction of all-trans retinoic acid with fluconazole in acute promyelocytic leukemia, J. Pediatr. Hematol. Oncol. 25 (2003) 403–404. M. Maden, N. Holder, Retinoic acid and development of the central nervous system, BioEssays 14 (1992) 431–438. J.D. Budroe, H.M. Schol, J.G. Shaddoct, D.A. Cassiano, Inhibition of 7,12-dimethylbenz[a]anthracene-induced genotoxicity in Chinese hamster ovary cells by retinol and retinoic acid, Carcinogenesis 9 (7) (1988) 1307–1311. A.F. McFee, R.R. Tice, Influence of treatment to sacrifice time and the presence of BrdUrd on chemically-induced aberration rates in mouse marrow cells, Mutat. Res. 241 (1990) 95–108. G. Ekstrom, M. Ingelman-Sundberg, Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1), Biochem. Pharmacol. 38 (1989) 1313–1319. S.S.T. Lee, J.T.M. Buters, T. Pineau, P. Fernandez-Salguero, F.J. Gonzalez, Role of CYP2E1 in the hepatotoxicity of acetaminophen, J. Biol. Chem. 271 (1996) 12063–12067. Y. Dai, J. Rashba-Step, A.I. Cederbaum, Stable expression of human cytochrome P4502E1 in HepG2 cells: characterization of catalytic activities and production of reactive oxygen intermediates, Biochemistry 32 (1993) 6928–6937. N. Larsson, U. Marklund, H.M. Gradin, G. Brattsand, M. Gullberg, Control of microtubule dynamics by oncoprotein 18: dissection of the regulatory role of multisite phosphorylation during mitosis, Mol. Cell. Biol. 17 (1997) 5530–5539.