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In vitro photochemical clastogenicity of quinolone antibacterial agents studied by a chromosomal aberration test with light irradiation
Mutation Research 517 (2002) 113–121
In vitro photochemical clastogenicity of quinolone antibacterial agents studied by a chromosomal aberration test with light irradiation Satoru Itoh a,∗ , Shiho Nakayama a , Hiroyasu Shimada b a b
Drug Safety Research Laboratory, Daiichi Pharmaceutical Co. Ltd., 16-13 Kita-Kasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan Drug Safety Administration Department, Daiichi Pharmaceutical Co. Ltd., 16-1 Kyobashi 2-Chome, Chuo-ku, Tokyo 104-8369, Japan Received 8 August 2001; received in revised form 4 March 2002; accepted 7 March 2002
Abstract The photochemical clastogenic potential of 12 quinolone antibacterial agents with or without light irradiation was assessed by an in vitro chromosomal aberration test using cultured CHL cells. Exposure to all test compounds, except for DK-507k, increased the incidence of cells with structural aberrations excluding gap (TA) following light irradiation. Test compounds used in the present study under light irradiation were divided into three groups based on their ED50 values, doses inducing chromosomal aberrations in 50% of cells. The first group with ED50 values below 30 g/ml includes sparfloxacin (SPFX), clinafloxacin (CLFX), gemifloxacin (GMFX), lomefloxacin (LFLX), sitafloxacin (STFX), grepafloxacin (GPFX) and fleroxacin (FLRX); the second group with ED50 values of 100 g/ml, enoxacin (ENX) and levofloxacin (LVFX); the third group with little or no potency, moxifloxacin (MFLX), trovafloxacin (TVFX) and DK-507k. The photochemical clastogenicity of these compounds correlates well with their reported in vivo phototoxic potentials. In the chemical structure and clastogenicity relationships, substitution of a methoxy group at the C-8 position in the quinolone nucleus was confirmed to reduce not only photochemical clastogenicity, but also the clastogenic potential of quinolone antibacterial agents. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Photochemical clastogenicity; Light irradiation; Chromosomal aberration test; Quinolone antibacterial agent
1. Introduction Quinolone antibacterial agents (quinolones) have been widely used in numerous diseases because of their broad spectrum activity against both gram-negative and gram-positive pathogens. One of the major adverse reactions due to quinolones is photosensitization, which has been reported in experimental and clinical studies [1,2]. Especially, concern ∗ Corresponding author. Tel.: +81-3-5696-8294; fax: +81-3-5696-8335. E-mail address: [email protected] (S. Itoh).
has been raised about photogenotoxicity induced by quinolones with ultraviolet A (UVA) irradiation. Namely, certain quinolones have been reported to be associated with irradiation induced gene mutation in bacteria [3] and cultured mammalian cells [4], chromosomal aberration or micronucleus in cultured mammalian cells [3,5,6], and DNA strand breakage in vitro and in vivo comet assays [3,7,8]. In photocarcinogenicity studies using Skh-1 hairless mice with UVA, lomefloxacin (LFLX) has been reported to cause cystic squamous cell carcinoma in the majority of animals treated [9,10]. The cause of photogenotoxicity induced by quinolones is thought to be the formation
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of reactive oxygen species, such as superoxide anion, hydrogen peroxide, hydroxy radical and singlet oxygen [11–14]. In chemical structure, quinolones, such as LFLX, fleroxacin (FLRX) and clinafloxacin (CLFX), possessing a halogen (fluorine or chlorine) at the C-8 position are known to generate a highly reactive carbene that causes strand breaks in DNA [15] at that position during photodegradation [6,7,16,18] and contributes to their phototoxicity and photogenotoxicity. It is, thus, important to elucidate the relationship between photogenotoxicity and chemical structure. To the best of our knowledge, there is no report dealing with comparative photogenotoxicity of more than ten quinolones under the same experimental conditions. In the present study, the photochemical clastogenicity of 12 quinolones was investigated based on their potentials for enhancing chromosomal aberrations. Accordingly, photochemical clastogenic activity was assessed by an in vitro chromosomal aberration test using Chinese hamster lung (CHL) cells in both the presence and absence of a sun-light simulator having a spectrum almost identical to that of solar irradiation.
8-Methoxypsoralen (8-MOP, CAS no. 298-81-7) as a positive control was purchased from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan) and dissolved in dimethyl sulfoxide. The solvent for all test compounds, 0.1 N NaOH, was used as the negative control. The present study consists of four experiments, and negative and positive controls were always set in each experiment.
2. Materials and methods
The same concentrations were used for all test compounds: 1, 3, 10, 30, 100 and 300 g/ml for light irradiation and 10, 30, 100, 300, 500 and 1000 g/ml for non-light irradiation. These dose levels were determined from the results of preliminary cytotoxicity and genotoxicity tests with STFX and LFLX. In the case of 8-MOP, a concentration of 0.02 g/ml was set for both light irradiation and non-irradiation [19]. One plate was used in each dose level.
2.1. Test chemicals Lomefloxacin (LFLX, CAS no. 98079-51-7) and enoxacin (ENX, CAS no. 74011-58-8) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Levofloxacin (LVFX, CAS no. 100986-85-4), clinafloxacin (CLFX, CAS no. 105956-97-6), fleroxacin (FLRX, CAS no. 79660-72-3), sparfloxacin (SPFX, CAS no. 110871-86-8), grepafloxacin (GPFX, CAS no. 119914-60-2), trovafloxacin (TVFX, CAS no. 147059-72-1), moxifloxacin (MFLX), gemifloxacin (GMFX), sitafloxacin (STFX) and DK-507k were synthesized in Daiichi Pharmaceutical Co. Ltd. (Tokyo, Japan). The chemical structures of these test compounds are shown in Fig. 1. Immediately prior to use, all test chemicals were dissolved or suspended in 0.1 N NaOH. The concentrations of 500 and 1000 g/ml in SPFX, STFX, MFLX, FLRX or CLFX treated group, and 300, 500 and 1000 g/ml in GPFX, LFLX or DK-507k treated group were suspended in 0.1 N NaOH.
2.2. Cell line and culture conditions CHL cells were purchased from Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan). Eagle’s minimum essential medium (MEM, Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 15% fetal bovine serum (FBS, Cancera International Inc., Canada) was used for cell culture. The cells were cultured in plastic petri dishes (Falcon, Becton Dickinson Labware, NJ, USA), 60 mm in diameter, in a humidified incubator with an atmosphere of 5% CO2 to 95% air at 37 ◦ C. 2.3. Dose levels
2.4. Light irradiation Light source and treatment procedures used in the present study were the same as those in the European Industry Association’s project on the neutral red assay with BALB/c 3T3 cells for validating in vitro methods to detect photocytotoxicity [20]. Namely, light irradiation was performed by a sun-light simulator SOL500 (Dr. Hönle, München, Germany) installed with an H1 filter (Dr. Hönle; 50% transmission of 335 nm wavelength) at room temperature. The irradiation spectrum was almost identical to that of solar irradiation, and the ratio of UVB to UVA was about 1:25 [21]. After
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pretreatment of CHL cells with each test compound in Earle’s balanced salt solution (EBSS; Gibco BRL) for 60 min in an incubator, culture dishes with lids were placed under the sun-light simulator and then irradiated at 0.8–1.0 mW/cm2 of UVA for 50 min (Irradiation dose of UVA, 2.4–3.0 J/cm2 , maximum dose not induce chromosomal aberrations). Emitted energy was measured by a simple UVA meter (type no. 37,
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Dr. Hönle) immediately before the light irradiation. The visible light intensity measured by a lux meter 3423 (Hioki E. E., Nagano, Japan) was 25,000–27,000 lx. For the non-irradiation group, on the other hand, the dishes were kept in the incubator for 50 min while light irradiation was performed on the light irradiation group. After light irradiation, the cells from all test groups, including the non-light
Fig. 1. Chemical structures of the quinolone antibacterial agents used in the present study.
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Fig. 1. (Continued ).
irradiation group, were washed with phosphate buffered saline followed by fresh 15% FBS-MEM, and then the cells were cultured for 22 h. 2.5. Specimen preparation At the end of the incubation period, 24 h after the commencement of treatment, chromosomal preparations were made. In order to collect the cells in metaphase, colcemid (GIBCO BRL, Life Technologies Inc., Rockville, MD, USA) at a final concentration of 0.05 g/ml was added to the medium 1.5 h prior to harvesting. After that, the cells resuspended in 1% sodium citrate were incubated for 15 min at room temperature and fixed by Carnoy’s solution (methanol:glacial acetic acid = 3:1, v/v). The fixative procedure was repeated more than three times. Chromosomal specimens were made by an air-dry technique and stained with Giemsa (Merck, Darmstadt, Germany). Three slides were prepared per plate.
(type and frequency) were scored. When the number of analyzable metaphase cells was less than 100 cells in three slides which corresponded to approximately 0.5% in mitotic index, cytotoxicity was judged to have occurred and we did not score it. Structural chromosomal aberrations were classified as gap, break, exchange, fragmentation, multiple-type aberrations and others. Any cell having chromosomal aberration(s) was recorded as an aberrant cell. In the present paper, chromosomal aberrations induced by the test compound is expressed as the incidence of cells with structural aberrations excluding gap (TA). The ED50 values (a dose inducing chromosomal aberrations in 50% cells, which was calculated from two values which are higher and lower than 50% induction points of chromosomal aberrations on figures) of the respective test compounds with or without light irradiation are presented. 2.7. Judgement for clastogenicity of test compounds
2.6. Observations One hundred well-spread metaphase cells per plate were analyzed, and the chromosomal aberrations
According to Ishidate’s criteria [22], the test compound was judged to be positive when 10% or more TA were observed.
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3. Results 3.1. Negative and positive controls TA values of the vehicle as the negative control were between 0 and 2% regardless of the presence or absence of light irradiation. The TA values of 8-MOP as the positive control in the presence and absence of light irradiation were 63–87 and 0–1%, respectively, suggesting that the test systems were properly functioning in the present study. 3.2. Test compounds The incidence of chromosomal aberrations (TA) induced by the 12 quinolones with or without light irradiation is shown in Fig. 2. CLFX, STFX, GPFX and GMFX increased TA, regardless of light irradiation compared with the negative control, for which increases were markedly greater with light irradiation Table 1 The relationship between ED50 and chemical structure of each test compound Compound
SPFX CLFX GMFX LFLX STFX GPFX FLRX ENX LVFX MFLX TVFX DK-507k
ED50 (g/ml) Light irradiation (+)
Light irradiation (−)
4 6 11 14 16 22 22 100 100 N.O.b N.O. –c
– 133 300 – N.O. N.O. – – – – – –
R5 a
X8 a
NH2 H H H H CH3 H H H H H H
CF CCl N CF CCl CH CF N COR COCH3 N COCH3
a X and R represent the following positions of quinolone 8 5 antibacterial agents.
b ED 50 value was not obtained since the TA value did not reach to 50% of the cells. c No chromosomal aberration was observed.
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than without light irradiation. ENX, LVFX, TVFX, SPFX, LFLX, MFLX and FLRX induced chromosomal aberrations only under light irradiation; whereas DK-507k induced no chromosomal aberration with or without light irradiation. The ED50 values of each test compound are indicated in Table 1. With light irradiation, the ED50 was <30 g/ml for SPFX, CLFX, GMFX, LFLX, STFX, GPFX and FLRX, and approximately 100 g/ml for ENX and LVFX. Without irradiation, CLFX and GMFX give ED50 values of 133 and 300 g/ml, respectively. MFLX and TVFX with light irradiation or STFX and GPFX without light irradiation have no ED50 value, because their TA values did not reach 50%.
4. Discussion The photochemical clastogenic potential of 12 quinolone antibacterial agents with or without light irradiation was assessed by an in vitro chromosomal aberration test using cultured CHL cells. Most of the test compounds caused increased TA with light irradiation, and their potential rank order from highest to lowest was shown to be SPFX > CLFX > GMFX > LFLX > STFX > GPFX = FLRX > ENX = LVFX > MFLX > TVFX, and DK-507k had no potential. It has been reported that several quinolones are classified as photogenotoxins by an in vitro chromosomal aberration assay with Chinese hamster V79 cells [3]. In this report, the clastogenic potential of LFLX was stated to be stronger than that of FLRX under UVA irradiation, which is in good agreement with our data. In the same way, the clastogenic potential of CLFX was reported to be stronger than that of LFLX in a chromosomal aberration test with UVA irradiation in CHO cells [6]; and this finding is also consistent with the present results. Additionally, the in vitro micronucleus test has also been used for investigating photochemical clastogenicity of quinolones [5]. The induction of micronucleated cells by GPFX was stronger in Chinese hamster V79 cells under UVA/UVB irradiation than that by LFLX. The reported results differed from our findings although the sensitivity to clastogens in the in vitro micronucleus test is almost identical to that in the chromosomal aberration test [23,24]. In summarizing the aforemen-
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Fig. 2. Incidence of chromosomal aberration excluding gap (TA) induced by each test compound. Closed circles and closed squares represent the incidence of TA in the presence and absence of light irradiation, respectively. The concentrations of the test compound used were 1, 3, 10, 30, 100 and 300 g/ml with light irradiation, and 10, 30, 100, 300, 500 and 1000 g/ml without light irradiation. Open circles and open squares represent that chromosome analysis could not be performed due to cytotoxicity of the test compound in the presence and absence of light irradiation, respectively. TA values of the negative and positive controls were described in Section 3.
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Fig. 2. (Continued ).
tioned results, although there were some differences in the experimental conditions including the light source, irradiation dose and cell line used, the results presented here seem to be similar to those of the other studies.
The 12 test compounds used in the present study were divided into three groups based on their ED50 values under light irradiation: the first, ED50 values <30 g/ml, included SPFX, CLFX, GMFX, LFLX,
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STFX, GPFX and FLRX; the second, ED50 values of 100 g/ml, ENX and LVFX; the third of little or no potency, MFLX, TVFX and DK-507k. The relationships between ED50 values and chemical structures of the quinolones used in the present study are also shown in Table 1. The chemical structure in vivo phototoxicity relationships are well summarized for quinolone antibacterial agents [1]. Namely, the in vivo phototoxic potential is mostly determined by the X8 substitution, and the highest phototoxicity is gained when the X8 is substituted by a halogen (fluorine or chlorine), and the least by COCH3 . The in vivo phototoxic potential of the X8 substitution is CF, CCl, N, CH, COCH3 in decreasing order. In addition, the R5 substitution also influences in vivo phototoxicity with an intensity order of CH3 > N > NH2 . In agreement with these, SPFX, CLFX, LFLX, STFX and FLRX having a halogen at the X8 position showed strong potentials for chromosomal aberration induction in the present study. In contrast, GMFX and GPFX, remainders of the first group mentioned earlier, do not have a halogen at the X8 position, instead, GMFX has N at the X8 position and also GPFX has CH3 at the R5 position. ENX and LVFX were classified as members of the second group quinolones with N and COR at the X8 position, respectively. In the third group, MFLX and DK-507k, but not TVFX, have COCH3 (the least in vivo phototoxicity) at the X8 position. TVFX has N at the X8 position similar to GMFX and ENX, which each have quite different potentials of causing chromosomal aberration induction under light irradiation. There may be other factors that affect their various potencies. In brief, the 12 quinolones used in the present study were classified into three groups based on their photochemical clastogenic potentials. In each group, the structure in the X8 and R5 substitutions resembled each other in principle and the photochemical clastogenic potential of these substitutions were almost identical to the data reported by Domaga [1]. It therefore appears that there is a good correlation between in vivo phototoxicity and in vitro photochemical clastogenicity in quinolones. SPFX, CLFX, LFLX, STFX and FLRX, which are classified in the first group, have a fluorine or chlorine at the C-8 position. LFLX and FLRX have been reported to generate a highly reactive carbene species which is known to cause strand breaks in DNA [15], at the C-8 position during photodegra-
dation [16–18]. It has been proposed that CLFX, chlorinated at the C-8 position, could follow a process similar to those two compounds [6]. SPFX and STFX have fluorine and chlorine at the C-8 position, respectively, and they showed low ED50 values in the present study. Considering the present results and these reports, the strong photochemical clastogenic potential of quinolones having a halogen at the C-8 position seems to be due to carbene generation at that position during photodegradation. Rosen et al. reported that Bayer Y3118, but not MFLX, produced dose-dependent increases in 8-oxo-7,8-dihydro-2 -deoxyguanosine in cultured liver epithelial cells after UVA irradiation [25]. MFLX contains a methoxy group substituent at the C-8 position instead of the chlorine of Bayer Y3118. In addition, Bayer Y3118 increased the mutation frequency on the HGPRT locus in Chinese hamster V79 cell line after UVA irradiation, whereas no photochemical mutagenicity was observed with MFLX [4]. In our study, the presence of a methoxy group substituent at the C-8 position in DK-507k protected against light-induced chromosomal aberrations, compared with chlorine in the potent STFX. The results obtained in the present study reproduced previous findings. Substitution from chlorine to methoxy at the C-8 position is reported to be effective for reducing the genotoxicity under non-light irradiation [1]. This is supported by the present results that STFX, but not DK-507k, induced chromosomal aberrations without cytotoxicity at 500 and 1000 g/ml in the absence of light irradiation. Therefore, a methoxy group substituent at the C-8 position may reduce not only photochemical clastogenicity but also the clastogenic potential of the quinolone antibacterial agents.
Acknowledgements We thank Dr. N. Tanaka (Food and Drug Safety Center, Kanagawa, Japan) for helpful advice about the light source and experimental conditions. References [1] J.M. Domagala, Structure-activity and structure-side-effect relationships for the quinolone antibacterials, J. Antimicrob. Chemother. 33 (1994) 685–706.
S. Itoh et al. / Mutation Research 517 (2002) 113–121 [2] J. Ferguson, Fluoroquinolone photosensitization: a review of clinical and laboratory studies, Photochem. Photobiol. 62 (1995) 954–958. [3] A.A. Chételat, S. Albertini, E. Gocke, The photomutagenicity of fluoroquinolones in tests for gene mutation, chromosomal aberration, gene conversion and DNA breakage (Comet assay), Mutagenesis 11 (1996) 497–504. [4] A.M. Jeffrey, L. Shao, S.Y. Brendler-Schwaab, G. Schlüter, G.M. Williams, Photochemical mutagenicity of phototoxic and photochemically carcinogenic fluoroquinolones in comparison with the photostable moxifloxacin, Arch. Toxicol. 74 (2000) 555–559. [5] B. Kersten, J. Zhang, S.Y. Brendler-Schwaab, P. Kasper, L. Müller, The application of the micronucleus test in Chinese hamster V79 cells to detect drug-induced photogenotoxicity, Mutat. Res. 445 (1999) 55–71. [6] S.J. Bulera, J.C. Theiss, T.A. Festerling, F.A. de la Iglesia, In vitro photogenotoxic activity of clinafloxacin: a paradigm predicting photocarcinogenicity, Toxicol. Appl. Pharmacol. 156 (1999) 222–230. [7] H.J. Reavy, N.J. Traynor, N.K. Gibbs, Photogenotoxicity of skin phototumorgenic fluoroquinolone antibiotics detected using the comet assay, Photochem. Photobiol. 66 (1997) 368– 373. [8] L. Marrot, J.P. Belaidi, C. Chaubo, J.R. Meunier, P. Perez, C. Agapakis-Causse, Fluoroquinolones as chemical tools to define a strategy for photogenotoxicity in vitro assessment, Toxicol. In Vitro 15 (2001) 131–142. [9] G. Klecak, F. Urbach, H. Urwyler, Fluoroquinolone antibacterials enhance UVA-induced skin tumors, J. Photochem. Photobiol. B: Biol. 37 (1997) 174–181. [10] M. Mäkinen, P.D. Forbes, F. Stenbäck, Quinolone antibacterials: a new class of photochemical carcinogens, J. Photochem. Photobiol. B: Biol. 37 (1997) 182–187. [11] N. Umezawa, K. Arakane, A. Ryu, S. Mashiko, Participation of reactive oxygen species in phototoxicity induced by quinolone antibacterial agents, Arch. Biochem. Biophys. 342 (1997) 275–281. [12] E. Gocke, S. Albertini, A.A. Chételat, S. Kirchner, W. Muster, The photomutagenicity of fluoroquinolones and other drugs, Toxicol. Lett. 102/103 (1998) 375–381. [13] L. Müller, P. Kasper, B. Kersten, J. Zhang, Photochemical genotoxicity and photochemical carcinogenesis—two side of a coin? Toxicol. Lett. 102/103 (1998) 383–387. [14] J.E. Rosen, A.K. Prahalad, G. Schlüter, D. Chen, G.M. Williams, Quinolone antibiotic photodynamic production of 8-oxo-7,8-dihydro-2 -deoxyguanosine in cultured liver epithelial cells, Photochem. Photobiol. 65 (1997) 990–996.
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[15] K. Nakatani, S. Maekawa, K. Tanabe, I. Saito, ␣-Diazo ketones as photochemical DNA cleavers: a mimic for the radical generating system of neocarzinostatin chromophore, J. Am. Chem. Soc. 117 (1995) 10635–10644. [16] L. Mart´ınez, C.F. Chignell, Photocleavage of DNA by the fluoroquinolone antibacterials, J. Photochem. Photobiol. B: Biol. 45 (1998) 51–59. [17] L.J. Mart´ınez, G. Li, C.F. Chignell, Photogeneration of fluoride by the fluoroquinolone antibacterial agents lomefloxacin and fleroxacin, Photochem. Photobiol. 65 (1997) 599–602. [18] L.J. Mart´ınez, R.H. Sik, C.F. Chignell, Fluoroquinolone antimicrobials: singlet oxygen, superoxide and phototoxicity, Photochem. Photobiol. 67 (1998) 399–403. [19] E. Gocke, L. Müller, P.J. Guzzie, S. Brendler-Schwaab, S. Bulera, C.F. Chignell, L.M. Henderson, A. Jacobs, H. Murli, R.D. Snyder, N. Tanaka, Considerations on photochemical genotoxicity: report of the international workshop on genotoxicity test procedures working group, Environ. Mol. Mutagen. 35 (2000) 173–184. [20] H. Spielmann, M. Balls, M. Brand, B. Döring, H.G. Holzhütter, S. Kalweit, G. Klecak, H.L. Eplattenier, M. Liebsch, W.W. Lovell, T. Maurer, F. Moldenhauer, L. Moore, W.J.W. Pape, U. Pfanenbecker, J. Potthast, O. De Silva, W. Steiling, A. Willshaw, EEC/COLIPA project on in vitro phototoxicity testing: first results obtained with a BALB/C 3T3 cell phototoxicity assay, Toxicol. In Vitro 8 (1994) 793– 796. [21] Y. Nakagawa, S. Wakuri, K. Sakamoto, N. Tanaka, The photogenotoxicity of titanium dioxide particles, Mutat. Res. 394 (1997) 125–132. [22] M. Ishidate Jr. (Ed.), Data Book of Chromosomal Aberration Test in vitro, Revised Edition, Elsevier, Amsterdam, 1988. [23] A. Mastuoka, N. Yamazaki, T. Suzuki, M. Hayashi, T. Sofuni, Evaluation of the micronucleus test using a Chinese hamster cell line as an alternative to the conventional in vitro chromosomal aberration test, Mutat. Res. 272 (1992) 223– 236. [24] B. Miller, F. Pötter-Locher, A. Seelbach, H. Stopper, D. Utesch, S. Madle, Evaluation of the in vitro micronucleus test as an alternative to the in vitro chromosomal aberration assay: position of the GUM working group on the in vitro micronucleus test, Mutat. Res. 410 (1998) 81–116. [25] J.E. Rosen, D. Chen, A.K. Prahalad, T.E. Spratt, G. Schluter, G.M. Williams, A fluoroquinolone antibiotic with a methoxy group at the 8 position yields reduced generation of 8-oxo-7,8-dihydro-2 -deoxyguanosine after ultraviolet—A irradiation, Toxicol. Appl. Pharmacol. 145 (1997) 381–387.
In vitro photochemical clastogenicity of quinolone antibacterial agents studied by a chromosomal aberration test with light irradiation Satoru Itoh a,∗ , Shiho Nakayama a , Hiroyasu Shimada b a b
Drug Safety Research Laboratory, Daiichi Pharmaceutical Co. Ltd., 16-13 Kita-Kasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan Drug Safety Administration Department, Daiichi Pharmaceutical Co. Ltd., 16-1 Kyobashi 2-Chome, Chuo-ku, Tokyo 104-8369, Japan Received 8 August 2001; received in revised form 4 March 2002; accepted 7 March 2002
Abstract The photochemical clastogenic potential of 12 quinolone antibacterial agents with or without light irradiation was assessed by an in vitro chromosomal aberration test using cultured CHL cells. Exposure to all test compounds, except for DK-507k, increased the incidence of cells with structural aberrations excluding gap (TA) following light irradiation. Test compounds used in the present study under light irradiation were divided into three groups based on their ED50 values, doses inducing chromosomal aberrations in 50% of cells. The first group with ED50 values below 30 g/ml includes sparfloxacin (SPFX), clinafloxacin (CLFX), gemifloxacin (GMFX), lomefloxacin (LFLX), sitafloxacin (STFX), grepafloxacin (GPFX) and fleroxacin (FLRX); the second group with ED50 values of 100 g/ml, enoxacin (ENX) and levofloxacin (LVFX); the third group with little or no potency, moxifloxacin (MFLX), trovafloxacin (TVFX) and DK-507k. The photochemical clastogenicity of these compounds correlates well with their reported in vivo phototoxic potentials. In the chemical structure and clastogenicity relationships, substitution of a methoxy group at the C-8 position in the quinolone nucleus was confirmed to reduce not only photochemical clastogenicity, but also the clastogenic potential of quinolone antibacterial agents. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Photochemical clastogenicity; Light irradiation; Chromosomal aberration test; Quinolone antibacterial agent
1. Introduction Quinolone antibacterial agents (quinolones) have been widely used in numerous diseases because of their broad spectrum activity against both gram-negative and gram-positive pathogens. One of the major adverse reactions due to quinolones is photosensitization, which has been reported in experimental and clinical studies [1,2]. Especially, concern ∗ Corresponding author. Tel.: +81-3-5696-8294; fax: +81-3-5696-8335. E-mail address: [email protected] (S. Itoh).
has been raised about photogenotoxicity induced by quinolones with ultraviolet A (UVA) irradiation. Namely, certain quinolones have been reported to be associated with irradiation induced gene mutation in bacteria [3] and cultured mammalian cells [4], chromosomal aberration or micronucleus in cultured mammalian cells [3,5,6], and DNA strand breakage in vitro and in vivo comet assays [3,7,8]. In photocarcinogenicity studies using Skh-1 hairless mice with UVA, lomefloxacin (LFLX) has been reported to cause cystic squamous cell carcinoma in the majority of animals treated [9,10]. The cause of photogenotoxicity induced by quinolones is thought to be the formation
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of reactive oxygen species, such as superoxide anion, hydrogen peroxide, hydroxy radical and singlet oxygen [11–14]. In chemical structure, quinolones, such as LFLX, fleroxacin (FLRX) and clinafloxacin (CLFX), possessing a halogen (fluorine or chlorine) at the C-8 position are known to generate a highly reactive carbene that causes strand breaks in DNA [15] at that position during photodegradation [6,7,16,18] and contributes to their phototoxicity and photogenotoxicity. It is, thus, important to elucidate the relationship between photogenotoxicity and chemical structure. To the best of our knowledge, there is no report dealing with comparative photogenotoxicity of more than ten quinolones under the same experimental conditions. In the present study, the photochemical clastogenicity of 12 quinolones was investigated based on their potentials for enhancing chromosomal aberrations. Accordingly, photochemical clastogenic activity was assessed by an in vitro chromosomal aberration test using Chinese hamster lung (CHL) cells in both the presence and absence of a sun-light simulator having a spectrum almost identical to that of solar irradiation.
8-Methoxypsoralen (8-MOP, CAS no. 298-81-7) as a positive control was purchased from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan) and dissolved in dimethyl sulfoxide. The solvent for all test compounds, 0.1 N NaOH, was used as the negative control. The present study consists of four experiments, and negative and positive controls were always set in each experiment.
2. Materials and methods
The same concentrations were used for all test compounds: 1, 3, 10, 30, 100 and 300 g/ml for light irradiation and 10, 30, 100, 300, 500 and 1000 g/ml for non-light irradiation. These dose levels were determined from the results of preliminary cytotoxicity and genotoxicity tests with STFX and LFLX. In the case of 8-MOP, a concentration of 0.02 g/ml was set for both light irradiation and non-irradiation [19]. One plate was used in each dose level.
2.1. Test chemicals Lomefloxacin (LFLX, CAS no. 98079-51-7) and enoxacin (ENX, CAS no. 74011-58-8) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Levofloxacin (LVFX, CAS no. 100986-85-4), clinafloxacin (CLFX, CAS no. 105956-97-6), fleroxacin (FLRX, CAS no. 79660-72-3), sparfloxacin (SPFX, CAS no. 110871-86-8), grepafloxacin (GPFX, CAS no. 119914-60-2), trovafloxacin (TVFX, CAS no. 147059-72-1), moxifloxacin (MFLX), gemifloxacin (GMFX), sitafloxacin (STFX) and DK-507k were synthesized in Daiichi Pharmaceutical Co. Ltd. (Tokyo, Japan). The chemical structures of these test compounds are shown in Fig. 1. Immediately prior to use, all test chemicals were dissolved or suspended in 0.1 N NaOH. The concentrations of 500 and 1000 g/ml in SPFX, STFX, MFLX, FLRX or CLFX treated group, and 300, 500 and 1000 g/ml in GPFX, LFLX or DK-507k treated group were suspended in 0.1 N NaOH.
2.2. Cell line and culture conditions CHL cells were purchased from Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan). Eagle’s minimum essential medium (MEM, Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 15% fetal bovine serum (FBS, Cancera International Inc., Canada) was used for cell culture. The cells were cultured in plastic petri dishes (Falcon, Becton Dickinson Labware, NJ, USA), 60 mm in diameter, in a humidified incubator with an atmosphere of 5% CO2 to 95% air at 37 ◦ C. 2.3. Dose levels
2.4. Light irradiation Light source and treatment procedures used in the present study were the same as those in the European Industry Association’s project on the neutral red assay with BALB/c 3T3 cells for validating in vitro methods to detect photocytotoxicity [20]. Namely, light irradiation was performed by a sun-light simulator SOL500 (Dr. Hönle, München, Germany) installed with an H1 filter (Dr. Hönle; 50% transmission of 335 nm wavelength) at room temperature. The irradiation spectrum was almost identical to that of solar irradiation, and the ratio of UVB to UVA was about 1:25 [21]. After
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pretreatment of CHL cells with each test compound in Earle’s balanced salt solution (EBSS; Gibco BRL) for 60 min in an incubator, culture dishes with lids were placed under the sun-light simulator and then irradiated at 0.8–1.0 mW/cm2 of UVA for 50 min (Irradiation dose of UVA, 2.4–3.0 J/cm2 , maximum dose not induce chromosomal aberrations). Emitted energy was measured by a simple UVA meter (type no. 37,
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Dr. Hönle) immediately before the light irradiation. The visible light intensity measured by a lux meter 3423 (Hioki E. E., Nagano, Japan) was 25,000–27,000 lx. For the non-irradiation group, on the other hand, the dishes were kept in the incubator for 50 min while light irradiation was performed on the light irradiation group. After light irradiation, the cells from all test groups, including the non-light
Fig. 1. Chemical structures of the quinolone antibacterial agents used in the present study.
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Fig. 1. (Continued ).
irradiation group, were washed with phosphate buffered saline followed by fresh 15% FBS-MEM, and then the cells were cultured for 22 h. 2.5. Specimen preparation At the end of the incubation period, 24 h after the commencement of treatment, chromosomal preparations were made. In order to collect the cells in metaphase, colcemid (GIBCO BRL, Life Technologies Inc., Rockville, MD, USA) at a final concentration of 0.05 g/ml was added to the medium 1.5 h prior to harvesting. After that, the cells resuspended in 1% sodium citrate were incubated for 15 min at room temperature and fixed by Carnoy’s solution (methanol:glacial acetic acid = 3:1, v/v). The fixative procedure was repeated more than three times. Chromosomal specimens were made by an air-dry technique and stained with Giemsa (Merck, Darmstadt, Germany). Three slides were prepared per plate.
(type and frequency) were scored. When the number of analyzable metaphase cells was less than 100 cells in three slides which corresponded to approximately 0.5% in mitotic index, cytotoxicity was judged to have occurred and we did not score it. Structural chromosomal aberrations were classified as gap, break, exchange, fragmentation, multiple-type aberrations and others. Any cell having chromosomal aberration(s) was recorded as an aberrant cell. In the present paper, chromosomal aberrations induced by the test compound is expressed as the incidence of cells with structural aberrations excluding gap (TA). The ED50 values (a dose inducing chromosomal aberrations in 50% cells, which was calculated from two values which are higher and lower than 50% induction points of chromosomal aberrations on figures) of the respective test compounds with or without light irradiation are presented. 2.7. Judgement for clastogenicity of test compounds
2.6. Observations One hundred well-spread metaphase cells per plate were analyzed, and the chromosomal aberrations
According to Ishidate’s criteria [22], the test compound was judged to be positive when 10% or more TA were observed.
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3. Results 3.1. Negative and positive controls TA values of the vehicle as the negative control were between 0 and 2% regardless of the presence or absence of light irradiation. The TA values of 8-MOP as the positive control in the presence and absence of light irradiation were 63–87 and 0–1%, respectively, suggesting that the test systems were properly functioning in the present study. 3.2. Test compounds The incidence of chromosomal aberrations (TA) induced by the 12 quinolones with or without light irradiation is shown in Fig. 2. CLFX, STFX, GPFX and GMFX increased TA, regardless of light irradiation compared with the negative control, for which increases were markedly greater with light irradiation Table 1 The relationship between ED50 and chemical structure of each test compound Compound
SPFX CLFX GMFX LFLX STFX GPFX FLRX ENX LVFX MFLX TVFX DK-507k
ED50 (g/ml) Light irradiation (+)
Light irradiation (−)
4 6 11 14 16 22 22 100 100 N.O.b N.O. –c
– 133 300 – N.O. N.O. – – – – – –
R5 a
X8 a
NH2 H H H H CH3 H H H H H H
CF CCl N CF CCl CH CF N COR COCH3 N COCH3
a X and R represent the following positions of quinolone 8 5 antibacterial agents.
b ED 50 value was not obtained since the TA value did not reach to 50% of the cells. c No chromosomal aberration was observed.
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than without light irradiation. ENX, LVFX, TVFX, SPFX, LFLX, MFLX and FLRX induced chromosomal aberrations only under light irradiation; whereas DK-507k induced no chromosomal aberration with or without light irradiation. The ED50 values of each test compound are indicated in Table 1. With light irradiation, the ED50 was <30 g/ml for SPFX, CLFX, GMFX, LFLX, STFX, GPFX and FLRX, and approximately 100 g/ml for ENX and LVFX. Without irradiation, CLFX and GMFX give ED50 values of 133 and 300 g/ml, respectively. MFLX and TVFX with light irradiation or STFX and GPFX without light irradiation have no ED50 value, because their TA values did not reach 50%.
4. Discussion The photochemical clastogenic potential of 12 quinolone antibacterial agents with or without light irradiation was assessed by an in vitro chromosomal aberration test using cultured CHL cells. Most of the test compounds caused increased TA with light irradiation, and their potential rank order from highest to lowest was shown to be SPFX > CLFX > GMFX > LFLX > STFX > GPFX = FLRX > ENX = LVFX > MFLX > TVFX, and DK-507k had no potential. It has been reported that several quinolones are classified as photogenotoxins by an in vitro chromosomal aberration assay with Chinese hamster V79 cells [3]. In this report, the clastogenic potential of LFLX was stated to be stronger than that of FLRX under UVA irradiation, which is in good agreement with our data. In the same way, the clastogenic potential of CLFX was reported to be stronger than that of LFLX in a chromosomal aberration test with UVA irradiation in CHO cells [6]; and this finding is also consistent with the present results. Additionally, the in vitro micronucleus test has also been used for investigating photochemical clastogenicity of quinolones [5]. The induction of micronucleated cells by GPFX was stronger in Chinese hamster V79 cells under UVA/UVB irradiation than that by LFLX. The reported results differed from our findings although the sensitivity to clastogens in the in vitro micronucleus test is almost identical to that in the chromosomal aberration test [23,24]. In summarizing the aforemen-
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Fig. 2. Incidence of chromosomal aberration excluding gap (TA) induced by each test compound. Closed circles and closed squares represent the incidence of TA in the presence and absence of light irradiation, respectively. The concentrations of the test compound used were 1, 3, 10, 30, 100 and 300 g/ml with light irradiation, and 10, 30, 100, 300, 500 and 1000 g/ml without light irradiation. Open circles and open squares represent that chromosome analysis could not be performed due to cytotoxicity of the test compound in the presence and absence of light irradiation, respectively. TA values of the negative and positive controls were described in Section 3.
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Fig. 2. (Continued ).
tioned results, although there were some differences in the experimental conditions including the light source, irradiation dose and cell line used, the results presented here seem to be similar to those of the other studies.
The 12 test compounds used in the present study were divided into three groups based on their ED50 values under light irradiation: the first, ED50 values <30 g/ml, included SPFX, CLFX, GMFX, LFLX,
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STFX, GPFX and FLRX; the second, ED50 values of 100 g/ml, ENX and LVFX; the third of little or no potency, MFLX, TVFX and DK-507k. The relationships between ED50 values and chemical structures of the quinolones used in the present study are also shown in Table 1. The chemical structure in vivo phototoxicity relationships are well summarized for quinolone antibacterial agents [1]. Namely, the in vivo phototoxic potential is mostly determined by the X8 substitution, and the highest phototoxicity is gained when the X8 is substituted by a halogen (fluorine or chlorine), and the least by COCH3 . The in vivo phototoxic potential of the X8 substitution is CF, CCl, N, CH, COCH3 in decreasing order. In addition, the R5 substitution also influences in vivo phototoxicity with an intensity order of CH3 > N > NH2 . In agreement with these, SPFX, CLFX, LFLX, STFX and FLRX having a halogen at the X8 position showed strong potentials for chromosomal aberration induction in the present study. In contrast, GMFX and GPFX, remainders of the first group mentioned earlier, do not have a halogen at the X8 position, instead, GMFX has N at the X8 position and also GPFX has CH3 at the R5 position. ENX and LVFX were classified as members of the second group quinolones with N and COR at the X8 position, respectively. In the third group, MFLX and DK-507k, but not TVFX, have COCH3 (the least in vivo phototoxicity) at the X8 position. TVFX has N at the X8 position similar to GMFX and ENX, which each have quite different potentials of causing chromosomal aberration induction under light irradiation. There may be other factors that affect their various potencies. In brief, the 12 quinolones used in the present study were classified into three groups based on their photochemical clastogenic potentials. In each group, the structure in the X8 and R5 substitutions resembled each other in principle and the photochemical clastogenic potential of these substitutions were almost identical to the data reported by Domaga [1]. It therefore appears that there is a good correlation between in vivo phototoxicity and in vitro photochemical clastogenicity in quinolones. SPFX, CLFX, LFLX, STFX and FLRX, which are classified in the first group, have a fluorine or chlorine at the C-8 position. LFLX and FLRX have been reported to generate a highly reactive carbene species which is known to cause strand breaks in DNA [15], at the C-8 position during photodegra-
dation [16–18]. It has been proposed that CLFX, chlorinated at the C-8 position, could follow a process similar to those two compounds [6]. SPFX and STFX have fluorine and chlorine at the C-8 position, respectively, and they showed low ED50 values in the present study. Considering the present results and these reports, the strong photochemical clastogenic potential of quinolones having a halogen at the C-8 position seems to be due to carbene generation at that position during photodegradation. Rosen et al. reported that Bayer Y3118, but not MFLX, produced dose-dependent increases in 8-oxo-7,8-dihydro-2 -deoxyguanosine in cultured liver epithelial cells after UVA irradiation [25]. MFLX contains a methoxy group substituent at the C-8 position instead of the chlorine of Bayer Y3118. In addition, Bayer Y3118 increased the mutation frequency on the HGPRT locus in Chinese hamster V79 cell line after UVA irradiation, whereas no photochemical mutagenicity was observed with MFLX [4]. In our study, the presence of a methoxy group substituent at the C-8 position in DK-507k protected against light-induced chromosomal aberrations, compared with chlorine in the potent STFX. The results obtained in the present study reproduced previous findings. Substitution from chlorine to methoxy at the C-8 position is reported to be effective for reducing the genotoxicity under non-light irradiation [1]. This is supported by the present results that STFX, but not DK-507k, induced chromosomal aberrations without cytotoxicity at 500 and 1000 g/ml in the absence of light irradiation. Therefore, a methoxy group substituent at the C-8 position may reduce not only photochemical clastogenicity but also the clastogenic potential of the quinolone antibacterial agents.
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