naphthyridone antibacterials

Mutation Research, 298 (1993) 227-236

227

© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1218/93/$06.00

MUTGEN 01848

High capacity in vitro micronucleus assay for assessment of chromosome damage: results with quinolone/naphthyridone antibacterials V. Ciaravino a M.J. Suto b and J.C. Theiss

a

a Department of Pathology and Experimental Toxicology and b Department of Chemistry, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, MI 48105, USA

(Received 8 June 1992) (Revision received7 August 1992) (Accepted 13 August 1992)

Keywords: Micronucleusassay, in vitro; Chinese hamster V79 cells; Quinolones; Naphthyridones; Chromosome damage

Summary A high capacity in vitro micronucleus assay was developed to evaluate the ability of selected 6-fluorinated quinolone and naphthyridone antibacterial compounds to induce micronuclei (MN) in vitro in V79 Chinese hamster lung cells. Log-phase cells in six-well cluster dishes were exposed for 3 h in the absence of $9 to 34 compounds. After treatment, cells were refed with media containing cytochalasin B, incubated for 16 h, and harvested for cell-cycle kinetics (CCK) and MN analyses. The quinolones tested were grouped according to the substituent at the 8-position. All 4 compounds having a halogen substitution at position 8, five of the six 8-trifluoromethyl quinolones, and all eight 8-methoxy-substituted compounds induced a significant increase in MN. Only 5 of the 10 naphthyridone compounds tested, having a variety of substituents at the 7-position, were inducers of MN and the overall magnitude of the response was less than with the quinolones. The minimum clastogenic concentration for the quinolones ranged from 4 to 4 0 0 / z g / m l and for the naphthyridones this range was from 22.5 to 1 0 0 / z g / m l . In the groups examined, naphthyridone compounds were less likely than quinolones to induce in vitro MN, particularly w h e n ' t h e substituent at the 7-position in the naphthyridone contains some bulk (methyl groups) around the amine side-chain. Most of the quinolones tested induced MN, irrespective of the substituents at positions 7 or 8.

One approach to determining the genotoxicity of potential therapeutic drug candidates is to test them in a battery of validated mammalian in vitro and in vivo gene-tox assays (e.g. in vitro and Correspondence: Dr. Victor Ciaravino, Department of Pathology and Experimental Toxicology,Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, MI 48105, USA.

bone-marrow chromosome aberration, in vitro mutation at the H G P R T or T K locus, in vitro and mouse micronucleus, etc,). However, this approach is very time-consuming and costly when testing of drug candidates is done one at a time. Another approach, the one taken in this study, was to evaluate a group of compounds in a single assay and identify suitable candidates for further development.

228 TABLE 1 SUMMARY OF MULTINUCLEATED AND MICRONUCLEATED BINUCLEATED CELLS Compound numbers

Structure

Solvent control Positive control 1 NH2 0

F

~

COzH

Group I, 8-halogenated substituted compound 2 0

F,v~COzH I-

II

NH20 F - , y ~ C Oz H '

N3c.

% MUN cells

% MNBN cells

10% 0,1 N NaOH 10% 0.1 N NaOH

79.50 88.00

1.30 0.80

6 12 24

90.00 90.00 84.00

2.25 6.25 * 20.75 *

6 12 24

64.50 73.50 63.00

2.25 8.75 * 30.25 *

25 50 100

87.00 86.00 57.00

1.75 7.75 * 23.25 *

II

3 I

Concentration (/xg/ml)

l

l

F

2

93.50

2.50

4 6

90.00 62.00

20.25 * 40.00 *

7 14 28

92.50 65.00 84.00

1.00 41.75 * 31.50 *

2 4 6

92.00 93.50 87.50

1.25 9.50 * 11.00 *

100 200 400

92.00 89.50 88.50

1.50 2.75 4.50 *

/,,

4 NHz 0 F ~ , , I ~

C0ZH

1.111.1 5 I

I

0

F ~ C 0 z H

Group II, 8-trifluoromethyl substituted compounds 6 0 F~~__,,ytC 02 H

1.1111

229 TABLE 1 (continued) Compound numbers

0 F~COzH

7

NHz

I

I[

% MNBN cells

25 50 100

54.50 87.00 74.00

1.50 7.25 * 39.00 *

50 100 200

90.00 91.00 50.00

3.25 8.75 * 47.00 *

250 500 1000

86.00 78.00 3.00

2.25 2.75 0.00

75 150 200

82.00 79.50 79.50

5.25 * 5.50 * 21.25 *

70 140 280

83.50 76.50 ,60.00

3.75 17.75 * 50.25 *

20 40 80

91.00 89.50 60.00

2.75 10.25 * 43.25 *

12.5 25 50

87.50 90.50 70.00

2.25 7.75 * 39.00 *

I[

8

F ~ EH t N~

% MUN cells

Concentration (/~g/ml)

Structure

0

COzH

CF3 ~

9

F ~

0

cOzH

iPrHN~N 7" X~ CF3/kk I0 0 EtHN F ~ cOzH 11

0

MeHN F~CO~H KX~

CF3 ~x

Group III, 8-methoxy substituted compounds 12 0

F~COzH

13

F ~

0

HzN'~'~N ~OMe~

COzH

230 TABLE 1 (continued) Compound numbers

Structure

14

0

15

0

16

HzN ~ i ~ 17

C02H

NH 2

0

18

0

%

%

MUN cells

MNBN cells

60 120 240

76.50 71.00 68.00

7.25 * 19.25 * 43,25 *

35 70 140

80.50 64,00 4.50

34,00 * 49.25 * 68,00 *

75 150 300

90.00 84.50 84.00

3.75 2.75 5.50 *

90 180 360

79.00 56.00 31.00

26.25 * 57.50 * 63.25 *

75 150 300

68.50 20.00 5.50

36.50 * 85.50 * 68.89 *

75 150 300

66.00 40,50 18.50

30.25 * 75,50 * 79.05 *

25 50 100

82.50 91.00 86.50

2.00 1.00 5.00 *

Concentration (~g/mD

ER~20M~e~ H tN CO~H 19

0

ER~20Me~~ H tN CO~H GOroup IV,napbtbyddones 2 HzN ~~COzH 0

231 TABLE 1 (continued) Compound numbers

Structure

21 0 I

F

Concentration (/zg/ml)

% MUN cells

% MNBN cells

12.5 50 200

92.00 91.00 38.50

1.75 6.50 * 1.50

50 75 100 150

82.50 64.00 87.00 49.00

5.25 * 6.00 * 5.25 * 1.50

5.6 22.5 90

96.00 90.50 86.00

2.00 9.75 * 36.75 *

12.5 50 200

93.00 87.50 53.00

0.25 3.25 1.00

50 75 100 150

88.00 86.50 86.50 65.50

3.50 1.25 2.00 2.00

12.5 50 200

93.00 92.50 29.50

1.25 2.50 1.00

50 75 100 150

84.00 85.00 76.00 63.00

3.25 4.00 2.25 0.75

12.5 50 200

87.50 90.00 41.50

2.00 1.00 0.50

I

~

NHz I II II ~'~N ~N ":"Z

C0zH

22

0 NHz F'~F~F~J~COzH

23 0 F~COzH HzN

24 0 MeHN F ~ COzH L~N ~ N"~"Z 25 0

MeHN F ~ c ~ ~COzH

232 TABLE 1 (continued) Compound numbers

Structure

26 0

F~COzH EtHN

I

II

0

I

F-~,~',,~e/~--.r~C0zH MezN

I

II

0 F., A ~ , ~ E C O 2 H I

% MNBN cells

60 120 240

92.00 76.50 36.50

1.75 2.25 2.00

12.5 50 200

81.00 85.00 21.00

2.25 1.25 3.00

12.5 50 200

83.50 77.00 86.50

2.50 1.75 3.50

12.5 50 200

84.50 89.50 23.50

2.50 10.50 * 14.50 *

115 230 460

90.50 86.50 93.00

2.50 0.75 1.25

100 200

89.50 89.00

1.75 3.25

400

84.00

2.50

II

28

NHz

% MUN cells

II

27 I

Concentration (/~ g/ml)

II

II

F 29 0

H

F , ~ C O z H

±. A[ ~ MeHN~N" ~ ~'N" Group V, quinolones with miscellaneous side chains 30

Me

F

~ L

CH3

0

C0zH II

II

y F

31

Me

S

0

F~~I*~C

H3C, , , , / ~

.j

HN

N" " ~ " ~ ' "

¢

OzH

N"

3.00

5.00

9.50

7.50 *

80o

F

g

400 600

150

2667.

233 TABLE 1 (continued) Compound numbers

Structure

Concentration (/xg/ml)

32 Me

NH2

0

F~C02 .L-._ .~

H

% MUN cells

% MNBN cells

160

2.50 3.50 0.00

9.09 * 24.00 * 0.00

95 190 380

89.00 90.00 69.00

3.00 2.50 0.75

35

85.00 63.50 3.50

2.00 3.25 20.00

40 80

F 33 Me

0

F ~ C O 2 H

cg3 34

Me2N

o

F~~.~CO2H I II II

140 70

MUN, multinucleated; MNBN, micronucleated binucleated; * Denotes significance p < 0.01.

Micronuclei are an indirect indicator of chromosomal damage formed during cell division. They originate from either acentric fragments, whole chromatids, or chromosomes which are not incorporated into daughter nuclei at the time of division and require one cell division to be expressed. When studied in an in vitro system, the micronucleus assay has been useful in assessing potential damage to Syrian hamster embryo fibroblasts (Schmuck et al., 1988; Schiffmann and De Boni, 1991), human lymphocytes (Countryman and Heddle, 1976; Hogstedt, 1984; Prosser et al., 1988), and Chinese hamster cells (Wakata and Sasaki, 1987; Krishna et al., 1989). The assay has also been used to determine the genotoxicity of various drugs (Dunn et al., 1987; Ujh~zy et al., 1988; Xing et al., 1989). With the introduction of the cytokinesis-block method in human lymphocytes for the analysis of micronuclei in culture (Fenech and Morley, 1985),

it was possible to conduct an analysis of micronuclei which was restricted to binucleated cells which have undergone one division, which is optimal for the expression of micronuclei (Wakata and Sasaki, 1987). This procedure also is useful to assess the effect treatment has upon the rate at which cells traverse through the cell cycle at the same time providing information on clastogenicity. In the present study, we have used the cytokinesis-block method in V79 Chinese hamster lung cells (Krishna et al., 1989) to evaluate a variety of quinolones/naphthyridones for cell-cycle effects as well as micronucleus induction. Quinolone drugs have previously been shown to readily induce micronuclei in this cell type in the absence of $9 (Kropko et al., 1991). A method was developed whereby cells were treated in six-well cluster dishes with 33 selected 6-fluorinated quinolone and naphthyridone antibacterial compounds. This

234 methodology replaced costly, time-consuming assays with a rapid screening assay which provided valuable information for further development of several of the anti bacterial compounds.

culture of approximately 11 h were used in the study. These cells maintain a high viability upon subculture and are free of mycoplasma contamination. Stock cultures are maintained in liquid nitrogen and periodically spot checked for mycoplasma contamination by fluorescent microscopy (Chen, 1977). V79 ceils were grown exponentially in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate (Gibco), and 50 /zg/ml gentamicin (M.A. Bioproducts). Cells were maintained in tissue-culture flasks at 37°C in a humidified atmosphere containing 5% CO 2 and were subcultured by treatment with trypsin-EDTA solution (Gibco) in phosphate-buffered saline (PBS).

continued for an additional 3 h. The total volume of the culture medium plus treatment drug solution was 5 ml per well. The drug concentrations were selected based on previously conducted clonogenic cytotoxicity assays and the highest concentration of each drug reduced the survival of the ceils by approximately 80%. Initially, 3 concentrations for each of 20 drugs were tested. A second trial was conducted utilizing additional concentrations of 4 of the compounds as well as testing an additional 13 compounds. At the end of the treatment, cells were rinsed with drug-free medium and refed with complete medium containing 3 /.~g/ml cytochalasin B (CYB). Cultures were incubated for an additional 16 h posttreatment. The cytokinesis-blocking agent CYB (Sigma Chemical Company) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 2 mg/ml, stored at -80°C, and diluted with PBS immediately prior to use. A concentration of 3/zg C Y B / ml culture medium was used in the experiments based on earlier reports (Fenech and Morley, 1985; Wakata and Sasaki, 1987; Krishna et al., 1989). The final concentrations of DMSO in culture was approximately 0.3%.

Test substances

Harvesting, slide preparation, and staining

The compounds and their structures used in the study are shown in Table 1. The solvent for all the test substances was 0.1 N NaOH and this solution at 10% in the media served as the negative control. The test substance, Compound 1, produced a well-defined response in micronucleus induction in previous experiments conducted in our laboratory and served as the positive control for this study.

At 16 h posttreatment, the cells were dislodged with trypsin-EDTA at 37°C and centrifuged at 100 x g for 6 min. The supernatant was removed and the pellet resuspended in the remaining solution. The cells were treated with 3 ml hypotonic solution (0.075 M KCI, dropwise) at 37°C for 3-5 rain and recentrifuged. The pellet was resuspended and two drops of cell suspension carefully spread on prelabeled dry slides angled at 45 °. The slides were then dipped in absolute methanol once and air-dried overnight. The time between hypotonic treatment and fixing is very critical, as cells continue to swell and may burst if cells are not fixed appropriately. The air-dried slides were stained with DiffQuik ® stain (American Scientific Products, Broadview Heights, OH), coverslipped, and scored blindly under 1000 x magnification. In each treatment group, the number of mononucleated, binucleated and polynucleated cells per 200 cells (100 cells/scorer)were quantitated for cell-

Materials and methods

Cell line V79 (lung, male Chinese hamster, Cricetulus griseus) cells with an average cell-cycle time in

Assay procedure V79 cells from an exponentially growing culture were planted in six-well cluster dishes at approximately 5 x 105 cells per well (approximately 9.5-cm 2 growth area per well) and incubated at 37°C for 18-24 h. Each control and treatment group consisted of single wells. At the time of treatment, cultures were refed with fresh medium, treated at the various drug concentrations as indicated in Table 1, and incubation was

235

cycle kinetic analysis and 400 binucleated cells (200 cells/scorer) were analyzed for the presence of micronuclei. The criteria for scoring micronuclei were similar to those of Countryman and Heddle (1976) with slight modification. The diameter of the micronuclei must be no larger than one-third the main nuclei (Roberts et al., 1986). They must be non-refractile, thus excluding small stain particles. The color must be the same as or brighter than the main nuclei. Micronuclei must be located within the cytoplasm but not in contact with the main nuclei. If there is any overlapping of the two main nuclei in a binucleated cell, it is not scored.

Statistical analysis Data for CCK (% MUN cells) were recorded but not statistically analyzed. The frequency of micronucleated binucleated (% MNBN) cells in the positive control as well as drug-treatment groups were compared with the solvent control group using Fisher's exact test. Results

Listed in Table 1 are the percentage of multinucleated cells (% MUN) (two or more nuclei) as well as % MNBN for the solvent control, positive control, and the 33 test compounds. Compounds with two sets of concentrations next to the structure were tested in each of two experiments. The results for the negative control indicate approximately 80% to 88% of the cells were MUN at the 16-h harvest. Approximately 1% of the binucleated cells contained MN. The positive control, Compound 1, demonstrated an effect on cell-cycle progression in the second experiment with a reduction of MUN cells to 63% at 24 /xg/ml. Values for % MNBN increased with increasing concentration in both trials with 12 and 2 4 / x g / m l showing statistical significance. The 33 compounds tested resulted in a wide range of responses both with respect to proliferation kinetics and MN induction. In many instances groups of compounds responded similarly, suggesting a structure-activity relationship. Group I represents 4 quinolone compounds with a halogen substitution at the 8-position. The

positive control is included in this category. The majority of the compounds produced a decrease in proliferation kinetics concomitant with an increase in MNBN cells as the concentration increased. In all cases, the MN response at the two high concentrations was statistically significant when compared to vehicle-control response. Additionally, the response of this group was independent of the halogen at position 8 (fluorine or chlorine) and independent of the substituent at position 7 (pyrrolidine or piperidine). Compounds listed in Group II have a trifluoromethyl substitution at position 8 and a pyrrolidine ring at the 7-position. The response in this group varied, although all but one (Compound 9) resulted in statistically significant increases in MNBN cells at one or more of the concentrations tested. In general, higher concentrations of these compounds were necessary to induce micronuclei relative to Group I compounds. Compound 9 showed a large decrease in multinucleated cells at 1000 /xg/ml and yet no MNBN cells were observed, probably due to a lack of cell proliferation. Compounds in Group III, which contained a methoxy substituent at the 8-position all induced MN. Several compounds (Compounds 15 and 18) caused a severe depression in proliferation kinetics at the high concentration yet MNBN frequency remained at high levels. The naphthyridones with their similarly substituted pyrrolidine side-chains which make up Group IV are relatively weak inducers of micronuclei, although proliferation was depressed in some cases. 3 of the 4 compounds which were statistically significant (Compounds 20, 21 and 22) have a primary amine group associated with the pyrrolidine. The other compounds evaluated in this group are N-alkyl derivatives, or in the case of Compound 23, the amine group is flanked by two methyl groups which provide bulk. Group V consisted of quinolones which were structurally diverse. Compound 31 did not induce an increase in MN frequency up to 4 0 0 / x g / m l in the first trial; however, after increasing the concentration in Trial 2 a significant increase in MNBN was demonstrated. Compound 32 had a profound effect on proliferation and at the same time induced MN at 80 /xg/ml. The other 3

236

compounds did not induce a significant MN response. Discussion

In the groups examined, naphthyridone compounds were less likely than quinolones to induce in vitro micronuclei in V79 Chinese hamster cells, particularly when the substituent at the 7-position contained some bulk (methyl groups) around the amine side-chain. Most of the quinolones tested appeared to induce micronuclei, irrespective of the substituents at position 7 or 8. The in vitro micronucleus assay proved to be a valuable tool in the genotoxicity testing of the quinolone and naphthyridone antibacterial compounds. The data derived provided valuable structure-activity information regarding the potential genotoxic hazard for these classes of antibacterial drugs and formed a basis for selection of compounds for further development. It would have been impractical to test this large number of compounds in the established in vitro or in vivo assays for assessing chromosome damage. At this point, those compounds which proved to have a reasonable genotoxic response can now be tested in one or several of the more standard assays. One of the advantages of the present methodology compared to classical metaphase chromosome analysis is that a smaller quantity of the test compound is required for treatment in the 6-well culture-dish system. During a period early in drug development, large quantities of drug are not available. Another advantage of the system is that more treatment groups can be handled in an experiment compared to the number of flasks needed in other assays. Finally, in the present assay, it is much easier and time efficient to assess micronuclei than it is chromosome aberrations. Therefore, less time and effort is spent in both conducting and scoring a study of this magnitude while gathering clastogenicity data on 34 compounds compared to a traditional chromosome-aberration assay of just one compound. This high capacity in vitro micronucleus assay has many potential applications. In addition to structure-activity studies and screening for po-

tential drug-development candidates, as demonstrated in this paper for quinolones and naphthyridones, this assay would be useful in mechanistic studies and interaction studies, where a variety of exposure conditions need to be assessed within a single experiment. References Chen, T.R. (1977) In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain, Exp. Cell Res., 104, 255-262. Countryman, P., and J. Heddle (1976) The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes, Mutation Res., 41, 321332. Dunn, T.L., R.A. Gardiner, G.J. Seymour and M.F. Lavin (1987) Genotoxicity of analgesic compounds assessed by an in vitro micronucleus assay, Mutation Res., 189, 299-306. Fenech, M., and A. Morley (1985) Measurement of micronuclei in lymphocytes, Mutation Res., 147, 29-36. Hogstedt, B. (1984) Micronuclei in lymphocytes with preserved cytoplasm: A method for assessment of cytogenetic damage in man, Mutation Res., 130, 63-72. Krishna, G., M.L. Kropko and J.C. Theiss (1989) Use of the cytokinesis-block method for the analysis of micronuclei in V79 Chinese hamster lung cells: Results with mitomycin C and cyclophosphamide, Mutation Res., 222, 63-69. Kropko, M.L., J.C. Theiss, V. Ciaravino and G. Krishna (1991) Clastogenic assessment of quinolone anti infective drugs, Environ. Mol. Mutagenesis, 17, 133 Abstr. Prosser, J.S., J.E. Moquet, D.C. Lloyd and A.A. Edwards (1988) Radiation induction of micronuclei in human lymphocytes, Mutation Res., 199, 37-45. Schiffmann, D., and U. De Boni (1991) Dislocation of chromatin elements in prophase induced by diethylstilbestrol: A novel mechanism by which micronuclei can arise, Mutation Res., 246, 113-122. Schmuck, G., G. Lieb, D. Weld, D. Schiffmann and D. Henschler (1988) Characterization of an in vitro micronucleus assay with Syrian hamster embryo fibroblasts, Mutation Res., 203, 397-404. Ujhhzy, E., I. Ohalupa, M. Bl~sko, J. Siraclo~, D. Zeljenkov~i, R. Noshr and L. Beni~ (1988) Genotoxicological study of the local anesthetic pentacaine in vitro micronucleus test, Pharmazie, 43, 8. Wakata, A., and M. Sasaki (1987) Measurement of micronuclei by cytokinesis-block method in cultured Chinese hamster cells: Comparison with types and rates of chromosome aberrations, Mutation Res., 190, 51-57. Xing, S.G., X.C. Shi, Z.L. Wu, W.-Z. Whong and T. Ong (1989) Effect of tetrandine on micronucleus formation and sister-chromated exchange in both in vitro and in vivo assays, Mutation Res., 224, 5-10.