Microbial mutagenicity and in vitro chromosome aberration induction by FK973, a new antitumor agent

Mutation Research Letters

Mutation Research 324 (1994) 43-50

ELSEVIER

Microbial mutagenicity and in vitro chromosome aberration induction by FK973, a new antitumor agent Osamu Hirai *, Youichi Miyamae, Yuko Hattori, Miyuki Takashima, Atsushi Miyamoto, Kazuyo Zaizen, Yasuhiro Mine Toxicology Research Laboratories, FujisawaPharmaceutical Co., Ltd,, 2-1-6, Kashima, Yodogawa-ku, Osaka 532, Japan (Received 22 November 1993; revision received 28 February 1994; accepted 4 March 1994)

Abstract The genotoxic activity of a new antitumor agent, FK973, was compared with that of mitomycin C (MMC) in eukaryotic and prokaryotic cells. In chromosome aberration tests using Chinese hamster fibroblast Don cells, FK973 induced a dose-related increase of aberrant cells after 6 h-pulse treatments, and the minimum effective concentrations with and without $9 were 0.625 and 0.0625/zg/ml, respectively. The compound increased revertant colonies in Salmonella typhimurium TA102 at the dose range of 10-5000/zg/plate with $9. Without $9, FK973 induced a small increase at the dose range of 500-5000 /zg/plate in two of three independent experiments, but the number of revertant colonies was less than double that of the vehicle control. The compound did not induce any revertant colonies in S. typhimurium TA100, TA98, TA1535 or TA1537 with or without $9. MMC was confirmed to increase both chromosome aberrations in Don cells and revertant colonies in TA102. The minimum clastogenic and mutagenic concentrations without $9 were 0.0156/xg/ml and 0.005/zg/plate, respectively. The results indicate that FK973 needs metabolic activation to induce reverse mutation in prokaryotic cells, but caused chromosome aberrations in mammalian cells without added $9.

Key words: FK973; Mitomycin C; Antitumor agents; Reverse mutation; Chromosome aberration; Bioactivation

I. Introduction FK973 (11-acetyl-8-carbamoyloxymethyl-4-formyl- 14-oxa- 1,11-diazatetracyclo-(7.4.1.O 2'7,0 10,12). tetradeca-2,4,6-trien-6,9-diyl diacetate) is a triacetylated derivative of a new antitumor antibiotic FR900482 which was recently isolated from Streptomyces sandaensis No. 6897 (Iwami et al.,

* Corresponding author.

1987; Shimomura et al., 1988; Uchida et al., 1987). FK973 was chemically modified from FR900482 to enhance its antitumor activity, and was at least as effective as mitomycin C in experimental murine tumor systems (Masuda et al., 1989; Shimomura et al., 1987, 1988). Since there is a close resemblance between the structures of FK973 and mitomycin C, in the present study we compared the genotoxic activity of FK973 with that of mitomycin C in the reverse mutation assay with Salmonella typhimurium

0165-7992/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165- 7992(94)00015- D

44

O. Hirai et aL / Mutation Research 324 (1994) 43-50

tester strains and chromosome aberration tests with the Chinese hamster cell line, Don.

2. Materials and methods

Chemicals

FK973 (CAS No. 113202-60-1)was synthesized in the Research Laboratories of Fujisawa Pharmaceutical Co. Ltd. Mitomycin C (MMC, CAS No. 50-07-7) was purchased from Kyowa Hakko Kogyo Co. Ltd. (Tokyo, Japan). The chemical structures of FK973 and MMC are shown in Fig. 1. Sodium azide (SAZ, CAS No. 26628-22-8), 2-aminoanthracene (2-AAnt, CAS No. 613-13-8) and 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF2, CAS No. 3688-53-7) were obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). 9-Aminoacridine HC1 (9-AAC, CAS No. 134-50-9) and dimethylnitrosamine (DMN, CAS No. 62-759) were from Nakarai Chemical Co. Ltd. (Kyoto, Japan). FK973, MMC and DMN were dissolved in saline and the other compounds were dissolved in DMSO. $9 mix

$9 was purchased from Nishin Seifun Co. Ltd. and stored at -80°C. The $9 preparation was obtained from phenobarbital- and 5,6-benzoflavone-treated SD male rats, and the protein content and NADPH-cytochrome c reductase activity were 22.6 mg/ml $9 and 248.5 nmol cytochrome c reduced/mg protein/min, respec-

tively. The $9 mix for the chromosome aberration tests contained (per ml) 5/zmol MgC12, 33/zmol KC1, 5 /zmol i>glucose-6-phosphate (G-6-P), 4 /zmol NADP, 4/xmol HEPES (pH 7.2) and 0.3 ml of $9. The $9 mix for Salmonella mutagenicity testing contained (per ml) 8 /zmol MgC12, 33 /zmol KC1,/xmol G-6-P, 4/~mol NADPH, 4/zmol NADH, 100 /zmol sodium phosphate (pH 7.4) and 0.1 ml of $9. The $9 mix was kept on ice until use.

Chromosome aberration test in uitro

Chinese hamster fibroblast Don cells were purchased from the Division of Laboratory Products, Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan). The test was carried out according to Ishidate and Odashima (1977). The cells (4 × 105/5 m l / dish) were cultured in Eagle's minimum essential medium (Gibco) supplemented with L-glutamine (Nissui Seiyaku Co. Ltd., 0.3/zg/ml), kanamycin (Meijiseika Co. Ltd., 60 ~g/ml) and fetal bovine serum (HyClone Lab., 10%) at 37°C in 5% CO 2 and 95% air. After 72 h of cultivation, the culture medium was changed for fresh medium containing the test chemicals with and without $9 mix and the cells were incubated for 6 h. After washing with Hanks' solution, the cultures were maintained in fresh medium for a further 18 h. Colcemid (Nakarai Chemical Co., final concentration 0.04 /zg/ml) was added 2 h before the end of culture. The cells were harvested by centrifugation at 4°C, 1000 rpm, resuspended in a hypotonic O

OCH3 O H C ~ N ~

FK973

H2N~,~~I~CH2OCONH2

NOCOCH3

MMC

Fig. 1. Chemical structures of FK973 and mitomycin C (MMC).

O. Hirai et al. / Mutation Research 324 (1994) 43-50

solution of 75 mM KCI at 37°C for 20 min, fixed 3 times in ice-cold Carnoy's solution (methanol : acetic acid; 3 : 1, v/v) and dropped on clean slides. The slides were air-dried and stained with Giemsa solution. One thousand cells per slide were observed for assessment of mitotic index and 100 metaphase cells were observed for analysis of chromosome aberrations. The chi-square test was used for the statistical analysis of the difference between treatment and solvent groups.

45

mm) containing 30 ml Vogel-Bonner's medium E minimal agar. The dishes were placed in an incubator at 37°C for 48 h (tester strains except TA102) or 72 h (S. typhimurium TA102). Revertant colonies were counted using a CA-7A II Automated Colony Counter (System Science Co., Tokyo, Japan). Induced cytotoxicity was indicated by the reduction in a thin lawn of bacterial growth relative to the negative control cultures.

3. Results and discussion

Mutagenicity testing Salmonella typhimurium tester strains TA1535,

Chromosome aberrations in Chinese hamster fibroblast Don cells were measured after a 6-h pulse-treatment with FK973 in the absence of $9 mix. The compound inhibited proliferation of the cells dose-dependently and the 50% inhibition dose was approximately 3 Izg/ml as estimated from the dose-response curve. FK973 induced a significant dose-related increase of aberrant cells in the culture, and the minimum effective concentration was 0.0625 tzg/ml (Table 1). FK973 produced principally chromatid-type as opposed to chromosome-type lesions; breaks and exchanges were the most frequent structural aberrations observed. The clastogenic and cytotoxic activities of FK973 were further evaluated in cultures with

TA1537, TA100 and TA98 were provided by Dr. B.N. Ames, Berkeley, CA. Strain TA102 was obtained from Dr. T. Nomi, NIHS, Tokyo, Japan. The reverse mutation test was carried out according to the preincubation method (Yahagi et al., 1977) with a minor modification of Ames et al. (1975). All tester strains used were examined for genetic characteristics by the method of Maron and Ames (1983). A mixture of 0.1 ml chemical solution, 0.5 ml phosphate buffer or $9 mix, and 0.1 ml tester strain culture was preincubated at 37°C for 20 rain followed by addition of 2.0 ml 0.6% agar containing 50 /zM L-histidine and Dbiotin. The reaction mixture was then vortexed gently and poured into petri dishes (100 x 15

Table 1 C h r o m o s o m e aberrations induced in D o n cells treated with FK973 and M M C for 6 h in the absence of $9 mix Treatment

Concentration

C h r o m o s o m e aberrations (%)

(izg/ml)

GT

Saline

0

FK973

MMC

BT

ET

0

2

0.0156 0.0625 0.25 1.0

0 0 0 0

0.0156 0.0625 0.25 1.0

0 2 3 .

.

Ta( - G)

MI

ML

(%)

(%)

0

0

2

100

0 0 0 0

1 1 3 2

0 0 2 6

5 10 * 19 ** 54 **

90 80 76 60

0 0 0 .

1 0 3

3 9 41

15 ** 41 ** 83 ** NS

92 57 27 11

GS

BS

ES

0

0

0

4 5 8 29

1 5 12 26

0 0 0 0

7 21 32

7 21 35

0 0 0

.

.

.

One h u n d r e d m e t a p h a s e cells were scored in each treatment group. MI in the saline control was 4.2%. GT, chromatid gap; BT, chromatid break; ET, chromatid exchange; GS, chromosome gap; BS, chromosome break; ES, chromosome exchange; ML, multiple aberration; T A ( - G), total aberrations without gaps. NS, m e t a p h a s e cells too few to evaluate chromosome structure. Significantly different from saline control at * p < 0.05 and ** p < 0.01.

46

o. Hirai et al./Mutation Research 324 (1994) 43-50

and without $9 mix. As shown in Table 2, FK973 induced a significant increase of aberrant cells in the culture with $9 mix, but the clastogenic and cytotoxic activities of FK973 decreased in comparison with those without $9. T h e s e data indicate that FK973 was metabolically detoxified by $9 in the culture conditions. T h e extracellular metabolic activation system used in the c h r o m o s o m e aberration tests was c o m p o s e d of $9 p r e p a r e d from p h e n o b a r b i t a l / 5,6-benzoflavone-treated rats. $9 has b e e n shown to activate m u t a g e n s and carcinogens from a variety of chemical classes (Matsushima et al., 1978; G a t e h o u s e and Delow, 1979; O n g et al., 1980). T h e N A D P H - c y t o c h r o m e c reductase activity in the $9 p r e p a r a t i o n used in the present experiments was e n o u g h to activate the positive control c o m p o u n d , D M N , for the induction of c h r o m o some aberrations in D o n cells (Tables 2 and 3). A l t h o u g h the metabolism of FK973 in the N A D P H - c y t o c h r o m e c system is not fully defined, the c o m p o u n d seems to be rapidly m e t a b o lized by rat liver $9 as assessed by high-perform a n c e liquid c h r o m a t o g r a p h y ( H P L C ) analysis of the incubation mixture (in a preliminary experiment, data not shown). FK973 was r e p o r t e d to be metabolized in the r o d e n t blood (hemolysate) to two diacetates in a few minutes, to a m o n o a c e t a t e

in a few hours and to a nonacetylated antitumor antibiotic FR900482 only after incubation for a long period ( M a s u d a et al., 1988). T h e m e t a b o lites had strong cytotoxicity on murine leukemia L1210 cells in vitro, but the cytotoxicity of FK973 was the most p o t e n t ( M a s u d a et al., 1988). These activities support the present results in which the clastogenicity and cytotoxicity of FK973 decreased in the culture with $9 (Table 2). T h e detoxification of FK973 in the c h r o m o s o m e aberration test with the metabolic activation system may occur by deacetylation of the c o m p o u n d by $9. M M C , which is a clinically useful antitumor agent, was confirmed to induce c h r o m o s o m e aberrations and mitotic inhibition in m a m m a l i a n cells and the m i n i m u m effective dose without $9 was 4 times lower than that of FK973 in the D o n cells (Table 1). T h e clastogenic and cytotoxic activities of M M C decreased in the culture with $9 in comparison with those without $9 (Table 3). N A D P H - c y t o c h r o m e c reductase is one of the enzymes responsible for the activation of M M C , especially in anaerobic conditions (Bachur et al., 1979; T o m a s z and Lipman, 1981; Bleigh et al., 1990; V r o m a n s et al., 1990; Krishna et al., 1991). In contrast, in vitro M M C is rapidly metabolized and inactivated by N A D P H - d e p e n d e n t enzymes

Table 2 Effect of $9 mix on the clastogenic activity of FK973 in Don cells Treatment Saline FK973

DMN Saline FK973

DMN

Concentration (~g/ml)

$9 mix

Chromosome aberrations (%) GT BT ET GS

BS

ES

ML

0 0.156 0.625 2.5 10 500

-

0 2 3 1

0 8 26 35

0 9 38 49

0 0 0 0

0 0 0 0

1 0 4 4

0 4 10 30

-

1

9

7

0

0

1

7

-

0

1

0

0

0

0

0 0.156 0.625 2.5 10 500

+ + + + + +

0 0 0 0 0 2

3 2 6 6 17 13

1 3 4 5 21 15

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

TA( - G)

MI

(%)

(%)

0

1 17 ** 61 ** 81 ** 19 ** 1

100 86 91 28 18 109

0 0 3 0 2 1

3 5 12 * 10 31 ** 23 **

100 87 102 96 87 63

One hundred metaphase cells were soared in each treatment group. MI in the saline control with and without $9 mix were 10.8 and 5.7%, respectively. For abbreviations see Table 1. Significantly different from saline control at * p < 0.05 and ** p < 0.01.

O. Hirai et al./ Mutation Research 324 (1994) 43-50

47

Table 3 Effect of $9 mix on the clastogenic activity of MMC in Don cells Treatment

Saline MMC

DMN Saline MMC

DMN

Concentration

$9

Chromosome aberrations (%)

TA( - G)

MI

(/zg/ml)

mix

GT

BT

ET

GS

BS

ES

ML

(%)

(%)

0 0.0156 0.0625 0.25 1 500

-

0 0 0 1 0 0

0 3 18 35 38 1

0 1 33 50 38 0

0 0 0 0 0 0

0 0 0 0 0 0

0 0 2 1 0 0

0 0 2 46 57 0

0 # 45 ** 98 ** 100 ** 1

100 106 88 48 12 118

0 0.0625 0.25 1 4 16 500

+ + + + + + +

0 0 0 1 1 0 0

0 1 1 23 47 58 17

0 0 3 32 68 79 28

0 0 0 0 0 0 0

0 0 0 0 0 0 0

0 0 0 2 3 1 2

0 0 0 0 24 12 2

0 1 4 46 98 95 38

** ** ** **

100 98 93 82 20 2 79

One hundred metaphase cells were scored in each treatment group. MI in the saline control with and without $9 mix were 9.5 and 6.6%, respectively. For abbreviations see Table 1. Significantly different from saline control at ** p < 0.01.

Table 4 Mutagenic activity of FK973 in Salmonella typhimurium TA102 with and without $9 mix Chemical

Amount (/zg/plate)

Saline

FK973

1 5 10 50 100 500 1000 5 000

MMC 2-AAnt

0.005 2.5 4.0

$9 mix

Number of revertant colonies per plate Exp. I

Exp. II

+

232 404

311 420

Exp. III 277 411

+ + + + + + + +

287 412 307 544 332 677 326 1 282 312 1 682 358 1 966 416 2 254 378 285 a

252 440 276 541 275 630 314 1 146 317 1481 362 1 646 378 1288 349 78 a

NT NT 281 554 306 698 321 1584 353 2 202 431 3 449 504 2 626 NT NT

+ +

1994 578 NT

2 197 801 NT

2858 NT 1 189

The mutagenic activity of FK973 was tested in three independent experiments with and without $9 mix. Number of revertant colonies observed after 72 h incubation shows the mean of duplicate plates. a Growth inhibition; NT, not tested.

O. Hirai et al. /Mutation Research 324 (1994) 43-50

48

in the rat liver microsomal fraction (Schwartz and Philips, 1961; Schwartz, 1962). The genotoxicity of FK973 was also tested in a reverse mutation assay using Salmonella typhimurium. FK973 induced dose-dependent increase of revertant colonies in S. typhimurium TA102 with $9 mix at the minimum concentration of about 10/xg/plate (Table 4). Without $9, the compound induced a small increase at the dose range of 500-5000 ~ g / p l a t e in two of three independent experiments, but the number of revertant colonies was less than double that of the vehicle control (Table 4). In other Salmonella tester strains, no significant increase of revertant colonies was obtained at concentrations of 1.6-50 /xg/plate with or without $9 mix (Table 5). On the contrary, FK973 with $9 mix showed clear cytotoxicity against both His + revertant cells and bacterial background lawn in the Salmonella strains except TA102 at the concentrations of 25 and 50/xg/plate. MMC was confirmed to induce an increase in

revertant colonies in S. typhimurium TA102 and the minimum effective concentrations with and without $9 mix were 0.005 and 0.1 ~g/plate, respectively (Fig. 2). MMC without $9 was 106 times more active than FK973 in TA102. MMC is well known to cross-link DNA and its mutagenicity in bacteria is known to depend upon an intact uvrB gene (Kondo et al., 1970; Murayama and Otsuji, 1973; Seino et al., 1982). MMC is very toxic and non-mutagenic towards bacterial strains which do not possess an intact uvrB gene (Murayama and Otsuji, 1973; Seino et al., 1978; Ferguson et al., 1988). The low cytotoxicity of FK973 against TA102 was identical to that of MMC. Since FK973 was reported to form interstrand DNA-DNA cross-links in murine tumor cells (Masuda et al., 1988), the probable explanation for the cytotoxicity in the Salmonella strains except TA102 is that, like MMC, the unrepaired FK973-induced cross-links between DNA strands are lethal and non-mutagenic. The first step in the interaction of MMC and

Table 5 Number of revertant colonies per plate induced by FK973 in the Ames test with and without metabolic activation Amount

$9 mix

(p~g/plate)

Number of revertant colonies per plate (mean 5: SEM) a TA1535

Saline

+

9 5:0.27 85:1.89

1.6

+ + + + + +

85:0.54 9 5:0.98 11 5 : 0 . 7 2 6 5:0.54 10 5 : 1 . 1 9 35:1.09 5 + 0.72 1 5:0.27 6 5: 1.78 05: 0.00c 3 5:0.27 05: 0.27c

3.1 6.3 12.5 25 50

Positive control

+

SAZ (0.5) b 122 5 : 3 . 5 6 2-AAnt (2) 185 5:12.73

TA1537 7 + 1.25 15 5 : 1 . 6 6 6 + 1.19 8 5:0.98 5 5:0.54 7 5:0.94 6 5:1.09 65:0.72 4 5:1.25 05:0.00 65: 1.52 05:0.00 c 05:0.27 05: 0.27c 9-AAC (80) 1546 5:70.04 2-AAnt (2) 192 5:12.06

a Mean of triplicate plates ( + standard error).

b Numbers in parentheses indicate the amount (/xg/plate) of positive control. c Growth inhibition.

TA100

TA98

106 5 : 6 . 8 7 159 5 : 8 . 4 9

22 5 : 0 . 7 2 20 + 1.52

111 + 3.54 112 5 : 7 . 2 5 111 5:10.87 104 5 : 9 . 4 6 98 5:10.92 80 5 : 6 . 8 0 69 5 : 0 . 7 2 23 5 : 1 . 2 5 48 5 : 4 . 3 2 05:0.00 1 + 0.54 05:0.00

8 + 0.54 18 + 2.36 17 + 1.41 15 5 : 2 . 8 7 8 + 2.13 9 5:1.66 10 + 2.05 5 _+ 0.27 5 + 1.91 0 5:0.00 c 1 _+ 0.27 05:0.00 c

AF-2 (0.05) 1297 5:16.04 2-AAnt (2) 981 + 9.44

AF-2 (0.05) 136 + 9.18 2-AAnt (0.5) 531 5:20.58

O. Hirai et al. / Mutation Research 324 (1994) 43-50

References

5000

4000 O,.

3000

3 c

2000

'5 1000 Z

49

! -.k

~

I"1

I I o"'~1

0 0.001

, ,..,d

0.01

Amount

. ,,,,,d

0.1

0 ,=,°d

1

, =,,°~1

10

100

, ,,.m

1000

( F g/plate)

Fig. 2. Comparison of mutagenic activity of FK973 and MMC in S. typhimurium TA102. The bacterial cells (3.5 x 108/plate) on agar plate were incubated at 37°C for 72 h after preincubation in a mixture of chemical solution and phosphate buffer or $9 mix at 37°C for 20 rain. Each point represents the mean of two plates, o o, FK973 without $9 mix; • •, FK973 with $9 mix; zx t,, MMC without $9 mix; • - , MMC with $9 mix. Similar results were obtained in independent experiments.

cellular DNA is thought to be the reductive activation of the quinone ring in MMC molecule by the cytochrome P450 reductase and xanthine oxidase (Bachur et al., 1979; Pan et al., 1984). Deacetylation of FK973, like that in blood cells (Masuda et al., 1989), might be one of the activating steps of the compound in Don cells. In murine tumor cells, monoamine oxidase, which is involved in metabolism of various other drugs (Blaschko, 1952; Gorkin, 1966), also was suggested to have a part in the metabolic activation of FK973 (Masuda et al., 1990). Further, the close resemblance of the structural features of FK973 and MMC makes it possible that an aziridinomitosen-like compound could be an active metabolite of FK973 (Fukuyama and Goto, 1989). S. typhimurium TA102 may lack a system which can metabolize FK973, or lack sufficient metabolic enzymes in the ceils. Further studies are needed to identify the mutagenic metabolite responsible for the genotoxic action of FK973.

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