Prophage λ induction test (inductest) of anti-tumor antibiotics

325

Mutation Research, 88 ( 1 9 8 1 ) 3 2 5 - - 3 3 5 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

PROPHAGE X INDUCTION TEST (INDUCTEST) OF ANTI-TUMOR ANTIBIOTICS 1

W A N N E E R O J A N A P O 2,3, M I N A K O N A G A O , T A K A S H I K A W A C H I a n d T A K A S H I SUGIMURA

Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104 (Japan) (Received 2 May 1980) ( R e v i s i o n r e c e i v e d 24 S e p t e m b e r 1 9 8 0 ) (Accepted 7 October 1980)

Summary A total of 20 compounds, consisting of anti-tumor antibiotics and their derivatives, were examined for their ability to induce prophage X in Escherichia coli GY5027. Mitomycin C, daunomycin, adriamycin, bleomycin and bleomycin analogs, induced prophage X without S9 mix. Aclacinomycin A and its monodemethyl derivative did not induce prophage X, but N-didemethylaclacinomycin A did. Actinomycin D, neothramycin, l-deoxy-pyrromycin, adriamycinone, aklavinone and daunomycinone did not induce prophage X either in the presence or absence of S9 mix.

During the past 10 years, many short-term assays have been developed for screening potential chemical carcinogens. The bacterial mutagenicity test described by Ames and his associates, known generally as the Ames test, is currently the standard test and is by far the most widely used (Ames et al., 1975). More than 100 carcinogens have been shown to be mutagenic to Salmonella typhimurium, and a fairly clear correlation has been found between carcinogenicity and mutagenicity (McCann et al., 1975; Nagao et al., 1978). Devoret and colleagues (Moreau et al:, 1976; Moreau and Devoret, 1977) modified the prophage X induction test, originally developed by Lwoff (1953), to detect DNAmodifying agents. This test might be used together with the Ames test to screen environmental DNA-modifying carcinogens. 1 Supported in part by Grant-in-Aid from the Ministry of Health and Welfar.e, Japan. 2 Received a fellowship from Japan International Cooperation Agency (under the Colombo plan) during the course of this work. 3 Present address: Research Division, National Cancer Institute, Rama VI Road, Bangkok 4, Thailand.

326 Anti-tumor antibiotics are widely used for clinical treatment of various kinds of cancer. Some of these agents are mutagenic (Benedict et al., 1977; Seino et al., 1978) and also induce chromosomal abberrations in cultured mammalian cells, and some are carcinogenic to laboratory animals. Bleomycins, which are not mutagenic to S. typhirnurium (Benedict et al., 1977; Seino et al., 1978), are suspected to be carcinogenic (Llombart, 1976). It seemed interesting, therefore, to examine whether c o m p o u n d s can induce prophage and also to study the correlation between their phage-inducing ability and their carcinogenicity. Recently, phage-inducing activities of antineoplastic agents were reported by Anderson et al. (1980). The results on some of the c o m p o u n d s which we tested confirmed their results. Materials and methods Chemicals

Adriamycin hydrochloride was obtained from K y o w a Hakko Kogyo Co. Ltd., T o k y o , Japan. Adriamycin, daunomycinone, aklavinone, aclacinomycin A and its demethylated derivatives, 1-deoxypyrromycin and N-didemethyl-1d e o x y p y r r o m y c i n were generously supplied by Sanraku-Ocean, Co. Ltd., Fujisawa, Japan. Daunomycin hydrochloride and neothramycin were generously supplied by Meiji-Seika Kaisha Ltd., T o k y o , Japan. Actinomycin D and mitomycin C were from PL Biochemicals Inc., Milwaukee, WI, and Sigma Chemical Company, resp. Bleomycin hydrochloride and its analogs (BLM-HCP, BLMPYP, BLM-A5033, BLM-A5196, BLM-BAPP and p e p l e o m y c i n ) w e r e kindly supplied from Nippon Kayaku Co. Ltd., T o k y o , Japan. NADPH was from Oriental Yeast, Co. Ltd., Tokyo, and G6P from Sigma Chemical Co. Dimethylsulfoxide (DMSO), spectrophotometric grade, was from Wako Pure Chemical Inc., Osaka, Japan. Bacterial strains

A lysogenic bacterium Escherichia coli K12 envA uvr (X) (strain GY5027) and a X indicator Amp R bacterium (strain GY4015) were kindly provided by Dr. R. Devoret, Laboratoire d'Enzymologie, C.N.R.S., Gif-sur-Yvette, France. Media and cultures

LB and LBE media and GT-amp plates were prepared as described by Moreau et al. (1976). LB: Difco yeast extract 5 g, Difco t r y p t o n e 10 g, NaC1 10 g, water 1 1. LBE: LB supplemented with 0.2% glucose and 20% medium E of Vogel and Bonner. GT-amp: peptone 8 g, Difco tryptone 5 g, NaC1 5 g, Difco agar 12 g, water 1 1, supplemented with 10 mg of D-ampicillin. The soft agar used consisted of 0.7% agar and 0.6% NaC1. The envA lysogen strain GY5027 was grown overnight in LBE medium at 37°C. A fresh culture was prepared by diluting 0.1 ml of an overnight culture with 10 ml of fresh LBE medium and incubating it at 37°C until the cell density became about 4 X 10 v viable cells per ml (OD6s0 = 0.3). The culture was then diluted with LBE medium to a density of a b o u t 5000 cells per ml and placed in ice until use.

327 The indicator bacterial strain GY4015 was grown overnight in LB medium at 37°C and then placed in ice until use. Inductest The inductest performed in this study was inductest III, a quantitative test in liquid m e d i u m developed by Moreau et al. (1976). The procedure used was essentially as described by Moreau and Devoret (1977 ) with slight modifications. Briefly, the test substance in 20 pl of DMSO was incubated with 0.5 ml of 0.1 M phosphate buffer (pH 7.4) or $9 mix and 0.1 ml of a fresh culture of lysogenic tester bacteria (about 5 X 103 cells/ml) in the dark at 37°C for 20 min. Then 0.3 ml of the culture of indicator bacteria and 2 ml of soft agar were added and the mixture was poured onto GT-amp plates. Prophage X induction was measured by counting infective centers after overnight incubation at 37 ° C. $9 mix was prepared as described by Ames et al. (1975): 0.5 ml of $9 mix contained 50 pl of liver $9 fraction prepared from PCB-treated rats. Potencies are expressed as the a m o u n t of chemical that induced 100 plaques over spontaneous ones. Spontaneous plaques were 10--28 t h r o u g h o u t the experiments.

Results 20 chemicals, namely anti-tumor antibiotics and their derivatives, were tested for prophage-inducing activity. The trivial names and structures of these compounds, the results of the inductest and information on the carcinogenicities and mutagenicities to S. t y p h i m u r i u m are shown in Table 1. In the inductest, an induced lysogen gives rise to an infective center, which

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Mitomycin C, bleomycin, daunomycin and adriamycin gave positive results, although their activities varied. At higher doses of bleomycin, phage production decreased. Actinomycin D, aclacinomycin A and neothramycin did not induce prophage, and the addition of S9 mix did not have any effect on the activities of these 3 antibiotics (results not shown). Fig. 2 shows the phage-inducing activities of aclacinomycin A, N-monodemethylaclacinomycin A and N-didemethylaclacinomycin A. N-Didemethylaclacinomycin A, but not the parental aclacinomycin A or its monodemethyl derivative, induced prophage. The induction by N-didemethylaclacinomycin A was observed only in the absence of $9 mix: the addition of $9 mix completely abolished the phage-inducing activity (result not shown), possibly owing to enzymatic inactivation or direct interaction of the chemical with protein in $9 mix. 1-Deoxypyrromycin is a hydrolysis product of aclacinomycin A. This compound and N-didemethyl-l-deoxypyrromyein did not induce phage production in the presence or absei-ee of $9 mix. Daunomyeinone, adriamycinone and aklavinone, the aglycones of daunomycin, adriamycin and aclacinomycin A, resp., had no phage-inducing ability at doses of up to 100 pg, either in the presence or absence of $9 mix.

TABLE

2

PROPHAGE-INDUCING

POTENCIES

Antibiotic

OF

ANTI-TUMOR

Inducing activity

ANTIBIOTICS

Amount of chemical inducing 100 plaques (~g) a

Actinomycin D Mitomycin C

--

(0.01--20)

+

0.032

Daunomycin Daunomycinone

+

1.4

--

(0.01--100)

Adriamycin Adriamycinonc

+

1.4

--

(0.01--100)

Aclacinomycin A

--

(0.01--100)

Aklavinone

--

(0.01--100)

N - M o n o d e m e t h y l a c l a c i n o m y cin A N-Dide mcthylaclacinomycin A

--

(0.01--100)

+

50

1-Deoxypyrromycin N-Didemethyl-l-deoxypyrromyein

--

(0.01--100)

--

(0.01--100)

Neothramycin Bleomycin

--

(0.01--100)

+

0.060

Bleomycin-HCP Bleomyein-PYP

+

0.045

+

0.17

Bleomycin-A5033

+

0.23

Bleomyein-A5196

+

0.008

Bleomycin-BAPP

+

0.014

Pepleomycin

+

0.056

a

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333

Various analogs of bleomycin differing in their terminal amine moiety have been studied for anti-tumor activity and toxicity to select a c o m p o u n d with a higher chemotherapeutic index than that for bleomycin (Matsuda et al., 1978). In this work, some of these bleomycin analogs were tested for phage-inducing ability. Fig. 3 shows that all the analogs tested were strong phage inducers in the absence of $9 mix. The addition of $9 mix decreased the induction activity (results n o t shown). BLM-BAPP and BLM-A5196 were the most active analogs, and BLM-A5033 was the least active. Table 2 shows the prophage-inducing potencies of the 20 anti-tumor antibiotics tested in this study. Mitomycin C and the bleomycin analogs BLM-A5196 and BLM-BAPP were the most active compounds. Discussion In t h e p r e s e n t study, 11 of 20 anti-tumor antibiotics induced production of bacteriophage. The 11 c o m p o u n d s were active in the absence of $9 mix, indicating that they may be direct-acting phage inducers or may have been activated by bacterial enzyme(s) before exerting their inducing activities. Of the 11 antibiotics that gave positive responses, 4 are known or suspected to be carcinogenic and 7 have n o t yet been tested for carcinogenicity. Of the c o m p o u n d s that gave negative responses in the inductest, only actinomycin D is reported to be carcinogenic in vivo (DiPaolo, 1960). The anthracyclin antibiotics, daunomycin and adriamycin, which are known to be useful in treatment of leukemia and some solid tumors, induce mammary and renal tumors when given in a single i.v. dose to rats (Marquardt et al., 1976) and they are also highly mutagenic to S. typhimuriurn TA98 and TA100 (Benedict et al., 1977; Seino et al., 1978). Anderson et al. (1980) reported that the addition of $9 enhanced the phage induction of daunomycin. Adriamycin and daunomycin cause chromosomal disintegration (Sieber and Adamson, 1975), break DNA strands in vivo (Schwartz, 1975) and intercalate with DNA (Zunino et al., 1972), resulting in inhibition of DNA-dependent DNA and RNA polymerases (Zunino et al., 1975). These compounds induced prophage (Fig. 1, Table 2). However, aclacinomycin A, which has a rather similar structure to daunomycin and adriamycin, b u t with 2 additional hexose rings and a dimethylated amino group on the sugar (Table 1) did not induce prophage ~ production (Fig. 1) or bacterial mutation (Umezawa et al., 1978). It is interesting that N-didemethylaclacinomycin A induced prophage, whereas N-monodemethylaclacinomycin A did not. Similar results have been Obtained in the Salmonella mutation system (Umezawa et al., 1978). Thus, the free amino group of anthracyclin antibiotics may be essential for the induction of both bacteriophage and bacterial mutation. Neither 1-deoxypyrromycin, a hydrolysis product of aclacinomycin A, nor its N-didemethylated product, induced prophage k production, b u t N - d i d e m e t h y l - l - d e o x y p y r r o m y c i n was mutagenic to S. typhimurium in the presence of metabolic activating enzymes (Umezawa et al., 1978). It is unk n o w n w h y N-didemethyl-l-deoxypyrromycin, which has a similar structure to N-didemethylaclacinomycin A, cannot induce prophage although it is mutagenic to S. typhimurium. The mutagenic activity of N-didemethyl-l-deoxypyr-

334

romycin is one-quarter of that of N-didemethylaclacinomycin A (Umezawa et al., 1978). The prophage-inducing property of mitomycin C (Moreau et al., 1976) was confirmed in this study. Mitomycin C is carcinogenic (Weisburger et al., 1975) and mutagenic to S. typhimurium TA92 (Seino et al., 1978). It also produces inter-strand cross-linking of double-strand DNA (Szybalski and Iyer, 1969). Actinomycin D, which binds preferentially to dG-dC pairs of DNA and inhibits DNA-directed RNA synthesis (Philips and Sternberg, 1975), but does not break DNA strands, did not induce prophage h production. Actinomycin D induces sarcomas, but only at the injection site (DiPaolo, 1960). Philips and Sternberg (1975) were unable to induce tumors in rats by repeated intra-gastric intubation of the maximally tolerated dose of actinomycin D. No mutagenic activity towards S. typhimurium was found in 2 separate laboratories (Benedict et al., 1977; Seino et al., 1978), although it is mutagenic to Neurospora crassa (Brockman et al., 1978). Bleomycin is now widely used in treatment of squamous cell carcinoma and Hodgkin's disease. In this study, bleomycin and its analogs differing in the terminal amine moiety were potent inducers of bacteriophage production. Bleomycin is blastogenic (Llombart, 1976), although no mutagenicity was observed (Abe et al., 1978; Benedict et al., 1977; Seino et al., 1978). Bleomycin inhibits the incorporation of [3H]thymidine into DNA in intact cells and also causes single-strand scission of DNA (Umezawa, 1975). The latter action may be responsible for the induction of prophage h. At the higher concentration of bleomycin used, the number of plaques decreased (Fig. 3), probably owing to fragmentation of the phage DNA, as suggested by Haidle et al. (1972). Recently, ~-galactosidase-inducing activity of bleomycin was reported for lysogenic E. coli, in which a lacZ gene was fused to an operon under h repressor control (Elespuru and Yarmolinsky, 1979). Among the bleomycin analogs tested in this study, BLM-BAPP and BLMA5196 were the most active in inducing prophage h. They are also the most potent drugs for inhibition of the growth of HeLa cells (A. Matsuda, personal communication). It is noteworthy that in this study bleomycin, which is carcinogenic but not mutagenic in the Salmonella mutation test, was a potent prophage h inducer. Together with the finding that nearly all anti-tumor antibiotics that are carcinogenic as well as mutagenic are phage inducers, the results of this work suggest that the inductest is useful in combination with the Salmonella mutagenicity test for identifying DNA-modifying agents. Acknowledgement We thank Dr. A. Matsuda, Nippon Kayaku Central Research Institute, Tokyo, for providing some of the chemicals used. References A b e , F., A. K o y u , H. I n o u e , T. Y a m a s h i t a , H. E z u r a , K. Y o s h i z a w a , K. T a k a h a s h i , O. Y o s h i o k a a n d A. M a t s u d a ( 1 9 7 8 ) S a f e t y e v a l u a t i o n of N K 631: A n t i g e n i e i t y , e f f e c t on d e l a y e d h y p e r s e n s i t i v i t y , irrita-

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