Mutagenicity of Several Classes of Antitumor Agents to Salmonella Typhimurium TA98, TA100, and TA921
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[CANCER RESEARCH 38, 2148-2156, July 1978] 0008-5472/78/0038-0000$02.00 Mutagenicity of Several Classes of Antitumor Agents to Salmonella typhimurium TA98, TA100, and TA921 Yuko Seino,2 Minako Nagao, Takie Yahagi, Akio Hoshi, Takashi Kawachi, and Takashi Sugimura Biochemistry [Y. S., M. N., T. Y., 7. K., T. S.] and Pharmacology [A. H.¡Divisions, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104, Japan ABSTRACT Chemicals. Adriamycin hydrochloride, 5-fluorouracil, mi tomycin C, and L-asparaginase were obtained from Kyowa The mutagenic activities of antitumor agents, including Hakko Kogyo Co., Ltd., Tokyo, Japan. Cyclophosphamide 5 antibiotics, 19 antimetabolites, 5 alkylating agents, 2 and vinblastine sulfate were purchased from Shionogi & alkaloids, 1 enzyme, and 1 adrenal steroid hormone, were Co., Ltd., Osaka, Japan; Methotrexate and busulfan were tested on Salmonella typhimurium TA100, TA98, and TA92. from Takeda Chemical Industries. Ltd., Osaka, Japan; 1-/3- Four of these, busulfan, carbazilquinone, 1-(4-amino-2- D-arabinofuranosylcytosine and cyclocytidine were from methy Ipy rim idine - 5 - yl)met hyI- 3-(2-ch loroethy l)-3 -n itroso- Kohjin Co., Ltd., Tokyo, Japan. 6-Mercaptopurine and 8- urea hydrochloride, and pipobroman were shown for the azaguanine were from Sigma Chemical Co., St. Louis, Mo.; first time to be mutagenic. Further, the known mutagenic- ACNU3 and carbazilquinone were from Sankyo Co., Ltd., ities of five others, daunomycin hydrochloride, Adriamy- Tokyo, Japan; and actinomycin D was from Nippon Merck- cin hydrochloride, mitomycin C, 6-mercaptopurine, and Banyu Co., Ltd., Tokyo, Japan. Bleomycin hydrochloride cyclophosphamide, were confirmed. was from Nippon Kayaku Co., Ltd., Tokyo, Japan; dauno mycin hydrochloride was from Meiji Seika Kaisha, Tokyo, INTRODUCTION Japan; and FT-207 was from Taiho Pharmaceutical Co., Ltd., Tokyo, Japan. Alkylcarbamoyl derivatives of 5-fluo Many chemotherapeutic agents are now widely used clinically for treatment of cancers and leukemias and some rouracil were synthesized at Mitsui Pharmaceuticals, Inc., times for prophylaxis of recurrent cancer after surgical Tokyo, Japan. Vincristine sulfate was from Eli Lilly & Co., treatment. Indianapolis, Ind., and pipobroman was from Dainippon However, a phenomenon known as Haddow's paradox Pharmaceutical Co., Ltd., Osaka, Japan. Prednisolone was from Sanwa Research Institute, Tokyo, Japan. Glucose 6- (5) is that some cancer chemotherapeutic agents are car phosphate, glucose-6-phosphate dehydrogenase (EC cinogenic to laboratory animals and also induce chromo somal aberrations in cultured mammalian cells. Thus, it is 1.1.1.49), and ATP were from Sigma; NADH and NADPH important to know whether chemotherapeutic agents are were from Oriental Yeast Co., Ltd., Tokyo, Japan; and carcinogenic. dimethyl sulfoxide, spectrophotometric grade, was from Recently, it has become widely accepted that many mu- Wako Pure Chemical, Inc., Osaka, Japan. tagens are carcinogens (15, 16, 24). Therefore, in this work Eight compounds for drug use contained some vehicle, we tested the mutagenic activities in S. typhimurium strains and the amounts of these compounds were expressed as TA100, TA98, and TA92 of various kinds of chemotherapeu fjiQof active principle. The names of these compounds and tic agents now in use or being tested for use in treatment of the amounts in 1 vial, 1 ampul, or 1 tablet were as follows: daunomycin-HCI (20 mg principle; actual weight, 120 mg/ human cancer. The compounds tested were antibiotics, vial), Adriamycin-HCI (10 mg principle; actual weight, 50 antimetabolites, alkylating agents, alkaloids, an enzyme, mg/vial), bleomycin-HCI (15 mg potency; actual weight, 8 and a hormone. mg/ampul), actinomycin D (0.5 mg principle; actual weight, MATERIALS AND METHODS 20.5 mg/vial), mitomycin C (0.996 mg principle; actual weight, 1 mg/vial), cyclophosphamide (500 mg principle; Microbes. S. typhimurium strains TA100, TA98, and TA92 actual weight, 725 mg/vial), L-asparaginase (10,000 ID; were kindly supplied by Dr. Bruce N. Ames, University of actual weight, 33 mg/vial), and prednisolone (5 mg princi California, Berkeley, Calif. TA100 and TA98 are uvrB and ple; actual weight, 153 mg/tablet). rfa (deep rough) mutants (1). TA92 has the capacity for Mutation Test. The mutation test was carried out by our excision repair and an intact lipopolysaccharide barrier modification (20) of the method of Ames ef al. (1). The test (LPS+) (1). TA100 and TA92 both contain the same base- chemical in 0.1 ml dimethyl sulfoxide or H,0 was placed in pair change mutation, and TA98 contains a frameshift a tube and mixed with 0.5 ml S-9 mix (150 /u.lof the S-9 mutation at his locus (1). All these strains carry R-factor fraction of rat liver pretreated with polychlorinated bi- plasmid pKM101 (1). phenyl, 2 /nmol NADPH, 2 ¿imolNADH,2.5 /¿molglucose 6- phosphate, 0.25 unit glucose-6-phosphate dehydrogenase, ' This work was supported ¡npart by Grants-in-Aid for Cancer Research 4 /Limol MgCIo, 16.5 /nmol KCI, and 50 /¿molsodium phos from the Ministry of Education, Science and Culture and the Ministry of phate buffer, pH 7.4 and 0.1 ml of culture of the bacterial Health and Welfare, Japan, and by the Princess Takamatsu Cancer Research Fund. 3 The abbreviations used are: ACNU, 1-(4-amino-2-methylpyrimidine-5- 2 To whom requests for reprints should be addressed. yl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride; FT-207, 1-(2-tetra- Received April 14, 1977; accepted March 22, 1978. hydrofuryl)-5-fluorouracil. 2148 CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1978 American Association for Cancer Research. Mutagenicity of Antitumor Agents tester strain (1 to 2 x 108 cells). Without metabolic activa in vivo and in vitro and mutation in mammalian cells in vitro tion 50 /¿molsodium phosphate buffer, pH 7.4, in 0.5 ml (14). Bleomycin hydrochloride was not mutagenic to TA100 were used instead of S-9 mix. The mixture was preincu- or TA98, with or without S-9 mix, but it is known to cause bated at 37°for 20 min and then mixed with 2 ml top agar DNA strand breakage (26). Actinomycin D, which binds (0.7% agar and 0.6% NaCI) at 45°andspread on a minimal- preferentially to dG-dC pairs of DNA (13) and inhibits DNA- glucose agar plate containing 0.1 /¿moleachL-histidine and dependent RNA synthesis (12), was not mutagenic to TA100 biotin. Plates were incubated at 37°for 2 days, and then or TA98 with or without S-9 mix. The in vivo carcinogenicity his+ revertant colonies were counted. of actinomycin D in rats has been reported (30). Actinomy cin D is also reported to have very weak mutagenic activity RESULTS AND DISCUSSION on mammalian cells cultured in vitro (14). Mitomycin C was not mutagenic to TA100 or TA98 but was weakly mutagenic The trivial names and structures of the 5 antibiotics, 19 to TA92, especially without S-9 mix, as shown in Table 2. antimetabolites, 5 alkylating agents, and other compounds Mitomycin C produces interstrand cross-linking of double- tested are shown in Tables 1,3,5, and 7, respectively, with stranded DNA (27). In this case uv repair-deficient strain the results of mutagenicity tests on them and information cells were killed and did not yield any mutants (19). TA92, on their carcinogenicities. which has uv repair capacity, gave more reversants than did Antibiotics. Results on the mutagenicities of the antibiot the spontaneous revertants. Mitomycin C produces tumors ics are listed in Table 2. The mutagenicities of daunomycin in vivo (30). Bleomycin hydrochloride and actinomycin D hydrochloride (16), Adriamycin hydrochloride (16), and mi- were not mutagenic but were toxic to TA100 and TA98, as tomycin C (17), which had been reported previously, were was mitomycin C. But these compounds were not muta confirmed. Daunomycin hydrochloride and Adriamycin hy genic to TA92 (Table 2). drochloride intercalate DNA (33), resulting in inhibition of Antimetabolites. Results on antimetabolites are listed in DMA-dependent DNA and RNA polymerases (32). These Table 4. 5-Fluorouracil, an inhibitor of thymidylate synthe- compounds were highly mutagenic to TA100 and TA98 tase, was not mutagenic to TA100 or TA98 with or without without S-9 mix, the latter showing more mutagenicity in S-9 mix, as shown in Table 4. 5-Fluorouracil had a strong TA98 than in TA100. Addition of S-9 mix reduced the lethal effect on cells at a concentration of 5 /¿g/plate activity. Clear dose-response activities of these compounds without S-9 mix. FT-207 also was not mutagenic to TA100 were obtained, as shown in Table 2. These compounds or TA98 with or without S-9 mix. These 2 compounds were have potent actions in producing malignant transformation also not mutagenic to TA92 with or without S-9 mix. Table 1 Trivial names, structures, mutagenicities, and carcinogenicities of antibiotics Trivial name Structure Mutagenicity Carcinogenicity Daunomycin-MCI + (14) Adriamycin-HOI + (14) OH NH2 Ft: —CH3, daunomycin —CHjOH, Adriamycin Bleomycin-MCI Glycopeptide Actinomycin D + (30) Peptide Peptide CO CO Mitomycin C° + (30) Mitomycin C is mutagenic to S. typhimurium TA92. JULY 1978 2149 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1978 American Association for Cancer Research. V. Se/no ef al. Table 2 Results of mutation tests on antibiotics his* revertants/plate TA100Drug"Daunomycin-HCIAdriamycin-HCIBleomycin-HCIActinomycin rowortantc/+ S-9mix144160 S-9mix286295 S-9mix24273362242827223155106423443252320221218172130212121221719-S-9mix340852 S-9mix4648494945282014484840806139-S-9 mix nmol*2800 (8,-)c3130(8, 0.420.830.020.050.10.20.5125100.941.884.79.418.82.46.112.20.011.02.5510+16314710913015215927030317018516899111125129106125100661471471107075103131-20214410630923498723620670190143100010390451321150000122TA98+1555136810232355610347762606445017187001815181626000017TA92fWo+ -)393732 02892434 DControl''Mitomycin 0323690120 CControl*"/itg/plate0.0830.17 43(2,-)1377226 " Drug contained some vehicle, and the amount of the drug was expressed as /*g of active principle.