Mutagenicity of Cancer Chemotherapeutic Agents in the Sa/Mone//A/Mi Erosome Test

[CANCER RESEARCH 37, 2209-2213, July 1977]

Mutagenicity of Cancer Chemotherapeutic Agents in the Sa/mon e//a/Mi erosome Test

William F. Benedict,1 Mary S. Baker, Lynne Haroun, Edmund Choi, and Bruce N. Ames2

Division ot Hematology-Oncology, Department of Medicine, Childrens Hospital, Los Angeles, California 90027 [W. F. B., M. S. B.Â¡,and Department oÃ Biochemistry, University of California, Berkeley, California 94720 Â¡L.H., E. C., B. N. A.Â¡

SUMMARY This study examines several of these agents in the Salmo- ne//a/microsome mutagenesis test system (3-5, 24, 27). Seventeen cancer Chemotherapeutic agents were tested This in vitro test makes use of a set of histidine mutants of S. for their ability to mutate Salmonella typhimurium tester typhimurium for detecting mutagens and a mammalian liver strains in the Sa/moneWa/microsome mutagenicity test. microsomal preparation for converting carcinogens to their There was a high correlation between the mutagenicity and active, mutagenic forms. The test system has been validated carcinogenicity of a given agent. Carcinogens positive in in several recent studies (23-25, 32, 43). the test were Adriamycin, daunomycin, 1-propanol-3,3'-iminodimethanesulfonate, cyclophosphamide, isophosphamide, hycanthone, chlornaphazin, nitrogen mustard, uracil MATERIALS AND METHODS mustard, melphalan, and thio-tepa. Two carcinogens, actinomycin D and bleomycin, were not detected as mutagens. Chemicals. The following drugs were obtained from The presumptive noncarcinogen, methotrexate, was nega Harry B. Wood, Drug Development Branch, Division of Can tive in the test. Tilorone and 6-mercaptopurine, tentatively cer Treatment, National Cancer Institute, Bethesda, Md.: classified as noncarcinogens, were mutagenic. The carci actinomycin D, Adriamycin, bleomycin, chlornaphazin, cy nogenicity of c/s-dichlorodiammineplatinum(ll), which was clophosphamide, isophosphamide, 6-mercaptopurine, positive in the test, has not been determined. methotrexate, 1-propanol-3,3'-iminodimethanesulfonate, thio-tepa [tris(1-aziridinyl)phosphine sulfide], and uracil mustard. Melphalan and hycanthone were obtained from INTRODUCTION Richard Adamson, Laboratory of Chemical Pharmacology, National Cancer Institute, Bethesda, Md., and tilorone was A number of short-term assays for screening potential received from Gerald Mayer, Merrell National Laboratories, chemical carcinogens/mutagens have been developed over Cincinnati, Ohio. Daunomycin-HCI was the gift of N. Ba- the last few years. Cancer Chemotherapeutic drugs are one chur, National Institutes of Health, Baltimore, Md. c/s-Di- group of agents that may be particularly suited for examin chlorodiammineplatinum(ll) was purchased from Strem ing the relationship among results from short-term in vitro Chemicals, Danvers, Maine, and nitrogen mustard was pur assays and in vivo carcinogenicity studies and chemical chased from Aldrich Chemical Co., Inc., Milwaukee, Wis. carcinogenesis in man. Several of these agents have been Mutagenesis Assay. Assays were performed as previ ously described (3-5). S. typhimurium tester strains TA1535, tested for their in vivo carcinogenicity, and some are sus TA1537, TA98, and TA100 were utilized (4, 27). The liver S-9 pected of being carcinogenic in man. The continued clinical fraction was prepared from male Sprague-Dawley rats (Hor- use of these agents may lead to further documentation of their carcinogenic potential. Recent studies on the ability of ton Animal Labs, Oakland, Calif.), given injections of various cancer Chemotherapeutic agents to produce mor Aroclor 1254 (Analabs, Inc., North Haven, Conn.) 5 days phological transformation in the C3H/10T/2 clone 8 mouse prior to sacrifice. The drugs were dissolved in water or cell line and rapid chromosomal damage in the A(T,)C1-3 dimethyl sulfoxide and added directly to the top agar at several concentrations ranging from nontoxic to highly cy- hamster cell line (6) have demonstrated that there is a high correlation between the ability of an agent to produce trans totoxic. formation or chromosomal damage and its capacity to be carcinogenic in vivo. RESULTS

Chemotherapeutic Drugs Mutagenic in the Salmonella 1 Recipient of Grant CA-14226 from the National Cancer Institute, NIH, and Test. Chart 1 indicates those drugs found positive for muta the Council for Tobacco Research, U.S.A., Inc., and Career Development Award CA-70996 from the National Cancer Institute. To whom requests for genesis in the Salmonella system. In cases where more than reprints should be addressed. 1 strain was reverted by the drug, that strain showing 2 Recipient of Energy Research and Development Administration Contract E (04-3)34, PA156. greatest activity was selected. Strain TA1535, sensitive to Received December 28, 1976; accepted April 15, 1977. chemicals causing base-pair substitutions, is reverted by

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0 4 8 10 048 0 800 0 200 400 0 800 1600

G H I J UJ cis-Dichlorodiamine- Hycanthone Chlornaphazin 6-Mercapto- Tilorone platinum purine

0 20 40 20 40 0 10 2O 30 2000 4000

1000 M N K Thio-TEPA 800 . Nitrogen Mustard Uracil Mustard . Melphalan 600 400 200 0 0 20 40 0 40 80 0 200 400 0 400 800 1200

jjg / PLATE

Chart 1. Dose-response curves of chemotherapeutic agents. Results shown in C toÂ£,/. and K to N are with strain TA1535; in A.B. and G with strain TA98; m F and H with TA100; and in J with TA1537 S-9 (20 Â¿Â¿I)wasadded for the activation of Chlornaphazin and melphalan: 300 M' were added for the activation of cyclophosphamide and isophosphamide. , without S-9; , with S-9. malphalan, thio-tepa, 1-propanol-3,3'-iminodimethanesul- phosphamide are active only in the presence of the S-9 fonate, and Â¡sophosphamide, and (as previously shown) by fraction. nitrogen mustard (2), uracil mustard (Ref. 25; H. Rosen Strain TA100 is the same as TA1535 but contains an R- kranz, personal communication), 6-mercaptopurine (16, factor plasmid (27). As previously reported, this strain is 33), and cyclophosphamide (Ref. 26 and M. Legator, per reverted by c/s-dichlorodiammineplatinum(ll) (29) and sonal communication). [Most of these results have been Chlornaphazin (25). Mutagenic activity with Chlornaphazin quoted in the test validation study (25)]. With the exception was increased in the presence of S-9. of 6-mercaptopurine, a purine analog, these drugs are all As previously shown, Adriamycin (Ref. 25; M. Legator, alkylating agents. The activity of melphalan is increased personal communication), daunomycin (5), and hycanthone approximately 3-fold by incubating in the presence of the (15, 25) revert the frameshift tester strain TA1538 and/or its liver S-9 preparation, while cyclophosphamide and iso R-factor derivative, strain TA98.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1977 American Association for Cancer Research. Mutagenicity of Chemotherapeutic Agents Tilorone at high concentrations (2 mg/plate) reverts the Sa//T7one//a/microsome system. In addition to being carci frameshift tester strain TA1537. nogenic in animal studies, some of the agents are sus Chemotherapeutic Drugs Not Mutagenic in the Salmo pected of being carcinogenic in man. Bladder tumors have nella Test. Actinomycin D, bleomycin, and (as previously been reported in patients receiving long-term treatment shown) methotrexate (16, 28, 48) were not mutagenic in with cyclophosphamide (46) [the urine of patients treated Salmonella. Each drug was tested at several concentrations with cyclophosphamide is mutagenic in Salmonella (28)] over a 100-fold range (actinomycin D, 0.01 to 50 /Â¿g/plate; and following treatment with Chlornaphazin (45). There is a bleomycin, 0.01 to 1.0 jug/plate; methotrexate, 10 to 1000 marked increase in acute myelogenous leukemia in patients /tg/plate), in which no plate was significantly higher than treated with melphalan for multiple myeloma (37), and sev controls, and no dose response was observed. All 4 tester eral cases of acute myeloblastic leukemia have been re strains were utilized, and each compound was tested both ported in patients receiving thio-tepa for the treatment of in the presence (20 /xl/plate) and absence of S-9. solid tumors (39). It will be important to determine whether Relation between Mutagenesis and Carcinogenesis. the other Chemotherapeutic agents that have been shown to The relationship between the ability of Chemotherapeutic be mutagenic in the Salmonella system also yield a high risk agents to mutate the Salmonella tester strains and their of primary or secondary cancers following exposure to known capacity to produce tumors in vivo is shown in Table these drugs. 1. In general, those agents that mutate the Salmonella tester A few of the carcinogenic cancer Chemotherapeutic strains are also carcinogenic. Only 2 agents, actinomycin D agents are not detected in the Salmonella test. Actinomycin and bleomycin, have been shown to be carcinogenic in vivo D and bleomycin [and also Natulan (Procarbazine) (25)] but were not mutagenic in Salmonella. Tilorone and 6- have not shown any mutagenic activity in the Salmonella mercaptopurine, which are mutagenic in Salmonella, are tester strains thus far. Actinomycin D was not cytotoxic to tentatively classified as noncarcinogens. the Salmonella at a concentration of 10 Â¿Â¿g/plate.There fore, it is likely that little actinomycin D is accumulated intracellularly in the bacteria. However, it has been shown DISCUSSION to be mutagenic in the dominant-lethal test in the mouse, to cause chromosomal damage, to be teratogenic (see Ref. Our studies show that 11 of 13 of the Chemotherapeutic 38), and to transform cells in culture (6). We are currently agents that are carcinogenic are also mutagenic in the examining the mutagenicity of these compounds in other Salmonella tester strains. Table 1 6-Mercaptopurine, tentatively classified as a noncarcinogen, is positive in the Salmonella test. In one study 6-mercap- Relationship between mutagenesis in Salmonella tester strains and Carcinogenesis in vivo produced by Chemotherapeutic agents topurine showed no carcinogenic activity when tested in Carcinogenic- rats (35), although, in studies with mice, lymphomas (11) Agent ity in vivo Ref. and lymphosarcomas (31) were observed. It is not certain if these tumors are due to the immunosuppressive effects of Positive for mutagenesis the drug or to a direct carcinogenic action. As the protocols Adriamycin 7, 22, 30 followed in these studies tend to reflect the clinical pattern Daunomycin 7, 22, 41 of administration and dosage, rather than to provide full Dimethanesulfonate 42 lifetime studies, further studies are needed to assess the full Cyclophosphamide 20, 36, 42, 47 carcinogenic potential of this compound. 6-Mercaptopurine Isophosphamide 42 c/s-Dichlorodiammineplat- ND" is mutagenic in the dominant-lethal assay in mice (13) and inum(ll) causes chromosome breakage in mouse bone marrow cells Hycanthone 14; D. Clayson, (17). However, exposure to 6-mercaptopurine in culture did Eppley Insti not transform cells or produce chromosome damage in a tute recent study by one of our groups (6). Also, 6-mercapto Chlornaphazin 19,45 Nitrogen mustard See Ref. 38 purine is, under certain conditions, a metabolite of azathio- Uracil mustard I, 42, 47 prine (Imuran). The latter drug is likewise used in cancer Melphalan 36,47 chemotherapy and is a mutagen in Salmonella (16, 40) and in Thio-tepa 35, 36, 42 mice, Drosophila, and Neurospora (8). It is presently un 6-Mercaptopurine I1, 31, 35 Tilorone R. Adamson, known whether the mutagenicity of azathioprine is direct or NCI a result of 1 or more of its metabolites. Tilorone, a presumptive noncarcinogen, is a very weak Negative for mutagenesis mutagen in Salmonella. Mutagenic activity is observed at a concentration approximately 200-fold higher than that which Actinomycin D 10, 18, 21, 44, 47 produces over a 90% decrease in plating efficiency after a Bleomycin 21 24-hr exposure to cell culture (6). The observed activity may Methotrexate6 34, 35 be due to an impurity present in the sample, although this " ND, not determined; -?, produces lymphomasand lymphosar- has not been determined. comas, possibly secondary to immunosuppression. Finally, the fact that the nitrogen mustard derivatives, * -, unknown at high-dose exposure (see Ref. 6). melphalan and Chlornaphazin, show increased activity in

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1977 American Association for Cancer Research. W. F. Benedict et al. the presence of S-9 is noteworthy, since these 2 alkylating 18. Kawamata. J.. Nakabayashi, N.. Kawai. A., and Ushida, T. Experimental Production of Sarcoma in Mice with Actinomycin. Med. J. Osaka Univ., agents are not considered to need liver activation to pro 8: 753-762, 1958. duce their cytotoxic effects. This has previously been 19. Koller. P. C. Dicentric Chromosomes in a Rat Tumor Induced by an shown for melphalan (28). Cyclophosphamide and isophos- Aromatic Nitrogen Mustard. Heredity, 6. 181-196, 1953. phamide, which are biologically inactive per se, are muta- 20. Kross, L. G., and Lavin, P. Effects of a Single Dose of Cyclophosphamide on Various Organs of the Rat. II. Response of Urinary Bladder Epithelium genie only in the presence of S-9. The metabolic pathway of According to Strain and Sex. J. Nati. Cancer Inst., 44: 1195-1200. 1970. 21. 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William F. Benedict, Mary S. Baker, Lynne Haroun, et al.

Cancer Res 1977;37:2209-2213.

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