Mutagenicity of Nitrosourea Compounds for Salmonella typhimurium: Brief Communication 1 ,2 Angela E. Auletta,3 Anne G. Martz,4 and Amar S. Parmar 5
ABSTRACT-The nitrosourea derivatives 1,3-bis{2-chloroethyl)1-nitrosou rea (NSC-409962), 1-{2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU; NSC-79037), 1-{2-chloroethyl)-3-{4-methylcyclohexyl)-1-nitrosourea (Me-CCNU; NSC-95441), and 1-methyl-1nitrosourea (MNU; NSC-23909) were screened for mutagenic potential with Salmonella typhimurium strains G46 and TA1530 in vitro and in vivo. All four agents were mutagenic in vitro; MNU, CCNU, and Me-CCNU were also mutagenic In the host-mediated assay. Four additional chemotherapeutic nitrosoureas were tested in vitro, three of which were mutagenic. The lack of activity of the fourth agent was probably a reflection of its stability in solution rather than a true indication of mutagenic potential. All eight agents were tested in a repair test with S. typhimurium strains TA1978 and TA1538. Results of this test were negative, reflecting the insensitivity of these strains to ethylating and methylating agents. The insensitivity of the host-mediated assay is also dlscussed.-J Natl Cancer Inst 60: 1495-1497, 1978.
The nitrosourea derivatives BCNU (NSC-409962), CCNU (NSC-79037), and Me-CCNU (NSC-95441) are potent antileukemic agents in mice (1) and have been used in the treatment of various neoplasms in man (24). MNU (NSC-23909), a known mutagenic and carcinogenic agent (5, 6), also has demonstrated therapeutic activity against experimentally induced tumors (7). These chemotherapeutic nitrosourea derivatives were tested for Salmonella typhimurium mutagenicity in vitro and in the host-mediated assay.
MATERIALS AND METHODS S. typhimurium strains G46 and TA 1530 were used for in vitro and in vivo testing. The in vitro mutagenesis assay was done according to the method of Ames et al. (8). The host-mediated assay was performed according to the method of Gabridge and Legator (9). BCNU was dissolved in a minimal amount of ethanol and brought to the volume necessary for testing with distilled water. All other drugs were suspended in HPC, the vehicle of administration for drug treatment in animals. For comparison, drugs were also dissolved in DMSO and used in the in vitro S. typhimurium assay. For the host-mediated assay, an 18-hour culture of S. typhimurium was diluted to give an optical density of 0.1 at 550 nm on a Beckman spectrophotometer. Then 2 ml of the diluted culture was administered ip to male Swiss albino mice weighing 20-25 g. Thirty, 60, and 120 minutes after inoculation of the bacteria, animals received im 25 mg drug/kg. Controls received a comparable amount of vehicle. Thirty minutes after the last im injection, animals were killed by cervical dislocation. The peritoneal VOL. 60. NO. 6. JUNE
Downloaded from https://academic.oup.com/jnci/article-abstract/60/6/1495/947874 by INSEAD user on 08 March 2018
fluid was removed and diluted from 10- 1 to 10- 7 in 0.85% saline. The three lowest dilutions were plated on Spizzen's minimal medium (10) with a plain agar overlay for the determination of the number of revertant colonies. The four highest dilutions were plated on minimal medium with an agar overlay that had been supplemented with 0.1 ml of 100 mM histidine for every 10 ml of overlay for the determination of the number of surviving bacteria. Viability was determined after 24 hours of culture at 37° C; the number of revertant colonies was scored after 48 hours at 37° C. The repair test was performed according to the method of Ames et al. (11) with S. typhimurium strains TAI978 and TA1538. Strain G46 was obtained from Dr. Errol Zeiger, N ational Institute of Environmental Health Sciences, Research Triangle Park, North Carolina. Strains TA1530, TA1538, and TAI978 were obtained from Dr. Bruce Ames, University of California, Berkeley, California. Drugs were obtained from the Drug Research and Development Branch, Division of Cancer Treatment, National Cancer Institute.
RESULTS AND DISCUSSION BCNU, CCNU, Me-CCNU, and MNU were tested in vitro and in vivo. Four other nitrosourea derivatives were tested in vitro only; all eight compounds were tested in a DNA repair test with S. typhimurium strains TAI538 and TA1978. Table I shows the results obtained in vitro with BCNU, CCNU, Me-CCNU, and MNU and S. typhimurium strains G46 and TA1530. The values in tables 1-3 are the averages of three plates per point. Background was not subtracted from the number of induced mutaABBREVIATIONS USED: BCNU = 1.3-bis(2-chloroethyl)-I-nitrosourea; CCNU = 1-(2-chloroethyl)-3-cyclohexyl-I-nitrosourea; DMSO = dimethyl sulfoxide; HPC = hydroxypropyl cellulose; LPS = lipopolysaccharide; Me-CCNU = 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-I-nitrosourea; MF = mutation frequency; MNU = I-methyl-I-nitrosourea.
I Received April 26.1977; revised January 11, 1978; accepted January 30, 1978, 2 Supported in part by Public Health Service contract NOI-CM33728 from the Division of Cancer Treatment, National Cancer Institute. 3 JRB Associates, Inc., 8400 Westpark Dr., McLean, Va. 22101. 4 The Johns Hopkins University Medical School, Baltimore, Md. 21205. 5 Mutagenesis and Transformation Screening Service, Microbiological Associates, Bethesda, Md. 20016.
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TABLE I.-In vitro mutagenicity ofnitrosourea derivatives for S. typhimurium Compound
Concentration, JLg/plate _b
_b
BCNU
CCNU
Me-CCNU
MNU
1 10 50 100 500 1,000 1 10 50 100 500 1,000 1 10 50 100 500 1,000 1 10 50 100 500 1,000
His+ revertants/plate"
G46
TA1530
3 3 4 7 82 135 160 3 2 8 82 110 120 1 3 18 110 130 140 5 20 380 947 2,500 4,800
35 47 56 65 89 210 250 52 54 57 83 150 140 49 60 66 140 205 250 50 87 200 420 4,000 9,950
" These values are the averages of three plates per point. b No compound was used: control or spontaneous reversion plates.
tions. Both strains had the same HisG46 mutation in the gene that was reverted by agents that caused base-pair substitutions. Strain TA1530 had a partially deleted LPS coat and lacked excision repair, whereas strain G46 had a full LPS coat and an excision repair system (12). The difference in the sensitivity of either strain to the action of these agents did not seem significant, although T Al530 did appear to be more sensitive to MNU at higher concentrations. The assay, as it was performed in this study, made no provision for determining cell kill, and the MF (No. of mutants/No. of survivors) could not be calculated for these experiments. It may be that, if the MF had been known and plotted, some differential strain sensitivity might have been noted. Greater mutagenicity was observed with either ethanol-water or HPC than with DMSO, perhaps due to the stability of the test agents in DMSO or to impurities in the solvent that may have had an adverse effect on the drug. MNU is known to be a highly reactive compound with increased stability at an acidic pH and, therefore, would be expected to be more stable in KH 2 P0 4 buffer (pH 4.0) than in DMSO. While this paper was in preparation, Zimmer and Bhuyan (13) published a report on the mutagenicity of nitrosourea derivatives in which they noted the high toxicity but low mutagenic activity of BCNU, CCNU, and a related agent, 2-[3-(2-chloroethyl)-3-nitrosoureido ]-2-deoxy-n-glucosopyranose, when these agents were dissolved in DMSO. This finding is in essential agreement with our observations on this solvent. Zimmer and Bhuyan also reported the increased sensitivity J NATL CANCER INST
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of log-phase cultures of bacteria to the action of streptozotocin and MNU. We have found this to be true of the nitrosoureas in general. Eighteen-hour cultures of bacteria were more sensitive to the action of these agents than were cultures that had been stored at 4° C for any length of time. Although no difference was apparent in the number of spontaneous revertants with 4° C storage of the culture, the number of induced mutants decreased proportionately with the age of the culture. Four additional nitrosourea derivatives, 3-(l-adamantyl)-I-(2-chloroethyl)-I-nitrosourea (NSC-86056), 3-( 1adaman tyl )-1-(2 -fl uoroeth yI)-1-nitrosourea (N SC-93161 ) , 1,1' -( 4 ,4'biphenylylenedimethylene)bis[3-(2-chloroethyl)-3-nitrosourea (NSC-116821), and l,l' -(1 ,4-cyclohexylenedimethylene)bis[3-(2-chloroethyl)-3-nitrosourea (NSC-120337) were tested for their in vitro mutagenic potential with strain G46 (table 2). Three of the four (NSC-86056, NSC-93161, and NSC-116821) were mutagenic for this strain to approximately the same extent as were BCNU, CCNU, and Me-CCNU. The fourth compound, NSC-120337, was inactive in this system. However, this may have been an indication of the instability of this compound in the test system rather than a true reflection of its mutagenic potential. The repair test, designed to measure differences in cell kill in strains with and without DNA repair enzymes, gave essentially negative results with the agents mentioned in this study; i.e., the zone of inhibition was not increased with strain TA1538, which lacks repair enzymes, over that seen with strain TA1978, which has no excision repair system. However, the repair test with these two strains was insensitive to ethylating and methylating agents (11), and all these agents possessed either or both a potentially reactive ethyl or methyl group. The nitrosoureas, especially BCNU, CCNU, and MeCCNU, show wide variation in their physicochemical properties, including differing carbamoylating and alkylating properties (14). In in vitro mammalian systems TABLE 2.-Mutagenicity of some nitrosourea derivatives for S. typhimurium strain G46 Compound _b
NSC-86056
NSC-93161
NSC-116821
NSC-120337
a b
Concentration, JLg/plate _b
1 10 100 1,000 1 10 100 1,000 1 10100 1,000 1 10 100 1,000
His+ revertants/plate for strain G46 a 1.5 1.5 9.5 47.5 125 5 31.5 47.5 101.5 13 31.5 65 137.5 2.5 1.5 4 1
These values are the averages of three plates per point. No compound was used: control or spontaneous reversion plates.
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and in animal model systems, various breakdown products of each agent are probably responsible for cell kill. In this system, however, all these agents appear to be behaving in a similar manner and this behavior is related to the presence of reactive ethyl or methyl groups. MNU has been reported to give positive results in a repair test with polA + and polA - strains of Escherichia coli (15). This system may, therefore, be especially suited to the testing of the nitrosourea derivatives mentioned here. Table 3 shows the results of the host-mediated assay with BCNU, CCNU, Me-CCNU, and MNU with strains G46 and TA1530. As reported in (16), MNU was clearly mutagenic for S. typhimurium in the host-mediated assay. CCNU was mutagenic for strain G46, and Me-CCNU exhibited some slight activity for strain T A1530. The apparent inactivity of BCNU in this system and the seeming discrepancy between strains G46 and TA 1530 with CCNU and Me-CCNU may have been more of an indictment of the test system than a true test of the in vivo mutagenic potential of these agents. The variables inherent in the in vivo system, e.g., drug concentration in the peritoneal cavity, animal strain insensitivity, less than optimum amounts of drug administered, failure of the drug or its active metabolites to reach the bacteria in effective amounts, or administration of either drug or bacteria by the wrong route, may have resulted in falsenegative or seemingly incongruous results with these agents. The data presented here indicate that these chemotherapeutic nitrosourea derivatives possess mutagenic TABLE 3.-Mutagenicity ofnitrosourea derivatives for S. typhimurium in the host-mediated assay Compound
S. typhimurium strain
Controls BC NU CCNU Me-CCNU MNU
G46 TA1530 G46 TA1530 G46 TA1530 G46 TA1530 G46 TA1530
No. of survivors
No. of revertants
No. of revertants/H)" survivors a
1x10" 1.6x 10" 2.4x 10" 3x10" 1.1 x 10" 1 x 10" 3x10" 4x10" 3x 10" 3 x 10"
100 0 100 0 800 0 100 100 1,200 3,000
100 0 41.7 0 727.3" 0 33.33 25" 400· 1,000·
These values are the averages of three plates per point. " Values significantly above those of the controls.
a
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potential in vivo and in vitro. In addition, we have recently demonstrated (manuscript in preparation) the transforming potential of one of these agents, MNU, a known carcinogen (6), for mammalian cells in culture. Due consideration should be given to these facts when a toxicologic evaluation of these agents is made.
REFERENCES (1) BURCHENAL JH, CARTER SK: New cancer chemotherapeutic agents. Cancer 30: 1639-1646, 1972 (2) DEVITA VT, CARBONE PP, OWENS AB JR, et al: Clinical trials with 1.,3-bis(2-chloroethyl)-I-nitrosourea, NSC-409962, Cancer Res 25:1876-1881,1965 (3) HANSON HH, SELAWAY OS, MUGGIA FM, et al: Clinical studies with 1-(2-chloroethyl)-3-cyclohexyl-I-nitrosourea (NSC-79037), Cancer Res 31:223-227,1971 (4) YOUNG RC, WALKER MD, CANELLOS GP, et al: Initial clinical trials with methyl-CCNU 1-(2-chloroethyl)-3-(4-methyl cyclohexyl)-I-nitrosourea (MeCCNU), Cancer 31: 1164-1169, 1973 (5) International Agency for Research on Cancer: The Evaluation of Carcinogenic Risk of Chemicals to Man. IARC Monogr I: 125134, 1972 (6) GICHNER T, VELEMINSKY J, POKORNY V: Recovery or increase of induced genetical effects dependent upon the moisture content of stored N-methyl-N-nitrosourea-treated barley seeds. Mutat Res 16:35-40, 1972 (7) HANSEN HH, MUGGIS FM, WALKER MD, et al: Treatment of malignant brain tumors with nitrosoureas. Cancer Chemother Rep 55:99-107,1971 (8) AMEs BN, McCANN J, YAMASAKI E: Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutat Res 31:347-364,1975 (9) GAB RIDGE MG, LEGATOR MS: A host-mediated microbial assay for the detection of mutagenic compounds. Proc Soc Exp BioI Med 130:831-834, 1969 (10) SPIZZEN J: Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc Natl Acad Sci USA 44:1072-1078,1958 (11) AMEs BN, LEE FD, DURSTON WE: An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc Natl Acad Sci USA 72:979-983, 1975 (12) AMEs BN: The detection of chemical mutagens with enteric bacteria. In Chemical Mutagens. Principles and Methods for Their Detection (Hollaender A, ed). New York-London: Plenum Press, 1971, pp 267-282 (13) ZIMMER DM, BHUYAN BK: Mutagenicity of streptozotocin and several other nitrosourea compounds in Salmonella typhimurium. Mutat Res 40:281-288, 1976 (14) OLIVERIO VT: Toxicology and pharmat:ology of the nitrosoureas. Cancer Chemother Rep 4: 13-20, 1973 (15) SLATER EE, ANDERsoN MD, ROSENKRANZ HS: Rapid detection of mutagens and carcinogens. Cancer Res 31 :970-973, 1971 (16) ROHRBoRN G: Nitrosomethylurea. In Chemical Carcinogenesis Essays (Montesano R, Tomatis L, eds). Lyon, France: IARC, 1974, pp 213-219
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ABSTRACT-The nitrosourea derivatives 1,3-bis{2-chloroethyl)1-nitrosou rea (NSC-409962), 1-{2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU; NSC-79037), 1-{2-chloroethyl)-3-{4-methylcyclohexyl)-1-nitrosourea (Me-CCNU; NSC-95441), and 1-methyl-1nitrosourea (MNU; NSC-23909) were screened for mutagenic potential with Salmonella typhimurium strains G46 and TA1530 in vitro and in vivo. All four agents were mutagenic in vitro; MNU, CCNU, and Me-CCNU were also mutagenic In the host-mediated assay. Four additional chemotherapeutic nitrosoureas were tested in vitro, three of which were mutagenic. The lack of activity of the fourth agent was probably a reflection of its stability in solution rather than a true indication of mutagenic potential. All eight agents were tested in a repair test with S. typhimurium strains TA1978 and TA1538. Results of this test were negative, reflecting the insensitivity of these strains to ethylating and methylating agents. The insensitivity of the host-mediated assay is also dlscussed.-J Natl Cancer Inst 60: 1495-1497, 1978.
The nitrosourea derivatives BCNU (NSC-409962), CCNU (NSC-79037), and Me-CCNU (NSC-95441) are potent antileukemic agents in mice (1) and have been used in the treatment of various neoplasms in man (24). MNU (NSC-23909), a known mutagenic and carcinogenic agent (5, 6), also has demonstrated therapeutic activity against experimentally induced tumors (7). These chemotherapeutic nitrosourea derivatives were tested for Salmonella typhimurium mutagenicity in vitro and in the host-mediated assay.
MATERIALS AND METHODS S. typhimurium strains G46 and TA 1530 were used for in vitro and in vivo testing. The in vitro mutagenesis assay was done according to the method of Ames et al. (8). The host-mediated assay was performed according to the method of Gabridge and Legator (9). BCNU was dissolved in a minimal amount of ethanol and brought to the volume necessary for testing with distilled water. All other drugs were suspended in HPC, the vehicle of administration for drug treatment in animals. For comparison, drugs were also dissolved in DMSO and used in the in vitro S. typhimurium assay. For the host-mediated assay, an 18-hour culture of S. typhimurium was diluted to give an optical density of 0.1 at 550 nm on a Beckman spectrophotometer. Then 2 ml of the diluted culture was administered ip to male Swiss albino mice weighing 20-25 g. Thirty, 60, and 120 minutes after inoculation of the bacteria, animals received im 25 mg drug/kg. Controls received a comparable amount of vehicle. Thirty minutes after the last im injection, animals were killed by cervical dislocation. The peritoneal VOL. 60. NO. 6. JUNE
Downloaded from https://academic.oup.com/jnci/article-abstract/60/6/1495/947874 by INSEAD user on 08 March 2018
fluid was removed and diluted from 10- 1 to 10- 7 in 0.85% saline. The three lowest dilutions were plated on Spizzen's minimal medium (10) with a plain agar overlay for the determination of the number of revertant colonies. The four highest dilutions were plated on minimal medium with an agar overlay that had been supplemented with 0.1 ml of 100 mM histidine for every 10 ml of overlay for the determination of the number of surviving bacteria. Viability was determined after 24 hours of culture at 37° C; the number of revertant colonies was scored after 48 hours at 37° C. The repair test was performed according to the method of Ames et al. (11) with S. typhimurium strains TAI978 and TA1538. Strain G46 was obtained from Dr. Errol Zeiger, N ational Institute of Environmental Health Sciences, Research Triangle Park, North Carolina. Strains TA1530, TA1538, and TAI978 were obtained from Dr. Bruce Ames, University of California, Berkeley, California. Drugs were obtained from the Drug Research and Development Branch, Division of Cancer Treatment, National Cancer Institute.
RESULTS AND DISCUSSION BCNU, CCNU, Me-CCNU, and MNU were tested in vitro and in vivo. Four other nitrosourea derivatives were tested in vitro only; all eight compounds were tested in a DNA repair test with S. typhimurium strains TAI538 and TA1978. Table I shows the results obtained in vitro with BCNU, CCNU, Me-CCNU, and MNU and S. typhimurium strains G46 and TA1530. The values in tables 1-3 are the averages of three plates per point. Background was not subtracted from the number of induced mutaABBREVIATIONS USED: BCNU = 1.3-bis(2-chloroethyl)-I-nitrosourea; CCNU = 1-(2-chloroethyl)-3-cyclohexyl-I-nitrosourea; DMSO = dimethyl sulfoxide; HPC = hydroxypropyl cellulose; LPS = lipopolysaccharide; Me-CCNU = 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-I-nitrosourea; MF = mutation frequency; MNU = I-methyl-I-nitrosourea.
I Received April 26.1977; revised January 11, 1978; accepted January 30, 1978, 2 Supported in part by Public Health Service contract NOI-CM33728 from the Division of Cancer Treatment, National Cancer Institute. 3 JRB Associates, Inc., 8400 Westpark Dr., McLean, Va. 22101. 4 The Johns Hopkins University Medical School, Baltimore, Md. 21205. 5 Mutagenesis and Transformation Screening Service, Microbiological Associates, Bethesda, Md. 20016.
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TABLE I.-In vitro mutagenicity ofnitrosourea derivatives for S. typhimurium Compound
Concentration, JLg/plate _b
_b
BCNU
CCNU
Me-CCNU
MNU
1 10 50 100 500 1,000 1 10 50 100 500 1,000 1 10 50 100 500 1,000 1 10 50 100 500 1,000
His+ revertants/plate"
G46
TA1530
3 3 4 7 82 135 160 3 2 8 82 110 120 1 3 18 110 130 140 5 20 380 947 2,500 4,800
35 47 56 65 89 210 250 52 54 57 83 150 140 49 60 66 140 205 250 50 87 200 420 4,000 9,950
" These values are the averages of three plates per point. b No compound was used: control or spontaneous reversion plates.
tions. Both strains had the same HisG46 mutation in the gene that was reverted by agents that caused base-pair substitutions. Strain TA1530 had a partially deleted LPS coat and lacked excision repair, whereas strain G46 had a full LPS coat and an excision repair system (12). The difference in the sensitivity of either strain to the action of these agents did not seem significant, although T Al530 did appear to be more sensitive to MNU at higher concentrations. The assay, as it was performed in this study, made no provision for determining cell kill, and the MF (No. of mutants/No. of survivors) could not be calculated for these experiments. It may be that, if the MF had been known and plotted, some differential strain sensitivity might have been noted. Greater mutagenicity was observed with either ethanol-water or HPC than with DMSO, perhaps due to the stability of the test agents in DMSO or to impurities in the solvent that may have had an adverse effect on the drug. MNU is known to be a highly reactive compound with increased stability at an acidic pH and, therefore, would be expected to be more stable in KH 2 P0 4 buffer (pH 4.0) than in DMSO. While this paper was in preparation, Zimmer and Bhuyan (13) published a report on the mutagenicity of nitrosourea derivatives in which they noted the high toxicity but low mutagenic activity of BCNU, CCNU, and a related agent, 2-[3-(2-chloroethyl)-3-nitrosoureido ]-2-deoxy-n-glucosopyranose, when these agents were dissolved in DMSO. This finding is in essential agreement with our observations on this solvent. Zimmer and Bhuyan also reported the increased sensitivity J NATL CANCER INST
Downloaded from https://academic.oup.com/jnci/article-abstract/60/6/1495/947874 by INSEAD user on 08 March 2018
of log-phase cultures of bacteria to the action of streptozotocin and MNU. We have found this to be true of the nitrosoureas in general. Eighteen-hour cultures of bacteria were more sensitive to the action of these agents than were cultures that had been stored at 4° C for any length of time. Although no difference was apparent in the number of spontaneous revertants with 4° C storage of the culture, the number of induced mutants decreased proportionately with the age of the culture. Four additional nitrosourea derivatives, 3-(l-adamantyl)-I-(2-chloroethyl)-I-nitrosourea (NSC-86056), 3-( 1adaman tyl )-1-(2 -fl uoroeth yI)-1-nitrosourea (N SC-93161 ) , 1,1' -( 4 ,4'biphenylylenedimethylene)bis[3-(2-chloroethyl)-3-nitrosourea (NSC-116821), and l,l' -(1 ,4-cyclohexylenedimethylene)bis[3-(2-chloroethyl)-3-nitrosourea (NSC-120337) were tested for their in vitro mutagenic potential with strain G46 (table 2). Three of the four (NSC-86056, NSC-93161, and NSC-116821) were mutagenic for this strain to approximately the same extent as were BCNU, CCNU, and Me-CCNU. The fourth compound, NSC-120337, was inactive in this system. However, this may have been an indication of the instability of this compound in the test system rather than a true reflection of its mutagenic potential. The repair test, designed to measure differences in cell kill in strains with and without DNA repair enzymes, gave essentially negative results with the agents mentioned in this study; i.e., the zone of inhibition was not increased with strain TA1538, which lacks repair enzymes, over that seen with strain TA1978, which has no excision repair system. However, the repair test with these two strains was insensitive to ethylating and methylating agents (11), and all these agents possessed either or both a potentially reactive ethyl or methyl group. The nitrosoureas, especially BCNU, CCNU, and MeCCNU, show wide variation in their physicochemical properties, including differing carbamoylating and alkylating properties (14). In in vitro mammalian systems TABLE 2.-Mutagenicity of some nitrosourea derivatives for S. typhimurium strain G46 Compound _b
NSC-86056
NSC-93161
NSC-116821
NSC-120337
a b
Concentration, JLg/plate _b
1 10 100 1,000 1 10 100 1,000 1 10100 1,000 1 10 100 1,000
His+ revertants/plate for strain G46 a 1.5 1.5 9.5 47.5 125 5 31.5 47.5 101.5 13 31.5 65 137.5 2.5 1.5 4 1
These values are the averages of three plates per point. No compound was used: control or spontaneous reversion plates.
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and in animal model systems, various breakdown products of each agent are probably responsible for cell kill. In this system, however, all these agents appear to be behaving in a similar manner and this behavior is related to the presence of reactive ethyl or methyl groups. MNU has been reported to give positive results in a repair test with polA + and polA - strains of Escherichia coli (15). This system may, therefore, be especially suited to the testing of the nitrosourea derivatives mentioned here. Table 3 shows the results of the host-mediated assay with BCNU, CCNU, Me-CCNU, and MNU with strains G46 and TA1530. As reported in (16), MNU was clearly mutagenic for S. typhimurium in the host-mediated assay. CCNU was mutagenic for strain G46, and Me-CCNU exhibited some slight activity for strain T A1530. The apparent inactivity of BCNU in this system and the seeming discrepancy between strains G46 and TA 1530 with CCNU and Me-CCNU may have been more of an indictment of the test system than a true test of the in vivo mutagenic potential of these agents. The variables inherent in the in vivo system, e.g., drug concentration in the peritoneal cavity, animal strain insensitivity, less than optimum amounts of drug administered, failure of the drug or its active metabolites to reach the bacteria in effective amounts, or administration of either drug or bacteria by the wrong route, may have resulted in falsenegative or seemingly incongruous results with these agents. The data presented here indicate that these chemotherapeutic nitrosourea derivatives possess mutagenic TABLE 3.-Mutagenicity ofnitrosourea derivatives for S. typhimurium in the host-mediated assay Compound
S. typhimurium strain
Controls BC NU CCNU Me-CCNU MNU
G46 TA1530 G46 TA1530 G46 TA1530 G46 TA1530 G46 TA1530
No. of survivors
No. of revertants
No. of revertants/H)" survivors a
1x10" 1.6x 10" 2.4x 10" 3x10" 1.1 x 10" 1 x 10" 3x10" 4x10" 3x 10" 3 x 10"
100 0 100 0 800 0 100 100 1,200 3,000
100 0 41.7 0 727.3" 0 33.33 25" 400· 1,000·
These values are the averages of three plates per point. " Values significantly above those of the controls.
a
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potential in vivo and in vitro. In addition, we have recently demonstrated (manuscript in preparation) the transforming potential of one of these agents, MNU, a known carcinogen (6), for mammalian cells in culture. Due consideration should be given to these facts when a toxicologic evaluation of these agents is made.
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