Mutation Research, 68 (1979) 1--8
© Elsevier/North-Holland Biomedical Press
THE MUTAGENICITY OF NITROSAMIDES IN S A L M O N E L L A TYPHIMURIUM
w. LIJINSKY and A.W. ANDREWS Chemical Carcinogenesis Program, Frederick Cancer Research Center, Frederick, MD 21701 (U.S.A.)
(Received 28 November 1978) (Revision received 6 April 1979) (Accepted 23 April 1979)
Summary 34 nitrosamides (10 nitrosoalkylcarbamates, 2 nitrosoalkylnitroguanidines, 12 nitrosoalkylureas, 6 substituted nitrosoalkylureas, and 4 cyclic nitrosoalkylureas) were tested for mutagenicity in Salmonella. All were direct-acting mutagens of varying potency.
The mutagenicity of a chemical is an index of its biological activity, as is carcinogenicity. It has been suggested that the mechanistic connection between the two types of activity is due to the formation of a reactive electrophilic species [10]. The nitrosamides are one large group of compounds which includes a n u m b e r o f highly active, direct-acting mutagens. Some of these have been tested for carcinogenic activity in rats [3,6]; others are being tested by longterm administration to mice. A comparison between the results of the animal and bacterial tests m a y lead to a better understanding o f the relationship between carcinogenicity and mutagenicity. An examination of the mutagenicity o f the large group o f nitrosamides reported here m a y shed considerable light on the nature of the mechanism o f nitrosamide mutagenesis in bacteria. Later, a comparison can be made with chemical properties, including the relative ease with which these compounds give rise to biologically active alkylating moieties. The nitrosamides in this study comprise nitrosoalkylcarbamate esters, m a n y o f which are derivatives of insecticides and have been shown to interact with h u m a n DNA [2], nitrosoalkylnitroguanidines and a large n u m b e r o f nitrosoalkylureas, including several with substituents in the alkyl group. A few of these
R e s e a r c h sponsored b y the N a t i o n a l C a n c e r Institute under Contract No. N O 1 - C O - 7 5 3 S 0 w i t h Litton Bionetics, I n c .
c o m p o u n d s have been tested previously for mutagenicity in Salmonella, and other organisms, by us and b y others [4,5], b u t it has been difficult to draw firm conclusions a b o u t mechanisms. For convenience of discussion, the c o m p o u n d s have been divided into 5 groups, each containing c o m p o u n d s of similar structure; the division is somewhat arbitrary, however. Materials and methods
Chemicals The preparation of the nitrosoalkylcarbamates b y nitrosation of the corresponding N-alkylcarbamate esters has been described [7]; N-methylphenylcarbamate was synthesized from methyl isocyanate and phenol. The nitrosoalkyinitroguanidines were prepared as previously described [9]. All of the nitrosoalkylureas were prepared by nitrosation o f the alkylureas synthesized b y a standard procedure from the appropriate amine hydrochloride and potassium cyanate. Details of the preparation and physical constants of those c o m p o u n d s which have not been described previously will be given elsewhere, b u t the general m e t h o d was as follows: 0.05 mole o f the amine hydrochloride (or of the amine neutralized with the equivalefit quantity of hydrochloric acid) was dissolved in 20 ml of water. Potassium cyanate, 4 g (0.05 mole) was added and the solution was boiled gently for 15--20 min on a h o t plate. After cooling, 10 ml of 5 N sulfuric acid was added to the mixture (in the case of phenyl-, cyclohexyl-, phenylethyl-, hexyl-, undecyl- and tridecylurea, 20 ml of acetic acid was added instead of sulfuric acid, because of their insolubility in dilute sulfuric acid). The solutions were cooled in ice and 3.5 g o f sodium nitrite added slowly with gentle stirring. After allowing the reaction to proceed for 20 min, nitrosourea formation was essentially complete. The isolation o f the p r o d u c t t o o k place in one of three ways (a) the nitrosourea was filtered off, washed with cold water and allowed to dry in air on filter paper for approximately 1 h, or (b) the acetic acid solutions were diluted with 2 vol. of water and the precipitate filtered off, washed with water and dried in air, or (c) the solutions of the soluble nitrosoalkylureas in dilute sulfuric acid, chloroethyl-, bromoethyl-, hydroxyethyl-, isopropanol-, 3-hydroxypropyl-, isopropyl- and sec-butyl-, were extracted with 60 ml ethyl acetate. The extract was shaken twice with a small volume o f water, dried with magnesium sulfate and evaporated to dryness in a rotary evaporator at 40 ° C. The residue was then crystallized in the freezer and squeezed dry on filter paper. The nitrosamides were stored at --20 ° C, at which temperature they were all fairly stable.
Mutagenicity tests In this study, all of the c o m p o u n d s were tested in the Salmonella mutagenicity test described b y Ames et al. [1]. Tester strain T A 1 5 3 5 was selected as the most suitable for dose-response studies. The one exception was nitrosophenylurea for which strain T A 1 5 3 7 was used. Although n o t essential for these direct-acting compounds, a microsomal liver fraction from rats stimulated with Aroclor 1254 was included in the tests.
The nitrosoureas were dissolved in dimethylsulfoxide (DMSO) and serially diluted so that each dose-response was carried o u t over a 1000-fold range at doses o f 1000, 500, 250, 100, 50, 25, 10, 5, 2.5 and 1/~g/0.1 ml. To 2 ml of molten t o p agar was added 0.2 ml of the tester strain, 0.1 ml of test c o m p o u n d solution and, when needed, 0.5 ml of $9 mix (75 ~1 of $9 fraction). This was then poured on to the surface of 20 ml o f VBE agar and the plates were incubated in the dark for 48 h. Revertant colonies were counted using a hand-held tally. F o r convenience, dose-response curves were plotted on a logarithmic scale. The revertant colonies per #mole were calculated by selecting from the ascending portion of the individual dose--response curves the n u m b e r of revertants at 100/~g. These figures and the molecular weight were then used to calculate the revertants per/~mole.
Carcinogenicity tests The nitrosoalkylcarbamates were administered to male and female SpragueDawley rats b y gavage in vegetable~il solution at approximately equimolar doses for a given time, after which they were allowed to die naturally. The pattern of death o f the rats with tumors was an index of carcinogenic potency, which was assumed to decrease as the survival increased. The nitrosoalkylureas are being painted on the skin of female Swiss mice as 0.04 M solutions in acetone, two applications a week for 50 weeks, after which the animals will be observed until skin tumors appear. These experiments are in progress. Results and discussion All of the nitrosamides tested were direct-acting and mutagenic in strain TA1535 o f Salmonella typhimurium, with a single exception, nitrosophenylurea; its mutagenicity was best demonstrated with strain TA1537. The effect o f incubation with the rat-liver microsomai preparation was to inactivate the nitrosamide and decrease mutagenicity or to leave it unaffected; there was never an increase in mutagenic activity. Representative dose--response curves are shown in Fig. 1 where the linear regression was done b y least-square analysis. Two examples are given for each group o f nitrosamides. Nitrosolandrin is representative of all the nitrosoalkylcarbamates except nitrosoethylurethane. Nitrosoethylurethane was n o t toxic at the highest dose tested while all other c o m p o u n d s were toxic or had given m a x i m u m reversion at 10--25 ~g. Maximum mutagenesis for nitrosomethylnitroguanidine was reached between 10 and 25 #g while nitrosoethylnitroguanidine was bacteriocidal at 50 #g. The nitrosoethylurea curve represents the nitrosoalkylureas with the exception of nitrosomethylurea. Nitrosoethylurea did n o t cause significant reversion until the dosage was between 100--200 ~g whereas nitrosomethylurea was mutagenic b e t w e e n 1 and 2.5/~g. For the substituted nitrosoalkylureas, nitrosochloroethylurea is representative except for nitroso-3-hydroxypropylurea. No toxicity was n o t e d for these c o m p o u n d s over the 1000-fold dose range we normally use. Nitroso-3-hydroxypropylurea did n o t cause reversion until the dose level was at least 2 5 #g. The other c o m p o u n d s
104
THE MUTAGENICITY OF NITROSAMIDES IN SALMONELLA TYPHIMURIUM STRAIN TA 1535 NITROSOCARBAMATES NITROSOLANDRIN NITROSOETHYLURETHANE
/..
J
103 102 1C 105
,
104 /
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o, 0 I,Z ,,¢ p,.
IM
Z
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101 NITROSOALKYLUREAS NITROSOETHYLUREA 104 NITROSOMETNYLUREA 103
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102
Z
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103
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,
NITROSOALKYLGUANIDINES NITROSOMETHYLNITROGUANIDINE NITROSOETHYLNITROGUANIDINE
102 101
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,
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L
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SUBSTITUTEDNITROSOALKYLUREAS 104 NITROSOCHLOROETHYLUREA NITROSO-3-HYDROXYPROPYLUREA
105 "
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102 10
CYCLICNITROSOALKYLUREAS REA NITROSOCYCLOHEXYLUREA 10~ NITROSOPHENYLETHYLU 103 102 10 100
101
102
1;3 100
101
102
103
DOSE (~g) Fig. I . T h e m u t a g e n i c i t y o f n i t r o s a m i d e s in SaZmonetia t y p h i m u r i u m strain T A 1 5 3 5 . T h e m e a n n u m b e r o f b a c k g r o u n d r e v e z t a n t s w a s 2 5 + 8 . 7 3 f o r T A 1 5 3 5 a n d 15 -+ 3 . 6 9 f o r T A 1 5 3 7 .
H c.J O-C-N/NO
CH~, NO-CARBARYL
II
~CH 3
0 NO--CARBOFURAN
~ 0 _ {ZH3)3
N
© Elsevier/North-Holland Biomedical Press
THE MUTAGENICITY OF NITROSAMIDES IN S A L M O N E L L A TYPHIMURIUM
w. LIJINSKY and A.W. ANDREWS Chemical Carcinogenesis Program, Frederick Cancer Research Center, Frederick, MD 21701 (U.S.A.)
(Received 28 November 1978) (Revision received 6 April 1979) (Accepted 23 April 1979)
Summary 34 nitrosamides (10 nitrosoalkylcarbamates, 2 nitrosoalkylnitroguanidines, 12 nitrosoalkylureas, 6 substituted nitrosoalkylureas, and 4 cyclic nitrosoalkylureas) were tested for mutagenicity in Salmonella. All were direct-acting mutagens of varying potency.
The mutagenicity of a chemical is an index of its biological activity, as is carcinogenicity. It has been suggested that the mechanistic connection between the two types of activity is due to the formation of a reactive electrophilic species [10]. The nitrosamides are one large group of compounds which includes a n u m b e r o f highly active, direct-acting mutagens. Some of these have been tested for carcinogenic activity in rats [3,6]; others are being tested by longterm administration to mice. A comparison between the results of the animal and bacterial tests m a y lead to a better understanding o f the relationship between carcinogenicity and mutagenicity. An examination of the mutagenicity o f the large group o f nitrosamides reported here m a y shed considerable light on the nature of the mechanism o f nitrosamide mutagenesis in bacteria. Later, a comparison can be made with chemical properties, including the relative ease with which these compounds give rise to biologically active alkylating moieties. The nitrosamides in this study comprise nitrosoalkylcarbamate esters, m a n y o f which are derivatives of insecticides and have been shown to interact with h u m a n DNA [2], nitrosoalkylnitroguanidines and a large n u m b e r o f nitrosoalkylureas, including several with substituents in the alkyl group. A few of these
R e s e a r c h sponsored b y the N a t i o n a l C a n c e r Institute under Contract No. N O 1 - C O - 7 5 3 S 0 w i t h Litton Bionetics, I n c .
c o m p o u n d s have been tested previously for mutagenicity in Salmonella, and other organisms, by us and b y others [4,5], b u t it has been difficult to draw firm conclusions a b o u t mechanisms. For convenience of discussion, the c o m p o u n d s have been divided into 5 groups, each containing c o m p o u n d s of similar structure; the division is somewhat arbitrary, however. Materials and methods
Chemicals The preparation of the nitrosoalkylcarbamates b y nitrosation of the corresponding N-alkylcarbamate esters has been described [7]; N-methylphenylcarbamate was synthesized from methyl isocyanate and phenol. The nitrosoalkyinitroguanidines were prepared as previously described [9]. All of the nitrosoalkylureas were prepared by nitrosation o f the alkylureas synthesized b y a standard procedure from the appropriate amine hydrochloride and potassium cyanate. Details of the preparation and physical constants of those c o m p o u n d s which have not been described previously will be given elsewhere, b u t the general m e t h o d was as follows: 0.05 mole o f the amine hydrochloride (or of the amine neutralized with the equivalefit quantity of hydrochloric acid) was dissolved in 20 ml of water. Potassium cyanate, 4 g (0.05 mole) was added and the solution was boiled gently for 15--20 min on a h o t plate. After cooling, 10 ml of 5 N sulfuric acid was added to the mixture (in the case of phenyl-, cyclohexyl-, phenylethyl-, hexyl-, undecyl- and tridecylurea, 20 ml of acetic acid was added instead of sulfuric acid, because of their insolubility in dilute sulfuric acid). The solutions were cooled in ice and 3.5 g o f sodium nitrite added slowly with gentle stirring. After allowing the reaction to proceed for 20 min, nitrosourea formation was essentially complete. The isolation o f the p r o d u c t t o o k place in one of three ways (a) the nitrosourea was filtered off, washed with cold water and allowed to dry in air on filter paper for approximately 1 h, or (b) the acetic acid solutions were diluted with 2 vol. of water and the precipitate filtered off, washed with water and dried in air, or (c) the solutions of the soluble nitrosoalkylureas in dilute sulfuric acid, chloroethyl-, bromoethyl-, hydroxyethyl-, isopropanol-, 3-hydroxypropyl-, isopropyl- and sec-butyl-, were extracted with 60 ml ethyl acetate. The extract was shaken twice with a small volume o f water, dried with magnesium sulfate and evaporated to dryness in a rotary evaporator at 40 ° C. The residue was then crystallized in the freezer and squeezed dry on filter paper. The nitrosamides were stored at --20 ° C, at which temperature they were all fairly stable.
Mutagenicity tests In this study, all of the c o m p o u n d s were tested in the Salmonella mutagenicity test described b y Ames et al. [1]. Tester strain T A 1 5 3 5 was selected as the most suitable for dose-response studies. The one exception was nitrosophenylurea for which strain T A 1 5 3 7 was used. Although n o t essential for these direct-acting compounds, a microsomal liver fraction from rats stimulated with Aroclor 1254 was included in the tests.
The nitrosoureas were dissolved in dimethylsulfoxide (DMSO) and serially diluted so that each dose-response was carried o u t over a 1000-fold range at doses o f 1000, 500, 250, 100, 50, 25, 10, 5, 2.5 and 1/~g/0.1 ml. To 2 ml of molten t o p agar was added 0.2 ml of the tester strain, 0.1 ml of test c o m p o u n d solution and, when needed, 0.5 ml of $9 mix (75 ~1 of $9 fraction). This was then poured on to the surface of 20 ml o f VBE agar and the plates were incubated in the dark for 48 h. Revertant colonies were counted using a hand-held tally. F o r convenience, dose-response curves were plotted on a logarithmic scale. The revertant colonies per #mole were calculated by selecting from the ascending portion of the individual dose--response curves the n u m b e r of revertants at 100/~g. These figures and the molecular weight were then used to calculate the revertants per/~mole.
Carcinogenicity tests The nitrosoalkylcarbamates were administered to male and female SpragueDawley rats b y gavage in vegetable~il solution at approximately equimolar doses for a given time, after which they were allowed to die naturally. The pattern of death o f the rats with tumors was an index of carcinogenic potency, which was assumed to decrease as the survival increased. The nitrosoalkylureas are being painted on the skin of female Swiss mice as 0.04 M solutions in acetone, two applications a week for 50 weeks, after which the animals will be observed until skin tumors appear. These experiments are in progress. Results and discussion All of the nitrosamides tested were direct-acting and mutagenic in strain TA1535 o f Salmonella typhimurium, with a single exception, nitrosophenylurea; its mutagenicity was best demonstrated with strain TA1537. The effect o f incubation with the rat-liver microsomai preparation was to inactivate the nitrosamide and decrease mutagenicity or to leave it unaffected; there was never an increase in mutagenic activity. Representative dose--response curves are shown in Fig. 1 where the linear regression was done b y least-square analysis. Two examples are given for each group o f nitrosamides. Nitrosolandrin is representative of all the nitrosoalkylcarbamates except nitrosoethylurethane. Nitrosoethylurethane was n o t toxic at the highest dose tested while all other c o m p o u n d s were toxic or had given m a x i m u m reversion at 10--25 ~g. Maximum mutagenesis for nitrosomethylnitroguanidine was reached between 10 and 25 #g while nitrosoethylnitroguanidine was bacteriocidal at 50 #g. The nitrosoethylurea curve represents the nitrosoalkylureas with the exception of nitrosomethylurea. Nitrosoethylurea did n o t cause significant reversion until the dosage was between 100--200 ~g whereas nitrosomethylurea was mutagenic b e t w e e n 1 and 2.5/~g. For the substituted nitrosoalkylureas, nitrosochloroethylurea is representative except for nitroso-3-hydroxypropylurea. No toxicity was n o t e d for these c o m p o u n d s over the 1000-fold dose range we normally use. Nitroso-3-hydroxypropylurea did n o t cause reversion until the dose level was at least 2 5 #g. The other c o m p o u n d s
104
THE MUTAGENICITY OF NITROSAMIDES IN SALMONELLA TYPHIMURIUM STRAIN TA 1535 NITROSOCARBAMATES NITROSOLANDRIN NITROSOETHYLURETHANE
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J
103 102 1C 105
,
104 /
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101 NITROSOALKYLUREAS NITROSOETHYLUREA 104 NITROSOMETNYLUREA 103
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NITROSOALKYLGUANIDINES NITROSOMETHYLNITROGUANIDINE NITROSOETHYLNITROGUANIDINE
102 101
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SUBSTITUTEDNITROSOALKYLUREAS 104 NITROSOCHLOROETHYLUREA NITROSO-3-HYDROXYPROPYLUREA
105 "
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102 10
CYCLICNITROSOALKYLUREAS REA NITROSOCYCLOHEXYLUREA 10~ NITROSOPHENYLETHYLU 103 102 10 100
101
102
1;3 100
101
102
103
DOSE (~g) Fig. I . T h e m u t a g e n i c i t y o f n i t r o s a m i d e s in SaZmonetia t y p h i m u r i u m strain T A 1 5 3 5 . T h e m e a n n u m b e r o f b a c k g r o u n d r e v e z t a n t s w a s 2 5 + 8 . 7 3 f o r T A 1 5 3 5 a n d 15 -+ 3 . 6 9 f o r T A 1 5 3 7 .
H c.J O-C-N/NO
CH~, NO-CARBARYL
II
~CH 3
0 NO--CARBOFURAN
~ 0 _ {ZH3)3
N
