Mutagenicity of Five Cyclic N-Nitrosamines: Assay With Salmonella fyphimurium' D. R. Stoltz and N. P. Sen 2 ABSTRACT-The mutagenicity of five cyclic N·nitrosamines was studied with the use of Salmonella typhimurium TA1535 in vitro with and without microsomal activation. The carcinogens nitrosopiperidine and nitrosopyrrolidine required metabolic activation before manifesting mutagenic activity. Nitrosoproline and nitrosohydroxyproline, noncarcinogens, were not mutagenic. Nitroso3-pyrrolidinol was mutagenic in the absence of microsomes, thereby suggesting a role of hydroxylation in the metabolic activation of nitrosopyrrolidine to an ultimate carcinogenic species. - J Natl Cancer Inst 58: 393-394, 1977.
The cyclic N -nitrosamine, nitrosopiperidine, has been shown to be a potent carcinogen (1, 2) and to possess mutagenic activity in vitro in the presence of a metabolic activation system (3). Nitrosopiperidine, like other nitrosamines, has been thought to require metabolic activation to a reactive form. Oxidation of nitrosopiperidine in the Undenfriend system and by rat liver microsomes yielded nitroso-4-piperidone and nitroso-4-piperidinol, respectively, although these compounds accounted for only a small proportion of the products (4). Nitroso-4-piperidone, nitroso-4-piperidinol, and nitroso-3-piperidinol have been shown to be potent carcinogens for rats (5). Thus these studies have demonstrated both the formation and tumorigenicity of oxygenated nitrosopiperidines. Two structurally related cyclic nitrosamines, nitrosopyrrolidine and nitrosoproline, have been tested for carcinogenicity. Whereas nitrosopyrrolidine was a potent carcinogen for rats (6) and a mutagen (7), nitrosoproline appeared to be inactive as a carcinogen in this species (8, 9). Nitroso-3-pyrrolidinol has been identified as a metabolite of nitrosopyrrolidine in rat urine (10). In our study, the mutagenicity of hydroxy derivatives of nitrosopyrrolidine and nitrosoproline was determined and correlated with reports of their carcinogenic activity.
scribed by Ames et al. (12). Briefly, mutation was detected by determining the reversion of Salmonella typhimurium T A1535 his- to his+ on minimal medium supplemented with biotin and a trace of histidine. The liver microsomal activation mixture was prepared from Arodor 1254-pretreated male Sprague-Dawley rats (12). Microsomal protein (2 mg) was added to each plate without a liquid preincubation. All compounds were tested in the plate incorporation assay at doses up to 500 /-Lg/plate and in spot tests where a few milligrams of chemical were placed directly on the agar surface. Diffusion from the central spot allowed a range of concentrations to be tested simultaneously (12). RESULTS AND DISCUSSION
The results (table 1) show that nitrosopiperidine, nitrosopyrrolidine, and nitroso-3-pyrrolidinol are mutaI.-Correlation of carcinogenicity and mutagenicity of nitrosamines for S. typhimurium TA1535 Mutagenicity b CarcinoNitrosamine structure a genicity b Without With S9M S9MC TABLE
o I NO
o
II
I
+(6)
+(3,7, P)
HOb
III
N
+
?
+(P)
~O
IV
-(8,9)
-(P)
-(8,9)
-(P)
VCOOH
N itrosopiperidine (Eastman Organic Chemicals, Rochester, N. Y.) and nitrosopyrrolidine (Adams Chemicals, Round Lake, Ill.) were obtained commercially and used without further purification. Nitrosoproline and nitrosohydroxyproline were gifts from Dr. W. Lijinsky of Oak Ridge National Laboratory, Oak Ridge, Tennessee, and Dr. W. Fiddler of the Agricultural Research Service, U.S. Dept. of Agriculture, Philadelphia, Pennsylvania. Nitroso-3-pyrrolidinol was synthesized by nitrosation of 3-pyrrolidinol (Aldrich Chemical Co., Milwaukee, Wis.) by a method similar to that of Kruger and Bertram (10). The compound was purified by chromatography on a silica gel column, and the purity was checked by thin-layer chromatography, mass spectrometry (probe sample), and nuclear magnetic resonance spectroscopy. The details will be published elsewhere (11). Procedures for the mutation assay have been deVOL. 58, NO.2, FEBRUARY 1977
+(3, P)
NO
MATERIALS AND METHODS
Downloaded from https://academic.oup.com/jnci/article-abstract/58/2/393/1012868 by UB Frankfurt/Main user on 05 March 2018
+(1,2)
393
~O HO
V
The cyclic N -nitrosamine, nitrosopiperidine, has been shown to be a potent carcinogen (1, 2) and to possess mutagenic activity in vitro in the presence of a metabolic activation system (3). Nitrosopiperidine, like other nitrosamines, has been thought to require metabolic activation to a reactive form. Oxidation of nitrosopiperidine in the Undenfriend system and by rat liver microsomes yielded nitroso-4-piperidone and nitroso-4-piperidinol, respectively, although these compounds accounted for only a small proportion of the products (4). Nitroso-4-piperidone, nitroso-4-piperidinol, and nitroso-3-piperidinol have been shown to be potent carcinogens for rats (5). Thus these studies have demonstrated both the formation and tumorigenicity of oxygenated nitrosopiperidines. Two structurally related cyclic nitrosamines, nitrosopyrrolidine and nitrosoproline, have been tested for carcinogenicity. Whereas nitrosopyrrolidine was a potent carcinogen for rats (6) and a mutagen (7), nitrosoproline appeared to be inactive as a carcinogen in this species (8, 9). Nitroso-3-pyrrolidinol has been identified as a metabolite of nitrosopyrrolidine in rat urine (10). In our study, the mutagenicity of hydroxy derivatives of nitrosopyrrolidine and nitrosoproline was determined and correlated with reports of their carcinogenic activity.
scribed by Ames et al. (12). Briefly, mutation was detected by determining the reversion of Salmonella typhimurium T A1535 his- to his+ on minimal medium supplemented with biotin and a trace of histidine. The liver microsomal activation mixture was prepared from Arodor 1254-pretreated male Sprague-Dawley rats (12). Microsomal protein (2 mg) was added to each plate without a liquid preincubation. All compounds were tested in the plate incorporation assay at doses up to 500 /-Lg/plate and in spot tests where a few milligrams of chemical were placed directly on the agar surface. Diffusion from the central spot allowed a range of concentrations to be tested simultaneously (12). RESULTS AND DISCUSSION
The results (table 1) show that nitrosopiperidine, nitrosopyrrolidine, and nitroso-3-pyrrolidinol are mutaI.-Correlation of carcinogenicity and mutagenicity of nitrosamines for S. typhimurium TA1535 Mutagenicity b CarcinoNitrosamine structure a genicity b Without With S9M S9MC TABLE
o I NO
o
II
I
+(6)
+(3,7, P)
HOb
III
N
+
?
+(P)
~O
IV
-(8,9)
-(P)
-(8,9)
-(P)
VCOOH
N itrosopiperidine (Eastman Organic Chemicals, Rochester, N. Y.) and nitrosopyrrolidine (Adams Chemicals, Round Lake, Ill.) were obtained commercially and used without further purification. Nitrosoproline and nitrosohydroxyproline were gifts from Dr. W. Lijinsky of Oak Ridge National Laboratory, Oak Ridge, Tennessee, and Dr. W. Fiddler of the Agricultural Research Service, U.S. Dept. of Agriculture, Philadelphia, Pennsylvania. Nitroso-3-pyrrolidinol was synthesized by nitrosation of 3-pyrrolidinol (Aldrich Chemical Co., Milwaukee, Wis.) by a method similar to that of Kruger and Bertram (10). The compound was purified by chromatography on a silica gel column, and the purity was checked by thin-layer chromatography, mass spectrometry (probe sample), and nuclear magnetic resonance spectroscopy. The details will be published elsewhere (11). Procedures for the mutation assay have been deVOL. 58, NO.2, FEBRUARY 1977
+(3, P)
NO
MATERIALS AND METHODS
Downloaded from https://academic.oup.com/jnci/article-abstract/58/2/393/1012868 by UB Frankfurt/Main user on 05 March 2018
+(1,2)
393
~O HO
V
