Takie Yahagi

Mutagenicities of N-nitrosamines on Salmonella.

The mutagenic activities of 11 N-nitrosamines were tested using Salmonella typhimurium TA100 and TA98. All the carcinogenic N-nitrosamines were mutagenic on TA100 with a drug-activating system from the rat liver, whereas N,N-diphenylnitrosamine, a non-carcinogen, was not mutagenic. None of the N-nitrosamines was mutagenic on TA98, except N,N-diethylnitrosamine which was weakly mutagenic. To detect the mutagenicity of N,N-dimethylnitrosamine, the pre-incubation of bacteria and N,N-dimethylnitrosamine with S-9 Mix before if was poured onto plates was obligatorily required. Dimethyl sulfoxide inhibited the mutagenic effect of N,N-dimethylnitrosamine.

Mutagenicities of smoke condensates and the charred surface of fish and meat.

Smoke condensates obtained from broiling fish showed mutagenic activity for Salmonella typhimurium TA100 and TA98. Metabolic activation was required to induce mutagenic activity of smoke condensates of some species of fish. The smoke condensate obtained during charcoal broiling of beefsteak was far less mutagenic than that of fish, with or without metabolic activation. Extracts of the charred surface of broiled fish and meat also contained mutagenic substances. These extracts needed metabolic activation to exhibit mutagenicities on TA98. The mutagenic activity of the smoke condensate obtained from one sardine weighing 100 g was equivalent to that of 132 micrograms benzo(a)pyrene and that of the charred surface of the sardine was equivalent to 358 micrograms benzo(a)pyrene. One piece of beefsteak weighing 190 g, contained mutagenic activity equivalent to that of 855 micrograns benzo(a)pyrene.

Mutagenicities of quinoline and its derivatives

Quinoline, recently reported to be carcinogenic in rats [12], was mutagenic to Salmonella typhimurium tester strains TA100 and TA98 in the presence of the metabolic activation system S-9 mix. 2-Chloroquinoline, a non-carcinogen [12], was non-mutagenic with or without S-9 mix. 8-Hydroxyquinoline, which is t known to be carcinogenic, was mutagenic with S-9 mix to both bacterial strains. The mutagenicities of 17 other quinoline derivatives that are not known to be carcinogenic were tested, and 12 of these compounds were mutagenic.

Mutagenicities of protein pyrolysates.

Smoke condensate obtained by pyrolysis of proteins, such as lysozyme and histone, was shown to be mutagenic to Salmonella typhimurium TA100 and TA98. In vitro metabolic activation by a mammaliam postmitochondrial enzyme preparation (S-9 Mix) was required. Smoke condensates obtained by pyrolysis of DNA, RNA, starch and vegetable oil were slightly mutagenic, whereas those from pyrolysis of L- and D-tryptophan and 5-hydroxy-D,L-tryptophan were very strongly mutagenic with metabolic activation by S-9 Mix. Because of the high correlation between mutagenicity and carcinogenicity, it is theorized that the cooking of proteinaceous foods might be an important cause of human cancers.

Differences in effects of norharman with various classes of chemical mutagens and amounts of S-9

The mutagenicities of aniline, o-toluidine and yellow OB were demonstrated only in the presence of the β-carboline compound, norharman. The effect of norharman increased linearly with increase in the amount of S-9. The mutagenicity of 4-dimethylaminoazobenzene was greatly enhanced by the presence of norharman, and again dose-dependency on the amount of S-9 was observed. In the presence of a large amount of S-9, norharman caused several fold enhancement of the mutagenicities of N-2-fluorenylacetamide, benzo(a)-pyrene, and 1,4-dimethyl-3-amino-5H-pyrido(4,3b) indole, isolated from a tryptophan pyrolysate. However, norharman suppressed the mutagenicities of these compounds in the presence of a small amount of S-9. The mutagenicity of kaempferol, a flavonoid, was inhibited by norharman with either a large or small amount of S-9.

Mutagenicity of smoke condensates from cigarettes, cigars and pipe tobacco.

Smoke condensates from cigarettes, cigars and pipe tobacco were mutagenic on Salmonella typhimurium TA100 and TA98 when activated with rat liver microsomal system. Mutagenicity of a unit weight of smoke condensate was rather high in cigars, low in pipe tobacco and intermediate in cigarettes. Specific mutagenic activity was almost comparable among smoke condensates from low- to high-tar cigarettes, although some variations were observed depending upon the country producing the cigarettes. Marked mutagenicity of cigarette smoke condensate could not be explained by the benzo (a) pyrene or nitroso compounds it actually contained, suggesting the presence of other very potent mutagens in tobacco smoke condensates.

Mutagenicities of nitrofuran derivatives on a bacterial tester strain with an R factor plasmid.

Many nitrofuran derivatives are known to be mutagenic on Escherichia coli WP2 but not on Salmonella typhimurium TA1535, TA1536, TA1537 or TA1538. Ames and coworkers recently obtained a new tester strain of S. typhimurium, TA100, by putting an R factor plasmid, pKM101, into TA1535. We found that all mutagenic nitrofuran derivatives previously found to be mutagenic on E. coli WP2 were mutagenic on this new strain (TA100).

Comutagenic actions of norharman derivatives with 4-dimethylaminoazobenzene and related compounds

Summary 4-Dimethylaminoazobenzene is only a weak mutagen on Salmonella typhimurium strains TA100 and TA98, although it is a fairly strong carcinogen. Norharman, which is present in tobacco smoke and tryptophan pyrolysate, enhanced the mutagenicity of 4-dimethylaminoazobenzene on strain TA98 about 40-fold. Norharman alone had no mutagenic activity on TA98 and so it is a ‘Comutagen’. The mutagenicities of 4-methylaminoazobenzene, 3′-methyl-4-dimethylaminoazobenzene, 4-aminoazobenzene and 4′-methoxycarbonyl- N -hydroxy-4-methylaminoazobenzene were all potentiated by norharman. Presence of a metabolic activation system was essential for comutagenic action of norharman with these compounds. Dihydronorharman, tetrahydronorharman, 9-methyl- β -carboline, harman and tetrahydroharman did not enhance the mutagenicity of 4-dimethylaminoazobenzene.

Identification of a mutagenic substance in a spice, sumac, as quercetin

The mutagenicity of a spice, sumac, was demonstrated on Salmonella typhimurium strain TA98. The active principle was purified and characterized by thin-layer chromatography, UV-absorption spectroscopy and mass spectrometry. All the mutagenic activity of sumac was found to be due to quercetin.

The effect of norharman on the metabolism of benzo[a]pyrene by rat-liver microsomes in vitro in relation to its enhancement of the mutagenicity of benzo[a]pyrene

The effect of norharman on the metabolism of benzo[alpha]pyrene by rat-liver microsomes was studied. Separation of the metabolites into hydrophilic and hydrophobic fractions showed that norharman inhibited the conversion of hydrophobic metabolites to hydrophilic ones. Analysis of the hydrophobic metabolites by high-pressure liquid chromatography showed that norharman also inhibited the disappearance of benzo[alpha]pyrene itself. However, large amounts of hydrophobic metabolites, such as phenol, quinones and diols, were formed in the presence of norharman, and formation of the strong mutagen 7,8-dihydroxybenzo[alpha]pyrene was increased 10-fold by norharman. The increase in formation of this compound may be one of the chief reasons why norharman enhances the mutagenicity of benzo[alpha]pyrene on Salmonella typhimurium.