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Salmonella tester strains which are reverted by base-pair substitution mutagens are relatively insensitive to the mutagenic effects of N-methyl-N-nitroso compounds. One reason for this insensitivity is the ability of these strains to withstand low doses of these compounds before they become sensitive to their mutagenic effects. In this report it is shown that mutagenesis induced by treatment of Salmonella typhimurium TA 1535 with N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) in buffer is biphasic with a low sensitivity range at low doses where little mutagenesis occurs, followed by a high sensitivity range whose onset begins after an apparent threshold dose has been exceeded. levels of O6-methylguanine (O6-MeG) in the DNA extracted from the bacteria follow a similar dose-response curve suggesting a dependency of mutagenesis on O6-MeG. In contrast, levels of 7-methylguanine (7-MeG) in the DNA increase linearly with dose. O6-MeG was undetectable at the lowest dose of MNNG whereas 7-MeG was readily detectable. Although such resistance to O6-alkylation has been demonstrated in MNNG- pretreated (adapted) E. coli, it has not been reported in unpretreated cells. Then isolated DNA was treated with MNNG a linear dose-response in the generation of O6-MeG was observed. The lack of O6-MeG in DNA isolated from MNNG treated cells after low doses is attributed to a saturable, constitutive repair activity in the bacteria. An attempt to observe the removal of O6-MeG from the bacteria after exposure to a short challenge dose of N-nitroso-N-methylurea (NMU) followed by a subsequent incubation in buffer was unsuccessful, probably because all the repair occurred within the time necessary to treat and lyse the cells.

The carcinogens N-nitrosodimethylamine (DMN) and N-nitrosodiethylamine (DEN) were detected in rat saliva by gas liquid chromatography or HPLC after their ip injections. Salivary levels were equal to or greater than blood levels. A fairly linear dose response in salivary levels was observed in the range of 1-50 mg/kg. Salivary levels of DMN and DEN decreased between 0.5 and 1.0 hr after administration with the decrease becoming more prominent at lower doses. Formaldehyde and bicarbonate, metabolites of DMN, were also detected in rat saliva.

Liver S-9 (9000 g supernatant) fractions isolated from 2-, 12- and 26-month untreated female Swiss-Webster mice were compared under different assay conditions as to their abilities to activate 2-acetylaminofluorene, benzo(a)pyrene, and dimethylnitrosamine to mutagens in Salmonella typhimurium tester strain TA 100. All fractions activated these compounds to mutagens, although 2-acetylaminofluorene was only weakly mutagenic. Some differences in the activating abilities of the three age groups were observed but they were for the most part relatively small.

The mutagenic activities of a diverse group of N-nitrosamines were measured in Salmonella typhimurium TA 100 under conditions designed to maximize metabolism of N-nitrosamines and enhance their mutagenic effects. These conditions were also chosen since some of the carcinogens were previously reported to be non-mutagenic or of questionable mutagenic activity and some only became mutagenic after the bacteria were exposed to a "threshold does" of metabolites. The mutagenic potencies spanned a range of 10(5)-fold and correlated well with semiquantitative carcinogenic potencies taken from the literature. This correlation appears to be the strongest yet reported for any particular class of compounds. In addition, the mutagenic activities of a number of carcinogens, previously reported to be non-mutagenic, were determined. Among the structural features necessary for high mutagenic activity in this group of compounds was a potential, unsubstituted methylating or ethylating group. Substitution of alkyl, hydroxyl, methoxyl, and cyano moieties at the alpha or beta carbon of these groups reduced mutagenic activity.

Mutagenesis induced by a number of N-nitrosamines in a liquid phase assay was enhanced 2-8 fold on going from a pH of 7.4 to 6.5, with the degree of enhancement dependent on the compound and the reaction conditions. In general, enhancement was greatest at lower concentrations of mutagen and with compounds of lower molecular weights. With dimethylnitrosamine (DMN) part of this effect was due to an enhanced rate of metabolism at low concentrations of DMN at a pH of 6.5. However, even at higher concentrations of DMN, mutagenesis was more effective at the lower pH despite the fact that metabolism of DMN was twice as rapid at a pH of 7.4 than 6.5. Directly acting mutagens such as N-nitroso-N-methylurea and N-methyl-N'-nitro-N-nitrosoguanidine were 2-10 times more potent at pH values of between 5.5 and 7.0 than at 7.4. No enhancement in the mutagenic activities of methyl methanesulfonate, benzo(a)pyrene or 3-methylcholanthrene was observed on going from a pH of 7.4 to 6.5.

The kinetics of benzo[a]pyrene induced mutagenesis in Salmonella typhimurium TA98 and TA100 were studied using a liquid-phase assay and were compared to kinetics of metabolism of benzo[a]pyrene in the same system. Mutagenesis was terminated by the addition of pyrene + menadione at selected times. Under conditions of the assay using hepatic microsomes from 3-methyl-cholanthrene pretreated rats, metabolism of benzo[a]pyrene was over 50% complete in 5 min, and metabolism of initial metabolites became appreciable after this time. The revertant frequency (revertants/10(8) survivors) and the production of tertiary metabolites increased sharply after 5 min and leveled off at 40 min.