Mutagenicity of nitric oxide and its inhibition by antioxidants

Nitric oxide (NO) is produced both by macrophages in vivo as a physiological response to infection and by a variety of cell types as an intercellular messenger. In addition, NO and nitrogen dioxide (NO₂) are significant components of many combustion processes. The ubiquitous exposure of humans to nitrogen oxides (NO_x), both endogenously and exogenously, may play a significant role in the carcinogenic process due to nitrosation of amines by NO_x. We report here that exposure to low concentrations of NO, alone or in combination with NO₂, results in significantly enhanced mutation in Salmonella typhimurium TA1535 using a modified Ames Salmonella reversion assay. The observed mutagenicity requires that the bacteria be actively dividing at the time of exposure to NO or NO₂, suggesting that the nitrogen oxides, or their reaction products, function as direct-acting mutagens and that the induced lesion is easily repairable by non-dividing cells. Exposure to NO resulted in a time- and dose-dependent increase in the number of revertants approximately proportional to the square of the NO concentration from 0 to 20 ppm. NO was a more effective mutagen relative to NO₂, however, the observed requirement for 0₂ suggests limited oxidation of NO (presumably to NO₂) is necessary. Numerous lipid- and aqueous-phase inhibitors of nitrosation, as well as a number of other general antioxidants and free-radical trapping agents, were examined for their effectiveness in blocking the mutagenic effects of NO. The mutagenic activity of NO was most effectively inhibited by β-carotene and tocopherols. BHT, dimethyl sulfoxide and mannitol also blocked the mutagenic effects of NO_x but appeared less effective than β-carotene or vitamin E, while ascorbate was ineffective as an inhibitor of mutation resulting from NO exposure.     

Nitration of angiotensin II by .NO2 radicals and peroxynitrite. .NO protects against .NO2 radical reaction.

To react with peptides, nitric oxide.NO has to be activated by oxidation, or by coupling with superoxide (O.-2) thereby producing peroxynitrite. In the course of.NO oxidation,.NO2 free radicals and N2O3 may be formed. Using gamma-irradiation methods, we characterized the products formed by these nitrogen oxides with angiotensin II. Angiotensin II is specifically nitrated at its tyrosinyl residue by.NO2 or peroxynitrite. Equimolecular amounts of each reagent in K+/Pi solutions at pH 7.4 led to 56% and 5% nitration yields, respectively. Nitrogen oxides produced by autoxidation of.NO, as well as.NO2 under.NO, reacted only with the arginine residue, giving a mixture of peptides containing citrulline, a N-(hydroxylamino-cyanamido-) instead of guanido group, and a conjugated diene derived from an arginine side-chain. However, nitrosation reactions by N2O3 occurred only when the initial concentration of.NO2 was 10 times that able to react with angiotensin II. Thus, in this case.NO appears to protect against.NO2 action.     

Mutagenicity of Maillard browning reaction products from various nitrosated amino acid-glucose mixtures

Ten different amino acid-glucose Maillard browning products before and after reaction with nitrite were evaluated by the Ames mutagenicity assay. No mutagenic response was observed in the methylene chloride extracts of any browning products tested before nitrosation. However, mutagenicity was showed in most of the browning mixtures, e.g., glycine-glucose, lysine-glucose (I), arginine-glucose, phenylalanine-glucose (II), and methionine-glucose after nitrosation when examined by Salmonella typhimurium strains TA98 and TA100 either with or without S-9 metabolic activation. Among the browning mixtures, (I) and (II) showed the greatest mutagenic activity after reaction with nitrite. The mutagenicity of lysine-glucose with nitrite was dependent on browning intensity, nitrosation pH, nitrosation time, nitrite level and blocking agents.     

The inhibitory effect of cysteine on the mutagenic activities of several carcinogens.

The Salmonella/microsome mutagenesis assay was used to determine the effect of cysteine (alpha-amino-beta-mercaptopropionic acid) on the mutagenic actions of several carcinogens: N-methyl-N'-nitro-N-nitrosoguanidine. N-acetoxy-2-acetylaminofluorene, N-hydroxy-2-acetylaminofluorene, 4-nitroquinoline-1-oxide, methyl methanesulfonate, 5-nitro-2-furaldehyde semicarbazone, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, aflatoxin B1 and the nitrosation products of methylurea and methylguanidine. Cysteine, at non-toxic concentrations, significantly decreased the frequency of reversion to histidine prototrophy when it was added to treatment mixtures. The extent of the inhibition of mutagenic action by cysteine depended on the carcinogen studied as well as the doses of cysteine and carcinogen employed. Cysteine (2.5--10 mM) completely inhibited the mutagenic actions of N-methyl-N'-nitro-N-nitrosoguanidine and methylguanidine nitrosation products while only partially preventing the mutagenic effects of the other carcinogens assayed. Inhibition of 5-nitro-2-furaldehyde semicarbazone-induced mutagenesis occurred only with higher cysteine concentrations (20--200 mM).     

Mutagenicity in lung of big Blue((R)) mice and induction of tandem-base substitutions in Salmonella by the air pollutant peroxyacetyl nitrate (PAN): predicted formation of intrastrand cross-links

Peroxyacetyl nitrate (PAN) is a ubiquitous air pollutant formed from NO(2) reacting with acetoxy radicals generated from ambient aldehydes in the presence of sunlight and ozone. It contributes to eye irritation associated with photochemical smog and is present in most urban air. PAN was generated in a chamber containing open petri dishes of Salmonella TA100 (gas-phase exposure). After subtraction of the background mutation spectrum, the spectrum of PAN-induced mutants selected at 3.1-fold above the background mutant yield was 59% GC-->TA, 29% GC-->AT, 2% GC-->CG, and 10% multiple mutations - primarily GG-->TT tandem-base substitutions. Using computational molecular modeling methods, a mechanism was developed for producing this unusual tandem-base substitution. The mechanism depends on the protonation of PAN near the polyanionic DNA to release NO(2)(+) resulting in intrastrand dimer formation. Insertion of AA opposite the dimerized GG would account for the tandem GG-->TT transversions. Nose-only exposure of Big Blue((R)) mice to PAN at 78ppm (near the MTD) was mutagenic at the lacI gene in the lung (mutant frequency +/-S.E. of 6.16+/-0.58/10(5) for controls versus 8.24+/-0.30/10(5) for PAN, P=0.016). No tandem-base mutations were detected among the 40 lacI mutants sequenced. Dosimetry with 3H-PAN showed that 24h after exposure, 3.9% of the radiolabel was in the nasal tissue, and only 0.3% was in the lung. However, based on the molecular modeling considerations, the labeled portion of the molecule would not have been expected to have been bound covalently to DNA. Our results indicate that PAN is weakly mutagenic in the lungs of mice and in Salmonella and that PAN produces a unique signature mutation (a tandem GG-->TT transversion) in Salmonella that is likely due to a GG intrastrand cross-link. Thus, PAN may pose a mutagenic and possible carcinogenic risk to humans, especially at the high concentrations at which it is present in some urban environments.     

Alcohol scavenges nitric oxide in gastric lumen.

The biological activities of endogenously produced nitric oxide (NO; nitrogen monoxide) can be affected not only by NO itself but also by relatively stable NO-derived substances or by-products of NO. It is well known that NO-derived nitrosation at the center of nitrogen, sulfur, and carbon (N-, S-, and C-nitrosation) have a great biological significance, while that at oxygen center (O-nitrosation) remains to be detected. During the course of a physiologic study on ethanol-induced gastric injury employing an ex vivo gastric chamber system of rats, we found that ethanol could be nitrosated by endogenously produced NO. Luminal nitrite and nitrate (NOx) levels in gastric lumen were decreased sharply about 70% during ethanol saline solution infusion to the chamber and then immediately returned to the basal levels by infusion of ethanol-free saline solution. On the other hand, NO levels in gastric mucosa were slightly increased during the infusion of ethanol saline solution, suggesting that ethanol never inhibits NO biosynthesis. These results demonstrate that ethanol perfused in gastric lumen can scavenge gastric tissue-derived NO and ethanol may react with NO-derived species (probably, N(2)O(3)) to form an ethylnitrite in oxygen-containing luminal solution. Alkyl nitrites including ethylnitrite have been not only widely used as a nitrovasodilator but also shown to act as a nitrosating agent. Thus, in vivo O-nitrosation also has important biological meaning in the same manner as N-, S-, and C-nitrosation.     

Formation of bacterial mutagens from the reaction of chewing tobacco with nitrite

Using the Salmonella/microsome assay system, the mutagenicity of chewing tobacco extracts (CTE) treated with and without sodium nitrite under acidic conditions was examined. Mutagenic activity was found only for nitrite-treated CTE in both tester strains, TA98 and TA100, and was independent of metabolic activation. Formation of mutagenic substances from CTE by nitrite was dependent on acidic pHs (the highest at pH 2) and could be inhibited by ascorbate. The mutagenic potency of CTE plus nitrite was proportional to the content of nitroso compounds generated in the reaction mixture, indicating that the nitrosation process was involved. The possible in vivo nitrosation and the potential health effect are discussed.     

2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potentiates nitrosation of a heterocyclic amine carcinogen by nitric oxide

Although nitrosation plays an important role in initiation of carcinogenesis, the reactive nitrogen oxygen species (RNOS) mediating this reaction by multiple pathways have not been determined. The heterocyclic amine carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was used as a target to investigate RNOS and pathways for potentiation of nitric oxide (NO)-mediated nitrosation. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) oxidizes NO to NO(2)(.) and was used as a tool to investigate NO(2)(.) potentiation of nitrosation. The IQ nitrosation product, 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline ((14)C-N-NO-IQ), was monitored by HPLC. Autoxidation of NO, generated by spermine NONOate (2.4 microM NO/min) for 7.5 min, did not convert 10 microM (14)C-IQ to N-NO-IQ. However, the presence of 15 muM CPTIO resulted in 3 microM N-NO-IQ formation. Potentiation by CPTIO occurred at low and high fluxes of NO, 0.075 to 1.2 microM/min, and over a range of IQ to CPTIO ratios of 0.5 to 10. A significant portion of N-NO-IQ formation was insensitive to azide (10 mM) inhibition, suggesting oxidative nitrosylation. NADH (0.02 mM) did not alter nitrosation by autoxidation, but effectively inhibited potentiation by CPTIO. Ascorbic acid (0.2 mM) and 5,5-dimethyl-1-pyrroline N-oxide (30 mM) inhibited nitrosation with or without CPTIO, while superoxide dismutase was not inhibitory. The RNOS produced by CPTIO had a 27-fold greater affinity for IQ than those produced by autoxidation. Results are consistent with NO(2)(.) or a RNOS like NO(2)(.) potentiating IQ oxidative nitrosylation. Nitrosation occurring at both low and high fluxes of NO can contribute to carcinogenesis.     

Genotoxic activity of 1,3-butadiene and nitrogen dioxide and their photochemical reaction products in Drosophila and in the mouse bone marrow micronucleus assay.

The genotoxic activity of a photochemical reaction mixture of 1,3-butadiene and nitrogen dioxide was investigated in vivo in the mouse bone marrow micronucleus assay and the somatic mutation and recombination test in Drosophila (the wing spot test). Butadiene alone was not mutagenic in Drosophila, but induced micronuclei in mice at 10 ppm after 23 h of exposure. Nitrogen dioxide was not genotoxic in either test system. The photochemical reaction products were toxic but probably not mutagenic in Drosophila and not genotoxic in mouse bone marrow. The in vivo results do not confirm earlier in vitro results that demonstrated a strong direct-acting mutagenic activity of the photochemical products in Salmonella.     

Mutagenicity of the reaction products of carbazole in the presence of nitrogen dioxide and nitrocarbazole

The mutagenicity of the photochemical reaction products of carbazole in the presence of nitrogen dioxide (NO2) and nitrocarbazole was investigated using a high-pressure mercury lamp (100 W). Samples extracted from the photochemical reaction products of carbazole with NO2 were more mutagenic than those of acridine and phenazine with NO2 for Salmonella typhimurium strain TA98 in the absence of S9 mix with a trend toward detoxification in the presence of the metabolic system. The mutagenicity of the photochemical reaction products of carbazole with NO2 were higher than those of the reaction products of carbazole with a mixture of NO2 and sulfur dioxide (SO2) and no irradiation. Mononitro- and dinitro-carbazole in the samples extracted from the reaction products were analyzed by mass spectrometry. It was suggested that mononitrocarbazole, which seemed to be weakly mutagenic, and dinitrocarbazole were readily formed by the reaction of carbazole with NO2, and that the other high-potency mutagens were formed by the photochemical reaction of carbazole with NO2 with irradiation by light.     

Mutagenicity of nitro derivatives induced by exposure of aromatic compounds to nitrogen dioxide

Mutagenic nitro derivatives were readily induced when 6 kinds of chemicals were exposed to 10 ppm of nitrogen dioxide (NO2). Single nitro derivatives were formed from pyrene, phenanthrene, fluorene or chrysene. Carbazole and fluoranthene each produced 2 derivatives substituted with nitro groups at different positions. The formation of nitro derivatives was enhanced by exposure of pyrene to NO2 containing nitric acid (HNO3, less than 100-fold enhancement) or sulphur dioxide (SO2, less than 15-fold enhancement). After 24 h of exposure the yields of the nitro derivative were 0.02% with 1 ppm of NO2 in air and 2.85% with NO2 (1 ppm) containing traces of HNO3. The nitro derivatives from all but phenanthrene and carbazole were chemically identified by means of gas chromatography (GC) and mass spectrometry (MS), and the mutagenicity of the 4 kinds of authentic nitro derivatives was tested by using Salmonella strains TA98 and TA1538 with or without the S9 fraction from rat liver treated with Aroclor 1254. The nitro derivative induced from pyrene was determined to be 1-nitropyrene; that of chrysene was 6-nitrochrysene; that of fluorene was 2-nitrofluorene; and those of fluoranthene were 3-nitrofluoranthene, and 8-nitrofluoranthene. Tested with strain TA98 in the absence of the S9 fraction, the first 4 of these derivatives yielded, respectively, 3050, 269, 433 and 13 400 revertants per nmole. Thus, each nitro derivative formed was potentially a direct-acting frameshift-type mutagen. Each compound exposed to NO2 showed a decreased mutagenic activity when tested in the presence of S9 mix. A possible explanation comes from experiments in which 1-nitropyrene was incubated with the S9 mix at 37 degree C for 10 min, and 1-aminopyrene was formed. The mutagenic activity of 1-aminopyrene was appreciable, but only about one-tenth of that of 1-nitropyrene in the Ames test.     

Novel metabolism of nitrogen in plants

Our previous study showed that approximately one-third of the nitrogen of 15N-labeled NO2 taken up into plants was converted to a previously unknown organic nitrogen (hereafter designated UN) that was not recoverable by the Kjeldahl method (Morikawa et al., 2004). In this communication, we discuss metabolic and physiological relevance of the UN based on our newest experimental results. All of the 12 plant species were found to form UN derived from NO2 (about 10-30% of the total nitrogen derived from NO2). The UN was formed also from nitrate nitrogen in various plant species. Thus, UN is a common metabolite in plants. The amount of UN derived from NO2 was greatly increased in the transgenic tobacco clone 271 (Vaucheret et al., 1992) where the activity of nitrite reductase is suppressed less than 5% of that of the wild-type plant. On the other hand, the amount of this UN was significantly decreased by the overexpression of S-nitrosoglutathione reductase (GSNOR). These findings strongly suggest that nitrite and other reactive nitrogen species are involved in the formation of the UN, and that the UN-bearing compounds are metabolizable. A metabolic scheme for the formation of UN-bearing compounds was proposed, in which nitric oxide and peroxynitrite derived from NO2 or endogenous nitrogen oxides are involved for nitrosation and/or nitration of organic compounds in the cells to form nitroso and nitro compounds, including N-nitroso and S-nitroso ones. Participation of non-symbiotic haemoglobin bearing peroxidase-like activity (Sakamoto et al., 2004) and GSNOR (Sakamoto et al., 2002) in the metabolism of the UN was discussed. The UN-bearing compounds identified to date in the extracts of the leaves of Arabidopsis thaliana fumigated with NO2 include a delta2-1,2,3-thiadiazoline derivative (Miyawaki et al., 2004) and 4-nitro-beta-carotene.     

Analysis of the neuroprotective effects of various nitric oxide donor compounds in murine mixed cortical cell culture

Nitric oxide (NO) has been implicated in both the pathogenesis of and protection from NMDA receptor-mediated neuronal injury. This apparent paradox has been attributed to alternate redox states of nitrogen monoxide, whereby, depending on the redox milieu, nitrogen monoxide can be neuroprotective via nitrosation chemistry or react with superoxide to form secondary toxic species. In our murine mixed cortical cell culture system, the NONOate-type NO donors diethylamine/NO complex sodium (Dea/NO), (Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium++ +-1,2-diolate (Papa/NO), and spermine/NO complex sodium (Sper/NO), as well as the S-nitrosothiols S-nitroso-L-glutathione (GSNO) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) (NO+ equivalents), decreased NMDA-induced neuronal injury in a concentration-dependent manner. 8-Bromo-cyclic GMP did not mimic the inhibitory effects of the donors, suggesting that the neuroprotection was not the result of NO-stimulated neuronal cyclic GMP production. Furthermore, neuronal injury induced by exposure of cultures to H2O2 was not altered by the presence of Dea/NO, indicating the absence of a direct antioxidant effect. NONOates did, however, reduce NMDA-stimulated uptake of 45Ca2+, whereas high potassium-induced 45Ca2+ accumulation, a measurement of entry via voltage-gated calcium channels, was unaffected. The parallel reduction of 45Ca2+ accumulation and NMDA neurotoxicity by NONOates mimicked that seen with an NMDA receptor antagonist. Electrochemical measurements of NO via an NO-sensitive electrode demonstrated that neuroprotective concentrations of all donors produced appreciable amounts of NO over the 5-min time frame. Determination of the formation of NO+ equivalents, as assessed by N-nitrosation of 2,3-diaminonaphthylene, revealed little or no observable N-nitrosation by Sper/NO, GSNO, and SNAP with significant N-nitrosation observed by Papa/NO and Dea/NO. However, addition of ascorbate (400 microM) effectively prevented the nitrosation of 2,3-diaminonaphthylene produced by Dea/NO and Papa/NO without altering their neuroprotective properties or their effects on 45Ca2+ accumulation. Present results indicate that the intrinsic NO/NO+ characteristics of NO donor compounds may not be a good predictor of their ability to inhibit NMDA receptor-mediated neurotoxicity at the cellular level.     

The mutagenicity analysis of imidapril hydrochloride and its degradant,diketopiperazine derivative,nitrosation mixtures by in vitro Ames test with two strains of Salmonella typhimurium

AimThe evaluation of mutagenic properties of imidapril hydrochloride (IMD) and its degradation impurity, diketopiperazine derivative (DKP), nitrosation mixtures was conducted in order to analyze the carcinogenic risk of IMD long-term treatment in patients. In this study an in vitro Ames test with Salmonella enterica serovar Typhimurium TA 98 and TA 100 strains was used.BackgroundIMD and DKP contain nitrogen atoms, which makes them theoretically vulnerable to in vivo nitrosation with the production of N-nitroso compounds (NOC). NOC, in turn, are known animal mutagens indicating that their endogenous production from nitrosable drugs constitutes a carcinogenic hazard.Materials and methodsPure IMD sample was exposed to forced degradation conditions of increased temperature and dry air in order to achieve a DKP sample. Both samples were then treated with a nitrosating agent and the obtained nitrosation mixtures were subjected to mutagenicity analysis by the Ames test with S. typhimurium TA 98 and TA 100 strains in the presence and absence of metabolic activation system (S9 mix) using a commercial Ames MPF 98/100 microplate format mutagenicity assay kit.ResultsNone of the six concentrations of the investigated nitrosation mixtures exhibited any mutagenic potential in both S. typhimurium strains. The addition of S9 mix did not alter the non-mutagenic properties of the studied compounds.ConclusionsThe nitrite treatment of both studied compounds has no impact on their mutagenic properties under the conditions of the present studies. Hence, IMD and DKP nitrosation mixtures are classified as non-mutagens in this test.     

Mutagenicity of coal-pyrolyzed products and their photochemical reaction products with nitrogen oxides in Salmonella typhimurium TA98 and TA100

The chemical class separation of coal-pyrolyzed products and the photochemical reaction of these fractions with nitrogen oxides in the experimental chamber, and the application of a short-term mutagenicity test were investigated. The altered products from the fraction hydroxy polycyclic aromatic compounds in a simulated atmosphere containing a small volume of nitrogen oxides under irradiation with a xenon lamp were the most potent mutagenic fraction among all the fractions tested against Salmonella typhimurium, both TA98 and TA100, with or without S9.     

Mutagenic effects of nitrogen dioxide combined with methylurea and ethylurea in Drosophila melanogaster

The standard sex-linked recessive lethal test was used to test whether NO2 induces lethal mutations in male germ cells of Drosophila in the presence or absence of alkylureas. Methylurea, ethylurea and NO2 alone did not enhance the mutation frequency significantly. However, highly significant enhancement in the mutation frequency was observed when adult flies were exposed to NO2 (150--280 ppm) for 3 h after ingestion of methylurea (0.1 M) or ethylurea (0.1 M) for 2 days. Oral administration of ethylnitrosourea and also of methylurea or ethylurea that had been exposed to NO2 in vitro were more effective in increasing the mutation frequency than methylurea or ethylurea combined in vivo with NO2. These results suggest that ingested alkylurea is converted in vivo by inhaled NO2 to highly mutagenic nitrosoalkylurea and/or other mutagens. No significant enhancement of the mutation frequency was observed when flies were fed on methylurea solution after they had been exposed to NO2.     

Nitro reaction in mice injected with pyrene during exposure to nitrogen dioxide

We have previously reported that the beta-glucuronidase-treated urine of mice injected intraperitoneally with pyrene during exposure to NO2 contained highly mutagenic compounds such as nitropyrene metabolites when tested by the Ames assay using Salmonella typhimurium strain TA98. In the present study, we found that the formation of these mutagens was dose-dependent between 10 and 200 mg of pyrene per kg of body weight at 5 and 10 ppm of NO2. Further, to elucidate the substrate of nitration in vivo, we injected 1-hydroxypyrene, which is the metabolite of pyrene, to mice intraperitoneally during exposure to NO2. Since the results were the same as those obtained by injection with pyrene, we suggest that the pyrene was not nitrated directly but after its hydroxylation.     

UV-irradiation potentiates the antimutagenicity of p-aminobenzoic and p-aminosalicylic acids in Salmonella typhimurium

UV-irradiation (254 nm, 10 or 20 J/cm2) of p-aminobenzoic acid (PABA) and p-aminosalicylic acid (NaPAS) potentiated their antimutagenicity towards N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis in Salmonella typhimurium. Their inhibitory action towards the formation of the mutagen N-methyl-N-nitrosourea from the nitrosation mixture of N-methylurea and nitrite was also increased by UV-irradiation. In contrast, UV-irradiated PABA exhibited no inhibitory effects towards the mutagenicity of sodium azide or 3-azidoglycerol. Neither PABA nor NaPAS nor their UV-irradiation products were themselves mutagenic in the Ames assay.     

Nitrosamine-induced mutagenesis in Escherichia coli K12 (343/113). 1. Mutagenic properties of certain aliphatic nitrosamines

The Escherichia coli K12 (343/113) test system developed by G. Mohn was used to detect the mutagenic activity induced by a group of aliphatic nitrosamines. Metabolic activation was incorporated into the assay by the addition of liver homogenates induced in either Sprague-Dawley rats or C3H mice with the addition of 0.1% phenobarbital to the drinking water. Nitrosodiethylamine (NDEA) was mutagenic upon metabolic activation and exhibited a preference to revert the missense mutation at the arginine locus. NDEA was also capable of inducing the forward mutation, selected as an ability to utilize galactose. NDEA was converted effectively into a mutagen in a time period of 30 min to 2 h. Metabolic activation with the mouse and rat liver preparations did not result in quantitative differences. Aliphatic nitrosamines that gave unexpected results with the Salmonella assay [4-10] were examined in the E. coli system. Nitrosodipropylamine (NDPA) and nitrosodiallylamine (NDAA) were mutagenic in both E. coli and Salmonella. Nitrosomethylethylamine (NMEA) was not mutagenic in Salmonella but was mutagenic in E. coli, and a strong carcinogen, nitrosomethylneopentylamine (NMNA), was not mutagenic in either assay. These results indicate the use of multiple genetic assays for the detection of genotoxic chemicals in our environment.     

The inhibitory effect of whole and deproteinized saliva on mutagenicity and clastogenicity resulting from a model nitrosation reaction

The objective of this study was to simulate in vitro at least some of the conditions that prevail in man during ingestion of nitrate and nitrosable compounds. Human saliva has been chosen because most chemicals ingested through food will interact with saliva. The nitrosation of methylurea was used as a model because the nitrosation products can be readily detected by their mutagenic (his+ revertants of S. typhimurium) and clastogenic (chromosome aberrations in CHO cells) properties. The results show that human saliva inhibits the formation of mutagenic and clastogenic nitrosation products when present during nitrosation. A 50% inhibition of mutagenicity results from the addition of a saliva sample diluted at 5% of the original concentration. In the test system used a similar inhibitory effect was obtained by 2.5 mM ascorbic acid or 2.0 mM chlorogenic acid. The main inhibitory agents seem to reside in a deproteinized fraction which was filtered through an ultrafilter UM2 (greater than 1000 MW). At strong acid levels (below pH 2) the saliva loses its inhibitory effect on the nitrosation of methylurea. The contribution of saliva to the inhibition of endogenous nitrosation within the oral cavity or stomach is discussed.