Mutagenicity studies on N-nitrosated products of the Maillard browning reaction: N-nitroso-fructose-amino acids

The N-nitroso derivatives of D-fructose-L-glycine, D-fructose-L-alanine, D-fructose-L-phenylalanine, D-fructose-L-serine, Dfructose-L-aspartic acid and D-fructose-L-tryptophan (a mixture of alpha-N-nitroso-D-fructose-L-tryptophan and 'indolyl-nitrosamine'-D-fructose-L-tryptophan) were tested for mutagenicity in five auxotrophic strains of Salmonella typhimurium with and without metabolic activation (S-9 mix). The alanine, phenylalanine and aspartic acid compounds were not mutagenic. The glycine and serine compounds showed a very low but reproducible increase in the numbers of his+ revertants in strain TA1535 without S-9 mix. The mixture containing both nitrosated D-fructose-L-tryptophan compounds was mutagenic in all five strains, with or without metabolic activation. The alpha-N-nitroso-D-fructose-L-tryptophan component of the mixture, which is nitrosated at the amino group, was isolated and tested without S-9 mix. It was mutagenic in three strains. Unnitrosated D-fructose-L-amino acids, D-fructose, and the individual L-amino acids were non-mutagenic when tested under those conditions for which a positive response had been obtained with the corresponding nitrosated compounds. These results indicate the potential value of developing analytical methods to identify alpha-N-nitroso-D-fructose-L-tryptophan in food or food extracts that are to be screened for mutagenic components.     

Mutagenicity assay in Salmonella for thirteen 2-substituted-1 H-phenanthro (9,10-d) imidazoles

Some 2-substituted-1 H-phenanthro [9,10-d] imidazole compounds synthesized as a predrugs were tested in mutagenicity assays in Salmonella strains TA97, TA98, and TA100 using a plate incorporation assay both with and without S9 mix. The 10 substances were mutagenic in TA97 and five of them were mutagenic only with metabolic activation, whereas one of them did not require the addition of S9. The eight substances were mutagenic in TA98 only with S9. For TA100, seven substances showed positive results both with and without S9, however another four required S9, whereas only one of them did not required metabolic activation. In summary, all of 13 substances derived from phenanthro [9,10-d] imidazole were found to be mutagenic for at least one or two of the three strains and their mutagenicity are discussed.     

Contribution of coffee aroma constituents to the mutagenicity of coffee

About 40 coffee aroma constituents belonging to the classes of dicarbonyls, sulphur-containing compounds, furfuryls, N-heterocyclics and others were systematically evaluated in three Ames tester strains. Only aliphatic dicarbonyl compounds showed notable direct mutagenic activity, which mainly affected 'base-pair substitution' in Ames tester strains TA100 and TA102. Very weak effects were also seen with some N-heterocyclics, mainly affecting frameshift tester strain TA98 upon metabolic activation. However, it was shown that these N-heterocyclics do not contribute substantially to the mutagenicity in coffee. The hydrogen peroxide and methylglyoxal contents of coffee were determined up to 26 hr after preparation. Their concentrations tended to decrease whereas mutagenic activity decreased significantly with time in tester strains TA100 and TA102. It is concluded that several highly labile coffee constituents contribute to the bacterial mutagenicity and also that the synergism between hydrogen peroxide and methylglyoxal is not the main factor. The absence of coffee mutagenicity/carcinogenicity in rodents with these highly reactive coffee aroma compounds can be explained in part by detoxification of microsomal enzyme systems.     

Biological and chemical studies on overheated brewed coffee

Vapour formed from overheated decaffeinated coffee was condensed and tested for mutagenicity using the Ames assay in Salmonella typhimurium strains TA98 and TA100. Vapour produced at 73 and 100 degrees C exhibited no mutagenicity. The basic fraction of vapour produced at 350 degrees C showed weak mutagenicity towards strains TA98 with metabolic activation. The chemical analysis of this fraction identified pyridines and pyrazines as the major constituents. None of the compounds identified in this fraction has been reported as mutagenic when tested in the Ames assay.     

Mutagenicity Assay in Salmonella for Thirteen 2-Substituted-1H-phenanthro (9,10-d) Imidazoles

Abstract

Some 2-substituted-1H-phenanthro [9,10-d] imidazole compounds synthesized as a predrugs were tested in mutagenicity assays in Salmonella strains TA97, TA98, and TA100 using a plate incorporation assay both with and without S9 mix. The 10 substances were mutagenic in TA97 and five of them were mutagenic only with metabolic activation, whereas one of them did not require the addition of S9. The eight substances were mutagenic in TA98 only with S9. For TA100, seven substances showed positive results both with and without S9, however another four required S9, whereas only one of them did not required metabolic activation. In summary, all of 13 substances derived from phenanthro [9,10-d] imidazole were found to be mutagenic for at least one or two of the three strains and their mutagenicity are discussed.     

Mutagenicity of 2-hydroxyalkyl-N-nitrosothiazolidines

The mutagenicity of 2-hydroxyalkyl-N- nitrosothiazolidines was tested using Salmonella typhimurium strains TA98 and TA100. The N- nitrosothiazolidines tested were unsubstituted N- nitrosothiazolidine (NT), N- nitrosothiazolidine -4-carboxylic acid ( NTC ), 2-hydroxymethyl-N- nitrosothiazolidine ( HMNT ), 2-(1,2,3,4- tetrahydroxybutyl )-N- nitrosothiazolidine , 2-(1,2,3,4- tetrahydroxypentyl )-N- nitrosothiazolidine , 2-(1,2,3,4,5- pentahydroxypentyl )-N- nitrosothiazolidine ( PHPNT ) and 2-(1,2,3,4,5- pentahydroxypentyl )-N- nitrosothiazolidine -4-car boxylic acid. Among the N- nitrosothiazolidines tested, only HMNT and PHPNT exhibited clear dose-response mutagenicity toward strain TA100 with or without metabolic activation. None of the 2-hydroxyalkyl-N- nitrosothiazolidines were mutagenic to strain TA98. NT exhibited much stronger mutagenicity than either HMNT or PHPNT . Mutagenic activities of NT and PHPNT were eliminated by carboxyl substitution in the position alpha to the N-nitroso group.     

Examination of some fragrance substances for mutagenic activity

We tested 64 perfumes and aftershaves for mutagenicity in Salmonella typhimurium strains TA98 and TA100; most were tested both with and without metabolic activation. None of the test compounds gave a positive mutagenic response. The available amounts of some test samples were very small, however, and the possibility that larger samples might have produced positive results cannot be altogether discounted. Some tests indicated the presence of cytotoxic substances which require chemical analysis.     

Mutagenicity and cytotoxicity of N-methyl-2-pyrrolidinone and 4-(methylamino)butanoic acid in the Salmonella/microsome assay

The industrial solvent N-methyl-2-pyrrolidinone (NMP) and its hydrolysis product, 4-(methylamino)butanoic acid (N-MeGABA), were examined for mutagenicity and cytotoxicity in the Ames Salmonella/microsome assay. In order to detect a broad range of possible mutagenic endpoints, the following strains were used in the assay: base-pair substitution strains TA100, TA102 and TA104; frameshift strains TA97 and TA98; and repair proficient strains TA2638, UTH8413 and UTH8414. In the standard plate incorporation assay, six log-linear doses of each compound were tested; doses ranged from 0.01 to 1000 mumol/plate for NMP, and 0.01 to 316 mumol/plate for N-MeGABA. Neither compound was detectably mutagenic when tested in the presence and absence of metabolic activation by Aroclor-induced rat liver S9. NMP did show significant responses with strains TA102 and TA104 that were less than two-fold over background, but no clear dose-response relationships were evident. A preincubation modification of the assay was also performed, using strains TA98 and TA104. Mutagenic activity was not observed for NMP, while N-MeGABA showed significant responses with TA104 but dose-related mutagenicity was not established. Preincubation testing revealed both NMP and N-MeGABA to be cytotoxic to the test population of Salmonella at the highest treatment doses.     

Mutagen formation in the reaction of maillard browning products, 2-acetylpyrrole and its analogues, with nitrite

Three 2-substituted pyrroles (2-acetylpyrrole, pyrrole-2-carboxaldehyde and pyrrole-2-carboxylic acid), which are products of the Maillard browning reaction, were reacted with nitrite in buffer solution (pH 3) at 50°C for 24 hr. The reaction mixtures were extracted with methylene chloride and the extracts were tested for mutagenicity using Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA104, with and without S-9 metabolic activation. The methylene chloride extract of the 2-acetylpyrrole-nitrite reaction mixture showed strong mutagenicity to all the tester strains, both in the presence and absence of S-9 mix. The reaction product of pyrrole-2-carboxaldehyde with nitrite only gave a weak mutagenic response with strain TA100, while the pyrrole-2-carboxylic acid-nitrite reaction product did not produce a mutagenic response in any of the tester strains. Two mutagenically active fractions, separated by thin-layer chromatography, were found in the reaction of 2-acetylpyrrole with nitrite. The formation of mutagenic products in the latter reaction was found to vary with reaction pH, time and temperature, with nitrite level and with 2-acetylpyrrole concentration.     

Mutagenicity and toxicity studies of p-phenylenediamine and its derivatives

The mutagenicity of p-phenylenediamine and its derivatives was tested using Ames Salmonella strains TA98 and TA100. p-Phenylenediamine was weakly mutagenic to TA98 with metabolic activation. 2-Nitro-p-phenylenediamine was directly mutagenic to both strains, while 2-methyl-p-phenylenediamine required S9 mix. All the test compounds induced a dose-related increase in chromosomal aberrations m Chinese hamster ovary (CHO) cells in the absence of the S9 mix. The mutagenicity and toxicity of these compounds did not correlate with their oxidation potentials, or any other tested physicochemical properties including the energy difference between the lowest unoccupied and the highest occupied molecular orbital, ionization potential, and dipole moment.     

Mutagenicity studies on dihydroergocristine

Dihydroergocristine (DHEC) is an ergot derivative used for the therapy of patients with cerebrovascular insufficiency. It was tested for mutagenicity by means of four tests. In the mutagenicity in vitro assay on Salmonella typhimurium, DHEC was checked at 10,000 micrograms/plate on TA98 and TA1538 strains and at 3000 micrograms/plate on TA1535, TA1537 and TA100 strains with and without metabolic activation. In a quantitative in vitro test for mutagenicity in V79 Chinese hamster cells, DHEC was studied at concentrations between 30 and 0.3 microgram/ml with and without metabolic activation. DHEC was tested for its ability to induce chromosomal damage in human lymphocyte cultures utilizing the concentrations of 10, 3 and 1 microgram/ml. In the in vivo mouse (Swiss strain) micronucleus assay, DHEC was orally administered at two dosages (50% and 16% of LD50) following the schedule of the test. Dihydroergocristine is a drug free of mutagenic activity on the basis of all the results obtained from the above in vitro and in vivo tests.     

Mutagenicity evaluation of phthalic acid esters and metabolites in Salmonella typhimurium cultures

The mutagenic potential of dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), and di-2-ethylhexyl phthalate (DEPH), as well as metabolites of DEHP--i.e., mono-2-ethylhexyl phthalate (MEHP), 2-ethylhexanol (2-EH), and phthalic acid (PA)--were tested in Salmonella typhimurium cultures using the Ames test procedure. The compounds were tested on strains TA98, TA100, TA1535, TA1537, TA1538, and TA2637 for base-pair substitution or frameshift-type mutations. Spot tests yielded negative responses for all compounds with the strains tested. Each compound was tested for a dose-effect relationship in the TA98, TA100, TA1535, and TA1538 systems. DEP and DBP exhibited a mildly positive response in both TA100 and TA1535 cultures, and DMP showed a similar response in TA1535. Normalization of the data for cytotoxicity of DMP suggests TA100 has a mildly positive effect. The higher doses of these compounds exhibited some cytotoxic effects. The mutagenic effects were apparently abolished by the addition of S9 fraction in TA100 and TA1535 cultures, while no effect, other than cytotoxicity, was observed in the TA98 and TA1538 systems. DEHP, MEHP, 2-EH, and PA exhibited no mutagenicity in any of the strains of Salmonella typhimurium tested, with or without S9 metabolic activation. MEHP and 2-EH, however, exhibited a moderate cytotoxic effect in most cultures.     

The Salmonella mutagenicity of water and sediments from the Porsuk River in Turkey

In this study, water and sediments from the Porsuk River were investigated for their potential mutagenicity in Salmonella typhimurium TA 98 and TA 100 strains by performing Ames test (plate incorporation assay) without metabolic activation. Different columns of XAD 4 and XAD 16 were used to fractionate the water samples. Positive results in XAD 4 extracts of water samples were obtained for TA 98 in two of these stations. Extracts of the sediment samples were assayed in five different doses of concentrations and mutagenic results were obtained for both of the two strains in different sampling sites. Total metal concentrations in the stream sediment samples were determined by atomic absorption spectrophotometry (AAS) in order to explain some of these mutagenic results. The presence of mutagens causing frameshift and base-pair substitution mutations in water and sediments of the Porsuk River was suggested by the results and this mutagenicity was discussed.     

Mutagenicity tests of cashewnut shell liquid, rice-bran oil and other vegetable oils using the Salmonella typhimurium/microsome system

In view of the shortage of edible oils in India, nutritional and toxicological evaluations have been carried out on some unconventional oils to determine whether they might be safe for human consumption. As part of these evaluations, eight unconventional oils were tested by the Ames mutagenicity assay, using Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation with S-9 mix prepared from the livers of rats pretreated with sodium phenobarbitone or Aroclor 1254. Of the oils tested, metsa oil (Hibiscus sabdariffa) and cashewnut shell liquid were mutagenic with and without metabolic activation with S-9 of either source. No mutagenic activity (with or without S-9 of either source) was observed with any of the other oils tested (rice-bran oil, Cleome viscosa oil, mango-kernel oil, mahua oil, kapok oil and neem oil).     

Mutagenicity of cysteine and penicillamine and its enantiomeric selectivity

We previously observed that postmitochondrial supernatant (S9) from rat liver and kidney homogenates transforms L-cysteine into a mutagen that reverts bacteria of the strain Salmonella typhimurium TA100 to histidine independence. In the present study the enantiomers of cysteine and penicillamine (beta, beta-dimethylcysteine) have been investigated for mutagenicity. The Salmonella typhimurium strain TA92 was found to be more sensitive than TA100 to the mutagenic action of L-cysteine and was therefore also included. This strain allowed the unambiguous realization of a (weak) mutagenic effect of L-cysteine even in the absence of mammalian enzyme preparations. D-cysteine did not show mutagenicity under any experimental conditions. However, it was strongly bacteriotoxic. On the other hand, both enantiomers of penicillamine exerted clear mutagenic effects. Qualitatively, their mutagenicity was similar to that of L-cysteine in the following respects: the penicillamines were directly mutagenic, their mutagenicity was enhanced by S9, kidney S9 enhanced the mutagenicity more than did liver S9, TA92 was more sensitive than TA100. Thereby it is noteworthy that the ratios of the specific mutagenicities in the two strains were virtually identical in the direct, kidney-S9-mediated and liver-S9-mediated tests suggesting that the ultimate mutagens under these different metabolic conditions were identical. On the other hand, substantial quantitative differences in the mutagenicity between the beta-thiol amino acids were observed. L-penicillamine was about eight times more mutagenic than the clinically used enantiomer, D-penicillamine. In the direct tests, the mutagenic potency of L-cysteine was equal to that of D-penicillamine. In the S9-mediated experiments, the mutagenic potency of L-cysteine was intermediate between those of L- and D-penicillamine.     

Ames mutagenicity tests on purified 3-nitropropionic acid

3- Nitropropionic acid is a toxic compound produced by several moulds involved in food fermentation or spoilage. An impure commercial sample of this compound was previously reported as being mutagenic to Salmonella typhimurium strains TA1535 and TA100. In the present study, a sample from the same lot of 3- nitropropionic acid was mutagenic in strain TA100 without metabolic activation, but this activity was diminished after recrystallization. This sample was not mutagenic in strain TA98, before or after recrystallization. A new, purer commercial sample was non-mutagenic in strains TA98, TA100 and TA1538, with or without metabolic activation. Therefore the mutagenicity reported to be due to 3- nitropropionic acid was considered to be due to the impurity(ies).     

Screening of tabacco smoke constituents for mutagenicity using the Ames' test

To clarify the mutagenic activity of individual smoke components, 239 compounds, representative of the gaseous and semivolatile phases of tobacco smoke, were assayed for mutagenicity towards 4 histidine-requiring mutants of Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537). All compounds were tested qualitatively both with and without metabolic activation using a liver fraction (S-9) from Aroclor 1254 or methylcholanthrene induced rats. Without S-9, only 2,3-dimethylindole and 2,3,5-trimethylindole showed mutagenic activity that was not enhanced by hte metabolic activation system. 2,6-Diaminotoluene and coronene, which like the above compounds are not documented carcinogens were found to be mutagenic for strain TA 98 with S-9. Mutagenic activity was also observed for the previously known mutagens benz[a]pyrene, chrysene, benz[a]-anthracene, perylene and β-naphthylamine, on exposure to strains TA 98 and/or TA 100 with S-9.     

The mutagenicities of seven coumarin derivatives and a furan derivative (nimbolide) isolated from three medicinal plants

Seven coumarin derivatives (imperatorin, heraclenin, xanthotoxin, marmesin, chelepin, oxypeucedanin, esculin) and a furan derivative (nimbolide) were screened on 6 Ames tester strains (TA92, TA94, TA97, TA98, TA100, TA102). The eight compounds are chemicals isolated from three Nigerian medicinal plants: Afraegle paniculata, Clausena anisata, and Azadirachta indica. Different preparations of the former are taken by Nigerians for gut disturbances, and a concoction of the latter called "Agbo" is taken as an antimalarial. Marmesin and imperatorin were mutagenic in all tester strains except TA94 and TA102. The mutagenicity potencies of marmesin and imperatorin were 20 and 0.2 respectively. Mutagenicity was highest in TA98 and TA100 in both compounds. Marmesin was optimally mutagenic at a dose of 1.04 micrograms, and imperatorin at 30.0 micrograms. Microsomal activation was not required for mutagenicity in both compounds.     

Mutagenicity of human urine after the consumption of fried salted salmon

Mutagenicity in the urine of four non-smoking individuals who had eaten salted salmon cooked at home for both lunch and supper was monitored by means of Salmonella/microsome mutagenicity tests. Extracts from fresh and salted salmon had the same level of mutagenicity after being cooked for 10 min at 200 degrees C, but no activity was detected before cooking. Salmonella strains TA98 and TA1538 were equally sensitive to the mutagens and required metabolic activation. No mutagenicity was shown with TA100 and TA1535. Urine samples were tested using a concentrate prepared by means of an XAD-2 resin column. Mutagenicity was detected mainly in urine excreted during 4-5 hr after the ingestion of cooked salmon, but only weak mutagenicity, or none at all, was detected in the urine after the ingestion of vegetables. The levels of urinary mutagenicity due to salmon consumption were not affected when cabbage was eaten simultaneously. The excretion of mutagenic substances was completed within about 20 hr, and there were almost no mutagens in the urine 24 hr after the ingestion of cooked salmon.     

Strong mutagenic effects of diesel engine emissions using vegetable oil as fuel

Diesel engine emissions (DEE) are classified as probably carcinogenic to humans. In recent years every effort was made to reduce DEE and their content of carcinogenic and mutagenic polycyclic aromatic compounds. Since 1995 we observed an appreciable reduction of mutagenicity of DEE driven by reformulated or newly designed fuels in several studies. Recently, the use of rapeseed oil as fuel for diesel engines is rapidly growing among German transportation businesses and agriculture due to economic reasons. We compared the mutagenic effects of DEE from two different batches of rapeseed oil (RSO) with rapeseed methyl ester (RME, biodiesel), natural gas derived synthetic fuel (gas-to-liquid, GTL), and a reference diesel fuel (DF). The test engine was a heavy-duty truck diesel running the European Stationary Cycle. Particulate matter from the exhaust was sampled onto PTFE-coated glass fibre filters and extracted with dichloromethane in a soxhlet apparatus. The gas phase constituents were sampled as condensates. The mutagenicity of the particle extracts and the condensates was tested using the Salmonella typhimurium/mammalian microsome assay with tester strains TA98 and TA100. Compared to DF the two RSO qualities significantly increased the mutagenic effects of the particle extracts by factors of 9.7 up to 59 in tester strain TA98 and of 5.4 up to 22.3 in tester strain TA100, respectively. The condensates of the RSO fuels caused an up to factor 13.5 stronger mutagenicity than the reference fuel. RME extracts had a moderate but significant higher mutagenic response in assays of TA98 with metabolic activation and TA100 without metabolic activation. GTL samples did not differ significantly from DF. In conclusion, the strong increase of mutagenicity using RSO as diesel fuel compared to the reference DF and other fuels causes deep concern on future usage of this biologic resource as a replacement of established diesel fuels.