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.     

Mutagenicity of cisplatin and carboplatin used alone and in combination with four other anticancer drugs

Mutagenicity of cisplatin and carboplatin was compared by using the drugs alone and in combination with bleomycin, 5-fluorouracil, vincristine and methotrexate in the Ames Salmonella assay employing the tester strains TA98, TA100 (excision deficient) and TA102 (excision proficient). Cisplatin showed the maximum yield of histidine revertants in TA98 and TA100 at 2 micrograms/plate followed by a decrease in the number of mutants/plate with increasing concentrations. In the excision proficient strain TA102, there was no decline in the number of mutants/plate even at a concentration of 8 micrograms/plate. Basically, similar results were also obtained with carboplatin but using higher concentrations of the drug. When cisplatin or carboplatin was combined with other anticancer drugs, there was no differential modification of mutagenicity of the 2 platinum compounds in any of the bacterial tester strains.     

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 of nitroxyl compounds: structure-activity relationships.

Three piperidinoxyl radicals were found to be directly mutagenic in Salmonella typhimurium TA 100, one pyrrolidinoxyl compound had weaker activity, and two other pyrrolidinoxyl derivatives did not produce an increase of the spontaneous revertants. The tester strain TA 100 was selected in preliminary tests for its higher sensitivity compared to TA 98 and TA 102. The mutagenic activity of the three active compounds was abolished by partial reduction with ascorbic acid, suggesting that the mutagenicity was linked to the free radical nature of these compounds, and reduced in the presence of a cofactor supplemented rat liver subcellular fraction. The mutagenicity of the tested compounds was correlated to the resistance of the nitroxyl spin labels to reduction: the more reactive radicals were found to possess higher mutagenic activity.     

Mutagenicity of the C-nitroso analog of fenitrothion

The chemicals fenitrothion, nitroso fenitrothion, amino fenitrothion and 3-methyl-4-nitrophenol were tested for mutagenicity to Salmonella typhimurium strains TA98 and TA100, both in the presence and absence of rat liver S-9 mix. The strong mutagenicity of nitroso fenitrothion to both strains either in the presence or absence of S-9 mix contrasted with the observation that fenitrothion displayed no mutagenicity in these tester strains. The results suggest that the normal nitroreductases present in TA98 and TA100 cannot metabolize fenitrothion to a mutagenic metabolite. This inability of the tester strains to effect partial nitroreduction results in the failure of this screening system to predict the potential genotoxicity of this pesticide.     

A notable antimutagenicity of two nonmutagenic novel oxadiazoles in Salmonella mutagenicity assay

The mutagenic activity of two newly synthesized oxadiazoles: 1,3-bis(5-benzylthio-1,3,4-oxadiazol-2-yl) benzene (M1) and 1,4-bis(5-benzylthio-1,3,4-oxadiazol-2-yl) benzene (M2) was studied in Salmonella typhimurium strains TA97, TA100, TA102 and TA1537 in the presence and absence of S9mix. The antimutagenicity of M1 and M2 against H2O2, sodium azide (SA) and 4-nitro-o-phenylene diamine (NPD) using the tester strains TA102, TA100 and TA97, respectively, was also investigated. The two compounds were found to be nonmutagenic using the four tester strains. However, they showed high mutagenic repression activity against hydrogen peroxide (95% and 97% for M1 and M2, respectively, at a concentration of 335 micrograms/plate). Moderate mutagenic repression against NPD (58% and 55% for M1 and M2, respectively, at a concentration of 167.5 micrograms/plate) and low mutagenic repression against SA (21% and 33% for M1 and M2 respectively, at a concentration of 335 micrograms/plate) was detected. The obtained results are very encouraging to test the above mentioned compounds as anticarcinogens.     

Mutagenicity evaluation of riot control agent o-chlorobenzylidene malononitrile (CS) in the Ames Salmonella/microsome test.

o-Chlorobenzylidene malononitrile (CS), a riot control agent, was evaluated for its possible mutagenic activity in the Ames Salmonella/mammalian microsome mutagenicity test. Five histidine-deficient (His-) mutant tester strains of Salmonella typhimurium--TA97a, TA98, TA100, TA102 and TA104--were used. The liquid preincubation procedure was used with metabolic activation (presence of S9 mixture) and without metabolic activation (absence of S9 mixture). For the experiments with metabolic activation, three different concentrations of S9 fraction (supernatant of Aroclor 1254-induced rat liver homogenate at 9000 g)--5%, 15% and 30% in S9 mixture--were used. Along with mutagenic activity, CS was also evaluated for cytotoxic activity in all the five tester strains of Salmonella typhimurium, both in the presence and absence of S9 mixture. The mutagenic and cytotoxic activities of CS were assessed by counting the His+ revertant colonies and by counting the microcolonies (His-, auxotrophs in the background lawn), respectively, and the respective mean values were compared with the relative negative (solvent) control. A dose range of 12.5-800 micrograms plate-1 for CS did not induce a mutagenic response either in the presence or absence of S9 mix. No change in the negative mutagenic response of CS has been observed even in the presence of an elevated level of S9 fraction in the S9 mix. A dose of 200 micrograms plate-1 for CS was found to be cytotoxic by decreasing the surviving cells as well as His+ revertant colonies; however, the effect was reduced in the presence of an elevated level of S9 fraction in the S9 mix.     

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.     

Mutagenic activity promoted by amentoflavone and methanolic extract of Byrsonima crassa Niedenzu

Byrsonima crassa is a plant pertaining to the Brazilian central savannah-like belt of vegetation and popularly used for the treatment of gastric dysfunctions and diarrhoea. The methanol extract contains catechin, tannins, terpenes and flavonoids; both mutagenic potential and antioxidant properties have been ascribed to flavonoids. The mutagenicity of some flavonoids is believed to be associated with the formation of reactive oxygen species and seems to depend on the number and position of hydroxyl groups. In the present study the mutagenic activity of the methanol, chloroform and 80% aqueous methanol extracts, as well as acetate and aqueous sub-fractions, of this medicinal plant were evaluated by Salmonella typhimurium assay, using strains TA100, TA98, TA102 and TA97a, and in mouse reticulocytes. The results showed mutagenic activity of the methanolic extract in the TA98 strain without S9, but no mutagenicity to mouse cells in any of the extracts. The acetate fraction showed strong signs of mutagenicity without S9, suggesting that in this enriched fraction were concentrated the compounds that induced mutagenic activity. The aqueous fraction showed no mutagenic activity. The TLC and HSCCC analyses of the acetate fraction with some standard compounds permitted the isolation of the quercetin-3-O-beta-D-galactopyranoside, quercetin-3-O-alpha-L-arabinopyranoside, amentoflavone, methyl gallate and (+)-catechin, of which only the amentoflavone exhibited positive mutagenicity to TA98 (+S9, -S9).     

Mutagenicity of some monoaromatic hydroxamic acids

The mutagenicity of some monoaromatic hydroxamic acids was tested in the presence and absence of rat liver S-9 with Salmonella typhimurium tester strains TA98 and TA100. Of the five N-(chlorophenyl)-substituted hydroxamic acids and seven N-arylformohydroxamic acids tested, 2 of the first and 4 of the latter series were mutagenic to both strains upon metabolic activation. None of the four N-acetyl-type hydroxamic acids was mutagenic to either strain, even upon activation. Because some of the N-acetyl-derived hydroxamic acids were inactive, whereas the same aromatic nucleus possessing a formyl group displayed significant activity, a consideration of the nature of the aryl group in hydroxamic acid mutagenicity is important.     

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.     

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.     

Effect of micronutrients,antioxidants and related compounds on the mutagenicity of 3,2′-dimethyl-4-aminobiphenyl,a colon and breast carcinogen

The possible antimutagenic effects of butylated hydroxytoluene (BHT), disulfiram, indole-3-carbinol, indole-3-acetonitrile, sodium selenite and α-tocopherol on 3,2′-dimethyl-4-aminobiphenyl-induced mutagenicity were studied using the Ames Salmonella/mammalian microsome assay system with strains TA98 and TA100. All seven compounds were nonmutagenic in both bacterial tester strains. The addition of 50–250 μg of sodium selenite, 5–50 mg of α-tocopherol of 50–250 μg of BHT per plate inhibited DMAB-induced mutagenicity in TA98 and/or TA100. Ethoxyquin, disulfiram and indole-3-carbinol increased DMAB-induced mutagenicity in TA100, whereas these compounds had little or no effect in TA98. Indole-3-acetonitrile had very little effect in either strain.     

N,N-diethylphenylacetamide, an insect repellent: absence of mutagenic response in the in vitro Ames test and in vivo mouse micronucleus test

N,N-diethylphenylacetamide (DEPA), a promising new insect repellent, was tested for mutagenicity in the in vitro Ames Salmonella/microsome mutagenicity test and the in vivo mouse micronucleus test. For the Ames test, DEPA was assayed both in the presence and absence of Aroclor 1254-induced rat-liver S-9 mix (5 and 20% S-9 fraction), using five tester strains of Salmonella typhimurium--TA97a, TA98, TA100, TA102 and TA104. For the micronucleus test, mice were exposed to DEPA through ip injection for 2 and 5 days in separate experiments, and bone marrow and peripheral blood were sampled 6 and 48 hr after the final injection, respectively. DEPA did not induce a mutagenic response in the Ames test, and mouse bone marrow and peripheral blood micronucleus tests. DEPA was not considered cytotoxic, as a depression of the percentage PCE was not observed at any dose in the range of 1 to 100 mg/kg body weight with either treatment protocol of the micronucleus test.     

Tofisopam--evaluation of mutagenic and genotoxic properties

The mutagenic properties of tofisopam, the member of the 2,3-benzodiazepine family, were evaluated on the basis of Ames test with Salmonella typhimurium TA1537, TA97, TA98, TA100 and TA102 strains. The genotoxic properties of tofisopam were estimated on L929 cell line with the cytokinesis-block technique. Under the experimental conditions, no mutagenic activity of tofisopam in tester bacteria strains was found, and no genotoxic activity was observed.     

Synthesis,characterization and mutagenicity of new cis-[Pt(2-substituted-benzimidazole)2Cl2] complexes

In this study, four new platinum(II) complexes with the structures cis-[Pt(Ligand)2Cl2] (ligand = 2-(p-methoxy-/or-p-chlorobenzyl or p-methoxyphenyl)benzimidazol (1, 2, 4 respectively) and 5(6)-methyl-2-phenoxymethylbenzimidazole (3) were synthesized and characterized by their elemental analysis, and IR and 1H NMR spectra. The potentials of the Pt(II) complexes for short-term bacterial mutagenicity were tested in reverse-mutation assays using Salmonella typhimurium frame-shift strain T 98 and S. typhimurium TA 100 and TA 102 strains, which carry mutations particularly sensitive to reversion by DNA base-pair substitution. The tests were performed in the absence of S9 rat liver fraction. Among the complexes tested 1 had no mutagenic activity. Complex 4 was found to be weakly mutagenic in TA 98 only. The Pt(II) complexes 2 and 3 were found to be mutagenic in TA 98, TA 100 and TA 102.     

Comparative mutagenicity studies of azo dyes and their reduction products in Salmonella typhimurium

The arabinose-resistant and Ames assay systems of Salmonella typhimurium were used to evaluate the mutagenic potential of azo dyes and their aromatic amine reduction products. Azo dyes, namely direct black 38, direct blue 15, and direct red 2, were mutagenic in the arabinose-resistant and Ames assays with both hamster and rat liver S9 activation. Both assays gave relatively higher mutagenic responses with hamster S9. Reduction products of these dyes, namely benzidine, o-dianisidine, and o-tolidine, were mutagenic in the Ames assay. Benzidine was weakly mutagenic and o-dianisidine and o-tolidine were nonmutagenic in the arabinose-resistant assay. These results indicate that both arabinose-resistant tester SV50 and Ames tester TA98 were sensitive in detecting mutagenicity of azo dyes. The use of the standard plate protocol with Ames tester TA98 is more efficient than the modified azo dye protocol in detecting mutagenicity of aromatic amine reduction products. Additional modifications in either the standard plate or modified azo dye protocols may improve detection of mutagenicity of these compounds in the arabinose-resistant assay system.     

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.     

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.     

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.