The effect of solvent and extraction methods on the bacterial mutagenicity of sidestream cigarette smoke

The mutagenic activity of sidestream cigarette smoke particles was estimated by testing sidestream cigarette smoke particles which had been collected under controlled burning conditions in the laboratory. Two different extraction methods (Soxhlet and ultrasonic agitation) and 3 different solvents (dichloromethane, methanol, and acetone) were compared for their efficiencies in the extraction of compounds from sidestream cigarette smoke particles which are mutagenic in the Ames test. The mutagenic activity of the sidestream smoke particles was estimated to be 15,000-20,000 revertants per cigarette in TA98 with metabolic activation and 12,000-17,000 revertants per cigarette in TA100 without metabolic activation. Only weak mutagenic activity was detected in TA98 without activation and in TA100 with activation. Under test conditions used, ultrasonic agitation produced the most consistent results and acetone extraction produced the highest levels of mutagenic activity.     

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.     

Ames mutagenicity tests of products from a heated potato-starch system

A charred sample was prepared from potato starch heated with ammonium carbonate at 600°C in a flask under a nitrogen stream. The water produced was collected and extracted with methylene chloride. The basic fraction obtained from the extract exhibited strong mutagenicity in Ames assays using Salmonella typhimurium strains TA98 or TA100 with metabolic activation (rat-liver S-9 mix). The basic fraction was further fractionated by silica gel column chromatography and subsequently by Scphadex column chromatography. Some of the resulting fractions exhibited strong mutagenic activities in S. typhimurium strain TA98 with S-9 mix.     

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.     

The mutagenicity of some of the proposed metabolites of direct black 38 and pigment yellow 12 IN THE Salmonella typhimurium assay system

The Ames Salmonella/mammalian microsome assay system was used to evaluate the mutagenic potential of some of the proposed metabolites of Direct Black 38 and Pigment Yellow 12. Direct Black 38, benzidine, 4-aminobiphenyl, monoacetylbenzidine, and diacetylbenzidine were mutagenic for at least one of the tester strains after metabolic activation with mouse liver S-9 fractions. The proposed metabolites of Pigment Yellow 12, namely dichlorobenzidine, monoacetyldichlorobenzidine and diacetyldichlorobenzidine were strongly mutagenic for Salmonella typhimurium TA 98.The major metabolite in the urine from hamsters given a single dose of Direct Black 38 (100 mg/kg) was found to be monoacetylbenzidine. Monoacetylbenzidine and the urine from the animals treated with Direct Black 38 were active in the Ames test only after S-9 fraction was added to the test mixture.     

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 of Mouriri pusa Gardner and Mouriri elliptica Martius

Mouriri pusa Gardner and Mouriri elliptica Martius are fruit-bearing plants of the Melastomataceae family, popularly known in Brazil as puçá-preto or jaboticaba-do-cerrado, and they are used in folk medicine for the treatment of gastric ulcers. In this study, we employ the Ames test to assess the mutagenicity of compounds obtained from the leaves of these species. The methanol extract of the M. pusa was mutagenic to the Salmonella typhimurium strains TA98, TA97a and TA100, with or without metabolic activation. The methanol extract of M. elliptica induced mutagenic activity in TA98 when metabolized with S9 fraction and TA97a with and without S9, but with lower mutagenicity index (MI) and potencies values than those for M. pusa. Enriched fractions of flavonoids and tannins of M. pusa were also evaluated and they demonstrated positive mutagenicity. The highest values of MI and potency were obtained with the flavonoid fraction, which contains large amounts of quercetin, quercetin glycosides and myricetin. These compounds are probably related to the mutagenicity observed in the Ames test. The dichloromethane extract was not mutagenic in any of the test conditions employed.     

In vitro mutagenicity of rubber chemicals and their nitrosation products

14 chemicals employed in rubber manufacture were assayed in the Salmonella reversion test with the strains TA98 and TA100. Mixed diaryl-p-phenylenediamines were weakly mutagenic in TA98 after metabolic activation; poly-p-dinitrosobenzene was active in TA98 without as well as with S9. After in vitro reaction with nitrite at low pH, mixed diaryl-p-phenylenediamines became directly mutagenic in both strains, whereas poly-p-dinitrosobenzene retained its activity unchanged. Furthermore, 4 of the remaining chemicals acquired mutagenic characteristics: in the presence of S9, N,N'-dimethylpentyl-p-phenylenediamine reverted TA98 and hexamethylenetetramine reverted both TA98 and TA100; N-isopropyl-N'-phenyl-p-phenylenediamine was mutagenic in TA98 with and without S9; N-nitrosodiphenylamine was active in both strains without S9 and weakly mutagenic in TA98 after metabolic conversion.     

Monitoring the rivers Rhine and Meuse in the Netherlands for mutagenic activity using the Ames test in combination with rat or fish liver homogenates

Organic concentrates of water of the rivers Rhine and Meuse and a control lake were tested for mulagenic activity with the Ames Salmonella/microsome assay at 3-mth intervals for more than 1 yr. The river samples were concentrated by adsorption on XAD (10³-fold) followed by elution with DMSO.Using strain TA 98, all Rhine water samples except one were found to contain both direct and indirect mutagens. No significant mutagenic activity was detected in the lake samples and most of the river Meuse samples. None of the samples were shown to be mutagenic when tested with strains TA 100 or TA 1535.On examining one Rhine location more frequently, the mutagenic activity was found to be persistent and to vary about 5- to 6-fold during one year.Finally, liver homogenates of bream (Abramis brama) from these waters were compared with the standard rat liver S-9 with regard to their ability to activate the indirect mutagens present in the water concentrates. Compared with the rat liver homogenates, the liver homogenates of Rhine fish were found to be equally active and those of Meuse fish somewhat less. No metabolic activation was observed with liver homogenates of the lake fish.     

Mutagenic activity of cytostatic methyl hydrazones with different strains of Salmonella typhimurium

Experiments are performed to ascertain the mutagenic properties of four new cytostatic methyl-hydrazones in the Ames test using different strains of Salmonella typhimurium. As could be demonstrated all four hydrazones are mutagenic per se without a metabolic activation through rat liver microsomes (S-9 fraction). Whereas the -chloroethyl hydrazones B1 and B2 cause a base-pair substitution with the strains TA100 and TA1535 the methyl-hydrazones EB4 and CyB4 both cause base-pair substitution with TA100 and frameshift mutation with TA98. At both strains the mutagenic activity of CyB4 ist powerful. Furthermore, no relation could be detected between the mutagenic properties of the methyl-hydrazones and their alkylating behaviour on 4-(4-nitrobenzyl)-pyridine.     

Effects of using liver fractions from different mammals, including man, on results of mutagenicity assays in Salmonella typhimurium

Twenty chemicals, including 16 aromatic amines, were studied in the Salmonella/mammalian-microsome mutagenicity test using the bacterial strains TA100 and TA98 to compare the activation potential of liver preparations from several mammalian species. The hepatic post-mitochondrial supernatants (S-9 fractions) of rat, mouse, hamster, dog, monkey and man were used for metabolic activation. Striking quantitative and even qualitative differences were apparent in the capacity of the different preparations to activate the compounds to mutagens. All compounds that gave positive results in the Ames test when activated with a liver preparation from Aroclor-pretreated rats were also identified as mutagens when tested in the presence of S-9 from one or more other species. Four substituted anilines, however, were converted to mutagenic metabolites only in the presence of a post-mitochondrial fraction of hamster liver. Three human carcinogens, 2-aminoanthracene, benzidine and cyclophosphamide were detected as mutagens under various experimental conditions, including metabolic activation by human or monkey liver S-9. There were no qualitative differences in the mutagenic responses obtained in assays with human and monkey liver S-9.     

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).     

In vitro antimutagenicity of curcumin against environmental mutagens

The effects of curcumin, the yellow pigment of the spice, turmeric (Curcuma longa) on the mutagenicity of several environmental mutagens were investigated in the Salmonella/microsome test with or without Aroclor 1254-induced rat-liver homogenate (S-9 mix). With Salmonella typhimurium strain TA98 in the presence of S-9 mix, curcumin inhibited the mutagenicity of bidi and cigarette smoke condensates, tobacco and masheri extracts, benzo[a]pyrne and dimethyl benzo[a]anthracene in a dose-dependent manner. Curcumin did not influence the mutagenicity without S-9 mix of sodium azide, monoacetylhydrazine and streptozocin in strain TA100 nor of 4-nitrophenylenediamine in strain TA98. Our observations indicate that curcumin may alter the metabolic activation and detoxification of mutagens.     

On the mutagenicity of metabolites derived from the mushroom poison gyromitrin

The hepatotoxic and carcinogenic hydrazine N-methyl-N-formyl hydrazine (MFH), which is formed from the mushroom poison gyromitrin by hydrolytic cleavage in vivo and in vitro during food processing is much more mutagenic for the strain TA 100 of Salmonella typhimurium in the presence of a metabolic activation system than in its absence. On the other hand, acetylated MFH (Ac-MFH) was not mutagenic for TA 100 in both test conditions. For the strain TA 98 neither MFH nor Ac-MFH were mutagenic both with and without metabolic activation. Therefore, a metabolic conversion of the free NH₂-moiety of MFH into a genotoxic metabolite of MFH is postulaed.     

Mutagenic activity at different stages of an industrial ammonia caramel process detected in Salmonella typhimurium TA100 following pre-incubation

Mutagenic activity of a commercial ammonia caramel colouring was demonstrated in Salmonella typhimurium TA100 without metabolic activation. The activity in strain TA100 was increased using a 10-min pre-incubation, and a clear dose-response relationship was seen using this method. Investigation of samples taken from the different stages in the industrial process showed a constant level of mutagenic activity in samples from the middle to the end of the heating process with a steep increase in the sample taken after the end of heating. No mutagenic activity was seen in assays with S. typhimurium strains TA1535 and TA98     

Mutagenicity of the products obtained from heated milk systems

Methylene chloride extracts of the browning reaction products prepared from model systems consisting of major milk components (casein and/or lactose, and non-fat dried milk) were tested for mutagenicity in the Ames Salmonella/microsome assay. Samples obtained by heating aqueous solutions of these components under either neutral or basic (pH 10) conditions exhibited no significant mutagenic activity when tested with or without S-9 mix. The addition of common food additives, such as sodium nitrite, butylated hydroxyanisole and butylated hydroxytoluene, to the aqueous solutions did not enhance the mutagenic activity of the browning samples. On the other hand, the tar samples prepared by heating the same milk components in the dry state exhibited strong mutagenicity, primarily to Salmonella typhimurium strain TA98 and only with S-9 mix. A casein/lactose mixture and non-fat dried milk were also heated with baking soda in the dry state. The presence of the baking soda enhanced the mutagencity of the browning products; the tar from the non-fat dried milk heated with baking soda was the most potently mutagenic of all the samples towards strain TA98 and also produced a positive response in strain TA100 in the presence of S-9 mix.     

Mutagenic activity of cytostatic methyl hydrazones with different strains of Salmonella typhimurium.

Experiments are performed to ascertain the mutagenic properties of four new cytostatic methyl-hydrazones in the Ames test using different strains of Salmonella typhimurium. As could be demonstrated all four hydrazones are mutagenic per se without a metabolic activation through rat liver microsomes (S-9 fraction). Whereas the beta-chloroethyl hydrazones B1 and B2 cause a base-pair substitution with the strains TA100 and TA1535 the methyl-hydrazones EB4 and CyB4 both cause base-pair substitution with TA100 and frameshift mutation with TA98. At both strains the mutagenic activity of Cy84 ist powerful. Furthermore, no relation could be detected between the mutagenic properties of the methyl-hydrazones and their alkylating behaviour on 4-(4-nitrobenzyl)-pyridine.     

Mutagenicity testing of 2,3,7,8-tetrachlorodibenzo-p-dioxin in histidine auxotrophs of Salmonella typhimurium

The mutagenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been examined using the Salmonella typhimurium histidine auxotrophs TA1535, TA100, TA1538, TA98, and TA1537. TCDD did not show mutagenicity in any of these auxotrophs in the presence of mammalian metabolic activating systems isolated from the livers of Arochlor 1254-treated rats, Arochlor 1254-treated hamsters, or TCDD-treated hamsters. Test run in the absence of NADP⁺-dependent metabolic activation failed to reveal any mutagenic activity of TCDD.     

Evaluation, using Salmonella typhimurium, of the mutagenicity of seven chemicals found in cosmetics

Six chemicals used as ingredients in cosmetics were evaluated for mutagenic activity in Salmonella typhimurium. Two of these ingredients, trans-4-phenyl-3-butene-2-one and 2,2′,4,4′-tetrahydroxybenzophenone, were mutagenic in the presence of rat liver S-9 towards strains TA100 and TA1537 respectively. An impurity found in some cosmetic products, N-nitrosodiethanolamine, was mutagenic to S. typhimurium stains TA1535 and TA100 in the presence of hamster-liver S-9 but not rat-liver S-9.