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.     

Mutagenicity of commercial hair dyes in Salmonella typhimurium TA98

Commercial permanent hair-dye formulations containing p-phenylenediamine, resorcinol and aminophenols were incubated with hydrogen peroxide and then tested for their ability to induce reverse mutations in Salmonella typhimurium TA98. Approximately half of the formulations (12 out of 25) gave positive results. The activity varied widely in degree and was observed only in the presence of an S-9 microsomal fraction from Aroclor-induced male rats. Five of the 12 positive formulations and one negative dye were administered topically to male rats; with one exception the urines of animals treated with the mutagenic hair dyes gave positive results in the presence of the S-9 mix.     

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 mutagenicity of butadiene towards salmonella typhimurium

Gaseous butadiene (BUT) was mutagenic towards S. typhimurium strain TA 1530 when the incubation mixture was supplemented with a NADPH-fortified rat liver microsomal preparation; mutagenicity increased with the dose.A significant mutagenic effect was similarly observed when the petri dishes, containing the bacteria but no metabolic activation system, were incubated in the presence of butadiene, in a desiccator in which plates containing the S-9 rat liver fraction had been placed. This indirect mutagenic effect was attributed to the formation, by the S-9 mix, of volatile intermediate(s) that migrated and induced mutations in neighbouring bacteria.     

Investigation of extracts from (Tunisian) Cyperus rotundus as antimutagens and radical scavengers

This study evaluates mutagenic and antimutagenic effects of aqueous, total oligomers flavonoïds (TOF), ethyl acetate and methanol extracts from aerial parts of Cyperus rotundus with the Salmonella typhimurium assay system.The different extracts showed no mutagenicity when tested with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1538 either with or without the S9 mix. On the other hand, our results showed that all extracts have antimutagenic activity against Aflatoxin B1 (AFB1) in TA100 and TA98 assay system, and against sodium azide in TA100 and TA1535 assay system. TOF, ethyl acetate and methanol extracts exhibited the highest inhibition level of the Ames response induced by the indirect mutagen AFB1. Whereas, ethyl acetate and methanol extracts exhibited the highest level of protection towards the direct mutagen, sodium azide, induced response. In addition to antimutagenic activity, these extracts showed an important free radical scavenging activity towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. TOF, ethyl acetate and methanol extracts showed IC₅₀ value of 15, 14 and 20 μg/ml, respectively.Taken together, our finding showed that C. rotundus exhibits significant antioxidant and antimutagenic activities.     

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.     

Ames mutagenicity tests of overheated brewed coffee

Five kinds of coffee samples were prepared from a commercial drip-grind coffee in order to examine the mutagenicity of brewed coffee using the Ames test. The samples prepared were a thick coffee syrup, coffee solid residues, dichloromethane and ethanol extracts of solid residues, a dichloromethane extract of a distillate from normally heated brewed coffee and dichloromethane extracts of distillates from overheated (150–300°C) brewed coffee. The samples were tested for mutagenicity towards Salmonella typhimurium strains TA98 and TA100 both with and without metabolic activation (S-9 mix). Only the extracts of the distillates obtained from coffee heated to 150° or 300°C exhibited mutagenicity towards strain TA98 with S-9 mix.     

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.     

Mutagenesis of certain benzo(a)pyrene phenols in vitro following further metabolism by mouse liver.

Low concentrations of 1-hydroxy- and 3-hydroxybenzo[a]pyrene in the presence of NADPH and liver S-9 fraction from 3-methylcholanthrene-treated C57BL/6N mice are as much as 3-fold more mutagenic than benzo[a]pyrene in the bacteria Salmonella typhimurium LT₂ tester strain TA98. The level of mutagenicity rises with increasing phenol or S-9 protein concentration. In this system, 9-hydroxy-benzo[a]pyrene is slightly mutagenic, while 2-hydroxy-, 7-hydroxy- and 12-hydroxybenzo[a]pyrene are not mutagenic at low concentrations. The S-9 fraction from 3-methylcholanthrene-treated DBA/2N mice or phenobarbital-treated C 5 7BL/6N mice does not support significant levels of mutagenesis. The high level of mutagenicity by 1-hydroxy- or 3-hydroxybenzo[a]pyrene is inhibited by α-naphthoflavone but is not inhibited by metyrapone, 1, 2-epoxy-3, 3, 3-trichloropropane or glutathione. The substrate for UDP-glucuron-osyltransferase, UDP-glucuronic acid, prevents more than half of the mutagenicity caused by the further metabolism of 1-hydroxy- and 3-hydroxybenzot alpyrene. The combination of UDP-glucuronic acid and UDP-N-acetylglucosamine provides an even higher level of protection. The addition of the substrate for sulfotransferase(s), 3'-phosphoadenosine 5'-phosphate sulfate, also prevents about half of the mutagenesis caused by 1-hydroxy- or 3-hydroxybenzo[a]pyrene.     

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.     

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.     

Mutagenic,antimutagenic and antioxidant potency of leaf extracts from Nitraria retusa

Four extracts were prepared from the leaves of Nitraria retusa; methanol, ethyl acetate, chloroform and hexane extracts. An assay for the ability of these extracts to prevent mutations induced by various oxidants in Salmonella typhimurium TA102 and TA 104 strains was conducted. These extracts from leaf parts of N. retusa showed no mutagenicity either with or without the metabolic enzyme preparation (microsome fraction). The highest protection against methylmethanesulfonate induced mutagenicity was observed with chloroform and methanol extracts with inhibition percentages of 44.93% (at 50 μg/plate in the presence of TA102 strain) and 38% (at 10 μg/plate in the presence of TA104 strain), respectively. Whereas Hexane and chloroform extracts reduced the mutagenicity induced by 2-aminoanthracene by 83.4% (using the S. typhimurium TA104 assay system) and 65.3% (using the S. typhimurium TA 102 assay system), respectively. Antioxidant activity of N. retusa extracts was determined by the ability of each extract to protect plasmid DNA against strand scission induced by hydroxyl radicals. Chloroform extract exhibited the highest ability to protect plasmid DNA against hydroxyl radical induced DNA damages and exhibited the highest antioxidant capacity, with 0.95 mM trolox equivalent when tested by the ferric reducing/antioxidant method.     

Isolation and chemical--structural identification of a novel aromatic amine mutagen in an ozonized solution of m-phenylenediamine

The mutagenicity of a m-phenylenediamine (m-PD) solution was markedly enhanced by oxidation with ozone. The ethyl acetate extracts from a m-PD solution ozonized at pH 10.7 were fractionated by normal-phase and reversed-phase column chromatography to isolate mutagens by monitoring mutagenic activities on Salmonella typhimurium TA98 in the presence of a mammalian metabolic activation system (S9 mix). From fraction 5-3-2, which exhibited the strongest mutagenicity (308000 revertants/mg), a major mutagenic compound was isolated. On the basis of the high-resolution EI-mass, (1)H NMR and (13)C NMR spectral, and X-ray crystallography data, the structure of this compound was determined to be 2-amino-5-[(3-aminophenyl)amino]-4-[(3-aminophenyl)imino]-2, 5-cyclohexadien-1-one (PDT-1), which is a novel compound. PDT-1 is a newly identified frame-shift type mutagen, inducing 65400 revertants and 295000 revertants of S. typhimurium TA98 and YG1024 per micromole, respectively, in the presence of S9 mix. When a m-PD solution was oxidized with 1 or 2 mol of ozone at pH 4.0, 7.0, and 10.7, the contribution of PDT-1 to the mutagenicity of ethyl acetate extracts from the ozonized m-PD solution was 5-23%.     

Possible mutagenic constituents in nitrite-treated soy sauce

Soy sauce was heated with 100, 500, 1000 or 2000 ppm sodium nitrite for 30 min at 80°C and pH 3. The reaction mixtures were extracted with dichloromethane followed by ethyl acetate. After removal of the solvents, the extracts were subjected to analysis (gas chromatograph-thermal energy analyser and gas chromatograph-mass spectrometer) and Ames mutagenicity tests. N-Nitrosodimethylamine and N-nitrosodiethylamine were found in the dichloromethane extract of the soy sauce treated with 2000 ppm nitrite at levels of 10 and 120 μg/ml, respectively. N-Nitrosoproline was identified in the ethyl acetate extract of the same sample at a level of 0.5 μg/ml. Both extracts exhibited dose-related mutagenicity in Salmonella typhimurium strain TA100 with S-9 mix. The dichloromethane extract showed much higher mutagenicity than did the ethyl acetate extract. The samples obtained from soy sauce treated with 100, 500 and 1000 ppm nitrite were not mutagenic, but N-nitrosodiethylamine was detected by thermal energy analysis in the soy sauce treated with 1000 ppm nitrite. The addition of 10,000 ppml-ascorbic acid, along with 2000 ppm nitrite, to soy sauce prevented the formation of mutagenic materials or detectable nitrosamines.     

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.     

Evaluation of acute and subacute toxicity and mutagenic activity of the aqueous extract of pecan shells [Carya illinoinensis (Wangenh.) K. Koch]

The infusion of pecan shells has been used to prevent and control hypercholesterolemia, diabetes and toxicological diseases. The aim of the present study was to evaluate toxicity and mutagenic effects of pecan shells aqueous extract (PSAE). Wistar rats were treated with a single dose of 300 or 2000 mg/kg of PSAE in the acute toxicity test. For the subacute test, the animals received 10 or 100 mg/kg of PSAE for 28 days. The mutagenicity was evaluated using Salmonella/microsome assay in TA1535, TA1537, TA98, TA100 and TA102 S. typhimurium strains in the presence and absence of metabolic activation (S9 mix) and micronucleus test in bone marrow. HPLC analyses indicated the presence of tannins, flavonoids, gallic and ellagic acids. Except for triglycerides, all treated groups presented normal hematological and biochemical parameters. Lower levels of triglycerides and weight loss were observed in the 100 mg/kg group. Mutagenic activities were not detected in S. typhimurium strains and by the micronucleus test. Based on these results, PSAE was not able to induce chromosomal or point mutations, under the conditions tested. The 100 mg/kg dose showed significant antihyperlipidemic action, with no severe toxic effects.     

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.     

Polyethylene sebacate: Genotoxicity,mutagenicity evaluation and application in periodontal drug delivery system

The objective of the present study is to evaluate Polyethylene sebacate (PES) for its toxicity profile including oral toxicity, genotoxicity and mutagenicity. PES was synthesised, and characterised by gel permeation chromatography, FTIR, ¹H-NMR, differential scanning calorimetry and X-ray diffraction. Oral toxicity studies revealed PES to be nontoxic up to 3000 mg/kg body weight with no significant changes in serum biochemistry. The standard battery of genotoxicity tests including micronucleus test, chromosomal aberration and comet assay revealed PES as nongenotoxic. Mutagenicity of PES was evaluated using the Ames microplate format mutagenicity assay sample kit using TA98 and TA100 strains of Salmonella typhimurium, both in presence and absence of Aroclor 1254 induced rat liver S9. Ames assay confirmed PES to be nonmutagenic. Periodontal implants of PES of varying roxithromycin/PES ratios and different diameter were prepared. A decrease in in vitro drug release was seen with increase in diameter of the implants. Release rates, however, increased with increase in PES concentration, and were attributed to decreased crystallinity of roxithromycin, confirmed by the DSC thermographs and XRD spectra. Roxithromycin release from the implants followed Higuchi kinetics and exhibited controlled release. The results suggest PES as a safe polymer for biomedical and pharmaceutical applications. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4781–4795, 2009     

The effects of butylated hydroxytoluene on the in vitro metabolism, DNA-binding and mutagenicity of aflatoxin B1 in the rat

Butylated hydroxytoluene pretreatment in the rat enhanced the total in vitro metabolism of aflatoxin B1 by the hepatic postmitochondrial fraction (S-9) and increased the formation of aflatoxin M1, aflatoxin Q1 and a metabolite tentatively identified as the aflatoxin-glutathione conjugate, the latter being the major metabolite produced. Addition of diethyl maleate, a glutathione depletor, to the incubation mix, reduced formation of the conjugate. No significant difference between treated and control animals was observed in the S-9-mediated binding of aflatoxin B1 to calf thymus DNA. However, the mutagenicity of aflatoxin B1 in Salmonella typhimurium TA98 was significantly lower in the presence of S-9 from BHT-treated rats than with S-9 from controls.     

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