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.     

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.     

3种促智药:石杉碱甲、茴拉西坦和吡乙酰胺的诱变作用和诱变协同作用

在鼠伤寒沙门菌/微粒体系统中测试了石杉碱甲、茴拉西坦和吡乙酸胺的诱变作用。结果表明,药物浓度从1μg-5mg/皿对TA97、TA98、TA100和TA1024个菌株,在无S_9代谢系统上所测的这3个药和有S_9代谢系统所测的石杉碱甲、茴拉西坦均未显示任何诱变作用。向拉西坦和吡乙酰胺在诱变协同实验中均不增加对-硝基喹啉在TA98上和甲基磺酸甲酯在TA100上诱发的回变数。ICR纯系小鼠骨髓微核试验,剂量高达1/2LD_(50)时不增加嗜多染红细胞的微核率,也无骨髓抑制作用.     

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.     

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.     

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.     

Mutagenicity studies of benzidine and its analogs: structure-activity relationships.

The Ames SALMONELLA:/microsome assay was employed to test the mutagenicity of benzidine and its analogs using strains TA98 and TA100 in the presence and absence of Aroclor 1254-induced rat S9 mix. 3,3'-Dichlorobenzidine-2HCl and 4,4'-dinitro-2-biphenylamine were directly mutagenic to TA98, while 4,4'-dinitro-2-biphenylamine was directly mutagenic to both TA98 and TA100 in the absence of S9 mix. 2-Aminobiphenyl, 3-aminobiphenyl, and 3,3'-5,5'-tetramethylbenzidine were not mutagenic in either strains in the presence or absence of S9. In the presence of S9 mix, 4-aminobiphenyl, benzidine, 3, 3'-dichlorobenzidine-2HCl, 3,3'-dimethoxybenzidine, 3,3'-4, 4'-tetraaminobiphenyl, o-tolidine, N, N-N', N'-tetramethylbenzidine, and 4,4'-dinitro-2-biphenylamine were mutagenic to TA98; 4-aminobiphenyl, 3,3'-dichlorobenzidine-2HCl, 3, 3'-dimethoxybenzidine, and 4,4'-dinitro-2-biphenylamine were mutagenic to TA100. Physicochemical parameters of these compounds including oxidation potentials, the energy difference between the lowest unoccupied molecular orbital and the highest occupied molecular orbital, ionization potentials, dipole moment, relative partition coefficient, and basicity did not correlate with their bacterial mutagenic activities.     

Mutagenicity and DNA-damaging activity caused by decomposed products of potassium sorbate reacting with ascorbic acid in the presence of Fe salt.

Although potassium sorbate (PS), ascorbic acid and ferric or ferrous salts (Fe-salts) are used widely in combination as food additives, the strong reactivity of PS and oxidative potency of ascorbic acid in the presence of Fe-salts might form toxic compounds in food during its deposit and distribution. In the present paper, the reaction mixture of PS, ascorbic acid and Fe-salts was evaluated for mutagenicity and DNA-damaging activity by means of the Ames test and rec-assay. Effective lethality was observed in the rec-assay. No mutagenicity was induced in either Salmonella typhimurium strains TA98 (with or without S-9 mix) or TA100 (with S-9 mix). In contrast, a dose-dependent mutagenic effect was obtained when applied to strain TA100 without S-9 mix. The mutagenic activity became stronger increasing with the reaction period. Furthermore, the reaction products obtained in a nitrogen atmosphere did not show any mutagenic and DNA-damaging activity. PS, ascorbic acid and Fe-salts were inactive when they were used separately. Omission of one component from the mixture of PS, ascorbic acid and Fe-salt turned the reaction system inactive. These results demonstrate that ascorbic acid and Fe-salt oxidized PS and the oxidative products caused mutagenicity and DNA-damaging activity.     

The effect of paraquat on the mutagenicity of benzo(a)pyrene

Elevated activities of superoxide dismutase (SOD) were detected in histidine-requiring strains of Salmonella typhimurium after the bacteria were preincubated for 1 h at 37 degrees C with S-9 mix and paraquat (methylviologen, PQ2+) at 10(-4) M. A fivefold increase in SOD level was found for strains TA 98 and TA 100. These elevated levels of SOD activity were correlated with a significant reduction of the mutagenicity of metabolically activated benzo(a)pyrene (B(a)P) in these tester bacteria when evaluated in a preincubation assay system. A 69.0-92.5% and 23.5-66.9% reduction was noticed when 0.5-4.0 micrograms per plate of B(a)P was used in TA 98 and TA 100, respectively. However, exogenous superoxide dismutase at 10-100 micrograms ml-1 added to top agar had no significant effect on the number of revertants produced by activated B(a)P. These data indicate a major role of intracellular superoxide anion in promoting mutagenicity of B(a)P.     

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.     

Ames试验检测十种中药有效成份和制剂的诱变性

本文以Ames实验室最新推荐的四株测试菌株(TA97、TA98、TA100、TA102)、用予培养法对斑蝥酸钠、去甲斑蝥素、三尖杉酯硷、高三尖杉酯硷、蒿甲醚、芫花酯甲、芫花酯乙等十种中药有效成份制剂进行了诱变性的研究。 结果表明,十种中药有效成份和制剂在加和不加S_9混合液情况下,均来显示诱变性。 用体外致突变试验方法对提取的中药有效成份进行检测,了解其潜在的遗传毒性是十分必要的。     

Salmonella/microsome mutagenicity tests of heat-processed milk samples

Samples of whole milk were heat treated by commercial heat-sterilization, by commercial heat-pasteurization or by a laboratory heat-pasteurization (65 degrees C for 30 min). Each sample was tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 with and without S-9 mix. Dichloromethane extracts of milk heated at 100, 135 and 150 degrees C for 5 hr were also tested for mutagenicity using the same assay. None of these samples exhibited mutagenicity in the Ames assay used.     

Mutagenicity study of nine monoalkyl phthalates and a dialkyl phthalate using Salmonella typhimurium and Escherichia coli

Nine monoalkyl (C1-C8) phthalates and di-(2-ethylhexyl) phthalate (DEHP) were assayed for mutagenicity in two strains of Salmonella typhimurium (TA98 and TA100) and two strains of Escherichia coli WP2 try- (uvrA+ and uvrA-) with and without metabolic activation with S-9 mix. The procedure of Ames et al. (Mutation Res. 1975, 31, 347) was used, with minor modifications. None of the compounds tested showed any mutagenic activity, but all the monoalkyl phthalates showed some lethality towards the S. typhimurium strains, the most toxic being monoheptyl phthalate. A marginally lethal effect on the Salmonella strains was shown by DEHP, but only at the highest concentration tested (2000 micrograms/plate) and in the absence of S-9 mix.     

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 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 acrylonitrile.

Incubation of Salmonella typhimurium strains in an atmosphere of 0.2% gaseous acrylonitrile increased the numbers of his+ revertants/plate only in the presence of a fortified S9 liver fraction. The mutagenic effect was particularly pronounced with strains TA1530, TA1535 and TA1950 and much weaker with strains TA100, TA98 and TA1978. The results of bacterial fluctuation tests confirmed the necessity of the presence of S9 mix and showed the particular sensitivity of TA1530. The reversion rate varied with the S9 mix composition, the animal species utilized and the type of pretreatments applied to the animals. The mutagenicity of acrylonitrile in S. typhimurium is therefore microsome-mediated and is particularly discernable with strains sensitive to base-substitution mutagens.     

Chemical fate and mutagenic formation potentials of phenothiazine and related compounds during water chlorination

The reactions of hetero-tricyclic aromatic hydrocarbons (H-TCAHs) with hypochlorite in an aqueous solution were investigated under conditions that simulate wastewater disinfection. H-TCAH-hypochlorite reaction products were determined by gas chromatographic-mass spectrometric (GC-MS) analyses. For 20 μM, 10H-phenothiazine, 10H-phenoxazine, and phenoxathiin reacted rapidly with active chlorine in neutral pH (7.0), but no phenazine-hypochlorite reaction was observed over pH values of 5-9 for 1 hr. The 10H-phenothiazine-hypochlorite reaction began by oxidation with active chlorine to form its dioxides, followed by chloro-substitution in water. The extent of the reactions depended on the chlorine dose, solution pH and compound structures. Ames assays for the chlorination byproducts of 10H-phenothiazine and 10H-phenoxazine also showed to be weak mutagenicity in TA98 and TA100 strains without S9 mix, but no chlorination byproducts of phenoxathiin exhibited any mutagenicity in both tester strains with and without S9 mix.     

Synthesis and mutagenicity of A-ring reduced analogues of 7,12-dimethylbenz[a]anthracene

The synthesis and mutagenicity of two derivatives of 7,12-dimethylbenz[a]anthracene (DMBA; 1), i.e., 1,2-H2DMBA (4) and 1,2,3,4-H4DMBA (5), are reported. These analogues (4 and 5) represent dihydro and tetrahydro A-ring reduced forms of DMBA, a region in the parent hydrocarbon (1) proposed to be involved in metabolism to the ultimate carcinogen. The synthesis for 4 without isolation of intermediates from the tosylhydrazone of 1,2,3,4-tetrahydrobenz[a]anthracene-4,7,12-trione (10) by successive reaction with 8 molar equiv of CH3Li, HI, and NaBH4 represents a novel approach to this hydrocarbon now available in sufficient quantity for biological studies. Interestingly, both of these reduced analogues 4 and 5 exhibited mutagenic activity in the Ames assay in the presence or absence of microsomal activation for strains TA98 and TA100. In these strains, DMBA was active only in the presence of S-9 fraction. In the plasmid-deficient strain TA1537, only tetrahydro analogue 5 exhibited mutagenic activity both in the absence and presence of S-9 fraction.     

Mutagenicity tests of lipid oxidation products in Salmonella typhimurium: Monohydroperoxides and secondary oxidation products of methyl linoleate and methyl linolenate

Nine hydroperoxy and hydroperoxy-epidioxy oxidation products derived from either autoxidation (AO) or photosensitized oxidation (PO) of methyl linoleate (MLo) or methyl linolenate (MLn) were tested for mutagenic activity by the Salmonella typhimurium his⁺ reversion assay using strains TA100, TA98, TA102, TA97 and TA1537. All nine oxidation products, monohydroperoxides from AO-MLn (I) or from PO-MLn (II), dihydroperoxides from PO-MLo (III), AO-MLn (IV) or PO-MLn (V), hydroperoxy epidioxides from PO-MLo (VI), AO-MLn (VII) or PO-MLn (VIII) and hydroperoxy bis-epidioxides from PO-MLn (IX), were weakly mutagenic in strains TA97 and/or TA100. The hydroperoxy epidioxides (VI–IX) exhibited significantly greater activity in strain TA97 than did the monohydroperoxides (I, II) or the dihydroperoxides (III–V). In strain TA100, all of the oxidation products tested exhibited similar activity. No major differences between products derived from autoxidized and photooxidized MLn (I v. II, IV v. V, VII v. VIII) were obtained. Rat-liver S-9 reduced the toxicity of all oxidation products to the tester strains. The greatest mutant yields were usually obtained in the presence of S-9, but mutagenic potency was sometimes greater without S-9. The structural feature common to all of the mutagenic oxidation products was the presence of a hydroperoxy group, suggesting that this characteristic is responsible for the observed mutagenicity, either directly or through a common degradative pathway to reactive products of lower molecular weight.     

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.