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.     

猫眼草水煎液体外致突变性的研究

目的检验猫眼草水煎液的致突变性;改进经典的A-mes试验体系使之适应于中药体外致突变性检验。方法通过经典的Ames试验检测猫眼草的体外致突变性;通过哺乳动物骨髓细胞染色体畸变试验检测猫眼草的致畸作用;改进的Ames试验通过增设含补充组氨酸(含量对应于猫眼草水煎液中组氨酸浓度)的阴性对照,排除样品中组氨酸成分对试验结果的影响。结果猫眼草水煎液在经典的Ames试验中为强阳性;对哺乳动物骨髓细胞染色体致畸作用为阴性;在改进的Ames试验中猫眼草水煎液的致突变性为阴性。结论经典的Ames试验不适合猫眼草水煎液致突变性检测,改进后的Ames实验体系适合。猫眼草水煎液在体外和体内均没有致突变性。     

Mutagenicity of N-nitrosodiethylamine in the Ames test with S. typhimurium TA1535 is due to volatile metabolites and is not dependent on cytochrome P4502E1 induction

N-Nitrosodiethylamine (NDEA) is carcinogenic in all investigated animal species at relatively low dosages. No threshold has been detected for these carcinogenic effects. The substance has been extensively investigated in various in vitro systems, revealing only weak mutagenicity at relatively high dosages. We reinvestigated NDEA in the Ames test with Salmonella typhimurium TA1535 to establish appropriate modifications of the standard Ames test protocol, to achieve a dose-dependent mutagenic response at a reasonably low dose range. Two main modifications were evaluated. Since the metabolism of dialkylnitrosamines is postulated to be mainly dependent on cytochrome P4502E1, a pyrazole-induced rat liver S9 was applied. The second modification involved a gastight preincubation, since metabolites of NDEA might evaporate from the incubation mixture. Cytochrome P4502E1 induction in Wistar rats was achieved by pyrazole treatment. For comparison, a rat liver S9-fraction produced by beta-naphtoflavone/phenobarbital induction was used. N-Nitrosopyrrolidine served as positive control for pyrazole-induced S9-mix with TA1535. NDEA showed no mutagenic response under all test conditions in the presence of pyrazole-induced S9-mix. A strong mutagenic response, exceeding the base rate up to 15-fold at a dose range of 25-1000 microg/plate, was observed using beta-naphtoflavone/phenobarbital-induced S9-mix, gastight preincubation and TA1535. In conclusion the Ames test with gastight preincubation can be useful for the testing of volatile compounds or substances leading to gaseous metabolites. The weak response of NDEA in the Ames test observed previously seems mainly to be due to the volatile character of its mutagenic metabolites. Our results do not support the hypothesis that cytochrome P4502E1 is a major toxifying enzyme for the formation of Ames-test-positive metabolites from NDEA.     

Mutagenicity in V79 cells does not correlate with carcinogenity in small rodents for 12 aromatic amines

The aim of this investigation was to study the correlation between carcinogenicity in small rodents and mutagenic potency of aromatic amines, as measured by the induction of 6-thioguanine resistance in V79 Chinese hamster cells. It has been previously shown that the carcinogenic potency of these compounds is not correlated to their ability to induce DNA breakage, SCEs, or point mutations in bacteria, but a correlation exists with autoradiographic DNA repair test (in primary hepatocyte cultures). Twelve aromatic amines were tested and the rat liver S9 fraction was routinely incorporated in the mutation assay; mouse liver and hamster liver S9 fractions were also used as metabolizing systems. The comparison of the ranks of mutagenic and oncogenic potencies by means of the Spearman test shows no correlation between carcinogenicity and V79 cell mutagenicity of the tested aromatic amines. There was a generally low mutagenicity seen for aromatic amines in V79 cells. In some cases this could be attributed to an insufficient metabolic activation by rat S9. For example, benzidine, which was inactive when assayed in the presence of rat S9, became mutagenic when in the presence of mouse S9. On the other hand, hamster S9, which has been shown to be the best activating system for 2-acetylaminofluorene in the Ames test, did not activate this compound in V79 cells. Inadequate metabolic activation of the standard system (rat S9) used in this work could explain the low mutagenicity and the lack of correlation observed between mutagenicity and carcinogenicity. A second possibility is that point mutation is not the essential end point for the initiating activity of aromatic amines during the carcinogenic process. A third possibility is that the activity of some aromatic amines is not restricted to the initiation step in carcinogenesis. Chronic treatments with the sublethal doses often result in significant promoting activities, which could mask efficiently the initiating potential of the same chemicals.     

Genotoxic evaluation of Halfenprox using the human peripheral lymphocyte micronucleus assay and the Ames test

The genotoxicity and mutagenicity of Halfenprox, a synthetic pyrethroid insecticide and acaricide, was assessed using two standard genotoxicity assays of the Salmonella typhimurium mutagenicity assay (Ames test) and in vitro micronucleus (MN) assay in human peripheral lymphocytes. In the Ames test, Salmonella strains TA98 and TA100 were treated with or without S9 fraction. The doses of Halfenprox were 6.25, 12.5, 25, 50, and 100?μg/plate and test materials were dissolved in DMSO. The concentrations of Halfenprox did not show mutagenic activity on both strains with and without S9 fraction. The MN assay was used to investigate the genotoxic effects of Halfenprox in human peripheral lymphocytes treated with 250, 500, 750, and 1000?μg/ml concentrations of Halfenprox for 24 and 48?h, and at 1000?μg/ml the concentration was significantly increased and the MN formation was compared with the negative control for both treatment periods. In addition, a significant decrease of the nuclear devision index (NDI) values at the higher concentrations of Halfenprox and at both treatment periods was observed.     

N-Nitrosodiethylamine mutagenicity at low concentrations

N-Nitrosodiethylamine (NDEA) requires metabolic activation by cytochrome P450 enzymes, leading to electrophile species that react in DNA. Although, carcinogenicity is not an end point in genotoxicity assays, NDEA has been considered a weak carcinogen. In this study, we carried out an analysis of the mutagenicity at low concentrations of NDEA. Using SOS chromotest in the presence of metabolic activation, we detected positive mutagenicity response for NDEA doses between 0.75 and 36.46 microg/ml. In Ames test, using more sensitive strains in the presence of S9 metabolic activation mixture (S9 mix), positive results were also detected for NDEA doses between 1.01 x 10(-3) and 50.64 x 10(-3 microg per plate. Our results indicate that NDEA mutagenicity can be detected at low concentrations when more sensitive conditions are used.     

False Positive Result for a Peptide Drug in the Gene Conversion Assay with Saccharomyces cerevisiae Strain D7

False Positive Result for a Peptide Drug in the Gene ConversionAssay with Saccharomyces cerevisiae Strain D7. DEPASS, L. R.,CHAN, R. L., JAGANNATH, D. R., AND HEYMAN, I. A. (1991). Fundam.Appl Toxicol. 17, 627–634. A battery of mutagenicity testswas performed with nafarelin, an agonist analogue of luteinizinghormone releasing hormone (LHRH) containing tryptophan (Trp)and histidine (His). Included were the Ames assay and the geneconversion assay with yeast strain D7. Both tests were negativewithout S9 activation, and the Ames test was negative with S9,but the yeast test was positive with S9 activation. Since theyeast test is based on conversion of cells to Trp independence,release of Trp by metabolism of the drug could account for thepositive result The test was repeated using Trp instead of thedrug. The result was positive even at the lowest Trp concentration.In another experiment with the drug, amino acid analysis ofthe incubation mixture revealed the presence of Trp but no detectableHis. Since the Ames test is based on mutation to His-independentcells, these data are completely consistent with the negativeresult in the Ames test and the false positive result in theyeast test. These data suggest the need for caution in interpretingthe results from mutagenicity assays with peptide drugs.     

Disposition of [14C]2,3-dichloropropene in Fischer-344 rats after oral or intraperitoneal administration

The genotoxicity of indigo has been assessed by two short-term tests. The mutagenicity of natural indigo was compared with that of synthetic indigo. Both chemicals were tested using the standard procedure of the Salmonella/microsome mutagenicity test as described by Ames. The substance exhibits mutagenicity towards strains TA1538 and TA98 when S9 preparations of rat liver induced with Aroclor 1254 were present in the medium. The clastogenic potential was evaluated by the micronucleus test in the bone marrow of male mice. The test compound was administered twice with an interval of 24 h, the animals were killed 30 h and 54 h after the first treatment. When the test compound was given by oral gavage as two equal dosages of 0.1, 1 and 1.2 g/kg body weight, no statistically significant increase in the percentage of polychromatic erythrocytes with micronuclei was observed for any group treated with natural indigo.     

o-Aminoazotoluene does induce the enzymes of its own mutagenic activation in mouse liver

The objective of this study was to investigate the CYP1A1 and CYP1A2 mRNAs and enzyme activities in mouse liver during induction with o-aminoazotoluene (OAT) as well as the capability of the hepatic S9-fraction from OAT-treated mice to induce its own activation to mutagens in the Ames test using S. typhymurium strain TA98. The data obtained indicate that when used at appropriate doses, OAT is a PAH-type inducer of mouse hepatic microsomal monooxygenases, which activity is not less than that of the known inducer 3,4-benzo[alpha]pyrene. In the absence of S9-fraction enzymes no OAT-mediated mutagenicity was observed in the Ames test. In the presence of the S9-fraction from OAT-pretreated mice, OAT induced as high revertant numbers, as it did in the presence of the S9 fraction from the liver of Aroclor 1254-treated mice. Thus, OAT does induce the enzymes of its own mutagenic activation in mouse liver.     

2-(N-nitroso-N-methylamino)propiophenone, a direct acting bacterial mutagen found in nitrosated Ephedra altissima tea

A new N-nitroso compound identified in a nitrosated tea extract made from the plant Ephedra altissima and shown to be formed under in vivo conditions was identified as 2-(N-nitroso-N-methylamino)propiophenone (NMAP). N-Nitrosoephedrine (NEP), another N-nitroso compound detected in nitrosated Ephedra altissima tea and NMAP are shown to exert mutagenic activity in the Salmonella/mammalian microsome mutagenicity (Ames) test. Base-pair substitution mutation-detecting strains (TA100 and TA1535) showed both compounds to be weak direct-acting mutagens without the addition of S9-mix. The identification, synthesis and mutagenicity of NMAP are discussed.     

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.     

百草胶囊的毒性研究

目的　研究百草胶囊的毒性。方法　采用最大耐受剂量 (MTD)试验 ,小鼠骨髓嗜多染红细胞微核试验 ,小鼠精子畸形试验 ,Ames试验和大鼠 30d喂养试验 ,分别观察百草胶囊的急性毒性 ,遗传毒性和亚急性毒性。结果　百草胶囊对小鼠经口MTD大于 16 0 0 0mg/kg ;对小鼠骨髓嗜多染红细胞微核试验无诱发微核增多作用 ;小鼠精子畸形试验未见导致精子畸形和畸形率增高 ;Ames试验在加与不加S9的条件下均无致突变性 ;大鼠 30d喂养试验未观察到中毒表现 ,百草胶囊各剂量组动物体重 ,食物利用率 ,血液学和血液生化学指标值 ,各脏器的脏 /体比值与空白对照组比较差异均无显著意义 (P >0 0 5 )。主要脏器在外观形态和组织学上均无异常变化。结论　百草胶囊对动物的生长发育、造血功能、肝肾功能、器官组织均无明显毒性。     

The toxicology and safety of apple polyphenol extract.

Apple polyphenol extract has strong antioxidant activity and various physiological functions, and is used in Japan as a food additive and nutritional supplements. Here, we tested the consumption safety of Applephenon, which is a polyphenol extract produced from unripe apples. The Ames test without S9 mixture revealed that Applephenon, had slight mutagenicity at a high concentration of 2500 microg/plate; however, both chromosomal aberration test and the micronucleus test found no significant mutagenicity. Furthermore, an acute oral-toxicity test, and a 90-day subchronic-toxicity test showed no significant hematological, clinical, chemical, histopathological, or urinary effects at a dose of 2000 mg/kg. These results confirm that Applephenon is safe and no toxic at average dietary level.     

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.     

Use of human liver S9 in the Ames test: assay of three procarcinogens using human S9 derived from multiple donors

The purpose of the present study was to examine the inter-individual variation in the mutagenicity of chemicals using a variety of human S9 fractions. For this purpose, three procarcinogens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), benzo[a]pyrene (BP), and dimethylnitrosamine (DMN), were selected for the Ames test and their mutagenicity was examined using human liver S9 fractions prepared from 18 different donors and one pooled liver S9 fraction prepared from 15 different donors. In addition, rat S9 fraction prepared from male rats pretreated with phenobarbital and 5,6-benzoflavone (PB/BF) was used as reference in order to examine the mutagenic differences between human and rat (PB/BF) S9 fractions. The data demonstrate a large inter-individual diversity in the mutagenic response to procarcinogens. The mutagenicity of IQ and BP in the presence of a human liver S9 fraction (lot HLS-014) was equal to that observed in the presence of rat (PB/BF) S9 fraction. The mutagenicity of IQ and BP in the presence of a pooled human liver S9 fraction was lower (90 and 95%, respectively) than that observed in the presence of rat (PB/BF) S9. On the contrary, the mutagenicity of DMN in the presence of either a selected human liver S9 fraction (lot HLS-014) or pooled fraction was 8-fold higher than that found in the presence of rat (PB/BF) S9 fraction. Human liver S9 fraction (lot HLS-014) had one of the highest cytochrome P450 enzyme activities among the 18 different donors and higher than the pooled human liver S9 fraction. These results suggest that the use of both selected human liver S9 fractions with high metabolic activity (e.g., lot HLS-014 as used in this study) and a pooled S9 fraction with moderate metabolic activity could be used as a means to evaluate the inter-individual variability in mutagenic response to chemicals and to confirm positive responses from studies completed with rodent S9.     

Safety evaluation of polyphenols extracted from hop bracts.

Hop bract polyphenols contain polyphenols as promising functional ingredients. To assess the safety of topical hop bract polyphenols, Hopsphenon, we examined acute, 14-day, 28-day and 90-day toxicity tests in rats, and mutagenicity tests using Ames test and micronucleus test in mice. The acute, 14-day, 28-day and 90-day toxicity tests revealed that Hopsphenon produced no symptoms of significant injury. The lethal dose of hop bract polyphenols is greater than 2000 mg/kg. The Ames test in the absence of S9 mix for TA98 and in the presence of S9 mix for TA1537 revealed that Hopsphenon had slight mutagenicity at a high dose of 5000 microg/plate; however, in the micronucleus test, Hopsphenon was negative. These tests demonstrated that hop bract polyphenols are safe and do not cause any detrimental effects in vivo under the conditions investigated in this study.     

Mutagenicity testing of organic extracts of diesel exhaust particles after spiking with polycyclic aromatic hydrocarbons (PAH)

In the present study, spiking was used as a strategy to evaluate the mutagenicity of individual compounds in a mixture. Mutagenicity of individual polycyclic aromatic hydrocarbons (PAH) was evaluated in an organic extract of diesel exhaust particles (DEP). The particles were extracted with dichloromethane (DCM). After replacing DCM with dimethylsulphoxide (DMSO), the extract was spiked with four individual PAH: benzo(a)pyrene, benzo(a)anthracene, pyrene and fluoroanthene. The PAH were added separately and in various combinations to the extract to determine the effects of each variable and to identify possible interactions between the individual PAH and between the PAH and the extract. The study was designed as a fractional factorial experiment with the five variables (the DEP extract and the four PAH), giving 16 (instead of 32) mixtures plus a triplicate centrepoint and background, i.e. a total of 20. The fractionated factorial design used in the present work supports a model with linear and interaction terms. The mixtures were tested for mutagenicity in the Ames assay using four strains of Salmonella typhimurium in the presence of rat liver xenobiotic enzymes (S9-mix). Projections to Latent Structures (PLS) was used to quantify the mutagenicity of each compound and possible interactions. The four individual PAH and the DEP extract acted additively in the Ames test with 10% S9-mix. Received: 2 March 1998 / Accepted: 1 July 1998     

Mutagenicity of aromatic amines and amides with chemical models for cytochrome P450 in Ames assay

Chemical models for cytochrome P450, consisting of water-insoluble or water-soluble iron porphyrin plus an oxidant, have been used to detect the mutagenicity of promutagens in genotoxicity assays. The procedure for using chemical models for cytochrome P450 as substitutes for the S9 mix in the Ames assay have been already established. Aromatic amines and amides require metabolic activation by cytochrome P450 when they exert their mutagenicity in Salmonella typhimurium strains. In this study, we optimized the conditions of the assay using a water-soluble chemical model, 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrinatoiron(III) pentachloride (4-MPy), plus tert-butyl hydroperoxide (t-BuOOH), magnesium monoperoxyphthalate, or iodosylbenzene, by comparing the mutagenicity of 2-aminofluorene (AF) in the Ames test. The model with 4-MPy/t-BuOOH showed the highest AF mutagenic potency. The chemical model activated 2-naphthylamine, 4-aminobiphenyl, and benzidine in S. typhimurium TA98. In aromatic amides, the model with 4-MPy/t-BuOOH weakly activated 2-acetylaminofluorene (AAF). To detect higher mutagenicity of aromatic amides, we used a higher concentration of 4-MPy/t-BuOOH by a factor of 5 over that used for aromatic amines, and then detected the mutagenicity of AAF, 2-acetylaminoanthracene, and 2-acetylamino-9-fluorenone. Furthermore, we concluded that the AAF mutagenicity in the presence of 4-MPy/t-BuOOH is derived from N-hydroxylacetylamino compounds.     

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.     

Evaluation of the metabolizing capacity of S9 liver preparations from rats and mice using a test with Salmonella typhimurium TA100

The S9 phenobarbital-induced preparations from Albino rats and 6 strains of inbred and outbred Pzh: SFISS mice were tested by an Ames test for their ability to metabolize the two promutagens 2-aminofluorene (2AF) and cyclophosphamide (CP), and to influence the mutagenic activity of the two directly acting mutagens methyl methanesulphonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). 2AF showed a mutagenic activity after incubation with all kinds of microsomal preparations. S9 from B10. mice (Ah+Ah+) was twice as active as that from D2.BN mice (Ah- Ah-). CP was mutagenic exclusively in the presence of S9 from Pzh: SFISS or B10. mice and from rats. Neither fraction deactivated significantly the mutagenicity of MMS. Microsomal preparations from Albino rats and outbred mice were most active in deactivating the mutagenicity of MNNG.