Biomonitoring of urine mutagenicity with the Ames test: improvement of the extraction/concentration method

Comparative extraction efficiency of the pre-packed Bakerbond-spe-SDB-1 resin and of Amberlite-AD2 (XAD-2) resin, for the preparation of urine extracts in biomonitoring studies. Urine extracts were prepared in parallel with the Bakerbond column and with the classical XAD-2 resin from urines (1) spiked with mutagenic chemicals, (2) collected from patients after chemotherapy, and (3) from smokers. Mutagenic activities were evaluated on Salmonella typhimurium tester strains TA97a, TA98, TA100 and TA102 with and without S9 mix. Mutagenic activities obtained with Bakerbond extracts were almost always higher or at least equivalent to those prepared on XAD-2 resin. Similar results were observed for the three urine sample groups. When fully validated, the use of the pre-packed columns will be more convenient and time-saving for large population studies.     

Mutagenic activity of group VIII metal-organic complexes in the Ames test: evaluation of potential glucose biosensor components

Five novel tris-(4,4-substituted-2,2-bipyridine) complexes of the group VIII metals [Fe(II), Ru(II) and Os(II)] (all having been previously examined electrochemically with respect to their ability to act as electron-transfer mediators for redox enzymes) were assayed in the Ames test to evaluate their mutagenic activity. For the bipyridine complexes, some complexes did exhibit mutagenic activity in the circumstances of this test, whereas others did not. this is comparable to the results known from the literature for ferrocene mediators. There was no specific correlation with the identities of the metal and ligand type; the possible explanations of the results are discussed in this paper. It seems probable that the penetration of the whole compound inside bacterial cells depends on the organic part of a complex, with only then the possible genotoxic activity of the complex being revealed. In view of our mutagenic test results, such complexes found to be mutagenic should not be taken into consideration as components of implanted glucose sensors in future in vivo experiments.     

蕨菜乙醇提取物的Ames试验研究

目的：研究蕨菜乙醇提取物的致突变作用,为蕨菜的开发利用提供一定依据;方法：制备蕨菜乙醇提取物,采用Ames试验方法,以50μg/皿敌克松作为诱变剂,分别添加提取物50、100、200、400、800μg/皿,统计回变菌落数;结果：受试物各剂量组回变菌落数均未超过自发回变菌落数的2倍,且无剂量-反应关系;结论：在本实验条件下,蕨菜乙醇提取物无致突变作用.     

Interactions of histidine-containing test substances and extraction methods with the Ames mutagenicity test

Free histidine as well as histidine in the form of dipeptides present in food samples or biological material interacted with the Ames mutagenicity test. This event becomes especially important for test compounds that exert only weak ‘mutagenic’ effects. Histidine bound to large molecules and methyl derivatives did not affect the Ames test. For the avoidance of misinterpretations, it is therefore indispensable to determine or eliminate the interacting forms of histidine when protein-containing samples are tested in the Ames test.Extraction methods using sorbants and solvents that contain contaminants or can induce chemical reactions with biological test substances should be well controlled or avoided. Solvents such as alcohols, esters and aliphatic hydrocarbons that would minimally react with such test material should therefore be used for extraction.     

Ames试验假阳性的判断方法

目的:确立一种判断Ames试验假阳性的可靠方法。方法:在Ames平皿掺入试验中发现经受试物处理的TA97和TA1535的菌落数相比溶媒对照组明显增加,为了证实其是因为受试物致突变性导致的回变菌落数增加还是由受试物毒性导致的菌落数增加,对分别经受试物和阳性对照物处理的Ames菌落进行增菌培养后接种于无组氨酸的培养基上,观察比较细菌的生长情况。结果:经受试物处理的菌株不能生长在无组氨酸的培养基中,而经阳性对照物处理的菌株则可以生长在无组氨酸的培养基上,说明经受试物处理的菌株没有发生突变。结论:此方法能可靠地验证菌株是否为突变菌株,由本研究可以发现经受试物处理的菌株没有发生突变,Ames平皿掺入试验中所观察到的菌落数增加是由于受试物毒性造成的假阳性。     

Reduction in Ames Salmonella mutagenicity of mainstream cigarette smoke condensate by tobacco protein removal

The mutagenic activity of cigarette smoke condensates (CSC) made from tobacco before and after removal of protein was assessed by the Ames Salmonella assay in bacterial strains TA98 and TA100. Removal of protein and peptides from flue-cured tobacco via water extraction followed by protease digestion reduced the mutagenicity of the resultant CSC by 80% in the TA98 strain and 50% in the TA100 strain. Similarly, reductions of 81% in TA98 and 54% in TA100 were seen following water extraction and protease digestion of burley tobacco. The significant reductions in Ames mutagenicity following protein removal suggest that protein pyrolysis products are a principal contributor to the genotoxicity of CSC as measured in this assay.     

Mutagenic activity of copper(II) chromate and dichromate complexes with polypyridines

Copper(II) chromate and dichromate complexes with 2,2'-bipyridyl and 1,10-phenathroline were tested for their mutagenic activity in the standard Ames test. All of six tested complexes exhibited markedly lower mutagenic activity than the reference compounds--potassium dichromate and sodium chromate. The blockage of Cr(VI) reduction capability in the presence of the complex Cu2+ ion and the competition between copper and chromium ions in the interaction with cellular components are discussed in the light of the results of our previous chemical study.     

Use of Salmonella typhimurium TA 98, YG 1024 and YG 1021 and deconjugating enzymes for evaluating the mutagenicity from smokers'' urine

Four smokers were chosen for their different smoking habits, and their declared cigarette consumption confirmed by urinary measurement of nicotine and its metabolites. The promutagenicity of their urine was evaluated by the Ames test, modified according to Kado et al. (Mutation Res., 31 (1983) 25–32) after extraction on XAD2 Amberlite resin. The different Salmonella typhimurium strains TA 98, YG 1021 and YG 1024 were compared to determine the presence of amino aromatic compounds in the urine of smokers of blond and black tobacco. The strain YG 1024 shows higher mutagenicity than TA 98 for extracts from the smoker's urine and more particularly from black tobacco smokers. In addition, the pretreatment of urine by external enzymatic systems (β-glucuronidase or arylsulfatase) reveals the presence in the urine of glucurono- and sulfoconjugated forms of promutagens, including amino aromatic compounds.     

Amine-free melanin-concentrating hormone receptor 1 antagonists: Novel non-basic 1-(2H-indazole-5-yl)pyridin-2(1H)-one derivatives and mitigation of mutagenicity in Ames test

To develop non-basic melanin-concentrating hormone receptor 1 (MCHR1) antagonists with a high probability of target selectivity and therapeutic window, we explored neutral bicyclic motifs that could replace the previously reported imidazo[1,2-a]pyridine or 1H-benzimidazole motif. The results indicated that the binding affinity of a chemically neutral 2H-indazole derivative 8a with MCHR1 (hMCHR1: IC₅₀ = 35 nM) was comparable to that of the imidazopyridine and benzimidazole derivatives (1 and 2, respectively) reported so far. However, 8a was positive in the Ames test using TA1537 in S9− condition. Based on a putative intercalation of 8a with DNA, we introduced a sterically-hindering cyclopropyl group on the indazole ring to decrease planarity, which led to the discovery of 1-(2-cyclopropyl-3-methyl-2H-indazol-5-yl)-4-{[5-(trifluoromethyl)thiophen-3-yl]methoxy}pyridin-2(1H)-one 8l without mutagenicity in TA1537. Compound 8l exerted significant antiobesity effects in diet-induced obese F344 rats and exhibited promising safety profile.     

Evaluation of the antimutagenic potential of chitosan oligosaccharide: Rec, Ames and Umu tests

Two types of chitosan oligosaccharides (COS), COS I (1-kDa < MW < 3-kDa) and COS II (3-kDa < MW < 5-kDa), were tested for antimutagenic activities against chemical mutagens using Umu gene expression, Ames, and Bacillus subtilis Rec mutagenicity tests. At the highest chitosan oligosaccharide dose (1 mg) tested, mutagenic activity of indirect-acting mutagen was inhibited by 50% in the Umu gene expression system and in the Ames test. Chitosan oligosaccharide (0.01, 0.1 and 1 mg) also suppressed 4-nitroquinoline-N-oxide (NQO)-induced mutagenicity in the B. subtilis Rec^– assay.     

Mutagenicities of 2,4- and 2,6-dinitrotoluenes and their reduced products in Salmonella typhimurium nitroreductase- and O-acetyltransferase-overproducing Ames test strains

Mutagenicities of 2,4- and 2,6-dinitrotoluene (2,4-and 2,6-DNT), and reduced metabolites formed by the incubation of 2,4- and 2,6-DNT with Salmonella typhimurium TA98, were tested using S. typhimurium YG strains possessing high level of nitroreductase (NR) and/or O-acetyltransferase (OAT) activities. All compounds tested showed greatest mutagenic activities toward strains YG1041 and YG1042, which possess high levels of NR and OAT activities. The relative mutagenic activities of 2,4-DNT and its related compounds toward YG1041 and YG1042 were aminonitrotoluenes (2A4NT, 4A2NT)<2,4-DNT<2,2′-dimethyl-5,5′-dinitroazoxybenzene (2,2′-DM-5,5′-DNAOB)4-hydroxylamino-2-nitrotoluene (4HA2NT)4,4′-dimethyl-3,3′-dinitroazoxybenzene (4,4′-DM-3,3′-DNAOB), and aminonitrotoluenes (2A4NT, 4A2NT)<2,4-DNT<4HA2NT4,4′-dimethyl-3,3′-dinitroazoxybenzene (4,4′-DM-3,3′-DNAOB)<2HA4NT, respectively. In addition, the relative mutagenic activities of 2,6-DNT and its related compounds toward YG1041 and YG1042 were 2,6-DNT<2-hydroxylamino-6-nitrotoluene (2HA6NT)<2,2′-dimethyl-3,3′-dinitroazoxybenzene (2,2′-DM-3,3′-DNAOB), and 2-amino-6-nitrotoluene (2A6NT)<2,6-DNT<2HA6NT, respectively. These results, together with previous findings, suggested that aminohydroxylamino dimethylazoxybenzenes or aminohydroxylamino dimethylazobenzenes produced either by the reduction of hydroxylaminonitrotoluenes or by the reduction of dimethyl dinitroazoxybenzenes are active metabolites responsible for the mutagenic activities of 2,4- and 2,6-DNT.     

Short Communication: Ames assay with Salmonella typhimurium TA102 for mutagenicity and antimutagenicity of metallic oxide powders having antibacterial activities

Metallic oxide powders (magnesium oxide, calcium oxide and zinc oxide) having antibacterial activity were examined for their mutagenicity and antimutagenicity by the Ames test with Salmonella typhimurium TA 102. These powders were not mutagenic to the tester strain and reduced the mutagenicity of methylglyoxal.     

浮游植物中叶绿素a提取方法的比较与改进

对水质监测中叶绿素a的提取方法进行改进,采用反复冻融-浸提方法提取叶绿素a,即将过滤水样的滤膜于-20℃冰箱内和室温下反复冻融3～5次后,放进90%丙酮溶液中浸提20h。与标准方法中的研磨法对比,该方法具有人为误差小、稳定性好、结果准确及操作简单安全等优点,适合运用于常规的水质监测。采用该方法时过滤水量对湖水水样的测定结果有影响(P<0.01),对于纯微囊藻液影响不明显(P>0.05)。因此,在对不同叶绿素水平或营养水平的水体采样时,应注意水样的水量。通过方差分析表明该方法中采用醋酸纤维滤膜、微孔滤膜和玻璃纤维滤膜对结果没有显著影响,对这3种滤膜可以进行自由选择。     

Comparison of Nuclease P1-Malonogalactan Complex with Nuclease P1

Properties of nuclease P₁-malonogalactan complex (P₁-MG) were compared with those of the polysaccharide-free nuclease P₁. Significant difference was not observed between them in phosphomonoester splitting activity, but marked differences were observed in nucleolytic activity as follows: (1) The pH optima of P₁-MG for RNA and heat-denatured DNA were lower than those of nuclease P₁. (2) At lower than 0.001 of ionic strength, RNA and heat- denatured DNA were attacked hardly by P₁-MG, but attacked well by nuclease P₁. (3) The increase in hydrolysis rate of RNA or heat-denatured DNA with an elevation of temperature from 37°C to 70°C was not so marked in P₁-MG as compared with nuclease P₁. (4) P₁-MG hydrolyzed polynucleotides, especially native DNA, much slower than nuclease P₁.

Influence of ionic strength, pH and temperature on the nucleolytic activity of nuclease P₁-galactan (P₁-G), which was prepared by demalonylating P₁-MG enzymatically, was similar to that of nuclease P₁, except that the activity of P₁-G for native DNA was much lower than nuclease P₁.     

Mutagenicity of blue rayon extracts of human bile in the Ames test

The mutagenicity of human bile was examined in the Ames Salmonella/microsome assay. Bile samples were obtained from the gallbladders resected from patients with cholelithiasis, choledocholithiasis, gallbladder cancer, extrahepatic bile duct cancer and other disease. For extraction of mutagenic components, the bile samples were treated with blue rayon and the adsorbed materials were assayed with Salmonella typhimurium TA98 in the presence of S9 mix. Twenty-four bile samples were tested and positive mutagenic activity was found in 14 samples. A 200-μl bile equivalent material gave 6.3 times as many revertant colonies as the solvent control. With several samples that had undergone two cycles of blue rayon extraction, clear dose-response relationships in mutagenicity were demonstrated.     

Ames test evaluation of two commercially available zero-valent nickel compounds

Zero-valent nickel compounds are organometallic chemicals that are used in synthetic applications and may also occur as intermediates in nickel-catalyzed hydrogenation reactions used in food processing. Few studies have been performed on their possible genotoxic actions. We have tested two commercially available examples of this class of compounds. Solubility and stability were examined. Mutagenicity testing did not confirm a previous report that bis(1,5-cyclooctadiene)nickel is positive in the Ames assay. No stimulation of lipid peroxidation was observed in studies of bovine erythrocytes exposed in vitro. Our results do not indicate that zero-valent nickel compounds have genotoxic effects.     

Detection of gentoxicity of benzidine and its derivatives with the Escherichia coli DJ 702 lacZ reversion mutagenicity assay

AIMS: The feasibility of Escherichia coli DJ 702 lacZ mutagenicity assay to detect genotoxicity of benzidine and its derivatives was evaluated. METHODS AND RESULTS: DJ 702 strain was grown overnight at 30 degrees C in Luria-Bertani (LB) medium containing some components, such as chloramphenicol, ampicillin, delta-aminolevulinic acid, isopropyl-beta-d-thiogalactoside, and trace element mix. The mixtures of a bacterial culture and tested chemical at indicated doses were incubated at 30 degrees C for 30 min. Subsequently, 2 ml of molten top agar was added and the resulting mixtures were immediately poured onto a minimal lactose (ML) plate. Plates were incubated at 30 degrees C for 48 h. The number of colonies was determined by visual scoring. In this study, results showed that all the tested chemicals were mutagenic to DJ 702 strain. CONCLUSIONS: E. coli lac mutagenicity assay using DJ 702 strain can detect the mutagenicity of benzidine and its derivatives. SIGNIFICANCE AND IMPACT OF THE STUDY: We detected the mutagenicity of benzidine and its derivatives in E. coli lac mutagenicity assay using DJ 702, indicating that this assay may be used to detect benzidine and its derivatives in a powerful, sensitive, and convenient mutagenesis assay.     

Evaluation of the mutagenicity of combustion particles from several common biomass fuels in the Ames/Salmonella microsome test

We have evaluated the mutagenicity of dichloromethane extracts of combustion particles from several biomass fuels that are commonly used in developing countries in Salmonella strains TA98 +/- S9 and TA100 +/- S9. Combustion-particle extracts from dried cow dung and crop residue exhibited mutagenic potencies similar to wood-smoke extracts (0.0-1.0 rev./microgram extract). However, extracts from coconut-shell-smoke particles showed relatively potent direct-acting mutagenicity (1.6 rev./micrograms, TA98-S9). Results from testing this sample in nitroreductase- and acetylase- deficient strains TA98NR and TA98 (1,8-DNP-6) revealed no contribution from nitroarenes.     

Synthesis and mutagenicity of 2-aryl-substitute ( o -hydroxy-, m -bromo-, o -methoxy-, o -nitro-phenyl or 4-pyridyl) benzothiazole derivatives on Salmonella typhimurium and human lymphocytes exposed in vitro

Derivatives of 2-aryl-substitute (o-hydroxy-, m-bromo-, o-methoxy-, o-nitro-phenyl or 4-pyridyl) benzothiazole were synthesized and tested for their mutagenicity in in vitro assays: (i) in the Ames test with Salmonella typhimurium TA98 and TA100 strains; and (ii) in the sister chromatid exchange (SCE) in cultured human lymphocytes. The four of compounds (BT-11, B-12, BT-14 and BT-15) caused statistically significant increase in revertant colonies of TA98 and TA100. Treatment of lymphocytes with compounds also caused a significant increase in SCE/cell in association with high levels and long exposure (300 μg/mL and 48 h) of the four compounds. It can be concluded that benzothiazole derivatives showed mutagenic activity and were also able to exert a genotoxic effect reducing both the replication index and mitotic index.