Chemopreventive effects of ellagic acid against genotoxicity induced by benzo(a)pyrene

Benzo(a)pyrene (B(a)P) is a commonly used indicator for polycyclic aromatic hydrocarbons (PAHs) contamination. The use of certain phytochemicals and some polyphenolic compounds proved to be effective in blocking PAH-induced effects. The aim of the present study was to examine the possible inhibitory effects of ellagic acid (EA), present in berries and nuts, against B(a)P-mediated toxicity using in vitro and in vivo test systems. In vitro method included the Ames test, using Salmonella typhimurium tester strain TA100. In vivo experiments were based on mouse bone marrow micronucleus (MN) assay and immunoblotting. EA produced a decrease in the number of revertant colonies against B(a)P in the Ames assay. Further, there was a reduction in the mean number of micronucleated polychromatic erythrocytes in the presence of EA in the co-treatment protocol of the MN test. Western blotting results showed downregulation of CYP450 isoform CYP1A1 which prevented bioactivation of B(a)P. Thus, the present study demonstrates antimutagenic role played by EA against the mutagenic activity of B(a)P in both in vitro and in vivo systems.     

Antimutagenic and bacteriostatic activities of Schiff‐base compounds derived from aminoguanidine,semicarbazone and thiosemicarbazone and a copper(II)‐coordination complex

The Schiff‐base aminoguanidine compounds including a resorcylidene aminoguanidine copper(II) complex that were synthesized at our laboratory posses non‐mutagenic properties when tested with Ames test using Salmonella typhimurium TA97, TA100 and TA102 bacterial strains. These compounds were tested as possible pharmacological agents in preventing a wide array of illnesses. Additionally, some of these compounds exhibit strong antimutagenic and bacteriostatic activity. Nitrovin, a known mutagen, was used in the antimutagenicity tests as the inducer. A three‐dimensional projection of the copper(II) complex was derived by the semi‐empirical method ZINDO/1 to obtain additional information about its structure, and to help elucidate a possible mechanism of action.     

采用生物发光检测法进行Ames试验的研究

将自行构建的含有青海弧菌荧光酶基因的质粒pACYCL184引入Ames试验的4个菌株TA97、TA98、TA100、TA102中,构建成能够生物发光的工程菌,分别命名为TAL97、TAL98、TAL100、TAL102.实验表明,该4株工程菌保持原出发菌株所具有的与Ames试验相关的性状,对各类致突变剂有良好的反应,且可用平皿计数法进行致突变试验.其发光值与致突变剂浓度存在良好的量效关系,故可用发光检测取代原Ames试验的平皿计数,使检测更简单、方便、快速.图3表3参11     

Analyses of PAHs (polycyclic aromatic hydrocarbons) and mutagenicity in raw and chlorinated water

Both raw water and chlorinated drinking water samples were collected from and the Liu‐Du water treatment plant in northern Taiwan from October 1990 to April 1992. The polycyclic aromatic hydrocarbons (PAHs) and mutagenicity in these water samples were analyzed by GC/MS and Ames test. The Mutagenicity/DMSO (Dimethyl Sulfoxide) ratio in S. typhimurium TA98 and TA100 with or without S9 mixture increased, even higher than 2, following the sequence of unit process. It was observed that the mutagenicity with TA98 (S9+) was highly related to most of PAHs in the raw water; while the mutagenicity with TA98 (S9+) was only correlated with DbA and BghiPr in the treated water. It could be expected that the mutagenicity level was controlled by other predominant components after the raw water was treated, for example, the chlorination process.     

饮用水遗传毒性测试中TA98和TA100的敏感度和可靠度比较

为了寻找适合我国水质特征的Ames试验简化方法,作者通过广泛的文献调研,对近30年以来,我国研究者用Ames试验评价饮用水和水源水的遗传毒性的实验数据进行了总结和分析。结果发现,TA98-S9测试体系的敏感度和可靠度最高,TA98+S9次之,TA100-S9与TA100+S9 2个测试体系对Ames试验结果的影响几乎可以忽略。只用1个TA98-S9测试体系的方法 1,仅用25%的工作量,可获得91%的致突变阳性检出率和74%的有效数据率;采用TA98-S9和TA98+S9 2个测试体系的方法 2,使用50%的工作量,可获得96%的阳性检出率和96%的有效数据率。据此,作者建议:采用Ames试验评价我国饮用水和水源水的遗传毒性时,可以将通常采用的4个测试体系的方法 4,简化为只采用TA98-S9与TA98+S9 2个测试体系的方法 2。在仅进行致突变性定性评价时,或者在费用或水样量不足等条件下,则可使用更为简化的方法 1,更利于Ames试验的广泛应用。     

蚕豆根尖微核技术的方法学新论

虽然蚕豆根尖微核技术已是一个成熟规范的检测污染物遗传毒性的方法,但其中仍涉及一些重要的方法学问题值得深入探讨和研究。本文在图示正确观测蚕豆根尖细胞内微核和染色体畸变,如染色体断裂、丢失及染色体桥等的基础上,提出应该对蚕豆根尖分裂相细胞及其染色体畸变进行观测,以便更加准确、细致地反映污染物作用的剂量-效应关系和分子机制。同时还就该方法的其他重要问题提出了自己的观点,供同行们深入探讨和研究,使之不断完善,更好地服务于环境监测和风险评价等领域。     

广州市某A2/O工艺城镇污水处理厂出水对大型溞的生殖毒性作用

城镇污水处理厂废水中存在大量生物毒性物质,但该废水对动物的生殖能力影响研究还十分缺乏。利用大型溞暴露实验,检测广州市某采用A~2/O工艺的城镇污水处理厂出水的急性毒性和生殖毒性。48 h急性毒性实验表明,所有水样均未表现出急性毒性效应。慢性生殖毒性(14 d)测试结果表明:(1)从进水到缺氧池出水均能显著提高第一胎产溞数量;(2)进水和沉砂池出水显著增加受试期总产溞数量;(3)从厌氧池到出水工艺段受试溞的第一胎产溞时间均推后;(4)好氧池出水的毒性显著降低,但仍然对大型溞具有生殖毒性。研究表明,好氧池(A~2/O)工艺能够显著去除具有大型溞生殖毒性的物质,但污水处理厂废水的生殖毒性仍需要引起关注。     

Synthesis and biological activities of new azacrown ether Schiff bases and spectrophotometric studies of their complexation with [60]fullerene

A series of novel azacrown ether Schiff bases 1–3 have been synthesized in good yield and in a simple way. Their host–guest interaction with [60]fullerene has been studied in toluene by absorption spectroscopic method. All the complexes are found to be stable with 1:1 stoichiometry. Because of their potential applications in industry, agriculture and medicine, they were investigated for their mutagenic and antimutagenic activities using the spot test and the plate incorporation assay of Ames. Compounds 1, 2 and 3 were found to be nonmutagenic in the Ames test using strains TA 1535, TA100 and TA97a of Salmonella typhimurium. However, using strain TA102 revealed that, although both compounds 1 and 2 were nonmutagenic, compound 2 gave a positive response indicating that it acts as an oxidative mutagen. The structure-activity relationship may throw some light on the biological activity of such series of compounds.     

Evaluation of the antimutagenic and antiradical potentials of extracts from the tubers of (Tunisian) Cyperus rotundus

The mutagenic potential of aqueous, total oligomers flavonoids (TOF), ethyl acetate and methanol extracts from tubers of Cyperus rotundus L. was assessed using Ames Salmonella tester strains TA98 and TA100, and SOS chromotest strain Escherichia coli PQ37 with and without metabolic activation (S9). None of the different extracts produced a mutagenic effect. Likewise, the antimutagenicity of the same extracts was tested using the “Ames test” and the “SOS chromotest”. Our results showed that C. rotundus extracts possess antimutagenic effects against S. typhimurium TA98 and TA100 strains towards the mutagen aflatoxin B1 (AFB1), similar to E. coli PQ37 strain against AFB1 and Nifuroxazide mutagens using the SOS chromotest assay. A free radical scavenging test was used in order to explore the antioxidant capacity of the extracts obtained from the tubers of C. rotundus. TOF, ethyl acetate and methanol extracts showed an important free radical scavenging activity towards the 1,1-diphenyl-2-picrylhydrazine (DPPH) free radical. These extracts showed an IC50 value of respectively 5, 20 and 65?µg?mL^?1. The beneficial effects of TOF, ethyl acetate and methanol extracts of C. rotundus were assessed by antioxidant and antimutagenic activities.     

Improvement of the assimilable organic carbon (AOC) analytical method for reclaimed water

Microbial growth is an issue of concern that may cause hygienic and aesthetic problems during the transportation and usage of reclaimed water. Assimilable organic carbon (AOC) is an important parameter which determines the heterotrophic bacterial growth potential of water. Pseudomonas fluorescens P17 and Spirillum sp. NOX are widely used to measure AOC in drinking water. The AOC values of various reclaimed water samples determined by P17 and NOX were compared with those determined by the new strains isolated from reclaimed water in this study. It showed that the conventional test strains were not suitable for AOC measurement of reclaimed water in certain cases. In addition to P17 and NOX, Stenotrophomonas sp. ZJ2, Pseudomonas saponiphila G3 and Enterobacter sp. G6, were selected as test strains for AOC measurement of reclaimed water. Key aspects of the bioassay including inoculum cell density, incubation temperature, incubation time and the pH of samples were evaluated for the newly selected test strains. Higher inoculum density (10⁴ CFU·mL^-1) and higher incubation temperature (25°C) could reduce the time required for the tests. The AOC results of various collected samples showed the advantages of the method proposed based on those five strains in evaluating the biologic stability of reclaimed water.     

磁性介孔硅胶萃取剂的制备及萃取性能研究

合成了C18基团修饰的磁性介孔硅胶材料,并利用该材料建立了磁性固相萃取-色谱分析方法,测定了几种环境水样中酞酸酯类(PAEs)污染物的含量.结果表明,该材料具有较大的比表面积(273 m.2g-1)和饱和磁通量(29 emu.g-1),对水样中痕量的PAEs有较强的萃取能力,而且萃取剂的磁分离特性使得萃取操作更为方便、快捷.在优化条件下,30 mg萃取剂在40 min内即可从500 mL水样中萃取痕量PAEs,回收率可达80%以上.此外,该萃取剂在处理复杂环境样品时仍能保持较强的萃取能力,环境水样中4种PAEs的检测限可达15.6—32.5 ng.L-1,加标回收率为62%—109%,相对标准偏差为2%—8%.     

The application of solid-phase micro-extraction (SPME) to the analysis of polycyclic aromatic hydrocarbons (PAHs)

There are many established extraction techniques regularly used in the isolation and analysis of PAHs and similar organic compounds from various phases. These include Soxhlet or ultrasonic extractions from solids, and liquid-liquid or solid-phase extraction from aqueous samples. However, these methods have some inherent disadvantages; most require large volumes of organic solvents, they can be time consuming and many involve multi-step processes that always present the risk of the loss of some analytes (Zhang et al., 1994). Solid-phase micro-extraction (SPME) is a relatively new technique that has been used with much success in the analysis of a variety of compounds including PAHs. Experiments are being carried out to determine the optimum range of conditions for the extraction of a range of PAHs. Parameters under investigation include temperature, equilibration time, salinity and compound concentration. Presented here are some preliminary experiments into the applicability of SPME for PAH analysis. Further work will investigate the reproducibility of the technique, limits of detection and matrix effects. When an optimised method has been developed the technique will be used in investigations into PAH profiles in sediment cores.     

Aliphatic and aromatic halocarbons as potential mutagens in drinking water

Chlorinated phenols are either products of industrial chemical processes or the result of chlorination of drinking water. Often, the formation of chlorinated phenols is based upon naturally occurring phenol. The following chlorinated phenols have been selected for testing in the Ames‐test for their mutagenic activity: 3‐chlorophenol, 4‐chlorophenol, 2,3,6‐, 2,4,5‐, 2,4,6‐trichlorophenol, 4‐chloro‐2‐methylphenol and 4‐chloro‐3‐methylchlorophenol. The tester strains TA97, TA98, TA100 and TA104 were employed. All tested compounds produced mutagenic activity at least in one tester strain. The highest numbers of revertants were detected for 2,3,6‐ and 2,4,6‐trichlorophenol. But in contrast to the other substances, these two induced only frameshift mutations in presence of a metabolizing system. The evidence of their presence in drinking water and of their mutagenic activity makes them to a potential health hazard.     

印染废水处理过程及排放水对草履虫遗传毒性评价

草履虫作为指示生物评估印染废水处理过程及排放水的毒性效应。急性毒性结果显示,原水、厌氧工段印染废水、好氧工段印染废水对草履虫的半数致死浓度(24h-LC₅₀)分别为54%、41%、98%;遗传毒性结果显示,印染废水原水、经处理的排放水在6.25%、12.25%稀释梯度下对草履虫造成的DNA损伤较对照组呈显著升高(p<0.01),而排放水对草履虫的DNA损伤较原水无显著性差异;草履虫增殖速率结果显示,印染废水排放水50%稀释梯度下对草履虫增殖速率有抑制作用。暴露于印染废水排放水中10%的草履虫其游动行为发生改变。以上结果表明,印染废水在经过处理后,理化指标均达到国家印染废水行业排放标准,但排放水仍对草履虫产生急性毒性,且遗传毒性较原水没有显著降低。水生生物毒性测试能有效补充理化指标评价,为印染废水行业全面达标排放提供依据。     

Assessment of mutagenic potency of source water treated by ozone and adsorption processes

This research utilized the Ames test to determine the mutagenicity of water treated by advanced processes, including ozonation and granular activated carbon (GAC). Raw water samples for this research included those obtained from the Pan Hsin waterworks as well as samples containing humic acids. Treated samples were collected from the pilot‐scale advanced treatment plant. The Ames test was used to measure the mutagenicity of the water after each treatment process. For the Pan Hsin raw water samples treated with ozone or GAC, it was indicated that, regardless of whether samples were preozonated or not, they all showed a mutagenic potency less than 2 once the S9 enzyme was added. This level of mutagenicity is insignificant. The prepared humic acid samples, on the other hand, demonstrated a significant reduction in mutagenicity after the pre‐ozonation process, indicating that preozonation can lower the degree of mutagenicity. Furthermore, the mutagenicity of the prepared humic acid samples gradually decreased after the advanced treatment process. However, when chlorine was added later to these samples, the mutagenicity increased again. This research shows that the use of O3/GAC processes to treat water can successfully lower mutagenicity, indicating a great potential for applications in the treatment of drinking water.     

Mutagenicity of diesel engine exhaust in the Ames / Salmonella assay using a direct exposure method

The aim of this study was to investigate the potential mutagenic activity of diesel engine exhaust in the Ames/Salmonella assay using a direct aerosol exposure system. So, TA 98 and TA 100 strains, with or without added S9 mix, were exposed to diesel emissions after varying degrees of filtration. Variants of these two strains, deficient in nitroreductase (TA 98NR and TA 100NR) or over-expressing O-Acetyl Transferase (YG 1024 and YG 1029), were also exposed to total (unfiltered) diesel exhaust to highlight the putative mutagenicity of any nitro-PAHs present in these emissions. Mutagenic activity of the diesel exhaust was demonstrated on Salmonella typhimurium, strains TA 100 and variants TA 100 NR and YG1029. The use of a particle filter did not modify the genotoxicity of the diesel emissions, indicating a major contribution of the gas phase to the mutagenicity of these diesel emissions. The prominent role of the particulate-associated nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) claimed by some authors working on diesel exhaust organic extracts was not confirmed by our results with native diesel exhaust exposure. Our results show that the gas phase is potentially more mutagenic than the particles alone.     

Genotoxicity evaluation of surface waters located in urban area of Xi’an City using Vicia faba bioassays

In this study, micronucleus （MCN） and chromosome aberration （CA） tests in Vicia faba root tip cells were carried out in order to assess the water quality and the comprehensive genotoxic potential of surface waters located in the urban area of Xi＇an City, China. For these evaluations, water samples from different surface waters （four rivers, two lakes, two biological treatment plant effluents） were collected, the ultra-pure water and methyl methanesulfonate solution was used as the negative and positive control, respectively. In our results, highly significant differences in MCN perrnillage （average number of micronuclei per 1000 cells）, CA frequencies and PI （pollution index） values were found among three rivers and two WWTP effluents, the tested samples from two rivers caused the decrease of mitotic index over 22% compared with the negative control. No significant changes were observed in micronuclei and chromosome aberrations frequencies at one river and two lakes during the period of test （wet season）. These results point out a poor state of the water quality and genotoxic activity of the main surface waters in Xi＇an City. It is recommended to establish a monitoring program for the presence of genotoxic agents in these surface waters.     

利用SOS/umu测试方法鉴定沙颍河河水中的遗传毒性物质

采用HPLC分割导向的SOS/umu测试方法鉴定了沙颍河河水中的遗传毒性物质。当采用TA1535/pSK1002菌株测定HPLC分割的各馏分时,如果不经大鼠肝微粒体酶(S9)代谢活化,只有馏分F10显示遗传毒性,加入大鼠肝微粒体酶代谢活化后,馏分F10和馏分F15都显示有遗传毒性,说明河水中存在某些需经过大鼠肝微粒体酶代谢活化才能显示出遗传毒性的物质。当采用过量表达O-乙酰转移酶(O-AT),对芳香胺类物质和硝基芳烃化合物有特殊响应的NM2009菌株测定时,馏分F8、F9和F10均呈现遗传毒性;特别是对于馏分F10,用NM2009菌株测定的遗传毒性远高于原始菌,说明这3个馏分中都含有芳香胺类或硝基芳烃类物质。     

SETAC GLB — Gründung und Entwicklung

Goal, Scope, and Background

In the context of the ISO-standardisation of the in vitro micronucleus test for (waste) water testing (ISO/DIS 21427-2), a national collaborative study was organized by the German Federal Institute of Hydrology (BfG) involving ten laboratories of private companies, universities and public authorities. The formation of micronuclei (MN) is a special kind of chromosomal aberration. To meet the standardisation requirements for this method, encoded waste water samples, some of them spiked with known genotoxins, had to be tested in a collaborative study. The study should demonstrate practicability of the in vitro micronucleus test for waste water testing and should provide validity data.

Material and Methods

The micronucleus assay was performed with the permanently growing Chinese hamster lung fibroblast cell line V79. Four encoded samples from one municipal and one industrial wastewater treatment plant were tested with and without metabolic activation by S9-mix. Two of these samples were spiked in advance with defined concentrations of the clastogenic substances cyclophosphamide and mytomycin C. The defined assessment criterion for genotoxicity was the lowest dilution of a sample that does not show any significant induction of micronuclei (LID; lowest ineffective dilution). Cytotoxicity was judged by determining the survival-index, i.e. the percentage growth rate of the cells compared with the respective negative controls. As supplementary qualitative criteria, the mitotic index and the proliferation index were assessed.

Results

Although some of the laboratories had little or even no experience with the protocol of the in vitro micronucleus test described in the ISO-draft, all participants suceeded in establishing the assay within few weeks and in generating viable test results. The two nongenotoxic samples were detected as negative by 90% or 100% of the participants. The mitomycin C-spiked sample (expected to be positive without S9 supplementation) was correctly evaluated as positive by all laboratories. The cyclophosphamide-spiked sample (expected to be positive with S9 supplementation) was evaluated correctly as genotoxic by 80% of the laboratories. A post-test analysis found evidence that the false-negative results were due to technical failure, but not of a methodological nature. The sample LID values varied by no more than one dilution step around the median LID-value for all samples investigated. The survival index was proven to be a robust measure for estimation of cytotoxicity.

Discussion

The measurement of micronuclei is an important parameter for the detection of cytogenetic damage. In contrast to the single-cell gel electrophoresis assay (Comet assay), which is used as an indicator test, the in vitro micronucleus test detects non-repairable and thus manifested genetic damage. Consequently, the in vitro micronucleus test can be regarded as the more significant test system. A more frequent occurrence of micronuclei in treated cells suggests a risk of severe genetic damage for subsequent cell generations. In the interest of a precautionary environmental protection and health protection no municipal or industrial waste-water samples should show any significant induction of micronuclei in the treated cell populations.

Perspectives

The presented collaborative study was the first interlaboratory comparison of the in vitro micronucleus test using wastewater samples. The test system is intended to complement the already DIN- and ISO-standardised bacterial tests, i.e. the umu-test and the Ames plate incorporation assay. The data generated in the course of this project justify the transformation of the draft standard into the final draft international standard (FDIS), the preliminary stage of an international norm, so that a valid, standardised test system for the detection of cukaryotic genotoxicity in water samples might become available.     

Mutagenicity of polyaromatic hydrocarbons by chemical models for cytochrome P450 in Ames assay

DNA damage is an important step in carcinogenesis. The Ames assay is a short-term screening of carcinogens that induce DNA damage. Most carcinogens require enzymatic activation through oxidation by cytochrome P450 (CYP450) in the presence of S9 mix. A combination of iron (Fe)(III) porphyrin and an oxidant is also able to oxidize compounds as an alternative metabolic pathway to CYP450. Previously it was reported that a chemical model containing a water-soluble 5,10,15,20-tetrakis(1-methylpyridinium4-yl)porphyrinatoiron(III) chloride (4-MPy) and tert-butyl hydroperoxide (t-BuOOH) activated aromatic amines and amides. In this study, a chemical model composed of an Fe porphyrin, water-insoluble 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride (F₅P) or water-soluble 4-MPy was optimized with an oxidant – t-BuOOH, magnesium monoperoxyphthalate (MPPT), or iodosylbenzene (PhIO). Subsequently the mutagenicity of benzo[a]pyrene (B[a]P) and chrysene in Salmonella typhimurium TA strains was compared. B[a]P was activated by a combination of F₅P or 4-MPy plus MPPT or PhIO in S. typhimurium TA1538. The B[a]P-induced mutagenicity with F₅P plus oxidant was higher than 4-MPy plus oxidant. Mutagenicity of chrysene, a tetracyclic aromatic hydrocarbon, was not detected in the presence of F₅P/PhIO in S. typhimurium TA98, but was activated in the presence of F₅P/MPPT. The F₅P/MPPT activated other polyaromatic hydrocarbons (PAH) in the S. typhimurium TA98 assay including dibenz[a,c]anthracene, dibenz[a,h]anthracene, 3-methylcholanthrene, and benzo[a]anthracene. The results indicated that the F₅P/MPPT was the most efficient model for detecting PAH-induced mutagenicity in the Ames assay.