体内碱性彗星试验法检测纳米氧化锌遗传毒性

目的利用体内碱性彗星试验方法,检测长期给予纳米氧化锌颗粒(ZnO NPs)对大鼠的遗传毒性作用。方法结合扩展一代生殖毒性试验,使用13周龄的亲代SD大鼠,经口每天灌胃给予ZnO NPs 0、7、50、350 mg/kg(纳米尺度分散状态最大浓度),记录体质量变化并观察。70 d后每个剂量组取亲代大鼠雌雄各10只处死后取乙二胺四乙酸抗凝全血,采用彗星试验试剂盒进行制片,用定量分析专业软件(Casp)进行彗星结果分析。结果与溶剂对照组比较,雄性大鼠高剂量组外周血DNA损伤细胞率和尾部DNA含量百分比明显上升,分别为28.60%和(36.38±5.84)%,差异有统计学意义(P0.05);雌性大鼠高剂量组外周血DNA损伤细胞率和尾部DNA含量百分比明显上升,分别为27.31%和(18.80±2.96)%,差异有统计学意义(P0.05)。结论在350 mg/kg剂量下ZnO NPs体内碱性彗星试验结果阳性。     

Baccharin Prevents Genotoxic Effects Induced by Methyl Methanesulfonate and Hydrogen Peroxide in V79 Cells

Baccharin is one of the major chemical compounds isolated from the aerial parts of Baccharis dracunculifolia DC (Asteraceae), a native plant of South America and the most important botanical source of the Brazilian green propolis that has been used in alternative medicine to treat inflammation, liver disorders, and stomach ulcers. The present study was carried out in V79 cells to determine the possible genotoxic and antigenotoxic activities of baccharin utilizing comet and micronucleus assays, where 2 known mutagenic agents with different mechanisms of DNA damage were used as positive controls. The V79 cells were treated with concentrations of baccharin (0.25, 0.5, 1.0, and 2.0 μg/mL) and for to investigate the antigenotoxicity these concentrations were associated with methyl methanesulfonate (MMS; 200 μM-comet assay and 400 μM-micronucleus assay) or hydrogen peroxide (H(2) O(2;) 50 μM-comet assay and 100 μM-micronucleus assay). Statistically significant differences in the rate of DNA damage were observed in cultures treated with the highest concentration of baccharin when compared to the control group, but this difference was not found in the micronucleus assay. The results also showed that the frequencies of DNA damage and micronuclei induced by MMS and H(2) O(2) were significantly reduced after treatment with baccharin. The baccharin showed a chemoprevention effect and can be the chemical compound responsible for the antigenotoxicity also demonstrated by the B. dracunculifolia. The antioxidant potential of baccharin may be related to its chemoprevention activity induced against both genomic and chromosomal damages.     

Control of imported irradiated frozen meat and poultry using the hydrocarbon method and the DNA comet assay

In Sweden import of irradiated food is not permitted (spices excepted, 1997). In this regard the Swedish National Food Administration (NFA) has adopted two analytical methods for controlling imported irradiated food: the DNA comet assay for screening and the hydrocarbon method as a confirmatory method. We report here the results of analyses of various samples imported to Sweden from different countries during the last 7 years. It was demonstrated that the control of irradiated food could be reliably performed using these methods. Received: 7 September 1999 / Revised version: 15 November 1999     

Detection of irradiated frozen food with the DNA comet assay: interlaboratory test

Ionising radiation induces DNA damage in the cells of the foodstuff. This fact was used to analyse DNA from single cells by agarose gel electrophoresis (DNA comet assay). The pattern formed by the DNA after electrophoresis (DNA comet) was visualised in a microscope, where hundreds of cells could be observed in a short time. Irradiated cells showed comets with long tails, while unirradiated cells showed no tail or very short ones. An estimation of the dose was made based on the shape of the comets. Nine laboratories, from Berlin, Berne, Chipping Campden, Karlsruhe, Copenhagen, Strasbourg, Uppsala and Warsaw, participated in a test to assess the validity of the method. The test material consisted of code-labelled cell suspensions made of irradiated and unirradiated chicken bone marrow, chicken and pork muscle tissue. The doses varied between 0 and 5 kGy. Samples of chicken bone marrow were used as references. A total of 162 test samples were sent for analysis. Results of analysis from 148 samples were reported. Of these, 138 were correctly identified. Six laboratories succeeded in identifying all the samples. In the group of 106 irradiated samples, 99 were correctly detected, while 39 out of 42 unirradiated samples were correctly detected. It is concluded that the comet assay can be used to detect frozen irradiated food. © 1998 SCI.     

Measuring oxidative DNA damage and DNA repair using the yeast comet assay

Chromosomal DNA damage can be a result of several processes and agents of endogenous or exogenous origin. These cause strand breaks or oxidized bases that lead to strand breaks, which relax the normally supercoiled genomic DNA and increase its electrophoretic mobility. The extent of DNA damage can be assessed by single cell gel electrophoresis, where the chromosomal DNA migration distance correlates with the extent of DNA damage. This technique has been used for a variety of applications with several organisms, but only a few studies have been reported for Saccharomyces cerevisiae. A possible reason for this absence is that low cellular DNA content could hamper visualization. Here we report an optimization of the comet assay protocol for yeast cells that is robust and sensitive enough to reproducibly detect background DNA damage and oxidative damage caused by hydrogen peroxide. DNA repair was observed and quantified as diminishing comet tail length with time after oxidative stress removal in a process well described by first‐order kinetics with a tail length half‐life of 11 min at 37 °C. This is, to our knowledge, the first quantitative measurement of DNA repair kinetics in S. cerevisiae by this method. We also show that diet antioxidants protect from DNA damage, as shown by a three‐fold decrease in comet tail length. The possibility of assessment of DNA damage and repair in individual cells applied to the model organism S. cerevisiae creates new perspectives for studying genotoxicity and DNA repair. Copyright © 2010 John Wiley & Sons, Ltd.     

基于脱细胞、细胞和体内彗星试验的全氟辛酸致DNA损伤研究

目的 本研究通过体内外联合试验探索全氟辛酸（PFOA）致DNA损伤情况。方法 以脱细胞核DNA作为模型,0.00、0.13、0.25、0.50 mmol/L PFOA染毒1 h后检测脱细胞核DNA损伤情况。以YAC-1细胞系为模型,采用CCK-8法测定不同染毒剂量对细胞活性的影响。在细胞彗星试验中,PFOA终浓度设定为0、1.0×10^-8、1.0×10^-7、1.0×10^-6 mol/L,连续暴露3 d,检测DNA损伤情况。0、10、20、40 mg/kg·BW PFOA分别灌胃给予大鼠两次,间隔24 h,末次给予受试物6 h后,采用肝脏、骨髓和外周血细胞开展中性和碱性彗星试验,利用骨髓细胞开展骨髓微核试验。结果 脱细胞碱性彗星试验各剂量组尾部DNA含量显著高于对照组（P<0.05）,呈剂量-反应关系,脱细胞中性彗星试验无显著差异（P>0.05）;细胞碱性彗星试验各剂量组尾部DNA含量显著高于对照组（P<0.05）,呈剂量-反应关系,细胞中性彗星试验无显著差异（P>0.05）;体内彗星联合微核试验表明,与对照组相比,肝脏、骨髓和外周血细胞碱性和中性彗星试验各剂量组尾部DNA含量,以及骨髓微核各剂量组无显著差异（P>0.05）。结论 PFOA在体外对DNA具有损伤作用,经口染毒未见对大鼠产生DNA损伤。     

Evaluation of the genotoxic and antioxidant effects of two novel feed additives (ethoxyquin complexes with flavonoids) by the comet assay and micronucleus test

The complexes of antioxidant ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline; EQ) with rutin or quercetin (EQ-R and EQ-Q, respectively) were studied in human lymphocytes for genotoxic and antioxidant activities with the use of the comet assay and micronucleus test. The study was undertaken to search for new potential antioxidants, and was motivated by reports of unfavourable side-effects observed in animals fed with feeds containing EQ, which is allowed up to 150 mg kg^-1 (0.015%) in complete animal feed. It was shown that EQ-R induced DNA damage in human lymphocytes when used at all the concentrations studied (1-25 µM), while after EQ-Q treatment, the genotoxic effect was observed mainly after higher doses (10 and 25 µM). An increase in the number of micronuclei was observed only for EQ-Q after a dose of 50 µM. The studied compounds decreased the degree of DNA damage induced by hydrogen peroxide (10 µM) in the comet assay. The results obtained in both tests showed that the antioxidant activity of EQ-Q was comparable with that of EQ, so further detailed studies are necessary to estimate its possible usefulness as a feed preservative.     

DNA degradation in chilled fresh chicken studied with the neutral comet assay

 DNA degradation in fresh chicken was studied with the neutral comet assay. Chicken legs were stored in a refrigerator at 2–4  °C. DNA from muscle tissue was analysed independently by the authors in Mol and Uppsala after 1–12 days. The cells were imbedded in agarose on a microscope slide, lysed and subjected to electrophoresis causing DNA and its fragments to migrate in the gel. The patterns ("comets") formed by the migration indicated the status of DNA. The comets were evaluated both with a fluorescence microscope by visual inspection (H.C.) and computer-assisted image analysis (G.K.). On day 1, a majority of the cells showed comets with very short tails, or no tail at all. A few cells showed advanced stages of DNA degradation. The number of these cells increased with time, as well as the degree of degradation. The bacterial contamination, which was rare on day 1, increased with time, and gave a characteristic background to day 10 samples. It was concluded that the neutral comet assay could be used as a method to rapidly screen fresh chicken in order to assess its quality. Received: 15 December 1997     

DNA degradation in chilled fresh chicken studied with the neutral comet assay

 DNA degradation in fresh chicken was studied with the neutral comet assay. Chicken legs were stored in a refrigerator at 2–4  °C. DNA from muscle tissue was analysed independently by the authors in Mol and Uppsala after 1–12 days. The cells were imbedded in agarose on a microscope slide, lysed and subjected to electrophoresis causing DNA and its fragments to migrate in the gel. The patterns ("comets") formed by the migration indicated the status of DNA. The comets were evaluated both with a fluorescence microscope by visual inspection (H.C.) and computer-assisted image analysis (G.K.). On day 1, a majority of the cells showed comets with very short tails, or no tail at all. A few cells showed advanced stages of DNA degradation. The number of these cells increased with time, as well as the degree of degradation. The bacterial contamination, which was rare on day 1, increased with time, and gave a characteristic background to day 10 samples. It was concluded that the neutral comet assay could be used as a method to rapidly screen fresh chicken in order to assess its quality. Received: 15 December 1997     

彗星图像智能分析软件的应用研究

目的 为提高彗星试验的自动化和标准化,对彗星智能分析软件(Comet A)的功能和可靠性进行研究和测试.方法 在3台配置不同的计算机安装Comet A,分别由3名分析员用Comet A软件分析30张图像,并随机选取部分图片用目测法对彗星评分,然后进行统计分析;同时对软件的自动识别、自动分析、结果储存和可溯源性进行测试....     

Preliminary evaluation of the genotoxic potential of a hydrophilic polymer with three preservation systems

This study compared the genotoxic potential of a polymeric associative thickener used in topically applied emulsions preserved with three different preservative systems. The method used for the assessment of genotoxicity is the in vitro micronucleus test [Organization for Economic Cooperation and Development (OECD) guideline number 487]. When changing an additive such as a preservation system in a raw material, it is crucial to re-evaluate its toxicity potential because this change may significantly alter its properties. This study shows that at the levels tested neither of the systems evaluated demonstrated any cytotoxic or genotoxic effects. Skin exposure must take into consideration factors such as duration, skin condition and metabolism, but most importantly concentration. Although preservatives can be toxic at high concentrations, they are usually safe at the concentrations used in cosmetic raw materials and formulations. If used to preserve raw materials, they undergo further dilution when added to the formulation.     

体外微核高内涵筛选法在食品毒理学遗传毒性评价中应用研究

目的探讨基于高内涵筛选(high-content screening, HCS)技术的体外微核(in vitro micronucleus, IVMN)检测方法应用于食品毒理学遗传毒性评价的可行性。方法采用IVMN和HCS法,分别对10种化合物进行遗传毒性评价,其中5种已知遗传毒性化合物(染色体断裂剂和非整倍体诱发剂)、1种已知非遗传毒性化合物以及4种食品原料,每个受试物至少设置3个剂量组,每个剂量组设2个复孔,同时设置阴性对照组(无血清最小必需培养基)和阳性对照组(+S9为环磷酰胺20μg/ml、-S9为丝裂霉素C 1.0μg/ml)。以中国仓鼠肺细胞为细胞模型,在有和/或无代谢活化系统条件下,依次对上述受试物采用短时处理(4 h)后进行微核检测,并分析微核细胞率。结果经IVMN和HCS法得到的IVMN试验结果显示:2.5～10μg/ml苯并芘[B(a)P]、5～20μg/ml甲磺酸甲酯(MMS)、0.01～0.04μg/ml 4-硝基喹啉-N-氧化物(4NQO)、0.25～1.0μg/ml秋水仙碱(COL)和0.5～2.0μg/ml硫酸长春碱(VB)在各自浓度范围内,在有或无代谢活化系统的条件下,诱导产生的微核细胞率随着受试物浓度的升高而增加,呈明显剂量-反应关系,且微核细胞率与阴性对照组比较,差异均有统计学意义(P0.05),试验结果为阳性;1 250～5 000μg/ml氯化钠(NaCl)、1 250～5 000μg/ml食品原料A、1 250～5 000μg/ml食品原料B、312.5～1 250μg/ml食品原料C和156.25～625μg/ml食品原料D在各自浓度范围内,在有和无代谢活化系统的条件下,微核细胞率虽呈现一定的剂量依赖性增加趋势,但均维持在较低的水平,且与阴性对照组比较,差异均无统计学意义(P0.05),试验结果为阴性。结论本试验条件下,两种方法对10种物质的检测结果均一致,提示将IVMN HCS法应用于食品毒理学遗传毒性评价具有一定的可行性。     

Mutagenicity assessment of food contact material migrates with the Ames MPF assay

ABSTRACT

A major challenge in the safety assessment of food contact materials (FCM) is the evaluation of unknown non-intentionally added substances (NIAS). Even though consumer exposure levels may be quantitatively low, these substances are considered to be of high toxicological concern if they act as DNA reactive mutagens. From a safety assessment perspective, it is therefore important to detect their presence in FCM migrates. The present study applied the Ames MPF assay to assess the mutagenicity of migrates obtained from 30 food contact material samples out of 3 categories: plastics, composite materials and coatings. As a food simulant, 95% ethanol (EtOH) had a superior performance to less volatile simulants when evaluating recovery rates of representative model substances in different volatility categories. To monitor possible interference of the FCM matrix with Ames MPF results, migrates were spiked with reference substances and recovery rates were established. Out of 30 samples tested, two caused significant inhibition of revertant formation in the presence of the spiking control. Overall detection limits of the applied test method were estimated by determination of the lowest effective concentrations (LEC) for 10 Ames-positive substances. Even though the current limits of detection are not sufficient to entirely fulfil regulatory and safety requirements, three out of 30 FCMs showed evidence of dose-dependent effects in the Ames MPF assay. Overall, the data obtained supported the relevance of testing FCM migrates for DNA reactive contaminants and showed the value of the Ames MPF assay for the safety assessment of FCMs.     

Antimutagenic and antigenotoxic effects of vegetable matrices on the activity of pesticides

Two in vitro tests, one to detect bacterial mutagenicity (Ames test) on Salmonella typhimurium TA98, TA100, and TA1535 and the other the primary DNA damage (SOS Chromotest) on Escherichia coli PQ37, were applied to determine the overall genotoxic activity of 12 pesticides (azinphos methyl, chlorothalonil, chlorphyriphos ethyl, chlorphyriphos methyl, λ-cyhalothrin, cypermethrin, cyprodinil, fenazaquin, fludioxonil, indoxacarb, iprodione and penconazol). These were detected by gas chromatography (GC) analysis with electron capture (ECD) and nitrogen phosphorus detection (NPD) in 18 samples of vegetables. Some extracts of vegetables, found positive for pesticides with GC, were subjected to the Ames test and SOS Chromotest to evaluate the possible antimutagenic and/or antigenotoxic effects of vegetable matrices. The same bioassays were also performed on the mixtures of pesticides found in these samples to evaluate whether interactions could occur between pesticides and be responsible for the possible antimutagenic and/or antigenotoxic effects of the contaminated matrices. Experiments were also carried out to compare the results found for contaminated vegetables with their content of antioxidant components. Significant differences in mutagenicity and genotoxicity were found among the pesticides selected for this study. Of the 12 pesticides tested, only azinphos methyl, cyprodinil, fludioxonil and iprodione were found to be positive for both S. typhimurium and E. coli. No mutagenic/genotoxic activity was found in the extracts of vegetables contaminated by pesticides. S. typhimurium TA1535 showed a strong positive mutagenic effect for the mixtures of pesticides while they were not able to induce the SOS system. The data concerning the content of polyphenols and the total reducing activity of the contaminated vegetables indicated high amounts of antioxidants that could explain the inhibitory effect on the activity of pesticides shown by vegetables.     

内蒙古不同种源山杏杏仁中苦杏仁苷含量差异分析

采用高效液相色谱法(HPLC),对内蒙古7个种源山杏杏仁中苦杏仁苷的含量进行差异性分析。结果表明:不同种源山杏杏仁中苦杏仁苷的含量存在显著差异(p<0.05);7个种源苦杏仁苷平均含量为5.43%,平均含量范围为4.34%~6.25%;平均变异系数为0.07,扎鲁特旗种源的变异系数较其它种源大;苦杏仁苷含量与经纬度呈极显著负相关,相关系数分别为0.806、0.722,与海拔呈极显著正相关(0.896);聚类分析将7个种源划分为2类。该研究结果为内蒙古地区山杏的深加工以及利用提供理论依据。      

Evaluation of skin cancer chemoprevention potential of sunscreen agents using the Epstein–Barr virus early antigen activation in vitro assay

In our continuing search for novel cancer chemopreventive compounds of natural and synthetic origin, we have evaluated 14 commonly used ultraviolet (UV) sunscreen agents (designated UV‐1 to UV‐14) for their skin cancer chemoprevention potential. They belong to 8 different chemical categories: aminobenzoate (UV‐5, UV‐7, UV‐8 and UV‐14), benzophenone (UV‐1, UV‐2, UV‐3 and UV‐13), benzotriazole (UV‐10), benzyloxyphenol (UV‐9), cinnamate (UV‐6), quinolone (UV‐4), salicylate (UV‐11) and xanthone (UV‐12). In the in vitro assay employed, the sunscreens were assessed by their inhibition of the Epstein–Barr virus early antigen (EBV‐EA) activation induced by the tumour promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in human lymphoblastoid Raji cells. All sunscreens tested were found to exhibit anti‐tumour promoting activity: listed in decreasing order, moderate (UV‐11, UV‐2, UV‐7, UV‐12, UV‐3, UV‐9 and UV‐14) to weak (UV‐1, UV‐6, UV‐8, UV‐16, UV‐5, UV‐4 and UV‐10) with octyl salicylate (UV‐11) as the most potent and drometrizole (UV‐10) as the least potent among the compounds evaluated. A plausible relationship between the antioxidant property of sunscreens and their ability to promote anti‐tumour activity was noted. The results call for a comprehensive analysis of skin cancer chemoprevention potential of currently used UV sunscreen agents around the globe to identify those with the best clinical profile.     

电子烟基因毒性评价方法概述

为了解电子烟的基因毒性及其评价方法,按照受试对象分类进行了综述。其中以细菌为受试对象的试验方法有细菌回复突变试验和DNA损伤分析等;以离体细胞为受试对象的试验方法有微核试验、DNA双链断裂试验、RNA转录测序试验、定向基因检测分析和Bhas细胞转化试验等;以模式动物为受试对象的试验方法有长期吸入毒性试验和生殖发育毒性试验等;以人体为受试对象的主要是临床试验和流行病学调查研究等。由于受试对象和试验方法的差异性,导致电子烟基因毒性结果的可信度和可比性较差,因此建议在评价电子烟基因毒性的过程中,应至少考虑细菌、离体细胞、模式动物和临床试验等4个层次的受试对象,通过回复突变试验、微核试验、长期吸入毒性试验和临床试验等多种试验方法获得多个基因毒性的试验终点,科学、客观和全面地综合评价电子烟的基因毒性。     

脱苦过程中苦杏仁苷含量的变化及其与苦味的关系

借助奎宁标准品进行苦味鉴评,探究苦杏仁中苦杏仁苷含量与苦味的关系。以脱皮苦杏仁为研究对象,进行水浴脱苦[脱苦温度70℃、料液比1:12(g/mL)、脱苦时间6h],每30min取样一次,对各样品进行感官评定,并采用高效液相色谱法(HPLC)测定样品中的苦杏仁苷含量。结果表明:苦杏仁中苦杏仁苷含量与苦味呈正相关关系,70℃水浴脱苦5h后,苦杏仁中苦杏仁苷含量低至0.91mg/g·干基,苦杏仁苦味等级降为Ⅰ级,品尝时已不苦。苦杏仁中苦杏仁苷含量与苦味密切相关,运用HPLC测定苦杏仁中苦杏仁苷的残留量即可准确判断其脱苦程度。