鉴别染色体断裂剂和非整倍体诱发剂的微核试验

非整倍体(aneuploidy)是指一个个体或细胞具有多于或少于正常n倍体数的染色体数。人的正常染色体数是46条,是二倍体。人体细胞出现多于或少于46条染色体,即非整倍体改变。它与自发性流产、先天性畸形、智力发育迟缓和致癌过程有关。动物和细胞的实验中观察到化学物、辐射、病毒诱发的肿瘤或自发的肿瘤以及恶性细胞转化均与     

非遗传毒物诱发非整倍体的检测方法研究

本研究采用完整中期细胞染色体计数和胞质分裂阻滞微核试验方法，对雌二醇、糖精、石棉、利血平４种非遗传毒致癌物诱发ＣＨＬ细胞非整倍体作用进行了研究，结果表明４种被检物均未诱发染色体结构畸变。但在中期细胞染色体计数试验中检查到明显的染色体数目异常，ＣＢ微核试验也获得阳性结果，表明４种被检物具有诱发非整倍体的作用。流式细胞术也可以检测到非整倍体的发生，但效果远不如前两种方法。为此，我们建议中期染色体计数和ＣＢ微核试验考虑作为检测非遗传毒致癌物的试验方法。     

第二代基因测序诊断产品批准上市

2014年6月30日,国家食品药品监督管理总局经审查,批准了BGISEQ-1000基因测序仪、BGISEQ-100基因测序仪和胎儿染色体非整倍体(T21、T18、T13)检测试剂盒(联合探针锚定连接测序法)、胎儿染色体非整倍体(T21、T18、T13)检测试剂盒(半导体测序法)的医疗器械注册。这是国家食品药品监督管理总局首次批准注册的第二代基因测序诊断产品。该批产品可通过对孕12周以上的高危孕妇外周血血浆中的游离基因片段进行基因测序,对胎儿染色体非整倍体疾病21-三体综合征、18-三体综合征和13-三体综合征进行无创产     

氯化汞诱发非整倍体效应的微核试验和CREST染色的研究

目的探讨低浓度氯化汞在体外试验中对V79细胞非整倍体诱发效应。方法体外培养条件下,低浓度氯化汞(0.005~10.0μmol/L)染毒V79细胞,然后进行微核形态学分析并计算微核率;同时用CREST染色计算微核着丝粒阳性率。本试验采用SPSS11.5进行统计分析。结果V79细胞经氯化汞染毒后,各剂量组的微核率均明显增加,呈现剂量-效应关系,也存在时间-效应关系;微核形状以圆形为主,与染色体断裂剂诱导的微核形状相似,但大微核发生率与VCR相似,即21.4%~27.9%。HgCl2各剂量组微核着丝粒阳性率为47.4~56.3%。结论氯化汞在低浓度下依然造成细胞染色体的损伤,具有一定的非整倍体毒性和染色体断裂效应,但以非整倍体毒性为主。     

非遗传毒物诱发整倍体的检测方法研究

本研究采用完整中期细胞染色体 计数和胞质分裂阻滞微核试验方法，对雌二醇、糖精、石棉、利血平４种非遗传毒致部物诱发ＣＨＬ细胞非整倍体作用进行了研究，结果表明４种被检物均未诱发染色体结构畸变。但在中期细胞染色体计数试验中检查到明显的染色体数目异常，ＣＢ微核试验也获得阳性结果，表明４种被检物具有诱发非整倍体的作用。流式细胞术也可以检测到非整倍体的发生，但效果远不如前两种方法。为此，我们建议中期染色体计数     

环磷酰胺对小鼠生殖细胞非整倍体诱变性研究

环磷酰胺对小鼠生殖细胞非整倍体诱变性研究孟全新，王菊凝，张月非整倍体是指细胞染色体数目不成倍数地增加或减少，从而形成不同于正常细胞核型的细胞。非整倍体对人类有巨大危害，当其发生在体细胞时可能引起癌变［１．２］，发生在生殖细胞可引起自然流产和先无缺陷［...     

荧光原位杂交技术分析不育男性精子X、Y及18号染色体

目的:应用荧光原位杂交技术分析不育男性精子的X、Y和18号染色体非整倍体情况.方法:15例接受卵细胞浆内单精子注射(ICSI)治疗的男性不育症患者中严重少弱精子症组(A组)、轻度少弱精子症组(B组)和弱精子症组(C组)各5例.选用CEPX/Y、CEP18染色体探针对其ICSI后剩余的精子进行荧光原位杂交实验,比较3组患者精子染色体的非整倍体发生率.结果:共计数精子3 000个,杂交率为97%.A、B、C组染色体非整倍体率分别为(6.72±2.64)%、(1.60±1.02)%和(4.03±1.50)%,差别有统计学意义(F=9.62,P<0.01).A组、B组之间和A组、C组之间差别均有统计学意义(均P<0.01),B组和C组差别无统计学意义(P>0.05).结论:对少弱精子症患者尤其是严重少弱精子症患者进行ICSI治疗时,应该在胚胎移植前进行胚胎染色体非整倍体筛查,防止染色体非整倍体胎儿出生.     

Crest染色法在毒理学检测中的应用

1980年,有学者发现具有CREST征的硬皮病人血清中含有一种能与人、小鼠、中国仓鼠等染色体着丝粒蛋白特异结合的抗体(ACA),又称CREST抗体。在现代毒理学检测中CREST抗体免疫荧光染色法在非整倍体检测中发挥着积极的作用。随着近年来对非整倍体研究的深入,对其危害的严重性认识也越来越清楚,可目前还没有一项有效、快速的检测非整倍体的方法。1988年,CREST抗体被用于微核(MN)试验中,有着丝粒的微核是在非整倍体毒剂作用下整条染色体丢失造成的,无着丝粒的微核则是在断裂剂的影响下染色体断裂形成。微核试验的普及程度高,这为CREST…     

ECRG2基因缺失导致HCT-116细胞中心体过度复制

目的:非整倍体是肿瘤细胞的典型特征,是人类肿瘤染色体不稳定性的普遍形式。中心体复制异常是肿瘤细胞中染色体不稳定性的主要原因。研究观察ECRG 2对中心体复制及染色体不稳定性的影响。方法:利用基因敲除和免疫荧光染色技术观察了ECRG 2基因对中心体复制和染色体不稳定性的影响。结果:s iRNA技术敲除ECRG 2基因导致中心体过度复制、多极纺锤体出现,最终导致染色体不稳定性和非整倍体细胞出现。结论:ECRG 2在中心体复制、纺锤体形成中起重要作用,对确保染色体稳定性必不可少;ECRG 2基因的缺失可能是肿瘤细胞染色体不稳定性和非整倍体细胞产生的重要原因。     

对比孕中期产前筛查、无创DNA检测及羊水诊断的临床价值

目的 对比染色体非整倍体疾病应用孕中期产前筛查、无创DNA检测、羊水诊断的临床价值,促使出生缺陷儿概率明显降低.方法 抽取2017年1月～2019年12月到本院产科接受产前筛查的孕中期孕产妇作为研究对象,共1580例,对21-三体综合征、18-三体综合征、13-三体综合征等风险进行评估,对于高风险的孕产妇要实施无创DN...     

Mechanisms and risk of chemically induced aneuploidy in mammalian germ cells

Aneuploidy is a pathological condition that affects 35% of human spontaneous abortions and 0.3% of livebirths. In spite of the increasing knowledge about molecular mechanisms of meiosis and chromosome segregation, maternal age remains the only ascertained aetiological factor. Genetically modified mouse models have been produced that show increased incidence of aneuploid gametes or abnormalities in meiotic recombination and synapsis. They suggest that genetic polymorphisms might also be involved in the aetiology of human germ cell aneuploidy. Experimental studies in the mouse have identified chemicals that can induce aneuploidy in male and female germ cells. Compounds affecting spindle assembly/dynamics are potent aneugens for oocytes and less so also for spermatocytes. They are active at acute doses during a short time interval preceding the metaphase-to-anaphase transition. Topoisomerase inhibitors are also meiotic aneugens which act on the recombination process; for the first time, the production of viable aneuploid mouse progeny was shown after paternal treatment with etoposide. A comparison between in vitro and in vivo effects of suspect aneugens demonstrates that there are biological mechanisms protecting mammalian oocytes from acute exposures to exogenous chemicals. Endocrine disruptors are a novel group of compounds that might affect chromosome segregation at meiosis. Data on bisphenol-A suggest that such chemicals could be active at low chronic exposure levels, but this hypothesis needs to be confirmed by further experiments. Experiments on cultured mouse oocytes treated with inhibitors of biochemical reactions involved in the regulation of chromosome segregation point to possible new mechanisms of action of environmental aneugens.     

The in vitro MN assay in 2011: origin and fate,biological significance,protocols, high throughput methodologies and toxicological relevance

Micronuclei (MN) are small, extranuclear bodies that arise in dividing cells from acentric chromosome/chromatid fragments or whole chromosomes/chromatids lagging behind in anaphase and are not included in the daughter nuclei at telophase. The mechanisms of MN formation are well understood; their possible postmitotic fate is less evident. The MN assay allows detection of both aneugens and clastogens, shows simplicity of scoring, is widely applicable in different cell types, is internationally validated, has potential for automation and is predictive for cancer. The cytokinesis-block micronucleus assay (CBMN) allows assessment of nucleoplasmic bridges, nuclear buds, cell division inhibition, necrosis and apoptosis and in combination with FISH using centromeric probes, the mechanistic origin of the MN. Therefore, the CBMN test can be considered as a “cytome” assay covering chromosome instability, mitotic dysfunction, cell proliferation and cell death. The toxicological relevance of the MN test is strong: it covers several endpoints, its sensitivity is high, its predictivity for in vivo genotoxicity requires adequate selection of cell lines, its statistical power is increased by the recently available high throughput methodologies, it might become a possible candidate for replacing in vivo testing, it allows good extrapolation for potential limits of exposure or thresholds and it is traceable in experimental in vitro and in vivo systems. Implementation of in vitro MN assays in the test battery for hazard and risk assessment of potential mutagens/carcinogens is therefore fully justified.     

Application of toxicogenomics to study mechanisms of genotoxicity and carcinogenicity

Specific genotoxic events such as gene mutations and/or chromosome damage are considered hallmarks of cancer. The genotoxicity testing battery enables relatively simple, rapid and inexpensive hazard identification, namely by assessing a chemical's ability to cause genetic damage in cells. In addition, the 2-year rodent carcinogenicity bioassay provides an assessment of a risk associated with the chemical to develop cancer in animals. Although the link between genotoxicity and carcinogenicity is well documented, this relationship is complicated due to the impact of non-genotoxic mechanisms of carcinogenesis and by character of the in vitro genotoxicity assays and specific endpoints making the interpretation of test results in light of human risk and relevance difficult. In particular, the specificity of test results has been questioned. Therefore, the development of novel scientific approaches bridging genotoxicity and carcinogenicity testing via understanding underlying mechanisms is extremely important for facilitating cancer risk assessment. In this respect, toxicogenomics approaches are considered promising as these have the potential of providing generic insight in molecular pathway responses. The goal of this report thus is to review recent progress in the development and application of toxicogenomics to the derivation of genomic biomarkers associated with mechanisms of genotoxicity and carcinogenesis. Furthermore, the potential for application of genomic approaches to hazard identification and risk assessment is explored.     

Genotoxicity of carcinogens in human hepatocytes: application in hazard assessment

Evaluation of chemical genotoxicity has been used in assessing human cancer hazard, based on the observation that most human carcinogens are known to be DNA-reactive. The availability of data on the DNA-reactivity of compounds in metabolically competent human cells would assist hazard assessment by providing direct information of human genotoxicity. To evaluate the reliability of human hepatocytes for this purpose, the induction of DNA repair by DNA-reactive carcinogens of several structural classes and related noncarcinogens was studied. All the carcinogens elicited DNA repair synthesis, whereas the noncarcinogens did not. These studies provide additional support for the use of human hepatocytes in a DNA repair test in the investigation of genotoxicity. The demonstration of genotoxicity in human cells is suggested to provide important information for hazard assessment.     

Selection of genotoxicity tests for risk assessment of effluents

Genotoxicity is one of the parameters within the whole effluent environmental risk procedure, an effect-based procedure developed in the Netherlands to supplement classical substance-specific risk assessment of effluents. To implement the genotoxicity parameter, one or more tests have to be selected for routine use on effluents. This paper deals with problems and considerations encountered during selection of genotoxicity tests. Tests were judged on: relevance, validation, detected genotoxic lesions, quantitative sensitivity, convenience, and cost-efficiency. Based on criterion detected genotoxic lesions and on criteria convenience and cost-efficiency, it is recommended to use at least a bacterial test which makes use of detection of the SOS pathway which is induced upon the occurrence of DNA damage. The criterion quantitative sensitivity is used in a laboratory study on effluent samples to select the most appropriate bacterial SOS pathway test. The range of detected genotoxic endpoints can be expanded by also including a more expensive test that detects clastogenesis and/or aneuploidy. In that case it seems wise first to establish the added value of such a test for the risk assessment of effluents, before deciding on further use. Furthermore, it is concluded that the presently available information on relevance and validation is of limited use for test selection. Finally, recommendations are made on test protocols and on pretreatment of effluent samples to optimize genotoxicity tests for effluent samples. Recommendations include data analysis, detection of the interference of cytotoxicity, extraction, the use of S9, concentration procedures, and filtration. © 2000 John Wiley & Sons, Inc. Environ Toxicol 15: 81–90, 2000     

Genetic toxicity assessment: employing the best science for human safety evaluation. Part II: Performances of the in vitro micronucleus test compared to the mouse lymphoma assay and the in vitro chromosome aberration assay.

The in vitro micronucleus test is commonly used in the early stages of pharmaceutical development as a predictive tool for the regulatory mouse lymphoma assay or in vitro chromosome aberration test. The accumulated data from this assay leads to the suggestion that it could be used as an alternative to the chromosome aberration test or the mouse lymphoma assay in the regulatory genotoxicity battery. In this paper, we present the results of the in vitro micronucleus test on L5178Y mouse lymphoma cells with 25 compounds from Servier research and have compared these results to those obtained in the genotoxicity regulatory battery. All the negative compounds were also negative in the in vitro micronucleus assay. Among the 14 positive compounds, two of them, positive in the mouse lymphoma assay, were found negative in the in vitro micronucleus test. However, this apparent discordance was likely to be due to cytotoxicity- or high concentration-related false positive responses in the mouse lymphoma assay. In addition, we confirmed that the in vitro micronucleus assay is useful for detecting aneugens, especially, when cells in metaphasis and multinucleated cells are also scored and when cells are allowed to recover after the long treatment. On this series of compounds, the in vitro micronucleus assay showed high sensitivity and possibly a better specificity than the mouse lymphoma assay. Thus, the in vitro micronucleus assay was shown to be at least as adequate as the mouse lymphoma assay or the in vitro chromosome aberration test to be used in the standard genotoxicity battery.     

Genotoxicity assessment of some cosmetic and food additives

α-Hexylcinnamaldehyde (HCA) and p-tert-butyl-alpha-methylhydrocinnamic aldehyde (BMHCA) are synthetic aldehydes, characterized by a typical floral scent, which makes them suitable to be used as fragrances in personal care (perfumes, creams, shampoos, etc.) and household products, and as flavouring additives in food and pharmaceutical industry. The aldehydic structure suggests the need for a safety assessment for these compounds. Here, HCA and BMHCA were evaluated for their potential genotoxic risk, both at gene level (frameshift or base-substitution mutations) by the bacterial reverse mutation assay (Ames test), and at chromosomal level (clastogenicity and aneuploidy) by the micronucleus test. In order to evaluate a primary and repairable DNA damage, the comet assay has been also included. In spite of their potential hazardous chemical structure, a lack of mutagenicity was observed for both compounds in all bacterial strains tested, also in presence of the exogenous metabolic activator, showing that no genotoxic derivatives were produced by CYP450-mediated biotransformations. Neither genotoxicity at chromosomal level (i.e. clastogenicity or aneuploidy) nor single-strand breaks were observed. These findings will be useful in further assessing the safety of HCA and BMHCA as either flavour or fragrance chemicals.     

Characterization of formaldehyde’s genotoxic mode of action by gene expression analysis in TK6 cells

Gene expression analysis has been established as a tool for the characterization of genotoxic mechanisms of chemical mutagens. It has been suggested that expression analysis is capable of distinguishing compounds that cause DNA damage from those that interfere with mitotic spindle function. Formaldehyde (FA) is known to be a DNA-reactive substance which mainly induces chromosomal damage in cultured mammalian cells. However, there has been concern that FA might also induce leukemia-specific aneuploidies, although recent cytogenetic studies excluded a relevant aneugenic potential of FA. We now investigated whether gene expression profiling can be used as a molecular tool to further characterize FA’s genotoxic mode of action and to differentiate between clastogenic and aneugenic activity. TK6 cells were exposed to FA for 4 and 24 h, and changes in gene expression were analyzed using a whole-genome human microarray. Results were compared to the expression profiles of two DNA-damaging clastogens (methyl methanesulfonate and ethyl methanesulfonate) and two aneugens (colcemid and vincristine). The genotoxic activity of FA, MMS and EMS under these conditions was confirmed by comet assay experiments. The gene expression profiles indicated that clastogens and aneugens induce discriminable gene expression patterns. Exposure of TK6 cells to FA led to a discrete gene expression pattern, and all toxicogenomics analyses revealed a closer relationship of FA with clastogens than with aneugens.     

An in vitro micronucleus assay with size-classified micronucleus counting to discriminate aneugens from clastogens

In the in vitro micronucleus (MN) assay, genotoxic chemicals can be characterized as aneugens and clastogens by the presence and absence of kinetochore protein or centromere regions in the micronuclei, respectively. Aneugens preferentially induce kinetochore- or centromere-positive micronuclei which can be detected by the immunofluorescence staining method or the fluorescence in situ hybridization (FISH) method. Both methods are robust and reliable; however, these assays require a definite period of time and cost to obtain a result that suggests that the genotoxic chemicals cause aneuploidy. This is why these methods are not adequate to evaluate dozens of chemicals which are mixtures of aneugens and clastogens. To evaluate a batch of chemicals, a quicker and more convenient assay is desirable. In the present study, we examined whether the size-classified counting of MN is as effective as the FISH method to characterize aneuploidy in the in vitro MN assay using Chinese hamster lung (CHL) cell lines. As aneugens, 9 substances (colcemid, vincristine sulfate, paclitaxel, thiabendazole, diethylstilbestrol, griseofulvin, bisphenol A, fisetin and okadaic acid) were used; as clastogens 6 substances (methylmethane sulfonate, N-methyl-N′-nitro-N-nitroso-guanidine, etoposide, mitomycin C, hydroxyurea and actinomycin D) were used. The size-classified counting revealed that all the 9 aneugens increased both the frequency and proportion of large-size MN as compared with the vehicle control. Although N-methyl-N′-nitro-N-nitroso-guanidine, etoposide and mitomycin C increased the frequency, no increase was observed in the proportion. Meanwhile, with the FISH method, all the aneugens induced centromere-positive micronuclei but the clastogens did not. Based on these results, it is considered that the frequency of large-size MN in the in vitro MN assay is an alerting index for aneugenic effects and that its proportion is a simple and reliable index which is as effective as the FISH analysis for discrimination of aneugens from clastogens.     

ReProComet: a new in vitro method to assess DNA damage in mammalian sperm.

The increasing request of chemical safety assessment demands for the validation of alternative methods to reduce the resort to animal experimentation. Methods that evaluate reproductive toxicity are among those requiring the largest use of animals. Presently, no validated in vitro alternative exists for the assessment of reproductive toxicity. Mammalian sperm are sensitive targets of DNA-reactive chemicals, which form premutagenic adducts. Here, we propose a new method based on comet assay to detect DNA damage induced by potential germ cell mutagens in bull sperm available from assisted reproduction practices. In somatic cells, chemical-induced adducts can be revealed by comet assay that detects DNA breaks produced during adduct repair. Mature sperm, however, are devoid of repair enzymes, and adducts are processed only after fertilization. For this reason, comet assay is not sensitive to detect DNA lesions induced in sperm by most chemicals. To overcome such limitation, we developed a modified comet assay based on the addition of a protein extract from HeLa cells to agarose-embedded sperm on microscopic slides. To test the method, sperm were treated in vitro with methyl methanesulfonate (MMS) or melphalan (MLP) and comet assay was conducted both with and without protein supplementation. No effect of MMS or MLP was detected without protein supplementation; on the contrary, a clear-cut dose-dependent effect was measured after addition of the cell extract. These results represent a proof of concept of a novel in vitro mutagenicity test on sperm that could offer a promising approach to complement previously validated in vivo germ cell genotoxicity assays.