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。     

荧光原位杂交技术产前诊断常见染色体非整倍体

目的:探讨荧光原位杂交(HSH)技术产前诊断常见染色体非整倍体的应用价值.方法:采用13、18、21、X和Y染色体特异性DNA探针对108例孕20～25周孕妇的未培养羊水细胞进行HSH检测,同时行羊水细胞染色体核型分析.结果:HSH检测108例成功104例(96.3%),其中染色体数目正常100例(58例为46,XX;42例为46,XY),染色体数目异常4例(3例47,XX,+21;1例45,X/46,XX).正常染色体数目中99例与传统核型分析结果一致,而另1例HSH结果为46,XX者传统核型分析显示为47,XX,+mar.染色体数目异常的4例与传统核型分析结果完全一致.核型分析为47,XX,+mar者因标记染色体来源不是13、18、21、X和Y染色体故HSH未能诊断.对于13、18、21、X和Y特异性探针来说,敏感性、特异性及与核型分析的符合率均为100%.结论:HSH技术用于产前诊断常见染色体非整倍体,具有简便、快速、特异性强、敏感性高、所用样本量少等优点.     

无创产前检测筛查胎儿性染色体非整倍体的临床效果评价

目的 探讨无创产前检测(NIPT)筛查胎儿性染色体非整倍体(SCA)的临床价值.方法 回顾性分析该院收集的2012年5月至2021年5月50972例孕妇的NIPT数据.从孕妇外周静脉血中提取游离胎儿DNA,在Illumina NextSeq 500高通量测序平台进行测序,NIPT结果通过羊水穿刺后经染色体微阵列分析进行...     

精液处理方法对精子染色体非整倍体率的影响

目的:分析不同精液处理方法对精子染色体非整倍体率是否有影响.方法:随机抽取10例接受卵胞浆内单精子注射(ICSI)治疗的轻度少弱精子症患者,分别采用上游法(A组)和Sperm-Grad双层密度梯度离心法(B组)处理其中5例患者精液,另外随机抽取于门诊就诊的5例轻度少弱精子症患者精液,不做处理,设为对照组.用CEPX/CEPY、CEPl8探针对这15例患者各1000个精子进行荧光原位杂交实验.比较3组精子中染色体非整倍体精子的比例.结果:性染色体非整倍体率,对照组、A组、B组分别为(4.21±249)%、(3.24±149)%和(2.62±0.89)%;18号染色体非整倍体率,对照组、A组、B组分别为(3.300±1.22)%、(2.00±1.22)%和(2.00±1.22)%,3组差异均无统计学意义(F分别为1.065和1.111,P＞0.05).结论:上游法和Sperm-Grad双层密度梯度离心法对精子的优选仅限于活力、畸形率等方面,而在精子染色体方面并无确定的优选作用.     

应用人体外周血淋巴细胞微核试验检测饮用水源水的致突变性

国内外流行病学调查提示,人类某些癌症死亡率和发病率与饮水质量存在一定相关性,至1981年,全世界饮水已鉴定出767种有机污染物。其中,确认为致癌物的20种,可疑致癌物26种,促癌物或助癌物18种,Ames试验致突变性物质48种。美国等国家对饮水诱变性也进行了大量的实验室研究,表明饮水存在致突变性物质。我国自七十年代后期开     

微核试验方法的研究进展

微核试验作为遗传毒性的检测方法,广泛应用于药品、化妆品、保健食品等领域的毒性评价,能够灵敏地反映出遗传毒性物质对染色体的损伤.随着检测技术的进步和新方法的应用,对于微核的检测手段也越来越多样.本文着重阐述了目前微核检测的技术方法,以期对现有的微核检测技术的应用提供参考依据.     

荧光原位杂交和抗着丝粒抗体染色对4种化合物诱发微核来源的分析

荧光原位杂交和抗着丝粒抗体染色对４种化合物诱发微核来源的分析曹佳胡斌１程天民程舸１（第三军医大学预防医学系分子毒理学实验室，重庆６３００３８；１广州师范学院生化教研室，广州５１０４００）微核分析可用于检测由染色体断裂剂诱发的无着丝粒断片，同时也可检测...     

体内淋巴细胞微核试验在肺及肠癌患者术前检测中的意义

目的 探讨体内淋巴细胞微核形成与肠癌和肺患者心栉肿瘤恶性程度程度的关系.方法 随机抽取大肠癌、肺癌患者各一组,术前取外周血进行体内淋巴细胞微核试验检测,比较癌症患者体内自发微核肜成与良性病谱及健康人群对照组的差异.结果 肺、大肠癌组微核率(MNF)与对照组相比,差异有统计学意义(P＜0.01);随着患癌组分化程度降低及淋巴结转移率的增加,MNF明显上升.结论 患者淋巴体内细胞微核形成与肺、大肠癌分化与转移密切相尖,为癌者患者的术前恶性程度削断和高危人群筛查提供了一个有用的生物学标志物.     

木糖醇的小鼠骨髓细胞微核试验

目的 采用小鼠骨髓细胞微核试验,对木糖醇进行体内致突变安全性评价.方法 小鼠骨髓细胞微核试验共设5组,其中受试物3个剂量组(高、中、低剂量分为10、5和2.5g· kg-1体重),阴性对照1组(灭菌注射用水,40mL·kg-1体重)和阳性对照1组(环磷酰胺400mg·kg-1体重),每组每种性别动物5只.给药采用30h给受试物法,即两次给受试物间隔为24h,5组小鼠均采用口服灌胃方式给药,给药量为0.04mL·g-1体重.在第二次给受试物后6h,全部小鼠颈椎脱臼处死,取股骨骨髓制片,固定、Gimesa染色和阅片.结果 受试物3个剂量组的微柱率(以千分率(‰)表示)分别与相应的阴性对照组进行比较,均无明显差异.结论 木糖醇的小鼠骨髓细胞微核试验结果为阴性.     

脱氢乙酸小鼠骨髓细胞微核试验

目的采用小鼠骨髓细胞微核试验,对脱氢乙酸进行体内致突变安全性评价。方法按照1/10 LD50、1/5 LD50和1/2 LD50设置三个剂量组,另设置阴性对照组和环磷酰胺(CP)阳性对照组(100 mg·kg-1)。每组10只小鼠,雌雄各半,灌胃给药,两次攻毒时间间隔24 h。在第二次灌胃后6 h,处死小鼠,制片,观察并计数嗜多染红细胞的微核率,并与阳性对照组和隐形对照组进行比较分析。结果 1/5 LD50、1/10 LD50剂量时,与正常对照组无显著差异;1/2 LD50剂量组与正常对照组有显著差异(P<0.05),但无剂量-效应关系。结论脱氢乙酸小鼠骨髓细胞微核试验结果为阴性。     

Statistical evaluation of the in vivo micronucleus assay

The statistical evaluation of the in vivo micronucleus assay is focused on multiple contrast tests for comparisons versus the negative control for count data taking the between-animals variability into account. For a possible claim the compound is not genotoxic in the micronucleus assay a proof of safety approach is proposed. For these statistical approaches user-friendly software is free available.     

微核试验和彗星试验检测雄黄的遗传毒性

目的用短期给药(小鼠骨髓细胞微核试验和彗星试验)和长期给药(利用生殖毒性Ⅰ段试验的大鼠做微核试验和彗星试验)检测雄黄的遗传毒性,探讨利用长期毒性试验或生殖毒性Ⅰ段试验用动物进行遗传毒性检测的可行性。方法小鼠ig给予雄黄0.25,0.5和1.0 g·kg-1,每天1次,2 d后,取骨髓细胞做微核试验和彗星试验;利用生殖毒性Ⅰ段大鼠ig给予0.125,0.25和0.55 g·kg-1,雄性连续给药42 d以上,交配成功后处死;雌性连续ig给药19 d以上,妊娠第15天,取骨髓细胞做微核试验和彗星试验,取血做外周血淋巴细胞微核试验。结果与阴性对照组比较,小鼠雄黄0.25,0.5和1.0 g·kg-1组微核试验微核率分别为3.0‰,4.40‰,7.01‰(P<0.05,P<0.01)和彗星试验拖尾率分别为6.3%,9.7%和11.3%(P<0.05,P<0.01)。与阴性对照组比较,生殖毒性Ⅰ段试验大鼠ig给予雄黄,雄黄0.55 g·kg-1组雄性大鼠的骨髓微核和外周血淋巴细胞微核率分别为2.83‰和6.67‰(P<0.05),雌性大鼠0.25和0.55 g·kg-1的骨髓微核和外周血淋巴细胞微核率分别为1.5‰,2.25‰以及2.58‰和4.40‰(P<0.05,P<0.01);雄黄使雄性和雌性大鼠彗星拖尾率明显升高(P<0.05)。结论利用生殖毒性Ⅰ段试验多次给药后取材做微核试验和彗星试验方法可行;外周血微核试验简便易行;在所观察的剂量下雄黄具有遗传毒性。     

Assessment of genome damage in a population of Croatian workers employed in pesticide production by chromosomal aberration analysis,micronucleus assay and Comet assay

The widespread use of pesticides suggests that the evaluation of their genotoxicity should be extended using the different assays available. In the present study we used two standard cytogenetic methods (chromosomal aberration analysis and micronucleus assay) and the Comet assay as a relatively new and powerful technique. The study included 10 workers occupationally exposed to a complex mixture of pesticides (atrazine, alachlor, cyanazine, 2,4-dichlorophenoxyacetic acid, malathion) during their production and 20 control subjects with no history of exposure to any physical or chemical agents. For the chromosomal aberration analysis, whole blood was cultivated for 48 h, whereas for the micronucleus assay, whole blood was cultivated for 72 h. For the comet assay whole blood was embedded in agarose on a microscope slide, lysed with detergent, denaturated and subjected to alkaline electrophoresis. Damage to DNA was evaluated by measuring tail length and calculating the tail moment. A significantly increased number of chromatid and chromosome breaks, as well as the presence of dicentric chromosomes and chromatid exchanges in exposed subjects compared with control subjects (P < 0.05), was found. There was also a statistically significant difference in frequency and distribution of micronuclei between the two groups examined. In the exposed subjects the Comet assay showed a statistically significant (P < 0.001) increase in DNA migration. Results suggest that long-term occupational exposure to pesticides could cause genome damage in somatic cells and therefore may represent a potential hazard to human health.     

Assessment of cytotoxic and genotoxic potential of pyracarbolid by Allium test and micronucleus assay

The present study evaluates the cytotoxic and genotoxic potential of pyracarbolid using both micronuleus (MN) assay, in human lymphocytes, and Allium cepa assay, in the root meristem cells. In Allium test, EC₅₀ value was determined in order to selecting the test concentrations for the assay and the root tips were treated with 25?ppm (EC₅₀/2), 50?ppm (EC₅₀) and 100?ppm (EC₅₀?×?2) concentrations of pyracarbolid. One percent of dimethyl sulphoxide (DMSO) and methyl methane sulfonate (MMS) were used as negative and positive controls, respectively. In the micronucleus assay, the cultures were treated with four concentrations (250, 500, 750 and 1000?µg/ml) of pyracarbolid for 24 and 48?h, negative and positive controls were also used in the experiment parallely. The results showed that mitotic index (MI) significantly reduced with increasing the pyracarbolid concentration at each exposure time. It was also obtained that prophase and metaphase index decreased significantly in all concentration at each exposure time. Anaphase index decreased as well and results were found to be statistically significant, except 24?h. A significant increase was observed in MN frequency in all concentrations and both treatment periods when compared with the controls. Pyracarbolid also caused a significant reduction in the cytokinesis block proliferation index (CBPI) in all concentration and both exposure time.     

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.     

大鼠肝彗星试验与骨髓微核试验评价结果比较研究

目的 梳理国家药物安全评价监测中心联合开展的大鼠多终点体内遗传毒性试验数据,比较大鼠肝彗星试验与骨髓微核试验结果的一致性和灵敏性。方法 试验分设阴性物质组、作用机制明确的遗传毒性阳性物质组、受试物组,阴性物质包括超纯水、0.9%氯化钠注射液、玉米油、0.5%羧甲基纤维素钠（CMC-Na）、5%蔗糖和聚山梨酯80,给药体积为10 mL·kg^-1;遗传毒性阳性物质包括200 mg·kg^-1甲磺酸乙酯（EMS）、40 mg·kg^-1N-乙基-N-亚硝基脲（ENU）、40 mg·kg^-1环磷酰胺、75 mg·kg^-1甲基苄肼、800 mg·kg^-1尿烷、75 mg·kg^-1对氯苯胺、40 mg·kg^-1 1,2-二溴-3-氯丙烷和10 mg·kg^-1秋水仙素;受试物包括100、300、1000 mg·kg^-1大黄素-8-O-β-D-葡萄糖苷,6.5、65.0、650.0 mg·kg^-1单蒽酮和6.5、65.0、650.0 mg·kg^-1大黄素甲醚。分别在实验0、24、45 hig给药1次,给药体积为10 mL·kg^-1。开展大鼠肝彗星试验和骨髓微核试验,计算每只动物的肝细胞刺猬细胞率和尾DNA百分含量（Tail% DNA）,以及每只动物的嗜多染红细胞（PCE）/总红细胞（ERY）比例和嗜多染红细胞微核（MNPCE）率。结果 大鼠肝彗星试验可有效检出DNA断裂剂,对多种烷化剂（甲磺酸乙酯、甲基苄肼和尿烷等）有较好的预测性,但对环磷酰胺和多倍体诱导剂不灵敏。大黄素-8-O-β-D-葡萄糖苷、单蒽酮和大黄素甲醚骨髓微核试验结果均为阴性。大黄素-8-O-β-D-葡萄糖苷在1 000 mg·kg^-1剂量下导致的肝Tail% DNA与0.5% CMC-Na组比较显著升高（P<0.05）;单蒽酮的肝彗星试验结果为明确阳性,剂量为650 mg·kg^-1时,单蒽酮可导致大鼠肝Tail% DNA显著升高（P<0.05）,且作用存在剂量相关性;大黄素甲醚的肝彗星试验结果为阴性。结论 大鼠肝彗星试验可与骨髓微核试验互补,有效检出主要作用于肝脏且亲电子性较强的遗传毒性化合物。     

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.     

流式细胞术在微核实验中的应用

微核实验检测终点明确、结果重复性好且易于开展,是广泛应用的遗传毒理学检测方法。由于微核的产生是小概率事件,而常规的人工显微镜计数方法即耗时、费力,且受主观因素影响较大,越来越难以满足大量遗传毒性评价任务的要求。流式细胞术作为一种高通量的快速自动化方法成为微核研究的一个重要方向。本文将对流式细胞仪检测体内微核的技术优化、方法验证及实际应用等最新进展进行综述,并对该技术在体外微核检测上的推广和广泛的实际应用前景进行展望。     

Antigenotoxicity of artepillin C in vivo evaluated by the micronucleus and comet assays

Artepillin C (3,5‐diprenyl‐p‐coumaric acid), a major compound found in Brazilian green propolis and Baccharis dracunculifolia, shows anti‐inflammatory, antibacterial, antiviral, antioxidant and antitumoral activities, among others. The aim of this study was to evaluate the genotoxic potential of artepillin C and its ability to prevent the chemically induced chromosome breakage or loss and the primary DNA damage using the micronucleus and comet assays in male Swiss mice, respectively. The animals were treated by gavage with different doses of artepillin C (0.4, 0.8 and 1.6 mg kg^?1 b.w.). For the antigenotoxicity assays, the different doses of artepillin C were administered simultaneously to doxorubicin (DXR; micronucleus test; 15 mg kg^?1 b.w.) and to methyl methanesulfonate (MMS; comet assay; 40 mg kg^?1 b.w.). The results showed that artepillin C itself was not genotoxic in the mouse micronucleus and comet assays. In the animals treated with artepillin C and DXR, the number of micronucleated reticulocytes was significantly lower in comparison with the animals treated only with DXR. Regarding antigenotoxicity, artepillin C at the tested doses significantly reduced the extent of DNA damage in liver cells induced by MMS. Copyright © 2011 John Wiley & Sons, Ltd.