Detection of environmental clastogens and aneugens in human fibroblasts by cytokinesis-blocked micronucleus assay associated with immunofluorescent staining of CENP-A in micronuclei

The cytokinesis-blocked micronucleus (CBMN) assay, in combination with fluorescent in situ hybridization (FISH) of human pan-centromeric DNA probes, or with CREST antibodies that specifically stain kinetochore proteins, is widely used on several cell types. It distinguishes micronuclei containing one or several whole chromosomes, which are positively labeled (centromere positive micronucleus, C+MN, due to aneugenic effect), or acentric chromosome fragments, which are unlabeled due to the absence of centromere (centromere negative micronucleus, C−MN, due to clastogenic effect). However, the very slight level of the centromeric signals obtained with the FISH technique on primary human fibroblasts, a cell type commonly used in environmental genetic toxicology, leads to great difficulties in distinguishing C+MN and C−MN. Furthermore, the CREST technique may lead to inappropriate results particularly with regards to variations in antibody composition between patient sera. Our results show that the in vitro CBMN, in combination with immunofluorescence staining of CENP-A (centromere protein A), efficiently screens genotoxicants for their ability to induce clastogenic and/or aneugenic effects. We propose the in vitro CBMN assay in combination with immunofluorescence staining of CENP-A as a suitable tool in environmental genotoxicity testing of primary human fibroblasts.     

Coking wastewater increases micronucleus frequency in mouse in vivo via oxidative stress

Coking wastewater has caused serious health risk in coal-producing areas of China, however its toxic effects have not been well understood. The genotoxicity induced by coking wastewater on mice in vivo and its possible oxidative mechanisms were investigated via observing the induction of micronuclei in polychromatic erythrocytes of mouse bone marrow, and subsequently determining the antioxidative enzyme activities （superoxide dismutase Cu, Zn-SOD, Se-dependent glutathione peroxidase, and catalase）, thiobarbituric acid reactive substance contents and protein carbonyl levels in brains and livers of mice. Results showed that the tested coking wastewater caused a significant increase of micronucleus frequencies in a concentration-dependent manner. Also, the sample increased lipid peroxidation and protein oxidation levels, which was accompanied by changes in antioxidative status. Interestingly, pre-treatment with an antioxidant （vitamin C） led to a statistical reduction in the micronucleus frequency caused by coking wastewater. This implies that coking wastewater induces evident genetic damage in mammalian cells, and exposure to polluted areas might pose a potential genotoxic risk to human beings; in the process, oxidative stress played a crucial role.     

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。     

nano-TiO_2与1,2,4-TCB联合作用对斑马鱼鳃组织结构及染色体损伤研究

采用组织病理切片法、微核及彗星试验研究nano-TiO_2和1,2,4-TCB联合染毒对斑马鱼鳃组织结构、细胞微核率及DNA损伤的毒性效应。结果表明,单独nano-TiO_2暴露不会导致斑马鱼鳃组织结构损伤,也不会引起明显的微核及染色体损伤。1,2,4-TCB(≥10 mg·L~(-1))会引起鳃组织和细胞染色体的损伤。nano-TiO_2与1,2,4-TCB联合作用会加重斑马鱼的鳃组织结构损伤,出现鳃小片排列严重紊乱,表皮细胞断失严重,鳃小片上皮细胞坏死脱落,鳃丝结构模糊等症状。两者联合作用会引起鳃细胞微核率增加和彗星细胞率的增加,呈现一定的剂量-效应关系,且两者间的联合作用具有协同作用。对彗星试验的尾部DNA百分比、尾长及尾距的分析发现,与对照组和单独作用相比,两者联合作用对彗星指标均有显著增强作用,这表明两者联合作用对染色体的损伤属于非特定片段损伤。     

重金属（Hg，As，Pb）污染对蚕豆根尖细胞微核的诱变研究

采用蚕豆根尖细胞微核检测技术,分别探讨不同浓度的HgCl2、As2O3、Pb（NO3）2对蚕豆根尖细胞微核率的影响。以蒸馏水处理作为阴性对照实验。结果表明不同浓度的HgCl2可使蚕豆根尖细胞微核率明显上升,说明HgCl2对蚕豆根尖具有明显的毒性效应——诱发突变（微核）。不同浓度As2O3可使蚕豆根尖细胞微核率明显上升。说明三氧化二砷（As2O3）对蚕豆根尖具有明显的毒性效应——诱发突变（微核）。蚕豆根尖微核率在铅离子浓度0—25mg／L范围内,随着硝酸铅处理浓度的升高而升高,说明硝酸铅是一种较强的诱变剂。     

土壤镉污染毒性效应的多指标综合评价

以草甸棕壤为供试土壤,以蚕豆幼苗根尖有丝分裂指数、染色体畸变率以及微核率,幼苗叶片内抗氧化酶活性、植物内源激素含量为指标,采用盆栽方法研究了0～10 mg·kg^-1镉胁迫对植物细胞的遗传和生态毒性效应.结果表明,在此浓度范围内,蚕豆根尖细胞有丝分裂指数、染色体畸变率以及微核率均随镉浓度增加呈显著的剂量-效应正相关关系,其中微核率变化最为明显,处理组微核率分别为对照组的1.43～3.22倍;蚕豆幼苗叶片内的SOD和POD活性变化呈先升高后下降的趋势;而CAT活性随镉浓度增加其变化规律与SOD、POD相反.此外,镉胁迫下植物激素脱落酸(ABA)、赤霉素(GA₃)与细胞分裂素类的玉米素和玉米素核苷总含量(Z&ZR)均表现出低浓度下诱导和较高浓度下诱导率降低的趋势,在镉浓度为2.5 mg·kg^-1时３种植物激素含量均达到最高值,分别比对照组增加了6.6%、 4.0%和12.6%.研究表明,各指标对污染物的毒性具有响应且响应的域值及其敏感度不同,将各指标综合使用可使土壤镉污染的遗传和生态毒性诊断更为有效.     

垃圾渗滤液的遗传毒性研究

应用蚕豆根尖和鲫鱼外周血细胞的微核测定技术对垃圾渗滤液遗传毒性进行了监测.用CODMn为1 415 mg/L的垃圾渗滤原液处理蚕豆根尖9 h,微核千分率(MCN)最高达到(25.62±0.87)‰,同时将垃圾渗滤原液分别稀释到CODm为710 mg/L、470 mg/L、280 mg/L和200 mg/L,在不同时间条件下处理蚕豆根尖,其MCN均呈现出随着处理时间的增长而逐渐增高的趋势;另外,用稀释后CODMn为1.5～14mg/L的垃圾渗滤液喂养鲫鱼,结果也显示,随渗滤液浓度的增高,鲫鱼外周血细胞MCN增大.垃圾渗滤液是一种毒性很强的液体,随着接触垃圾渗滤液的时间增长,会有导致生物体细胞分裂受阻和造成细胞内遗传物质损伤的潜在危害.     

阿特拉津对弹琴蛙（Rana adenopleura）蝌蚪微核

分别在d1、d3、d7、d14,观察空白对照(CK-)、水溶助剂0.1mL/L二甲基亚砜对照(CK )、阿特拉津0.01mg/L(ρ1)、0.1mg/L(ρ2)、1mg/L(ρ3)和10mg/L(ρ4)溶液中弹琴蛙(Ranaadenopleura)蝌蚪血红细胞微核和核异常细胞数.结果表明,d14时,ρ1、ρ2、ρ3和ρ4组蝌蚪的微核率分别是CK-的2.25、2.50、4.25、5.11倍(P<0.05),说明阿特拉津可引起蝌蚪血红细胞微核率升高;且与d1、d3、d7相比较,微核率随着时间的延长有升高的趋势.d14时,ρ2、ρ3和ρ4组蝌蚪的总核异常率(含微核率)分别是CK-的2.59、2.17、1.96倍(P<0.05),说明阿特拉津也可引起蝌蚪血红细胞总核异常率升高.图2表1参17     

氟化钠与强致突变物联合诱致的高等植物细胞微核效应

氟化钠在与强致突变物的联合实验中均发生较强的颉颃(减毒)作用,例如非常显著地抑制由平阳霉素(染色体断裂剂)或秋水仙素(有丝分裂毒素)诱致的微核效应(F＇相似文献   

利用微核技术评价水质重金属污染的指标探讨

利用蚕豆根尖细胞的微核技术和正交试验法,对含Pb、Cd、Cr和As的不同浓度组合的模拟废水进行监测,结果表明,各处理的徽核率与对照的微核率的差异均达到极显著或显著水平,当污染指数<1.68时,水质基本没有受重金属污染物污染;当污染指数≥1.68-<2.98时,“废水”中重金属污染物含量未超标;当污染指数≥2.98-<4.90时,污染物含量亦未超标,但已有1种以上污染物含量达到农用灌溉水质二类标准;当污染指数≥4.90-<5.38时,有1种或2种污染物含量超标;当污染指数>5.38时,至少有3种污染物含量超标.