The Cytokinesis-Block Micronucleus Assay on Peripheral Blood Lymphocytes as a Prospective Biological Test-System to Estimate the Influence of Radon on the Human Organism: Recent Progress and Future Prospects

This paper discusses the problem of assessing the negative after-effects of low doses of ionising radiation exposure in humans. Radon and its decay daughter products are the most widespread source of such irradiation. Miners (in both uranium and non-uranium mines) as well as laypeople in domestic life may be exposed to radon, making the problem of assessing the cytogenetic effects of exposure extremely crucial. One of the more promising test systems to assess the effect of radon is the cytokinesis-block micronucleus assay (CBMN) on peripheral blood lymphocytes, which has a number of advantages over other cytogenetic techniques. Recent progress and future prospects of this cytogenetic method are discussed here.

Detection of Cytogenetic Effects in Peripheral Lymphocytes of Students Exposed to Formaldehyde with Cytokinesis-Blocked Micronucleus Assay

Cytokinesis-blocked micronucleus assay was applied as a biological dosimeter to detect abnormalities in human peripheral lymphocytes of thirteen students exposed to formaldehyde (FA) during a 12-week (10 h per week) anatomy class. Breathing-zone air samples colleeted during dissection procedures showed a mean concentration of 2. 37 ppm (3. 17mg/m3 ). Ten students from the same school but without FA exposure served as controls. Chromosome aberrations (CA) and sister chromatid exchanges (SCE) were detected in both groups. The micronuclei (MN) rate (6. 38 ± 2. 50‰ ) and CA rate (5. 92 ±2. 40‰ ) in the FA-exposed group showed a significant increase (P< 0. 01 ) when compared with those of the controls (3. 15 ±1. 46‰and 3. 40 ± 1. 57 % respectively). A correlation between MN and CA in individuals was observed. SCE in the exmpd group were also increased (P< 0. 05), but not so greatly as MN or CA. The results indicated that FA might damage the chromosomes of human lymphocytes.

Study on the Genetic and Physiological Toxicity of Wastewater from a Pharmaceutical Factory Using Root Tip Micronucleus Technology of Vicia faba

ObjectiveThe aim was to assess genetic and physiological toxicity of wastewater from a pharmaceutical factory using root tip micronucleus technology of Vicia faba. MethodThe pollution of wastewater from a pharmaceutical factory was detected by using root tip micronucleus technology of Vicia faba, and the genetic and physiological toxicity of the wastewater to Vicia faba was assessed. ResultNon-processed wastewater had an extremely high level of biological toxicity; the cells were unable to live with the wastewater at a high concentration; the cells were able to grow with the wastewater at a low concentration, though the micronucleus ratio was extremely high. The processed wastewater had no significant impact on cell growth, but the micronucleus ratio was extremely high, showing that the processed water also had a high pollution index. ConclusionThe research could provide scientific references for the national treatment of wastewater from a pharmaceutical factory.

Effect of various drinking water on human micronucleus frequency in high-risk population of PHC

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Micronucleus analysis in patients with colorectal adenocarcinoma and colorectal polyps

AIM:To determine,by counting micronucleus (MN) frequencies,whether chromosomal or DNA damage have an effect on the pathogenesis of early colorectal adenocarcinoma (CRC). METHODS:We analyzed MN frequencies in 21 patients with CRC,24 patients with colon polyps [10 neoplastic polyps (NP) and 14 non-neoplastic polyps (NNP)] and 20 normal controls. RESULTS:MN frequency was significantly increased in CRC patients and in NP patients compared with controls (3.72 ± 1.34,3.58 ± 1.21 vs 1.97 ± 0.81,P < 0.001). However,there was no difference in the MN frequency between CRC patients and NP patients (P > 0.05). Similarly,there was no difference in the MN frequency between NNP patients (2.06 ± 0.85) and controls (P > 0.05). CONCLUSION:Our results suggest increased chromosome/DNA instabilities may be associated with the pathogenesis of early CRC.

Joannesia princeps： Evaluation of Aqueous Extracts Genotoxicity Utilizing Allium cepa Assay and Micronucleus Test

Evaluation of the genotoxic and cytotoxic potentials is a part of the analysis process where plant products with therapeutic properties must be submitted for being safely employed, mainly through long time points, doannesia princeps, also known as ＂cotieira＂ has been used on popular medicine as laxative and several diseases treatment. Aiming to analyze genotoxic potential ofd. princeps leaves extract, Allium cepa assay and micronucleus test were employed. No genotoxic activity presented J. princeps leaves extract; however, extracts cytotoxic activity over A. cepa meristematic cells and mice marrow cells was observed. J. princeps leaves extract presented antiproliferative activity at concentrations and systems employed indicating their therapeutic potential for cellular cycle inhibition. Moreover, results showed that in regard to its mutagenic effect, usage of J. princeps leaves tea on popular medicine has been a safe procedure, as well.

TESTING THE MUTAGENICITY OF FIVE PESTICIDES WITH MICRONUCLEUS TECHNIQUE OF TRADESCANTIA PALUDOSA

Applying the micronucleus technique T.adescanlia paludosa, the mutagenicity of five pesticides, Decis, Sumicid, Cypermethrin, Trichlorphon and Diazinon were tested. The testing results showed that 0.64%, 0.32%, 0.16%, 0.08% of Decis, 0.64%, 0.32%, 0. 16%, 0.04% of Trichlorphon and 0.32%, 0.16%, 0.08%, 0.04% of Diazinon all had mutagenicity on Tradescantia paludosa, but Sumicid and Cypermethrin had no such characteristic.

Application of Micronucleus Technique in Testing River Pollution in the Qinhuang River, Binzhou City

To figure out water pollution of the Qinhuang River in Binzhou City,broad bean root-tip micronucleus technique was applied to test water quality,water sample from different sampling points was taken to detect the contamination of broad bean root tips,so as to reflect water pollution using the pol ution indexes.The results showed that water in the Qinhuang River was polluted in different degrees,and water pollution grew more serious from the upper to the lower reaches.Water pollution sources include domestic sewage and solid waste,thus pollution discharge into the river must be strictly controlled to restore the polluted water and the ecological functions of waterscapes.

NASOPHARYNGEAL CARCINOMA RADIOSENSITIVITY PREDICTION BY CYTOKINESIS－BLOCK MICRONUCLEUS ASSAY

Cytokinesis-block micronucleus method is used to evaluate the radiosensitivity of a nasopharyngeal carcinoma cell line (CNE-1) and biopsies obtained from 31 patients with nasopharyngeal carcinoma. The number of micronuclei increases with theradiation dose. A good correlation was found between the radiosensitivity determined by the micro-nucleus assay and that measured by the colony-forming assay in CNE-1 cell line (r=-0.998). Moreover, the results of micronucleus assay for tumor cells from biopsies of patients with primary carcinoma look promising for the prediction of tumor radiosensitivity. These results are encouraging but fleed to be confirmed with a larger number of patients with a longer follow-up.

The Chromosomal Effect of Birchen Dust as Determined by the Micronucleus Test

In a wood processing factory, the measured air concentration of birchen dust was 1. 26 ±0. 41 mg/m3, and the micronucleus frequency of peripheral blood lymphocytes in 83 workersexposed to wood dust was 1. 13 ± 2. 83%, which was significantly higher (P < 0.01 ) thanthat of control group (0. 51 ± 1. 41% ). The number of exposed workers with positive mi-cronucleus test was 9. 6 %, which was higher than that of control group (4. 5 % ), but thedifference was not significant (P >0. 05 ). The micronucleus test in mice treated with waterextracts of unsteamed and unbaked birchen dust showed that the micronucleus frequencies inall treated groups were significantly higher than that of contro group (P < 0. 01 ) and therewas also a doseresponse correlation (r = 0. 96, P < 0. 0005 ). The results of steamed andbaked birchen dust extracts were significantly lower than those of the unsteamed and unbakedones at the same doses (P< 0. 001 ). This suggests that when the birchen dust is steamed atthe temperature of 100℃ for 24h or baked at the temperature of 80℃, its inducing effect inmicronucleus test could be lowered

STABLE EXPRESSION OF HUMAN CYTOCHROME CYP2B6 AND CYP1A1 IN CHINESE HAMSTER CHL CELLS:THEIR USE IN MICRONUCLEUS ASSAYS

With specific designed prmers. CYP2B6 and CYP1A1 cDNA were generatecl by reverse transcrlI7tion-Polymerase chain reaction(RT-PCR )technlque Performed on total RNAs isolated frorn hum1ln liver and 3-rnethylch(,lanthrene(3-Mtt)induc human amnion FL, cells. Cell llnes (CHL, 2B6 and CtHL-1A1 ) capableof expressing hunlan cytochome P 15O (CYP ) 2B6 and 1A1 were establishecl after transfection of corre-sponding eukaryotic reconlbinant expression plasmid with human CYP2ll6 and 1A1 cDNA lnserts respectlvely. These cell lines stably expressed the mRNAs and the enzymatic activltles cc)rresI’onding to ttYP2B6and CYP1A1, respectively’ Compared with Chinese hamster lung (CHL) cells, the n1icr()nucleus frecluencyin CHl,-2B6 cells is markedly lncreased when exPosed to nitrosamines,aflatoxln B, (AFB1) and cyclophos-Phamide (CPA). Thls is also in CHL-1A1 cells,when exposed to carcinogenic polycycllc aromatic hydrocar-bons.

Evaluation of Protective Effects of Bioactive Phytochemicals Against Methotrexate in Salmonella typhimurium TA1535/pSK1002 Coupled with Micronucleus Assay

We evaluated the antimutagenic effects kinds of bioactive phytochemicals and of 10 some phytochemical combinations against methotrexate （MTX）-induced genotoxicity by the umu test in Salmonella typhimurium TA1535/rpSK1002 combined with a micronucleus assay. We observed that allicin, proanthocyanidins, polyphenols, eleutherosides, and isoflavones had higher antimutagenic activities than the other five types of bioactive phytochemicals. At the highest dose tested, MTX-induced genotoxicity was inhibited by 25%-75%. Kunming mice treated by MTX along with bioactive phytochemical combinations showed significant reduction in micronucleus induction and sperm abnormality rate （P〈0.01）. These results indicate that bioactive phytochemical combinations can be potentially used as new cytoprotectors.

蚕豆根尖细胞微核检测方案与数据分析

微核检测技术在世界范围内被广泛应用于环境监测以及食品安全性检测,由于其可操作性强,亦是大学开展课外活动,培养学生科研能力的有效方法。本文针对传统方法中本底微核数变异大和检验效率低的问题,提出了新的设计方案及分析方法。对师生进行课外研究及环境因素的遗传安全性分析与鉴定具有一定指导意义。

微管抑制及尾桥蛋白磷酸化促进骨肉瘤细胞U2OS微核形成

目的探讨微管及微管相关蛋白尾桥蛋白在肿瘤细胞微核形成中的作用。方法利用DAPI染色检测不同类型细胞自发微核率;利用诺考达唑(nocodazole)抑制微管聚集后,DAPI染色和胞质分裂阻滞(cytokinesis-block,CB)微核实验观察人骨肉瘤细胞株U2OS中微核形成情况;免疫荧光多重染色检测诺考达唑处理不同时间对U2OS细胞中微管及尾桥蛋白的影响;在U2OS细胞中过表达尾桥蛋白或利用collybistin使尾桥蛋白磷酸化,再利用免疫荧光及DAPI染色检测其对微管及微核形成的影响。结果相对于人胚肾HEK293细胞和原代神经细胞,U2OS自发微核率更高;诺考达唑处理使U2OS细胞微核数量增加,且与释放时间相关;诺考达唑处理使尾桥蛋白分布改变,逐渐聚集于细胞核;U2OS细胞中磷酸化尾桥蛋白主要表达于分裂期细胞;过表达尾桥蛋白和(或)增加其磷酸化水平并不影响U2OS细胞中微管蛋白的表达,但过表达尾桥蛋白并增加其磷酸化水平可使微核数量增多。结论肿瘤细胞内微核形成与微管聚集能力关系密切,可形成于细胞周期不同时期,具有积累效应。尾桥蛋白磷酸化后细胞中微核数量增多,提示其可能参与调节肿瘤细胞微核形成。

252名医疗放射工作人员的血象及淋巴细胞微核率检测

目的对贵州省252名放射工作医务人员的血象及淋巴细胞微核率进行检测,并分析可能的影响因素。方法选取接受职业健康体检的252名从事医疗放射工作人员为研究对象,根据其工种分为诊断放射学组(n=68)、介入放射学组(n=15)及其他放射工作组(n=169);使用全自动生化分析仪检测外周血象常规指标,采用微量全血培养法检测其外周血淋巴细胞微核率,并根据不同工种、年龄以及接害工龄分组对结果进行Kruskal-Wallis H秩和检验与χ^(2)检验,采用Spearman秩相关进行相关性分析。结果252名放射工作医务人员的外周血象结果均在正常范围内,不同工种、年龄组被检者白细胞、红细胞、血红蛋白及血小板计数比较,差异无统计学意义(P>0.05);不同接害工龄被检者的白细胞、红细胞、血红蛋白比较,差异无统计学意义(P>0.05);不同接害工龄被检者的血小板计数比较、差异有统计学意义(P<0.05),血小板数与接害工龄呈负相关关系(r=-0.170,P=0.007);微核率分析显示,不同工种被检者外周血淋巴细胞微核率、微核率异常率比较,差异无统计学意义(P>0.05);不同年龄组被检者的微核率、微核率异常率比较,差异有统计学意义(P<0.05),微核率与年龄呈正相关关系(r=0.179,P=0.004);不同接害工龄被检者微核率比较、差异无统计学意义(P>0.05),但微核率异常率比较、差异有统计学意义(P<0.05)。结论医疗放射工作人员的接害工龄与血小板负相关,年龄与淋巴细胞微核率、微核异常率正相关,且工龄增加、微核异常率也增加。

铜和锌对泥鳅外周血红细胞微核率的影响初步研究

为了解铜和锌对泥鳅(Misgurnus anguillicaudatus)的遗传毒性,在通过急性毒性试验测定铜和锌对泥鳅48 h的半致死浓度并求得对应的安全浓度基础上,分别设置6个铜处理组(Cu^(2+)浓度为0.00、0.02、0.04、0.08、0.10、0.20 mg/L的硫酸铜溶液)和7个锌处理组(Zn^(2+)浓度为0.0、0.2、0.6、1.1、3.0、5.0、7.0 mg/L的硫酸锌溶液)对泥鳅进行染毒,并分别在染毒后第1天、第3天和第6天时采血制片,研究了铜、锌对泥鳅外周血红细胞微核率的影响。结果表明,泥鳅经硫酸铜和硫酸锌染毒后,其红细胞微核率较对照组都有一定变化,硫酸铜和硫酸锌都能不同程度地引起泥鳅红细胞微核率的升高,而且具有明显的剂量和时间效应。在一定的浓度范围内,微核率与硫酸铜和硫酸锌的浓度呈正相关;但当浓度过高时,微核率反而随浓度的升高而降低。

微核激活cGAS-STING信号通路的机制及其肿瘤免疫功能

环鸟苷酸-腺苷酸合成酶(cGAS)-干扰素基因刺激因子(STING)信号通路可监测微生物入侵和组织损伤等生理病理异常状态,是天然免疫系统的重要组成之一。作为DNA感受器,cGAS主要识别异常定位于细胞质的双链DNA(dsDNA),通过催化合成二级信使环鸟苷酸-腺苷酸启动由STING介导的Ⅰ型干扰素和炎症信号通路。微核是有丝分裂后期染色体错误分离的产物,也是细胞质dsDNA的重要来源之一。作为一类不稳定的亚细胞器结构,微核核膜倾向于不可逆的破裂,导致微核基因组DNA暴露在细胞质中。暴露的微核基因组DNA招募并激活cGAS-STING信号通路,诱导STING下游信号通路活化,包括Ⅰ型干扰素信号通路和经典核因子κB(NF-κB)信号通路,导致细胞衰老、细胞凋亡和细胞自噬的发生,从而介导免疫系统的活化以清除肿瘤细胞,或者直接诱导肿瘤细胞死亡。另外,STING持续激活诱导的内质网应激,以及慢性Ⅰ型干扰素信号通路和非经典NF-κB信号通路的活化,营造了免疫抑制的肿瘤微环境,导致肿瘤细胞免疫逃逸,促进肿瘤转移和肿瘤细胞存活。因此,在肿瘤的发生发展和治疗过程中,活化的cGAS-STING免疫通路扮演着抑制或促进肿瘤的双重作用。本文阐述了肿瘤微环境中微核诱导cGAS-STING免疫通路活化的机制研究进展,探讨了其在肿瘤发生发展和治疗中的潜在重要作用。

口腔黏膜微核细胞计数及血清、唾液中CYFRA21-1水平与口腔上皮癌变进程的相关性研究

目的 探讨口腔黏膜微核细胞计数及血清、唾液中细胞角蛋白19片段(Cytokeratin-19-fragment, CYFRA21-1)水平与口腔上皮癌变进程的相关性。方法 对口腔疾病患者102例进行回归性分析,包括舌白斑57例(白斑组),口腔鳞状细胞癌45例(鳞癌组),另选同期检查口腔黏膜正常人员44例(正常组),对比各组人员的口腔黏膜微核细胞计数及血清、唾液中CYFRA21-1水平,分析各指标与口腔上皮癌变进程之间的关系。结果 三组的口腔黏膜微核细胞计数及血清、唾液CYFRA21-1表达水平:鳞癌组>白斑组>正常组,三组对比差异具有统计学意义(P<0.05);组间两两对比差异具有统计学意义(P<0.05)。鳞癌患者中晚期组的口腔黏膜微核细胞计数及血清、唾液CYFRA21-1表达水平均高于早期组,两组对比差异具有统计学意义(P<0.05)。根据单因素及多因素统计分析得,分化程度、淋巴结转移情况、口腔黏膜微核细胞计数、血清CYFRA21-1、唾液CYFRA21-1均为口腔鳞状细胞癌的癌症进程独立危险因素(P<0.05)。口腔黏膜微核细胞计数、血清CYFRA21-1、唾液CYFRA21-1单项检测的敏感度分别为84.0%、72.0%、64.0%,特异度为78.9%、68.4%、73.7%;联合检测的敏感度和特异度分别为88.0%、84.2%,其中联合检测的敏感度和特异度最高,对早期鳞癌和癌前病变的诊断效能最好。结论 口腔黏膜微核细胞计数及血清、唾液CYFRA21-1与口腔鳞癌的疾病进程存在密切联系,可以帮助预测诊断早期口腔鳞癌及癌前病变。

基于4%中性甲醛固定肝组织的大鼠肝微核实验方法的建立与验证

目的建立并验证基于4%中性甲醛固定肝组织制备肝细胞的大鼠肝微核实验(LMNT)方法(甲醛固定-LMNT法)。方法(1)SD大鼠分为雌性和雄性组,然后分别随机分为溶剂对照组和肝微核阳性化合物N-亚硝基二乙胺(DEN)12.5 mg·kg^(-1)组,每组5只,分别ig给予生理盐水和DEN,每天1次,连续14 d后取肝组织,同时用胶原酶消化-LMNT法和甲醛固定-LMNT法检测微核化肝细胞数量和处于有丝分裂期肝细胞数,分别计算肝细胞微核率和有丝分裂指数,肝细胞微核率>0.07%判定为阳性结果。(2)雄性SD大鼠分为喹啉(30,60和120 mg·kg^(-1))组、N-亚硝基吡咯烷(NPYR,25,50和100 mg·kg^(-1))组、各自溶剂对照组(NPYR,去离子水;喹啉,玉米油)及阳性对照DEN(12.5 mg·kg^(-1))组,每组5~6只,连续ig 15 d。每日记录体重,实验结束取肝记录肝总重,计算肝系数。自动生化分析仪检测肝功能相关血清生化指标谷丙转氨酶(GPT)、谷草转氨酶(GOT)活性和血清总胆红素(T-BIL)、直接胆红素(D-BIL)的水平。采用胶原酶消化-LMNT法和甲醛固定-LMNT法测定肝细胞微核率,用外周血微核实验和肝彗星实验评价喹啉和NPYR遗传毒性。结果(1)与各自溶剂对照组的肝细胞微核率(0.069%和0.030%)相比,甲醛固定-LMNT法测得的DEN组雄性大鼠肝细胞微核率为1.10%,雌性大鼠为0.82%,均显著升高(P<0.05);与各自溶剂对照组(0.060%和0.030%)相比,胶原酶消化法-LMNT法测得的DEN组雄性大鼠肝细胞微核率为1.45%,雌性大鼠为0.46%,均显著升高(P<0.05),判定为阳性。2种方法中雄性大鼠的肝细胞微核率均显著高于雌性(P<0.05)。与各自溶剂对照组相比,2种方法测得的DEN组雄性和雌性大鼠肝细胞有丝分裂指数均无明显差异。(2)与溶剂对照组相比,NPYR 50和100 mg·kg^(-1)组大鼠体重在ig第7~14天显著降低(P<0.01),DEN组在ig第8~14天显著降低(P<0.01);喹啉120 mg·kg^(-1)组大鼠在ig第4~14天显著降低(P<0.01),DEN组在ig第10~14天显著降低(P<0.05)。与溶剂对照组相比,NPYR 100 mg·kg^(-1)组(P<0.01)和DEN组(P<0.05)的肝重和肝系数均显著降低;喹啉60和120 mg·kg^(-1)组肝重(P<0.01)和肝系数(P<0.05)均显著增加。与溶剂对照组相比,DEN组大鼠血清T-BIL水平显著升高(P<0.01),NPYR 100 mg·kg^(-1)组GPT、GOT活性和D-BIL、T-BIL水平均显著升高(P<0.01),NPYR 25,50 mg·kg^(-1)和喹啉各剂量组则均无显著差异。甲醛固定-LMNT法测得的NPYR组肝细胞微核率较胶原酶消化-LMNT法略高,均判定为阳性;与溶剂对照组相比,2种方法测得的NPYR组肝细胞微核率均显著增加(P<0.05),喹啉组结果相当,均判定为阳性;2种方法测得的喹啉组肝细胞微核率均显著增加(P<0.05)。2种方法测得的NPYR和喹啉组肝细胞微核率相关性较好(R2分别为0.8614和0.9279)。NPYR组外周血微核实验为阴性,彗星实验结果为阳性;喹啉组外周血微核实验和彗星实验结果均为阴性。结论初步建立并验证了甲醛固定-LMNT法,且该方法检测肝致癌物的灵敏度与胶原酶-LMNT法相近。