合成洗涤剂对人和哺乳动物细胞的诱变性研究

各种合成洗涤剂(洗衣粉,洗发膏,餐具洗涤剂等)产量日增。在日常生活中使用越来越普遍。洗涤剂直接或间接通过环境污染对人类健康产生影响,特别是潜在的致突变性引起公众的普遍注意。而现有的研究结果并不一致。本研究选用三种型号的合成洗涤剂,以小鼠生殖细胞染色体畸变和骨髓细胞微核率及离体的人类细胞和中国仓鼠细胞的染色体畸变和姐妹染色单体交换(SCE)为测定指标,系统地对合成洗涤剂的诱变活     

频繁使用染发剂对小鼠染色体畸变率影响的研究

研究了多次接触７ 种染发剂对不同组小鼠骨髓和生殖细胞染色体畸变率的影响。结果发现, ７ 种染发剂均导致出现较高的染色体畸变率,３ 种能引起小鼠骨髓细胞染色体畸变率显著上升, 其中以粉末状染发剂的影响最为严重。４ 种染发剂对进行生殖细胞染色体畸变实验的小鼠均产生显著影响, 尤以氧化型染发剂最为明显。     

频繁使用染发剂对小鼠染色体畸变影响的研究

研究了多次接触７种染发剂对不同小鼠骨髓和生殖细胞染色体畸变率的影响。结果发现,７种染发剂均导致出现较高的染色体畸变率,３种能引起小鼠骨髓细胞染色体畸变率显著上升,其中以粉末状染发剂的影响最为严重。４种染发剂对进行生殖细胞染色体畸变实验的小鼠均产生显著影响,尤以氧化型染发剂最为明显。     

重铬酸钾对蚕豆根尖细胞致畸效应的研究

以蚕豆根尖为材料,研究重铬酸钾对蚕豆根尖细胞的致畸效应。采用蚕豆根尖细胞的微核试验和染色体畸变试验方法,以不同浓度的重铬酸钾为诱变剂,测定蚕豆根尖细胞的微核率和染色体畸变率。结果表明:重铬酸钾能诱发较高频率的微核率,即在一定浓度范围内,其微核率随重铬酸钾处理浓度的升高而增加,但高于一定浓度后反而呈下降趋势;不同浓度的重铬酸钾均使蚕豆根尖细胞有丝分裂指数增大;重铬酸钾还能诱导蚕豆根尖细胞产生较高频率的染色体畸变,且产生多种类型的染色体畸变。结论是重铬酸钾对蚕豆根尖细胞具有明显的致畸效应。     

大葱提取液对蚕豆根尖细胞的致突作用研究

以蚕豆根尖分生区细胞为实验材料,从微核、染色体指标研究大葱提取液对分裂旺盛细胞的致突作用,为开发大葱提取液的药用价值提供新思路。结果表明:与对照相比,20%和50%大葱提取液均可诱导蚕豆根尖细胞产生微核、染色体发生畸变,其中20%诱发微核数目最多,微核率及染色体畸变率最高。一定浓度的大葱提取液可干扰正在分裂细胞的染色体行为,最终诱导细胞发生凋亡。     

氟化钠致突变作用的研究

以昆明种大鼠和小鼠分别进行氟化钠诱发染色体畸变和微核试验,结果表明,在本实验条件下,未 见组化钠诱发动物骨髓细胞染色体畸变率及微核率明显增高。     

稀土元素钬对蚕豆的细胞毒性和遗传毒性研究

运用氧化钬与稀硝酸反应制备结晶,以去离子水溶解并且稀释成梯度溶液,对蚕豆根尖染毒6 h,分别修复培养22h和24h,观察根尖变化,统计微核率、染色体畸变率及有丝分裂指数。结果表明,4mg/L（以氧化钬质量体积浓度计）以下剂量对根尖生长具有促进作用;随着浓度的递增,微核率、染色体畸变率逐步上升,有丝分裂指数逐步下降,表现出明显的剂量-效应关系,说明稀土元素钬具有一定的细胞毒性和遗传毒性。同时,不同修复组在微核率、染色体畸变率及有丝分裂指数上也存在一定差异,表现为微核率22h修复组低于24 h 修复组,而染色体畸变率和分裂指数均高于24h修复组。微核检测应在染色体畸变检测之后进行。      

乙酸铜对蚕豆根尖细胞致畸效应

采用蚕豆根尖细胞的微核试验和染色体畸变试验方法,以不同浓度的乙酸铜为诱变剂,选择不同的处理时间,测定蚕豆根尖细胞的有丝分裂指数、微核率和染色体畸变率。结果表明：乙酸铜能诱发较高频率的微核率,处理6h、12h时微核率均随着乙酸铜浓度的升高而增加,具有明显的剂量效应;处理24h时在实验浓度范围内,其微核率随乙酸铜浓度的升高而增加,但高于一定浓度后反而呈下降趋势。不同浓度的乙酸铜在不同处理时间均使蚕豆根尖细胞有丝分裂指数增大。乙酸铜还能诱导蚕豆根尖细胞产生较高频率的染色体畸变,且产生多种类型的染色体畸变。因此,乙酸铜对蚕豆根尖细胞具有明显的致畸效应。     

提高辐射诱变育种效果及辐射遗传效应的研究：Ⅲ作物辐射遗传效应 …

试验结果证明：１．γ辐射处理可导致水稻和蚕豆芽中可溶性蛋白质与同工酶谱的变化;２．γ射线诱发蚕豆根尖细胞畸变率、断片率、微核率、染色体畸变率与辐照剂量的关系均符合模式Ｙ＝ａ＋ｂｘ＋ｃｘ＾２,呈抛物线变化趋势;３．微核率、断片率与核畸变率、染色体畸变率呈正相关。从而作者认为微核率、断片率可以作为检测染色体辐射效应的可靠指标;４．γ射线照射可引起蚕豆根尖细胞染色体带型的变化。     

四硼酸钠诱导蚕豆根尖细胞微核及染色体畸变的实验研究

本文以蚕豆为实验材料,用不同浓度的四硼酸钠处理蚕豆根尖。通过对蚕豆根尖细胞镜检观察结果表明:四硼酸钠可诱导微核的形成及染色体畸变的产生,实验组与对照组差异极为显著,即细胞微核率随处理时间的延长有所增加,50与200ppm浓度诱导细胞微核率没有明显差异。此外,本文对微核的形成与染色体畸变的相关性进行了讨论。     

DNA adducts, strand breaks and micronuclei in mice exposed to styrene by inhalation

Genotoxic and clastogenic effects of styrene were studied in mice. Male NMRI mice were exposed by inhalation to styrene in concentrations of 750 and 1500 mg/m3 for 21, 7, 3 and 1 days (6 h/day, 7 days/week). Followed parameters included styrene in blood, specific styrene oxide (SO) induced DNA adducts, DNA strand breaks and micronuclei. The formation of SO induced 7-SO-guanines and 1-SO-adenines in DNA was analysed from lung tissues by two versions of the 32P-postlabeling technique. In lungs after 21 days of exposure to 1500 mg/m3 the level of 7-SO-guanine was 23.0+/-11.9 adducts/10(8) normal nucleotides, while 1-SO-adenine was detected at the levels of 0.6+/-0.2 adducts/10(8) normal nucleotides. Both 7-SO-guanines and 1-SO-adenines strongly correlated with exposure parameters, particularly with styrene concentration in blood (r=0.875, P=0.0002 and r=0.793, P=0.002, respectively). DNA breaks were measured in peripheral lymphocytes, bone marrow cells and liver cells using comet assay. To discern oxidative damage and abasic sites, endonuclease III was used. In bone marrow of exposed mice slight increase of strand breaks can be detected after 7 days of inhalation. A significant increase was revealed in the endonuclease III-sensitive sites after 21 days of inhalation in bone marrow. In the liver cells inhalation exposure to both concentrations of styrene did not virtually affect either levels of DNA single-strand breaks or endonuclease III-sensitive sites. The inhalation of 1500 mg/m3 of styrene induced significant increase of micronuclei after 7 days of exposure (10.4+/-2.5/1000 cells, i.e. twice higher micronuclei frequency than in controls). After 21 days of inhalation no significant difference between the control group and the two exposed groups was observed. Whether the decrease of micronuclei after 21 days of inhalation was due to the inhibition of cell proliferation caused by styrene or due to the natural elimination of chromatide fragments, remains to be clarified. An interesting link has been found between DNA single-strand breaks in bone marrow and frequencies of micronuclei (r=0.721, P=0.028).     

Chromothripsis in Hepatocarcinogenesis: The Role of a Micronuclear Aberration and Polyploidy

In this study, we used the mouse model of chemically induced hepatocarcinogenesis to investigate the chromosomal aberrations in hepatic cells. The model was obtained by combined treatment of mice with Dipin (radiomimetic drug) followed by partial hepatectomy. Cytological analysis of isolated liver cells treated with Dipin has demonstrated a number of hepatocytes with structural nuclear abnormalities and multiple micronuclei. Karyotype analysis of polyploid hepatocytes has shown numerous chromosomal aberrations including alleged morphological manifestations of chromothripsis, a special type of genomic reorganization characterized by the local disintegration of chromosomes. Micronuclei with chromosomal fragments have developed as a result of double-strand DNA breaks and might serve as the initial substrate for chromothripsis. The emergence of micronuclei containing chromosomal fragments is the most important result of the treatment employed. Therefore, the presented model of liver cancer (hepatocarcinogenesis) can be used to study the process of chromothripsis in the future.     

Protection of cellular DNA from γ-radiation-induced damages and enhancement in DNA repair by troxerutin

The effect of troxerutin on γ-radiation-induced DNA strand breaks in different tissues of mice in vivo and formations of the micronuclei were studied in human peripheral blood lymphocytes ex vivo and mice blood reticulocytes in vivo. Treatments with 1 mM troxerutin significantly inhibited the micronuclei induction in the human lymphocytes. Troxerutin protected the human peripheral blood leucocytes from radiation-induced DNA strand breaks in a concentration dependent manner under ex vivo condition of irradiation (2 Gy). Intraperitoneal administration of troxerutin (175 mg/kg body weight) to mice before and after whole body radiation exposure inhibited micronuclei formation in blood reticulocytes significantly. The administration of different doses (75, 125 and 175 mg/kg body weight) of troxerutin 1 h prior to 4 Gy γ-radiation exposure showed dose-dependent decrease in the yield of DNA strand breaks in murine blood leucocytes and bone marrow cells. The dose-dependent protection was more pronounced in bone marrow cells than in blood leucocytes. Administration of 175 mg/kg body weight of the drug (i.p.) 1 h prior or immediately after whole body irradiation of mice showed that the decrease in strand breaks depended on the post-irradiation interval at which the analysis was done. The observed time-dependent decrease in the DNA strand breaks could be attributed to enhanced DNA repair in troxerutin administered animals. Thus in addition to anti-erythrocytic, anti-thrombic, fibrinolytic and oedema-protective rheological activity, troxerutin offers protection against γ-radiation-induced micronuclei formation and DNA strand breaks and enhances repair of radiation-induced DNA strand breaks. (Mol Cell Biochem xxx: 57–68, 2005)     

Kinetochore identification in micronuclei in mouse bone-marrow erythrocytes: an assay for the detection of aneuploidy-inducing agents

An in vivo micronucleus assay using mouse bone marrow for identifying the ability of chemicals to induce aneuploidy and/or chromosome breaks is described. Micronucleus formation in bone-marrow erythrocytes of mice is commonly used as an index for evaluating the clastogenicity of environmental agents. However, micronuclei may also originate from intact lagging chromosomes resulting from the effect of aneuploidy-inducing agents. We have used immunofluorescent staining using anti-kinetochore antibodies to classify micronuclei for the presence or absence of kinetochores. Micronuclei positive for kinetochores are assumed to contain intact chromosomes and result from induced aneuploidy; while those negative for kinetochores contain acentric chromosomal fragments and originate from clastogenic events. The assay was evaluated using X-irradiation (a known clastogen) and vincristine sulfate (an aneuploidy-inducing agent). A dose-related response for the induction of micronuclei was observed for both agents. Micronuclei induced by X-irradiation were negative for kinetochores while the majority of the micronuclei resulting from vincristine treatment contained kinetochores. Thus, the micronucleus assay in combination with immunofluorescent staining for kinetochores may provide a useful method to simultaneously assess the ability of chemicals to induce aneuploidy and/or chromosome breaks.     

Enhancement of radiation-induced DNA double-strand breaks and micronuclei in human colon carcinoma cells by N-methylformamide

The differentiation-inducing agent N-methylformamide (NMF) enhances the sensitivity of some cell lines to ionizing radiation. To elucidate the mechanism of NMF-mediated radiosensitization, we examined the effects of this agent on gamma-ray-induced DNA double-strand breaks and micronuclei in two cell lines, clone A (human colon carcinoma) and HCA-1 (murine hepatocarcinoma). Both cell lines form a better differentiated phenotype upon exposure to NMF, yet only clone A is radiosensitized. The neutral (pH 9.6) elution assay was used to evaluate the effects of this maturational agent on radiation-induced double-strand breaks in these cell lines. Exposure of HCA-1 cells to NMF had no effect on the level of DNA double-strand breaks induced by gamma rays. In clone A cells, however, exposure to NMF enhanced the initial formation of gamma-ray-induced double-strand breaks at each dose tested. The repair of double-strand breaks in both cell lines was not influenced by NMF. As a measure of chromosome fragmentation after irradiation, we evaluated micronuclei using the cytokinesis block method. Exposure to NMF had no effect on radiation-induced micronuclei formation in HCA-1 cells yet significantly enhanced the frequency of micronuclei induced by radiation in clone A cells. In clone A cells, the increases in radiation-induced double-strand breaks and micronuclei as a function of NMF exposure time reached maximums by approximately 72 h. These data suggest that NMF-mediated radiosensitization is the result of an increase in the initial level of radiation-induced DNA double-strand breaks.     

Genotoxic effects of butadiene in mouse lung cells detected by an ex vivo micronucleus test

Lung fibroblasts from BD-exposed mice have been analysed for the occurrence of micronuclei. Primary cultures set up 24h after the end of exposure were treated with cytochalasin B and micronuclei scored in binucleate cells. A three-fold statistically significant increase of micronucleated cells was detected after exposure to 500ppm, the lowest tested concentration. A linear dose effect relationship was observed between 500 and 1300ppm. Immunofluorescent staining of kinetochore proteins was applied to distinguish between acentric micronuclei produced by chromosome breaks and micronuclei containing a centromeric region, most likely induced by chromosome loss. A statistically significant increase of both types of MN in 1300ppm-exposed females and a significant increase in centromeric MN in 500ppm-exposed males were detected. These data demonstrate that an intermediate of BD metabolism with a potential for clastogenic and aneugenic effects is active in lung cells after inhalation exposure. These effects can play a role in the initiation and promotion of BD-induced lung tumours.     

Transmission of radiation-induced acentric chromosomal fragments to micronuclei in normal human fibroblasts

A simplifying assumption made when calculating the probability of a chromosomal aberration resulting in a micronucleus is that virtually all radiation-induced micronuclei result from acentric fragments. In the present study we used antibodies to chromosomal centromeres (kinetochores) to determine the frequency of centric versus acentric micronuclei in normal human fibroblasts exposed to 6 Gy of 60Co gamma rays while they were in density-inhibited growth. Up to 14% of the micronuclei induced by this exposure contained one or more kinetochores; i.e., they were not composed of acentric chromatin. By deleting kinetochore-positive micronuclei from the analysis, and by reconstructing micronucleus frequencies based on the fraction of cells that had divided following radiation exposure, a direct comparison between micronuclei and acentric chromosome fragments was made. On that basis, the probability of an acentric fragment becoming a visible micronucleus in either daughter cell of a dividing pair was estimated to be about 0.6. The distribution of acentric fragments among mitotic cells conformed to Poisson expectation, while the distribution of micronuclei among daughter cells was significantly overdispersed. The phenomenon of overdispersion is discussed in connection with proposed cellular processes that effect a nonrandom segregation of acentric fragments.     

Phosphorylation of histone H2AX in radiation-induced micronuclei

DNA double-strand breaks are thought to precede the formation of most radiation-induced micronuclei. Phosphorylation of the histone H2AX is an early indicator of DNA double-strand breaks. Here we studied the phosphorylation status of the histone H2AX in micronuclei after exposure of cultured cells to ionizing radiation or treatment with colchicine. In human astrocytoma SF268 cells, after exposure to gamma radiation, the proportion of gamma-H2AX-positive to gamma-H2AX-negative micronuclei increases. The majority of the gamma-H2AX-positive micronuclei are centromere-negative. The number of gamma-H2AX-positive micronuclei continues to increase even 24 h postirradiation when most gamma-H2AX foci in the main nucleus have disappeared. In contrast, in normal human fibroblasts (BJ), the proportion of gamma-H2AX-positive to gamma-H2AX-negative micronuclei remains constant, and the majority of the centromere-negative cells are gamma-H2AX-negative. Treatment of both cell lines with colchicine results in mostly centromere-positive, gamma-H2AX-negative micronuclei. Immunostaining revealed co-localization of MDC1 and ATM with gamma-H2AX foci in both main nuclei and micronuclei; however, other repair proteins, such as Rad50, 53BP1 and Rad17, that co-localized with gamma-H2AX foci in the main nuclei were not found in the micronuclei. Combination of the micronucleus assay with gamma-H2AX immunostaining provides new insights into the mechanisms of the formation and fate of micronuclei.     

Clastogenic effect of the human T-cell leukemia virus type I Tax oncoprotein correlates with unstabilized DNA breaks

Expression of the human T-cell leukemia virus type I (HTLV-I) Tax oncoprotein rapidly engenders DNA damage as reflected in a significant increase of micronuclei (MN) in cells. To understand better this phenomenon, we have investigated the DNA content of MN induced by Tax. Using an approach that we termed FISHI, fluorescent in situ hybridization and incorporation, we attempted to characterize MN with centric or acentric DNA fragments for the presence or absence of free 3'-OH ends. Free 3'-OH ends were defined as those ends accessible to in situ addition of digoxigenin-dUTP using terminal deoxynucleotidyl transferase. MN were also assessed for centromeric sequences using standard fluorescent in situ hybridization (FISH). Combining these results, we determined that Tax oncoprotein increased the frequency of MN containing centric DNA with free 3'-OH and decreased the frequency of MN containing DNA fragments that had incorporation-inaccessible 3'-ends. Recently, it has been suggested that intracellular DNA breaks without detectable 3'-OH ends are stabilized by the protective addition of telomeric caps, while breaks with freely detectable 3'-OH are uncapped and are labile to degradation, incomplete replication, and loss during cell division. Accordingly, based on increased detection of free 3'-OH-containing DNA fragments, we concluded that HTLV-I Tax interferes with protective cellular mechanism(s) used normally for stabilizing DNA breaks.     

Kinetics of micronucleus formation in relation to chromosomal aberrations in mouse bone marrow

A simulation analysis of the kinetics of micronucleus formation in polychromatic erythrocytes in mouse bone marrow was performed after a single administration of 3 chemicals--mitomycin C (MMC), 6-mercaptopurine (6-MP) and 1-beta-D-arabinofuranosylcytosine (Ara-C)--with different modes of action. The time-response patterns in the incidence of chromosomal aberrations and micronuclei after treatment with each chemical were compared and subjected to the simulation study with 3 parameters. Two of them, the time between the final mitotic metaphase of the erythroid series and nucleus expulsion (T1), and the duration of the polychromatic erythrocyte (PCE) stage in the bone marrow (T2), were almost identical for the 3 chemicals. However, the coefficients of formation rate of micronucleated cells resulting from cells with chromosomal aberration(s) (k) differed: Ara-C differed from the other two. These results indicate that chromosomal aberrations, especially chromatid breaks and probably gaps, induced by this chemical, effectively contribute to micronucleus formation. The DNA content of micronuclei was also compared to the length of acentric fragments induced by Ara-C and it was found that their distributions were comparable. These findings strongly suggest that chromosomal aberrations induced by chemicals are essential events for the induction of micronuclei in the PCE of bone marrow.