环境污染物对着床前小鼠胚胎的DNA甲基转移酶活性的影响

目的探讨环境污染物对着床前小鼠胚胎发育的直接影响,以及对基因组DNA甲基化的影响。方法着床前小鼠1细胞期胚被放入含有不同的环境污染物的培养液中进行体外培养;观察1细胞期胚发育至胚泡期胚的发育率;测定胚泡期胚的DNA甲基转移酶(DNA methyltransferase)活性。结果小鼠胚胎着床前期在含有环境污染物的培养液里发育的过程中,其形态没有发生显著的异常变化,各实验组的胚胎发育率在61%～67%。然而,DNA甲基转移酶活性应环境污染物种类而异发生不同变化。与对照组相比,二英2,3,7,8tetrachlorodibenzopdioxin(TCDD)使着床前胚的DNA甲基转移酶活性显著升高;而二乙烯二苯乙烯雌酚Diethylstilbestrol(DES)和多氯联苯中的2,2′,3,3′4,4′polychlorinatedbiphenyl(PCB153)使着床前胚的DNA甲基转移酶活性显著下降。不过,二氯联苯二氯乙烯p,p′dichlorodiphenelethylene(DDE)和苯二甲酸二丁酯dibutylphthalate(DBP)对着床前胚的甲基转移酶活性的影响未达到统计显著水平。结论环境污染物可在体外培养中对着床前胚DNA甲基转移酶活性产生作用,进而可能影响基因组甲基化模式的变化。     

Participation of glucose in the synthesis of glycoproteins in preimplantation mouse embryos

Electrophoretic separation of solubilized embryos incubated for 24 h in the presence of [U-14C]glucose indicated incorporation of glucose carbon into a number of protein bands. Treatment of nitrocellulose blots of electrophoretograms with glucosidases had no effect on incorporated counts, confirming that the labelled bands were not due to protein bound glycogen. Furthermore, addition of 0.1 microgram mL-1 tunicamycin to the incubation medium virtually eliminated incorporation of glucose into the protein bands but had no effect on the pattern or rate of incorporation of labelled amino acids in parallel experiments. Also the pattern of labelling of protein by glucose was reflected in the pattern of binding of Con A to the nitrocellulose blots. There were quantitative and qualitative changes in labelling as development progressed. For embryos cultured from the 2-cell stage, a small amount of label was incorporated in two major bands at relative mobility (Mr) 69 and 97 K. With culture from the 8-cell stage, three additional major bands (33, 44 and 56 K) were labelled. Embryos cultured from the morula stage showed a different profile of incorporation; there was much more active labelling, and eight major and a number of minor radioactive bands were identified. Whilst tunicamycin suppressed glucose incorporation into glycoproteins and inhibited compaction of embryos, it had little effect on other parameters of metabolism during incubation in its presence for 24 h. No significant effects of the metabolite on protein synthesis, glycogen storage, lactate production or overall macromolecular synthesis were evident. By contrast, the anabolic metabolism of embryos decompacted by long periods of exposure to tunicamycin was severely reduced although glycolysis was still unaffected. Amphomycin at very high concentration (500 micrograms mL-1) was toxic to embryos but at concentrations up to 250 micrograms mL-1 had no effect on compaction and development of blastocysts. Addition of monensin to the incubation medium [16 micrograms mL-1] did not interfere with the development of either 2-cell or 8-cell embryos to blastocysts.     

尼古丁对L-929细胞毒性作用

目的 评价尼古丁对L-929细胞毒性及作用机制。方法 采用(MTT)法检测尼古丁对L-929细胞增殖的影响,采用流式细胞术观察尼古丁对L-929细胞周期的影响。结果 不同浓度尼古丁作用于L-929细胞24,48和72 h后,均能抑制L-929细胞生长。0.01,0.1μg/mL尼古丁作用24,48 h细胞毒性为1级,作用72 h细胞毒性则为2级。10 000μg/mL尼古丁作用24 h细胞毒性为3级,作用48,72 h细胞毒性则为4级。随着尼古丁浓度增加,G₀G₁期细胞比例逐渐增高,而S期和G2期细胞比例则呈下降趋势。结论 尼古丁对体外培养L-929细胞具有明显细胞毒性,且阻遏细胞周期。     

Obtention of a photo-induced adduct between a vitamin and an essential aminoacid. Binding of riboflavin to tryptophan

Studies in animals have suggested that the products of the irradiation of tryptophan in the presence of riboflavin may play a role in the development of hepatic dysfunction during parenteral nutrition. In this paper we describe the formation of an adduct between tryptophan and riboflavin obtained as a consequence of an anaerobic irradiation of these compounds. Through the use of molecular sieves and of an ion-exchange resin it was possible to separate the photo-adduct from the dimer riboflavin and other reaction products. The various fractions were characterized on the basis of their absorption and emission spectra. Also used were measures of anisotropy of fluorescence emission in order to characterize the derived adduct.     

微囊藻毒素-LR和二甲苯对斑马鱼胚胎的毒性作用

目的 研究二甲苯和微囊藻毒素-LR(MC-LR)对斑马鱼胚胎的毒性作用.方法 将斑马鱼胚胎分为对照(培养液)组和MC-LR单独染毒(25～1 000 μg/L)组及二甲苯单独染毒(2.5～40 mg/L)组以及MC-LR(25～400 μg/L)+二甲苯(2.5～40 mg/L)联合染毒组,每组36个.测定斑马鱼胚胎的死亡情况、孵化情况以及畸形情况,并测定斑马鱼幼鱼平均速度.结果 当MC-LR和二甲苯的浓度分别达到100 μg/L和5 mg/L时,会对胚胎造成致死作用.随着二甲苯和(或)MC-LR染毒浓度的升高,斑马鱼胚胎的孵化率呈下降趋势,死亡率和畸形率均呈升高趋势,而幼鱼的运动速度减慢.结论 二甲苯和MC-LR对斑马鱼胚胎具有致死、致畸作用,并影响斑马鱼的发育.     

The effect of extracellular matrix molecules on mouse preimplantation embryo development in vitro

The extracellular matrix (ECM) molecules, laminin (LN), chondroitin sulfate (CS), fibronectin (FN), hyaluronic acid (HA), mucin (MUC) and heparan sulfate proteoglycan (HS), were investigated as supplements to culture medium to improve the in vitro development of mouse 1-cell zygotes to blastocysts. Development was also compared with that in medium supplemented with bovine serum albumin (BSA) to determine the potential for ECM molecules as suitable alternatives to serum albumin in culture medium. Supplementation of sequential culture media with LN at all concentrations examined failed to result in more than 70% of zygotes developing to blastocysts; therefore, LN was considered unsuitable as a replacement for BSA and was not examined further. The optimal concentration of the remaining ECM molecules was used to supplement sequential culture media and the effect on blastocyst quality was assessed by determining the differential cell numbers of blastocysts grown in BSA-supplemented medium. Development to blastocyst was similar, regardless of the macromolecule used. The number of inner cell mass cells was significantly higher in HS-supplemented medium compared with controls. Trophectoderm cell numbers were similar to control values for all ECM molecules examined except CS for which there were fewer trophectoderm cells. It is concluded that ECM molecules, FN, HA, MUC and HS may be used as substitutes for serum protein supplementation of culture media EG0/G2 for mouse preimplantation embryo development. Heparan sulfate proteoglycan increases inner cell mass numbers and this may be due to interactions with the growth factors fibroblast growth factor 4 (FGF-4) and granulocyte-macrophage colony-stimulating factor.     

丙烯酰胺对人神经母细胞瘤NB-1细胞的毒性

分析丙烯酰胺(ACR)对人神经母细胞瘤NB-1细胞毒作用的剂量-效应和时间-效应关系,进一步探讨丙烯酰胺的神经毒性机制。将NB-1细胞诱导成熟后,设置正常对照及染毒浓度组、时间组,采用MTT法和LDH法检测各组细胞存活和生长情况,分析ACR对NB-1细胞的毒性作用。MTT结果表明,>60μg/ml的ACR对NB-1细胞开始表现出明显的抑制作用,浓度越大,抑制作用越强。LDH结果表明,24 h、72 h时间点,ACR浓度>60μg/ml,LDH释放率明显增高;而48 h则在ACR浓度>40μg/ml,LDH释放率明显增高,且均随浓度的加大,LDH释放率逐渐升高。时间上,LDH结果与MTT一致,均以48 h表现最为显著。提示在一定浓度范围内,ACR显示出对NB-1细胞的毒性作用。     

Angiopoietin-1 and -2 mRNA and protein expression in mouse preimplantation embryos and uteri suggests a role in angiogenesis during implantation

After attachment and migration through the endometrial epithelium, the embryo must induce angiogenesis within the endometrial stroma to successfully complete the implantation process. Growth factors have been shown to play an important role in embryo implantation and placentation. The aim of the study was to investigate the expression of angiopoietin-1 and -2 (Ang-1 and -2) mRNA and protein expression during the development of single preimplantation mouse embryos and of possible complementary expression in mouse uteri. Angiopoietin-1 mRNA was expressed throughout development in 78% of zygotes, 66% of 2-cell-embryos, 71% of 4-cell-embryos, 70% of 8-cell-embryos, 60% of morula stages, 48% of early blastocysts and 78% of late blastocysts. The number of Ang-1-expressing embryos in the early-blastocyst group was significantly different in comparison with zygotes, 4-cell-embryos, 8-cell-embryos and late blastocysts. Angiopoietin-2 mRNA and protein expression could not be detected in preimplantation embryos. Examination of the uteri revealed Ang-2 mRNA and protein expression in the oestrogen-dominated cycling phase and the progesterone-dominated mated phase, whereas Ang-1 expression was restricted to the mated phase. Herein, Ang-1 expression in preimplantation mouse embryos as well as Ang-1 and -2 expression in mouse uteri is demonstrated, suggesting a possible role for angiopoietins in the embryo-maternal dialogue of the implantation process via an enhancement of the vascular remodelling in favour of an implanting conceptus.     

镍对大鼠生殖细胞毒性及一氧化氮合酶的影响

目的 探讨镍对雌性大鼠生殖细胞的毒作用及其机制。方法 健康性成熟Wistar雌性大鼠腹腔注射硫酸镍(NiSO4)1．25，2．50，5．00mg／kg染毒，1次／d．连续21d。染毒结束次日进行超数排卵和卵细胞体外培养，观察排卵数和存活情况．同时酶法测卵巢一氧化氮合酶(NOS)活力和一氧化氮(NO)含量。结果 染毒组动物超排卵总数和卵母细胞体外培养过程的各观察时相卵母细胞存活数均减少。卵巢NOS酶活力和NO量升高。结论 卵巢NOS活性增加、NO水平升高可能是致卵巢功能损害，卵细胞质量下降的机制之一。     

PCB153对INS-1细胞毒性作用及机制

目的探讨2,2',4,4',5,5'-六氯联苯(PCB153)对体外培养胰岛β细胞株(INS-1)细胞毒性作用及机制。方法PCB153设3个剂量组(1、3、6 μmol/L),二甲基亚砜(DMSO)为溶剂对照组,染毒INS-1细胞24 h后,检测细胞存活率、凋亡、活性氧(ROS)水平、Caspase 3 和Caspase 12等凋亡相关基因表达水平。结果3、6 μmol/L PCB153剂量组细胞存活率分别为(80.9±8.7)% 和(42.2±4.3)%,与对照组比较均有下降(P<0.05);3 μmol/L PCB153剂量组ROS为(9.2±0.4)、凋亡率为(30.7±3.4)%,6 μmol/L PCB153剂量组ROS为(13.7±1.6)、凋亡率为(40.4±1.3)%,与对照组比较,ROS水平和细胞凋亡率均增加(P<0.05);与对照组比较,3 μmol/L PCB153剂量组Caspase 3及3、6 μmol/L PCB153剂量组Caspase 12基因表达水平均有增加(P<0.05)。结论PCB153可诱导INS-1细胞凋亡,氧化应激和内质网信号通路可能参与PCB153对INS-1细胞的毒性作用。     

The effect of RU486 on transport, development and implantation of mouse embryos

Pregnancy was interrupted in Swiss-Rockefeller mice by a single subcutaneous injection of the antiprogesterone RU486 given postcoitally. A dose of 0.1 mg/animal injected on day 1, produced partial inhibition of pregnancy, a notable delay in embryonic development and a slight retention of embryos in the oviducts. When the same dose was injected on day 2, 3 or 4, no implantation sites were seen at autopsy performed on day 12 of pregnancy. Treatment with 0.5 or 1 mg/animal on day 1 produced the loss of a large proportion of the embryos from the genital tract by day 4 of pregnancy and suppressed implantation completely. These observations indicate that preimplantation phenomena in mice are highly dependent on progesterone action.     

In vivo gene transfer in mouse preimplantation embryos after intraoviductal injection of plasmid DNA and subsequent in vivo electroporation

The aim of this study was to investigate whether direct injection of nonviral DNA into the oviductal lumen and subsequent in vivo electroporation leads to in vivo gene transfer in mouse preimplantation embryos present within an oviduct, as an alternative to the pre-existing pronuclear microinjection-based transgenesis. With this technique, effects of expression of the gene of interest (GOI) on mouse preimplantation development can be monitored with relative ease. Superovulated 4-week-old B6C3F1 female mice (hybrids between C57BL/6N and C3H/HeN) were mated with adult B6C3F1 male mice. Two days later, females that had been identified as pregnant, based on the presence of copulation plugs, were injected with 1?µl of a solution containing an enhanced green fluorescent protein (EGFP) expression plasmid (0.5?µg) and 0.05% trypan blue. The entire oviduct was then electroporated using tweezer-type electrodes with 8 square-wave pulses of 50 V each with 50-ms duration. The next day, the 8-cell stage embryos were collected, and their number, morphology, and EGFP-derived fluorescence were recorded. Of the 12 oviducts (6 females used) examined, 3 contained fluorescent 8-cell stage embryos (33%, 19/58 tested), but the intensity of fluorescence varied among the embryos. In total, 10% (19/192 tested) of the embryos were fluorescent and the fluorescence was maintained in these embryos after 1 day of culture. However, the fluorescence disappeared in the late gestational stage fetuses, and the transgenes could not be detected. Our results indicate that it is possible to transfect in vivo preimplantation embryos, although the success rate appears to be relatively low and gene expression is transient. This technology may provide a new method for manipulating preimplantation embryos in vivo, by using, for example, Cre-mediated conditional DNA recombination.     

人蛔虫体腔液对人肠上皮细胞的毒性作用

目的探讨人蛔虫体腔液（Ascaris body fluid，ABF）对人肠上皮细胞株（HCT-8）的毒性作用及作用浓度和时间的关系。方法以体外细胞培养方法观察ABF对HCT-8细胞的毒性作用．比较不同浓度ABF作用下不同时间HCT-8细胞的死亡率和形态结构。结果HE染色显示，当ABF浓度为800μg／ml时，HCT-8细胞的毒性作用最大，除浓度为12．5μm/ml的实验组，各实验组（25～800μg／ml）的毒性均高于对照组。结论ABF对HCT-8细胞所产生毒性作用在一定程度上表现出浓度和作用时间的依赖关系，并诱导细胞凋亡的发生。     

In vivo gene transfer in mouse preimplantation embryos after intraoviductal injection of plasmid DNA and subsequent in vivo electroporation

The aim of this study was to investigate whether direct injection of nonviral DNA into the oviductal lumen and subsequent in vivo electroporation leads to in vivo gene transfer in mouse preimplantation embryos present within an oviduct, as an alternative to the pre-existing pronuclear microinjection-based transgenesis. With this technique, effects of expression of the gene of interest (GOI) on mouse preimplantation development can be monitored with relative ease. Superovulated 4-week-old B6C3F1 female mice (hybrids between C57BL/6N and C3H/HeN) were mated with adult B6C3F1 male mice. Two days later, females that had been identified as pregnant, based on the presence of copulation plugs, were injected with 1?μl of a solution containing an enhanced green fluorescent protein (EGFP) expression plasmid (0.5?μg) and 0.05% trypan blue. The entire oviduct was then electroporated using tweezer-type electrodes with 8 square-wave pulses of 50 V each with 50-ms duration. The next day, the 8-cell stage embryos were collected, and their number, morphology, and EGFP-derived fluorescence were recorded. Of the 12 oviducts (6 females used) examined, 3 contained fluorescent 8-cell stage embryos (33%, 19/58 tested), but the intensity of fluorescence varied among the embryos. In total, 10% (19/192 tested) of the embryos were fluorescent and the fluorescence was maintained in these embryos after 1 day of culture. However, the fluorescence disappeared in the late gestational stage fetuses, and the transgenes could not be detected. Our results indicate that it is possible to transfect in vivo preimplantation embryos, although the success rate appears to be relatively low and gene expression is transient. This technology may provide a new method for manipulating preimplantation embryos in vivo, by using, for example, Cre-mediated conditional DNA recombination.     

Comparison of 4', 6'-diamidino-2-phenylindole and Giemsa stainings in preimplantation mouse embryos micronucleus assay including a triple dose study

Analysis of micronuclei (MN) in preimplantation embryos is a good method for the evaluation of cytogenetic damage induced by occupational and environmental mutagen during early pregnancy. To examine whether conventional Giemsa staining produced the same accuracy of micronuclei as the DNA-specific 4', 6'-diamidino-2-phenylindole (DAPI) staining in preimplantation embryo induced by maternal exposure to chlorpyrifos, we conducted assays on 469 mouse (3 groups) preimplantation embryos micronucleus. Slides were stained with DAPI. After DAPI staining, the slides were de-stained and restained with Giemsa. Giemsa staining showed similar frequencies in MN to DNA-specific DAPI staining in all three groups. Both staining techniques revealed significant increases in frequency of MN in the treated group in comparison to the control group. Both methods showed a statistically significant correlation between MN frequency and the dose of chlorpyrifos. Compared with DAPI staining, the sensitivity of Giemsa staining was 85.0%, 86.0% and 90.9% for control, 40 mg/kg, and 80 mg/kg chlorpyrifos treated group, respectively. The specificity was 97.9%, 91.4% and 96.5% for control, 40 mg/kg, and 80 mg/kg chlorpyrifos treated group, respectively. Thus, we recommend that Giemsa staining technique be a standard staining method in detecting MN of preimplantation embryos induced by occupational or environmental hazards.     

纳米二氧化硅和纳米氧化锌颗粒对人肝癌细胞的毒性作用

目的探讨纳米二氧化硅和纳米ZnO颗粒对人肝癌(HepG2)细胞毒性的影响。方法将处于对数生长期的细胞分别暴露于含终浓度分别为0(对照)~200μg/ml纳米二氧化硅、纳米ZnO颗粒的无血清培养基中培养24 h。检测细胞活性及细胞膜的完整性。结果与对照组比较,各浓度纳米二氧化硅及25~200μg/ml纳米ZnO染毒组HepG2细胞的存活率均较低,差异有统计学意义(P0.05);且随着染毒浓度的升高,纳米二氧化硅和纳米ZnO染毒HepG2细胞的存活率均呈下降趋势。与相同浓度纳米二氧化硅相比,50~200μg/ml纳米ZnO染毒组HepG2细胞的存活率均较高,差异有统计学意义(P0.05)。与对照组比较,各浓度纳米二氧化硅及纳米ZnO染毒组HepG2细胞上清液中LDH的活力均较高,差异有统计学意义(P0.05);且随着纳米二氧化硅和纳米ZnO染毒浓度的升高,HepG2细胞上清液中LDH的活力均呈上升趋势。与相同浓度纳米二氧化硅相比,各浓度纳米ZnO染毒组HepG2细胞上清液中LDH的活力均较低,差异有统计学意义(P0.05)。结论两种纳米颗粒均可对HepG2细胞产生毒性作用,且纳米二氧化硅比纳米ZnO颗粒的细胞毒性作用更强。     

In vitro effects of cadmium chloride on preimplantation rat embryos

Preimplantation rat embryos (8-cell, morulae, blastocysts) were incubated for 24 hr in vitro in a Brinster medium with or without cadmium chloride (1 microgram/ml). CdCl2 arrested the development of both the 8-cell and morulae into blastocysts. Morphologic observations revealed the 8-cell embryo to have shrunken cells and pyknotic nuclei. Morulae appeared smaller in size with fewer cells than comparable controls. CdCl2 was clearly cytotoxic to the early blastocysts, inner mass cells were most damaged with cell death, pyknosis, and accumulation of debris.     

Toxic effects of nicotinamide methylation on mouse brain striatum neuronal cells and its relation to manganese

Objective

It is well known that manganese (Mn) exposure is involved in parkinsonism. The aim of our study was to test the hypotheses that Mn affects nicotinamide N-methyltransferase (NNMT) activity, increases the metabolism of nicotinamide (NA) to 1-methylnicotinamide (MNA), and leads to neurocytotoxicity.

Methods

Following demonstration of the effects of Mn concentrations on the survival rate of Mouse CD1 brain striatum neuronal cells (MS cells), the effect of Mn on NNMT activity was investigated by comparing the difference in the amount of MNA produced after various Mn concentrations were added to mouse brain cytosol fractions as an enzyme solution. Toxicity induced by MNA and its precursor NA on MS cells was measured.

Results

The survival rate of MS cells decreased significantly with increasing concentrations of Mn in the culture medium. With respect to the influence of Mn on NNMT activity, NNMT activity increased significantly at Mn concentrations of 1 μmol/mg protein. MNA and NA neurotoxicity were compared by comparing cell survival rate. Cell survival rate dropped significantly when the cells were cultivated with 10 mM of MNA. There was also a tendency for the survival rate to fall following the addition of 10 mM NA; however, the difference with the control was not significant.

Conclusions

Our study suggests the possibility that Mn causes increased NNMT activity, thereby increasing MNA levels in the brain and bringing about neuron death. Daily absorption of Mn and NA may thus contribute to idiopathic Parkinson’s disease.     

A simple rapid 4.5 M dimethyl-sulfoxide freezing technique for the cryopreservation of one-cell to blastocyst stage preimplantation mouse embryos.

This study applies a 4.5 M dimethyl-sulfoxide freezing procedure, developed for 2-cell mouse embryos, to pronuclear to hatched blastocyst stage mouse embryos. The embryos were plunged into liquid nitrogen after 3 min equilibration at room temperature, or 3-60 min equilibration at 0 degrees C. Equilibration at 0 degrees C gave survival rates as high as or higher than rates after equilibration at room temperature. Optimal blastocyst formation, or re-expansion, rates for embryos frozen after equilibration at 0 degrees C were 76% for pronuclear stage embryos and 96-100% for 2-cell to mid-blastocyst stage embryos. The optimal rates of fetus formation, per embryo frozen, ranged from 62 to 88% for pronuclear to mid-blastocyst stage embryos. These results compared favourably with non-frozen control embryos (80-100% blastocyst formation, and 67-78% fetus formation).