高分子避孕复合材料在哺乳动物体内外遗传毒性检测

发布时间：2018-09-18 18:02

【摘要】：目的检测高分子避孕复合材料和金属铜在体外对哺乳动物细胞致遗传毒性的作用，评价该材料的安全性。方法采用小鼠淋巴瘤实验（Mouse Lymphoma Assay, MLA）和彗星实验即单细胞凝胶电泳实验（SCGE）检测各材料组对tk基因致突变能力和导致单个细胞DNA损伤程度。各材料RMPI-1640浸提液经火焰原子吸收分光光度法测定铜离子浓度，预实验后选择金属铜组50%、25%、12.5%浸提液浓度，含铜复合材料组100%、50%、25%浸提液浓度，无铜复合材料组100%、50%、25%浸提液浓度分别测定致突变能力和DNA损伤程度，溶剂对照作为阴性对照，小鼠淋巴瘤实验中在有无S9mix时分别选择环磷酰胺和甲磺酸甲酯作为阳性对照。小鼠淋巴瘤实验通过计数处理当天平板、表达后平板和TFT拮抗平板的集落数测定PE0、PE2和MF。彗星实验以Tail DNA%和Olive尾距评价细胞DNA损伤程度。结果在含有铜离子的浸提液中，细胞均出现了不同程度的突变，突变频率较阴性对照组有明显升高，其中金属铜50%、25%和含铜复合材料组100%组可以致遗传毒性，含铜复合材料50%组为可疑致遗传毒性。彗星实验结果表明，各含铜复合材料组和金属铜组均可导致细胞DNA损伤，Tail DNA%和OTM与阴性对照组相比有显著性差异，损伤程度与浸提液中铜离子浓度有一定关系。无铜复合材料在各浓度下突变频率、Tail DNA%和OTM均无明显增高结论含铜复合材料、金属铜组由于铜离子的存在体外可以致DNA损伤和细胞突变，具有一定的体外遗传毒性。损伤程度与铜离子浓度存在剂量反应关系。目的研究高分子避孕复合材料和金属铜哺乳动物体内致遗传毒性的能力。方法KM小鼠（体重：25-30g）作为实验动物，各材料组生理盐水浸提，火焰原子吸收分光光度法测定铜离子浓度，选择金属铜组50%、25%、12.5%浸提液浓度,含铜复合材料组100%、50%、25%浸提液浓度，无铜复合材料组100%、50%、25%浸提液浓度，阳性对照选择环磷酰胺，浓度为40mg/kg体重，阴性对照为生理盐水。各组浸提液按照1.5ml/100g体重腹腔注射染毒动物，24小时后颈椎脱臼处死动物，胸骨骨髓涂片Giemsa染色计数含微核的嗜多染红细胞数量评定各材料体内遗传毒性。结果染毒24小时胸骨骨髓微核计数结果显示，无铜材料组各染毒浓度下微核数量无明显增加。含铜复合材料组100%、50%和25%组微核数目（每1000PCE）分别为16.0、14.6和13.4，金属铜组50%、25%和12.5%分别为11.1、14.1和12.1。与阴性对照组相比均有显著性差异（P0.05），但各组微核数量与染毒浓度并未表现出明显的剂量反应关系。结论含铜复合材料和金属铜组微核数目与阴性对照组相比均有显著性差异，但并未表现出明显的剂量反应关系，按照OECD474指导标准，尚不能确定此两组材料具有体内遗传毒性。无铜材料组无体内遗传毒性。目的检测体外含铜复合材料浸提液处理细胞后,去除处理物细胞DNA损伤修复情况。方法用含铜复合材料50%和25%浸提液浓度处理小鼠淋巴瘤细胞3小时，溶剂作为阴性对照。处理后取部分细胞采用彗星实验检测细胞DNA损伤程度，其余细胞在去除处理物后继续培养3小时，同样采用彗星实验测定细胞DNA损伤程度。彗星实验结果经CASP软件分析后，Tail DNA%、OTM作为DNA损伤观测指标。含铜复合材料25%浸提液处理后以及继续培养3小时后检测细胞内ROS荧光强度，Image ProPlus分析其平均荧光强度。结果细胞经两个不同含铜复合材料浸提液浓度处理3小时后，，细胞DNA受损，继续培养3小时后，彗星实验结果表明，细胞受损程度较处理后有所增加，Tail DNA%和OTM与处理后相比有显著性差异(P0.05)，但继续培养3小时后细胞间DNA损伤程度差异增大，表明受损少的细胞得到一定的修复。细胞内ROS含量在处理后和继续培养3小时平均荧光强度与阴性对照组相比有明显升高，分别为79.84、86.29和49.74。结论细胞经处理后胞内ROS含量较处理前明显增高并持续存在，继续培养后由于细胞内ROS的升高可以使细胞DNA继续受损。损伤修复不明显。目的机体对高浓度铜离子染毒造成DNA损伤修复能力检测，以及与血浆SOD含量的关系。方法用金属铜100%浸提液连续7天按1.5ml/100g体重腹腔注射染毒小鼠，分别在染毒前以及每次染毒后24小时断尾取血，全血细胞进行彗星实验检测血细胞DNA损伤程度，血浆用WST法测定超氧化物歧化酶活力。彗星实验结果经CASP软件分析后，Tail DNA%、OTM作为DNA损伤观测指标。结果金属铜100%浸提液浓度染毒连续染毒小鼠7天彗星实验结果显示，雌性小鼠于染毒后第2、3、4天DNA损伤程度最严重（Tail DNA%为分别为55.41%、51.05%和51.99%），雄性小鼠于染毒后第4天DNA损伤最严重（57.20%）。雌性第小鼠第5天DNA损伤有明显降低（18.91%），雄性小鼠第6天明显降低（30.89%）。第7天雌雄小鼠血细胞Tail DNA%分别为11.25%和10.81%。WST法测定血浆内SOD含量表明，雄性小鼠体内SOD含量于第4天和第7天显著升高，雌性小鼠于第3天达到高峰。结论连续染毒处理小鼠后，金属铜100%浸提液可以导致小鼠体内血细胞DNA明显受损。同时，体内的损伤修复机制可以使受损细胞修复并抑制铜离子进一步对细胞的损伤，SOD在损伤修复中发挥一定的作用。
[Abstract]:Objective To investigate the genotoxic effect of polymer contraceptive composites and metal copper on mammalian cells in vitro and evaluate the safety of the materials.
Methods the Mouse Lymphoma Assay (MLA) and comet assay (SCGE) were used to detect the mutagenic ability of TK gene and the degree of DNA damage in each cell group. The concentrations of 50%, 25%, 12.5% extract, 100%, 50%, 25% extract, 100%, 50%, 25% extract of copper-containing composite, 100%, 50%, 25% extract of copper-free composite, and 25% extract of copper-free composite were used to determine the mutagenicity and DNA damage degree respectively. Solvent control was used as negative control. Cyclophosphamide and methyl methylsulfonate were used as the choices of mice lymphoma experiment in the presence or absence of S9 mix. The colony number of PE0, PE2 and MF. Comet assay was used to evaluate the degree of cell DNA damage by Tail DNA% and Olive tail distance.
Results In the extract containing copper ions, the mutation frequency of the cells was significantly higher than that of the negative control group. 50%, 25% and 100% of the copper-containing composite materials could cause genetic toxicity, and 50% of the copper-containing composite materials could be suspected to cause genetic toxicity. There was a significant difference between Tail DNA% and OTM and negative control group, and the degree of damage was related to the concentration of copper ion in the extract.
Conclusion Copper-containing composites can cause DNA damage and cell mutation in vitro due to the presence of copper ions. The degree of damage is dose-dependent on the concentration of copper ions.
Objective to study the genetic toxicity of polymer contraceptive composite materials and metallic copper lactating animals.
Methods KM mice (body weight: 25-30g) were used as experimental animals. The concentration of copper ion was determined by flame atomic absorption spectrophotometry. The concentration of copper ion was 50%, 25%, 12.5% in copper group, 100%, 50%, 25% in copper composite group, 100%, 50%, 25% in non-copper composite group, 50%, 25% in copper composite group, and positive control group. Cyclophosphamide, 40 mg/kg body weight, negative control was normal saline. The animals in each group were injected intraperitoneally at the weight of 1.5 ml/100 g. The animals were sacrificed after cervical dislocation 24 hours. The number of micronucleated polychromatic erythrocytes in the sternal bone marrow smears was counted by Giemsa staining.
Results The number of micronuclei in sternal bone marrow of non-copper group did not increase significantly at different concentrations after 24 hours exposure. The number of micronuclei in 100%, 50% and 25% copper-containing composite group (16.0, 14.6 and 13.4 per 1000 PCE), 50%, 25% and 12.5% copper group (11.1, 14.1 and 12.1 respectively) were significantly higher than those in negative control group. Differences (P0.05), but the number of micronucleus in each group did not show a dose-response relationship with the concentration.
Conclusion The number of micronuclei in copper-containing composites and copper-containing metals group was significantly different from that in negative control group, but there was no significant dose-response relationship. According to OECD474 standard, it was not determined that the two materials had genotoxicity in vivo.
Objective To detect the DNA damage and repair of cells treated with copper-containing composite extract in vitro.
Methods Mouse lymphoma cells were treated with 50% and 25% copper-containing composite extract for 3 hours, and the solvent was used as negative control. Some of the treated cells were tested for DNA damage by comet assay. The remaining cells were cultured for 3 hours after removal of the treated materials. The degree of DNA damage was also determined by comet assay. Results After analysis by CASP software, Tail DNA and OTM were used as the indexes of DNA damage observation. The ROS fluorescence intensity was measured after 25% extract of copper-containing composite material was treated and cultured for 3 hours. The average fluorescence intensity was analyzed by Image ProPlus.
Results After treated with two different concentrations of copper-containing composite extracts for 3 hours, the DNA damage of the cells was observed. After continued culture for 3 hours, the comet assay showed that the damage degree of the cells was increased. There was significant difference between Tail DNA% and OTM (P 0.05), but the damage degree of DNA between the cells was different after 3 hours of continuous culture. Compared with the negative control group, the average fluorescence intensity of ROS content in the treated and cultured cells after 3 hours was significantly higher, 79.84, 86.29 and 49.74 respectively.
Conclusion The content of ROS in the treated cells was significantly higher than that before treatment and persisted. After continuous culture, the DNA of the cells could continue to be damaged due to the increase of ROS.
Objective To study the DNA damage repair ability of human body exposed to high concentration of copper ion and its relationship with plasma SOD content.
Methods The mice were injected intraperitoneally with 100% copper extract for 7 days at a weight of 1.5ml/100g. Blood samples were taken at the tail of the mice before and 24 hours after each exposure. The whole blood cells were tested for DNA damage by comet assay and the activity of superoxide dismutase (SOD) by WST in plasma. L DNA% and OTM were used as indicators of DNA damage observation.
Results The results of comet assay in mice exposed to 100% copper extract for 7 days showed that the DNA damage of female mice was the most serious (55.41%, 51.05% and 51.99% respectively) on the 2nd, 3rd and 4th day after exposure. The DNA damage of male mice was the most serious (57.20%) on the 4th day after exposure. On the 7th day, Tail DNA content in blood cells of male and female mice was 11.25% and 10.81%, respectively. The content of SOD in plasma of male mice increased significantly on the 4th and 7th days, and reached the peak on the 3rd day.
Conclusion After continuous exposure to copper, 100% extract of copper can induce DNA damage of blood cells in mice. At the same time, the mechanism of damage repair in vivo can repair damaged cells and inhibit the further damage of copper ions to cells. SOD plays a certain role in the repair of injury.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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