电子垃圾污染物对人类遗传毒性的初步研究

目的： 对长期居住在天津市某镇电子垃圾处理较集中的3个村庄的居民进行细胞遗传学研究,分析电子垃圾污染物对人体的遗传损伤效应。方法：在电子垃圾处理区居民中随机选择171位村民作为暴露组,进行染色体畸变 (CA)和胞浆阻滞微核 (CBMN)分析,并进行单细胞凝胶电泳检测DNA损伤。选取与该镇毗邻区域且从未接触电子垃圾处理的30位村民 (男女各半)作为对照组。暴露组按照性别和年龄分组,分别观察不同性别和年龄组的CA、CBMN和DNA损伤水平。结果：暴露组染色体总畸变率为5.50%,与对照组(1.70%)差别有统计学意义 (P<0.01);暴露组微核率为16.99‰,对照组为3.47‰,差异有统计学意义 (P<0.01);单细胞凝胶电泳检测显示暴露组尾部DNA百分含量 (TDNA,%)、尾矩 (TM)、Olive尾矩 (OTM)较对照组均显著升高 (P<0.01)。染色体畸变率、微核率和DNA损伤水平女性均明显高于男性 (P<0.01),而在各不同年龄组间未见明显差异 (P>0.05)。结论：电子垃圾污染物是潜在的遗传诱变剂,能够造成污染地区人群的细胞遗传学损伤,不能忽视其对当代及其子代健康的有害影响。     

β_胡萝卜素对丝裂霉素C致小鼠骨髓细胞遗传损伤的保护作用

背景与目的： 探讨β_胡萝卜素(β_carotene,βC)对丝裂霉素C(mitomycin_C,MMC)诱导的小鼠骨髓细胞遗传损伤的保护作用。 材料与方法： 分别用3 mg/kg和30 mg/kg的βC连续饲喂小鼠8 d后,以2 mg/kg MMC进行腹腔注射染毒,另设阳性对照组(用生理盐水)和阳性对照组(一次性腹腔注射MMC 2 mg/kg)。各组于末次给药后12、24 h处死小鼠,分别采用单细胞凝胶电泳技术和染色体畸变分析检测骨髓细胞DNA损伤及染色体畸变情况。 结果： 两个不同剂量的βC与MMC联合作用组小鼠骨髓细胞拖尾率、平均尾长及染色体畸变率均低于阳性对照组(单独MMC),但均高于阴性对照组,差异均有统计学意义(P<0.01)。 结论： βC对MMC诱导的小鼠骨髓细胞遗传损伤有明显的保护作用,但在实验所给剂量范围内(3～30 mg/kg)并不能完全抑制MMC所造成的遗传损伤。     

五种氯化有机物对V79细胞DNA损伤作用的研究

本文采用近年来发展的单细胞凝胶电泳试验检测了二氯甲烷、三氯甲烷、四氯化碳及一氯化酸和三氯乙酸对Ｖ７９细胞的ＤＮＡ损伤作用。结果显示：五种受试物均可引起体外培养的Ｖ７９细胞ＤＮＡ损伤,但其作用强度差异很大。其顺序为四氯化碳〉三氯甲烷〉二氯甲烷〉一氯乙酸〉三氯乙酸。该结果还提示：五种氯化有机物对Ｖ７９细胞的毒性及ＤＮＡ损伤作用的大小顺序是一致的,氯化烷基类化合物的细胞毒性及ＤＮＡ损伤作用均大于氯乙酸类     

单细胞微凝胶电泳技术的研究和应用

在研究辐射生物效应中, 检测DNA 损伤的方法很多。例如: 细胞遗传学方法染色体畸变、微核、姐妹染色单体互换等等。但上述方法费时费力, 并有一定的局限性。八十年代由O st ing 等首次提出的用微板电泳技术检测单细胞水平DNA 损伤。后经Singh 等进一步改进和完善了单细胞微凝胶电泳Single Cellm icrogel elect ropho resis 简称SCGE) 技术, 又称彗星检测法(Comet assay)。该方法突破了传统局限性, 即设备、操作简便, 所需细胞少、无需放射性示踪剂, 而且灵敏、快速, 整个实验过程从来样到结果分析可在一个工作日完成的新技术。因此, 该技术在国外有关研究领域已得到广泛应用。近年来, 国内一些研究领域也引进和建立此方法。主要用于环境诱突变剂或诱癌剂引起的DNA 损伤ö修复的研究; 癌症病人放、化疗方案的最佳选择及科学预后的研究; 作为生物标记进行生物监测及流行病学、毒理遗传学等诸多领域的研究。目前, 也有一些专家学者将此技术应用于植物细胞的研究。SCGE 技术在本实验室已建立, 其方法是使包埋于琼脂糖中的细胞经裂解去掉细胞浆, 在碱性环境下DNA 双链螺旋成为单链, 在电泳电场作用下DNA 断链离开细胞核向正极迁移出现“彗星”状拖尾现象, 再通过荧光染料溴化乙啶结合DNA 显色检测其损伤程度。为将此技术推广于辐射生物效应体内外研究中, 本实验选用在辐射生物效应中经常用的人血淋巴细胞(HBL ) , 中国仓鼠肺细胞(CHL ) 和兔血淋巴细胞这3 种细胞探索利用此技术检测DNA 损伤的可行性。实验结果表明: SCGE 技术能够对单个细胞DNA 损伤进行研究, 荧光测定核DNA 直径和迁移DNA (即彗星尾) 长度, 而尾长度和尾部荧光强度与DNA 链的断裂呈正相关。由于“彗星”成像是因损伤DNA 在电泳中从核内迁出, 因此它的尾长和密度等是评价与量化电泳结果的重要因素。我们选用上述3 种细胞接受60Co2·线照射后, 经SCGE 技术检测DNA 损伤, 照射组与未照射组DNA 迁移距离经统计学检验, 差异显著性水平A= 0. 5。细胞经照射处理后可引起DNA 电泳迁移, 而迁移长度随照射剂量的增加而延长。此项技术可从受照人员外周血淋巴细胞DNA 电泳迁移长度来估算受照剂量, 从而为评价急性照射和慢性小剂量长期照射的损伤程度提供科学依据。     

液体石蜡的毒性研究

山西省研究用石蜡涂膜法在常温下贮存保鲜鸡蛋的技术，为此，测试了液体石蜡的性毒，小鼠ＬＤ５０为４．６４ｇ／ｋｇ，大鼠ＬＤ５０为６．８１ｇ／ｋｇ，液体石蜡对大鼠胄髓细胞染色体畸变试验三个剂量组即６．０，６０．０，６００．０ｍｇ／ｋｇ均未观察到染色体的损害；同时以３．０，３０．０，３００．０ｍｇ／ｋｇ观察小鼠染色体畸变率为阴性Ａｍｅｓ试验五个浓度、ＴＡ９８、ＴＡ１００的回变菌     

彗星实验检测紫外线诱导的K562细胞DNA损伤

背景与目的研究紫外线(Ultraviolet,UV)诱导K562细胞DNA损伤情况,评价彗星实验改良方法及分析参数的可靠性。材料与方法采用0.3mW/cm2UV以用不同时间(3、5、10、40、60、120、180、240s)照射K562细胞,诱导细胞DNA损伤,用彗星实验检测,CASP软件分析。结果尾长、彗星长、尾矩、Olive尾矩4个参数与紫外线照射时间有良好的时间依赖关系。结论0.3mW/cm2的紫外线照射K562细胞3s即可造成细胞DNA损伤,CASP分析软件可以用于彗星检测分析,其中尾长、彗星长、尾矩、Olive尾矩可作为彗星实验的分析指标,所改良的彗星实验系统可靠性较强,且基本上解决了实验中常见的脱胶现象,采图更方便。     

极替沙星遗传毒性及致突变作用的研究

目的与方法: 采用Ames试验,小鼠骨髓嗜多染红细胞(PEC)微核试验及中国仓鼠肺成纤维细胞(CHL)染色体畸变试验,检测受试物极替沙星是否具有潜在的遗传毒理学效应.结果:TA97、TA100、TA102 3株菌的Ames试验结果为阴性;TA98菌株的4 μg/ml和3 μg/ml剂量组在非活化法(-S9 )和活化法(+S9)试验中诱发的回变菌落数均高于自发回变菌落数10倍以上.PEC微核试验在中低剂量组试验均为阴性,而高剂量组与各自对照组相比,差异均具有显著性.在CHL染色体畸变试验中:无S9时,药物组在0.05 mg/ml～0.025 mg/ml范围内染色体畸变率在正常范围,在0.4 mg/ml～0.1 mg/ml范围内染色体畸变率为40.5%～69.0%;有S9时,药物组在0.1 mg/ml～0.025 mg/ml范围内染色体畸变率在正常范围,在0.4 mg/ml～0.2mg/ml范围内染色体畸变率为61%～91%.结论:极替沙星具有一定的遗传毒性及致突变作用.     

用单细胞凝胶电泳技术检测甲醛对小鼠睾丸细胞的DNA损伤

背景与目的：研究甲醛对离体和体内小鼠睾丸细胞DNA的损伤作用。材料与方法：以6、30、60、120 μmol/L甲醛浓度处理离体小鼠睾丸生精细胞,以0.2、2、20 mg/kg的甲醛腹腔暴露小鼠,利用单细胞凝胶电泳检测睾丸生精细胞DNA的损伤作用。结果：甲醛在6μmol/L时就可以使小鼠离体睾丸细胞产生DNA损伤,与阴性对照不同 (除 tail moment外),并随浓度增加DNA迁移也相应增加,到30 μmol/L时DNA损伤最为明显,但随浓度升高DNA迁移反而减少,当甲醛浓度为120 μmol/L时DNA迁移与阴性对照相似。腹腔注射0.2、2 mg/kg的甲醛组小鼠睾丸细胞DNA迁移高于对照组,0.2 mg/kg组DNA迁移最为明显。 结论：甲醛对小鼠睾丸细胞具有遗传性损伤,低剂量时是以细胞DNA断裂损伤为主,而高剂量时可能引起其他方式的损伤。     

稀土元素镨对蚕豆的遗传毒性和细胞毒性研究

背景与目的:研究稀土元素镨对蚕豆的遗传毒性和细胞毒性。材料与方法:蚕豆初生幼苗浸入7个PrCl3浓度(1,2,4,8,16,32,64μg/ml)系列溶液,25℃恒温培养6h,之后双蒸水中修复培养24h,切取根尖。进行蚕豆根尖细胞微核试验,计数有丝分裂指数、染色体畸变率。结果:PrCl3浓度在≥8μg/ml时可损伤蚕豆根尖细胞,影响根尖的生长;在1～32μg/ml浓度范围内根尖细胞微核率呈剂量_效应关系(r=0.948,P<0.05);在2～64μg/ml浓度范围内有丝分裂指数呈剂量_效应关系(r=-0.789,P<0.05);在1～8μg/ml浓度范围内染色体畸变率呈剂量_效应关系(r=0.992,P<0.05)。结论:稀土元素镨对蚕豆根尖细胞具有一定的遗传毒性和细胞毒性。     

用SCGE分析甲氨蝶呤对小鼠体内多个组织器官DNA损伤作用

背景与目的:为进一步了解甲氨蝶呤(Methotrexate,MTX)的作用机制,探测其对不同组织器官作用的敏感性。材料与方法:用单细胞凝胶电泳技术(Singlecellgelelectrophoresisassay,SCGE)检测小鼠腹腔注射MTX染毒1、3、6、12、24h后对肝、脾、骨髓、胸腺、肾、睾丸、胃和外周血淋巴细胞的DNA损伤作用及其与MTX剂量间的关系。结果:腹腔注射1.25~5mg/kgMTX可诱发小鼠脾细胞、骨髓细胞、胸腺细胞和外周血淋巴细胞的DNA单链断裂;核DNA损伤程度与用药剂量呈正相关。结论:MTX可致小鼠体内多脏器细胞的DNA单链断裂,不同脏器细胞对MTX的易感性不同,脾、骨髓、胸腺、外周血淋巴细胞可考虑为MTX的遗传毒性靶细胞。     

Aspirin Intake Suppresses MMC-induced Genotoxicity in Mice

The genotoxicity induced by mitomycin C (MMC) was found to be decreased by aspirin on alkaline single cell gel electrophoresis (SCG) assay in multiple organs of mice. Aspirin at doses of 0.5, 5 and 50 mg/kg and MMC at 2 mg/kg were administered and then liver, lung, kidney, spleen, colon and bone marrow were sampled after 3 h. Significant protective effects of aspirin against MMC-induced genotoxicity were observed in all but the bone marrow, where no change was evident. The results suggest that the radical scavenging ability of aspirin prevents damage by MMC-induced reactive oxygen species (ROS) in multiple organs.     

两种人类肝细胞系评价饮水氯化消毒副产物MX致DNA损伤作用的敏感性

背景与目的:研究两种人类来源的肝细胞在评价饮水氯化消毒副产物3-氯-4-二氯甲基-5-羟基-2(5氯)-呋喃酮(MX)DNA损伤作用的敏感性.材料与方法:应用单细胞凝胶电泳试验(彗星试验),研究人肝癌细胞株HepG2和人胚肝细胞L-02在评价饮水氯化消毒副产物MX DNA损伤作用的敏感性.结果:随着MX剂量增加,可引起HepG2(＞ 30 μmol/L)和L-02细胞(＞100μmol/L)DNA迁移度显著增加;中度以上损伤所占百分比在同剂量组中HepG2细胞大于L-02细胞,经比较分析,差异有统计学意义(χ2=20.985;8.0;11.97,P＜0.05).结论:应用单细胞凝胶电泳试验评价MX的DNA损伤作用,人肝癌细胞株HepG2比人胚肝细胞L-02更为敏感.     

低剂量甲氨蝶呤对DNA的损伤及甲酰四氢叶酸对其保护作用

背景与目的：研究多次低剂量应用甲氨蝶呤所致BALB/c小鼠淋巴细胞及骨髓细胞DNA的损伤,并探讨甲酰四氢叶酸对其损伤的保护作用。材料与方法：应用甲氨蝶呤0.5 mg/kg腹腔注射,每周1次,共8周;用单细胞凝胶电泳方法检测其外周血淋巴细胞及骨髓细胞DNA的损伤情况。结果：单用甲氨蝶呤组细胞尾长及拖尾频率与对照组相比均有显著差异,而同时应用等量的甲酰四氢叶酸,其细胞尾长及拖尾频率与对照组相比无显著差异。结论：SCGE技术可敏感地检测多次应用低剂量甲氨蝶呤造成的DNA损伤,并能反映甲酰四氢叶酸对甲氨蝶呤所致的DNA损伤的保护作用。     

体外硫芥对人外周血淋巴细胞DNA的损伤

目的与方法：利用碱性单细胞凝胶电泳（ＳＣＧＥ）技术在体外检测硫芥对人外周血淋巴细胞的损伤情况。结果与结论：结果表明：在硫芥的作用下细胞ＤＮＡ的迁移率和迁移长度增加,且呈显著剂量效应关系。     

Study on the DNA damage induced by coal tar pitch fume extracts in rat alveolar macrophage and it‘s mechanism

The carcinogenic mechanism of coal tar pitch (CTP) as a recognized carcinogen has been studying. It is widely believed that the carcinogenicity of CTP is based on the genotoxicity of CTP. In the process of carcinogenesis caused by extrinsic chemical substance, the DNA damage mainly occurred in the initiation phase. UP to now, the most sensitive detecting endpoint for DNA damage is to detect DNA single strand breaks. The single cell gel electrophoresis has been rapidly becoming a widely used analytical procedure during the last few years, which can detect DNA strand breaks. The method is a fast, relatively inexpensive, easy to perform, non-radioactive, and very sensitive method. This method suits to different tests in vitro or in vivo. Virtually any eukaryotic cell, which could be made into single cell suspensions, can be processed for analysis of DNA damage using the single cell gel electrophoresis. The aim of the study is to investigate the role of DNA damage induced by CTP fume in rat AM, to examine the changes of ROS, MDA and SOD, and to explore the mechanism of DNA damage by CTP fume. The present study is in favor of studying the mechanism of mutagenesis and carcinogenesis induced by CTP. Method: The healthy male Wistar rats were anesthetized intraperitoneally with 40 mg pentobarbital sodium per kilogram of body weight. The animals were exanguinated by excising femoral, and collected the rat alveolar macrophage by Joseph's method. The concentration of AM had been regulated to 1.5×106 cell/ml. AMs, which had been cultured in 24-well culture plate, were divided into 4 groups. These cells were exposured to 5.0 μg/ml extracts of coal tar pitch fume, and contacted with 500 μM, 1 000 μM, and 2 000 μM of GSH respectively. These cells were divided into 4 groups. After incubation 24 hours, the indexes that had been used above were measured. Results: ①The DNA strand breaks induced by coal tar pitch fume extracts: After undergoing electrophoresis, the ‘comet’ cells are seldom in control group, but the ‘comet’ cells increased significantly in experimental groups. When the concentration of CTP fume extracts increased from 0 to 10.0 μg/ml, the incidences of ‘comet’ cell increased from 6.0% to 92.0%. There were significant differences in all groups (P<0.001). There was a dose-response relationship between the concentrations of CTP fume extracts and the incidences of ‘comet’ cell. There were correlation relationship between the grade of DNA damage and the concentration of CTP fume extracts (P<0.001). ②The level of ROS induced by CTP fume extracts in AM: After incubation 24 h, the concentration of ROS increased. The difference between control group and experimental groups were significant (P<0.001). ③ The changes of MDA and SOD in AM induced by CTP fume extracts: As the concentration of coal tar pitch fume extracts increased, the concentration of MDA increased too. The differences between experimental groups and control group were significant. But the difference of SOD activity in all groups weren’t significant. ④The role of GSH interfere: GSH could protect the DNA of AM from DNA-damage induced by CTP fume extracts. With the increasing of the concentration of GSH, the incidences of ‘comet’ cell decreased from 77.0% to 16.0%. The differences of incidences of ‘comet’ cell in all groups were different significantly. There was a dose-response relationship between the concentrations of GSH and the incidences of ‘comet’ cell. There was correlation relationship between the grade of DNA damage and the concentration of GSH (P<0.001). GSH could inhibit the increasing of ROS and MDA. The differences of SOD activity in all groups weren't significant (P>0.05). Conclusion: ①Coal tar pitch fume extracts can induce DNA single strand breaks in rat alveolar macrophage; the single cell gel electrophoresis appears to be suitable to detect DNA strand breaks in rat alveolar macrophage. ②Coal tar pitch fume extracts can induce the rat alveolar macrophage to produce reactive oxygen species (ROS), and can induce lipid peroxidation: the DNA strand breaks induced by coal tar pitch fume extracts in AM are associated with the producing of ROS. ③GSH can inhibit coal tar pitch fume extracts-induced DNA damage in rat alveola r macrophage.     

不同浓度的一氧化氮对食管癌细胞核DNA的损伤作用

目的: 研究不同浓度的一氧化氮(nitric oxide,NO)对食管癌细胞核DNA损伤作用的特征性规律.方法: 以硝普钠(sodium nitroprusside,SNP)作为NO的体外供体,以食营癌细胞系EC109作为细胞模型,采用单细胞凝胶电泳法检测不同浓度的SNP作用一定时间后,食管癌细胞核DNA被损伤的情况.结果:无论SNP的作用时间是8 h,还是16 h,彗星状细胞核所占的百分率均越来越高,经χ2检验差异有显著性意义(P＜0.01).曲线回归分析结果表明,食管癌细胞彗星状细胞核生成的百分率与SNP的浓度之间明显相关.结论:在一定的条件下,NO对食管癌细胞核DNA的损伤作用具有明显的浓度依赖性特征.     

煤焦沥青对作业工外周血淋巴细胞DNA的损伤

背景与目的:了解煤焦沥青接触工人外周血淋巴细胞DNA损伤情况.材料与方法:应用外周血淋巴细胞单细胞凝胶电泳(SCGE)试验及微核(MN)试验,分别检测43名煤焦沥青接触工人和21名对照工人外周血淋巴细胞DNA损伤情况.结果:接触组外周血淋巴细胞中彗星出现率及微核发生率分别为(3.26±1.12)×10-2和(1.23±0.27)×10-3,高于对照组,且这种损伤效应可随接触时间的延长而增强.结论:长期接触煤焦沥青可明显地导致作业工外周血淋巴细胞DNA损伤.