基于微孔板的人HepG2细胞体外彗星试验方法的研究

目的运用微孔板体外培养技术,建立人HepG2细胞的体外彗星试验方法,为进一步建立体外高通量筛选方法提供依据。方法选用人HepG2细胞,运用96孔板体外细胞培养技术,建立微孔板的彗星试验方法,结合中性红检测细胞毒性的方法,选择20种化合物对所建方法进行验证。结果阳性和阴性对照物用所建方法与体内试验所获结果一致,验证结果显示,8种遗传毒物在体外彗星试验中均获得阳性结果,灵敏度为100%,且当细胞的相对活力>50%时,拖尾率及尾长均与剂量呈正相关。12种非遗传毒物中,11种物质呈阴性反应,1种物质呈阳性反应,特异性为91%。结论运用微孔板体外细胞培养技术,可在1块96孔板上同时检测4~5种受试物,并在无S9的情况下能检测出直接与间接诱变剂,并可界定出引起遗传毒性的作用剂量,提高了筛选的速度,减少了受试物等的用量,具有发展成为体外高通量筛选方法的良好条件。     

HepG2细胞中性红染色法测定化合物急性毒性的研究

目的探索Hep G2细胞中性红染色法检测化合物毒性的可行性,为建立化合物急性毒性快速筛选方法提供依据。方法采用人肝癌Hep G2细胞中性红染色法检测秋水仙碱、环磷酰胺、吡蚜酮等30种化合物的细胞毒性IC50、IC45、IC40、IC35和IC30值。采用线性回归模型拟合ICx值对大鼠经口半数致死剂量(LD50)的回归方程,利用回归方程预测LD50值。采用LD50实际值和预测值对化合物毒性进行分级,比较两种方法的一致性。结果 lg(IC50)、lg(IC45)、lg(IC40)、lg(IC35)、lg(IC30)与lg(LD50)均有相关性(P0.01),相关系数(r)为0.779~0.815;lg(IC50)与lg(LD50)的相关性最高,lg(LD50)=0.887×lg(IC50)+0.414。LD50实际值将30种化合物划分为高毒、中等毒和低毒各10种,LD50预测值划分出高毒12种、中等毒10种、低毒8种;两种方法的划分结果差异无统计学意义(P0.05),一致率为86.7%。结论人肝癌Hep G2细胞中性红染色法可作为快速检测化合物急性毒性的方法。     

雷公藤红素体内与体外急性毒性试验结果的比较

[目的]研究雷公藤红素对3T3细胞的毒性作用,获得的细胞毒性结果预测急性毒性半数致死量(LD50),并与采用小鼠上下法(UDP)和Bliss法测定的急性毒性数值进行比较. [方法]以已验证的3T3细胞中性红摄取为细胞毒性模型,以0.01～2.05 mg/L的8个浓度(剂间比为2.15)的雷公藤红素进行处理,分别于处理后48h进行中性红摄取试验,计算半数抑制浓度(IC50).以RC数学模型预测其全身急性毒性LD50.参考此LD50值设定起始剂量进行小鼠上下法实验.取70只ICR小鼠,雌雄各半,以3、4、5、6、7、8mg/kg的雷公藤红素溶液和0mg/kg[4％二甲亚砜(DMSO)氯化钠注射液]静脉给药,观察14d,根据动物死亡率采用Bliss法计算LD50. [结果]雷公藤红素3T3细胞中性红摄取试验的IC50值为(0.1197 ±0.0187)mg/L(n=2),决定系数R2＞ 0.96,预测其全身急性毒性LDs0为5.375 mg/kg.上下法起始剂量设为3.3 mg/kg,测得雷公藤红素在ICR小鼠的LD50值为3.157 mg/kg,95％可信区间为3.1～5.5 mg/kg.小鼠Bliss法测定急性毒性LD50为4.899mg/kg,95％可信区间为4.483～5.354 mg/kg. [结论]雷公藤红素的体外3T3细胞毒性预测急性毒性与体内方法所得数值一致.     

细胞毒试验对香烟毒性检测的研究

目的通过体外细胞毒试验研究快速检测香烟综合毒性的技术。方法采用细胞毒试验(中性红法),选用仓鼠肺细胞(V79)与受试物作用,经处理、培养和仪器测试判定待检样品是否对细胞有毒性作用。结果在各不同时间段可以看出,不同浓度组香烟提取物(CSE)对细胞毒性作用的强弱不同,显色变化明显。低毒香烟组显色浅,半数抑制浓度(IC50)值大,细胞毒性小;高毒香烟组显色深,IC50值小,细胞毒性大。结论香烟的综合毒性越强,细胞毒试验显色越明显,反之亦然,证实细胞毒试验可作为快速检测香烟综合毒性的初筛方法。     

抗流感病毒天然化合物体外筛选模型的建立及应用

目的建立抗流感病毒天然化合物体外筛选模型。方法以扎那米韦为阳性药物,运用酶联免疫吸附实验(ELISA)和三磷酸腺苷荧光检测(ATPlite)技术,建立基于细胞培养的天然化合物抗流感病毒体外筛选模型,测定205种天然化合物的细胞毒性及其抗流感病毒活性。结果成功构建抗流感病毒天然化合物细胞体外筛选模型,以扎那米韦验证,流感病毒被有效抑制,对A型流感病毒的IC50为0.002 8μmol/L,对B型流感病毒的IC50为0.057μmol/L。205种天然提取化合物中,有143种无显著细胞毒性,应用体外模型筛选,未检出有抗流感活性的化合物。结论成功建立基于ELISA法和ATP荧光法的体外抗流感天然化合物细胞筛选模型,可用于抗流感药物大规模筛选。     

乙型肝炎病毒体外感染HepG2细胞的初步研究

目的建立HBV阳性血清体外感染HepG2细胞的实验模式。方法在六孔板上培养HepG2细胞,用HBV阳性血清体外感染HepG2细胞,设立感染组和对照组。用HBV阳性血清和感染组细胞共孵育24 h,0.01 mol/L PBS清洗细胞后,每孔板中加入4 ml DMEM细胞培养液,每隔12 h收集细胞上清液至PBS洗后84 h。收集完最后一次细胞上清液,收集细胞培养板中的细胞。ELSIA检测细胞培养上清中HBsAg;PCR方法检测细胞培养上清和细胞中HBV DNA;免疫荧光定量PCR检测细胞培养上清中HBV DNA的含量。结果HBV感染组细胞培养上清,在PBS洗后第12 h,ELISA即可检测到HBsAg并持续到84 h,PCR检测感染组细胞培养上清和感染组细胞的HBV DNA均阳性。荧光定量PCR检测感染组细胞培养上清中HBV DNA的结果:感染组PBS洗后(0 h)的HBV定量为阴性,而在PBS洗后36 h8、4 h细胞培养上清中HBV的量达到5×105,3×106copies/ml)。结论用HBV阳性血清体外感染HepG2细胞的实验是可行的。     

体外MTT法评价化妆品光毒性试验的建立与应用

目的 应用体外MTT法评价化妆品光毒性.方法 分别以MTT法、中性红试验评价5种物质的体外3T3细胞光毒性,并与动物试验结果相比较.结果 MTT法与中性红法均能正确判断光毒阳性物与光毒阴性物,对3种育发液的检测结果一致.上述结果与动物实验结果相符.结论 体外MTT法可作为光毒性动物试验的替代方法.     

利用体外3T3细胞中性红摄取试验检测化学物质光毒性作用

目的建立体外Balb/c3T3细胞中性红摄取法作为检测化学物光毒性的试验方法,初步探讨体外BaLb/c3T3细胞中性红摄取法作为豚鼠光毒性试验的一种替代方法。方法采用体外培养的BaLb/c3T3细胞建立反应的最佳条件。结果中性红浓度、孵育时间、pH值、性别浓度等是影响试验结果的因素。结论体外BaLb/c3T3细胞中性红摄取试验可作为动物光毒性试验的替代方法。     

3T3细胞光毒替代实验的建立及在化妆品评价中的应用

目的 通过建立3T3中性红成纤维细胞光毒替代实验,研究体外替代方法替代动物实验及在化妆品安全性评价中取代动物光毒性实验的可行性。方法 选用24种已知光毒性化学物质（15种阳性、9种阴性）和20种待检特殊用途化妆品,测量Bal／c3T3细胞经化学物质和紫外线照射联合作用后的平均光效应强度和光刺激因子,与整体动物皮肤光毒性结果比较。结果 3T3体外替代实验对24种已知光毒性化学物质检测结果为14种阳性和10种阴性,20种化妆品为阴性,经校正χ^2检验与既往光毒性结果比较,差异无统计学意义。结论 本次研究成功建立了3T3中性红成纤维细胞光毒替代实验方法,该方法可取代豚鼠皮肤光毒性实验对化妆品终产品进行安全性评价。     

急性经口毒性评价试验方法的研究进展

急性经口毒性试验是毒理学安全性评价第一阶段最基础性的试验，LD50是其最为常用的评价指标。本文评述了传统的LD50测试法以及3种主要替代方法（上下增减剂量法，固定剂量法，急性毒性等级法）的优缺点及其实际应用前景。     

Toxicological assessment of industrial solvents using human cell bioassays: assessment of short-term cytotoxicity and long-term genotoxicity potential

There is an increasing demand for simple toxicological screening methods to assess the human health risk associated with exposure to environmental toxicants. Such screening tools should allow for risk evaluation in terms of both short-term/acute toxicity and longer-term genetic damage, which may lead to mutagenicity and carcinogenicity. We employed a battery of human cell bioassays using the human hepatoma cell-line, HepG2, to assess the cytotoxic and genotoxic potential of environmental pollutants. Here, we demonstrate direct application of these human cell bioassays to the toxicological assessment of a number of industrial solvents that are in common use worldwide. HepG2 cells were exposed to various solvents at concentrations ranging from 25 to 500 ppm. The cells were then analysed using specific protocols for four different adverse effects: cell death/acute cytotoxicity using a neutral red uptake assay, altered enzyme function (often an indicator of cell stress) using the ethoxyresorufin O-deethylase (EROD) bioassay, DNA single strand breaks (SSB), and DNA repair induction, which evaluates mutagenic activity. Using the positive controls, linear dose-response curves were achieved for all four bioassays. The high sensitivity of the tests allowed for environmentally meaningful assessments, and precision studies showed excellent reproducibility for all four bioassays. Comparing the results of the four bioassays on each of 16 industrial solvents allowed for ranking of the anticipated relative human toxicity of these solvents, which were comparable with data from standard toxicity tests and human occupational data. Overall, the study clearly supports the application of the HepG2 cell bioassay system for rapid toxicological screening of many candidate toxicants for both short- and long-term toxicity potential.     

顺铂对大鼠肝细胞毒性及谷胱甘肽的保护作用

目的探讨顺铂对原代培养的大鼠肝细胞的毒性及谷胱甘肽对其影响.方法从大鼠的肝脏分离培养肝实质细胞接种于96孔培养板,培养3h后加入一系列浓度的顺铂,或在加入顺铂前16和4h,分别加入谷胱甘肽(glutathione,GSH)合成抑制剂DL-buthionine-(S,R)-sulfoximine(BSO)和GSH的前体物半胱氨酸,继续培养,分别在8,24和48 h 3个时间点用噻唑蓝(MTT)方法检测细胞存活率.结果顺铂对大鼠肝细胞有明显的毒性,在不同的时间点有各自的剂量-反应关系,顺铂对大鼠肝细胞在8,24和48 h 3个时间点半数抑制浓度(IC50)分别为1.13,0.21和0.15 mmol/L;BSO能使3组IC50均降低,分别为0.017,0.011和0.013 mmol/L,而半胱氨酸则可使3组IC50均升高,均大于5mmol/L.结论顺铂对大鼠肝细胞具有明显的毒性作用,且呈时间和剂量依赖关系;BSO可增强顺铂的肝细胞毒性,而半胱氨酸对顺铂引起的肝细胞毒性有保护作用,提示细胞内GSH对顺铂所致肝细胞毒性有保护作用.     

In vitro cytotoxicity of organic pollutants to bluegill sunfish (BF-2) cells

BF-2 cells, an established cell line derived from bluegill sunfish, (Lepomis macrochirus), were exposed to 18 organic toxicants, with cytotoxicity being assayed by the neutral red (NR) technique. Based on the concentration of toxicant that reduced lysosomal uptake of neutral red by 50% (NR50), the rank order of cytotoxicity was methyl mercury greater than pentachlorophenol greater than 2,3,5,6-tetrachlorophenol greater than 2,3,5-trichlorophenol greater than 2,3-dinitrotoluene greater than 2,4,6-trichlorophenol greater than 2,4-dichlorophenol greater than 2,4-dichlorotoluene greater than 6-chloro-3-hydroxytoluene greater than o-chlorotoluene greater than 4-chlorophenol greater than 2-chlorophenol, 2,4-dimethylphenol greater than 2,4-dinitrophenol greater than 4-nitrophenol greater than 3-methylphenol greater than phenol greater than toluene. Published in vivo LC50 values were identified for 11 of the 18 test agents and, with the exception of 2,4-dinitrophenol, there was a good correlation between the in vitro cytotoxicity of the test agents and their in vivo waterborne acute toxocity. The potencies of the substituted phenolics and chlorinated toluenes as determined by the in vitro cytotoxicity assay correlated strongly with their log octanol/water partition coefficients (log P). However, the toxicity of 2,3-dinitrotoluene in vitro, and apparently also in vivo, was not a function of its log P value.     

纳米红色元素硒的急性毒性和生物利用性

纳米红色元素硒是以蛋白质为分散剂的元素硒的纳米粒子,粒径在60nm 以内。体外研究证实,纳米红色元素硒能够与谷胱甘肽反应,其反应能力约为亚硒酸钠的1/20至1/10。急性毒性实验证实以口服硒元素的量计,纳米红色元素硒的LD50 = 112.98(89.95～141.90)m g/kgBW,亚硒酸钠的LD50= 15.72(13.38～18.47)m g/kgBW,显示出纳米红色元素硒的毒性低。生物利用性试验证实:小鼠分别口服纳米红色元素硒和亚硒酸钠(Se 50μg/kg BW)30天,与对照组比,2种硒都能显著提高小鼠血硒和肝硒浓度,血中谷胱甘肽过氧化物酶活性升高。这说明纳米红色元素硒能被小鼠较好利用     

In vitro cytotoxicity testing of airborne formaldehyde collected in serum-free culture media

The purpose of this study was to identify a suitable sampling model for on-site toxicity assessment of soluble air contaminants such as formaldehyde, a well known industrial and indoor air contaminant. The in vitro cytotoxicity of formaldehyde, the selected model for soluble air contaminants, was studied using the MTS (tetrazolium salt) assay in two carcinoma cell lines, A549 epithelial lung and HepG2 hepatocarcinoma, and in skin fibroblasts. The cytotoxic effects of airborne formaldehyde were evaluated using test atmospheres in concentrations below 10 ppm (12.3 mg/m3), generated by a dynamic diffusion method and bubbled (0.3 L/min) through serum-free culture media for one or four hours. Human cells were treated with formaldehyde air samples, and cell viability was determined after four hours incubation. In parallel, the concentration of airborne formaldehyde was monitored, using the 3500 NIOSH method. Cell viability of the HepG2 cells exposed to formaldehyde air samples (8.75 ppm x 4 h) was reduced to less than 50% (31.6 +/- 1.24%). The HepG2 cell lines were found to be more sensitive (IC50 = 103.79 +/- 23.55 mg/L) to formaldehyde than both A549 cell lines (IC50= 198.36 +/- 9.54 mg/L) and skin fibroblasts (IC50 = 196.68 +/- 36.73 mg/L) (P < 0.01). An average of 96.8% was determined for collection efficiency of formaldehyde in serum-free culture media. The results of this study suggest that absorption of soluble air contaminants, such as formaldehyde, in serum-free culture media can be used as a suitable sampling model for on-site toxicity assessments.     

Rapid determination of glutathione peroxidase and thioredoxin reductase activities using a 96-well microplate format: comparison to standard cuvette-based assays

Gluthatione peroxidase and thioredoxin reductase are selenocysteine-containing enzymes that are constituents of the cellular antioxidant defense system. Conventional cuvette-based assays for glutathione peroxidase and thioredoxin reductase enzymes are laborious and time consuming. The ability to assay their activities rapidly in multiple samples would aid efforts focused on understanding the impact of these enzymes on the cellular antioxidant defense system. High throughput can be achieved with assays adapted to work in a clinical analyzer but require expensive equipment. Assays designed to work in a 96-well microplate reader provide an alternative methodology for high throughput with reduced instrumentation cost. However, due to differences in the light pathlength when using a 96-well format, the values obtained cannot be compared directly with those obtained using a 1-cm cuvette. Described here are assays for glutathione peroxidase and thioredoxin reductase modified to work in a 96-well format that incorporates light pathlength determinations into the assays. The values obtained using a high throughput 96-well format in conjunction with pathlength determinations are in agreement with those obtained using a standard 1-cm cuvette. While spectrophotometrically derived pathlengths are the most accurate, calculated pathlengths based on assay volume and well size can be used with only a small amount of error introduced. This method can also be applied to many other enzyme assays, thus allowing the rapid analysis of large numbers of samples without the need for expensive equipment.     

四种细胞毒性试验测定柴油机尾气颗粒物提取物急性毒性的比较

目的比较四种体外细胞毒性试验检测柴油机尾气颗粒物(DEP)提取物急性毒性的能力。方法用浓度分别为5、10、20、40、80和160μg/ml的DEP提取物作用于16HBE细胞,于12、24及48h后,通过MTT法、中性红法、乳酸脱氢酶(LDH)法及蛋白测定法测细胞存活率。结果 MTT法在检测急性毒性方面与其它三种方法相比,染毒12h时就表现出了很高的敏感性。随着染毒时间的增加,MTT法与中性红法的敏感性逐渐提高,染毒24h后与对照组相比,各剂量组细胞存活率明显降低(P<0.05)。LDH法测定DEP提取物染毒24h后细胞存活率有所降低,但继续增加染毒时间,存活率不再发生变化。蛋白法测定染毒12h和24h的细胞存活率,仅高剂量组显著降低,染毒48h后,各剂量组细胞存活率均显著降低。结论四种不同的细胞毒性试验测定结果存在差异,MTT法和中性红法比蛋白法和LDH法更为敏感。结合各试验检测的毒性指标可以看出,DEP提取物主要影响16HBE细胞内各细胞器的功能,如线粒体、溶酶体;对于细胞贴壁的影响和细胞膜损伤程度的影响较弱。     

丙戊酸钠两次LD50测定结果的对比研究

在１９９１年召开的第一届国际ＩＣＨ会议上,参加协调的３方（欧盟、美国、日本）同意在申报新药时取消对ＬＤ５０资料的要求〔１〕。尽管如此,我国和一些国家在药品安全性评价中仍延用ＬＤ５０〔２,３〕。本文结合丙戊酸钠的两次ＬＤ５０的测定结果,就这一问题进行探讨。材料和方法　（１）试剂：丙戊酸钠,法国Ｓａｎｏｆｉ生产。（２）动物：ＳＰＦ级ＫＭ种小鼠,由中国药品生物制品检定所实验动物中心提供,合格证号为京动管质字〔１９９４〕０７９号。（３）实验分组：①将体重１６－５～１７－９ｇ的５０只小鼠随机分为５组,每组…