PT-141染色体畸变试验

目的采用中国仓鼠肺成纤维细胞(CHL)进行染色体畸变试验,评价PT-141在体外有无致突变性,为临床应用提供安全依据。方法CHL细胞用含质量浓度为20%的小牛血清和双抗(青霉素、链霉素的总浓度分别为100U/ml、100μg/ml)的1640培养液于37℃,恒温培养。(1)细胞毒性试验:采用苔盼蓝染色检测CHL细胞接触PT-14148h的半数细胞生长抑制浓度(IC50),作为剂量选择的依据。(2)染色体畸变试验:以CHL细胞进行加与不加S9的染色体畸变试验。不加S9时,培养瓶内加入4.95ml细胞培养液,50μl受试物,培养24和48h;加S9时,培养瓶内加入4.45ml细胞培养液,50μl受试物,0.5mlS9混合物,培养6h后,弃培养液,PBS洗涤3遍,再加入1640细胞培养液5ml,继续培养至24和48h。分别于收获前4h加入终浓度为0.2μg/ml的秋水仙碱。实验设PT-141高,中,低3个剂量组、并设阴性对照组,阳性对照组分不加S9试验阳性对照组(MMC0.25μg/ml)和加S9试验阳性对照组(CP20μg/ml,作用6h)。收获细胞后,常规低渗、固定、制片和Giemsa染色,每个剂量组油镜下观察分裂中期相细胞100个,记录畸变类型,计算细胞染色体畸变率(%)。各试验剂量均作平行样3份。结果细胞毒性实验显示,PT-141各剂量组,细胞数未发生显著差异,说明PT-141在高剂量5mg/ml未显示毒性反应。故正式实验选择高剂量为5mg/ml、按等比倍量稀释法,中、低剂量分别设定为1mg/ml、200μg/ml。染色体畸变结果显示,阳性对照组的畸变率明显高于阴性对照组,差异具有统计学意义(P<0.01),畸变类型以断裂、缺失、交换为主;CHL细胞接触PT-1412448h,加S9还是不加S9,各剂量组的染色体畸变率与阴性对照组比较差异均无统计学意义(P>0.05)。结论PT-141在体外CHL细胞染色体畸变试验中结果为阴性,在本实验条件下,未发现其具有潜在的致突变性。     

石房蛤毒素致突变性研究

摘 要：目的 通过Ames试验以及体外哺乳类细胞染色体畸变试验探索石房蛤毒素是否存在致突变性。 方法 通过Ames试验,选用组氨酸营养缺陷型鼠伤寒沙门氏菌TA97、TA98、TA100、TA102菌株,经过菌株鉴定及菌株毒性试验,在有无代谢活化系统存在的情况下分别对石房蛤毒素0.04、0.2、1.0 μg/皿三个剂量组进行试验,了解其是否具有致突变性。通过体外哺乳类细胞染色体畸变试验,采用MTT法检测石房蛤毒素对CHL细胞毒性,依据IC50,确定试验剂量后染毒,制片、吉姆萨染色,镜下观察染色体畸变数量及类型,计算畸变率,评价其致突变性。 结果 在有无S9活化系统条件下,在0.04、0.2、1.0 μg/皿测试浓度范围内,石房蛤毒素对测试菌株诱发产生的回变菌落数未达到溶剂对照组回变菌落数的2倍,且无剂量反应关系。石房蛤毒素对CHL细胞毒性测定结果显示,有S9时,其IC50值为1.20 μg/mL;无S9时,其IC50值为1.34 μg/mL。石房蛤毒素对CHL细胞染色体畸变作用的研究结果显示,各剂量组分别在加入S9混合液和未加S9混合液条件下染色体畸变数及畸变率与阳性对照组比较,存在显著性差异（P＜0.05）;与阴性对照组比较,均无显著性差异（P＞0.05）。结论 在本试验条件下,石房蛤毒素未见致突变性。     

中药提取物RX对染色体畸变作用的实验研究

目的观察中药提取物RX对染色体畸变作用。方法采用指数生长期CHL细胞,对受试药6个剂量组进行测试,观察其诱发CHL细胞染色体畸变率。结果中药提取物RX在试验所设6个剂量条件下,经24h、48h条件培养,未见有明显染色体畸变。结论中药提取物RX在试验剂量范围内,未发现有致突变作用。     

降血糖药格利齐特应用染色体畸变试验检测的诱变性

以培养的中国仓鼠肺成纤维细胞(CHL)染色体畸变试验,检测降血糖药格列齐特的诱变性。药物在87.5～350μg/ml 时与 CHL 细胞接触24～48h,不论肝微粒体酶复合因子(S_9)是否存在,CHL 细胞的染色体畸变率均为2～4%。     

地红霉素对中国仓鼠肺细胞体外染色体畸变的影响

目的:检测地红霉素对中国仓鼠肺细胞(CHL细胞)染色体致畸变的影响.方法:用体外方法检测地红霉素在12.50～50.0μg·mL-1浓度范围内,代谢活化组(含S9mix)及非活化组(不含S9mix) CHL细胞的染色体畸变率.结果:地红霉素的代谢活化组(含S9mix)及非活化组(不含S9mix)的染色体畸变率均低于3%.结论:地红霉素对CHL细胞染色体畸变实验结果为阴性.     

毛蚶提取物的遗传毒性试验研究

目的 评价毛蚶提取物的遗传毒性,为评价其安全性提供毒理学依据。方法 选用体外细菌回复突变(Ames)试验、中国仓鼠肺细胞(CHL)体外染色体畸变试验、小鼠骨髓嗜多染红细胞微核试验,进行毛蚶提取物的遗传毒性研究。Ames试验设5000.0、2500.0、1250.0、625.0、312.5 μg/皿5个剂量;体外CHL细胞染色体畸变试验,设立5.00、2.50、1.25 mg/mL 3个剂量,毛蚶提取物与CHL细胞分别接触6、24 h;小鼠骨髓微核试验,设立5000、2500、1250 mg/kg 3个剂量,给药1次。结果 Ames试验,毛蚶提取物在加与不加S9时各剂量组回变菌落数均未超过自发回变菌落数的2倍,亦无剂量相关性,结果为阴性;CHL染色体畸变试验,毛蚶提取物代谢活化组和非代谢活化组的染色体畸变率均低于5%,染色体畸变试验结果为阴性。小鼠骨髓微核试验,各剂量组的微核率与阴性对照组比较,差异不显著,结果为阴性。结论 在本试验条件下,毛蚶提取物未见潜在的遗传毒性。     

补血益母丸遗传毒性研究

目的 对补血益母丸干膏粉进行遗传毒性试验,为临床安全用药提供依据。方法 采用组氨酸营养缺陷型鼠伤寒沙门氏菌TA97a、TA98、TA100、TA102、TA1535进行细菌回复突变（Ames）试验,采用中国仓鼠肺成纤维（CHL）细胞进行体外染色体畸变试验,采用ICR小鼠进行骨髓细胞微核试验综合评估补血益母丸干膏粉的遗传毒性。其中,Ames试验设50、150、500、1 500、5 000μg·皿-1 5个剂量;体外细胞染色体畸变试验设125、250、500μg·mL-1 3个剂量;小鼠骨髓细胞微核试验设500、1 000、2 000 mg·kg-1 3个剂量,每天给药1次,连续给药3 d。结果 在代谢及非代谢活化条件下（+S9/-S9）,Ames试验结果显示,补血益母丸干膏粉对各菌株均无明显或可重复的诱变性及抑菌性;体外CHL细胞染色体畸变试验结果显示,补血益母丸干膏粉对CHL细胞染色体结构畸变率未见有意义的升高;小鼠骨髓细胞微核试验结果显示,补血益母丸干膏粉对ICR小鼠骨髓细胞微核率无明显影响。结论 补血益母丸干膏粉未见潜在的遗传毒性。     

NatA-50的遗传毒性试验

目的检测NatA-50是否具有潜在的遗传毒性,为临床应用提供安全性依据。方法采用小鼠骨髓嗜多染红细胞微核试验、Ames试验和中国仓鼠肺成纤维细胞(CHL)染色体畸变试验,分别从基因水平和细胞水平进行评价。结果小鼠微核试验显示,小鼠经口按5.0、2.5和1.25g/kg连续7d灌胃给予NatA-50,未见小鼠骨髓嗜多染红细胞微核数目增加;NatA-50的5个剂量组5000、2500、500、250和25μg/皿的Ames试验结果表明,加和不加大鼠干微粒体酶(S9)代谢活化剂,都未引起鼠伤寒沙门氏菌TA97、98、100和1024菌株突变菌落数目的增加或减少;体外CHL染色体畸变试验采用11.40、5.70、2.85、1.43和0.71μg/ml4个浓度,有无S9代谢活化剂参与,对CHL细胞均未发现明显的染色体损伤效应,结果均为阴性。结论在本试验条件下,未观察到NatA-50有遗传毒性。     

宫炎平片的遗传毒性试验研究

目的在体外条件下研究宫炎平片(GYP)是否具有遗传毒性,为临床用药安全提供依据。方法对GYP设不同剂量组,选用Ames试验、体外CHL细胞染色体畸变试验和小鼠骨髓嗜多染红细胞微核试验进行遗传毒性试验。结果Ames试验各组平皿均未见细菌毒性表现,结果为阴性。CHL试验中24 h后采样以及48 h后采样,所有剂量的细胞染色体畸变率均未显著增高,GYP无抑制有丝分裂或诱导染色体数目畸变的能力,微核试验结果为阴性无统计学意义(P>0.05)。结论综合以上3个试验结果,认为在实验条件下,GYP无遗传毒性。     

氟苯咪唑的致突变性试验

通过诱发 CHL 细胞染色体畸变的体外试验和小鼠骨髓细胞染色体的体内试验这两条途径来检测该药的致突变性,结果显示:在体内试验中,氟苯咪唑(640—1280mg/kg)与阴性对照组(N.S)相比,有非常显著的差异(p<0.01);在体外试验中,氟苯咪唑(0.39—1.56μg/ml)培养24h 和48h 对 CHL 细胞所诱发的染色体变化率显阳性。     

Genotoxicity study of bojungchisup-tang, an oriental herbal decoction-in vitro chromosome aberration assay in Chinese hamster lung cells andin vivo supravital-staining micronucleus assay with mouse peripheral reticulocytes

The toxicity evaluation of oriental herbal drugs is of great concern at present. Bojungchisuptang (BCST, in Korean), a decocted medicine of oriental herbal mixture, is now well used in clinic at oriental hospitals for the treatment of edema of several diseases in practice. However, the toxicity of the oriental herbal decocted medicines such as genetic toxicity is not well defined until now. In this respect, to clarify the genetic toxicity of BCST,in vitro chromosome aberration assay with Chinese hamster lung (CHL) fibroblasts andin vivo supravital micronucleus assay with mouse peripheral reticulocytes were performed in this study. In the chromosome aberration assay, we used 5,000 μg/ml BCST as maximum concentration because no remarkable cytotoxicity in CHL cells was observed both in the presence and absence of S-9 metabolic activation system. No statistical significant differences of chromosome aberrations were observed in CHL cells treated with 5,000, 2,500 and 1,250 μg/ml BCST for 6 hour both in the presence and absence of S-9 metabolic activation. However, very weak positive result (6.5∼8.0% aberration) of BCST was obtained in the absence of S-9 metabolic activation system at 5,000 μg/ml BCST when treated for 24 hour, i.e. 1.5 normal cell cycle time. And also,in vivo clastogenicity of BCST was studied by acridine orange-supravital staining micronucleus assay using mouse peripheral reticulocytes. We used 2,000 mg/kg as the highest oral dose in this micronucleus assay because no acute oral toxicity of BCST was observed in mice. The optimum induction time of micronucleated reticulocytes (MNRETs) was determined as 36 hours after oral administration of 2,000 mg/kg BCST. No significant differences of MNRETs between control and BCST treatment groups were observedin vivo micronucleus assay. From these results, BCST revealed very weak positive result in chromosome aberration assayin vitro with CHL cells and no clastogenicity in micronucleus assayin vivo.     

Effect of temperature on the frequency of chromosome aberrations and micronuclei in cultured Chinese hamster cells

We previously reported that both hyperthermia and hypothermia induced micronuclei in mouse bone marrow cells (Asanami and Shimono, 1997a, 1997b, 1999). To investigate the effects of temperature on chromosome aberration in vitro, we conducted chromosome aberration and micronucleus tests under hyper- and hypothermic conditions using Chinese hamster lung (CHL) cells. In the chromosome aberration test, we observed positive responses at 40 degrees C and 41 degrees C for 24 hr, and at 42 degrees C for 6 hr and over. In the micronucleus test, we observed positive responses at 31 degrees C, 33 degrees C, and 40 degrees C for 24 hr, and at 42 degrees C for 2 hr. The results suggest that in CHL cells, hypothermic conditions can induce micronuclei while hyperthermic conditions can induce both chromosome aberrations and micronuclei.     

大鼠尿中人参皂苷Rd及其代谢物的LC-MS研究

目的探讨人参皂苷Rd在大鼠体内的代谢产物及转化途径。方法选择SD大鼠6只，单剂量口服和静脉给予人参皂苷Rd，分段收集给药前和给药后0～24 h尿样，将尿样分时段合并后采用旋转薄膜蒸发浓缩，以固相萃取小柱纯化处理，采用高效液相色谱-串联飞行时间质谱进行检测。结果通过比较给药前后的TOF总离子流图，对尿中推测的代谢物和标准物质的出峰时间及相关化合物选择离子扫描二级质谱图进行了比较分析，结果在尿中发现了7种代谢产物，系统分析了这些代谢产物的代谢转化规律及可能结构。结论大鼠尿中人参皂苷Rd的主要转化途径为氧化、水解、结合及异构化代谢反应。     

Studies of the toxicological potential of tripeptides (L-valyl-L-prolyl-L-proline and L-isoleucyl-L-prolyl-L-proline): VIII. Assessment of cytotoxicity and clastogenicity of tripeptides-containing casein hydrolysate and Lactobacillus helveticus-fermented milk powders in Chinese hamster lung cells

The objective of this chromosomal aberration test was to assess the mutagenic potential of tripeptides by determining their ability to induce chromosomal aberrations in cultured Chinese hamster lung (CHL) cells. The test agents used in these experiments were (1) powdered casein hydrolysate (CH) and (2) powdered Lactobacillus helveticus-fermented milk (FM). Both test agents contain two tripeptides, L-valyl-L-prolyl-L-proline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP). CHL cells were cultured and exposed in the presence or absence of a rat hepatic metabolizing system (S9); CH or FM (1250, 2500, 5000 microg/ml of incubation medium); or positive-control agents, mitomycin C (0.1 or 0.05 microg/ml) or benzo(a)pyrene (20 microg/ml). In experiments with CH, cells were incubated for 6 h (either in the presence or absence of S9) or for 24 h (without S9). In experiments with FM, the cells were incubated for 6 h (either in the presence or absence of S9) or for 24 or 48 h (without S9). Neither short-term nor continuous exposure to either CH or FM caused the induction of significant changes in cell growth indices, incidences of chromosomal aberrations or the incidence of polyploids. Exposures of cells to mitomycin C and benzo(a)pyrene consistently resulted in the induction of the anticipated aberrant cells after either short-term or continuous exposures. The results of these assays support the conclusions that, under the conditions of these experiments, neither CH nor FM possesses demonstrable potential for the induction of cytotoxicity or clastogenesis.     

中药浮海石致突变的实验研究

目的研究中药浮海石致突变性。方法本试验采用鼠伤寒沙门菌回复突变试验(Ames试验),哺乳动物培养细胞(CHL)染色体畸变试验和小鼠骨髓细胞微核试验观察了浮海石的致突变作用。结果浮海石生理盐水浸提液0.5~5000μg/皿剂量下Ames试验结果阴性;250~1000μg/mL剂量下对CHL细胞无损伤作用,10~40g/kg剂量下对小鼠骨髓细胞染色体无致畸变作用。结论浮海石无致突变作用,用于临床是安全的。     

聚乙二醇修饰降纤酶的遗传毒性评价

目的检测聚乙二醇修饰降纤酶的遗传毒性。方法应用鼠伤寒沙门菌回复突变试验(Ames试验)、体外培养CHO细胞染色体畸变试验和小鼠骨髓微核试验检测聚乙二醇修饰降纤酶的遗传毒性。结果 Ames试验结果显示每平皿100、20、4、0.8、0.16 U各个剂量组,在加或不加S9代谢活化系统时对组氨酸缺陷型鼠伤寒沙门菌TA97、TA98、TA100、TA102及TA1535所诱发的回复突变菌落数均与溶剂对照的突变菌落数相近。体外培养CHO细胞染色体畸变试验结果显示2.5、5.0和10.0 U.mL-1各个剂量组在加S9代谢活化系统于24 h和不加S9代谢活化系统于24 h、48 h培养的CHO细胞染色体畸变率与溶剂对照组比较均无显著差异(P>0.05)。小鼠骨髓微核试验显示425、850、1700 U.kg-1各个剂量组对ICR小鼠的微核诱发率与溶剂对照组比较均无显著差异(P>0.05)。结论聚乙二醇修饰降纤酶对鼠伤寒沙门菌无致突变性,对哺乳动物培养细胞的染色体无致畸变作用,对ICR小鼠无诱发骨髓嗜多染红细胞微核的效应。表明聚乙二醇修饰降纤酶在本实验条件下无遗传毒性。     

Safety evaluation of Elsholtzia splendens extracts: assessment of acute toxicity and mutagenicity.

Much attention is recently gained for Elsholtzia splendens extracts and issue on their usage is raised due to their biological properties. However, there is no sufficient background information on toxicological evaluation of E. splendens extracts to give an assurance of safety for developing dietary supplements and functional foods. The objective of this study was to evaluate safety on E. splendens extracts using acute oral toxicity, bacterial reverse mutation, and chromosome aberration test. Total flavonoids within E. splendens were extracted with 80% of methanol by a reflux condenser. Both female and male mice were orally administrated E. splendens extracts at the dose of 0, 500, 1000, and 2000 mg/kg body weight/day. Mutagenicity of the extracts was evaluated in a bacterial reverse mutation assay using histidine requiring Salmonella typhimurium (TA 98, TA 100, TA 1535, and TA 1537) and tryptophan-requiring Escherichia coli (WP2uvrA). In vitro chromosome aberration assay in Chinese Hamster Lung (CHL) was conducted to evaluate genotoxicity. Single administration of dose levels of 500, 1000, and 2000 mg/kg body weight/day to mice for 15 days did not produce any significant mortality, clinical signs, body weight loss, and gross findings. E. splendens extracts in the range of 156.3-5000 microg/plate did not induce mutagenicity in S. typhimurium and E. coli with and without metabolic activation system. Any significant chromosomal aberration was not observed in CHL cells 6h after treating with the extract at the concentrations of 1250, 2500, and 5000 microg/mL in absence and presence of metabolic activation system. However, frequency of chromosomal aberration in 22 h after treatment without metabolic activation system was increased with showing a pattern of dose-response relationship. The highest concentration of 5000 microg/mL significantly induced chromosomal aberration. E. splendens extracts may induce chromosomal structure abnormality in CHL cells. This study suggests that further study is needed to assess the potential genotoxic effects of E. splendens extracts.     

Ginsenoside Rd prevents and rescues rat intestinal epithelial cells from irradiation-induced apoptosis

Panax ginseng has been shown to have a protective effect for irradiated animals or cells. Ginsenosides are the most active components isolated from ginseng, and ginsenoside Rd has been identified as one of the effective compounds responsible for the pharmaceutical actions of ginseng. In the present study, we studied the molecular mechanisms for the radio-protective action of ginsenoside Rd in rat intestinal epithelial IEC-6 cells. Cells were irradiated with gamma-ray, and apoptosis was examined using Hoechst staining and Western blot analysis. Treatment with ginsenoside Rd before gamma-irradiation inhibited irradiation-induced apoptosis in IEC-6 cells. Administration of Rd after irradiation also inhibited apoptosis in these cells. Irradiation of IEC-6 cells resulted in inactivation of Akt phosphorylation that was abrogated by Rd. On the other hand, irradiation activated phosphorylation of ERK1/2 but did not affect that of p38 MAPK. Inhibition of Akt phosphorylation prevented the reduction of apoptosis by Rd following irradiation. Pretreatment with an inhibitor of the MEK pathway further decreased the number of apoptotic cells. Rd decreased the ratios of Bax/Bcl-2 and Bax/Bcl-xL, the levels of cytochrome c, and the cleaved form of caspase-3 in irradiated IEC-6 cells. Our results suggest that ginsenoside Rd protects and rescues rat intestinal epithelial cells from irradiation-induced apoptosis through a pathway requiring activation of PI3K/Akt, inactivation of MEK, and also inhibition of a mitochondria/caspase pathway.