TK6和WTK1细胞tk位点突变敏感性的比较研究

目的比较研究2种起源于人类的tk^ /-杂合子细胞TK6和WTK1细胞对化合物——甲基磺酸甲酯(MMS)诱发tk位点突变的敏感性，为tk位点突变敏感细胞株的筛选提供实验依据。方法用标准诱变剂MMS处理TK6和WTK1细胞，对培养物进一步作tk位点突变测试，以及细胞p53基因蛋白表达水平的检测。结果MMS可诱导TK6和WTK1细胞tk位点的突变，其诱发突变分别是自发突变的2—7倍和3～10倍。WTK1细胞对MMS的细胞毒作用具有较大的抗性。MMS的作用下，WTK1细胞的突变频率分别是TK6细胞的15．7、19．0和20．4倍。在tk位点诱发了2种不同表型的突变集落，但以慢生长突变体为主。无论是自发突变还是MMS的诱发突变，WTK1细胞的突变频率均显著地高于TK6细胞。经MMS处理后，TK6细胞p53蛋白的表达水平增高更为明显。结论WTK1细胞是更为敏感的tk基因突变试验检测细胞株。MMS诱发的突变有染色体畸变和基因突变，但以染色体畸变为主。     

TK6细胞tk基因突变试验检测甲基磺酸甲酯的诱变性

本研究通过用标准诱变剂甲基磺酸甲酯(MMS)处理TK6细胞 ,对培养物进一步作tk位点突变测试 ,以及细胞p5 3基因蛋白表达水平的检测 .结果表明 ,MMS可诱导TK6细胞tk位点的突变 ,诱发突变是自发突变的 2～ 7倍 .在tk位点诱发了两种不同表型的突变集落 :即正常生长突变体 (tk NGmutant)和慢生长突变体 (tk SGmutant) .但以慢生长突变体为主 .MMS处理后 ,TK6细胞P5 3蛋白的表达水平增高 .本研究为将TK6细胞应用于我国tk基因突变的毒理学评价和机理的研究提供了实验依据 .     

骨水泥的遗传毒性研究

目的检测医疗器械进口产品骨水泥的遗传毒性。方法以骨水泥粉体浸提液与其液体混合液为供试液,采用细菌回复突变试验(Ames试验)和小鼠淋巴瘤细胞试验(MLA),在非代谢活化(-S9)和代谢活化(+S9)条件下检测。Ames试验检测了25、50、100μL/皿3个剂量组诱发鼠伤寒沙门氏菌(his-)TA98、TA100、TA1535、TA1537及大肠杆菌(trp-)WP2uvrA的回变菌落数;MLA检测了0.125、0.25、0.5%(V/V)3个浓度组,在+S9/3 h、-S9/3 h、-S9/24 h三种处理条件下诱发的L5178Y细胞tk-+/-基因突变频率(MF)和小集落突变百分率(SC%)。结果 +/-S9条件下,剂量组诱发五株菌的回变菌落数对比DMSO溶媒(阴性)对照组均无明显增加;-S9/24 h处理条件下0.5%(V/V)浓度组诱发的MF明显增加(P<0.01),但未达到阳性评判标准。结论骨水泥对试验菌的his-/trp-无明显诱变作用,对测试细胞的tk+/-及染色体无明显损伤作用。     

马兜铃酸诱导小鼠淋巴瘤细胞突变的机制

目的为研究马兜铃酸(Aristolochic Acid,AA)的致突变机制提供实验依据。方法挑取对照组和100μg/ml AA处理小鼠淋巴瘤L5178Y tk /--3.7.2-细胞得到的胸苷激酶(Thymidine kinase,tk)基因突变克隆,提取DNA,用基于PCR的杂合性缺失(Loss of heterozygosity,LOH)的分析方法检测含功能性tk基因(tk )的11号染色体(11 b)上的断裂情况。结果100μg/ml AA诱导的突变体中tk 基因缺失率为99.01%,其中所有的小克隆(Small Clone,SC)都失去了tk 基因,而大克隆(Large Clone,LC)的LOH发生率为96.6%。进一步分析11号染色体上的分布的3个微卫星位点(D11Mit42,D11Mit29,D11Mit74)的LOH状况如下:6 cm长度的DNA断裂频率为76%,38 cm长度DNA即超过染色体一半长度的DNA断裂频率为34%,整条染色体的缺失率为16%。另外LC在3个微卫星位点(D11Mit42,D11Mit29,D11Mit74)上的LOH发生率分别是:88%,60%和24%;SC的分别为:64%,8%和8%。结论AA是一个强染色体断裂剂。在11b染色体上LC的染色体断裂情况比LC严重。     

杜仲水煎剂的遗传毒性研究

目的 研究杜仲水煎剂的遗传毒性.方法 采用小鼠淋巴瘤细胞试验(MLA)和小鼠骨髓微核试验(MNT).在MLA中,2.5、5、10、20 mg·mal-1生药4个浓度组在非代谢活化(-S9))和代谢活化(+S9)条件下与15178Y细胞作用3 h,表达2 d,制备基因突变频率平板并培养12 d,计数大、小突变细胞集落的孔数,计算总突变率(MF)和小集落突变百分率(SC%);在MNT中,12.8、25.6、51.2 g·kg-1生药3个剂量组间隔24 h灌胃给药2次,制作骨髓涂片,计数每只小鼠两千个嗜多染红细胞中含微核的嗜多染红细胞数,计算每组动物嗜多染红细胞的平均微核率.结果 4个浓度组在+/-S9条件下诱发的MF呈现剂量相关性增加,与阴性对照组比较有统计学意义(P<0.05),-S9条件下的SC%与阳性对照组相仿,+S9条件下的SC%随浓度增加而升高.各剂量组未显示骨髓抑制作用,诱发的微核率与阴性对照组比较未见明显增加.结论 杜仲水煎剂在+/-S9条件下均可诱发L5178Y细胞tk位点突变并导致染色体损伤,提示对人体具有潜在的遗传毒性;但供试品对小鼠骨髓细胞染色体无损伤,经体内代谢活化后未显示遗传毒作用.     

对二甲基亚砜在小鼠淋巴瘤细胞实验中适宜浓度的探索

目的:通过采用小鼠淋巴瘤细胞实验微孔法,检测二甲基亚砜(DMSO)的细胞毒性及遗传毒性,探索其在检测系统中的适宜浓度。方法:在非代谢活化(-S9)条件下,检测美国Sigma和Tedi A两家公司生产的2个批号DMSO,终浓度0.5%,1.0%,2.0%,4.0%处理L5178Y细胞3 h后产生的细胞毒性及诱发的TK基因突变频率(MF)。结果:2个批号DMSO的细胞毒性均以1%浓度组为最低(<20%),4%浓度组为最高(>37%);1%浓度组诱发的MF与空白对照组相近,而其他3组则明显增加(P<0.05或0.01)。结论:当以DMSO为溶媒、采用L5178Y细胞进行MLA微孔法实验时,采用1%作为检测系统中受试物的体积比浓度较为适宜。     

基于L5178Y细胞的Pig-a基因突变试验方法学研究

目的:采用不同作用机制化合物研究基于L5178Y细胞体外Pig-a基因突变试验方法。方法:使用不同浓度范围的马兜铃酸I(aristolochic acid I,AAI)、N-亚硝基二乙胺(N-nitrosodiethylamine,NDEA)、甲磺酸甲酯(methyl methanesulfonate,MMS)、环磷酰胺(cyclophosphamide,CP)、秋水仙素(colchicine,COL)、丝裂霉素C(mitomycin C,MMC)、糖精、乙烯利、苯并芘[benzoa pyrene,B(a)P]和4-硝基喹啉-N-氧化物(4-nitroquinoline N-oxide,4-NQO)共10种化合物,分别与L5178Y细胞作用一段时间后表达8 d,细胞经APC抗CD45与PE抗CD90.2抗体孵育后使用流式细胞术收集100万个细胞并识别表型为CD90-/CD45⁺突变细胞,并计算突变率。结果:致突变剂AAI、NDEA、MMS、MMC、B(a)P、4-NQO分别在无或有代谢活化条件下导致L5178Y细胞Pig-a基因突变率显著性升高,而需代谢活化的致突变剂CP、整倍体诱变剂COL、非遗传毒性致癌物乙烯利和糖精的试验结果为阴性。结论:基于L5178Y细胞的体外Pig-a基因突变检测方法可在无及有代谢活化条件下有效检出致突变剂,特异性和可靠性较高,在药物基因突变风险评价及机制研究领域具有不可或缺的价值。     

金橙II及金胺O的体外遗传毒性评价

目的 使用基于鼠伤寒沙门氏菌的Ames波动试验和小鼠淋巴瘤细胞（L5178Y）的体外微核试验评价2种染料金橙Ⅱ和金胺O的遗传毒性风险。方法 在-/+S9处理下,不同质量浓度的金橙Ⅱ和金胺O（0.625~10.000 μg·mL^-1）分别与鼠伤寒沙门氏菌组氨酸营养缺陷型菌株TA98和TA100混合后接种于96孔板中,37℃下孵育72 h后判断其对细菌回复突变的影响;在体外微核试验中,在-/+S9处理下,金橙Ⅱ（10~50 μg·mL^-1）和金胺O（0.6~1.2 μg·mL^-1）分别与L5178Y细胞作用4 h或24 h,并在给药后24 h收集细胞进行流式细胞术检测。结果 金橙II自2.50 μg·mL^-1起可引起-S9和+S9处理组的TA98回复性突变孔数显著增加（P<0.05、0.01）,代谢活化后增加幅度更为明显;自5 μg·mL^-1起可引起-S9处理组的TA100回复性突变孔数显著性增加（P<0.01）,作用均呈浓度相关性。10 μg·mL^-1金胺O可引起-S9处理组的TA98回复性突变孔数显著增加（P<0.01）;自0.625 μg·mL^-1起即可引起+S9处理组的TA98回复性突变孔数显著增加（P<0.05、0.01）,且存在浓度相关性;自2.5 μg·mL^-1起可引起+S9处理组的TA100回复性突变孔数显著性增加（P<0.05、0.01）,且存在浓度相关性。在-S9处理组中,30~50 μg·mL^-1金橙Ⅱ可引起L5178Y细胞微核率显著增加（P<0.01）,且存在浓度相关性; 0.9~1.2 μg·mL^-1金胺O可引起L5178Y细胞微核率显著增加（P<0.01）,且存在浓度相关性。在+S9处理组中,10~50 μg·mL^-1金橙Ⅱ可导致L5178Y细胞微核率显著增加（P<0.05、0.01）。30~50 μg·mL^-1金橙Ⅱ可导致S期的L5178Y细胞比例增加;金胺O自0.6 μg·mL^-1起即可导致L5178Y细胞亚二倍体细胞核率增加。结论 金橙Ⅱ和金胺O均存在一定的遗传毒性风险。     

血清灭活对小鼠淋巴瘤试验结果的影响

目的 探索小鼠淋巴瘤胸腺激酶（tk）位点基因突变试验中血清灭活对结果的影响。方法 取对数生长期的小鼠淋巴瘤细胞（L5178Y3.7.2c-tk^+/-）设立阴性对照组和阳性对照组,阴性对照组加入100 μL DMSO,阳性对照组加入100 μL 10 μg/mL 4-硝基喹啉（4-NQO）诱导突变,测定细胞悬浮增长率（SG）、相对悬浮增长率（RSG）、平板效率（PE）、相对存活率（RS）和相对总生长率（RTG）等指标。根据血清灭活与否和选择突变剂三氟胸苷（TFT）的含量,在筛选突变集落过程中,将阴性对照组和阳性对照组各分为：灭活组、未灭活+1.5倍TFT组、未灭活组,测定突变频率（MF）,对试验结果进行评价。结果 阴性对照组SG在8~32,细胞倍增速度符合试验要求;PE在65%~120%,符合试验成立条件。阳性对照的灭活组、未灭活+1.5倍TFT组、未灭活组中MF均比阴性对照组至少增加300×10^-6且小克隆突变频率至少占40%,符合试验要求。阴性对照灭活组的MF数值在标准范围内,未灭活组的MF数值明显高于灭活组,超过标准数值范围,且96孔板中的大小克隆不易区分;阴性对照未灭活+1.5倍TFT组使得MF数值在标准范围内,但远高于灭活组。结论 在小鼠淋巴瘤试验中,测定MF的平板一定要用灭活的血清,未灭活血清可能通过抵消TFT作用对结果产生很大影响。     

芥子气体内生物标志物的检测及其代谢与分布研究进展

芥子气（sulfurmustard,HD）是一种糜烂性化学战剂,能造成皮肤、眼及呼吸系统等的损伤。虽然芥子气作用机制的研究开展较早,但迄今尚未完全阐明。芥子气中毒后可产生多种生物标志物,主要包括水解、氧化产物,谷胱甘肽、蛋白及DNA加合物,本文重点围绕这些生物标志物的检测技术、体内分布及代谢行为等研究进展进行评述。     

秋水仙碱和长春新碱诱导L5178Y小鼠淋巴瘤细胞tk基因杂合性缺失分析

目的探讨秋水仙碱(Col)和长春新碱(V in)诱导tk基因分子突变类型及机制。方法挑选经Col,V in诱导的tk基因突变子(tk-/-)和自发突变体,提取基因组DNA,经等位基因特异性PCR扩增,杂合性缺失(LOH)分析等技术,分析其tk基因杂合性缺失。结果Col和V in诱导突变体的tk基因LOH发生率分别为91.5%和92.1%。由LOH分析结果显示,诱导突变体的LOH发生率在自发突变与诱导突变之间,大集落与小集落之间差异均没有显著性。结论Col和V in诱导的tk基因是以功能性等位基因缺失为主的分子突变。     

Genotoxicity studies on green tea catechin

The beneficial effects of tea catechins are well documented. We evaluated the genotoxic potential of a green tea catechin preparation using established genotoxicity assays, including a bacterial reverse mutation assay (Ames test), a chromosomal aberration assay in cultured Chinese hamster lung cells (CHL/IU), a mouse lymphoma L5178Y/tk assay, and a bone marrow micronucleus (MN) assay in ICR CD mice and SD rats. No significant increases in the number of revertant colonies were observed in the Ames test, but positive responses were observed in two in vitro assays: the chromosomal aberration assay and mouse lymphoma L5178/tk assay. However, the in vivo study demonstrated no significant increase in micronucleated polychromatic erythrocytes (MNPCE) in the bone marrow of both ICR CD mice and SD rats administered a high dose of the green tea catechin preparation up to 2000 mg/kg. Combined with favorable epidemiological information suggesting a chemopreventive effect of tea catechins on carcinogenesis, we conclude that green tea catechin presents no significant genotoxic concern under the anticipated conditions of use. These results are consistent with other genotoxicity studies of tea catechins, which show minimal, if any, genotoxic potential.     

Evaluation of the butter flavoring chemical diacetyl and a fluorochemical paper additive for mutagenicity and toxicity using the mammalian cell gene mutation assay in L5178Y mouse lymphoma cells

Diacetyl (2,3-butanedione) is a yellowish liquid that is usually mixed with other ingredients to produce butter flavor or other flavors in a variety of food products. Inhalation of butter flavoring vapors was first associated with clinical bronchiolitis obliterans among workers in microwave popcorn production. Recent findings have shown irreversible obstructive lung disease among workers not only in the microwave popcorn industry, but also in flavoring manufacture, and in chemical synthesis of diacetyl, a predominant chemical for butter flavoring. It has been reported that perfluorochemicals utilized in food packaging are migrating into foods and may be sources of oral exposure. Relatively small quantities of perfluorochemicals are used in the manufacturing of paper or paperboard that is in direct contact with food to repel oil or grease and water. Because of recent concerns about perfluorochemicals such as those found on microwave popcorn bags (e.g. Lodyne P208E) and diacetyl in foods, we evaluated both compounds for mutagenicity using the mammalian cell gene mutation assay in L5178Y mouse lymphoma cells. Lodyne P208E was less toxic than diacetyl and did not induce a mutagenic response. Diacetyl induced a highly mutagenic response in the L5178Y mouse lymphoma mutation assay in the presence of human liver S9 for activation. The increase in the frequency of small colonies in the assay with diacetyl indicates that diacetyl causes damage to multiple loci on chromosome 11 in addition to functional loss of the thymidine kinase locus.     

Mutagenicity of the mycotoxin alternariol in cultured mammalian cells

The mycotoxin alternariol (AOH) is an important contaminant of fruit and cereal products. Concern about exposure to low levels of AOH was raised after the disclosure that contamination of food with the AOH-producing species Alternaria alternata is associated with oesophagal cancer. Previously we have reported that AOH induces kinetochore-negative micronuclei in cultured Chinese hamster V79 cells. The present study investigates the mutagenicity of AOH at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene locus in V79 cells and at the thymidine kinase (TK) gene locus in mouse lymphoma L5178Y tk(+/-) cells (MLC). Concentrations of 10 microM AOH and more gave rise to a significant and concentration-dependent induction of HPRT and TK mutations in V79 cells and in MLC, respectively. The mutagenic potency of AOH was about 50-fold lower than that of the established mutagen 4-nitroquinoline-N-oxide in both cell lines. Discrimination between small and large colonies in the TK assay revealed the predominant induction of small colonies, which are indicative for extensive chromosomal deletions and which correlated with the induction of micronuclei in MLC. The mutagenicity of AOH may have a bearing on the carcinogenicity of this mycotoxin.     

Bromodeoxyuridine resistance induced in mouse lymphoma cells by microsomal activation of dimethylnitrosamine.

Many chemicals are not mutagenic per se, but when metabolized by mammalian tissues yield mutagenic products. Dimethylnitrosamine (DMN) is such a promutagen. It has no effect on cell growth or mutant frequency when incubated alone with L5178Y mouse lymphoma cells, but exerts both mutagenic and toxic effects when incubated in a microsome reaction mixture. Microsomes were prepared from C3H/f We 16-wk-old male mice by the calcium preciptation technique. L5178Y continuously cultured mouse lymphoma cells heterozygous for thymidine kinase (TK+/-) were incubated for 15 min with calcium-precipitated microsomes and various concentrations of DMN in appropriate reaction mixtures. After a 48-hr expression time, treated cells were cloned in soft agar with and without bromodeoxyuridine (BUdR) (50 mug/ml); 10 days later colonies grown to greater than about 200 mum diameter were counted. The frequency of BUdR-resistant (mutant) colonies increased linearly with the DMN concentration. A reconstruction experiment showed that the assay conditions did not significantly alter the relationship between parent and BUdR-resistant cells in growth and cloning efficiency. The smallest dose of DMN used in these experiments was 100mumol/liter, the one-sided (100 mumol greater than control frequency) -p value is 0.036. The locus is extremely sensitive to mutagenesis by DMN compared with other known mutagens at similar levels of cell survival.     

Evaluation of ochratoxin A for mutagenicity in a battery of bacterial and mammalian cell assays

Ochratoxin A (OA), a nephrotoxic mycotoxin, was evaluated for genotoxic potential in a battery of in vitro and in vivo assays. OA was not mutagenic to Salmonella typhimurium, either with or without metabolic activation, in the plate incorporation (Ames) test at concentrations of 50-600 micrograms OA/plate or in the gradient plate assay at concentrations of 0.1-1000 micrograms OA/ml. No induction of unscheduled DNA synthesis was evident in primary cultures of rat hepatocytes exposed to concentrations of OA ranging from 0.000025 to 500 micrograms/ml. In the mouse lymphoma forward mutation assay, exposure of L5178Y TK+/- mouse lymphoma cells to OA did not increase the numbers of L5178Y TK-/- mutants. There was no significant difference between the numbers of sister-chromatid exchanges in cells from OA-treated Chinese hamsters and those in cells from the negative-control animals.     

Mutagenicity of chromium picolinate and its components in Salmonella typhimurium and L5178Y mouse lymphoma cells.

Chromium picolinate is one of the most commonly used chromium dietary supplements available in the United States, and it has been marketed to consumers for use in weight loss, increasing muscle mass, and lowering serum cholesterol. Chromium picolinate is a synthetic compound that provides a bioavailable form of Cr(III) that is absorbed better than dietary chromium. However, there are several reports that it can have adverse effects. In order to study the mechanism of observed cellular toxicity and mutagenicity, chromium picolinate and its component compounds, chromium (III) chloride and picolinic acid, were evaluated in Salmonella typhimurium and L5178Y mouse lymphoma cells. Neither chromium picolinate nor chromium chloride induced a mutagenic response in S. typhimurium. However, in the L5178Y mouse lymphoma mutation assay, chromium picolinate induced mutagenic responses without and with the addition of S9.     

New studies on the in vitro genotoxicity of sodium molybdate and their impact on the overall assessment of the genotoxicity of molybdenum substances

The potential of molybdenum substances to cause genotoxic effects has been studied previously. However, a review of existing in vitro data, including an assessment of relevance and reliability, has shown that inconsistent results have been observed in the past. To resolve the inconsistencies, new studies were performed with the highly soluble sodium molybdate dihydrate according to OECD test guidelines. In a bacterial reverse mutation assay sodium molybdate dihydrate did not induce reverse mutations in five strains of Salmonella typhimurium. No mutagenic or clastogenic effect was observed at the tk locus of L5178Y mouse lymphoma cells. In a micronucleus test in cultured human peripheral blood lymphocytes no clastogenic or aneugenic effects were seen. These results can be read across to other inorganic molybdenum substances, that all release the molybdate ion [MoO₄]²⁻ under physiological conditions as the only toxicologically relevant species. In summary, a weight of evidence assessment of all available in vitro data shows no evidence of genotoxicity of molybdenum substances.