人类磷脂酰肌醇聚糖A类基因突变在遗传毒性研究中应用进展

基因突变是遗传毒性损伤的重要检测终点。国际人用药物注册技术协调会议(ICH)M7指南中将啮齿类动物体内磷脂酰肌醇聚糖A类(Pig-a)基因突变试验列为药物杂质遗传毒性试验阳性结果的追加试验。啮齿类动物Pig-a基因突变试验作为一项新的检测体内基因突变的方法,相对于转基因动物等其他体内基因突变试验,具有成本低、方法简单和检测快速的优点。基于Pig-a基因的物种保守性,近年来开发了检测人外周血以及体外人细胞的PIG-A基因突变试验的方法,以期用于临床基因突变监测以及非临床遗传毒性评价。本文主要对PIG-A基因突变试验研究常用人外周血成熟红细胞、网织红细胞和白细胞及TK6细胞和MCL-5细胞检测方法等进行简要综述。     

Pig-a基因突变检测方法在药物遗传毒性评价中的应用及研究进展

ICH修订技术指导原则将动物实验的3Rs原则(减少、替代、优化)引入到遗传毒性试验中,对新试验方法体系提出了更高的要求。Pig-a基因突变检测是近年建立的遗传毒性体内试验方法,其特点是使用流式细胞仪对外周血红细胞进行分析,所需血液样品量较少,同时具有无创、连续、动态监测和检测窗口期长等优点。此外,该方法能够结合重复给药毒性试验进行多终点检测,符合动物实验3Rs原则,适应当前毒理学研究的发展趋势。本文对Pig-a基因突变检测方法及研究进展作一综述。     

Pig-a基因突变试验研究与应用进展

Pig-a基因位于人类X染色体短臂,参与糖基磷脂酰肌醇(GPI)连接蛋白的早期合成。Pig-a基因突变后不能正常合成GPI连接蛋白,致使细胞表型缺失,是阵发性睡眠性血红蛋白尿症发病的分子机制。该特性被应用于一项新的体内致突变试验——Pig-a基因突变试验:动物经过一次或多次染毒,一段时间后收集少量目标细胞,即可采用流式细胞术或有限稀释法对细胞突变频率进行定量检测,从而判定化学物的致突变性。Pig-a基因突变试验具有相对快捷、样品需求量小、可与重复毒性试验相结合的优势,能够更为全面可靠地评价受试物遗传毒性,被认为具有良好的应用前景。     

大黄素体内遗传毒性风险评价

目的 评价连续28 d灌胃给予小鼠大黄素,小鼠体内遗传毒性风险。方法 KM小鼠连续28 d灌胃重复给予大黄素并设28 d恢复期。分别于给药前、首次给药后第14、 28、 42和56天采集外周血检测网织红细胞和总红细胞的突变率,以及网织红细胞占总红细胞的百分率;末次给药后采集肝、肾、外周血开展彗星试验,分析每只动物至少100个细胞的尾DNA百分含量;末次给药后制备骨髓细胞样本,计算嗜多染红细胞占总红细胞的百分率以及嗜多染红细胞的微核发生率。结果 与溶媒对照组（0.5%羧甲基纤维素钠）相比,300 mg·kg-1及以下给药组连续给药28 d未见Pig-a基因突变率增加。肾细胞彗星试验结果显示,与溶媒对照组（0.5%羧甲基纤维素钠）相比,所设浓度范围内各组尾DNA百分含量显著升高（P<0.01,P<0.001）。骨髓微核试验显示所设浓度范围内未见微核率的升高。结论 连续28 d经口灌胃给予小鼠大黄素后,主要作用部位为肾脏;当前试验条件下,大黄素未导致小鼠体内Pig-a基因突变发生率和骨髓微核发生率增加,但可导致肾脏细胞DNA损伤。     

含银离子烧伤敷料的遗传毒性研究

目的：采用哺乳动物细胞和细菌对含银离子烧伤敷料进行遗传毒性研究.方法：通过鼠伤寒沙门菌回复突变试验（Ames试验）、体外哺乳动物细胞染色体畸变试验和体外哺乳动物细胞基因突变试验,观察该敷料浸提液是否引起细胞的基因突变、染色体结构的改变或DNA和基因的改变.结果：该试样在3.0 cm2/ml（浸提原液剂量）样品的二甲基亚砜（DMSO）浸提液对试验所用鼠伤寒沙门菌有抑制作用,3.0 cm2/ml样品浸提液对中国仓鼠肺成纤维细胞（CHL）具有毒性作用,对体外培养的V79细胞无诱导HGPRT基因突变作用.结论：结果显示在一定的剂量下,该含银离子烧伤敷料未见引起明显的遗传毒性作用,而浸提原液剂量组具有一定的遗传毒性作用.     

体内基因突变试验研究进展

遗传毒性研究是新药非临床安全性评价的重要内容之一。由于遗传毒性体外细胞试验不能完全反映整体动物系统对受试物的吸收、分布、代谢和排泄等情况,而且试验结果的假阳性率过高,因此采用体外细胞试验进行药物安全性评价尚存在一定局限性。为了进一步完善对致突变风险的控制,尚需建立更加灵敏的体内基因突变试验方法。本文主要就体内基因突变试验的基本原理、优缺点及基本应用等研究进展进行综述,并对这些体内试验的发展和完善提出一些展望。     

体内彗星试验在药物遗传毒性评价中的研究方法

体内彗星试验是采用单细胞凝胶电泳的方法检测体内DNA损伤的技术。具有灵敏度高、操作简便、经济快速等优点。随着遗传毒性研究的发展,体内彗星试验已经成为重要的药物遗传毒性评价方法。对动物试验阶段和彗星试验阶段各操作步骤进行了归纳总结,以期对相关方法学建立提供参考,并提出采用简化和标准化的研究方法将有利于体内彗星试验在药物遗传毒性评价的应用。     

抗早孕植物HG的致突变试验

对抗早孕植物HG提取物进行致突变试验。结果表明,HG提取物经体外试验——Ames试验和染色体畸变试验及休内试验——小鼠微核试验检测均呈阴性,提示HG提取物不引起体外基因突变和染色体畸变,也不引起体内骨髓细胞染色体的损伤,存在遗传毒性的可能性较小。     

小鼠淋巴瘤细胞试验用于突变检测的研究进展

自从1972年小鼠淋巴瘤细胞试验（mouse lymphoma assay MLA）建立以来，该试验已经广泛地用于各种理化因素的遗传毒性评价。其使用的细胞株小鼠淋巴瘤L5178Ytk^＋/-，-3．2．7c细胞在11号染色体上存在￡南等位基因。由于只含有1个有活性的峨基因，因此可以检测出以耐药性为指标的基因突变（如tk^＋/-→tk^＋/-），该方法的优点是不仅可以检测点突变等小的基因突变，     

现行药物、保健食品和化妆品Ames试验方法的比较分析

Ames试验广泛应用于药物、食品、化妆品及医疗器械的遗传毒性研究,是应用最广泛的检测基因突变的体外试验.药物、保健食品和化妆品Ames试验方法有一定的差异,该文就药物、保健食品和化妆品Ames试验方法的主要异同点作一比较分析.     

Genetic toxicity assessment: employing the best science for human safety evaluation part IV: Recommendation of a working group of the Gesellschaft fuer Umwelt-Mutationsforschung (GUM) for a simple and straightforward approach to genotoxicity testing.

Based on new scientific developments and experience of the regulation of chemical compounds, a working group of the Gesellschaft fuer Umweltmutationsforschung (GUM), a German-speaking section of the European Environmental Mutagen Society, proposes a simple and straightforward approach to genotoxicity testing. This strategy is divided into basic testing (stage I) and follow-up testing (stage II). Stage I consists of a bacterial gene mutation test plus an in vitro micronucleus test, therewith covering all mutagenicity endpoints. Stage II testing is in general required only if relevant positive results occur in stage I testing and will usually be in vivo. However, an isolated positive bacterial gene mutation test in stage I can be followed up with a gene mutation assay in mammalian cells. If this assay turns out negative and there are no compound-specific reasons for concern, in vivo follow-up testing may not be required. In those cases where in vivo testing is indicated, a single study combining the analysis of micronuclei in bone marrow with the comet assay in appropriately selected tissues is suggested. Negative results for both end points in relevant tissues will generally provide sufficient evidence to conclude that the test compound is nongenotoxic in vivo. Compounds which were recognized as in vivo somatic cell mutagens/genotoxicants in this hazard identification step will need further testing. In the absence of additional data, such compounds will have to be assumed to be potential genotoxic carcinogens and potential germ cell mutagens.     

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 )、甲磺酸甲醋(methyl methanesulfonate,MMS ...     

A battery of in vivo and in vitro tests useful for genotoxic pollutant detection in surface waters

Since the 1980s, stricter water quality regulations have been promulgated in many countries throughout the world. We discuss the application of a battery of both in vivo and in vitro genotoxicity tests on lake water as a tool for a more complete assessment of surface water quality. The lake water concentrated by adsorption on C18 silica cartridges were used for the following in vitro biological assays: gene conversion, point mutation, mitochondrial DNA mutability assays on the diploid Saccharomyces cerevisiae D7 strain, with or without endogenous P450 complex induction; DNA damage on fresh human leukocytes by the comet. Toxicity testing on yeast and human cells was also performed. In vivo genotoxicity was determined by the comet assay on two well-established bio-indicator organisms of water quality (Cyprinus carpio erythrocytes and Dreissena polymorpha haemocytes) exposed in situ. The in vivo experiments and the water samplings were carried out during different campaigns to detect seasonal variations of both the water contents and physiological state of the animals. Temperature and oxygen level seasonal variations and different pollutant contents in the lake water appeared to affect the DNA migration in carp and zebra mussel cells. Seasonal variability of lake water quality was also evident in the in vitro genotoxicity and cytotoxicity tests, with regards to water pollutant quantity and quality (direct-acting compounds or indirect-acting compounds on yeast cells). However, the measured biological effects did not appear clearly related to the physical-chemical characteristics of lake waters. Therefore, together with the conventional chemical analysis, mutagenicity/genotoxicity assays should be included as additional parameters in water quality monitoring programs: their use could permit the quantification of mutagenic hazard in surface waters.     

Investigating the relationship between in vitro-in vivo genotoxicity: derivation of mechanistic QSAR models for in vivo liver genotoxicity and in vivo bone marrow micronucleus formation which encompass metabolism

Strategic testing as part of an integrated testing strategy (ITS) to maximize information and avoid the use of animals where possible is fast becoming the norm with the advent of new legislation such as REACH. Genotoxicity is an area where regulatory testing is clearly defined as part of ITS schemes. Under REACH, the specific information requirements depend on the tonnage manufactured or imported. Two types of test systems exist to meet these information requirements, in vivo genotoxicity assays, which take into account the whole animal, and in vitro assays, which are conducted outside the living mammalian organism using microbial or mammalian cells under appropriate culturing conditions. Clearly, with these different broad experimental categories, results for a given chemical can often differ, which presents challenges in the interpretation as well as in attempting to model the results in silico. This study attempted to compare the differences between in vitro and in vivo genotoxicity results, to rationalize these differences with plausible hypothesis in concert with available data. Two proof of concept (Q)SAR models were developed, one for in vivo genotoxicity effects in liver and a second for in vivo micronucleus formation in bone marrow. These "mechanistic models" will be of practical value in testing strategies, and both have been implemented into the TIMES software platform ( http://oasis-lmc.org ) to help predict the genotoxicity outcome of new untested chemicals.     

Current regulatory perspectives on genotoxicity testing for botanical drug product development in the U.S.A.

Genotoxicity testing is an important part of preclinical safety assessment of new drugs and is required prior to Phase I/II clinical trials. It is designed to detect genetic damage such as gene mutations and chromosomal aberration, which may be reflected in tumorigenic or heritable mutation potential of the drug. Botanical new drugs in the U.S. are entitled to a waiver for preclinical pharmacology/toxicology studies, including genotoxicity testing, in support of an initial clinical trial under IND, contingent on previous human experience. Recently, ethical concerns have been raised over conducting Phase I/II clinical trials of new drugs with positive genotoxicity findings in healthy volunteers. Although the relevance of this issue to patients, as opposed to healthy volunteers, depends on the drug’s indication, duration of treatment, and specific findings related to the assays, the regulatory view is to avoid exposing patients to genotoxic compounds unnecessarily in clinical trials. This philosophy may impact on herbal supplement marketing and botanical drug development, in that genotoxicity data are often lacking while consumers are exposed to the herbal supplement, or healthy volunteers are tested in an initial Phase I/II clinical trial on the botanical drug. This paper presents results of a survey conducted on genotoxicity data in botanical INDs submitted to the Agency and discusses the significance of this information. The information presented indicates that the sponsors of botanical INDs have increasingly recognized the importance of genotoxicity information and may have prioritized its acquisition in their strategic drug development programs.     

The value and limitations of short-term genotoxicity assays and the inadequacy of current criteria for selecting chemicals for cancer bioassays

The Salmonella gene mutation assay is established as the primary in vitro test for genotoxicity. It does not, however, detect all known genotoxic carcinogens, and several mammalian cell genotoxicity assays are now used to complement the Salmonella test--the most usual being cytogenetic analysis in vitro. Use of these two tests provides an adequate screen for genotoxins, but leaves undetected a variety of weak, DNA-unreactive animal carcinogens such as DDT, saccharin and diethylhexyl phthalate. A reappraisal of the present confused situation examines the underlying problems and options for action. It is suggested that the most effective way of focussing resources on the detection of possible new human carcinogens involves in vitro evaluation of genotoxicity, followed where appropriate by further evaluation in vivo, with urgent attention being paid to the methods currently used to select chemicals for cancer bioassays and to the resolution of the current debate on the validity of some recent classifications of carcinogenicity.     

Genotoxicity studies OF p-dimethylaminoazobenzene (DAB)

The potential genotoxicity of the rodent liver carcinogen p-dimethylaminoazobenzene (DAB) was evaluated in compliance with the guidelines for genotoxicity studies of drugs (Notification No. 1604, Nov. 1, 1999, Ministry of Health and Welfare, Japan) and the OECD guidelines for testing chemicals. DAB was clearly positive in both the bacterial reverse mutation test (Ames test) and in vitro chromosomal aberration test in the presence of metabolic activation, whereas it was weakly positive at toxic doses in the rat bone marrow micronucleus test. It has been reported that DAB was clearly positive in in vivo genotoxicity tests, i.e., a mouse alkaline single cell gel electrophoresis (comet) assay and a young rat liver micronucleus test. These results suggest that the test system using the liver is effective for in vivo genotoxicity assessment of chemicals that show mutagenicity in in vitro genotoxicity tests in the presence of metabolic activation.     

Gene toxicity studies on titanium dioxide and zinc oxide nanomaterials used for UV-protection in cosmetic formulations

Abstract

Titanium dioxide and zinc oxide nanomaterials, used as UV protecting agents in sunscreens, were investigated for their potential genotoxicity in in vitro and in vivo test systems. Since standard OECD test methods are designed for soluble materials and genotoxicity testing for nanomaterials is still under revision, a battery of standard tests was used, covering different endpoints. Additionally, a procedure to disperse the nanomaterials in the test media and careful characterization of the dispersed test item was added to the testing methods. No genotoxicity was observed in vitro (Ames' Salmonella gene mutation test and V79 micronucleus chromosome mutation test) or in vivo (mouse bone marrow micronucleus test and Comet DNA damage assay in lung cells from rats exposed by inhalation). These results add to the still limited data base on genotoxicity test results with nanomaterials and provide congruent results of a battery of standard OECD test methods applied to nanomaterials.     

Surfactants: a survey of short-term genotoxicity testing

The available results for tests on over 200 surfactants in nine short-term genotoxicity assay systems were reviewed. These tests included the Salmonella/microsome mutation assay, bacterial DNA repair tests, mitotic recombination in Saccharomyces cerevisiae, the mouse lymphoma cell-mutation assay, unscheduled DNA synthesis and sister chromatid exchange assays in mammalian cells, mammalian chromosome damage tests in vitro and in vivo, the dominant lethal test in rodents, and mammalian cell-transformation tests. The collected data cover all four major classes of surfactants: anionic, cationic, nonionic and amphoteric. The results of these genotoxicity tests were overwhelmingly negative. Although there were occasional positive results in bacterial or cell-transformation systems, the testing performed to date indicates that surfactants have negligible potential to cause genetic damage. The available data also indicate that none of the assays were incompatible with testing surfactants for genotoxicity.