食品中典型有机污染物和重金属的毒性通路研究进展

食品污染是影响食品安全和人群健康的重要风险因素。《21世纪毒性测试愿景与战略》提出未来的毒性测试应充分考虑人群暴露可能，基于体外人源性细胞系、高通量筛选和毒性通路的毒性测试战略，试图解决传统毒性测试成本高昂、耗时费力、种属差异方面的挑战。本综述围绕我国食品污染问题突出的典型重金属和有机污染物，分析归纳了有关毒性通路研究现况，并展望未来的研究方向。     

三种细胞形态学分析方法在体外微核检测中的比较研究

【目的】探讨基于流式细胞术(FCM)和细胞高内涵筛选技术(HCS)的两种高通量体外微核试验方法应用于化学品遗传毒性评价的可行性。【方法】参考经济合作与发展组织(OECD)TG487推荐的程序方法,采用化学品遗传毒性评价的典型阳性剂环磷酰胺(CP)、丝裂霉素C(MMC)为受试物,CP剂量范围为5~20 mg/L,MMC剂量范围为0.25~1.0 mg/L,分别设3个剂量组,短时处理(4 h)体外培养的CHL细胞模型,分别建立基于FCM和HCS的高通量体外微核测试方法。平行采用传统胞质分裂阻断微核法,在加/不加代谢活化系统条件下,检测各剂量组细胞的微核形成情况,并分析微核细胞率。实验同步设置阴性对照组(无血清MEM培养液)。【结果】经常规人工显微镜阅片和高通量方法的FCM及HCS得到的CP、MMC体外微核试验结果均为阳性:经CP诱导产生的微核细胞率(CP浓度由低到高)依次为1.9%、7.6%、10.4%(人工阅片),2.8%、2.6%、7.8%(FCM),2.8%、6.2%、9.1%(HCS);经MMC诱导产生的微核细胞率(MMC浓度由低到高)依次为5.9%、11.4%、16.7%(人工阅片),3.2%、3.7%、5.1%(FCM),7.9%、10.1%、10.2%(HCS)。受试物各剂量组与阴性对照组相比,差异均有统计学意义(P<0.05),且呈现剂量-反应关系。【结论】在本试验条件下,流式细胞术、高内涵体外微核检测的方法与传统人工阅片的方法得到的检测结果均一致,提示体外微核高通量方法可以实现细胞微核形成的自动化检测,提高检测效率,可为将高通量体外微核测试方法纳入化学品遗传毒性评价相关标准提供数据支持。     

急性毒性体外筛选方法的比较

目的比较4种急性毒性的体外筛选方法,为建立高通量的急性毒性筛选方法提供依据。方法选用人HepG2细胞,运用96孔板体外细胞培养技术,对47种已知体内急性毒性的化合物用4种细胞毒性的检测方法及5个评价指标进行检测,并将体外评价指标与体内评价指标LD50进行分析比较。结果MTT、中性红、LDH及刃天青4种检测方法中,以中性红法检测结果与体内LD50的相关性最好,评价指标中4种检测方法均以IC45与LD50的相关性最高。结论选用人HepG2细胞,运用微孔板体外细胞培养技术,4种细胞毒性检测方法中以中性红法方法最好,评价指标以IC45为宜,可作为建立高通量的急性毒性筛选方法的依据。     

胚胎毒性体外试验的研究进展

胚胎毒性体外试验已广泛应用于胚胎生殖发育毒物筛选和致畸机制研究等方面,现已有三十余种不同类型的毒性测试方法。欧洲替代方法研究中心推荐的有效性较高的3个胚胎毒性体外筛选试验,即体外全胚胎培养试验、胚胎细胞微团培养试验和胚胎干细胞试验具有良好的应用价值,就以上试验的研究方法、应用价值及研究进展综述。     

胚胎毒性体外试验的研究进展

胚胎毒性体外试验已广泛应用于胚胎生殖发育毒物筛选和致畸机制研究等方面,现已有三十余种不同类型的毒性测试方法。欧洲替代方法研究中心推荐的有效性较高的3个胚胎毒性体外筛选试验,即体外全胚胎培养试验、胚胎细胞微团培养试验和胚胎干细胞试验具有良好的应用价值,就以上试验的研究方法、应用价值及研究进展综述。     

高内涵筛选技术应用于药物毒理方面的研究进展

高内涵筛选技术(HCS)是基于高效新药筛选需求发展起来的一项新技术,基于活细胞、多参数、低成本、高通量及高灵敏度优点,能够快速实现对化合物多种生物活性及毒性的早期快速检测,并且对药物毒理机制研究提供帮助,是药物细胞毒理学研究优秀的技术手段。目前,HCS已用于多种靶器官细胞毒性、遗传毒性、神经毒性等的检测以及毒理学分子机制的研究。     

肾毒性评价体外替代模型研究进展

随着《21世纪毒性测试:愿景与策略》的提出,毒性测试的重点开始从整体动物实验转向基于人类细胞或细胞组分等替代方法的测试策略。由于肾毒性物质的靶器官选择性,且药物诱导的肾损伤是新药开发时需解决的重要问题,故需良好的体外替代模型来评价包括药物在内的外源性化合物的肾脏毒性。然而,现有的体外模型由于缺少肾小管上皮细胞在体内的形态及功能,难以预测外源性化合物的肾脏毒性。肾体外替代模型的细胞来源、特点、培养条件以及检测终点是在建立体外替代模型时重点考虑的问题。多功能干细胞诱导分化肾细胞的出现、3D培养及肾脏芯片技术的发展、组学技术、高内涵筛选的应用为体外模型的建立及模型预测结果从体外到体内的外推提供了新思路。     

创新药物体外ADMET的高通量筛选技术研究进展

目前的新药研发策略已从传统的以活性为主导的筛选模式,转变为在测定药效的同时平行评价化合物的ADMET性质,并建立了系列体外ADMET高通量筛选技术.本文综述了近年来药物体外ADMET高通量筛选技术的研究进展.     

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。     

在Tox21化学库筛查乙酰胆碱酯酶抑制剂

随着Tox21的持续发展, 体外测试系统优化和新兴替代方案开发已经成为当前化学物毒性评价的研究重点。美国国立卫生研究院(National Institutes of Health, NIH)的一项研究成果于2021年4月发表在《Environmental Health Perspectives》杂志, 该研究基于定量高通量筛查系统(quantitatively high-throughput screening, qHTS)高效、迅速筛查Tox21 10K文库中具有乙酰胆碱酯酶(acetylcholinesterase, AChE)抑制能力的化学物, 为化学物毒性评价的新兴体外实验流程提供范例。     

Application of chemical toxicity distributions to ecotoxicology data requirements under REACH

The European Union's REACH regulation has further highlighted the lack of ecotoxicological data for substances in the marketplace. The mandates under REACH (registration, evaluation, authorization, and restriction of chemicals) to produce data and minimize testing on vertebrates present an impetus for advanced hazard assessment techniques using read-across. Research in our group has recently focused on probabilistic ecotoxicological hazard assessment approaches using chemical toxicity distributions (CTDs). Using available data for chemicals with similar modes of action or within a chemical class may allow for selection of a screening point value (SPV) for development of environmental safety values, based on a probabilistic distribution of toxicity values for a specific endpoint in an ecological receptor. Ecotoxicity data for acetylcholinesterase inhibitors and surfactants in Daphnia magna and Pimephales promelas were gathered from several data sources, including the U.S. Environmental Protection Agency's ECOTOX and Pesticides Ecotoxicity databases, the peer-reviewed literature, and the Human and Environmental Risk Assessment (HERA) project. Chemical toxicity distributions were subsequently developed, and the first and fifth centiles were used as SPVs for the development of screening-predicted no-effect concentrations (sPNECs). The first and fifth centiles of these distributions were divided by an assessment factor of 1,000, as recommended by REACH guidance. Use of screening values created using these techniques could support the processes of data dossier development and environmental exposure assessment, allowing for rigorous prioritization in testing and monitoring to fill data gaps.     

Chemical genomics profiling of environmental chemical modulation of human nuclear receptors

Background: The large and increasing number of chemicals released into the environment demands more efficient and cost-effective approaches for assessing environmental chemical toxicity. The U.S. Tox21 program has responded to this challenge by proposing alternative strategies for toxicity testing, among which the quantitative high-throughput screening (qHTS) paradigm has been adopted as the primary tool for generating data from screening large chemical libraries using a wide spectrum of assays.Objectives: The goal of this study was to develop methods to evaluate the data generated from these assays to guide future assay selection and prioritization for the Tox21 program.Methods: We examined the data from the Tox21 pilot-phase collection of approximately 3,000 environmental chemicals profiled in qHTS format against a panel of 10 human nuclear receptors (AR, ERα, FXR, GR, LXRβ, PPARγ, PPARδ, RXRα, TRβ, and VDR) for reproducibility, concordance of biological activity profiles with sequence homology of the receptor ligand binding domains, and structure–activity relationships.Results: We determined the assays to be appropriate in terms of biological relevance. We found better concordance for replicate compounds for the agonist-mode than for the antagonist-mode assays, likely due to interference of cytotoxicity in the latter assays. This exercise also enabled us to formulate data-driven strategies for discriminating true signals from artifacts, and to prioritize assays based on data quality.Conclusions: The results demonstrate the feasibility of qHTS to identify the potential for environmentally relevant chemicals to interact with key toxicity pathways related to human disease induction.     

3D细胞模型在化学物质毒性测试中的应用研究进展

在化学物质毒性预测及风险评估中,体外细胞模型具有较高的实际应用价值。随着3D生物打印、类器官培养、器官芯片等技术的迅速发展,体外细胞模型取得了长足的进步。相比起2D细胞模型,3D细胞模型在结构、功能及生理环境上与在体组织、器官更为接近。基于3D细胞模型进行化学物质的危害识别及剂量-反应关系分析,可以获得较为准确的毒性数...     

Cytochromes P450 and species differences in xenobiotic metabolism and activation of carcinogen.

The importance of cytochrome P450 isoforms to species differences in the metabolism of foreign compounds and activation of procarcinogens has been identified. The possible range of P450 isozymes in significant variations in toxicity exhibited by experimental rodent species may have a relevance to chemical risk assessment, especially as human P450s are likely to show changes in the way they metabolize xenobiotics. Consequently, in the safety evaluation of chemicals, we should be cautious in extrapolating results from experimental animal models to humans. This paper focuses on examples in which species differences in P450s lead to significant alterations in carcinogenic response, and includes a discussion of the current procedures for toxicity screening, with an emphasis on short-term tests.     

Innovative non-animal testing strategies for reproductive toxicology: the contribution of Italian partners within the EU project ReProTect

Reproductive toxicity, with its many targets and mechanisms, is a complex area of toxicology; thus, the screening and identification of reproductive toxicants is a main scientific challenge for the safety assessment of chemicals, including the European Regulation on Chemicals (REACH). Regulatory agencies recommend the implementation of the 3Rs principle (refinement, reduction, replacement) as well as of intelligent testing strategies, through the development of in vitro methods and the use of mechanistic information in the hazard identification and characterization steps of the risk assessment process. The EU Integrated Project ReProTect (6th Framework Programme) implemented an array of in vitro tests to study different building blocks of the mammalian reproductive cycle: methodological developments and results on male and female germ cells, prostate and placenta are presented.     

A tiered approach for assessing children's exposure

Recently, intense attention has been given to children's health issues, particularly in the use of consumer products. Because of this attention, researchers have been planning and initiating studies specifically aimed at developing both toxicology data and exposure data directed to improve our understanding of industrial and consumer product chemical impacts on children's health. To ensure that this research is focused on the highest priority chemicals, we present a methodology for determining and prioritizing the higher hazard chemicals and scenarios for which children could be disproportionately or highly exposed. This tiered approach includes a screening step for initial chemical selection, a hazard assessment based on no- or lowest-observed-adverse-effect levels, and a margin of exposure (MOE) calculation. The initial chemical screen focuses on the chemical presence in specific media that are special to children, such as foods children regularly eat and drink, residential or school air, products children use, and soil and dust in and around residences. Data from the literature or from models serve as the initial exposure estimate. This methodology would allow us to focus on those chemicals to which children are most exposed that are also associated with, potentially, the highest risk. Use of the MOE calculation allows for comparison among chemicals, prioritization of chemicals for evaluation and testing, and identification of significant data gaps.     

化学物血管毒性评价的斑马鱼成像分析方法

目的利用转基因斑马鱼实时观测方法进行化学物血管毒性评价。方法分别使用激光共聚焦显微成像和高内涵成像分析技术对暴露化学物后斑马鱼血管形态的时空变化进行观察及定量分析。结果采用激光共聚焦显微成像技术采集斑马鱼血管形态指标的方法操作相对简便,图片解析度高,但通量和效率较低;应用高内涵成像筛选系统采集高分辨血管图像方法,可高通量获取高质量全血管毒性数据,但对操作和实验条件要求较高。结论两种斑马鱼血管成像方法各有优劣,激光共聚焦显微成像法适合少量化学物对局部血管损害的高精度测量,高内涵成像方法适合大规模化学物血管毒性测试及筛选。     

化学物血管毒性评价的斑马鱼成像分析方法

目的利用转基因斑马鱼实时观测方法进行化学物血管毒性评价。方法分别使用激光共聚焦显微成像和高内涵成像分析技术对暴露化学物后斑马鱼血管形态的时空变化进行观察及定量分析。结果采用激光共聚焦显微成像技术采集斑马鱼血管形态指标的方法操作相对简便,图片解析度高,但通量和效率较低;应用高内涵成像筛选系统采集高分辨血管图像方法,可高通量获取高质量全血管毒性数据,但对操作和实验条件要求较高。结论两种斑马鱼血管成像方法各有优劣,激光共聚焦显微成像法适合少量化学物对局部血管损害的高精度测量,高内涵成像方法适合大规模化学物血管毒性测试及筛选。     

Mechanistic approaches for evaluating the toxicity of reactive organochlorines and epoxides in green algae

Reactive electrophilic chemicals, such as reactive organochlorine compounds or epoxides, react specifically with a broad spectrum of nucleophilic biomolecules, including proteins and DNA. Conventional toxicity tests for algae, involving the observation of growth inhibition, i.e., the inhibition of cell multiplication, after several days, yield unreliable information for risk assessment because reactive compounds hydrolyze to different extents during the exposure period. The diversity of their modes of toxic action further complicates effect assessment and calls for methods yielding additional information on the mechanisms of toxicity. One of the primary targets of reactive chemicals in cells is the tripeptide glutathione (GSH), which is important for detoxification but can also be regarded as a toxicity sensor because changes in glutathione levels indicate stress. A vital system for algae is the photosynthetic system, which is indirectly affected by reactive chemicals. The test systems developed in this study for the assessment of reactive toxicity toward algae were therefore based not only on nonspecific toxicity indicators like growth inhibition but also on indicators for disturbance of photosynthesis (inhibition of photosystem II quantum yield) and glutathione metabolism. The application of the developed test systems on Scenedesmus vacuolatus after short-term exposure of 2 h showed that these tests can be used as fast screening tests for algal toxicity and in mode-of-action-based test batteries.