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Polypharmacy in chick embryos

Polypharmacy in chick embryos

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2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者： Yuji Yoshiyama Center for Clinical Pharmacy and Clinical Sciences Kitasato University School of Pharmacy

It is not surprising that the administration of one drug may modify the action of another drug given *** there are not very many reports of multiple drug *** all of the studies involve just two *** the present study,we investigated the multiple drug interaction to take amitriptyline,fluconazole and disopyramide in chick *** eggs of White Leghorns were incubated and amitriptyline,fluconazole and disopyramide were injected into the fertilized *** were recorded 60 minutes after the injection,and heart rate was determined from R R *** have reported that toxic interactions involve just two drugs were demonstrated in chick *** combination with disopyramide modified the pharmacological effects of amitriptyline or fluconazole in chick embryos and led to an arrhythmia of the *** this study,the heart rate was significantly decreased by combination with amitriptyline,fluconazole and *** addition,arrhythmia was produced by combination with amitriptyline,fluconazole and *** findings indicate that the multiple drug interaction to take amitriptyline,fluconazole and disopyramide has a marked influence on the heart rate in chick *** the exact mechanism underlying the influence of the multiple drug interaction on the pharmacological effects of the drug remains to be clarified,the multiple drug interaction seems to enhance the toxicity of the drug in chick *** conclusion,our in ovo recording system for ECG of chick embryos may be useful for investigating the multiple drug interaction of amitriptyline,fluconazole and disopyramide.

关键词： polypharmacy multiple drug interaction amitriptyline fluconazole disopyramide chick embryo electrocardiogram

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A Systems Model for High-throughput Drug-induced Arrhythmogenicity Assessment

A Systems Model for High-throughput Drug-induced Arrhythmoge...

引用

2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者： Pan Li Li Zhang Ling Chao Chao Wang Yu Yuan Center for Public Health Informatics School of Public HealthXinxiang Medical University Coronary Care Unit First Affiliated HospitalXinxiang Medical University

Drug-induced cardiotoxicity,*** potentially lethal arrhythmia Torsade de Pointes(TdP),has been a critical issue since the early stage of drug design and ***,unexpected post-marketing occurrence of cardiotoxic effects remains a leading cause of drug withdrawn and *** studies raised the question that if the in vitro hERG block assay can provide sufficient information to accurately discriminate "safe" from "dangerous" ***,a novel systems model is presented for high-throughput drug-induced arrhythmogenicity assessment with better predictive *** model is designed to achieve a balance between systems-level mechanistic details of ventricular excitation and a computational efficiency feasible for high-throughput drug *** of evaluating drug effects at molecular or cellular level,we adopted a system-level approach to quantitatively investigate the frequency-and dose-dependent profile of drug-induced electric conduction blocks,which is a direct and less-biased marker for ventricular arrhythmias such as *** results suggest that the model may serve as a promising arrhythmogenicity assessment tool for both academic and industrial communities.

关键词： A Systems Model for High-throughput Drug-induced Arrhythmogenicity Assessment Model

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MAM结构和功能调节在SPIO纳米颗粒诱导肝细胞凋亡中的作用研究

MAM结构和功能调节在SPIO纳米颗粒诱导肝细胞凋亡中的作用研究

引用

2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者：姚欢张力引杨川丽蒋盛威何承勇林忠宁林育纯分子疫苗学和分子诊断学国家重点实验室厦门大学公共卫生学院

目的:线粒体关联性内质网膜（MAM）是线粒体和内质网（ER）特定位点之间特化的膜关联性偶联结构,有几十种蛋白质富集于其间,作为交互作用的生物功能平台与钙信号传导、脂质代谢、ER应激和细胞死亡等密切相关,成为细胞活性和转归调控的枢... 详细信息

目的:线粒体关联性内质网膜（MAM）是线粒体和内质网（ER）特定位点之间特化的膜关联性偶联结构,有几十种蛋白质富集于其间,作为交互作用的生物功能平台与钙信号传导、脂质代谢、ER应激和细胞死亡等密切相关,成为细胞活性和转归调控的枢纽,影响外源物诱导的细胞生理和毒性损伤状态。我们前期发现在生物医用领域（特别是药物传输）中有重要应用的新型纳米材料超顺磁氧化铁纳米颗粒（SPIO-NPs）可诱发ER应激、以及经由线粒体自噬诱导肝细胞线粒体损伤。提示SPIO-NPs作用于线粒体和ER之间的关联性值得研究,拟探讨MAM结构和功能调节在诱导肝细胞凋亡中的作用。方法:PEG包被的相容性SPIO-NPs（2.5、7.5、12.5λg/ml）作用于永生化人正常肝L02细胞建立暴露模型。采用透射电镜（TEM）、邻位连接技术（PLA）等分析MAM结构;采用Ca探针亚细胞定位、高内涵细胞分析（HCA）等检测ER-线粒体间Ca转运,作为MAM功能分析;qRT-PCR和蛋白免疫印迹检测MAM相关蛋白水平。结果:（1）SPIO-NPs组细胞MAM结构相关蛋白IP3R、GRP75、VDAC1水平存在差异;PLA显示IP3R和VDAC1位置邻近;TEM发现SPIO-NPs组细胞MAM区域间距显著缩短（P<0.05）;表明SPIO-NPs可诱导线粒体-ER相互作用增强,提示介导MAM结构的调节作用。（2）HCA检测发现SPIO-NPs组细胞胞浆Ca和线粒体Ca信号增高、内质网Ca降低;表明从ER流向线粒体的Ca转运增强,提示介导MAM功能增强的调节。（3）SPIO-NPs组细胞中Cyt C、Bax、Bcl-2、caspase 3等蛋白水平明显升高;提示线粒体依赖性凋亡的发生,与MAM调节线粒体Ca过载有关。（4）采用siHSPA9干预GRP75表达模型,结果显示SPIO-NPs组细胞Ca转运抑制、线粒体依赖性凋亡明显降低;验证了MAM结构干预可调控SPIO-NPs诱导的肝细胞转归。结论:SPIO-NPs可经由靶向MAM结构和功能的调控,介导肝细胞凋亡转归,为纳米材料诱导肝细胞毒性机制和安全性评价提供依据。

关键词：线粒体关联性内质网膜(MAM) 超顺磁氧化铁纳米颗粒线粒体依赖性凋亡肝细胞毒性

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原代细胞3D系统的技术进展和应用

原代细胞3D系统的技术进展和应用

引用

2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者：蒋敏齐来俊江苏齐氏生物科技有限公司美国CHI Scientific Inc.

细胞的3D培养系统是基于细胞和器官组织乃至生物体的一种特殊的科研工具,近几年得以飞速发展,从原始的缺陷型细胞株到如今的原代细胞,从当初的胶原基质培养到如今的高分子材料的三维支架,细胞3D系统的培养技术也经历了很大的革新,在应... 详细信息

细胞的3D培养系统是基于细胞和器官组织乃至生物体的一种特殊的科研工具,近几年得以飞速发展,从原始的缺陷型细胞株到如今的原代细胞,从当初的胶原基质培养到如今的高分子材料的三维支架,细胞3D系统的培养技术也经历了很大的革新,在应用方面,也从当初的单一的细胞之间的信息传递研究到如今在药代动力学,毒理学以及靶向药物的测试等方面广泛应用。就3D细胞和2D细胞系统的比较和应用优势而言,2D细胞培养的研究在扩宽我们对发育生物学、组织形态发生、疾病机制、药物发现、大批量蛋白生产、组织工程和再生医学方面的知识方面扮演了至关重要的角色。其操作简单、易重复等特点曾经在新药研发中占据重要的地位,然后近20年来,超过90%的候选药物通过了体外临床前研究,却在临床实验中未能达到预期的疗效或者安全指数,癌症药物的失败率更高,导致人们开始怀疑2D细胞系统的有效性。事实上,诸多研究表明,2D细胞培养试验中,细胞无法实现类似于生物体内的结构组织和连接,这使得细胞形态、存活力、增值能力、分化、基因和蛋白表达、对刺激的反应、药物代谢及一般的细胞功能受到了限制或减弱。而在体内,几乎所有的细胞都分布在一个复杂三维(3D)结构的细胞外基质(ECM)中,它们和相邻的细胞通过信号传导或者直接接触而产生相互作用。如此细胞-细胞和细胞-细胞外基质间的相互作用构建成了一个用于维持组织特异性和稳态的3D通讯网络。进而调控着细胞生命周期的各项活动。3D细胞培养创立了一个近乎体内细胞生长微环境的系统,相较于2D培养,3D细胞系统的生理相关性更加贴近体内组织的特异性。近年来,3D细胞系统被广泛应用于干细胞培养和分化、肿瘤生物学、药物和毒性筛选及组织工程等方面。在3D细胞系统的培养方面,目前以细胞三维支架(胶原、水凝胶、琼脂糖、人工合成高分子等)和悬浮培养为主要技术手段。而在细胞选择方面,逐步以原代细胞培养取代过去具有基因缺陷型的细胞株。3D原代细胞系统在新药研发、毒理学以及环境科学等领域的应用取得的很大进展,主要表现在以下几个方面:3D培养技术在干细胞研究中的应用(间充质干细胞、神经干细胞、胚胎干细胞、肿瘤干细胞等);3D培养技术在肿瘤药物研发中的应用(乳腺癌、肺癌、肝癌等);3D细胞系统在毒性、毒理研究中的应用(皮肤细胞);3D细胞系统在环境监测研究中的应用(肝实质细胞系统、肺上皮细胞系统)。本综述试图总结细胞3D系统的培养技术和应用,以及当下面临的挑战。系统地阐述3D细胞培养技术的发展;全面比较2D细胞和3D细胞系统的优势和缺点;详细描述3D原代细胞培养技术在新药研发、毒理学和环境科学等方面科研的应用。

关键词：新药研发生物体三维支架原代细胞培养细胞株

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氧化应激介导PFOS诱导人胚胎肝细胞自噬和凋亡的研究

氧化应激介导PFOS诱导人胚胎肝细胞自噬和凋亡的研究

引用

2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者：潘小元郭辛鑫陈聪曾怀才南华大学公共卫生学院

目的:探讨氧化应激在PFOS诱导L-02肝细胞自噬和凋亡中的作用及其机制,为PFOS的肝毒性提供理论依据。方法:将实验设为空白组(DMSO)和PFOS处理组(50、100、150、200和250μM),MTT法检测不同处理组在不同时间点(24h、48h)对L-02肝细胞存活... 详细信息

目的:探讨氧化应激在PFOS诱导L-02肝细胞自噬和凋亡中的作用及其机制,为PFOS的肝毒性提供理论依据。方法:将实验设为空白组(DMSO)和PFOS处理组(50、100、150、200和250μM),MTT法检测不同处理组在不同时间点(24h、48h)对L-02肝细胞存活率的影响,紫外和荧光发光光度法检测PFOS对L-02细胞氧化应激损伤的影响;荧光探针法检测L-02细胞内活性氧(ROS)水平,MDC染料检测自噬泡生成情况,Annexin-V FITC/PI检测细胞凋亡情况,Rh123染料检测线粒体膜电位变化情况。Western Blot检测凋亡相关蛋白Bcl-2、Bax、Cleaved-Caspase3,自噬相关蛋白LC3、p62的表达情况,Q-PCR检测Caspase-3 mRNA的表达情况。结果:不同浓度PFOS(0、50、100、150、200、250μM)处理L-02细胞24、48h后,细胞存活率降低,48h时间点较24h时间点下降更多;紫外和分光光度法结果显示PFOS引起L-02细胞中超氧化物歧化酶(SOD)和谷胱甘肽还原酶(GSH)水平下降,同时引起总活性氧(ROS)和丙二醛(MDA)水平上升,氧化抑制剂NAC可以抑制PFOS诱导L-02细胞生成ROS;MDC结果显示PFOS可引起L-02细胞内自噬泡生成增加,自噬抑制剂3-MA和氧化抑制剂NAC均可分别抑制其增加;此外,PFOS能诱导L-02细胞凋亡,NAC、3-MA能分别抑制PFOS导致的L-02细胞凋亡。罗丹明染料检测结果显示PFOS可使L-02细胞线粒体膜电位降低,NAC、3-MA都可缓解PFOS导致的L-02细胞线粒体膜电位的降低。Western Blot结果显示50、100、200μM PFOS作用L-02细胞6h后,与对照组相比,自噬相关蛋白LC3-Ⅱ表达增加,p62表达降低;200μM PFOS作用L-02细胞12h,凋亡相关蛋白Bcl-2表达降低,Bax、Cleaved-Caspase3表达增加,Bax与Bcl-2比值降低,自噬相关蛋白LC-Ⅱ表达增加,p62表达降低,NAC预处理后可抑制PFOS导致的凋亡相关蛋白Bax,Cleaved-Caspase3和自噬相关蛋白LC3-Ⅱ的表达。结论:PFOS暴露刺激L-02细胞ROS产生增加,损伤细胞线粒体,降低细胞活性,诱导细胞凋亡;PFOS诱导的L-02细胞自噬扮演着促进细胞凋亡的角色,可能是由于PFOS激活细胞自噬,导致细胞线粒体膜通透性改变,进而促进了凋亡。

关键词：全氟辛烷磺酸盐自噬凋亡活性氧线粒体膜电位

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Tissue engineering-based approaches tophysiological-relevant cell-based assays

Tissue engineering-based approaches tophysiological-relevant...

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2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者： Yasuyuki SAKAI Japanese Society for Alternatives to Animal Experiments(JSAAE) Department of Chemical System Engineering Graduate School of Engineering International Research Center on Integrative Biomedical Systems(CIBiS)Institute of Industrial Science Max Planck-University of Tokyo Center for Integrative InflammologyUniversity of Tokyo

Anima-free and mechanistic understanding and prediction of human body responses is the ultimate goal of alternatives to animal *** human cell-based assays are its fundamental but existing assays do not always have high physiological *** solve one of the most serious problems in current plate-based culture systems,we used oxygen-permeable silicone membrane that enables direct oxygen supply to the cells and their in vivo-like aerobic respiration as opposed to the dominancy of anaerobic respiration in plastic *** also enables in vivo-like complete 3 D hierarchical cocultures of liver parenchymal and non-parenchymal cells at the bottom surfaces of the *** microenvironments such as mechanical stimulations or continuous perfusion of culture medium may be beneficial and be realized by organ-on-a-chip ***/organs-on-a-chip technologies may be a key to this because they enables finding of unknown organ-to-organ *** such better physiologically-relevant cell-based assays are emerging but we have to keep in mind that they will never be built up to a complete human ***,integration of such advanced cell-based assays with various omics and numerical methods arerequired in better understanding of human body responses and in quantitative in vitro-in vivo extrapolation.

关键词： Tissue engineering-based approaches tophysiological-relevant cell-based assays

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Hepatotoxic drug assessment on a 3D liver co-culture model

Hepatotoxic drug assessment on a 3D liver co-culture model

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2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者： Yaqing Wang Li Wang Jianhua Qin Division of Biotechnology Dalian Institute of Chemical PhysicsChinese Academy of Sciences University of Chinese Academy of Sciences

Preservation of hepatic phenotype and functions in vitro has always been a great challenge for the reconstruction of liver tissue *** induced hepatocytes(hiHeps)from fibroblasts represent nearly normal levels of liver specific functions,which are considered as a new source of hepatocytes resource for applications in vitro and in *** this work,we established a simple paper-based 3D scaffold for the long-term culture hiHeps and human umbilical vein endothelial cells(HUVECs),and creating a 3D liver co-culture *** found that hiHeps co-cultured with HUVECs on paper presented a 3D-like morphology by self-assembling and maintained the normal liver specific functions for up to 2 months,as measured by the production of albumin and *** liver model maintained higher expression of cytochrome P450 genes compared with conventional monolayer culture of hiHeps,revealing a marked enhancement of hepatic ***,upon incubation with hepatotoxic drugs including acetaminophen and pioglitazone,the liver co-culture model better evaluated in vivo-like drug-induced toxicity compared with hepatocyte monolayer *** together,the established long lasting 3D liver model provides a potential platform for drug-induced toxicity testing and even for disease modeling applications.

关键词： Hepatotoxic drug assessment on a 3D liver co-culture model

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Metabobsm-dependent drug testing on an organs-on-chip

Metabobsm-dependent drug testing on an organs-on-chip

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2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者： Zhongyu Li Fangchao Yin Jianhua Qin Division of Biotechnology Dalian Institute of Chemical PhysicsChinese Academy of Sciences University of Chinese Academy of Sciences

Pharmaceutical development is greatly hindered by the poor predictive power of existing in vitro models for drug efficacy and toxicity *** this work,we present a new organs-on-a-chip device that allows for the assessment of drug metabolism,and its resultant drug efficacy and cytotoxicity in different organ-specific cells *** cell lines representing the liver,tumor(breast cancer and lung cancer),and normal tissue(gastric cells) were cultured in the compartmentalized micro-chambers of the multilayer *** adopted the prodrug capecitabine was used as a model *** on this device,the intermediate metabolites5-deoxy-5-fluorocytidine of capecitabine that were metabolized from liver and its active metabolite 5-fluorouracil from the targeted cancer cells and normal tissue cells were identified using mass *** exhibited strong cytoxicity on breast cancer and lung cancer cells,but not in normal gastric ***,the drug-induced cytotoxicity on cells varied in various target tissues,suggesting the metabolism-dependent drug efficacy in different tissues as exisits in *** in vitro model can not only allow for characterizing the metabolism of anti-cancer drugs in different tissues simultaneously,but also facilitate the assessment of drug bioactivity on various target tissues in a simple way,indicating the utility of this organs-on-chip for applications in drug efficacy and toxicity testing.

关键词： Organs-on-a-chip Metabolism Drug testing

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State of the art of read-across

State of the art of read-across

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2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者： Renjun Gao Nicholas Ball The Dow Chemical Company

Read-across plays an important role in hazard assessment of chemicals under numerous internal and external regulatory *** is used in association with the formation of categories or small groups of analogous chemicals,and these are typically developed based on structural similarity and,increasingly often,also on mechanistic(biological) *** global acceptance of read-across is *** instance read-across and analogous techniques are widely used as part of the US EPA"s Pre-Manufactory Notification *** the European Union,more understanding of the acceptance of read-across for toxicity prediction in the regulatory context is ***,the European Union REACH legislation explicitly calls for the use of non-animal alternative methods and thus opens up the use of read-across,and in 2015 the European Chemicals Agency(ECHA) published their Read-Across Assessment Framework(RAAF) as a structured approach to the scientific evaluation of read-across justifications included in REACH registration *** RAAF details the crucial scientific aspects that should be considered and discussed in the rationale provided to support a SAR-based read-across assessment.A summary of the analysis of the RAAF,the utility of the RAAF in preparing and evaluating the read-across justification and available supporting evidence to minimize the uncertainty will be presented.A list of the key aspects that should be considered in any scientifically robust SAR-based read-across assessment will be *** the end,ideas about how can we overcome the barriers inhibiting application of read-across methodologies in regulatory decision-making today will be also explained.

关键词： read-across Read-Across Assessment Framework structural similarity biological similarity

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Can in vitro tipping points inform human risk? Application of PBPK modelling for chemical risk assessment with case studies.

Can in vitro tipping points inform human risk? Application o...

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2017（第三届）毒性测试替代方法与转化毒理学（国际）学术研讨会

作者： Jin Li Unilever Safety and Environmental Assurance Center

Novel alternative chemical safety assessmentswithout traditional animal data rely on human cells-relevant,toxicity pathway-based in vitro approaches,coupled with in silico *** of the key challenges is how information gleaned from relevant cell assays,for example toxicity pathway-perturbing concentrations of chemicals(i.e.,in vitro tipping points),can be used to inform the risk of human health with sound scientific *** talk will first introduce some principal concepts of non-animal approaches to risk assessment,including exposure,in-vitro tipping points,physiologically-based pharmacokinetics(PBPK) modelling,In vitro-in vivo extrapolation(IVIVE).Then we will describe the procedure of application of PBPK modelling to extrapolate in-vitro tipping dose to human relevant equivalent *** studies of chemicals will be discussed to illustrate that in vitro tipping points do show some merits of indicating human risk when an adequate reverse dosimetry PBPK modelling is applied and a proper PBPK metric is used,though with some uncertainty factors associated,including in vitro free concentrations kinetics,active transporters and metabolism,*** framework in this talk paves a way to reduce the need of animal testing in our novel human risk assessment.

关键词： Application of PBPK modelling for chemical risk assessment with case studies Can in vitro tipping points inform human risk

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