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Assessment of Bile Salt Export Pump (BSEP) Inhibition in Membrane Vesicles Using Radioactive and LC/MS-Based Detection Methods 利用放射性和LC/ ms检测方法评价胆汁盐出口泵(BSEP)对膜泡的抑制作用
Pub Date : 2017-02-01 DOI: 10.1002/cptx.15
Lisa D. Marroquin, Paul D. Bonin, Julie Keefer, Thomas Schroeter

The bile salt export pump (BSEP, ABCB11) belongs to the ATP-binding-cassette superfamily of transporters and is predominately found in the liver. BSEP is an efflux transporter that plays a critical role in the secretion of bile salts into the bile. Inhibition of BSEP function by drugs can result in the buildup of bile salts in the liver and eventually leads to cholestasis and drug-induced liver injury (DILI). DILI is a major cause of withdrawal of drugs from the pharmaceutical market and accounts for >50% of acute liver failures. Therefore, early detection of BSEP inhibition by drugs can help to mitigate the possibility of BSEP-associated liver injury. This unit describes two assays that investigate the relationship between drug interference with BSEP function and liver injury using membrane vesicles prepared from Hi5 insect cells transfected with human BSEP. Comprehensive protocols for assessing BSEP inhibition in a 384-well format using radiolabeled and liquid chromatography/mass spectrometry (LC/MS)–based detection methods are described. © 2017 by John Wiley & Sons, Inc.

胆盐输出泵(BSEP, ABCB11)属于atp结合盒转运蛋白超家族,主要存在于肝脏。BSEP是一种外排转运蛋白,在胆盐分泌到胆汁中起关键作用。药物抑制BSEP功能可导致肝脏胆盐积聚,最终导致胆汁淤积和药物性肝损伤(DILI)。DILI是药品撤出市场的一个主要原因,占急性肝衰竭的50%。因此,早期发现药物对BSEP的抑制有助于降低BSEP相关性肝损伤的可能性。本单元描述了用转染人BSEP的Hi5昆虫细胞制备的膜泡研究药物干扰BSEP功能与肝损伤之间关系的两种检测方法。描述了在384孔格式中使用放射性标记和液相色谱/质谱(LC/MS)为基础的检测方法评估BSEP抑制的综合方案。©2017 by John Wiley &儿子,Inc。
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引用次数: 8
Preparation of Specific Compartments of the Lungs for Pathologic and Biochemical Analysis of Toxicologic Responses 制备用于毒理学反应病理和生化分析的特定肺室
Pub Date : 2017-02-01 DOI: 10.1002/cptx.18
Laura S. Van Winkle, Jacklyn S. Kelty, Charles G. Plopper

This unit focuses on protocols for assessing microenvironment-specific responses in the thoracic lung tissues. Aspects of the entire respiratory system serve as potential targets for candidate toxicants, but each candidate toxicant may impact distinct sites due to differential distribution of either the toxicant or the target cells. Within the conducting airways, the composition of resident cell populations and the metabolic capabilities of the cell populations vary greatly. Thus, studies of this region of the lung require unique, site-selective methods to clearly define the toxic response. Without site-specific sampling, as described in this chapter, the experimental limit of detection for toxicant effects in conducting airways is weakened because differences unrelated to treatment, but related to location, may dominate the response. The protocols included here allow assessment of toxicological responses in the tracheobronchial airways and the gas exchange area of the lung, with specific application to laboratory mammals. © 2017 by John Wiley & Sons, Inc.

本单元侧重于评估胸肺组织中微环境特异性反应的方案。整个呼吸系统的各个方面都是候选毒物的潜在靶点,但由于毒物或靶细胞的不同分布,每种候选毒物可能影响不同的部位。在传导气道内,驻留细胞群的组成和细胞群的代谢能力变化很大。因此,对肺这一区域的研究需要独特的、部位选择性的方法来明确定义毒性反应。如本章所述,如果没有特定地点的采样,在传导气道中检测毒性效应的实验极限就会被削弱,因为与治疗无关但与位置有关的差异可能会主导反应。这里包括的方案允许评估气管支气管和肺气体交换区域的毒理学反应,并具体应用于实验室哺乳动物。©2017 by John Wiley &儿子,Inc。
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引用次数: 5
Fluorescence-Based Microplate Assays for In Vitro Assessment of Mitochondrial Toxicity, Metabolic Perturbation, and Cellular Oxygenation 荧光微孔板法测定线粒体毒性,代谢扰动和细胞氧化的体外评估
Pub Date : 2016-11-01 DOI: 10.1002/cptx.3
James Hynes, Conn Carey, Yvonne Will

High-throughput in vitro cell metabolism assays are of particular use for identification and delineation of mitochondrial toxicity and related metabolic perturbation. Here, a panel of fluorescence-based metabolism assays are described for measuring oxygen consumption, glycolytic flux, and cellular oxygenation. They can be applied to analysis of both isolated mitochondria and cell models. Sample data are presented illustrating how these protocols can be used to examine drug treatment, the interplay between oxidative and glycolytic ATP generation, and the impact of cell oxygenation on this balance. Descriptions are provided on how these measurements can be applied to 3D systems and how they can be multiplexed with other relevant metabolic readouts. Mitochondrial isolation and cell permeabilization protocols are also provided. © 2016 by John Wiley & Sons, Inc.

高通量体外细胞代谢测定特别用于鉴定和描述线粒体毒性和相关代谢扰动。在这里,一组基于荧光的代谢测定被描述为测量氧气消耗,糖酵解通量和细胞氧合。它们可以应用于分离线粒体和细胞模型的分析。样本数据展示了这些方案如何用于检查药物治疗,氧化和糖酵解ATP生成之间的相互作用,以及细胞氧合对这种平衡的影响。描述了如何将这些测量应用于3D系统,以及如何将它们与其他相关的代谢读数进行多路复用。还提供了线粒体分离和细胞渗透方案。©2016 by John Wiley &儿子,Inc。
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引用次数: 8
Basic Techniques in Mammalian Cell Tissue Culture 哺乳动物细胞组织培养的基本技术
Pub Date : 2016-11-01 DOI: 10.1002/cptx.13
Katy Phelan, Kristin M. May

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2016 by John Wiley & Sons, Inc.

培养的哺乳动物细胞在细胞生物学研究中被广泛使用。它需要一些特殊的技能,以便能够在培养中保存细胞的结构、功能、行为和生物学。本单元描述了维持和保存细胞培养所需的基本技能:保持无菌技术,准备具有适当特性的培养基,传代,冷冻和储存,恢复冷冻库存,计数活细胞。©2016 by John Wiley &儿子,Inc。
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引用次数: 55
In Vivo Determination of Mitochondrial Function Using Luciferase-Expressing Caenorhabditis elegans: Contribution of Oxidative Phosphorylation, Glycolysis, and Fatty Acid Oxidation to Toxicant-Induced Dysfunction 用表达荧光素酶的秀丽隐杆线虫在体内测定线粒体功能:氧化磷酸化、糖酵解和脂肪酸氧化对毒物诱导功能障碍的贡献
Pub Date : 2016-08-01 DOI: 10.1002/cptx.10
Anthony L. Luz, Cristina Lagido, Matthew D. Hirschey, Joel N. Meyer

Mitochondria are a target of many drugs and environmental toxicants; however, how toxicant-induced mitochondrial dysfunction contributes to the progression of human disease remains poorly understood. To address this issue, in vivo assays capable of rapidly assessing mitochondrial function need to be developed. Here, using the model organism Caenorhabditis elegans, we describe how to rapidly assess the in vivo role of the electron transport chain, glycolysis, or fatty acid oxidation in energy metabolism following toxicant exposure, using a luciferase-expressing ATP reporter strain. Alterations in mitochondrial function subsequent to toxicant exposure are detected by depleting steady-state ATP levels with inhibitors of the mitochondrial electron transport chain, glycolysis, or fatty acid oxidation. Differential changes in ATP following short-term inhibitor exposure indicate toxicant-induced alterations at the site of inhibition. Because a microplate reader is the only major piece of equipment required, this is a highly accessible method for studying toxicant-induced mitochondrial dysfunction in vivo. © 2016 by John Wiley & Sons, Inc.

线粒体是许多药物和环境毒物的靶标;然而,毒物诱导的线粒体功能障碍如何促进人类疾病的进展仍然知之甚少。为了解决这个问题,需要开发能够快速评估线粒体功能的体内检测方法。在这里,我们使用模式生物秀丽隐杆线虫,描述了如何使用表达荧光素酶的ATP报告菌株,快速评估有毒物质暴露后电子传递链、糖酵解或脂肪酸氧化在能量代谢中的体内作用。毒物暴露后线粒体功能的改变可以通过抑制线粒体电子传递链、糖酵解或脂肪酸氧化来消耗稳态ATP水平来检测。短期抑制剂暴露后ATP的差异变化表明毒性诱导的抑制部位的改变。因为微孔板读取器是唯一需要的主要设备,这是一种非常容易获得的方法来研究体内毒物诱导的线粒体功能障碍。©2016 by John Wiley &儿子,Inc。
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引用次数: 27
Multielectrode Array (MEA) Assay for Profiling Electrophysiological Drug Effects in Human Stem Cell-Derived Cardiomyocytes 多电极阵列(MEA)分析分析电生理药物对人干细胞源性心肌细胞的影响
Pub Date : 2016-05-04 DOI: 10.1002/cptx.2
Mike Clements

More relevant and reliable preclinical cardiotoxicity tests are required to improve drug safety and reduce the cost of drug development. Human stem cell-derived cardiomyocytes (hSC-CMs) provide a potential model for the development of superior assays for preclinical drug safety screening. One such hSC-CM assay that has shown significant potential for enabling more predictive drug cardiac risk assessment is the MEA assay. The Multi-electrode Array (MEA) assay is an electrophysiology-based technique that uses microelectrodes embedded in the culture surface of each well to measure fluctuations in extracellular field potential (FP) generated from spontaneously beating hSC-CMs. Perturbations to the recorded FP waveform can be used as an unbiased method of predicting the identity of ion channel(s) impacted on drug exposure. Here, a higher throughput MEA assay using hSC-CMs in 48-well MEA plates is described for profiling compound-induced effects on cardiomyocyte electrophysiology. Techniques for preparing hSC-CM monolayers in MEA plates and methods to contextualize MEA assay experimental results are also covered. © 2016 by John Wiley & Sons, Inc.

需要更多相关和可靠的临床前心脏毒性试验来提高药物安全性并降低药物开发成本。人干细胞来源的心肌细胞(hSC-CMs)为临床前药物安全性筛选提供了一个潜在的模型。一种这样的hSC-CM分析已经显示出实现更多预测性药物心脏风险评估的重大潜力,即MEA分析。多电极阵列(MEA)检测是一种基于电生理学的技术,它使用嵌入每孔培养表面的微电极来测量自发加热hSC-CMs产生的细胞外场电位(FP)的波动。对记录的FP波形的扰动可以作为预测影响药物暴露的离子通道身份的无偏方法。在这里,使用hSC-CMs在48孔MEA板上进行高通量MEA分析,描述了化合物对心肌细胞电生理的影响。在MEA板中制备hSC-CM单层的技术以及将MEA分析实验结果置于背景下的方法也被涵盖。©2016 by John Wiley &儿子,Inc。
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引用次数: 29
Investigating the Effects of Particulate Matter on House Dust Mite and Ovalbumin Allergic Airway Inflammation in Mice 颗粒物质对小鼠屋尘螨和卵清蛋白过敏性气道炎症的影响
Pub Date : 2016-05-04 DOI: 10.1002/cptx.5
Alejandro R. Castañeda, Kent E. Pinkerton
Particulate matter (PM), a component of air pollution, has been shown to enhance allergen-mediated airway hypersensitivity and inflammation. Surprisingly, exposure to PM during the sensitization to allergen is sufficient to produce immunological changes that result in heightened inflammatory effects upon future allergen exposures (challenge) in the absence of PM. This suggests that PM has the ability to modulate the allergic immune response, thereby acting as an adjuvant by enhancing the immunological memory formed during the adaptive immune response; however, the mechanisms through which this occurs remain elusive. Establishing a reproducible animal model to study the PM-mediated immunotoxicological effects that enhance allergy, may provide insights to understand how air pollution activates the immune system and thereby modulates the pathophysiology of asthma. The basic protocol can be used to study various characteristics of air pollution, such as PM size, source, or chemical composition, to help elucidate how such features may affect the allergic response in a mouse model of asthma. Using a BALB/c model of acute exposure (14 days), mice are first sensitized with allergen and PM, and then subsequently challenged with allergen only. The endpoints of this basic protocol include the assessment of inflammation via cells recovered from broncho-alveolar lavage (BAL), histopathological analysis, gene expression profiles, and protein quantification of inflammatory markers. © 2016 by John Wiley & Sons, Inc.
颗粒物(PM)是空气污染的一个组成部分,已被证明会增强过敏原介导的气道过敏和炎症。令人惊讶的是,在过敏原致敏过程中暴露于PM足以产生免疫学变化,导致在没有PM的情况下,在未来的过敏原暴露(挑战)中炎症效应加剧。这表明PM具有调节过敏性免疫反应的能力,从而通过增强适应性免疫反应中形成的免疫记忆而起到佐剂的作用;然而,这种情况发生的机制仍然难以捉摸。建立一个可重复的动物模型来研究pm介导的免疫毒理学效应,从而增强过敏,可能为了解空气污染如何激活免疫系统从而调节哮喘的病理生理提供见解。基本方案可用于研究空气污染的各种特征,如PM大小,来源或化学成分,以帮助阐明这些特征如何影响哮喘小鼠模型的过敏反应。采用急性暴露(14天)BALB/c模型,首先用过敏原和PM致敏小鼠,然后仅用过敏原致敏小鼠。该基本方案的终点包括通过支气管肺泡灌洗(BAL)恢复的细胞来评估炎症、组织病理学分析、基因表达谱和炎症标志物的蛋白质定量。©2016 by John Wiley &儿子,Inc。
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引用次数: 15
Determination of Metabolic Viability and Cell Mass Using a Tandem Resazurin/Sulforhodamine B Assay 利用瑞唑脲/硫代丹胺B串联测定代谢活力和细胞质量
Pub Date : 2016-05-04 DOI: 10.1002/cptx.1
Filomena S.G. Silva, Irina G. Starostina, Vilena V. Ivanova, Albert A. Rizvanov, Paulo J. Oliveira, Susana P. Pereira

The identification of rapid, reliable, and highly reproducible biological assays that can be standardized and routinely used in preclinical tests constitutes a promising approach to reducing drug discovery costs and time. This unit details a tandem, rapid, and reliable cell viability method for preliminary screening of chemical compounds. This assay measures metabolic activity and cell mass in the same cell sample using a dual resazurin/sulforhodamine B assay, eliminating the variation associated with cell seeding and excessive manipulations in assays that test different cell samples across plates. The procedure also reduces the amount of cells, test compound, and reagents required, as well as the time expended in conventional tests, thus resulting in a more confident prediction of toxic thresholds for the tested compounds. © 2016 by John Wiley & Sons, Inc.

确定快速、可靠和高度可重复的、可标准化并常规用于临床前试验的生物测定方法,是减少药物发现成本和时间的一种有希望的方法。本单元详细介绍了一种串联、快速、可靠的细胞活力方法,用于化合物的初步筛选。该检测方法使用瑞祖啉/硫代丹胺B双重检测,测量同一细胞样品中的代谢活性和细胞质量,消除了与细胞播种相关的变化,以及在检测不同细胞样品时跨板进行的过度操作。该程序还减少了所需的细胞、测试化合物和试剂的数量,以及在传统测试中花费的时间,从而导致对测试化合物的毒性阈值更有信心的预测。©2016 by John Wiley &儿子,Inc。
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引用次数: 37
The Use of Induced Pluripotent Stem Cells for the Study and Treatment of Liver Diseases 诱导多能干细胞在肝脏疾病研究和治疗中的应用
Pub Date : 2016-02-01 DOI: 10.1002/0471140856.tx1413s67
Marc C. Hansel, Julio C. Davila, Massoud Vosough, Roberto Gramignoli, Kristen J. Skvorak, Kenneth Dorko, Fabio Marongiu, William Blake, Stephen C. Strom

Liver disease is a major global health concern. Liver cirrhosis is one of the leading causes of death in the world and currently the only therapeutic option for end-stage liver disease (e.g., acute liver failure, cirrhosis, chronic hepatitis, cholestatic diseases, metabolic diseases, and malignant neoplasms) is orthotropic liver transplantation. Transplantation of hepatocytes has been proposed and used as an alternative to whole organ transplant to stabilize and prolong the lives of patients in some clinical cases. Although these experimental therapies have demonstrated promising and beneficial results, their routine use remains a challenge due to the shortage of donor livers available for cell isolation, variable quality of those tissues, the potential need for lifelong immunosuppression in the transplant recipient, and high costs. Therefore, new therapeutic strategies and more reliable clinical treatments are urgently needed. Recent and continuous technological advances in the development of stem cells suggest they may be beneficial in this respect. In this review, we summarize the history of stem cell and induced pluripotent stem cell (iPSC) technology in the context of hepatic differentiation and discuss the potential applications the technology may offer for human liver disease modeling and treatment. This includes developing safer drugs and cell-based therapies to improve the outcomes of patients with currently incurable health illnesses. We also review promising advances in other disease areas to highlight how the stem cell technology could be applied to liver diseases in the future. © 2016 by John Wiley & Sons, Inc.

肝病是一个主要的全球健康问题。肝硬化是世界上导致死亡的主要原因之一,目前治疗终末期肝病(如急性肝功能衰竭、肝硬化、慢性肝炎、胆汁淤积性疾病、代谢性疾病和恶性肿瘤)的唯一选择是正异性肝移植。在一些临床病例中,肝细胞移植已被提出并用作整个器官移植的替代方案,以稳定和延长患者的生命。尽管这些实验性疗法已经证明了有希望和有益的结果,但由于可用于细胞分离的供体肝脏短缺、这些组织的质量多变、移植受体可能需要终身免疫抑制以及成本高,它们的常规使用仍然是一个挑战。因此,迫切需要新的治疗策略和更可靠的临床治疗方法。干细胞发展的最新和持续的技术进步表明它们在这方面可能是有益的。在这篇综述中,我们总结了干细胞和诱导多能干细胞(iPSC)技术在肝分化背景下的历史,并讨论了该技术在人类肝脏疾病建模和治疗中的潜在应用。这包括开发更安全的药物和基于细胞的疗法,以改善目前无法治愈的疾病患者的预后。我们还回顾了在其他疾病领域的有希望的进展,以强调干细胞技术在未来如何应用于肝脏疾病。©2016 by John Wiley &儿子,Inc。
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引用次数: 31
A Simple Light Stimulation of Caenorhabditis elegans 秀丽隐杆线虫的简单光刺激
Pub Date : 2016-02-01 DOI: 10.1002/0471140856.tx1121s67
Kun He Lee, Michael Aschner

Response via noxious stimulus can be an important indicator of sensory neuron function and overall health of an organism. If the stimulation is quick and simple, and the animal can be rescued afterwards, such a method not only allows for assays pertaining to changed sensory ability after various treatments, but also increases the reliability of the statistical relationships that are established. This protocol demonstrates a stimulation assay in Caenorhabditis elegans, using blue light from common laboratory equipment: the fluorescent microscope. The nematode detects blue light using a set of amphid ciliary sensory neurons, and blue light is detrimental to its overall health after a prolonged exposure. However, under brief exposure, blue light stimulation provides a rapid and easy method for quantifying sensory functions and health without harming the animal. © 2016 by John Wiley & Sons, Inc.

对有害刺激的反应是生物体感觉神经元功能和整体健康状况的重要指标。如果刺激是快速和简单的,并且动物可以在之后获救,这种方法不仅允许对各种治疗后感觉能力的变化进行分析,而且还增加了所建立的统计关系的可靠性。本方案演示了秀丽隐杆线虫的刺激试验,使用来自常见实验室设备:荧光显微镜的蓝光。线虫使用一组两栖动物纤毛感觉神经元来检测蓝光,长时间暴露在蓝光下对其整体健康有害。然而,在短时间照射下,蓝光刺激提供了一种快速简便的方法来量化动物的感觉功能和健康状况,而不会伤害动物。©2016 by John Wiley &儿子,Inc。
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引用次数: 6
期刊
Current protocols in toxicology
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