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Current protocols in toxicology最新文献

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Toxicology in the Super-Resolution Era 超分辨率时代的毒理学
Current protocols in toxicology Pub Date : 2019-04-18 DOI: 10.1002/cptx.77
Richard Cole
{"title":"Toxicology in the Super-Resolution Era","authors":"Richard Cole","doi":"10.1002/cptx.77","DOIUrl":"10.1002/cptx.77","url":null,"abstract":"<p>Light microscopy has played a central role in science for the past couple of hundred years and will continue to do so. Multiple super-resolution microscopy techniques have been in the headlines for smashing what for more than 100+ years was believed to be the limits of optical microscopy. This resolution improvement enables the visualization of molecular structures and processes on the nano scale. While certain scientific questions in toxicology can benefit from modalities within the super-resolution suite, due diligence is required for efficiency and to achieve optimal results. For a given hypothesis being tested, there are biophysical issues that need to be considered before heading down the super-resolution road. All commercially available super-resolution modalities, along with cautions and tips, will be discussed. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":72743,"journal":{"name":"Current protocols in toxicology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cptx.77","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37165079","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Generation of Macrophages from Cynomolgus-Monkey Bone Marrow as a Model to Evaluate Effects of Drugs on Innate Immunity 以食蟹猴骨髓巨噬细胞生成为模型评价药物对先天免疫的影响
Current protocols in toxicology Pub Date : 2019-04-14 DOI: 10.1002/cptx.74
Nianyu Li, Susan A. Ludmann, Lisa Anest, Ching He, Padma Kumar Narayanan
{"title":"Generation of Macrophages from Cynomolgus-Monkey Bone Marrow as a Model to Evaluate Effects of Drugs on Innate Immunity","authors":"Nianyu Li,&nbsp;Susan A. Ludmann,&nbsp;Lisa Anest,&nbsp;Ching He,&nbsp;Padma Kumar Narayanan","doi":"10.1002/cptx.74","DOIUrl":"10.1002/cptx.74","url":null,"abstract":"<p>Macrophages are innate immune cells that play important roles in various physiological and pathological processes. Evaluation of pro-inflammatory effects of drugs on macrophages has become commonplace in preclinical drug development prior to human clinical trials. Despite their body-wide distribution, tissue macrophages are often difficult to collect from large animals and humans in a noninvasive manner. Therefore, <i>in vitro</i>–differentiated macrophages are important tools to facilitate cross-species analysis of macrophage function. Although cynomolgus monkeys are an essential non-rodent species for preclinical research, <i>in vitro</i> differentiation of cynomolgus-monkey macrophages has been poorly characterized. In the present unit, we describe a protocol to differentiate cynomolgus-monkey macrophages from isolated bone marrow mononuclear cells (BMMCs). In contrast to monocytes, cynomolgus-monkey BMMCs show robust expansion in the presence of macrophage colony–stimulating factor <i>in vitro</i>, which allows expansion of many cells from a single animal donor. Macrophages differentiated from BMMCs retain many of the macrophage phenotypes and functions, including phagocytosis and cytokine release, and therefore can be used as a surrogate to assess effects of drugs on cynomolgus-monkey macrophages. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":72743,"journal":{"name":"Current protocols in toxicology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cptx.74","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37313411","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Analysis of Human Mitochondrial DNA Content by Southern Blotting and Nonradioactive Probe Hybridization 人线粒体DNA含量的Southern印迹和非放射性探针杂交分析
Current protocols in toxicology Pub Date : 2019-04-14 DOI: 10.1002/cptx.75
Joel H. Wheeler, Carolyn K. J. Young, Matthew J. Young
{"title":"Analysis of Human Mitochondrial DNA Content by Southern Blotting and Nonradioactive Probe Hybridization","authors":"Joel H. Wheeler,&nbsp;Carolyn K. J. Young,&nbsp;Matthew J. Young","doi":"10.1002/cptx.75","DOIUrl":"10.1002/cptx.75","url":null,"abstract":"<p>A single cell can contain several thousand copies of the mitochondrial DNA genome or mtDNA. Tools for assessing mtDNA content are necessary for clinical and toxicological research, as mtDNA depletion is linked to genetic disease and drug toxicity. For instance, mtDNA depletion syndromes are typically fatal childhood disorders that are characterized by severe declines in mtDNA content in affected tissues. Mitochondrial toxicity and mtDNA depletion have also been reported in human immunodeficiency virus–infected patients treated with certain nucleoside reverse transcriptase inhibitors. Further, cell culture studies have demonstrated that exposure to oxidative stress stimulates mtDNA degradation. Here we outline a Southern blot and nonradioactive digoxigenin-labeled probe hybridization method to estimate mtDNA content in human genomic DNA samples. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":72743,"journal":{"name":"Current protocols in toxicology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cptx.75","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37313410","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 9
Mitochondrial Electron Transfer Cascade Enzyme Activity Assessment in Cultured Neurons and Select Brain Regions 线粒体电子转移级联酶在培养神经元和特定脑区的活性评估
Current protocols in toxicology Pub Date : 2019-04-05 DOI: 10.1002/cptx.73
Abdulbaki Agbas, Partha Krishnamurthy, Mary L. Michaelis, Elias K. Michaelis
{"title":"Mitochondrial Electron Transfer Cascade Enzyme Activity Assessment in Cultured Neurons and Select Brain Regions","authors":"Abdulbaki Agbas,&nbsp;Partha Krishnamurthy,&nbsp;Mary L. Michaelis,&nbsp;Elias K. Michaelis","doi":"10.1002/cptx.73","DOIUrl":"10.1002/cptx.73","url":null,"abstract":"<p>Measurement of the electron transfer cascade (ETC) enzyme activities and their kinetic profiles is important in assessing mitochondrial function in the nervous system in health and disease or following exposure to toxic agents. The optimization of enzymatic assays for brain tissues and neurons is critical to the development of high-throughput assay formats. This article describes a step-by-step protocol for reliable and reproducible assessment of ETC enzyme kinetics (Complex I-IV) for mitochondria from small quantities of tissue from different brain regions, such as the hippocampus, cerebellum, and frontal cortex, or from neurons in culture. Methods for differential and density gradient centrifugation are detailed for isolating cell body and synaptic mitochondria from brain, as well as measurement of ETC activities in microwell plate or single-cuvette format using spectrophotometric methods. Easy-to follow assay layouts and useful tips are presented, allowing the user to perform these assays in under 3 hr. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":72743,"journal":{"name":"Current protocols in toxicology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cptx.73","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9358910","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 9
The Use of Intracerebral Microdialysis to Elucidate Environmentally Induced Neurotoxic Mechanisms 利用脑内微透析阐明环境诱导的神经毒性机制
Current protocols in toxicology Pub Date : 2019-04-02 DOI: 10.1002/cptx.72
Stephen M. Lasley
{"title":"The Use of Intracerebral Microdialysis to Elucidate Environmentally Induced Neurotoxic Mechanisms","authors":"Stephen M. Lasley","doi":"10.1002/cptx.72","DOIUrl":"10.1002/cptx.72","url":null,"abstract":"<p>The technique of microdialysis permits the assessment of neurotransmitter activity and the monitoring of other cellular entities in tissue extracellular fluid. The method is widely used for quantifying biogenic amine and amino acid transmitters, peptides, administered drugs, and other molecules in response to various experimental treatments. This article provides an overview of the manner in which the methodology of intracerebral microdialysis is utilized in the field of neurotoxicology to elucidate the actions of environmental agents. The technique is employed in a variety of creative ways to address specific experimental goals involving myriad toxicants. With appropriate consideration of method parameters, investigators have also been able to address mechanistic issues in their studies. These investigations consist of sampling of neurotransmitters in extracellular fluid after various protocols of environmental metal exposure as well as assessments of blood–brain barrier permeability, the detection of reactive oxygen species, and description of the toxicodynamics of environmental agents. The purpose of this examination is not to review the investigational findings, per se, but to highlight the various approaches utilized with this methodology and the experimental questions that have been addressed. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":72743,"journal":{"name":"Current protocols in toxicology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cptx.72","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37114861","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Isolation of Alveolar Type II Cells from Adult Bovine Lung 成年牛肺ⅱ型肺泡细胞的分离
Current protocols in toxicology Pub Date : 2019-03-15 DOI: 10.1002/cptx.71
Diane Frances Lee, Mark Andrew Chambers
{"title":"Isolation of Alveolar Type II Cells from Adult Bovine Lung","authors":"Diane Frances Lee,&nbsp;Mark Andrew Chambers","doi":"10.1002/cptx.71","DOIUrl":"10.1002/cptx.71","url":null,"abstract":"<p>Alveolar type II (ATII) cells play a key role as part of the distal lung epithelium, including in the innate immune response and as self-renewing progenitors to replace alveolar type I (ATI) cells during epithelial regeneration. Their secretion of surfactant protein helps maintain homeostasis and exerts protective, antimicrobial properties. ATII cells remain difficult to study, partly due to inefficient and expensive isolation methods, a propensity to differentiate into ATI cells, and susceptibility to fibroblast contamination. Published methods of isolation often require specialized technology, negatively impacting the development of <i>in vitro</i> models of disease, including bovine tuberculosis. Presented here is a simple and cost-effective method for generation of bovine primary ATII cells. These cells exhibit an ATII phenotype in 2D and 3D culture and are conducive to further study of the role of ATII cells in bovine respiratory diseases. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":72743,"journal":{"name":"Current protocols in toxicology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-03-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cptx.71","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37221245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
Preparation and Measuring of Brain GABAAR-coupled Cl-/HCO3-- Activity for Integral Assessment of Aquatic Toxicity 脑gabaar偶联Cl-/HCO3-活性的制备和测定及其在水生毒性综合评价中的应用
Current protocols in toxicology Pub Date : 2019-03-07 DOI: 10.1002/cptx.70
Sergey A. Menzikov
{"title":"Preparation and Measuring of Brain GABAAR-coupled Cl-/HCO3-- Activity for Integral Assessment of Aquatic Toxicity","authors":"Sergey A. Menzikov","doi":"10.1002/cptx.70","DOIUrl":"10.1002/cptx.70","url":null,"abstract":"<p>The wide use of aromatic hydrocarbons in various industries is having a negative effect on the environment and human health. Therefore, a key focus of current toxicology is the development and use of protein reporters with high sensitivity to various aromatic hydrocarbons (including phenolics and drugs). One molecular target for a wide range of pharmacology drugs and aromatic hydrocarbons (including phenol) is the neuronal GABA<sub>A</sub>R-coupled Cl<sup>-</sup>/HCO<sub>3</sub><sup>-</sup>-ATPase. In this study, we present a protocol for isolation of the membrane-bound Cl<sup>-</sup>/HCO<sub>3</sub><sup>-</sup>-ATPase from neuronal cells of animal brain. We then describe an uncomplicated <i>in vitro</i> method for measuring this ATPase activity for assessment of toxicity after interaction of this protein with an aquatic sample. This assay offers new avenues for using the Cl<sup>-</sup>/HCO<sub>3</sub><sup>-</sup>-ATPase as a biomarker of water toxicity. This biotest is efficient, requires very little of the enzyme, and retains its sensitivity at low levels of various compounds. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":72743,"journal":{"name":"Current protocols in toxicology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cptx.70","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37033183","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Inducing a Mucosal Barrier–Sparing Inflammatory Response in Laboratory-Grown Primary Human Nasal Epithelial Cells 在实验室培养的原代人鼻上皮细胞中诱导粘膜屏障保护炎症反应
Current protocols in toxicology Pub Date : 2019-02-04 DOI: 10.1002/cptx.69
Mahnaz Ramezanpour, Harrison Bolt, Alkis Psaltis, Peter-John Wormald, Sarah Vreugde
{"title":"Inducing a Mucosal Barrier–Sparing Inflammatory Response in Laboratory-Grown Primary Human Nasal Epithelial Cells","authors":"Mahnaz Ramezanpour,&nbsp;Harrison Bolt,&nbsp;Alkis Psaltis,&nbsp;Peter-John Wormald,&nbsp;Sarah Vreugde","doi":"10.1002/cptx.69","DOIUrl":"10.1002/cptx.69","url":null,"abstract":"<p>Here we use the toll-like receptor (TLR) 3 agonist poly I:C (LMW) to induce an inflammatory response in cells of submerged and/or air-liquid interface (ALI) cultures of human nasal epithelial cells (HNECs). The inflammatory response is determined by measuring interleukin-6 (IL-6) protein levels by enzyme-linked immunosorbent assay (ELISA). The mucosal barrier integrity is determined by measuring transepithelial electrical resistance (TEER) and passage of fluorescently labeled dextrans. Stimulation with poly (I:C) LMW induces a 15- to 17-fold increase in IL-6 production by HNEC-ALI cells. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":72743,"journal":{"name":"Current protocols in toxicology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cptx.69","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36925607","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 12
Issue Information TOC 发布信息TOC
Current protocols in toxicology Pub Date : 2019-01-25 DOI: 10.1002/cptx.61
{"title":"Issue Information TOC","authors":"","doi":"10.1002/cptx.61","DOIUrl":"10.1002/cptx.61","url":null,"abstract":"","PeriodicalId":72743,"journal":{"name":"Current protocols in toxicology","volume":"79 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-01-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cptx.61","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49138619","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
In Vitro Monocyte/Macrophage Phagocytosis Assay for the Prediction of Drug-Induced Thrombocytopenia 体外单核细胞/巨噬细胞吞噬试验预测药物性血小板减少症
Current protocols in toxicology Pub Date : 2019-01-23 DOI: 10.1002/cptx.68
Padma Kumar Narayanan, Nianyu Li
{"title":"In Vitro Monocyte/Macrophage Phagocytosis Assay for the Prediction of Drug-Induced Thrombocytopenia","authors":"Padma Kumar Narayanan,&nbsp;Nianyu Li","doi":"10.1002/cptx.68","DOIUrl":"10.1002/cptx.68","url":null,"abstract":"<p>Phagocytosis of platelets by monocytes and macrophages is a primary mechanism of platelet clearance <i>in vivo</i> and has been increasingly implicated in playing an important role in thrombocytopenia mediated by monoclonal antibodies intended for therapeutic purposes. In the present article, we describe an <i>in vitro</i> flow cytometry assay to assess the effect of antibody-mediated platelet phagocytosis by monocytes. Freshly isolated platelets were labeled with a fluorescent probe, 5-chloromethylfluorescein diacetate (CMFDA) and then co-cultured with isolated peripheral blood mononuclear cells (PBMCs) from the same donor in the presence of increasing concentrations of a monoclonal antibody drug. After incubation, an increase in CMFDA fluorescence intensity of CD14 positive monocytes was evaluated by flow cytometry as an assessment for drug-mediated platelet phagocytosis by monocytes. The assay has been evaluated using both human and cynomolgus monkey cells for the prediction of drug-induced thrombocytopenia. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":72743,"journal":{"name":"Current protocols in toxicology","volume":"79 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cptx.68","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36889315","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 5
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