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Current Protocols in Toxicology

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https://www.readbyqxmd.com/read/28777444/intracellular-cytokine-detection-by-flow-cytometry-in-surface-marker-defined-human-peripheral-blood-mononuclear-t-cells
#1
Fredine T Lauer, Jesse L Denson, Ellen Beswick, Scott W Burchiel
In a recent unit in this series, protocols for the isolation, cryopreservation, thawing, and immunophenotyping of HPBMC isolated from peripheral whole blood using cell surface marker (CSM) staining and multi-color flow cytometry analysis were presented. The current procedure describes the detection and quantification of CSM and intracellular markers (ICM), including transcription factors and cytokines, following activation and differentiation of CD4+ T-cells using multi-color flow cytometry. Results indicated that repeatable and robust detection of ICM could be obtained in surface marker-defined T cells that identify functional subsets of cells...
August 4, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28777443/use-of-human-pluripotent-stem-cell-derived-cardiomyocytes-to-study-drug-induced-cardiotoxicity
#2
Agnes Maillet, Kim Peng Tan, Liam R Brunham
Drug-induced cardiotoxicity is the one of the most common causes of drug withdrawal from market. A major barrier in managing the risk of drug-induced cardiotoxicity has been the lack of relevant models to study cardiac safety. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great potential in drug discovery and cardiotoxcity screens as they display many characteristics of the human myocardium and offer unlimited supply. This unit describes how to use pluripotent stem cells derived cardiomyocytes to study drug-induced cardiotoxicty using doxorubicin as an example...
August 4, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28777442/microphysiological-systems-to-assess-nonclinical-toxicity
#3
Kirk P Van Ness, Shih-Yu Chang, Elijah J Weber, Danielle Zumpano, David L Eaton, Edward J Kelly
The liver and the kidney are key toxicity target organs during drug development campaigns, as they typically carry the burden of drug transport and metabolism. Primary hepatocytes and proximal tubule epithelial cells grown in traditional in vitro 2-D culture systems do not maintain transporter and metabolic functions, thus limiting their utility for nonclinical toxicology investigations. We have developed a renal and hepatic microphysiological system (MPS) platform that uses a commercially available MPS device as the core cell culture platform for our methodologies...
August 4, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28777441/optimizing-dual-fluorescent-analysis-to-investigate-the-toxicity-of-agnps-in-e-coli
#4
Wei Hong, Shaopeng Chen
The ever-increasing use of silver nanoparticles (AgNPs) carries potential ecotoxicological risks. For full risk assessment, E. coli cells harboring a plasmid with a constitutively expressed GFP gene under control of lac promoter (lac::GFP) are extensively utilized. Flow cytometry is an advanced technology usually applied to toxicological research for rapid, efficient, multi-parameter analysis of single cells. However, it is difficult to accurately and sensitively detect the toxicity of nanoparticles with flow cytometry due to the interference of aggregated nanoparticles...
August 4, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28777440/3d-differentiation-of-luhmes-cell-line-to-study-recovery-and-delayed-neurotoxic-effects
#5
Georgina Harris, Helena Hogberg, Thomas Hartung, Lena Smirnova
Current neurotoxicity testing and the study of molecular mechanisms in neurodegeneration in vitro usually focuses on acute exposures to compounds. 3D Lund human mesencephalic (LUHMES) cells allow long-term treatment or pulse exposure in combination with compound washout to study delayed neurotoxic effects as well as recovery and neurodegeneration pathways. In this unit we describe 3D LUHMES culture and characterization. Characterization of the model involves immunocytochemistry, flow cytometry, and qPCR measurements...
August 4, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28777439/combining-cytotoxicity-assessment-and-xenopus-laevis-phenotypic-abnormality-assay-as-a-predictor-of-nanomaterial-safety
#6
Karamallah Al-Yousuf, Carl A Webster, Grant N Wheeler, Francesca Baldelli Bombelli, Victoria Sherwood
The African clawed frog, Xenopus laevis, has been used as an efficient pre-clinical screening tool to predict drug safety during the early stages of the drug discovery process. X. laevis is a relatively inexpensive model that can be used in whole organism high-throughput assays whilst maintaining a high degree of homology to the higher vertebrate models often used in scientific research. Despite an ever-increasing volume of biomedical nanoparticles (NPs) in development, their unique physico-chemical properties challenge the use of standard toxicology assays...
August 4, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28463420/behavioral-phenotyping-for-autism-spectrum-disorders-in-mice
#7
Yu-Chi Chang, Toby B Cole, Lucio G Costa
Autism spectrum disorder (ASD) represents a heterogeneous group of disorders characterized by alterations in three behavioral symptom domains: Social interactions, verbal and nonverbal communication, and repetitive behaviors. Increasing prevalence of ASD in recent years suggests that exposure to environmental toxicants may be critical in modulating etiology of this disease. As clinical diagnosis of autism still relies on behavioral evaluation, it is important to be able to assess similar behavioral traits in animal models, to provide biological plausibility of associations between environmental exposures and ASD...
May 2, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28463419/micropatterned-co-cultures-of-human-hepatocytes-and-stromal-cells-for-the-assessment-of-drug-clearance-and-drug-drug-interactions
#8
Christine Lin, Salman R Khetani
Drug clearance rates from the body can determine drug exposure that can affect efficacy or toxicity. Thus, accurate prediction of drug clearance during preclinical development can help guide dose selection in humans, but animal testing is not always predictive of human outcomes. Because hepatic drug metabolism is a rate-limiting step in the overall clearance of many drugs, primary human hepatocytes (PHHs) in suspension cultures or monolayers are used for drug clearance predictions. Yet, the precipitous decline in drug metabolism capacity can lead to significant underestimation of clearance rates, particularly for low turnover compounds that have desirable one-pill-a-day dosing regimens...
May 2, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28463418/use-of-primary-rat-hepatocytes-for-prediction-of-drug-induced-mitochondrial-dysfunction
#9
Cong Liu, Shuichi Sekine, Binbin Song, Kousei Ito
Mitochondrial dysfunction plays a central role in drug-induced liver injury. To evaluate drug-induced mitochondrial impairment, several isolated mitochondria- or cell line-based assays have been reported. Among them, culturing HepG2 cells in galactose provides a remarkable method to assess mitochondrial toxicity by activating mitochondrial aerobic respiration. We applied this assay to primary rat hepatocytes by culturing cells in galactose and hyperoxia to enhance the evaluation of metabolism-related drug-induced mitochondrial toxicity...
May 2, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28463417/a-multi-parametric-fluorescent-assay-for-the-screening-and-mechanistic-study-of-drug-induced-steatosis-in-liver-cells-in-culture
#10
Laia Tolosa, M José Gómez-Lechón, M Teresa Donato
Human hepatic cells have been used for drug safety risk evaluations throughout early development phases. They provide rapid, cost-effective early feedback to identify drug candidates with potential hepatotoxicity. This unit presents a cell-based assay to evaluate the risk of liver damage associated with steatogenic drugs. Detailed protocols for cell exposure to test compounds and for the assessment of steatosis-related cell parameters (intracellular lipid content, reactive oxygen species production, mitochondrial impairment, and cell death) are provided...
May 2, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28463416/quantification-of-lactate-dehydrogenase-for-cell-viability-testing-using-cell-lines-and-primary-cultured-astrocytes
#11
Simon Kaja, Andrew J Payne, Yuliya Naumchuk, Peter Koulen
Drug discovery heavily relies on cell viability studies to assess the potential toxicity of drug candidates. L-Lactate dehydrogenase (LDH) is a cytoplasmic enzyme that catalyzes the concomitant interconversions of pyruvate to L-lactate and NADH to NAD(+) during glycolysis, and the reverse reactions during the Cori cycle. In response to cellular damage, induced by endogenous cellular mechanisms or as a result of exogenously applied insults, LDH is released from the cytoplasm into the extracellular environment...
May 2, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28463415/using-chromatin-immunoprecipitation-in-toxicology-a-step-by-step-guide-to-increasing-efficiency-reducing-variability-and-expanding-applications
#12
Shaun D McCullough, Doan M On, Emma C Bowers
Histone modifications work in concert with DNA methylation to regulate cellular structure, function, and response to environmental stimuli. More than 130 unique histone modifications have been described to date, and chromatin immunoprecipitation (ChIP) allows for the exploration of their associations with the regulatory regions of target genes and other DNA/chromatin-associated proteins across the genome. Many variations of ChIP have been developed in the 30 years since its earliest version came into use, which makes it challenging for users to integrate the procedure into their research programs...
May 2, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28146282/preparation-of-specific-compartments-of-the-lungs-for-pathologic-and-biochemical-analysis-of-toxicologic-responses
#13
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...
February 1, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28146281/flow-cytometry-based-method-to-detect-escherichia-coli-and-shigella-spp-using-16s-rrna-based-probe
#14
Yong Xue, Jon G Wilkes, Ted J Moskal, Anna J Williams, Willie M Cooper, Dan A Buzatu
Detection of microbial contamination in foods before they go on to the market can help prevent the occurrence of foodborne illness outbreaks. Current methods for the detection of Escherichia coli are limited by time-consuming procedures, which include multiple culture incubation steps, and require several days to get results. This unit describes the development of an improved rapid flow-cytometry-based detection method that has greater sensitivity and specificity. This method requires less time-to-results (TTR) and can detect a small number of E...
February 1, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28146280/assessment-of-bile-salt-export-pump-bsep-inhibition-in-membrane-vesicles-using-radioactive-and-lc-ms-based-detection-methods
#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...
February 1, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28146279/detecting-protein-sulfenylation-in-cells-exposed-to-a-toxicant
#16
Phillip A Wages
Protein sulfenylation is a post-translational modification that is linked to many cell signaling networks and specific protein functions, thus the detection of any sulfenylated protein after a toxicological exposure is of importance. Specifically, the detection of protein sulfenylation can provide multiple levels of mechanistic insight towards understanding the impact of a toxicological exposure. For instance, sulfenylation is caused by only a handful of reactive chemical species. Any altered sulfenylation suggests a change in cellular health, and the elucidation of the specific protein target that undergoes sulfenylation can help ascertain downstream targets and associated adverse outcomes...
February 1, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28146278/detection-and-manipulation-of-the-stress-response-protein-metallothionein
#17
Sadikshya Bhandari, Clare Melchiorre, Kristen Dostie, Debby Laukens, Lindsey Devisscher, Ariel Louwrier, Amy Thees, Michael A Lynes
Metallothioneins (MTs) are small molecular weight stress response proteins that play a central role as reservoir of essential divalent heavy metal cations such as zinc and copper, and also can diminish the effects of toxic heavy metals such as mercury and cadmium. Historically, MT has been considered to be an intracellular protein with roles to play in the management of heavy metals, as a regulator of cellular redox potential, and as a buffer of free radicals. Our recent studies have highlighted immunomodulatory role of MT in inflammatory diseases and also in the progression of metastatic cell movement...
February 1, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/27801935/fluorescence-based-microplate-assays-for-in-vitro-assessment-of-mitochondrial-toxicity-metabolic-perturbation-and-cellular-oxygenation
#18
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...
November 1, 2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/27801934/basic-techniques-in-mammalian-cell-tissue-culture
#19
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.
November 1, 2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/27479365/a-high-throughput-screening-assay-to-identify-kidney-toxic-compounds
#20
Susanne Ramm, Melanie Adler, Vishal S Vaidya
Kidney toxicity due to drugs and chemicals poses a significant health burden for patients and a financial risk for pharmaceutical companies. However, currently no sensitive and high-throughput in vitro method exists for predictive nephrotoxicity assessment. Primary human proximal tubular epithelial cells (HPTECs) possess characteristics of differentiated epithelial cells, making them a desirable model to use in in vitro screening systems. Additionally, heme oxygenase 1 (HO-1) protein expression is upregulated as a protective mechanism during kidney toxicant-induced oxidative stress or inflammation in HPTECs and can therefore be used as a biomarker for nephrotoxicity...
August 1, 2016: Current Protocols in Toxicology
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