3D Differentiation of LUHMES Cell Line to Study Recovery and Delayed Neurotoxic Effects.
Authors: Harris G, Hogberg H, Hartung T, Smirnova L
Abstract
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. Studying the delayed effects ...
Source: Current Protocols in Toxicology - August 6, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Optimizing Dual Fluorescent Analysis to Investigate the Toxicity of AgNPs in E. coli.
Authors: Hong W, Chen S
Abstract
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. I...
Source: Current Protocols in Toxicology - August 6, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Microphysiological Systems to Assess Nonclinical Toxicity.
We describe protocols for isolating and propagating human proximal epithelial cells and how to seed and culture a renal MPS to recapitulate the human proximal tubule. We present two methods to culture hepatocytes within an MPS and the steps required to connect a renal MPS to a liver MPS. © 2017 by John Wiley & Sons, Inc.
PMID: 28777442 [PubMed - in process] (Source: Current Protocols in Toxicology)
Source: Current Protocols in Toxicology - August 6, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Use of Human Pluripotent Stem Cell Derived-Cardiomyocytes to Study Drug-Induced Cardiotoxicity.
We present a workflow that explains procedure for editing hPSC using the CRISPR/Cas9 system and for differentiation of hPSC into cardiomyocytes. We also report protocols to study drug effect on ROS production, intracellular calcium concentration, formation of DNA double strand breaks, gene expression and electrophysiological properties of hPSC-CMs. © 2017 by John Wiley & Sons, Inc.
PMID: 28777443 [PubMed - in process] (Source: Current Protocols in Toxicology)
Source: Current Protocols in Toxicology - August 6, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Intracellular Cytokine Detection by Flow Cytometry in Surface Marker-Defined Human Peripheral Blood Mononuclear T Cells.
Authors: Lauer FT, Denson JL, Beswick E, Burchiel SW
Abstract
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...
Source: Current Protocols in Toxicology - August 6, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Using Chromatin Immunoprecipitation in Toxicology: A Step-by-Step Guide to Increasing Efficiency, Reducing Variability, and Expanding Applications.
Authors: McCullough SD, On DM, Bowers EC
Abstract
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 th...
Source: Current Protocols in Toxicology - May 5, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Quantification of Lactate Dehydrogenase for Cell Viability Testing Using Cell Lines and Primary Cultured Astrocytes.
Authors: Kaja S, Payne AJ, Naumchuk Y, Koulen P
Abstract
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. Its stability in cell culture medium make...
Source: Current Protocols in Toxicology - May 5, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
A Multi-Parametric Fluorescent Assay for the Screening and Mechanistic Study of Drug-Induced Steatosis in Liver Cells in Culture.
Authors: Tolosa L, Gómez-Lechón MJ, Donato MT
Abstract
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 provi...
Source: Current Protocols in Toxicology - May 5, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Use of Primary Rat Hepatocytes for Prediction of Drug-Induced Mitochondrial Dysfunction.
Authors: Liu C, Sekine S, Song B, Ito K
Abstract
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. Conventional culture ...
Source: Current Protocols in Toxicology - May 5, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Micropatterned Co-Cultures of Human Hepatocytes and Stromal Cells for the Assessment of Drug Clearance and Drug-Drug Interactions.
Authors: Lin C, Khetani SR
Abstract
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 underesti...
Source: Current Protocols in Toxicology - May 5, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Behavioral Phenotyping for Autism Spectrum Disorders in Mice.
Authors: Chang YC, Cole TB, Costa LG
Abstract
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 ...
Source: Current Protocols in Toxicology - May 5, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Detection and Manipulation of the Stress Response Protein Metallothionein.
Authors: Bhandari S, Melchiorre C, Dostie K, Laukens D, Devisscher L, Louwrier A, Thees A, Lynes MA
Abstract
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 imm...
Source: Current Protocols in Toxicology - February 3, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Detecting Protein Sulfenylation in Cells Exposed to a Toxicant.
Authors: Wages PA
Abstract
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 ...
Source: Current Protocols in Toxicology - February 3, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Assessment of Bile Salt Export Pump (BSEP) Inhibition in Membrane Vesicles Using Radioactive and LC/MS-Based Detection Methods.
Authors: Marroquin LD, Bonin PD, Keefer J, Schroeter T
Abstract
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,...
Source: Current Protocols in Toxicology - February 3, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Flow-Cytometry-Based Method to Detect Escherichia coli and Shigella Spp. Using 16S rRNA-Based Probe.
Authors: Xue Y, Wilkes JG, Moskal TJ, Williams AJ, Cooper WM, Buzatu DA
Abstract
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 ...
Source: Current Protocols in Toxicology - February 3, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
