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Current Protocols in Toxicology

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https://www.readbyqxmd.com/read/29512128/use-of-human-embryoid-bodies-for-teratology
#1
Anthony Flamier, Supriya Singh, Theodore P Rasmussen
Human birth defects are relatively common and can be caused by exposure to environmental teratogens or to pharmaceuticals with teratogenic activities. Human embryonic stem cells (hESCs), by virtue of their pluripotent nature, provide an excellent cellular platform for teratogen detection and risk assessment. This unit describes detailed protocols for the preparation and validation of highly pluripotent hESCs, the production of large quantities of aggregated multicellular spheroids composed of hESCs, and these spheroids' differentiation into embryoid bodies (EBs)...
February 21, 2018: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/29512127/determination-of-mycophenolic-acid-and-mycophenolic-acid-glucuronide-using-liquid-chromatography-tandem-mass-spectrometry-lc-ms-ms
#2
Uttam Garg, Ada Munar, Clinton Frazee
Mycophenolic acid (MPA) is an immunosuppressant that is used in renal, liver, and heart transplantation. Due to its narrow therapeutic range, monitoring of MPA levels is essential to avoid toxicity and organ rejection. Although immunoassays are available for the determination of MPA, due to their higher specificity, mass spectrometry methods are preferred. In this unit, we describe a liquid chromatography tandem mass spectrometry (LC/MS/MS) method utilizing positive ionization electrospray and multiple reaction monitoring (MRM) for the quantification of levels of MPA and its conjugate MPA glucuronide (MPAG)...
February 21, 2018: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/29512126/a-real-time-image-based-approach-to-distinguish-and-discriminate-apoptosis-from-necrosis
#3
Asha Lekshmi, Shankara Narayanan Varadarajan, Santhik Subhasingh Lupitha, Mydhily Nair, Aneesh Chandrasekharan, T R Santhoshkumar
Recent cell biology studies reveal that a cell can die through multiple pathways via distinct signaling mechanisms. Among these, apoptosis and necrosis are two distinct cell death pathways, and their detection and discrimination is vital in the drug discovery process and in understanding diverse biological processes. Although sensitive assays for apoptosis and necrosis are available, it is extremely difficult to adapt any of these methods to discriminate apoptosis-inducing stimuli from necrosis-inducing stimuli because of the acquisition of secondary necrosis by apoptotic cells when they are not phagocytosed...
February 21, 2018: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/29512125/evaluation-of-marijuana-compounds-on-neuroimmune-endpoints-in-experimental-autoimmune-encephalomyelitis
#4
Barbara L F Kaplan
Cannabinoid compounds refer to a group of more than 60 plant-derived compounds in Cannabis sativa, more commonly known as marijuana. Exposure to marijuana and cannabinoid compounds has been increasing due to increased societal acceptance for both recreational and possible medical use. Cannabinoid compounds suppress immune function, and while this could compromise one's ability to fight infections, immune suppression is the desired effect for therapies for autoimmune diseases. It is critical, therefore, to understand the effects and mechanisms by which cannabinoid compounds alter immune function, especially immune responses induced in autoimmune disease...
February 21, 2018: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/29512124/development-of-an-in-vitro-method-to-estimate-the-sensitization-induction-level-of-contact-allergens
#5
Valentina Galbiati, Sue Gibbs, Erwin Roggen, Emanuela Corsini
Recently, an in vitro procedure, which combines the epidermal equivalent potency assay with assessment of IL-18 to provide a single test for identification and classification of skin sensitizers, was developed and validated. This unit will describe a simple in vitro method for estimation of the expected sensitization induction level interpolating in vitro EC50 and IL-18 SI2 values to predict LLNA EC3 and/or human NOEL from standards curves generated using reference contact allergens, based on the use of Reconstituted human Epidermis (RhE)...
February 21, 2018: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/29512123/preparation-of-human-primary-colon-tissue-derived-organoid-using-air-liquid-interface-culture
#6
Tatsuya Usui, Masashi Sakurai, Koji Umata, Hideyuki Yamawaki, Takashi Ohama, Koichi Sato
In vitro analysis of intestinal epithelium has been hindered by a lack of suitable culture systems useful for gastrointestinal research. To overcome the problem, an air liquid interface (ALI) method using a collagen gel was established to culture three-dimensional primary cells containing both primary epithelial and mesenchymal components from mouse gastrointestinal tissues. ALI organoids accurately recapitulate organ structures, multilineage differentiation, and physiology. Since ALI organoids from human tissues have not been produced, we modified the previous protocol for mouse ALI organoid culture to establish the culture system of ALI organoids from normal and tumor colorectal tissues of human patients...
February 21, 2018: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/29512122/phenotypic-and-functional-evaluation-of-hematopoietic-stem-and-progenitor-cells-in-toxicology-of-heavy-metals
#7
Qian Li, Zhengli Yang, Yifan Zhao, Xiaodong Jia, Zhijun Zhou, Yubin Zhang
All blood cells are differentiated from hematopoietic stem and progenitor cells (HSPCs), a process known as hematopoiesis that can be influenced by a variety of environmental factors. In this unit, we introduce a couple of protocols including phenotypic analysis, mixed bone marrow (BM) chimera and rescue assays, colony-forming unit (CFU), and in vitro OP9 stromal cell differentiation assays for evaluating HSPCs in the BM of mice, and provide some examples of their implications in mouse models treated with heavy metals...
February 21, 2018: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/29117438/metallic-nano-composite-toxicity-evaluation-by-zebrafish-embryo-toxicity-test-with-identification-of-specific-exposure-biomarkers
#8
Roberta Pecoraro, Antonio Salvaggio, Fabio Marino, Gianfranco Di Caro, Fabiano Capparucci, Bianca Maria Lombardo, Giuseppina Messina, Elena Maria Scalisi, Maurizio Tummino, Francesco Loreto, Giusi D'Amante, Roberto Avola, Daniele Tibullo, Maria Violetta Brundo
Nanomaterials (NM) have different shapes and can be composed of different materials such as carbon, silicon, and some metals like gold, silver, and titanium. They are used as fillers, catalysts, semiconductors, cosmetics, drug carriers in medicine, energy storage systems, and antifriction coatings. NM are the parent compounds of nanoparticles (NPs), which may be divided into two groups: fullerenes and engineered nanoparticles (ENPs). After crossing the cell membrane, NPs may be stored in vesicles, mitochondria, and additional organelles within epithelial cells...
November 8, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/29117437/measuring-%C3%AE-galactosidase-activity-in-gram-positive-bacteria-using-a-whole-cell-assay-with-mug-as-a-fluorescent-reporter
#9
Norman H L Chiu, Amanda L Watson
The use of β-galactosidase enzyme as a biomarker has the potential to determine activity levels of the microbiome of a variety of organisms due to its common presence in both eukaryotes and prokaryotes. Completing the assay in a whole-cell format facilitates the monitoring of β-galactosidase activity in its actual cellular environment. This unit describes an optimized fluorescent assay for β-galactosidase that has enough sensitivity to detect the enzymatic activity despite the thick gram-positive bacterial cellular membrane...
November 8, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/29117436/isolation-cryopreservation-and-immunophenotyping-of-human-peripheral-blood-mononuclear-cells
#10
Fredine T Lauer, Jesse L Denson, Scott W Burchiel
This unit describes procedures for the isolation, cryopreservation, and thawing of human peripheral blood mononuclear cells (HPBMC) and analysis of cell surface markers (CSM) for immunophenotyping using polychromatic flow cytometry. This methodology can be used to ensure that cell integrity and phenotype stability are not altered through cryopreservation and extended storage. For this analysis, HPBMC were isolated from 7 healthy individuals, and 11-color flow cytometry was performed on freshly isolated samples as well as samples cryopreserved for short- and long-term periods...
November 8, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/29117435/overview-of-neurotoxicology
#11
Lucio G Costa
The nervous system has a central and primary function in the body, and its relevance and complexity make it a target for a large number of toxic substances. The most common forms of neurotoxicity are the death of neurons (neuronopathy), the degeneration of axons (axonopathy), damage to glial cells (e.g., myelinopathy), and interference with the axonal membrane or neurotransmission. Important neurotoxicants are found among pesticides, metals, solvents, natural substances, and industrial chemicals. Environmental chemicals may also contribute to the etiopathogenesis of neurodevelopmental, neuropsychiatric, and neurodegenerative disorders...
November 8, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/29117434/co-culture-of-neurons-and-microglia
#12
Pamela J Roqué, Lucio G Costa
Microglia, the resident immune cells of the brain, have been implicated in numerous neurodegenerative and neurodevelopmental diseases. Activation of microglia by a variety of stimuli induces the release of factors, including pro- and anti-inflammatory cytokines and reactive oxygen species, that contribute to modulating neuro-inflammation and oxidative stress, two crucial processes linked to disorders of the central nervous system. The in vitro techniques described here will provide a set of protocols for the isolation and plating of primary cerebellar granule neurons, primary cortical microglia from a mixed glia culture, and methods for co-culturing both cell types...
November 8, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/29117433/inter-and-intra-laboratory-standardization-of-tunel-assay-for-assessment-of-sperm-dna-fragmentation
#13
Sajal Gupta, Rakesh Sharma, Ashok Agarwal
The functional aspects of sperm activity such as sperm chromatin integrity and ability to fertilize cannot be characterized by routine semen parameters. Men with unexplained infertility and idiopathic infertility, as well as men with normozoospermic semen profiles, show high DNA fragmentation. Molecular anomalies in the sperm can be detected by a sperm DNA fragmentation (SDF) assay which can be used in adjunct to conventional semen analysis. While the sperm chromatin structure assay (SCSA) remains the "gold standard," the TUNEL assay using flow cytometry is becoming popular among the different tests that are currently available to measure sperm DNA fragmentation...
November 8, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/29117432/gas-chromatography-mass-spectrometry-gc-ms-for-identification-of-designer-stimulants-including-2c-amines-nbome-compounds-and-cathinones-in-urine
#14
Hemamalini Ketha, Milad Webb, Larry Clayton, Sean Li
Phenethylamine derivatives are being increasingly exploited for recreational use as "designer" stimulants designed to mimic psychostimulant properties of amphetamine or other illicit substances like 3,4-methylenedioxymethamphetamine (MDMA [ecstasy]). Clandestine operations meticulously design phenethylamines so the user can bypass legal action when detected, as many of these are yet to be regulated by government authorities. Substituted phenethylamines or 2C amines, N-methoxybenzyl derivatives of the corresponding 2C amines commonly known as NBOMe compounds, and cathinones are among the most commonly abused phenethylamines...
November 8, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28777444/intracellular-cytokine-detection-by-flow-cytometry-in-surface-marker-defined-human-peripheral-blood-mononuclear-t-cells
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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