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Current Protocols in Toxicology

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https://www.readbyqxmd.com/read/29117438/metallic-nano-composite-toxicity-evaluation-by-zebrafish-embryo-toxicity-test-with-identification-of-specific-exposure-biomarkers
#1
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
#2
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
#3
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
#4
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
#5
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
#6
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
#7
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
#8
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
#9
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
#10
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
#11
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
#12
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
#13
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
#14
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
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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