Current protocols in toxicology最新文献

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. The use of a smaller fluorometric substrate, namely 4-methylumbelliferyl β-D-galactopyranoside (MUG), has facilitated its penetration into the cells as well as its direct detection without any extra steps. This assay provides an improved technique for measuring a well-studied reporter enzyme and offers new avenues for using β-galactosidase as a biomarker. © 2017 by John Wiley & Sons, Inc.

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. A few representative results that illustrate the utility of this procedure for the screening of drug-induced steatosis are shown. © 2017 by John Wiley & Sons, Inc.

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. These methods allow the study of how microglia and the factors they release in this shared environment mediate the effects of toxicants on neuronal function and survival. The protocols presented here allow for flexibility in experimental design, the study of numerous toxicological endpoints, and the opportunity to explore neuroprotective strategies. © 2017 by John Wiley & Sons, Inc.

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. They may generate reactive oxygen species, oxidative stress, and cytotoxicity, as well as denaturation of proteins, apoptosis, and necrosis. Nowadays, new toxicological data are required to assess the potential exposure of the environment and human beings to pollutants. The aim of the present investigation is to evaluate the toxicity of the metallic nano-composite by the zebrafish embryo toxicity test (ZFET). The methods described here can be easily adapted to other nanocomposites or nanomaterials with some variations. © 2017 by John Wiley & Sons, Inc.

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. Furthermore, the differences in ChIP protocols can confound efforts to increase reproducibility across studies. The streamlined ChIP procedure presented here can be readily applied to samples from a wide range of in vitro studies (cell lines and primary cells) and clinical samples (peripheral leukocytes) in toxicology. We also provide detailed guidance on the optimization of critical protocol parameters, such as chromatin fixation, fragmentation, and immunoprecipitation, to increase efficiency and improve reproducibility. Expanding toxicoepigenetic studies to more readily include histone modifications will facilitate a more comprehensive understanding of the role of the epigenome in environmental exposure effects and the integration of epigenetic data in mechanistic toxicology, adverse outcome pathways, and risk assessment. © 2017 by John Wiley & Sons, Inc.

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. Current FDA-approved assays used in screening for illicit drug use lack the sensitivity needed to detect designer stimulants making it challenging for toxicologists to accurately identify these compounds. Gas chromatography mass spectrometry (GC-MS) is a sensitive method for identifying designer stimulants. This unit describes and compares two qualitative GC-MS methods for identifying 2C amines, NBOMe compounds, and cathinones in urine. © 2017 by John Wiley & Sons, Inc.

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. In this unit, we describe the inter-laboratory and intra-laboratory standardization of the TUNEL assay using a benchtop cytometer. The article also provides a step-by-step protocol for measuring sperm DNA fragmentation using the TUNEL assay and a bench-top flow cytometer, and also points out the inherent challenges with this test. © 2017 by John Wiley & Sons, Inc.

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. 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.

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. In this protocol, dual-fluorescence detection with a propidium iodide (PI)-lac::GFP assay is used to determine the toxicity of AgNPs and successfully discriminate the dead or fragilized bacteria from living bacteria and aggregated nanoparticles. © 2017 by John Wiley & Sons, Inc.