Current protocols in toxicology最新文献

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. Therefore, this unit will describe induction and assessment of the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), and its potential alteration by cannabinoid compounds. The unit includes three approaches to induce EAE, two of which provide correlations to two forms of MS, and the third specifically addresses the role of autoreactive T cells in EAE. © 2018 by John Wiley & Sons, Inc.

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). Blood collected in a plain, EDTA, or heparin-containing tube is centrifuged to separate the serum or plasma. Proteins are precipitated using a solution containing zinc sulfate and acetonitrile that has been spiked with deuterated internal standards. The resulting protein-free supernatant is injected into the LC/MS/MS system for analysis. The chromatography involves the use of a C18 column and ammonium acetate/water/formic acid and ammonium acetate/methanol/formic acid mobile phases. Quantification of MPA and MPAG levels is achieved by comparing the MRM peak area ratios of analytes and internal standards, consisting of specific precursor/product pairs, with those of calibrators at various concentrations. Calibration curves are constructed from the MRM peak area ratios of calibrators and internal standards versus concentration. © 2018 by John Wiley & Sons, Inc.

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). EBs contain a variety of cells of endodermal, ectodermal, and mesodermal origin and can be subjected to compound exposure in vitro. Hence, they are useful for the detection of chemicals with teratogenic activities. Beyond describing protocols to assemble and culture EBs, this unit details methods to exploit the EB system for teratological assessment. In addition, strategies to distinguish compounds with bona fide teratogenic activity versus simple toxicity are discussed. © 2018 by John Wiley & Sons, Inc.

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. The protocols for evaluating hematopoietic stem cells (HSCs) comprise phenotypic analysis and functional transplantation assays. The protocols for assessing hematopoietic progenitor cells (HPCs) consist of phenotypic analysis and in vitro differentiation assays. The main techniques applied in these protocols include flow cytometry (phenotypic analysis and sorting), irradiation, transplantation, and cell culture. © 2018 by John Wiley & Sons, Inc.

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. Essentially, any assay for discriminating apoptosis and necrosis needs to be carried out in real-time kinetic mode. Caspase 3 or 7 activation is observed in the majority of apoptotic cell death. Similarly, the absence of caspase 3/7 activation and cell membrane leakage are the two prominent indicators for necrotic cell death or necroptosis. The programmed form of necrosis, called pyroptosis, is also accompanied by membrane leakage and most often associated with activation of specific caspases such as caspase 1, 4, or 11, but not through caspase 3/7 activation. Here, a robust and sensitive real-time method is described to distinguish and discriminate apoptosis from necrosis. The assay utilizes stable integration of a genetically encoded fluorescence resonance energy transfer (FRET) probe for caspase 3/7 activation and the mitochondrion-targeted DsRed to identify necrotic cells. Caspase activation is determined by cleavage of the FRET probe; loss of soluble FRET probe with retention of mitochondrial red fluorescence indicates necrosis. This unit describes an important protocol for the generation of sensor cells expressing both probes, followed by detailed analysis of apoptosis and necrosis by microscopy imaging, confocal imaging, high-throughput imaging, and flow cytometry. © 2018 by John Wiley & Sons, Inc.

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. The current unit presents a protocol for preparation of the ALI organoid culture from normal and tumor colorectal tissues of human patients. ALI organoid culture from human tissues might be useful for examining not only resistance to chemotherapy in a tumor microenvironment but also toxic effects on organoids. © 2018 by John Wiley & Sons, Inc.

The protocols described in this unit are designed to measure the intragastric pressure in anesthetized rats by a water-filled low-compliance rubber balloon. The balloon is introduced into the stomach either orally (by passing the balloon down the esophagus) or directly via a small incision of the fundus after laparotomy. The effects of both stimulatory (e.g., carbachol) and inhibitory (e.g., oxymetazoline) agents can be evaluated on the gastric tone and phasic contractions. The model allows the evaluation of dose-response curves and also the time-course of the effects. Furthermore, by combining centrally or peripherally acting agents the site of action can also be determined. Curr. Protoc. Toxicol. 57:21.12.1-21.12.11. © 2013 by John Wiley & Sons, Inc.

Glutathionylation is a posttranslational modification that results in the formation of a mixed disulfide between glutathione and the thiol group of a protein cysteine residue. Glutathionylation of proteins occurs via both nonenzymatic mechanisms involving thiol/disulfide exchange and enzyme-mediated reactions. Protein glutathionylation is observed in response to oxidative or nitrosative stress and is redox-dependent, being readily reversible under reducing conditions. Such findings suggest that glutathionylation plays an important role in mediating redox-sensitive signaling. Indeed, glutathionylation can affect protein function by altering activity, protein-protein interactions, and ligand binding. Glutathionylation may also serve to prevent cysteine residues from undergoing irreversible oxidative modification. Thus, determining the ability of a given protein to become glutathionylated can provide insight into its redox regulation and putative role in dictating cellular response to oxidative and nitrosative stress. Methods to measure protein glutathionylation using immunoblotting and mass spectrometry are described. Curr. Protoc. Toxicol. 57:6.17.1-6.17.18. © 2013 by John Wiley & Sons, Inc.

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. Specific testing guidelines exist to assess potential neurotoxicity and developmental neurotoxicity in particular, and novel alternative testing approaches are being developed. © 2017 by John Wiley & Sons, Inc.

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. There is no significant difference in the percentage of cells expressing the CSM CD3, CD4, CD8, CD45RO, CD16, CD19, or CD56 between freshly isolated and cryopreserved HPBMC. Hence, cryopreservation of HPBMC does not influence the phenotype of distinct cellular subsets in isolated mononuclear cells. This protocol for HPBMC isolation, cryopreservation, and thawing of HPBMC is intended for long-term studies of large cohorts requiring sample shipment and subsequent batch analysis. © 2017 by John Wiley & Sons, Inc.