Simultaneous, untargeted metabolic profiling of polar and nonpolar metabolites by LC-Q-TOF mass spectrometry.
Authors: Kirkwood JS, Maier C, Stevens JF
Abstract
At its most ambitious, untargeted metabolomics aims to characterize and quantify all of the metabolites in a given system. Metabolites are often present at a broad range of concentrations and possess diverse physical properties complicating this task. Performing multiple sample extractions, concentrating sample extracts, and using several separation and detection methods are common strategies to overcome these challenges but require a great amount of resources. This protocol describes the untargeted, metabolic profiling of polar and nonpolar metabolites wi...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
In vivo measurement of intragastric pressure with a rubber balloon in the anesthetized rat.
Authors: Zádori ZS, Gyires K
Abstract
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...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Methods to detect protein glutathionylation.
Authors: Poerschke RL, Fritz KS, Franklin CC
Abstract
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 redo...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Measurement of arginine metabolites: regulators of nitric oxide metabolism.
Authors: Augustine MS, Rogers LK
Abstract
Arginine is the substrate for nitric oxide synthases (NOS), and arginine availability regulates the production of nitric oxide. Through the activity of methyltransferases, arginine can be methylated to form monomethylarginine (NMMA), asymmetrical dimethylarginine (ADMA), and symmetrical dimethylarginine (SDMA). NMMA and ADMA directly inhibit NOS, whereas SDMA inhibits the cellular import of arginine through the cationic amino acid transporter. Increased levels of methylarginine compounds have been associated with many diseases including atherosclerosis, renal failu...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
In vitro assays of chemotaxis as a window into mechanisms of toxicant-induced immunomodulation.
Authors: Pietrosimone KM, Bhandari S, Lemieux MG, Knecht DA, Lynes MA
Abstract
Dysregulated cell movement can lead to developmental abnormalities, neoplasia, and immune system disorders, and there are a variety of contexts in which xenobiotics (and biologic) effects on this movement are of interest. Many toxins and toxicants have been shown to disrupt controlled cell movement. Identification of compounds that affect cell movement is crucial to drug discovery. Drug components may have unexpected consequences with respect to cell motility, which would exclude these compounds in drug development. Finally, the...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Multi-parametric analysis of cell death pathways using live-cell microscopy.
Authors: Joshi GN, Knecht DA
Abstract
Programmed cell death is a complex process with new forms being discovered with regularity. Each pathway has a distinct and overlapping biochemical and physiological change occurring as the cell prepares for death. Detection of these changes can be facilitated by the availability of various fluorescent probes and advances in microscope systems. By analyzing these probes over time using fluorescence microscopy, the changes that occur in each cell en route to death can be analyzed on a cell-by-cell basis. While the timing of events varies considerably from cell to cell, ...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Assessment of drug transporter function using fluorescent cell imaging.
Authors: Bircsak KM, Gibson CJ, Robey RW, Aleksunes LM
Abstract
ATP-binding cassette (ABC) proteins, including the breast cancer resistance protein (BCRP) and multidrug resistance proteins (MDRs), actively transport structurally diverse chemicals from a number of tissues and are being increasingly cited as mediators of clinically relevant drug-drug interactions. The potential outcomes of concomitantly administering two drugs that interact at the same transporter include altered disposition and toxicity and/or efficacy of one or both of the drugs. Research demonstrating the role of transporters in clinical ...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Developmental toxicity assays using the Drosophila model.
Authors: Rand MD, Montgomery SL, Prince L, Vorojeikina D
Abstract
The fruit fly (Drosophila melanogaster) has long been a premier model for developmental biologists and geneticists. In toxicology studies, Drosophila has only recently gained broader recognition as a tool to elaborate molecular genetic mechanisms of toxic substances. In this article, two practical applications of Drosophila for developmental toxicity assays are described. The first assay takes advantage of newly developed methods to render the fly embryo accessible to small molecules, toxicants, and drugs. The second assay engages straightfo...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
High-Content Imaging Assays for Identifying Compounds that Generate Superoxide and Impair Mitochondrial Membrane Potential in Adherent Eukaryotic Cells.
Authors: Billis P, Will Y, Nadanaciva S
Abstract
Reactive oxygen species (ROS) are constantly produced in cells as a result of aerobic metabolism. When there is an excessive production of ROS and the cell's antioxidant defenses are overwhelmed, oxidative stress occurs. The superoxide anion is a type of ROS that is produced primarily in mitochondria but is also generated in other regions of the cell including peroxisomes, endoplasmic reticulum, plasma membrane, and cytosol. Here, a high-content imaging assay using the dye dihydroethidium is described for identifying compounds that generate superoxide in euk...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Synaptic structure quantification in cultured neurons.
Authors: Roqué PJ, Guizzetti M, Costa LG
Abstract
Behavioral problems (e.g., learning and memory) following developmental exposure to toxicants suggests that dysregulation of the process of synapse formation and function may occur. The ability to assess these changes is thus of value. This unit describes a method to investigate toxicant-induced changes to synaptic structure formation in primary hippocampal neurons using immunocytochemical labeling of the pre- and post-synaptic markers synaptophysin and PSD-95, confocal imaging, and three-dimensional object analysis. Protocols for the long-term culturing o...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Measuring Mitochondrial Function in Permeabilized Cells Using the Seahorse XF Analyzer or a Clark-Type Oxygen Electrode.
Authors: Divakaruni AS, Rogers GW, Murphy AN
Abstract
Measurements of mitochondrial respiration in intact cells can help define metabolism and its dysregulation in fields such as cancer, metabolic disease, immunology, and neurodegeneration. Although cells can be offered various substrates in the assay medium, many cell types can oxidize stored pools of energy substrates. A general bioenergetic profile can therefore be obtained using intact cells, but the inability to control substrate provision to the mitochondria can restrict an in-depth, mechanistic understanding. Mitochondria can be isolated from intact...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Assessment of Fatty Acid Beta oxidation in cells and isolated mitochondria.
Authors: Rogers GW, Nadanaciva S, Swiss R, Divakaruni AS, Will Y
Abstract
Fatty acid beta oxidation is a major pathway of energy metabolism and occurs primarily in mitochondria. Drug-induced modulation of this pathway can cause adverse effects such as liver injury, or be beneficial for treating heart failure, type 2 diabetes, and obesity. Hence, in vitro assays that are able to identify compounds that affect fatty acid oxidation are of value for toxicity assessments, as well as for efficacy assessments. Here, we describe two high-throughput assays, one for assessing fatty acid oxidation in cells and the ot...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Identifying Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore in Isolated Rat Liver Mitochondria.
Authors: Marroquin L, Swiss R, Will Y
Abstract
The mitochondrial permeability transition pore (MPTP) is a protein pore that forms in the inner mitochondrial membrane and allows the membrane to be permeable to all molecules of less than 1500 Da. Ca(2+) , numerous reactive chemicals, and oxidative stress induce MPTP opening, whereas cyclosporin A (CsA) or bongkrekic acid block it. In addition, several drugs have been shown to induce MPTP opening, leading to the loss of mitochondrial membrane potential, swelling of the matrix because of water accumulation, rupture of the outer mitochondrial membrane, and rele...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Comparative human and rat "neurosphere assay" for developmental neurotoxicity testing.
Authors: Baumann J, Barenys M, Gassmann K, Fritsche E
Abstract
The developing nervous system is highly vulnerable to the adverse effects of chemical agents. Currently, there is an increasing need for testing and regulating chemical compounds in general use and, due to the lack of available data, to identify those which are developmental neurotoxicants. In this context, alternative testing strategies are needed in order to allow fast and cost-efficient screening and to reduce the number of animal experiments usually required. In this unit we present an in vitro three-dimensional model for developmental neur...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Using the cytosensor microphysiometer to assess ocular toxicity.
Authors: Nash JR, Mun G, Raabe HA, Curren R
Abstract
Measuring in vitro cytotoxicity is one method currently used to estimate damage to the eye after chemical exposure. The Cytosensor Microphysiometer method evaluates cytotoxicity by measuring the test material-induced reduction in the metabolic rate of L929 cells. Changes in metabolic rate are measured indirectly as a function of changes in the extracellular acidification rate of the cells. During exposure to increasing concentrations of a cytotoxic material, there is a decrease in the release of acid byproducts into the surrounding medium as the cells di...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
