High-Throughput Assessment of Mechanistic Toxicity of Chemicals in Miniaturized 3D Cell Culture.
Authors: Joshi P, Kang SY, Datar A, Lee MY
Abstract
High-content imaging (HCI) assays on two-dimensional (2D) cell cultures often do not represent in vivo characteristics accurately, thus reducing the predictability of drug toxicity/efficacy in vivo. On the other hand, conventional 3D cell cultures are relatively low throughput and possess difficulty in cell imaging. To address these limitations, a miniaturized 3D cell culture has been developed on a micropillar/microwell chip platform with human cells encapsulated in biomimetic hydrogels. Model compounds are used to validate human cell microarrays for hig...
Source: Current Protocols in Toxicology - November 4, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Assessment of Altered Cholesterol Homeostasis by Xenobiotics Using Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.
Authors: Herron J, Hines KM, Xu L
Abstract
Cholesterol and cholesterol-derived oxysterols are critical for embryonic development, synapse formation and function, and myelination, among other biological functions. Indeed, alterations in levels of cholesterol, sterol precursors, and oxysterols result in a variety of developmental disorders, emphasizing the importance of cholesterol homeostasis. The ability of xenobiotics to reproduce similar phenotypes by altering cholesterol homeostasis has increasingly become of interest. Therefore, the ability to quantitatively assess alterations in cholesterol homeostasi...
Source: Current Protocols in Toxicology - October 16, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Simultaneous Determination of Uranium and Depleted Uranium Isotopic Ratio Using Inductively Coupled Mass Spectrometry.
Authors: Velez-Quinones MA, Xu H, Vo N, Gaitens JM, McDiarmid MA, Lewin-Smith MR, Strathmann FG
Abstract
We have developed and validated a method for the simultaneous quantitative measurement of total uranium (TU) and uranium 235 U/238 U isotopic ratio (UIR) in urine by inductively coupled plasma mass spectrometry (ICP-MS) using a Thermo Scientific iCAP-Q instrument. The performance characteristics of the assay were determined to be in compliance with clinical laboratory standards. The assay was linear in the concentration range of 1.0 to 500.0 ng/liter TU. The method was precise and accurate with limits o...
Source: Current Protocols in Toxicology - October 5, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Determination of Thirteen Trace and Toxic Elements in Urine Using Inductively Coupled Mass Spectrometry.
Authors: Velez-Quinones MA, Xu H, Vo N, Gaitens JM, McDiarmid MA, Lewin-Smith MR, Strathmann FG
Abstract
We developed and validated a method for the assessment of thirteen separate trace and toxic elements using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). Included elements were as follows: aluminum, chromium, manganese, iron, cobalt, nickel, copper, zinc, arsenic, molybdenum, cadmium, tungsten, and lead. The measurements of all elements in urine samples were conducted using ICAP-Q ICP-MS in a single method. The performance characteristics of the assay were determined according to clinical labora...
Source: Current Protocols in Toxicology - September 22, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Structural and Functional Analysis of the Gut Microbiome for Toxicologists.
Authors: Nichols RG, Cai J, Murray IA, Koo I, Smith PB, Perdew GH, Patterson AD
Abstract
Characterizing the reciprocal interactions between toxicants, the gut microbiota, and the host, holds great promise for improving our mechanistic understanding of toxic endpoints. Advances in culture-independent sequencing analysis (e.g., 16S rRNA gene amplicon sequencing) combined with quantitative metabolite profiling (i.e., metabolomics) have provided new ways of studying the gut microbiome and have begun to illuminate how toxicants influence the structure and function of the gut microbiome. Developing a standardize...
Source: Current Protocols in Toxicology - September 21, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Analysis of Mitochondrial Protein Synthesis: De Novo Translation, Steady-State Levels, and Assembled OXPHOS Complexes.
Authors: Hilander T, Konovalova S, Terzioglu M, Tyynismaa H
Abstract
Mitochondria are multifunctional organelles with their own genome and protein synthesis machinery. The 13 proteins encoded by mitochondrial DNA (mtDNA) are core subunits of the oxidative phosphorylation (OXPHOS) system producing the majority of cellular ATP. Yet most mitochondrial proteins are encoded by nuclear genes, synthesized by cytosolic ribosomes, and imported into mitochondria. Therefore, disturbances in cytosolic proteostasis have consequences on the gene expression and synthesis of mtDNA-encoded proteins and overall on mitochond...
Source: Current Protocols in Toxicology - August 2, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Microbiome Analyses for Toxicological Studies.
Authors: Benjamino J, Beka L, Graf J
Abstract
Microbiomes can be thought of as the integration of biotic and abiotic factors, including the microbial communities and the distinct physio-chemical properties that are present in a habitat. The microbes within a microbiome can influence the chemical environment by degrading medications inside patients or contaminants in the environment, such as hydrocarbons released after the Deepwater Horizon oil spill. Identifying and monitoring the relative abundance of microbes can help to elucidate variations in their response to toxins and should be considered as a varia...
Source: Current Protocols in Toxicology - July 27, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Quantification of Serum Voriconazole, Isavuconazole, and Posaconazole by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS).
Authors: Smith A, Dowis J, French D
Abstract
Voriconazole, isavuconazole, and posaconazole are triazole anti-fungal drugs commonly used to treat invasive fungal infections. Therapeutic drug monitoring is commonly carried out when patients are prescribed these drugs to ensure that the serum concentrations are sufficiently high for efficacy, but do not reach toxic concentrations. Mass spectrometric (MS) assays have been successfully utilized to determine the serum concentrations of these drugs. This protocol describes a sample preparation procedure and a liquid chromatography-tandem mass spectrometry assay f...
Source: Current Protocols in Toxicology - July 26, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
An In Vitro Model of Hematotoxicity: Differentiation of Bone Marrow-Derived Stem/Progenitor Cells into Hematopoietic Lineages and Evaluation of Lineage-Specific Hematotoxicity.
Authors: Mahalingaiah PK, Palenski T, Van Vleet TR
Abstract
Hematotoxicity is a significant issue for drug safety and can result from direct cytotoxicity toward circulating mature blood cell types as well as targeting of immature blood-forming stem cells/progenitor cells in the bone marrow. In vitro models for understanding and investigating the hematotoxicity potential of new test items/drugs are critical in early preclinical drug development. The traditional method, colony forming unit (CFU) assay, is commonly used and has been validated as a method for hematotoxicity screening. The CFU assay has multipl...
Source: Current Protocols in Toxicology - July 26, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
In Vitro Methods to Characterize the Effects of Tobacco and Nontobacco Products on Human Platelet Function.
Authors: Loelius SG, Spinelli SL, Lannan KL, Phipps RP
Abstract
In this document, we describe methods for the isolation, treatment, and functional testing of human blood platelets in vitro. Functional assays for inflammatory function include flow cytometry and immunoassays for platelet release of platelet factor 4, soluble CD40L, prostaglandin E2 , and thromboxane. Assays for platelet hemostatic function described here examine platelet spreading, aggregation using platelet-rich plasma, and thromboelastography. Also described here are methods for testing cigarette smoke on primary human platelets in vitro, ...
Source: Current Protocols in Toxicology - July 26, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Determining Food Allergens by Skin Sensitization in Mice.
Authors: Tamehiro N, Adachi R, Kimura Y, Sakai S, Teshima R, Kondo K
Abstract
A food allergy is a chronic inflammatory disease against dietary antigens with high prevalence in industrialized countries. Because there is currently no cure for food allergies, avoiding the allergen is crucial for the prevention of an allergic reaction. Therefore, a further understanding of the pathogenesis and risk factors that augment the sensitization to food allergens is required. We have previously developed a food allergy mouse model using transdermal sensitization, which influences the susceptibility to food allergies. I...
Source: Current Protocols in Toxicology - July 26, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Accidental and Programmed Cell Death in Investigative and Toxicologic Pathology.
This article describes tissue-based immunohistotechnical protocols used for assessing PCD and necrosis in formalin-fixed tissues obtained from preclinical species used in investigative and toxicologic pathology. Two commonly employed protocols for the identification of PCD and necrosis are described in this article: immunohistochemistry (IHC) for cleaved caspase 3, and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). TUNEL has been used to detect DNA fragmentation by labeling the terminal ends of nucleic acids in necrotic and apoptotic cells. © 2018 by John Wiley & Sons, Inc.
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Source: Current Protocols in Toxicology - July 26, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Newly Revised Quantitative PCR-Based Assay for Mitochondrial and Nuclear DNA Damage.
This article describes a method based on a long-amplicon quantitative PCR-based assay that does not require a separate mitochondrial isolation step, which can often be labor-intensive and generate artifacts. The detailed basic protocol presented here is newly revised, with particular attention to application in Homo sapiens, Rattus norvegicus, and Caenorhabditis elegans resulting from changes in availability of PCR reagents. Optimized extraction support protocols are also described for high-quality DNA from multiple rat tissues for which these procedures had not previously been described. © 2018 by John Wiley & Sons, ...
Source: Current Protocols in Toxicology - July 26, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Effects of Rodent Diet Choice and Fiber Type on Data Interpretation of Gut Microbiome and Metabolic Disease Research.
Authors: Pellizzon MA, Ricci MR
Abstract
Poor diet reporting and improperly controlling laboratory animal diet continues to reduce our ability to interpret data effectively in animal studies. In order to make the best use of our resources and improve research transparency, proper reporting methods that include a diet design are essential to improving our understanding of the links between gut health and metabolic disease onset. This unit will focus on the importance of diet choice in laboratory animal studies, specifically as it relates to gut health, microbiome, and metabolic disease development. The two ...
Source: Current Protocols in Toxicology - July 23, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Generating Germ-Free Drosophila to Study Gut-Microbe Interactions: Protocol to Rear Drosophila Under Axenic Conditions.
Authors: Kietz C, Pollari V, Meinander A
Abstract
As several diseases have been linked to dysbiosis of the human intestinal microflora, manipulation of the microbiota has emerged as an exciting new strategy for potentially treating and preventing diseases. However, the human microbiota consists of a plethora of different species, and distinguishing the impact of a specific bacterial species on human health is challenging. In tackling this challenge, the fruit fly Drosophila melanogaster, with its far simpler microbial composition, has emerged as a powerful model for unraveling host-microbe interactions. To...
Source: Current Protocols in Toxicology - June 24, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
