A Functional Assay to Assess Toxicity During Murine B Cell Development In Vitro.
Authors: Guilbert C, Chou H, Bolt AM, Wu TH, Luo VM, Orthwein A, Mann KK Abstract B lymphocytes, or B cells, are important players in immunity that produce antigen-specific immunoglobulins. As a result, they are involved in various immune-linked pathologies. To better understand, prevent, or treat B cell-associated disease and immunotoxicity, we developed an in vitro assay to model early murine B cell differentiation within the bone marrow. This model uses sorted B cell precursors cultured on a supporting stromal cell layer, which over time acquire markers of further differentiated B cells, such as surface antigens...
Source: Current Protocols in Toxicology - December 19, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Analysis of Protein-Protein Interactions by Split Luciferase Complementation Assay.
This article describes a split luciferase complementation (SLC) method designed to discover inhibitors of protein-protein interaction. Different fusion proteins with split luciferase are constructed, expressed, and purified, and then assessed to determine the best pair that generates the strongest luminescence. SLC specificity and affinity are further confirmed. Step-by-step instructions are provided for performing these assays using the NS2B-NS3 interaction as an example. NS2B is an essential cofactor for flaviviral NS3 protease function. Advantages and disadvantages of these assays are further discussed. © 2019 by J...
Source: Current Protocols in Toxicology - December 6, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Culturing Rat Whole Ovary for UV Filter Benzophenone-3 Treatment.
We describe a detailed protocol to establish a newborn rat whole ovary culture, which enables the study of direct effects (independent of hypothalamic-pituitary-gonadal axis) of endocrine disrupting chemicals (EDCs), such as benzophenone-3 (BP-3). This method is useful to understand changes in follicle formation, primordial to primary transition, and expression of regulatory molecules linked to these processes and also provides an alternative to animal models. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Rat ovarian surgery Basic Protocol 2: Whole organ/ovarian culture Basic Protocol 3: RNA isolation and qu...
Source: Current Protocols in Toxicology - November 27, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Adaptation of Mammalian Cells to Chemically Defined Media.
This article focuses on the gradual adaptation of a mammalian suspension cell culture to a chemically defined medium. The first step is to transfer the cells cultured in medium supplemented with fetal bovine serum (FBS) to a chemically defined medium of your choice, containing the same amount of FBS. The next steps consist of progressively reducing the amount of FBS, while monitoring cell growth and viability up to the complete elimination of FBS. This protocol has been successfully used to adapt THP-1 cells to a chemically defined medium with similar maximum specific growth rate as those cultured with FBS. © 2019 by ...
Source: Current Protocols in Toxicology - November 24, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Assessment of Sertoli Cell Proliferation by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide and Sulforhodamine B Assays.
Authors: Martins AD, Oliveira PF, Alves MG Abstract The correct functioning of Sertoli cells (SCs) is pivotal for successful spermatogenesis. They are major targets for hormones, endocrine disruptors, and other substances that men are subjected to every day. One of the main SC functions that quickly responds to a deleterious stimulus is proliferation. This is directly related to the in vivo capacity of these cells to sustain a good number of developing germ cells. The protocols in this article can be tested on SCs of different origin. For the case of human SCs from small human testicular biopsies, a short and simpl...
Source: Current Protocols in Toxicology - September 19, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Assembly and Application of a Three-Dimensional Human Corneal Tissue Model.
Authors: McKay TB, Ford A, Wang S, Cairns DM, Parker RN, Deardorff PM, Ghezzi CE, Kaplan DL Abstract The cornea provides a functional barrier separating the outside environment from the intraocular environment, thereby protecting posterior segments of the eye from infection and damage. Pathological changes that compromise the structure or integrity of the cornea may occur as a result of injury or disease and can lead to debilitating effects on visual acuity. Over 10 million people worldwide are visually impaired or blind due to corneal opacity. Thus, physiologically relevant in vitro approaches to predict corneal t...
Source: Current Protocols in Toxicology - September 19, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Preparation of Primary Rat Hepatocyte Spheroids Utilizing the Liquid-Overlay Technique.
We describe the liquid-overlay technique which provides an ultra-low attachment surface on which PRHs can be cultured as spheroids. © 2019 The Authors. Basic Protocol 1: Preparation of agarose-coated plates Basic Protocol 2: Primary rat hepatocyte isolation procedure Basic Protocol 3: Primary rat hepatocyte spheroid culture Basic Protocol 4: Immunofluorescent analysis of PRH spheroids. PMID: 31529797 [PubMed - in process] (Source: Current Protocols in Toxicology)
Source: Current Protocols in Toxicology - September 19, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Generation and Validation of Tissue-Specific Knockout Strains for Toxicology Research.
Authors: Taylor CA, Shawlot W, Ren JX, Mukhopadhyay S Abstract Tissue-specific knockout mice are widely used throughout scientific research. A principle method for generating tissue-specific knockout mice is the Cre-loxP system. Here, we give a detailed description of the steps required to generate and validate tissue-specific knockout mice using the Cre-loxP system. The first protocol describes how to use gene targeting in mouse embryonic stem cells to generate mice with conditional alleles. Subsequent protocols describe how to recover Cre transgenic mice from cryopreserved sperm using in vitro fertilization and p...
Source: Current Protocols in Toxicology - September 19, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Isolating Rat Intestinal Explants for In Vitro Cultures.
Authors: Kothari A, Rajagopalan P Abstract The small intestine is an important organ primarily involved in digestion of food and absorption of nutrients. In vitro intestinal models are being developed to study this organ in health and disease. Intestinal explants can be used in such investigations since they contain all the major intestinal cell types. A detailed procedure to isolate intestinal explants from the rat jejunum is described. A protocol for culturing them in vitro for up to 24 hr is also provided. © 2019 by John Wiley & Sons, Inc. PMID: 31120607 [PubMed - as supplied by publisher] (Source: ...
Source: Current Protocols in Toxicology - May 25, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Acute Metabolic Switch Assay Using Glucose/Galactose Medium in HepaRG Cells to Detect Mitochondrial Toxicity.
Authors: Kamalian L, Douglas O, Jolly CE, Snoeys J, Simic D, Monshouwer M, Williams DP, Park BK, Chadwick AE Abstract Using galactose instead of glucose in the culture medium of hepatoma cell lines, such as HepG2 cells, has been utilized for a decade to unmask the mitochondrial liability of chemical compounds. A modified glucose-galactose assay on HepG2 cells, reducing the experimental period for screening of mitochondrial toxicity to 2 to 4 hr, has been previously reported. HepaRG cells are one of the few cell lines that retain some of the important characteristics of human hepatocytes, offering advantages of work...
Source: Current Protocols in Toxicology - May 8, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

A Reliable Preclinical Model to Study the Impact of Cigarette Smoke in Development and Disease.
Authors: Aedo G, Miranda M, Chávez MN, Allende ML, Egaña JT Abstract The World Health Organization has estimated that, worldwide, cigarette smoking has caused more than 100 million deaths in the last century, a number that is expected to increase in the future. Understanding cigarette smoke toxicity is key for research and development of proper public health policies. The current challenge is to establish a reliable preclinical model to evaluate the effects of cigarette smoke. In this work, we describe a simple method that allows for quantifying the toxic effects of cigarette smoke using zebrafish. He...
Source: Current Protocols in Toxicology - May 8, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Toxicology in the Super-Resolution Era.
Authors: Cole R Abstract Light microscopy has played a central role in science for the past couple of hundred years and will continue to do so. Multiple super-resolution microscopy techniques have been in the headlines for smashing what for more than 100+ years was believed to be the limits of optical microscopy. This resolution improvement enables the visualization of molecular structures and processes on the nano scale. While certain scientific questions in toxicology can benefit from modalities within the super-resolution suite, due diligence is required for efficiency and to achieve optimal results. For a given...
Source: Current Protocols in Toxicology - April 20, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Analysis of Human Mitochondrial DNA Content by Southern Blotting and Nonradioactive Probe Hybridization.
Authors: Wheeler JH, Young CKJ, Young MJ Abstract A single cell can contain several thousand copies of the mitochondrial DNA genome or mtDNA. Tools for assessing mtDNA content are necessary for clinical and toxicological research, as mtDNA depletion is linked to genetic disease and drug toxicity. For instance, mtDNA depletion syndromes are typically fatal childhood disorders that are characterized by severe declines in mtDNA content in affected tissues. Mitochondrial toxicity and mtDNA depletion have also been reported in human immunodeficiency virus-infected patients treated with certain nucleoside reverse transcr...
Source: Current Protocols in Toxicology - April 15, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Generation of Macrophages from Cynomolgus-Monkey Bone Marrow as a Model to Evaluate Effects of Drugs on Innate Immunity.
Authors: Li N, Ludmann SA, Anest L, He C, Narayanan PK Abstract Macrophages are innate immune cells that play important roles in various physiological and pathological processes. Evaluation of pro-inflammatory effects of drugs on macrophages has become commonplace in preclinical drug development prior to human clinical trials. Despite their body-wide distribution, tissue macrophages are often difficult to collect from large animals and humans in a noninvasive manner. Therefore, in vitro-differentiated macrophages are important tools to facilitate cross-species analysis of macrophage function. Although cynomolgus mo...
Source: Current Protocols in Toxicology - April 15, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Mitochondrial Electron Transfer Cascade Enzyme Activity Assessment in Cultured Neurons and Select Brain Regions.
This article describes a step-by-step protocol for reliable and reproducible assessment of ETC enzyme kinetics (Complex I-IV) for mitochondria from small quantities of tissue from different brain regions, such as the hippocampus, cerebellum, and frontal cortex, or from neurons in culture. Methods for differential and density gradient centrifugation are detailed for isolating cell body and synaptic mitochondria from brain, as well as measurement of ETC activities in microwell plate or single-cuvette format using spectrophotometric methods. Easy-to follow assay layouts and useful tips are presented, allowing the user to perf...
Source: Current Protocols in Toxicology - April 7, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

The Use of Intracerebral Microdialysis to Elucidate Environmentally Induced Neurotoxic Mechanisms.
This article provides an overview of the manner in which the methodology of intracerebral microdialysis is utilized in the field of neurotoxicology to elucidate the actions of environmental agents. The technique is employed in a variety of creative ways to address specific experimental goals involving myriad toxicants. With appropriate consideration of method parameters, investigators have also been able to address mechanistic issues in their studies. These investigations consist of sampling of neurotransmitters in extracellular fluid after various protocols of environmental metal exposure as well as assessments of blood-b...
Source: Current Protocols in Toxicology - April 4, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Isolation of Alveolar Type II Cells from Adult Bovine Lung.
Authors: Lee DF, Chambers MA Abstract Alveolar type II (ATII) cells play a key role as part of the distal lung epithelium, including in the innate immune response and as self-renewing progenitors to replace alveolar type I (ATI) cells during epithelial regeneration. Their secretion of surfactant protein helps maintain homeostasis and exerts protective, antimicrobial properties. ATII cells remain difficult to study, partly due to inefficient and expensive isolation methods, a propensity to differentiate into ATI cells, and susceptibility to fibroblast contamination. Published methods of isolation often require speci...
Source: Current Protocols in Toxicology - March 16, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Preparation and Measuring of Brain GABAA R-coupled Cl- /HCO3 - - Activity for Integral Assessment of Aquatic Toxicity.
In this study, we present a protocol for isolation of the membrane-bound Cl- /HCO3 - -ATPase from neuronal cells of animal brain. We then describe an uncomplicated in vitro method for measuring this ATPase activity for assessment of toxicity after interaction of this protein with an aquatic sample. This assay offers new avenues for using the Cl- /HCO3 - -ATPase as a biomarker of water toxicity. This biotest is efficient, requires very little of the enzyme, and retains its sensitivity at low levels of various compounds. © 2019 by John Wiley & Sons, Inc. PMID: 30843667 [PubMed - as supplied by publisher] (Source...
Source: Current Protocols in Toxicology - March 9, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Inducing a Mucosal Barrier-Sparing Inflammatory Response in Laboratory-Grown Primary Human Nasal Epithelial Cells.
Authors: Ramezanpour M, Bolt H, Psaltis A, Wormald PJ, Vreugde S Abstract Here we use the toll-like receptor (TLR) 3 agonist poly I:C (LMW) to induce an inflammatory response in cells of submerged and/or air-liquid interface (ALI) cultures of human nasal epithelial cells (HNECs). The inflammatory response is determined by measuring interleukin-6 (IL-6) protein levels by enzyme-linked immunosorbent assay (ELISA). The mucosal barrier integrity is determined by measuring transepithelial electrical resistance (TEER) and passage of fluorescently labeled dextrans. Stimulation with poly (I:C) LMW induces a 15- to 17-fold ...
Source: Current Protocols in Toxicology - February 6, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

In Vitro Monocyte/Macrophage Phagocytosis Assay for the Prediction of Drug-Induced Thrombocytopenia.
Authors: Narayanan PK, Li N Abstract Phagocytosis of platelets by monocytes and macrophages is a primary mechanism of platelet clearance in vivo and has been increasingly implicated in playing an important role in thrombocytopenia mediated by monoclonal antibodies intended for therapeutic purposes. In the present article, we describe an in vitro flow cytometry assay to assess the effect of antibody-mediated platelet phagocytosis by monocytes. Freshly isolated platelets were labeled with a fluorescent probe, 5-chloromethylfluorescein diacetate (CMFDA) and then co-cultured with isolated peripheral blood mononuclear c...
Source: Current Protocols in Toxicology - January 24, 2019 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Screening for Neurotoxicity with Microelectrode Array.
This article describes the preparation and use of a 48-well microelectrode array (MEA) platform along with custom data analysis algorithms and commercially available analysis tools to screen for neurotoxic liabilities and seizurogenic effects using recorded spike file data generated from cryogenically preserved rat cortical neurons. © 2018 by John Wiley & Sons, Inc. PMID: 30575314 [PubMed - as supplied by publisher] (Source: Current Protocols in Toxicology)
Source: Current Protocols in Toxicology - December 25, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

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 high-through...
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 homeostasis resulti...
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 of detecti...
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 laboratory stan...
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 standardized protoco...
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 mitochondrial func...
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 variable in st...
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 for quanti...
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 multiple limitat...
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, which our...
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. In this mo...
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. ...
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 & So...
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 most comm...
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 study th...
Source: Current Protocols in Toxicology - June 24, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Phenotypic and Functional Evaluation of Hematopoietic Stem and Progenitor Cells in Toxicology of Heavy Metals.
Authors: Li Q, Yang Z, Zhao Y, Jia X, Zhou Z, Zhang Y Abstract 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 hemat...
Source: Current Protocols in Toxicology - March 9, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Preparation of Human Primary Colon Tissue-Derived Organoid Using Air Liquid Interface Culture.
Authors: Usui T, Sakurai M, Umata K, Yamawaki H, Ohama T, Sato K Abstract 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 mo...
Source: Current Protocols in Toxicology - March 9, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Development of an In Vitro Method to Estimate the Sensitization Induction Level of Contact Allergens.
Authors: Galbiati V, Gibbs S, Roggen E, Corsini E Abstract Recently, an in vitro procedure, which combines the epidermal equivalent potency assay with assessment of IL-18 to provide a single test for identification and classification of skin sensitizers, was developed and validated. This unit will describe a simple in vitro method for estimation of the expected sensitization induction level interpolating in vitro EC50 and IL-18 SI2 values to predict LLNA EC3 and/or human NOEL from standards curves generated using reference contact allergens, based on the use of Reconstituted human Epidermis (RhE).© 2018 by Joh...
Source: Current Protocols in Toxicology - March 9, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Evaluation of Marijuana Compounds on Neuroimmune Endpoints in Experimental Autoimmune Encephalomyelitis.
Authors: Kaplan BLF Abstract 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 ...
Source: Current Protocols in Toxicology - March 9, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

A Real-Time Image-Based Approach to Distinguish and Discriminate Apoptosis from Necrosis.
Authors: Lekshmi A, Varadarajan SN, Lupitha SS, Nair M, Chandrasekharan A, Santhoshkumar TR Abstract 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 beca...
Source: Current Protocols in Toxicology - March 9, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Determination of Mycophenolic Acid and Mycophenolic Acid Glucuronide Using Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS).
Authors: Garg U, Munar A, Frazee C Abstract 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 ...
Source: Current Protocols in Toxicology - March 9, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Use of Human Embryoid Bodies for Teratology.
Authors: Flamier A, Singh S, Rasmussen TP Abstract 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). E...
Source: Current Protocols in Toxicology - March 9, 2018 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Gas Chromatography Mass Spectrometry (GC-MS) for Identification of Designer Stimulants Including 2C Amines, NBOMe Compounds, and Cathinones in Urine.
Authors: Ketha H, Webb M, Clayton L, Li S Abstract 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 compoun...
Source: Current Protocols in Toxicology - November 10, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Inter-and Intra-Laboratory Standardization of TUNEL Assay for Assessment of Sperm DNA Fragmentation.
Authors: Gupta S, Sharma R, Agarwal A Abstract 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 b...
Source: Current Protocols in Toxicology - November 10, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Co-Culture of Neurons and Microglia.
Authors: Roqué PJ, Costa LG Abstract 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, pri...
Source: Current Protocols in Toxicology - November 10, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Overview of Neurotoxicology.
Authors: Costa LG Abstract 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, ne...
Source: Current Protocols in Toxicology - November 10, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Isolation, Cryopreservation, and Immunophenotyping of Human Peripheral Blood Mononuclear Cells.
Authors: Lauer FT, Denson JL, Burchiel SW Abstract 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-te...
Source: Current Protocols in Toxicology - November 10, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Measuring β-Galactosidase Activity in Gram-Positive Bacteria Using a Whole-Cell Assay with MUG as a Fluorescent Reporter.
Measuring β-Galactosidase Activity in Gram-Positive Bacteria Using a Whole-Cell Assay with MUG as a Fluorescent Reporter. Curr Protoc Toxicol. 2017 Nov 08;74:4.44.1-4.44.8 Authors: Chiu NHL, Watson AL Abstract 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 a...
Source: Current Protocols in Toxicology - November 10, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research

Metallic Nano-Composite Toxicity Evaluation by Zebrafish Embryo Toxicity Test with Identification of Specific Exposure Biomarkers.
Authors: Pecoraro R, Salvaggio A, Marino F, Caro GD, Capparucci F, Lombardo BM, Messina G, Scalisi EM, Tummino M, Loreto F, D'Amante G, Avola R, Tibullo D, Brundo MV Abstract 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 cro...
Source: Current Protocols in Toxicology - November 10, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research