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Current Protocols in Toxicology

Diane Frances Lee, Mark Andrew Chambers
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...
March 15, 2019: Current Protocols in Toxicology
Sergey A Menzikov
The wide use of aromatic hydrocarbons in various industries is having a negative effect on the environment and human health. Therefore, a key focus of current toxicology is the development and use of protein reporters with high sensitivity to various aromatic hydrocarbons (including phenolics and drugs). One molecular target for a wide range of pharmacology drugs and aromatic hydrocarbons (including phenol) is the neuronal GABAA R-coupled Cl- /HCO3 - -ATPase. In this study, we present a protocol for isolation of the membrane-bound Cl- /HCO3 - -ATPase from neuronal cells of animal brain...
March 7, 2019: Current Protocols in Toxicology
Mahnaz Ramezanpour, Harrison Bolt, Alkis Psaltis, Peter-John Wormald, Sarah Vreugde
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 increase in IL-6 production by HNEC-ALI cells...
February 4, 2019: Current Protocols in Toxicology
Jenifer A Bradley, Christopher J Strock
Neurotoxicity and seizurogenic liabilities are difficult to detect using currently available in vitro cytotoxicity assays. This is primarily due to the inherent limitations of these assays to predict adverse neural network disruptions and chemically induced perturbations. Many of these detrimental effects are detected with in vivo studies after substantial time and monetary resources have already been invested. Due to these late-stage unforeseen side effects, the implementation of a reliable high throughput in vitro method for assessing seizure-inducing and neurotoxic compound effects early in the drug discovery process would be ideal...
December 21, 2018: Current Protocols in Toxicology
Padma Kumar Narayanan, Nianyu Li
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 cells (PBMCs) from the same donor in the presence of increasing concentrations of a monoclonal antibody drug...
February 2019: Current Protocols in Toxicology
Pranav Joshi, Soo-Yeon Kang, Akshata Datar, Moo-Yeal Lee
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-throughput assessment of mechanistic toxicity...
November 2, 2018: Current Protocols in Toxicology
Josi Herron, Kelly M Hines, Libin Xu
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 resulting from exposure to xenobiotics is of value...
November 2018: Current Protocols in Toxicology
Maria A Velez-Quinones, Hanna Xu, Nhu Vo, Joanna M Gaitens, Melissa A McDiarmid, Michael R Lewin-Smith, Frederick G Strathmann
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 detection of 2...
November 2018: Current Protocols in Toxicology
Maria A Velez-Quinones, Hanna Xu, Nhu Vo, Joanna M Gaitens, Melissa A McDiarmid, Michael R Lewin-Smith, Frederick G Strathmann
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 standards...
November 2018: Current Protocols in Toxicology
Robert G Nichols, Jingwei Cai, Iain A Murray, Imhoi Koo, Philip B Smith, Gary H Perdew, Andrew D Patterson
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 protocol is important for establishing robust, reproducible, and importantly, comparative data...
November 2018: Current Protocols in Toxicology
Taru Hilander, Svetlana Konovalova, Mügen Terzioglu, Henna Tyynismaa
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 function...
July 31, 2018: Current Protocols in Toxicology
Jacquelynn Benjamino, Lidia Beka, Joerg Graf
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 statistical analyses...
July 25, 2018: Current Protocols in Toxicology
Michael A Pellizzon, Matthew R Ricci
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...
July 20, 2018: Current Protocols in Toxicology
Christa Kietz, Vilma Pollari, Annika Meinander
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...
June 22, 2018: Current Protocols in Toxicology
Laurie H Sanders, Jeremy P Rouanet, Evan H Howlett, Tess C Leuthner, John P Rooney, J Timothy Greenamyre, Joel N Meyer
Given the crucial role of DNA damage in human health and disease, it is important to be able to accurately measure both 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...
May 2018: Current Protocols in Toxicology
Zaher A Radi, Zachary S Stewart, Shawn P O'Neil
Cellular development and homeostasis are regulated via programmed cell death (PCD; apoptosis), which is a genetically regulated cellular process. Accidental cell death (ACD; necrosis) can be triggered by chemical, physical, or mechanical stress. Necrosis is the presence of dead tissues or cells in a living organism regardless of the initiating process and can be observed in infectious and non-infectious diseases and toxicities. 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...
May 2018: Current Protocols in Toxicology
Norimasa Tamehiro, Reiko Adachi, Yoshie Kimura, Shinobu Sakai, Reiko Teshima, Kazunari Kondo
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...
May 2018: Current Protocols in Toxicology
Shannon G Loelius, Sherry L Spinelli, Katie L Lannan, Richard P Phipps
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 lab developed to address a major knowledge gap regarding how cigarette smoke dysregulates platelets and how this platelet dysregulation contributes to cardiovascular disease...
May 2018: Current Protocols in Toxicology
Prathap Kumar Mahalingaiah, Tammy Palenski, Terry R Van Vleet
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...
May 2018: Current Protocols in Toxicology
Andrew Smith, Justin Dowis, Deborah French
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 quantifying serum voriconazole, isavuconazole, and posaconazole in a single method...
May 2018: Current Protocols in Toxicology
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