Preparation of Specific Compartments of the Lungs for Pathologic and Biochemical Analysis of Toxicologic Responses.
Authors: Van Winkle LS, Kelty JS, Plopper CG
Abstract
This unit focuses on protocols for assessing microenvironment-specific responses in the thoracic lung tissues. Aspects of the entire respiratory system serve as potential targets for candidate toxicants, but each candidate toxicant may impact distinct sites due to differential distribution of either the toxicant or the target cells. Within the conducting airways, the composition of resident cell populations and the metabolic capabilities of the cell populations vary greatly. Thus, studies of this region of the lung require unique, site-selective methods...
Source: Current Protocols in Toxicology - February 3, 2017 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Basic Techniques in Mammalian Cell Tissue Culture.
Authors: Phelan K, May KM
Abstract
Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2016 by John Wiley & Sons, Inc.
PMID: 27801934 [PubMed - in process] (Source: Current Protocols in Toxicology)
Source: Current Protocols in Toxicology - November 2, 2016 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Fluorescence-Based Microplate Assays for In Vitro Assessment of Mitochondrial Toxicity, Metabolic Perturbation, and Cellular Oxygenation.
Authors: Hynes J, Carey C, Will Y
Abstract
High-throughput in vitro cell metabolism assays are of particular use for identification and delineation of mitochondrial toxicity and related metabolic perturbation. Here, a panel of fluorescence-based metabolism assays are described for measuring oxygen consumption, glycolytic flux, and cellular oxygenation. They can be applied to analysis of both isolated mitochondria and cell models. Sample data are presented illustrating how these protocols can be used to examine drug treatment, the interplay between oxidative and glycolytic ATP generation, and the impact of ...
Source: Current Protocols in Toxicology - November 2, 2016 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Methods for the Detection of Autophagy in Mammalian Cells.
Authors: Zhang Z, Singh R, Aschner M
Abstract
Macroautophagy (hereafter referred to as autophagy) is a degradation pathway that delivers cytoplasmic materials to lysosomes via double-membraned vesicles designated autophagosomes. Cytoplasmic constituents are sequestered into autophagosomes, which subsequently fuse with lysosomes, where the cargo is degraded. Autophagy is a crucial mechanism involved in many aspects of cell function, including cellular metabolism and energy balance; alterations in autophagy have been linked to various human pathological processes. Thus, methods that accurately measure autoph...
Source: Current Protocols in Toxicology - August 2, 2016 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
In Vivo Determination of Mitochondrial Function Using Luciferase-Expressing Caenorhabditis elegans: Contribution of Oxidative Phosphorylation, Glycolysis, and Fatty Acid Oxidation to Toxicant-Induced Dysfunction.
Authors: Luz AL, Lagido C, Hirschey MD, Meyer JN
Abstract
Mitochondria are a target of many drugs and environmental toxicants; however, how toxicant-induced mitochondrial dysfunction contributes to the progression of human disease remains poorly understood. To address this issue, in vivo assays capable of rapidly assessing mitochondrial function need to be developed. Here, using the model organism Caenorhabditis elegans, we describe how to rapidly assess the in vivo role of the electron transport chain, glycolysis, or fatty acid oxidation in energy metabolism following toxicant exposure, using a luciferase...
Source: Current Protocols in Toxicology - August 2, 2016 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
A High-Throughput Screening Assay to Identify Kidney Toxic Compounds.
Authors: Ramm S, Adler M, Vaidya VS
Abstract
Kidney toxicity due to drugs and chemicals poses a significant health burden for patients and a financial risk for pharmaceutical companies. However, currently no sensitive and high-throughput in vitro method exists for predictive nephrotoxicity assessment. Primary human proximal tubular epithelial cells (HPTECs) possess characteristics of differentiated epithelial cells, making them a desirable model to use in in vitro screening systems. Additionally, heme oxygenase 1 (HO-1) protein expression is upregulated as a protective mechanism during kidney toxicant-indu...
Source: Current Protocols in Toxicology - August 2, 2016 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Investigating the Effects of Particulate Matter on House Dust Mite and Ovalbumin Allergic Airway Inflammation in Mice.
Authors: Castañeda AR, Pinkerton KE
Abstract
Particulate matter (PM), a component of air pollution, has been shown to enhance allergen-mediated airway hypersensitivity and inflammation. Surprisingly, exposure to PM during the sensitization to allergen is sufficient to produce immunological changes that result in heightened inflammatory effects upon future allergen exposures (challenge) in the absence of PM. This suggests that PM has the ability to modulate the allergic immune response, thereby acting as an adjuvant by enhancing the immunological memory formed during the adaptive immune response; however, ...
Source: Current Protocols in Toxicology - May 5, 2016 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Determination of Metabolic Viability and Cell Mass Using a Tandem Resazurin/Sulforhodamine B Assay.
Authors: Silva FS, Starostina IG, Ivanova VV, Rizvanov AA, Oliveira PJ, Pereira SP
Abstract
The identification of rapid, reliable, and highly reproducible biological assays that can be standardized and routinely used in preclinical tests constitutes a promising approach to reducing drug discovery costs and time. This unit details a tandem, rapid, and reliable cell viability method for preliminary screening of chemical compounds. This assay measures metabolic activity and cell mass in the same cell sample using a dual resazurin/sulforhodamine B assay, eliminating the variation associated with cell seeding a...
Source: Current Protocols in Toxicology - May 5, 2016 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Multielectrode Array (MEA) Assay for Profiling Electrophysiological Drug Effects in Human Stem Cell-Derived Cardiomyocytes.
Authors: Clements M
Abstract
More relevant and reliable preclinical cardiotoxicity tests are required to improve drug safety and reduce the cost of drug development. Human stem cell-derived cardiomyocytes (hSC-CMs) provide a potential model for the development of superior assays for preclinical drug safety screening. One such hSC-CM assay that has shown significant potential for enabling more predictive drug cardiac risk assessment is the MEA assay. The Multi-electrode Array (MEA) assay is an electrophysiology-based technique that uses microelectrodes embedded in the culture surface of each well to measure...
Source: Current Protocols in Toxicology - May 5, 2016 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
A Simple Light Stimulation of Caenorhabditis elegans.
Authors: Lee KH, Aschner M
Abstract
Response via noxious stimulus can be an important indicator of sensory neuron function and overall health of an organism. If the stimulation is quick and simple, and the animal can be rescued afterwards, such a method not only allows for assays pertaining to changed sensory ability after various treatments, but also increases the reliability of the statistical relationships that are established. This protocol demonstrates a stimulation assay in Caenorhabditis elegans, using blue light from common laboratory equipment: the fluorescent microscope. The nematode detects blue...
Source: Current Protocols in Toxicology - February 2, 2016 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
The Use of Induced Pluripotent Stem Cells for the Study and Treatment of Liver Diseases.
Authors: Hansel MC, Davila JC, Vosough M, Gramignoli R, Skvorak KJ, Dorko K, Marongiu F, Blake W, Strom SC
Abstract
Liver disease is a major global health concern. Liver cirrhosis is one of the leading causes of death in the world and currently the only therapeutic option for end-stage liver disease (e.g., acute liver failure, cirrhosis, chronic hepatitis, cholestatic diseases, metabolic diseases, and malignant neoplasms) is orthotropic liver transplantation. Transplantation of hepatocytes has been proposed and used as an alternative to whole organ transplant to stabilize and prolong the lives of patients ...
Source: Current Protocols in Toxicology - February 2, 2016 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Associating Changes in the Immune System with Clinical Diseases for Interpretation in Risk Assessment.
Authors: DeWitt JC, Germolec DR, Luebke RW, Johnson VJ
Abstract
This overview is an update of the unit originally published in 2004. While the basic tenets of immunotoxicity have not changed in the past 10 years, several publications have explored the application of immunotoxicological data to the risk assessment process. Therefore, the goal of this unit is still to highlight relationships between xenobiotic-induced immunosuppression and risk of clinical diseases progression. In immunotoxicology, this may require development of models to equate moderate changes in markers of immune functions to potential c...
Source: Current Protocols in Toxicology - February 2, 2016 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Evaluation of Toxicity in Mouse Bone Marrow Progenitor Cells.
Authors: Ezeh PC, Xu H, Wang SC, Medina S, Burchiel SW
Abstract
Development of blood cells through hematopoiesis occurs in the bone marrow (BM), and can be adversely impacted by various substances and/or conditions ranging from known therapeutic, intentionally administered xenobiotics to unintentional food additives and exposure to environmental chemicals. The principles underlying the techniques for evaluating toxicity to BM progenitors (erythroid, myeloid, and lymphoid) exploit changes in the normal hematopoietic process, biochemical cell surface and intracellular markers, as well as components of the BM...
Source: Current Protocols in Toxicology - February 2, 2016 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage.
Authors: Gonzalez-Hunt CP, Rooney JP, Ryde IT, Anbalagan C, Joglekar R, Meyer JN
Abstract
Because of the role that DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit, we...
Source: Current Protocols in Toxicology - February 2, 2016 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Measurement of phenylalanine monooxygenase (PAH) activities.
Authors: Steventon GB, Mitchell SC
Abstract
Mammalian phenylalanine monooxygenase (phenylalaninase, phenylalanine hydroxylase, PAH; EC 1.14.16.1) is a member of the large aromatic amino acid hydrolase cohort of enzymes that include tyrosine monooxygenase and tryptophane monooxygenase. PAH is a non-heme-iron-dependent protein that normally catalyzes the C-oxidation of phenylalanine (Phe) to tyrosine (Tyr) in the presence of BH(4), utilizing molecular dioxygen as an additional substrate. However, over recent years, the presumed narrow substrate specificity of PAH has been questioned and catalytic activity to...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
