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Current Protocols in Toxicology

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https://www.readbyqxmd.com/read/27801935/fluorescence-based-microplate-assays-for-in-vitro-assessment-of-mitochondrial-toxicity-metabolic-perturbation-and-cellular-oxygenation
#1
James Hynes, Conn Carey, Yvonne Will
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 cell oxygenation on this balance...
November 1, 2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/27801934/basic-techniques-in-mammalian-cell-tissue-culture
#2
Katy Phelan, Kristin M May
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.
November 1, 2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/27479365/a-high-throughput-screening-assay-to-identify-kidney-toxic-compounds
#3
Susanne Ramm, Melanie Adler, Vishal S Vaidya
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-induced oxidative stress or inflammation in HPTECs and can therefore be used as a biomarker for nephrotoxicity...
2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/27479364/in-vivo-determination-of-mitochondrial-function-using-luciferase-expressing-caenorhabditis-elegans-contribution-of-oxidative-phosphorylation-glycolysis-and-fatty-acid-oxidation-to-toxicant-induced-dysfunction
#4
Anthony L Luz, Cristina Lagido, Matthew D Hirschey, Joel N Meyer
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-expressing ATP reporter strain...
2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/27479363/methods-for-the-detection-of-autophagy-in-mammalian-cells
#5
Ziyan Zhang, Rajat Singh, Michael Aschner
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...
2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/27145112/multielectrode-array-mea-assay-for-profiling-electrophysiological-drug-effects-in-human-stem-cell-derived-cardiomyocytes
#6
Mike Clements
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 fluctuations in extracellular field potential (FP) generated from spontaneously beating hSC-CMs...
2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/27145111/determination-of-metabolic-viability-and-cell-mass-using-a-tandem-resazurin-sulforhodamine-b-assay
#7
Filomena S G Silva, Irina G Starostina, Vilena V Ivanova, Albert A Rizvanov, Paulo J Oliveira, Susana P Pereira
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 and excessive manipulations in assays that test different cell samples across plates...
2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/27145110/investigating-the-effects-of-particulate-matter-on-house-dust-mite-and-ovalbumin-allergic-airway-inflammation-in-mice
#8
Alejandro R Castañeda, Kent E Pinkerton
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, the mechanisms through which this occurs remain elusive...
2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/26828332/pcr-based-analysis-of-mitochondrial-dna-copy-number-mitochondrial-dna-damage-and-nuclear-dna-damage
#9
Claudia P Gonzalez-Hunt, John P Rooney, Ian T Ryde, Charumathi Anbalagan, Rashmi Joglekar, Joel N Meyer
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 provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays...
2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/26828331/evaluation-of-toxicity-in-mouse-bone-marrow-progenitor-cells
#10
Peace C Ezeh, Huan Xu, Shu Chun Wang, Sebastian Medina, Scott W Burchiel
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 microenvironment...
2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/26828330/associating-changes-in-the-immune-system-with-clinical-diseases-for-interpretation-in-risk-assessment
#11
Jamie C DeWitt, Dori R Germolec, Robert W Luebke, Victor J Johnson
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 changes in incidence or severity of infectious diseases...
2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/26828329/the-use-of-induced-pluripotent-stem-cells-for-the-study-and-treatment-of-liver-diseases
#12
Marc C Hansel, Julio C Davila, Massoud Vosough, Roberto Gramignoli, Kristen J Skvorak, Kenneth Dorko, Fabio Marongiu, William Blake, Stephen C Strom
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 in some clinical cases...
2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/26828328/a-simple-light-stimulation-of-caenorhabditis-elegans
#13
Kun He Lee, Michael Aschner
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...
2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/26523475/use-of-ciliogenesis-to-detect-aneugens-the-role-of-primary-cilia
#14
Kathyayini V Divi, Yvona Ward, Miriam C Poirier, Ofelia A Olivero
Primary cilia arise from the centrosomes of quiescent or post-mitotic cells, and serve as sensory organelles that communicate mechanical and chemical stimuli from the environment to the interior of the cell. Cilium formation may, therefore, become a useful end point signaling exposure to genotoxins or aneugens. Here we have used the aneugen, zidovudine (AZT), an antiretroviral drug that induces DNA replication arrest and centrosomal amplification (>2 centrosomes per quiescent cell), to evaluate cilia formation in retinal epithelial (pigmented) cells...
November 2, 2015: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/26523474/seahorse-xfe-24-extracellular-flux-analyzer-based-analysis-of-cellular-respiration-in-caenorhabditis-elegans
#15
Anthony L Luz, Latasha L Smith, John P Rooney, Joel N Meyer
Mitochondria are critical for their role in ATP production as well as multiple nonenergetic functions, and mitochondrial dysfunction is causal in myriad human diseases. Less well appreciated is the fact that mitochondria integrate environmental and intercellular as well as intracellular signals to modulate function. Because mitochondria function in an organismal milieu, there is need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XF(e) 24 Extracellular Flux Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide (DCCD, ATP synthase inhibitor), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, mitochondrial uncoupler), and sodium azide (cytochrome c oxidase inhibitor), we describe how to obtain in vivo measurements of the fundamental parameters [basal oxygen consumption rate (OCR), ATP-linked respiration, maximal OCR, spare respiratory capacity, and proton leak] of the mitochondrial respiratory chain in the model organism Caenorhabditis elegans...
November 2, 2015: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/26523473/measuring-p66shc-signaling-pathway-activation-and-mitochondrial-translocation-in-cultured-cells
#16
Mariusz R Wieckowski, Cláudia M Deus, Renata Couto, Monika Oparka, Magdalena Lebiedzińska-Arciszewska, Jerzy Duszyński, Paulo J Oliveira
The adaptor protein p66Shc links membrane receptors to intracellular signaling pathways, with downstream consequences on mitochondrial metabolism and reactive oxygen species production. Moreover, p66Shc has also been implicated in cancer development, progression, and metastasis. Increased phosphorylation of serine 36 residue of p66Shc very often correlates with oxidative stress-associated pathologies. The pro-oxidative role of p66Shc also appears to be involved in chemical toxicity, being an important component of stress responses triggered by xenobiotics...
November 2, 2015: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/26523472/caenorhabditis-elegans-as-a-model-for-toxic-effects-of-nanoparticles-lethality-growth-and-reproduction
#17
Laura L Maurer, Ian T Ryde, Xinyu Yang, Joel N Meyer
The nematode Caenorhabditis elegans is extensively utilized in toxicity studies. C. elegans offers a high degree of homology with higher organisms, and its ease of use and relatively inexpensive maintenance have made it an attractive complement to mammalian and ecotoxicological models. C. elegans provides multiple benefits, including the opportunity to perform relatively high-throughput assays on whole organisms, a wide range of genetic tools permitting investigation of mechanisms and genetic sensitivity, and transparent bodies that facilitate toxicokinetic studies...
November 2, 2015: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/26523471/immune-cell-phenotyping-using-flow-cytometry
#18
A Graham Pockley, Gemma A Foulds, Julie A Oughton, Nancy I Kerkvliet, Gabriele Multhoff
Fluorescent immunophenotyping uses fluorescently-conjugated antibodies to identify, characterize and quantify distinct subpopulations of cells within heterogeneous single-cell populations, either in the context of tissue (using fluorescence and imaging microscopy) or in a single-cell suspension (using multiparameter imaging microscopy, imaging cytometry, and/or flow cytometry). Flow cytometry is an optical, laser-based technology which analyzes the physical and fluorescent properties of cells in suspension in real-time as they flow through the instrument...
November 2, 2015: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/26309451/high-resolution-multi-photon-imaging-of-morphological-structures-of-caenorhabditis-elegans
#19
M Gabriele Bixel, Stephanie J B Fretham, Michael Aschner
In this protocol, we combine two-photon excitation fluorescence to visualize in caenorhabditis elegans (C. elegans) dopaminergic (DAergic) neurons and their processes with non-linear optical measurements to reconstruct the three-dimensional architecture of the pharyngeal region and the muscular system of the anterior and mid-body region. Femto second laser pulses excite second-harmonic generation (SHG) and third harmonic generation (THG) signals, which show detailed structural information regarding the organization of myofibrils that are arranged around the central pharynx region...
May 2015: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/25645244/methods-to-measure-olfactory-behavior-in-mice
#20
Junhui Zou, Wenbin Wang, Yung-Wei Pan, Song Lu, Zhengui Xia
Mice rely on the sense of olfaction to detect food sources, recognize social and mating partners, and avoid predators. Many behaviors of mice, including learning and memory, social interaction, fear, and anxiety are closely associated with their function of olfaction, and behavior tasks designed to evaluate those brain functions may use odors as cues. Accurate assessment of olfaction is not only essential for the study of olfactory system but also critical for proper interpretation of various mouse behaviors, especially learning and memory, emotionality and affect, and sociality...
February 2, 2015: Current Protocols in Toxicology
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