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Current Protocols in Toxicology

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https://www.readbyqxmd.com/read/28463420/behavioral-phenotyping-for-autism-spectrum-disorders-in-mice
#1
Yu-Chi Chang, Toby B Cole, Lucio G Costa
Autism spectrum disorder (ASD) represents a heterogeneous group of disorders characterized by alterations in three behavioral symptom domains: Social interactions, verbal and nonverbal communication, and repetitive behaviors. Increasing prevalence of ASD in recent years suggests that exposure to environmental toxicants may be critical in modulating etiology of this disease. As clinical diagnosis of autism still relies on behavioral evaluation, it is important to be able to assess similar behavioral traits in animal models, to provide biological plausibility of associations between environmental exposures and ASD...
May 2, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28463419/micropatterned-co-cultures-of-human-hepatocytes-and-stromal-cells-for-the-assessment-of-drug-clearance-and-drug-drug-interactions
#2
Christine Lin, Salman R Khetani
Drug clearance rates from the body can determine drug exposure that can affect efficacy or toxicity. Thus, accurate prediction of drug clearance during preclinical development can help guide dose selection in humans, but animal testing is not always predictive of human outcomes. Because hepatic drug metabolism is a rate-limiting step in the overall clearance of many drugs, primary human hepatocytes (PHHs) in suspension cultures or monolayers are used for drug clearance predictions. Yet, the precipitous decline in drug metabolism capacity can lead to significant underestimation of clearance rates, particularly for low turnover compounds that have desirable one-pill-a-day dosing regimens...
May 2, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28463418/use-of-primary-rat-hepatocytes-for-prediction-of-drug-induced-mitochondrial-dysfunction
#3
Cong Liu, Shuichi Sekine, Binbin Song, Kousei Ito
Mitochondrial dysfunction plays a central role in drug-induced liver injury. To evaluate drug-induced mitochondrial impairment, several isolated mitochondria- or cell line-based assays have been reported. Among them, culturing HepG2 cells in galactose provides a remarkable method to assess mitochondrial toxicity by activating mitochondrial aerobic respiration. We applied this assay to primary rat hepatocytes by culturing cells in galactose and hyperoxia to enhance the evaluation of metabolism-related drug-induced mitochondrial toxicity...
May 2, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28463417/a-multi-parametric-fluorescent-assay-for-the-screening-and-mechanistic-study-of-drug-induced-steatosis-in-liver-cells-in-culture
#4
Laia Tolosa, M José Gómez-Lechón, M Teresa Donato
Human hepatic cells have been used for drug safety risk evaluations throughout early development phases. They provide rapid, cost-effective early feedback to identify drug candidates with potential hepatotoxicity. This unit presents a cell-based assay to evaluate the risk of liver damage associated with steatogenic drugs. Detailed protocols for cell exposure to test compounds and for the assessment of steatosis-related cell parameters (intracellular lipid content, reactive oxygen species production, mitochondrial impairment, and cell death) are provided...
May 2, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28463416/quantification-of-lactate-dehydrogenase-for-cell-viability-testing-using-cell-lines-and-primary-cultured-astrocytes
#5
Simon Kaja, Andrew J Payne, Yuliya Naumchuk, Peter Koulen
Drug discovery heavily relies on cell viability studies to assess the potential toxicity of drug candidates. L-Lactate dehydrogenase (LDH) is a cytoplasmic enzyme that catalyzes the concomitant interconversions of pyruvate to L-lactate and NADH to NAD(+) during glycolysis, and the reverse reactions during the Cori cycle. In response to cellular damage, induced by endogenous cellular mechanisms or as a result of exogenously applied insults, LDH is released from the cytoplasm into the extracellular environment...
May 2, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28463415/using-chromatin-immunoprecipitation-in-toxicology-a-step-by-step-guide-to-increasing-efficiency-reducing-variability-and-expanding-applications
#6
Shaun D McCullough, Doan M On, Emma C Bowers
Histone modifications work in concert with DNA methylation to regulate cellular structure, function, and response to environmental stimuli. More than 130 unique histone modifications have been described to date, and chromatin immunoprecipitation (ChIP) allows for the exploration of their associations with the regulatory regions of target genes and other DNA/chromatin-associated proteins across the genome. Many variations of ChIP have been developed in the 30 years since its earliest version came into use, which makes it challenging for users to integrate the procedure into their research programs...
May 2, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28146282/preparation-of-specific-compartments-of-the-lungs-for-pathologic-and-biochemical-analysis-of-toxicologic-responses
#7
Laura S Van Winkle, Jacklyn S Kelty, Charles G Plopper
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 to clearly define the toxic response...
February 1, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28146281/flow-cytometry-based-method-to-detect-escherichia-coli-and-shigella-spp-using-16s-rrna-based-probe
#8
Yong Xue, Jon G Wilkes, Ted J Moskal, Anna J Williams, Willie M Cooper, Dan A Buzatu
Detection of microbial contamination in foods before they go on to the market can help prevent the occurrence of foodborne illness outbreaks. Current methods for the detection of Escherichia coli are limited by time-consuming procedures, which include multiple culture incubation steps, and require several days to get results. This unit describes the development of an improved rapid flow-cytometry-based detection method that has greater sensitivity and specificity. This method requires less time-to-results (TTR) and can detect a small number of E...
February 1, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28146280/assessment-of-bile-salt-export-pump-bsep-inhibition-in-membrane-vesicles-using-radioactive-and-lc-ms-based-detection-methods
#9
Lisa D Marroquin, Paul D Bonin, Julie Keefer, Thomas Schroeter
The bile salt export pump (BSEP, ABCB11) belongs to the ATP-binding-cassette superfamily of transporters and is predominately found in the liver. BSEP is an efflux transporter that plays a critical role in the secretion of bile salts into the bile. Inhibition of BSEP function by drugs can result in the buildup of bile salts in the liver and eventually leads to cholestasis and drug-induced liver injury (DILI). DILI is a major cause of withdrawal of drugs from the pharmaceutical market and accounts for >50% of acute liver failures...
February 1, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28146279/detecting-protein-sulfenylation-in-cells-exposed-to-a-toxicant
#10
Phillip A Wages
Protein sulfenylation is a post-translational modification that is linked to many cell signaling networks and specific protein functions, thus the detection of any sulfenylated protein after a toxicological exposure is of importance. Specifically, the detection of protein sulfenylation can provide multiple levels of mechanistic insight towards understanding the impact of a toxicological exposure. For instance, sulfenylation is caused by only a handful of reactive chemical species. Any altered sulfenylation suggests a change in cellular health, and the elucidation of the specific protein target that undergoes sulfenylation can help ascertain downstream targets and associated adverse outcomes...
February 1, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/28146278/detection-and-manipulation-of-the-stress-response-protein-metallothionein
#11
Sadikshya Bhandari, Clare Melchiorre, Kristen Dostie, Debby Laukens, Lindsey Devisscher, Ariel Louwrier, Amy Thees, Michael A Lynes
Metallothioneins (MTs) are small molecular weight stress response proteins that play a central role as reservoir of essential divalent heavy metal cations such as zinc and copper, and also can diminish the effects of toxic heavy metals such as mercury and cadmium. Historically, MT has been considered to be an intracellular protein with roles to play in the management of heavy metals, as a regulator of cellular redox potential, and as a buffer of free radicals. Our recent studies have highlighted immunomodulatory role of MT in inflammatory diseases and also in the progression of metastatic cell movement...
February 1, 2017: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/27801935/fluorescence-based-microplate-assays-for-in-vitro-assessment-of-mitochondrial-toxicity-metabolic-perturbation-and-cellular-oxygenation
#12
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
#13
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/27479363/methods-for-the-detection-of-autophagy-in-mammalian-cells
#14
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...
August 1, 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
#15
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...
May 4, 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
#16
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...
February 1, 2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/26828331/evaluation-of-toxicity-in-mouse-bone-marrow-progenitor-cells
#17
REVIEW
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...
February 1, 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
#18
REVIEW
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...
February 1, 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
#19
REVIEW
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...
February 1, 2016: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/26828328/a-simple-light-stimulation-of-caenorhabditis-elegans
#20
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...
February 1, 2016: Current Protocols in Toxicology
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