Aldehyde reduction by cytochrome P450.
Authors: Amunom I, Srivastava S, Prough RA
Abstract
This protocol describes the procedure for measuring the relative rates of metabolism of the α,β-unsaturated aldehydes 9-anthracene aldehyde (9-AA) and 4-hydroxy-trans-2-nonenal (4-HNE). More specifically, these assays measure the aldehyde reduction reactions of cytochrome P450s (CYPs). They can be performed using liver microsomal or other tissue fractions, spherosome preparations of recombinant CYPs, or recombinant CYPs from other sources. The method for reduction of 9-AA (a model α,β-unsaturated aldehyde) by CYPs was adapted from an assay for 9-anthr...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Assessment of mitochondrial toxicity in HepG2 cells cultured in high-glucose- or galactose-containing media.
Authors: Swiss R, Will Y
Abstract
Drug-induced mitochondrial toxicity has been recognized as contributing to a variety of organ toxicities, such as liver, heart, kidney, and CNS, and has been found to contribute to drug attrition and black box warnings. Here, we describe a cell-based assay that can detect direct drug-induced mitochondrial toxicity, providing protocols for screening in 96- and 384-well format. Cultured cells grown in glucose media produce their ATP by glycolysis, largely bypassing the mitochondria, and hence are fairly resistant to drugs that affect mitochondrial function. However, when gro...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Measuring intracellular hsp70 in leukocytes by flow cytometry.
Authors: Agnew LL
Abstract
Heat shock or stress proteins are constitutively expressed redox-sensitive proteins, the synthesis of which is induced in almost all organisms exposed to a range of stressors, including heat shock, oxidative stress, free radicals, UV radiation, and heavy metals. This unit details a method, with supporting protocols, for the measurement of their expression in peripheral blood leukocytes by flow cytometry.
PMID: 21818752 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Toxicology)
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Measurement of peroxiredoxin activity.
Authors: Nelson KJ, Parsonage D
Abstract
Peroxiredoxins are cysteine-dependent peroxidases that react with hydrogen peroxide, larger hydroperoxide substrates, and peroxynitrite. Protocols are provided to measure Prx activity with peroxide by (1) a coupled reaction with NADPH, thioredoxin reductase, and thioredoxin, (2) the direct monitoring of thioredoxin oxidation, (3) competition with horseradish peroxidase, and (4) peroxide consumption using the FOX assay.
PMID: 21818753 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Toxicology)
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Overview of peroxiredoxins in oxidant defense and redox regulation.
Authors: Poole LB, Hall A, Nelson KJ
Abstract
Peroxiredoxins are important hydroperoxide detoxification enzymes, yet have only come to the fore in recent years relative to the other major players in peroxide detoxification, heme-containing catalases and peroxidases and glutathione peroxidases. These cysteine-dependent peroxidases exhibit high reactivity with hydrogen peroxide, organic hydroperoxides, and peroxynitrite and play major roles not only in peroxide defense, but also in regulating peroxide-mediated cell signaling. This overview focuses on important peroxiredoxin features that have emerged over th...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Methods to assess neuroinflammation.
Authors: Monnet-Tschudi F, Defaux A, Braissant O, Cagnon L, Zurich MG
Abstract
Neuroinflammation is observed in many brain pathologies: in neurodegenerative diseases and multiple sclerosis as well as in chemically induced lesions. It is characterized by the reactivity of microglial cells and astrocytes, activation of inducible NO-synthase (i-NOS), and increased expression and/or release of cytokines and chemokines. Clearly, cell-to-cell signaling between the different brain cell types plays an important role in the initiation and propagation of neuroinflammation, but despite the growing list of known molec...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Murine embryonic stem cell derivation, in vitro pluripotency characterization, and in vivo teratoma formation.
Authors: Chou YF, Yabuuchi A
Abstract
The derivation of embryonic stem (ES) cells represents one of the most important breakthroughs in mammalian developmental biology. In addition to their utility in a wide array of in vitro studies, ES cells are also one of the most useful starting materials for the generation of mutants by homologous recombination in mice (Thomson and Solter, 1988). When ES cells are injected into host blastocysts and transferred to the uterus of a pseudo-pregnant mouse, they can contribute to different types of tissues in chimeric mice, including the germ line (Bradley et al., 1984). H...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Preparation of epithelial and mesenchymal stem cells from murine mammary gland.
Authors: Guest I, Ilic Z, Ma J
Abstract
The mammary gland is a complex organ consisting of multiple cell types that undergo extensive remodeling during pregnancy and involution, cyclical changes that suggest the existence of a resident stem cell population that is responsible for remarkable tissue regeneration. The basic functional unit of the mammary gland is the terminal duct lobular unit, which invades the stromal tissue (fat, connective tissue, blood vessels, etc.). Luminal epithelial cells line the ducts while outer myoepithelial cells secrete the basal lamina that separates the mammary gland parenchy...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
High-throughput, multiplexed analysis of 3-nitrotyrosine in individual proteins.
Authors: Jin H, Zangar RC
Abstract
Reactive nitrogen species (RNS) and reactive oxygen species (ROS) are derived as a result of inflammation and oxidative stress and can result in protein modifications. As such, these protein modifications are used as biomarkers for inflammation and oxidative stress. In addition, modifications in single-tissue-associated proteins released into blood can provide insight into the tissue localization of the inflammation or oxidative stress. We have developed an enzyme-linked immunosorbent assay antibody microarray platform to analyze the levels of 3-nitrotyrosine in specific ...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Developmental immunotoxicity (DIT): assays for evaluating effects of exogenous agents on development of the immune system.
Authors: DeWitt JC, Peden-Adams MM, Keil DE, Dietert RR
Abstract
Developmental immunotoxicity (DIT) occurs when exposure to environmental risk factors prior to adulthood, including chemical, biological, physical, or physiological factors, alters immune system development. DIT may elicit suppression, hyperactivation, or misregulation of immune responses and may present clinically as decreased resistance to pathogens, allergic and autoimmune diseases, and inflammatory diseases. Immunotoxicity testing guidelines established by the Environmental Protection Agency for adult animals (OPPTS 8703.7800) require fun...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
The local lymph node assay (LLNA).
Authors: Rovida C, Ryan C, Cinelli S, Basketter D, Dearman R, Kimber I
Abstract
The murine local lymph node assay (LLNA) is a widely accepted method for assessing the skin sensitization potential of chemicals. Compared with other in vivo methods in guinea pig, the LLNA offers important advantages with respect to animal welfare, including a requirement for reduced animal numbers as well as reduced pain and trauma. In addition to hazard identification, the LLNA is used for determining the relative skin sensitizing potency of contact allergens as a pivotal contribution to the risk assessment process. The LLNA...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
CYP1B1 detection.
Authors: Divi RL, Luch A, Verma M, Mahadevan B
Abstract
This unit describes procedures for measuring CYP1B1 gene expression by reverse transcription real-time PCR (qRT-PCR), CYP1B1 protein levels by western blotting, and CYP1B1 enzyme activity through conversion of 7-ethoxyresorufin substrate. To achieve specific measurement of CYP1B1 activity in the presence of CYP1A1 and CYP1A2, CYP1B1 inhibition and a subtractive approach have been adopted. 2,4,3',5'-Tetramethoxystilbene (TMS) is a potent and selective competitive inhibitor of CYP1B1 with an IC₅₀ of 3 nM for EROD and ~90 nM for E2 4-hydroxylation. B...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Morphological assessment of neurite outgrowth in hippocampal neuron-astrocyte co-cultures.
Authors: Giordano G, Costa LG
Abstract
Neurite outgrowth is a fundamental event in brain development, as well as in regeneration of damaged neurons. Astrocytes play a major role in neuritogenesis, by expressing and releasing factors that facilitate neurite outgrowth, such as extracellular matrix proteins, and factors that can inhibit neuritogenesis, such as the chondroitin sulfate proteoglycan neurocan. In this unit we describe a noncontact co-culture system of hippocampal neurons and cortical (or hippocampal) astrocytes for measurement of neurite outgrowth. Hippocampal pyramidal neurons are plated on glas...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Measuring altered disposition of xenobiotics in experimental models of liver disease.
Authors: Hardwick RN, Cherrington NJ
Abstract
Understanding the metabolic pathway and excretion mechanisms governing the disposition of a compound is essential to the safe use of pharmaceutical agents. Because the liver is the primary organ responsible for the metabolism and elimination of xenobiotics, chronic liver disease can have a significant effect on the disposition of many xenobiotics due to changes in the expression or function of drug metabolizing enzymes and transporters. Liver disease can result in increased retention of a xenobiotic within the body, causing greater exposure of the individual to...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Use of zinc finger nuclease technology to knock out efflux transporters in C2BBe1 cells.
Authors: Pratt J, Venkatraman N, Brinker A, Xiao Y, Blasberg J, Thompson DC, Bourner M
Abstract
A limitation of the traditional Caco-2 cell assay for measuring transporter-mediated efflux of a given substrate is that it is not possible to determine which specific transporter is involved. The methods in this unit describe an approach for generating specific transporter knockout cell lines that can be used to test efflux with any desired substrates. In this approach, human C2BBe1 cells (a subclone of Caco-2 cells) are nucleofected with specific zinc finger nucleases (ZFN), which can be designed to target any...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
