Assessment of metabolic stability using the rainbow trout (Oncorhynchus mykiss) liver S9 fraction.
Authors: Johanning K, Hancock G, Escher B, Adekola A, Bernhard MJ, Cowan-Ellsberry C, Domoradzki J, Dyer S, Eickhoff C, Embry M, Erhardt S, Fitzsimmons P, Halder M, Hill J, Holden D, Johnson R, Rutishauser S, Segner H, Schultz I, Nichols J
Abstract
Standard protocols are given for assessing metabolic stability in rainbow trout using the liver S9 fraction. These protocols describe the isolation of S9 fractions from trout livers, evaluation of metabolic stability using a substrate depletion approach, and expression of the result as in vivo intrinsic clearance. Additional guidance is provided on the care and ...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Assessing cell fusion and cytokinesis failure as mechanisms of clone 9 hepatocyte multinucleation in vitro.
Authors: Simic D, Euler C, Thurby C, Peden M, Tannehill-Gregg S, Bunch T, Sanderson T, Van Vleet T
Abstract
In this in vitro model of hepatocyte multinucleation, separate cultures of rat Clone 9 cells are labeled with either red or green cell tracker dyes (Red Cell Tracker CMPTX or Vybrant CFDA SE Cell Tracer), plated together in mixed-color colonies, and treated with positive or negative control agents for 4 days. The fluorescent dyes become cell-impermeant after entering cells and are not transferred to adjacent cells in a population, but are inherited by daughter cells after fusion. The mixed-color cult...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Mining the salivary proteome with grating-coupled surface plasmon resonance imaging and surface plasmon coupled emission microarrays.
We describe a high-content microarray assay that employs both grating-coupled surface plasmon resonance imaging and surface plasmon-coupled emission modalities in a highly sensitive assay with a large dynamic range. This powerful approach provides the tools to map the proteome of saliva, which in turn should greatly enhance the utility of salivary biomarker profiles in personalized medicine.
PMID: 22896008 [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
Evaluation of gastric ulcerogenic and healing impairment effects of bisphosphonates: adverse gastric reactions of bisphosphonate.
Authors: Takeuchi K, Amagase K
Abstract
Bisphosphonates (BPPs) were developed as antiresorptive drugs capable of treating diseases related to bone remodeling; however, they have untoward effects including ulceration in the upper gastrointestinal tract and worsen the healing-impairment action of nonsteroidal anti-inflammatory drugs, prescribed in patients with arthritis or osteoporosis. We produced ulcers in the antrum by administering BPPs to fasted rats, followed by refeeding, and confirmed their healing-impairment action on pre-existing gastric ulcers; the ulcerogenic effect is due to direct mucosal irri...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Renal transporters in drug disposition, drug-drug interactions, and nephrotoxicity.
Authors: Feng B, El-Kattan AF, Radi ZA
Abstract
This unit describes in detail the in vitro methods for measuring the interaction of new chemical entities (NCEs) with human renal transporters (hOAT1, hOAT2, and hOCT2) as both a substrate and inhibitor. Renal transporter substrate assays help in the identification of renal secretion mechanisms and assessment of the potential renal drug-drug interactions (DDIs) for NCE as a target, as well as to predict its renal clearance in humans. Human renal transporter (hOAT1, hOAT2, and hOCT2) inhibition assays characterize the inhibition potency of NCE and predict the ...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
The NCTC 2544 IL-18 assay for the in vitro identification of contact allergens.
Authors: Galbiati V, Corsini E
Abstract
Assessment of allergenic potency of low-molecular-weight compounds is generally performed using animal models, such as the murine Local Lymph Node Assay (LLNA) and the Guinea Pig Maximization Test (GMT). Progress in understanding the mechanism of skin sensitization, including effects on the production of cytokines by different cell types within the skin, provides the opportunity to develop in vitro tests as an alternative to in vivo sensitization testing. This unit will describe a method for differentiating contact allergens from low-molecular-weight respiratory alle...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Assessment of intestinal peristalsis in vitro.
Authors: Pozzoli C, Poli E
Abstract
The protocol detailed in this unit is designed to assess intestinal peristaltic motility in the isolated small intestine in vitro and to measure the effects of drugs able to interfere with gut propulsive activity. The procedure is based on Trendelenburg's classic technique, described at the beginning of the 20th century in the isolated guinea pig ileum and, later on, extended to other intestinal preparations from the same animal and other animal species. This unit illustrates the basic procedures for setting up the intestinal preparation, recording peristalsis under near...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
The vesicular transport assay: validated in vitro methods to study drug-mediated inhibition of canalicular efflux transporters ABCB11/BSEP and ABCC2/MRP2.
Authors: Herédi-Szabó K, Kis E, Krajcsi P
Abstract
The canalicular membrane of hepatocytes contains several transport proteins that use the energy of ATP to efflux potentially toxic molecules to the bile. Probably the two most important proteins at this location are MRP2 and BSEP, which transport phase II conjugates of xenobiotics and endobiotics and conjugated bile salts, respectively. The impaired function of either of these transporter proteins reduces the clearance of the toxic conjugates, resulting in their accumulation in the hepatocytes and eventually the plasma. Conjugated bile salts and phase II...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Membrane vesicle ABC transporter assays for drug safety assessment.
Authors: van Staden CJ, Morgan RE, Ramachandran B, Chen Y, Lee PH, Hamadeh HK
Abstract
The use of plasma membrane vesicles that overexpress the bile salt export pump (BSEP) or multidrug resistance-associated protein 2, 3, or 4 (MRP2-4) with an in vitro vacuum filtration system offers a rapid and reliable means for screening drug candidates for their effects on transporter function in hepatocytes and thus their potential for causing drug-induced liver injury (DILI). Comparison of transporter activity in the presence and absence of ATP allows for determination of a specific assay window for each transporter....
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
High-throughput gene silencing and mRNA expression analysis in hepatocyte sandwich cultures.
Authors: Hollingshead BD, Gauthier LM, Burdick AD
Abstract
Primary hepatocyte sandwich cultures are useful for a variety of research applications where maintenance of metabolic competency is essential. To ensure an optimal hepatocellular phenotype, cells are seeded on collagen-coated dishes and embedded with an overlay of Matrigel. This culturing condition makes gene silencing by traditional reagent-mediated transfection methods challenging. Here, an siRNA delivery method in primary mouse hepatocytes that allows cells to be cultured with Matrigel overlay is described. This method delivers >80% mRNA sile...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Preparation of rodent testis co-cultures.
Authors: Wegner S, Hong S, Yu X, Faustman EM
Abstract
Male reproductive development is a complex process that is sensitive to disruption by a range of toxicants. There is a great need for in vitro models that can evaluate potential male reproductive toxicants. The current unit presents a protocol for preparation of a three-dimensional in vitro model of male reproductive development that reduces the number of animals required for evaluation of toxicants. A Matrigel overlay provides a three-dimensional extracellular matrix that improves cell attachment, viability, and communication, and makes the model more ...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
High-throughput, cell-based screens to identify small-molecule inhibitors of ricin toxin and related category b ribosome inactivating proteins (RIPs).
Authors: Wahome PG, Mantis NJ
Abstract
Ricin is a member of the ubiquitous ribosome-inactivating protein (RIP) family of toxins. The Centers for Disease Control and Prevention (CDC) classify ricin and related toxins as Category B biothreat agents. There are currently no antidotes or therapeutics to counteract RIPs in humans. The discovery of effective small-molecule inhibitors of RIPs is increasingly possible, however, due to the availability and accessibility of diverse small-molecule chemical libraries coupled with robust robotics and automated screening methodologies. In this article, we describe a cell...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
In situ measurement of vapor uptake in the rodent upper respiratory tract.
Authors: Morris JB, Cichocki JA, Smith GJ
Abstract
The response of respiratory tract tissue target sites to inhaled vapors depends on the amount of inhaled vapor that is delivered to those sites. Direct measurement of vapor absorption within a specific airway of the living animal requires that the airway be isolated, which currently can only be performed on the upper airways. Towards this end, the upper respiratory tract (all airways anterior to the larynx) is surgically isolated in the anesthetized rat by tracheostomy and insertion of two endotracheal tubes, one leading anteriorly and the other posteriorl...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
The use of fish-derived cell lines for investigation of environmental contaminants: an update following OECD's fish toxicity testing framework No. 171.
Authors: Dayeh VR, Bols NC, Tanneberger K, Schirmer K, Lee LE
Abstract
Protocols for evaluating chemical toxicity at the cellular level using fish cell lines are described in this unit. Routine methodologies for growing salmonid cell lines, and using them in aquatic toxicology studies that support the mandate of the Organization for Economic Co-operation and Development (OECD) to reduce the use of whole animals in toxicity testing, are presented. Rapid, simple, cost-effective tests evaluating viability of cells with three indicator dyes per sample provides a broad overview of the sensitivity of cells to ch...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
Assessment of adult neurogenesis in mice.
Authors: Pan YW, Wang W, Xia Z
Abstract
Adult neurogenesis is a lifelong developmental process that occurs in two discrete regions in the adult mammalian brain: the subgranular zone of the dentate gyrus (DG) and the subventricular zone (SVZ) along the lateral ventricles. Despite immense interest in the therapeutic potential of adult neural stem cells (aNSCs) residing along these two neurogenic regions, molecular and cellular mechanisms regulating this process are not fully defined. Defining the regulatory mechanisms responsible for the genesis of new neurons in the adult brain is integral to understanding ...
Source: Current Protocols in Toxicology - December 21, 2015 Category: Toxicology Tags: Curr Protoc Toxicol Source Type: research
