Current protocols in toxicology最新文献

Drug-induced cardiotoxicity is the one of the most common causes of drug withdrawal from market. A major barrier in managing the risk of drug-induced cardiotoxicity has been the lack of relevant models to study cardiac safety. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great potential in drug discovery and cardiotoxcity screens as they display many characteristics of the human myocardium and offer unlimited supply. This unit describes how to use pluripotent stem cells derived cardiomyocytes to study drug-induced cardiotoxicty using doxorubicin as an example. We present a workflow that explains procedure for editing hPSC using the CRISPR/Cas9 system and for differentiation of hPSC into cardiomyocytes. We also report protocols to study drug effect on ROS production, intracellular calcium concentration, formation of DNA double strand breaks, gene expression and electrophysiological properties of hPSC-CMs. © 2017 by John Wiley & Sons, Inc.

Current neurotoxicity testing and the study of molecular mechanisms in neurodegeneration in vitro usually focuses on acute exposures to compounds. 3D Lund human mesencephalic (LUHMES) cells allow long-term treatment or pulse exposure in combination with compound washout to study delayed neurotoxic effects as well as recovery and neurodegeneration pathways. In this unit we describe 3D LUHMES culture and characterization. Characterization of the model involves immunocytochemistry, flow cytometry, and qPCR measurements. Studying the delayed effects of compounds is more relevant to human exposures and neurodegenerative diseases with a strong genetic or environmental component. Most assays for molecular endpoints have been developed for monolayer cell culture and therefore need to be adapted for 3D models. In this unit, we further describe toxicological assays for molecular endpoints such as ATP levels, mitochondrial viability, and neurite outgrowth, which have been adapted for use in 3D LUHMES cultures. © 2017 by John Wiley & Sons, Inc.

The African clawed frog, Xenopus laevis, has been used as an efficient pre-clinical screening tool to predict drug safety during the early stages of the drug discovery process. X. laevis is a relatively inexpensive model that can be used in whole organism high-throughput assays whilst maintaining a high degree of homology to the higher vertebrate models often used in scientific research. Despite an ever-increasing volume of biomedical nanoparticles (NPs) in development, their unique physico-chemical properties challenge the use of standard toxicology assays. Here, we present a protocol that directly compares the sensitivity of X. laevis development as a tool to assess potential NP toxicity by observation of embryo phenotypic abnormalities/lethality after NP exposure, to in vitro cytotoxicity obtained using mammalian cell lines. In combination with conventional cytotoxicity assays, the X. laevis phenotypic assay provides accurate data to efficiently assess the safety of novel biomedical NPs. © 2017 by John Wiley & Sons, Inc.

In a recent unit in this series, protocols for the isolation, cryopreservation, thawing, and immunophenotyping of HPBMC isolated from peripheral whole blood using cell surface marker (CSM) staining and multi-color flow cytometry analysis were presented. The current procedure describes the detection and quantification of CSM and intracellular markers (ICM), including transcription factors and cytokines, following activation and differentiation of CD4+ T-cells using multi-color flow cytometry. Results indicated that repeatable and robust detection of ICM could be obtained in surface marker-defined T cells that identify functional subsets of cells. There were no observed differences between fresh and cryopreserved HPBMC in eight phenotypes analyzed (T-CD3, Th-CD4, Tmem-CD45RO, activated T-CD3/CD25, Treg- Foxp3/CD25, Th1-IFNγ, Th2- IL-4, Th17-IL-17A). There was an observed difference in activated T- CD3/CD69 in the short term (30-90 days) cryopreserved samples as compared to the freshly isolated samples, which may have resulted from the variance in controls or small sample size. © 2017 by John Wiley & Sons, Inc.

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. Conventional culture of primary hepatocytes under high-glucose and hypoxic conditions could force cells to switch energy generation to glycolysis. By contrast, cells cultured in galactose and hyperoxia could maintain energy generation from mitochondrial aerobic respiration, which is consistent with physiological conditions, and consequently improve the susceptibility of cells to mitochondrial toxicants. Measuring the toxicities of test compounds in primary rat hepatocytes cultured in modified conditions provides a useful model to identify mitochondrial dysfunction-mediated drug-induced hepatotoxicity. © 2017 by John Wiley & Sons, Inc.

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. In contrast, micropatterned co-cultures (MPCCs) of PHHs and fibroblasts display phenotypic stability for several weeks and can help mitigate the limitations of conventional cultures. Here, we describe protocols to create and use MPCCs for drug clearance predictions, and for modeling clinically-relevant drug-drug interactions that can affect drug clearance. © 2017 by John Wiley & Sons, Inc.

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. Its stability in cell culture medium makes it a well-suited correlate for the presence of damage and toxicity in tissues and cells. We herein present protocols for a reproducible and validated LDH assay optimized for several cell types. In contrast to commercially available LDH assays, often associated with proprietary formulations and high cost, our protocols provide ample opportunities for experiment-specific optimization with low variability and cost. © 2017 by John Wiley & Sons, Inc.

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. coli in the presence of large numbers of other bacteria. Clear step-by-step protocols for cell concentration determination, sample preparation, and flow cytometric analysis are provided. © 2017 by John Wiley & Sons, Inc.

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. This document describes straightforward approaches to detect protein sulfenylation of total protein as well as individual proteins of interest with a focus on immunoblotting approaches. © 2017 by John Wiley & Sons, Inc.

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. Hence, manipulation and detection of MT is essential for its possible use as a diagnostic and in therapeutic interventions of chronic inflammation. This review describes procedures used to detect MT using techniques such as western immunoblot, competition ELISA, flow cytometry and immunohistochemistry. Additionally, it also describes the use of a colorimetric cell proliferation assay (CellTiter 96 AQ_ueous One Solution/MTS) to study the proliferative effect of MT. © 2017 by John Wiley & Sons, Inc.