Current protocols in toxicology最新文献

Neurotoxicity and seizurogenic liabilities are difficult to detect using currently available in vitro cytotoxicity assays. This is primarily due to the inherent limitations of these assays to predict adverse neural network disruptions and chemically induced perturbations. Many of these detrimental effects are detected with in vivo studies after substantial time and monetary resources have already been invested. Due to these late-stage unforeseen side effects, the implementation of a reliable high throughput in vitro method for assessing seizure-inducing and neurotoxic compound effects early in the drug discovery process would be ideal. We have developed an in vitro screening tool to identify chemical entities that cause neurotoxic and seizurogenic effects. This article describes the preparation and use of a 48-well microelectrode array (MEA) platform along with custom data analysis algorithms and commercially available analysis tools to screen for neurotoxic liabilities and seizurogenic effects using recorded spike file data generated from cryogenically preserved rat cortical neurons. © 2018 by John Wiley & Sons, Inc.

High-content imaging (HCI) assays on two-dimensional (2D) cell cultures often do not represent in vivo characteristics accurately, thus reducing the predictability of drug toxicity/efficacy in vivo. On the other hand, conventional 3D cell cultures are relatively low throughput and possess difficulty in cell imaging. To address these limitations, a miniaturized 3D cell culture has been developed on a micropillar/microwell chip platform with human cells encapsulated in biomimetic hydrogels. Model compounds are used to validate human cell microarrays for high-throughput assessment of mechanistic toxicity. Main mechanisms of toxicity of compounds can be investigated by analyzing multiple parameters such as DNA damage, mitochondrial impairment, intracellular glutathione level, and cell membrane integrity. IC₅₀ values of these parameters can be determined and compared for the compounds to investigate the main mechanism of toxicity. This paper describes miniaturized HCI assays on 3D-cultured cell microarrays for high-throughput assessment of mechanistic profiles of compound-induced toxicity. © 2018 by John Wiley & Sons, Inc.

Cholesterol and cholesterol-derived oxysterols are critical for embryonic development, synapse formation and function, and myelination, among other biological functions. Indeed, alterations in levels of cholesterol, sterol precursors, and oxysterols result in a variety of developmental disorders, emphasizing the importance of cholesterol homeostasis. The ability of xenobiotics to reproduce similar phenotypes by altering cholesterol homeostasis has increasingly become of interest. Therefore, the ability to quantitatively assess alterations in cholesterol homeostasis resulting from exposure to xenobiotics is of value. This unit describes methods for the quantitative assessment of altered post-squalene cholesterol biosynthesis and subsequent oxysterol formation in various sample types using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Understanding alterations in cholesterol homeostasis resulting from xenobiotic exposure can provide key insight into the toxicant's mechanism of action and resulting phenotype. © 2018 by John Wiley & Sons, Inc.

We have developed and validated a method for the simultaneous quantitative measurement of total uranium (TU) and uranium ²³⁵U/²³⁸U isotopic ratio (UIR) in urine by inductively coupled plasma mass spectrometry (ICP-MS) using a Thermo Scientific iCAP-Q instrument. The performance characteristics of the assay were determined to be in compliance with clinical laboratory standards. The assay was linear in the concentration range of 1.0 to 500.0 ng/liter TU. The method was precise and accurate with limits of detection of 2.5 ng/liter for TU and 9.8 ng/liter for UIR. The accuracy was >93% and the coefficient of variation (% CV) was <5.0% for both TU and UIR. All results were within established guidelines and agreed-upon criteria, and the results fell within the certified range for the reference controls. The method has thus been shown to be effective as a simple, precise, and sensitive analytical technique for testing urine samples. © 2018 by John Wiley & Sons, Inc.

We developed and validated a method for the assessment of thirteen separate trace and toxic elements using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). Included elements were as follows: aluminum, chromium, manganese, iron, cobalt, nickel, copper, zinc, arsenic, molybdenum, cadmium, tungsten, and lead. The measurements of all elements in urine samples were conducted using ICAP-Q ICP-MS in a single method. The performance characteristics of the assay were determined according to clinical laboratory standards. The assay was linear in the concentration range of 1.0 to 1000.0 µg/liter for all elements. The method was precise and accurate with limits of quantitation of 1 µg/liter for chromium, manganese, cobalt, nickel, copper, cadmium, tungsten, and lead; 2 µg/liter for iron and arsenic; 5 µg/liter for aluminum; and 50 µg/liter for zinc. This method has successfully been used for the assessment of all thirteen elements included in urine and has been shown to be effective as a simple, precise, and sensitive analytical technique for biological monitoring of urine samples. © 2018 by John Wiley & Sons, Inc.

Characterizing the reciprocal interactions between toxicants, the gut microbiota, and the host, holds great promise for improving our mechanistic understanding of toxic endpoints. Advances in culture-independent sequencing analysis (e.g., 16S rRNA gene amplicon sequencing) combined with quantitative metabolite profiling (i.e., metabolomics) have provided new ways of studying the gut microbiome and have begun to illuminate how toxicants influence the structure and function of the gut microbiome. Developing a standardized protocol is important for establishing robust, reproducible, and importantly, comparative data. This protocol can be used as a foundation for examining the gut microbiome via sequencing-based analysis and metabolomics. Two main units follow: (1) analysis of the gut microbiome via sequencing-based approaches; and (2) functional analysis of the gut microbiome via metabolomics. © 2018 by John Wiley & Sons, Inc.

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. Thus, methods that accurately measure autophagic activity are necessary. In this unit, we introduce several approaches to analyze autophagy in mammalian cells, including immunoblotting analysis of LC3 and p62, detection of autophagosome formation by fluorescence microscopy, and monitoring autophagosome maturation by tandem mRFP-GFP fluorescence microscopy. Overall, we recommend a combined use of multiple methods to accurately assess the autophagic activity in any given biological setting. © 2016 by John Wiley & Sons, Inc.

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. In this article, we describe two different methods to screen for HO-1 increase: A homogeneous time resolved fluorescence (HTRF) assay and an immunofluorescence assay. The latter provides lower throughput but higher sensitivity due to the combination of two readouts, HO-1 intensity and cell number. The methods described in the protocol are amendable for other cell types as well. © 2016 by John Wiley & Sons, Inc.

Mitochondria are multifunctional organelles with their own genome and protein synthesis machinery. The 13 proteins encoded by mitochondrial DNA (mtDNA) are core subunits of the oxidative phosphorylation (OXPHOS) system producing the majority of cellular ATP. Yet most mitochondrial proteins are encoded by nuclear genes, synthesized by cytosolic ribosomes, and imported into mitochondria. Therefore, disturbances in cytosolic proteostasis have consequences on the gene expression and synthesis of mtDNA-encoded proteins and overall on mitochondrial function. Internal and environmental factors such as mutations, aging, oxidative stress, and toxic agents can affect the translation and the stability of mitochondrial proteins and lead to OXPHOS dysfunction. Here, methods for analysis of mitochondrial translation rate and protein stability using radioactive and non-radioactive technique as well as the methods for studying steady-state levels and assembly of OXPHOS complexes are described. © 2018 by John Wiley & Sons, Inc.