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Evaluation of Mitochondrial Respiration in Cultured Rat Hepatocytes

Mitochondrial dysfunction is a major mechanism whereby drugs can induce liver injury and other serious side effects, such as lactic acidosis and rhabdomyolysis, in some patients. Several in vitro and in vivo investigations can be performed in order to determine if drugs can disturb mitoch ...

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Statistical Analysis of Quantitative RT-PCR Results

Real-time reverse transcription polymerase chain reaction (RT-PCR) represents a benchmark technology in the detection and quantification of mRNA. Yet, accurate results cannot be realized without proper statistical analysis of RT-PCR data. Here, we examine some of the issues conce ...

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Protocols and Applications of Cellular Metabolomics in Safety Studies Using Precision-Cut Tissue Slices and Carbon 13 NMR

Numerous xenobiotics are toxic to human and animal cells by interacting with their metabolism, but the precise metabolic step affected and the biochemical mechanism behind such a toxicity often remain unknown. In an attempt to reduce the ignorance in this field, we have developed a new approa ...

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Protocols of Two-Dimensional Difference Gel Electrophoresis to Investigate Mechanisms of Toxicity

In recent years, several global omics technologies have been increasingly used to better understand the molecular mechanisms of drug toxicity. Two-dimensional difference gel electrophoresis (2D-DIGE) is a large-scale proteomics high-resolution gel-based quantitative m ...

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Generation and Analysis of Transcriptomics Data

Transcript profiling (“Transcriptomics”) is a widely used technique that obtains information on the abundance of multiple mRNA transcripts within a biological sample simultaneously. Therefore, when a number of such samples are analysed, as in a scientific experiment, large and com ...

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Manual Whole-Cell Patch-Clamping of the HERG Cardiac K+ Channel

Delayed ventricular repolarization, as measured by a prolongation of the QT interval on the electrocardiogram, is a major safety issue in the drug development process. It is now recognized that most cases of drug-induced QT prolongation arise from direct pharmacological inhibition of t ...

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The Use of Bacterial Repair Endonucleases in the Comet Assay

The comet assay is a sensitive electrophoretic method for measuring DNA breaks at the level of single cells, used widely in genotoxicity experiments, biomonitoring, and in fundamental research. Its sensitivity and range of application are increased by the incorporation of an extra step, ...

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Micronucleus Assay and Labeling of Centromeres with FISH Technique

The cytokinesis-block micronucleus (CBMN) assay has since many years been applied for in vitro genotoxicity testing and biomonitoring of human populations. The standard in vitro/ex vivo micronucleus test is usually performed on human lymphocytes and has become a comprehensive met ...

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Tissue Microarrays and Digital Image Analysis

Tissue microarrays (TMAs) have recently emerged as very valuable tools for high-throughput pathological assessment, especially in the cancer research arena. This important technology, however, has yet to fully penetrate into the area of toxicology. Here, we describe the creation of T ...

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Principles and Methods of Immunohistochemistry

Immunohistochemical techniques detect antigens in tissue sections by means of immunological and chemical reactions. This technique is highly sensitive and specific and can detect a wide variety of antigens in multiple animal species. This chapter reviews common immunohistoch ...

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Histopathology Procedures: From Tissue Sampling to Histopathological Evaluation

Histological procedures aim to provide good quality sections that can be used for a light microscopic evaluation of human or animal tissue changes in either spontaneous or induced diseases. Routinely, tissues are fixed with neutral formalin 10%, embedded in paraffin, and then manually se ...

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Necropsy and Sampling Procedures in Rodents

Necropsy is a major step of most studies using laboratory animals. During necropsy, tissue and organ changes noticeable grossly can be recorded, and important tissue samples can be stored for subsequent evaluation. It is therefore important that the personnel in charge of this key experime ...

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Preclinical Evaluation of Juvenile Toxicity

A pediatric assessment is now a required component of every New Drug Application in North America or Marketing Authorization Application in Europe, unless a waiver has been granted previously. Nonclinical juvenile toxicity studies are usually required as part of this assessment. The p ...

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Developing Combination Drugs in Preclinical Studies

Although combination drugs have been available for many years, it is only recently that preclinical guidelines have been released by the Food and Drugs Administration (FDA) and EMEA and as yet they are not part of the ICH process. In addition, the World Health Organisation and FDA have issued guidel ...

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One-Dimensional Western Blotting Coupled to LC-MS/MS Analysis to Identify Chemical-Adducted Proteins in Rat Urine

The environmental toxicant hydroquinone (HQ) and its glutathione conjugates (GSHQs) cause renal cell necrosis via a combination of redox cycling and the covalent adduction of proteins within the S3 segment of the renal proximal tubules in the outer stripe of the outer medulla (OSOM). Follo ...

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Identification of Chemical-Adducted Proteins in Urine by Multi-dimensional Protein Identification Technology (LC/LCMS/MS)

Recent technological advancements in mass spectrometry facilitate the detection of chemical-induced posttranslational modifications (PTMs) that may alter cell signaling pathways or alter the structure and function of the modified proteins. To identify such protein addu ...

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Optimization of SELDI for Biomarker Detection in Plasma

The surface-enhanced laser desorption ionization (SELDI) technology is a promising approach not only for the research of biomarkers in the blood of patients in clinical applications but also in preclinical studies to assess the drug-induced toxicities. The optimization of the SELDI p ...

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Differential Proteomics Incorporating iTRAQ Labeling and Multi-dimensional Separations

Considerable effort is currently being expended to integrate newly developed “omics”-based approaches (proteomics, transcriptomics, and metabonomics) into preclinical safety evaluation workflows in the hope that more sensitive prediction of toxicology can be achieved ...

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NMR and MS Methods for Metabonomics

Metabonomics, also often referred to as “metabolomics” or “metabolic profiling,” is the systematic profiling of metabolites in bio-fluids or tissues of organisms and their temporal changes. In the last decade, metabonomics has become increasingly popular in drug development, mole ...

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Absolute Quantification of Toxicological Biomarkers by Multiple Reaction Monitoring

With the advent of “–omics” technologies, there has been an explosion of data generation in the field of toxicology, as well as in many others. As new candidate biomarkers of toxicity are being regularly discovered, the next challenge is to validate these observations in a targeted manner. Tradit ...

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