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Traditionally, chemical toxicity is determined by in vivo animal studies, which are low throughput, expensive, and sometimes fail to predict compound toxicity in humans. Due to the increasing number of chemicals in use and the high rate of drug candidate failure due to toxicity, it is imperative to develop in vitro, high-throughput screening methods to determine toxicity. The Tox21 program, a unique research consortium of federal public health agencies, was established to address and identify toxicity concerns in a high-throughput, concentration-responsive manner using a battery of in vitro assays. In this article, we review the advancements in high-throughput robotic screening methodology and informatics processes to enable the generation of toxicological data, and their impact on the field; further, we discuss the future of assessing environmental toxicity utilizing efficient and scalable methods that better represent the corresponding biological and toxicodynamic processes in humans. [ABSTRACT FROM AUTHOR]

DNA chips are glass surfaces that represent thousands of DNA fragments arrayed at discrete sites. Hybridization of RNA or DNA-derived samples to DNA chips allows us to monitor expression of mRNAs or the occurrence of polymorphisms in genomic DNA. The technology holds great promise for identifying gene polymorphisms that predispose man to disease, gene regulation events involved in disease progression, and more-effective disease treatments.

The cloning and characterization of topoisomerase 1 (TOP1) gene from Candida albicans were described. A conditional TOP1 disruption mutant strain was constructed causing the deletion of one copy of TOP1 which resulted in slight attenuation of virulence. Findings showed that TOP1 gene is not necessary for in vitro growth of Candida albicans but its expression is significant for germ-tube formation, normal cellular structure and virulence in a mouse model system.

Abstract Toxicogenomics can measure the expression of thousands of genes to identify changes associated with drug induced toxicities. It is expected that toxicogenomics can be an alternative or complementary approach in preclinical drug safety evaluation to identify or predict drug induced toxicities. One of the major concerns in applying toxicogenomics to diagnose or predict drug induced organ toxicity, is how generalizable the statistical classification model is when derived from small datasets? Here we presented that a diagnosis of kidney proximal tubule toxicity, measured by pathology, can successfully be achieved even with a study design of limited number of training studies or samples. We selected a total of ten kidney toxicants, designed the in life study with multiple dose and multiple time points to cover samples at doses and time points with or without concurrent toxicity. We employed SVM (Support Vector Machine) as the classification algorithm for the toxicogenomic diagnosis of kidney proximal tubule toxicity. Instead of applying cross validation methods, we used an independent testing set by dividing the studies or samples into independent training and testing sets to evaluate the diagnostic performance. We achieved a Sn (sensitivity) = 88% and a Sp (specificity) = 91%. The diagnosis performance underscores the potential application of toxicogenomics in a preclinical lead optimization process of drugs entering into development.

BACKGROUND: A central challenge in toxicity testing is the large number of chemicals in commerce that lack toxicological assessment. In response, the Tox21 program is re-focusing toxicity testing from animal studies to less expensive and higher throughput in vitro methods using target/pathway-specific, mechanism-driven assays. OBJECTIVES: Our objective was to use an in-depth mechanistic study approach to prioritize and characterize the chemicals affecting mitochondrial function. METHODS: We used a tiered testing approach to prioritize for more extensive testing 622 compounds identified from a primary, quantitative high-throughput screen of 8,300 unique small molecules, including drugs and industrial chemicals, as potential mitochondrial toxicants by their ability to significantly decrease the mitochondrial membrane potential (MMP). Based on results from secondary MMP assays in HepG2 cells and rat hepatocytes, 34 compounds were selected for testing in tertiary assays that included formation of reactive oxygen species (ROS), upregulation of p53 and nuclear erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE), mitochondrial oxygen consumption, cellular Parkin translocation, and larval development and ATP status in the nematode Caenorhabditis elegans. RESULTS: A group of known mitochondrial complex inhibitors (e.g., rotenone) and uncouplers (e.g., chlorfenapyr), as well as potential novel complex inhibitors and uncouplers, were detected. From this study, we identified four not well-characterized potential mitochondrial toxicants (lasalocid, picoxystrobin, pinacyanol, and triclocarban) that merit additional in vivo characterization. CONCLUSIONS: The tier-based approach for identifying and mechanistically characterizing mitochondrial toxicants can potentially reduce animal use in toxicological testing., Introduction Humans are exposed daily to numerous naturally occurring and man-made chemicals, some of which may adversely affect health. Furthermore, each year, hundreds of new chemicals are synthesized and introduced [...]

Abstract Background Neurons in the dorsal spinal cord play important roles in nociception and pain. These neurons receive input from peripheral sensory neurons and then transmit the signals to the brain, as well as receive and integrate descending control signals from the brain. Many molecules important for pain transmission have been demonstrated to be localized to the dorsal horn of the spinal cord. Further understanding of the molecular interactions and signaling pathways in the dorsal horn neurons will require a better knowledge of the molecular neuroanatomy in the dorsal spinal cord. Results A large scale screening was conducted for genes with enriched expression in the dorsal spinal cord using DNA microarray and quantitative real-time PCR. In addition to genes known to be specifically expressed in the dorsal spinal cord, other neuropeptides, receptors, ion channels, and signaling molecules were also found enriched in the dorsal spinal cord. In situ hybridization and immunohistochemistry revealed the cellular expression of a subset of these genes. The regulation of a subset of the genes was also studied in the spinal nerve ligation (SNL) neuropathic pain model. In general, we found that the genes that are enriched in the dorsal spinal cord were not among those found to be up-regulated in the spinal nerve ligation model of neuropathic pain. This study also provides a level of validation of the use of DNA microarrays in conjunction with our novel analysis algorithm (SAFER) for the identification of differences in gene expression. Conclusion This study identified molecules that are enriched in the dorsal horn of the spinal cord and provided a molecular neuroanatomy in the spinal cord, which will aid in the understanding of the molecular mechanisms important in nociception and pain.

BACKGROUND: A central challenge in toxicity testing is the large number of chemicals in commerce that lack toxicological assessment. In response, the Tox21 program is re-focusing toxicity testing from animal studies to less expensive and higher throughput in vitro methods using target/pathway-specific, mechanism-driven assays. OBJECTIVES: Our objective was to use an in-depth mechanistic study approach to prioritize and characterize the chemicals affecting mitochondrial function. METHODS: We used a tiered testing approach to prioritize for more extensive testing 622 compounds identified from a primary, quantitative high-throughput screen of 8,300 unique small molecules, including drugs and industrial chemicals, as potential mitochondrial toxicants by their ability to significantly decrease the mitochondrial membrane potential (MMP). Based on results from secondary MMP assays in HepG2 cells and rat hepatocytes, 34 compounds were selected for testing in tertiary assays that included formation of reactive oxygen species (ROS), upregulation of p53 and nuclear erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE), mitochondrial oxygen consumption, cellular Parkin translocation, and larval development and ATP status in the nematode Caenorhabditis elegans. RESULTS: A group of known mitochondrial complex inhibitors (e.g., rotenone) and uncouplers (e.g., chlorfenapyr), as well as potential novel complex inhibitors and uncouplers, were detected. From this study, we identified four not well-characterized potential mitochondrial toxicants (lasalocid, picoxystrobin, pinacyanol, and triclocarban) that merit additional in vivo characterization. CONCLUSIONS: The tier-based approach for identifying and mechanistically characterizing mitochondrial toxicants can potentially reduce animal use in toxicological testing. [ABSTRACT FROM AUTHOR]

More than 75 000 man-made chemicals contaminate the environment; many of these have not been tested for toxicities. These chemicals demand quantitative high-throughput screening assays to assess them for causative roles in neurotoxicities, including Parkinson's disease and other neurodegenerative disorders. To facilitate high throughput screening for cytotoxicity to neurons, three human neuronal cellular models were compared: SH-SY5Y neuroblastoma cells, LUHMES conditionally-immortalized dopaminergic neurons, and Neural Stem Cells (NSC) derived from human fetal brain. These three cell lines were evaluated for rapidity and degree of differentiation, and sensitivity to 32 known or candidate neurotoxicants. First, expression of neural differentiation genes was assayed during a 7-day differentiation period. Of the three cell lines, LUHMES showed the highest gene expression of neuronal markers after differentiation. Both in the undifferentiated state and after 7 days of neuronal differentiation, LUHMES cells exhibited greater cytotoxic sensitivity to most of 32 suspected or known neurotoxicants than SH-SY5Y or NSCs. LUHMES cells were also unique in being more susceptible to several compounds in the differentiating state than in the undifferentiated state; including known neurotoxicants colchicine, methyl-mercury (II), and vincristine. Gene expression results suggest that differentiating LUHMES cells may be susceptible to apoptosis because they express low levels of anti-apoptotic genes BCL2 and BIRC5/ survivin, whereas SH-SY5Y cells may be resistant to apoptosis because they express high levels of BCL2, BIRC5/ survivin, and BIRC3 genes. Thus, LUHMES cells exhibited favorable characteristics for neuro-cytotoxicity screening: rapid differentiation into neurons that exhibit high level expression neuronal marker genes, and marked sensitivity of LUHMES cells to known neurotoxicants. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The first guidance on Good Cell Culture Practice (GCCP) dates back to 2005. This document expands this to include aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice that can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance to facilitate the generation of reliable data from cell culture systems, and is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when it is necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and the safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered a first step toward a revised GCCP 2.0. [ABSTRACT FROM AUTHOR]