NCGC qHTS assays test chemicals at multiple concentrations - as many as 15 per chemical. The range of low to high concentrations allows concentration-response curves to be generated, which provide more information about and better prediction of toxicity for each chemical.
NCGC uses a 1536-well microtiter plate format, which allows for the testing of 1408 samples along with concurrent negative and positive controls on each plate. Each plate represents a different concentration. Processing 100 of these plates per day, the HTS robots can test up to one million samples in one week.
Toxicogenomics as a field encompasses the collection, interpretation, and storage of information about global gene expression levels of messenger RNAs (transcriptomics), proteins (proteomics) and metabolites (metabolomics) within a cell or tissue in response to a chemical exposure. Toxicogenomics combines RNA, protein, and metabolite profiling with conventional toxicology to investigate the interaction between genes and the environment in relation to toxicity and disease causation.
Toxicogenomic transcriptomics (or gene expression) approaches include using microarrays with complementary DNA (cDNA) or oligonucleotide platforms and Next Generation (NextGen) RNA sequencing. These approaches may help improve classification of the exposure-response relationship and enable scientists to better understand the genetic profile of a cell or tissue when it is exposed to an environmental substance.
Data from all phases of Tox21, along with full chemical characterization and assay protocol details, are being deposited into publicly available databases, which are accessible through the Tox21 Toolbox.
In the first phase of Tox21 research (2005 – 2010), the partners utilized the high throughput screening (HTS) technologies of the National Human Genome Research Institute's National Chemical Genomics Center (NCGC, now a part of the National Center for Advancing Translational Sciences, NCATS) and performed quantitative HTS (qHTS) testing (see box). NCGC screened approximately 2800 compounds in more than 75 qHTS assays. Concurrently, the Environmental Protection Agency’s National Center for Computational Toxicology screened 309 unique compounds across more than 500 biochemical- and cell-based assays through its ToxCast™ program. These compounds were also tested in NTP's "WormTox" laboratory, where toxicity screens used the roundworm, Caenorhabditis elegans.
In Phase II of Tox21 research (2011 – 2016), the partners are testing a chemical library, expanded from Phase I to greater than 10,000 compounds, at NCATS using a HTS robotics system sponsored by NTP. More than 200 databases of chemicals and drugs in the United States and abroad were analyzed to select the compounds for testing. This chemical library includes industrial and consumer products, food additives, drugs, and mixtures. Tox21 partners are establishing a full spectrum of cell-based assays to further define and characterize activities identified in the initial qHTS tests. The results will provide information to help evaluate biological processes that may lead to adverse health effects in humans.
Phase III of Tox21 research (2014 – present) aims to better represent human health and disease by increasing the biological diversity of human cell lines used in the HTS assays. Phase III plans to include three-dimensional organoid (or organ-like) model cultures and simple model organisms to address chemical metabolism and assess the effects of chemicals in more complex biological systems. Phase III research also adds medium to high throughput transcriptomics assays to test for effects of chemicals on gene expression. These Phase III screening assays will look at multiple endpoints and processes in order to better identify chemicals that may cause adverse human health effects.
During Phase III, Tox21 partners have identified sets of genes in human cells and tissues that respond to toxic chemicals (i.e., toxicogenomics - see box). The resulting human S1500+ gene set comprises approximately 2750 genes that will be used to evaluate gene expression changes from chemical exposures. Similar gene sets are being developed for screening cells and tissues from rats, mice, and zebrafish. The human S1500+ gene set should provide transcriptomic information on cellular responses that are independent of cell type or species, and also those that are specific by organ and/or cell type.