GEMS ANNUAL SPRING MEETING

High Throughput Screening for Chemical Genomics and Computational Toxicology

The Genetics and Environmental Mutagenesis Society (GEMS) Spring Meeting was held on May 17, 2010 at the North Carolina Biotechnology Center. The theme of the meeting was "High Throughput Screening for Chemical Genomics and Computational Toxicology". The Spring Meeting brought together five speakers from NTP/NIEHS, the U.S. EPA, the Hamner Institutes for Health Sciences, and the NIH Chemical Genomics Center (NCGC) who presented a collective progress report on the TOX21 approach to screening potential toxicants. GEMS also presented a lifetime achievement award to to its founding President, Dr. Larry Claxton.  (Use this link to see more about the award with a message from Dr. Claxton).

The first speaker was Menghang Xia, Ph.D., NCGC, NHGRI, NIH, who introduced an application of quantitative high throughput screening (qHTS) in toxicological studies at NCGC. Dr. Xia presented slides showing the qHTS laboratory, including a short video of laboratory robotics at work: loading multi-well plates into incubators, liquid handling equipment, and sample readers. NCHS is the lead laboratory in the TOX21 project, and they are currently doing phase I testing on 320 substances across a limited set of endpoints and human cell lines. The qHTS laboratory can process up to one hundred 1536-well culture plates a day, which provides the capacity to screen approximately 140,000 samples per week. Phase II testing is scheduled to begin later this year with an additional 1000 substances and with a potential consideration of toxicological endpoints from 500 cell lines, and increasing to a capacity of 10,000 substances by the end of 2011. The qHTS can also do continuous sample readings at increments as frequent as every 5 minutes throughout the study duration (2-4 days), which permits finding the optimum toxicity kinetics of a tested substance.

Windy Boyd, Ph.D., Biomolecular Screening Branch, NTP, NIEHS presented, Caenorhabditis elegans in Medium-Throughput Toxicological Testing. The roundworm, C. elegans, is one of a limited number of in vivo assays being used for Tox21. Roundworms, although microscopic in size, require 96 chamber or larger multi-well plates for processing, which is considered medium throughput screening (MTS) rather than HTS. C. elegans has been used in laboratory studies for many years, so there is much historical information about toxic response in the species. Since it is an invertebrate species, there are no animal use issues related to this organism. The four larval stages of C. elegans are easily stained to differentiate both reproductive and developmental endpoints for toxicity studies. Automated counters can easily enumerate the stained transparent worms. The C. elegans assay was added to the Tox21 project in 2008, and it is being used to test the 320 Phase I substances. Since most of these substances are pesticides, C. elegans has identified a high number of positive outcomes, and it is especially effective among those substances used to eradicate the common agriculture nematode, a related worm species.

Steven Simmons, Ph.D., ISTD, NHEERL, EPA, presented Integrated Pathway Approach to High-Throughput Toxicant Identification and Characterization. Dr. Simmons described a systems biology approach to the analysis of the bioinformatics being generated in the qHTS and MTS data that is being collected by ToxCast for the Tox21 project. Dr. Simmons focused on metabolic pathways associated with adaptive stress, such as oxidative stress, heat shock, genotoxic stress, etc. An integrated approach is being used to relate various qHTS data to activity in molecular pathways. Various types of toxic response kinetics, such as time, pathways, cell types, and metabolism, are some of the possible endpoints to be combined with QSAR descriptors in cluster analysis to identify chemically related substances. Dr. Simmons suggested that cluster analysis might allow a few assays to discriminate specific toxicities that may not necessarily be directly related. For example an in vitro apoptosis assay and a cell toxicity assay together have identified a specific pathway cluster related to hormonal toxicity. Although the current data is limited, when the database of substances and endpoints increases, such an integrated approach may identify many toxicity pathway relationships that can be used to regulate chemical toxicity using data from in vitro or ex vivo test results.

In the afternoon Russell Thomas, Ph.D. from The Hamner Institutes for Health Sciences presented his talk on Experimentally Defining Toxicity Pathways Using In Vitro High-Content Screening of Embryonic Fibroblasts from the Mouse Diversity Panel. Toxicity pathways are being identified from the traditional metabolic regulatory pathways. Since much of the existing toxicity information has been collected from rodent studies, it is important to focus on metabolic pathways that are highly conserved across species, and that are relevant to humans. The initial study is focused on the discovery of differences in inter-strain phenotypes in cell signaling pathways from 32 inbred strains of mice. Four toxicity endpoints are being examined using high content imaging techniques for replicate samples at multiple time points. Custom software is being developed to model the different toxicity endpoints. The study is currently incomplete; 38 of 100 substances have been tested across the battery of tests. Future studies are also being planned to screen the 100 substances using primary fibroblasts.

Richard Judson, Ph.D., NCCT, EPA concluded the seminar with his presentation of ToxCast/ACToR HTS Informatics for Computational Toxicology Models. In 2007, EPA launched its ToxCast Program to develop an approach for prioritizing the toxicity testing of thousands of chemicals. ACToR is the EPA database being used to collect HTS and MTS data from several laboratories participating in the Tox21 program. The initial Phase I study will provide data on 320 substances (primarily pesticides), and Phase II will add another 1000 or more substances by next year. Various multivariate computational models will be utilized and new ones developed to identify patterns of toxicity pathways, gene regulation, and other biological associations related to toxicology. Dr. Judson also mentioned other integrated databases being developed and utilized by Tox21. EPA/NCCT also links to various toxicity informatics sources through its ACToR database with over 500 sources (toxicity, exposure, etc.) and over 500 substances. The NCCT has links to various informatics tools, such as ToxMiner, a Virtual Tissue Knowledgebase, and integrated toxicity pathways and reverse toxicokinetics, which can be utilized to provide information and analysis for the rapidly growing Tox21 project.