Keith A. Houck

In Vitro Screening of Environmental Chemicals for Targeted Testing Prioritization: The ToxCast Project

Background Chemical toxicity testing is being transformed by advances in biology and computer modeling, concerns over animal use, and the thousands of environmental chemicals lacking toxicity data. The U.S. Environmental Protection Agency’s ToxCast program aims to address these concerns by screening and prioritizing chemicals for potential human toxicity using in vitro assays and in silico approaches. Objectives This project aims to evaluate the use of in vitro assays for understanding the types of molecular and pathway perturbations caused by environmental chemicals and to build initial prioritization models of in vivo toxicity. Methods We tested 309 mostly pesticide active chemicals in 467 assays across nine technologies, including high-throughput cell-free assays and cell-based assays, in multiple human primary cells and cell lines plus rat primary hepatocytes. Both individual and composite scores for effects on genes and pathways were analyzed. Results Chemicals displayed a broad spectrum of activity at the molecular and pathway levels. We saw many expected interactions, including endocrine and xenobiotic metabolism enzyme activity. Chemicals ranged in promiscuity across pathways, from no activity to affecting dozens of pathways. We found a statistically significant inverse association between the number of pathways perturbed by a chemical at low in vitro concentrations and the lowest in vivo dose at which a chemical causes toxicity. We also found associations between a small set of in vitro assays and rodent liver lesion formation. Conclusions This approach promises to provide meaningful data on the thousands of untested environmental chemicals and to guide targeted testing of environmental contaminants.

Predictive Model of Rat Reproductive Toxicity from ToxCast High Throughput Screening

The U.S. Environmental Protection Agencys ToxCast research program uses high throughput screening (HTS) for profiling bioactivity and predicting the toxicity of large numbers of chemicals. ToxCast Phase I tested 309 well-characterized chemicals in more than 500 assays for a wide range of molecular targets and cellular responses. Of the 309 environmental chemicals in Phase I, 256 were linked to high-quality rat multigeneration reproductive toxicity studies in the relational Toxicity Reference Database. Reproductive toxicants were defined here as having achieved a reproductive lowest-observed-adverse-effect level of less than 500 mg kg−1 day−1. Eight-six chemicals were identified as reproductive toxicants in the rat, and 68 of those had sufficient in vitro bioactivity to model. Each assay was assessed for univariate association with the identified reproductive toxicants. Significantly associated assays were linked to gene sets and used for the subsequent predictive modeling. Using linear discriminant analysis and fivefold cross-validation, a robust and stable predictive model was produced capable of identifying rodent reproductive toxicants with 77% ± 2% and 74% ± 5% (mean ± SEM) training and test cross-validation balanced accuracies, respectively. With a 21-chemical external validation set, the model was 76% accurate, further indicating the models potential for prioritizing the many thousands of environmental chemicals with little to no hazard information. The biological features of the model include steroidal and nonsteroidal nuclear receptors, cytochrome P450 enzyme inhibition, G protein-coupled receptors, and cell signaling pathway readouts—mechanistic information suggesting additional targeted, integrated testing strategies and potential applications of in vitro HTS to risk assessment.

Increased AKT Activity Contributes to Prostate Cancer Progression by Dramatically Accelerating Prostate Tumor Growth and Diminishing p27Kip1 Expression

The PTEN tumor suppressor gene is frequently inactivated in human prostate cancers, particularly in more advanced cancers, suggesting that the AKT/protein kinase B (PKB) kinase, which is negatively regulated by PTEN, may be involved in human prostate cancer progression. We now show that AKT activation and activity are markedly increased in androgen-independent, prostate-specific antigen-positive prostate cancer cells (LNAI cells) established from xenograft tumors of the androgen-dependent LNCaP cell line. These LNAI cells show increased expression of integrin-linked kinase, which is putatively responsible for AKT activation/Ser-473 phosphorylation, as well as for increased phosphorylation of the AKT target protein, BAD. Furthermore, expression of the p27Kip1 cell cycle regulator was diminished in LNAI cells, consistent with the notion that AKT directly inhibits AFX/Forkhead-mediated transcription of p27Kip1. To assess directly the impact of increased AKT activity on prostate cancer progression, an activated hAKT1 mutant was overexpressed in LNCaP cells, resulting in a 6-fold increase in xenograft tumor growth. Like LNAI cells, these transfectants showed dramatically reduced p27Kip1 expression. Together, these data implicate increased AKT activity in prostate tumor progression and androgen independence and suggest that diminished p27Kip1expression, which has been repeatedly associated with prostate cancer progression, may be a consequence of increased AKT activity.

The Toxicity Data Landscape for Environmental Chemicals

Objective Thousands of chemicals are in common use, but only a portion of them have undergone significant toxicologic evaluation, leading to the need to prioritize the remainder for targeted testing. To address this issue, the U.S. Environmental Protection Agency (EPA) and other organizations are developing chemical screening and prioritization programs. As part of these efforts, it is important to catalog, from widely dispersed sources, the toxicology information that is available. The main objective of this analysis is to define a list of environmental chemicals that are candidates for the U.S. EPA screening and prioritization process, and to catalog the available toxicology information. Data sources We are developing ACToR (Aggregated Computational Toxicology Resource), which combines information for hundreds of thousands of chemicals from > 200 public sources, including the U.S. EPA, National Institutes of Health, Food and Drug Administration, corresponding agencies in Canada, Europe, and Japan, and academic sources. Data extraction ACToR contains chemical structure information; physical–chemical properties; in vitro assay data; tabular in vivo data; summary toxicology calls (e.g., a statement that a chemical is considered to be a human carcinogen); and links to online toxicology summaries. Here, we use data from ACToR to assess the toxicity data landscape for environmental chemicals. Data synthesis We show results for a set of 9,912 environmental chemicals being considered for analysis as part of the U.S. EPA ToxCast screening and prioritization program. These include high-and medium-production-volume chemicals, pesticide active and inert ingredients, and drinking water contaminants. Conclusions Approximately two-thirds of these chemicals have at least limited toxicity summaries available. About one-quarter have been assessed in at least one highly curated toxicology evaluation database such as the U.S. EPA Toxicology Reference Database, U.S. EPA Integrated Risk Information System, and the National Toxicology Program.

Integration of Dosimetry, Exposure and High-Throughput Screening Data in Chemical Toxicity Assessment

High-throughput in vitro toxicity screening can provide an efficient way to identify potential biological targets for chemicals. However, relying on nominal assay concentrations may misrepresent potential in vivo effects of these chemicals due to differences in bioavailability, clearance, and exposure. Hepatic metabolic clearance and plasma protein binding were experimentally measured for 239 ToxCast Phase I chemicals. The experimental data were used in a population-based in vitro-to-in vivo extrapolation model to estimate the daily human oral dose, called the oral equivalent dose, necessary to produce steady-state in vivo blood concentrations equivalent to in vitro AC(50) (concentration at 50% of maximum activity) or lowest effective concentration values across more than 500 in vitro assays. The estimated steady-state oral equivalent doses associated with the in vitro assays were compared with chronic aggregate human oral exposure estimates to assess whether in vitro bioactivity would be expected at the dose-equivalent level of human exposure. A total of 18 (9.9%) chemicals for which human oral exposure estimates were available had oral equivalent doses at levels equal to or less than the highest estimated U.S. population exposures. Ranking the chemicals by nominal assay concentrations would have resulted in different chemicals being prioritized. The in vitro assay endpoints with oral equivalent doses lower than the human exposure estimates included cell growth kinetics, cytokine and cytochrome P450 expression, and cytochrome P450 inhibition. The incorporation of dosimetry and exposure provide necessary context for interpretation of in vitro toxicity screening data and are important considerations in determining chemical testing priorities.

Zebrafish developmental screening of the ToxCast™ Phase I chemical library

Zebrafish (Danio rerio) is an emerging toxicity screening model for both human health and ecology. As part of the Computational Toxicology Research Program of the U.S. EPA, the toxicity of the 309 ToxCast™ Phase I chemicals was assessed using a zebrafish screen for developmental toxicity. All exposures were by immersion from 6-8 h post fertilization (hpf) to 5 days post fertilization (dpf); nominal concentration range of 1 nM-80 μM. On 6 dpf larvae were assessed for death and overt structural defects. Results revealed that the majority (62%) of chemicals were toxic to the developing zebrafish; both toxicity incidence and potency was correlated with chemical class and hydrophobicity (logP); and inter-and intra-plate replicates showed good agreement. The zebrafish embryo screen, by providing an integrated model of the developing vertebrate, compliments the ToxCast assay portfolio and has the potential to provide information relative to overt and organismal toxicity.

Endocrine Profiling and Prioritization of Environmental Chemicals Using ToxCast Data

Background The prioritization of chemicals for toxicity testing is a primary goal of the U.S. Environmental Protection Agency (EPA) ToxCast™ program. Phase I of ToxCast used a battery of 467 in vitro, high-throughput screening assays to assess 309 environmental chemicals. One important mode of action leading to toxicity is endocrine disruption, and the U.S. EPA’s Endocrine Disruptor Screening Program (EDSP) has been charged with screening pesticide chemicals and environmental contaminants for their potential to affect the endocrine systems of humans and wildlife. Objective The goal of this study was to develop a flexible method to facilitate the rational prioritization of chemicals for further evaluation and demonstrate its application as a candidate decision-support tool for EDSP. Methods Focusing on estrogen, androgen, and thyroid pathways, we defined putative endocrine profiles and derived a relative rank or score for the entire ToxCast library of 309 unique chemicals. Effects on other nuclear receptors and xenobiotic metabolizing enzymes were also considered, as were pertinent chemical descriptors and pathways relevant to endocrine-mediated signaling. Results Combining multiple data sources into an overall, weight-of-evidence Toxicological Priority Index (ToxPi) score for prioritizing further chemical testing resulted in more robust conclusions than any single data source taken alone. Conclusions Incorporating data from in vitro assays, chemical descriptors, and biological pathways in this prioritization schema provided a flexible, comprehensive visualization and ranking of each chemical’s potential endocrine activity. Importantly, ToxPi profiles provide a transparent visualization of the relative contribution of all information sources to an overall priority ranking. The method developed here is readily adaptable to diverse chemical prioritization tasks.

Incorporating Human Dosimetry and Exposure into High-Throughput In Vitro Toxicity Screening

Many chemicals in commerce today have undergone limited or no safety testing. To reduce the number of untested chemicals and prioritize limited testing resources, several governmental programs are using high-throughput in vitro screens for assessing chemical effects across multiple cellular pathways. In this study, metabolic clearance and plasma protein binding were experimentally measured for 35 ToxCast phase I chemicals. The experimental data were used to parameterize a population-based in vitro-to-in vivo extrapolation model for estimating the human oral equivalent dose necessary to produce a steady-state in vivo concentration equivalent to in vitro AC(50) (concentration at 50% of maximum activity) and LEC (lowest effective concentration) values from the ToxCast data. For 23 of the 35 chemicals, the range of oral equivalent doses for up to 398 ToxCast assays was compared with chronic aggregate human oral exposure estimates in order to assess whether significant in vitro bioactivity occurred within the range of maximum expected human oral exposure. Only 2 of the 35 chemicals, triclosan and pyrithiobac-sodium, had overlapping oral equivalent doses and estimated human oral exposures. Ranking by the potencies of the AC(50) and LEC values, these two chemicals would not have been at the top of a prioritization list. Integrating both dosimetry and human exposure information with the high-throughput toxicity screening efforts provides a better basis for making informed decisions on chemical testing priorities and regulatory attention. Importantly, these tools are necessary to move beyond hazard rankings to estimates of possible in vivo responses based on in vitro screens.

ACToR - Aggregated Computational Toxicology Resource

ACToR (Aggregated Computational Toxicology Resource) is a database and set of software applications that bring into one central location many types and sources of data on environmental chemicals. Currently, the ACToR chemical database contains information on chemical structure, in vitro bioassays and in vivo toxicology assays derived from more than 150 sources including the U.S. Environmental Protection Agency (EPA), Centers for Disease Control (CDC), U.S. Food and Drug Administration (FDA), National Institutes of Health (NIH), state agencies, corresponding government agencies in Canada, Europe and Japan, universities, the World Health Organization (WHO) and non-governmental organizations (NGOs). At the EPA National Center for Computational Toxicology, ACToR helps manage large data sets being used in a high-throughput environmental chemical screening and prioritization program called ToxCast.

Estimating toxicity-related biological pathway altering doses for high-throughput chemical risk assessment.

We describe a framework for estimating the human dose at which a chemical significantly alters a biological pathway in vivo, making use of in vitro assay data and an in vitro-derived pharmacokinetic model, coupled with estimates of population variability and uncertainty. The quantity we calculate, the biological pathway altering dose (BPAD), is analogous to current risk assessment metrics in that it combines dose-response data with analysis of uncertainty and population variability to arrive at conservative exposure limits. The analogy is closest when perturbation of a pathway is a key event in the mode of action (MOA) leading to a specified adverse outcome. Because BPADs are derived from relatively inexpensive, high-throughput screening (HTS) in vitro data, this approach can be applied to high-throughput risk assessments (HTRA) for thousands of data-poor environmental chemicals. We envisage the first step of HTRA to be an assessment of in vitro concentration-response relationships across biologically important pathways to derive biological pathway altering concentrations (BPAC). Pharmacokinetic (PK) modeling is then used to estimate the in vivo doses required to achieve the BPACs in the blood at steady state. Uncertainty and variability are incorporated in both the BPAC and the PK parameters and then combined to yield a probability distribution for the dose required to perturb the critical pathway. We finally define the BPADL as the lower confidence bound of this pathway-altering dose. This perspective outlines a framework for using HTRA to estimate BPAD values; provides examples of the use of this approach, including a comparison of BPAD values with published dose-response data from in vivo studies; and discusses challenges and alternative formulations.