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There is a need for fast, efficient, and cost-effective hazard identification and characterization of chemical hazards. This need is generating increased interest in the use of zebrafish embryos as both a screening tool and an alternative to mammalian test methods. A Collaborative Workshop on Aquatic Models and 21st Century Toxicology identified the lack of appropriate and consistent testing protocols as a challenge to the broader application of the zebrafish embryo model. The National Toxicology Program established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative to address the lack of consistent testing guidelines and identify sources of variability for zebrafish-based assays. This report summarizes initial SEAZIT information-gathering efforts. Investigators in academic, government, and industry laboratories that routinely use zebrafish embryos for chemical toxicity testing were asked about their husbandry practices and standard protocols. Information was collected about protocol components including zebrafish strains, feed, system water, disease surveillance, embryo exposure conditions, and endpoints. Literature was reviewed to assess issues raised by the investigators. Interviews revealed substantial variability across design parameters, data collected, and analysis procedures. The presence of the chorion and renewal of exposure media (static versus static-renewal) were identified as design parameters that could potentially influence study outcomes and should be investigated further with studies to determine chemical uptake from treatment solution into embryos. The information gathered in this effort provides a basis for future SEAZIT activities to promote more consistent practices among researchers using zebrafish embryos for toxicity evaluation.

Changes in gene expression can help reveal the mechanisms of disease processes and the mode of action for toxicities and adverse effects on cellular responses induced by exposures to chemicals, drugs and environment agents. The U.S. Tox21 Federal collaboration, which currently quantifies the biological effects of nearly 10,000 chemicals via quantitative high-throughput screening(qHTS) in in vitro model systems, is now making an effort to incorporate gene expression profiling into the existing battery of assays. Whole transcriptome analyses performed on large numbers of samples using microarrays or RNA-Seq is currently cost-prohibitive. Accordingly, the Tox21 Program is pursuing a high-throughput transcriptomics (HTT) method that focuses on the targeted detection of gene expression for a carefully selected subset of the transcriptome that potentially can reduce the cost by a factor of 10-fold, allowing for the analysis of larger numbers of samples. To identify the optimal transcriptome subset, genes were sought that are (1) representative of the highly diverse biological space, (2) capable of serving as a proxy for expression changes in unmeasured genes, and (3) sufficient to provide coverage of well described biological pathways. A hybrid method for gene selection is presented herein that combines data-driven and knowledge-driven concepts into one cohesive method. Our approach is modular, applicable to any species, and facilitates a robust, quantitative evaluation of performance. In particular, we were able to perform gene selection such that the resulting set of "sentinel genes" adequately represents all known canonical pathways from Molecular Signature Database (MSigDB v4.0) and can be used to infer expression changes for the remainder of the transcriptome. The resulting computational model allowed us to choose a purely data-driven subset of 1500 sentinel genes, referred to as the S1500 set, which was then augmented using a knowledge-driven selection of additional genes to create the final S1500+ gene set. Our results indicate that the sentinel genes selected can be used to accurately predict pathway perturbations and biological relationships for samples under study.

SUMMARY : Access to high-quality reference data is essential for the development, validation, and implementation of in vitro and in silico approaches that reduce and replace the use of animals in toxicity testing. Currently, these data must often be pooled from a variety of disparate sources to efficiently link a set of assay responses and model predictions to an outcome or hazard classification. To provide a central access point for these purposes, the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods developed the Integrated Chemical Environment (ICE) web resource. The ICE data integrator allows users to retrieve and combine data sets and to develop hypotheses through data exploration. Open-source computational workflows and models will be available for download and application to local data. ICE currently includes curated in vivo test data, reference chemical information, in vitro assay data (including Tox21 TM /ToxCast™ high-throughput screening data), and in silico model predictions. Users can query these data collections focusing on end points of interest such as acute systemic toxicity, endocrine disruption, skin sensitization, and many others. ICE is publicly accessible at https://ice.ntp.niehs.nih.gov. https://doi.org/10.1289/EHP1759.

Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, "organotypic" cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomic data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics. On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data.

Background: The Next Generation (NexGen) of Risk Assessment effort is a multi-year collaboration among several organizations evaluating new, potentially more efficient molecular, computational, and systems biology approaches to risk assessment. This article summarizes our findings, suggests applications to risk assessment, and identifies strategic research directions., Objective: Our specific objectives were to test whether advanced biological data and methods could better inform our understanding of public health risks posed by environmental exposures., Methods: New data and methods were applied and evaluated for use in hazard identification and dose-response assessment. Biomarkers of exposure and effect, and risk characterization were also examined. Consideration was given to various decision contexts with increasing regulatory and public health impacts. Data types included transcriptomics, genomics, and proteomics. Methods included molecular epidemiology and clinical studies, bioinformatic knowledge mining, pathway and network analyses, short-duration in vivo and in vitro bioassays, and quantitative structure activity relationship modeling., Discussion: NexGen has advanced our ability to apply new science by more rapidly identifying chemicals and exposures of potential concern, helping characterize mechanisms of action that influence conclusions about causality, exposure-response relationships, susceptibility and cumulative risk, and by elucidating new biomarkers of exposure and effects. Additionally, NexGen has fostered extensive discussion among risk scientists and managers and improved confidence in interpreting and applying new data streams., Conclusions: While considerable uncertainties remain, thoughtful application of new knowledge to risk assessment appears reasonable for augmenting major scope assessments, forming the basis for or augmenting limited scope assessments, and for prioritization and screening of very data limited chemicals. Citation: Cote I, Andersen ME, Ankley GT, Barone S, Birnbaum LS, Boekelheide K, Bois FY, Burgoon LD, Chiu WA, Crawford-Brown D, Crofton KM, DeVito M, Devlin RB, Edwards SW, Guyton KZ, Hattis D, Judson RS, Knight D, Krewski D, Lambert J, Maull EA, Mendrick D, Paoli GM, Patel CJ, Perkins EJ, Poje G, Portier CJ, Rusyn I, Schulte PA, Simeonov A, Smith MT, Thayer KA, Thomas RS, Thomas R, Tice RR, Vandenberg JJ, Villeneuve DL, Wesselkamper S, Whelan M, Whittaker C, White R, Xia M, Yauk C, Zeise L, Zhao J, DeWoskin RS. 2016. The Next Generation of Risk Assessment multiyear study-highlights of findings, applications to risk assessment, and future directions. Environ Health Perspect 124:1671-1682; http://dx.doi.org/10.1289/EHP233., Competing Interests: K.B. is an occasional expert consultant for chemical and pharmaceutical companies, including Boehringer Ingelheim and Sangamo, and owns stock in and is a consultant for CytoSolv, an early stage biotechnology company. D.K. (D. Krewski) and G.M.P. are subcontractors to ICF International, Durham, NC, USA. G.P. is employed by Grant Consulting Group, Washington, DC, USA. D.K. (D. Krewski) is a Principal at Risk Sciences International (RSI), a Canadian company established in partnership with the University of Ottawa in 2006. RSI contributed to the development of the U.S. EPA NexGen framework under a subcontract with ICF International. M.T.S. has received consulting and expert testimony fees from lawyers representing both plaintiffs and defendants in cases involving claims related to exposure to benzene. These activities are unrelated to the current work. The remaining authors declare they have no actual or potential competing financial interests.

Background: Diabetes affects an estimated 346 million persons globally, and total deaths from diabetes are projected to increase > 50% in the next decade. Understanding the role of environmental chemicals in the development or progression of diabetes is an emerging issue in environmental health. In 2011, the National Toxicology Program (NTP) organized a workshop to assess the literature for evidence of associations between certain chemicals, including inorganic arsenic, and diabetes and/or obesity to help develop a focused research agenda. This review is derived from discussions at that workshop., Objectives: Our objectives were to assess the consistency, strength/weaknesses, and biological plausibility of findings in the scientific literature regarding arsenic and diabetes and to identify data gaps and areas for future evaluation or research. The extent of the existing literature was insufficient to consider obesity as an outcome., Data Sources, Extraction, and Synthesis: Studies related to arsenic and diabetes or obesity were identified through PubMed and supplemented with relevant studies identified by reviewing the reference lists in the primary literature or review articles., Conclusions: Existing human data provide limited to sufficient support for an association between arsenic and diabetes in populations with relatively high exposure levels (≥ 150 µg arsenic/L in drinking water). The evidence is insufficient to conclude that arsenic is associated with diabetes in lower exposure (< 150 µg arsenic/L drinking water), although recent studies with better measures of outcome and exposure support an association. The animal literature as a whole was inconclusive; however, studies using better measures of diabetes-relevant end points support a link between arsenic and diabetes.

Trichloroethylene (TCE), an environmental contaminant of National concern, is the focus of a new health risk assessment process incorporating the Proposed Cancer Risk Assessment Guidelines. This paper describes not only how TCE became an environmental problem for the Air Force, but also details the new Risk Assessment process envisioned by the Environmental Protection Agency's (EPA) National Center for Environmental Assessment (NCEA). Insights on epidemiological evaluations, both past and future, and their impact on the cancer classification of TCE are discussed. Examples of how physiologically based pharmacokinetics and dose-response characterization described in the new Cancer Guidelines are applied to TCE are provided. In addition, a variety of modeling techniques are discussed for the development of reference doses (oral exposure) and reference concentrations (inhalation exposures) for TCE. Finally, the role of risk communication is included. This new process provides an example of how interagency (EPA, Department of Defense. Department of Energy) and extramural (industry, academia) partnerships can provide greater gains to the nation, as a whole, than any of the parts on their own.

A panel of recombinant virus genomes was constructed by exchanging homologous genome fragments between the potent T-cell lymphoma inducer Moloney murine leukemia virus (MoMuLV) and its closely related but significantly less virulent relative MoMuLV-TB. Testing of these recombinant viruses in BALB/c mice established that only nucleotide changes within the Clal(-590)-Kpnl(36) fragment altered virulence. Fine analysis of this fragment showed that while mutations within the enhancer of MoMuLV-TB attenuated the latency period most, mutations within the MoMuLV-TB fragments flanking the enhancer also helped reduce the virulence of MoMuLV. The present study also suggests that the small difference in the relative number of lymphomas that developed primarily in the spleens of MoMuLV- or MoMuLV-TB-infected mice may correlate with nucleotide differences between the Clal-Kpnl fragments of the two viruses. However, the significantly greater proportion of premature death observed in MoMuLV-TB-relative to MoMuLV-infected mice could not be correlated with nucleotide differences in a specific genome fragment.

The mycotoxin, ochratoxin A (OA), is a potent in vivo teratogen. Studies were performed to determine the in vitro effects of OA on postimplantation rat embryos. Embryos were explanted from pregnant Sprague-Dawley rats on day 10 of gestation and were cultured (within the yolk sac) for 45 hr in gassed rat serum containing OA at concentrations between 0 and 300 micrograms/mL. Gross morphology, histopathology and protein and DNA content of embryos were evaluated. An OA concentration-dependent reduction in yolk sac diameter, crown-rump length, somite number count, and protein and DNA content was observed. Ochratoxin A treatment also resulted in an increase in the incidence of defective embryos. Malformations included: growth retardation, hypoplasia of the telencephalon, poor flexion, stunted limb bud development, underdeveloped sensory primordia and decreased mandibular and maxillary size. Histological examination demonstrated extensive OA-induced necrosis of embryonal mesodermal structures and neuroectoderm. Thus, the rat embryo in culture is a sensitive indicator of OA toxicity and may be useful for predicting developmental hazards associated with this mycotoxin.

Gastric mucin glycoproteins form an adherent gel over the surface epithelium that is thought to protect the stomach against chemical and physical damage. The purpose of this study was to measure the release of mucin glycoproteins from rat stomach after treatment with cysteamine and prostaglandin F2 beta, two structurally unrelated drugs that have been shown to protect the stomach against the noxious effects of alcohol and other damaging agents. Gastric mucin was separated into soluble (washout) and insoluble (adherent) phases before colorimetric quantitation of total mucin, protein-bound hexose, and sialic acid. Cysteamine produced a dose-dependent increase in release of soluble and gel mucin. Prostaglandin F2 beta caused a dose-dependent release of hexose-containing mucin but had no effect on sialic acid-containing glycoproteins. Sepharose 4B chromatography of both the soluble and adherent mucus revealed that greater than 90% was a high molecular weight glycoprotein fraction. N-Ethylmaleimide, a known inhibitor of cytoprotection by cysteamine, had no effect on mucin secretion. Similarly, indomethacin inhibited mucin secretion by cysteamine but did not significantly influence cytoprotection. Thus the secretion of mucin by cytoprotective agents is unlikely by itself to explain the ability of the stomach to resist chemical or physical damage.

Pretreatment of rats with the polychlorinated biphenyl (PCB) Aroclor 1254 or phenobarbital markedly increased the duodenal ulcerogenic action of acrylonitrile. The extent of forestomach and hepatic lesions in these rats, on the other hand, was not modified. The duodenal ulcers produced by Aroclor 1254 and acrylonitrile morphologically resembled the ulcers induced in other animal models of the human duodenal ulcer disease. The possible mechanisms of this potentiation of acrylonitrile action are discussed.

Occupational stress occurs in the working environment where the stressors may be physical, chemical, biologic or psychosocial in nature. This review especially emphasizes the most novel and probably most prevalent type of work-related factors and response to them: psychologic stress. A brief historical introduction concerning the development of the (biologic) stress concept underlines the duality of stressors (i.e., extremely unpleasant and pleasant events, too much work and work stagnation) equally cause stress. A section on 'Recognition and manifestation' emphasizes the need for understanding the origins and nature of occupational stress before considering its manifestations (e.g., nonspecific behavioral changes and specific hormone level measurements). Under 'Prevention and treatment' reduction or avoidance of psychosocial stressors coupled with minimizing predisposing and promoting protective intervening variables are discussed. Thus, although occupational stress might be a major complication at the workplace, understanding this stress could lead to its partial or complete prevention.

A single dose of acrylonitrile can produce fatal adrenal apoplexy within approximately 2 h. Our previous studies also indicate that multiple injections of the chemical cause acute hemorrhagic and occasional nonperforating duodenal ulcers. Other authors have reported increase in gut and lung neoplasia after chronic exposure. The present study was designed to elucidate the subacute and chronic actions of acrylonitrile on the adrenals, stomach and duodenum by correlating biochemical, functional and morphologic investigations, as well as to gain insight into the mechanisms of action of acrylonitrile. Rats were exposed to 0, 0.0001% (1 ppm), 0.002%, 0.01%, 0.05% or 0.2% acrylonitrile in drinking water, or to the same amount of the chemical given through daily gavage, for 7, 21 or 60 days. Acrylonitrile caused a time- and dose-dependent decrease in plasma corticosterone levels; aldosterone was affected only by the 'high' dose and prolonged time of exposure. Young rats were more susceptible than adults to this action of acrylonitrile. The adrenal cortex, especially the zona fasciculata, was atrophic in rats that had ingested the nitrile through drinking water. At 0.05% and 0.2%, it also caused decreased food intake and body weight gain. The adrenals were enlarged with a hyperplastic zona fasciculata after daily doses of a bolus of acrylonitrile. Ingestion of the chemical did not interfere with compensatory enlargement of the adrenal gland following unilateral adrenalectomy. On the other hand, the ACTH-induced elevation of corticosterone plasma concentration was significantly attenuated by acrylonitrile in drinking water. Electron microscopy of the adrenal glands revealed no consistent changes in the steroid-producing cells. We thus postulate that accelerated turnover of circulating corticoids and/or interference with the secretion or action of ACTH may primarily be responsible for the decreased plasma levels of corticosterone and aldosterone in rats that ingest acrylonitrile. The mucosa in the stomach at the junction of the forestomach and glandular region of animals that had ingested acrylonitrile was hyperplastic. The corpus also showed regional mucosal hyperplasia with the appearance of 'cobble-stoning'. These changes were preceded and associated with an elevated concentration of non-protein sulfhydryls mostly in the mucosa of the glandular stomach. A similar, less prominent elevation also occurred in the proximal duodenum. These alterations may resemble the preneoplastic combination of elevated glutathione and focal hyperplasia described in the liver with hepatocarcinogens.(ABSTRACT TRUNCATED AT 400 WORDS)