Epigenetic Applications in Adverse Outcome Pathways and Environmental Risk Evaluation.

BACKGROUND: The epigenome may be an important interface between environmental chemical exposures and human health. However, the links between epigenetic modifications and health outcomes are often correlative and do not distinguish between cause and effect or common-cause relationships. The Adverse Outcome Pathway (AOP) framework has the potential to demonstrate, by way of an inference- and science-based analysis, the causal relationship between chemical exposures, epigenome, and adverse health outcomes. OBJECTIVE: The objective of this work is to discuss the epigenome as a modifier of exposure effects and risk, perspectives for integrating toxicoepigenetic data into an AOP framework, tools for the exploration of epigenetic toxicity, and integration of AOP-guided epigenetic information into science and risk-assessment processes. DISCUSSION: Organizing epigenetic information into the topology of a qualitative AOP network may help describe how a system will respond to epigenetic modifications caused by environmental chemical exposures. However, understanding the biological plausibility, linking epigenetic effects to short- and long-term health outcomes, and including epigenetic studies in the risk assessment process is met by substantive challenges. These obstacles include understanding the complex range of epigenetic modifications and their combinatorial effects, the large number of environmental chemicals to be tested, and the lack of data that quantitatively evaluate the epigenetic effects of environmental exposure. CONCLUSION: We anticipate that epigenetic information organized into AOP frameworks can be consistently used to support biological plausibility and to identify data gaps that will accelerate the pace at which epigenetic information is applied in chemical evaluation and risk-assessment paradigms. https://doi.org/10.1289/EHP2322.

Adverse outcome pathway networks I: Development and applications.

Based on the results of a Horizon Scanning exercise sponsored by the Society of Environmental Toxicology and Chemistry that focused on advancing the adverse outcome pathway (AOP) framework, the development of guidance related to AOP network development was identified as a critical need. This not only included questions focusing directly on AOP networks, but also on related topics such as mixture toxicity assessment and the implementation of feedback loops within the AOP framework. A set of two articles has been developed to begin exploring these concepts. In the present article (part I), we consider the derivation of AOP networks in the context of how it differs from the development of individual AOPs. We then propose the use of filters and layers to tailor AOP networks to suit the needs of a given research question or application. We briefly introduce a number of analytical approaches that may be used to characterize the structure of AOP networks. These analytical concepts are further described in a dedicated, complementary article (part II). Finally, we present a number of case studies that illustrate concepts underlying the development, analysis, and application of AOP networks. The concepts described in the present article and in its companion article (which focuses on AOP network analytics) are intended to serve as a starting point for further development of the AOP network concept, and also to catalyze AOP network development and application by the different stakeholder communities. Environ Toxicol Chem 2018;37:1723-1733. © 2018 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Adverse outcome pathway networks II: Network analytics.

Toxicological responses to stressors are more complex than the simple one-biological-perturbation to one-adverse-outcome model portrayed by individual adverse outcome pathways (AOPs). Consequently, the AOP framework was designed to facilitate de facto development of AOP networks that can aid in the understanding and prediction of pleiotropic and interactive effects more common to environmentally realistic, complex exposure scenarios. The present study introduces nascent concepts related to the qualitative analysis of AOP networks. First, graph theory-based approaches for identifying important topological features are illustrated using 2 example AOP networks derived from existing AOP descriptions. Second, considerations for identifying the most significant path(s) through an AOP network from either a biological or risk assessment perspective are described. Finally, approaches for identifying interactions among AOPs that may result in additive, synergistic, or antagonistic responses (or previously undefined emergent patterns of response) are introduced. Along with a companion article (part I), these concepts set the stage for the development of tools and case studies that will facilitate more rigorous analysis of AOP networks, and the utility of AOP network-based predictions, for use in research and regulatory decision-making. The present study addresses one of the major themes identified through a Society of Environmental Toxicology and Chemistry Horizon Scanning effort focused on advancing the AOP framework. Environ Toxicol Chem 2018;37:1734-1748. © 2018 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Standardization of the collection of exhaled breath condensate and exhaled breath aerosol using a feedback regulated sampling device.

Exhaled breath condensate (EBC) and associated exhaled breath aerosols (EBA) are valuable non-invasive biological media used for the quantification of biomarkers. EBC contains exhaled water vapor, soluble gas-phase (polar) organic compounds, ionic species, plus other species including semi- and non-volatile organic compounds, proteins, cell fragments, DNA, dissolved inorganic compounds, ions, and microbiota (bacteria and viruses) dissolved in the co-collected EBA. EBC is collected from subjects who breathe 'normally' through a chilled tube assembly for approximately 10 min and is then harvested into small vials for analysis. Aerosol filters without the chilled tube assembly are also used to separately collect EBA. Unlike typical gas-phase breath samples used for environmental and clinical applications, the constituents of EBC and EBA are not easily characterized by total volume or carbon dioxide (CO2) concentration, because the gas-phase is vented. Furthermore, EBC and associated EBA are greatly affected by breathing protocol, more specifically, depth of inhalation and expelled breath velocity. We have tested a new instrument developed by Loccioni Gruppa Humancare (Ancona, Italy) for implementation of EBC collection from human subjects to assess EBC collection parameters. The instrument is the first EBC collection device that provides instantaneous visual feedback to the subjects to control breathing patterns. In this report we describe the operation of the instrument, and present an overview of performance and analytical applications.

Editor's Highlight: Mechanistic Toxicity Tests Based on an Adverse Outcome Pathway Network for Hepatic Steatosis.

Risk assessors use liver endpoints in rodent toxicology studies to assess the safety of chemical exposures. Yet, rodent endpoints may not accurately reflect human responses. For this reason and others, human-based invitro models are being developed and anchored to adverse outcome pathways to better predict adverse human health outcomes. Here, a networked adverse outcome pathway-guided selection of biology-based assays for lipid uptake, lipid efflux, fatty acid oxidation, and lipid accumulation were developed. These assays were evaluated in a metabolically competent human hepatocyte cell model (HepaRG) exposed to compounds known to cause steatosis (amiodarone, cyclosporine A, and T0901317) or activate lipid metabolism pathways (troglitazone, Wyeth-14,643, and 22(R)-hydroxycholesterol). All of the chemicals activated at least one assay, however, only T0901317 and cyclosporin A dose-dependently increased lipid accumulation. T0901317 and cyclosporin A increased fatty acid uptake, decreased lipid efflux (inferred from apolipoprotein B100 levels), and increased fatty acid synthase protein levels. Using this biologically-based evaluation of key events regulating hepatic lipid levels, we demonstrated dysregulation of compensatory pathways that normally balance hepatic lipid levels. This approach may provide biological plausibility and data needed to increase confidence in linking invitro-based measurements to chemical effects on adverse human health outcomes.

Tipping the Balance: Hepatotoxicity and the 4 Apical Key Events of Hepatic Steatosis.

Hepatic steatosis is a condition were fat accumulates in the liver and it is associated with extra-hepatic diseases related to metabolic syndrome and systemic energy metabolism. If not reversed, steatosis can progress to steatohepatitis and irreversible stages of liver disease including fibrosis, cirrhosis, hepatocellular carcinoma, and death. From a public health standpoint, identifying chemical exposures that may be factors in steatosis etiology are important for preventing hepatotoxicity and liver disease progression. It is therefore important to identify the biological events that are key for steatosis pathology mediated by chemical exposure. In this review, we give a current overview of the complex biological cascades that can disrupt lipid homeostasis in hepatocytes in the context of 4 apical key events central to hepatic lipid retention: hepatic fatty acid (FA) uptake,de novoFA and lipid synthesis, FA oxidation, and lipid efflux. Our goal is to review these key cellular events and visually summarize them using a network for application in pathway-based toxicity testing. This effort provides a foundation to improve next-generation chemical screening efforts that may be used to prevent and ultimately reverse the growing incidence of fatty liver disease in our population.

Taxonomic applicability of inflammatory cytokines in adverse outcome pathway (AOP) development.

Cytokines, low-molecular-weight messenger proteins that act as intercellular immunomodulatory signals, have become a mainstream preclinical marker for assessing the systemic inflammatory response to external stressors. The challenge is to quantitate from healthy subjects cytokine levels that are below or at baseline and relate those dynamic and complex cytokine signatures of exposures with the inflammatory and repair pathways. Thus, highly sensitive, specific, and precise analytical and statistical methods are critically important. Investigators at the U.S. Environmental Protection Agency (EPA) have implemented advanced technologies and developed statistics for evaluating panels of inflammatory cytokines in human blood, exhaled breath condensate, urine samples, and murine biological media. Advanced multiplex, bead-based, and automated analytical platforms provided sufficient sensitivity, precision, and accuracy over the traditional enzyme-linked immunosorbent assay (ELISA). Thus, baseline cytokine levels can be quantified from healthy human subjects and animals and compared to an in vivo exposure response from an environmental chemical. Specifically, patterns of cytokine responses in humans exposed to environmental levels of ozone and diesel exhaust, and in rodents exposed to selected pesticides (such as fipronil and carbaryl), were used as case studies to generally assess the taxonomic applicability of cytokine responses. The findings in this study may aid in the application of measureable cytokine markers in future adverse outcome pathway (AOP)-based toxicity testing. Data from human and animal studies were coalesced and the possibility of using cytokines as key events (KE) to bridge species responses to external stressors in an AOP-based framework was explored.

Integrating Publicly Available Data to Generate Computationally Predicted Adverse Outcome Pathways for Fatty Liver.

Newin vitrotesting strategies make it possible to design testing batteries for large numbers of environmental chemicals. Full utilization of the results requires knowledge of the underlying biological networks and the adverse outcome pathways (AOPs) that describe the route from early molecular perturbations to an adverse outcome. Curation of a formal AOP is a time-intensive process and a rate-limiting step to designing these test batteries. Here, we describe a method for integrating publicly available data in order to generate computationally predicted AOP (cpAOP) scaffolds, which can be leveraged by domain experts to shorten the time for formal AOP development. A network-based workflow was used to facilitate the integration of multiple data types to generate cpAOPs. Edges between graph entities were identified through direct experimental or literature information, or computationally inferred using frequent itemset mining. Data from the TG-GATEs and ToxCast programs were used to channel large-scale toxicogenomics information into a cpAOP network (cpAOPnet) of over 20 000 relationships describing connections between chemical treatments, phenotypes, and perturbed pathways as measured by differential gene expression and high-throughput screening targets. The resulting fatty liver cpAOPnet is available as a resource to the community. Subnetworks of cpAOPs for a reference chemical (carbon tetrachloride, CCl4) and outcome (fatty liver) were compared with published mechanistic descriptions. In both cases, the computational approaches approximated the manually curated AOPs. The cpAOPnet can be used for accelerating expert-curated AOP development and to identify pathway targets that lack genomic markers or high-throughput screening tests. It can also facilitate identification of key events for designing test batteries and for classification and grouping of chemicals for follow up testing.

Immunochemistry for high-throughput screening of human exhaled breath condensate (EBC) media: implementation of automated Quanterix SIMOA instrumentation.

Immunochemistry is an important clinical tool for indicating biological pathways leading towards disease. Standard enzyme-linked immunosorbent assays (ELISA) are labor intensive and lack sensitivity at low-level concentrations. Here we report on emerging technology implementing fully-automated ELISA capable of molecular level detection and describe application to exhaled breath condensate (EBC) samples. The Quanterix SIMOA HD-1 analyzer was evaluated for analytical performance for inflammatory cytokines (IL-6, TNF-α, IL-1ß and IL-8). The system was challenged with human EBC representing the most dilute and analytically difficult of the biological media. Calibrations from synthetic samples and spiked EBC showed excellent linearity at trace levels (r(2) > 0.99). Sensitivities varied by analyte, but were robust from ~0.006 (IL-6) to ~0.01 (TNF-α) pg ml(-1). All analytes demonstrated response suppression when diluted with deionized water and so assay buffer diluent was found to be a better choice. Analytical runs required ~45 min setup time for loading samples, reagents, calibrants, etc., after which the instrument performs without further intervention for up to 288 separate samples. Currently, available kits are limited to single-plex analyses and so sample volumes require adjustments. Sample dilutions should be made with assay diluent to avoid response suppression. Automation performs seamlessly and data are automatically analyzed and reported in spreadsheet format. The internal 5-parameter logistic (pl) calibration model should be supplemented with a linear regression spline at the very lowest analyte levels, (<1.3 pg ml(-1)). The implementation of the automated Quanterix platform was successfully demonstrated using EBC, which poses the greatest challenge to ELISA due to limited sample volumes and low protein levels.

Probe molecule (PrM) approach in adverse outcome pathway (AOP) based high-throughput screening (HTS): in vivo discovery for developing in vitro target methods.

Efficient and accurate adverse outcome pathway (AOP) based high-throughput screening (HTS) methods use a systems biology based approach to computationally model in vitro cellular and molecular data for rapid chemical prioritization; however, not all HTS assays are grounded by relevant in vivo exposure data. The challenge is to develop HTS assays with unambiguous quantitative links between in vitro responses and corresponding in vivo effects, which is complicated by metabolically insufficient systems, in vitro to in vivo extrapolation (IVIVE), cross-species comparisons, and other inherent issues correlating IVIVE findings. This article introduces the concept of ultrasensitive gas phase probe molecules (PrMs) to help bridge the current HTS assay IVIVE gap. The PrM concept assesses metabolic pathways that have already been well-defined from intact human or mammalian models. Specifically, the idea is to introduce a gas phase probe molecule into a system, observe normal steady state, add chemicals of interest, and quantitatively measure (from headspace gas) effects on PrM metabolism that can be directly linked back to a well-defined and corresponding in vivo effect. As an example, we developed the pharmacokinetic (PK) parameters and differential equations to estimate methyl tertiary butyl ether (MTBE) metabolism to tertiary butyl alcohol (TBA) via cytochrome (CYP) 2A6 in the liver from human empirical data. Because MTBE metabolic pathways are well characterized from in vivo data, we can use it as a PrM to explore direct and indirect chemical effects on CYP pathways. The PrM concept could be easily applied to in vitro and alternative models of disease and phenotype, and even test for volatile chemicals while avoiding liquid handling robotics. Furthermore, a PrM can be designed for any chemical with known empirical human exposure data and used to assess chemicals for which no information exists. Herein, we propose an elegant gas phase probe molecule-based approach to in vitro toxicity testing.

Comparative analysis of temporal and dose-dependent TCDD-elicited gene expression in human, mouse, and rat primary hepatocytes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-elicited time- and dose-dependent differential gene expression was compared in human, mouse, and rat primary hepatocytes. Comprehensive time course (10 nM TCDD or dimethyl sulfoxide vehicle control for 1, 2, 4, 8, 12, 24, and 48h) studies identified 495, 2305, and 711 differentially expressed orthologous genes in human, mouse, and rat hepatocytes, respectively. However, only 16 orthologs were differentially expressed across all three species, with the majority of orthologs exhibiting species-specific expression (399 human, 2097 mouse, and 533 rat), consistent with species-specific expression reported in other in vitro and in vivo comparative studies. TCDD also elicited the dose-dependent induction of 397 human, 100 mouse, and 443 rat genes at 12h and 615 human, 426 mouse, and 314 rat genes at 24h. Comparable EC50 values were obtained for AhR battery genes including Cyp1a1 (0.1 nM human, 0.05 nM mouse, 0.08 nM rat at 24h) and Tiparp (0.97 nM human, 0.63 nM mouse, 0.14 nM rat at 12h). Overrepresented functions and pathways included amino acid metabolism in humans, immune response in mice, and energy homeostasis in rats. Differentially expressed genes functionally associated with lipid transport, processing, and metabolism were overrepresented in all three species but exhibited species-specific expression consistent with the induction of hepatic steatosis in mice but not in rats following a single oral gavage of TCDD. Furthermore, human primary hepatocytes showed lipid accumulation following 48h of treatment with TCDD, suggesting that AhR-mediated steatosis in mice more closely resembles human hepatic fat accumulation compared with that in rats. Collectively, these results suggest that species-specific gene expression profiles mediate the species-specific effects of TCDD despite the conservation of the AhR and its signaling mechanism.

Aryl hydrocarbon receptor-mediated induction of Stearoyl-CoA desaturase 1 alters hepatic fatty acid composition in TCDD-elicited steatosis.

2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD) induces hepatic dyslipidemia mediated by the aryl hydrocarbon receptor (AhR). Stearoyl-CoA desaturase 1 (Scd1) performs the rate-limiting step in monounsaturated fatty acid (MUFA) synthesis, desaturating 16:0 and 18:0 into 16:1n7 and 18:1n9, respectively. To further examine the role of Scd1 in TCDD-induced hepatotoxicity, comparative studies were performed in Scd1(+/+) and Scd1(-/-) mice treated with 30 µg/kg TCDD. TCDD induced Scd1 activity, protein, and messenger RNA (mRNA) levels approximately twofold. In Scd1(+/+) mice, hepatic effects were marked by increased vacuolization and inflammation and a 3.5-fold increase in serum alanine aminotransferase (ALT) levels. Hepatic triglycerides (TRGs) were induced 3.9-fold and lipid profiling by gas chromatography-mass spectroscopy measured a 1.9-fold increase in fatty acid (FA) levels, consistent with the induction of lipid transport genes. Induction of Scd1 altered FA composition by decreasing saturated fatty acid (SFA) molar ratios 8% and increasing MUFA molar ratios 9%. Furthermore, ChIP-chip analysis revealed AhR enrichment (up to 5.7-fold), and computational analysis identified 16 putative functional dioxin response elements (DREs) within Scd1 genomic loci. Band shift assays confirmed AhR binding with select DREs. In Scd1(-/-) mice, TCDD induced minimal hepatic vacuolization and inflammation, while serum ALT levels remained unchanged. Although Scd1 deficiency attenuated TCDD-induced TRG accumulation, overall FA levels remained unchanged compared with Scd1(+/+) mice. In Scd1(-/-) mice, TCDD induced SFA ratios 8%, reduced MUFA ratios 13%, and induced polyunsaturated fatty acid ratios 5% relative to treated Scd1(+/+) mice. Collectively, these results suggest that AhR regulation of Scd1 not only alters lipid composition but also contributes to the hepatotoxicity of TCDD.