Adverse outcome pathway (AOP) discovery and definition
The use of mechanistic or pathway-based data (e.g., Quantitative Structure-Activity Relationships [QSARs], in vitro, biomarkers) in risk assessment and regulatory applications is limited by the lack of well-established linkages between endpoints measured, or predicted, at molecular and cellular levels of organization, and adverse outcomes that manifest at higher levels of biological organization (e.g., organ function in humans; survival, growth/development, and reproduction in wildlife). In their vision and strategy for toxicity testing in the 21st century, the National Research Council cited the linkage of adverse effects to specific toxicity pathways (i.e., development of Adverse Outcome Pathways [AOPs]), as a critical research activity to support development and regulatory application of pathway-based testing. Research under this task aims to formalize and expand the description, inference, and dissemination of AOPs and AOP knowledge through a systems-level approach that identifies critical molecular interactions that initiate biological perturbation, and links those perturbations to adverse outcomes at levels of biological organization relevant to risk assessment. The primary outcome of this research will be the population and annotation of an AOP knowledge-base (e.g., www.aopwiki.org) with both new and existing AOP knowledge. Initial efforts will focus on AOPs relevant to reproductive and developmental toxicity in fish and the application of AOP knowledge to support hazard extrapolation across taxa.
Rationale and Research Approach:
Research aimed at elucidating adverse outcome pathways (AOPs) provides a scientific foundation to address a number of critical challenges outlined in the Chemical Safety for Sustainability (CSS) framework. These include: (1) identification of critical pathways perturbed by environmental chemicals that lead to adverse effects, (2) ways to apply the knowledge gained in human health risk assessment to ecotoxicology, and (3) enhancing interpretation of biomarkers. However, while there is a long history of research relevant to the elucidation of AOPs, well defined systematic approaches to the discovery, definition, and dissemination of AOP knowledge to regulators and other environmental decision-makers have been lacking. This research aims to address those needs.
We propose to employ a multi-step approach to AOP discovery and definition. The first step involves scoping the problem with relevant program office partners: for example, identifying a specific guideline toxicity test for which it would be desirable to develop more cost effective/efficient alternatives, or developing an AOP relevant to a specific pesticide registration, water quality criteria development, etc. Once the scope has been defined, the second step is to assemble the relevant expertise within Office of Research and Development (ORD) and/or via external partners and develop a conceptual model of the system of interest which includes critical biological functions and their regulation at the molecular level (to the extent it is understood). The conceptual model is then used to identify points/targets within the system that are potentially vulnerable to perturbation by the chemical(s) of interest.
This assessment of putative molecular initiating events serves as the basis for hypothesized AOP formulation. Hypothesized AOPs are first evaluated against the extant literature to identify whether sufficient supporting evidence linking the molecular perturbation with an adverse outcome relevant to the regulatory context exists. Where gaps are identified or supporting evidence is lacking, targeted research is then conducted to fill data gaps, generate supporting evidence, and/or reject the hypothesized AOP. Where feasible, this targeted research will employ “omic” approaches (e.g., transcriptomics, metabolomics, proteomics) which will facilitate and accelerate the discovery of other biological pathways/functions impacted by chemical perturbations and provide important insights into the function of AOPs within a broader systems biology context.
Once supporting information for an AOP has been generated and/or assembled, that information will then be deposited into a curated, accessible, searchable, and adaptable AOP knowledge-base (e.g., www.aopwiki.org). The first product of this research program (Product 1) will involve working with relevant partners (e.g., World Health Organization Mode of Action steering committee, Organisation for Economic Cooperation and Development [OECD], Effectopedia developers, International Life Sciences Institute-Health and Environmental Sciences Institute [ILSI-HESI]) to develop a set of example AOP descriptions that will be used to start populating an AOP knowledge-base. ORD scientists will also engage with program office partners to define AOP quality criteria that can be used to tag information in the knowledge-base relative to its suitability for various types of EPA applications.
In addition to populating the AOP knowledge-base, aspects of this research will develop tools and approaches for evaluating the conservation of AOPs across taxa. These will be used to evaluate whether key molecular targets are conserved, whether they respond similarly to chemical perturbation, and to understand how differences in the conservation and/or physiological roles of those targets among taxa may influence both the susceptibility to adverse outcomes and the type(s) of adverse outcomes that occur in different taxa as a result of chemical exposure. This will facilitate annotation of that AOP knowledge-base with relevant information regarding the taxonomic domain of applicability for each AOP. Products 2 and 3 will focus on these aims.
Product 2 will use iterative targeted testing approaches to evaluate comparability of AOPs and responses between fish, rodent, and potentially invertebrate models. Product 3 will consist of a web-based tool that will provide quantitative characterization of the molecular conservation of any protein target of interest among a diversity of taxa that may be relevant to a risk management decision. Finally, as AOPs are developed, the investigators will also identify specific QSAR, toxicity pathway assays (e.g., in vitro, high throughput assays), targeted tests, and/or biomarkers with scientifically defensible relevance to specific adverse outcomes. This information will be mapped into the AOP knowledge-base to serve as a resource for identifying data that could be useful for assessments concerning a particular AOP (Product 4).
Initial research under this task will build on established efforts within ORD related to the development of AOPs for reproductive toxicity and developmental neurotoxicity in small fish models. Consequently, the initial AOPs developed are expected to have relevance to the evolution of the Endocrine Disruptor Screening Program (e.g., EDSP21), identification of potential alternatives to fish early life stage toxicity tests (e.g., OCSPP 850.1400), and/or potential use of fish data for predicting developmental neurotoxicity in mammals (in support of Office of Pollution Prevention [OPP’s] Targeted Testing and Priority Setting workplans). However, as the research program evolves, the intent is for this line of research to mature into an active partnership between CSS scientists and risk assessors within specific program offices. The aim is to develop an efficient process whereby the programs can obtain ORD support in developing and/or applying AOP knowledge in support of specific program office activities. For example, to support an OPP ruling under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), points of contact within ORD could recruit CSS scientists with scientific expertise relevant to endpoints of concern identified for a chemical in question. These scientists would work with the OPP risk assessor to map the toxicity data submitted by the registrant to existing AOPs in the AOP knowledgebase and/or to develop new AOPs as needed. Critical data gaps identified through this process would be candidates for research under the experimental arm of this task. Over the course of these interactions, CSS scientists will work with the program office partners to develop a series of best-practices recommendations regarding the use of pathway-based data (e.g., in silico, in vitro, “omics”, and/or biomarkers), in conjunction with the AOP knowledge-base, to support the prediction of reproductive or developmental hazards associated with chemical exposures (Product 5).
Ankley, G.T. 2013. AOP Wiki: A new tool for developing and documenting adverse outcome pathways. Globe (SETAC newsletter) 14:2.
Ankley, G.T. and L.E. Gray. 2013. Cross-species conservation of endocrine pathways: A critical analysis of tier 1 fish and rat screening assays with 12 model chemicals. Environmental Toxicology and Chemistry 32:1084-1087.
Ankley, G.T. and C.R. Tyler. 2013. Development of methods to detect occurrence and effects of endocrine-disrupting chemicals: Fueling a fundamental shift in regulatory ecotoxicology. Environmental Toxicology and Chemistry 32:2661-2662.
Brooks, B.W., G.T. Ankley, A.B. Boxall, and M.A. Rudd. 2013. Towards sustainable environmental quality: A call to prioritize global research needs. Integrated Assessment and Management 9:179-180.
Hooper, M.J., G.T. Ankley, D.A. Cristol, L.A. Maryoung, P.D. Noyes, and K.E. Pinkerton. 2013. Interactions between chemical and climate stressors: A role for mechanistic toxicology in assessing climate change risks. Environmental Toxicology and Chemistry 32:32-48.
LaLone, C.A., D.L. Villeneuve, L.D. Burgoon, C.L. Russom, H.W. Helgen, J.P. Berninger, J.E. Tietge, M.N. Severson, J.E. Cavallin, and G.T. Ankley. 2013. Molecular target sequence similarity as a basis for species extrapolation to assess the ecological risk of chemicals with known modes of action. Aquatic Toxicology 144-145:141-154.
LaLone, C.A., D.L. Villeneuve, J.E. Cavallin, M.D. Kahl, E.J. Durhan, E.A. Makynen, K.M. Jensen, K.E. Steven, M.N. Severson, C.A. Blanksma, K.M. Flynn, P. Hartig, J. Woodard, J.P. Berninger, T.J. Norberg-King, R.D. Johnson, and G.T. Ankley GT. 2013. Cross species sensitivity to a novel androgen receptor agonist of potential environmental concern, spironolactone. Environmental Toxicology and Chemistry 32:2528-2541.
Leet, J.K., K. Lesteberg, H.L. Schoenfuss, A.W. Olmstead, J.J. Amberg, G.T. Ankley, and M.S. Sepulveda. 2013. Sex-specific gonadal and gene expression changes throughout development in fathead minnow. Sexual Development 7:303-307.
Meyer, J.L., S. Rogers-Burch, J.K. Leet, D.L. Villeneuve, G.T. Ankley, and M.S. Sepúlveda. 2013. Reproductive physiology in eastern snapping turtles (Chelydra serpentina) exposed to runoff from a concentrated animal feeding operation. Journal of Wildlife Diseases 49:996-999.
Perkins, E.J., G.T. Ankley, K.M. Crofton, N. Garcia-Reyero, C.A. LaLone, M.S. Johnson, J.E. Tietge, and D.L. Villeneuve. 2013. Current perspectives on the use of alternative species in human health and ecological hazard assessments. Environmental Health Perspectives 121:1002-1010.
Ralston-Hooper, K.J., M.E. Turner, E.J. Soderblom, D.L. Villeneuve, G.T. Ankley, M.A. Moseley, R.A. Hoke, and P.L. Ferguson. 2013. Application of a label-free, gel-free quantitative proteomics method for ecotoxicological studies of small fish species. Environmental Science & Technology 47:1091-1100.
Skolness, S.Y., C.A. Blanksma, J.E. Cavallin, J.J. Churchill, E.J. Durhan, K.M. Jensen, R.D. Johnson, M.D. Kahl, E.A. Makynen, D.L. Villeneuve, and G.T. Ankley. 2013. Propiconazole inhibits steroidogenesis and reproduction in the fathead minnow (Pimephales promelas). Toxicological Sciences 132:284-297.
Villeneuve, D.L., M. Breen, D.C. Bencic, J.E. Cavallin, K.M. Jensen, E.A. Makynen, L.M. Thomas, L.C. Wehmas, R.B. Conolly, and G.T. Ankley. 2013. Developing predictive approaches to characterize adaptive responses of the reproductive endocrine axis to aromatase inhibition: I. Data generation in a small fish model. Toxicological Sciences 133:225-233.
Villeneuve, D.L., D.C. Volz, M.R. Embry, G.T. Ankley, S.E. Belanger, M. Léonard, K. Schirmer, R. Tanguay, L. Truong, and L. Wehmas. 2013. Investigating alternatives to the fish early life-stage test: a strategy for discovering and annotating adverse outcome pathways for early fish development. Environmental Toxicology and Chemistry 33:158-169.
Adedeji, O.B., E.J. Durhan, M.D. Kahl, K.M. Jensen, C.A. LaLone, E.A. Makynen, L. Thomas, D.L. Villeneuve, and G.T. Ankley. 2012. Short-term study investigating the estrogenic potency of diethylstilbesterol in the fathead minnow (Pimephales promelas). Environmental Science & Technology 46:7826-7835.
Ankley, G.T., J.E. Cavallin, E.J. Durhan, K.M. Jensen, M.D. Kahl, E.A. Makynen, L. Thomas, L.C. Wehmas, and D.L. Villeneuve. 2012. A time-course analysis of effects of the steroidogenesis inhibitor ketoconazole on components of the hypothalamic-pituitary-gonadal axis of fathead minnows. Aquatic Toxicology 114-115:88-95.
Ekman, D., P. Hartig, M. Cardon, D.M. Skelton, Q. Teng, E. Durhan, K.M. Jensen, M.D. Kahl, D.L. Villeneuve, L. Gray, T. Collette, and G.T. Ankley. 2012. Metabolite profiling and a transcriptional activation assay provide direct evidence of androgen receptor antagonism by bisphenol A in fish. Environmental Science & Technology 46:9673-9680.
LaLone, C.A. , D.L. Villeneuve, A.W. Olmstead, E.K. Medlock , M.D. Kahl, K.M. Jensen, E.J. Durhan, E.A. Makynen, C.A. Blanksma, J.E. Cavallin, L.M. Thomas, S. Seidl, S.Y. Skolness, L.C. Wehmas, R.D. Johnson, and G.T. Ankley. 2012. Effects of a glucocorticoid receptor agonist, dexamethasone, on fathead minnow reproduction, growth, and development. Environmental Toxicology and Chemistry 31:611-622.
Skolness, S.Y., E.J. Durhan, K.M. Jensen, M.D. Kahl, E.A. Makynen, D.L. Villeneuve, and G.T. Ankley. 2012. Effects of gemfibrozil on lipid metabolism, steroidogenesis, and reproduction in the fathead minnow (Pimephales promelas). Environmental Toxicology and Chemistry 31:2615-2624.
Villeneuve, D.L., N. Garcia-Reyero, B.L. Escalon, K.M. Jensen, J.E. Cavallin, E.A. Makynen, E.J. Durhan, M.D. Kahl, L.M. Thomas, E.J. Perkins, and G.T. Ankley. 2012. Ecotoxicogenomics to support ecological risk assessment: A case study with bisphenol A in fish. Environmental Science & Technology, Ecogenomics Focus Issue 46:51-59.
Villeneuve, D.L., N. Garcia-Reyero, D. Martinovic-Weigelt, Z. Li, K.H. Watanabe, E.F. Orlando, C.A. LaLone, S.W. Edwards, L.D. Burgoon, N.D. Denslow, E.J. Perkins, and G.T. Ankley. 2012. A graphical systems model and tissue-specific functional gene sets to aid transcriptomic analysis of chemical impacts on the female teleost reproductive axis . Mutation Research, Special Issue 746:151-162.
Wang, R.-W., D. Bencic, A. Biales, R. Flick, J. Lazorchak, D.L. Villeneuve, and G.T. Ankley. 2012. Discovery and validation of gene classifiers for endocrine-disrupting chemicals in zebrafish (Danio rerio). BMC Genomics 13:358.
Ankley, G.T., J.E. Cavallin, E.J. Durhan, K.M. Jensen, M.D. Kahl, E.A. Makynen, D. Martinovic-Weigelt, L. C. Wehmas, and D.L. Villeneuve. 2011. Temporal evaluation of effects of a model 3β-hydroxysteroid dehydrogenase inhibitor on endocrine function in the fathead minnow. Environmental Toxicology and Chemistry 30:2094-2102.
Biales, A.D. , D.C. Bencic, D.L. Villeneuve, G.T. Ankley, and D.L. Lattier. 2011. Proteomic analysis of zebrafish brain tissue following exposure to the pesticide prochloraz. Aquatic Toxicology 105:618-628.
Ekman, D.R., D.L. Villeneuve, Q. Teng, K.J. Ralston-Hooper , D. Martinovic-Weigelt, M.D. Kahl, K.M. Jensen, E.J. Durhan, E.A. Makynen, G.T. Ankley, and T.W. Collette. 2011. Use of gene expression, biochemical and metabolite profiles to enhance exposure and effects assessment of the model androgen 17b-trenbolone in fish. Environmental Toxicology and Chemistry 30:319-329.
Kramer, V.J., M.A. Etterson, M. Hecker, C.A. Murphy, G. Roesijadi, D.J. Spade, J.A. Spromberg, M. Wang, and G.T. Ankley. 2011. Adverse outcome pathways and ecological risk assessment: Bridging to population-level effects. Environmental Toxicology and Chemistry 30:64-76.
Martinovic-Weigelt, D., R.-L. Wang, D.L. Villeneuve, D.C. Bencic, J. Lazorchak, and G.T. Ankley. 2011. Gene expression profiling of androgen receptor antagonists flutamide and vinclozolin in zebrafish (Danio rerio) gonads. Aquatic Toxicology 101:447-458.
Skolness, S.Y., E.J. Durhan, N. Garcia-Reyero, K.M. Jensen, M.D. Kahl, E.A. Makynen, D. Martinovic, E. Perkins, D.L. Villeneuve, and G.T. Ankley. 2011. Effects of a short-term exposure to the fungicide prochloraz on endocrine function and gene expression in female fathead minnows (Pimephales promelas). Aquatic Toxicology 103:170-178.
Villeneuve, D.L. and N. Garcia-Reyero. 2011. A vision and strategy: Predictive ecotoxicology in the 21st century. Environmental Toxicology and Chemistry 30:1-8.
Volz, D.C., S. Belanger, M. Embry, S. Padilla, H. Sanderson, K. Schirmer, S. Scholz, and D.L. Villeneuve. 2011. Adverse outcome pathways during early fish development: A framework for identifying and implementing alternative chemical prioritization strategies. Toxicological Sciences 123:349-358.
Wang, R.-L., D. Bencic, J. Lazorchak, D. Villeneuve, and G.T. Ankley. 2011. Transcriptional regulatory dynamics of the hypothalamic-pituitary-gonadal axis and its peripheral pathways as impacted by the 3-beta HSD inhibitor trilostane in zebrafish (Danio rerio). Ecotoxicology and Environmental Safety 74:1461-1470.
Ankley, G.T., R.S. Bennett, R.J. Erickson, D.J. Hoff, M.W. Hornung, R.D. Johnson, D.R. Mount, J.W. Nichols, C.L. Russom, P.K. Schmieder, J.A. Serrano, J.E. Tietge, and D.L. Villeneuve. 2010. Adverse outcome pathways: A conceptual framework to support ecotoxicology research and risk assessment. Environmental Toxicology and Chemistry 29:730-741.
Ankley, G.T., K.M. Jensen, M.D. Kahl, E.J. Durhan, E.A. Makynen, J.E. Cavallin, D. Martinovic, L.C. Wehmas, N.D. Mueller, and D.L. Villeneuve. 2010. Use of chemical mixtures to differentiate mechanisms of endocrine action in a small fish model. Aquatic Toxicology 99:389-396.
Collette, T.W., Q. Teng, K.M. Jensen, M.D. Kahl, E.A. Makynen, E.J. Durhan, D.L. Villeneuve, D. Martinovic-Weigelt, G.T. Ankley, and D.R. Ekman. 2010. Impacts of an anti-androgen and an androgen/anti-androgen mixture on the metabolite profile of male fathead minnow urine. Environmental Science & Technology 44:6881-6886.
Villeneuve, D.L., N. Garcia-Reyero, D. Martinovic, N.D. Mueller, J.E. Cavallin, E.J. Durhan, E.A. Makynen, K.M. Jensen, M.D. Kahl, L.S. Blake, E.J. Perkins, and G.T. Ankley. 2010. I. Effects of a dopamine receptor antagonist on fathead minnow, Pimephales promelas, reproduction. Ecotoxicology and Environmental Safety 73:472-477.
Villeneuve, D.L., N. Garcia-Reyero, D. Martinovic, N.D. Mueller, J.E. Cavallin, E.J. Durhan, E.A. Makynen, K.M. Jensen, M.D. Kahl, L.S. Blake, E.J. Perkins, and G.T. Ankley. 2010. II. Effects of a dopamine receptor antagonist on fathead minnow dominance behavior and ovarian gene expression in the fathead minnow and zebrafish. Ecotoxicology and Environmental Safety 73:478-485.
Wang, R.-L., D. Bencic, D.L. Villeneuve, G.T. Ankley, J. Lazorchak, and S. Edwards. 2010. A transcriptomics-based biological framework for studying mechanisms of endocrine disruption in small fish species. Aquatic Toxicology 98:230-244.
Ankley, G.T., D.C. Bencic, M.S. Breen, T.W. Collette, R.B. Conolly, N.D. Denslow, S.W. Edwards, D.R. Ekman, N. Garcia-Reyero, K.M. Jensen, J.M. Lazorchak, D. Martinovic, D.H. Miller, E.J. Perkins, E.F. Orlando, D.L. Villeneuve, R.-L. Wang, and K.H. Watanabe. 2009. Endocrine disrupting chemicals in fish: Developing exposure indicators and predictive models of effects based on mechanism of action. Aquatic Toxicology 92:168-178.
Ankley, G.T., D. Bencic, J.E. Cavallin, K.M. Jensen, M.D. Kahl, E.A. Makynen, D. Martinovic, N. Mueller, L.C. Wehmas, and D.L. Villeneuve. 2009. Dynamic nature of alterations in the endocrine system of fathead minnows exposed to the fungicide prochloraz. Toxicological Sciences 112:344-353.
Ekman, D.R., Q. Teng, D.L. Villeneuve, M.D. Kahl, K.M. Jensen, E.J. Durhan, G.T. Ankley, and T.W. Collette. 2009. Profiling lipid metabolites yields unique information on sex- and time-dependent responses of fathead minnows (Pimephales promelas) exposed to 17 a -ethynylestradiol. Metabolomics 5:22-32.
Garcia-Reyero, N., K.J. Kroll, L. Liu, E.F. Orlando, K.H. Watanabe, M.S. Sepulveda, D.L. Villeneuve, E.J. Perkins, G.T. Ankley, and N.D. Denslow. 2009. Gene expression responses in male fathead minnows exposed to binary mixtures of an estrogen and antiestrogen. BMC Genomics 10:doi:10.186/1471-2164-10-308.
Garcia-Reyero, N., D.L. Villeneuve, K.J. Kroll, L. Liu, E. Orlando, K. Watanabe, M. Sepúlveda, G.T. Ankley, and N.D. Denslow. 2009. Expression signatures for a model androgen and antiandrogen in the fathead minnow (Pimephales promelas) ovary. Environmental Science & Technology 43:2614-2619.
Martinovic, D., D.L. Villeneuve, M.D. Kahl, L.S. Blake, J. Brodin, and G.T. Ankley. 2009. Hypoxia alters gene expression in the gonads of zebrafish (Danio rerio). Aquatic Toxicology 95:258-272.
Martyniuk, C.J., S. Alvarez, S. McClung, D.L. Villeneuve , G.T. Ankley, and N.D. Denslow. 2009. Quantitative proteomic profiles of androgen receptor signaling in the liver of fathead minnows (Pimephales promelas). Journal of Proteome Research 8:2186-2200.
Petkov, P.I. , S. Temelkov, D.L. Villeneuve, G.T. Ankley, and O.G. Mekenyan. 2009. Mechanism-based categorization of aromatase inhibitors: A potential discovery and screening tool. SAR and QSAR in Environmental Research 20:657-678.
Villeneuve, D.L., N.D. Mueller, D. Martinovic, E.A. Makynen, M.D. Kahl, K.M. Jensen, E.J. Durhan, J.E. Cavallin, D. Bencic, and G.T. Ankley. 2009. Direct effects, compensation, and recovery in female fathead minnows exposed to a model aromatase inhibitor. Environmental Health Perspectives 117:624-631.
Villeneuve, D.L., R.-L. Wang, D.C. Bencic, A. Biales, D. Martinovic, J. Lazorchak, G. Toth, and G.T. Ankley. 2009. Altered gene expression in the brain and ovaries of zebrafish (Danio rerio) exposed to the aromatase inhibitor fadrozole: microarray analysis and hypothesis generation. Environmental Toxicology and Chemistry 28:1767-1782.
|Sep 30, 2013||(1) Example AOP descriptions in formats consistent with OECD guidance on developing and assessing AOPs and compatible with nascent AOP knowledge-base (e.g., Effectopedia) development. Examples will focus on AOPs related to reproductive toxicity and developmental toxicity and their relevance to EDSP21 and Priority Setting workplans developed with OCSPP and OW.||Daniel Villeneuve|
|Sep 30, 2014||2) Comparative AOP knowledge: AOP descriptions comparing linkages (e.g., causal) between specific pathway perturbations and reproductive or developmental outcomes in multiple species (e.g., rodents, fish, invertebrates) (reports). These will provide data that support the development of tools and guidance cross-species extrapolation of effects and hazard.||Daniel Villeneuve|
|Sep 30, 2014||(3) Molecular target homology tool: Web-based tool for evaluating cross-species conservation of key molecular targets associated with molecular initiating events and/or key events represented in AOPs as a means for predicting the relative sensitivity or susceptibility of various species to adverse effects associated with exposure to chemicals acting through those AOPs.||Carlie Lalone|
|Sep 30, 2016||4) Mapping alternative assays to AOPs: Annotation of an AOP knowledge-base with information identifying specific QSARs (e.g., those being developed under the Inherency Topic), pathway-based in vitro assays (including high throughput), and medium throughput in vivo endpoints (e.g., with fish embryo assays, biomarkers, metabolite changes) relevant to specific AOPs for reproductive or developmental toxicity.||Daniel Villeneuve|