Ecotoxicology – part 4 New topics and future issues Ludek Blaha + ecotox colleagues Current approaches (black box of apical endoints) vs Future (mechanistic understanding & AOPs) OrganismChemical Adverse Effects Death Altered Reproduction Inhibition of Growth Tumorigenicity Skin irritation … + Traditionally – Evaluation of adverse effects using the whole organism models REGULATORY FOCUS (APICAL ENDPOINTS) Hazard assessment OrganismChemical Adverse Effects Death Inhibition of Growth Altered Reproduction Tumor Skin irritation … + Traditionally – Evaluation of adverse effects using the whole organism models +10^4 Chemicals HTS High-Throughput-Screening Chemical-biological interactions, Mechanistic Toxicological Data Hazard assessment New – Ex vivo / in vitro / In chemico / In silico Methods Key task/question: How to link MECHANISTIC INFORMATION with APICAL ENDPOINTS ? MoA and omics are supported by strategic documents Toxicity Testing in the 21st Century: A Vision and a Strategy US National Academies of Sciences http://www.nap.edu/catalog/11970.html Adverse Outcome Pathways The EXISTING KNOWLEDGE is used to link the two anchor points: Molecular Initiating Event (MIE) and Adverse Outcome (AO) via a series of intermediate steps: Key Events Chemical Macro- Molecular Interaction Cellular Response Organ Response Organism Response Population Response Molecular Initiating Event Key Event 1 Key Event 2 Adverse Outcome Tissue Effect Key Event 3 Chemical Property Receptor/Ligand Interactions DNA Binding Protein Oxidation Gene Activation Protein Production Altered Signaling Altered Physiology Disrupted Homeostasis Altered Development / Function Lethality Impaired Development Impaired Reproduction Altered Sex Ratio Extinction Adverse Outcome Pathway Toxicity Pathway Mode of Action In silico, In chemico, In Vitro, Ex vivo In vivo Ankley, G. T., R. S. Bennett, et al. (2010). "Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment." Environmental Toxicology and Chemistry 29(3): 730-741. AOP = Global strategy with support from OECD, EU, USA http://www.oecd.org/chemicalsafety/testing/projects-adverse-outcome-pathways.htm http://aopkb.org/ Key documents OECD Guidance document and a template for developing and assessing adverse outcome pathways (Series No. 184, Series on Testing and Assessment) Handbook for AOP developers AOP Wiki • https://aopkb.org/aopwiki/index.php/Main_Page • Wiki-based platform for development of AOPs • Only members of an OECD AOP development project can create / edit AOPs What AOPs are now in AOP Wiki (autumn 2017?) OECD Endorsed (WNT and TFHA) 6 1x ecotoxicology: Aromatase inhibition leading to reproductive dysfunction (in fish) EAGMST Approved 2 1x Ecotox - Androgen receptor agonism leading to reproductive dysfunction EAGMST Under Review + open for comment 17 (9+8) Under Development 131 • OECD Extended Advisory Group on Molecular Screening and Toxicogenomics (EAG MST) • The Working Group of the National Coordinators of the Test Guidelines Programme (WNT) https://aopwiki.org/aopsCheck online: AOP Example: MIE aromatase inhibition Environmental Toxicology and Chemistry, Vol. 30, No. 1, pp. 64–76, 2011 Aromatase inhibition leading to reproductive dysfunction (in fish) https://aopwiki.org/wiki/index.php/Aop:25 AOP Example from RECETOX: Modulation of RAR/RXR  developmental toxicity in fish Jonáš et al. 2014 Aquatic Toxicology http://dx.doi.org/10.1016/j.aquatox.2014.06.022 Activation of RAR/RXR in P19/A15 cells by atRA and cyanobacterial metabolites ZF exposed to ATRA and cyanobacterial (120 hpf) - Control (A), exudates of C. raciborskii 3.3× (B) and 10× (C), M. aeruginosa 10× (D) and D. quadricaudatus 17× (E). ATRA 4 μg/L (13.3 nM) (F), 12 μg/L (40 nM) ((G) and (H)), 36 μg/L (I) and 108 μg/L (J). atRA other RAs in cyanos • http://www.effectopedia.org/ -> link to program download • Visually Expresses AOPs in their biological context: – Life-stage, Taxonomy, Gender, Time-to-effect.. • Quantitative Relationships • ADME (Absorption, Distribution, Metabolism, Excretion) • Open-knowledge, crowd-sourcing • Formal approval not required to enter / modify • Credit to authors / reviewers • Even fragments of information are welcome (any contribution) • Export<->Import from/to AOP Wiki & others Related Projects & Studies & Databases • TOXNET - http://toxnet.nlm.nih.gov/ – searching databases on toxicology, hazardous chemicals, environmental health, and toxic releases • Tox21 - http://www.epa.gov/ncct/Tox21/ – 10,000 chemicals – 14 concentrations, 4 logs, 3 replicates – 1536 well plates, 2-8 uL volumes – 50+ assays • ToxCast - http://www.epa.gov/ncct/toxcast/ – App. 2000 chemicals – 700+ assay, 300 signaling pathways – DATA AVAILABLE iCSS Dashboard • http://actor.epa.gov/dashboard • http://ww.epa.gov/ncct/toxcast/data.html Related Projects & Studies & Databases • ToxRefDB (Toxicity Reference Database) – in vivo toxicological data – http://actor.epa.gov/toxrefdb/faces/Home.jsp • ExpoCast – information on human exposures – http://www.epa.gov/ncct/expocast/ • Human Toxome Project – information on human exposures – http://www.ewg.org/sites/humantoxome/ • Agriculture Health Study – Occupational Exposure to Pesticides – a cohort study – http://aghealth.nih.gov/ Summary • Toxicology is about doses – The goal is LD(LC)50 or NOAEL/NOEC • Legislation defines … what assays and how to do them – About 30 assays – The most widely used standard - OECD Guidelines for Testing of Chemicals • Replacing „black box“ in traditional testing – Synthesis of mechanistic and omics data – Adverse Outcome Pathways – Strategically supported by OECD, EU, USA Do we need testing with animals? Are there alternatives 3Rs REDUCTION REFINEMENTREPLACEMENT „Alternatives“ to toxicity testing … 3R rules Why doing replacement, reduction, refinement? •Because activists put pressure to do so? •Because animal welfare is a concern for EU citizen? •Because animal testing is “bad” and “alternatives” are good? •Because I will get “better” results? •Because it is cutting edge technologies? •Because I have to? E.g. EU law directive 2010/63/eu, ban on animal testing for cosmetics 3Rs are driven by EU laws, little by Member States. Scientific agenda is not driven enough by scientists itself… Academia is in general more reactive than proactive e.g. stop vivisection’s ECI European Policies on 3Rs Use of animals in EU (2011) • >60 3Rs Tests submitted to ECVAM since 2008 (update 01/2015) • 10 validated or ongoing validation => Prioritisation! •VALIDATION •MoA •Reliable •Relevant Substance Tested e.g. endocrine disrupters receptor binding Validation Prevalidation Development Research Independent Review Implementation Regulatory Acceptance Alternative Methods – R&D to Implementation ESAC Industry Regulators Academia PARERE ESTAF Demanding process: 7-8 YEARS COMPUTATIONAL (ECO)TOXICOLOGY PBPK models PBPK (PBTK) Physiologically based pharmacokinetic (toxicokinetic) models Fragmentation of a complex systém to „boxes“  All Processes described by arrows (mathematical equations) Example – computational toxicology for EDCs Li (2011) BMC Systems Biology Conceptual model EE2 – ethinylestradiol ER, AR atd. – receptors VTG – vitellogenin (marker of toxicity) Arrows – differential equations Li (2011) BMC Systems Biology Results: MODELLED (white) Vs MEASURED (grey) …good comparable Update – quantitative mechanistic/computational toxicology PLoS Comput Biol. 2016 Apr 20;12(4):e1004874. Update – quantitative mechanistic/computational toxicology Fig 1. The HPOL signaling network in rainbow trout as formulated in our model. Arrows and symbols on graph follow CellDesigner vs. 4.4 notation (www.celldesigner.org). GnRH is secreted from the hypothalamus into the pituitary stimulating the production of mFSH and mLH, which then leads to formation of FSH and LH, respectively. FSH, which is being continuously secreted from the pituitary, travels to the ovaries to stimulate production of E2. E2 then travels to the liver to bind with E2 receptors (R; translated from mR) to form ER. ER then stimulates the production of mVTG, which produces VTGL. Secreted VTG then travels from the liver to the ovaries via the plasma (VTGP) where it is absorbed by follicles in stages 3 through 6 (the proportion of follicles in these stages are denoted by Sj, j = 3, 4, 5, and 6) during vitellogenesis, the rate of which is affected by FSHP, to promote oocyte growth (OAvg). Oocyte growth then progresses the oocytes through the stages using a Weibull distribution created from OAvg together with OVar. In the later stages LHP stimulates the oocytes to produce DHP. Finally, oocytes undergo final maturation (SFOM) and combined with DHP, determine when the fish ovulates PLoS Comput Biol. 2016 Apr 20;12(4):e1004874. Update – quantitative mechanistic/computational toxicology Fig 3. HPOL model predictions for (A) pituitary levels of FSHβ subunit mRNA, (B) pituitary levels of LHβ subunit mRNA, (C) Hepatic levels of E2 receptor mRNA and (D) Hepatic levels of VTG mRNA Observed data (dark grey circles; mean ±TG mRn = 3) PLoS Comput Biol. 2016 Apr 20;12(4):e1004874. Closing remarks • Ecotoxicology is exciting science! • Interface: science and society • Many opportunities • Sometimes hard work 10% inspiration and 90% „perspiration“ • Be creative: move frontiers • Keep the purpose in mind • Be critical: do not accept perceptions as facts • Speak up: you have something to say!