1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 1212570_28446780.jpg logo_mu_cerne.gif trojlogo.gif Ecotoxicology – part 4 New topics and future issues Ludek Blaha + ecotox colleagues 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Current approaches (black box of apical endoints) vs Future (mechanistic understanding & AOPs) 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif http://3.bp.blogspot.com/-dokK9_P3A4I/UZ38uN8AoUI/AAAAAAAAAJc/d1N8vDjsmAw/s1600/what+is+a+chemical+ reactions.jpg http://upyourtalent.com/wp-content/uploads/2012/04/white-rat.jpg https://encrypted-tbn3.gstatic.com/images?q=tbn:ANd9GcS52E4zw_1IWgj6a-zMCfagRjY0GxrrXUj7RPtc7h6W2mD _trk2 http://www.lfu.bayern.de/analytik_stoffe/biol_analytik_toxizitaetstests/pic/159498_gr.jpg Organism Chemical 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) Výsledek obrázku pro government icon Hazard assessment 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif http://3.bp.blogspot.com/-dokK9_P3A4I/UZ38uN8AoUI/AAAAAAAAAJc/d1N8vDjsmAw/s1600/what+is+a+chemical+ reactions.jpg http://upyourtalent.com/wp-content/uploads/2012/04/white-rat.jpg https://encrypted-tbn3.gstatic.com/images?q=tbn:ANd9GcS52E4zw_1IWgj6a-zMCfagRjY0GxrrXUj7RPtc7h6W2mD _trk2 http://www.lfu.bayern.de/analytik_stoffe/biol_analytik_toxizitaetstests/pic/159498_gr.jpg Organism Chemical Adverse Effects Death Inhibition of Growth Altered Reproduction Tumor Skin irritation … + Traditionally – Evaluation of adverse effects using the whole organism models http://www.tdi.ox.ac.uk/_asset/image/tdi-barcoded-plates.jpeg/fit/300/300/FF0000 http://wallpoper.com/images/00/27/10/25/rainbows-vials_00271025.jpg + http://ehp.niehs.nih.gov/wp-content/uploads/118/4/ehp.0901392.g001.png 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 ? 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 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 https://encrypted-tbn3.gstatic.com/images?q=tbn:ANd9GcTAlr7MmMMxBP1Gffwd_0Gk4X2faiMuSVai0zxTF1WtiV1 DANMq https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcQyQs5sVs1TzKletUOC20iLNg93Liu4X5Q_XF_I6rQWsL4 04Y83 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 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. 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif AOP = Global strategy with support from OECD, EU, USA http://www.oecd.org/chemicalsafety/testing/projects-adverse-outcome-pathways.htm 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 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 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 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 • • https://aopkb.org/aopwiki/images/thumb/c/c9/FiveLogos.gif/780px-FiveLogos.gif 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 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/aops https://aopkb.org/aopwiki/images/thumb/c/c9/FiveLogos.gif/780px-FiveLogos.gif Check online: 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif AOP Example: MIE aromatase inhibition Environmental Toxicology and Chemistry, Vol. 30, No. 1, pp. 64–76, 2011 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Aromatase inhibition leading to reproductive dysfunction (in fish) https://aopwiki.org/wiki/index.php/Aop:25 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif AOP Example from RECETOX: Modulation of RAR/RXR à developmental toxicity in fish Jonáš et al. 2014 Aquatic Toxicology http://dx.doi.org/10.1016/j.aquatox.2014.06.022 Concentration-response curves of the retinoid-like activity (expressed as % of ... Activation of RAR/RXR in P19/A15 cells by atRA and cyanobacterial metabolites Comparison of phenotypes of control and zebrafish embryos exposed to ... 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 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •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 • • Effectopedia 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 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 • • • • • http://epa.gov/research/annualreport/2012/images/toxcast1.jpg 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 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/ – • • 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 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 – Organisation for Economic Co-operation and Development EU%2520Flag 2 https://aopkb.org/aopwiki/images/thumb/c/c9/FiveLogos.gif/780px-FiveLogos.gif 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •3Rs REDUCTION REFINEMENT REPLACEMENT „Alternatives“ to toxicity testing … 3R rules 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •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 Ø3Rs are driven by EU laws, little by Member States. ØScientific agenda is not driven enough by scientists itself… ØAcademia is in general more reactive than proactive e.g. stop vivisection’s ECI 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •European Policies on 3Rs 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Use of animals in EU (2011) 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif • >60 3Rs Tests submitted to ECVAM since 2008 (update 01/2015) • 10 validated or ongoing validation => Prioritisation! 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •VALIDATION •MoA •Reliable •Relevant •Substance Tested • • •e.g. endocrine disrupters receptor binding 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •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 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif •COMPUTATIONAL •(ECO)TOXICOLOGY 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif PBPK models •PBPK (PBTK) •Physiologically based pharmacokinetic (toxicokinetic) models http://upload.wikimedia.org/wikipedia/en/thumb/2/22/WholeBody_wiki.svg/220px-WholeBody_wiki.svg.png https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcS1Xr-zyM_wH8apSCpkALlZltgIBwQ4IcylkdluXxtRsQN mocsqJw •Fragmentation of •a complex systém •to „boxes“ • •à All Processes described by arrows (mathematical equations) 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Example – computational toxicology for EDCs 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Li (2011) BMC Systems Biology •Conceptual model •EE2 – ethinylestradiol •ER, AR atd. – receptors • •VTG – vitellogenin (marker of toxicity) • •Arrows – differential equations 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Li (2011) BMC Systems Biology •Results: • •MODELLED (white) •Vs •MEASURED (grey) • •…good comparable 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Update – quantitative mechanistic/computational toxicology PLoS Comput Biol. 2016 Apr 20;12(4):e1004874. 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Update – quantitative mechanistic/computational toxicology http://journals.plos.org/ploscompbiol/article/figure/image?size=large&id=10.1371/journal.pcbi.10048 74.g001 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. 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif Update – quantitative mechanistic/computational toxicology http://journals.plos.org/ploscompbiol/article/figure/image?size=large&id=10.1371/journal.pcbi.10048 74.g003 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. 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 1212569_21823227.jpg logo_mu_cerne.gif trojlogo.gif 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! • Výsledek obrázku pro sweat