1212569_21823227.jpg logo_mu_cerne.gif 1212570_28446780.jpg logo_mu_cerne.gif Luděk Bláha, PřF MU, RECETOX www.recetox.cz BIOMARKERS AND TOXICITY MECHANISMS 01 - INTRODUCTION OPVK_MU_stred_2 1212569_21823227.jpg logo_mu_cerne.gif Course summary •1) Introduction • - Intro and overview of the mechanisms beyond the toxicity • (with special respect to environmental contaminants) • - Intro and concept of biomarkers • •2) Details on selected important toxicity mechanisms • - Membrane toxicity, enzyme inhibitions, oxidative stress, genotoxicity, Nuclear Receptors (AhR, ER, AR ….) etc. • - Methods to determine toxicity mechanism • •3) Biomarkers • - What it is and how to find (identify) suitable biomarker(s)? • - The overview of the most important biomarker classes • - Methods of biomarker assessment • 1212569_21823227.jpg logo_mu_cerne.gif The importance of understanding to toxicity mechanisms 1212569_21823227.jpg logo_mu_cerne.gif 1962 © Patuxent Wildlife Refuge, MA, USA http://www2.ucsc.edu/scpbrg/ 1212569_21823227.jpg logo_mu_cerne.gif In vivo: shell thinning In situ: bioaccumulation -> bird population decline Bitman et al. Science 1970, 168(3931): 594 Biochemistry discovered in 1970s: Bird carbonate dehydratase 1212569_21823227.jpg logo_mu_cerne.gif Thalidomide http://pubs.acs.org/cen/img/83/i25/8325thalidomide.gif •Originally marketed in 1957 as sedative / hypnotic –also curing anxiety, gastritis, tension –against nausea and morning sickness of pregnant •TERATOGENICITY à Develoment of phocomelia = limb malformations (10 000 children worldwide / 40% survived) • http://blogs.smithsonianmag.com/smartnews/files/2012/09/thalidomide-image1.jpg http://stealthepidemic.com/wp-content/uploads/2014/06/ThalidomideTiming-for-Sophomore.png http://blogs.smithsonianmag.com/smartnews/files/2012/09/thalidomide-image1.jpg •Currently still in use - completely different targets : anticancer (multiple myeloma), antileprosis, immunosupression 1212569_21823227.jpg logo_mu_cerne.gif Thalidomide http://pubs.acs.org/cen/img/83/i25/8325thalidomide.gif http://upload.wikimedia.org/wikipedia/commons/1/11/Chronology-TLP.png 1212569_21823227.jpg logo_mu_cerne.gif Thalidomide ... mechanisms of action http://pubs.acs.org/cen/img/83/i25/8325thalidomide.gif http://www.springerimages.com/img/Images/Springer/JOU=10238/VOL=2007.7/ISU=3/ART=2007_134/MediaObje cts/MEDIUM_10238_2007_Article_134_Fig1.jpg (2) Teratogenicity (3) Anticancer http://yamaguchi.bio.titech.ac.jp/english/images/thalidomide_en.jpg (1) Sedative effects ... mechanism unknown 1212569_21823227.jpg logo_mu_cerne.gif MECHANISMS of chronic toxicity •Various chronic effects have uniform biochemical basis – – – – – –principle studies with mechanistically based in vitro techniques –estimation of in vitro effects of individual compounds HORMONE Biochemical effects TOXIN In vivo effects RECEPTOR Understanding MoA ... may predict higher-level effects Organism Population & beyond 1212569_21823227.jpg logo_mu_cerne.gif Basics and keywords from toxicology 1212569_21823227.jpg logo_mu_cerne.gif Toxicity - concept • Escher, B. I., Behra, R., Eggen, R. I. L., Fent, K. (1997), "Molecular mechanisms in ecotoxicology: an interplay between environmental chemistry and biology", Chimia, 51, 915-921. MoA = mode of action 1212569_21823227.jpg logo_mu_cerne.gif From mechanisms (or modes of action) to biomarkers -Chemical enters organism + may be metabolized/detoxified, transported, released ... -Chemical reacts with target (e.g. DNA) and changes a specific nucleotide (e.g. G à de-oxo-G) - -Elevated de-oxo-G in blood à Toxicokinetics - - à Toxicodynamics = toxicity mechanisms (MoA) and following toxic effects (e.g. mutation, cancer ...) à (Selective) biochemical marker (biomarker) = information about exposure and/or effect 1212569_21823227.jpg logo_mu_cerne.gif Paracelsus (1493 - 1541) Toxicity – the cause-effect paradigm ‘What is there which is not a poison? lAll things are poison and nothing without poison. lSolely the dose determines that a thing is not a poison. l lToxicology – the science of doses 2 1212569_21823227.jpg logo_mu_cerne.gif What processes are beyond toxicokinetics? ADME caption Toxicokinetics ... ... EXPOSURE phase à Determines the final dose 1212569_21823227.jpg logo_mu_cerne.gif E3 Toxicokinetics in fish 1212569_21823227.jpg logo_mu_cerne.gif ToxicoDYNAMICS http://4.bp.blogspot.com/-MebbujGDXi0/UAu26D7WxII/AAAAAAAAACk/StePoxIb3Go/s1600/2.png Dynamic simulation of processes causing toxicity and their grouping into toxicokinetics and toxicodynamics, illustrated on the example of the aquatic invertebrate Gammarus pulex. MoA ... and measurable EFFECTS TARGETS = macromolecules (DNA/RNA, proteins, membrane lipids) 1212569_21823227.jpg logo_mu_cerne.gif What is toxicity? What are the types of effects? •Toxicity –degree to which a substance (at certain dose) can damage an organism • •Exposure & toxicity –acute (immediate, high doses, days) –chronic (sublethal / low doses, long-term) • •Effect & toxicity –lethal (acute) •mortality – definitive endpoint / high doses •easy to determine (single endpoint – death) –nonlethal, sublethal (chronic) •endocrine disruption, reproduction toxicity, immunotoxicity, tumor induction etc. •difficult to determine (multiple endpoints) •more specific – low concentrations / longer exposures •often reflected by specific biochemical changes (biomarkers) • •Systems and organ & toxicity –Systemic lethal toxicity –Organ-specific toxicity (neurotoxicity, hepatotoxicity, nefrotoxicity ...) –Developmental toxicity –Reproduction toxicity – 1212569_21823227.jpg logo_mu_cerne.gif Principles of toxicity testing 1)Define and know biological target (molecule, cell, organism, population) and its properties 2) •2) Define and know chemical and its properties • •3) Define exposure of biological system to a chemical -variable concentrations -defined or variable duration (time) -conditions (T, pH, life stage ....) - •4) Assess effects, i.e. Changes in measurable parameter in relationship to variable doses • •5) Dose-response evaluation & estimation of the toxicity value (i.e. concentration or dose): • LDx, ICx, ECx, LOEC/LOEL, MIC ... • • 1212569_21823227.jpg logo_mu_cerne.gif auto0 AcuteToxScheme_RM HaF-adult arenic~4 Daphnia Dania pict Cu addition Effect concentrations expressed in total/dissolved Cu Effect assessment - procedure 1212569_21823227.jpg logo_mu_cerne.gif How to study (chronic) toxicity ? •In vitro studies (biochemical mechanisms) –+ easy to perform, short-term - ecotoxicological relevancy –+ highly controlled conditions - mostly with vertebrate cells –+ lower amounts of chemicals needed (new cmpnds screening) – •In vivo biotest testing –+ unique whole organisms - only few (ecologically –+ controlled conditions nonrelevant) organisms used –+ better ecological interpretation - mostly ACUTE assays – - chronic: long exposures • •Field and in situ observations, epidemiological studies 1212569_21823227.jpg logo_mu_cerne.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 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 1212569_21823227.jpg logo_mu_cerne.gif Mode of Action (omics) toxicity testing http://www.omics.jp/about/img/omics_01.jpg And other „omes“ •Lipidome •Microbiome … 1212569_21823227.jpg logo_mu_cerne.gif http://systemsbiology.usm.edu/SystemsBiologyCenter/EL_ERDC/ERDC_img/Earthworm-Toxicogenomics.jpg Omes is not only for humans … 1212569_21823227.jpg logo_mu_cerne.gif MoA and omics are supported by strategic documents & organizations 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 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 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 Concept of “Adverse Outcome Pathway” (AOP) http://www.ufz.de/export/data/1/60568_Bionalytical%20Tools.jpg Mechanisms or modes of action à à Effects 1212569_21823227.jpg logo_mu_cerne.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 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 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 What AOPs are now in AOP Wiki – Feb 2020 ? OECD Endorsed (WNT and TFHA) 14 •Alkylation of DNA in male pre-meiotic germ cells leading to heritable mutations •Aromatase inhibition leading to reproductive dysfunction EAGMST Under Review & for comments 20 EAGMST Under Development 34 SAAOP AOP Under Development 130+ •Total 269 AOPs •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 1212569_21823227.jpg logo_mu_cerne.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 Aromatase inhibition leading to reproductive dysfunction (in fish) https://aopwiki.org/wiki/index.php/Aop:25 1212569_21823227.jpg logo_mu_cerne.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 Kidd, K.A. et al. 2007. Collapse of a fish population following exposure to a synthetic estrogen. Proceedings of the National Academy of Sciences 104(21):8897-8901 Controls +Ethinylestradiol 5 ng/L (!) 7 years http://upload.wikimedia.org/wikipedia/commons/8/8d/Ethinylestradiol-2D-skeletal.png 1212569_21823227.jpg logo_mu_cerne.gif Keywords to remember and understand •What is meant by the “mechanism of action” (or “mode of action”) in toxicology? •Why is it necessary to understand MoAs? What is the AOP concept? •What is toxicokinetics? What is ADME? •What is toxicodynamics? •What is the relationship between the exposure and the effect? •What are the different types of toxicity? •How can the (toxic) effect be measured / assessed? •What types of “bioassays” are available to study toxicity and/or MoA? •How is the result (i.e. „toxicity“) described in numbers? •