Biomarkers and mechanisms of toxicity Course summary –1) Introduction – - Overview of toxicity mechanisms – (with special respect to environmental contaminants) – - Concept of biomarkers - overview – –2) Details on selected important toxicity mechanisms – - Membrane toxicity, enzyme inhibitions, Oxidative stress, Genotoxicity, Detoxification, Nuclear Receptors (AhR, ER, AR ….), Neurotoxins – –3) Biomarkers – - In vitro and in vivo biomarkers / assays – - Applications in environmental studies The importance of understanding mechanisms DDT story 1962 © Patuxent Wildlife Refuge, MA, USA http://www2.ucsc.edu/scpbrg/ Možné negativní důsledky chemického znečištění pro organismy poprvé přinesla do širšího povědomí kniha S.S. 1962 - spojila zřejmý úbytek populací ptáků („charismatické druhy“) s používáním DDT - pesticid proti komárům (a přenášeným chorobám) - i přes celosvětový zákaz (tzv. Stockholmská úmluva OSN - obdoba Kjotského protokolu) je jeho výroba velmi levná: užití dodnes v Africe, Indii … od 40.let považován za zcela neškodný a zázračný („netoxický“) …. Bitman et al. Science 1970, 168(3931): 594 Biochemistry bird carbonate dehydratase In vivo: shell thining In situ: bioaccumulation -> bird population decline Možný mechanismus byl navržen až po řadě let - inhibice ptačí karbonátdehydratázy v žláze tvořící skořápku vajíčka (BTW - enzym, který v našich krvinkách zajišťuje přeměnu CO2 na HCO3z tkání - in vivo -> in situ Proč tento obrázek? - 1) až za 30 let od uvedení na trh se poznalo co vlastně DDT dělá …, 2) ukázka jak se malá změna na biochemické úrovni promítne až na úroveň ekosystémů … 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. Mechanisms vs. 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 -Toxicokinetics - - -Toxicodynamics + toxicity mechanism - -(Selective) biochemical marker = information about exposure and/or effect) Biomarkers lChanges in biological systems l … induced by various stressors, stimuli etc., l … with a sufficently long half-life (so they can be measured), l … which allow location where in the biological system change occur and to quantify the change. l lExamples: lproducts of metabolism/toxicity reaction (small MW molecules) lchanges in enzymatic activities lnew presence (or absence) of certain proteins lstructural (histopathological) changes l lApplications in medicine: Hippocrates – urine colour ~ health status lBiomarkers in present - identification (and prediction) of long-term risks : humans – carcinogenesis : ecotoxicology – early markers of toxic effects l l l Introduction to general toxicology l- Toxicokinetics l- Toxicodynamics l l- Toxicity = effects l l- Toxicity testing l l Cause – effect paradigm: nothing new…. ‘What is there which is not a poison? lAll things are poison and nothing without poison. l lSolely the dose determines that a thing is not a poison. l 2 Paracelsus (1493 - 1541) Toxicokinetics l- Processes involved in the fate of toxicant after entering the organism: l l : adsorbtion / membrane transport l : transport in body fluids : distribution in body (fat / specific organs) l : transformation (liver / kidney ...) l & elimination (urine / bile / sweat) l E3 Toxicokinetics Toxicokinetics - membrane - proteins Toxicokinetics - membrane transport - Toxicodynamics lCharacterization of specifity & affinity: l homeostatic constants / coefficents (Ki; Kd): l Xen + Biol -> XenBiol (v1) l XenBiol -> Xen + Biol (v2) l l K ~ v1 / v2 ~ often expressed as concentrations (e.g. IC50) l lAs lower is ICx as stronger is the binding to specific receptor and related toxic effect Toxicodynamics one compound - more targets Targets (=receptors in toxicodynamics) ANY BIOMOLECULE Toxicity ? lExposure & toxicity l - acute / chronic (exposure) l lEffect & toxicity l - lethal (acute) l : mortality – definitive endpoint : high concentrations l : easy to determine (single endpoint – death) l l - nonlethal (chronic) : organisms do not die - "less dangerous" (?) (endocrine disruption, reproduction toxicity, immunotoxicity, cancerogenesis) l : difficult to determine (multiple endpoints) l : more specific – low concentrations / longer exposures : reflected by specific biochemical changes (biomarkers) Chronic toxicity lChronic toxicity is difficult to study and predict – –time and cost consuming experiments – –limited number of species (laboratory vs. natural species) – –effect = combination of chemical exposure and life style, habits ... – –metabolites or derivatives (not parent compounds) are often the active substances – MECHANISMS of chronic toxicity of POPs lVarious chronic effects have uniform biochemical basis – –principle studies with mechanistically based in vitro techniques – – – – – –estimation of in vitro effects of individual compounds lunderstanding the mechanisms, prediction of hazard – –application for risk assessment or monitoring lderivation of relative potencies ("toxic equivalents") -> RA lin vitro biomarkers - direct characterization of complex samples HORMONE Biochemical effects TOXIN In vivo effects RECEPTOR Estrogen receptor activation 1)female reproduction disorders 2) 2)male feminisation 3) 3)tumor promotion 4) 4)immunomodulations 5) 5)developmental toxicity SINGLE mechanism -> SEVERAL effects => understanding to mechanisms may predict effects Understanding mechanisms ... ... explains the effects at higher levels Organism Population & beyond 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 Toxicity assessment l1) Biological target (molecule, cell, organism, population) l2) Chemical definition l3) Exposure of biological system to chemical l - variable concentrations l - defined or variable duration (time) l - conditions (T, pH, life stage ....) l4) Effect assessment l - changes in relationship to concentrations l5) Dose-response evaluation & estimation of toxicity value (! concentration): LDx, ICx, ECx, LOEC/LOEL, MIC ... l How to study (chronic) toxicity ? lIn 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) – lIn vivo biotest testing –+ unique whole organisms - only few (ecologically –+ controlled conditions nonrelevant) organisms used –+ better ecological interpretation - mostly ACUTE assays – - chronic: long exposures l lField and in situ observations, epidemiological studies Effect assessment - procedure auto0 AcuteToxScheme_RM HaF-adult arenic~4 Daphnia Dania pict Cu addition Effect concentrations expressed in total/dissolved Cu Extrapolation = PNECs or EQCs expressed in total / dissolved Cu Effect assessment - results 50 100 LC50 [concentration] in mg/L or % effluent Threshold: No Observed Effect Concentration (NOEC)