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 - AhR & "dioxin-like" toxicity (Vondráček) - ER & xenoestrogenicity (Sovadinová) - Other nuclear receptors & toxicity (Janošek+Bláha) 3) Biomarkers - In vitro and in vivo biomarkers / assays - Applications in environmental studies Toxicity - concept - Toxicokinetics & Toxicodynamics - Evaluation of toxicity (design) - Expression of toxicity (ICx, exposure time ...) - Acute vs. chronic toxicity vs. mechanisms - Mechanisms of toxicity: concept - cellular & biochemical events -> general "species-independent" in vivo effects Toxicokinetics - Processes involved in the fate of toxicant after entering the organism: : adsorbtion / membrane transport : transport in body fluids : distribution in body (fat / specific organs) : transformation (liver / kidney ...) : elimination (urine / bile / sweat) Toxicodynamics - Interaction of toxicant with biological molecules : membrane phospholipids, DNA, proteins ... : covalent / non-covalent binding : specific domains in proteins, DNA ... / general reactivity What affects the specificity and affinity of interaction ? ~ toxicokinetics - concentration of both xenobiotic / biol. molecule ~ affinity - structure, physico-chemical parameters Toxicodynamics Characterization of specifity & affinity: homeostatic constants / coefficents (Ki; Kd): Xen + Biol -> XenBiol (v1) XenBiol -> Xen + Biol (v2) K ~ v1 / v2 ~ often expressed as concentrations (e.g. IC[50]) As lower is ICx as stronger is the binding to specific receptor and related toxic effect Toxicity assessment 1) Biological target (molecule, cell, organism, population) 2) Chemical definition 3) Exposure of biological system to chemical - variable concentrations - defined or variable duration (time) - conditions (T, pH, life stage ....) 4) Effect assessment - changes in relationship to concentrations 5) Dose-response evaluation & estimation of toxicity value (! concentration): LDx, ICx, ECx, LOEC/LOEL, MIC ... Toxicity ? Exposure & toxicity - acute / chronic (exposure) Effect & toxicity - lethal (acute) : mortality – definitive endpoint : high concentrations : easy to determine (single endpoint – death) - nonlethal (chronic) : animal doesn´t die - "less dangerous" (?) (endocrine disruption, reproduction toxicity, immunotoxicity, cancerogenesis) : difficult to determine (multiple endpoints) : more specific – low concentrations / longer exposures : reflected by specific biochemical changes (biomarkers) Mechanisms of toxicity - overview - What is the "toxicity mechanism" - interaction of xenobiotic with biological molecule - induction of specific biochemical events - in vivo effect - Biochemical events induce in vivo effects (mechanisms) - Changes of in vivo biochemistry reflect the exposure and possible effects (biomarkers) Factors affecting the toxicity Xenobiotic - physico-chemical characteristics - solubility / lipophilicity - reactivity and redox-characteristics - known structural features related to toxicity (organophosphates) - structurally related molecules act similar way - bioavailability & distribution (toxicokinetics) Biological targets (receptors) - availability (species- / tissue- / stage- specific effects) - natural variability (individual susceptibility) Concentration of both Xenobiotic and Receptor Mechanisms of toxicity - specificity - Tissue-specific mechanisms - hepatotoxicity; neurotoxicity; nefrotoxicity; haematotoxicity - toxicity to reproduction organs; - embryotoxicity, teratogenicity, immunotoxicity - Species-specific mechanisms - photosynthetic toxicity vs. teratogenicity - endocrine disruption – invertebrates vs. vertebrates - Developmental stage-specific mechanisms - embryotoxicity: toxicity to cell differenciation processes BIOMARKERS Biomarkers - markers in biological systems with a sufficently long half-life which allow location where in the biological system change occur and to quantify the change. Applications in medicine: Hippocrates – urine colour ~ health status Toxicology – present status: - identification of markers of long-term risks : humans – carcinogenesis : ecotoxicology – early markers of toxic effects Cellular toxicity mechanisms - overview 1 Membrane nonspecific toxicity (narcosis) 2 Inhibition of enzymatic activities 3 Toxicity to signal transduction 4 Oxidative stress – redox toxicity 5 Toxicity to membrane gradients 6 Ligand competition – receptor mediated toxicity 7 Mitotic poisons & microtubule toxicity 9 DNA toxicity (genotoxicity) 10 Defence processes as toxicity mechanisms and biomarkers - detoxification and stress protein induction NARCOSIS / nonspecific toxicity - All organic compounds are narcotic in particular ("high") concentrations - Compounds are considered to affect membranes; nonspecific disruption of fluidity and protein function - Related to lipophilicity (logP, Kow): tendency of compounds to accumulate in body lipids (incl. membranes) Narcotic toxicity to fish: log (1/LC50) = 0.907 . log Kow - 4.94 - The toxic effects occur at the same "molar volume" of all narcotic compounds (volume of distribution principle) Volume of distribution Enzyme inhibition - toxicity mechanism - Millions of enzymes (vs. millions of compounds) : body fluids, membranes, cytoplasm, organels - Compound - an enzyme inhibitor ? - Enzymology: interaction of xenobiotics with enzymes - Competitive vs. non-competitive: active site vs. side domains - Specific affinity – inhibition (effective) concentration - What enzymes are known to be selectively affected ? Enzyme inhibition - toxicity mechanism Enzyme inhibition - examples Acetylcholinesterase (organophosphate pesticides) Microsomal Ca^2+-ATPase (DDE) Inhibition of hemes – respiratory chains (cyanides) d-Aminolevulinic Acid Dehydratase (ALAD) inhibition (lead - Pb) Inhibition of proteinphosphatases (microcystins) Non-competitive inhibition – changes in terciary structure (metals: toxicity to S-S bonds) Acetylcholinesterase inhibition by organophosphate pesticides Inhibition of Ca^2+-ATPase by DDE Ca2+: general regulatory molecule contractility of muscles calcium metabolism in bird eggs stored in ER (endo-/sarcoplasmatic reticulum) concentrations regulated by Ca^2+-ATPase Inhibition of hemes by cyanide oxidations in respiratory chains; Hemoglobin ALAD inhibition by lead (Pb) PPase inhibitions by microcystins Microcystins – produced in eutrophied waters by cyanobacteria; kg – tons / reservoir