Mechanisms of toxicity - overview l- What is the "toxicity mechanism" - interaction of xenobiotic with biological molecule l - induction of specific biochemical events l - in vivo effect l l- Biochemical events induce in vivo effects (mechanisms) l- Changes of in vivo biochemistry reflect the exposure and possible effects (biomarkers) Factors affecting the toxicity lXenobiotic - physico-chemical characteristics – - solubility / lipophilicity - reactivity and redox-characteristics - known structural features related to toxicity (organophosphates) – - structurally related molecules act similar way l - bioavailability & distribution (toxicokinetics) l lBiological targets (receptors) –- availability (species- / tissue- / stage- specific effects) –- natural variability (individual susceptibility) l lConcentration of both Xenobiotic and Receptor Mechanisms of toxicity - specificity l- Tissue-specific mechanisms (& efffects) l - hepatotoxicity; neurotoxicity; nefrotoxicity; haematotoxicity l - toxicity to reproduction organs; l - embryotoxicity, teratogenicity, immunotoxicity l l- Species-specific mechanisms l - photosynthetic toxicity vs. teratogenicity l - endocrine disruption – invertebrates vs. vertebrates l l- Developmental stage-specific mechanisms l - embryotoxicity: toxicity to cell differenciation processes Cellular toxicity mechanisms - overview lMembrane nonspecific toxicity (narcosis) lInhibition of enzymatic activities lToxicity to signal transduction lOxidative stress – redox toxicity lToxicity to membrane gradients lLigand competition – receptor mediated toxicity lMitotic poisons & microtubule toxicity lDNA toxicity (genotoxicity) lDefence processes as toxicity mechanisms and biomarkers - detoxification and stress protein induction Two principal „types“ of toxic action Non-specific toxicity - nonpolar (narcotic) toxicity / basal toxicity - polar narcosis - reactive toxicity Specific toxicity - enzyme inhibition, interaction with specific receptor… Toxicity mechanisms in general 1) All ORGANIC compounds affect membrane phospholipids (organic/lipids attract organics) = nonpolar narcotic toxicity (membrane toxicity) (effects at relatively high concentrations, depends on Kow) 2) Besides the nonpolar narcosis, more polar compounds may affect also „nonspecifically“ affect membrane proteins (polar narcosis) (effects at lower concentrations than expected from Kow, molecular mechanisms not fully clear) 3) Further, some compounds with reactive properties may directly - and nonspecifically (nonselectively) - react and modify any biological macromolecule (lipids, proteins, nucleic acids) (effects at even lower concentrations than 1+2; reactive chemicals are mostly „electrophiles“ reacting with „nucleophiles“ in cells – i.e. electrone-rich sites (nucleotides, -NH2, -SH and others) 4) Only certain specific compounds selectively affect specific targets causing „specific“ toxicity (enzyme inhibitions – e.g. drugs, insecticides; receptor interactions – e.g. estrogens; effects at very low concentrations) 1-3 = nonspecific (large groups of chemicals, no specific target – reacts with „all“ biomolecules) Vs. 4 = specific toxicity - General concept – toxicity mechanisms Membrane and membrane toxicity Cell membrane lMany key functions for life -Primary barrier / separation of „living“ inside from „abiotic“ outside -Semipermeability for nutrients / signals -Reception of chemical signals & regulatory molecules -Keeping gradients necessary for life -H+ - ATP synthesis(mitochondria / bacterial emambrane) -K+/Na+ - neuronal signals -Proteosynthesis (ribosomes) depends on membranes -Many other enzymes bound to membranes (e.g. signaling, detoxification, post-translational modifications) -Etc…. Note: cholesterol – strucutral/size similartity to toxic organics e.g. PAHs NARCOSIS / nonspecific toxicity l- All organic compounds are narcotic in particular ("high") concentrations l l- Compounds are considered to affect membranes; nonspecific disruption of fluidity and protein function l l- 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 l l- The toxic effects occur at the same "molar volume" of all narcotic compounds (volume of distribution principle) l Volume of distribution principle 001 001 Acute basal toxicity Direct correlation logP vs EC50 at aquatic organisms (Daphnia, fish) Narcotic toxicity in ecotoxicology Example: Neutral organics à Nonpolar narcosis à Amines, phenols à Polar narcosis (similar logP à higher toxicity, i.e. higher Values of 1/EC50 in comparison to neutral organics) à Enzyme inhibition as a toxicity mechanism Enzyme inhibition - toxicity mechanism l- Millions of enzymes (vs. millions of compounds) l : body fluids, membranes, cytoplasm, organels l l- Compound - an enzyme inhibitor ? l - Enzymology: interaction of xenobiotics with enzymes l - Competitive vs. non-competitive: l active site vs. side domains l - Specific affinity – inhibition (effective) concentration l l- What enzymes are known to be selectively affected ? l l- Nonspecific inhibitions (!) l Compound affects high osmomolarity or pH … Enzyme inhibition - toxicity mechanism 1 Enzyme inhibition - toxicity mechanism 1 Enzyme inhibition – few examples lAcetylcholinesterase (organophosphate pesticides) l lMicrosomal Ca2+-ATPase (DDE) l lInhibition of hemes – respiratory chains (cyanides) l ld-Aminolevulinic Acid Dehydratase (ALAD) inhibition (lead - Pb) l lInhibition of proteinphosphatases (microcystins) l lGlyphosate (roundup) action l l(Enzyme inhibitions are beyond many others à see e.g. REGULATIONS etc.) l Acetylcholinesterase inhibition by organophosphate pesticides 1 E3 Inhibition of Ca2+-ATPase by DDE lCa2+: lgeneral regulatory molecule lcontractility of muscles lcalcium metabolism in bird eggs lstored in ER (endo-/sarcoplasmatic reticulum) lconcentrations regulated by Ca2+-ATPase l Inhibition of hemes by cyanide oxidations in respiratory chains; Hemoglobin (also CYP450) 1 1 ALAD inhibition by lead (Pb) 1864 PPase inhibitions by microcystins (liver !) 1 1b lMicrocystins – produced in eutrophied waters by cyanobacteria; kg – tons / reservoir Glyphosate lN-(phosphonomethyl)glycine lBroad-spectrum herbicide („RoundUp“) lSelective inhibition of ESPs 5-enolpyruvylshikimate-3-phosphate synthase; l(synthesis of aromatic aa – Tyr, Trp, Phe) lUptake via leafs - only to growing plants l„Non-toxic“ to other organisms l(no ESPs in animals, aa-like chemical - rapid degradation)