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 04 – Mechanisms @membranes OPVK_MU_stred_2 1212569_21823227.jpg logo_mu_cerne.gif Major mechanisms (modes of action) to be discussed in detail •Proteins and inhibition of enzymatic activities •Mitotic poisons & microtubule toxicity • •Membrane nonspecific toxicity (narcosis) •Toxicity to membrane gradients • •DNA toxicity (genotoxicity) • •Complex mechanisms –Detoxificiation •defence processes as toxicity mechanisms –Oxidative stress – redox toxicity –Toxicity to signal transduction –Ligand competition – receptor mediated toxicity – – – 1212569_21823227.jpg logo_mu_cerne.gif Cell membrane •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…. 1212569_21823227.jpg logo_mu_cerne.gif Note: cholesterol – structural/size similarity to toxic organics e.g. Benzo[a]pyrene Benzo[a]pyren 1212569_21823227.jpg logo_mu_cerne.gif Nonspecific (basal, narcotic) toxicity •- All organic compounds tend to accumulate in membranes, being “narcotic” at relatively "high“ concentrations • •- Compounds then affect membranes à nonspecific disruption of fluidity à and/or disruption of membrane proteins • •- Related to lipophilicity (Kow): tendency of compounds to accumulate in body lipids (incl. membranes) E.g. 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) • 1212569_21823227.jpg logo_mu_cerne.gif Volume of distribution principle 001 BCF – bioconcentration factor * Depends on hydrophobicity (i.e. Kow) * Higher BCF à lower concentration is sufficient for bioconcentration to the same “tissue concentration” à lower external concentration (IC50) will induce toxic effect * Confirmed by chemical analyses (same molar concentrations of different compounds accumulated in membranes) 1212569_21823227.jpg logo_mu_cerne.gif Acute basal toxicity Direct correlations between logKow (=logP) and EC50 for aquatic organisms (e.g. Daphnia magna) Narcotic toxicity in ecotoxicology Example: Neutral organics à Nonpolar narcosis Amines, phenols à Polar narcosis (similar logP à higher toxicity, i.e. higher Values of 1/EC50 in comparison to neutral organics) à More specific ... In addition to membrane accumulation, direct interactions with proteins are anticipated 1212569_21823227.jpg logo_mu_cerne.gif Disruption of membrane gradients 1212569_21823227.jpg logo_mu_cerne.gif Toxicity to membrane gradients and transport -Semipermeability is essential for membranes key functions • - cytoplasmic membrane: signalling, neural cells Na+/K+ gradient - mitochondrial membrane: electrone flow à ATP synthesis - endoplasmatic reticulum Ca2+ signalling • -Disruptions can be either through nonspecific narcotic toxicity (above) or via specific effects of toxicants à discussion further • • 1212569_21823227.jpg logo_mu_cerne.gif Direct membrane gradient disruption •Ion transfer ("ionofores") e.g. antibiotics (K+, Ca2+, Mg2+) • • • • • • • C:\Documents and Settings\Ludek Blaha\Obrázky\1.gif C:\Documents and Settings\Ludek Blaha\Obrázky\1.jpg C:\Documents and Settings\Ludek Blaha\Obrázky\1.gif 1212569_21823227.jpg logo_mu_cerne.gif Mitochondrial membrane 1212569_21823227.jpg logo_mu_cerne.gif http://www.qub.ac.uk/schools/SchoolofBiologicalSciences/People/DrAGalkin/Research/Image1,172487,en. jpg Mitochondria (= energy metabolism!) -- membrane processes: H+ formation -- 1212569_21823227.jpg logo_mu_cerne.gif Role of membrane: gradient of H+ à ATP generation & its disruption http://classconnection.s3.amazonaws.com/64/flashcards/266064/png/poison1355459598482.png 1212569_21823227.jpg logo_mu_cerne.gif Gradient of H+ à ATP generation & its disruption Rotenone Oligomycin CO (carbon monoxide) CN (cyanide) à Binding to haem structures 1212569_21823227.jpg logo_mu_cerne.gif Receptors/Channels & membrane gradients 1212569_21823227.jpg logo_mu_cerne.gif C:\Documents and Settings\Ludek Blaha\Obrázky\1.jpg C:\Documents and Settings\Ludek Blaha\Obrázky\2.jpg Principal types of channel activation 1212569_21823227.jpg logo_mu_cerne.gif Various membrane channels - examples 1212569_21823227.jpg logo_mu_cerne.gif Activation of AcChol receptors à Disruption of membrane gradients http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-13/13_20.jpg http://philschatz.com/anatomy-book/resources/1216_Ligand-gated_Channels.jpg Reminder: AcCholEsterase & inhibition by OPs is another related toxicity mechanism 1212569_21823227.jpg logo_mu_cerne.gif Activation / inhibition of ligand-gated channels https://courses.washington.edu/chat543/cvans/images/sfp/large_format/achrecs.gif Agonist vs Antagonist Concentration-dependent action 1212569_21823227.jpg logo_mu_cerne.gif EXAMPLE: related biological effects http://www.atsdr.cdc.gov/csem/cholinesterase/images/nicotinic_muscarinic.png 1212569_21823227.jpg logo_mu_cerne.gif Environmentally relevant toxins - ion channel activators •Neurotoxins (cyanobacterial) • • • • • Toxins 02 02359 g005 1024 https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcQHRpAgIeIPwnv_Z0rdf0lvw1uSuGlyI5UAd8_akQHeZy4 mLGrf http://microbiology.science.oregonstate.edu/files/micro/theo%20photo%202.jpg 1212569_21823227.jpg logo_mu_cerne.gif Environmentally relevant toxins - ion channel activators SAXITOXINS •Produced by dinoflagelates and cyanobacteria •(toxic blooms, „red tides“) Výsledek obrázku pro saxitoxin 1212569_21823227.jpg logo_mu_cerne.gif Roles of membranes in the release of neurotransmitters 1212569_21823227.jpg logo_mu_cerne.gif Botulinum and Tetanus toxins (Clostridium botulinum, Clostridium tetani) C:\Documents and Settings\Ludek Blaha\Obrázky\1.gif ((Complex MoA - mediated through both proteins and membranes)) Toxins = enzymes - proteases (!) - direct cleavage of proteins involved in vesicle formation - selective inhibition of neutrotransmitter release BOTULINISM à neurotoxicity (paralysis) http://ssrl.slac.stanford.edu/research/highlights_archive/bont-stevens_fig1.jpg 1212569_21823227.jpg logo_mu_cerne.gif TETANUS TOXIN (tetanospasmin) blocks release of INHIBITORY NEUROTRANSMITERS (γ-aminobutyric acid (GABA) in CNS à neurotoxicity – permanent muscle contraction https://encrypted-tbn1.gstatic.com/images?q=tbn:ANd9GcStx6ZbOIqeRwoQiN90IdLcl0UtWWVcyk74FiwYbUwJA9i _hrnOiA http://www.atsu.edu/faculty/chamberlain/Website/Lects/toxin3.jpg Botulinum and Tetanus toxins (Clostridium botulinum, Clostridium tetani)