1212569_21823227.jpg logo_mu_cerne.gif 1212570_28446780.jpg logo_mu_cerne.gif Luděk Bláha, PřF MU, RECETOX www.recetox.cz MECHANISMS OF TOXICITY OVERVIEW OPVK_MU_stred_2 1212569_21823227.jpg logo_mu_cerne.gif Different categorizations of Mechanisms of Action (MoA) •According to target molecules (next slide) –Mechanisms primarily targeting different •BIOLOGICAL MACROMOLECULES –i.e. PROTEINS and/or NUCLEIC ACIDS and/or PHOSPHOLIPIDS •SMALL BIOLOGICAL (ORGANIC) MOLECULES –E.g. Antioxidants or scavengers (vit.E, GSH) • •According to INTERACTION between toxicant/target (next slide) –Non-covalent interactions •Partitioning (v d Waals, H-bonds, hydrophobic interactions) à [1] below •Partitioning with specific steric fit à [3] below –Formation of covalent bonds •... with proteins / DNA-RNA / P-lipids / small molecules à [2] below • •According to “STERIC SPECIFICITY” of the interaction –NON-SPECIFIC MECHANISMS •the interaction between the toxicant and the target occurs “generally” with any target of certain general properties (e.g. toxicant is able to bind to ANY protein having e.g. SH- group), it does not require specific steric (structural) properties of the target –mechanisms [1] and [2] below –SPECIFIC MECHANISMS •the toxicant interacts only with certain and specific structural properties (e.g. specific binding of a pesticide into the active site of enzyme acetylcholinesterase) –mechanism [3] • 1212569_21823227.jpg logo_mu_cerne.gif Target (receptor) in MoA / toxicodynamic = BIOMOLECULE [1] [3] [2] 1212569_21823227.jpg logo_mu_cerne.gif Categorizations of MoAs •[1] non/specific membrane toxicity –Involves ALL ORGANIC compounds –Affinity to non-polar environment (membrane phospholipids) –Two types can be discriminated •nonpolar basal / narcotic toxicity ( –effects observed at relatively high concentrations, depends on hydrophobicity (Kow) •polar narcosis –more polar compounds may affect also membrane proteins (effects at lower concentrations than expected from Kow) • •[2] nonspecific reactive toxicity –some compounds with “reactive” properties may directly modify biological macromolecule (lipids, proteins, nucleic acids) causing thus toxic effects –reactive chemicals are mostly „electrophiles“ (reacting with „nucleophiles“ in cells – i.e. electrone-rich sites - nucleotides, -NH2, -SH and others) • •[3] specific steric interactions –only certain specific compounds selectively affect specific targets –E.g. enzyme inhibitions (drugs, insecticides); receptor interactions (e.g. Estrogens) –Can be non-covalent as well as covalent –Effects at very low concentrations All organics à membranes Reactive 1212569_21823227.jpg logo_mu_cerne.gif Categorizations of MoA •Species-specific mechanisms, examples –photosynthetic toxicity (only in plants) vs. teratogenicity (only in vertebrates) –Endocrine disruption •different hormonal systems in invertebrates vs vertebrates à different toxicity mechanisms Growth in invertebrates ecdysis (moulting) - ecdysteroids http://img.docstoccdn.com/thumb/orig/8860991.png Growth in humans several hormones 1212569_21823227.jpg logo_mu_cerne.gif Categorizations of MoA •- Tissue-specific mechanisms (& effects) • - hepatotoxicity; neurotoxicity; nephrotoxicity; haematotoxicity • - toxicity to reproduction organs; • - immunotoxicity • • • • • • • • • •Developmental stage-specific mechanisms -- embryotoxicity/teratogenicity: toxicity to cell differenciation processes http://img2.allvoices.com/thumbs/image/609/480/103574038-diclofenac-drug.jpg http://www.painstopanswers.com/images/diclofenac.jpg Thalidomide Cyanobacterial metabolites Fig2C Fig2B Malformations in frog tadpoles 1212569_21823227.jpg logo_mu_cerne.gif Keywords to remember and understand •What is it MoA? •Can you give examples of species-specific MoA? •What are the biological targets for toxicants? How can they be classified? •What are the possible interactions between toxicants and biological targets? •What is it specific and non-specific toxicity mechanism? •What biological molecules are likely to be affected (usually at relatively high concentrations) by ALL ORGANIC COMPOUNDS? • • • .... and now let’s look in detail on major MoAs and their toxic consequences 1212569_21823227.jpg logo_mu_cerne.gif •Student is expected to know principles and some examples of the following main types of toxicity mechanisms • •Membrane nonspecific toxicity (narcosis) • •Proteins and inhibition of enzymatic activities •Ligand competitions – receptor mediated toxicity • •DNA toxicity (genotoxicity) • •Complex mechanisms –Oxidative stress – redox toxicity: discussed in the presentation on cell and organismal effects – Toxicity mechanisms - overview 1212569_21823227.jpg logo_mu_cerne.gif DNA as target to toxicants 1212569_21823227.jpg logo_mu_cerne.gif DNA as target to toxicants -principal molecule for life -structure and function carefully checked -changes rapidly repaired -irreversible changes à cell death (physiologically by apoptosis) • •Mutagenesis à MUTATIONS • à variability and evolution • or à damage to DNA (structure or coding) •… naturally billions of nucleotides/day à most are repaired •… stress-induced à toxicity http://upload.wikimedia.org/wikipedia/commons/thumb/e/e4/DNA_chemical_structure.svg/800px-DNA_chemi cal_structure.svg.png 1212569_21823227.jpg logo_mu_cerne.gif DNA damage and its effects Cellular changes à Health and evolutionary consequences 1212569_21823227.jpg logo_mu_cerne.gif •Damage of DNA is carefully controlled • constitutively expressed repair systems • •Sudden changes in DNA • à induction of additional repair enzymes (e.g."SOS-repair“ in bacteria - biomarker of DNA damage) DNA repair 1212569_21823227.jpg logo_mu_cerne.gif Various types of molecular changes in DNA ... and corresponding repair systems Note! •Not all nucleotides are affected in the same rate (mutations occur only at specific sites due to physicochemical properties) Most common patterns: • G - the most frequent target (highly nucleophilic character) • T=T at the same strand • G=G crosslinks 1212569_21823227.jpg logo_mu_cerne.gif http://academic.pgcc.edu/%7Ekroberts/Lecture/Chapter%207/07-21_PointMutations_L.jpg Examples – point mutations and their IMPACT à (a) silent, (b) missense, (c) nonsense, (d) frameshift 1212569_21823227.jpg logo_mu_cerne.gif •PHYSICAL FACTORS • •Ionizating radiation • - direct interactions with NA • - interactions with water à formation of OH* (and other oxygen radical species – ROS) • à Various impacts on bases and strands • •UV radiation • - interaction with aromatic cycles (bases) à base dimerization (T=T) • What are the agents inducing mutations? MUTAGENS 1212569_21823227.jpg logo_mu_cerne.gif Ionizing radiation effects on DNA 1212569_21823227.jpg logo_mu_cerne.gif •CHEMICALS • •1) Small electrophilic molecules (attracted by nucleophilic/basic sites … e.g. in DNA) •2) Other reactive molecules * alkylating and arylating agents – covalent adducts * specifically intercalating agents •3) Base analogs • inserted during replication instead of nucleotides • •Some compounds may require “activation” by metabolism pro-mutagen (pro-carcinogen) à mutagen (carcinogen) What are the agents inducing mutations? MUTAGENS 1212569_21823227.jpg logo_mu_cerne.gif •HNO2, HSO3- Hydroxylamine (HO-NH2), Methoxyamine (CH3-O-NH2) • •Example: oxidation (deamination) à CG to à TA shift • Small molecules à deamination of bases 1212569_21823227.jpg logo_mu_cerne.gif •Covalent binding to NA (alkylation of bases, crosslinks in dsDNA) •Alkylsulphates, Nitro-urea, N-nitroso-alkyles, cis-platinum cisplatin cyclophosphamide ALKYLating compounds Nitrourea 1212569_21823227.jpg logo_mu_cerne.gif •Covalent binding, aromatic „adducts“ with bases (see also discussion at biomarkers) • •Mycotoxins (Aflatoxins) – requires activation • •PAHs (benzo[a]pyrene) – requires activation •PAH derivatives - 2-AA, 2-AF (grill products) - NQO – model mutagen in experiments • •... many others • ARYLating compounds http://www.uoguelph.ca/%7Edjosephy/lab/images/mutagens.gif 1212569_21823227.jpg logo_mu_cerne.gif Bioactivation of benzo[a]pyrene à genotoxicity BaP is oxidized to epoxides and OH-derivatives during detoxification (CYP450) à increased reactivity (including binding to bases ... primarily G or A) (Similar bioactivation e.g. at aflatoxin) 1212569_21823227.jpg logo_mu_cerne.gif Bioactivation of aflatoxin à genotoxicity Výsledek obrázku pro aflatoxin activation Výsledek obrázku pro aflatoxin producer AFLATOXIN sources Výsledek obrázku pro aflatoxin source Výsledek obrázku pro aflatoxin source 1212569_21823227.jpg logo_mu_cerne.gif •INTERCALATORS Compounds with characteristic structures “fitting” into DNA à both noncovalent and covalent intercalation Intercalating agents http://what-when-how.com/wp-content/uploads/2011/05/tmp32C166_thumb1.jpg Example 1 – ETHIDIUMBROMIDE - experimental dye – visualization of DNA - intercalation à sharing of electrones with bases à high fluorescence 1212569_21823227.jpg logo_mu_cerne.gif •Structure similarity with natural bases à Incorporation into DNA during replication à Base exchange mutations • •Example •5-Br-Uracil (anticancer drug) –AT à GC shift Base analogs 1212569_21823227.jpg logo_mu_cerne.gif Mutations (alleles) and evolution http://www.anselm.edu/homepage/jpitocch/genbi101/13_03bPesticideResist-L%20copy.jpg 1212569_21823227.jpg logo_mu_cerne.gif MEMBRANES AS TARGETS TO TOXICANTS 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 Toxicity to membrane gradients and transport -Semipermeability of membranes and key functions • •à DISRUPTIONS AND RELATED TOXIC EFFECTS - cytoplasmic membrane: signalling, neural cells Na+/K+ gradient - mitochondrial membrane: electrone flow à ATP synthesis - endoplasmatic reticulum Ca2+ signalling 1212569_21823227.jpg logo_mu_cerne.gif PROTEINS AS TARGETS OF ECOTOXICANTS 1212569_21823227.jpg logo_mu_cerne.gif •Structure of proteins • – primary (sequence of aminoacids, AA), • - secondary, tertiary, quarternary (folding – important for functions) • •Proteins - large/long – key target for number of toxicants! • = polypeptides - tens to thousands of AA •Peptides (small, “πεπτός, "digested“, 2x AA to e.g. 20x AA) • may have various functions (e.g. protective - glutathione) • •Key functions of proteins •STRUCTURE and PROTECTION •CATALYSIS (enzymes) •TRANSFER (information and mass) • - receptors, channels, transporters • Proteins as targets to toxicants https://www.mun.ca/biology/scarr/iGen3_06-04_Figure-L.jpg 1212569_21823227.jpg logo_mu_cerne.gif Most common interactions (and some examples) Hydrogen bond disruption alcohols, amines Ion bonds acids (COOH), alkalic compounds (amines) toxic metals Hg+2, Pb+2, Cd+2 , Ag+1 Tl+1, carbonyls S-S bonds toxic metals See also http://www.elmhurst.edu/~chm/vchembook/568denaturation.html http://www.elmhurst.edu/%7Echm/vchembook/images/568denathbond.gif http://www.elmhurst.edu/%7Echm/vchembook/images/568denatdisul.gif Non-specific interactions & denaturation 1212569_21823227.jpg logo_mu_cerne.gif Specific effects of environmental toxicants on proteins - examples •ENZYME INHIBITIONS • Acetylcholinesterase (organophosphate pesticides) • Inhibition of hemes – respiratory chains (cyanides) • Glyphosate (roundup) action • •EFFECTS ON RECEPTORS • membrane receptors (neurotoxicants) • nuclear receptors (endocrine disrupters) 1212569_21823227.jpg logo_mu_cerne.gif 1 Acetylcholinesterase inhibition by organophosphates http://s2.hubimg.com/u/4316027_f520.jpg 1212569_21823227.jpg logo_mu_cerne.gif Acetylcholinesterase inhibition by organophosphates (and carbamates) http://www.scielo.br/img/revistas/jbchs/v15n6/22667f1.gif Insecticides - OPs http://www2.mcdaniel.edu/Biology/eh01/pesticides/carbamates.gif Insecticides - Carbamates Nerve gases (warfare agents) http://www.darkgovernment.com/news/wp-content/uploads/2012/12/sarin.jpg http://1.bp.blogspot.com/-vJjcfxZOpBY/UghViEI7QXI/AAAAAAAADdw/0EuZdLLJHRw/s1600/nerve.tif Novichok 1212569_21823227.jpg logo_mu_cerne.gif 1 1 Inhibition of hemes – e.g. Haemoglobin, Mitchochondria, CYP450 etc. (cyanide HCN, carbon monooxide – CO) http://uvahealth.com/Plone/ebsco_images/7351.jpg 1212569_21823227.jpg logo_mu_cerne.gif 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 •N-(phosphonomethyl)glycine •Broad-spectrum herbicide („RoundUp“) •Selective inhibition of ESPs 5-enolpyruvylshikimate-3-phosphate synthase; •(synthesis of aromatic AAs – Tyr, Trp, Phe) •Uptake via leafs - only to growing plants •„Non-toxic“ to other organisms (no ESPs in animals, AA-like chemical - rapid degradation) Glyphosate action 1212569_21823227.jpg logo_mu_cerne.gif EFFECTS on „receptors“ – part 1 / membranes receptors http://www.uic.edu/classes/bios/bios100/lectures/hormone_types.jpg 1212569_21823227.jpg logo_mu_cerne.gif Environmentally relevant 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 ion channel activators SAXITOXINS •Produced by dinoflagelates and cyanobacteria •(toxic blooms, „red tides“) Výsledek obrázku pro saxitoxin 1212569_21823227.jpg logo_mu_cerne.gif EFFECTS OF CHEMICALS on „receptors“ à nuclear receptors http://www.uic.edu/classes/bios/bios100/lectures/hormone_types.jpg 1212569_21823227.jpg logo_mu_cerne.gif 46 Receptor Blood plasma Protein Lipophilic hormones mRNA DNA Hormone response element 1. Hormone passes through plasma membrane 2. Inside target cell the hormone binds to a receptor protein in the cytoplasm or nucleus 3. Hormone-receptor complex binds to hormone response element on DNA, regulating gene transcription 4. Protein synthesis 5. Change in protein synthesis is cellular response Cytoplasm Plasma membrane Nucleus Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1212569_21823227.jpg logo_mu_cerne.gif NUCLEAR (Intracellular) RECEPTORS in summary •Important physiological functions, and •All NRs share similar structure and mechanisms of action –Act as direct transcription factors on DNA •Natural ligands are small lipophilic hormones (steroids, thyroids, retinoids) –Role in toxicity – NR are modulated (activated/inhibited) by structurally close xenobiotics •Important roles in pathologies and chemical toxicity –Endocrine disruption •à effects on reproduction as well as other hormone-regulated processes (immune-, neuro-, metabolism – obesity etc.) –Dioxin-like toxicity •immunosuppression, cancer – • • The most studied NRs: ER – estrogenic receptor à xenoestrogens AhR – Arylhydrocarbon receptor („dioxin“ receptor) 1212569_21823227.jpg logo_mu_cerne.gif Natural ligands of NR •Small, lipid-soluble molecules –Diffuse through plasma and nuclear membranes and interact directly with the transcription factors they control. –STEROID HORMONES: •sex steroids (estrogen, progesterone, testosterone) •corticosteroids (glucocorticoids and mineralcorticoids) –OTHER HORMONES and ligands Thyroid hormone, vitamin D3, retinoic acid, ligands of AhR –Small molecules - gases e.g. NO (signaling for immune reactions) • • 1212569_21823227.jpg logo_mu_cerne.gif Natural products genistein naringenin coumestrol zearalenone Various POPs DDT and its metabolites (DDE) kepone PCBs/OH-PCBs PAHs and dioxins Industrial chemicals Bisphenol A Nonionic surfactants Pthalate esters (eg. DEHP) Endosulfan (pesticide) Pharmaceuticals Ethinyl estradiol Diethylstilbestrol gestodene norgestrel endosulfan_obr dehp DEHP >> Highly diverse group of substances >> Do not necessarily share structural similarity to the prototypical estrogen 17b-estradiol >> may act as AGONISTS and/or ANTAGONISTS (depending on situation and concentration!) Ligands of ER – ESTROGEN RECEPTOR Environmental estrogens (xenoestrogens, exoestrogens) Consequences * Toxicity to reproduction 1212569_21823227.jpg logo_mu_cerne.gif AhR (Arylhydrocarbon receptor) AhR structure 2,3,7,8-TCDD (dioxin) bound to AhR 1212569_21823227.jpg logo_mu_cerne.gif AhR •Ligand-activated transcription factor –Similar to all NRs •AhR has effects on many different genes • •important mediator of toxicity of POPs – primary target of planar aromatic substances –regulator of xenobiotic metabolism and activation of promutagens • •Crossactivation/crosstalk with other NRs • •Strongest known ligand - TCDD –(not endogeneous !) 1212569_21823227.jpg logo_mu_cerne.gif AhR regulated genes •Many genes contain xenobiotic response elements (XRE) or dioxin responsive elements (DRE) in their promoter region: • –Detoxification genes phase I enzymes (CYP 1A1, CYP 1A2, CYP 1B1) and phase II enzymes (UDP-glucuronosyltransferase, GST-Ya, NADP(H):oxidoreductase) •à Detoxification after toxicant exposure … also with possible toxic consequences (oxidative stress, activation of promutagens accelerated clearance of hormones) – –Other genes - regulation of cell cycle and apoptosis •Bax (apoptosis control), p27Kip1, Jun B (MAP-kinase), TGF-b (tumor growth factor) • à Various adverse toxic effects • • 1212569_21823227.jpg logo_mu_cerne.gif Denison & Nagy, Annu. Rev. Pharmacol. Toxicol. 43:309 Classical and “non-classical” AhR ligands Classical = planar structures à direct binding to AhR “Non-classical” Diverse compounds known to activate AhR 1212569_21823227.jpg logo_mu_cerne.gif Schmidt & Bradfield, Annu. Rev. Cell Dev. Biol. 12:55 Biological responses to TCDD (via AhR)