1212569_21823227.jpg logo_mu_cerne.gif 1212570_28446780.jpg logo_mu_cerne.gif Luděk Bláha, SCI MUNI Toxicokinetics OPVK_MU_stred_2 1212569_21823227.jpg logo_mu_cerne.gif Take home messages of this lecture What processes can a chemical compound undergo inside the ORGANISM? What is TOXICOKINETICS and what processes does it describe? • ADME •Absorption - Uptake •Distribution •Metabolism (transformations) •Excretion 1212569_21823227.jpg logo_mu_cerne.gif TOXICOKINETICS Fate of compounds inside an organism (uptake / transformations / excretion) E3 1212569_21823227.jpg logo_mu_cerne.gif Processes in toxicokinetics = ADME ADME Absorption Distribution Metabolism Elimination caption Toxicokinetics ... ... EXPOSURE phase à Determines the final dose 1212569_21823227.jpg logo_mu_cerne.gif Toxico“kinetics“ vs „dynamics“ http://4.bp.blogspot.com/-MebbujGDXi0/UAu26D7WxII/AAAAAAAAACk/StePoxIb3Go/s1600/2.png Dynamic simulation of processes causing toxicity and their grouping into toxicokinetics and toxicodynamics, illustrated on the example of the aquatic invertebrate Gammarus pulex. Interaction with target molecules (Mode of Action) ... Measurable EFFECTs TARGETS = macromolecules (DNA/RNA, proteins, membrane lipids) 1212569_21823227.jpg logo_mu_cerne.gif UPTAKE ~ ELIMINATION (equilibrium, homeostasis) - compound is maintained in the body in a concentration lower than harmful - organism has to invest energy to maintain this equilibrium (elimination processes, metabolism ...) UPTAKE > ELIMINATION - the concentration of the compound increases - it is a matter of time until it exceeds the threshold level When limits of homeostatic processes are exceeded à transition of an individual from the state of resistance (or adaptation) to the state of detectable negative effects à negative effects at higher levels of organization (tissue, organism, etc.) Toxicity = imbalance between UPTAKE and EXCRETION 1212569_21823227.jpg logo_mu_cerne.gif „Fate“ (transport) of a chemical through body (from gut, through blood / organ (liver) / blood again to kidney/urine Blood Blood 20 Circulatory Systems Blood 1212569_21823227.jpg logo_mu_cerne.gif A screenshot of a cell phone Description automatically generated „Fate“ (transport) of a chemical through body (from gut, through blood / organ (liver) / blood again to kidney/urine http://upload.wikimedia.org/wikipedia/commons/thumb/0/0e/Benzo%28a%29pyrene_metabolism.svg/540px-Be nzo%28a%29pyrene_metabolism.svg.png http://upload.wikimedia.org/wikipedia/commons/thumb/0/0e/Benzo%28a%29pyrene_metabolism.svg/540px-Be nzo%28a%29pyrene_metabolism.svg.png http://upload.wikimedia.org/wikipedia/commons/thumb/0/0e/Benzo%28a%29pyrene_metabolism.svg/540px-Be nzo%28a%29pyrene_metabolism.svg.png 1212569_21823227.jpg logo_mu_cerne.gif Uptake of compounds in various organisms 1) unicellular organisms - passive diffusion through a membrane - „selective“ input through present transport systems 2) multicellular organisms / algae - diffusion of the toxicant through membrane and between the cells 3) terrestrial plants - compounds dissolved in water/soil – uptake via roots/leafs - gaseous toxicants – uptake via leaf stomata - lipophilic compounds (some herbicides) – penetration of the waxy cuticle - into the cell à through the membrane TOXICOKINETICS 1: uptake of compounds into the organism 1212569_21823227.jpg logo_mu_cerne.gif Uptake of compounds into the organism: 4) animals - 3 main uptake pathways - food/drinking water - passage through the digestive system, changes/transformation dependent on pH, gut microflora, e.g. cycasin: nontoxic – conversion in the gut à strong mutagen) - via respiration - tracheae of insect, gills of aquatic organisms, lungs - large surface for exchange/entry of compounds (often 25times larger than body surface) - via body surface -higher importance for smaller organisms (relatively larger area) and aquatic organisms - in any case à transfer through membranes TOXICOKINETICS 1: uptake of compounds into the organism 1212569_21823227.jpg logo_mu_cerne.gif Regardless of the type of the organism or uptake pathway (into higher organisms) the toxicant has to cross the plasmatic membrane barrier (or as well the cell wall). Membranes – essential barrier for toxic compounds proteins 1212569_21823227.jpg logo_mu_cerne.gif Toxicants crossing the membranes Most common (all compounds) - passive diffusion Selected ompounds with special/certain properties (e.g. alike to nutrients or natural compounds) – co-transport / active transport Large molecules + particles - pinocytosis 1212569_21823227.jpg logo_mu_cerne.gif Toxicants crossing the membranes PASSIVE DIFFUSION -random movement of molecules down a concentration gradient -process characterized by the first order kinetics - depends on: - concentration gradient - membrane and cell wall area and thickness - compound‘s solubility in fat and its ionization - lipophilic and neutral compounds – good diffusion - charged compounds – diffusion more difficult - molecular weight: - small molecules (<0.4 nm) water soluble (CO, HCN, N2O, NO) good diffusion TOXICOKINETICS 1 - uptake of compounds into the organism - 1212569_21823227.jpg logo_mu_cerne.gif Toxicants crossing the membranes CO-TRANSPORT - transmembrane proteins bind extracellular compounds and facilitate transmembrane transport : toxic compound - interference (Ca2+ / calmodulin, Fe2/3+ / transferrin) ACTIVE TRANSPORT - „pumps“ down/up the concentration gradient - compound binds to a receptor / ATP powered membrane transport coupled transports Na+/K+ ATPases - toxic compounds/ interference These special biological processes occur rarely with xenobiotics – exceptionally with compounds alike to nutrients and such (e.g. cyanobacterial toxins: peptides) TOXICOKINETICS 1 - uptake of compounds into the organism - 1212569_21823227.jpg logo_mu_cerne.gif A screenshot of a cell phone Description automatically generated „Fate“ (transport) of a chemical through body (from gut, through blood / organ (liver) / blood again to kidney/urine Figure shows transporters involved in the transfer (including excretion) of structurally specific compounds to and from the organism Alternative (passive diffusion) route for benzo(a)pyrene is added http://upload.wikimedia.org/wikipedia/commons/thumb/0/0e/Benzo%28a%29pyrene_metabolism.svg/540px-Be nzo%28a%29pyrene_metabolism.svg.png http://upload.wikimedia.org/wikipedia/commons/thumb/0/0e/Benzo%28a%29pyrene_metabolism.svg/540px-Be nzo%28a%29pyrene_metabolism.svg.png http://upload.wikimedia.org/wikipedia/commons/thumb/0/0e/Benzo%28a%29pyrene_metabolism.svg/540px-Be nzo%28a%29pyrene_metabolism.svg.png 1212569_21823227.jpg logo_mu_cerne.gif PINOCYTOSIS - transport of larger molecules via endocytosis - e.g. entry of airborne toxicants with dust particles (< 1 mm) into alveolar cells, entry of asbestos fibers into alveolar macrophages TOXICOKINETICS 1 - uptake of compounds into the organism - entry of airborne toxicants with dust particles into alveolar cells, entry of asbestos fibers into alveolar macrophages 1212569_21823227.jpg logo_mu_cerne.gif Transport in animals - blood, lymph, haemolymph - transport of dissolved compounds - transport after binding to proteins (albumin, specific proteins) ! Many organic (nonpolar) compounds can be bound TOXICOKINETICS 2 - transport of compounds in the organism - 1212569_21823227.jpg logo_mu_cerne.gif Transport in plants - water stream in xylem - plasmodesms in phloem - - -processes dependent on environmental conditions (t, humidity, light...) TOXICOKINETICS 2 - transport of compounds in the organism - 1212569_21823227.jpg logo_mu_cerne.gif Affinity to different tissues - affinity is determined by chemical properties -> target tissues - bioconcentration seashells - Cd/Pb - gonads mammals Cd – brain/bones, Pb – kidneys/bones Hg – in mammals: kidneys > liver > spleen > gut > heart... lipophilic compounds -> fatty tissues (liver, brain) TOXICOKINETICS 2 - Distribution of compounds in the organism - 1212569_21823227.jpg logo_mu_cerne.gif Example – metals in tissues of fish: Nové Mlýny (Kenšová et al. ACTA VET. BRNO 2010, 79: 335-345) 1212569_21823227.jpg logo_mu_cerne.gif Transformation of xenobiotics in organisms - all organisms have genetically fixed old conservative systems for transformation of xenobiotics: - in the past - transformation of biotoxins (moulds, plants, bacteria...) - combustion products (PAHs) TOXICOKINETICS 3 - transformation of compounds in the organism - 1212569_21823227.jpg logo_mu_cerne.gif Basic detoxification strategy - Removal from the organism = exposure limitation - Most excretion organs: aqueous solutions :transformation = increasing water solubility - production of more polar, less hydrophobic (more hydrophilic) products - Two (2) … or 3 with elimination … main phases of detoxification - well examined in animals (mammals) Note: in vertebrates (esp. mammals – warm-blooded = higher speed of reactions) >> detoxication more active than in fish or invertebrates (è bivalves accumulate PAHs x mammals less: oxidation/excretion) In plants – transformation with oxidative enzymes: cytochrome oxidase, phenol oxidase, peroxidase, ascorbate oxidase TOXICOKINETICS - transformation of compounds in the organism - production of more polar, less hydrophobic / more hydrophilic products 1212569_21823227.jpg logo_mu_cerne.gif TOXICOKINETICS – rate of detoxification reactions Rate of transformations depends on • overall metabolic rate (indirectly also on body size) • temperature (the higher the temperature – the higher the rate of reactions) 1212569_21823227.jpg logo_mu_cerne.gif A picture containing screenshot Description automatically generated A close up of a map Description automatically generated A screenshot of a cell phone Description automatically generated 1212569_21823227.jpg logo_mu_cerne.gif - MFO enzymes (mixed function oxidase, mixed function oxygenase) - membrane enzymes bound to ER, extractable as membrane vesicles (= microsomes = S-9 fraction = microsomal oxidase) - Conserved – in all plants and animals TOXICOKINETICS - transformation – PHASE I 1212569_21823227.jpg logo_mu_cerne.gif - based on enzymes containing heme as cofactor = cytochromes P450 (CYP) = superfamily with more than 150 genes - in vertebrates mostly in liver parenchyma = main detoxifying organ (but also in – gut epithelium, gills...) - in invertebrates in hepatopancreas and digestive glands - main reaction – reaction with oxygen + other reactions (hydrolysis / epoxidation / dehalogenation / hydroxylation / deamination / dealkylation) TOXICOKINETICS Phase I transformation – CYP450 based on enzymes containing heme as cofactor = cytochromes 1212569_21823227.jpg logo_mu_cerne.gif E3 http://upload.wikimedia.org/wikipedia/commons/thumb/0/0e/Benzo%28a%29pyrene_metabolism.svg/540px-Be nzo%28a%29pyrene_metabolism.svg.png http://upload.wikimedia.org/wikipedia/commons/thumb/0/0e/Benzo%28a%29pyrene_metabolism.svg/540px-Be nzo%28a%29pyrene_metabolism.svg.png 1212569_21823227.jpg logo_mu_cerne.gif E3 Phase I biotransformation reactions – examples 1 1212569_21823227.jpg logo_mu_cerne.gif E3 Phase I biotransformation reactions – examples 2 1212569_21823227.jpg logo_mu_cerne.gif Phase I biotransformation reactions – examples 3 E3 1212569_21823227.jpg logo_mu_cerne.gif - many compounds after metabolization with detoxification enzymes turn into more toxic metabolites = BIOACTIVATION (simplified as Procarcinogen à Carcinogen activation; the process is GENERAL – not only carciongens(!)) Example – POLYCYLIC AROMATIC HYDROCARBONS E.g. epoxidation of benzo[a]pyrene (BaP) -> reaction with guanosine residues in DNA - mutation / activation of oncogenes BUT BaP without activation -> acutely nontoxic compound - strong induction of detoxification enzymes after exposition to xenobiotics can have also other negative effects (dioxin type toxicity – see further) TOXICOKINETICS: Detoxification à (BIO)ACTIVATION 1212569_21823227.jpg logo_mu_cerne.gif E3 TOXICOKINETICS – Bioactivation of Procarcinogen Metabolism/oxidation à formation of more toxic/carcinogenic products BaP intercalated in DNA 1212569_21823227.jpg logo_mu_cerne.gif Detoxification – Phase II •Key reactions = conjugations –Reactive xenobiotics or metabolites formed in phase I – with endogenous substrates •saccharides and their derivatives – glucuronic acid, •aminoacids (glycine) •peptides: glutathione (GSH) •Forming water soluble AND “nontoxic” products (conjugates) • •Phase II enzymes (“transferases”): –glutathion S-transferase (GST) –UDP-glucuronosyltransferase (UDP-GTS) –sulfotransferase (ST) • http://www.nature.com/tpj/journal/v3/n3/images/6500171f1.jpg 1212569_21823227.jpg logo_mu_cerne.gif C:\Documents and Settings\Ludek Blaha\Plocha\E3.GIF C:\Documents and Settings\Ludek Blaha\Plocha\E3.GIF Phase II - Examples of conjugation reactions 1212569_21823227.jpg logo_mu_cerne.gif - tripeptide structure - Phase II conjugation reactions + general scavenger/antioxidant -major donor of SH (thiol) groups in cells (MW ~ 300 g/mol) - concentrations in tissues and blood up to 5 mM (1.5 g/L) - the major „antioxidant“ which can be synthesized by animals (other antioxidants – e.g. vitamin C, E - food antioxidants) Glutathione (GSH) 1212569_21823227.jpg logo_mu_cerne.gif C:\Documents and Settings\Ludek Blaha\Plocha\E3.GIF Xenobiotic conjugations with GSH 1212569_21823227.jpg logo_mu_cerne.gif Phase III – elimination / membrane transport •Phase III transporters –Transporting toxic molecules / metabolites / intermediates / conjugates from inside the cell to extracellular matrix (blood etc) – –ATP-binding cassette transporters (ABC transporters) –protein superfamily (one of the largest, and most ancient in all extant phyla from prokaryotes to humans) –transmembrane proteins - transport across extra- and intracellular membranes (metabolic products, lipids, sterols, drugs) • 1212569_21823227.jpg logo_mu_cerne.gif - MRP (MDR) - multidrug resistance-associated protein family - OATP - Organic Anion Transporting Polypeptide - P-glycoprotein ABC transporters - examples Bile channel in the liver à GIT 1212569_21823227.jpg logo_mu_cerne.gif ABC one of the resistance mechanisms of bacteria to antibiotics http://www.nature.com/nature/journal/v406/n6797/images/406775ac.2.jpg 1212569_21823227.jpg logo_mu_cerne.gif Extent of xenobiotic elimination à extent of possible toxicity longer exposure > higher probability of effects TERRESTRIAL ORGANISM - most soluble non-gaseous and nonvolatile compounds - urine glomerulus : filtration / active transcellular excretion / transcellular diffusion / also resorption (!) - significant/relevant excretion also - bile active transport of conjugates at excretion / further transformation by microflora in the gut / event. resorption - gaseous compounds (NH3) and volatiles (alcohols) – lungs/breathing EXCRETION 1212569_21823227.jpg logo_mu_cerne.gif AQUATIC ANIMALS - main excretion organ are gills (NH3) + bile (kidneys to a lesser extent) PLANTS à SEQUESTRATION – storage in vacuoles (leaves), excretion of gaseous toxicants EXCRETION 1212569_21823227.jpg logo_mu_cerne.gif Sequestration of xenobiotics in inert tissues à limits circulation in a body (reduction of internal exposure) Plants - vacuoles, leafs, bark (à autumn fall off) Animals - fat (organochlorine compounds) - teeth, hair, horns (metals) - in invertebrates: for example metals (Zn-granules) gut of leech Release from storage later during life •PCBs and other organochlorine compounds stored in fat during life •Rapid energy demand (egg production in fish, starvation, milk production) à release from storage à rapid peak exposure •(Also – exposures of babies from milk stored in mothers: but(!) – benefits of breastfeeding overweight temporal risks!) SEQUESTRATION Giant pet leech feeds on person's arm, video creeps people out | Trending - Hindustan Times 1212569_21823227.jpg logo_mu_cerne.gif Metallothioneins (MTs, MT-like proteins) - cytoplasmic low molecular weight proteins (6-10 kD) rich on Cys - recognized in most eukaryotes - bind metals: Zn, Cd, Hg ... => reduce exposure and toxicity - long half-life of proteins (~ 25 days) SEQUESTRATION – example mechanism Molecular functions of metallothionein and its role in hematological malignancies | Journal of Hematology & Oncology | Full Text Structure of metallothionein and its bound polynuclear metal clusters.... | Download Scientific Diagram Structure of MT (1-7: metal atoms bound to the structure) 1212569_21823227.jpg logo_mu_cerne.gif TK processes (Phase I – MFO / Phase II - Ts / Phase III) are inducible Presence of substrates (of enzymatic reactions)à de novo synthesis (induction) of enzymes / proteins - MFO enzymes are induced by a number of (lipophilic/toxic) compounds - organochlorine compounds, PCDDs/Fs, PAHs, PCBs ... - Phase II and Phase III enzymes/proteins are induced by - increased metabolites („activated“ substrates from the Phase I) - occurrence of oxidants and reactive toxicants in cells (ROS, ox. stress) - long-term exposure to sublethal doses à induction of detoxification enzymes à increase of tolerance to toxicant (physiological adaptation) à too long exposure: energy depletion à death INDUCTION OF transformation / metabolism / elimination „Physiological“ adaptation to toxicants 1212569_21823227.jpg logo_mu_cerne.gif Induction of TKs: biomarker of exposure and effects - previous exposure to xenobiotics can be deduced from the measurement of activity of detoxification enzymes = biomarkers (up to 100+ times increase compared to background activities) - often discussed is the induction of CYP1A (cytochromes P450 1A1) - after binding and activation of AhR (aryl hydrocarbon receptor) à transcription and translation of new CYP enzymes - experimental assessment of AhR/CYP1A1activation –EROD (ethoxyresorufin-O-deethylase) - good correlation with organic (+ chlorine) pollution (Note: AhR is also very important mediator of toxicity à discussed elsewhere during the course) 1212569_21823227.jpg logo_mu_cerne.gif 1212569_21823227.jpg logo_mu_cerne.gif E3 Seasonal changes in EROD activity of fish / carps (males vs. females) from two rivers (Anoia, Cardener) upstream and downstream (2 stations) from the waste water treatment plants outlets. 1212569_21823227.jpg logo_mu_cerne.gif Induction of MTs in fish exposed to arsenic (As) 1212569_21823227.jpg logo_mu_cerne.gif What are the main processes that a compound undergoes in the organism? What are the main products formed during the metabolism? What enzymes are involved in different phases of the biotransformation? What chemical reactions are the most common during biotransformation processes? What is glutathione? What is the first and the second phase of detoxification? In which organism will the biotransformation (detoxification) processes be faster? In fish or in human? What would be the most probable products of transformation in an organism exposed to benzene? Name a model compound that can be “bioactivated” in the organism. Explain bioactivation. In what form and by which organ(s) fish excrete toxic compounds and their metabolites? Wrap up questions