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 07 – Mechanisms Metabolism & Detoxification OPVK_MU_stred_2 1212569_21823227.jpg logo_mu_cerne.gif What processes are beyond toxicokinetics? ADME caption Toxicokinetics ... EXPOSURE à Determines the final dose & eventual toxicity 1212569_21823227.jpg logo_mu_cerne.gif Metabolism and detoxification •Chemicals enter body ... mostly via food •Pass directly through liver à main metabolism organ • 1212569_21823227.jpg logo_mu_cerne.gif Detoxification •Basic principle of detoxification – elimination of hydrophobic compounds from bodyà formation of more polar & soluble products – •Two principal phases in metabolism (Phase I & II) –well studied in vertebrates (mammals) –liver: major organ involved in detoxification •Plants –similar oxidating enzymes as described (cytochrom oxidase, phenol oxidase, peroxidase...) • •Phase III - elimination - both from cell & body • C:\Documents and Settings\Ludek Blaha\Plocha\E3.GIF 1212569_21823227.jpg logo_mu_cerne.gif Importance of nutrients and vitamins in detoxification 1212569_21823227.jpg logo_mu_cerne.gif Phase I •Key enzymes – MFOs = mixed function oxidases / oxygenases •Membrane bound to Endoplasmic Reticulum –membrane vesicles "microsomes" = S-9 fraction can be extracted from cells http://www.xenotechllc.com/Images/Products/Subcellular-Fractions/Subcellular-Fraction-Preparation-M ethod.aspx?width=400&height=374 S9 microsomes used for in vitro metabolization (e.g.during genotoxicity testing) 1212569_21823227.jpg logo_mu_cerne.gif Detoxification - Phase I •Key principle enzymes are cytochromes P450 (CYPs) –Haem (porfyrin) - containing enzymes –superfamily of more than 150 genes - several classes and subclasses •different substrate specificity; structure ... • •Some examples ... Diverse functions –Cytochrome P450 1A (CYP1A) •basic for detoxification of hydrophobic environmental contaminants –Cytochrome P450 19A (CYP19) •"aromatase" involved in synthesis of estradiol (aromatization of testosterone) • http://www.nature.com/mp/journal/v18/n3/images/mp201242f1.jpg DME = Drug Metabolism Enzymes 1212569_21823227.jpg logo_mu_cerne.gif CYPs and their functions http://people.bu.edu/djw/images/D%20Waxman%20Slide1.jpg 1212569_21823227.jpg logo_mu_cerne.gif Types of reactions catalyzed by CYPs (and Phase II enzymes) Highlighted = will be discussed also later 1212569_21823227.jpg logo_mu_cerne.gif CYPs - example: steroid hormone synthesis 1212569_21823227.jpg logo_mu_cerne.gif CYP450 overview 1212569_21823227.jpg logo_mu_cerne.gif C:\Documents and Settings\Ludek Blaha\Plocha\E3.GIF Hydroxylation (oxidation) mechanism – key in “detoxification” 1212569_21823227.jpg logo_mu_cerne.gif C:\Documents and Settings\Ludek Blaha\Plocha\E3.GIF Examples of CYP mediated reactions 1212569_21823227.jpg logo_mu_cerne.gif C:\Documents and Settings\Ludek Blaha\Plocha\E3.GIF Examples of CYP mediated reactions 1212569_21823227.jpg logo_mu_cerne.gif C:\Documents and Settings\Ludek Blaha\Plocha\E3.GIF Examples of CYP mediated reactions 1212569_21823227.jpg logo_mu_cerne.gif C:\Documents and Settings\Ludek Blaha\Plocha\E3.GIF Benzo[a]pyrene CYPs and BIOACTIVATION pro-mutagen (procarcinogen) à mutagen (carcinogen) 1212569_21823227.jpg logo_mu_cerne.gif CYPs and BIOACTIVATION of procarcinogen 1212569_21823227.jpg logo_mu_cerne.gif CYPs and BIOACTIVATION – AFLATOXIN-A 1212569_21823227.jpg logo_mu_cerne.gif CYPs and BIOACTIVATION – ethanol http://pubs.niaaa.nih.gov/publications/arh301/images/Seitz-Figure.gif 1212569_21823227.jpg logo_mu_cerne.gif CYPs and toxicity of drugs •Example - PARACETAMOL toxicity http://upload.wikimedia.org/wikipedia/commons/thumb/1/12/Paracetamol_metabolism.svg/1024px-Paraceta mol_metabolism.svg.png 1212569_21823227.jpg logo_mu_cerne.gif Detoxification – Phase II •Key reactions = conjugations –Reactive xenobiotics or metabolites formed in phase I – with endogeneous 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) –epoxid hydrolase (EH) –sulfotransferase (ST) • http://www.nature.com/tpj/journal/v3/n3/images/6500171f1.jpg 1212569_21823227.jpg logo_mu_cerne.gif 1212569_21823227.jpg logo_mu_cerne.gif - 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) Glutathione 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 Examples of conjugation reactions 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 –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 1212569_21823227.jpg logo_mu_cerne.gif http://www.nature.com/nrc/journal/v2/n6/images/nrc823-f3.gif ABC one of the resistance mechanisms of tumour cells to anticancer drugs 1212569_21823227.jpg logo_mu_cerne.gif ABC: one of the resistance mechanisms of bacteria to antibiotics [both via Physiological and Evolutionary adaptation mechanisms] http://www.nature.com/nature/journal/v406/n6797/images/406775ac.2.jpg 1212569_21823227.jpg logo_mu_cerne.gif Mechanism of evolutionary adaptation 1. Random mutation à 2. Population change (selection) by the environment http://my.clevelandclinic.org/PublishingImages/Disorders/6260_gene_transfer2_nih.gif 1212569_21823227.jpg logo_mu_cerne.gif Example of evolutionary adaptation: Development of resistance to pesticides http://econjsun.files.wordpress.com/2011/11/pest.gif 1212569_21823227.jpg logo_mu_cerne.gif Constitutive vs Induced detoxification •Detoxification enzymes expression –Constitutive – low background levels (always present) –Presence of substrates à INDUCTIONS of levels/activities • •CYP1A – inductin via Ah-receptor (AhR) –Substrate: hydrophobic organochlorine compounds (PCDDs/Fs, PAHs PCBs ...) [see also: lectures on nuclear receptors] •Other CYPs –Drugs à inductions of specific CYP classes • •Phase II enzymes –Substrates = reactive toxicants, metabolites from Phase I • •ABC transporters –Induction by respective chemicals (drugs etc) • •INDUCED DETOXIFICATION: –„Physiological adaptation“ to toxicants –Measurements used as „Biomarkers“ (of exposure, susceptibility, effect) • 1212569_21823227.jpg logo_mu_cerne.gif CYP1A induction – role of AhR (discussed later) 1212569_21823227.jpg logo_mu_cerne.gif Summary – “toxic consequences” of detoxification •BIOACTIVATION –activation of pro-mutagens/pro-carcinogens etc. –increasing side adverse effects of certain drugs • •Increase in oxidative reactions – oxidative stress –production of Reactive Oxygen Species (ROS) •(see oxidative damage and stress lectures) •Side toxic effects (see nuclear receptor lectures) –e.g. increased degradation of endogeneous compounds (retinoids – regulatory molecules degraded by CYP1A –Crosstalk with other mechanisms & receptors • •Energy (ATP) depletion –chronic inductions of detox enzymes à permanent extra energetic demand – •Development of resistence - physiological adaptation or evolutionary adaptation to toxic compounds –Loss of efficiency of anticancer drugs, antibiotics etc. –Resistance of pests • • • • 1212569_21823227.jpg logo_mu_cerne.gif img_7180 Toxic effects associated with increased detoxification (metabolism)