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 10 – Mechanisms Nuclear Receptors OPVK_MU_stred_2 1212569_21823227.jpg logo_mu_cerne.gif Various signalling types … now focus on nuclear receptors http://www.uic.edu/classes/bios/bios100/lectures/hormone_types.jpg 1212569_21823227.jpg logo_mu_cerne.gif NUCLEAR (Intracellular) RECEPTORS in summary •Important physiological functions, and •Important roles in pathologies and chemical toxicity –Endocrine disruption –Dioxin-like toxicity,etc. •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 • 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 5 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 6 Fate and action of HORMONES activating NRs •Circulation in the blood bound to transport proteins •Dissociation from carrier at target cells •Passing through cell membrane •Binding to an intracellular receptor (either in the cytoplasm or the nucleus) •Hormone-receptor complex binds to hormone responsive elements in DNA • à Regulation of gene expression • •à De-regulation at any level described above = TOXICITY • • 1212569_21823227.jpg logo_mu_cerne.gif NR signalling is complex … examples of complexity (1) 1. Receptor activation is dependent not only on „ligand“ (glucocorticoid) but also on „inhibitor“ protein (HSPs) 2. Dimerization (after the activation) is often needed for proper action (binding to GREs – glucocorticoid responsive elements) 3. Receptor with ligand can activate its own targets (GREs) as well as „repress“ other binding sites (TFREs) http://brainimmune.com/wp-content/uploads/2010/08/The-Glucocorticoid-Receptor3.jpg 1212569_21823227.jpg logo_mu_cerne.gif NR signalling is complex … examples of complexity 2 15_013 4. „Co-activator“ proteins are needed for proper action on DNA 5. Nuclear receptor action are (also) controlled - stimulated / suppressed - by other signalling pathways (e.g. phosphorylation by protein kinases) http://www.nature.com/nrc/journal/v3/n11/images/nrc1211-f7.jpg 1212569_21823227.jpg logo_mu_cerne.gif 6. Interaction (crosstalk) among various NRs •“antiestrogenicity” of AhR ligands •fast clearance of retinoids after AhR activation •Immunosuprresions after ER activations hsp90 hsp90 P RAR hsp90 hsp90 P P RAR P ? RAR RAR NR signalling is complex … examples of complexity 3 1212569_21823227.jpg logo_mu_cerne.gif Details - specificities of NRs • •Regulation of transcription activity - mechanisms may vary –Steroid receptors often dimerize with a partner to activate gene transcription –Receptors for vitamin D, retinoic acid and thyroid hormone form heterodimers and then bind to responsive elements on DNA •Second component of the heterodimer is RXR monomer (i.e, RXR-RAR; RXR-VDR) • •NR dimers –Heterodimeric receptors - exclusively nuclear; •without ligand represses transcription (by binding to their cognate sites in DNA) –Homodimeric receptors •mostly cytoplasmic without ligands à hormone binding leads to nuclear translocation of receptors • • 1212569_21823227.jpg logo_mu_cerne.gif STEROIDs - most studied ligands detailed view 1212569_21823227.jpg logo_mu_cerne.gif 1212569_21823227.jpg logo_mu_cerne.gif STEROID HORMONE biosynthesis 1212569_21823227.jpg logo_mu_cerne.gif Why are NR important? à common mediators of Endocrine Disruption 1212569_21823227.jpg logo_mu_cerne.gif Endocrine disruption •Interference of xenobiotics with normal functioning of hormonal system • •Known consequences •à Disruption of homeostasis, reproduction, development, and/or behavior (and other hormone-controlled processes), such as –Shift in sex ratio, defective sexual development –Low fecundity/fertility –Hypo-immunity, carcinogenesis –Malformations –etc. 1212569_21823227.jpg logo_mu_cerne.gif Endocrine disrupters in the environment? • • EDCs... •Persistent Organic Compounds (POPs and their metabolites) •steroid hormones and their derivatives from contraception pills •alkylphenols •organometallics (butyltins) •pharmaceuticals •Pesticides •+ number of unknowns … • Tributyl-tin alkylphenols 2,3,7,8-TCDD estradiol 1212569_21823227.jpg logo_mu_cerne.gif Toxicants interact with hormonal system at different levels Synthesis Transport Metabolization Interaction with receptors Stimulation Suppression Consequences (both negative!) Possible mechanisms of endocrine disruption - Disruption of the „master“ hormones (FSH/LH) - Decrease of HR cellular levels - Nonphysiological activation of hormone receptor (HR) - Binding to HR without activation - Changes in hormone metabolism (clearance) 1212569_21823227.jpg logo_mu_cerne.gif biosynthesis and release of hormones binding to plasmatic transport proteins binding to nuclear hormonal receptor (HR) activation of HR (dissociation of associated heat shock proteins, formation of homodimers) binding of the activated receptor complex to specific DNA motifs - HREs chromatin rearrangement and transcription of estrogen-inducible genes effects at the cellular, tissue, organ, organism, and/or population level e.g. modulation of CYP11A and/or CYP19 activities e.g. down-regulation of receptor levels Direct interference (activation / inhibition) e.g. steroidogenesis Mechanisms of toxicant effects in detail à various MoAs of endocrine disruption e.g. modulation of other nuclear receptors (PPAR/RXR, RXR/TR) 1212569_21823227.jpg logo_mu_cerne.gif Examples – modulations of (synthetic) enzyme activities http://erc.endocrinology-journals.org/content/13/4/995/F2.large.jpg Phytoestrogens promote synthesis of estrogens à feminization http://icb.oxfordjournals.org/content/45/2/321/F5.large.jpg Crosstalk with other signalling pathways (such as cAMP), which can be target to toxicants 1212569_21823227.jpg logo_mu_cerne.gif ESTROGEN RECEPTOR – ER the most studied target of EDCs 1212569_21823227.jpg logo_mu_cerne.gif Estrogens •Synthesis in ovaries • •Functions –key roles in female hormone regulation and signalling –responsible for metabolic, behavioural and morphologic changes occurring during stages of reproduction –involved in the growth, development and homeostasis in a number of tissues –control the bone formation, regulation of homeostasis, cardiovascular system and behaviour –regulate production, transport and concentration of testicular liquid and anabolic activity of androgens in males • • DISRUPTION OF ESTROGEN SIGNALLING à many documented effects in aquatic biota & laboratory organisms 1212569_21823227.jpg logo_mu_cerne.gif Kidd, K.A. et al. 2007. Collapse of a fish population following exposure to a synthetic estrogen. Proceedings of the National Academy of Sciences 104(21):8897-8901 Controls +Ethinylestradiol 5 ng/L (!) 7 years 1212569_21823227.jpg logo_mu_cerne.gif ER-a (in breast, ovary, brain, liver, bone and cardiovascular system, adrenals, testis and urogenital tract) ER-b (in kidneys, prostate and gastrointestinal tract) (ER-g in fish) ESTROGEN RECEPTORS - subtypes 1212569_21823227.jpg logo_mu_cerne.gif Natural products genistein naringenin coumestrol zearalenone Various POPs DDT 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!) Environmental estrogens (xenoestrogens, exoestrogens) 1212569_21823227.jpg logo_mu_cerne.gif Exoestrogens - Relative Potencies to bind to ERa (REPs) REP – a measure of toxic potency of a compound (similar also at other NRs) 1212569_21823227.jpg logo_mu_cerne.gif Janošek, J., Hilscherová, K., Bláha, L., and Holoubek, I. (2006). Environmental xenobiotics and nuclear receptors-Interactions, effects and in vitro assessment. Toxicology in Vitro 20, 18-37. How to assess ESTROGENICITY? Number of in vivo and in vitro methods available 1212569_21823227.jpg logo_mu_cerne.gif • uterotropic assay • vaginal cornification assay • • • • •production of estrogen-inducible proteins (e.g. vitellogenin and zona radiata protein) à also discussed at “biomarkers” part • •standard (in vivo) test procedures for reproductive and developmental toxicity •using mice, rats, fish, amphibians etc. • IN VIVO ASSAYS FOR ESTROGENICITY http://www.nature.com/nm/journal/v18/n12/images/nm.3009-F2.jpg Rat uterus Control Estrogen exposure 1212569_21823227.jpg logo_mu_cerne.gif In vitro assays for estrogenicity •Level 1 – interaction of toxicant with the protein (receptor) –INTERACTION (BINDING) to the receptor •competitive ligand binding assays –Various variants (e.g. displacement of radioactive substrate, fluorescence resonance energy transfer (FRET) techniques etc. àinformation only about “binding potency” but the effect remains unknown (? Activation / suppression / no effect ?) – http://jbx.sagepub.com/content/15/3/268/F1.large.jpg 1212569_21823227.jpg logo_mu_cerne.gif In vitro assays for estrogenicity •Level 2 - effects at cellular level –à interference with receptor biological activity • •Cell proliferation assays –Estrogens induce proliferation • • • • http://2.bp.blogspot.com/-IYTZf1bSORs/UtDTVo3194I/AAAAAAAAGBQ/-Hz4uu-mlZA/s1600/pharmatutor-art-210 7-1.png 1212569_21823227.jpg logo_mu_cerne.gif In vitro assays for estrogenicity •Level 2 - effects at cellular level –à interference with receptor biological activity • •Endogenous protein expression (or enzyme activity) assays –reporter gene assays • • • • Cell assays in vitro •Cells (e.g. breast carcinoma) naturally carrying functional ER. •Genetic modification - stable transfection with firefly luciferase gene: under the control of ER •Estrogens in media à light induction Výsledek obrázku pro firefly luciferase 1212569_21823227.jpg logo_mu_cerne.gif 96 microwell plate cultivation of transgenic cell lines ER: breast carcinoma MVLN cells Cell lysis à extraction of induced luciferase Exposure (6 – 24 h) standards / samples Lumino Luminescence determination (microplate luminescence reader) Luciferase reporter assay for estrogenicity in brief Výsledek obrázku pro firefly luciferase Similar principle for other NRs activities Mammalian cells * AhR – H4IIE.luc cells (CALUX) * AR – MDA.kb2 cells * RAR/RXR - P19/A15 cells Yeast models * Luciferase based * Also beta-galactosidase etc. 1212569_21823227.jpg logo_mu_cerne.gif schema_princip_biosenzoru.jpg Bioassay (biosensor) for NR-modulator based on yeast cells Jarque, S., M. Bittner, L. Bláha and K. Hilscherová (2016). "Yeast biosensors for detection of environmental pollutants: current state and limitations." Trends in Biotechnology 34(5): 408-419 (doi:10.1016/j.tibtech.2016.01.007). 1212569_21823227.jpg logo_mu_cerne.gif ANDROGEN RECEPTOR (AR) role in toxicity confirmed ... but less explored than ER 1212569_21823227.jpg logo_mu_cerne.gif Androgens -Role in males similar to the of estrogens in females -development of male sexual characteristics -stimulating protein synthesis, growth of bones -cell differenciation, spermatogenesis -male type of behaviour • https://www.netterimages.com/images/vpv/000/000/058/58054-0550x0475.jpg 1212569_21823227.jpg logo_mu_cerne.gif -Endogenous ligands – androgen hormones -Two key androgens -testosterone (T) -dihydrotestosterone (DHT) -Other androgens – androstanediol, dehydroepiandrosterone, androstenedione - •T: synthesis in testis (Leydig cells) –in lesser extent in adrenals •DHT: Formed extratesticulary from T -In several tissues (seminal vesicles, prostate, skin) higher affinity to androgen receptor than T -Daily production 5-10% of testosterone • Testosterone Androgens 1212569_21823227.jpg logo_mu_cerne.gif Mechanisms of androgen signalling disruption •1) Binding to AR –Mostly competitive inhibition –Xenobiotics mostly DO NOT activate AR-dependent transcription – •Only few compounds able to activate AR in the absence of androgen hormones but they are anti-androgenic in the presence of strong androgens like T or DHT • - metabolites of fungicide vinclozoline, some PAHs • • • • •2) FSH/LH (gonadotropins) signalling disruption – less explored –FSH/LH expression - regulation via negative feedback by testosterone – Suppression à alterations of spermatogenesis • vinclozoline 1212569_21823227.jpg logo_mu_cerne.gif •3) Alterations of testosterone synthesis –Inhibition of P450scc needed for side chain cleavage of cholesterol or inhibitions of 17-beta-hydroxylase and other CYPs •fungicide ketoconazol • • • •4) Testosterone metabolic clearance –Induction of detoxification enzymes (UDP-glucuronosyltransferase or monooxygenases CYP1A, 1B) •Pesticides endosulfan, mirex, o-p´-DDT • Mechanisms of androgen signalling disruption 1212569_21823227.jpg logo_mu_cerne.gif Effects of male exposure to antiandrogens •Exposure during prenatal development: – malformations of the reproductive tract •reduced anogenital distance •hypospadias (abnormal position of the urethral opening on the penis) •vagina development •undescendent ectopic testes •atrophy of seminal vesicles and prostate gland • •Exposure in prepubertal age: –delayed puberty – reduced seminal vesicles – reduced prostate • •Exposure in adult age: –oligospermia –azoospermia –loss of sexual libido • 1212569_21823227.jpg logo_mu_cerne.gif AR-binding – potencies - reference DHT: EC50 ~ 0.1 µM) Compound IC50 (µM) Benz[a]anthracene 3.2 Benzo[a]pyrene 3.9 Dimethylbenz[a]anthracene 10.4 Chrysene 10.3 Dibenzo[a,h]anthracene activation in range 0.1-10µM Bisphenol A 5 vinclozolin metabolites 9.7 hydroxyflutamide 5 Aroclor typical values 0.25-1.11 Individual PCBs typical values 64 - 87 tris-(4-chlorophenyl)-methanol 0.2 1212569_21823227.jpg logo_mu_cerne.gif Antiandrogenic compound •tris-(4-chlorophenyl)-methanol –Ubiquitous contaminant of uncertain origin –Probable metabolite of DDT-mixtures –Levels in human blood serum cca. 50nM –antiAR potency - EC50 – cca. 200nM • • 1212569_21823227.jpg logo_mu_cerne.gif (Anti)androgenicity assessment •In vivo Hershberger assay –castrated rats treated with examined substance –Endpoint – after 4-7 days – seminal vesicles and ventral prostate weight •In vivo measurement of testosterone blood levels • •In vitro cell proliferation assays –cells with androgen-dependent growth: mammary carcinoma cell lines –prostatic carcinoma cell lines – •Receptor-reporter assays –Gene for luciferase (or GFP) under control of AR •AR-CALUX (human breast carcinoma T47D) •PALM (human prostatic carcinoma PC-3) •CHO515 (Chinese hamster ovary CHO) –Yeast transfected cells •beta-galactosidase reporter • • 1212569_21823227.jpg logo_mu_cerne.gif THYROID SIGNALLING 1212569_21823227.jpg logo_mu_cerne.gif hypoTHYROID 1 hyperthyroid HYPOTHYROIDISM HYPERTHYROIDISM •Crucial roles in metabolism, development and maturation –Regulation of metabolism •increasing oxygen consumption •modulating levels of other hormones (insulin, glucagon, somatotropin, adrenalin) –Important in cell differenciation –Crucial role in development of CNS, gonads and bones – •EDC compounds interfering with thyroid signalling –“GOITROGENS” •Many food (vegetables) contain goitrogens http://jeevalifestyle.com/wp-content/uploads/2013/11/Goitrogen-Food.jpg Thyroid hormones 1212569_21823227.jpg logo_mu_cerne.gif Thyroid hormones T4 – prohormone 5´-deiodination à active form, T3 Thyroxine (T4) Also called tetraiodothyronine Contains 4 iodide ions Triiodothyronine (T3) Contains 3 iodide ions -Most T3 produced by deiodination in target tissues (deiodinases) 1212569_21823227.jpg logo_mu_cerne.gif Disruption of enzymes involved in Thyroid metabolism „outer“ „inner“ •Thyroid peroxidases – iodination of tyrosyl residues – coupling of iodinated tyrosyl residues – •Thyroid deiodinases –D1, D2 - activation of T4 into T3 via deiodination on „outer“ ring –D3 - deactivation into rT3 via deiodination on „inner“ ring • • •Many goitrogens affect expression, activities and outcomes of these key enzymes –PTU – propylthiouracil – àeffect deiodinases – – –Thiocyanate ([SCN]−) or perchlorate (NaClO4) – àeffect on iodine uptake • http://www.frontiersin.org/files/Articles/118995/fendo-05-00188-HTML/image_m/fendo-05-00188-g001.jp g 1212569_21823227.jpg logo_mu_cerne.gif Disruption of transport of thyroid hormones in blood •SPECIFIC TRANSPORTERS in blood –regulating free T4 and T3 levels –3 types : •Thyroid-binding prealbunin (transthyretin) (20-25%) •Albumin (5-10%) •Thyroid binding globulin (TBP, 75%) • •NUMBER OF EDCs à act on transport proteins –OH-PCBs, brominated and chlorinated flame retardants, DDT, dieldrin –OH-PCBs – equal affinity to TBP as T4 and T3 (!!!) • •Increased levels of “free T4” in blood –negative feedback to TSH release à increased depletion à increased weight, changes in thyroid gland –Documented after exposures to POPs in vertebrates Hydroxylated PCB formation Polybrominated diphenyl ethers (PBDEs) – flame retardants 1212569_21823227.jpg logo_mu_cerne.gif Effects of thyroid disruption cretin1 •Exposures during prenatal stages –severe damage of CNS (cretenism, delayed eye opening, cognition) –Megalotestis –Histological changes in thyroid gland (goitre) • •Exposures during development –nervous system fails to develop normally –mental retardation –skeletal development • 1212569_21823227.jpg logo_mu_cerne.gif RAR/RXR receptors - vitamin A and its derivatives: RETINOIDS - & their role in toxicity http://www.nature.com/nature/journal/v508/n7494/images/nature13158-sf9.jpg 1212569_21823227.jpg logo_mu_cerne.gif Retinol (vitamin A) Bond cleavage Retinoic Acid b-karoten RETINOIDS Sources: from diet - dietary hormones Retinyl esters – animal sources Plant carotenoids 1212569_21823227.jpg logo_mu_cerne.gif Retinoids and their functions •Regulation of development and homeostasis in tissues of vertebrates and invertebrates •Development of embryonic, epithelial cells (gastrointestinal tract, skin, bones) •Necessary for vision •Suppressive effects in cancer development •Important for cell growth, apoptosis and differenciation •Antioxidative agent •Affect nervous and immune function • 1212569_21823227.jpg logo_mu_cerne.gif schémata pro přednášku o retinoidech - 2 RE: Retinol-Ester R: Retinol RBP: Retinol Binding Protein (LMW) TTR: Transthyrethin (HMW) Retinoid transport 1212569_21823227.jpg logo_mu_cerne.gif schémata pro přednášku o retinoidech - 1 CRBP – cellular retinol binding protein - binding of retinol, immediate decrease of retinol concentration CRBAP – cellular retinoic acid binding protein - Controlling the ratio free retinol/free retinoic acid Retinoid binding proteins Retinoid fate in the cells Gene expression 1212569_21823227.jpg logo_mu_cerne.gif •Isoforms of RAR a RXR –Formation of homo- and heterodimers –48 possible RAR-RXR heterodimers –à sensitive regulation of gene expression •RXR – heterodimers with other receptors –VDR, TR, PPAR ... à see crosstalk • •RETINOIC ACID (RA) •3 basic subtypes –all-trans- (ATRA) –9-cis- and 13-cis-retinoic acid •All-trans RA (ATRA) binds selectively to RAR •Cis RA bind to both receptor types • • RAR/RXR and RA 1212569_21823227.jpg logo_mu_cerne.gif Disruption of retinoid signalling by xenobiotics •Possible modes of action – disruption of retinoid signalling: –Metabolization of retinoids by detoxication enzymes –Disruption of binding retinoids to transport proteins –Retinoids as antioxidants may be consumed by oxidative stress induced by xenobiotics – Interference during binding to RAR/RXR • •Effects –Decreased retinoid levels in organisms •Downregulation of growth factors • Xerophtalmia, night blindness •Embryotoxicity, developmental abnormalities –Increased ATRA concentration •teratogenic effects • • 1212569_21823227.jpg logo_mu_cerne.gif •Polluted areas –mostly decrease of retinoid levels •Documented in aquatic birds, mammals and fish – •Disruption of retinoid transport: PCBs • •Effects on retinoid receptors: –RAR, RXR binding and/or transactivation •pesticides (chlordane, dieldrin, methoprene, tributyltin…) •Effect on ATRA mediated response – TCDD, PAHs • •Disruption of retinoid metabolism: –PCDD/Fs, PAHs, PCBs, pesticides – changes of serum concentrations of retinol and RA – mobilization of hepatic storage forms • Disruption of retinoid signalling by xenobiotics https://encrypted-tbn1.gstatic.com/images?q=tbn:ANd9GcSONXqyh_VVzL77Bnzx_gaG-dJo0ur_N94l61b6So_OJMJ PgwCwk4-BFmU 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 Physiological role of AhR •Physiological role for AhR still not known (?) –Most likely – “protection” against toxicants à induction of detoxification • •Many adverse effects documented in AhR-deficient mice –significant growth retardation; – defective development of liver and immune system; – retinoid accumulation in liver; – abnormal kidney and hepatic vascular structures. –resistant to BaP-induced carcinogenesis and TCDD-induced teratogenesis; –no inducible expression of CYP 1A1 and 2. • • à this implies presence of natural endogeneous ligand(s) (not only exogeneous toxicants can bind AhR) • 1212569_21823227.jpg logo_mu_cerne.gif What is the natural (endogenous) physiological ligand of AhR ? Potential candidate: 6-formylindolo[3,2-b]carbazole (FICZ) 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 1212569_21823227.jpg logo_mu_cerne.gif „Non-classical“ AhR ligands – various structures “Classical” ligand 1212569_21823227.jpg logo_mu_cerne.gif Schmidt & Bradfield, Annu. Rev. Cell Dev. Biol. 12:55 Biological responses to TCDD 1212569_21823227.jpg logo_mu_cerne.gif Toxic equivalency factors (TEF)/TEQ concept •Toxicity of compounds with similar toxicological properties as TCDD (activating AhR) may be evaluated by TEF/TEQ concept –TEF = Toxic Equivalency Factor (“characteristic” of the Chemical) –TEQ = Toxic Equivalent (sum of TEFs x concentrations) • •TEFs are consensus values based on REPs (relative potencies) across multiple species and/or endpoints. –TEFs are based upon a number of endpoints, from chronic in vivo toxicity to in vitro toxicity with the former having the greatest importance in determining overall TEF. • •TEQs provide a simple, single number that is indicative of overall toxicity of a sample (water, sediment, food) containing a mixture of dioxins and dioxin-like compounds. • •The total potency of a mixture can be expressed in TCDD TEQ concentration –i.e. TEQ = concentration corresponding to the effect that would be induced by TCDD • • 1212569_21823227.jpg logo_mu_cerne.gif Eljarrat & Barceló, Trends Anal. Chem.22: 655 Final concentration is expressed as „Equivalents of TCDD“ (e.g. ng TEQ / kg = ng TCDD / kg) Toxic equivalency factors for PCDDs, PCDFs and PCBs: 1212569_21823227.jpg logo_mu_cerne.gif Biomarkers/bioanalytical methods for AhR toxicity •In vivo studies –liver enlargement, reduction of thymus weight, wasting syndrome, reproductive and developmental disorders •In vivo biomarkers –EROD activity, CYP 1A1 and 1B1 expression (discussed in biomarker section) • • in vitro assessment of chemical potencies –EROD (ethoxyresorufin-O-deethylase activity) in cell cultures; –CALUX/CAFLUX assays (luciferase expression – reporter gene assays) –GRAB assay (AhR-DNA binding) –yeast bioassay; –immunoassays; –detection of CYP1A mRNA (qPCR) or AhR protein (western blotting) • 1212569_21823227.jpg logo_mu_cerne.gif CALUX – Chemical Assisted Luciferase Expression DR-CALUX (Dioxin Responsive CALUX) (i.e. Luciferase Reporter Gene Assay with H4IIE.luc cells) In vitro CALUX/CAFLUX assays 1212569_21823227.jpg logo_mu_cerne.gif DETECTION of EROD activity - example 1212569_21823227.jpg logo_mu_cerne.gif Comparing toxicity of compounds à Application in Risk Assessment •Quantification of effects (EC50) •Comparison with the effect of reference toxicant (2,3,7,8-TCDD) • à relative potencies (REPs) to TCDD (= in vitro “Toxic Equivalency Factors” ~ TEFs) 0 20 40 60 80 100 120 1.E-07 1.E-04 1.E-01 1.E+02 TCDD 4´-OH-PCB 79 4´-OH-PCB 3 concentration ( m M) IC50 = 10 pM IEC50 = 2 m M B[a]P B[e]P TCDD: IC50 PAH: IEC50 Relative Potency (REP) = Induction Equivalency Factor IEF = IC50 / IEC50 REP interpretation: How many times is the compound "weaker" inducer than TCDD ? 1212569_21823227.jpg logo_mu_cerne.gif Example - relative potencies of PAHs (two exposure periods) 1212569_21823227.jpg logo_mu_cerne.gif 1212569_21823227.jpg logo_mu_cerne.gif Summary – Nuclear receptors •Important physiological functions, •Important roles in pathologies and chemical toxicity (ENDOCRINE DISRUPTION) • •NRs with well studied roles in toxicity: ER and AhR –Other NRs (AR, RAR/RXR, ThR) – important but less explored • •All NRs share similar structure and mechanisms of action –Act as direct transcription factors on DNA •Natural ligands of NRs are small lipophilic hormones –steroids, thyroids, retinoids –Various regulatory functions –Role in toxicity: NR interact with structurally similar xenobiotics •Various mechanisms beyond the toxicity –Adverse are both STIMULATIONS and INHIBITIONS directly at the receptor site (e.g. “anti-androgenicity) –Additional mechanisms – transport of hormones in blood (Thyroids), metabolism (Thyroids) clearance (Retinoids), heterodimerization and “crosstalk” – •Other key information to remember –REPORTER GENE ASSAYS (principle, use, what is CALUX?) –Characterization of chemical “toxic potentials” •General concept of “REPs” (valid for activation of all NRs) •Specifically for AhR - concept of TEFs / TEQs •