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 09b –Nuclear Receptors AHR – Arylhydrocarbon Receptor OPVK_MU_stred_2 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 •Also known as „dioxin-receptor“ (and its modulation leads to so called „dioxin-like“ activity or toxicity) • •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 completely (?) –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) Tryptofan – Wikipedie 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 & AhR ligands 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 Výsledek obrázku pro benzo a pyrene Výsledek obrázku pro benzofluoranthene Výsledek obrázku pro acenaphthylene 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) „CALUX“ assay Longer period: lower induction due to (partial) metabolization of PAHs (CYPs, oxidation) to products less potent to AhR 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 –in blood (Thyroids), metabolism (Thyroids) clearance (Retinoids), heterodimerization and transport of hormones, “crosstalk” of different NRs – •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 •