Nuclearreceptors核受体.ppt

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Toxic equivalency factors for PCDDs, PCDFs and PCBs: Eljarrat Barceló, Trends Anal. Chem.22: 655 Final concentration is expressed as ?Equivalents of TCDD“  (e.g. ng TEQ / kg = ng TCDD / kg) Biomarkers/bioanalytical methods for AhR toxicity in vivo: liver enlargement, reduction of thymus weight, wasting syndrome, reproductive and developmental disorders in vivo biomarkers: EROD activity, CYP 1A1 and 1B1 expression in vitro: EROD (ethoxyresorufin-O-deethylase activity)  in H4IIE rat hepatoma cells; CALUX/CAFLUX assays; GRAB assay (AhR-DNA binding) yeast bioassay; immunoassays; detection of CYP1A mRNA or protein AhR-mediated effects luciferase reporter assay - H4IIE.luc cells Light Light Luciferase Nuclear Factors 1 P + AhR HSP90 HSP90 Src HSP90 HSP90 Adapted from Blankenship (1994) DRE - Luc AhR ARNT Modulation of Gene Expression ARNT Src “Activated” 2 P P Cytosolic Proteins Membrane Proteins Increased Protein Phosphorylation Ligand (TCDD) Light Light Luciferase Nuclear Factors 1 P + AhR HSP90 HSP90 Src Src HSP90 HSP90 Adapted from Blankenship (1994) DRE - Luc AhR ARNT Modulation of Gene Expression ARNT Src “Activated” 2 P P Cytosolic Proteins Membrane Proteins Increased Protein Phosphorylation Estrogens [AhR - ER] In vitro CALUX/CAFLUX assays Detection of EROD activity: Comparing compounds- Application in Risk Assessment  Quantification of effects (EC50) - relative potencies Comparison with the effect of reference toxicant (2,3,7,8-TCDD) Expression as 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 AhR-mediated activity (%TCDD-max) concentration ( m M) IC50 = 10 pM IEC50 = 2 m M B[a]P B[e]P TCDD: IC50 PAH: IEC50 Induction Equivalency Factor IEF = IC50 / IEC50 How many times is the compound weaker inducer than TCDD ? Crosstalk in signalling of nuclear receptors Nuclear Recept