1、INTRACELLULAR RECEPTORS,The intracellular (nuclear) receptor superfamily Steroid hormones, thyroid hormones, retinoids and vitamin D,Intracellular receptor,HYDROPHOBIC: Non-polar molecules Gases Steroids,Regulation of transcription activity,Regulatory mechanisms vary Heterodimeric receptors - exclus

2、ively nuclear; without ligand, repress transcription by binding to their cognate sites in DNA Homodimeric receptors - mostly cytoplasmic (without ligands) RXR-VDR),Specificities of some receptors ,Intracellular signal molecules small, lipid-soluble molecules such as steroid hormones, retinoids, thyr

3、oid hormones, Vitamin D. (made from cholesterol) These molecules diffuse through plasma and nuclear membranes and interact directly with the transcription factors they control.,6,Lipophilic Hormones,Circulation in the blood bound to transport proteins Dissociation from carrier at target cells Action

4、 in the cell - Pass through the cell membrane and bind to an intracellular receptor, either in the cytoplasm or the nucleus - Hormone-receptor complex binds to hormone response elements in DNA - Regulate gene expression,7,Steroid Hormones,STEROID HORMONES: - sex steroids (estrogen, progesterone, tes

5、tosterone) - corticosteroids (glucocorticoids and mineralcorticoids) OTHER HORMONESThyroid hormone, vitamin D3, and retinoic acid have different structure and function but share the same mechanism of action with the other steroids.,BIOSYNTHESIS OF STEROIDS,Endocrine disruption,Interference of xenobi

6、otics with normal function of hormonal system Possible consequences: Disruption of homeostasis, reproduction, development, and/or behavior (and other hormone-controlled processes). Shift in sex ratio, defective sexual development Low fecundity/fertility Hypo-immunity, carcinogenesis Malformations,To

7、xicants interact with hormonal system at different levels,Transport,Metabolization,Interaction with receptors,Stimulation,Suppression,biosynthesis and release of hormones,binding to plasmatic transport proteins,binding to nuclear hormonal receptor (HR),activation of HR (dissociation of associated he

8、at 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 a

9、ctivities,e.g. down-regulation of receptor levels,e.g. modulation of other nuclear receptors (PPAR/RXR, RXR/TR),STEROIDOGENESIS,Mechanisms of steroid hormones signalling disruption,- Nonphysiological activation of hormone receptor (HR) Binding to HR without activation Decrease of HR cellular levels

10、Disruption of the master“ hormones (FSH/LH) Changes in hormone metabolism,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

11、Pesticides + number of unknowns ,ESTROGEN RECEPTOR ERthe most studied target of EDCs,Estrogens: play a key role in female hormone regulation and signalling are responsible for metabolic, behavioural and morphologic changes occurring during stages of reproduction are involved in the growth, developme

12、nt and homeostasis of 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 Synthesis in ovaries DISRUPTION - investigated in aqu

13、atic biota phase II enzymes - UDP-glucuronosyltransferase, GST-Ya, NADP(H):oxidoreductase; other genes - Bax, p27Kip1, Jun B, TGF-b - regulation of cell cycle and apoptosis;,6-formylindolo3,2-bcarbazole (FICZ) potent endogenous physiological (natural) ligand of AhR,Denison defective development of l

14、iver 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.,Schmidt CALUX/CAFLUX assays; GRAB assay (AhR-DNA binding) yeast bioassay; immunoa

15、ssays; detection of CYP1A mRNA or protein,AhR-mediated effects luciferase reporter assay - H4IIE.luc cells,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 wi

16、th the effect of reference toxicant (2,3,7,8-TCDD) Expression as Equivalency Factors ( TEFs),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,?,Cross-talk between estrogen signalling pathways and other receptors,estrogen signalling pathways and other members of n