1、INTRACELLULAR RECEPTORS,The intracellular (nuclear) receptor superfamily Steroid hormones, thyroid hormones, retinoids and vitamin D,Intracellular receptor,HYDROPHOBIC:Non-polar moleculesGasesSteroids,Regulation of transcription activity,Regulatory mechanisms vary Heterodimeric receptors - exclusive
2、ly nuclear; without ligand, repress transcription by binding to their cognate sites in DNA Homodimeric receptors - mostly cytoplasmic (without ligands) & hormone binding leads to nuclear translocation of receptorsWithout ligand - aggregation of receptor with inhibitor proteins (eg Hsp90),Steroid hor
3、mones are often required to dimerize with a partner to activate gene transcription Receptors for vitamin D, retinoic acid and thyroid hormone bind to responsive elements as heterodimers Second component of the heterodimer is RXR monomer (i.e, RXR-RAR; RXR-VDR),Specificities of some receptors ,Intrac
4、ellular signal molecules small, lipid-soluble molecules such as steroid hormones, retinoids, thyroid 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,Circ
5、ulation in the blood bound to transport proteins Dissociation from carrier at target cells Action 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 g
6、ene expression,7,Steroid Hormones,STEROID HORMONES:- sex steroids (estrogen, progesterone, testosterone)- corticosteroids (glucocorticoids and mineralcorticoids)OTHER HORMONES Thyroid hormone, vitamin D3, and retinoic acid have different structure and function but share the same mechanism of action
7、with the other steroids.,BIOSYNTHESIS OF STEROIDS,Endocrine disruption,Interference of xenobiotics 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, defecti
8、ve sexual development Low fecundity/fertility Hypo-immunity, carcinogenesis Malformations,Toxicants interact with hormonal system at different levels,Transport,Metabolization,Interaction with receptors,Stimulation,Suppression,biosynthesis and release of hormones,binding to plasmatic transport protei
9、ns,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 cellula
10、r, tissue, organ, organism, and/or population level,e.g. modulation of CYP11A and/or CYP19 activities,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 activati
11、on of hormone receptor (HR)Binding to HR without activationDecrease of HR cellular levelsDisruption 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 deriva
12、tives from contraception pills alkylphenols organometallics (butyltins) pharmaceuticals Pesticides + number of unknowns ,ESTROGEN RECEPTOR ER the most studied target of EDCs,Estrogens: play a key role in female hormone regulation and signallingare responsible for metabolic, behavioural and morpholog
13、ic changes occurring during stages of reproductionare involved in the growth, development and homeostasis of a number of tissues control the bone formation, regulation of homeostasis, cardiovascular system and behaviourregulate production, transport and concentration of testicular liquid and anaboli
14、c activity of androgens in malesSynthesis in ovariesDISRUPTION - investigated in aquatic biota & laboratory organisms (see notes on EDCs),ESTROGEN RECEPTORS - ER- & ER-:subtype: ER- (in breast, ovary, brain, liver, bone and cardiovascular system, adrenals, testis and urogenital tract)ER- (in kidneys
15、, prostate and gastrointestinal tract)(ER- in fish),Natural products genistein naringenin coumestrol zearalenone,Environmental pollutant DDT kepone PCBs/OH-PCBs PAHs and dioxins,Industrial chemicals Bisphenol A Nonionic surfactants Pthalate esters endosulfan,Pharmaceuticals Ethinyl estradiol Diethyl
16、stilbestrol gestodene norgestrel,DEHP,Environmental estrogens (xenoestrogens, exoestrogens),a diverse group of substances that do not necessarily share structural similarity to the prototypical estrogen (17-estradiol) May act as AGONISTS and/or ANTAGONISTS,Exoestrogens - Relative Potencies to bind t
17、o ERa (REPs),Toxicity assessment number of in vivo and in vitro methods,Janoek, J., Hilscherov, K., Blha, L., and Holoubek, I. (2006). Environmental xenobiotics and nuclear receptors-Interactions, effects and in vitro assessment. Toxicology in Vitro 20, 18-37.,In vitro assays,INTERACTION (BINDING) t
18、o the receptorcompetitive ligand binding assay Effect unknown (? Activation / suppression / no effect ?)Testing the effect at cellular level (interference with receptor biological activity)cell proliferation assayendogenous protein expression (or enzyme activity) assayreporter gene assay,In vitro ER
19、- mediated effects luciferase reporter assay,ER- mediated effects luciferase reporter assay,96 microwell plate cultivation of transgenic cell linesER: breast carcinoma MVLN cellsSIMILAR DESIGN FOR OTHER RECEPTORS (discussed below): AhR (H4IIE.luc cells) AR (MDA cells) RAR/RXR (P19 cells),Cell lysis
20、- extraction of induced luciferase,Exposure (6 24 h) standards / samples,Luminescence determination (microplate luminescence reader),In vivo assays,uterotropic assayvaginal cornification assaystandard test procedures for reproductive and developmental toxicity (e.g. FETAX)production of estrogen-indu
21、cible proteins(e.g. vittelogenin and zona radiata protein),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,ANDROGEN RECEPTOR (AR) effects
22、known but less explored than ER,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,Androgens,Endogenous ligands androgen hormones t
23、estosterone (T) dihydrotestosterone (DHT) 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 Dai
24、ly production 5-10% of testosterone,Testosterone,Mechanisms of androgen signalling disruption,1) Binding to AR Mostly competitive inhibition xenobiotics mostly DO NOT activate AR-dependent transcription Only few compounds are able to activate AR in the absence of androgen hormones, and these are als
25、o anti-androgenic in the presence of T/DHT (metabolites of fungicide vinclozoline, some PAHs),2) FSH/LH (gonadotropins) signalling disruption less exploredFSH/LH expression - regulation via negative feedback by testosteroneSuppression leads to alterations of spermatogenesis,Mechanisms of androgen si
26、gnalling disruption,3) Alterations of testosterone synthesisInhibition of P450scc needed for side chain cleavage of cholesterol (fungicide ketoconazol)Inhibition of 17-hydroxylase and other CYPs - enzymes needed for testosterone synthesis (ketoconazol),4) Testosterone metabolic clearanceInduction of
27、 UDP-glucuronosyltransferase or monooxygenases CYP1A, 1B involved in androgen catabolismPesticides endosulfan, mirex, o-p-DDT,Effects of male exposure to antiandrogens,Exposure during prenatal development:malformations of the reproductive tract- reduced anogenital distance - hypospadias (abnormal po
28、sition of the urethral opening on the penis)- vagina development - undescendent ectopic testes - atrophy of seminal vesicles and prostate gland,Exposure in prepubertal age: delayed pubertyreduced seminal vesiclesreduced prostate,Exposure in adult age:oligospermia azoospermia libido diminution,Antian
29、drogenic compound,tris-(4-chlorophenyl)-methanol- Ubiquitous contaminant of uncertain origin- Probable metabolite of DDT-mixture contaminant- Levels in human blood serum cca. 50nM- EC50 cca. 200nM,AR-binding - potencies (Ref: DHT EC50 0.1 uM),(Anti)androgenicity assessment,In vivo Hershberger assay
30、- castrated rats treated with examined substance- Endpoint after 4-7 days seminal vesicles and ventral prostate weight In vivo measurement of testosterone blood levelsIn vitro cell proliferation assays cell lines with androgen-dependent growth - mammary carcinoma cell lines- prostatic carcinoma cell
31、 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 cellsbeta-galactosidase reporter,Treatment: tested chemical only - androgenicity Cotreatm
32、ent with DHT - antiandrogenicity,Thyroid hormones,Thyroid hormones,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,Play cru
33、cial roles in stimulating metabolism, development and maturation,HYPOTHYROIDISM,HYPERTHYROIDISM,Thyroid hormones,T4 prohormone5-deiodination leads to active form, T3,Thyroxine (T4),Also called tetraiodothyronine Contains 4 iodide ions,Triiodothyronine (T3),Contains 3 iodide ions Most T3 produced by
34、deiodination in target tissues (deiodinases),Enzymes involved in thyroid metabolism,Thyroid peroxidases iodination of tyrosyl residuescoupling of iodinated tyrosyl residues Thyroid deiodinasesD1, D2 - activation of T4 into T3 via deiodination on outer“ ringD3 - deactivation into rT3 via deiodination
35、 on inner“ ringEDCs - may affect metabolism of these key enzymes,outer“,inner“,Thyroid hormones are transported in the blood by thyroid binding proteins,Regulating free T4 and T3 levels in blood 3 types : Thyroid-binding prealbunin (transthyretin) (20-25%) Albumin (5-10%) Thyroid binding globulin (7
36、5%)NUMBER OF ENVIRONMENTAL TOXICANTS act at transport proteins OH-PCBs, brominated and chlorinated flame retardants, DDT, dieldrin OH-PCBs equal affinity to TBP as T4 and T3 (!)More of free T4 in blood negative feedback to TSH release = increased depletion = increased weight, histological changes in
37、 thyroid gland Observed after exposure to POPs in mammals, birds, fish,Competitive binding to TR,Probably less important than binding to TBP Chemicals that affect thyroid signalling in vivo mostly dont bind to TR (DDT, PCBs) or bind with much lesser affinity than T3 (OH-PCBs 10000x),Accelerated depl
38、etion of TH,UDP-glucuronosyltransferase detoxication enzyme (II.biotransformation phase)Induced by PCBs, dioxinsKey enzyme in thyroid catabolism Increased by disruption of TBP binding,Other possible effects of EDCs on Thyroid signalling,Effects of thyroid disruption,Disruption during prenatal develo
39、pment - severe damage of CNS (cretenism, delayed eye opening, cognition) Megalotestis Histological changes in thyroid gland (goitre),nervous system fails to develop normally mental retardation skeletal development,Assessment of effects,In vivo approaches TH serum levels simple, nondestructive x vari
40、ation within time of day, age, sensitive to other than biochemical stresses Thyroid gland weight and folicular cells number Developmental toxicity assays - delayed eye opening, abnormalities in brain development and cognition, increased testis weight and sperm counts Perchlorate discharge test (TH s
41、ynthesis) Hepatic UDP-glucuronosyltransferase activity (marker of enhanced TH clearance from serum)In vitro Enzyme inhibition assays (thyroid peroxidase, deiodinases) assessment of thyroid metabolism Competitive binding assays with TBP TH- dependent proliferation assay (pituitary tumor GH3, thyroid
42、tumors like FRTL-5 cell line) or TSH-dependent proliferation assay (thyroid tumors) Receptor-reporter gene assays with luciferase (monkey kidney CV-1, chinese hamster ovary CHO or insect Sf9 cell lines),Retinoids Vitamin A and its derivatives Toxicants affect retinoid action but effects are much les
43、s explored,Suppressive effects in cancer development,Retinoids,Necessary for vision,Important for cell growth, apoptosis and differenciation,Development of embryonic, epithelial cells (gastrointestinal tract, skin, bones),Antioxidative agent,Affect nervous and immune function,Regulation of developme
44、nt and homeostasis in tissues of vertebrates and invertebrates,Retinoids,Retinol (vitamin A),Bond cleavage,Retinoic Acid,-karoten,Sources: from diet (dietary hormones) Retinyl esters animal sources Plant carotenoids,RE: Retinol-EsterR: RetinolRBP: Retinol Binding Protein (LMW)TTR: Transthyrethin (HM
45、W),TRANSPORT OF RETINOIDS,RAL - Retinal,CRBP cellular retinol binding protein - binding of retinol, immediate decrease of retinol concentrationCRBAP cellular retinoic acid binding protein - Controlling ratio free retinol/free retinoic acid,Retinoid binding proteins,Mode of action,Isoforms of RAR a R
46、XR Both have isoforms a, b and g, each of them several subtypes Formation of homo- and heterodimers 48 possible RAR-RXR heterodimers =sensitive regulation of gene expression RXR heterodimers even with other receptors like VDR, TR, PPAR,3 basic subtypes all-trans-, 9-cis- and 13-cis-retinoic acid All
47、-trans RA binds selectively to RAR Cis RA bind to both receptor types,Retinoic acid,Gene expression,Disruption of retinoid signalling by xenobiotics,Relatively little known Possible modes of action:Metabolization of retinoids by detoxication enzymesDisruption of binding retinoids to retinoid binding
48、 proteinsRetinoids as antioxidants may be consumed cause of oxidative stress caused by xenobioticsInterference of chemicals (binding to RAR/RXR),Consequences of retinoid signalling disruption,Decreased retinoid levels in organisms- Downregulation of growth factors- Xerophtalmia, night blindness- Emb
49、ryotoxicity, developmental abnormalities X Increased ATRA concentration teratogenic effect,Change may cause severe developmental anomalies (both excess and deficiency),Disruption of retinoid signalling by xenobiotics,Polluted areas mostly decrease of retinoid levels in aquatic birds, mammals and fis
50、h 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 RAmobilization of hepatic storage formsin kidney, concentration of all forms elevated,
