Natural and environmental estrogens Estrogen receptor: • a member of the nuclear hormone receptor superfamily • a ligand - inducible trancription factor • subtype: ER-a (in breast, ovary, brain, liver, bone and cardiovascular system, adrenals, testis and urogenital tract) ER-ß (in kidneys, prostate and gastrointestinal tract) ER-y (in fish) Ä - -- - G>-ĽZ3 "NNN< ERE h Domains Transactivation Dimerization Nuclear localization Binding of H sp90 Page 1 Estrogens: OH 17-ß-estradiol estriol • 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, development 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 Mechanism of action of the estrogen hormones TARGET CELL plasmatic transport protein ŕ í biological 1 I response I •\= protein new synthesis, proteins ESTROGEN RECEPTORS - ER-a & ER-ß: Cemral nervous system: ERah ER ß Breast: ERüt, ERp Liver :EftQ Bauer ERff. ERp Cardiovascular system: ERah ERp Gast rointestinaJ tract: ERp Uťůgejlitil IŕACll ERüf, ERp Synthesis and metabolism of estrogens CYP11A (20,22-lyaza) mezipraluliy ■ 17a-hydroxypregnenolon-------—■ Dehydroepiandrosteron CYP17 (17a-hya"0!yeza) CYP17 (17,20-lyáza) 1 17a-hydroxyprogesteron - CYP21 -— (21 c^hydroxyláza)—■ 11-deoxykortikostercn 11-deoxykortisol CYP11P -— (11P-hydroxyláza)—■ Kortikosteron 18-hydroxyláza Aldosteron Estradiol ■/ sulfotrasrferází Estron u u Estradiol 17P-HSD 2-OH-estradiol CYP1A1 iYPlgVoH- estradiol fr I Stero i dní sulfatáza Estrcn sulfä \ 16-OH-estradiol biosynthesis and release of estrogens e.g. modulation of CYP11A and/or CYP19 activities binding to plasmatic transport proteins e.g. down-regulation of ER protein levels binding to nuclear estrogenic receptor (ER) activation of ER (dissociation of associated heat shock proteins, formation of homodimers) \ e.g. modulation of orher nuclear receptors (PPAR/RXR, RXR/TR) binding of the activated receptor complex to specific DNA motifs - EREs \ chromatin rearrengement and transcription of estrogen-inducible genes effects at the cellular, tissue, organ, organism, and/or population level Cross-talk between estrogen signalling pathways ____________and other receptors________________ • estrogen signalling pathways and other members of nuclear receptor superfamily • estrogen signalling pathways and AhR • estrogen signalling pathways and receptors for EGF and insuline Page 2 Environmental estrogens (xenoestrogens, exoestrogens) • are a diverse group of substances that do not necessarily share any structural resemblance to the prototypical estrogen (17ß-estradiol) but evoke effects resembling those of estrogen • estrogenic substances (estrogen agonist) • estrogon-like substances • ANTI-estrogenic substances Exoestrogens - Relative Potencies to bind to ERa (REPs) Chemical group Substance REP Endogenous hormones Estradiol 1 Estriol 6,3.10"3 restosteron 9,6.10"" Phytoestrogens Cuomestrd 6,8.10"3 Genistein 4,9.10"4 Pesticides o,p'-DDT 1,110 6 PCB s 2,4,6-trichlorbiphenyl-4 '-d 1.10"2 2,5-dichlorobiphenyl-4 '-d 6,2.10"3 3,3',5,5'tetrachlorobiphenyl-4,4'-diol 1,6.10"4 alkylphenoles 4-tert- oktylphend 3,6.10e phthalates butylbenzylphthalate 4.10e REP (RElative Potencies) of selected compounds related to 17-ß-estradiol derived from reporter yeast assay Page 3 Exoestrogens - examples (1) Natural products genistein naringenin coumestrol zearalenone Industrial chemicals BisphenolA m / :"' Nomonic surfactants / ,1,^^- Pthalate esters y enjjosulfan \ CH, b-iiphcn»! A Environmental pollutant DDT kepone PCBs/OH-PCBs PAHs and dioxins Ô Ctfe CH3 DEHP Pharmaceuticals Ethinyl estradiol Diethylstilbestrol gestodene norgestrel Toxicity assessment - in vivo and in vitro methods Away íreF-i Exposure [J|íc Detect* ť ň-JcprrtdL-nt ,i|_ľi ii ■■ ' Mwls ivm. E R-dependent agrntí? CH st Ingush« ■t"_■■■»■«■ PhumULrokiiKEi inj rnutilMJlisi included0 tt-cťeplcw-hii^íd avmjí. Kcctptor binding n-izay (27> KůOťpKirüctivaiKin nu; [íl-H) ln vinei ííKťisen-icruljtcJ renins: t^nyn Ml.'J-ľ lľII pľoliľu'diiii-i ,ir-'.i\ i-l!'-InduCliim asüjy* (4ťL4B) DNA tryrtlus^ assay* \41\ In vivo ch[ri)^iMVŕcuulu.kd r^.i.iľiie jmjv;. UtiTulraphn: rcspunw iusny Ct9> Vŕi^ihal conilfcnäoti ai^íiy I5»~i> Ulrríne ľlmd imbibilinn (M] Cell lys&le Cells Ír ihm Cells in tíiio CtLľi in vili« tells in y\W Whole nnimul WlhíL- :........| Whnle íiflĹnuil Whnle sníma] Whulé «linul Milt«J lt*3LH Winsle iinima Ves ťci ťu Yl". Yw ťcs ÍL'-.;■, Yea Yt-i \i: X,: Nm Vi Ltmtod I ■ m i ■■_ lI Limned I i n- i :■_■.! Ü |tl It'll Liiiiied ■-;-. Vft Ves" Ver Ves" Ves" V-V-s ■i .-i-V.'.' ^^^ 1- Ne Nu fíc Níi No Ves V« Yei Y« Y«s [ffo Y/« |n viifo ovacím steiuM assay <55i Fn tňaovBiian ;itertwd ussay ^^t^) 1 ľiL-i. i-.....i m" : r:r;i;'iuii-.<\ rc ľ| L11 r ^ -. iinc iir' :l.1i1i[Íiim.iI _u.rn.Lps wich iľ:i Ili:i1erií|l + ŕ-i.irjjiľit In vitro assay • competitive ligand binding assay • cell proliferation assay • endogenous protein expression (or enzyme activity) assay • reporter gene assay In vivo assay • uterotropic assay • vaginal cornification assay • standard test procedures for reproductive and developmental toxicity (e.g. FETAX) • production estrogen-inducible proteins (e.g. vittelogenin and zona radiata protein) Page 4