INTRACELLULAR RECEPTORS MECHANISMS of chronic toxicity Various chronic effects have uniform biochemical basis WĚ ď(ÄT5) ^^ Biochemical effects In vivo effects * ľ 111 rLlĽClIlI III I - Coll S urine c INTRACELLULAR RECEPTORS (for lipid soluble messengers) function in the nucleus as transcription factors to alter the rate of transcription of particular genes >ligand-activated transcription factors >crucial role in cell signaling >activation of different responsive elements (genes) SINGLE mechanism - > SEVERAL effects => understanding to mechanisms may predict effects Estrogen receptor activation J 1) female reproduction disorders 2) malefeminisation 3) tumor promotion 1 4) immunomodulations 5) developmental toxicity Types of signaling in multicellular organisms Secretory cell unit f1 /-""NSecret Gap Junction . "■*' Adjacent target cells (a) Direct contact (b) Paracrine signaling Hormone secretion into blood by endocrine gland Neurotransmitter V/Í.;p Target Nerve cell W 9 Bloodvessel - f _ Distant target cells Synaptic gap (c) Endocrine signaling (d) Synaptic signaling Modes of cell-cell signaling 1. Direct cell-cell or cell-matrix 2. Secreted molecules. A. Endocrine signaling. The signaling molecules are hormones secreted by endocrine cells and carried through the circulation system to act on target cells at distant body sites. B. Paracrine signaling. The signaling molecules released by one cell act on neighboring target cells. C. Autocrine signaling. Cells respond to signaling molecules that they themselves produce (response of the immune system to foreign antigens, and cancer cells). Intracellular signal molecules are 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. .3s 1-0 Retinole ■ i:. ■■. O-i /-°-\ /-CHľ-CH-c' The intracellular (nuclear) receptor superfamily Steroid hormones, thyroid hormones, retinoids and vitamin D Cortisol ......Dim estrogen receptor progesterone receptor vim minO receptor thyroid hormone receptor rňlirVJiC ícid rícíplOr ONA Ijindiní] důmSi inhibitory"" proteins ,B> INACTIVE R EC EPTOH trsriKripiiciftol target genes Kl ACTIVE RECEPTOR : ■•■'• i-- •- ■■■ " ■■___i_________________;________Li_l_Li_____ Intracellular receptor ' Cortisol Transcription InhibitdKt activating nt)(2-^^W Signal molecule binding domain DNA binding site blocked domain DNA binding site exposed^^- HYDROPHOBIC: - Non-polar molecules Intracellular Receptors alter gene expression ľ;/í'l'; :;.TP-Iň HOFMntiF HhíWjr; }FlFASFF; ňlMTWfi Ol ůCTrťĚ flFCĚPTOP COMtíí IM CYTOSOL hip« ANO EXPOSES NUCLEAR TO DNA ACTIVATES TfiANÍCAVNC N LĎ0UI2A'|QN SIGNAL Sequence similarities and three functional regions 1 J 1 ■■'■■ ■ 1 _JMt t| .M 1 \rl, ■ | _J \VM N-l II 1 r- / / \ Estroůen *sesů[or (Efl h Frúg«BMrů*i6 rnůnp[ůr í PR| GlucKcriioaid racaplcr iGflf Thyroiťina icfepioi ÍTHJ RaEmaic acid rocoploT IňAHI ]— Ľ Gnnnral primarv slrurturo Amino acid DNA-bii^diUů Uoind-Mfldlno. riísmiiirnee na| tlcmíHn L22S-2Í5 vol 42-34% IB-B7% ■ N-terminal region of variable length; in some receptors portions of this region act as activation domain ■ At the center, DNA binding domain, made of a repeat of C4-zinc finger motif ■ Near the C-terminal end, hormone binding domain, which may act as an activation or repression domain. • Steroid hormones 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 heterdimer is RXR monomer (i.e, RXR-RAR; RXR-VDR) Regulation of transcription activity Regulatory mechanisms differ for hetero-dimeric and homodimeric receptors Heterodimeric receptors are exclusively nuclear; without ligand, they repress transcription by binding to their cognate sites in DNA Homodimeric receptors are mostly cytoplasmic in the absence of ligands Hormone binding leads to nuclear translocation of receptors Absence of hormone causes the aggregation of receptor as a complex with inhibitor proteins, such as Hsp90 Endocrine System Endocrine System-; The endocrine system includes all the organs and tissues that produce hormones • Includes endocrine glands, which are specialized to secrete hormones • Also organs, like the liver, that secrete hormones in addition to other functions A hormone is a chemical that is secreted into extracellular fluid and carried by the blood - can therefore act at a distance from source - only targets with receptor can respond Hypothalamus-pituitary-gonad Axis (Control System) Gonadotropin- Anterior Hypothalamus —* releasing------•" pituitary —.. Negative* feedback Q Negative { ^feedback hormone (GnRH) Inhibits secretion of GnRH Inhibits responsiveness to GnRH Gonadotropins (FSH and LH) Sex steroid------------- hormones (estrogens and androgens} Gonads Steroid hormones synthesis - upstrearr s.gnals Luteinizing Hormone (LH) - stimulates progesterone and testosterone Adrenocorticotropic hormone (ACTH) - stimulates Cortisol Follicle Stimulating Hormone (FSH) -stimulates estradiol Angiotensin - stimulates aldosterone Hypothalamo-pituitary axis - Regulation of hormone synthesis - Hypothalamus -Gonadotropin releasing hormone (GnRH) - Pituitary-folicle stimulating (FSH) and luteineising hormone (LH) ^ oestrogais pragestsonis-Iiiliibin Feedback Mechanisms • For hormone secretion regulated .«> hypothalamus J ^ - - - - by the negative feedback loop: when hypothalamic hormone gland X releases + hormone X, this H ,. stimulates target cells to release hormone Y. When anterior piltiitary liormone there is an excess of hormone Y, gland X píripľwiďl endocrine \ "senses" this and v. gland J inhibits its release of peripheral gland hormone hormone X. 1 targets Lip Lipophilic ho hormones the thyroic -As well as Cortisol (Hydrocortisone) CH2OH C 0 HO^Ä^s-OH J3ť ophilic Hor rmones include (derived from c hormones (tyre > the retinoids, c Testosterone OH H3C 1 nones the steroid holesterol) and )sine + iodine) )r vitamin A Thyroxine 1 1 COOH HO -\f-°-\ )-CH2-CH 1 1 NH2 18 Lipophilic Hormones These hormones circulate in the blood bound to transport proteins - Dissociate from carrier at target cells - 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 1. Hormone passes through plasma membrane I Lipophilic hormones Plasma membrane 2. Inside target cell the hormone binds to a iptor p rote i the cytoplas Steroid hormones - a ■S:er:j I -:_<'rcres. sre <:-.&.' ve;l fľ:jT ■:."■:: e&:eľ:jl neta ::>■:: \s.n in n t^hüiv: n.: Cortisol awn TheünminantglLoaGortiHncl p tmy if la: in. e Elites h bcc o^sŕ^w1^ Estradiol Ar EE-lrc : en p" rc.p.i :4 í: cpa fenalE Hí T jduce-d n the f'-šLAw Progesterone -i:: . ::il" : r c-e :ne■"c :■"& Testosterone An androgen, mile sa> ■:rr:-e *>-!-« :;: ■ -.-■= lEEtG-E from progeElerrne. ^6 5 k ■■ : e:; ■iicinc-.r/ rale : + ■:■■■?■:■* stira. Pregnenolone l.'ľ.c-í d -ťc: /-■:■ ■ :li-: =■;:; ■: . ifi= f=;- = ;r ->: =■■:. Is :-: ■; Steroid Hormones They include sex steroids (estrogen, progesterone, testosterone) corticosteroids (glucocorticoids and mineralcorticoids) Thyroid hormone, vitamin D3, and retinoic acid have different structure and function but share the same mechanism of action with the other steroids. ■ Steroid hormones and thyroid hormone diffuse easily into their target cells ■ Once inside, they bind and activate a specific intracellular receptor ■ The hormone-receptor complex travels to the nucleus and binds a DNA-associated receptor protein ■ This interaction prompts DNA transcription to produce mRNA ■ The mRNA is translated into proteins, which bring about a cellular effect - xxSé xä-^í* „«s0 Endocrine disruption • Interference of xenobiotics with normal function of hormonal system Possible consequences: Disruption of homeostasis, reproduction development, and/or behavior. Shift in sex ratio, defective sexual development Low fecundity/fertility Hypo-immunity, carcinogenesis Malformations ^__^_^_^_^^_ 4 Interaction with hormone system Synthesis * Transport Stimulation Interaction with receptors Suppression * Metabolization biosynthesis and release of hormones STEROIDOGENESIS Leg. modulation ofCYPHA and/or CYP19 activities | binding to plasmatic transport proteins | e.g. down-regulation of receptor levels binding to nuclear hormonal receptor (HR) activation of HR (dissociation of associated heat shock proteins, formation of homodimers) e.g. modulation of other nuclear receptors ______(PPAR/RXR, RXR/TR)______ 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) Mechanisms of steroid hormones signalling disruption - Illegitimate activation of hormonal receptor (HR) - Binding to HR without activation - Decrease of HR cellular levels - FSH/LH signalling disruption - Changes in hormone metabolism Endocrine disrupters in the environment? EDCs., POPs and their metabolites steroid hormones and their derivatives from contraception pills alkylphenols organometallics (butyltins) pharmaceuticals pesticides Estrogens: 17 - fi-estrad i o I 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 Estrogen receptor: • a member of the nuclear hormone receptor superfamily • a ligand - inducible transcription 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 - - - - ÍIW <^> TV ans activation = Dimerization ■^ Nuclear localization i ■ Binding of Hsp90 ESTROGEN RECEPTORS - ER-a & ER-ß: Central act vous system: ERtih ER p ürcaa; ERůr, ERß ^>^^ Cardiovascular synem: ERah ERß G asi r «intestinal traa: ERp Bane: ERof. ERß i -líc í. i: r i =■ Mechanism of action of the estrogen hormones estradiol TARGET CELL plasmatic transport (T biological response ^ protein new synthesis, Synthesis and metabolism of estrogens ľŕPiľ i17o-hydro*/pregnendon -----—■ Dehydroepandrateron CYP17 ■ 17a-hyct"oxyprogesteron - . Androstendion CYP21 - (21ouhydro»/láza) —■ 11-deoxykortkosteron 11-deoxykortisol CYPľP ■— (11P-hydro>yláza)—- Kotli kost era n 8-hydroxyláz Strado. 1, 1"«° iLlfotrasrferázav' .L. „l. Íl Steroicťii sulfataza Estron siJfä I CYpTM Estradol 'CYHftohiestradid \ 16-OH-sstradiol 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) • ANTI-estrogenie substances Exoestrogens - examples 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 o^y- $0- Pharmaceuticals Ethinyl estradiol Diethylstilbestrol gestodene norgestrel Exoestrogens - Relative Potencies to bind to ERa (REPs) Chemical group Substance REP Endogenous hormones Estradiol 1 Estriol 6,3.10"3 ľestosteron 9,6.10"6 Phytoestrogens Cuomestrd 6,8. ÍO"3 Genistein 4,9.10"4 Pesticides o,p'-DDT 1,1.10"6 PCB s 1,4,6 - tri chl orbi ph enyl - 4' - d 1.10"2 2,5-dichlorobiphenyl-4'-ol 6,2.10"3 i.3',5,5'tetrachlorobiphenyl-4,4'-diol 1,6.10"4 alkyl phenol e s 4 - ter t-okty lphen d 3,6.10"e shthalates Dutylbenzy lphtha lat e 4.10"6 REP (RElative Potencies) of selected compounds related to 17-ß-estradiol derived from reporter yeast assay Toxicity assessment - in vivo and in vitro methods tav M] !:>|TiiMin ijpe Flt-ili-|HiuL ' ER Jcpfndejit .ii l.u.in'.M wJ FneiibcLi« iruluLW K.e«pwr h«iJii-.Ľ iissiy [27l BswpKír uclivaurm iwuy |Í2-M] lii «iiii>*íii(íiísii-itřuljtcd fe^mw Mfiayí MCF-7 ľcI] prdifeniliurí uttuy Cil) InJuCiiun aiKii,-. (4MS) PNA sviuhésis asijijh |J7| Iľi u^i L-.lľii^ĽII-rťUllIiil-Jil l\js.>.ilľii: js:,.i^ L=:lr-.>lrop>ni■,■ rci-p'iiv: ,'.---í.i> l-ľ-l■ Sf jl ■.■• ■•!■: • -■. •-. . '« . Vaginal opening <] h Uteri« Hui.iimbit.ili™ íll| Cell lysHlt Cell« in vilKi CellHirivino {elk il v It r-, Cell« in vHm Whole unimul Wlwfc ai-iiriiil tfHOliillilV.il Whnk unimul Whole HnlrtUl Mined liaaiu «v-i,.|,' jľiiva Yen Vei Ves Vim V«. Ye« Ye* V.-. Ye > Yen \i> V 1 in ik-il .....ilL-ll .....'I'll LimitSiJ Y& Tí-.-Yes Y«." Yti" Vn-■,,■■; ■,.■■;' V.-.1 Ya 1,- ho Nu No Yb Yct V eh Ves Yrs Vrh Inhibition of ^rmjij synthesis assay« lii vino sr-arlan Miiiiitl assay I5.li F* vivo overran steroid assay l^fil 'Ceietľíinťi nf aniapifiiaH irquires lnč uk :iddi umal gmmjis *iiri if ■WwJW. In vitro assay • competitive ligand binding assay • cell proliferation assay • endogenous protein expression (or enzyme activity) assay • reporter gene assay In vitro ER- mediated effects luciferase reporter assay or x en oestrogen ----------y^^ © í y leb LebJo® ER- mediated effects luciferase reporter assay Exposure (6 - 24 h) standards / samples 96 microwell plate cultivation of transgenic cell lines ER: breast carcinoma MVLN cells SIMILAR DESIGN FOR OTHER RECEPTORS: AhR (H4IIE.1UC cells) AR (MDA cells) RAR/RXR(P19cells) Cell lysis -> extraction of induced luciferase —| i i L Luminescence determination 'Lutnino (microplate luminescence reader) In vivo assay • uterotropic assay • vaginal cornification assay • standard test procedures for reproductive and developmental toxicity (e.g. FETAX) • production of estrogen-inducible proteins (e.g. vittelogenin and zona radiata protein) ANDROGEN RECEPTOR (AR) Androgen rwepior DNA ending domain. -Ä- 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 r ormones -testosterone - dihydrotestosterone (DHT) -androstanediol - dehydroepiandrosterone -androstenedione Testosterone Hypothalamo-pituitary axis ■ Folicle stimulating hormone - Stimulates synthesis of androgen binding proteins and spermatogenesis in Sertoli cells (testis) ■ Luteineizing hormone - Stimulates testosterone production in Leydig cells ^ oestrogais ftfŮTjKtetOIW?— Inlilbin Testosterone Testicular Biosynthetic Pathway of Testosterone Cholesterol 1 PJMscc Pregnenolone ------► Progesterone 1 ITa-Hydioaylise 17a-Hydroxypregnenolone— -----► ^a-Hytíro^yprogesterorie Dehydro epjandrostersne Androstenedione 1 1 T|UHy-d rasysterotd T dehydrogenise | Sjí-HydroKysteroid dehydrogenase - synthetized in testis (Leydig cells) - in lesser extent in adrenals Dihydrotestosterone ■ The most important derivative of testosterone ■ Formed extrátesticularv from testosterone ■ 5a-reductase 8 Dihydrotestosterone - In several tissues (seminal vesicles, prostate, skin) higher affinity to androgen receptor than testosterone - Daily production 5-10% of testosterone Mechanisms of androgen signalling disruption Binding to AR - Mostly competitive inhibition - xenobiotics do mostly NOT activate AR-dependent transcription - Few compounds are able to activate AR in absence of androgen hormones x in presence of T/DHT antiandrogenic (metabolites of fungicide vinclozoline, some PAHs) FSH/LH (gonadotropins) signalling disruption - FSH/LH expression - regulation via negative feedback by testosterone - Suppressing leads to alterations of spermatogenesis Mechanisms of androgen signalling disruption Alterations of testosterone synthesis - Inhibition of P450scc needed for side chain cleavage of cholesterol (fungicide ketoconazol) - Inhibition of 17- a-hydroxylase and other CYPs — enzymes needed for testosterone synthesis (ketoconazol) Testosterone metabolic clearance - Induction of UDP-glucuronosyltransferase or mono oxygenases CYP1A, IB involved in androgen catabolism - Pesticides 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 position of the urethral opening on the penis) - vagina development - undescendent ectopic testes - atrophy of seminal vesicles and prostate gland Effects of male exposure to antiandrogens Exposure in prepubertal age: - delayed puberty - reduced seminal vesicles - reduced prostate Exposure in adult age: - oligospermia - azoospermia - libido diminution AR-binding - potencies (Ret DHT EC50 ~ 0.1 uM) Compound IC„,(nM) B enz [a] anthr ac ene 3.2 Benzo[a]pyrene 3.9 Dimethylbenz[a]anthracene 10.4 Chrysene 10.3 Dibenzo[a,h] anthracene activation in range 0.1-1 OuM Bisphenol A 5 vinclozolin metabolites 9.7 hy droxy flutam ide 5 Aroclor typical values 0.25-1.11 Individual PCBs typical values 64-87 ŕhs-(4-chlorophenyl)-methanol 0.2 Antiandrogenic compounds /rá-(4-chlorophenyl)-meťhanol - Ubiquitous contaminant of uncertain origin - Probable metabolite of DDT-mixture contaminant - Levels in human blood serum cca. 50nM - EC50 - cca. 200nM In vivo antiandrogenicity assessment Hershberger assay - castrated rats treated with examined substance - Endpoint - after 4-7 days - seminal vesicles and ventral prostate weight Measurement of testosterone concentration in serum In vitro antiandrogenicity assessment Most often employed - prostatic cell lines Cell proliferation assays - cell lines with androgen-dependent growth; - Treatment with tested chemical only (androgenicity) or cotreatment with DHT (antiandrogenicity) - mammary carcinoma cell lines - prostatic carcinoma cell lines In vitro antiandrogenicity assessment Receptor-reporter assays - Gene for luciferase or GFP synthesis under transcriptional control of AR - Luciferase: - AR-CALUX (human breast carcinoma T47D) - PALM (human prostatic carcinoma PC-3) - CH0515 (Chinese hamster ovary CHO) In vitro antiandrogenicity assessment GFP - Possibility of nondestructive measurement (fluorescence of intact cells) X Less sensitive - lack of enzymatic amplification - Human prostatic cell lines Yeast assays - Mostly B-galactosidase as reporter enzyme - Easy cultivation and experimental design X - Cell wall may obstruct transport of chemical into cell=> => false negatives 10 Thyroid hormones Thyroid hormones Thyroxine Triiodothyronine Calcitonin Play crucial roles in stimulating metabolism and influencing development and maturation 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 The Thyroid Gland Thyroid hormones bind to nuclear receptors - regulate carbohydrate & lipid metabolism - adults with hypothyroidism have low production of thyroxine - reduced metabolism and overweight - adults with hyperthyroidism have high production (excessive secretion) of thyroid hormones (thyroxine) - high metabolism and weight loss - trigger metamorphosis in amphibians Effects of thyroid disruption Thyroid hormones - if absent during fetal development or for first year: - nervous system fails to develop normally - mental retardation results In prenatal development - severe damage of CNS (cretenism, delayed eye opening, cognition) Megalotestis Histological changes in thyroid gland (goitre) if T4 concentrations decline before puberty: - normal skeletal development will not continue Thyroid hormones Thyroxine (T4) Triiodothyronine (T3) Also called tetraiodothyronine Contains 3 iodide ions Contains 4 iodide ions i - T4 - prohormone - 5'-deiodination leads to active form, T3 Thyroid hormones Enter target cells by transport system Affect most cells in body T4 and small amount of T3 produced in thyroid gland Most T3 produced by deiodination in target tissues (deiodinases) - T4 synmesis - iodination of tyrosin residues on tyreoglobulin - coupling of two iodotyrosines conducted by thyroid peroxidase 11 r Pituitary-thyroid axis - Regulation of thyroid synthesis - Control the secretion of thyroid T T3+T4 A -co hormones through two negative feedback loops - Pituitary TSH (thyroid stimulating hormone) stimulates both I" uptake and iodination of tyrosine resides on Tg Enzymes involved in thyroid metabolism „outer" ■ Thyroid peroxidases - iodination of tyrosyl residues - coupling of iodinated tyrosyl residues ■ Thyroid deiodinases „inner - D1, D2 - activation of T4 into T3 via deiodination on „outer" ring (formation of T3) - D3 - deactivation into rT3 via deiodination on „inner" ring Thyroid receptors - Mechanism of action Thyroid hormones bind to receptors in: > cytoplasm > surfaces of mitochondria > nucleus Alike other nuclear receptors - 5 isoform s of TR - After activation formation of homo- andheterodimers - Binding to thyroid responsive elements (TRE) - Gene expression 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 (75%) Competitive binding to thyroid binding 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 thyroid gland (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 don't bind to TR (DDT, PCBs) or bind with much lesser affinity than T3 (OH-PCBs - lOOOOx) Accelerated depletion of TH >UDP-glucuronosyltransferase - detoxication enzyme (II.biotransformation phase) > Induced by PCBs, dioxins > Key enzyme in thyroid catabolism Mncreased by disruption of TBP binding 12 In vivo assessment ■ TH serum levels - simple, nondestructive x variation wimin time of day, age, sensitive to other man 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 synthesis) ■ Hepatic UDP-glucuronosyltransferase activity (marker of enhanced TH clearance from serum) In vitro assessment Enzyme inhibition assays (thyroidperoxidase, deiodinases) — assessment of myroid metabolism Competitive binding assays with TBP TH- dependent proliferation assay (pituitary tumor GH3, myroid tumors like FRTL-5 cell line) or TSH-dependent proliferation assay (myroid tumors) Receptor-reporter gene assays wim luciferase (monkey kidney CV-1, Chinese hamster ovary CHO or insect Sfô cell lines) Retinoids Vitamin A and its derivatives Retinoids Regulation of development Important for cell growth, and homeostasis in tissues of apoptosis and vertebrates and invertebrates differenciation Development of embryonic, epithelial cells (gastrointestinal tract, skin, bones) Necessary for vision Suppressive effects in cancer development Antioxidative agent Affect nervous and immune function Retinoids Sources: from diet (dietary hormones) Retinyl esters - animal sources Plant carotenoids ß-karoten Bond cleavage Retinol (vitamin A) Retinoic Acid RE: Retinol-Ester R: Retinol RBP: Retinol Binding Protein (LMW) TTR: Transthyretin (HMW) CHBP !-R RBP RE BE re 13 Retinoid binding proteins CRBP- cellular retinol binding protein - binding of retinol, immediate decrease of retinol concentration CRBAP - cellular retinoic acid binding protein RAL - Retinal - Controlling ratio free retinol/free retinoic acid v Mode of action w ■ - IsoformsofRARaRXR w X - Both have isoforms a, ß and y, each of them several subtypes Exprese - Formation of homo- and heterodimers genů - 48 possible RAR-RXR heterodimers =>sensitive regulation of gene expression - RXR - heterodimers even with other receptors like VDR, TR, PPAR Retinoic acid - 3 basic subtypes - all-trans-, 9-cis- and 13-cis-retinoic acid - All-trans RA binds selectively to RAR - Cis RA bind to both receptor types Disruption of retinoid signalling by xenobiotics ■ Relatively little is known ■ Possible modes of action: - Metabolization of retinoids by detoxication enzymes - Disruption of binding retinoids to retinoid binding proteins - Retinoids as antioxidants may be consumed cause of oxidative stress caused by xenobiotics - Interference of chemicals (binding to RAR/RXR) Consequences of retinoid signalling disruption Decreased retinoid levels in organisms - Downregulation of growth factors - Xerophtalmia, night blindness - Embryotoxicity, 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 fish Disruption of retinoid transport: PCBs Effects on retinoid receptors: - RAR, RXR binding and/or trans activation - pesticides (chlordane, dieldrin, memoprene, tributyltin...) - Effect on ATRA mediated response - TCDD, PAHs Disruption of retinoid metabolism: - PCDD/Fs, PAHs, PCBs, pesticides - changes of serum concentrations of retinol and RA - mobilization of hepatic storage forms - in kidney, concentration of all forms elevated Tests to assess retinoid signalling disruption In vivo - Mostly derived from classical toxicity tests, particularly of developmental toxicity - Direct measurements of various retinoid forms in living organisms (laboratory and wildlife) In vitro - Mostly epithelial cell lines (keratinocytes) - Mouse embryonic cell lines P19 pluripotent cells differentiation dependent on circumstances, triggered by ATRA - reporter gene assay PI 9/A15 - Other cell lines - rainbow trout gonads, human salivary gland, breast or prostatic carcinomas etc. AhR (Arylhydrocarbon receptor) AhR structure S ?? Physiological role for AhR —► Effects 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. AhR • ligand-activated transcription factor • activation of different responsive elements (genes) • important mediator of toxicity of POPs - primary target of coplanar aromatic substances • regulator of xenobiotic metabolism and activation of promutagens • crossactivation/crosstalk with other receptors • strongest known ligand TCDD Biological responses to TCDD Schmidt & Bradfield, Annu. Rev. Cell Dev. Biol. 12:55 ľpilhtlijl h ypc rp I nx i d I Tumor ptrimnCin« | Mick 11k lign ill-: IVirpfivril Lŕ« r*»H!j «mm llivroJLioe — W*HÍIIE - Míliih^lijiň (ir^ŕ*í}iU«iit *tid or> tufilflgiciElt i: Iti c pniducci totelifecfedby rCDD, zzfak: Pcrsdcn' iJij hiľI bornior Kcepuf aelLvaiiiMi I- C i P rrtrpuir il.^mi-rĽjjul.iiii* Lipid cvraiiiibliún 1 W I------- J 1_J *......| IcraLMť^r ĽUlUállÚľt ť ľ tfC*iH iJippM LÍS5UC fJJÍtlHHI ťjfuUÍ d> S fHilt lifiň I>1> A wide «*lfetV rf Ľclhdiir iin AhR = cytosolic helix-loop-helix/PAS protein PAS proteins: lkL«f( n__ r *ui _"T. t_lÜB BUH Basa ^^M »«'■E3ZI m '■'«hn» , 1- — 41 1 V * . AhR domain structure: iHLlC PAfi Dcoftiü 1 11— 1 HB2 1 |q| m@H '|0 tich COOK HTÄ NES umetiviLiún Bad/. Interact. 141: 3 Binding AhR: Kŕ r. t j ř"o™ DRB »t ion Tí VK ÉQimBLt ion ■it. ľ. Demiia mcaie nif the A h K Denison et a/., Chem. 15 AhR activation: K1 ťr-Vf HV I> AhR regulated genes: contain xenobiotic response elements (XRE) or dioxin responsive elements (DRE) in their promoter region: • phase I enzymes - CYP 1A1, CYP 1A2, CYP 1B1; • phase II enzymes - UDP-glucuronosyltransferase, GST-Ya, NADP(H):oxidoreductase; • other genes - Bax, p27K'1, Jun B, TGF-ß - regulation of cell cycle and apoptosis; "< -1 minl-AI.fi l.i...niK.ndl VPHI Inrincrrs 1 J.TJ-I w nrMw^lh^iijft-imiMja :i.4.v.4.'ř-P*mwhtof aNMwtthl j.M,>r I HrWlltrrtflHffBriRiriH i «waryMwiih»*w-imto p$ o$$> %o .^MrtrUWrM^ftlhfrrW Denison &Nagy, Annu. Rev. Pharmacol. Toxicol. 43:309 „Non-classical" AhR ligands .IfA ľVŕŕj ihr rr dŕ. i Om GRAB assay (AhR-DNA binding) ■> yeast bioassay; ■> immunoassays; ■» detection of CYP1A m RNA or protein Detection of EROD activity: V. T'H -t ill. í rrnlm-ii-|fThF,l,-..riiMJ in.i.v.ji Jn.^h (f? lltVT) JJtJ J"Ü. 4 J 1* £4 40 4S e-j 72 ľtf. 1 Tiitw Ľnuru «f tnJuclinn Lif CVPlA I | * in.|^iiiLj|> i,.|M| 1,-h ul i:k lK|Tiii^>irH. u|iti :^. Írom fsu*r ikícfvnpJiiiL LArvriiiK-nr.-.. 16 AhR-mediated effects luciferase reporter assay - H4IIE.luc cells Estrogens [AhR <-> ER] 0- ® (jispuij) ChŠpsí> (g) I Increased Proteirl\ Modulation of Gene \ Phosphorylation V^ Expression Ackjrted from Bknteiisliip (1994) CALUX/CAFLUX assays 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) Ö Induction Equivalency Factor IEF = ICsn / IECsn How many times is the compound "weaker" inducer than TCDD ? Toxic equivalency factors (TEFJ/TEQ concept: TEFs provide a simple, single number that is indicative of overall toxicity of a sample containing a mixture of dioxins and dioxin-like compounds. TEFs are consensus values based on REPs 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. The total potency of a mixture can be expressed in TCDD TEQ concentration: ThX? — £ {com pound i x Tlíl-1 + , -f- compound,, x TfiF0) Toxic equivalency factors for PCDDs, PCDFs and PCBs: TkMt 4, |.m Ei^4t1 ii i-" ™ľ-v; * rtOBCo^O" toHtym n M r «p» WHQ-lff ŕt k t „r«™. IwHOTV ZÍ7J-ICDD i J : ii n Ol Dl V.....■!.. 1.M7APi«CC ■. i hh-in MM. ;1 |fi acixrt lľMľfrrtKLUO d.l JJJ7»F*CDŕ II-, 11 T.n:- 1 1-1. .-.. . ii.i ,i|. .l! _.: .....: Ml Mill n m» DĽl ll.\7m\t«.Ůt> Ml mffHkOf ■■■ KMMt Ml \2Vb-*l%X 013 am 1 M»+M w ■■■ \*»iii.i"ll.i oa» U I.MfUMIÍ Hli ni.iif, iíj*w*mnijf ..... HVlii IWWI ■-■: ---lljH. Ijl Mill -T ;.^l .-. MOCI '* III r(iii;i KI#15É ÍÍWÍ Eý'arraf & Barcetó, Trends /Ina/. Cnem.22:655 In vitro assays for nongenotoxic effects YiVEL Nuclear Receptors & Signalling Crosstalk poorly characterized (toxicity) mechanisms Nuclear receptors (AhR, ER, RAR/RXR...) = Transcription factors with numerous cofactors and interactions (crosstalk) Cross-talk between estrogen signalling pathways and other receptors ' estrogen signalling pathways and other members of nuclear receptor superfamily ' eslrogen signalling pathways and AhR ' eslrogen signalling pathways and receptors for EGF and insuline 17 In vitro assays for nongenotoxic effects Li>-■■*_= Modulation of RAR/RXR : retinoic acid signalling ATRA - important regulatory molecule : cellular differentiation (embiyotoxicity, teratogenicity), other biological events Concentrations of retinoids are known to be modulated by PCBs (? mechanism) In vitro assay for modulation of ATRA - RAR/RXR effects Luciferase reporter gene assay (embryonic P19/A15) RAR- dependent gene transcription 18