INTRACELLULAR RECEPTORS MECHANISMS of chronic toxicity Various chronic effects have uniform biochemical basis <^^^ TOXIN H^RMO^E 2 Types of Receptors Intrac e llular Cell Surface Biochemical effects ^> In vivo effects 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 1) female reproduction disorders 2) male feminisation 3) tumor promotion 4) immunomodulations 5) developmental toxicity Types of signaling in multicellular organisms Gap junction Secretory cell (a) Direct contact Adjacent target cells (b) Paracrine signaling Hormone secretion into blood by endocrine gland Neurotransmitter htm Target Blood vessel Distant target cells (c) Endocrine signaling Nerve cell W£_* Synaptic gap (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. CH2OH Cortisol Retinole acid NH 2

. hypothalamic hormone 4 anterior pituitary hormone peripheral endocrine gland peripheral gland hormone I targets Lipophilic Hormones Lipophilic hormones include the steroid hormones (derived from cholesterol) and the thyroid hormones (tyrosine + iodine) -As well as the retinoids, or vitamin A 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 19 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Blood plasma 1. Hormone passes through plasm| membrane Lipophilic hormones Plasma membrane Receptor 5. Change in protein synthesis is cellular response ~-% 2. Inside target cell the hormone binds to a receptor protein in the cytoplas or nucleus 4. Protein synthesis Cytoplasm Nucleus m RNA |3. Hormone-receptor complex binds hormone response element on DN> regulating gene transcription \ Hormone response element ormones - a review Steroid hormones ore derived from cholesterol metabolism in mitochondria Cortisol The com na^tglucocort :c d r h., r 3 - s Iffr.hs zě: -■:•" :rogestehne in the zojľa •3£;i';u,'?rä oŕthe adrenal cortex, /ruolvec in s ves s adapta: on, elevates h nm: Dressure and NaJ uptake. Inmunomodulaton. Aldosterone Principal nineraloccrtiioic. Frocucedfrom prcgestero-e in the zena giom erutosa of aďena cortex, rases blood pre s su^e a-11 d flu d w unr e, increases Na1 uptake. Estradiol An es tragen ,prnc pa renale s es horn one, produced n:hE oßry, responsible for secondaryrenale sex characteristics. After ti enopau s e estrogen is ř-roduLec iron ^eslos:erone r the adrenal glands. Progesterone Procucedfrom pregnenolone anc se^eled rrcm She corpus lu"eum. Responsible for changes assoc aied with lu^era p*ase o: the mensirual cycle, difle'enr ation factor forma nroary glands Testosterone An a" dreg en, male sex hormone synthesized in he lesw-sfrom progesterone. Responsible for secondary m ale sex character s tics. Pregnenolone rVade directly fron che lessen: the pressor nc ecule Per al CUl C,B and C;, steroids 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 Cholesterol HO &--Methyl group Major Pathways in Steroid. Biosynthesis CH3 Pregnenolone 9=0 17-hydroxy ch3 pregnenolone c=° Dehydroepiandrosterone HO CYP17 3JÖHSD CH3 Progesterone 9=0 17-hydroxy progesterone %% CYP21A2 os CH2OH Deoxv- c=0 corticosterone 11-deoxycortisol c=o CTTP21A2 CH2OH CTP11B1 HO CH-.OH HO Androstenedione 3JÖHED Estrone HSD JCYP19 o Testosterone oh HO Aldosterone HO, Major progestagen Major mineralocorticoid Major glucocorticoid (^species variation} Major gonadal estrogens Major gonadal androgen 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 WPO Hypothalamus I Gn RH pituitary FSH,LH i I gonads / \ gametes sex hormones Interaction with hormone system Synthesis Transport Stimulation Suppression Interaction with receptors Metabolization biosynthesis and release of hormones STEROIDOGENESIS i e.g. modulation of CYP11A and/or CYP19 activities binding to plasmatic transport proteins T e.g. down-regulation of receptor levels binding to nuclear hormonal receptor (HR) i activation of HR (dissociation of associated heat shock proteins, formation of homodimers) I e.g. modulation of other nuclear receptors (PPAR/RXR, RXR/TR) binding of the activated receptor complex to specific DNA motifs - HREs i chromatin rearrangement and transcription of estrogen-inducible genes i effects at the cellular, tissue, organ, organism, and/or population level Mechanisms of steroid hormones siqnallin 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 ESTROGEN RECEPTOR - ER OH Estrogens: 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 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) A/B _________ D_____________ F Ě ^H ^H ^h ^h J c ^"J e ^^h Domains Ä W^!> W& NNN <- V J DNA Transactivation = Dimerization ^ Nuclear localization pop •»•■ ■«■•■> Binding of Hsp90 ESTROGEN RECEPTORS - ER-a & ER-ß: CcniraJ act vúus ^stem:ERtíP !:R|1 Breast: ERür, ERp Liver Efta Boner ER«, ERß Cardiovascular system: EROt. ERß Gastrointestinal tract: ERß Urogenital iraci. HRiX ERp Mechanism of action of the estrogen hormones estradiol plasmatic transport protein biological response estradiol new proteins TARGET CELL NUCLEUS protein synthesis Synthesis and metabolism of estrogens Cholesterol CYP11A (20,22-lyáza) Pregnendon 17a-hydroxypregnendon CYP17 (17a-hydroxyláza) Progesteron meziprodukty CYP17 (17,20-lyáza) Deh yd roepi and rosteron 17a -hy droxyprogesteron Androstendion Testosteron CYP21 (21a-hydroxyláza) I . CYP19 (aromatáza) 2-OH-estradiol 11 -deoxykortikosteron 11-deoxykortisol CYPľP (11ß-hydroxylaza) Estron cypTm Estradiol ^PlSVoH-estradid 17ß-HSD Kortikosteron Kortisd Estradiol 1/ sulfofrasitferázaV // Steroidní sulfatáza Estron sdfát \ 16-OH-estradiol 18-hydroxyláza Aldosteron 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-estrogenic substances Exoestrogens - examples Natural products genistein ^^ oh narmgenm coumestrol zearalenone Environmental pollutant DDT kepone PCBs/OH-PCBs PAHs and dioxins Gl Cl Industrial chemicals Bisphenol A Nonionic surfactants Pthalate esters endosulfan =x CH3 ----- CH3 ----- l>is|>henol A 9H3 C-O-CH.r CH-CH^Q HaCH^CH3 f-O-CH^-CH-ChtCH^CHjCHj Q CH2 CH2 DEHP 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. KT3 Testosteron 9,6.10"6 Phytoestrogens Cuomestrol 6,8.10"3 Genistein 4,9. KT4 Pesticides o,p'-DDT 1,1.10"6 PCB s 2,4,6-trichlorbiphenyl-4 -ol 1.10"2 2,5-dichlorobiphenyl-4 -ol 6,2.10"3 3,3 ,5,5 tetrachlorobiphenyl-4,4'-diol 1,6. KT4 alkylphenoles 4-tert-oktylphenol 3,6.10"6 phthalates butylbenzylphthalate 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 Detects; rtelfťlk I WW Uisti npuisftvs. Phanruiookinetn tiR-dĽpfndcnt EIK-dep^ndem agonal víf^x and rn^tubĽ-Lisn Assuyíľíf.) EupOSyn; (Vpe iigents? iiEeiiK1 anUifiuŕirft'1 irtctuded0 Rc^ept^r-tvised a&mys Receptor binding nissav (27} Lell ly^nle Yen No Mu No RíMplOr Ktivalkin iivtfj (12-34) Cd h in vitŕO Vo, No Ves" Nu viiío-íHif-oiín-ncsulatc^ r^innsti íim-i.-iv:l MCF-7 cell piKjlifcrjtMxi usMiy (41) Cells in viiio Y.- LI mined Yes" IVo iniĽuHitm assays í-lo.4Ä) Cfclls in vitnn Ves Li mi lad VehJ r-,.-. DVA x^fflfH&ifc iiVJ-A |J7t I'líLU i IL V|[]J Whole nnimul Ves Limited Ye*:' Y« Vysmál arfniíkiJiiiurl a^y f5fl) Whole iirmml Ves Limiled Ves" Y«* V;L|ľÍiľi;il i^Ik: i-iiľ.^ {Ill Whole íiíiiľinil Vl-, LimilŕLl Ves" Yes Uti.TiJie fluid initubhion (Ll| Whole Uli i itillI Voh Limiied. Ves* Y« Utenne ep-ilheliuh h\pertnoph\ L í l > Whole anintíl Ves Limiled Ves3 Y*s hioiunm u>f ^ertnd 5i.yrtChnŕsLs assays In viiro ovitfiiAii ^cunid assay í5Ji VHilcaJ ÜSMJe N» Yes Yes No Ei vivo murijin steroid assay (5ťi* Whole ani mul No Vfs v« Yes ■------ ^M------- ^Tl---- ^-------- ■■-------- ^M------ ■ !"■ Wt---- -^— ■ — '^— "Deuecfinri of aisia^onijjs require* iu€ ol" additional group* wich ícsr maieria! + CMrjdiiJ, 5 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 Estrogen or xenoestrogen Nuclear Factors Protein Phosphorylation of ER: Ligand-Independent Activation ERE-Luc DNA Binding \ i mRNA / "Estrogenic Effects" ER-Responsive Genes 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 Cell lysis -> extraction of induced luciferase SIMILAR DESIGN FOR OTHER RECEPTORS: AhR (H4IIE.1UC cells) AR (MDA cells) RAR/RXR(P19cells) : _ iLumino Luminescence determination (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 receptor DNA binding domain. Androgens - Role in males similar to the of estroqens 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 - testosterone -dihvdrotestosterone f DHT) r -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 negative feedback long loop inhibition 1 FSH& testosterone ~ inhlbln " short loop inhibition oestrogen* progesterone^—' Inhibin Testosterone Testicular Biosynthetic Pathway of Testosterone Cholesterol PÍ5UECC Pregnenolone X 17a-Hydrr»tyla5e 17a-Hydro xypreg nenol o ne Clľ.aQ-Lyjse Dehydroepiantirosŕerone 1 7|^Hyd růxysterůtd r dehydrogenase Androstenedioí Progesterone 17 a-Hy tfroxy progestero ne Androsfenedione TESTOSTERONE 3]5-Hydrů)íysterůid dtliydrůíjOiidSi - synthetized in testis (Leydig cells - in lesser extent in adrenals Dihydrotestosterone - The most important derivative of testosterone - Formed extratesticularv from testosterone - 5a-reductase Metabolism of Testosterone to 5\ m i \ uma á j ii i y rcnra m \ ím t am 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 monooxygenases CYP1A, IB involved in androgen catabolism - Pesticides endosulfan, mirex, o-d'-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-bindin otencies (Ref: DHT EC50 ~ 0.1 uM) Compound Benz [a] anthracene Benzo[a]pyrene Dimethylbenz[a]anthracene Chrysene Dibenzo [a,h] anthracene Bisphenol A vinclozolin metabolites IC, 10.4 10.3 activation in range 0.1-lOuM hydroxyflutamide Aroclor typical values Individual PCBs typical values rm-(4-chlorophenyl)-methanol 0.25-1.11 64-87 Antiandrogenic compounds ŕrzs-(4-chlorophenyl)-methanol - 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 liSž^IRTOrtTHTiHRI^RMií^iltliggggi^ - Easy cultivation and experimental design X - Cell wall may obstruct transport of chemical into cell=> => false negatives Thyroid hormones Play crucial roles in stimulating metabolism and influencing development and maturation yro. inyrori 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) Also called tetraiodothyronine Contains 4 iodide ions Triiodothyronine (T3) Contains 3 iodide ions - T4 - prohormone - 5'-deiodination leads to active form, T3 Thyroxine (T4) 3,5,3'-Triiodothyronine (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 rdeiodinases - T4 synthesis - iodination of tyrosin residues on tyreoglobuhn - coupling of two iodotyrosines conducted by thyroid peroxidase Hypothalamus 4 Hypothalamus TRH Pituitary-thyroid axis - Regulation of thyroid synthesis - Control the secretion of thyroid hormones through two negative feedback loops - Pituitary TSH (thyroid stimulating hormone) stimulates both I" uptake and iodination of tyrosine resides on Tg T3+T4 Thyroid Enzymes involved in thyroid metabolism - Thyroid peroxidases - iodination of tyrosyl residues - coupling of iodinated tyrosyl residues - Thyroid deiodinases outer' inner - D1, D2 - activation of T4 into T3 via deiodination on „outer" ring (formation of T3) - D3 - deactivation into rT3 via deiodination on „inner' nn< Thyroid receptors - Mechanism of action Thyroid hormones bind to receptors in: > cytoplasm > surfaces of mitochondria > nucleus Alike other nuclear receptors - 5 isoforms of TR - After activation formation of homo- and heterodimers - Binding to thyroid responsive elements (TRE) - Gene expression STEPI Transport across cell membrane Increased production / Alteration of cellular activity 1 m STEPS Binding to receptúra at mitochondria and nucleus STEP e Translation and protein synthesis Transcription and mRNA production Receptor STEP 4 Gene activation y STEP 3 Binding of honnone- receptor complex to DMA Thvroid bindin - 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 In vivo assessment - TH serum levels - simple, nondestructive x variation 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 synthesis) - Hepatic UDP-glucuronosyltransferase activity (marker of enhanced TH clearance from serum) In vitro assessment - Enzyme inhibition assays (thyroidperoxidase, deiodinases) -assessment of thyroid metabolism - Competitive binding assays with TBP - TH- dependent proliferation assay (pituitary tumor GH3? thyroid 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 Retinoids Regulation of development and homeostasis in tissues of vertebrates and invertebrates Development of embryonic, epithelial cells (gastrointestinal tract, skin, bones) Necessary for vision Suppressive effects in cancer development Important for cell growth, apoptosis and differenciation 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 Bindin] Protein (LMW) TTR: Transthyrethin (HMW) RBP RE P^\ chylomicrons RĽ Kb TTR-RBP-R blood vessel CKCTt nr CRBP f}R ¥A CRBPI-RkL r^ - V atRA J^ CRABP l/lt \ target tissui 4-0 H RA 4-oxo RA 13 c/s RA 9-ds RA Retinoid binding proteins RAL - Retinal CRBP - cellular retinol binding protein - binding of retinol, immediate decrease of retinol concentration CRBAP - cellular retinoic acid binding protein - Controlling ratio free retinol/free retinoic acid ^ ? Retinoidy [RAR | ► r---------j _/ (rar ^ v7 ; rxr] i JDC Mode of action - IsoformsofRARaRXR - Both have ísoforms a, p and y, each of them several subtypes -----------------------iTľi - 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 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 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 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 PI9 pluripotent cells differentiation dependent on circumstances, triggered by ATRA - reporter gene assay P19/A15 - Other cell lines - rainbow trout gonads, human salivary gland, breast or prostatic carcinomas etc. AhR (Ary I hydrocarbon receptor) AhR structure Denison et al., Chem. Biol. Interact. 141: 3 Thr343j ?? 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 Epithelial hyperplasia Tumor promotion Induction of drug-metabolizing enzymes Altered ER signaling Porphyria Deregulated lipid metabolism Decreased serum thyroxine Wasting - Metabolism of arachldonic acid to biologically active products Persistent thyroid hormone receptor activation EGF receptor down-regulation Lipid peroxidation Immunosuppression Inhibition of gluconeogenic si s Teratogenesis/em bry ©toxicity Utilization of brown adipose tissue Vitamin A depletion Cardiac dysfunction Figure I Biological responses to TCDD, A wide variety of cellular processes have been shown to be affected by TCDD. AhR = cytosolic helix-loop-helix/PAS protein PAS proteins: R. J. Kew/ey et a/./The International Journal of Biochemistry & Cell Biology 36 (2004) 1H9-204 a. bHLH b. bHLH/Zip c. b H LH/P AS Class I bHLH dimerization DNA binding T E47, Myo D Zip secondary DMLrl dimerization TT - Max, Myc bHLH dimerization DNA binding PAS secondary dimerization ___I___ AhR AhRR HIF-a IPAS 5IM1&2 Class II ARNT1 & 2 B B B B ■ B Transactivation ^m Xenobiotic response M 22J Hypoxia signalling Neurogenesis Repression ^ General partner factor BMAL1 & 2 B Circadian rhythm Fig. 1. Schematic representation of the domain structure of some bHLH transcription factor family members. AhR domain structure: bHLf 1 1 I PAS Domain 1 1 NH2 1 ■ ^ 1 B |Q rich COOH NLS NES L J L AhR:Arnt:DRE Complex Formation Ligand & hsp90 Transactivation Binding ________I L Trans format ion Fig. 2. Domain structure of the AhR. Denison etal., Chem. Biol. Interact. 141: 3 AhR activation: RJ. Kew/ey et al./The Internation u I Journal of Biochemistry & Cell Biology 36 (2004) 189-204 193 ► Xenobiotic response genes 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'P1, Jun B, TGF-ß - regulation of cell cycle and apoptosis; CI 'O ^Classicflľ'AhR lÁyanáK and CVP1 Al Inducers CI -CI ,CI CI CI -CI CI 2,3,7.8-Tetrachloťodibtti zootoxin 3,-UT Af5-Pental>iphenvl 2J.7,»-letraclilorodibeMzofuran 3-Mťthykliolanlhrťnť Benzora^pvrene fl-ľManlirhoflavonc Denison & Nagy, Annu. Rev. Pharmacol. Toxicol. 43:309 „Non-classical" AhR ligands M.S. Denison et al. í Chemico-Biological interact ions J 41 (2002) 3 24 CH, 2^8-TctracMorodibcnzo-p-diosiii 2-fMethvlmerwmtiťianiWi.B CF, CH2^ CH3- O-^n-O 2-f4'-Chlorophcnyl)beiiaotliiazolc ■N=N—N-CN SKF71739 CH3 CH2 Bilirubin NHŽ 1 ^-THflminonaphthaleiie OCONHCH, "*> CH. ChU Carbaryl Omeprazole OCH, O Indirubin ar"- C6-MethTtenedioxvbenzene H Trvptamine CH, Biomarkers/bioanalytical methods: • 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 in H4IIE rat hepatoma cells; ■» CALUX/CAFLUX assays; • GRAB assay (AhR-DNA binding) -» yeast bioassay; -» immunoassays; ■* detection of CYP1A mRNA or protein Detection of EROD activity: 140 M. 77// et at. / Chemico-Biological Interactions 1 17 (1999) 135-150 c 800 c 1 i— o. O) E x o E Q. ■> 600 400 « 200 - O O a: m 0 0 8 16 24 40 48 Incubation time (h) 64 72 Fig. 2. Time course of induction of CYPlAl-catalyzed 7-ethoxyiesorufin O-deethylase (EROD) activity in primary cultures of rat hepatocytes, after addition of 1.7 x 10 ~5 M benzo[a]pyrene (-T-), 1.9 x 10 ~6 M benzo[A]m.ioranthene (-A-J or 9.4 x 10_1 M acenaphthylene (-O-). EROD activity was determined in cell homogenates. The data represent means + S.D. from four independent experiments. In vitro assays for nongenotoxic (epigenetic) effects AhR-mediated effects luciferase reporter assay - H4IIE.luc cells Ligand (TCDD) Estrogens [AhR <-> Light ARNT HSP9I HSP9I Nuclear Factors kHSP9l mime* 'Activated" \ / DRE-Luc Increased Protein^ Modulation of Gene X Phosphorylation \ Expression Membrane Proteins Cytosolic v. Proteins Luciferase, Adapted from Blankenship (1994) CALUX/CAFLUX assa 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) X TO E ô o o _> o (0 ■o o .5 'S o E QĹ 120 100 TCDD B[a]P B[e]P TCDD: IC 50 P AH: IEC 50 Induction Equivalency Factor IEF = IC50 / IEC50 How many times is the compound "weaker" inducer than TCDD ? 1.E-07 1.E-04 1.E-01 1.E+02 concentrationjj(M) Toxic equivalency factors (TEF)/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: TEQ = I {compound! x TEFj + ... + compound x TEFn} Toxic equivalency factors for PCDDs, PCDFs and PCBs: Table 4. Toxic Equivak TU Factors established by the WHO (WHO-TEFs) for dioxins and dioxin-lilce PCSs [41 PC D D Congener WHO-TDF PC D F Congener WHO-TEF PCB Congener WHO-TEF 2A7>TCDD 1 2,3^8-TCDF 0.1 Nan-anha 12,3,7,fi-PeCDD 1 12,37,8-PeCDF 0.05 PCB#Í11 00005 12347ß~HxCDD 0.1 2347^ Pe CD F 0.5 PCB#77 00005 12367ß^HxCDD 0.1 12347flHxCDF 0.01 PCB#126 0.1 12,37,89'HxCDD 0.1 l23R7ß^HxCDr 01 PCB#1&9 0.01 1234n7ß~HpCDD 0.01 234ů7ftHxCDr 0.1 Mono-ortho OCDD 00001 1237^-HxCDF 0.1 PCB#105 00001 1234678^ H pC D ľ 0.01 PCB#114 00005 12347A9 HpCDF 0.01 PCB#11ß 0 0001 OCDF 0.0001 PCB#123 PCB#156 PCB#157 PCB#1&7 PCB#1ß9 0 0001 0.0005 00005 0 00001 00001 Eljarrat & Barceló, Trends Anal. Chem.22: 655 In vitro assays for nongenotoxic effects 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 • estrogen signalling pathways and AhR • estrogen signalling pathways and receptors for EGF and insuline In vitro assays for nongenotoxic effects Modulation of RAR/RXR : retinoic acid signalling ATRA - important regulatory molecule : cellular differentiation (embryotoxicity, 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