Fyziologie působení f armak a toxických látek Přednáška č.4 Endokrinní dísrupce u obratlovců I. ER, AR, pR, GR Endocrine disruptor: A chemical that interferes with the synthesis, secretion, transport, binding, action or elimination of any hormone in the body Endocrine Disrupting Chemicals DES Nonylphenol ■EU Vinclozolin PCB-153 Dioxin Výzkum endokrinní disrupce je soustředěn do dvou oblastí: • obratlovci, kteří alespoň část svého životního cyklu tráví ve vodném prostředí - ryby, obojživelníci -expozice vodou, potravou; • terestričtí obratlovci - expozice především v rámci potravního řetězce; Nejohroženější skupina - vrcholoví konzumenti - dravci. hŤŤp://www.epa.qov/endo/ Biomagnif ikace a bioakumulace DDT LuncL-ntíJliůrt; irtilrédůe Of 10 million, li ave s DDTki fish-Ľrtliny biiáa 25 ppn* DDTirL larg« fíih. j2pf» TT . DDT i* ■ . mull fldh 0,5 ppm DDT lil lůů-^Lartktiin OL 04 |t pni DDT JrtWdlťi Ü.Q0Ü0Ü3 pprti Effects of DDT (Dichlorodiphenyltrichloroethane) •Invertebrates •Fish •Birds •Mammals Endocrine Disruption in Wildlife - Eggshell thinning in raptors from DDT - Beak, skeletal, reproductive abnormalities from PCBs (bald eagles, gulls, cormorants) - Intersex fish below UK sewage effluents from estradiol, alkylphenols Decreased plasma sex steroids, egg and gonadal size; delayed sexual maturity from dioxin below paper mills (Great Lakes white suckers) Poorly developed testes, small penises, low testosterone; abnormal ovaries; males with high estradiol; poor hatchling success from DDE (Lake Apopka alligators) Endocrine Disruption in Lab - Masculinization of females by kepone, DDT, methoxychlor - Disruption of estrous cycle by atrazine, choroquine - Hypospadias, vaginal pouches, reduced sperm production in males exposed to vinclozolin in utero - Impaired testosterone synthesis, and spermatogenesis; decreased anogenital distance, delayed testis decent, impaired and feminized behavior of rats by dioxin -Acceleration of puberty and loss of fertility in females by many estrogenic chemicals - Delay of puberty, binding to androgen receptor; nipple retention in males by many estrogenic chemicals -Atrophy of the thymus by PCBs and dioxin Evidence for ED in Humans • Genital malformation (boys), vaginal cancer, infertility (girls) exposed in utero to DES • Neurological effects, decreased growth, developmental abnormalities (e.g., penis size) in children exposed in utero to PCBs • Altered girl/boy ratio after population exposure to dioxin (Saveso, Italy) • Shortened lactation associated with DDE • Decreased sperm count and quality • Increased prostate, testicular, breast cancer Human Breast Cancer • Breast cancer has increased but • Epidemiological studies are conflicting - It is not possible to assign a specific chemical or physical cause at this time • Better animal models are needed to predict human risk Human Sperm Counts Carisenetal, 1992 meta-analysis: 61 studies Suggests 50% decline in count, volume Decline seen in both Europe and US but Large geographic variation among studies Potential selection bias, other confounders A large, carefully controlled prospective study is needed for confirmation Testicular Cancer • Increase in testicular cancer observed in most countries • Affects mostly ages 15-45 • Year of birth, birth weight, genital tract abnormalities are risk factors • Evidence suggests high estrogen environment during fetal life may be involved but • No increase in testicular cancer in DES sons TDS = testicular dysgenesis syndrome Environmental factors incl. endocrine disruption Genetic defects / polymorphisms e.g. 46 XY/45X0 Abnormal Testis Development i.e. testicular dysgenesis Impaired germ cell differentiation Altered leydig cell Altered Sertoli cell Androgen insufficiency differentiation /function differentiation / function Human Syndrome] inefficient spermatogenesis cryptorchidism hypospadias testicular germ cell tumours rigure 1 Schemalic represenlalion of I he polenlial pathogenic links between leslis developmenl and Ihe clinical manifesto I ions of leslicular dysgenesis syndrome (JDS). The similarilies in Ihe palhologies induced by in uferodibulyl phlhalale (DBP) adminislralion and human TDS are compared. Endocrine (hormonal) system regulates Metabolic function and equilibrium Reproduction Growth/development Hypothalamus Pituitary Thyroid Parathyroid? Thymus. Adrenals Pariere» Ovary There are over 50 different hormones Environmental estrogens (xenoestrogens) ■ Sources - pesticides - plastics - pharmaceuticals - some cleansers - contraception ■ vs. phytoestrogens - antiherbivore compounds in many plant species - lignans (many fruits, vegetables), isof lavones (soy) Možnosti účinku environmentálních estrogenu na buněčné úrovni Decision Network Growth Factar Receptor Ligands 1 -J - I c i°i 3 4 ----1 flP 1/SP 1 |-| TATA AP1/SP1 lelheretJ cell cycle activation DNA Interaction TARGET GENE ACTIVATION (MODULATION] Environmentálni estrogeny: OCH3 ■r'X OH OCH3 p.p'-Meihoxychlor H -G C C H i OH Bisphenol-A OH p-Alkylphenclä ^COjCHjCeHj KC02(CHľ}3CH3 Dietŕiylstilbestrol Benzyl bulyIphthalate Figure 2 Structures of some xc n oestrogen s. Environmentálni antiandrogeny: 5a~DHT* Hydroxyflutamide* p.p'-DDE HC / b- >/ / Fenitrothion Linuron Vinclozolin Fl c;. 2. Structural diversity among environmental chemicals reported to be antiandrogenic. The steroidal androgen, Sa-dihydroxytestosterone (5q-DHT! and its pharmaceutical antagonist, hydroxyflutamide, are shown for comparison. p,//-DDE is a persistent contaminant, while the remaining are currently used pesticides: fenitrothion, an insecticide; linuron, an herbicide; and vinclozolin, a fungicide. Steroids 17pV£stra(iiol Pharmaceuticals Diethylstilbestrol Ethynyl Estradiol Fungal Products OH ö | AAA/-, Zearalenone Pollutants í 1 i DDT PCB ^ Bisphenol A Ncmylphenol Kepone Plant Products Genisteir» (isoflavone) I T Luteolin (flavone) /Ar° Resveratrol (stilbene) Coumestrol (coumarin) Fig. 5. Chemicals found Ln the environment reported to be estrogenic. This list is not comprehensive, but illustrates representative structures of estrogenic compounds from various sources. Information on these compounds is contained in the text. Xenoestrogens and Mimic or partly mimic the sex steroid hormones estrogens and androgens (the male sex hormone) by binding to hormone receptors or influencing cell signaling pathways. Those that act like estrogen are called environmental estrogens. Modify the making and function of hormone receptors. xenoandrogens can: ■ Block, prevent and alter hormonal binding to hormone receptors or influencing cell signaling pathways. Chemicals that block or antagonize hormones are labeled anti-estrogens or anti-androgens. ■ Alter production and breakdown of natural hormones. 76 Interakce AhR a ER: XCCC" 2,3,7,11-10 D D ^VM'^pentaCB CHaOH I3C BaP Figure 5 2JJ,S-'['dmL:h[orüdilvn/o-p-dioxin (TCDD) and related compounds thai bind to the AhR. E2 H0-E2 CYP1A1 ÍCYP1B1 t **■ Decreased E2 Tf(-> ■*■ Inhibition of E2-indJCGd gcnos **i B üC Coaciivator binding E2 Dire-ct inhibition of E2-indjcedge ií!4 Limiting levels ol c□ activators Ě2 #1 #2 #3 «4 ERc dp^nreguUilpii ^ Limiting 4 pro tea somes | * levels of ERa *5 Figure 3. Proposed mechanisms of inhibitory AhR—ERa crosstalk {123-126). Hormonální přípravky jako EĎs - ethinylestradiol ■ Male fish living near municipal sewage outlets in England had both male and female sex characteristics and their livers produced vitellogenin, a female egg-yolk protein not normally found in males ■ cancers of the female and male reproductive tract ■ malformed Fallopian tubes, uterus and cervix ■ altered bone density and structure ■ abnormal blood hormone levels ■ reduced fertility ■ altered sexual behavior ■ modified immune system Biosyntéza steroidních hormonů a endokrinní disrupce: HO I 7(ji-OH-Prcgncnolonc I Cholesterol Cliŕiiiiiferul iidt-chain íltirLjgfc 1 CH Pregnenolone 17<*-OH-Progcstcr(řnc Androgens /\ Testosterone fr EiCro^cns Cortisol I____________________________I I________ Progesterone Corticestcranc I Aldosterons __________________________I (+ Prugrstsrane) Gonadal steroids Adrenal steroids Androstcncdionc DihjfdrotciCCíteronc OH 2 (oř 4)-h)idrox)íesmone I ůci-hjrdroxyesirone S-mtchaxysiďone ■JJJ-JIJJJJ.LIJ tJ UJ 9 La 9 Lt 9 O O O O O ~^—i—i—i—i 1 Z* H* * * o p — 'V O DO i—■ — Si ^J1 ö Kí II 31 B jp ** B > -c O" L) c o Ü 1Ü0- r1 50- i A. I x 1 S 10 concentration of t r: st substances (M M) CľZľl mety rapoine I I MeSCV D DE ketoconazflle 150 o <£ lüo o o o ^ 50- 1 -9 -B -7 -S -5 concentration öltest substances (log M) ■ me-tyrapone * ketoconazole * 3-MeSOrDDE Effects of test substances on Cortisol and 11-deoxycortisol formation in H295R cells, assumed to represent CYP11B and CYP21 activity. 300 sr* 250 c o u a* 200 > (O a> u> fíS ns E o s 150 100 50 D1 uM ■ 10 uM ,o* ^ / # # <ŕ / FIG. 2. Effect of 4-hydroxyandrostenedione (4-H A; 1 /xM), DDT, three of its metabolites (1 or 10 jllM) or 8-bromo-cyclic adenosine monophosphate (8Br-cAMP; 300 jllM) on aroniatase activity in H295R cells. Exposures were for 24 h, in quadruplicate, * Significantly lower than control. Testy estrogenity a antiestrogenity In vitro ass av Measured end point Advantages Limitations E-Scrccn Ligand-Mnding (EDSTAC)a LR-hindin» to ERE GSTpull-down/ľRLT/ two-hvbrid assay Trunsactivation assay in yeast or mammalian eefls(LDSTAC)a Analysis o t" gene expression Analysis of enzyme activity Analysis o ť steroidogenesis íl-D ST Ada Proliferation of I:Rot-positive cells Binding affinilv to ERa or" ER p Binding affinity of Er« or E R ß" to ERI-' Ligand-dependent association of ERa or LRp with co-activators ERa or ER p mediated activation of reporter Expression of ER-regulated genes Activity of I: R-regulated enzymes Induction/inhibition of estroiien biosynthesis Measures physiological endpoint oi estrogen action, measures estrogens and ant iestrogens Simple, high-throughput method High-throughput method, various LRLs can be used Analysis of molecular interaction, defined ER subtype or LR domain as well as co-activators can be used, measures estrogens and antiestro- gens 11 igh-throughput method, measures estrogens and an t i estrogens, can be done in metabolic competent cells to account for (anti)-estrogenic metabolites Analysis of physiological response, versatile, measures estrogens and antiestrogens Analysis of physiological response, measures estrogens and antiestrogens Analysis of physiological response, measures E R-inde pendent pathways No defined ER expression, no mechanistic data Does not measure ER activation, does not measure physiological response Does not measure LR activation, low sensitivity. does not measure physiological response Does not measure direct ER activation, low throughput, does not measure physiological response Does not measure physiological response Low throughput Cell lines or primary cell c u! t u res with active marker enzymes suitable only Cells with active steroidogenesis suitable onlv Mikrobiální syntéza androgenů?? Stigmasterol Plant Sterol from Wood Pulp Pregnenolone DHEA Androstenedione Steroids Released In River Metabolism Within Bacteria in the River Testosterone Fl«. 3. The production of androgenic compounds by bacteria. Stigmasterol, a major plant sterol found in wood pulp, is efficiently metabolized to androgenic steroids such as androstenedione by the bacteria, Mycobacterium smegmatis. M. smegmaäs form extensive colonies, or "bacterial mats," at the effluent site of pulp and paper mills. The natural plant sterol, stigmasterol, contained in the pulp effluent is converted by M. smegmatis into androstenedione, which is released into the river or stream. Female mosquito fish exposed to these androgens develop male structures. (See Refs. 34, 35, and 36 for details.j Většina látek narušujících androgenní dráhy jsou antiandrogeny!!!! Anti- androgenic compounds in the environment There are a number of commonly used environmental chemicals that have been identified as having anti-androgenic properties. These chemicals have been administered to pregnant rodents during the period of reproductive tract development. When the male pups were examined, they displayed many of the abnormalities associated with f lutamide administration. Some chemicals (vinclozolin, procymidone, linuron, p,p'-DDE (1,1,1-dichloro-2,2-bis(pchlorophenyl)ethane) act as androgen receptor antagonists, others (phthalate esters) reduce androgen synthesis, but it is likely that other modes of action are also involved in the toxicity induced by these compounds. There are major problems in comparing the published studies of the effects of anti-androgenic compounds / inconsistent protocols. Human impact???? Polycyklické aromatické uhlovodíky mají antiandrogenní účinek: B 10 DHT - + + ARE (+> + + -ARE(-> - - + Ař # éŕJ> ^ 4?<ŕ<ŕ^ <ŕ ^ «* 1 Ě B bound free re oj I— 4-r