Fyziologie působení farmak a toxických látek Přednáška č.7 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 T™6 / C9Ul9----(Oy^OCH2CH2^OH 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. Biomagnifikace a bioakumulace DDT L-un(.L'ntriÉlJuh: Littríiůr of 10 itti]lii>rt llďi«fl DDT in Liah-ĽrtLtn^ birds 25 p p m DDT in lär£.e íísh 2 pfltl m MU J__k DDT in za tvp Lank E cm 0,04 p-pni DDT i±i 4UlůJl fl«h JpTll DDT irt WJlŕr 0,000003 ppm 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) I 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 Carisen et al, 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 XV / 45X0 Impaired germ cell differentiation Altered leydig cell Altered Sertoli cell Androgen insufficiency differentiation /function differentiation / function Figure 1 Schemalic represenlalion of (he polenlial palhogenic links between leslis developmenl and (he clinical manifes-lalions of leslicular dysgenesis syndrome (TDS). The similarilies in I he palhologies induced by in uiero dibulyl phlhalale (DBP) adminislralion and human TDS are compared. Endocrine (hormonal) system regulates • Metabolic function and equilibrium ■ Reproduction • Growth/development Pineal Hypothalamus Pituitary Thyroid Parathyroid* Thymus Adrenal! Pancreas 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 Factor Receptor Ligands Nuclear ER *f, 1 r«J_ UM IcCOH ~.I~I

Estrogens I____________________________I (+ Pnagísterane) Gonadal steroids Cortisol I________ Progesterone Corticostcnonc I Aldosterons ____________I Adrenal steroids Androstcncdionc EZSE333 Di h)«J roten« tenons OH HO HC 1 2 (or 4J'h)«írox)fle!tnjne loff-hydroxyBsn-one HO' OH (Cacecholastroge ns) 2-mtthoicyeitfon* i Eltriol ■JJ-!JJJJJ.l,IJ ^ c=o Prcgnancdiol y 20«-h) Hsimi í ň Ě o lít 10 PCB126(M) íl 10 < (C) 12 r 1.(1 G O « O.K vi a, 0.6 K OJ 3 0.4 0,2 k 0,0 CYPI7 □ basal DcAMP ŕ* äi o 10' 10' PCB126(M) * * íl 10 I IP 3 re "J O 2 I n 3 B 6! +" S 3 »5 T-3 x | o fold expression JC - - K» tJ U Ö W * J£ « ^ » Ln © O* O O O O © O fold expression o i--------n 1. m b K) Q O 1 £ - o hH \ V * * T—I------1—I------1 K» * * b p — — LA O L* n to ro b II M 3H HH** Tp** > "B o- 100- O 50- JL I 1 5 10 concent rat ion of teat substances metyraporie WeSQj-DDE 150 o £ 100 o o O & 50 ketoconazole -9 -B concentration of test substances (log M) ■ mety na pone * ketoconazole * 3-MeSQrDDE Effects of test substances on Cortisol and 11-deoxycortisol formation in H295R cells, assumed to represent CYP11B and CYP21 activity. 300 cP* Ji* 4 / / S sŕ # ? é FIG. 2. Effect of 4-hydroxyandrostenedione (4-HA; 1 ju.M), DDT, three of its metabolites (1 or 10 ju.M) or 8-bromo-cyclic adenosine monophosphate (8Br-cAMP; 300 (jlM) on aromatase activity in H295R cells. Exposures were for 24 h, in quadruplicate. * Significantly lower than control. Testy estrogenity a antiestrogenity In vitro [iss a v Measured endpoint Advantages Limitations I i-Screen Ligand-binding (EDSTAC)a ER-binding to ERE GST pull-daw n/1-'RI LT/ two-hvbrid assay Transactivation assay in yeas L or mam mul i a n cells (EDSTAC)a Analysis of gene expression Analysis of enzyme activity Analysis o\" steroidogenesis (EDSTAC)a Proliferation oť ľ! Rö-positive cells Binding affinity to ERct or ER p Einding afľiniLy of lira orERp to ERE Ligand-dependent association of ERa or ERß with ea-aetivators ERa or ERß mediated act i valia n of reporter Expression of ER-reaulaled tie n es Activity o( ER-regulated enzymes Induction/inhibition ot estrogen biosynthesis Measures physiological endpoint o\" estrogen action, measures estrogens and antiestrogens Simple, high-throughput method High-throughput method, various ER Es can be used Analysis of molecular interaction, defined ER subtype or ER domain as well us co-activators can he used, measures estrogens and antiestrogens High-throughput method, measures estrogens and antiestrogens. can he done in metabolic competent cells to account for (anti)-estrogenic metabolites No defined ER expression. no mechanistic data Does not measure ER activation, does not measure physiological response Does not measure ER 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 Analysis of physiological response, Low throughput versatile, measures estrogens and antiestrogens Analysis of physiological response, measures estrogens and antiestrogens Analysis of physiological response. measures ER-independent pathways Cell lines or primary cell cultures with active marker enzymes suitable only Cells with active steroidogenesis suitable only Mikrobiální syntéza androgenů?? Stigmasterol Plant Sterol from Wood Pulp Pregnenolone □ HEA Androstenedione Steroids Released In River Metabolism Within Bacteria in the River Testosterone Fig. S. 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 smegma&s. M. smegmatis 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.) 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: A DHT - + + ARE (+) + + - ARE(-) - - + bound free B yv« ^ <č 10 ra v a) q. S I < w Q. 2 ■ ŕ, X nd nd n 1 SKF-525A - +■ - + - + I_______I ■ ■ ■ ■ blank DHT DHT control + Chr _ + DHT + BkF m l£q <& V* DHT + BaP Antiandrogenní účinky PCB: Effect of PCB Congeners on Androgen Receptor Activity 0 J0Q1 -01 1 10-IŮOnU 0 1 3 S 9 18>JM C I ii 5 9 'BpM 0 13 5 3 ISjM 0 3f3-3gM Rlflíl PíľDl.íft PCliUvS PCB180 PCBiiilx Interakce polutantů s endokrinní dráhou = velmi složitý proces: PCDD/Fs, COPLANAR AND MONO-ORTHO-CHLORINATED PCBs NONCOPLANARPCBs y | STEROIDOGENESIS \ ENHANCED METABOLISM LEADING TO DECREASED CONCENTRATIONS OF ESTROGENS IN BLOOD ANTIESTROGENICITY MEDIATED THROUGH DIRECT INTERACTIONS OF THE AhR WITH iDRE, COMPETITION OF AhR WITH ER FOR COFACTORS, INDUCTION OF INHIBITORY FACTORS OR INDUCTION OF ER DEGRADATION ANTIESTROGENICITY MEDIATED THROUGH DIRECT SUPRESSION OF ER ACTIVATION Brómované zpomalovače hoření - nový typ endokrinních disruptorů?? Structure compared to PCBs, dioxin, thyroxin °^f*^ PCB BDE-47 2,3,7,8-TCDD (dioxin) h h h ö Thyroxin (T4)