Fyziologie působení f armak a toxických látek Přednáška č.9 Endokrinní disrupce u obratlovců II. Modulace funkcí RAR/RXR, TR a PPAR - deregulace vývoje organismu, modulace endokrinních signálu a karcinogenní účinky; Efekty spojené s deregulací hladiny retinoidů: • Funkce RA; • Vznik končetin; • Vývoj nervové soustavy; • Vývojové abnormality obojživelníků; • Narušení hladin vitamínu A; ^^ 3,4-didehydro-RA ^^ 3,4^didehydra-retinol / 14-hydroxy-reí/u-retingl / 4 18 retínol □ ^-"^ retina! --------------------(—^- OH retinoic acid (RA) Enz? 18-OH-RA ^OH Struktura a syntéza kyseliny retinové retiny! palmitate 0-C-fCH3)7: rr^ji - RCHo-cRBPd.) ^"ROH,CRBPŕ„ _ ra intestine |l-carotône örV 0^ OH 9-eŕs-RA CRBP-RCHO TTR-RSP-ROH CR8P-ROH It RE FIG. 3. Absorption, distribution, and metabolism of naturally occurring retinoids. 9,13-di-dS-RA FIG. 2. Structures of naturally occurring retinoids. A A j B C D £ F Transcriptional activation domains (AF-1) DNA binding domain (RARE) Hinge region Ligand-binding Helerodimerization Transcription activation (AF-2) N-Cor?SMRT binding domains ; ? B 5' R X AGGTCA - AGGTCA- -AGGTCA-AGGTCA 1\W RARE ^ Early response genes ^ Secondary response genes 3' Gene product (e.g. transcription factors STATs, RARs, c/EBP, etc.) Inhibition of cell growth, Induction of differentiation, apoptosis Fig. 1 - Structure and functions of retinoid receptors. A) Schematic representation of retinoid receptor protein depicting various functional domains. B) A molecular model for retinoid action. The liganded RAR forms heterodimer with RXR, binds to specific regulatory sequences (RARE) in the promoter region of target genes. Transactivation of such early response genes is a primary event of retinoid action. In addition to this, the products of early response genes can activate the transcription of secondary genes. Transactivation of these genes therefore represents secondary action of retinoids since their transcription requires protein synthesis. This cascade of gene events leads to secondary and tertiary events that eventually produce a phenotype that is characteristic of retinoid action. • Abnormalities caused by exogenous agents (certain chemicals or viruses, radiation, or hyperthermia) are called disruptions. The agents responsible for these disruptions are called teratogens. Most teratogens produce their effects only during certain critical periods of development. The most critical time for any organ is when it is growing and forming its structures. Different organs have different critical periods, but the time from period from day 15 through day 60 of gestation is critical for many human organs. • Retinoic acid is important in forming the anterior-posterior axis of the mammalian embryo and also in forming the limbs. In these instances, retinoic acid is secreted from discrete cells and works in a small area. However, if retinoic acid is present in large amounts, cells that normally would not receive such high concentrations of this molecule will respond to it. Inside the developing embryo, vitamin A and 13-c/s-retinoic acid become isomerized to the developmental^ active forms of retinoic acid, all- trans-retinoic acid and 9-c/5-retinoic acid. Some of the Hox genes have retinoic acid response elements in their promoters. • The disastrous consequences of exposure to exogenous retinoids during human pregnancy were underscored in the early 1980s when the drug Accutane® (the trade name for isoretinoin, or 13-<ľ/5-retinoic acid) was introduced as a treatment for severe acne. Women who took this drug during pregnancy had an increased number of spontaneous abortions and children born ...:iL__________-L L:„.iL _!_.£___a-. RA reguluje vznik a vývoj končetin There are discrete positions where limb fields are generated. Researchers have precisely localized the limb fields of many vertebrate species. Interestingly, in all land vertebrates, there are only four limb buds per embryo, and they are always opposite each other with respect to the midline. Although the limbs of different vertebrates differ with respect to which somite level they arise from, their position is constant with respect to the level of Hox gene expression along the anterior-posterior axis. For instance, in fishes (in which the pectoral and pelvic fins correspond to the anterior and posterior limbs, respectively), amphibians, birds, and mammals, the forelimb buds are found at the most anterior expression region of Hoxc-6, the position of the first thoracic vertebra. Retinoic acid appears to be critical for the initiation of limb bud outgrowth, since blocking the synthesis of retinoic acid with certain drugs prevents limb bud initiation, suggested that a gradient of retinoic acid along the anterior-posterior axis might activate certain homeotic genes in particular cells and thereby specify them to become included in the limb field. Legs regenerating from retinoic acid-treated tadpole tail. (A) The tail stump of a balloon frog tadpole treated with retinoic acid after amputation will form limbs from the amputation site. (B) Normal tail regeneration in a Rana temporäriatadpole 4 weeks after amputation. A small neural tube can be seen above a large notochord, and the muscles are arranged in packets. No cartilage or bone is present. (C) A retinoic acid-treated tadpole tail makes limb buds (arrows) as well as pelvic cartilage and bone. The cartilaginous rudiment of the femur can be seen in the right limb bud. unter ľt postsricr Drosophils Hox complex leb pb |ni V Dfa Scr ifo) Anlp Uhx ebd-A Ahd-B T 1 I í í í ľtiiM LTj*v3 Uflul Unv4 U,:. Moni Hok2 Mox3 WmJ Hojc5 WojlS ťosn-trB^ ancestral I I I I j I : !__[■_ Hox Complex ^^-T n^J-"LJ-"UJ- Hox7 íposloriorí a- Bi B2 B3 Bi BS B6 B? BS B9 HOX tí HOXC B13 -O C4 C5 CB CS CS CIO Cil C!J y*J3 □í 03 D4 noxo -n-------d—O hindbrain and gpirial cord mBsodBrm anterior DB DS D10 011 012 D13 posterior The Hox complex of an insect and the Hox complexes of a mammal compared and related to body regions. dorsal view sidevisw úcusa'. view side-view Expression domains of Hox genes in a mouse. The photographs show whole embryos displaying the expression domains of two genes of the HoxB complex (blue stain). These domains can be revealed by in situ hybridization or, as in these examples, by constructing transgenic mice containing the control sequence of a Hox gene coupled to a LacZ re porter gene, whose product is detected histochemically. Each gene is expressed in a long expanse of tissue with a sharply defined anterior limit. The earlier the position of the gene in its chromosomal complex, the more anterior the anatomical limit of its expression. Thus, with minor exceptions, the anatomical domains of the successive genes form a nested set, ordered according to the ordering of the genes in the chromosomal complex. Teratogenesis in frogs. (A) Wild green frog {Rana clamitans) with an eye deformity, collected in New Hampshire in 1999 by K. Babbitt. (B) Xenopustadpole with eye deformities caused by incubating newly fertilized eggs in water containing methoprenic acid, a by-product of methoprene. (C) One of several pathways by which methoprene can decay into teratogenic compounds such as methoprenic acid. (D) Ar\ isomer of retinoic acid showing the structural similarities to guluje vývoj CNS Top panel: At left, retinoic acid activates gene expression in a subset of cells in the normal developing forebrain of a midgestation mouse embryo (blue areas indicate ß-galactosidase reaction product, an indicator of gene expression in this experiment); at right, after maternal ingestion of a small quantity of retinoic acid (0.00025 mg/g of maternal weight), gene expression is ectopically activated throughout the forebrain. Bottom panel: At left, the brain of a normal mouse at term; at right, the grossly abnormal brain of a mouse whose mother ingested this same amount of retinoic acid at m id-gestation. Neural trest. Neural tube Senk hedgŕlicg, retjfiůif 4f id, fiůggifi and choř din in rloofplate and ŕiotoťhoŕd Rctinok acid Location of some inductive signals in the developing neural tube. Inductive signals are provided by either the notochord, the f loorplate, the roof plate and dorsal ectoderm, or the somites. These signals act locally on either the ventral or dorsal neuroepithelium of the developing spinal cord and hindbrain to elicit distinct patterns of gene expression and, ultimately, differentiation of specific classes of neurons. The peptide hormone sonic hedgehog (shh) is the most important ventral signal and is produced by both the notochord and f loorplate. In addition, noggin, chordin, and retinoic acid are produced either by the notochord or f loorplate. In contrast, a variety of signals including dorsalin and other members of the TGF family as well as noggin and retinoic acid are provided by the roof plate and dorsal ectoderm. These signals influence the differentiation of several dorsal cell types including the neural crest PHAHs a PAHs narušují funkci a strukturu štítné žlázy a hladiny thyroidních hormonů a retinoidů CI- CI 2,3,7,8-TCDD 2,2 A A', 5-pentaCB CH2OK I3C Ba P Figure 5 23,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds that bind to the AbR. TABLE I. Alterations in thyroid inland morphology, thyroid hormones and retinoid levels in marine mammals associated with exposure In poh/halogenated aromatic hydrocarbons. Associated Tissue Species Study location ThyroitvVetinoitl changes References 1 [arbor seal8 North Sea PCBsb ili\raid J- m. 1 ihvroid colloid depletion Sch.....acheret al.. 11993) (Phoca ťit uhliu • inlerlollicnlar fibrosis Harbor porpoise (Phocoena phocoaia) Beluga wliale SI. Lawrence Estuary, l'( Us thyroid i;li lll<[ thvroid ahscosscs J>, Cniseelal.. I'm Deiphlnaptena leucas) ynehec other (>Csľ Ihvroid adenoma Norlliorn olopliaiil seal California n lis plasma 1 retino! Beckmen el al., (I9£i7) (Mlrotmga angusHrvstris) p.p'-nni ■ I Tit'. IT:;1 1 [arbor seal captive PCBs plasma l retino! Brouwerel al., (1989) {Phoca rii nliti/i * p.p'-DDF. I IT,. IT.,*. TT3 Harbor seal captive \'i Ms pl.lMII.I 1 retino! IV Swart ft nl.. (199 I. 199.it (Phoca oittdiiia) dioxin TF.Q'sJl I TT4, ITs1 <:re\ sealJ Norwa) PCBs plasma J TT.,. l"ľ. Jenssen el al. 1 [995) (Hallchoenis grypia) Grey seď1 United Kingd..... ľi l; lli() plasma J.tt3:TT4 Hall h al.,(1998) (Halichoerm grypia) TABLE 3. Alterations in thyroid gland morphology and retinoid levels in fish associated with exposure ii> polyhalogenated aromatic hydrociiľbons and polymideaľ aromatic hydrocarbons. AssíKÍiitul 11 "[■ Species Study Inc-alinn contaminants s: imp In I TIimoiiI r, limíil cliiuiiiis Hi-Ii-ivikvs Sonstegard el al.. (1976) Spear el al.. (1992) Branchaudel al.. 1995 Ndayibagira el al.. (1994) Doyon et al., (1999) Arcand-Hoyet al.. (1999) Salmon species f ill Ml Likes unknown factor thyroid gland thyroid hypertrophy, h (Oneorhyachus sp.) While sucker Montreal. Qilelxr cop lunar liver pi ľplasia . retino! (Catostonmi coiwitenoni ľi IV 1 retinyl palmilale hike sturgeon Montreal. ynelxi- K ľ- Intestine J retinyl palmitate (Aclpenier fiilvacent 1 dehydroretlny] p.lllPlil.lli-T li.V' metabolism hike sturgeon Si Lawrence River, coplanar PCBs liver (Aclpeiiw fiilvt acen s) Brown bullheads Anuiurus nebidoim) Quebec Great Lakes PA lis' liver I retinoids ■I retinyl palmitate I dehydroretiny] palmilale 52 C-8D 94 52 09 5193 TABLE i. Alterations in thyroid gland morphology, aromatic hydrocarbons. lliyroid licinncint's iind retinoid levels in free-ranging avian species associated with exposure to polyhalogenated Sp, Study locatii Associated contaminants "11 ssi iľ sampled Thryoid/retinoid d.....ges thyroid T thyroid mass thyroid hyperplasia liver . retiny] palm State liver i retinal i retiny! pal mi t ate egg yolk T retinol: retiny] palm it ate Helen Herring gulls {Lana argeiitatus) Urning trills (Lana argeiifafi« Herring gulls (Lan« argeiiíaíi«) Herring gulls (Larus argeutatut) Great blue herons (Area heradlas) < cormorants (Phalacrocorax carba) Herring ijiills (Larus argeutatut) Caspian terns (Sterna oaspla) Cd.......)ll I ill is Sterna hlrtmdo) ][crriiii{ ijiills (Ĺarus argpitatus) Tree swallowsP ;Tiiihijiiinii> bicolor Great l^ikt-s Great Lakes Great Lakes Lakes Huron, Erie, Ontario St Lawrence River (jiit-lnf N ('Midlands Great Likes Hi-li;inni Ni-lluT-lands Great Ijiki-s Great Lakes Si. Lawrence River basin i'HAlls-' PI 1A1 Is 2.3.7.S-TCDD1, üat.s-tcpp iPCDDs + PCDFŕ dioxln TEQsd ÜPCHs Klí + HS" XPCBs IDS + 1IS TEQsf PCBsS ľi DO«, PCDFs ľ< :iw mcinci-ciiilin PClis PCDDs II in-•- egg yolk i retinyl palmltote . !gg ) . .Ik liver 1 IT,'' T EROD1 plasma plasma11 i retinol egg yolk plasma liver PCBs, DDF.." dieldrin, mirex liver All-inducing ilii-niitals'l liver I retiny] palmitate1 i TT3m, TT.,". !"ľ.| T plasma retino] Id yolk s.ii-retinyl palmitate i retin; I palmitate I retinol T EROD Mocda et al.. t lOSti) Govemnieiil ol Canada. 1001 Spear et al., (LSWi. 1002) Speareta1.,(1990) Boil) e1 al., 1994 van den Berg ei al. <19tM) Grasman et al.. (1996) Mink .-l ;il.. t lööß) Fox e1 al. ..l!)!)S> Bishop et al.. (1999) 65 PHAHs modulují hladiny retinoidu - mobilizace zásob vitaminu A v játrech c o o o 300 250 200 - 150 •S 100 o. X 50 i« - - - ■ :"-'- Ť ::-:!ív;-> ■ ■Čiŕllicäl Dams Falus Male Female Male Female GD20 PND21 PND90 FIG. 3. Hepatic retinal concentrations, expressed as percentage of control values, mean ± SEM, from dams, their fetuses (jV = 6), male and female neonates (A' = 8-10), and adult offspring (N = 10) following maternal exposure to 0 O, 5 M, or 25 O mg Aroclor l254/kg on Days 10-16 of gestation. *lndicates a significant difference from controls, p < 0.05. GD20, Gestation Day 20; PND21, Postnatal Day 21; PND90, Postnatal Day 90, BPA moduluje hladiny retinoidních receptoru v průběhu embryogenéze - myši o,p'-DDT OH p-Alkylphenols OCH3 Cf H—C—C—Cl Cl OCH3 'j.p'-Methüxychlor Hydroxy-PCB CO^CHjCeHj C02{CHz)3CH3 Diethylstilbestrol Benxylbutylplttfialat« Figure 2 Structures of some xen oestrogen s. Funkce thyroidních hormonů v ontogenezí a vliv organických polutantů: • Funkce thyroidních hormonů v metamorfóze • Funkce thyroidních hormonů ve vývoji nervové soustavy; Hypotéza - environmentálni polutanty jako kauzální faktor neurologických poruch (autismus, poruchy učení, hyperaktivita, nádorová onemocnění, juvenilní formy diabetes); • Toxické látky narušující thyroidní regulace; a i h m aj i zasaani vyznám pro iniciaci metamorfózy obojživelníků Temp Light Nutr. etc. ^ 4% jť ---------Hypothalamus + v e Embryo Fig. 1. Schematic representation of the hormonal regulation of amphibian metamorphosis. In response to environmental cues, the dormant thyroid gland of the tadpole is activated to produce the thyroid hormones T4 and T3 by the hypothalamic and pituitary hormones TR1\ CRF and TSIl. Thyroid hormone (TH) is obligatorily required to initiate and maintain the metamorphosis, its action being potentiated by glucocorticoid hormone and retarded by prolactin. Nutr., nutritional factors; TRE1. thyrotrophin-releasing hormone; CRE\ cortcotrophin-releasing factor; TSH, thyo i d-stí mulati n g hormone; T4, l-thyroxine; T3, triíodo-L-thyroníne; GC, glucocorticoid hormone. Tahle 1 Diversity of morphological and biochemical responses to thyroid hormone during amphibian metamorphosis issue Response Morphologies 1 Biochemical lírai n . i ver Eye Skin Limb bud, lung Tail, gills Intestine, pancreas Immune system M u sel e Restructuring; axon guidance and growth; cell turnover Functional differentiation; restructuring Repositioning; new retinal neurones; altered lens Restructuring; keratinisation; granular gland formation De novo morphogenesis of bone, skin, muscle, nerve, etc. Total tissue regression and removal Major remodelling of tissues Redistribution of immune cell populations Growth, differentiation, apoptosis Cell division; apoptosis; protein synthesis Induction of albumin and urea cycle enzymes; larval adult haemoglobin switch Visual pigment switch; induction of ß-crystallin Induction of collagen, 63 kDa keratin, magainin Cell proliferation; gene expression Programmed cell death; induction of lytic enzymes New structural and functional constituents Aquisition of new immunocompetence Induction of myosin heavy chain Conception 12 weeks 24 weeks Birth Maternal thyroid hormone _________—-----------—— " Thyroid receptors _____-------------- — Fetal thyroid hormone ]n ifjaa\rf-------------- 4 weeks Hypothalamus Gweeks Cochlea 1& weeks 7weeks Hippocampus 5 weeks Synaptogenesis, myelinogenesis, gliogenesis 7weeks Sexual differentiations—urogenitalia Figure 1. Role of thyroid hormones in fetal neurologic development in relation to timing of several landmark stages of dey/elopment. Figure adapted from Howdeshell (2002). Although it has been known for a century that hypothyroidism leads to retardation and other serious developmental effects, the role of thyroid hormones in brain development is still not completely understood. It is also accepted that thyroid hormones transferred from the mother to the embryo and fetus are critical for normal brain development, even though the thyroid gland of a fetus starts producing thyroid hormones at about 10 weeks. We now recognize that only a slight difference in the concentration of thyroid hormones during pregnancy can lead to significant changes in intelligence in children. Možné mechanismy disrupce funkce thyroidních hormonů • Inhibition of active transport of inorganic iodide into the follicular cell • Interference with the sodium/iodide transporter system • Inhibition of thyroid peroxidases to convert inorganic iodide into organic iodide to couple iodinated tyrosyl moieties into thyroid hormone • Damage to follicular cells • Inhibition or enhancement of thyroid hormone release into the blood • Inhibition or activation of the conversion of T4 to T3 by 5'-monodeiodinase at various sites in the body, for example, the fetal brain • Enhancement or interference of the metabolism and excretion of thyroid hormone by liver uridine diphosphate • Interference with transport of thyroid hormones • Vitamin A (retinol) disturbances • Blocking of or interfering with thyroid receptors 22 Mechanisms of Action of Thyroid-Disrupting Chemicals The complexity of the development of both the neurologic and thyroid systems offers numerous opportunities for chemicals to interfere as the systems develop, mature, and function. Briefly, there are chemicals that interfere with iodine uptake (the herbicides 2,4-D and mancozeb, several PCB congeners, and thiocyanates) and peroxidation at the molecular level (the herbicides aminotriazole and thioureas, the insecticides endosulfan and malathion, and PCBs). They also interfere with the protein transporter that provides a pathway for iodine to enter the cell (military and aerospace chemicals, Perchlorates). Certain antagonists (PCBs, the herbicides aminotriazole and dimethoate, and the insecticide fenvalerate) prevent the release of thyroid hormone from the cell and inhibit conversion of T4 to triiodothyronine (T3). Various chemicals enhance excessive excretion of thyroid hormones, some through activation of the cytochrome P450 system (dioxin, hexachlorobenzene, and fenvalerate). Some PCBs, phthalates, and other widely used chemicals compete for sites on the thyroid transport proteins that deliver thyroid hormones throughout the body. New research focuses on the role of chemicals as they interfere with vitamin A (retinols). retinols, a process essential for thyroid hormone expression. There is still no evidence that environmental chemicals directly block the thyroid receptor. Hydroxylované PCB During normal enzyme detoxification of PCBs in the maternal liver, certain PCB congeners are hydroxylated. This metabolic step enhances the binding affinity of the hydroxylated PCBs to TTR. Through their high-affinity binding the hydroxylated ci congeners displace essential f T4 that must get to the fetal brain to be converted to fT3. Hydroxylated PCBs also interfere with the normal excretion of thyroid hormones by inhibiting their sulfation. PCB hydroxylates also have estrogenic and antithyroid properties. Thyroidni disrupce u volně žijících obratlovců: Obojživelníci Gutleb and co-workers did a series of exposure studies with Xenopus laevisar\d Rana femporaria. They found increased incidence of mortality in tadpoles weeks after they ceased dosing the animals. Over an 80-day period, 47.5% of the tadpoles died. The X. laevis exposed to 7.7 pM and 0.64 nM PCB 126 exhibited swimming disorders prior to death. Both increased mortality and reduced T4 concentrations occurred in a dose- response manner in X laevis. Severe eye and tail malformations increased in the froglets in a dose-response manner after approximately 60-68 days. Ptáci Thyroid hormones in birds have been investigated for their role in migration and courtship. Preventing migrating species from breeding out of season is especially critical for their survival. From the 1950s through to the 1970s, fish-eating birds in the Great Lakes were experiencing very poor reproductive success. Keith suggested that the high embryo mortality and low chick survival in herring gulls nesting in upper Green Bay in the mid 1960s was both the result of a) the effects of the chemical residues from the mother on the embryo and b) the effects of the adult's contamination on its parental Ryby Migration of salmonids is linked with THs effecting a sequence of behaviors. In the laboratory, increases in T4 led to less display of aggressive behavior such as territoriality. Elevated concentrations of both T3 and T4 reduced the fishes' preference for shade to more open areas (phototaxis). T3 treatment caused the fish to swim with the current rather than against the flow (rheotaxis). Savci PCBs and dioxins have been shown to alter thyroid function in rodents by multiple mechanisms, including direct toxic effects on the thyroid gland, induction of thyroid hormone metabolism via the UDP-glucuronyl transferases, and interactions with thyroid hormone plasma transport proteins, particularly transthyretin. A number of investigators have evaluated the effects of maternal PCB exposure on thyroid function of rat pups. Pup serum thyroxine (T4) levels are markedly reduced by PCB or dioxin exposure, but the levels of the active form of the hormone, triiodothyronine (T3), are generally unchanged, or only slightly reduced. A relationship between exposure to dioxins and PCBs and alterations in thyroid hormones has also been reported in human infants. Infants exposed to higher levels of PCBs and dioxins had lower free T4 levels and higher thyroid-stimulating hormone levels. 22 PPAR • Deregulace PPAR a reprodukce • PPAR a karcinogenita Li gaud- índepend ent activation domain (AFI) I DBD Li gand-dependent activation domain (AF-2) ^--------------- LRD-Dim. DNA Binding Domain (2 zinc fingers) Ligand Binding and Dimerization Domains n PPAR I RXR LJ 9-ciVretinoic acid AGGTCANAGGTCA TCCAGTNTCCAGT r ch, CiglitEizone Ml TX NU % Pioglitaone JQCT % ch Troglitflironc CT MI Rosiglitazone FIG. 1. General structure and mechanism of action of PPARs, PPAR isoforms share a common domain structure and molecular mechanism of action. Membrane phospholipids I Phospholipase A2 Arachidonic acid Linoleic acid OxLDL Cycloxygenase Lipoxygenases Lipoxygenases 15-deq)cy PS-J2 LTB4, S-HETE, 15-HETE 9-HODE, 13-HODE Glitazones PPAR COj,H Hnoleic acid 303H 15-deoxy-A1Jli-PGJ2 CO,H HO 9-HODE COjH eicosapentaenoic acid H i— OH 8(S)-HETE COsH OH 13-HODE Insulin N L\ Secnetŕgn^ INSULIN ■V...VL......J___V.._ 7.......~l~......^ Genetic Factors Hyperglycemia FFAs Actlp»cylokln*s Envirgnment ZJZ. —.=:(' Insulin Resistance ) "f?........X I Upolysis A \ FATTY a ACIDS * Glucose A Producliori GIucůsě Uptake GLUCOSE * Figure 1. Insulin secretion and resistance. Organ manifestations of insulin resistance and metabolic consequences: adipose tissue — increased lipolysis and excessive FFA release; liver (and kidney) — increased glucose production; muscle — decreased glucose uptake. The role of insu lin resistance in brain is theoretical at present since evidence is available only from a brain-specific insulin receptor knockout mouse (4). ■ Key messages * With the thíazolídínedíones rosíglítazone and píoglítazone, a novel treatment modality for type 2 diabetes has become available. * The mechanism of action of these compounds involves binding to the peroxisome proliferator-activated receptor (PPAR) gamma , a transcription factor that regulates the expression of specific genes especially in fat cells but also in other tissues. * As monotherapy, glycosylated hemoglobin (HbA1c) on average can be improved by almost 1%. * Thiazolidinediones reduce insulin resistance not only in type 2 diabetes but also in non-diabetic conditions associated with insulin resistance such as obesity. Mo no ethyl (M EP) Monobutyl(MBP) Monopentyl(MPP) Monomethyli'MMP'i O Monohexyl(MHP) Mo no p no pyl (MPrP) Mono-(2-ethylhexyľj(MEHP) Fiyure 1. Structurally related phthalate monoesters. Diesters of o-phthalic acid are quickly metabolized in vivo to their active metabolites, the monesters. The length and structure of the side chain is important for toxicity. Ftaláty jako Ugandy PPAR - efekty na samčí a samicí reprodukční systém TABLE 1 A Summary of Eft'eets of in Utero Exposure to Fht nu hit es on the PPARiRXR E R: E R Developing MiiIľ Reprot uctiv ľ Tract Phthalates-* n DD Endpoint measured" DBP DEHP B BP DIN P u LJLJ Up- oi down- Testis regulation I Weigh! i i i i i i ~~ r i I 1 T Sperm numbeT L- i | | Degenerations atrophy of i i i ERE Regulated Gene seminiferous lubules Leydigcell hyperplasia/aggregales i i i D Phťhalates Leydigcell adenoma i i Cryptorchidism i i i i RXR Sex organs Epididymis: . wt, agenesis/malformed i i i _ PPAR 0- •0-0 \ TR or RAR Penis: de laved/incomplete preputial i i i - s separation, hypospadias, . wl of g Ian s x Down- Prostate: ... wt, agenesis l l l - S Regulation Seminal vesicle: J Wt i i i i |- te i j j 1 T 1 ŕ" li1 1 > ■ TR | | | V as deferens: . wt, maliormed/agenesis Miscellaneous E or RARE Regulated Gene Anogenilal disLance Q) l l l I Nipple retention i i i 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 Figure 1 Schemalic represenlalion of I he polenlial palhogenic links belween leslis developmenl and Ihe clinical manifes-lalions of leslicular dysgenesis syndrome (TDS). The similarilies in Ihe palhologies induced by in utero dibulyl phlhalale (DBP) adminislralion and human TDS are compared. Ftaláty modulují expresi enzymů kontrolujících syntézu a odpourávání steroidních hormonů 1 HDL LJ SR-BllT1- Ľ-4 TLS a AXA> iju y«, j ijy Vi&____/y u vätftít AÄ Aaí y y i. I StAR 4T1- ' PBR -It« Tl" 1 Sot-Reductase Tľ Dih ydrote st o sterone I6P-OHT Estrone Sp-OH T 1CYP2B1 TL14 CYP.^A2 TT' Vs 4-0 H E2 17B-HSD1V L* CYPIBI iL1» Testosterone 2(i-OH T I60C-OHT TYP2C11 liMr> CYP2C7TL;i CYP19 lO» ni! TbI3t5 Estradiol CYP2C11 Tl.'", F ACTAT tL" 2-OH E2 16«-OHE2 .aceatTl17 ES J-L'f E2-FA Esters T-FA Esters E2- Sulfate Basement membrane HOjC' ü hany acid ,^m '■■■ico^.....,----, ^ö»..........T^- ..:.iMF ATP esterol Pregnenolone Progesterone Testost e no n e r Aromatase ^f r Estradiol ^WfrHSDtV Ír Estrone U Cholesterol Pregnenolone 3ff-ws0 ý ^ Progesterone iJa-OHase 17,20-Lyase Androstenedione iLcn I Granulosa cel Tlieca cell Figure 4. Proposed model of MEHP action in the granulosa cell, MEHP interferes with two points of the steroid hormone pathway, Abbreviations: ER, estrogen receptor; 17«-0Hasef 17«-hydroxylase; SCC, P450 side chain cleavage enzyme, First, MEHP suppresses FSH-stimulated cAMP, possibly by inhibiting binding of FSH to its receptor or altering activation of adenylate cyclase (Grasso et al, 1993), MEHP also activates PPARs, possibly by release of fatty acids, endogenous activators of PPAR, The activation of either P PAR« or PPARy decreases aromatase m RNA. PPAR« activation also causes an increase in the transcript level of 17p-HSD IV, which metabolizes estradiol to estrone. Finally, both PPAR« and y increase levels of FABP in the cell, which is able to transport MEHP and fatty acids through the cell, delivering these ligands to the PPAR receptors. 25 ~^ 20 o ^ £ => J_ .-"-. ts ■^- !■ O ,_ +^ S FADH O I R — CH,— CH = CH—C — SCoA H,O Hydratage O I R—CH, —CH—CH, —C—SCo A OH Dehydro 9 er NAD* NADH + H O I R —CH,—C—CH,—C —SCoA II O II- ,-.. Ihiolysc R —CHj—C —SCoA + H,C — C — SCoA At styl Co A Aeyl CůA shartanad by twoearban atoms Oxidation of fatty acids by peroxisomes. Peroxisomes degrade fatty acids with more than 12 carbon atoms by a series of reactions similar to those used by liver mitochondria. In peroxisomes, however, the electrons and protons transferred to FAD and NAD+ during the oxidation reactions an subsequently transferred to oxygen, forming H202 PPARa a karcinogeneze S. Bosgra et ai / Toxicology 206 (2005) 309-323 PAtó TGACCTnTGACCT PPRE \ gene regulation ■ peroxisome proliferation ■ ß-oxidation (ACO, BFE, THLJ ■co-oxidation (CYP4A) cell proliferation apo ptosis | ion f\ H2O2 initiated hepatocytes i liver tumours Peroxisome proliferation ■ Liver growth - hypertrophy - hyperplasia ■ Induction of liver enzymes - peroxisomal enzymes (peroxisome proliferation) - P450 - the CYP4 genes ■ Proliferation of the Endoplasmic Reticulum and peroxisomes ■ Hypolipidaemia Oxidative Theory Fatty acids increase in peroxisomal ß-oxidation without catalase (<2 fold) Increase H202 ^^^^^^ genotoxic DNA damage Cancer Úloha Kupfférových buněk Kupffer cells Hep a to c yt es TGACCTnTGACCT PPRE I gens regulation peroxisome proliferation ^oxidation (ACO,BFE,THL] d »oxidation f CYP4 A) ■-*■ HřO? liferation + l sis j J cell proliferation apoptosis ? : initiated hepatocytes "i liver tumours Species Differences and Human Risk Assessment ■ PPARa exists m mouse, rat, guinea pig and human ■ Low hepatic levels \r\ human and g-pig ■ Human liver - No peroxisome proliferation - No induction of liver growth ■ If PPARs cause cancer m rats, do they cause cancer in humans? Therefore, no risk of cancer???