Literatura académica sobre el tema "Endokriner Disruptor"

ZusammenfassungPestizide, Schwermetalle, Weichmacher, Schadstoffe aus dem Tabakrauch und andere Substanzen wirken als Endokrine Disruptoren und können chronische Erkankungen und Tumorwachstum fördern sowie Alterungsprozesse von Geweben beschleunigen. Aminosäuren sind in der Lage, neoplastischen Prozessen entgegenzuwirken, beeinflussen neurochemische Resilienzfaktoren und ZNS-Faktoren positiv, beugen Insulinresistenz und damit Diabetes vor. Auch für die Detoxifikation eliminationspflichtiger lipophiler Substrate ist die ausreichende Versorgung mit Aminosäuren notwendig. Endokrine Disruptoren können immunologische und neurologische Regelkreise stören, den Aminosäurestoffwechsel empfindlich beeinträchtigen und zu einem deutlich erhöhten Bedarf an Aminosäuren führen. Eine gezielte individuelle laborkontrollierte Aminosäurezufuhr kann ein effektives therapeutisches Element in der angewandten Präventivmedizin sein.

Abstract: The endocrine disruptors are natural or arteficial molecules wich are present in the animal (human) environment and entering into the organism. They are bound by hormone receptors, simulating or inhibiting the normal hormonal message. This way they are able to stimulate or hinder the function of the given cell, as well as the synthesis and transport of hormones or receptors. They can cause faulty hormonal imprinting in critical periods of development with lifelong consequences, as alteration of hormone-influenced cell functions, inclination to or manifestation of diseases, so they have medical importance. The number of endocrine disruptors as well as their amount are large and continously growing. Numerous, in adult age manifested disease (e.g. malignant tumors) can be deduced to perinatal harms. Their long-lasting effect can cause the alteration of basal human developmental characteristics (e.g. start of menarche). Vitamins A and D are hormones (exohormones) and could be endocrine disruptors. Perinatal imprinting caused by endocrine disruptors is transmitted to the progenies epigenetically, which also can influence the drug-sensitivity of offspring’ receptors. If the epigenetic change is continuously transmitted to the progeny generations, this could have human-evolutionary importance. Orv Hetil. 2017; 158(37): 1443–1451.

Das Schilddrüsenhormon Triiodthyronin (T3) ist ein essenzieller Regulator physiologischer Prozesse der Entwicklung, des Wachstums und im Intermediärstoffwechsel. Täglich werden zahlreiche natürliche und synthetische Stoffe aufgenommen, die mit dem endokrinen System interferieren und deshalb als Endokrine Disruptoren (ED) bezeichnet werden. Zur Untersuchung einer direkten Interferenz von ED mit den Schilddrüsenhormonrezeptoren (TR) und ihrer transkriptionellen Aktivität wurde im Rahmen dieser Arbeit ein neues Luziferase-basiertes T3 Reportergensystem mit TRalpha1-transfizierten humanen Leberzellen aufgebaut. Durch Validierung mit dem synthetischen TR-Agonisten GC-1 und dem Antagonisten NH-3 konnte nachgewiesen werden, dass dieser Assay ein hoch-sensitives System zur Analyse von agonistischen sowie antagonistischen Effekten von Testsubtanzen darstellt. Zur Bestimmung der endokrinen Aktivitäten einiger humanrelevanter Vertreter aus den Stoffkategorien der Nahrungsmittel, Kosmetika, Pestizide und Industriechemikalien wurden Dosis-Wirkungskurven in Aktivierungs- und T3-Kompetitionsexperimenten ermittelt. In mikromolaren Konzentrationen wirkten von insgesamt 21 Testsubstanzen einige als reine Agonisten oder Antagonisten während andere gemischt agonistische/antagonistische Effekte hatten. Aufgrund ihrer hier beobachteten Effekte und der gegebenen Humanexposition wird eine eingehendere Analyse von 4-Methylbenzyliden Campher, 4-Nonylphenol, Acetochlor, Benzophenon 2, Benzophenon 3, Bisphenol A, Genistein, Octylmethoxycinnamat, Tetrabromobisphenol A und Xanthohumol empfohlen. Außerdem erwiesen sich einige Metaboliten von Schilddrüsenhormonen als potente Agonisten im T3-Reportergenassay und bedürfen weiterer Aufmerksamkeit. Für die molekulare Charakterisierung der Einflüsse solcher Substanzen auf die T3-regulierte Transaktivierung konnte mit dem hier etablierten Bioassay ein zuverlässiges neues Testsystem für reproduzierbare Screeningserien geschaffen werden.
Triiodothyronine (T3) is a crucial regulator of many physiological processes during development, growth and metabolism. A variety of natural and synthetic substances, which are collectively termed endocrine disrupters (ED) due to their interference with the endocrine system, is taken up on a daily base. A novel luciferase-based T3-responsive reporter gene system employing a human liver cell line transfected with thyroid hormone receptor (TR) alpha1 was established in this work to elucidate the potential molecular interference of certain ED with TR and their transcriptional activity. This assay was validated to be a highly sensitive and reliable tool for analyzing agonistic and antagonistic effects of test compounds using the synthetic TR agonist GC-1 and the antagonist NH-3. Dose-response data of test compounds contained in food, cosmetics, pesticides, plasticizers and other industrial chemicals were obtained after applying the substances alone in activation assays or in combination with T3 in competition assays. In total 21 test compounds were screened of which some acted as pure agonists or antagonists while others were mixed agonists/antagonists in the micromolar concentration range and only one was without effect. Follow-up studies are recommended for some of these substances with regard to their effects as determined in this bioassay and in light of information known on human exposure, i.e., 4-methylbenzyliden camphor, 4-nonylphenol, acetochlor, benzophenone 2, benzophenone 3, bisphenol A, genistein, octylmethoxycinnamate, tetrabromobisphenol A and xanthohumol. In addition some endogenous metabolites of thyroid hormones were surprisingly potent agonists in the T3 reporter gene assay and merit further attention. The novel bioassay established here represents a reliable tool for the screening and molecular characterization of substances interfering with T3-mediated transactivation of gene expression.

Due to the current rise of exposure to natural and synthetic compounds in our daily life, the debate concerning the safety of many substances is becoming increasingly relevant. The estrogenic activity of various compounds, described as xenoestrogens, is the major part of this debate. Humans beings are exposed to these substances from different environmental contaminations ranging from conscious intake of estrogenic substances, as in contraception or in hormone replace therapy (HRT), to unconscious exposure, from food, the use of synthetic material in daily life and air and water pollution. At this point the need for methods to investigate the activity and the safety of these substances is becoming increasingly important. Classical methods for the analysis of the estrogenic activity of substances, like batteries of in vivo test systems on the rat uterotrophic assay are not able to describe the different pathways of action of recently discovered estrogenic substances. This evidence was already shown by the Organization for Economic Cooperation and Development (OECD), introducing new test guidelines for the investigation of effects of endocrine disruptors (according to enhanced Test Guideline 407). As reviewed by Nilsson (Nilsson et al., 2001), after the interaction of the estrogens with the Estrogen Receptor (ER) in the cells, the mechanism of activation possible is not only via direct binding of the ER to the Estrogen Responsive Elements (EREs) present in the promoter region of the target gene, very well described for many target genes, but that also other mechanisms are used: the interaction of the ER with the AP 1, Sp 1 and NFkB modes, that are discovered but not yet comprehensively described. The aim of my work is to produce a microarray DNA chip for the investigation of the estrogenic activity of different compounds present in the environment. The chip will consist of a selection of 100 genes that are estrogen responsive and it will cover the spectrum of activities of estrogenic compounds in various organs of the body. In the gene selection, genes were chosen that are estrogen responsive in the classical target tissues of estrogens, linked to reproduction, like uterus and mammary gland, and also in tissues not related to reproduction like liver, bones and capillars. In addition, other genes are included to monitor different pathways that are related to disease states; control of cell proliferation, apoptosis or cancer related genes. Currently these kinds of investigations are already in process, but by other methods which are more time consuming and with a lower throughput e.g. the gene expression profiling using the real time RT-PCR. The use of microarray’s satisfies the need for a less time consuming, high throughput method, to obtain a fast characterization of the gene expression finger print of the candidate substances and their mechanism of action in the organism. In my work I investigated the estrogenic potency of different Xenoestrogens that commonly occur in our daily life, in rat cells and tissue using well known estrogen sensitive genes like C3, Clu, IGFBP1 and CaBP9k. I focused on their effect on cell proliferation, studying PCNA expression. For the first time sensitivity of the gene CA2 was proofed in liver and uterus. A new identified mRNA sequence, r52, was characterized for its sensitivity to estrogenic exposure. This sequence was investigated at the molecular level expanding the known nucleic sequence. I produce a microarray chip with 16 genes to investigate the estrogenic potency of different compounds. As proof of principle of the microarray method completely produced in house I compared the result of gene expression obtained by the chip to that obtained by real time RT PCR finding a similarity of results. This new established method is less sensitive than the real-time RT PCR but allows a high throughput of gene expression analysis producing at the end a more complete picture of the expression signature of a compound.

Due to the current rise of exposure to natural and synthetic compounds in our daily life, the debate concerning the safety of many substances is becoming increasingly relevant. The estrogenic activity of various compounds, described as xenoestrogens, is the major part of this debate. Humans beings are exposed to these substances from different environmental contaminations ranging from conscious intake of estrogenic substances, as in contraception or in hormone replace therapy (HRT), to unconscious exposure, from food, the use of synthetic material in daily life and air and water pollution. At this point the need for methods to investigate the activity and the safety of these substances is becoming increasingly important. Classical methods for the analysis of the estrogenic activity of substances, like batteries of in vivo test systems on the rat uterotrophic assay are not able to describe the different pathways of action of recently discovered estrogenic substances. This evidence was already shown by the Organization for Economic Cooperation and Development (OECD), introducing new test guidelines for the investigation of effects of endocrine disruptors (according to enhanced Test Guideline 407). As reviewed by Nilsson (Nilsson et al., 2001), after the interaction of the estrogens with the Estrogen Receptor (ER) in the cells, the mechanism of activation possible is not only via direct binding of the ER to the Estrogen Responsive Elements (EREs) present in the promoter region of the target gene, very well described for many target genes, but that also other mechanisms are used: the interaction of the ER with the AP 1, Sp 1 and NFkB modes, that are discovered but not yet comprehensively described. The aim of my work is to produce a microarray DNA chip for the investigation of the estrogenic activity of different compounds present in the environment. The chip will consist of a selection of 100 genes that are estrogen responsive and it will cover the spectrum of activities of estrogenic compounds in various organs of the body. In the gene selection, genes were chosen that are estrogen responsive in the classical target tissues of estrogens, linked to reproduction, like uterus and mammary gland, and also in tissues not related to reproduction like liver, bones and capillars. In addition, other genes are included to monitor different pathways that are related to disease states; control of cell proliferation, apoptosis or cancer related genes. Currently these kinds of investigations are already in process, but by other methods which are more time consuming and with a lower throughput e.g. the gene expression profiling using the real time RT-PCR. The use of microarray’s satisfies the need for a less time consuming, high throughput method, to obtain a fast characterization of the gene expression finger print of the candidate substances and their mechanism of action in the organism. In my work I investigated the estrogenic potency of different Xenoestrogens that commonly occur in our daily life, in rat cells and tissue using well known estrogen sensitive genes like C3, Clu, IGFBP1 and CaBP9k. I focused on their effect on cell proliferation, studying PCNA expression. For the first time sensitivity of the gene CA2 was proofed in liver and uterus. A new identified mRNA sequence, r52, was characterized for its sensitivity to estrogenic exposure. This sequence was investigated at the molecular level expanding the known nucleic sequence. I produce a microarray chip with 16 genes to investigate the estrogenic potency of different compounds. As proof of principle of the microarray method completely produced in house I compared the result of gene expression obtained by the chip to that obtained by real time RT PCR finding a similarity of results. This new established method is less sensitive than the real-time RT PCR but allows a high throughput of gene expression analysis producing at the end a more complete picture of the expression signature of a compound.

In der vorliegenden Arbeit wurde untersucht, inwieweit die Reproduktionsbiologie von aquatischen Lebewesen durch die Gewässerbelastung mit endokrin wirksamen Substanzen (endocrine disruptors, ED) beeinflusst wird. Mit dem Amphib Xenopus laevis steht ein etabliertes Studienmodell zur Untersuchung der Wirkungen von ED auf die Reproduktionsbiologie zur Verfügung, das für die vorliegenden Studien modifiziert und erweitert sowie mit gewässeranalytischen Methoden verknüpft wurde. Die Gefährdung wasserlebender Tiere durch Gewässerbelastung mit ED kann erfasst werden, indem die Wirkung einer ausgewählten Einzelsubstanz auf die Reproduktionsbiologie untersucht wird. Anschließend erfolgt ein Nachweis der Substanz in Umweltproben. Durch Expositionsversuche, histologische Untersuchungen und Expressionsnachweis eines molekularen östrogenen Biomarkers konnte festgestellt werden, dass Bisphenol A (BPA) in Kaulquappen von Xenopus laevis verweiblichend wirkt und seine Effekte über eine Bindung an den Östrogenrezeptor vermittelt. Chemische Analysen während der Expositionsversuche zeigten, dass BPA von den Kaulquappen aufgenommen wird und dass eine geringe Abbaubarkeit der Substanz während eines Zeitraums von 48 Stunden besteht. Die Analyse von BPA in Wasserproben, die aus dem Fluss Alb oder aus Kläranlagenausläufen stammten, zeigte, dass BPA im Gewässer in relevanten Konzentrationen vorhanden ist und hauptsächlich durch eine Kläranlage in den Fluss eingeleitet wird. In einem Gewässer liegt allerdings ein Gemisch aus unterschiedlichen ED mit verschiedenen Wirkmechanismen vor. Die Gewässerbelastung mit endokrin wirksamen Substanzen kann weiterhin untersucht werden, indem Gewässerextrakte fraktioniert und in in vitro-Screeningmethoden getestet werden. Als Screeningmethoden dienten Rezeptorbindungsstudien an Östrogen- und Androgenrezeptoren sowie die Behandlung von Leberzellkulturen mit den Gewässerextrakten aus der Alb, um eine Regulation der Expression bestimmter Biomarkergene durch potenzielle ED nachzuweisen. Dazu wurde als neuer (anti)androgener und (anti)östrogener Biomarker das Retinol-binding Protein eingeführt. Die Untersuchung der Gewässerextrakte mit diesen Methoden zeigte, dass die Alb eine Belastung mit endokrin wirksamen Substanzen aufweist und dass hauptsächlich östrogen wirksame Substanzen vorkommen. Die Proben aus den Kläranlagenausläufen weisen die höchste endokrine Aktivität auf.
The present study examined the influence of endocrine active compounds (endocrine disruptors, ED), which are present in surface waters, on reproductive biology of aquatic organisms. The amphibian Xenopus laevis is a well-established model organism for the study of effects of ED on reproduction. It has been modified and broadened for the purpose of this study, and it was combined with chemical methods for water analyses. It is possible to assess water pollution with ED by detecting effects on repro-ductive biology of one particular substance, and then by looking for this substance in environmental water samples. We showed the feminizing potency of Bisphenol A (BPA) in conducting exposure experiments with tadpoles, in examining histological samples of gonads and in detecting the induction of the expression of a molecular estrogenic biomarker. BPA was recognized to mediate its effects via binding to the estrogen receptor. Moreover, analysis of BPA during exposure experiments revealed that BPA is taken up by tadpoles and is not readily degradable during a time period of 48 hours. Chemical analyses of environmental water samples from the river Alb or samples from sewage treatment works (STW) showed that BPA is released into the environment by STW effluents. In surface waters, there are different kinds of ED with different modes of action. Thus, it is another possibility to assess water pollution with ED by fractionating environmental water samples and by testing these fractions in rapid in vitro-screening methods. In the present work, receptor binding assays were carried out, both examining the binding to estrogen and androgen receptors. Furthermore, Xenopus laevis hepatocyte cultures were treated with fractions of environmental samples and biomarker expression was detected. A new biomarker to assess (anti)androgenic or (anti)estrogenic modes of action, respectively, was established. This new biomarker was the Retinol-binding Protein. The results obtained by these methods revealed that the river Alb is mainly polluted with estrogenic ED. Samples from STW effluents possessed the highest endocrine activity.

Organotin compounds are one of the most produced and most used organometallic compounds. Some of these substances are endocrine disruptors, persistent organic polutants and their high toxic effects are observed. That’s why their presence in the environment caused by human activity could endanger many organisms. The aim of this thesis is summarize their properties and their occurrence in the environment. Then the quick, easy and relatively cheap method for determination of trialkyltin compounds in heavily poluted aquatic sediments using capillary zone electrophoresis is developed.

Nespušteni testis predstavlja odsustvo testisa u skrotumu sa jedne ili obe strane. Faktori rizika za pojavu nespuštenog testisa obuhvataju genetsku predispoziciju, prevremeno rođenje, nisku porođajnu masu i prenatalnu izloženost endokrinim disruptorima ili duvanskom dimu. Endokrini disruptori se definišu kao egzogene supstance koje imaju uticaj na homeostazu organizma i proizvodnju reproduktivnih hormona. U ovoj grupi nalaze se organofosforni pesticidi koji se široko upotrebljavaju u poljoprivredi. Većina organofosfornih pesticida ima antiandrogeni uticaj i uz činjenicu da živimo u pretežno agrarnoj sredini predmet su našeg interesovanja. Cilj istraživanja je da se utvrdi razlika u izloženosti organofosfornim pesticidima korišćenjem upitnika kreiranog po modelu standardizovanog Evropskog upitnika QLK 4-1999-01422 kod osoba koje su rodile zdravu mušku decu i osoba koje su rodile decu sa nespuštenim testisom. Pored toga, cilj istraživanja je i da se odredi i uporedi vrednost metabolita organofosfornih pesticida (dimetilfosfat, dimetilditiofosfat, dietilfosfat, dietiltiofosfat i dietilditiofosfat) u urinu majki koje su rodile mušku decu sa nespuštenim testisima i majki koje su rodile zdravu mušku decu. Metodologija: Rad je radomizovano, prospektivno, kliničko istraživanje sprovedeno na Klinici za ginekologiju i akušerstvo Kliničkog centra Vojvodine i Katedri za farmakologiju i toksikologiju Medicinskog fakulteta, Univerziteta u Novom Sadu. U kliničko istraživanje uključeno je 50 porodilja koje su rodile mušku decu sa nespuštenim testisima (eksperimentalna grupa) i 53 porodilje koje su rodile zdravu mušku decu (kontrolna grupa) u periodu od oktobra 2012. godine do aprila 2018. godine. Tokom boravka u porodilištu ispitanice su popunjavale upitnik o navikama nakon čega im je uzet uzorak urina radi analiziranja nivoa metabolita OF pesticida. Uzorci urina su pripremljeni metodom koju su opisali Wu i saradnici 2010. godine, a potom analizirani na gasnom hromatografu masenom spektrofotometaru marke Agilent 7890A. Rezultati: Ispitivane grupe se ne razlikuju po starosti ispitanica (prosečna starost kontrolne grupe 29,41 ± 5,58 godina, a eksperimentalne 30,54 ± 4,87 godina). U obe grupe prosečno je ispitanicama ovo bila druga trudnoća. Ispitanice se nisu razlikovale ni po načinu porođaja. Prosečna gestacijska nedelja trudnoće na porođaju iznosila je 39,45 ± 1,38 nedelja za kontrolnu grupu i 39,20 ± 1,38 nedelja za eksperimentalnu grupu, a porođajna masa novorođenčeta 3527,30 ± 470,16 g u kontrolnoj grupi i 3404,37 ± 508,20 g u eksperimentalnoj grupi. Statistički značajna razlika postoji u odnosu na mesto stanovanja (50,9 % ispitanica kontrolne grupe i 77,6 % ispitanica eksperimentalne grupe žive u gradu), jedinicu stanovanja (67,9 % kontrolne i 45,7 % ispitanica eksperimentalne grupe žive u kući) i načinu začeća (6 % ispitanica eksperimentalne i 1,9 % ispitanica kontrolne grupe prijavilo je IVF kao način začeća). Skoro polovina ispitanica obe grupe su pušači, a njih 32,7 % kontrolne grupe i 38,8 % eksperimentalne pušile su i tokom trudnoće. Izloženost pesticidima prijavilo je 50,9 % ispitanica kontrolne i 44 % ispitanica eksperimentalne grupe, a profesionalnu izloženost prijavilo je 3 ispitanice kontrolne i 2 ispitanice eksperimentalne grupe. Ispitanice se ne razlikuju ni po poreklu voća i povrća koje konzumiraju, kao ni po vrsti voća koje su konzumirale tokom trudnoće. Prosečne izmerene vrednosti DMF u kontrolnoj grupi iznose 5,604 ± 6,103 ug/L, a u eksperimentalnoj 4,815 ± 6,729 ug/L. Izmerene vrednosti DEF u kontrolnoj grupi su 0,408 ± 0,447 ug/L, a u eksperimentalnoj 0,461 ± 0,593 ug/L. Nivo DMDTF u kontrolnoj grupi bio je 0,431 ± 0,508 ug/L, a u eksperimentalnoj 0,547 ± 0570 ug/L, a DETF 0,403 ± 0,606 ug/L u kontrolnoj i 0,529 ± 0,725 ug/L u eksperimentalnoj grupi. Ni jedan metabolit ne pokazuje statistički značajnu razliku u ispitivanim grupama. Slične vrednosti dobijene su i za vrednosti korigovane za nivo kreatinina. Univarijantna regresiona analiza pokazala je da ispitanice koje žive u gradu imaju 3,3 puta veće šanse da rode dete sa nespuštenim testisom, a one koje žive u stanu imaju 2,5 puta veće šanse za isti ishod. Statistički značajna razlika primećena je u nivou DEDTF u zavisnosti od starosti ispitanica i jedinici stanovanja. Više vrednosti DETF dobijene su kod ispitanica koje su bile na hormonskoj terapiji tokom trudnoće. Ispitanice koje su prijavile da su bile izložene pesticidima tokom trudnoće u urinu su imale statistički značajno više vrednosti DMDTF u odnosu na ispitanice koje su se izjasnile da nisu bile izložene pesticidima. Slični rezultati za vrednost DEDTF dobijeni su kod ispitanica koje su se izjasnile da poseduju kućne ljubimce. Statistički više vrednosti DEF i DETF korigovano za nivo kreatinina dobijene su kod ispitanica koje nisu konzumirale jabuke, a više vrednosti DEF i DEDTF dobijene su kod ispitanica koje su konzumirale maline i kupine tokom trudnoće. Ostale grupe nisu pokazale statistički značajnu razliku među ispitivanim grupama. Zaključci: Izloženosti trudnica OF pesticidima nije značajno veća u grupi majki koje su rodile decu sa nespuštenim testisom u odnosu na izloženost OF pesticidima kod majki zdrave muške dece. Vrednosti metabolita OF pesticida (dimetilfosfat, dimetilditiofosfat, dietilfosfat, dietiltiofosfat, dietilditiofosfat) u urinu majki koje su rodile mušku decu sa nespuštenim testisima nije viša u odnosu na vrednosti metabolita OF pesticida (dimetilfosfat, dimetilditiofosfat, dietilfosfat, dietiltiofosfat, dietilditiofosfat) izmerene u jutarnjem urinu majki koje su rodile zdravu mušku decu.
Undescended testis is the absence of testis in the scrotum on one or both sides. Risk factors for the occurrence of undescended testis include genetic predisposition, premature birth, low birth weight and prenatal exposure to endocrine disruptors or tobacco smoke. Endocrine disruptors are defined as exogenous substances that can affect homeostasis of the organism and the production of reproductive hormones. In this group are organophosphorus pesticides that are widely used in agriculture. Most of organophosphorus pesticides have anti-androgenic effect and with the fact that we live in a predominantly agricultural area, they are the focus of our interest. The aim of the research: The aim of this study is to determine the difference in exposure to organophosphorous pesticides using questionnaires created by standardized European model questionnaire QLK 4-1999-01422 in individuals who gave birth to a healthy male children and women who gave birth to children with undescended testis. In addition, the aim of this study is to determine and compare the value of metabolites of organophosphorus pesticides (dimethylphosphate, dimethyldithiophosphate, diethylphosphate, diethylthiophosphate and diethyldithiophosphate) in the urine of mothers who gave birth to male children with undescended testis and mothers who gave birth to healthy male children. Methodology: This work is randomized, prospective, clinical research conducted at the Clinic for gynaecology and obstetrics of the Clinical center of Vojvodina and the Department of Pharmacology and toxicology of the Faculty of Medicine, University of Novi Sad. This clinical research includes 50 new mothers that gave birth to male children with undescended testes (experimental group) and 53 new mothers that gave birth to healthy male children (control group) in the period from October 2012 to April 2018. During their stay at the maternity hospital the subjects were asked to fill out a questionnaire about habits and to give a urine sample for analyzing the level of metabolites of organophosphus pesticides. Urine samples were then prepared using the method described by Wu and associates 2010, and analyzed on gas chromatograph with a mass spectrophotometer Agilent 7890A brand. Results: Study groups do not differ according to the age of women (average age of control group is 29.41 ± 5.58 years, and experimental 30.54 ± 4.87 years). In both groups this was second pregnancy on average. The subjects did not distinguish either by the way of delivery. The average gestational weeks of pregnancy to childbirth was 39.45 ± 1.38 weeks for the control group and 39.20 ± 1.38 weeks for the experimental group, and birth weight of newborn was 3527.30 ± 470.16 g in control group and 3404.37 ± 508.20 g in the experimental group. There is no statistically significant difference in relation to the place of residence (50.9 % of the control group and 77.6 % of experimental live in the city), the living unit (67.9 % and 45.7 % of the control and experimental groups are living in the house) and the way of conception (6 % of experimental and 1.9 % of the control group reported IVF as a way of conception). Nearly half of both groups are smokers, and 32.7 % of women in the control group and 38.8 % in experimental smoked during pregnancy. Exposure to pesticides reported 50.9 % of mothers in control and 44 % of mothers in the experimental group. Professional exposure was reported by 3 control subjects and 2 subjects in experimental group. The subjects did not differ according to the origin of fruits and vegetables they were consuming, neither regarding the type of fruits they consumed during pregnancy. Average level of dimethylphosphate in control group was 5.604 ± 6.103 ug/L, and in experimental 4.815 ± 6.729 ug/l. Levels of diethylphosphate in control group were 0.408 ± 0.447 ug/L, and in experimental 0.461 ± 0.593 ug/l. DMDTP level in the control group was 0.431 ± 0.508 ug/L, and in experimental 0.547 ± 0570 ug/L, and the DETP was measured 0.403 ± 0.606 ug/L in control, and 0.725 ± 0.529 ug/L in the experimental group. These metabolites showed no statistically significant difference in the examined groups. Similar values are obtained for the adjusted values for creatinine level. Univariate regression analysis showed that the subjects who live in town are 3.3 times more likely to have child with undescended testis, and those who live in the apartment are 2.5 times more likely for the same outcome. Statistically significant difference was noticed in DEDTP level depending on the age of the subject and the living unit. Higher levels of DETP metabolites were detected in subjects that have been on hormonal therapy during pregnancy. The subjects who reported being exposed to pesticides during pregnancy had statistically significantly higher DMDTP values in relation to the subjects that were not exposed to pesticides. Similar results are obtained for the DEDTP level with higher levels in subjects owning pets. Statistically higher levels of DEP and DETP adjusted for creatinine were obtained in subjects that were not reporting eating apples, and higher levels of DEP and DEDTP were obtained in subjects that consumed raspberries and blackberries during pregnancy. Other groups showed no statistically significant difference between the study groups. Conclusion: Exposure of pregnant women to OP pesticides is not significantly greater in the group of mothers who gave birth to children with undescended testis in relation to exposure to OP pesticides in mothers of healthy male children. The level of OP metabolites (dimethylphosphate, dimethyldithiophosphate, diethylphosphate, diethylthiophosphate and diethyldithiophosphate) in the urine of mothers who gave birth to children with undescended testis is not higher in relation to the levels of OP metabolites (dimethylphosphate, dimethyldithiophosphate, diethylphosphate, diethylthiophosphate and diethyldithiophosphate) recorded in urine of mothers who gave birth to healthy male children.

Die vorliegende Arbeit befasst sich mit der Erweiterung des etablierten Stu-dienmodells Xenopus laevis zur Untersuchung der Wirkung von endocrine disruptors auf die Reproduktionsbiologie von Amphibien. Um einen Einblick in die grundlegenden Mechanismen der sexuellen Differenzierung von Amphibien zu gewinnen, wurden die Konzentrationen bestimmt, mit denen androgene und estrogene Sexualsteroide während der larvalen Entwicklung in verschiedenen Stadien von Xenopus vorliegen. Parallel wurde das Auftreten der korrespondierenden Rezeptoren im Verlauf der Entwicklung untersucht, über welche die hormonelle Wirkung vermittelt wird. Auf der Basis der gewonnenen Erkenntnisse konnte eine neue Hypothese zur sexuellen Differenzierung von Amphibien entwickelt und vorgestellt werden. Sie stellt das Enzym 5alpha-Reduktase, das die Umwandlung von Testosteron in das potentere und nicht weiter aromatisierbare Androgen Dihydrotestosteron (DHT) bewerkstelligt, in den Mittelpunkt des Prozesses der Geschlechtsdifferenzierung. Abhängig von der genetisch bedingten Expression dieses Enzyms kommt es zu einem höheren oder niedrigeren Auftreten des DHT und damit zu Unterschieden im Verhältnis von DHT zu Estradiol (E2). Der Charakter dieses Verhältnisses scheint der entscheidende Auslöser für die Entwicklung eines weiblichen oder männlichen Phänotyps zu sein. In einem zweiten, anwendungsorientierten Teil wurde untersucht, in wie weit die bislang auf Laboruntersuchungen beschränkte Arbeit mit X. laevis auf Feldstudien erweiterbar ist und ob sich auf diese Weise gewonnene Daten auf die Situation heimischer Amphibien übertragen lassen. Parallele Expositionen des Krallenfrosches einerseits und des Grasfrosches (Rana temporaria) andererseits gegenüber realen Medien unter Freilandbedingungen bestätigten die hervorragende Eignung des Studienmodells X.laevis zur Beurteilung endokriner Belastungssituationen. Darüber hinaus konnte gezeigt werden, dass sich durch die Verwendung von Festphasenextrakten die endokrinen Wirkungen komplexer Matrizes unter standardisierten Laborbedingungen charakterisieren lassen. Rezeptorbindungsstudien sowie Untersuchungen zur Genexpression spezifischer Marker, histologische Betrachtungen von Gonadengewebe und die Bestimmung von Geschlechterverhältnissen ermöglichten Aussagen auf vielfältigen Nachweisebenen. Auf diese Weise konnte das Potenzial, mit dem Proben jeder Art, sowohl durch kurz- als auch durch langfristige Exposition, adverse Effekte auf das amphibische Hormonsystem hervorrufen können, umfassend und differenziert analysiert werden.
The presented work aims to contribute to the various opportunities of studying the effects of endocrine disruption on sexual differentiation in amphibians provided by the well established model Xenopus laevis. In order to gain insight into the basic mechanisms underlying the sexual differentiation in amphibians, the concentrations of androgen and estrogen sexual steroids during several stages of the larval development of Xenopus were determined. In parallel, the ocurrence of the corresponding receptors, which mediate the effects of the respective hormones, was observed. Based on the results of the studies described, a new hypothesis regarding sexual differentiation in amphibians is presented, which assignes the enzyme 5alpha-reductase as the central element of sexual development. This enzyme converts the androgen testosterone into dihydrotestosterone, which can not be aromatized into estradiol. Depending on the genetic sex of the indivual, genexpression of 5a-reductase may differ and therefore lead to a characteristic ratio of androgens and estrogens. We suggest, that this ratio might be the essential trigger for amphibians to develop into a male or a female. A second part aimed to enlarge the Xenopus model to the use in field studies and to proof the transferability of such data to the situation of endemic amphibians. Exposure in parallel of Xenopus on one hand and the green frog Rana temporaria on the other to the effluent of a bavarian wastewater treatment plant revealed the exceeding suitability of the model to asess the endocrine charge of the environment. Furthermore, the use of solid phase extracts derived from natural samples allowed the characterization of the respective endocrine potential under standardized laboratory conditions. Rezeptor binding studies, detection of genexpression of specific biomarkers, histological examination of gonadal tissue and the determination of sex ratios provided the evaluation of effects on several levels of investigation. By this means the Xenopus model offers the opportunity to assess the ability of any kind of sample to cause endocrine impacts on amphibians after short time as well as after long time exposure in a broad and at the same time differentiated way.