Tesis sobre el tema "Vinclozolin"

Vinclozolin (VCZ), an antiandrogenic fungicide, is an endocrine disrupting chemical that is known to possess high affinity for the androgen receptor (AR) and modulate expression of critical androgen-dependant genes in the prostate. In this study, viability and expression of AR, NKX3.1 and CYP3A4 genes were measured in androgen-sensitive prostate cells LNCaP after exposure to VCZ and VCZ treated with S9 microsomes in a time and dose dependent manner. NKX3.1 is an androgen regulated gene that plays a vital role in prostate development. CYP3A4 is involved in xenobiotic metabolism. VCZ decreased the viability at high doses after 48 hours which was slightly mitigated by treatment with S9 metabolites. Expression of NKX3.1 and CYP3A4 was upregulated while an initial downregulation of AR was observed. NKX3.1 upregulation corroborates with possibility of antiandrogens to act as androgens in LNCaP. The results illustrate that VCZ can interfere with the expression of critical prostate genes.

Les polluants environnementaux, en particulier les perturbateurs endocriniens (PE), agissent à très faibles doses sur des cibles multiples. Les effets rapportés portent en majorité sur les organes de reproduction. Très peu d'études ont porté sur le squelette alors que le cartilage et l'os sont sous un puissant contrôle hormonal, depuis le stade fœtal où le système hormonal se met en place jusqu'au vieillissement, en passant par la naissance (hormones thyroïdiennes, hormone de croissance), la puberté et la ménopause chez la femme (stéroïdes sexuels). L'objectif de ce travail est d'étudier les effets de polluants anti-androgènes (vinclozoline, V et métabolite actif M2) ou xenestrogènes (génistéine, G; bisphénol A, BPA), in vivo sur le développement du squelette du rat Wistar et in vitro sur les marqueurs de différenciation chondrogéniques. Les effets in vivo ont été étudiés à des doses inférieures aux "No Observed Adverse Effect Levels " (NOAEL) fixés par les instances européennes (EFSA) et internationales (US EPA). Des rattes ont été exposées à V, G seuls, combinés (GV) et/ ou associés au BPA (BGV), et ce de la conception des petits jusqu'à leur sevrage (J30) ou leur sacrifice (J30, J110). Les effets ont été recherchés sur des petits de mères et portées différentes, quatre pour chaque traitement, âge et sexe. Les effets in vitro du métabolite M2 de la Vinclozoline, associé ou non avec G et BPA, ont été étudiés sur l'expression de marqueurs chondrogéniques en utilisant : 1) un modèle murin de cellules souches inductibles vers la voie chondrogénique (C1) pour les effets sur la différenciation chondrogénique précoce et 2) des chondrocytes de souriceaux nouveau-nés, différenciés en culture primaire ou dédifférenciés (passages répétés). Comparaison avec les effets du bFGF, facteur de dédifférenciation chondrogénique. Résultats : In vivo, l'exposition à V, seule ou associée à G ou au BPA induit chez les rattes F1 exposées, une cannelure de la queue, discrète mais perceptible à la palpation en regard de chaque articulation intervertébrale. Les xénestrogènes tendent à réduire cet effet de V. Les rats et les animaux F2 ne sont pas atteints. L'examen par micro CT-scan montre une augmentation significative de la largeur des apophyses transverses (ITA) des vertèbres, et une diminution de la hauteur des corps vertébraux chez les rattes F1 exposées en regard des contrôles. Ces modifications anatomiques rappellent certaines pathologies génétiques des collagènes (dysplasies épiphysaires) chez l'homme Elles sont absentes chez les rats F1 et les animaux F2. Elles sont en partie transitoires car présentes à J30 (effets sur ITA et longueur) quand seul l'effet sur l'ITA perdure à J110. L'examen histologique des cartilages de croissance des corps vertébraux montre un déséquilibre entre les zones de prolifération et d'hypertrophie qui évoque une modification de la maturation du cartilage de type estrogénique. Ces effets sont ici aussi transitoires et majoritairement observés chez les rattes. L'effet plus prononcé du BPA lisse toutes les autres activités. Cela suggère que les PE pourraient moduler la différenciation du cartilage de croissance. C'est ce qui a été étudié in vitro. In vitro. Le premier objectif était d'évaluer les effets des PE sur la dynamique d'induction chondrogénique (cellules C1). Nous montrons que l'addition de M2 seul ou avec G ou BPA modifie le processus de maturation du collagène2 sans effet sur les autres marqueurs (SOX9, Agrécane, Col10). M2 prolonge l'expression de COL2A immature et retarde son remplacement par l'isoforme COL2B. Le second objectif était d'étudier les effets des PE sur la régulation de l'expression de COL2A au cours du processus de dé-différenciation des chondrocytes in vitro. L'expression de COL2A augmente avec le degré de dédifférenciation cellulaire (passages successifs) et double en présence de M2, G et BPA. Cet effet dépend des récepteurs aux estrogènes (ER) et des voies p38-MAPK. (...)

Substanzen, die durch ihr hormonell wirksames Potenzial mit dem Hormonsystem interagieren und adverse Effekte auf die Reproduktion von Invertebraten und Vertebraten ausueben koennen, erlangten in den letzten Jahrzehnten große Aufmerksamkeit. Viele dieser Substanzen reduzieren die Fertilitaet oder die Fekunditaet, fuehren zu Abnormalitaeten in der Ontogenese oder im Verhalten der Tiere und haben Einfluss auf die Geschlechterverhaeltnisse. In der vorliegenden Arbeit wurden verschiedene Aspekte dieses Themengebietes bearbeitet. Das in Europa endemisch vorkommende Rotauge (Rutilus rutilus), ein Sueßwasserfisch, wurde als Modelltier fuer den Nachweis von (anti)androgenen Effekten auf aquatisch lebende Organismen etabliert. Zum Nachweis der (anti)androgenen Wirkmechanismen wurden die Tiere mit Modellsubstanzen aus drei verschiedenen Gruppen exponiert. Aus der Gruppe der Substanzen mit potenziell androgener Wirkung wurden Triphenylzinn (TPT) und Methyltestosteron (MT) verwendet, aus der Gruppe der Antiandrogene Vinclozolin (VIN) und Cyproteronazetat (CYP) und aus der Gruppe der Aromatasehemmer, und somit potenziell androgener Wirkung, Letrozol (LET) und Fenarimol (FEN). Feedbackmechanismen auf die Hypothalamus-Hypophysen-Gonaden-Achse (mRNA-Expression des Luteinisierenden Hormons, des Follikel stimulierenden Hormons und der Aromatase), mRNA-Expression potentieller Biomarker in der Leber (Androgen-Rezeptor-mRNA, Oestrogen-Rezeptor-mRNA), Sexsteroidspiegel im Blutplasma (17beta-Oestradiol und 11-keto-Testosteron), Enzymaktivitaeten im Gehirn (Aromatase), Histologie der Gonaden, Totallaenge, Gewicht und Geschlechterverteilung wurden als Endpunkte analysiert, um adverse Effekte auf die Reproduktionsbiologie von R. rutilus zu zeigen. Die untersuchten Endpunkte eigneten sich sehr gut zum Nachweis verschiedener Wirkmechanismen.
Substances that are able to interact with the endocrine system and cause adverse effects on the reproduction of invertebrates and vertebrates have gained much attention over the last few decades. Many of these substances reduce fertility or fecundity, lead to developmental abnormalities or abnormalities in the behaviour of animals and have an impact on sex ratios. The present study examines various aspects of these topics. The roach (Rutilus rutilus), a freshwater fish endemic in Europe, was established as a model animal for the detection of (anti)androgenic effects on aquatic organisms. For examination of the (anti)androgenic action, the animals were exposed to model compounds from three different groups: triphenyltin (TPT) and methyltestosterone (MT) from the group of substances with potentially androgenic effect, vinclozolin (VIN) and cyproteronacetate (CYP) from the group of antiandrogens, and letrozol (LET) and fenarimol (FEN) from the group of aromatase inhibitors, which thus have a potentially androgenic effect. Feedback mechanisms on the hypothalamus-pituitary-gonad-axis (mRNA expression of luteinising hormone, follicle stimulating hormone and aromatase), mRNA expression of potential biomarkers in the liver (androgen receptor mRNA, oestrogen receptor mRNA), steroid levels in the blood plasma (17beta-oestradiol and 11-ketotestosterone), enzyme activity in the brain (aromatase), histology of the gonads, total length, weight and sex ratios were analysed as endpoints to show adverse effects on the reproductive biology of R. rutilus. The studied endpoints are suitable for the detection of different modes of action. The histological examination of the gonads proved to be especially sensitive with the exposure to AACs to resulting in fundamental adverse damages to the gonads. It was ascertained that - in the early stages of ontogeny - androgens play as crucial of a role in the development of the gonads as previously attributed primarily to oestrogens.

Notre environnement contient de plus en plus de polluants dont les perturbateurs endocriniens (PE), associés à une augmentation de la prévalence de pathologies graves et à l'émergence de nouvelles pathologies. Récemment, une nouvelle pathologie amélaire appelée hypominéralisation des molaires et des incisives (MIH) a été décrite. Cette pathologie, dont l'étiologie est inconnue, est devenue un véritable problème de santé publique avec une prévalence d'environ 18%. L'objectif de ce travail fut de rechercher des relations potentielles entre l'exposition aux PE et le MIH. Pour cela, des rats Wistar ont été exposés à de faibles doses de trois PE, le bisphénol A (BPA), la génistéine et la vinclozoline, seuls ou en association, de la conception jusqu'au sacrifice. 75% des rats traités au BPA seul, présentaient des taches d'hypominéralisation amélaire dont les caractéristiques structurales et biochimiques étaient comparables à celles des dents humaines atteintes par le MIH analysées en parallèle. Ainsi, nous proposons d'utiliser les dents MIH comme biomarqueur précoce d'exposition aux PE agissant comme le BPA. Par ailleurs, nos résultats ont montré que l'action du BPA sur la prolifération des précurseurs améloblastiques et les modulations d'expression de deux gènes cibles clé, l'énaméline et la kallikréine 4, ne semble emprunter qu'en partie la voie œstrogénique suggérant que dans l'épithélium dentaire, le BPA interagit avec d'autres récepteurs que ERα et ERß. La combinaison du BPA avec les deux autres PE impacte apparemment moins l'amélogenèse. Les trois PE étudiés ici modulent chacun différemment l'expression des gènes codant les protéines matricielles amélaires et les protéases spécifiques de l'émail, réduisant l'effet des autres PE. Ceci explique, au moins en partie, l'impact différentiel sur l'émail de substances exogènes hypominéralisantes dont la relation avec le développement de certaines pathologies sera intéressante à étudier dans le futur
Our environment has become increasingly contaminated by pollutants including endocrine disruptor Chemicals (EDCs), associated with an increased prevalence of serious diseases and the emergence of new diseases. Recently, a new dental pathology called molar incisor hypomineralization (MIH) has been described. This pathology, whose etiology is unknown, has become a real public health problem with a prevalence of roughly 18%. The aim of this work was to investigate potential relationships between exposure to EDCs and MIH. For this purpose, Wistar rats were treated from the conception to the sacrifice, with low doses of three EDCs, bisphenol A (BPA), genistein and vinclozolin, alone or in combination. 75% of rats treated with BPA alone have shown enamel hypomineralized spots sharing similar biochemical and structural characteristics with human teeth affected by MIH analyzed in parallel. Thus, we propose to use MIH teeth as an early biomarker o exposure to EDCs acting as BPA. The effects of BPA on pre-ameloblast prolifération and enamelin and klk4 expression seem to use the estrogenic pathway only in part suggesting that BPA could interact with other receptors than ERa and ERp in dental epithelial cells. Each combination of BPA with other EDCs affects specifically the amelogenesis explaining the lower impact of the combination compared with BPA alone. This explains, at least in part, the différentiel impact of exogenous hypomineralizing substances on enamel whose relationship with the development of certain diseases will be interesting to study in the future

L'iprodione est transformee par voie chimique et par voie microbiologique dans les sols. La transformation microbiologique de ce fongicide est minoritaire dans les sols non adaptes et majoritaire dans les sols adaptes. Elle entraine l'hydrolyse au niveau de la fonction uree de la chaine laterale du cycle dicarboximide de l'iprodione conduisant au n-(3,5-dichlorophenyl)-2,4-dioxoimidazolidine: metabolite (ii). Ce metabolite subit ensuite une ouverture du cycle dicarboximide pour conduire a l'acide 3,5-dichlorophenyluree acetique: metabolite (iii). L'hydrolyse du metabolite (iii) conduit a la formation de la 3,5-dichloroaniline. Trois souches bacteriennes impliquees dans la degradation de l'iprodione ou de ses metabolites ont ete isolees. Pseudomonas sp. Et pseudomonas fluorescens degradent l'iprodione en metabolite (ii) et dans certaines conditions le metabolite (ii) en metabolite (iii). Pseudomonas paucimobilis degrade successivement le metabolite (ii) en metabolite (iii) puis en 3,5-dichloroaniline. Le denombrement de la souche d'arthrobacter par marquage immunofluorescent a montre que la proliferation de cette souche est une des causes de la degradation acceleree de l'iprodione dans un sol adapte. La vinclozoline est microbiologiquement degradee en 3',5'-dichlorophenyl-2-hydroxy-2-methylbut-3-enanilide et en 3,5-dichloroaniline ou chimiquement hydrolysee en enanilide et en acide n-(2-hydroxy-2-methyl-1-oxobuten-3-yl)-3,5-dichlorophenyl-1-carbamique. Une souche appartenant au genre corynebacterium ou a un genre voisin degrade l'acide n-carbamique en enanilide et en compose d non identifie mais sa capacite a degrader la vinclozoline n'a pas pu etre clairement demontree. Un sol adapte a la vinclozoline degrade l'iprodione de facon acceleree mais l'inverse n'a pas ete observee. Ces deux fongicides presentent donc un phenomene d'adaptation croisee dans un seul sens

In the last two decades, there have been growing scientific concern, public debate, and media attention over the possible deleterious effects in humans and wildlife that may result from exposure to substances that have the potential to interfere with the endocrine system. Endocrine disrupting compounds (EDCs) encompass a variety of substance classes, including natural and synthetic hormones, plant constituents, pesticides, substances used in industry and in consumer products, pollutant. It is well documented that EDCs targets are mainly the nuclear receptors (NRs) such as the sexual hormones estrogen receptors ERs. ER is involved in a broad spectrum of physiological processes in different organs and tissues as well as in several diseases, such as breast and endometrial cancer, osteoporosis, and prostate hypertrophy, neurodegenerative diseases or in immune system activation. The regulatory agencies for the protection of human health and wildlife have been issuing the necessity to investigate and clarify the mode of action of these exogenous substances by the development of alternative toxicological methods such as in silico model and in vitro testing in order to predict the toxicity of these EDCs. This research had the ambitious aim to develop an integrated toxicological strategy based on the combination of available information of endocrine disrupting activity retrieved from the scientific literature, in silico model, imaging methodologies applied to reporter systems in vitro and in vivo and ex vivo to predict among a set of chemicals those with an endocrine disrupting activity or ability to activate other toxicological pathways such as inflammation and oxidative stress measured in the male reproductive organs and in the genital and abdominal area of mice. The selected molecules range from known (DES) to suspected (BPA) endocrine disruptors and included both synthetic (DEHP) and natural (genistein) compounds. The first step used was in silico analysis with evaluation of the possible binding of selected substances to the estrogen alpha receptor to support the hypothesis that their hormonal activity occurred through a receptorial mechanism. This approach is commonly used and is also part of the first level of investigation suggested by EFSA/ECHA in the recognition of EDCs. The computational methodology estimated different values of affinity of each ligand to hER Ligand Binding Domain (LBD). The use of two different approaches (XP GLIDE SCORE and MMGBSA dG Bind) also allowed for solvation to be taken into account. That meant that MMGBSA protocol considered both the interactions of some water molecules with the LBD and the solvent-ligand ones. The estradiol showed the best affinity values in both approaches as being the endogenous hormone was able to contrast the solvation effect. The dissociation constant (Ki) values calculated from the XP GLIDE SCORE fitted well with the Ki experimentally determined in vitro binding assay by other research groups. Thus, the computed Ki has been chosen as parameter for the prediction of putative endocrine disruptor activity. However, the lack of correlation between the (computed and experimental) Ki and in vivo experimental observed ED activity (from literature data) for all chemicals (only estradiol and zearalenone have similar affinity to ER and literature evidences of ED activity; genistein has a good Ki values but not ED activity such as BPA and methoxychlor), did not allow a prioritization of the investigated chemicals for ED activity through the results of their affinity. In the next step, to check if the receptor binding well correlated with the hormonal activity, the ER Reporter gene assay was performed, based on the ability of a compound to stimulate ER-dependent transcriptional activity in genetically engineered mammalian cells. The cell lines are MCF-7 cells which express human endogenous ERα. The cells are transformed (transfected) by introducing vectors containing DNA sequences for the receptor, along with EREs linked to a reporter gene, and the reporter gene itself. The reporter gene used in human cancer cells usually codes for luciferase (CALUX, chemically activated luciferase expression). In the transactivation the EDCs show their estrogenic potency calculated as EC50, in respect to the positive control, 17β-estradiol. This system has enabled us to evaluate the kinetic and the biological consequences of cellular activation in the same cell monolayer by bioluminescence imaging of photon emissions that were pictures of activated ER status at 6 and 48 hours after the initial treatments. From this in vitro assay three factors were taken into consideration, power, efficacy and trend over time. The in vitro dynamic ER activation showed that for some chemicals (genistein, BPA, methoxychlor), the potency (EC50) and the efficacy (fold induction) changed over time, but not for others (estradiol, zearalenone and DES). Considering that estradiol, zearalenone and DES certainly have an activity in the animal and in man as endocrine disruptors, the duration of effect parameter combined with power and efficacy were likely to be associated in predicting the activity. Together with receptor affinity and the ability to induce a biological response, it also seemed relevant how long the response was lasting. By consequence, the combination of the variation of the potency response and the efficacy, “normalized” respect to the efficacy values quantify at 48 hours, was successfully used in discriminating positive and negative compounds for their endocrine disrupting activity. By means of this analysis 17β-estradiol, DES and zearalenone were put at the top of the list (also supported by their known ED activity), genistein resulted to represent a putative threshold of no-concern for ED effect, in supporting published data, while methoxychlor and BPA were definitely not considered a priority in terms of ED activity. This in vitro classification fitted well with the in silico outcomes, since the strongest estrogen receptor binders were ranked in the first positions (17β-estradiol, DES and zearalenone). Besides, was no possible to calculate EC50s for 4-nonylphenol, DEHP and vinclozolin, not making possible to classify them as ED, totally in agreement with in silico results and in line with literature data (vinclozolin is mainly an androgen antagonist). The third step of our stepwise approach was intended to verify in vivo the interaction of selected chemicals with the estrogen receptor and in addition the activation of their pathways triggering primary harmful effects. We used three reporter mice designed in order to evaluate the effect of selected compounds to activate ER and causing oxidative stress and inflammation. We have chosen to test zearalenone (well-known endocrine disruptor and clearly identified as such by our in silico-in vitro approach), and BPA for which there are controversial data in the literature and that our approach has negatively classified as ED. In our experiments zearalenone showed to be active on ER pathway in the abdominal area and significantly activated the inflammatory pathway in the genitals (in this specific case in the prostate, result of ex vivo bioluminescence analysis). These results were perfectly in line with the literature reports, in which prostate inflammation and metaphase were detected in both mice and rats. Bisphenol A did not produce a significant activation in both the areas and in the ex vivo analysis, again in agreement with in silico/in vitro results. We used a combination of innovative approaches that led to a conclusion that in silico screening cannot be used as a stand-alone procedure due to its intrinsic lack of biological meaning, although it can be successfully used as a first prioritizing step in a tier approach. The second mandatory check for the in silico positive hits should be an in vitro evaluation procedure, in which the affinity of the positive hits is measured through a reference cellular assay. Our results showed that integrating the time variable in the evaluation of the potency, the tested compounds could be classified as ED or no-ED. The in vivo experiment highlighted that a potent estrogenic compound, as zearalenone, could also raise concern for the activation of other toxicological pathway such as the inflammatory ones. We are aware that this indication of procedure must be evaluated and validated on dozens of molecules whose in vivo activity is already known before arriving at its use to predict the possible activity of ED of unknown molecules, but we think that this approach deserves to be implemented.

L'incidence de cancers hormono-dépendants ne cesse d'augmenter dans les pays occidentaux ; une infertilité accrue et des anomalies du système reproducteur ont également été notées dans ces mêmes pays. L'hypothèse a été émise selon laquelle des composés faisant partie de notre environnement, appelés perturbateurs endocriniens (PE), pourraient être liés à ces phénomènes. Afin d'évaluer le risque de PE sur le développement de la glande mammaire, nous avons développé un modèle original d'exposition, où un phyto-œstrogène de soja (génistéine) et un fongicide anti-androgénique retrouvé dans les légumes et les fruits (vinclozoline), sont administrés à de faibles doses (1 mg/kg/jour) chez le rat. Nous avons mis en avant que l'exposition in utero et pendant la lactation à ces composés altère le développement de la glande mammaire chez les animaux pubères [augmentation du branchement et de prolifération épithéliale, hyperplasie ductale et changement de la réceptivité hormonale] ainsi que chez les animaux cycles [augmentation de la surface mammaire et développement des structures lobulo-alvéolaires sécrétrices]. L'analyse transcriptomique montre que cette exposition in utero altère également le profil d'expression génique de la glande mammaire ; ces altérations moléculaires sont compatibles avec les modifications histologiques. Par ailleurs, l'exposition du sevrage jusqu'à l'âge adulte à la génistéine et à la vinclozoline induit des altérations morphologiques de la glande mammaire des animaux adultes. Ce travail apporte de nouvelles connaissances concernant les effets d'un androgène/anti-androgène sur le développement de la glande mammaire chez la femelle. Cette étude montre pour la première fois que l'exposition à un mélange de deux perturbateurs endocriniens, appartenant à deux familles différentes, perturbe le développement de la glande mammaire chez le rat femelle. Les conséquences d'une exposition au mélange apparaissent plus sévères comparées à celles observées avec les molécules isolées, ce qui souligne le risque de la multi-exposition
The incidence of hormone-dependent cancers is increasing in Western countries; increased infertility and reproductive abnormalities were also noted in these same countries. This has raised the hypothesis that compounds found in our environment, called endocrine disruptors (EDs), could be related to these pathologies. This thesis aims at identifying the effects and mechanisms of EDs on the development of rat mammary gland following the administration of two xeno-hormones having distinct endocrine properties: genistein (soy phytoestrogen with major estrogen receptor affinity) and vinclozolin (anti-androgenic fungicide found in vegetables and fruit). The particularises of this project were: 1) a study following in utero and gestational exposure at peri-pubertal period (PND35 and PND50); 2) a long life exposure study on adult rats; 3) doses relevant overall human exposure (1 mg/kg/day). We show that exposure in utero and during lactation to these compounds alter the development of the mammary gland in pubertal animals [increase in branching and epithelial proliferation, ductal hyperplasia and changes in hormonal receptivity] and in cycled animals [increase of the mammary gland area and development of lobulo-alveolar structures]. Using a transcriptomic approach, we also evaluated gene expression changes in mammary gland. Molecular changes are dependent on the period of postnatal development and the compound considered. The molecular data concerning in utero and lactational exposure are consistent with the histological observations. Furthermore, exposure from weaning to adulthood induces different alterations of the mammary gland at adulthood. In summary, this work provides new observations on the effects of a chemical anti-androgen on the development of the mammary gland in females. These studies also show for the first time that exposure of a mixture of two endocrine disruptors, belonging to two different families, disrupts the development of the mammary gland in female rats with more severe effects compared to those observed with the isolated molecules; these results highlight the risk of multiple exposure

Cette etude analytique a ete orientee sur trois pesticides, l'iprodione, la vinclozoline et la cymoxanil, couramment employes dans le cadre de la production des framboises et des laitues de la region rhone-alpes. Nous presentons la mise au point de methodes d'analyse repetables et reproductibles avec une comparaison sur les dosages effectues en chromatographies en phase liquide sur colonne (clhp) et sur couche mince (ccm) ainsi qu'en phase gazeuse (cpg). Nous avons suivi l'evolution de la disparition de ces trois fongicides sur les framboises et les laitues traitees selon differentes modalites. Il ressort de notre etude que la cinetique de disparition de l'iprodione et de la vinclozoline suit un modele de pseudo-premier ordre. Par ailleurs, les parametres meteorologiques et agronomiques ont une influence sur le devenir des residus de pesticides

Les glandes salivaires sont des glandes mixtes : la salive (produit exocrine) estimpliquée dans le maintien de l'homéostasie buccale alors que les secrétions endocrines (ex :facteurs de croissance) ont un rôle physiologique (gamétogénèse, ostéogenèse,hypertension...) Chez les mammifères, elles affichent un dimorphisme sexuel qui laisseentrevoir une sensibilité éventuelle à des xeno-hormones.Ce mémoire présente l'action de la génistéine (phyto-oestrogène) et/ou de la vinclozoline(anti-androgène) sur la glande submandibulaire (SM) de rat lors d'une exposition précoce viala mère (gestation-lactation) et lors d'une exposition pendant la période de croissance (dusevrage à l'âge adulte). Les glandes SM, prélevées au stade immature et jeune adulte, ont faitl'objet d'une analyse histologique et d'une étude de marqueurs moléculaire des fonctionsendocrines et exocrines associées aux processus gustatifs. L'exposition précoce ralenti ledéveloppement de la glande SM et augmente sélectivement la préférence au sucré des malesimmatures mais pas des adultes ; l'analyse moléculaire révèle une action sélective sur lesfonctions exocrines corrélée à celle sur les préférences, ainsi qu'une action sur les fonctionsendocrines (facteurs de croissances) qui s'inverse avec l'âge. L'exposition à partir du sevrageperturbe seulement les mâles qui présentent des altérations des structures sécrétrices coupléesà des modifications d'expression des récepteurs hormonaux et facteurs de croissance, maisaussi au taux sérique de l'EGF.Cette étude identifie la glande submandibulaire comme cible de perturbateurs endocriniens etpose la question des conséquences physiologiques à terme

碩士
國立臺灣大學
園藝學研究所
87
Fresh cabbage was fermented 18 days or 5 days after immersed carbofuran、vinclozolin、chlorpyrifos and permethrin 1000 or 1500 dilution, were extracted with acetonitrile, cleaned up with Florisil cartridge. Four pesticides were determined by HPLC between uv 230nm~330nm, simultaneously. Results indicated that the 5th pesticide residues of 5 days fermentation and the 18th pesticide residues of 18 days fermentation were 37.1~61.4﹪、33.2~75.9﹪, respectively. The maximums of PCA culture were log 11.7 and log 7.2, respectively. The experiments were studied on pesticide degradation by microorganisms of cabbage in different sugar concentration, different temperature and different inoculation. Results found that suitable concentration of sugar could help the growth of lactobacillus, and the four pesticide residues in 3﹪sugar fortification, 37℃ incubation and 0.5﹪inoculation were 14.2~76.7﹪,1.3~91.2﹪and 52.4~89.4﹪, respectively. The total results indicated that carbofuran、vinclozolin、chlorpyrifos and permethrin were degraded acceleratively by microorganism onto cabbage. Sugar concentration and incubation temperature all could effect species of microorganism growth. Therefore, the degradation on various pesticides showed in different rates .

碩士
國立臺灣大學
農業化學研究所
98
Dicarboximide fungicide vinclozolin was found to have the anti-androgen effects, and its metabolites, M1 (2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid) and M2 (3’,5’-dichloro-2-hydroxy-2-methylbut-3-enanilide), were found to have more affinity to androgen receptors than vinclozoline. In this study, the effect of vinclozolin metabolites in soil on its persistence and soil microbial populations diversity were investigated. The toxicity of vinclozolin on Daphnia pulex, were compared with 2 kinds of its metabolites. First, we extract vinclozolin from commmerical products and then perform chemical hydrolysis to collect the metabolites. The soil sample were collected from Taoyuan District Agricultural Research (Pu) and Extension Center and Hualien District Agricultural Research (Wl). The experimental concentration were 20.0 mg kg-1 for M1 and 5.0 mg kg-1 for M2. We used solvent phase extraction to extract residues from soil, and utilized HPLC-DAD to detect them. In sterilized soil, M1 and M2 have longer half-lives (10.9-126.6 and 87.1-296.5 day) than vinclozolin (8.7-35.0 day). The impact of vinclozolin metabolites on soil microbial community were analysed by PCR-DGGE. In the DGGE patterns, we could discover some original superior populations with the resisence to toxicity of metabolites, such as Acidobacterium sp. and Pseudomonas sp. in Pu soil, and Ammoniphilus oxalaticus in Wl soil had the resistence to M1 or M2. But some populations were disappeared or enlarged by metabolites treatment, such as Pseudomonas sp. and Bacillus sp. did enlarge their population in treated soil. We conducted 48 hr acute toxicity assay on daphnia. The result of toxicity are in the sequence of M2 >vinclozolin>M1. The metabolites from degradation of vinclozolin, still have the hazard on environment. Due to vinclozolin metabolites have characteristics of persistence and toxicity. When we assess the impact of vinclozolin on environment, the effect of metabolites should be taken into account.

Androgeny to kluczowe związki biorące udział w regulacji procesów rozrodczych u samicy, działające głównie przez receptor androgenowy (AR), należący do nadrodziny receptorów jądrowych. Androgeny są jednymi z ważniejszych związków wpływających na proces folikulogenezy. Z jednej strony modulują funkcje pęcherzyka poprzez interakcje z różnymi czynnikami i wspierają różnicowanie komórek ziarnistych. Z drugiej strony, mogą hamować rozwój pęcherzyka poprzez indukcję procesu apoptozy komórek ziarnistych, co w konsekwencji może prowadzić do atrezji pęcherzyka jajnikowego. W jajnikach ssaków większość pęcherzyków ulega atrezji przed owulacją. Coraz częściej mówi się, że substancje występujące w środowisku, mogą w sposób zasadniczy wpływać na procesy życiowe wielu organizmów, w tym na różnicowanie płci u ssaków. Związki te, to Endocrine Disrupting Chemicals (EDCs), czyli m. in. związki powstające w wyniku działalności człowieka, przedostające się do środowiska i w efekcie zaburzające regulację hormonalną poprzez interakcje z receptorami hormonów steroidowych. Taki mechanizm działania negatywnie wpływa na szereg procesów zachodzących w układzie rozrodczym samicy. Celem prezentowanej pracy było określenie wpływu androgenów, testosteronu (T) i dihydrotestoseronu (DHT) oraz fungicydu o właściwościach antyandrogennych, winklozoliny (Vnz) na ekspresję AR na poziomie mRNA i białka, aktywność steroidogenną i mechanizm apoptozy zarówno w komórkach ziarnistych jak całych pęcherzykach jajnikowych świni (Ø 4-6mm). Komórki ziarniste izolowane z pęcherzyków jajnikowych hodowano przez 24 godziny i następnie dodawano: T (10-7M), DHT (10-7M) i Vnz (1,4×10-5M) osobno lub w kombinacji: T i Vnz lub DHT i Vnz na kolejne 24 godziny. Całe pęcherzyki jajnikowe hodowano narządowo przez 24 godziny, stosując warianty hodowli takie jak w przypadku hodowli pierwotnych. Androgeny (T, DHT) i antyandrogen środowiskowy winkolzolina (Vnz) wpływają na ekspresję AR na poziomie mRNA i białka w komórkach ziarnistych pęcherzyków jajnikowych świni. Wykazano lokalizację cytoplazmatyczną i około jądrową AR w komórkach ziarnistych traktowanych Vnz. Stwierdzono wzrost ekspresji AR na poziomie mRNA i białka w komórkach ziarnistych pod wpływem Vnz oraz kombinacji T+Vnz lub DHT+Vnz. W przypadku pęcherzyków jajnikowych hodowanych w obecności androgenów i antyandrogenu jednocześnie (T+Vnz i DHT+Vnz) obserwowano wzrost ekspresji mRNA AR z jednoczesnym spadkiem ekspresji na poziomie białka. W komórkach ziarnistych i pęcherzykach jajnikowych świni androgeny (T i DHT) oraz antyandrogen (Vnz) wpływają na aktywność steroidogenną. Zmiany poziomu hormonów steroidowych, tak w medium hodowlanym jak w płynie pęcherzykowym, świadczą o zaburzonym przebiegu steroidogenezy w komórkach ziarnistych i całych pęcherzykach jajnikowych świni. Androgeny (T, DHT) i antyandrogen środowiskowy winkolzolina (Vnz) wpływają w sposób znaczący na mechanizm apoptozy. Obserwowano zmiany morfologii komórek ziarnistych i całych pęcherzyków jajnikowych świni, co świadczy o destrukcyjnym wpływie tych związków na prawidłową strukturę komórki i pęcherzyka. Vnz powoduje nekrozę hodowanych komórek ziarnistych oraz indukuje apoptozę komórek ziarnistych w całych pęcherzykach jajnikowych. Wykazano, że dodanie T, DHT lub Vnz do medium hodowlanego komórek ziarnistych lub pęcherzyków powoduje uruchomienie procesu programowanej śmierci komórki, ponieważ obserwowano wzrost ekspresji i aktywności kaspazy-3. Vnz zakłóca klasyczny receptorowy oraz mitochondrialny szlak apoptozy. Ponadto wykazano, że traktowanie komórek ziarnistych Vnz powoduje spadek ekspresji białek antyapoptotycznych, z jednoczesnym brakiem wzrostu ekspresji białek proapoptotycznych. Prawdopodobnie stosowanie Vnz wpływa na prawidłowe podziały komórki (zatrzymuje cykl komórkowy), gdyż obserwowano spadek ekspresji białka p53 i surwiwiny. Co więcej, dodanie Vnz do medium hodowlanego komórek ziarnistych skutkuje wzrostem fosforylacji kinazy ERK1/2 i Akt. Sugeruje to pozagenomowy mechanizm działania Vnz. Co ważniejsze, prezentowane badania wykazały zdecydowanie antyapoptotyczne działanie wysokiego stężenia androgenów stosowanych jednocześnie z Vnz, o czym m. in. świadczy spadek ilości komórek apoptotycznych i aktywności kaspazy-3. Podsumowując uzyskane wyniki, można stwierdzić, że winklozolina może zaburzać prawidłowe funkcje jajnika, ponieważ działając za pośrednictwem AR uruchamia niegenomowe szlaki przekazywania sygnału i zakłóca fizjologiczne procesy programowanej śmierci komórkowej. W konsekwencji może to prowadzić do obniżenia potencjału rozrodczego samicy.
Androgens are the key regulators in female reproductive physiology. They act primarily via the androgen receptor (AR), a member of the nuclear receptor superfamily, which operates as ligand-regulated transcription factor. Androgens are one of the most important agents influencing folliculogenesis. They can modulate follicular function by interactions with various factors and promote granulosa cells differentiation. On the other hand, androgens can antagonize follicular development by inducing apoptosis in granulosa cells and in this way they can promote follicular atresia. In mammalian ovaries the majority of follicles are lost at various stages before ovulation by atresia. Recently, there has been an increasing concern about potential for Environmental Endocrine Disrupting Chemicals (EDCs) to alter sexual differentiation in mammals. They originate as a result of human industrial activities, enter the natural environment and then disturb hormonal regulation by interaction with steroid hormone receptors. Such a mechanism of action negatively influences on many process taking place in the reproductive tract of a female. The aim of this study was to investigate whether the androgens testosterone (T) and dihydrotestosterone (DHT) and the antiandrogenic fungicide vinclozolin (Vnz) influence AR expression on mRNA and protein level, steroidogenic activity, and mechanism of apoptosis both in granulosa cells and in whole porcine ovarian follicles (Ø 4-6mm). Granulosa cells isolated from pig follicles were cultured for 24 hours, and then were exposed to T (10-7M), DHT (10-7M), Vnz (1,4×10-5M) or the equivalent concentrations of T and Vnz or DHT and Vnz for further 24 hours. Whole porcine follicles were also exposed to the same compounds and combinations of compounds for 24 hours. Androgens (T and DHT) and an antiandrogen (Vnz) influence on AR mRNA and protein expression in porcine ovarian granulosa cells and whole follicles. The immunostaining for AR was detected mainly in the cytoplasm and assembled in the perinuclear region after exposure to Vnz. Treatment with Vnz and combinations of T+Vnz and DHT+Vnz, granulosa cells resulted in an increase in the expression of AR on both mRNA and protein levels. In the whole follicles treatment with T+Vnz and DHT+Vnz resulted in an increased expression of mRNA for AR with simultaneous decrease of AR protein. In porcine granulosa cells and whole ovarian follicles androgens (T and DHT) and an antiandrogen (Vnz) affected their steroidogenic activity. Incorrect or disturbed process of steroidogenesis was observed in all experimental groups of cells and follicles. Besides all compounds tested caused the disturbance of hormonal balance in the follicular fluid. Androgens (T and DHT) and an antiandrogen (Vnz) in a significant manner influenced on the mechanism of apoptosis. Morphological changes in granulosa cells and ovarian follicles indicated destructive effects of this agents. Vnz caused massive necrosis of granulosa cells and induced apoptosis of granulosa cells in whole follicles. Androgens induced apoptosis in granulosa cells of ovarian follicles . It was found out that the addition of T, DHT or Vnz to the culture media of granulosa cells or follicles caused the induction of the process of programmed cell death, because an increased caspase-3 expression and activity have been observed. It seems that Vnz disrupted classical receptor and mitochondrial signaling pathways of apoptosis. Also, it was shown that Vnz treatment caused a decrease in the expression of antiapoptotic proteins, but without an increase in that of proapoptotic ones. Probably supplementation with Vnz altered normal cell division (cell-cycle arrest), because a decrease in p53 and survivin proteins expression has been observed. Moreover, the addition of Vnz to the culture media of granulosa cells increased phosphorylation of ERK1/2 and Akt kinases. It is suggested that Vnz may interfere with the nongenomic pathway of androgens signaling. What is more important, the presented data clearly showed that Vnz applied together with high concentration of androgens (T and DHT) exerted an antiapoptotic effect, as evidenced by the decreased number of apoptotic cells and caspase-3 activity. In conclusion, these results suggest that normal function of the ovary may be destroyed by the environmental antiandrogenic fungicide vinclozolin. It can activate the nongenomic signaling pathway used by androgens or act through the AR. This mechanism of action disturbs the physiological process of programmed cell death. It seems that selective destruction of porcine follicles is a serious consequence of their exposure to vinclozolin leading to premature ovarian failure in the affected organism.

In the present study, both in vitro and in vivo experiments were performed to investigate modes of action of selected EDCs including mercury chloride (HgCl2), bisphenol A (BPA), vinclozolin (VZ) and Di-(2-ethylhexyl)-phthalate (DEHP) on male reproductive physiology in fish. Results obtained from in vitro studies showed that EDCs decrease sperm motility via disruption in energetics and damage to spermatozoa; however the effective doses are higher than reported in the environmentally relevant concentrations. In contrast, in vivo studies showed adverse effects of BPA, VZ, and DEHP on sperm quality at environmentally relevant concentrations. Decrease in sperm quality is associated with disruption in testicular steroidogenesis, particularly inhibition of androgens. Only BPA induced vitellogenin production, associated with increase in transcripts of estrogen receptor, androgen receptor and cytochrome P450 mRNA at the high dose approaching environmental level. Inhibition of androgen production is due to disruption in transfer of cholesterol into the steroidogenesis pathway, because transcript of steroidogenic acute regulatory protein mRNA was decreased. In addition, BPA acts through sex steroid-mediated mechanism while DEHP mode of action is sex-steroid independent mechanism.

Pollution of the aquatic environment with xenobiotics has become a serious health concern in recent years. In the present study the effect of DQ, TBBPA, BPA and VIN on sperm quality parameters, DNA integrity and oxidative stress indices in sterlet (Acipenser ruthenus) sperm and sperm from brook trout (Salvenilus fontinalis) were investigated. To do this, an in vitro spermatozoa motility assay was used by a computer-aided Motion-Analysis system. The sperm of sterlet (Acipenser ruthenus) was diluted to obtain the spermatozoa density of 5×108 cells×ml?1 and then exposed for 2 h to final concentrations of xenobiotics: DQ - 25, 50, 100 and 150 ?M, TBBPA - 0.5, 1.75, 2.5, 5, 7.5 and 10 ?g/l, BPA - 0.5, 1.75, 2.5, 5 and 10 ?g/l and Vin - 0.5, 1.75, 2.5, 5 and 10 ?g/l. Spermatozoa velocity and percentage of motile sperm were significantly decreased at each time post-activation compare to control. The level of DNA damage expressed as a % DNA in Tail and Olive Tail moment significantly increased when spermatozoa were exposed to higher concentrations of xenobiotics. The level of oxidative stress indices lipid peroxidation (LPO) and carbonyl derivatives of proteins (CP) and antioxidant activity of superoxide dismutase (SOD) increased significantly with increasing concentration of xenobiotics. On the other hand the intracellular ATP content in sperm samples had a significantly decreasing effect. In short, xenobiotics can induce reactive oxygen species (ROS) stress in fish spermatozoa, which could impair the sperm DNA integrity, quality and antioxidant defense system. The present study confirms that environmental concentrations of xenobiotics are capable to induce oxidative stress, leading to impaired sperm quality, DNA fragmentation and intracellular ATP content Obtained results also suggested that the use of fish spermatozoa in vitro assays may provide a novel and efficiently means for monitoring residual pharmaceutical in aquatic environment.