Indiana University-Purdue University Indianapolis (IUPUI) / Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants suspected to act as endocrine disruptors. We tested the commercial PBDE mixture DE-71 and its in vivo metabolites for estrogenic activity. MCF-7 breast cancer cells culture, ERE-luciferase gene expression, 3H-β-estradiol displacement from recombinant ERα, and ovariectomized (OVX) mice served as bioassays. Although DE-71 did not bind ERα, it was able to increase MCF-7 cell proliferation and this was prevented by the antiestrogen fulvestrant. DE-71 co-treatment reduced the effect of estradiol in MCF-7 cells. In the OVX mouse (BALB/c) 3-day assay, DE-71 administered alone had no effect on uterine or vaginal tissues but when administered subcutaneously potentiated estradiol’s effect on uterine weight in a dose-dependent manner. DE-71 administered SQ to BALB/c mice for 34 days slightly increased uterine epithelial height (UEH), vaginal epithelial thickness (VET) and mammary ductal lumen area, and attenuated the estradiol-induced increase in UEH; these effects were not seen in C57BL/6 mice. DE-71 increased liver weight in BALB/c, C57BL/6 and estrogen receptor-alpha knockout (ERαKO) mice. Liver cytochrome P450 1A (CYP1A) and CYP2B activities increased 2.5-fold and 7-fold respectively when DE-71 was administered PO, but only CYP2B increased (5-fold) after SQ treatment. Six OH-PBDE metabolites were found in mice after 34-day DE-71 treatment and all were able to bind recombinant ERα. Para-hydroxylated metabolites displayed a 10- to 30-fold higher affinity for ERα compared to ortho-hydroxylated PBDEs. Para-OH-PBDEs induced ERE-luciferase and produced an additive effect when coadministered with β-estradiol. DE-71 was also additive with β-estradiol. At high concentrations (≥ 5x10-5 M), ortho-OH-PBDEs were antiestrogenic in the ERE-luciferase assay. In conclusion, DE-71 behaves as a weak estrogen in both MCF-7 breast cancer cells and ovariectomized adult mice. Mice strain, treatment route and duration determined if DE-71 was estrogenic. BALB/c mice are more susceptible to DE-71 effects in estrogen target tissues than C57BL/6 mice. DE-71 increased liver weight, 5%-51% depending on mouse strain and treatment regime, independently of ERα. The observations that the DE-71 mixture does not displace 3H-β-estradiol from ERα while the hydroxylated metabolites do, suggest that the cellular and tissue effects were due to a metabolic activation of individual congeners.

No description available.

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.

Background. Endocrine disruptors (EDC) are exogenous compounds that mimic the action of natural hormones and alter the normal endocrine system. Life-long chronic exposure to Bisphenol A (BPA), a putative EDC, has been linked with risk of metabolic disorders in epidemiological studies. Objectives. The aim was to study the human health effects of exposure to BPA, using an integrated approach combining environmental epidemiology and toxicology. Methods. Urinary levels of BPA exposure were measured in participants of the InChianti longitudinal study, a representative population-based study of Italian adults, at the Baseline (1998-00) and nine years later (3rd Wave, 2007-09). Hormones levels and the gene expression of specific target genes were the end points considered. Results were validated in laboratory studies on a human leukemic T-cell line (Jurkat cells). Results. In general, urinary BPA (uBPA) concentrations were higher among men and younger respondents, and within subjects uBPA concentrations were correlated (r=0.58; p=0.013, model adjusted for age, sex, urinary creatinine). At baseline, uBPA concentration were associated with higher total testosterone concentrations in men (β = 0.05; 95% CI, 0.02–0.08). In the 3rd wave, gene expression analysis revealed positive associations between uBPA concentrations and ESR2 (estrogen receptor beta) expression (β=0.18; 95% CI: 0.04, 0.32) and ESRRA (estrogen related receptor alpha) expression (β= 0.17; 95% CI: 0.02, 0.32). In a following in vitro study, BPA exposure (0.001-1 micro molar) led to enhanced expression of ESRRA and ESR2 in Jurkat cells over a period of 72 hours. Conclusions. Results indicate that BPA is bioactive in the human body and is able to alter circulating hormone concentrations and estrogen receptor/estrogen-related receptr gene expression. In particular, given the role of ERRα as a major control point for oxidative metabolism and heart development, this research provides indications on the possible molecular mechanisms of action of BPA in metabolic diseases.

A new complex aspect in the matter of water quality is the occurrence of emerging organic pollutants and contaminants in waste water. The currently low extent to which treatment of waste water is performed in Brazil, and in the world as a whole, there is a considerable need for development of cheap and accurate in-situ sampling methods for far-reaching studies of surface water quality. The lack of such methods today makes the maintenance and establishing of sanitary safety difficult. This diploma work gives a brief introduction to the basic principles of the passive sampling method known as Diffusive Gradient in Thin-films (DGT). A method that could be useful for such monitoring of quality in water bodies world wide. The aim of this study is to develop a method, for the detection of organic emerging pollutants and contaminants – i.e. compounds, which usually are present at very low concentrations when found in the environment as a result of human activity. More specifically, this work investigates the potential and usefulness of the application of DGT devices in detection of organic compounds that can affect human health and ecosystems, even at low concentrations, however, their effects still are in need of further investigations. This study focuses on both purely technical as well as practical points of views. The efficiency of organic DGT (o-DGT) with biochar as the adsorbing agent is examined targeting the detection of organic pollutants and contaminants in surface water. In this sense, the specific aim of the work is to evaluate the performance of biochar as adsorbing agent. This work showed that the performance of biochar as the adsorbing agent in binding layers in o-DGT sample devices can be considered as satisfactory since all compounds of interest in this study was successfully detected, quantified an identified. Further investigations in the future are needed to determine the effects of varying pH, temperature and ion concentration in the deployment media, as well as the properties of the binding layer in relation to concentration of biochar and the thickness of the layer. These in order to optimize the method for in-situ water sampling, aiming conventional use of biochar as the adsorbing agent in the future.

Neste estudo foi realizada a avaliação de seis substâncias sintéticas resultantes de processos industriais e de atividades antropogênicas dietilftalato, dibutilftalato, nonilfenol, pentaclorofenol, bisfenol A e benzo[a]pireno os quais possuem capacidade de interferir negativamente no funcionamento normal do sistema endócrino de animais e de seres humanos (xenoestrógenos), em águas tratada e bruta de quatro municípios do Vale do Paraíba paulista. Para tanto, utilizou-se um método analítico de alta seletividade e sensibilidade, sendo a determinação por cromatografia gasosa acoplada ao detector de espectrometria de massas (GC/MS), precedida da concentração das amostras pelo método SPE (extração em fase sólida). Como não são suficientes apenas boas técnicas analíticas para garantir a qualidade dos dados gerados e a confiabilidade dos resultados, a metodologia desenvolvida foi submetida ao processo de validação, onde foram avaliados os parâmetros: seletividade, especificidade, linearidade, faixa linear de trabalho, limites de detecção e quantificação, precisão, exatidão, recuperação e robustez, além da incerteza na medição. Os resultados demonstraram que o método proposto é adequado para quantificação das amostras do rio Paraíba do Sul, e pela sua aplicação foi constatada a presença de todos os poluentes estudados tanto na água bruta quanto na potável em pelo menos um dos três períodos de amostragem, sendo as maiores concentrações mensuradas nas águas brutas e no período seco. Estes resultados evidenciam que as atividades antrópicas na região influenciam a qualidade da água do recurso hídrico estudado, bem como a qualidade da água de distribuição. / The evaluation of six synthetics substances from industrials process and anthropogenic activities - diethyl phthalate, dibuthyl phthalate, nonylphenol, pentachlorophenol, bisphenol A and benzo[a]pyrene which one can interfer negatively in normal function of the endocrine system from animals and humans (xenoestrogens), was carried out in this study in drinking water and raw water on four cities at region of Paraíba do Sul River. An analytical method with high selectivity and sensitivity, with determination by gas chromatography coupled to mass spectrometry detector (GC/MS), preceded of samples concentration through the SPE technique was used. Good analytical techniques are not enough to ensure quality and reliability of the generated data results. For this, the methodology was submitted to the validation process, where parameters evaluated were: selectivity, specificity, linearity, work range, detection and quantification limits, precision, accuracy, robustness, percentage of recovery and uncertainty in measurement. The validation results showed that proposed method is suitable to quantify Paraíba do Sul river samples, and by its application could observe presence of all studied pollutants in drinking and raw water in one of the three sampling periods at least, and the highest measured concentrations were in raw water at dry periods. These results evidence that the anthropic activities at region are influencing the water quality of the studied water resource, as well as the quality of water for public supply.

Durant ma thèse, j’ai étudié l’implication d’un variant du récepteur aux œstrogènes α, ERα36, dans l’initiation et la progression du cancer du sein. Au laboratoire, son expression dans les cancers testiculaires avait été montrée comme étant inductrice de la prolifération cellulaire in vitro et in vivo après une exposition à un mélange de polluants environnementaux, considérés comme perturbateurs endocriniens oestrogéno-mimétique : les alkylphénols. Une analyse rétrospective d’échantillons de tumeurs mammaires a montré, par la modélisation de réseaux d’interactions géniques, que l’expression d’ERα36 était corrélée avec l’expression de marqueurs de migration cellulaire, caractéristiques de la progression tumorale. La surexpression d’ERα36 par transfection in vitro et dans un modèle unique de souris Knocked In exprimant ERalpha36 dans la glande mammaire ont montré qu’ERα36 est suffisant pour altérer le phénotype épithélial des cellules mammaires saines. Une exposition aux alkylphénols qui stimulent son expression endogène accentue les altérations cellulaires observées et contribue à l’acquisition transgénérationnelle de propriétés relatives à une transformation tumorale. Les analyses de ce projet pluridisciplinaire se sont appuyées sur des expertises biologiques, mathématiques et bioinformatiques et ont permis de mettre en évidence pour la première fois le rôle potentiel d’ERα36 dans l’initiation tumorale et de confirmer son implication dans la progression du cancer du sein. Enfin, nous avons montré que l’exposition à des doses environnementales d’alkylphénols lors de la période de périnatalité peut conduire à une modification transgénérationnelle de la différenciation de la glande mammaire sous le contrôle d’ERα36 et ainsi augmenter le risque de cancer mammaire / This work was dedicated to study how a variant of estrogen receptor α, ERα36, acts in initiation and progression of breast cancer. In the laboratory, his expression in testicular cancer was shown to stimulate cell proliferation in vitro and in vivo after environmental pollutant exposure. The compounds studied, the alkylphenols, are endocrine disruptors, interfering with normal estrogen signaling. Gene interaction network modelling from retrospective analysis of breast cancer samples showed that ERα36 expression was correlated with the expression of cell migration markers, typical of tumor progression. In vitro ERα36 overexpression and in a unique mouse Knocked In model, expressing ERα36 in the mammary gland, showed that ERα36 is sufficient to alter epithelial phenotype of normal breast cells. Alkylphenols exposure, that stimulated ERα36 endogenous expression, increased cellular alterations and contributed to transgenerational acquisition of properties related to neoplastic transformation. Analysis of this multidisciplinary project were based on biological expertise, mathematics and bioinformatic tools. These results enabled to highlight for the first time the potential role of ERα36 in tumor initiation and confirmed his involvement in breast cancer progression. Finally, we showed that exposure to environmental doses of alkylphenols during the perinatal period can lead to transgenerational modification of mammary gland differentiation under ERα36 control and eventually may increase breast cancer risk

Alguns compostos orgânicos como interferentes endócrinos e ativos farmacêuticos estão sendo encontrados em águas superficiais e vêm chamando a atenção de orgãos públicos e da comunidade científica. Dentre esses compostos, foram enfatizados nesse trabalho, o hormônio sintético 17α-etinilestradiol, o surfactante (4-nonilfenol), bisfenol A, o antibiótico β-lactâmico amoxicilina e a cafeína como traçador de atividade antrópica. Estes compostos são continuamente introduzidos em corpos d\'água pelo lançamento de esgoto in natura o que afeta a qualidade da água, a saúde dos ecossistemas e impacta o suprimento de água potável. Considerando a baixa taxa de remoção desses contaminantes pelo tratamento de água atualmente utilizado no Brasil e o estado de degradação dos mananciais de água para abastecimento público, esse trabalho apresenta o desenvolvimento de metodologia analítica para determinação dos compostos citados e a avaliação da eficiência de remoção quando o tratamento convencional de água é aplicado. A determinação foi baseada em métodos já estabelecidos e envolveram as etapas de limpeza e concentração de amostra usando extração em fase sólida (SPE) e análise por cromatografia a líquido de alta eficiência (HPLC) com detector de arranjo de diodos (DAD) e espectrometria de massas. As amostras do Reservatório do Guarapiranga foram utilizadas para ensaios em escala laboratorial que simulavam o tratamento convencional de água em planta real. Os resultados mostraram a adequação dos métodos utilizados para a separação e determinação desses compostos em amostras ambientais. Além disso, apontaram para a necessidade de se avaliar a utilização de metodologias não convencionais que otimizem a remoção pelo tratamento de água atualmente utilizado. / Some organic compounds, such as endocrine disruptors and pharmaceuticals, are being found in the surface water, and are concerning both the public organs and the scientific community. Among these compounds, the synthetic hormone 17α-ethinylestradiol, the surfactant (4-nonylphenol), bisphenol-A, the β-lactam antibiotic amoxicillin and the marker of human activity caffeine were highlighted in the present work. These compounds are continuously drained to the water bodies by sewers, affecting not only the water quality itself, but also the ecosystems, and impacting the drinking water availability. Currently in Brazil, the removal rate of these contaminants is low, whereas the water sources for public supply are highly degraded. In this context, the present work develops an analytical method to detect the aforementioned compounds, and to evaluate the efficiency of its removal by conventional water treatments. Detection was based on pre-established methods and steps involved clean up and samples concentration by solid phase extraction (SPE), high performance liquid chromatography with diode array detection (DAD), and mass spectrometry. Recovery tests were done using environmental samples from Guarapiranga Reservoir (São Paulo, SP). This samples were further used in laboratorial tests, simulating real scale water conventional treatments. Current results revealed the adequacy of the chosen methods in the separation and determination of the abovementioned compounds from natural samples. Also, they highlight the need for the evaluation of non-conventional methodologies that optimize removal by current water treatment.

Huang, Hui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 141-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Les perturbateurs endocriniens (PE) font l’objet d’une controverse scientifique à l’heure actuelle car, pour certains scientifiques, ces molécules n’auraient pas de seuil de toxicité et agiraient à de très faibles concentrations. Cette particularité proviendrait du fait que les PE, quelles que soient leurs origines, agissent sur les hormones et leurs récepteurs. A ce titre, ils seraient assujettis aux mêmes règles que les hormones endogènes. Leurs effets à de très faibles doses s’expliqueraient par la sensibilité des récepteurs hormonaux aux faibles fluctuations de concentrations. L’absence de seuil d’effet pour les PE s’expliquerait par différents mécanismes, dont des différences d’affinité suivant la concentration entre substrat et récepteur, qui engendreraient des effets doses non monotones. Un certain nombre de revues remettent en cause ces hypothèses et les observations qui les accompagnent. Elles indiquent notamment le fait que ces données manquent de répétitions, qu’elles sont principalement issues d’études in vitro ou sur des modèles d’organismes en développement et non-nécessairement misent en perspective avec des effets toxiques pour l’organisme. Ainsi ces données ne seraient pas suffisantes pour statuer dans ce débat.Dans ce contexte, afin d’apporter de nouvelles données expérimentales au débat, nous avons cherché à caractériser les effets aux faibles doses d’un PE de référence, le flutamide (FLU), sur l’appareil reproducteur du rat mâle adulte. Pour ce faire, nous avons conduit trois études successives au cours desquelles nous avons exposé des rats Wistar de 7 semaines à une large gamme de doses de FLU. La dose la plus forte est connue pour induire des lésions testiculaires et une atrophie de la prostate. Les autres doses correspondent à la NOAEL définie dans une étude publiée précédemment, et à cette valeur divisée par 10, 100 et 1000, ce qui correspond à des faibles doses selon la définition de l’OMS. Pour chaque étude, chaque groupe était composé de 16 animaux gavés quotidiennement pendant 28 jours. Ces études ont permis d'explorer différents paramètres. Dans le cadre de la caractérisation du mode d'action du flutamide sur le testicule (Exposition --> blocage des récepteurs aux androgènes de l'hypophyse --> augmentation de la LH circulante --> prolifération des cellules de Leydig --> Hyperplasie), nous avons montré que, pour chaque évènement clé, l’effet dose est monotone et qu’il est possible de définir un seuil. Par ailleurs, nous proposons le Pdgfd, un facteur de croissance, comme médiateur de l’effet mitogène de la LH sur les cellules de Leydig.Pour la prostate, le mode d'action du flutamide n’étant pas complètement élucidé à ces niveaux de doses, le travail a été plus exploratoire. Afin d'expliquer, aux niveaux moléculaire et cellulaire, l'origine de la baisse du poids des prostates induites par le flutamide dès 1 mg/kg/j, nous avons exploré trois hypothèses: (1) l'augmentation de l'apoptose, avec un test d'activité des caspases 3 et 9 qui montre une activité supérieure pour le groupe forte dose de 10 mg/kg/j de flutamide, (2) la diminution de la prolifération, avec un marquage ki67 qui ne montre pas de différence entre le groupe témoin et la forte dose, et enfin (3) la baisse de la fonction de sécrétion de la prostate, évaluée par un western blot dirigé contre la probasine qui montre une baisse de sa production. Dans le même temps, nous avons montré, au niveau des transcrits hépatiques (RT-qPCR), que le flutamide exerce un effet-dose monotone sur certaines enzymes impliquées dans le métabolisme des xénobiotiques. En conclusion, dans ce modèle, pour l'ensemble des paramètres explorés, sur le testicule, l’hypophyse, la prostate, le sang et le foie, nous n'avons observé que des effets à seuil et aucun effet non monotone. / The dose-response characterization of Endocrine disrupting chemicals (EDCs) toxicity is an on-going debate which is controversial when exploring the nature of the dose-response curve and the effect at the low concentration. A part of the scientific community, suggests these molecules have no toxicity threshold and act at very low concentrations. This assumption stems from the fact that EDCs, whatever their origins, act on hormones and their receptors. As such, they would be subject to the same rules as the endogenous hormones. Effects at very low doses could be explained by the sensitivity of the hormone receptor to small fluctuations in concentrations. The lack of threshold for EDCs may be explained by different mechanisms, including affinity differences according to concentrations between substrate and receptor, which would generate non-monotonic dose-responses. A number of reviews challenge this assumption and the linked observations. They argue that the data are missing replication; they are mainly from in vitro studies or conducted in developing models and not-necessarily associated with toxic effects on the organism. Thus, these data are not sufficient to rule in this debate.In this context, in order to provide new experimental data to this debate, we worked to characterize the effects at low doses of a reference EDCs, flutamide (FLU) on the reproductive system of adult male rats. In that aim, we conducted three successive studies exposing Wistar rats of 7 weeks at a wide range of Flu doses. The highest dose is known to induce testicular damage and atrophy of the prostate gland. Other doses correspond to the defined NOAEL in a study published earlier, and to this value divided by 10, 100 and 1000, corresponding to low doses according to the WHO definition. For each study, each group consisted of 16 animals exposed daily by gavage for 28 days. During characterization of flutamide mode of action on the testis (Exposition -> androgen receptors blocking in the pituitary gland -> increase in circulating LH -> Leydig cells proliferation -> Leydig cells Hyperplasia), we have shown that for every key event, dose-response curve is monotonous and we were able to define a threshold. Furthermore, we suggest that the Pdgfd, a growth factor, mediates the mitogenic effect of LH on Leydig cells.In the prostate gland, the mode of action of flutamide is not completely elucidated at these dose levels, so the work was more exploratory. To explain, at the molecular and cellular levels, the origin of prostate atrophy induced by flutamide from 1 mg/kg/day, we explored three hypotheses: (1) the increase in apoptosis, with a caspases 3 and 9 activity test, which shows a higher activity for the high dosage group of 10 mg/kg/day of flutamide, (2) the decreasing of epithelium proliferation, with Ki67 marker which shows no difference between the control group and the high dose, and finally (3) the decrease in prostate secretory function, assessed by western blot against the Probasin which shows a drop in production.At the same time, we have shown in liver that flutamide has an effect on enzymes involved in the metabolism of xenobiotics with monotonic dose-response.In conclusion, in this model, for all investigated parameters on the testis, pituitary, prostate, blood and liver, we only observed threshold effects and no non-monotonic dose-responses.