Orientador: José Teixeira Filho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Agrícola / Made available in DSpace on 2018-08-25T00:46:54Z (GMT). No. of bitstreams: 1 Campos_JulyenneMeneghetti_M.pdf: 7123166 bytes, checksum: da20e59802401636508451e97ba79194 (MD5) Previous issue date: 2014 / Resumo: Leitos cultivados tem sido citados como um método alternativo de redução da concentração de alguns agrotóxicos e hormônios levemente hidrofóbicos, como o 17-?-etinilestradiol, que é um dos principais responsáveis pela causa da alteração no sistema endócrino de humanos e animais. Neste contexto, o presente projeto visou avaliar a eficiência da redução de pH, 17-?-etinilestradiol, oxigênio dissolvido (OD), cor aparente, turbidez, nitrogênio total Kjeldahl (NTK), nitrogênio amoniacal (NH3), nitrito (NO2-), nitrato (NO3-), demanda química de oxigênio (DQO) e fósforo total (PT), utilizando-se três leitos tendo brita como meio suporte ¿ dois deles cultivados com a macrófita Eichhornia crassipes (aguapé), com 43 dias de diferença de cultivo entre eles, e outro leito sem cultivo apenas com a brita para controle e comparação dos resultados. A vazão de entrada nos leitos cultivados variou entre 312 e 1.059 L.dia-1, o TDH médio permaneceu entre 2 e 3 dias, as médias máximas de retenção da concentração de DQO, cor, turbidez, NTK, NH3 e PT pelos leitos foram 94,2%, 76,7%, 90,0%, 42,7%, 39,6% e 51,0%, respectivamente. A biomassa dos dois leitos cultivados com aguapé absorveram juntos, 163 g.kg-1 de nitrogênio e 47,2 g.kg-1 de fósforo, durante o período monitorado. Dentre os parâmetros de eficiência analisados o leito cultivado com Eichhornia crassipes com maior grau de desenvolvimento foi o mais eficiente na retenção da maioria dos parâmetros avaliados com nível 5% de significância. A metodologia adaptada para análise do hormônio 17-?-etinilestradiol por Cromatografia Líquida de Alta Eficiência acoplada à Detector de Arranjo de Diodos obteve limite de detecção de 1,26 µg.L-1, e limite de quantificação de 2,52 µg.L-1, porém as amostras analisadas ficaram abaixo do nível de detecção, não sendo possível a determinação das concentrações das amostras. Conclui-se que a macrófita Eichhornia crassipes fixada em meio suporte obteve retenções de nutrientes consideradas satisfatórias em tratamento de esgoto doméstico, e as informações obtidas por este trabalho poderão contribuir com a melhoria e desenvolvimento de novas tecnologias para retenção e métodos analíticos para quantificação de 17-?-etinilestradiol em águas residuárias, diminuindo o lançamento destes interferentes endócrinos nos corpos hídricos / Abstract: Constructed wetlands have been quoted as an alternative method for the removal of slightly hydrophobic pesticides and hormones, such as 17-?-ethinylestradiol, which is one of the main hormones responsible for humans and animals endocrine systems changes. In this context, this project tried to evaluate the reduction efficiency with regards to pH, 17-?-ethinylestradiol, dissolved oxygen (DO), apparent color, total Kjeldahl nitrogen (NTK), ammonia nitrogen (NH3), nitrite (NO2-), nitrate (NO3-), chemical oxygen demand (COD) total phosphorus (PT), using three constructed wetlands having gravel as support media ¿ two of them cultivated with Eichhornia crassipes (water hyacinth), with 43 growth days gap between, and another with gravel only for control and results comparison. The entrance flow rate in the constructed wetlands ranged between 312 to 1,059 L.dia-1, the average HRT stayed between 2 and 3 days, the maximum chemical oxygen demand retention averages, color, turbidity, NTK, ammonia nitrogen and total phosphorus by the constructed wetlands were 94,2%, 76,7%, 90,0%, 42,7%, 39,6% and 51,0%, respectively. The biomass of both water hyacinth constructed wetlands absorbed together 163 g.kg-1 of nitrogen and 47,2 g.kg-1 of phosphorous during the monitored period. Among the analyzed efficiency parameters, the constructed wetland cultivated with Eichhornia crassipes, also 43 days older, was the most efficient in the majority of the parameters evaluated at a 5%significance level. The suitable methodology for analyzing 17-?-ethinylestradiol by high efficiency liquid chromatography attached to a Diode Arrange Detector, had a detection limit of 1.26µg.L-1, and a quantification limit of 2.53 µg.L-1, but the analyzed samples stayed below the detection level, becoming impossible determine the samples¿ concentrations. The conclusion is that the macrophyte Eichhornia crassipes fixed in a support media had satisfactory nutrients retention from domestic sewage, and the obtained information by this work can contribute for an improvement and development of new technologies for retention and analytical methods for 17-?-ethinylestradiol wastewater quantification, decreasing the discharge of these endocrine disruptors in water bodies / Mestrado / Agua e Solo / Mestra em Engenharia

Orientador: Hernandes Faustino de Carvalho / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T07:59:39Z (GMT). No. of bitstreams: 1 Rosa-Ribeiro_Rafaela_M.pdf: 2991640 bytes, checksum: 1c3d98622022906218832c47353b3c63 (MD5) Previous issue date: 2012 / Resumo: O desenvolvimento, a fisiologia e o câncer de próstata dependem de balanços entre os níveis de andrógenos e estrógenos, que agem via receptor de andrógenos (AR) e receptores de estrógeno (ER'alfa' e ER'beta'), respectivamente. Sabe-se que a cinética de morte das células epiteliais da próstata ventral de ratos (PV) após castração (grupo Cas), administração de alta dose de 17'beta'-estradiol (grupo E2) e combinação de ambos (grupo Cas+E2) é diferenciada, com um nítido efeito aditivo nesta última situação (Garcia-Florez et al, 2005). Neste trabalho, procuramos investigar elementos comuns e exclusivos a estas diferentes condições hormonais, empregando análises morfológicas, estudo de componentes da via AKT/PTEN, e análise da expressão diferencial de genes (utilizando microarranjos de DNA) combinada com identificação de fatores de transcrição (FT) a ela relacionados. O peso relativo da PV foi significativamente diminuído nos grupos Cas e Cas+E2. A castração promove um padrão de descamação das células epiteliais que deve contribuir para redução do número de células epiteliais. No grupo E2, foi marcante a presença agregados protéicos citoplasmáticos e proliferação das células epiteliais com freqüente estratificação do epitélio. Outro aspecto importante foi o descolamento das células musculares lisas do epitélio quando este apresentava dobras epiteliais. No grupo Cas+E2, foram observados os diferentes aspectos observados em cada grupo individual. A análise bioquímica mostrou redução significativa na fosforilação de 4EBP2 nos grupos E2 e Cas+E2 (tendência similar foi observada no grupo Cas). Há uma grande quantidade de genes que são diferencialmente expressos em relação ao controle e que são comuns aos diferentes tratamentos. Surpreendentemente, não houve redução da expressão de probasina no grupo E2. A análise de ontologias e de termos enriquecidos mostrou a ausência de termos enriquecidos no conjunto de genes exclusivos do grupo Cas+E2, o que se concilia bem com a idéia de que os eventos observados neste grupo são aqueles observados nos dois grupos individuais. Em conclusão, a busca por sítios de ligação de FT na região promotora dos genes mais regulados em cada grupo e a identificação de FT nas rede de dos termos enriquecidos indicou os FT Evi-1, NF-Y, HNF-4, Elk-1, GATA-2, c-Rel, v-Myb e NFkB como candidatos a atuarem na regulação dos eventos associados à regressão prostática / Abstract: Prostate development, physiology and cancer depend on a fine balance between the levels of androgens and estrogens acting via the androgen receptor (AR) and the estrogen receptors (ER'alpha' e ER'beta'), respectively. It is known that the kinetics of apoptosis are different in the rat ventral prostate (VP) of castrated rats (Cas group) and in rats subjected to 17'beta'-estradiol high dose (group E2) or their combination (group Cas+E2), with an evident additive effect in the latter situation (Garcia-Florez et al, 2005). In this work, we investigated elements in common and exclusive to each of these hormonal conditions, employing morphological analysis, components of the AKT/PTEN pathway and analyses of differential gene expression using DNA microarrays combined with a search for transcription factors (TF). The VP weight was significantly reduced in the Cas and Cas+E2 groups. In the E2 group, the remarkable effects were the presence of protein aggregates in the cytoplasm, and cell proliferation and layering of the epithelium. Another important aspect was the detachment of the smooth muscle cells from the epithelium when it showed infolds. In the Cas+E2 group, the different aspects observed in the individual groups were observed, except by less frequent epithelial layering. The biochemical analysis showed a significant reduction in 4EBP phosphorylation in the groups E2 and Cas+E2 (similar tendency was observed in the Cas group). There is a large number of differentially expressed genes as compared to the controls and shared by the different treatments, Surprisingly, there was no reduction in the expression of the probasin gene in the E2 group. The recovered gene onthologies and enrichment terms revealed the absence of enrichment terms among the genes exclusive to the Cas+E2 group, what conciliates with the Idea that the observed changes in this group are identical or at least very similar to those occurring in the individual treatments. In conclusion, the search for TF binding sites in the promoter region of the regulated genes and the identification of TF in the regulatory pathways obtained for the enrichement terms indicated the TF Evi-1, NF-Y, HNF-4, Elk-1, GATA-2, c-Rel, v-Myb and NFkB as candidates to regulate the events associated with prostate regression / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

O letrozol é usado como terapêutico em desordens reprodutivas provocadas pelos altos níveis de estrógenos. A enzima citocromo P450 aromatase biossintetiza estrógenos a partir dos andrógenos em tecidos com capacidade esteroidogênica, como o cérebro, testículo e ovário. O objetivo do nosso estudo foi avaliar se o letrozol afeta o desenvolvimento das principais vias de controle reprodutivo de preás machos e fêmeas. Para tanto, consideramos o ganho de peso corporal, do testículo, do ovário e do cérebro, a progressão morfológica da espermatogênese e da foliculogênese, bem como a atividade enzimática da citocromo P450 aromatase nesses tecidos, comparado-os entre os grupos experimentais de machos e fêmeas. Os preás receberam 0,01 g/kg-1 de letrozol diluído, via oral, semanalmente, até as idades de 30, 45, 90 e 120 dias. O letrozol aumentou o ganho de peso corporal, das gônadas e do cérebro. Também, prejudicou a formação do epitélio germinativo testicular e estratificou o epitélio de revestimento do ovário. Ainda, o inibidor pode alterar os campos neurais relacionados às zonas de aromatização. E alterou os sítios de atuação da aromatase nas gônadas. Concluímos que o uso prolongado do letrozol pode ocasionar efeito anabólico, infertilidade de machos, induzir a displasia ovariana em fêmeas e alterar os sítios de atuação da aromatase. / Letrozole is used as a therapeutic in reproductive disorders caused by high estrogen levels. The enzyme cytochrome P450 aromatase biosynthesizes estrogens from androgens on tissues with steroidogenic capacity such as brain, testis and ovary. The objective of our study was to evaluate whether letrozole affects the development of the main reproductive control pathway of male and female spixs yellow-toothed cavy. For this, we considered body weight, testis, ovary and brain gain, spermatogenesis and folliculogenesis, as well as the enzymatic activity of cytochrome P450 aromatase in these tissues compared to experimental groups of males and females. The cavies received dilute letrozole orally (0,01 g/kg-1), once a week, until 30, 45, 90 and 120 days. Letrozole increased body weight, gonad and brain gain. Also, it impaired the formation of the testicular germinal epithelium and epithelium of ovary. Furthermore, the inhibitor may alter the neural fields related to the aromatization zones and changed the sites of aromatase in the gonads. We concluded that prolonged use of letrozole may result in an anabolic effect, male infertility, induce ovarian dysplasia in females, and alter the sites of aromatase activity.

Les œstrogènes sont impliqués dans la prolifération des cellules épithéliales du sein normal et l'exposition prolongée à ces hormones s'accompagne d'une augmentation du risque de développement de cancer du sein. Les œstrogènes exercent leurs effets via les récepteurs des œstrogènes (REs). L'activité de ces récepteurs est finement régulée par un grand nombre de cofacteurs transcriptionnels, mais également par les modifications post-traductionnelles. Mon travail de thèse a eu pour objectif la compréhension de l'impact de ces deux niveaux de régulation sur la signalisation œstrogénique. Il a été récemment décrit que la sumoylation affectait de manière drastique l'activité du RE. La sumoylation est une modification dont le caractère réversible est assuré par des isopeptidases appelé SENPs (SENtrin Proteases). Dans une première étude nous avons montré que SENP2 pouvait fortement réprimer l'activité transcriptionnelle dépendante des œstrogènes ainsi que la prolifération cellulaire. Dans une seconde étude, nous nous sommes attelés à mieux caractériser les mécanismes d'action à l'origine du caractère répresseur du cofacteur transcriptionnel LCoR (Ligand-dependent Corepressor). Nous nous sommes plus précisément intéressés aux relations existant entre LCoR et un autre cofacteur du RE, RIP140 (Receptor Interacting Protein of 140 kDa) répresseur majeur de l'activité œstrogénique. Nous avons pu caractériser, outre les modes de recrutement des deux protéines, les modulations d'expression exercées par les deux cofacteurs. L'ensemble de nos travaux identifie de nouveaux cofacteurs des REs et contribue à une meilleure compréhension de la signalisation œstrogénique. / Estrogens are involved in the proliferation of normal breast epithelial cells. The prolonged exposure to these hormones comes along with an increase of the risk of breast cancer.development. Estrogen receptors (ERs) mediate the effects of estrogens. The activity of these receptors is finely tuned by a large number of transcriptional cofactors, but also by post-translational modifications. This work aimed at understanding the impact of these regulations on estrogenic signalling. It was recently described that sumoylation could strongly affect ER-dependent activity. SUMO conjugation is a dynamic process which is reversed by SUMO specific proteases also known as SENtrin Proteases (SENPs). In a first study, we investigated the role of SENP2, in ER-dependent transcriptional activity. We showed that SENP2 could acts as a transcriptional cofactor independently of its catalytic activity by strongly repressing ER-dependent transcriptional activity. We also provided evidence for a role in in breast cancer cell line proliferation. In a second part of the work we investigated the mechanism of action of the transcriptional cofactor LCoR (Ligand-dependent Corepressor) with a specific emphasis on the relationship between LCoR and another ER cofactor, RIP140 (Receptor Interacting Protein of 140 kDa). We characterized a crossed expression modulation of the two transcription cofactors. We also depicted an interaction between these two corepressors and a regulation of LCoR activity by RIP140. Our work provides new insights in identifying new coregulators of ER and contributes to a better understanding of both LCoR and RIP140 mechanism of action, and therefore of estrogenic signalling.

Orientador: Sebastião Roberto Taboga / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T21:06:44Z (GMT). No. of bitstreams: 1 Santos_FernandaCristinaAlcantarados_D.pdf: 8013059 bytes, checksum: 981f820c48d983ff59f0fae9c64ba43d (MD5) Previous issue date: 2006 / Resumo: A próstata feminina é uma glândula funcionalmente ativa encontrada em diversas espécies de mamíferos, incluindo humanos e roedores. Em fêmeas adultas de gerbilos, a próstata apresenta localização parauretral, exibindo íntimo contato com a parede da uretra mediana e distal. Esta glândula é homóloga a próstata ventral de roedores machos, sendo formada por um conjunto de glândulas e ductos inseridos em um estroma fibromuscular. Em machos, a fisiologia prostática é regulada por hormônios esteróides, principalmente andrógenos e estrógenos. Em fêmeas, os fatores que influenciam a atividade prostática são pouco conhecidos, embora existam indícios de que alterações hormonais decorrentes da senescência estejam associadas à instalação de lesões prostáticas. Assim, o objetivo deste trabalho foi avaliar os fatores que promovem a regulação hormonal da próstata feminina do gerbilo (Meriones unguiculatus) em condições de hiperandrogenismo e de supressão da atividade estrogênica. Os resultados obtidos com as análises estruturais, ultra-estruturais, sorológicas e imunocitoquímicas permitiram concluir que a próstata feminina do gerbilo é sensível à ação de andrógenos e de agentes anti-estrogênicos. O estímulo androgênico provocou crescimento anormal da próstata, aumento da atividade secretória, além de causar displasia prostática e síndrome de ovário policístico. O tratamento com letrozol resultou em aumento dos níveis séricos de testosterona, hiperplasia glandular, incremento da atividade secretória e crescimento displásico, simulando os efeitos causados por andrógenos exógenos. Os efeitos causados pelo tamoxifeno indicam que este agente endócrino atuou como agonista estrogênico na próstata, causando hipertrofia glandular, diminuição da atividade secretória e desenvolvimento de lesões prostáticas, tais como prostatites e adenocarcinoma. Deste modo, pode-se concluir que a utilização de drogas hormonalmente ativas resulta em uma série de efeitos complexos que comprometem a fisiologia de órgãos hormônio-dependentes, como a próstata feminina e os ovários. O desequilíbrio hormonal provocado pela administração destas drogas causa profundas alterações na morfologia prostática, de maneira muito similar ao que ocorre durante o desenvolvimento de lesões espontâneas em mulheres no período pós-menopausa. Assim, essas terapias devem ser utilizadas com cautela, visto que longos períodos de tratamento podem resultar em lesões malignas da próstata feminina / Abstract: The female prostate is a functionally active gland found in several species of mammals, including humans and rodents. In adult female gerbils, the prostate presents a paraurethral location, showing close contact with the wall of urethra in its median and distal portions. This gland is homologue to the ventral prostate of male rodents and it is formed by a cluster of glands and ducts inserted into a fibermuscular stroma. In males, the prostatic physiology is regulated by steroid hormones, mainly androgen and estrogen. In females, the factors that influence the prostatic activity are unclear, although there are evidences that the hormonal alterations caused by aging are associated with the installation of prostatic lesions. Thus, the objective of this work is to evaluate the factors that promote the hormonal regulation of the gerbil (Meriones unguiculatus) female prostate in hyperandrogenic conditions and estrogenic activity suppression. The results obtained with the structural, ultrastructural, serologic and immunocytochemical analyses showed that the gerbil female prostate is responsive to androgenic and the anti-estrogenic action. The androgenic stimulus has caused an abnormal prostatic growth, increase in secretory activity, and has also caused prostatic dysplasia and polycystic ovary syndrome. The letrozole treatment has stimulated an increase in testosterone serum levels, glandular hyperplasia, increment of the secretory activity and dysplasic growth, simulating the effects provoked by exogenous androgens. The effects caused by tamoxifen indicate that this endocrine agent has acted as an estrogenic agonist on the prostate, causing glandular hypertrophy, decrease in secretory activity and prostatic lesions. Hence, it is possible to conclude that the use of hormonally active drugs results in a series of complex effects that endanger the physiology of hormone-dependent organs, like female prostate and ovaries. The hormonal unbalance caused by the administration of such drugs results in alterations in prostatic morphology similar to what occurs during the development of spontaneous lesions in post-menopausal women. Thus, the utilization of such therapies must occur in a careful manner because a long-term treatment can cause malignant lesions in female prostate / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Fertilization is a unique biological event where male and female gametes fuse together to produce a new organism. Before the gametes are able to fuse, however, they must undergo a series of controlled changes. For the male gamete, capacitation and acrosome reaction (AR) must occur, which take place during the sperm migration through the female genital tract. Unfortunately, while the process of capacitation has been known for over half a century, the molecular basis and influential factors behind it are not fully understood. Although estrogens have been considered mainly female reproductive hormones, there is increasing evidence suggesting that these steroids have an important role also in regulation of male reproductive functions. Sperm come into the contact with estrogens during their formation in the male and female genital tract, indicating that the hormone may play an important role in sperm maturation. In this study, we examined the importance of three endogenous estrogens (E1 -estron, E2 - 17β estradiol, E3 - estriol) and one synthetic estrogen (EE2 - 17α ethinylestradiol) on sperm maturation during capacitation and AR. Stimulatory effect were observed with all tested estrogens on both capacitation and zona pellucida induced AR. Moreover, we have determinied that the stimulatory effect on...

There are an increasing amount of compounds in the environment that can have a negative effect on reproductive parameters in both male and female organism. There has been a worldwide decline of sperm quality during past decades and this fact lead to an increase of unnatural ways of conception through assisted reproduction techniques in the specialised centres. Natural estrogens are one of these compounds and they get into waste water after being excluded from the body by the urine. They get back into the human body from drinking water or from the food, and they can interfere with function of endogenous hormones in very low concentrations. For these reasons it is up to date to deal with the influence of these compounds on mammalian sperm. For many years, estrogens have been considered typically female sex hormones. It is now certain that they are also very important in the regulation of male reproduction. Endogenous estrogens in mammalian males are an important part of the endocrine system. Estrogens play an important role in the development of germ cells, spermatogenesis and processes leading to successful egg fertilization such as a capacitation or acrosomal reaction. Tyrosine phosphorylation is one of the essential steps for the properly ongoing process of capacitation in sperm followed by a...

Endometrial prostaglandins (PGs) play a role in menstruation in primates and in luteolysis in nonprimates. Their biosynthesis is regulated by estrogen (E) and progesterone (P) in a manner not fully understood. The purpose of this thesis research was to (1) study the effects of E and P, both in vivo and in vitro, on basal endometrial PG output in vitro during the course of the artificial menstrual cycle in the rhesus monkey, and (2) further to examine the cellular mechanisms of P action in vivo on PG output using an ovine model system. To carry out the first objective, ovariectomized rhesus monkeys (n=39) were maintained on either a standard or manipulated artificial menstrual cycle (SAMC and MAMC, respectively) and endometrial biopsies were obtained at precise times in separate cycles on: cycle day 9 (mid-proliferative), 13 (mid-cycle E peak), 14 (one day post E peak), and 23 (mid-secretory). PGF2α was the most abundant PG produced in vitro by endometrial organ cultures, the levels of which changed most dramatically throughout the SAMC. Within the first 24 hours of organ culture, PGF2α accumulation was low on day 9 and rose significantly (p<0.01) on day 13, indicating a stimulatory effect of E in vivo. However, E added in vitro, at either physiologic or supraphysiologic concentrations, to endometrial cultures did not stimulate PGF2α accumulation on any cycle day examined. On day 14, just one day post E peak, there was a dramatic fall in PGF2α accumulation which appeared to be due to both a decline in stimulatory E in vivo and a rise in inhibitory P in vivo. Basal PGF2α accumulation in vitro by day 23 endometrial cultures was 10-fold higher (p<0.01) compared to days 9 and 14. This high level of PGF2α output on day 23 appeared to be caused by a paradoxical priming effect of P in vivo and also a slight enhancement by the mid-cycle peak of E in vivo. Padded in vitro, at a physiologic concentration, to day 23 endometrial cultures markedly inhibited (p<0.01) the high level of PGF2α accumulation, suggesting that P withdrawal in vivo promotes the rise in endometrial PGF2α production in vivo at the time of menstruation in primates. An ovine model system was further used to investigate the cellular mechanisms of P action in vivo. Ovariectomized sheep (n=8) were administered an infusion regimen of either E and P, or E and P vehicle alone, to examine the effects of P in vivo on PGF2α production in vitro by endometrial explants during short-term incubations. P in vivo increased the mass amount of stimulated PGF2α output by both physiologic and pharmacologic mechanisms. In addition, P did not appear to significantly alter the sensitivity of the endometrium to stimulatory levels of oxytocin in vitro indicating that the cellular events accounting for the P priming effect, in part, may occur independent of the oxytocin receptor closer to the PG biosynthetic pathway. In P-primed endometrium, the mass amount of PGF2α stimulated by a calcium-ionophore (A23l87) was less than that stimulated by OT suggesting the involvement of calcium-insensitive mechanisms in PGF2α synthesis.

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.

Indiana University-Purdue University Indianapolis (IUPUI) / The hormonal and cellular mechanisms controlling bone formation are not completely understood. The proline-rich tyrosine kinase 2 (Pyk2) is important for osteoblast (OB) activity and bone formation. However, female mice lacking Pyk2 (Pyk2-KO) exhibit elevated bone volume/total volume. Previously, our laboratory found ovariectomized Pyk2-KO mice supplemented with 17β-estradiol (E2) exhibited a greater increase in bone volume than WT mice treated with E2. The overall hypotheses of our studies are that Pyk2 regulates OB activity by modulating the E2-signaling cascade and that a Pyk2-inhibitor will promote OB activity and be suitable for bone regeneration applications. In Aim1, we determined the mechanism of action of Pyk2 and E2 in OBs. Pyk2-KO OBs showed significantly higher proliferation, matrix formation, and mineralization than WT OBs. In the presence of E2 or raloxifene, a selective estrogen receptor (ER) modulator, both matrix formation and mineralization were further increased in Pyk2-KO OBs, but not WT OBs. Consistent with a role of Pyk2 in E2 signaling, Pyk2-depletion led to the proteasome-mediated degradation of ERα, but not ERβ. Finally, we found Pyk2-depletion and E2 have an additive effect on ERK phosphorylation, known to increase cell differentiation and survival. In Aim2, we developed a Pyk2-inhibitor loaded hydrogel and evaluated its viscosity, gelation time, swelling, degradation, and release behavior. We found that a hydrogel composed of PEGDA1000 plus 10% gelatin exhibited viscosity and shear-thinning behavior suitable for use as an injectable-carrier. Importantly, the Pyk2-inhibitor-hydrogel was cytocompatible, retained its inhibitory activity against Pyk2 leading to an increase in OB activity. In conclusion, therapeutic strategies targeting Pyk2 may improve systemic bone formation, while Pyk2-inhibitor loaded hydrogels may be suitable for targeted bone regeneration in craniofacial and/or the other skeletal defects.