Orientador: José Luiz Moraes Vasconcelos / Resumo: O objetivo deste trabalho foi o testar a combinação de tratamentos hormonais com progesterona (CIDR), gonadorelina (análogo de GnRH), prostaglandina F2a (PGF2a) e cipionato de estradiol (ECP), com a finalidade de avaliar tratamentos hormonais de sincronização da ovulação e viabilizar a inseminação artificial em tempo fixo (IATF), em vacas mestiças lactantes Holandês-Zebu mantidas a pasto no verão. O trabalho foi dividido em 2 experimentos. Experimento I foi realizado entre Dezembro (2001) a Fevereiro (2002), em Fernandópolis, SP, Brasil. Os animais foram divididos aleatoriamente em 4 tratamentos: Controle (n=51): inseminação artificial (IA) 12 horas após detecção do estro; GP + CIDR (n=50): GnRH - 6dias - PGF2a e IA 12 horas após detecção do estro; ECPsynch + CIDR (n=52): GnRH - 6dias - PGF2a - 24hs - ECP - 48hs - IATF e Ovsynch + CIDR (n=53): GnRH - 6dias - PGF2a - 36hs - GnRH - 12hs - IATF. A análise estatística foi procedida por CATMOD (com nível de significância de P<0,05). A eficiência na detecção de estro foi de 50,9%. A taxa de ciclicidade foi influenciada por ordem de lactação (P<0,001; sendo de 41,4% nas vacas primíparas e de 72,6% nas multíparas) e por escore de condição corporal (ECC; P<0,001). A resposta ovulatória ao 1° GnRH foi influenciada por ECC (P< 0,01). A taxa de sincronização de ovulação não foi influenciada por tratamentos (P=0,45, sendo de 75,4% no protocolo Ovsynch + CIDR e de 73,0% no protocolo ECPsynch + CIDR), porém condição corporal (P<0,05) proporcionou efeito sobre a taxa de sincronização. Na taxa de prenhez a IATF, foi verificada interação entre os tratamentos e ordem de lactação (P<0,01), sendo o protocolo ECPsynch + CIDR mais eficiente nas multíparas (50,0%) em relação ao Ovsynch + CIDR (16,7%), e nas primíparas o protocolo Ovsynch + CIDR (37,9%) foi superior...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this experiment was to evaluate the combination of hormonal treatments with progesterone (CIDR), gonadorelin (GnRH analogue), prostaglandin F2a (PGF2a) and estradiol cypionate (ECP), in protocols that allow fixed time artificial insemination (TAI) in crossbred cows managed in a grazing based dairy system during the summer. The experiment was divided in two trials. Trial I was conducted from December (2001) to February (2002), in Fernandópolis, SP, Brazil. The animals were randomly assigned to one of 4 treatment groups: Control (n=51) AI 12 hours after estrus detection; GP + CIDR (n=50): GnRH - 6day - PGF2a - AI 12 hours after estrus detection; ECPsynch + CIDR (n=52): GnRH - 6day - PGF2a - 24h - ECP - 48h - AI and Ovsynch + CIDR (n=53): GnRH - 6day - PGF2a - 36h - GnRH - 12h - AI. Data were analyzed by CATMOD procedure of SAS. Efficiency in the heat detection was 50,9%. The cyclicity was influenced by parity (P<0,001; 41,4% in primiparous cows and of 72,6% in multiparous cows) and by body condition score (BCS, P<0,001). The ovulatory response to the first GnRH injection was influenced by BCS (P<0,01). Synchronization rate was not influenced by treatments (P=0,45, 75,4% in the Ovsynch+CIDR and of 73,0% in the ECPsynch+CIDR), but it was influenced by BCS (P<0,05). Interactions were observed between treatments and parity (P <0,01) in the pregnancy rate at TAI. Treatment ECPsynch+CIDR was more efficient in multiparous cows (50,0%) when compared with Ovsynch+CIDR (16,7%). In primiparous cows the Ovsynch+CIDR (37,9%) was more efficient then ECPsynch+CIDR (25,0%). Pregnancy rate in groups with TAI was influenced by BCS (P<0,01). The service rate in GP+CIDR, evaluated for 5 day after the application of PGF2a, was influenced by cyclic status (P<0,05). Conception rate of the animals that were inseminated after the heat detection (GP+CIDR and Control) was influenced by BCS (P<0,05)...(Complete abstract click electronic access below) / Mestre

As novas biotécnicas são ferramentas inovadoras no estudo da fisiologia da reprodução de cães, bem como na preservação do material genético de espécies ameaçadas de extinção. Tendo em vista, pois, a relevância de tais ferramentas, emprestou-se a este estudo o objetivo maior de avaliar os efeitos do 17-ß estradiol, progesterona e do co-cultivo em células da tuba uterina na maturação in vitro de oócitos de cães. Ovários de cadelas em anestro foram fatiados em PBS com 10% SFB. Os oócitos foram selecionados e divididos em 8 grupos: grupo 1 (sem suplementação hormonal), grupo 2 (20 µg/ml de 17-ß estradiol), grupo 3 (20 µg/ml de progesterona), grupo 4 (20 µg/ml de 17-ß estradiol e 20 µg/ml de progesterona), grupo 5 (co-cultivo em células da tuba uterina sem suplementação hormonal), grupo 6 (co-cultivo em células da tuba uterina + 20 mg/ml de 17-ß estradiol), grupo 7 (co-cultivo em células da tuba uterina + 20 µg/ml de progesterona) e grupo 8 (co-cultivo em células da tuba uterina + 20 µg/ml de 17-ß estradiol + 20 µg/ml de progesterona). O cultivo das células da tuba uterina foi realizado por no mínimo 48 horas previamente à adição dos oócitos. Para a maturação, utilizou-se TCM 199 suplementado com 2,5 µg/ml de FSH e 5 µg/ml de LH. Decorridas 48, 72 e 96 horas em estufa a 39&ordm;C, 5% de CO2 em ar e alta umidade, os oócitos foram fixados em paraformaldeído a 4% com Triton 10X e corados com Hoescht 33342 (10 µg/ml) em glicerol. O estágio de maturação nuclear (vesícula germinativa - VG, quebra da vesícula germinativa – QVG, metáfase I – MI, metáfase II – MII, degenerados ou não passíveis de determinação) foi avaliado em microscópio invertido equipado com luz ultravioleta e filtro de 365-420 de excitação/emissão. Os dados foram analisados mediante o PROC NPAR1WAY de análise de variância não paramétrica, sendo o efeito dos tratamentos verificado por meio do teste de Kruskal-Wallis, e as comparações múltiplas do teste de Wilcoxon com os tratamentos comparados dois a dois, considerando as diferenças significativas quando p<0,05. Verificou-se influência positiva da suplementação hormonal no desenvolvimento oocitário in vitro, particularmente com referência à adição de progesterona, associada ou não ao estrógeno. Não houve influência significativa do co-cultivo de células da tuba uterina na maturação oocitária em comparação aos meios de cultivo, sem presença de células epiteliais da tuba uterina, suplementados com hormônios esteróides. Conquanto sem significância estatística, verificou-se tendência de os oócitos, obtidos de cadelas em anestro, desenvolverem-se ao estágio de metáfase II após o período de maturação de 96 horas. Demonstrou-se, portanto, ser plenamente exeqüível o estudo da maturação de oócitos de cães em sistemas de cultivo in vitro a partir de ovários obtidos por ovário-histerectomia, tal estudo propicia, ademais, a pesquisa de características reprodutivas fisiológicas da espécie. / Recent biotechniques are innovative tools for the canine reproductive physiology research, as well as for assisted reproduction of endangered species. The aim of this study was to evaluate the effects of estradiol-17ß, progesterone and the co-culture with oviductal ephitelial cells on in vitro maturation. Ovaries from anestrous bitches were sliced in PBS with 10% FCS. Oocytes were selected and distributed in 8 groups: group 1 (no hormonal suplementation), group 2 (estradiol-17ß at 20 µg/ml), group 3 (progesterone at 20 µg/ml), group 4 (estradiol-17ß at 20 µg/ml + progesterone at 20 µg/ml), group 5 (co-culture in oviductal ephitelial cells without hormonal suplementation), group 6 (co-culture in oviductal ephitelial cells + estradiol-17ß at 20 µg/ml), group 7 (co-culture with oviductal ephitelial cells + progesterone at 20 µg/ml) and group 8 (co-culture with oviductal ephitelial cells + estradiol-17ß at 20 µg/ml + progesterone at 20 µg/ml). Oviductal ephitelial cells culture was performed at least 48 hours prior to oocyte co-culture. For in vitro maturation, TCM 199 supplemented with FSH (2.5 &micro;g/ml) and LH (5 &micro;g/ml) was utilized. After periods of 48, 72 and 96 hour at 39ºC in humidified atmosphere of 5% CO2 in air, oocytes were fixed with Triton-X10 in paraformaldehyde at 4% and and stained with Hoescht 33342 (10 µg/ml) in glycerol. Oocytes were examined through an inverted fluorescence microscope equipped with a 365-420 nm wavelength excitation/emisson filter. Nuclear maturation was classified according to chromatin configuration as: germinal vesicle stage (GV), germinal vesicle break down (GVBD), metaphase I (MI), metaphase II (MII) and degenerated and unidentifiable oocytes. Data was evaluated through PROC NPAR1WAY of non parametrical variance analysis and treatment was verified by the Kruskal-Wallis test and the Wilcoxon test for multiple comparisions. Differences were considered significant at p<0.05. Positive influence of hormonal supplementation for oocyte development rates was verified, particularly with respec to progesterone. No significant influence of oviductal cells co-culture was however verified. In addition, a tendency of the oocytes reaching metaphase II in the 96 hour period was observed. Therefore, the study of canine in vitro oocyte maturation is feasible and provides an important tool for reproductive physiology research.

O presente trabalho teve por objetivo investigar a variação na secreção de LH em resposta ao tratamento com neurotransmissores, na presença ou não de esteróides gonadais e desta forma gerar informações sobre os mecanismos neuroendócrinos envolvidos na maturação sexual de novilhas da raça Nelore. A concentração de LH foi quantificada por radioimunoensaio e as novilhas ovariectomizadas (OVX) apresentaram maior concentração basal de LH (P&le;0,05), que as novilhas inteiras (INT). A administração de um antagonista gabaérgico (picrotoxina, 0,18 mg/kg, iv, amostras 15 min por 10 h) aos 8, 10, 14 e 17 meses de idade, aumentou (P&le;0,05) a concentração média e área total de secreção de LH nas novilhas INT tratadas aos 14 meses, a área total dos picos e área do maior pico de secreção de LH foi maior (P&le;0,05) nas novilhas OVX aos 14 e 17 meses de idade . A administração de um antagonista dopaminérgico (sulpiride, 0,59 mg/kg, sc, amostras 15 min por 10 h) aos 8, 12 e 16 meses de idade diminuiu (P&le;0,05) a secreção de LH (concentração média, área total, área total dos picos e área do maior pico secreção de LH) nas novilhas OVX aos 8 meses de idade. A administração de um estimulador alfa-adrenérgico (clonidina, 10 µg/kg, iv, amostras 15 min por 4 h) aos 8, 12 e 15 meses de idade, diminuiu (P&le;0,05) o número de picos nas novilhas OVX com 8 meses de idade. A administração do 17&beta;-estradiol (2 µg/kg, iv, amostra 15 min por 3 h, 1 h por 7 h e 3 h por 22 h) aos 10, 13 e 17 meses de idade diminuiu a diferença (P&ge;0,05) entre os grupos OVX e INT em relação ao número de picos, área total de picos, área do maior pico e tempo necessário para acontecer o maior pico. Foi avaliada a secreção de LH da desmama à primeira ovulação em novilhas INT e OVX. A concentração de LH aumentou durante a maturação sexual, tanto nas novilhas INT quanto nas OVX. O número de picos de secreção de LH e amplitude máxima de secreção de LH foi maior (P&le;0,05) nas novilhas OVX com o aproximar da primeira ovulação. Os resultados indicam uma diminuição da sensibilidade do hipotálamo aos esteróides gonadais durante o processo de maturação sexual nas novilhas da raça Nelore e a participação alternada de neurotransmissores, inibindo e estimulando a secreção de LH. Concluímos que, em novilhas pré-púberes da raça Nelore o desenvolvimento da retroalimentação positiva aos esteróides gonadais no hipotálamo foi importante para aumentar a secreção de LH antes da primeira ovulação, com a participação de neurotransmissores estimulando ou inibindo a secreção de LH. / The variation on LH secretion after neurotransmitter administration, on the presence or absence of gonadal steroid, was investigated, generating information about the mechanisms evolved on sexual maturation in Nellore heifers. LH concentration was quantified by RIA. As expected ovariectomized heifers higher basal LH concentration (P&le;0,05) than intact heifers. The picrotoxin administration (GABA antagonist, 0,18 mg/kg, iv, samples 15 min for 10 h) at 8, 10, 14 and 17 months of age increased (P&le;0,05) average concentration and total secretion area on intact treated heifers at 14 months and peak total area and area of highest peal on ovariectomized heifers at 14 and 17 months of age. The dopaminergic antagonist (sulpiride, 0.59 mg/kg, sc, sample 15 min for 10 h) administrated at 8, 12 and 16 months of age decreased (P&le;0,05) LH secretion (average levels, total peak area and area of the highest secretion peak) on ovariectomized heifers at 8 months of age. The administration of an alfa-adrenérgic stimulatory (clonidine 10 µg/kg, iv, samples 15 min for 4 h) at 8, 12 and 15 months of age decreased (P&le;0,05) decreased the number of peaks at 8 months of age. 17&beta;-estradiol administration (2 µg/kg, iv, samples every 15 min for 3 h, every 1 h for 7 h and every 3 h for 22 h) decreased the differences between ovariectomized and intact heifers on number of peaks, total peak area, highest peak area and time to highest peak occurrence. The LH secretion from weaning to first ovulation in non treated intact and ovariectomized heifers was also evaluated. LH concentration increase during sexual maturation in both ovariectomized and intact heifers. The number of peaks, and maximum LH secretion amplitude was higher in ovariectomized heifer closest to first ovulation, when compared to intact heifers. The results suggested a decrease on hipothalamus sensitivity to gonadal steroid during the sexual maturation in Nelore heifer associated with neurotransmitter participation either stimulating or inhibiting LH secretion. It was possible to conclude that the decrease of negative feedback associated with the increase on positive feedback of gonadal steroids over hipothalamus was necessary to increase LH secretion before first ovulation, that was associated with neurotransmitter participation on LH secretion.

In an effort to elucidate the mechanism by which indomethacin (IN) attenuates the stimulatory effect of estradiol (E$\sb2$) on rabbit splenic red pulp macrophages (RPM), thirty-nine female New Zealand White rabbits were divided into 10 groups: ovariectomized (OVX), OVX/IN at 0.1 and 5.0 mg/kg body weight (bw)/day; sham OVX (SOVX), SOVX/IN at 0.1 and 5.0 mg/kg bw/day; OVX/25 mg E2, OVX/25 mg E$\sb2$/IN at 0.1 and 5.0 mg/kg bw/day; intact Control. Quantitative changes in RPM population in response to the treatments were measured using a 0 to 4 histologic grading scale. Estradiol treatment resulted in increased RPM grade when compared to the OVX non-E$\sb2$ groups. Indomethacin addition decreased mean RPM grade in the SOVX/IN 5.0 group when compared to its E$\sb2$ control group. Indomethacin administration had no significant effect on levels of PGE$\sb2$ in the spleen, blood or urine (p $>$.05). Hematocrits were reduced in both OVX and OVX/E$\sb2$ groups and this decrease was exacerbated by the high IN dose. The results from this study suggest that the effect of IN on E$\sb2$-induced RPM activation may be mediated through a non-prostaglandin pathway. The observed hematocrit changes are possibly the result of direct action of IN and E$\sb2$ on erythrocytes. To further investigate whether a direct interaction of IN and E$\sb2$ with rabbit erythrocytes may be responsible for the decreases in hematocrit observed in vivo, an in vitro study was conducted to determine the effect of these drugs on erythrocyte fragility characteristics. Two ml aliquots of treated New Zealand White rabbit whole blood were assayed as; Control, IN (9.6 $\mu$g/ml), E$\sb2$ (500 pg/ml) and IN plus E$\sb2$, for changes in erythrocyte fragility. Osmotic (OF) and mechanical (MF) fragility were evaluated under approximate physiological conditions by measurement of hemoglobin release at 545 nm. Blood samples at 39.5$\sp\circ$C were assayed immediately after drug addition (initial) and again 4 hours after incubation (final). Eight replicates of each experiment were run. Results of the OF assays showed a significant increase (p $<$.05) in mean 50% hemolysis point between IN (final) and IN plus E$\sb2$ (final) when compared to their mean initial values and to the mean final Control value. The OF hemolysis dispersion was increased by IN and IN plus E$\sb2$ treatment when final values were compared to initial values. The mean final values for MF increased with IN, E$\sb2$ and IN plus E$\sb2$ treatment versus the mean final Control value (p $<$.05). While the increase in MF from IN was greater than that from E$\sb2$, the MF from the combination (IN plus E$\sb2$) was not greater than from IN alone (p $>$.05). The IN-induced increases in both OF and MF indicate a difference in degrees of interaction with the erythrocyte from that of E$\sb2$, which only affected MF and whose effect was not additive or synergistic with that of IN. The in vitro experimental results demonstrate that the increased fragility produced by IN and E$\sb2$ on rabbit erythrocytes may account for the observed in vivo reduction in hematocrit. Increased erythrocyte fragility would also lead to their accelerated clearance from the circulation by splenic RPM and subsequent increases in activity of these macrophages. This elevation in splenic RPM population may also be enhanced by direct E$\sb2$ stimulation of macrophage proliferation.

Ketoconazole is a broad-spectrum antifungal with oral activity and is documented to block steroid biosynthesis in fungi and mammals. Estradiol-primed ovariectomized rats lose the estradiol-induced, cornified layer of the vaginal epithelium as a result of ketoconazole treatment. This effect is not explained by current knowledge of ketoconazole's mechanism of action. Ketoconazole's effect on estradiol's binding to either receptors in target tissues (target-cell receptors) or serum-estradiol-carrying proteins in the aforementioned rats was investigated. The uterus is responsive to estradiol treatment, which causes an increase in uterine size and weight. This tissue is a sensitive means of studying the effects of ketoconazole on target-cell estradiol receptors. Tamoxifen, documented to block the effects of estradiol by binding to target-cell estradiol receptors, was used as a control drug. Uterine weight and horn diameter were measured in estradiol-treated, ovariectomized rats after 7 days of oral, twice-a-day, drug treatment. Ketoconazole had no effect on mean uterine weights and uterine horn diameters at any of the doses tested; however, tamoxifen at a dose of 10 mg/kg caused a significant reduction in uterine weight and horn diameter. Vaginal histopathology showed that ketoconazole and tamoxifen caused decornification. The percentage of unbound estradiol in the serum was measured in estradiol-treated, ovariectomized rats treated with ketoconazole orally, twice-a-day for 3 days to determine the effects of ketoconazole on the serum binding of estradiol. The results of vaginal cytology showed that ketoconazole caused normal decornification. In comparison with control animals, the results of the serum percent-unbound estradiol assays showed that ketoconazole caused a dose-related increase in the percent-unbound estradiol in the blood. The dose required to cause a 5 percent increase in serum unbound estradiol was approximately 6 times greater than the minimum dose required to cause decornification. Ketoconazole's effects on target-cell estradiol receptors and serum estradiol-carrying proteins do not appear to have any relationship to its effects in causing vaginal decornification in this model.

Der brustgewebsspezifische Metabolismus des weiblichen Sexualhormons 17β-Estradiol (E2) spielt eine wichtige Rolle bei der Brustkrebsentstehung. In der Brust wird E2 durch die humanen Cytochrom P450-abhängigen Monooxygenasen (CYP) Isoenzyme 1A1 (hCYP1A1) und 1B1 (hCYP1B1) zu 2-Hydroxy (2-OH) und 4-HO-E2 oxidiert und vorrangig durch die Catechol-O-Methyltransferase (COMT) entgiftet. Bei unzureichender O-Methylierung können diese Catecholestrogene zu elektrophilen Chinonen oxidiert werden, welche mit der DNA reagieren und somit Mutationen induzieren können. Eine niedrige COMT-Aktivität, durch Polymorphismen und/oder durch Nahrungsbestandteile, die mit dem Enzym selbst oder seiner Genexpression wechselwirken, könnte daher die Mutagenität von E2 und dessen Catecholestrogenen beeinflussen. Im Rahmen der vorliegenden Arbeit wurde der Einfluss der Hemmung der COMT-Aktivität auf die Mutagenität von E2 und dessen Catecholestrogenen untersucht. Zu diesem Zweck wurden der Hypoxanthin-Guanin-Phosphoribosyltransferase (HPRT)-Test und der Mikrokern-Test eingesetzt. Die Untersuchungen erfolgten in kultivierten Lungenfibroblasten des Chinesischen Hamsters (V79-Zellen) und in V79-Zellen, die mit hCYP1A1 transfiziert wurden. Begleitend zu den Mutagenitätsuntersuchungen wurde das Metabolitenprofil der Testsubstanzen anhand von Gaschromatographie gekoppelt mit Massenspektrometrie bestimmt. Nach Inkubation mit 0,08 µM 2 HO-E2 wurde dieses vollständig zu dessen Methylcatecholen umgesetzt, ab 2,5 µM hingegen war zusätzlich 2 HO-E2 im Medium nachweisbar. Demnach war die Hemmung der COMT-Aktivität bei der Inkubation mit 0,08 µM 2 HO-E2 vollständig und ab 2,5 µM partiell. Mit und ohne Hemmung der COMT-Aktivität war 2 Methoxyestradiol der Hauptmetabolit. 2-HO-E2 induzierte im Konzentrationsbereich 0,08 µM - 5 µM, mit und ohne Hemmung der COMT-Aktivität, keine Erhöhung der Mutantenfrequenz im hprt-Lokus von V79-Zellen. Im Gegensatz hierzu induzierte 2 HO-E2 ab 2,5 µM, mit und ohne Hemmung der COMT-Aktivität, mindestens eine Verdreifachung der Mikrokernrate im Vergleich zur Kontrollpopulation, wobei dieser Effekt ohne Inhibierung der COMT-Aktivität stärker ausgeprägt war. Über den gesamten getesteten Konzentrationsbereich (5 - 40 µM) wurde 4 HO-E2 zu dessen beiden Methylcatecholen umgesetzt, wobei 4-Methoxyestradiol den größten Anteil der detektierten Verbindungen (≥ 86%) ausmachte. Nach der Behandlung mit 3,5-Dinitrocatechol waren keine Methylierungsprodukte mehr nachweisbar, weswegen von einer vollständigen Hemmung der COMT-Aktivität im gesamten getesteten Konzentrationsbereich auszugehen war. 4-HO-E2 induzierte über den gesamten getesteten Konzentrationsbereich keine Genmutationen im hprt-Lokus. Erst nach Hemmung der COMT-Aktivität und Behandlung mit 20 µM 4 HO-E2 wurde eine Verdreifachung der Mutantenfrequenz im Vergleich zur Kontrollpopulation beobachtet. Mit und ohne Hemmung der COMT-Aktivität induzierte 4 HO E2 ab 20 µM eine Verdopplung der Mikrokernrate im Vergleich zur Kontrollpopulation. Im Kulturmedium der V79 hCYP1A1-Zellen, die mit 0,1 und 1 µM E2 für bis zu drei Wochen behandelt wurden, machten über die gesamte Versuchsdauer E2 (> 86%) und Estron (> 10%, bezogen auf die Summe aller Peakflächen) den größten Anteil der detektierten Verbindungen aus. Wie erwartet, waren nach Hemmung der COMT-Aktivität keine Methylierungsprodukte mehr nachweisbar. Die durchgehende, zwei- und dreiwöchige Behandlung mit jeweils 0,1 und 1 µM E2 bewirkte keine Induktion von Genmutationen im hprt-Lokus. Demgegenüber erhöhte sich die Mutantenfrequenz nach Hemmung der COMT-Aktivität und dreiwöchiger Behandlung mit 0,1 µM E2 um Faktor 4 im Vergleich zur Kontrollpopulation. Was die Mikrokerninduktion betrifft, so wurde nach 24-stündiger Inkubation mit 0,1 und 1 µM E2, mit und ohne Hemmung der COMT-Aktivität, keine Erhöhung der Mikrokernrate im Vergleich zur Kontrollpopulation beobachtet. Über die gesamte Dauer der Mutagenitätstests von E2 und dessen Catecholestrogenen unterschieden sich die Zellzyklusverteilung, die Wachstumskurven und die Koloniebildungsfähigkeit zum Zeitpunkt der Selektion, mit und ohne Hemmung der COMT-Aktivität, nicht statistisch signifikant von denjenigen der Kontrollpopulationen. Demnach war von einer sicheren Detektion von Mutationen im HPRT-Test und im Mikrokerntest auszugehen. Zusammenfassend bestätigen die durchgeführten Untersuchungen, dass die zelluläre COMT-Aktivität eine essentielle Rolle zur Entgiftung mutagener Catecholestrogene spielt. Eine hundertprozentige Inhibierung der Aktivität dieses Enzyms führt zur Induktion von Genmutationen durch 4 HO-E2 in V79-Zellen ohne CYP-Aktivität und durch E2 in V79-Zellen, die hCYP1A1 exprimieren. Demnach könnte eine Reduktion der COMT-Aktivität durch Polymorphismen und/oder durch Nahrungsbestandteile, die mit dem Enzym selbst oder seiner Genexpression wechselwirken, die Induktion von Genmutationen durch E2 und dessen Catecholestrogenen begünstigen. / Oxidative metabolism of the female sex hormone 17β-estradiol (E2) is considered to play a major role in the initiation of hormone-induced carcinogenesis. In extrahepatic tissues, E2 undergoes metabolic activation by human cytochrome P450-dependent monooxygenase (CYP) isozymes 1A1 (hCYP1A1) and 1B1 (hCYP1B1) to 2-hydroxy (HO)- and 4-HO-E2. If not conjugated these catecholestrogens can further oxidize to electrophilic quinones, which may react with DNA and induce thereby mutations. Conjugation of these catechols in extrahepatic tissues is mainly catalyzed by catechol-O-methyltransferase (COMT). A low COMT activity, caused for example by polymorphisms and certain food components, which influence the enzyme activity or its gene expression, may therefore enhance quinone formation and thereby the induction of mutations. The aim of the present work was to determine the effect of inhibition of COMT on the mutagenic potential of a) the catechols 2- and 4-HO-E2 in V79 wild type cells without CYP activity and b) E2 in V79 cells expressing hCYP1A1. 3,5-dinitrocatechol (20 μM) served as COMT inhibitor. Gene and chromosomal mutations were assessed using the hypoxanthine-guanine-phosphoribosyltransferase (HPRT) and the micronuclei assay. In addition, the metabolite profile of E2 and its catechols in the culture medium was analyzed via gas chromatography/mass spectrometry. After incubation of V79 cells with 2-HO-E2, 2-methoxyestradiol was the major metabolite, whereas in the presence of the COMT inhibitor, disappearance (0.08 μM) and a decreased amount (2.5 μM or more) of methylated inactivation products was observed. Treatment of V79 cells with 0.08 μM – 5 μM 2-HO-E2, neither with nor without inhibition of COMT activity induced a significant increase in mutant frequency at the hprt locus. In contrast, at concentrations above or equal to 2.5 μM 2-HO-E2, at least a 3-fold increase of the micronuclei rate compared to control cells was observed with and without inhibition of COMT activity. Over the entire concentration range (5-40 μM) 4-HO-E2 was mainly converted to its methylation products, 4-methoxyestradiol being the major metabolite (≥ 86%) of the detected compounds. After treatment with 3,5-dinitrocatechol no methylation products were detected, thus indicating a complete inhibition of COMT over the entire concentration range. 4-HO-E2 did not induce gene mutations at the hprt locus in V79 cells with active COMT. Yet, after inhibition of COMT and treatment with 20 μM 4-HO-E2, a 3-fold increase in the mutant frequency was observed in comparison to control cells. Like 2-HO-E2, induction of micronuclei by 20 µM 4-hydroxyestradiol and more, was not affected by inhibition of COMT. In the culture medium of V79 cells expressing hCYP1A1, which were incubated with 0.1 and 1 μM E2 for up to three weeks, over the entire assay duration, E2 and estrone (86% and 10% of the sum of all peak areas, respectively) were the major metabolites. As expected, no methylation products were detected after inhibition of COMT. Treatment of V79 cells expressing hCYP1A1, for two and three weeks with 0.1 and 1 μM E2 did not induce gene mutations at the hprt locus. In contrast, a 4-fold increase in mutant frequency was observed in comparison to control cells after inhibition of COMT and treatment with 0.1 μM E2 for three weeks. With and without inhibition of COMT, no increase in micronuclei rate compared to control cells was observed after incubation with 0.1 and 1 μM E2 for 24 hours. Over the entire duration of the mutagenicity assays of E2 and its catechols, cell cycle distribution, cell growth and plating efficiency at the time of mutant selection, with and without inhibition of COMT, did not differ statistically significant from control cells; therefore a reliable detection of mutations in the micronuclei and the HPRT assay can be assumed. The present work confirms that cellular COMT is essential for the inactivation of mutagenic catechols. Complete inhibition of its enzyme activity results in the induction of gene mutations by 4-HO-E2 in V79 wildtype cells without CYP activity and also by E2 in cells expressing hCYP1A1. Polymorphisms and food components lowering COMT activity could therefore mediate the potential of E2 and its metabolites to induce gene mutations.

The project involved the study of 12 Victorian municipal wastewater treatment plant discharges. These included lagoon-based plants and those with activated sludge based processes. Permission was obtained from all the relevant water authorities to collect samples of final effluent at point of discharge to the environment, whether that was to a creek, a river, the ocean, or the land. Samples were collected in November 2003, and then again in April and June 2004, and subjected to a number of biological and chemical analyses, including toxicity tests, measurement of hormonal (estrogenic) activity using yeast-based bioassays, and the measurement of specific hormonal concentrations (17-estradiol) using enzyme-linked immunosorbent assays (ELISA). Almost all of the effluents examined showed estrogenic activity, to a greater or lesser extent (no response to 55 ng/L 17β-estradiol equivalents). On the whole, the levels of estrogenic activity observed were to the lower end of the range observed overseas in the northern hemisphere, and comparable with that recently reported in Australia and New Zealand using similar, human-estrogen receptor based assays (no response to ~ 10 ng/L 17β-estradiol equivalents). The reassuring low/no assay response is bolstered by the chemical assessment of estradiol concentrations by ELISA, which returned concentrations of these compounds for the most part in the range 2-5 ng/L. From an aquatic environmental perspective, it is difficult to say with any certainty what the potential risk to aquatic organisms in waters receiving these effluents will be. Typically, in environmental risk assessment one first looks to agreed national or international guideline or trigger values for the type of waters being assessed. In this case, there are as yet no guideline values. Without guideline values to drive the assessment, then one compares a chemical’s concentration in a sample (in this case a WWTP effluent) with data obtained from toxicological experiments in which the concentration known to elicit a specific effect has been determined. In this case, levels of 17β-estradiol were typically between the lowest reported level to induce the production of Female-indicative proteins in male fish (plasma vitellogen; 1 ng/L), and the lowest concentration of known to induce intersex in fish (8 ng/L). Consequently, such levels in a WWTP discharge are likely to be an environmental risk if there is little or no dilution of the discharge by the receiving water, i.e. discharge represents major component of stream flow. In short, to truly assess the risk (hormonal impact) of these WWTP effluents, in vivo testing needs to be undertaken, ideally with a representative native species but failing that with a ‘standard’ species such as the fathead minnow. When this programme began, the ‘watching brief’, being held in Australia on the topic of endocrine disrupting chemicals and their potential effects on aquatic wildlife was considered too passive by many. It still is, by some. Despite the assurance the results may provide (of minimal impact in most cases if there is significant dilution), there is still a need for further extensive on-ground, reassurance research to provide data for higher-level risk assessment by industry and government agencies.

Sex steroid exposure constitutes a risk factor for breast cancer, but little is known about the effects of sex steroids on factors mediating angiogenesis, the development of new blood vessels, in normal and malignant breast tissue. In this thesis we have investigated the effects of estradiol, progesterone, and the nonsteroidal anti-estrogen tamoxifen on vascular endothelial growth factor (VEGF) and its receptors (VEGFR-1 and VEGFR-2) in normal human breast tissue, endothelial cells, and breast cancer. We have applied the technique of microdialysis to provide in situ sampling of estradiol and VEGF in tumors and normal breast tissue of breast cancer patients in vivo. Furthermore, we present a novel method of culturing normal human breast tissue ex vivo. Our results suggest a pro-angiogenic effect of estradiol and an anti-angiogenic effect of tamoxifen in the breast. Estradiol increased extracellular levels of VEGF in normal human breast tissue and breast cancer cells in vitro. In addition, estradiol decreased sVEGFR-1 in breast cancer cells and indirectly increased VEGFR-2 in endothelial cells. Compared to estradiol treatment alone, estradiol + tamoxifen increased sVEGFR-1 and decreased VEGF in breast cancer cells in vitro. Furthermore, estradiol + tamoxifen decreased tumor VEGF levels and tumor vasculature in human breast cancer xenografts in vivo. In breast cancer patients, a significant correlation was found between in vivo levels of estradiol and VEGF sampled by microdialysis in normal human breast tissue, suggesting that estradiol may be a potent regulator of VEGF in the breast in vivo. Tumor levels of VEGF were significantly higher than in normal breast tissue in vivo, supporting the role of VEGF in tumor angiogenesis. For studies of normal human breast, whole breast tissue may be cultured in vitro for up to one week with preserved morphology. Using this method, estradiol, and not progesterone, appears to be the main sex steroid regulator of extracellular VEGF in normal breast tissue. In conclusion, the data suggest that sex steroids and tamoxifen exert pro- and anti-angiogenic effects in normal breast tissue and breast cancer.

The aim of this thesis was to determine any association between plasma oxytocin and vasopressin concentrations and renal water and sodium excretion during normal pregnancy. In addition to investigate changes in concentrations of estradiol, progesterone, oxytocin, cortisol, and glucose in the blood before and in the nearest hours after delivery and if treatment with oxytocin affected these concentrations and the fluid balance during the different stages of labour. Oxytocin, vasopressin, estradiol, progesterone, and cortisol were analysed in blood plasma or serum by radioimmunoassay or ELISA: serum glucose, and osmolality, and sodium in plasma and urine were  analysed by standard laboratory techniques. Fifty-seven women were studied during pregnancy and fifty-one during parturition and post partum. The low plasma vasopressin and increasing plasma oxytocin concentrations with unchanged water and sodium excretion indicate that oxytocin assists vasopressin in concentrating urine during pregnancy. Plasma vasopressin concentration continued to be low during parturition and post partum. Urine flow and concentration was unrelated to changes in plasma sodium concentration, indicating regulation of fluid balance during parturition was different to the non-gravid state. Women with weak myometrial contractions during parturition (slow progress of labour) reacted differently than women with normal parturition and a group of women with fast progress of labour. The group with slow labour had lower serum estradiol concentration in the latency phase and became hyponatremic. Pulsatile and continuous oxytocin infusions were both effective in the treatment of slow progress of labour. A lower amount of oxytocin was needed to affect delivery when given as pulsatile infusion. Serum cortisol and glucose concentrations were high during labour and cortisol level remained elevated after delivery and glucose concentration reached the highest levels (12 mmol/L) at the same time. Insulin resistance together with the long time of elevated cortisol concentration partly explained the high glucose concentration. In conclusion, fluid balance is not regulated according to the usual sensitive osmotic and volumetric influence on vasopressin release from the neurohypophysis during pregnancy and parturition. Parturition involves a change from one demanding condition, pregnancy, to another, lactation. Parturition and the hours directly after delivery are a turbulent period involving considerable stress.

Several studies suggest profound effects on mood and cognition by neuroactive steroids. Estrogen alone or in combination with antidepressant drugs affecting the serotonin system has been used to treat mood disorders. On the other hand, progesterone is related to negative effects on mood and memory. A major part of the progesterone effects on the brain can be mediated by its metabolite allopregnanolone, which is also de novo synthesized in the brain, and affects the GABAA receptors. It would be of great importance to find a substance that antagonize allopregnanolone adverse effects. To investigate how long term supplementation of estradiol and progesterone, resembling postmenopausal hormone replacement therapy, affects serotonin receptors in different brain areas important for mood and memory functions, we used ovariectomized female rats. After 2 weeks of supplementation with 17β-estradiol alone or in combination with progesterone, or placebo pellets, estradiol alone decreases but estradiol supplemented together with progesterone increases 5HT1A mRNA expression in the hippocampus. Estradiol decreases the 5HT2C receptor gene expression, while estradiol in combination with progesterone increases the 5HT2A mRNA expression in the ventral hippocampus. Thus, estradiol alone has opposite effects compared to the estradiol/progesterone combination. To detect if acute tolerance develops to allopregnanolone, an EEG method was used where male rats by continuous allopregnanolone infusion were kept on anesthesia level of the silent second (SS). After different time intervals (first SS, 30 min or 90 min of anesthesia) several GABAA receptor subunit mRNAs were measured for detecting if changed expression of any GABAA receptor subunits is involved in development of acute tolerance. There is development of acute tolerance to allopregnanolone and brain regions of importance are hippocampus, thalamus and hypothalamus. The GABAA receptor alpha4 subunit in thalamus and alpha2 subunit in the dorsal hippocampus are related to development of acute tolerance. For assessing allopregnanolone behavioral effects, we studied how this neurosteroid affects spatial learning in the Morris water maze task Allopregnanolone inhibits spatial learning short after the injection and shows a specific behavioral pattern with swimming close to the pool wall. The steroid UC1011 can inhibit the increase in chloride ion uptake induced by allopregnanolone. UC1011 decreases allopregnanoloneinduced impairment of spatial learning in the water maze, as well as the specific behavioral swim pattern. In conclusion, the present work demonstrates that neuroactive steroids affect the 5HT and GABA systems in a brain region specific way. GABAA receptor subunit changes in hippocampus and thalamus are related to acute allopregnanolone tolerance. Allopregnanolone induces cognitive deficits, like spatial learning impairment and UC1011 can inhibit allopregnanolone-induced effects in vitro and in vivo. Key words: Estradiol, progesterone, HRT, allopregnanolone, UC1011, serotonin receptor, GABAA receptor, mRNA, Morris water maze, silent second, tolerance.