Chan, Ming Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 92-106). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / 摘要 --- p.iv / LIST OF ABBREVIATIONS --- p.vi / TABLE OF CONTENTS --- p.viii / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Aromatase --- p.1 / Chapter 1.2 --- Tissue Specific Promoter for Aromatase Expression --- p.4 / Chapter 1.3 --- Signaling Pathway --- p.7 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.9 / Chapter 2.1 --- Chemicals And Materials --- p.9 / Chapter 2.2 --- Mammalian Cell Culture --- p.9 / Chapter 2.2.1 --- Maintenance of Cells --- p.10 / Chapter 2.2.2 --- Preparation of Cells Stock --- p.10 / Chapter 2.2.3 --- Cell Recovery from Liquid Nitrogen Stock --- p.11 / Chapter 2.3 --- Tritiated Water Release Assay --- p.11 / Chapter 2.3.1 --- Aromatase Activity in Intact Cell --- p.11 / Chapter 2.3.2 --- Aromatase Assay on Recombinant Supersomes --- p.12 / Chapter 2.4 --- RNA Isolation and cDNA Synthesis --- p.13 / Chapter 2.5 --- Semi-Quantitative PCR Reaction --- p.13 / Chapter 2.6 --- Quantitative Real Time PCR Using Taqman Probe --- p.15 / Chapter 2.7 --- Western Blotting --- p.17 / Chapter 2.8 --- Measurement of Promoter Activity --- p.18 / Chapter 2.8.1 --- Plasmid Preparation --- p.18 / Chapter 2.8.2 --- Transient Transfection and Dual Luciferase Assay --- p.18 / Chapter 2.9 --- Statistical Methods --- p.19 / Chapter CHAPTER 3 --- Genistein up-regulate aromatase in Estrogen receptor alpha-transfected HepG2 cells --- p.21 / Chapter 3.1 --- Introduction --- p.21 / Chapter 3.1.1 --- Cardiovascular Disease (CVD) --- p.21 / Chapter 3.1.2 --- Phytoestrogen --- p.21 / Chapter 3.1.3 --- Estrogen Receptor --- p.24 / Chapter 3.1.4 --- Protective Mechanism Against CVD Protection --- p.25 / Chapter 3.1.5 --- Effects of genistein on LDL Receptor and Apolipoprotein A-I --- p.26 / Chapter 3.1.6 --- Effects of estradiol on LDL Receptor and Apolipoprotein A-I　 --- p.26 / Chapter 3.1.7 --- Aim of study and hypothesis --- p.27 / Chapter 3.2 --- Result --- p.29 / Chapter 3.2.1 --- ERa increased Aromatase Activity in HepG2 cells --- p.29 / Chapter 3.2.2 --- Genistein increased Aromatase Activity in HepG2 cells --- p.29 / Chapter 3.2.3 --- Differential Effect of MAP kinase Inhibitors --- p.35 / Chapter 3.2.4 --- "Role of MAP Kinase, PKA and PKC in Genistein Induced Aromatase Activity in ERa-transfected HepG2 cells" --- p.35 / Chapter 3.2.5 --- Genistein Increased Aromatase Protein Expression in ERa-transfected HepG2 cells --- p.38 / Chapter 3.2.6 --- Genistein Induced Aromatase mRNA Expression Attributed to Induction of Exon ̐ơ.1 Expression --- p.40 / Chapter 3.2.7 --- Genistein Induced Promoter 1.1 Transcriptional Activity in ERa- transfected HepG2 cells --- p.44 / Chapter 3.2.8 --- Genistein Increased ERE and AP-1 Reporter Activity Through Interaction with ERa --- p.47 / Chapter 3.3 --- Discussion --- p.51 / Chapter CHAPTER 4 --- "Effect of 2,3,7,8-tetrachlorodibenzo- para-TCDD (TCDD) on aromatase in MCF-7 cells" --- p.54 / Chapter 4.1 --- Introduction --- p.54 / Chapter 4.1.1 --- Breast Cancer --- p.54 / Chapter 4.1.2 --- TCDD --- p.54 / Chapter 4.1.3 --- CYP Enzymes --- p.55 / Chapter 4.1.4 --- TCDD and Breast Cancer --- p.56 / Chapter 4.1.5 --- Aim of Study --- p.56 / Chapter 4.2 --- Result --- p.57 / Chapter 4.2.1 --- Effect of TCDD on Aromatase Activity in Different Cell Lines --- p.57 / Chapter 4.2.2 --- TCDD Increased Aromatase Activity in MCF-7 Cells --- p.62 / Chapter 4.2.3 --- Effect of TCDD on Human CYP 19 Recombinant Supersomes® and MCF-7aro Cells --- p.66 / Chapter 4.2.4 --- TCDD Increased Aromatase Protein Expression in MCF-7 Cells --- p.66 / Chapter 4.2.5 --- Effect of TCDD in Aromatase mRNA Expression in MCF-7 Cells --- p.70 / Chapter 4.2.6 --- Effect of TCDD in CYP 19 Promoter and AP-1 Promoter Activity in MCF-7 Cells --- p.70 / Chapter 4.2.7 --- Effect of TCDD in CYP 19 mRNA Half-life --- p.75 / Chapter 4.2.8 --- "Role of MAP Kinase, PKA and PKC in Genistein Induced Aromatase Activity in MCF-7 Cells" --- p.78 / Chapter 4.2.9 --- TCDD induced ERK1/2 Activation --- p.78 / Chapter 4.2.10 --- Induction of aromatase activity in MCF-7erk cells --- p.78 / Chapter 4.3 --- Discussion --- p.87 / Chapter CHAPTER 5 --- Summary --- p.90 / BIBLIOGRAPHY --- p.92

Na avicultura industrial, diferenças no desempenho zootecnico de machos e femeas de linhagens pesadas forcam o setor a alojar separadamente os sexos com o propósito de facilitar manejo, uniformizar lotes, reduzir custos e otimizar a producao. Desse modo, meios que reduzam ou eliminem a disparidade entre sexos no ambito zootecnico podem resultar em ganhos gerenciais e econômicos no mercado avicola. A masculinizacao de femeas objetivando aproximar sua performance aquela manifestada por machos pode ser considerado um recurso interessante. Sendo assim a aplicacao de inibidores ou bloqueadores da aromatase P450 durante o desenvolvimento embrionario pode induzir graus variados de masculinizacao das femeas sem a utilizacao de protocolos hormonais, em um fenomeno chamado reversao sexual. Assim, os objetivos desse projeto foram: (1) comparar a acao de inibidores de aromatase de terceira geracao (Droga B e droga C) em relacao ao fadrozole (farmaco amplamente citado em literatura) sobre a taxa e o grau de reversao sexual em frangas de corte; e (2) estimar o impacto desses tratamentos sobre parametros de incubacao (mortalidade embrionaria e eclodibilidade) e indices zootecnicos (peso ao nascer, ganho de peso, conversao alimentar e peso final aos 42 dias). Os resultados, deste estudo, mostraram que o inibidor de aromatase droga B obteve maior proporção machos quando comparado aos outros IAs testados. Contudo, quando avaliamos as aves tratadas por este fármaco, em relação aos índices zootécnicos, estas apresentaram resultados similares ao grupo misto e inferior quando comparadas aos machos genéticos. Na sexagem morfológica aos 42 dias de idade foi observado que no grupo tratado com a droga B, 58% das aves apresentavam ovário ou ovostestis ao invés de testículos o que impactou negativamente no ganho de peso deste grupo. / Performance diferences between male and female broilers compel the poultry industry to rear sexes separately in order to favor management, uniform flocks, reduce costs and optimize production. Notwithstanding, this practice has logistical implications that create additional expenses, and in some cases, broiler companies encounter producer preferences for a particular sex due to its productive traits. Thus, methods that reduce or eliminate gender disparity in meat production efficiency can result in operational and economical benefits to poultry market. Masculinization of females aiming to bring performance closer to those expressed by males may be viewed as an interesting alternative. Therefore, the application of aromatase inhibitors or blockers during embryonic development can induce varying degrees of female masculinization without the use of hormonal protocols, in a process called sex reversal. Therefore, our objectives herein were: (1) to compare the action of third generation aromatase inhibitors (Drug B and Drug D) in relation to fadrozole (a drug widely used in literature) on the rate and degree of sexual reversal in broiler pullets; and (2) to estimate the impact of these treatments on incubation (embryonic mortality and hatchability) and performance parameters (birth weight, weight gain, feed conversion and final weight at 42 days). The results, from this study, showed that the aromatase inhibitor drug B obtained a higher rate of sexual reversion when compared to the other AIs tested. However, when we evaluated the birds treated by this drug, in relation to the zootechnical indexes, these presented similar results to the mixed and inferior group when compared to the genetic males. In the morphological sexing at 42 days of age, it was observed that in the group treated with AI, 58% of the birds presented ovary or ovostestis instead of testicles, which impacted the weight gain of this group.

Indiana University-Purdue University Indianapolis (IUPUI) / Aromatase is the cytochrome P450 enzyme that converts androgens to estrogens. Aromatase is the target of the aromatase inhibitor class of drugs widely used to treat estrogen-mediated conditions including breast cancer. Little is known about the role of this enzyme in drug metabolism or in drug interactions. Since this lack of knowledge has been an impediment to optimal therapy, it is important to understand these roles of aromatase. Therefore, a comprehensive series of studies was carried out to characterize its ability to metabolize drugs and its susceptibility to inhibition by xenobiotics. The overall objective of this work was to better understand the interactions of small molecules with aromatase and to use this new knowledge to predict aromatase-mediated drug interactions and anticipate novel molecular structures that interact with the enzyme. Aromatase was shown to be a drug metabolizing enzyme able to metabolize methadone both in vitro (Km of 314 μM) and in vivo (22% of methadone clearance). A number of novel aromatase inhibitors that employ diverse kinetic mechanisms were identified. These include a potent competitive inhibitor: norendoxifen (Ki of 35 nM), two non-competitive inhibitors: endoxifen (Ki of 4.0 μM) and N-desmethyl-tamoxifen (Ki of 15.9 μM), a mechanism-based inhibitor: methadone (KI of 40.6 ± 2.8 μM; kinact of 0.061 ± 0.001 min-1), and a stereoselective inhibitor: naringenin (IC50s of 2.8 μM for (R)-enatiomer and 1.4 μM for (S)-enatiomer). Through investigation of the structure-potency relationships so discovered, a series of new biochemical structures to be exploited as aromatase inhibitors were identified. These studies have identified new roles for aromatase as a catalyst for methadone metabolism and as a mediator of the effects of tamoxifen by demonstrating that a number of its metabolites can act as aromatase inhibitors. This work also provides a new mechanistic framework for the design of novel aromatase inhibitors that can be used in breast cancer. Overall, the data suggest ways to more consistently treat breast cancer with current medications, to better anticipate drug interactions, and therefore to improve the quality of life of patients in ways that minimize side effects, while optimizing therapeutic benefits, in each person treated.

Foram estudadas 20 pacientes na menacme portadoras de leiomioma uterino, sintomáticas, que utilizaram anastrozol na dose 1 mg/dia por três meses consecutivos. Durante o tratamento acompanhou-se o volume do conjunto úteroleiomiomas com ultra-sonografia, no momento inicial e após um mês e três meses do início do uso da medicação. Além disso, foi observada a evolução da sintomatologia relacionada ao leiomioma (hipermenorréia, dismenorréia e metrorragia), presença de sintomas ligados ao hipoestrogenismo e dosagens séricas de FSH, estradiol, colesterol total e frações, triglicérides e glicose. Obtivemos redução média do volume do conjunto útero-leiomiomas de 9,32% até 31%. Houve diminuição significante dos sintomas relacionados ao leiomioma do útero. Não ocorreram sintomas ligados ao hipoestrogenismo. Não se comprovou modificação significante dos demais parâmetros estudados. Concluiu-se que o inibidor da aromatase, o anastrozol, na dosagem utilizada, foi efetivo na redução volumétrica do conjunto útero-leiomiomas, além de proporcionar significativa melhora dos sintomas relacionados a essa doença, sem alterar, no entanto, os valores de FSH, estradiol, colesterol total e frações, triglicérides e glicose. / We studied 20 patients in menacme with leiomyoma in the uterus, referring symptoms, using anastrozol 1 mg per day for three months. During this treatment we monitored the uterus-leiomyoma volume using ultrasound, at the beginning, one month after and three months after the beginning of the use of the medication. We also observed the leiomyoma symptoms evolution, presence of usual symptoms of hipoestrogenism and seric values of FSH, estradiol, cholesterol and its fractions triglycerides and glicemia. We obtained reduction in uterus-leiomyoma volume of 9.32% in average, in maximum 31%. There was significative reduction in symptoms related with leiomyoma. There were not symptoms of hipoestrogenism. We not observed significative modifications in the other studied parameters. This study concluded that the aromatase inhibitor, anastrozol, in this dosage, is effective in uterus-leiomyomas reduction, cause significative reduction of the symptoms related with this illness, without change the seric dosage of FSH, estradiol, total cholesterol and fractions, triglycerides and glicemia.

published_or_final_version / Public Health / Master / Master of Public Health

Five studies were designed to characterize the effects of a non-steroidal aromatase inhibitor, letrozole, on ovarian function in cattle. The general hypothesis was that non-steroidal aromatase inhibitors have potential as a steroid-free option for the control of ovarian function for the purposes of fixed-time artificial insemination and embryo production. The specific objectives were to determine the effect of route and vehicle, type of aromatase inhibitor, and duration of aromatase inhibitor treatment (short vs prolonged) on ovarian follicles in cattle, and to test the efficacy of an aromatase inhibitor-based protocol to synchronize ovulation in cattle. In the first experiment, heifers were treated with letrozole intravenously (n=10) or intramuscularly (n=10) or allocated in iv and im control groups (n=5/group). During the second experiment, heifers were divided randomly into two groups (n=15/group) and an intravaginal device containing 1 g of letrozole or a blank device (control) was inserted. The third experiment was designed with the goal of formulating and testing an intravaginal device that provides biologically active circulating concentrations of an aromatase inhibitor for a minimum of 4 days. The biological significance of the pharmacokinetic differences between the letrozole intravaginal devices resulting from the third study was evaluated during the fourth study. A final study was designed to determine the effect of stage of the estrous cycle on the proportion of animals that ovulated and the synchrony of ovulation of heifers treated with an aromatase inhibitor-based ovulation-synchronization protocol and to determine subsequent pregnancy outcomes. In all the studies, the effects of aromatase inhibitor on ovarian function were assessed by transrectal ultrasound examination of the ovaries, and blood samples were collected for hormone concentration determination. Results demonstrated that route of administration, or more precisely, the nature of iii the vehicle used for the administration of letrozole (intravenous, intramuscular depot, short release intravaginal or prolonged release intravaginal) has an impact on the effects of letrozole on hormonal profiles and ovarian dynamics. The intramuscular route appeared to provide a prolonged release of letrozole from the injection site which had a marked effect on estradiol production, dominant follicle lifespan, and CL form and function. Letrozole treatment during the ovulatory follicle wave by means of a gel-based intravaginal releasing device during the second study resulted in more rapidly growing dominant follicles and larger ovulatory follicles, delayed ovulation (by 24 h) of a single follicle and formation of a CL that secreted higher levels of progesterone. A wax-based vehicle allowed for a steady and continuous delivery of the active compound over the treatment period. During the third study, the addition of a letrozole-containing gel coating increased the rate of initial absorption and hastened the increase on plasma concentrations of the active ingredient, while the letrozole-containing wax-based vehicle prolonged drug-delivery from the intravaginal device. When tested in vivo during the fourth study, we confirmed that letrozole-impregnated intravaginal devices formulated with a wax base plus a gel coat vehicle was most suitable for the application of a letrozole-based protocol for the synchronization of ovulation in cattle, since it effectively delivered elevated concentrations of letrozole, and reduced estradiol production resulting in increased follicular growth and lifespan, without adversely affecting progesterone production. The application of a letrozole-impregnated intravaginal device for 4 days, combined with PGF treatment at device removal and GnRH 24 h post-device removal increased the percentage of ovulations and synchrony of ovulation in cattle, regardless the stage of the estrous cycle at initiation of treatment. As observed in previous studies, the effects observed could be associated with an increase in circulating LH iv concentrations. However, the effects of treatment on gonadotropin concentrations are inconclusive, possibly due to inadequate sampling frequency. The impact of letrozole treatment of oocyte fertility remains unknown. The results of the five experiments support our general hypothesis that non-steroidal aromatase inhibitors have potential as a steroid-free option for the control of ovarian function in cattle. However, further research is needed in order to elucidate the effects of letrozole treatment during the proestrous on oocyte competence and fertility of the resulting ovulations in cattle.

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Mar 3

Thesis (M.S.Ed.)--Baylor University, 2005. / Includes bibliographical references (p. 64-69).

Introdução: Miomas são tumores benignos extremamente comuns em mulheres em idade reprodutiva. Constituem um importante problema de saúde pública, uma vez que atualmente o tratamento cirúrgico é o mais eficaz. Os estrógenos parecem ter importante participação no desenvolvimento e crescimento dos miomas. Dentre as hipóteses estudadas para explicar esta influência, destacamse o aumento da concentração de receptores de estrogênio no tecido miomatoso e a habilidade deste de converter androgênios em estrogênio in situ, via aumento da expressão da aromatase. Métodos: Foi realizado um estudo caso-controle. Foram obtidas amostras de miomas e miométrios normais de 14 pacientes em idade reprodutiva que se submeteram a histerectomia por miomatose. Resultados: Foram avaliadas amostras de leiomioma e miométrio normal de 12 pacientes. Não foi encontrada diferença estatisticamente significativa entre a expressão protéica dos receptores REα, REβ e da aromatase (P= 0,239, P = 0,695 e P = 0,203, respectivamente). Conclusão: Não foi encontrada diferença estatisticamente significativa na expressão protéica dos receptores de estrógeno alfa e beta e na expressão protéica da aromatase. Os estudos realizados até o momento em relação a este assunto são controversos e o assunto está longe de ser esclarecido. / Introduction: Leiomyomas are common benign tumors of the female reproductive tract. They are a major public health problem since nowadays surgical treatment is the most effective. Evidence suggests that estrogens regulate cell proliferation and myoma growth. This estrogen-mediated effect might be due to different amounts of estrogen receptors (ERα and ERß) in normal and myoma tissues and overexpression of aromatase P450 in myomas, which is responsible for conversion of androgens to estrogens in situ. Methods: Case-control study. Samples were collected from 14 premenopausal women admitted for abdominal hysterectomy due to fibroids. Results: We analyzed samples from myoma and normal myometrium from 12 patients. The protein expression of ERα, ERβ and aromatase had no statistical difference between leiomyoma and normal myometrium (P = 0.239, P = 0.695 and P = 0.203, respectively). Conclusions: The protein expression of ERα, ERβ and aromatase was similar in leiomyoma and normal myometrium. Studies accomplished so far reveal conflicting data and this subject still needs to be elucidated.

Introdução: Miomas são tumores benignos extremamente comuns em mulheres em idade reprodutiva. Constituem um importante problema de saúde pública, uma vez que atualmente o tratamento cirúrgico é o mais eficaz. Os estrógenos parecem ter importante participação no desenvolvimento e crescimento dos miomas. Dentre as hipóteses estudadas para explicar esta influência, destacamse o aumento da concentração de receptores de estrogênio no tecido miomatoso e a habilidade deste de converter androgênios em estrogênio in situ, via aumento da expressão da aromatase. Métodos: Foi realizado um estudo caso-controle. Foram obtidas amostras de miomas e miométrios normais de 14 pacientes em idade reprodutiva que se submeteram a histerectomia por miomatose. Resultados: Foram avaliadas amostras de leiomioma e miométrio normal de 12 pacientes. Não foi encontrada diferença estatisticamente significativa entre a expressão protéica dos receptores REα, REβ e da aromatase (P= 0,239, P = 0,695 e P = 0,203, respectivamente). Conclusão: Não foi encontrada diferença estatisticamente significativa na expressão protéica dos receptores de estrógeno alfa e beta e na expressão protéica da aromatase. Os estudos realizados até o momento em relação a este assunto são controversos e o assunto está longe de ser esclarecido. / Introduction: Leiomyomas are common benign tumors of the female reproductive tract. They are a major public health problem since nowadays surgical treatment is the most effective. Evidence suggests that estrogens regulate cell proliferation and myoma growth. This estrogen-mediated effect might be due to different amounts of estrogen receptors (ERα and ERß) in normal and myoma tissues and overexpression of aromatase P450 in myomas, which is responsible for conversion of androgens to estrogens in situ. Methods: Case-control study. Samples were collected from 14 premenopausal women admitted for abdominal hysterectomy due to fibroids. Results: We analyzed samples from myoma and normal myometrium from 12 patients. The protein expression of ERα, ERβ and aromatase had no statistical difference between leiomyoma and normal myometrium (P = 0.239, P = 0.695 and P = 0.203, respectively). Conclusions: The protein expression of ERα, ERβ and aromatase was similar in leiomyoma and normal myometrium. Studies accomplished so far reveal conflicting data and this subject still needs to be elucidated.