Thesis--Wisconsin. / Vita. Includes bibliographical references (leaves 103-109).

Abstract 17β-Hydroxysteroid dehydrogenases (17HSDs) catalyze the reactions between 17-hydroxy and 17-keto steroids. In the present study, the enzyme activities and tissue distribution of 17HSD type 1, type 2 and type 4 were characterized. Furthermore, the role of 17HSD type 1 in estrogen-dependent growth was studied in MCF-7 breast cancer cells which were stably transfected with type 1 cDNA. Endogenous oxidative 17HSD activity found in COS-m6 monkey kidney cells was first compared with that of human placental 17HSD. Cultured COS-m6 cells exclusively possessed oxidative 17HSD activity, converting estradiol (E2) to less active estrone (E1). When placental 17HSD was transfected into these cells, highly reductive activity appeared. The 17HSD enzyme in COS-m6 cells also catalyzed the conversion of testosterone to androstenedione, whereas the placental enzyme was estrogen-specific. These results further proved the existence of different 17HSD isoenzymes. The enzymatic properties and cell- and tissue-specific expression of 17HSD type 1, type 2 and oxidative type 4 were further characterized. The data confirmed that in cultured cells the direction of 17HSD activity is determined by the expression of different isoenzymes and not by the intracellular environment. In addition, the 17HSD type 1 gene expresses two mRNA signals, 1.3 kb and 2.3 kb in size. The expression of 1.3 kb mRNA, but not 2.3 kb mRNA was related to enzyme concentration in all the cell types studied. The type 1 enzyme was expressed in the placenta, ovary and in some breast cancer specimens and in the cell lines originated from these tissues. 17HSD type 2 was more widely expressed in both steroidogenic and in target tissues of steroid action. 17HSD type 4 was expressed in almost all cell lines and in all tissues studied, but no correlation with 17HSD activity was detected. These results suggest that 17HSD type 1 is involved in E2 production in females and 17HSD type 2 is responsible for inactivation of sex steroids. However, the oxidation of 17β-hydroxysteroids seems not to be the primary activity of 17HSD type 4. The mRNAs for 17HSD type 1, type 2 and type 4 were found to be expressed in human mammary epithelial cells. In breast tissue samples both 17HSD type 1 and type 2 were detected by in situ hybridization. Despite the presence of 17HSD type 1 mRNA in human mammary epithelial cells, only oxidative 17HSD activity was detected. The reason for the lack of reductive activity is not yet known. Finally, MCF-7 breast cancer cells were stably transfected with 17HSD type 1 cDNA in order to study the effect of 17HSD type 1 on estrogen-dependent growth. In wild type MCF-7 cells, very low 17HSD activity was detected and E1 did not have any effect on cell growth. In the cells expressing 17HSD type 1, E1 was rapidly converted to E2. Hence in these cells E1 had a similar growth-promoting effect as E2 as a result of the action of 17HSD type 1. The presence of 17HSD type 1 in breast cancer cells may thus be an important factor regulating estrogen exposure and the estrogen-responsive growth of breast cancer tissue.

Steroid and eicosanoid metabolism occurs in two phases and primarily within hepatic tissues, but localized metabolism has been examined in several extra-hepatic tissues in humans and rodents. Phase I of metabolism is performed by Cytochrome P450s (CYP) that add hydroxyl groups to the carbon ring structure which is further metabolized by phase II UDP-glucuronosyltransferase (UGT). The overall objectives of the following experiments were to: 1) determine the amount of extra-hepatic steroid metabolism within reproductive tissues of cattle across the estrous cycle; 2) determine the amount of extra-hepatic steroid metabolism and an oxylipin profile within reproductive tissues of cattle based on pregnancy status; and 3) determine the amount of endometrial blood perfusion in cattle using a novel laser Doppler technique. Activity of CYP1A was found within corpora lutea (CL) tissues of both pregnant and non-pregnant cattle, but not within endometrial tissues. Endometrial perfusion, measured using a novel laser Doppler technique, was also validated by measuring angiogenic factors in close proximity to the location of perfusion. A positive correlation (r = 0.28; P = 0.04) was observed between endometrial perfusion and nitrite concentration, an angiogenic factor. Endometrial blood perfusion was affected by the proximity to the CL, but not by the proximity of the dominant follicle. In addition, UGT was categorized across the estrous cycle and the activity was dependent upon the proximity of the CL. Oxylipins, including eicosanoids, were also profiled in CL of cattle that were non-pregnant and pregnant with 5 out of 39 oxylipins differentially expressed. The activity and oxylipin products of steroid and eicosanoid enzymes were not correlated with serum or luteal progesterone. Through these experiments, we have verified that there is localized metabolism of steroids and eicosanoids within reproductive tissues of cattle as well as fetal tissues. Also, we have achieved a full oxylipin profile of non-pregnant and pregnant cattle CL with five oxylipins contained in various amounts between pregnancy status.

The metabolism of androgenic anabolic steroids has been studied in the racing greyhound. Various drug preparations have been investigated utilising different derivatisation techniques, coupled with gas chromatographic analysis, to enable the identification of key metabolites in canine post administration samples. This has led to an increased understanding of some of the generic routes of steroid metabolism that take place in the greyhound. This valuable information can help to support metabolism studies in the future. The identification of specific metabolites for each compound investigated, has provided a means for controlling the misuse of these compounds, and contributed valuable enhancements to screening protocols utilised in the canine sports drug testing industry. Utilisation of the techniques described, resulted in the identification of specific major metabolites of the anabolic steroid methyltestosterone, namely 17??-methyl-5??- androstan-3??-17??-diol and 17??-methyl-5??-androstan-3??,16??,17??-triol. 16??- hydroxylation was shown to be a major phase I metabolic pathway in the canine along with phase II conjugation with glucuronic acid. Similar results were obtained during the metabolism study of the progestatgenic steroid norethisterone. Several di- and trihydroxy metabolites were detected in the glucuronic acid fraction of the post administration urines from this study. The norethisterone metabolism study also provided some insight, into the area of trace contaminants of pharmaceutical preparations. Low levels of nandrolone metabolites were also detected in the norethisterone post administration urine samples, leading to the discovery that the administered pharmaceutical tablets contained small quantities of nandrolone and 19- norandrostenedione, albeit below FDA approved contaminant levels. Modern methods of drug screening employ such highly sensitive techniques, that they allow for the detection of metabolites of such trace contaminants, following administration of the drug preparation to the greyhound. It is therefore important to have a broad understanding of the metabolism of various drug preparations, both banned and permitted substances alike; as detection of a trace amount of a banned substance metabolite, arising from the administration of a permitted medication, whose iii metabolite profile is unknown, and therefore potentially not detected, could present an interesting case. In conjunction with research into controlling the use of banned substances for the purposes of suppressing oestrus in the greyhound bitch, an investigation into normal/reference levels of endogenous hormones has been carried out. The endogenous steroid levels in a population of 212 greyhound bitches have been studied with a view to establishing a method for the detection of the exogenous administration of the endogenous anabolic steroid testosterone. The major urinary metabolites investigated were epiandrosterone, 5??-androstane-3??,17??-diol and 5??-androstane-3??,17??-diol. Statistical evaluations have been carried out to support the implementation of a suitable threshold for the key testosterone metabolites, namely 5??-androstane-3??,17??-diol and epiandrosterone. The detection of 5??-androstane-3??,17??-diol was found to be a very good indicator of the exogenous administration of testosterone to the greyhound bitch, when compared with the reference population data for this metabolite. However, further statistical/analytical data evaluation was deemed necessary before an absolute threshold could be implemented for this analyte, for the purposes of controlling the misuse of testosterone in the racing greyhound bitch. To support the understanding of endogenous steroid levels in the female greyhound, yet further, the endogenous reproductive steroid profiles were measured throughout the entire oestrus cycle of a cohort of 33 racing bitches. The results of the study clearly indicate a surge in androgen metabolites during the first 7-10 days of the oestrus cycle, in particular epiandrosterone and 5??-androstane-3??,17??-diol. This unique set of data has provided detailed information regarding the fluctuating concentrations of androgen and progesterone metabolites (following ovulation), at key stages of the canine oestrus cycle. The information obtained from this research can be used to support regulatory decisions regarding the misuse of testosterone in the racing greyhound bitch.

The human endometrium is a complex multicellular tissue, located within the cavity of the uterus. Its luminal surface is defined by a layer of epithelial cells supported on a multicellular stroma containing fibroblasts, glands (lined by a secretory epithelium), blood vessels (lined with endothelial cells) and several populations of immune cells; the latter includes a unique population of natural killer (uNK) cells. The endometrium undergoes dynamic remodelling across the menstrual cycle in response to fluctuating levels of sex steroids secreted by ovarian cells. The phases of the endometrial cycle include an oestrogendominated proliferative phase, a progesterone-dominated secretory phase and menses (endometrial shedding precipitated by falling levels of progesterone). A key feature of the secretory phase is differentiation (decidualisation) of endometrial stromal fibroblasts (ESC) an event characterised by transformation of cell shape, secretion of growth factors/cytokines, angiogenesis/vascular remodelling and an increase in the numbers of resident immune cells. Decidualisation ensures an appropriate nutritional and hormonal environment exists during the establishment of pregnancy. Studies in mice suggest that de novo biosynthesis of oestrogen within the uterus may play an essential role in regulation of decidualisation but no data exist for human. Endometrial endothelial and uNK cells both contain oestrogen receptors but the impact of oestrogens on their function has not been explored. In the current studies three questions have been addressed: 1. Is oestrogen biosynthesis a feature of human endometrial stromal cell decidualisation? 2. What is the impact of oestrogen on uNK cell function? 3. What role (if any) does oestrogen play in the interplay between decidual, immune and vascular cells within the human endometrial stroma? Results obtained provide the first evidence that de novo biosynthesis of oestrogens occurs during decidualisation of human ESC. This was attributed to changes in expression patterns of mRNAs encoding proteins that play a critical role in regulation of oestrogen biosynthesis (STAR, CYP11A1, CYP19A1 [aromatase], HSD17B2 [17βHSD2] and STS [steroid sulphatase]). Changes in the pattern of metabolism were confirmed using thin layer chromatography and analysis of concentrations of oestrone (E1) and oestradiol (E2) in culture media. Secretion of E1 and E2 was reduced by addition of an aromatase inhibitor. Data derived from studies described within this thesis also show for the first time that incubation of uNK cells with E2 not only enhanced cell migration but also stimulated secretion of factors that had a significant impact on endothelial cell angiogenesis. These findings were supported by novel evidence that E2 had a significant impact on expression of genes associated with cell motility and angiogenesis. In addition, factors, including E1/E2, secreted by decidualised stromal cells, stimulated chemotaxis of uNK cells. Future experiments will focus on determining the identity of the angiogenic factors secreted by uNK cells in response to E2 and the mechanisms responsible for uNK cell movement. In summary, new data presented in this thesis provide evidence that local biosynthesis of oestrogens within the endometrial stroma may play a previously unrecognised role in regulating the function of uNK cells and endometrial endothelial cells in women. These results have implications for treatment of disorders such as infertility, heavy menstrual bleeding and endometriosis.

This study (1) quantitatively compared steroid production in cultured Y-l mouse adrenal tumor cells exposed to Camel and Carlton-smoke derived residues, and (2) localized the effects in the cell. Basal steroid production was increased by Camel residues but not by Carlton, while ACTH stimulation was interfered with by both residues. Camel basal stimulation was comparable to that of cAMP, and was abolished by Cytochalasin D. The stimulation was also comparable to that of cholera toxin, which activates adenyl cyclase. Results indicate that residue components dissolve in the membrane stimulating adenyl cyclase at a point similar to or before that utilized by cholera toxin for its stimulating effect.

The enzymes CYP27A1 and CYP7B1 are widely expressed in various human tissues and perform catalytic reactions in cholesterol homeostasis and endocrine signaling. We have investigated the metabolism of a synthetic oxysterol. In this study, we show that CYP27A1 is the enzyme responsible for a 28-hydroxylation of this oxysterol and that the rate of CYP27A1-mediated metabolism is relatively slow. This may give an explanation for the prolonged inhibitory effects on cholesterol biosynthesis that have been shown for this oxysterol. The current study contributes to the knowledge of synthetically produced oxysterols and their potential use as cholesterol lowering drugs. In two studies we investigated CYP7B1-mediated metabolism of different sex hormones. Our data indicate that CYP7B1 may carry out a previously unknown catalytic reaction involving an androgen. Taken together the data suggest that varying steroid concentrations in cells and tissues may be important for CYP7B1-dependent metabolism of sex hormones and sex hormone precursors. CYP7B1-mediated hydroxylation of sex hormones may influence the cellular levels of these steroids and may be a potential pathway for elimination of the steroids from the cell. Some known CYP7B1 substrates are agonists for ERα and ERβ but the reported role(s) of CYP7B1 for ER action are not fully understood. In the last study we investigated the role(s) of CYP7B1-mediated metabolism for ER-mediated action. Our data indicate that CYP7B1-mediated conversion of steroids that affect ER-mediated response into their 7α-hydroxymetabolites will result in loss of action. This indicates that CYP7B1 may have an important role for regulation of ER-mediated processes in the body. In summary, results from this thesis contribute to the knowledge on the metabolism of synthetic oxysterols of potential use as cholesterol lowering drugs and the role(s) of CYP7B1-mediated metabolism for processes related to the functions of sex hormones. / Disputationsordförande;Professor Eva Brittebo, Inst. för Biovetenskap, Avd. för Toxikologi, Uppsala Universitet, UppsalaBetygsnämndens ledamöten; Docent Lena Ekström, Inst. för Laboratoriemedicin, Avd. för Klinisk Kemi, Karolinska Universitetssjukhuset, HuddingeDocent Ulf Diczfaluzy, Inst. för Laboratoriemedicin, Avd. för Klinisk Kemi, Karolinska Universitetssjukhuset, HuddingeProfessor Agneta Oskarsson, Inst. BVF, Avd. för farmakologi och toxikologi, SLU, Uppsala