Regulation of sex steroid formation by interleukin-4 and interleukin-6 in breast cancer cells

Sex steroids play a predominant role in the development and differentiation of normal mammary gland as well as in the regulation of hormone-sensitive breast cancer growth. There is evidence suggesting that local intracrine formation of sex steroids from inactive precursors secreted by the adrenals namely, dehydroepiandrosterone (DHEA) and 4-androstenedione (4-dione) play an important role in the regulation of growth and function of peripheral target tissues, including the breast. Moreover, human breast carcinomas are often infiltrated by stromal/immune cells secreting a wide spectra of cytokines. These might in turn regulate the activity of both immune and neoplastic cells. The present study was designed to examine the action of cytokines on 17β-hydroxysteroid dehydrogenase (17β-HSD) and 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) activities in human breast cancer cells. The various types of human 17β-HSD (five types) and 3β-HSD (two types), because of their tissue- and cell-specific expression and substrate specificity, provide each cell with necessary mechanisms to control the level of intracellular active androgens and estrogens. We first investigated the effect of exposure to IL-4 and IL-6 on reductive and oxidative 17β-HSD activities in both intact ZR-75-1 and T-47D human breast cancer cells. In ZR-75-1 cells, a 6 d exposure to IL-4 and IL-6 decreased E₂-induced cell proliferation, the half maximal inhibitory effect being exerted at 88 and 26 pM, respectively. In parallel, incubation with IL-4 and IL-6 increased oxidative 17β-HSD activity by 4.4- and 1.9-fold, respectively, this potent activity being observed at ₅₀ values of 22.8 and 11.3 pM, respectively. Simultaneously, reductive 17β-HSD activity leading to E₂ formation was decreased by 70 and 40% by IL-4 and IL-6, respectively. Moreover, IL-4 and IL-6 exerted the same regulatory effects on 17β-HSD activities when testosterone and 4-dione were used as substrates, thus strongly suggesting the expression of the type 2 17β-HSD ZR-75-1 cells. In contrast, in T-47D cells, IL-4 increased the formation of E₂, whereas IL-6 exerts no effect on this parameter. However, we found that T-47D cells failed to convert testosterone efficiently into 4-DIONE, thus suggesting that there is little or no expression of type 2 17β-HSD in this cell line. The present findings demonstrate that the potent regulatory effects of IL-4 and IL-6 on 17β-HSD activities depend on the cell-specific gene expression of various types of 17β-HSD enzymes. We have also studied the effect of cytokines on the regulation of the 3β-HSD expression in both ZR-75-1 and T-47D human breast cancer cells. Under basal culture conditions, there is no 3β-HSD activity detectable in these cells. However, exposure to IL-4 caused a rapid and potent induction of 3β-HSD activity, whereas IL-6 failed to induce 3β-HSD expression. Our data thus demonstrate that cytokines may play a crucial role in sex steroid biosynthesis from inactive adrenal precursors in human breast cancer cells.     

Two non-reactive ternary complexes of estrogenic 17beta-hydroxysteroid dehydrogenase: crystallization and preliminary structural analysis.

Human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1, EC1.1.1.62) is an important enzyme that catalyses the last step of active estrogen formation. 17β-HSD1 plays a key role in the proliferation of breast cancer cells. The three-dimensional structures of this enzyme and of the enzyme-estradiol complex have been solved (Zhu et al., 1993, J. Mol. Biol. 234:242; Ghosh et al., 1995, Structure 3:503; Azzi et al., 1996, Nature Struct. Biol. 3:665). The determination of the non-reactive ternary complex structure, which could mimic the transition state, constitutes a further critical step toward the rational design of inhibitors for this enzyme (Ghosh et al. 1995, Structure 3:503; Penning, 1996, Endocrine-Related Cancer, 3:41).

To further study the transition state, two non-reactive ternary complexes, 17β-HSD1–EM519-NADP⁺ and 17β-HSD1–EM553-NADP⁺ were crystallized using combined methods of soaking and co-crystallization. Although they belong to the same C2 space group, they have different unit cells, with a=155.59 Å, b=42.82 Å, c=121.15 Å, β=128.5° for 17β-HSD1–EM519-NADP⁺, and a=124.01 Å, b=45.16 Å, c=61.40 Å, β=99.2° for 17β-HSD1–EM553-NADP⁺, respectively. Our preliminary results revealed that the inhibitors interact differently with the enzyme than do the natural substrates.     

3D-structure of human estrogenic 17β-HSD1: binding with various steroids

Human estrogenic dehydrogenase (17β-HSD1) catalyses the last step in the biosynthesis of the active estrogens that stimulate the proliferation of breast cancer cells. While the primary substrate for the enzyme is estrone, the enzyme has some activity for the non-estrogenic substrates. To better understand the structure–function relationships of 17β-HSD1 and to provide a better ground for the design of inhibitors, we have determined the crystal structures of 17β-HSD1 in complex with different steroids.The structure of the complex of estradiol with the enzyme determined previously (Azzi et al., Nature Structural Biology 3, 665–668) showed that the narrow active site was highly complementary to the substrate. The substrate specificity is due to a combination of hydrogen bonding and hydrophobic interactions between the steroid and the enzyme binding pocket. We have now determined structures of 17β-HSD1 in complex with dihydrotestosterone and 20α-OH-progesterone. In the case of the C19 androgen, several residues within the enzyme active site make some small adjustments to accommodate the increased bulk of the substrate. In addition, the C19 steroids bind in a slightly different position from estradiol with shifts in positions of up to 1.4 Å. The altered binding position avoids unfavorable steric interactions between Leu 149 and the C19 methyl group (Han et al., unpublished). The known kinetic parameters for these substrates can be rationalized in light of the structures presented. These results give evidence for the structural basis of steroid recognition by 17β-HSD1 and throw light on the design of new inhibitors for this pivotal steroid enzyme.     

The type I and type II 11beta-hydroxysteroid dehydrogenase enzymes.

Local tissue concentrations of glucocorticoids are modulated by the enzyme 11β-hydroxysteroid dehydrogenase which interconverts cortisol and the inactive glucocorticoid cortisone in man, and corticosterone and 11-dehydrocorticosterone in rodents. The type I isoform (11β-HSD1) is a bidirectional enzyme but acts predominantly as a oxidoreductase to form the active glucocorticoids cortisol or corticosterone, while the type II enzyme (11β-HSD2) acts unidirectionally producing inactive 11-keto metabolites. There are no known clinical conditions associated with 11β-HSD1 deficiency, but gene deletion experiments in the mouse indicate that this enzyme is important both for the maintenance of normal serum glucocorticoid levels, and in the activation of key hepatic gluconeogenic enzymes. Other important sites of action include omental fat, the ovary, brain and vasculature. Congenital defects in the 11β-HSD2 enzyme have been shown to account for the syndrome of apparent mineralocorticoid excess (AME), a low renin severe form of hypertension resulting from the overstimulation of the non-selective mineralocorticoid receptor by cortisol in the distal tubule of the kidney. Inactivation of the 11β-HSD2 gene in mice results in a phenotype with similar features to AME. In addition, these mice show high neonatal mortality associated with marked colonic distention, and remarkable hypertrophy and hyperplasia of the distal tubule epithelia. 11β-HSD2 also plays an important role in decreasing the exposure of the fetus to the high levels of maternal glucocorticoids. Recent work suggests a role for 11β-HSD2 in non-mineralocorticoid target tissues where it would modulate glucocorticoid access to the glucocorticoid receptor, in invasive breast cancer and as a mechanism providing ligand for the putative 11-dehydrocorticosterone receptor. While previous homologies between members of the SCAD superfamily have been of the order of 20–30% phylogenetic analysis of a new branch of retinol dehydrogenases indicates identities of >60% and overlapping substrate specificities. The availability of crystal structures of family members has allowed the mapping of conserved 11β-HSD domains A–D to a cleft in the protein structure (cofactor binding domain), two parallel β-sheets, and an -helix (active site), respectively.     

Steroid regulation of progesterone synthesis in a stable porcine granulosa cell line: a role for progestins

The objective of this investigation was to determine the effect of steroid hormones on the synthesis of progesterone in a stable porcine granulosa cell line, JC-410. We also examined the effect of steroid hormones on expression of the genes encoding the steroidogenic enzymes, cytochrome P450-cholesterol side chain cleavage (P450scc) and 3β-hydroxy-5-ene steroid dehydrogenase (3β-HSD). We observed that 48 h exposure of the JC-410 cells to estradiol-17β (estradiol), androstenedione, 5-dihydrotestosterone, levonorgestrel, and 5-cholesten-3β, 25-diol (25-hydroxycholesterol) resulted in stimulation of progesterone synthesis. 25-Hydroxycholesterol augmented progesterone synthesis stimulated by estradiol, 5-dihydrotestosterone, levonorgestrel and 8-bromoadenosine 3′:5′-cyclic monophosphate (8-Br-cAMP). This increase in progesterone synthesis was additive with estradiol, 5-dihydrotestosterone and levonorgestrel, and synergistic with 8-Br-cAMP. Cholera toxin, progesterone, levonorgestrel and androstenedione increased P450scc mRNA levels, whereas estradiol had no effect. Cholera toxin, progesterone and levonorgestrel increased 3β-HSD mRNA levels, but estradiol and androstenedione had no effect. The results were interpreted to mean that estrogens, androgens and progestins regulate progesterone synthesis in the JC-410 cells. The effect of androgens appears to be mediated by stimulation of P450scc gene expression while progestins stimulate both P450scc and 3β-HSD gene expression. Our results support the concept that progesterone is an autocrine regulator of its own synthesis in granulosa cells.     

The role of 11β-hydroxysteroid dehydrogenase in steroid hormone specificity

11β-hydroxysteroid dehydrogenase (11β-HSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the mineralocorticoid receptor (MR) from occupancy by endogenous glucocorticoids. In aldosterone target cells the type 2 11β-HSD is present, which, in contrast to the type 1 11β-HSD, has very high affinity for its substrate, is unidirectional and prefers NAD as cofactor. cDNAs encoding 11β-HSD2 have been recently cloned from different species, and the cell-specific expression of its mRNA and protein were determined. 11β-HSD2 is expressed in every aldosterone target tissue. Northern analysis revealed that the rabbit 11β-HSD2 is expressed at high levels in the renal collecting duct and at much lower levels in the colon. RT-PCR experiments demonstrated that 11β-HSD2 mRNA is present only in aldosterone target cells within the kidney. We determined the subcellular localization of the rabbit 11β-HSD2 using a chimera encoding 11β-HSD2 and the green fluorescent protein (GFP). This construct was stably transfected into CHO and MDCK cells. The expressed 11β-HSD2/GFP protein retained high enzymatic activity, and its characteristics were undistinguishable from those of the native enzyme. The intracellular localization of this protein was determined by fluorescence microscopy. 11β-HSD2-associated fluorescence was observed as a reticular network over the cytoplasm whereas the plasma membrane and the nucleus were negative, suggesting endoplasmic reticulum (ER) localization. Co-staining with markers for ER proteins, the Golgi membrane, mitochondria and nucleus confirmed that 11β-HSD2 is localized exclusively to the ER. To determine what structural motifs are responsible for the ER localization, we generated deletion mutants missing the C-terminal 42 and 118 amino acids, and fused them to GFP. Similarly as with the intact 11β-HSD2, these mutants localized exclusively to the ER. Both C-terminal deletion mutants completely lost dehydrogenase activity, independently whether activity was determined in intact cells or homogenates. These results indicate that 11β-HSD2 has a novel ER retrieval signal which is not localized to the C-terminal region. In addition, the C-terminal 118 amino acids are essential for NAD-dependent 11β-HSD activity.     

Cortisol as a mineralocorticoid in human disease

The type 2 isozyme of 11β-hydroxysteroid dehydrogenase inactivates cortisol to cortisone and enables aldosterone to bind to the MR. Congenital deficiency of the enzyme results in cortisol-mediated mineralocorticoid excess and arises because of inactivating mutations in the HSD11B2 gene. Inhibition of the enzyme following licorice or carbenoxolone ingestion results in a similar, though milder phenotype and the enzyme is overwhelmed in ectopic ACTH syndrome. Loss of 11β-HSD2 expression may be important in sodium balance and blood pressure control in some patients with renal disease. Finally, while some studies demonstrate impaired 11β-HSD activity in broader populations of patients with hypertension, further studies are required to clarify the role of 11β-HSD2 in ‘essential’ hypertension.     

Tissue- and temporal-specific regulation of 11beta-hydroxysteroid dehydrogenase type 1 by glucocorticoids in vivo.

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) catalyses the interconversion of active corticosterone and inert 11-dehydrocorticosterone. Short-term glucocorticoid excess upregulates 11β-HSD-1 in liver and hippocampus leading to suggestions that 11β-HSD-1 ameliorates the deleterious effects of glucocorticoid excess by its 11β-dehydrogenase activity. However the predominant activity of 11β-HSD-1 in vivo is 11β-reduction, thus generating active glucocorticoid. We have re-examined the time-course of glucocorticoid regulation of 11β-HSD-1 in the liver, hippocampus and kidney of adult male rats in vivo.

Sham operation markedly reduced 11β-HSD-1 mRNA expression in all tissues, and reduced 11β-HSD bioactivity in liver and hippocampus when compared to untouched controls. Adrenalectomy reduced 11β-HSD-1 expression in all tissues in the short-term (7 days), followed by subsequent recovery of enzyme activity by 21 days in liver and hippocampus. Dexamethasone replacement of adrenalectomised rats attenuated the initial decrease in hepatic 11β-HSD-1 activity, but by 21 days dexamethasone reduced activity compared to control levels.

Thus glucocorticoids regulate 11β-HSD-1 in a complex tissue- and temporal-specific manner. This pattern of regulation suggests glucocorticoids repress 11β-HSD-1 at least in the liver, a pattern of regulation more consistent with the evidence that 11β-HSD-1 is an 11β-reductase in vivo. Operational stress per se down-regulates 11β-HSD-1 which has implications for interpretation and design of in vivo studies of 11β-HSD-1.     

Cannulation in situ of equine umbilicus. Identification by gas chromatography-mass spectrometry (GC-MS) of differences in steroid content between arterial and venous supplies to and from the placental surface

Equine umbilicus was cannulated in utero and a series of cord plasma samples removed for analysis. After steroid extraction and derivatisation, gas chromatographic-mass spectrometric (GC-MS) analysis demonstrated large differences in steroid content between the plasma samples obtained from the umbilical artery and vein, the blood supplies leading to and from the placental surface, respectively. 3β-Hydroxy-5,7-androstadien-17-one, dehydroepiandrosterone, pregnenolone, 3β-hydroxy-5-pregnan-20-one, 5-pregnene-3β,20β-diol and 5β-pregnane-3β,20β-diol were identified as major constituents in extracts from umbilical arterial plasma samples, mostly as unconjugated steroids. Together with 5-pregnane-3,20-dione, these steroids were identified in extracts from umbilical venous plasma samples but at significantly reduced levels to those determined in arterial plasma samples. Oestradiol-17, dihydroequilin-17 and dihydroequilenin-17 were identified in extracts (mostly sulphate-conjugated) from both umbilical arterial and venous plasma samples, much larger amounts being detected in the plasma sampled from, rather than to, the placental surface. Equilin, equilenin, oestrone, oestradiol-17β, dihydroequilin-17β and dihydroequilenin-17β were not detected in the present studies. Isomers of 5(10)-oestrene-3,17β-diol together with 5(10),7-oestradiene-3,17β-diol and its possible oxidative artifact, 5(10),7,9-oestratriene-3,17β-diol, were tentatively identified only in sulphate-conjugated extracts from umbilical venous plasma samples. No glucuronic acid-conjugated steroids could be detected. The implications of this work in the elucidation of the biosynthetic pathways leading to both the formation of oestrogens and C₁₈ neutral steroids at the placental surface are discussed.     

Transformation of steroids by Bacillus strains isolated from the foregut of water beetles (Coleoptera:Dytiscidae): I. Metabolism of androst-4-en-3,17-dione (AD)

Two Bacillus strains were isolated from the foregut of the water beetle Agabus affinis (Payk.) and tested for their steroid transforming ability. After incubation with androst-4-en-3,17-dione (AD), 13 different transformation products were detected. AD was hydroxylated at C₆, C₇, C₁₁ and C₁₄, resulting in formation of 6β-, 7-, 11- and 14-hydroxy-AD. One strain also produced small amounts of 6β,14-dihydroxy-AD. Partly, the 6β-hydroxy group was further oxidized to the corresponding 6-oxo steroids. In addition, a specific reduction of the Δ⁴-double bond was observed, leading to the formation of 5-androstane derivatives. In minor yields the carbonyl functions at C₃ and C₁₇ were reduced leading to the formation of 3ξ-OH or 17β-OH steroids. EI mass spectra of the trimethylsilyl and O-methyloxime trimethylsilyl ether derivatives of some transformation products are presented for the first time.     

Crystallization and preliminary crystal structure of the complex of 17beta-hydroxysteroid dehydrogenase with a dual-site inhibitor

Human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1) catalyzes the synthesis of 17β-estradiol (E₂) from estrone, in the ovary and peripheral tissues. While the structures of 17β-HSD1 alone and in complex with E₂ have been determined (D. Ghosh, V. Pletnev, D.-W. Zhu, Z. Wawrzak, W.-L. Duax, W. Pangborn, F. Labrie, S.-X. Lin, Structure of human 17β-hydroxysteroid dehydrogenase at 2.20 Å resolution, Structure 3 (1995) 503–513; A. Azzi, P.H. Rhese, D.-W. Zhu, R.L. Campbell, F. Labrie, S.-X. Lin, Crystal structure of human estrogenic 17β-hydroxysteroid dehydrogenase complexed with 17_β-estradiol, Nature Struct. Biol. 3 (1996) 665–668, no structures of inhibitor/enzyme complex, either modeled or from crystallography, have been reported before the submission of the present paper. The best available inhibitors are among the ‘dual-site inhibitors’, blocking estrogenic 17β-HSD and the estrogen receptor. These compounds belong to a family of estradiol analogues having an halogen atom at the 16 position and an extended alkyl-amide chain at the 7 position (C. Labrie, G. Martel, J.M. Dufour, G. Levesque, Y. Merand, F. Labrie, Novel compounds inhibit estrogen formation and action, Cancer Res. 52 (1992) 610–615). We now report the crystallization of this enzyme/inhibitor complex. The complex of the best available dual-site inhibitor, EM-139, with 17β-HSD1 has been crystallized using both cocrystallization and soaking methods. Crystals are isomorphous to the native crystals grown in the presence of 0.06% β-octyl-glucoside and polyethyleneglycol 4000, with a monoclinic space group C2. Data at 1.8 Å have been collected from a synchrotron source. Even though the size of the inhibitor is greater than that of the substrate, our preliminary X-ray-diffraction study shows that EM-139 fits into the active site in a position similar to that of estrogen. The availability of such structural data will help design more potent inhibitors of estrogenic 17β-HSD.     

19-oxygenations of 3-deoxy androgens, potent competitive inhibitors of estrogen biosynthesis, with human placental aromatase

Aromatase is a cytochrome P450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione (AD) to estrone through three sequential oxygenations of the 19-methyl group. To gain insight into the ability of 3-deoxy derivative of AD, compound 1, and its 5-ene isomer 4, which are potent competitive inhibitors of aromatase, to serve as a substrate, we studied their 19-oxygenation by human placental aromatase and the metabolites isolated were analyzed by gas chromatography–mass spectrometry. Inhibitors 1 and 4 were found to be oxygenated with aromatase to produce the corresponding 19-hydroxy derivatives 2 and 5 and 19-oxo derivatives 3 and 6 as well as the 17β-reduced 19-hydroxy compounds 7 and 8. Kinetic studies indicated that the 5-ene steroid 4 was surprisingly a good substrate for the aromatase-catalyzing 19-oxygenation with the V_max value of 45 pmol/min per mg prot which was approx. four times higher than that of the other. The relative K_m value for steroids 1 and 4 obtained in this study is opposite from the relative K_i value obtained previously in the inhibition study. The results reveal that there is a difference between a binding suitable for serving as an inhibitor of aromatase and a binding suitable for serving as a substrate of the enzyme in the 3-deoxy steroid series and the C-3 carbonyl group of AD is essential for a proper binding as a substrate to the active site of aromatase.     

17-Hydroxylase/C17,20-lyase (CYP17) is not the enzyme responsible for side-chain cleavage of cortisol and its metabolites

The question addressed in this study was the nature of the enzyme required to remove the side-chain of 17-hydroxycorticosteroids, leading in the case of cortisol to the excretion of 11β-hydroxyandrosterone, 11-oxo-androsterone and the corresponding etiocholanolones. We questioned whether it could be CYP17, the 17-hydroxylase/17,20-lyase utilized in androgen synthesis. The conversion of exogenous cortisol to C₁₉ steroids in patients with complete 17-hydroxylase deficiency (17HD) was studied rationalizing that if CYP17 was involved no C₁₉ steroids would be formed. The urinary excretion of the four 11-oxy-C₁₉ steroids as well as many of the major C₂₁ cortisol metabolites were measured by GC/MS. Our results showed that the conversion of cortisol to C₁₉ steroids was normal in 17HD indicating that a currently unidentified enzyme must be responsible for this transformation.

A secondary goal was to determine to what extent 11-oxy-C₁₉ steroids were metabolites of cortisol or adrenal synthesized 11β-hydroxyandrostenedione. Since cortisol-treated 17HD patients cannot produce androstenedione, all C₁₉ 11-oxy-metabolites excreted must be derived from exogenous cortisol. The extent to which 17HD patients have lower relative excretion of C₁₉ steroids should reflect the absence of 11β-hydroxyandrostenedione metabolites. Our results showed almost all of 11-oxo-etiocholanolone and 11β-hydroxyetiocholanolone were cortisol metabolites, but in contrast the excretion of 11β-hydroxyandrosterone was less than 10% that of normal individuals, indicating that in excess of 90% must be a metabolite of 11β-hydroxyandrostenedione.     

Estrogen-stimulated glucuronidation of dihydrotestosterone in MCF-7 human breast cancer cells

The non-aromatizable androgen dihydrotestosterone (DHT) has been shown to exert a potent inhibitory effect on the proliferation of some human breast cancer cell lines. DHT, however, has little or no significant inhibition on MCF-7 cell proliferation in either the presence or absence of estradiol (E₂). Since the metabolism of DHT into non-active compounds may be responsible for the observed lack of androgenic effect in this cell line, we have investigated the metabolic fate of labeled DHT in MCF-7 cells. A time course incubation was performed with 1 nM [³H]DHT and analysis of the various metabolites formed revealed a time-dependent increase in glucuronidated steroids which was stimulated more than 4-fold by 0.1 nM E₂. The major glucuronidated steroid was androstane-3, 17β-diol in both control and E₂-stimulated cells, comprising 22 ± 1.2% and 30 ± 0.6% of the total radioactivity in the medium, respectively. Other steroid glucuronides observed included DHT, androstane-3β, 17β-diol, and androsterone, all of which were elevated in the E₂-treated cells relative to control values. The present data show that E₂ exerts a stimulatory effect on the glucuronidation of androgens and their metabolites in the estrogen-dependent breast cancer celll line MCF-7. Since glucuronidation is an effective means of cellular elimination of active steroids, such a pathway may be considered as a possible site of regulation of breast cancer cell growth by hormones.     

Role of 17β-hydroxysteroid dehydrogenase type 1 in endocrine and intracrine estradiol biosynthesis

Enzymes with 17β-hydroxysteroid dehydrogenase (17β-HSD) activity catalyse reactions between the low-active female sex steroid, estrone, and the more potent estradiol, for example. 17β-HSD activity is essential for glandular (endocrine) sex hormone biosynthesis, but it is also present in several extra-gonadal tissues. Hence, 17β-HSD enzymes also take part in local (intracrine) estradiol production in the target tissues of estrogen action. Four distinct 17β-HSD isozymes have been characterized so far, and the data strongly suggests that different 17β-HSD isozymes have distinct roles in endocrine and intracrine metabolism of sex steroids. Current data suggest that 17β-HSD type 1 is the principal isoenzyme involved in glandular estradiol production both in humans and rodents. During ovarian follicular development and luteinization, rat 17β-HSD type 1 is regulated by gonadotropins, and the effects of gonadotropins are modulated by steroid hormones and paracrine growth factors. Human 17β-HSD type 1 favors the reduction reaction, thereby converting estrone to estradiol both in vitro and in cultured cells. Hence, the enzymatic properties of the enzyme are also in line with its suggested role in estradiol biosynthesis. Interestingly, 17β-HSD type 1 is also expressed in certain target tissues of estrogen action such as normal and malignant human breast and endometrium. Hence, 17β-HSD type 1 could be one of the factors leading to a relatively high tissue/plasma ratio of estradiol in breast cancer tissues of postmenopausal women. We conclude that 17β-HSD type 1 has a central role in regulating the circulating estradiol concentration as well as its local production in estrogen target cells.     

Crystal structures of D-psicose 3-epimerase from Clostridium cellulolyticum H10 and its complex with ketohexose sugars

D-Psicose 3-epimerase (DPEase) is demonstrated to be useful in the bioproduction of D-psicose, a rare hexose sugar, from D-fructose, found plenty in nature. Clostridium cellulolyticum H10 has recently been identified as a DPEase that can epimerize D-fructose to yield D-psicose with a much higher conversion rate when compared with the conventionally used DTEase. In this study, the crystal structure of the C. cellulolyticum DPEase was determined. The enzyme assembles into a tetramer and each subunit shows a (β/α)₈ TIM barrel fold with a Mn²⁺ metal ion in the active site. Additional crystal structures of the enzyme in complex with substrates/ products (D-psicose, D-fructose, D-tagatose and D-sorbose) were also determined. From the complex structures of C. cellulolyticum DPEase with D-psicose and D-fructose, the enzyme has much more interactions with D-psicose than D-fructose by forming more hydrogen bonds between the substrate and the active site residues. Accordingly, based on these ketohexosebound complex structures, a C3-O3 proton-exchange mechanism for the conversion between D-psicose and D-fructose is proposed here. These results provide a clear idea for the deprotonation/protonation roles of E150 and E244 in catalysis.     

A preliminary study of steroid reproductive hormones in human hair

Nowadays research and clinical studies of human reproductive endocrinology are generally carried out using human blood reproductive hormone assays. However the acquisition of human blood samples has some shortcomings. In search of new approaches, we paid attention to the fact that progesterone can be detected in cow's hair. Consequently we investigated whether or not steroid hormones are measurable in human hair. The results showed that the levels of steroid hormones in hair are not affected by shampoo and do not significantly vary between different segments of hair (i.e. top, middle and basal segments). The menstrual estradiol and progesterone rhythm of female hair is similar to that of female serum. The ratio of hair estradiol to serum estradiol in the female is 41.2% and that of hair progesterone to serum progesterone is 59.0%; the ratio of hair testosterone to serum testosterone in male is 116%. There are significant correlations between hair and serum steroid hormones of healthy human adult: γ (estradiol)=0.395 (n=20), p<0.05; γ (progesterone)=0.440 (n=22), p<0.025 and γ (testosterone)=0.395 (n=25), p<0.05.     

Spectrophotometric study of cytochrome P-450 (11 beta) interaction with physiological effectors

Using the optical absorbance spectroscopy method, the interaction of a number of biospecific ligands (steroids, adrenodoxin) with homogeneous cytochrome P-450 (11 beta) from bovine adrenal mitochondria was investigated. The parameters of the steroid-protein interaction in a number of substrates and products of the 11 beta- and 18 (19)-hydroxylation with the active site of cytochrome P-450 (11 beta) were determined. A sharp decrease in the cytochrome affinity for steroids upon the insertion of the first hydroxy group was observed, which provides for a predominant formation of monohydroxylated products from the substrate and minimum amounts of dihydroxylated ones, despite the presence of more than one position for the substrate hydroxylation by cytochrome P-450 (11 beta). Some structural elements of the steroid molecule were determined as any alterations in these strongly affect the enzyme affinity for the steroid. These structures are: 1) delta 4-3-oxo structure; 2) either 21-hydroxy group of pregnen steroids or the one fulfilling its functions, 17 beta-hydroxy or 17-oxo group of androsten steroids, and 3) the 11th position of all the substrates under study. It was shown that the binding of various substrates into stoichiometric (1:1) steroid-protein complexes provides a transition to high spin state from 30-40% (cortisol, corticosterone) to 90-95% (11-deoxycorticosterone) of hemoprotein iron. Using the experimental system containing individual cytochrome P-450 (11 beta) and adrenodoxin, as well as the steroid and nonionic detergent Tween 20, it was shown that the parameters of substrate binding and hemoprotein spin equilibrium did not differ from the corresponding parameters of the cytochrome-adrenodoxin dienzyme complex. The peculiarities of the multiligand interactions in the 11 beta-hydroxylase system, involving cytochrome, substrates and ferredoxin demonstrate some analogy with a bacterial camphor hydroxylase system and some differences from the mitochondrial system for the side chain cleavage of cholesterol.