Production of 11-Oxygenated Androgens by Testicular Adrenal Rest Tumors.

CONTEXT: Testicular adrenal rest tumors (TART) are a common complication in males with classic 21-hydroxylase deficiency (21OHD). TART are likely to contribute to the androgen excess in 21OHD patients, but a direct quantification of steroidogenesis from these tumors has not been yet done. OBJECTIVE: We aimed to define the production of 11-oxygenated 19-carbon (11oxC19) steroids by TART. METHODS: Using liquid chromatography-tandem mass spectrometry, steroids were measured in left (n = 7) and right (n = 4) spermatic vein and simultaneously drawn peripheral blood (n = 7) samples from 7 men with 21OHD and TART. For comparison, we also measured the peripheral steroid concentrations in 5 adrenalectomized patients and 12 age- and BMI-matched controls. Additionally, steroids were quantified in TART cell- and adrenal cell-conditioned medium, with and without adrenocorticotropic hormone (ACTH) stimulation. RESULTS: Compared with peripheral blood from 21OHD patients with TART, the spermatic vein samples displayed the highest gradient for 11ß-hydroxytestosterone (11OHT; 96-fold) of the 11oxC19 steroids, followed by 11-ketotestosterone (47-fold) and 11ß-hydroxyandrostenedione (11OHA4; 29-fold), suggesting production of these steroids in TART. TART cells produced higher levels of testosterone and lower levels of A4 and 11OHA4 after ACTH stimulation compared with adrenal cells, indicating ACTH-induced production of testosterone in TART. CONCLUSION: In patients with 21OHD, TART produce 11oxC19 steroids, but in different proportions than the adrenals. The very high ratio of 11OHT in spermatic vs peripheral vein blood suggests the 11-hydroxylation of testosterone by TART, and the in vitro results indicate that this metabolism is ACTH-sensitive.

Testosterone Metabolite 6ß-Hydroxytestosterone Contributes to Angiotensin II-Induced Abdominal Aortic Aneurysms in Apoe-/- Male Mice.

Background Sex is a prominent risk factor for abdominal aortic aneurysms (AAAs), and angiotensin II (Ang II) induces AAA formation to a greater degree in male than in female mice. We previously reported that cytochrome P450 1B1 contributes to the development of hypertension, as well as AAAs, in male mice. We also found that a cytochrome P450 1B1-generated metabolite of testosterone, 6ß-hydroxytestosterone (6ß-OHT), contributes to Ang II-induced hypertension and associated cardiovascular and renal pathogenesis in male mice. The current study was conducted to determine the contribution of 6ß-OHT to Ang II-induced AAA development in Apoe-/- male mice. Methods and Results Intact or castrated Apoe-/-/Cyp1b1+/+ and Apoe-/-/Cyp1b1-/- male mice were infused with Ang II or its vehicle for 28 days, and administered 6ß-OHT every third day for the duration of the experiment. Abdominal aortas were then evaluated for development of AAAs. We observed a significant increase in the incidence and severity of AAAs in intact Ang II-infused Apoe-/-/Cyp1b1+/+ mice, compared with vehicle-treated mice, which were minimized in castrated Apoe-/-/Cyp1b1+/+ and intact Apoe-/-/Cyp1b1-/- mice infused with Ang II. Treatment with 6ß-OHT significantly restored the incidence and severity of AAAs in Ang II-infused castrated Apoe-/-/Cyp1b1+/+ and intact Apoe-/-/Cyp1b1-/- mice. However, administration of testosterone failed to increase AAA incidence and severity in Ang II-infused intact Apoe-/-/Cyp1b1-/- mice. Conclusions Our results indicate that the testosterone-cytochrome P450 1B1-generated metabolite 6ß-OHT contributes to Ang II-induced AAA development in Apoe-/- male mice.

Brain Testosterone-CYP1B1 (Cytochrome P450 1B1) Generated Metabolite 6ß-Hydroxytestosterone Promotes Neurogenic Hypertension and Inflammation.

Previously, we showed that peripheral administration of 6ß-hydroxytestosterone, a CYP1B1 (cytochrome P450 1B1)-generated metabolite of testosterone, promotes angiotensin II-induced hypertension in male mice. However, the site of action and the underlying mechanism by which 6ß-hydroxytestosterone contributes to angiotensin II-induced hypertension is not known. Angiotensin II increases blood pressure by its central action, and CYP1B1 is expressed in the brain. This study was conducted to determine whether testosterone-CYP1B1 generated metabolite 6ß-hydroxytestosterone locally in the brain promotes the effect of systemic angiotensin II to produce hypertension in male mice. Central CYP1B1 knockdown in wild-type (Cyp1b1+/+) mice by intracerebroventricular-adenovirus-GFP (green fluorescence protein)-CYP1B1-short hairpin (sh)RNA attenuated, whereas reconstitution of CYP1B1 by adenovirus-GFP-CYP1B1-DNA in the paraventricular nucleus but not in subfornical organ in Cyp1b1-/- mice restored angiotensin II-induced increase in systolic blood pressure measured by tail-cuff. Intracerebroventricular-testosterone in orchidectomized (Orchi)-Cyp1b1+/+ but not in Orchi-Cyp1b1-/-, and intracerebroventricular-6ß-hydroxytestosterone in the Orchi-Cyp1b1-/- mice restored the angiotensin II-induced: (1) increase in mean arterial pressure measured by radiotelemetry, and autonomic imbalance; (2) reactive oxygen species production in the subfornical organ and paraventricular nucleus; (3) activation of microglia and astrocyte, and neuroinflammation in the paraventricular nucleus. The effect of intracerebroventricular-6ß-hydroxytestosterone to restore the angiotensin II-induced increase in mean arterial pressure and autonomic imbalance in Orchi-Cyp1b1-/- mice was inhibited by intracerebroventricular-small interfering (si)RNA-androgen receptor (AR) and GPRC6A (G protein-coupled receptor C6A). These data suggest that testosterone-CYP1B1-generated metabolite 6ß-hydroxytestosterone, most likely in the paraventricular nucleus via AR and GPRC6A, contributes to angiotensin II-induced hypertension and neuroinflammation in male mice.

6ß-Hydroxytestosterone, a metabolite of testosterone generated by CYP1B1, contributes to vascular changes in angiotensin II-induced hypertension in male mice.

BACKGROUND: Previously, we showed that 6ß-hydroxytestosterone (6ß-OHT), a cytochrome P450 1B1 (CYP1B1)-derived metabolite of testosterone, contributes to angiotensin II (Ang II)-induced hypertension in male mice. This study was conducted to test the hypothesis that 6ß-OHT contributes to increased vascular reactivity, endothelial dysfunction, vascular hypertrophy, and reactive oxygen species production associated with Ang II-induced hypertension. METHODS: Eight- to 10-week-old intact or castrated C57BL/6 J (Cyp1b1+/+ and Cyp1b1-/-) mice were anesthetized for implantation of a micro-osmotic pump which delivered Ang II (700 ng/kg/day) or saline for 14 days. Mice were injected with 6ß-OHT (15 µg/g b.w every third day), flutamide (8 mg/kg every day), or its vehicle. Blood pressure was measured via tail-cuff. Vascular reactivity, endothelial-dependent and endothelial-independent vasodilation, media to lumen ratio, fibrosis by collagen deposition, and reactive oxygen species production by dihydroethidium staining were determined in the isolated thoracic aorta. RESULTS: The response of thoracic aorta to phenylephrine and endothelin-1 was increased in Ang II-infused Cyp1b1+/+ mice compared to intact Cyp1b1-/- or castrated Cyp1b1+/+ and Cyp1b1-/- mice; these effects of Ang II were restored by treatment with 6ß-OHT. Ang II infusion caused endothelial dysfunction, as indicated by decreased relaxation of the aorta to acetylcholine in Cyp1b1+/+ but not Cyp1b1-/- or castrated Cyp1b1+/+ and Cyp1b1-/- mice. 6ß-OHT did not alter Ang II-induced endothelial dysfunction in Cyp1b1+/+ mice but restored it in Cyp1b1-/- or castrated Cyp1b1+/+ and Cyp1b1-/- mice. Ang II infusion increased media to lumen ratio and caused fibrosis and reactive oxygen species production in the aorta of Cyp1b1+/+ mice. These effects were minimized in the aorta of Cyp1b1-/- or castrated Cyp1b1+/+ and Cyp1b1-/- mice and restored by treatment with 6ß-OHT. Treatment with the androgen receptor antagonist flutamide reduced blood pressure and vascular hypertrophy in castrated Ang II-infused mice injected with 6ß-OHT. CONCLUSIONS: 6ß-OHT is required for the action of Ang II to increase vascular reactivity and cause endothelial dysfunction, hypertrophy, and increase in oxygen radical production. The effect of 6ß-OHT in mediating Ang II-induced hypertension and associated hypertrophy is dependent on the androgen receptor. Therefore, CYP1B1 could serve as a novel target for the development of therapeutics to treat vascular changes in hypertensive males.

Androgen- and estrogen-receptor mediated activities of 4-hydroxytestosterone, 4-hydroxyandrostenedione and their human metabolites in yeast based assays.

4-Hydroxyandrost-4-ene-3,17-dione, also named formestane, is an irreversible aromatase inhibitor and therapeutically used as anti-breast cancer medication in post-menopausal women. Currently, no therapeutical indication led to approval of its 17-hydroxylated analog 4-hydroxytestosterone, an anabolic steroid. However, it is currently investigated in a clinical trial for breast cancer. In context with sports doping, aromatase inhibitors are administered to reduce estrogenic side effects of misused anabolic substances or their metabolites. Therefore, both substances are prohibited in sports by the World Anti-Doping Agency (WADA). Analysis of urinary phase I and phase II metabolites showed similar results for both compounds. In the current investigation, 4-hydroxyandrost-4-ene-3,17-dione, 4-hydroxytestosterone and seven of their described urinary metabolites as well as 2α-hydroxyandrostenedione were tested in the yeast androgen screen and the yeast estrogen screen. Androgenic effects were observed for all tested substances, except for one, which showed anti-androgenic properties. With regard to the yeast estrogen screen, estrogenic effects were observed for only two metabolites at rather high concentrations, while six out of the ten substances tested showed anti-estrogenic properties. In terms of the strong androgenic effect observed for 4-hydroxytestosterone (10-8 M), 4-hydroxyandrost-4-ene-3,17-dione (10-8 M) and two more urinary metabolites, the yeast androgen assay may also be used to trace abuse in urine samples.

Multiple Chemical Inducible Tal Effectors for Genome Editing and Transcription Activation.

Inducible modulation is often required for precise investigations and manipulations of dynamic biological processes. Transcription activator-like effectors (TALEs) provide a powerful tool for targeted gene editing and transcriptional programming. We designed a series of chemical inducible systems by coupling TALEs with a mutated human estrogen receptor (ERT2), which renders them 4-hydroxyl-tamoxifen (4-OHT) inducible for access of the genome. Chemical inducible genome editing was achieved via fusing two tandem ERT2 domains to customized transcription activator-like effector nuclease (TALEN), which we termed "Hybrid Inducible Technology" (HIT-TALEN). Those for transcription activation were vigorously optimized using multiple construct designs. Most efficient drug induction for endogenous gene activation was accomplished with minimal background activity using an optimized inducible TALE based SunTag system (HIT-TALE-SunTag). The HIT-SunTag system is rapid, tunable, selective to 4-OHT over an endogenous ligand, and reversible in drug induced transcriptional activation. Versatile systems developed in this study can be easily applied for editing and transcriptional programming of potentially any genomic loci in a tight and effective chemical inducible fashion.

11-Oxygenated Androgens Are Biomarkers of Adrenal Volume and Testicular Adrenal Rest Tumors in 21-Hydroxylase Deficiency.

Context: Patients with 21-hydroxylase deficiency (21OHD) have long-term complications, resulting from poor disease control and/or glucocorticoid overtreatment. Lack of optimal biomarkers has made it challenging to tailor therapy and predict long-term outcomes. Objective: To identify biomarkers of disease control and long-term complications in 21OHD. Setting and Participants: Cross-sectional study of 114 patients (70 males), ages 2 to 67 years (median, 15 years), seen in a tertiary referral center. Methods: We correlated a mass-spectrometry panel of 23 steroids, obtained before first morning medication, with bone age advancement (children), adrenal volume (adults), testicular adrenal rest tumors (TART), hirsutism, menstrual disorders, and pituitary hormones. Results: Total adrenal volume correlated positively with 18 steroids, most prominently 21-deoxycortisol and four 11-oxygenated-C19 (11oxC19) steroids: 11ß-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11ketoA4), 11ß-hydroxytestosterone (11OHT), and 11-ketotestosterone (11ketoT) (r ≈ 0.7, P < 0.0001). Nine steroids were significantly higher (P ≤ 0.01) in males with TART compared with those without TART, including 11OHA4 (6.8-fold), 11OHT (4.9-fold), 11ketoT (3.6-fold), 11ketoA4 (3.3-fold), and pregnenolone sulfate (PregS; 4.8-fold). PregS (28.5-fold) and 17-hydroxypregnenolone sulfate (19-fold) levels were higher (P < 0.01) in postpubertal females with menstrual disorders. In males, testosterone levels correlated positively with all 11oxC19 steroids in Tanner stages 1 and 2 (r ≈ 0.7; P < 0.001) but negatively in Tanner stage 5 (r = -0.3 and P < 0.05 for 11ketoA4 and 11ketoT). In females, testosterone level correlated positively with all four 11oxC19 steroids across all Tanner stages (r ≈ 0.8; P < 0.0001). Conclusion: 11oxC19 steroids and PregS might serve as clinically useful biomarkers of disease control and long-term complications in 21OHD.

Profiling adrenal 11ß-hydroxyandrostenedione metabolites in prostate cancer cells, tissue and plasma: UPC2-MS/MS quantification of 11ß-hydroxytestosterone, 11keto-testosterone and 11keto-dihydrotestosterone.

Adrenal C19 steroids serve as precursors to active androgens in the prostate. Androstenedione (A4), 11ß-hydroxyandrostenedione (11OHA4) and 11ß-hydroxytestosterone (11OHT) are metabolised to potent androgen receptor (AR) agonists, dihydrotestosterone (DHT), 11-ketotestosterone (11KT) and 11-ketodihydrotestosterone (11KDHT). The identification of 11OHA4 metabolites, 11KT and 11KDHT, as active androgens has placed a new perspective on adrenal C11-oxy C19 steroids and their contribution to prostate cancer (PCa). We investigated adrenal androgen metabolism in normal epithelial prostate (PNT2) cells and in androgen-dependent prostate cancer (LNCaP) cells. We also analysed steroid profiles in PCa tissue and plasma, determining the presence of the C19 steroids and their derivatives using ultra-performance liquid chromatography (UHPLC)- and ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). In PNT2 cells, sixty percent A4 (60%) was primarily metabolised to 5α-androstanedione (5αDIONE) (40%), testosterone (T) (10%), and androsterone (AST) (10%). T (30%) was primarily metabolised to DHT (10%) while low levels of A4, 5αDIONE and 3αADIOL (≈20%) were detected. Conjugated steroids were not detected and downstream products were present at <0.05µM. Only 20% of 11OHA4 and 11OHT were metabolised with the former yielding 11keto-androstenedione (11KA4), 11KDHT and 11ß-hydroxy-5α-androstanedione (11OH-5αDIONE) and the latter yielding 11OHA4, 11KT and 11KDHT with downstream products <0.03µM. In LNCaP cells, A4 (90%) was metabolised to AST-glucuronide via the alternative pathway while T was detected as T-glucuronide with negligible conversion to downstream products. 11OHA4 (80%) and 11OHT (60%) were predominantly metabolised to 11KA4 and 11KT and in both assays more than 50% of 11KT was detected in the unconjugated form. In tissue, we detected C11-oxy C19 metabolites at significantly higher levels than the C19 steroids, with unconjugated 11KDHT, 11KT and 11OHA4 levels ranging between 13 and 37.5ng/g. Analyses of total steroid levels in plasma showed significant levels of 11OHA4 (≈230-440nM), 11KT (≈250-390nM) and 11KDHT (≈19nM). DHT levels (<0.14nM) were significantly lower. In summary, 11ß-hydroxysteroid dehydrogenase type 2 activity in PNT2 cells was substantially lower than in LNCaP cells, reflected in the conversion of 11OHA4 and 11OHT. Enzyme substrate preferences suggest that the alternate pathway is dominant in normal prostate cells. Glucuronidation activity was not detected in PNT2 cells and while all T derivatives were efficiently conjugated in LNCaP cells, 11KT was not. Substantial 11KT levels were also detected in both PCa tissue and plasma. 11OHA4 therefore presents a significant androgen precursor and its downstream metabolism to 11KT and 11KDHT as well as its presence in PCa tissue and plasma substantiate the importance of this adrenal androgen.

11-Oxygenated C19 Steroids Are the Predominant Androgens in Polycystic Ovary Syndrome.

Context: Androgen excess is a defining feature of polycystic ovary syndrome (PCOS), but the exact origin of hyperandrogenemia remains a matter of debate. Recent studies have highlighted the importance of the 11-oxygenated C19 steroid pathway to androgen metabolism in humans. In this study, we analyzed the contribution of 11-oxygenated androgens to androgen excess in women with PCOS. Methods: One hundred fourteen women with PCOS and 49 healthy control subjects underwent measurement of serum androgens by liquid chromatography-tandem mass spectrometry. Twenty-four-hour urinary androgen excretion was analyzed by gas chromatography-mass spectrometry. Fasting plasma insulin and glucose were measured for homeostatic model assessment of insulin resistance. Baseline demographic data, including body mass index, were recorded. Results: As expected, serum concentrations of the classic androgens testosterone (P < 0.001), androstenedione (P < 0.001), and dehydroepiandrosterone (P < 0.01) were significantly increased in PCOS. Mirroring this, serum 11-oxygenated androgens 11ß-hydroxyandrostenedione, 11-ketoandrostenedione, 11ß-hydroxytestosterone, and 11-ketotestosterone were significantly higher in PCOS than in control subjects, as was the urinary 11-oxygenated androgen metabolite 11ß-hydroxyandrosterone. The proportionate contribution of 11-oxygenated to total serum androgens was significantly higher in patients with PCOS compared with control subjects [53.0% (interquartile range, 48.7 to 60.3) vs 44.0% (interquartile range, 32.9 to 54.9); P < 0.0001]. Obese (n = 51) and nonobese (n = 63) patients with PCOS had significantly increased 11-oxygenated androgens. Serum 11ß-hydroxyandrostenedione and 11-ketoandrostenedione correlated significantly with markers of insulin resistance. Conclusions: We show that 11-oxygenated androgens represent the majority of circulating androgens in women with PCOS, with close correlation to markers of metabolic risk.

Inhibitory Effects of Vegetable Juices on CYP3A4 Activity in Recombinant CYP3A4 and LS180 Cells.

It is thought that eating habits induces individual variation in intestinal absorption and metabolism of drugs. The objective of this research was to clarify the influence of vegetables juices on CYP3A4 activity, which is an important enzyme in intestine. Five vegetables juices (VJ-o, Kagome Original(®); VJ-g, Kagome 30 kinds of vegetables and fruits(®); VJ-p, Kagome Purple vegetables(®); VJ-r, Kagome Sweet Tomato(®); and VJ-y, Kagome Fruity Salada(®); KAGOME Co., Ltd., Aichi, Japan) were centrifuged (1630×g, 10 min) and filtered using filter paper and 0.45-µm membrane filters. In this study, recombinant CYP3A4 and LS180 cells were used for the evaluation of CYP3A4 activity. The metabolisms to 6ß-hydroxytestosterone by recombinant CYP3A4 were significantly inhibited by VJ-o, VJ-g, and VJ-y in a preincubation time-dependent manner, and CYP3A4 activity in LS180 cells were significantly inhibited by VJ-o and VJ-y. These results show that the difference in ingestion volume of vegetable juices and vegetables might partially induce individual difference in intestinal drug metabolism.

6ß-hydroxytestosterone, a cytochrome P450 1B1 metabolite of testosterone, contributes to angiotensin II-induced hypertension and its pathogenesis in male mice.

Previously, we showed that Cyp1b1 gene disruption minimizes angiotensin II-induced hypertension and associated pathophysiological changes in male mice. This study was conducted to test the hypothesis that cytochrome P450 1B1-generated metabolites of testosterone, 6ß-hydroxytestosterone and 16α-hydroxytestosterone, contribute to angiotensin II-induced hypertension and its pathogenesis. Angiotensin II infusion for 2 weeks increased cardiac cytochrome P450 1B1 activity and plasma levels of 6ß-hydroxytestosterone, but not 16α-hydroxytestosterone, in Cyp1b1(+/+) mice without altering Cyp1b1 gene expression; these effects of angiotensin II were not observed in Cyp1b1(-/-) mice. Angiotensin II-induced increase in systolic blood pressure and associated cardiac hypertrophy, and fibrosis, measured by intracardiac accumulation of α-smooth muscle actin, collagen, and transforming growth factor-ß, and increased nicotinamide adenine dinucleotide phosphate oxidase activity and production of reactive oxygen species; these changes were minimized in Cyp1b1(-/-) or castrated Cyp1b1(+/+) mice, and restored by treatment with 6ß-hydroxytestoterone. In Cyp1b1(+/+) mice, 6ß-hydroxytestosterone did not alter the angiotensin II-induced increase in systolic blood pressure; the basal systolic blood pressure was also not affected by this agent in either genotype. Angiotensin II or castration did not alter cardiac, angiotensin II type 1 receptor, angiotensin-converting enzyme, Mas receptor, or androgen receptor mRNA levels in Cyp1b1(+/+) or in Cyp1b1(-/-) mice. These data suggest that the testosterone metabolite, 6ß-hydroxytestosterone, contributes to angiotensin II-induced hypertension and associated cardiac pathogenesis in male mice, most probably by acting as a permissive factor. Moreover, cytochrome P450 1B1 could serve as a novel target for developing agents for treating renin-angiotensin and testosterone-dependent hypertension and associated pathogenesis in males.

11ß-Hydroxydihydrotestosterone and 11-ketodihydrotestosterone, novel C19 steroids with androgenic activity: a putative role in castration resistant prostate cancer?

Adrenal C19 steroids, dehydroepiandrostenedione (DHEA(S)) and androstenedione (A4), play a critical role in castration resistant prostate cancer (CRPC) as they are metabolised to dihydrotestosterone (DHT), via testosterone (T), or via the alternate 5α-dione pathway, bypassing T. Adrenal 11OHA4 metabolism in CRPC is, however, unknown. We present a novel pathway for 11OHA4 metabolism in CRPC leading to the production of 11ketoT (11KT) and novel 5α-reduced C19 steroids - 11OH-5α-androstanedione, 11keto-5α-androstanedione, 11OHDHT and 11ketoDHT (11KDHT). The pathway was validated in the androgen-dependent prostate cancer cell line, LNCaP. Androgen receptor (AR) transactivation studies showed that while 11KT and 11OHDHT act as a partial AR agonists, 11KDHT is a full AR agonist exhibiting similar activity to DHT at 1nM. Our data demonstrates that, while 11OHA4 has negligible androgenic activity, its metabolism to 11KT and 11KDHT yields androgenic compounds which may be implicated, together with A4 and DHEA(S), in driving CRPC in the absence of testicular T.

Effect of androgens on different breast cancer cells co-cultured with or without breast adipose fibroblasts.

About 70% of breast tumors express androgen receptors. In addition, there is clinical evidence suggesting that androgens can inhibit mammary epithelial proliferation. Vice versa, there is also significant evidence indicating that androgens can increase the risk of breast cancer via multiple mechanisms, e.g. direct conversion to estrogens that can bind to the estrogen receptor and thereby stimulate cell proliferation. We examined the effect of testosterone (T) and dihydroxytestosterone (DHT) on cell proliferation, pS2 and Ki-67 expression in three different breast cancer cell lines alone or in co-culture with primary human breast adipose fibroblasts (BAFs) obtained from breast cancer patients. In the co-cultures, T induced cell proliferation, pS2 and Ki-67 expression in the estrogen receptor positive (ER(+)) MCF-7 and T47D cells. This was not observed in the (ER(-)) MDA-MB-231 cells. The differences might be explained by the high expression of aromatase, which converts androgens to estrogens in BAFs followed by ER-mediated cell proliferation. In line with this absence of increased cell proliferation, pS2 and Ki-67 expression was observed in the presence of DHT, which is not a substrate for aromatase. In contrast, DHT caused a significant suppression of cell proliferation (68% and 38%), pS2 and Ki-67 expression in the (ER(+)) MCF-7 and T47D cells. More importantly, DHT decreased cell proliferation in (ER(-)) MDA-MB-231 cells by 38%. The results suggest that androgens that cannot be aromatized, like DHT, may provide a perspective for treatment of breast cancer patients, especially those with triple negative breast cancer.

The ligands of estrogen receptor α regulate cytochrome P4502C9 (CYP2C9) expression.

Cytochrome P4502C9 (CYP2C9) is an important drug-metabolizing enzyme responsible for the metabolism of approximately 16% of all clinically relevant drugs. It was shown previously that the activity of CYP2C9 in vivo is inhibited by oral contraceptives. The mechanisms of this effect have not been elucidated. We hypothesize that this may occur because of the sex steroid-dependent activation of estrogen receptor α (ERα) with further transactivation of the CYP2C9 gene. Here, we show that the CYP2C9 promoter indeed contains a functionally relevant estrogen responsive element (ERE) half-site at position -149/-145. Its ERα binding activity was tested by the luciferase gene reporter assay. Promoter constructs bearing this site were cotransfected with ERα into Huh7 hepatoma cells and treated with various ERα ligands including 4-hydroxytamoxifen (4-OHT), raloxifene (R), 17ß-estradiol (EE), and 17α-ethinylestradiol (ETE). The luciferase activity driven by the wild-type CYP2C9 promoter construct was up-regulated by 4-OHT and R and significantly or marginally suppressed by ETE and EE, respectively. An identical effect was observed in primary hepatocytes treated with these compounds. Mutations introduced into the ERE half-site abolished the observed effects in the Huh7 cells. Electrophoretic mobility-shift assay revealed sequence-specific binding of a nuclear protein to the oligonucleotide containing the ERE half-site, which was identified as ERα by antibody supershift analysis. In addition, the association of ERα with CYP2C9 promoter was strongly supported by chromatin immunoprecipitation data. Taken together, these results indicate that ERα and its ligands play an important role in the regulation of CYP2C9 expression.

Dose-response relationship of 15alpha-hydroxylated sex steroids to gonadotropin-releasing hormones and pituitary extract in male sea lampreys (Petromyzon marinus).

The sea lamprey (Petromyzon marinus) is one of the earliest extant vertebrates for which the hypothalamic-pituitary-gonadal (HPG) axis has been shown to control and regulate reproduction in a similar fashion to gnathostome vertebrates. While the two forms of gonadotropin-releasing hormones in the sea lamprey (GnRH I and GnRH III) have been studied extensively, their in vivo effect on synthesis of 15alpha-hydroxytestosterone (15alpha-T) and 15alpha-hydroxyprogesterone (15alpha-P) have only been partially characterized. In the present study, plasma concentrations of 15alpha-T and 15alpha-P were measured in prespermiating sea lampreys that were given a single injection of either GnRH I or GnRH III in doses ranging from 5 to 100 microg/kg, or of pituitary extract (as a source of gonadotropin). Plasma was sampled at 1-6h and 6-48 h post-injection, in separate experiments, in order to characterize the peak and duration of responses. 15alpha-T plasma concentrations increased slightly in response to all three treatments, but not in a dose-dependent manner, and the timing of peak concentrations varied between doses. However, 15alpha-P plasma concentrations showed a greater range of response (between 1 and 100 ng/ml) and were clearly correlated with the injection dose. Plasma concentrations of 15alpha-P also responded to far lower doses of GnRH I and GnRH III than any other steroid previously investigated in lampreys. The plasma concentrations of 15alpha-P peaked at 6h after injection for all three treatments, and levels reached a mean of 53.1 ng/ml. In female lampreys that were injected twice with 50 microg/ml GnRH I or III, 15alpha-T concentrations did not exceed 0.5 ng/ml and 15alpha-P concentrations did not exceed 1 ng/ml. These results lend further support to the hypothesis that 15alpha-P plays an important role in the reproductive endocrinology of male lampreys.

Marijuana extracts possess the effects like the endocrine disrupting chemicals.

The progesterone 17alpha-hydroxylase activity, which is one of the steroidogenic enzymes in rat testis microsomes, was significantly inhibited by crude marijuana extracts from Delta(9)-tetrahydrocannabinolic acid (THCA)- and cannabidiolic acid (CBDA)-strains. Delta(9)-Tetrahydrocannabinol, cannabidiol and cannabinol also inhibited the enzymatic activity with relatively higher concentration (100-1000 microM). Testosterone 6beta- and 16alpha-hydroxylase activities together with androstenedione formation from testosterone in rat liver microsomes were also significantly inhibited by the crude marijuana extracts and the cannabinoids. Crude marijuana extracts (1 and 10 microg/ml) of THCA strain stimulated the proliferation of MCF-7 cells, although the purified cannabinoids (THC, CBD and CBN) did not show significant effects, such as the extract at the concentration of 0.01-1000 nM. These results indicate that there are some metabolic interactions between cannabinoid and steroid metabolism and that the constituents showing estrogen-like activity exist in marijuana.

Specific deletion of focal adhesion kinase suppresses tumor formation and blocks malignant progression.

We have generated mice with a floxed fak allele under the control of keratin-14-driven Cre fused to a modified estrogen receptor (CreER(T2)). 4-Hydroxy-tamoxifen treatment induced fak deletion in the epidermis, and suppressed chemically induced skin tumor formation. Loss of fak induced once benign tumors had formed inhibited malignant progression. Although fak deletion was associated with reduced migration of keratinocytes in vitro, we found no effect on wound re-epithelialization in vivo. However, increased keratinocyte cell death was observed after fak deletion in vitro and in vivo. Our work provides the first experimental proof implicating FAK in tumorigenesis, and this is associated with enhanced apoptosis.

The role of progesterone metabolism and androgen synthesis in renal blood pressure regulation.

11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a crucial role in converting hormonally active cortisol into inactive cortisone, conferring specificity onto the human mineralocorticoid receptor (MR). Progesterone binds with even higher affinity to the MR, but acts as an MR antagonist. How aldosterone is able to keep its function as predominant MR ligand in clinical situations with high progesterone concentrations, such as pregnancy, is not clear. We have shown in vitro that the human kidney possesses an effective enzyme system that metabolizes progesterone to inactive metabolites in a process similar to the inactivation of cortisol by 11beta-HSD2. In studies on patients with adrenal insufficiency, we have shown that the in vivo anti-mineralocorticoid activity of progesterone is diminished by inactivating metabolism of progesterone, local formation of the deoxycorticosterone mineralocorticoid from progesterone, and inhibition of 11beta-HSD2 by progesterone and its metabolites resulting in decreased inactivation of cortisol and hence increased MR binding by cortisol. The enzymes involved in progesterone metabolism are also responsible for the capability of the human kidney to convert pregnenolone to DHEA and androstenedione leading to the formation of active androgens, testosterone and 5alpha-DH-testosterone. Locally produced androgens might be responsible for the observed difference in blood pressure between men and women and higher susceptibility to hypertension in men.

Evidence for 15alpha- and 7alpha-hydroxylase activity in gonadal tissue of the early-life stages of sea lampreys, Petromyzon marinus.

Gonads of premetamorphosing larval (PML), transforming (TL) and newly metamorphosed (juvenile) sea lampreys (JL) (Petromyzon marinus) were incubated in vitro with tritiated pregnenolone ([(3)H]P(5)), progesterone ([(3)H]P(4)), and androstenedione ([(3)H]A(4)) to identify the major products of steroidogenesis in early developmental stages. Reverse-phase high-performance liquid chromatography, using two mobile phase gradients, was used to separate the radioactive steroid metabolites. 7alpha-Hydroxylase activity was evident, based on the loss of radioactivity from [(3)H]P(5) labelled at position 7, appearing as tritiated water, and on the appearance of radiolabelled 7alpha-hydroxypregnenolone in the incubation medium. In addition, there was evidence of the synthesis of 15alpha-hydroxylated steroids from the three steroid precursors used. For the progestogen precursors, one of the major 15alpha -hydroxylated metabolites synthesized by both testis and ovarian tissue co-eluted with authentic 15alpha-hydroxyprogesterone, and for [(3)H]A(4), the product was predominantly [(3)H]15alpha-hydroxyandrostenedione. Additional polar steroids were produced, some of which co-eluted with authentic 15alpha-hydroxytestosterone and 15alpha-hydroxyestradiol, whereas others could not be correlated with the authentic 15alpha- or 15beta-hydroxylated steroids available. Ovarian tissues from PML and TL developmental stages synthesized several very non-polar compounds, some of which were present as unconjugated compounds, and others only in the conjugated fraction. These molecules had retention times consistent with pregnanes, and their presence in the incubation medium was therefore indicative of the presence of 5alpha-reductase. These metabolites were not present in the incubation medium from testis, or the JL ovary, suggesting that there is no expression of 5alpha-reductase activity in these tissues. Traces of 17beta-estradiol were found in the incubation medium from ovarian tissue incubated with P(5), but not following incubation with P(4) or A(4). Testosterone was not present in the incubation medium from either ovarian or testis fragments incubated with any of the substrates used.