A relationship in adrenal androgen levels between mothers and their children from a dioxin-exposed region in Vietnam.

Over the past decades, southern Vietnam has been burdened by dioxins from contaminated herbicides sprayed during the Vietnam War. In a previous study, we found that dioxin exposure decreased levels of salivary dehydroepiandrosterone (DHEA), an adrenal androgen, in 3-year-old children. In present study, to assess the relationship between adrenal hormones disruption in lactating mothers and in children, we compared mother-child pairs from dioxin- and nondioxin-contaminated regions. In 2010 and 2011, mother-child pairs from a dioxin hotspot region (n=37) and a non-contaminated region (n=47) were recruited and donated breast milk and serum samples for dioxin and steroid hormones determination. Mothers were 20-30years old and had given birth to their first child between 4 and 16weeks previously. One year later, saliva samples were collected from the children. Dioxin levels in breast milk were determined by gas chromatography/high-resolution mass spectrometry. Salivary DHEA, cortisol in children and androstenedione (A-dione), estradiol, cortisol, and DHEA in maternal serum were analyzed by liquid chromatography/tandem mass spectrometry. Concentrations of dioxin congeners in the hotspot region were 2- to 5-fold higher than in samples from the non-contaminated region. Salivary DHEA levels in children and serum A-dione levels in mothers were significantly higher in the hotspot region; no difference was found in the levels of other hormones. Moreover, there was a significant positive correlation between the elevated hormone levels in mothers and children (r=0.62, p<0.001). Several dioxin congeners exhibited strong significant dose-response relationships with salivary DHEA and serum A-dione levels. Our findings suggest that dioxin disrupts adrenal androgens in mothers and breastfeeding children through the same mechanism.

The relationship between dioxins exposure and risk of prostate cancer with steroid hormone and age in Vietnamese men.

Although Vietnam's massive herbicide exposure in 1960s and 1970s was clearly injurious to health, not all causal relationships have been clarified. We therefore explored associations among dioxins, steroid hormones, age and prostate cancer risk in men. We compared serum levels of dioxin, steroid hormones and prostate specific antigen (PSA) in men aged 56-81years from herbicide-exposed hotspots (n=50) with those from non-sprayed regions (n=48). Mean serum levels of dioxin congeners in the hotspot group were 1.5-11.3 times higher than the non-sprayed group depending on specific compound. Levels of testosterone, estradiol and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) activity in the hotspot group were also significantly higher than in non-sprayed group. Estradiol levels were significantly related to levels of several specific dioxin derivatives in both group. Significant positive correlations were also found between DHT and 1234678-HpCDD or 1234678-HpCDF; and between 3ß-HSD activity and 123678-HxCDD, 123478-HxCDF, 123678-HxCDF, or HxCB#169. After adjusting for age, body mass index, and tobacco use, multiple linear regressions showed levels of dihydrotestosterone (DHT), estradiol, testosterone and 3ß-HSD activity were not associated with dioxins in the two groups; however, levels of DHT, testosterone and 3ß-HSD activity increased significantly with age in the hotspot group. The hotspot and non-sprayed groups did not significantly differ in PSA levels. But six of the hotspot subjects had PSA levels >3ng/mL, 3 of whom were suspected to have prostate cancer (PC) after digital rectal examination. Our findings suggest that dioxin exposure can lead to increased levels of several sex steroid hormones with age. The correlation of dioxin with steroid hormone levels and prostate cancer risk should be studied further.

Influence of dioxin exposure upon levels of prostate-specific antigen and steroid hormones in Vietnamese men.

Most studies on the relationship between Agent Orange and prostate cancer have focused on US veterans of the Vietnam War. There have been few studies focusing on the relationship between levels of prostate-specific antigen (PSA) and dioxins or steroid hormones in Vietnamese men. In 2009-2011, we collected blood samples from 97 men who had resided in a "dioxin hotspot" and 85 men from a non-sprayed region in Vietnam. Then levels of PSA, dioxins, and steroid hormones were analyzed. Levels of most dioxins, furans, and non-ortho polychlorinated biphenyls were higher in the hotspot than those in the non-sprayed region. Levels of testosterone, dehydroepiandrosterone, and estradiol differed significantly between the hotspot and the non-sprayed region, but there were no correlations between levels of PSA and steroid hormones and dioxins in either of the two regions. Our findings suggest that PSA levels in Vietnamese men are not associated with levels of dioxin or steroid hormones in these two regions.

Low serum dehydroepiandrosterone examined by liquid chromatography-tandem mass spectrometry correlates with poor prognosis in hormone-naïve prostate cancer.

BACKGROUND: There is no consensus on blood adrenal androgen concentrations in men with different stages and pathological grades of prostate cancer. In this study, dehydroepiandrosterone (DHEA) concentrations in blood were examined by ultrasensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS). We analyzed the correlation between DHEA concentrations in blood and clinicopathological findings of prostate cancer. METHODS: We analyzed 196 men (mean age 70 years) with prostate cancer. The patients underwent systematic needle biopsy, and peripheral blood sampling was conducted for measurement of DHEA. DHEA concentrations in blood were determined using LC-MS/MS method. Patient age, serum prostate-specific antigen, prostate volume measured by ultrasound, and DHEA levels in blood were compared with Gleason score and clinical stage by multivariate analyses. RESULTS: Median value of PSA and prostate volume were 11.5 ng/ml and 27.7 ml, respectively. Median concentration of DHEA in blood was 1,506.4 pg/ml. There was no correlation between serum DHEA and clinical variables such as age, serum PSA, and prostate volume. In multivariate analysis, low serum DHEA levels in prostate cancer patients were significantly related to high Gleason score and advanced clinical stage. Serum PSA levels in prostate cancer patients were also significantly associated with high Gleason score and advanced clinical stage. High serum PSA and low serum DHEA levels were significantly associated with poor prognosis factors in men with hormone-naïve prostate cancer. CONCLUSIONS: DHEA concentrations in blood were examined by newly developed ultrasensitive LC-MS/MS. We confirmed that low serum DHEA levels in prostate cancer patients were related to high Gleason score and advanced clinical stage. These results suggest that serum DHEA level may be a useful prognostic factor in prostate cancer patients.

Measurement of peripheral plasma 18-oxocortisol can discriminate unilateral adenoma from bilateral diseases in patients with primary aldosteronism.

Adrenal venous sampling is currently the only reliable method to distinguish unilateral from bilateral diseases in primary aldosteronism. In this study, we attempted to determine whether peripheral plasma levels of 18-oxocortisol (18oxoF) and 18-hydroxycortisol could contribute to the clinical differentiation between aldosteronoma and bilateral hyperaldosteronism in 234 patients with primary aldosteronism, including computed tomography (CT)-detectable aldosteronoma (n=113) and bilateral hyperaldosteronism (n=121), all of whom underwent CT and adrenal venous sampling. All aldosteronomas were surgically resected and the accuracy of diagnosis was clinically and histopathologically confirmed. 18oxoF and 18-hydroxycortisol were measured using liquid chromatography tandem mass spectrometry. Receiver operating characteristic analysis of 18oxoF discrimination of adenoma from hyperplasia demonstrated sensitivity/specificity of 0.83/0.99 at a cut-off value of 4.7 ng/dL, compared with that based on 18-hydroxycortisol (sensitivity/specificity: 0.62/0.96). 18oxoF levels above 6.1 ng/dL or of aldosterone >32.7 ng/dL were found in 95 of 113 patients with aldosteronoma (84%) but in none of 121 bilateral hyperaldosteronism, 30 of whom harbored CT-detectable unilateral nonfunctioning nodules in their adrenals. In addition, 18oxoF levels below 1.2 ng/dL, the lowest in aldosteronoma, were found 52 of the 121 (43%) patients with bilateral hyperaldosteronism. Further analysis of 27 patients with CT-undetectable micro aldosteronomas revealed that 8 of these 27 patients had CT-detectable contralateral adrenal nodules, the highest values of 18oxoF and aldosterone were 4.8 and 24.5 ng/dL, respectively, both below their cut-off levels indicated above. The peripheral plasma 18oxoF concentrations served not only to differentiate aldosteronoma but also could serve to avoid unnecessary surgery for nonfunctioning adrenocortical nodules concurrent with hyperplasia or microadenoma.

High testosterone levels in prostate tissue obtained by needle biopsy correlate with poor-prognosis factors in prostate cancer patients.

BACKGROUND: There is currently no consensus on the correlations between androgen concentrations in prostate tissue and blood and stage and pathological grade of prostate cancer. In this study, we used a newly-developed ultra-sensitive liquid-chromatography tandem mass spectrometry method to measure testosterone (T) and dihydrotestosterone (DHT) concentrations in blood and needle biopsy prostate specimens from patients with prostate cancer. METHODS: We analyzed androgen levels in 196 men diagnosed with prostate cancer. All patients had undergone systematic needle biopsy, and an additional needle biopsy from the peripheral zone was conducted for the simultaneous determination of T and DHT. We analyzed the relationships between T and DHT levels in tissue and blood and Gleason score, clinical stage, and percentage of positive biopsy cores, using multivariate analysis. RESULTS: The median T and DHT levels in blood were 3551.0 pg/mL and 330.5 pg/mL, respectively. There was a strong correlation between serum T and DHT. The median T and DHT levels in prostate tissue were 0.5667 pg/mg and 7.0625 pg/mg, respectively. In multivariate analysis, serum prostate-specific antigen and tissue T levels were significantly associated with poor prognosis; high T levels in prostate tissue were significantly related to high Gleason score (p = 0.041), advanced clinical stage (p = 0.002), and a high percentage of positive biopsy cores (p = 0.001). CONCLUSIONS: The results of this study indicate that high T levels in prostate tissue are related to high Gleason score, advanced clinical stage, and a high percentage of positive biopsy cores in patients with prostate cancer. T level in needle biopsy specimens may therefore be a useful prognostic factor in prostate cancer patients.

High cortisol and cortisone levels are associated with breast milk dioxin concentrations in Vietnamese women.

OBJECTIVE: Dioxin (polychlorinated dibenzodioxins+polychlorinated dibenzofurans) is one of the most toxic chemical substances known. Although it is suspected to cause endocrine disruption, very few epidemiological studies have been carried out on its effects on human steroid hormones. The aim of this study was to elucidate the association of dioxin exposure with steroid hormone levels in the saliva and serum of Vietnamese women. STUDY DESIGN: Two areas, namely Phu Cat (hot spot) and Kim Bang (nonexposed area), were selected for the study. The study subjects consisted of 51 and 58 women respectively. Saliva, blood, and breast milk samples were collected from the subjects in both the areas. METHODS: Cortisol, cortisone, DHEA, androstenedione, estrone, and estradiol levels in serum and saliva were determined by liquid chromatography-tandem mass spectrometry; dioxin concentrations in breast milk were measured by gas chromatography-mass spectrometry. RESULTS: Dioxin concentrations in the breast milk of women from the dioxin hot spot were three to four times higher than those in the breast milk of women from the nonexposed area. Good correlations were found between the levels of six steroid hormones in saliva and those in serum respectively. Salivary and serum cortisol and cortisone levels in women from the dioxin hot spot were significantly higher than those in women from the nonexposed area (P<0.001) and those in all the subjects were positively associated with dioxin concentrations in Vietnamese women (P<0.01). CONCLUSION: These results suggest that dioxin influences steroidogenesis in humans. Saliva samples can be used for hormone analysis and are therefore excellent specimens in epidemiological studies.

Liquid chromatography-tandem mass spectrometry analysis of human adrenal vein 19-carbon steroids before and after ACTH stimulation.

CONTEXT: A broad analysis of adrenal gland-derived 19-carbon (C19) steroids has not been reported. This is the first study that uses liquid chromatography-tandem mass spectrometry to quantify 9 C19 steroids (androgens and their precursors), estrone, and estradiol in the adrenal vein (AV) of women, before and after ACTH stimulation. OBJECTIVE: The objective of this study was to define the adrenal androgen metabolome in women before and after ACTH infusion. DESIGN: This was a retrospective study. PATIENTS: Seven women, aged 50.4 ± 5.4 years, with suspected diagnosis of an adrenal aldosterone-producing adenoma were included in the study. METHODS: AV and iliac serum samples were collected before and after administration of ACTH (15 minutes). AV samples were analyzed using for concentrations of 9 unconjugated C19 steroids, estrone, and estradiol. Dehydroepiandrosterone sulfate (DHEA-S) was quantified by radioimmunoassay. RESULTS: AV levels of DHEA-S were the highest among the steroids measured. The most abundant unconjugated C19 steroids in AV were 11ß-hydroxyandrostenedione (11OHA), dehydroepiandrosterone (DHEA), and androstenedione (A4). ACTH significantly increased the adrenal output of 9 of the 12 steroids that were measured. ACTH increased the mean AV concentration of DHEA-S by 5-fold, DHEA by 21-fold, A4 by 7-fold, and 11OHA by 5-fold. 11ß-Hydroxytestosterone and testosterone were found to be potent androgen receptor agonists when tested with an androgen-responsive cell reporter model. CONCLUSION: The current study indicates that the adrenal gland secretes primarily 3 weak androgens, namely DHEA, 11OHA, and A4. Active androgens, including testosterone and 11ß-hydroxytestosterone, are also produced but to a lesser degree.

Liquid chromatography-tandem mass spectrometry analysis of human adrenal vein corticosteroids before and after adrenocorticotropic hormone stimulation.

CONTEXT: Although steroid hormones produced by the adrenal gland play critical roles in human physiology, a detailed quantitative analysis of the steroid products has not been reported. The current study uses a single methodology (liquid chromatography-tandem mass spectrometry, LC-MS/MS) to quantify ten corticosteroids in adrenal vein (AV) samples pre- and post-adrenocorticotropic hormone (ACTH) stimulation. DESIGN/METHODS: Three men and six women with a diagnosis of an adrenal aldosterone-producing adenoma (APA) were included in the study. Serum was collected from the iliac vein (IV) and the AV contralateral to the diseased adrenal. Samples were collected, before and after administration of ACTH. LC-MS/MS was then used to quantify serum concentrations of unconjugated corticosteroids and their precursors. RESULTS: Prior to ACTH stimulation, the four most abundant steroids in AV were cortisol (90%), cortisone (4%), corticosterone (3%) and 11-deoxycortisol (0.8%). Post-ACTH administration, cortisol remained the major adrenal product (79%); however, corticosterone became the second most abundantly produced adrenal steroid (11%) followed by pregnenolone (2.5%) and 17α-hydroxypregnenolone (2%). ACTH significantly increased the absolute adrenal output of all ten corticosteroids measured (P < 0.05). The four largest post-ACTH increases were pregnenolone (300-fold), progesterone (199-fold), 17α-hydroxypregnenolone (187-fold) and deoxycorticosterone (82-fold). CONCLUSION: Using LC-MS/MS, we successfully measured 10 corticosteroids in peripheral and AV serum samples under pre- and post-ACTH stimulation. This study demonstrates the primary adrenal steroid products and their response to ACTH.

18-oxocortisol measurement in adrenal vein sampling as a biomarker for subclassifying primary aldosteronism.

CONTEXT: 18-Oxocortisol (18-oxoF) is a derivative of cortisol (F) that is produced by aldosterone synthase (CYP11B2). The potential for this steroid as a biomarker for differentiating patients with aldosterone-producing adenoma (APA) from those with idiopathic hyperaldosteronism (IHA) has not been examined. OBJECTIVES: We measured 18-oxoF, aldosterone, and F in plasma from adrenal vein sampling (AVS) of patients with primary aldosteronism. We compared 18-oxoF levels and 18-oxoF/F ratios for their potential to differentiate APA from IHA. DESIGN, SETTING, AND SUBJECTS: This study measured 18-oxoF, F, and aldosterone in AVS obtained from patients with unilateral APA (14 cases) or bilateral IHA (seven cases, 14 samples total) using liquid chromatography-tandem mass spectrometry and RIA analyses. RESULTS: The levels of 18-oxoF and the ratios of 18-oxoF/F, before and after ACTH stimulation, were significantly higher in blood-draining APA than in those from the contralateral adrenal glands and from adrenal glands with IHA. CONCLUSIONS: The 18-oxoF levels and ratios of 18-oxoF/F in AVS samples can be a clinically useful biomarker for differentiating APA from IHA and for determining the localization or lateralization of APA in patients with primary aldosteronism.

Effect of castration monotherapy on the levels of adrenal androgens in cancerous prostatic tissues.

The mechanism accounting for the development of castration-resistant prostate cancer (CRPC) remains unclear. Studies in CRPC tissues suggest that, after androgen deprivation therapy (ADT), the adrenal androgens may be an important source of testosterone (T) and 5-alpha dihydrotestosterone (DHT) in CRPC tissues. To clarify the role of adrenal androgens in the prostatic tissues (prostatic tissue adrenal androgens) during ADT, we developed a high sensitive and specific quantification method for the levels of androgens in prostatic tissue using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Human prostatic tissues were purified using mixed-mode reversed-phase, strong anion exchange Oasis cartridges (Oasis MAX). Analysis of steroids was performed using LC-MS/MS after picolinic acid derivatization. The validation tests showed that our method of quantitative analysis was precise and sensitive enough for the quantification of dehydroepiandrosterone (DHEA), androstenedione, androstenediol, T, and DHT in the prostatic tissue. The levels of adrenal androgens in prostate cancer tissues after ADT were similar to those in untreated PCa. Especially, DHEA was the most existing androgen precursor in PCa tissues after ADT. The levels of DHEA were high in PCa tissues, irrespective of ADT. We assumed that DHEA played a significant role in the synthesis of T and DHT in PCa tissues after ADT.

Testosterone-lowering activity of canola and hydrogenated soybean oil in the stroke-prone spontaneously hypertensive rat.

Canola and some other types of oil unusually shorten the survival of stroke-prone spontaneously hypertensive rats (SHRSP), compared with soybean oil, perilla oil and animal fats. Since differential effects of canola and soybean oil on steroid hormone metabolism were suggested by a preliminary DNA microarray analysis as a reason for this, the steroid hormone levels in the serum and tissues of SHRSP fed different oils were investigated. The testosterone levels in the serum and the testes were found to be significantly lower in the canola oil group than in the soybean oil group, while no significant differences were detected in the corticosterone and estradiol levels in tissues. In a second experiment, it was found that hydrogenated soybean oil, with a survival-shortening activity comparable to that of canola oil, also decreased the testosterone level in testes to a similar degree. The testosterone-lowering activity of canola and hydrogenated soybean oil observed in SHRSP was considered in relation to other factors possibly affecting the physiology of SHRSP.

Immunolocalization of estrogen-producing and metabolizing enzymes in benign breast disease: comparison with normal breast and breast carcinoma.

It is well known that estrogens play important roles in the cell proliferation of breast carcinoma. Benign breast disease (BBD) contains a wide spectrum of diseases, and some are considered an important risk factor for subsequent breast carcinoma development. However, the significance of estrogens in BBD has remained largely unknown. Therefore, in this study, we examined tissue concentrations of estrogens and immunolocalization of estrogen-producing/metabolizing enzymes in BBD, and compared these findings with those in the normal breast and ductal carcinoma in situ (DCIS). Tissue concentration of estradiol in BBD (n = 9) was significantly (3.4-fold) higher than normal breast (n = 9) and nearly the same (0.7-fold) as in DCIS (n = 9). Immunoreactivity of estrogen sulfotransferase in BBD was significantly lower (n = 82) than normal breast (n = 28) but was not significantly different from DCIS (n = 28). Aromatase and steroid sulfatase immunoreactivities tended to be higher (P = 0.07) in BBD than in normal breast, and 17ß-hydroxysteroid dehydrogenase type 1 immunoreactivity was significantly higher in BBD than normal breast in the postmenopausal tissues. Immunoreactivity of estrogen and progesterone receptors was also significantly higher in BBD than normal breast. These results suggest that tissue concentration of estradiol is increased in BBD at a level similar to DCIS, which is considered mainly due to loss of estrogen sulfotransferase expression. Increased local estradiol concentration in BBD due to aberrant expression of estrogen-producing/metabolizing enzymes may play important roles in the accumulation of estradiol-mediated growth and/or subsequent development of breast carcinoma.

5alphaDH-DOC (5alpha-dihydro-deoxycorticosterone) activates androgen receptor in castration-resistant prostate cancer.

Prostate cancer often relapses during androgen-depletion therapy, even under the castration condition in which circulating androgens are drastically reduced. High expressions of androgen receptor (AR) and genes involved in androgen metabolism indicate a continued role for AR in castration-resistant prostate cancers (CRPCs). There is increasing evidence that some amounts of 5alpha-dihydrotestosterone (DHT) and other androgens are present sufficiently to activate AR within CRPC tissues, and enzymes involved in the androgen and steroid metabolism, such as 5alpha-steroid reductases, are activated in CRPCs. In this report, we screened eight natural 5alphaDH-steroids to search for novel products of 5alpha-steroid reductases, and identified 11-deoxycorticosterone (DOC) as a novel substrate for 5alpha-steroid reductases in CRPCs. 11-Deoxycorticosterone (DOC) and 5alpha-dihydro-deoxycorticosterone (5alphaDH-DOC) could promote prostate cancer cell proliferation through AR activation, and type 1 5alpha-steroid reductase (SRD5A1) could convert from DOC to 5alphaDH-DOC. Sensitive liquid chromatography-tandem mass spectrometric analysis detected 5alphaDH-DOC in some clinical CRPC tissues. These findings implicated that under an extremely low level of DHT, 5alphaDH-DOC and other products of 5alpha-steroid reductases within CRPC tissues might activate the AR pathway for prostate cancer cell proliferation and survival under castration.

New quantification method for estradiol in the prostatic tissues of benign prostatic hyperplasia using liquid chromatography-tandem mass spectrometry.

Estrogen is suspected to play a role in the pathogenesis of benign prostatic hyperplasia (BPH) and prostate cancer. To clarify the role of estradiol (E2) in the prostatic tissues (prostatic tissue E2) during the development of prostatic disorders, we developed a new sensitive and specific quantification method for prostatic tissue E2 using liquid chromatography-tandem mass spectrometry (LC-MS/MS). For the solid-phase extraction, E2 was purified by anion-exchange through an Oasis MAX cartridge. In addition, after the formation of 3-pentaflurobenzyl-17beta-pyridinium-estradiol derivative (E2-PFBPY), E2-PFBPY was purified by cation-exchange through an Oasis WCX cartridge. These processes in the LC-MS/MS method improved the specificity and sensitivity for prostatic tissue E2 measurement, compared to the radioimmunoassay (RIA) method. The validation tests showed that intra-day and inter-day precisions were both within +/-15% (except for 15.5% of the inter-day precision of the lowest concentration), with the accuracy ranging from 88 to 110%. The quantification limit of this assay was 0.15pg/tube in our method, which was 80-fold more sensitive than that of the RIA method. With the use of our present method, the median E2 levels in the prostatic tissues in patients with BPH (n=20, median age: 71 years) were 12.0pg/g tissue (95% confidence interval=9.1-22.6pg/g tissue). Furthermore, the E2 levels increased significantly with aging. These results showed that our present method would be useful for elucidating the role of prostatic tissue E2 in the development of prostatic disorders with a small amount of tissue samples.

Prostate cancer stromal cells and LNCaP cells coordinately activate the androgen receptor through synthesis of testosterone and dihydrotestosterone from dehydroepiandrosterone.

One of the mechanisms through which advanced prostate cancer (PCa) usually relapses after androgen deprivation therapy (ADT) is the adaptation to residual androgens in PCa tissue. It has been observed that androgen biosynthesis in PCa tissue plays an important role in this adaptation. In the present study, we investigated how stromal cells affect adrenal androgen dehydroepiandrosterone (DHEA) metabolism in androgen-sensitive PCa LNCaP cells. DHEA alone had little effect on prostate-specific antigen (PSA) promoter activity and the proliferation of LNCaP cells. However, the addition of prostate stromal cells or PCa-derived stromal cells (PCaSC) increased DHEA-induced PSA promoter activity via androgen receptor activation in the LNCaP cells. Moreover, PCaSC stimulated the proliferation of LNCaP cells under physiological concentrations of DHEA. Biosynthesis of testosterone or dihydrotestosterone from DHEA in stromal cells and LNCaP cells was involved in this stimulation of LNCaP cell proliferation. Androgen biosynthesis from DHEA depended upon the activity of various steroidogenic enzymes present in stromal cells. Finally, the dual 5alpha-reductase inhibitor dutasteride appears to function not only as a 5alpha-reductase inhibitor but also as a 3beta-hydroxysteroid dehydrogenase inhibitor in LNCaP cells. Taken together, this coculture assay system provides new insights of coordinate androgen biosynthesis under the microenvironment of PCa cells before and after ADT, and offers a model system for the identification of important steroidogenic enzymes involved in PCa progression and for the development of the corresponding inhibitors of androgen biosynthesis.

Intratumoral estrogens and estrogen receptors in human non-small cell lung carcinoma.

PURPOSE: The possible involvement of gender-dependent factors has been suggested in human non-small cell lung carcinomas (NSCLC), but their precise roles remain largely unclear. Therefore, we examined intratumoral estradiol concentrations in NSCLC to examine local actions of estrogens in NSCLC. EXPERIMENTAL DESIGN: Fifty-nine frozen specimens of NSCLC were available for liquid chromatography/electrospray tandem mass spectrometry to study intratumoral estradiol concentrations. In addition, A549 NSCLC cells stably expressing estrogen receptor (ER) alpha (A549 + ERalpha) or ERbeta (A549 + ERbeta) were used in vitro studies. RESULTS: Forty-three (73%) of 59 NSCLC showed higher concentration of estradiol in carcinoma tissues than the corresponding nonneoplastic lung tissues from the same patient, and intratumoral estradiol concentrations were significantly (P = 0.0002 and 2.2-fold) higher than the corresponding nonneoplastic lungs. The intratumoral concentration of estradiol was positively correlated with aromatase expression, tumor size, and Ki-67 status in ERalpha- or ERbeta-positive cases. In in vitro studies, estradiol significantly increased cell proliferation of A549 + ERalpha or A549 + ERbeta, which was significantly suppressed by selective ER modulators, tamoxifen or raloxifene. Both A549 + ERalpha and A549 + ERbeta cells expressed aromatase. The cell proliferation level in these cells was significantly increased under treatment with testosterone, and it was inhibited by addition of the aromatase inhibitor letrozole. CONCLUSIONS: These results suggest that estradiol is locally produced in NSCLC mainly by aromatase and plays an important role in the growth of ERalpha- or ERbeta-positive NSCLC. Therefore, use of selective ER modulators and/or aromatase inhibitors may be clinically effective in NSCLC that are positive for both ER and aromatase.

Novel 5 alpha-steroid reductase (SRD5A3, type-3) is overexpressed in hormone-refractory prostate cancer.

Prostate cancer often relapses during androgen-depletion therapy, even under conditions in which a drastic reduction of circulating androgens is observed. There is some evidence that androgens remain present in the tissues of hormone-refractory prostate cancers (HRPC), and enzymes involved in the androgen and steroid metabolic pathway are likely to be active in HRPC cells. We previously carried out a genome-wide gene expression profile analysis of clinical HRPC cells by means of cDNA microarrays in combination with microdissection of cancer cells and found dozens of transactivated genes. Among them, we here report the identification of a novel gene, SRD5A2L, encoding a putative 5 alpha-steroid reductase that produces the most potent androgen, 5 alpha-dihydrotestosterone (DHT), from testosterone. Liquid chromatography-tandem mass spectrometry analysis following an in vitro 5 alpha-steroid reductase reaction validated its ability to produce DHT from testosterone, similar to type 1 5 alpha-steroid reductase. Because two types of 5 alpha-steroid reductase were previously reported, we termed this novel 5 alpha-steroid reductase 'type 3 5 alpha-steroid reductase' (SRD5A3). Reverse transcription-polymerase chain reaction and northern blot analyses confirmed its overexpression in HRPC cells, and indicated no or little expression in normal adult organs. Knockdown of SRD5A3 expression by small interfering RNA in prostate cancer cells resulted in a significant decrease in DHT production and a drastic reduction in cell viability. These findings indicate that a novel type 3 5 alpha-steroid reductase, SRD5A3, is associated with DHT production and maintenance of androgen-androgen receptor-pathway activation in HRPC cells, and that this enzymatic activity should be a promising molecular target for prostate cancer therapy.

Neonatal exposure to estrogen in the Wistar rat decreases estrogen receptor-beta and induces epithelial proliferation of the prostate in the adult.

BACKGROUND/AIMS: The effects of environmental endocrine disruptors on the male reproductive system have received much attention. We attempted to assess the responsible reproduction period vulnerable to decrease in ER beta mRNA by exposing neonate rats to disruptors. METHODS: Each of 64 male Wistar rats was given an injection of estradiol at a dose of 25 mug or oil on days 1, 3 and 5 after birth. These rats were sacrificed on days 80, 120, 160 and 180 and then subjected to measurements of both serum and tissue testosterone levels. Real-time quantitative polymerase chain reaction (PCR) was used to measure the levels of androgen receptor (AR) and estrogen receptor-beta (ER beta) mRNA of the ventral prostate. Histological compositions were analyzed by quantitative morphometry. Immunohistochemical analysis of proliferating cell nuclear antigen (PCNA) was determined. RESULTS: The incidence of epithelial components by histomorphometry and the expression of PCNA was significantly higher in the estrogen group in the mature adult (day 160). Levels of AR and ER beta mRNAs in the estrogen group were significantly lower in middle-aged rats (day 180). CONCLUSION: Estrogen exposure in the neonatal period to Wistar rats decreases the number of ER beta in the mature adult and accelerates cell proliferation.

Highly sensitive determination of estrone and estradiol in human serum by liquid chromatography-electrospray ionization tandem mass spectrometry.

A highly sensitive and specific quantification method of estrone and estradiol in human serum was described based upon the use of picolinoyl derivatization and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) in a positive mode. Estrogens were treated with picolinoyl chloride hydrochloride or picolinic acid and 2-methyl-6-nitrobenzoic anhydride followed by a solid-phase extraction with ODS cartridge. Picolinoyl derivatization proceeded quantitatively even in a microscale, and the picolinoyl esters provided simple positive ESI-mass spectra showing [M+H](+) as base peaks for these estrogens. The picolinoyl derivatives of these estrogens showed 100-fold higher detection response compared to underivatized intact molecules by LC-ESI-MS (selected reaction monitoring). Using this derivatization, estrogens spiked in the charcoal treated human serum samples were analyzed with limit of quantification (LOQ), intra-day accuracy and precision of 1.0pg/ml, 96.0% and 9.9% for estrone, and 0.5pg/ml, 84.4% and 12.8% for estradiol, respectively. Estrone and estradiol added to the crude serum samples were recovered with comparable LOQ and accuracy obtained for the charcoal treated serum samples as well.