Estrone Sulfate Research Articles

Organic anion-transporting polypeptide (OATP) 2B1 contributes to the cellular uptake of theaflavin

Organic anion-transporting polypeptide (OATP) 2B1 has been reported in the apical membranes of the human small intestinal epithelium, where it contributes to the intestinal absorption of pharmacologically active drugs. To investigate the potential for OATP2B1-mediated drug–food interactions, the effects of several polyphenolic compounds on OATP2B1-mediated estrone-3-sulfate (E3S) transport were studied by using OATP2B1-expressing HEK293 cells. Our results showed that some compounds, especially theaflavin, were strong inhibitors of OATP2B1-mediated E3S uptake. Theaflavin showed a significantly higher uptake into the OATP2B1-expressing HEK293 cells than the control cells. The concentration dependence of the uptake of theaflavin was determined over a range of concentrations (0.5–100 μM) and the kinetic parameters (Km and Vmax) of theaflavin uptake were found to be 5.12 ± 0.67 μM and 41.6 ± 1.3 pmol/mg protein/min, respectively. The OATP2B1-mediated theaflavin uptake was inhibited by known OATP2B1 substrates such as E3S, bromsulphthalein (BSP), dehydroepiandrosterone-3-sulfate (DHEAS), and fluvastatin. Our results indicate that theaflavin is a novel substrate of OATP2B1. The results of this study might be helpful to predict the potential OATP2B1-mediated drug–theaflavin interactions and to avoid undesirable clinical consequences.

Counter-flow suggests transport of dantrolene and 5-OH dantrolene by the organic anion transporters 2 (OAT2) and 3 (OAT3).

Dantrolene is the only available drug for the treatment of malignant hyperthermia, a life-threatening inborn sensitivity of the ryanodine receptor (RyR1) in skeletal muscles to volatile anesthetics. Dantrolene is metabolized in the liver to 5-OH dantrolene. Both compounds are zwitterions or net negatively charged. Here, we investigated interactions of dantrolene and 5-OH dantrolene with solute carrier (SLC) family members occurring in skeletal muscle cells, hepatocytes, and renal proximal tubule cells. SLC22A8 (organic anion transporter 3, OAT3) was very sensitive to both compounds exhibiting IC50 values of 0.35±0.03 and 1.84±0.34μM, respectively. These IC50 concentrations are well below the plasma concentration in patients treated with dantrolene (3-28μM). SLC22A7 (OAT2) was less sensitive to dantrolene and 5-OH dantrolene with IC50 values of 15.6±2.1 and 15.8±3.2μM, respectively. SLCO1B1 (OATP1B1), SLCO1B3 (OATP1B3), and SLCO2B1 (OATP2B1) mainly interacted with 5-OH dantrolene albeit with higher IC50 values than those observed for OAT2 and OAT3. Dantrolene and 5-OH dantrolene failed to inhibit uptake of 1-methyl-4-phenylpyrimidinium (MPP) by OCT1 and of carnitine by OCTN2. In counter-flow experiments on OAT3, dantrolene and 5-OH dantrolene decreased pre-equilibrated cellular [3H]estrone-3-sulfate (ES) content as did the transported substrates glutarate, furosemide, and bumetanide. With OAT2, dantrolene and 5-OH dantrolene slightly decreased the pre-equilibrated [3H]cGMP content. If no other transporter markedly contributes to uptake or release of ES or cGMP, respectively, these data suggest that OAT3 and OAT2 may be involved in absorption, metabolism, and excretion of dantrolene and its metabolite 5-OH dantrolene.

Roles of organic anion transporters (OATs) in renal proximal tubules and their localization.

Organic anions (OAs) are secreted in renal proximal tubules in two steps. In the first step, OAs are transported from the blood through basolateral membranes into proximal tubular cells. The prototypical substrate for renal organic anion transport systems, para-aminohippurate (PAH), is transported across basolateral membranes of proximal tubular cells via OAT1 (SLC22A6) and OAT3 (SLC22A8) against an electrochemical gradient in exchange for intracellular dicarboxylates. In the second step, OAs exit into urine through apical membranes of proximal tubules. This step is thought to be performed by multidrug efflux transporters and a voltage-driven organic anion transporter. However, the molecular nature and precise functional properties of these efflux systems are largely unknown. Recently, we characterized an orphan transporter known as human type I sodium-phosphate transporter 4, hNPT4 (SLC17A3), using the Xenopus oocyte expression system. hNPT4 acts as a voltage-driven efflux transporter ("human OATv1") for several OAs such as PAH, estrone sulfate, diuretic drugs, and urate. Here, we describe a model for an OA secretory pathway in renal tubular cells in which OAs exit cells and enter the tubular lumen via hOATv1 (hNPT4). Additionally, hOATv1 functions as a common renal secretory pathway for both urate and drugs, indicating that hOATv1 may be a leak pathway for excess urate that is reabsorbed via apical URAT1 to control the intracellular urate levels. Therefore, we propose a molecular mechanism for the induction of hyperuricemia by diuretics: the diuretics enter proximal tubular cells via basolateral OAT1 and/or OAT3 and may then interfere with the NPT4-mediated apical urate efflux in the renal proximal tubule.

Tryptophan Residue Located at the Middle of Putative Transmembrane Domain 11 Is Critical for the Function of Organic Anion Transporting Polypeptide 2B1

Organic anion transporting polypeptide 2B1 (OATP2B1), which is highly expressed in enterocytes and hepatocytes could be a key determinant for the intestinal absorption and hepatic uptake of its substrates, most of which are amphipathic organic anions. Tryptophan residues may possess a multitude of functions for a transport protein through aromatic interactions, such as maintaining the proper protein structure, guiding the depth of membrane insertion, or interacting directly with substrates. There are totally six tryptophan residues in OATP2B1. However, little is known about their role in the function and expression of OATP2B1. Our results show that, while W272, W276, and W277 located at the border of extracellular loop 3 and transmembrane domain 6 exhibit a moderate effect on the surface expression of OATP2B1, W611 located at the middle of transmembrane domain 11 plays a critical role in the function of OATP2B1. The tryptophan-to-alanine mutation of W611 changes the kinetic characteristics of OATP2B1-mediated estrone-3-sulfate (E3S) transport radically, from a monophasic saturation curve (with Km and Vmax values being of 7.1 ± 1.1 μM and 182 ± 7 pmol/normalized mg/min, respectively) to a linear curve. Replacing alanine with a phenylalanine will rescue most of OATP2B1's function, suggesting that the aromatic side chain of residue 611 is very important. However, hydrogen-bond forming and positively charged groups at this position are not favorable. The important role of W611 is not substrate-dependent. Molecular modeling indicates that the side chain of W611 faces toward the substrate translocation pathway and might interact with substrates directly. Taken together, our findings reveal that W611 is critical for the function of OATP2B1.

Influence of SULT1A1 genetic variation on age at menopause, estrogen levels, and response to hormone therapy in recently postmenopausal white women.

Objective:Onset and symptoms of menopause, and response to hormone therapy (HT) show large interindividual variability. SULT1A1 encodes for a highly expressed enzyme that metabolizes estrogens. We evaluated the relationship between genetic variation in SULT1A1, menopause age, symptoms, and response to HT.Methods:Women enrolled in the Kronos Early Estrogen Prevention Study at Mayo Clinic were randomized to 48 months of treatment with oral conjugated equine estrogen (n = 34), transdermal 17β-estradiol (E2) (n = 33), or placebo (n = 35). Linear regression models and ANOVA were used to test for association of SULT1A1 copy number, rs3760091, rs750155, and rs9282861 (SULT1A1∗2), with age at menopause and symptoms, levels of estrogens (estrone [E1], estrone sulfate [E1S], E2, and estradiol sulfate [E2S]), before and after HT.Results:SULT1A1 gene copy number affected the minor allele frequency for each single nucleotide polymorphisms tested. Before administration of exogenous hormones, increasing number of G alleles at rs9282861 was associated with earlier age at menopause (P = 0.014), lower frequency of night sweats (P = 0.009), and less severe insomnia (P = 0.046). After 48 months of treatment, SULT1A1 genotype was not associated with the presence of menopausal symptoms. In women randomized to oral conjugated equine estrogen, increasing number of the A allele at rs750155 was associated with lower E1S and E2S (P = 0.004 and 0.017), whereas increasing number of the C allele at rs3760091 was associated with lower E2S/E2 (P = 0.044).Conclusions:Interindividual variability in onset of menopause and symptoms before initiation of HT is explained in part by genetic variation in SULT1A1 and may represent a step toward individualizing HT treatment decisions.

Reversible downregulation of the hypothalamic-pituitary-gonadal axis in stallions with a novel GnRH antagonist

The GnRH antagonist, acyline, has not yet been investigated in the stallion. Our study aimed to: (1) evaluate the downregulation of the stallion hypothalamic-pituitary-gonadal axis by acyline through assessment of seminal parameters, testicular volume, and sexual behavior; (2) assess hormonal response of acyline-treated stallions to GnRH stimulation; and (3) verify reversibility after treatment. Stallions were assessed pretreatment and subsequently treated (every five days) for 50 days: acyline (n = 4; 330 μg/kg acyline) or control (n = 4, vehicle). The stallions were then monitored for 62 days after the last day of treatment. Treatment-induced declines (P < 0.05) in FSH, LH, testosterone, and estrone sulfate. Gonadotropins and testosterone returned to control values within 9 days, and estrone sulfate by 14 days, after discontinuation of treatment. Acyline-treated stallions failed to respond with a rise in FSH, LH, and testosterone after exogenous GnRH stimulation (gonadorelin) at Day 46 of treatment compared to pretreatment stimulation and control stallions. Decreases (P < 0.05) were observed in total sperm numbers and motility (week 2) in acyline-treated stallions, as well as total seminal plasma protein (week 2) and testicular volume (week 5). Over the course of the study, the time to erection, time to ejaculation, and number of mounts increased (P < 0.0001) across both groups of stallions; however, there was no effect of treatment or treatment by time interactions on these parameters. Testicular volume, and most seminal parameters regained normal levels within 62 days after treatment ended; on follow-up, sperm output of acyline-treated stallions was regained within 7 months after the end of experiment. In conclusion, acyline reversibly suppresses the stallion hypothalamic-pituitary-gonadal axis.

Abstract 651: Male mouse model of canine and human inflammatory breast cancer (IBC)

Abstract Inflammatory breast cancer (IBC) is an aggressive type of breast cancer with poor survival in women. Canine IBC has been proposed as a good surrogate model for the study of the human disease. Male breast cancer is a rare disease, accounting for less than 1% of all malignancies in men. Although rarely, IBC has been also found in men. The aim was to evaluate the capacity of male SCID mice to develop IBC tumors after the injection of canine and human IBC cell lines (IPC-366 and SUM149, respectively). Eighty SCID mice: 40 male and 40 females, 6-8 weeks old were used. Suspensions of 106 IPC-366 and SUM149 cells were implanted subcutaneously into the fourth inguinal mammary gland in 20 male and 20 female mice (of each cell line), respectively. Mice were inspected twice a week for the development of tumors. Tumors were monitored by palpation and measured by calipers. Mice were sacrificed when tumors reached 1.5 cm3 and tumors and organs were removed for immunohistochemical (IHC) characterization. A portion of tumor was used for hormonal analysis (Progesterone, Estradiol, Estrone sulphate, Testosterone, Androstenedione). Results revealed that IPC-366 reproduced tumors in 90% (male) vs 100% (female) after 2 weeks p.i. SUM149 reproduced tumors in 40% (male) vs 80% (female) at 4 weeks p.i. Both cell lines produced metastasis in lungs. Higher metastatic rates found in females than in males (IPC-366: 90% vs 20%; SUM149: 80% vs 50%). EIA hormonal analysis revealed that male tumors had higher T and SO4E1 concentrations compared to female tumors (p&amp;lt; 0.05). IHC analysis revealed that male and female tumors shared immunophenotipic characteristics. All xenografts were highly proliferative (high Ki-67 indexes), triple negative (ER-, PR-, HER-2-), vimentin positive and contained less than 10% of cells positive to AE1-AE3 and CK-14. In conclusion, IPC-366 and SUM149 cell lines can reproduce tumors in male mice capable of metastasize. Male and female xenograft models shared immunophenotypic characteristics. Male mice model would be useful for future IBC research using a different endocrine environment that could help to better understand the androgens function in canine and human IBC. Citation Format: Sara Caceres, Laura Peña, Maria J. Illera, Gema Silvan, Paloma J. de andres, Hui Gao, Wendy A. Woodward, James M. Reuben, Juan C. Illera. Male mouse model of canine and human inflammatory breast cancer (IBC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 651.

Abstract 2600: Improving breast cancer risk prediction models: the addition of a genetic risk score, mammographic density, and endogenous hormones

Abstract Background: Risk prediction models have important public health implications for targeted disease prevention by identifying women at higher risk who would most likely benefit from screening, chemoprevention, or other risk-reducing regimens. Among such models, the Gail and Rosner-Colditz models have been well validated and used to identify high-risk women. Each of these models generally includes only traditional breast cancer risk factors (e.g., parity and family history of breast cancer). No prior study has examined the joint contribution of a genetic risk score (GRS), mammographic density (MD), and postmenopausal endogenous hormone levels to these models. Method: We conducted a nested-case control study within the Nurses’ Health Study (NHS, follow-up 1990-2010) and NHS II (1997-2009). We created a breast cancer GRS based on 67 single-nucleotide polymorphism (SNPs). We measured percent MD as well as circulating testosterone, estrone sulfate, and prolactin levels in prediagnostic plasma samples. Using receiver-operating-characteristic curve analyses, we calculated the area under the curve (AUC), adjusting for age, as a measure of discrimination for the 5-year risk of invasive breast cancer and estrogen receptor (ER) and progesterone receptor (PR) positive disease (ER+PR+) by menopausal status, after adding GRS, MD, and/or circulating hormones to the modified Gail and Rosner-Colditz models. Results: We documented 11,880 women (4,006 cases/7,874 controls; age range: 34-80 yr) for Gail model and 8,210 women (2,665 cases/5,455 controls) for Rosner-Colditz model analyses. In both models, about 45% women were premenopausal, 25% were postmenopausal women not using postmenopausal hormone (HT), and 30% were postmenopausal women using HT at blood draw. The AUC was statistically significantly improved by 7.0 units in premenopausal women (Gail+GRS+MD), 11.8 units in postmenopausal women not using HT (Gail+GRS+MD+hormones), and 6.1 units in postmenopausal women using HT (Gail+GRS+MD+prolactin, all p-value&amp;lt;0.001). For the Rosner-Colditz model, the corresponding AUC improved by 5.4 units, 7.9 units, and 5.4 units (all p-value&amp;lt;0.001). For ER+PR+ tumors specifically, among postmenopausal women not using HT, the AUC was improved by 13.2 units for the Gail model and 9.9 units for the Rosner-Colditz model (all p-values&amp;lt;0.001). Conclusion: While each of genetic markers, percent MD, and hormones independently improved risk prediction for invasive breast cancer and ER+PR+ disease, incorporating all of these factors simultaneously improved models the most, especially among postmenopausal women not using HT. If validated in independent populations, our findings could help identify women at a higher risk, who would most benefit from chemoprevention, other risk-reducing regimens or screening. Citation Format: Xuehong Zhang, Megan Rice, Shelley S. Tworoger, Bernard A. Rosner, A. Heather Eliassen, Rulla M. Tamimi, Jing Qian, Graham A. Colditz, Walter C. Willett, Susan E. Hankinson. Improving breast cancer risk prediction models: the addition of a genetic risk score, mammographic density, and endogenous hormones. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2600.

Metabonomic analysis of the protective effect of quercetin on the toxicity induced by mixture of organophosphate pesticides in rat urine.

The present study aims to investigate the protective effect of quercetin against the joint toxic action induced by the mixture of four organophosphate pesticides (mixture-OPs) (dimethoate, acephate, dichlorvos, and phorate) at their corresponding no observed adverse effect level (NOAEL) using metabonomics. Rats were randomly divided into control, quercetin-treated, mixture-OPs-treated, and quercetin plus mixture-OPs-treated groups. Mixture-OPs and quercetin were given to the rats daily through drinking water and intragastric administration, respectively, for 90 days. The metabonomic profiles of rat urine were analyzed using ultra-performance liquid chromatography-mass spectrometry (UPLC/MS). The 14 metabolites significantly changed in the treatment groups compared with the control group, including the biomarkers of OPs exposure (dimethylphosphate, dimethyldithiophosphate, diethylphosphate) and the metabolites of quercetin (quercetin and isorhamnetina). The intensities of gentisic acid, creatinine, suberic acid, hippuric acid, uric acid, and citric acid significantly decreased, whereas the intensities of 7-methylguanine, estrone sulfate, and cholic acid significantly increased, in the mixture-OPs-treated group compared with the control group ( p < 0.01). The variation tendency of the aforementioned metabolites was significantly ameliorated in the high-dose quercetin (50 mg/(kg bw day)) plus mixture-OPs-treated group compared with the mixture-OPs-treated group ( p < 0.05). However, the intensities of these metabolites in the high-dose quercetin plus mixture-OPs-treated group were still significantly different from those of the control group ( p < 0.05). Results indicated that high dose of quercetin elicits a partial protective effect on the toxicity induced by mixture-OPs, including fatty acid and energy metabolism, antioxidant defense system, DNA damage, and liver and kidney function.

Contribution of Estrone Sulfate to Cell Proliferation in Aromatase Inhibitor (AI) -Resistant, Hormone Receptor-Positive Breast Cancer.

Aromatase inhibitors (AIs) effectively treat hormone receptor-positive postmenopausal breast cancer, but some patients do not respond to treatment or experience recurrence. Mechanisms of AI resistance include ligand-independent activation of the estrogen receptor (ER) and signaling via other growth factor receptors; however, these do not account for all forms of resistance. Here we present an alternative mechanism of AI resistance. We ectopically expressed aromatase in MCF-7 cells expressing green fluorescent protein as an index of ER activity. Aromatase-overexpressing MCF-7 cells were cultured in estrogen-depleted medium supplemented with testosterone and the AI, letrozole, to establish letrozole-resistant (LR) cell lines. Compared with parental cells, LR cells had higher mRNA levels of steroid sulfatase (STS), which converts estrone sulfate (E1S) to estrone, and the organic anion transporter peptides (OATPs), which mediate the uptake of E1S into cells. LR cells proliferated more in E1S-supplemented medium than did parental cells, and LR proliferation was effectively inhibited by an STS inhibitor in combination with letrozole and by ER-targeting drugs. Analysis of ER-positive primary breast cancer tissues showed a significant correlation between the increases in the mRNA levels of STS and the OATPs in the LR cell lines, which supports the validity of this AI-resistant model. This is the first study to demonstrate the contribution of STS and OATPs in E1S metabolism to the proliferation of AI-resistant breast cancer cells. We suggest that E1S metabolism represents a new target in AI-resistant breast cancer treatment.

Twelve-Month Estrogen Levels in Premenopausal Women With Hormone Receptor-Positive Breast Cancer Receiving Adjuvant Triptorelin Plus Exemestane or Tamoxifen in the Suppression of Ovarian Function Trial (SOFT): The SOFT-EST Substudy.

To describe estradiol (E2), estrone (E1), and estrone sulfate (E1S) levels during the first year of monthly triptorelin plus exemestane or tamoxifen and to assess possible suboptimal suppression while receiving exemestane plus triptorelin. Premenopausal patients with early breast cancer on the Suppression of Ovarian Function Trial who selected triptorelin as the ovarian suppression method and were randomly assigned to exemestane plus triptorelin or tamoxifen plus triptorelin were enrolled until the target population of 120 patients was reached. Blood sampling time points were 0, 3, 6, 12, 18, 24, 36, and 48 months. Serum estrogens were measured with a highly sensitive and specific assay. This preplanned 12-month analysis evaluated E2, E1, E1S, follicle-stimulating hormone, and luteinizing hormone levels in all patients and the proportion of patients with E2 levels greater than 2.72 pg/mL at any time point during treatment with exemestane plus triptorelin. One hundred sixteen patients (exemestane, n = 86; tamoxifen, n = 30; median age, 44 years; median E2, 51 pg/mL; 55% prior chemotherapy) started triptorelin and had one or more samples drawn. With exemestane plus triptorelin, median reductions from baseline E2, E1, and E1S levels were consistently ≥ 95%, resulting in significantly lower levels than with tamoxifen plus triptorelin at all time points. Among patients on exemestane plus triptorelin, 25%, 24%, and 17% had an E2 level greater than 2.72 pg/mL at 3, 6, and 12 months, respectively. Baseline factors related to on-treatment E2 level greater than 2.72 pg/mL were no prior chemotherapy (P = .06), higher body mass index (P = .05), and lower follicle-stimulating hormone and luteinizing hormone (each P < .01). During the first year, most patients on exemestane plus triptorelin had E2 levels below the defined threshold of 2.72 pg/mL, consistent with levels reported in postmenopausal patients on aromatase inhibitors, but at each time point, at least 17% of patients had levels greater than the threshold.

Atorvastatin improves renal organic anion transporter 3 and renal function in gentamicin-induced nephrotoxicity in rats.

What is the central question of this study? This study was designed to determine the renoprotective effects of atorvastatin treatment in an experimental model of gentamicin-induced nephrotoxicity through modulating the Nrf2 pathway by decreasing the oxidative stress. What is the main finding and its importance? Atorvastatin exerts a nephroprotective effect by attenuating oxidative stress, protecting renal function and renal organic anion transporter 3 function from the effects of gentamicin. Atorvastatin might protect the tissues via its antioxidant property and by modulating the antioxidant enzymes through the Nrf2 signalling pathway, which may be the underlying mechanisms of these protective effects. Recent evidence demonstrates that statins, 3-hydroxy-3-methylglutaryl coenzymeA reductase inhibitors, exert not only lipid-lowering effects but also antioxidant, anti-inflammatory and anti-apoptotic effects. Nephrotoxicity, a serious side-effect of gentamicin, is related to an increase in reactive oxygen species in the kidney. This study was designed to determine the renoprotective effects of atorvastatin treatment in an experimental model of gentamicin-induced nephrotoxicity. Male Sprague-Dawley rats were used. Nephrotocixity was induced by i.p. injection of gentamicin, 100mgkg(-1) day(-1) , for 15days. Atorvastatin, 10mgkg(-1) day(-1) , was administered by gavage 30min before gentamicin injection (pretreatment) for 15days or only on days 10-15 (post-treatment). Renal function and renal organic anion transporter 3 (Oat3) function and expression were examined. Gentamicin-treated rats demonstrated impaired renal function by attenuation of creatinine clearance and increased oxidative stress. Gentamicin treatment also decreased renal Oat3 function and expression as shown by decreased [(3) H]estrone sulfate uptake into renal cortical slices and decreased expression. The protein expressions of protein kinaseC, Nrf2, NAD(P)H:quinone oxidoreductase1, haeme oxygenase 1 and glutamate-cysteine ligase were markedly increased in gentamicin-treated rats, indicating the increase in oxidative stress. Administration of atorvastatin improved renal function and alleviated oxidative stress, and atorvastatin pretreatment had a greater ability to decrease oxidative stress than atorvastatin post-treatment. These effects helped to preserve renal function and Oat3 function and expression. These results indicate that atorvastatin has a renoprotective effect against gentamicin-induced nephrotoxicity by decreasing overoxidation in the kidney, and might be used in conjunction with gentamicin to protect against renal damage.

Alcohol-related breast cancer in postmenopausal women - effect of CYP19A1, PPARG and PPARGC1A polymorphisms on female sex-hormone levels and interaction with alcohol consumption and NSAID usage in a nested case-control study and a randomised controlled trial.

BackgroundAlcohol consumption is associated with increased risk of breast cancer (BC), and the underlying mechanism is thought to be sex-hormone driven. In vitro and observational studies suggest a mechanism involving peroxisome proliferator-activated receptor gamma (PPARγ) in a complex with peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) and interaction with aromatase (encoded by CYP19A1). Use of non-steroidal anti-inflammatory drugs (NSAID) may also affect circulating sex-hormone levels by modifying PPARγ activity.MethodsIn the present study we assessed whether genetic variation in CYP19A1 is associated with risk of BC in a case-control study group nested within the Danish “Diet, Cancer and Health” cohort (ncases = 687 and ncontrols = 687) and searched for gene-gene interaction between CYP19A1 and PPARGC1A, and CYP19A1 and PPARG, and gene-alcohol and gene-NSAID interactions. Association between the CYP19A1 polymorphisms and hormone levels was also examined among 339 non-HRT users. Incidence rate ratios were calculated based on Cox’ proportional hazards model. Furthermore, we performed a pilot randomised controlled trial to determine the effect of the PPARG Pro12Ala polymorphism and the PPARγ stimulator Ibuprofen on sex-hormone levels following alcohol intake in postmenopausal women (n = 25) using linear regression.ResultsGenetic variations in CYP19A1 were associated with hormone levels (estrone: Prs11070844 = 0.009, estrone sulphate: Prs11070844 = 0.01, Prs749292 = 0.004, Prs1062033 = 0.007 and Prs10519297 = 0.03, and sex hormone-binding globulin (SHBG): Prs3751591 = 0.03) and interacted with alcohol intake in relation to hormone levels (estrone sulphate: Pinteraction/rs2008691 = 0.02 and Pinteraction/rs1062033= 0.03, and SHBG: Pinteraction/rs11070844 = 0.03). CYP19A1/rs3751591 was both associated with SHBG levels (P = 0.03) and with risk of BC (Incidence Rate Ratio = 2.12; 95 % Confidence Interval: 1.02–4.43) such that homozygous variant allele carriers had increased levels of serum SHBG and were at increased risk of BC. Acute intake of alcohol decreased blood estrone (P = <0.0001), estrone sulphate (P = <0.0001), and SHBG (P = 0.009) levels, whereas Ibuprofen intake and PPARG Pro12Ala genotype had no effect on hormone levels.ConclusionsOur results suggest that genetically determined variation in CYP19A1 is associated with differences in sex hormone levels. However, the genetically determined differences in sex hormone levels were not convincingly associated with BC risk. The results therefore indicate that the genetically determined variation in CYP19A1 contributes little to BC risk and to alcohol-mediated BC risk.Trial registrationNCT02463383, June 3, 2015.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2317-y) contains supplementary material, which is available to authorized users.

Examination of Duct Physiology in the Human Mammary Gland

BackgroundThe human breast comprise several ductal systems, or lobes, which contain a small amount of fluid containing cells, hormones, proteins and metabolites. The complex physiology of these ducts is likely a contributing factor to the development of breast cancer, especially given that the vast majority of breast cancers begin in a single lobular unit.MethodsWe examined the levels of total protein, progesterone, estradiol, estrone sulfate, dehydroepiandrosterone sulfate, and macrophages in ductal fluid samples obtained from 3 ducts each in 78 women, sampled twice over a 6 month period. Samples were processed for both cytological and molecular analysis. Intraclass correlation coefficients and mixed models were utilized to identify significant data.ResultsWe found that the levels of these ductal fluid components were generally uncorrelated among ducts within a single breast and over time, suggesting that each lobe within the breast has a distinct physiology. However, we also found that estradiol was more correlated in women who were nulliparous or produced nipple aspirate fluid.ConclusionsOur results provide evidence that the microenvironment of any given lobular unit is unique to that individual unit, findings that may provide clues about the initiation and development of ductal carcinomas.

Fate of Conjugated and Free Estrogens in Swine Manure Collected from areas Housing Piglets, Pregnant Sows and Finisher Pigs

Occurrence and fate of estrogens and their metabolites were studied in pig manure collected during winter and summer seasons from sites housing piglets (Nursery (Nur)-manure), pregnant and nursing sows (PS manure) and finisher pigs (FPig manure). The liquid and solid fractions of manure were analyzed for (1) total solids (TS), volatile solids (VS) and total organic carbon (TOC), (2) E coli and M fujisawaense enumeration, (3) total and individual estrogens and (4) estrogenic activity. This study showed that VS and OC values, E coli and M fujisawaense enumerations, estrogen concentrations and estrogenic activity exhibited the following pattern: Nur-manure &lt;&lt; FPig-manure &lt; PS-manure. The values for summer and winter Nur-manure did not differ significantly, while the values in summer PS-manure or FPig manure samples were significantly higher than values in corresponding winter samples. Although, estrogens distributed between liquid and solid fractions of manure, concentrations of free, but not conjugated, estrogens depended on manures’ TOC values: an increase in TOC associated with an increase in free estrogen concentrations in liquid manure. However, an increase in TOC decreased the bacterial population in manure liquid by increasing their translocation from liquid into the in solids and ensuing bacterial stabilization. This may increase estrogens’ deconjugation and/or degradation. Estrone (E1) sulfate (sE1), free E1 (fE1), E1 glucuronide (gE1) and E1 metabolites were major steroids present in Nur- and PS-manure, while fE2 and gE2 were predominant estrogens in FPig-manure. In total, the winter estrogen load in liquid was 5.1 mg/L and the load in solid was 4.93 mg/kg. Assuming that 2.3 x 108 kg of manure is produced in the USA per day, approximate hormone load will be 2.3 tons/day. However, manure contains microorganisms that hydrolyze estrogens, thus actual hormone load will be considerably lower.

Transport of Estrone Sulfate by Human Placental Plasma Membrane Vesicles

Transport of Estrone Sulfate by Human Placental Plasma Membrane Vesicles

Characterization of Transporters in the Hepatic Uptake of TAK-475 M-I, a Squalene Synthase Inhibitor, in Rats and Humans.

TAK-475 (lapaquistat acetate) is a squalene synthase inhibitor and M-I is a pharmacologically active metabolite of TAK-475. Preclinical pharmacokinetic studies have demonstrated that most of the dosed TAK-475 was hydrolyzed to M-I during the absorption process and the concentrations of M-I in the liver, the main organ of cholesterol biosynthesis, were much higher than those in the plasma after oral administration to rats. In the present study, the mechanism of the hepatic uptake of M-I was investigated.The uptake studies of (14)C-labeled M-I into rat and human hepatocytes indicated that the uptakes of M-I were concentrative, temperature-dependent and saturable in both species with Km values of 4.7 and 2.8 μmol/L, respectively. M-I uptake was also inhibited by cyclosporin A, an inhibitor for hepatic uptake transporters including organic anion transporting polypeptide (OATP). In the human hepatocytes, M-I uptake was hardly inhibited by estrone 3-sulfate as an inhibitor for OATP1B1, and most of the M-I uptake was Na(+)-independent. Uptake studies using human transporter-expressing cells revealed the saturable uptake of M-I for OATP1B3 with a Km of 2.13 μmol/L. No obvious uptake of M-I was observed in the OATP1B1-expressing cells.These results indicated that M-I was taken up into hepatocytes via transporters in both rats and humans. OATP1B3 would be mainly involved in the hepatic uptake of M-I in humans. These findings suggested that hepatic uptake transporters might contribute to the liver-selective inhibition of cholesterol synthesis by TAK-475. This is the first to clarify a carrier-mediated hepatic uptake mechanism for squalene synthase inhibitors.

Hormonal profiles of captive Galapagos tortoises (Chelonoidis nigra).

Monthly blood samples, daily mating observations from Galapagos tortoises (Chelonoidis nigra), and local rainfall and temperature were collected at the Honolulu Zoo as part of a fertility evaluation. Testosterone concentrations were measured for males (n = 6), two of which were seen copulating and were considered sexually active. Estrone sulfate and progesterone concentrations were measured for female tortoises (n = 9), two of which nested and only one had laid eggs. Testosterone profiles were similar for both sexually active and sexually inactive males, both of which were positively correlated with temperature but not rainfall. Peak testosterone concentrations (12.0 ± 1.4 ng/ml sexually active animals vs. 14.4 ± 2.4 ng/ml sexually inactive animals) occurred at the end of the nesting season, from April to July. Estrone sulfate concentrations were similar for nesting (n = 2) and non-nesting (n = 7) female tortoises, rising from non-detectable concentrations (September), and increasing to peak concentrations during the nesting season. Progesterone concentrations remained low and spiked (9.44 ng/ml) only for the female that nested and laid eggs. Testosterone was negatively correlated with mating behavior, and the male tortoises were likely capable of spermatogenesis even though only two of them engaged in mating behavior. The female tortoises were not senescent, as the estrone sulfate concentrations likely reflected waves of ovarian follicular activity. Endocrine parameters were not in synchrony with rainfall, and a disconnect between the timing of reproductive events and the environmental milieu may help to explain the poor fertility of these tortoises. Zoo Biol. 35:237-245, 2016. © 2016 Wiley Periodicals, Inc.

Serum steroid concentrations remain within normal postmenopausal values in women receiving daily 6.5 mg intravaginal prasterone for 12 weeks

This study integrates all data obtained in women aged 40–80years enrolled with moderate to severe symptoms of vulvovaginal atrophy (VVA) who received daily intravaginal administration of 0.50% (6.5mg) dehydroepiandrosterone (DHEA; prasterone) for 12weeks (n=723; ITT-S population) as compared with placebo (n=266; ITT-S population). To this end, serum steroid levels (DHEA, DHEA-sulfate (DHEA-S), androst-5-ene-3β, 17β-diol (5-diol), testosterone, dihydrotestosterone (DHT), androstenedione (4-dione), estrone (E1), estradiol (E2), estrone sulfate (E1-S), androsterone glucuronide (ADT-G), and androstane-3α, 17β-diol 17-glucuronide (3α-diol-17G)) were measured at Day 1 and Week 12 by liquid chromatography-tandem mass spectrometry (LC–MS/MS) following validation performed according to the FDA guidelines [1–6]. In agreement with the mechanisms of intracrinology where DHEA is exclusively transformed intracellularly into active sex steroids which act and are inactivated locally before being released as glucuronided or sulfated metabolites for elimination by the kidneys and liver, all sex steroids remained well within normal postmenopausal values following administration of intravaginal DHEA. Serum estradiol, the most relevant sex steroid, was measured after 12weeks of treatment at 3.36pg/ml (cITT-S population) or 19% below the normal postmenopausal value of 4.17pg/ml. On the other hand, serum E1-S, the best recognized marker of global estrogenic activity, shows an average value of 209pg/ml at 12 weeks compared to 220pg/ml in normal postmenopausal women. Moreover, serum ADT-G, the main metabolite of androgens, also remains well within normal postmenopausal values. The present data shows that a low daily intravaginal dose (6.5mg) of DHEA (prasterone) which is efficacious on the symptoms and signs of VVA, permits to achieve the desired local efficacy without systemic exposure, in agreement with the stringent mechanisms of menopause established after 500 million years of evolution where each cell in each tissue is the master of its sex steroid exposure.

Changes in maternal androgens and oestrogens in mares with experimentally-induced ascending placentitis.

While advanced stages of ascending placentitis can be diagnosed by transrectal ultrasonography and clinical signs, early stages can be missed. Thus, additional tools could enhance assessment of placental health. To characterise peripheral dehydroepiandrosterone sulphate (DHEA-S) and testosterone concentrations in mares carrying normal pregnancies (Study 1) and compare plasma concentrations of DHEA-S, testosterone, oestradiol 17-β (oestradiol) and oestrone sulphate (OES) in mares with or without placentitis (Study 2). Longitudinal cohort study of healthy mares (Study 1) and controlled experiment (Study 2). In Study 1, mares had serum samples collected from 100 days of gestation to term. In Study 2, pregnant mares (260-280 days gestation) were assigned to a control group or a group with placentitis. Placentitis was induced via intracervical inoculation of Streptococcus equi ssp. zooepidemicus. Blood was collected at inoculation/commencement for control mares (day = 0) and daily for 12 days post inoculation (DPI) or until abortion. Steroid concentrations were determined by immunoassays. Concentrations of steroids in Study 2 were also evaluated relative to days from abortion (DFA -8 days to 0). In Study 1, DHEA-S peaked by 180 days gestation, while testosterone concentrations were progressively increased from Days 100 to 180 with a plateau until ~240 days and a progressive decline until 290 days of gestation. In Study 2, concentrations of DHEA-S and testosterone were not significantly different between groups. There were significant effects of time (oestradiol P = 0.0008, OES P = 0.01) and time-by-group interactions (oestradiol P<0.001, OES P<0.0001) for oestrogen concentrations. For mares with experimental placentitis, concentrations of oestradiol were significantly reduced at -6, -2, -1 and 0 DFA, while OES concentrations were significantly reduced on the day before abortion (0 DFA). Testosterone and DHEA-S were increased and varied through pregnancy. Oestrogens but not androgens decreased significantly in mares with experimentally-induced ascending placentitis.