Conversion of estriol to estrone: A bacterial strategy for the catabolism of estriol.

Natural estrogens, including estrone (E1), 17ß-estradiol (E2), and estriol (E3), are potentially carcinogenic pollutants commonly found in water and soil environments. Bacterial metabolic pathway of E2 has been studied; however, the catabolic products of E3 have not been discovered thus far. In this study, Novosphingobium sp. ES2-1 was used as the target strain to investigate its catabolic pathway of E3. The metabolites of E3 were identified by high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) combined with stable 13C3-labeling. Strain ES2-1 could almost completely degrade 20 mg∙L-1 of E3 within 72 h under the optimal conditions of 30°C and pH 7.0. When inoculated with strain ES2-1, E3 was initially converted to E1 and then to 4-hydroxyestrone (4-OH-E1), which was then cleaved to HIP (metabolite A6) via the 4, 5-seco pathway or cleaved to the B loop via the 9,10-seco pathway to produce metabolite with a long-chain ketone structure (metabolite B4). Although the ring-opening sequence of the above two metabolic pathways was different, the metabolism of E3 was achieved especially through continuous oxidation reactions. This study reveals that, E3 could be firstly converted to E1 and then to 4-OH-E1, and finally degraded into small molecule metabolites through two alternative pathways, thereby reducing E3 pollution in water and soil environments.

Strong Protection by 4-Hydroxyestrone against Erastin-Induced Ferroptotic Cell Death in Estrogen Receptor-Negative Human Breast Cancer Cells: Evidence for Protein Disulfide Isomerase as a Mechanistic Target for Protection.

Ferroptosis is a recently identified form of regulated cell death, characterized by excessive iron-dependent lipid peroxidation. Recent studies have demonstrated that protein disulfide isomerase (PDI) is an important mediator of chemically induced ferroptosis and also a new target for protection against ferroptosis-associated cell death. In the present study, we identified that 4-hydroxyestrone (4-OH-E1), a metabolic derivative of endogenous estrogen, is a potent small-molecule inhibitor of PDI, and can strongly protect against chemically induced ferroptotic cell death in the estrogen receptor-negative MDA-MB-231 human breast cancer cells. Pull-down and CETSA assays demonstrated that 4-OH-E1 can directly bind to PDI both in vitro and in intact cells. Computational modeling analysis revealed that 4-OH-E1 forms two hydrogen bonds with PDI His256, which is essential for its binding interaction and thus inhibition of PDI's catalytic activity. Additionally, PDI knockdown attenuates the protective effect of 4-OH-E1 as well as cystamine (a known PDI inhibitor) against chemically induced ferroptosis in human breast cancer cells. Importantly, inhibition of PDI by 4-OH-E1 and cystamine or PDI knockdown by siRNAs each markedly reduces iNOS activity and NO accumulation, which has recently been demonstrated to play an important role in erastin-induced ferroptosis. In conclusion, this study demonstrates that 4-OH-E1 is a novel inhibitor of PDI and can strongly inhibit ferroptosis in human breast cancer cells in an estrogen receptor-independent manner. The mechanistic understanding gained from the present study may also aid in understanding the estrogen receptor-independent cytoprotective actions of endogenous estrogen metabolites in many noncancer cell types.

16α-OHE1 alleviates hypoxia-induced inflammation and myocardial damage via the activation of ß2-Adrenergic receptor.

OBJECTIVE: Myocardial injuries resulting from hypoxia are a significant concern, and this study aimed to explore potential protective strategies against such damage. Specifically, we sought to investigate the cardioprotective effects of 16α-hydroxyestrone (16α-OHE1). METHODS: Male Sprague‒Dawley (SD) rats were subjected to hypoxic conditions simulating high-altitude exposure at 6000 m in a low-pressure chamber for 7 days. Before and during hypoxic exposure, estradiol (E2) and various doses of 16α-OHE1 were administered for 14 days. Heart weight/body weight (HW/BW), myocardial structure, Myocardial injury indicators and inflammatory infiltration in rats were measured. H9C2 cells cultured under 5% O2 conditions received E2 and varying doses of 16α-OHE1; Cell viability, apoptosis, inflammatory infiltration, and Myocardial injury indicators were determined. Expression levels of ß2AR were determined in rat hearts and H9C2 cells. The ß2AR inhibitor, ICI 118,551, was employed to investigate ß2AR's role in 16α-OHE1's cardioprotective effects. RESULTS: Hypoxia led to substantial myocardial damage, evident in increased heart HW, CK-MB, cTnT, ANP, BNP, structural myocardial changes, inflammatory infiltration, and apoptosis. Pre-treatment with E2 and 16α-OHE1 significantly mitigated these adverse changes. Importantly, the protective effects of E2 and 16α-OHE1 were associated with the upregulation of ß2AR expression in both rat hearts and H9C2 cells. However, inhibition of ß2AR by ICI 118,551 in H9C2 cells nullified the protective effect of 16α-OHE1 on myocardium. CONCLUSION: Our findings suggest that 16α-OHE1 can effectively reduce hypoxia-induced myocardial injury in rats through ß2ARs, indicating a promising avenue for cardioprotection.

A randomized, double-blind, placebo-controlled, cross-over trial to evaluate the effect of EstroSense® on 2-hydroxyestrone:16α-hydroxyestrone ratio in premenopausal women.

OBJECTIVES: Some estrogen metabolites are associated with increased breast cancer risk, while others are protective. Research efforts have focused on modifiable factors, including bioactive compounds found in food or supplements, promoting estrogen profiles with anti-cancer properties. EstroSense® is a nutraceutical product with bioactive compounds, including Indole-3-carbinol and green-tea catechins, which may favourably affect estrogen profiles. This study was conducted to determine if EstroSense use, compared to placebo, promotes a higher urinary 2-hydroxyestrone:16α-hydroxyestrone ratio (2-OHE1:16α-OHE1), a biomarker associated with a lowered risk of breast cancer. METHODS: A total of 148 premenopausal women were recruited from British Columbia, Canada to participate in a randomized, double-blind, cross-over, multicentre, placebo-controlled study in which women were randomized to a treatment sequence that consisted of either EstroSense®, followed by placebo or vice-versa. The women were instructed to consume three capsules per day of EstroSense® or the placebo for three menstrual cycles (∼12 weeks). The primary outcome was the measurement of 2-OHE1:16α-OHE1 in casual samples at baseline and after each treatment phase. RESULTS: After 12 weeks of intervention, the mean (95% CI) urinary 2-OHE1:16α-OHE1 was 4.55 (2.69, 6.42) (p<0.001) higher following EstroSense than placebo adjusted for baseline values. CONCLUSIONS: EstroSense use led to markedly higher urinary 2-OHE1:16α-OHE1 than the placebo, a biomarker associated with a lower risk of breast cancer. REGISTRATION: http://clinicaltrials.gov (NCT02385916).

Establishment and validation of a sensitive LC-MS/MS method for quantification of urinary estrogens in women with gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is not only a threat to the health of pregnant women, but also has profound effects on the health of offspring. Studies have shown that the imbalance of estrogen metabolism is associated with an increased risk of GDM. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established and validated for simultaneous quantification of thirteen estrogens in the urine of GDM women, including estrone (E1), estradiol (E2), estriol (E3), and their hydroxylated and methylated metabolites. The method was achieved on a Waters CORTECS C18 column (2.1 mm × 150 mm, 1.6 µm) within 8.5 min. The linear range of thirteen estrogens in urine was 2-1000 pg·mL-1. Both intra- and inter-day precision for each analyte were less than 15%, with accuracies ranging from 8.3% to 7.3%. The extraction recoveries rate were between 86% and 111%, and stability verification results met the requirements for determination of biological samples. The results suggested that the concentrations of estrogens in all urine samples range from 0.08 to 134.06 (pg·mg-1 creatinine). The mean levels of E1, E2 and most estrogen metabolites in the urine of GDM women were higher than those in healthy pregnant women. Notably, the mean level of 2-hydroxyestrone (2-OHE1) in GDM women was 13.2-fold lower than that in healthy pregnant women. The types of estrogens with the highest mean levels in the urine of GDM and healthy pregnant women were obviously different, which are 2-methoxyestrone (2MeOE1) and E3, respectively. Our results demonstrated that this specific and sensitive method is suitable for quantifying estrogens in human urine and could provide support for further research on estrogen-related pathological mechanisms in GDM and other diseases.

Identification of an important function of CYP123: Role in the monooxygenase activity in a novel estradiol degradation pathway in bacteria.

Nowadays, 17ß-estradiol (E2) biodegradation pathway has still not been identified in bacteria. To bridge this gap, we have described a novel E2 degradation pathway in Rhodococcus sp. P14 in this study, which showed that estradiol could be first transferred to estrone (E1) and thereby further converted into 16-hydroxyestrone, and then transformed into opened estrogen D ring. In order to identify the genes, which may be responsible for the pathway, transcriptome analysis was performed during E2 degradation in strain P14. The results showed that the expression of a short-chain dehydrogenase (SDR) gene and a CYP123 gene in the same gene cluster could be induced significantly by E2. Based on gene analysis, this gene cluster was found to play an important role in transforming E2 to 16-hydroxyestrone. The function of CYP123 was unknown before this study, and was found to harbor the activity of 16-estrone hydratase. Moreover, the global response to E2 in strain P14 was also analyzed by transcriptome analysis. It was observed that various genes involved in the metabolism processes, like the TCA cycle, lipid and amino acid metabolism, as well as glycolysis showed a significant increase in mRNA levels in response to strain P14 that can use E2 as the single carbon source. Overall, this study provides us an in depth understanding of the E2 degradation mechanisms in bacteria and also sheds light about the ability of strain P14 to effectively use E2 as the major carbon source for promoting its growth.

Association of microbial dynamics with urinary estrogens and estrogen metabolites in patients with endometriosis.

Endometriosis is an estrogen dependent gynecological disease associated with altered microbial phenotypes. The association among endogenous estrogen, estrogen metabolites, and microbial dynamics on disease pathogenesis has not been fully investigated. Here, we identified estrogen metabolites as well as microbial phenotypes in non-diseased patients (n = 9) and those with pathologically confirmed endometriosis (P-EOSIS, n = 20), on day of surgery (DOS) and ~1-3 weeks post-surgical intervention (PSI). Then, we examined the effects of surgical intervention with or without hormonal therapy (OCPs) on estrogen and microbial profiles of both study groups. For estrogen metabolism analysis, liquid chromatography/tandem mass spectrometry was used to quantify urinary estrogens. The microbiome data assessment was performed with Next generation sequencing to V4 region of 16S rRNA. Surgical intervention and hormonal therapy altered gastrointestinal (GI), urogenital (UG) microbiomes, urinary estrogen and estrogen metabolite levels in P-EOSIS. At DOS, 17ß-estradiol was enhanced in P-EOSIS treated with OCPs. At PSI, 16-keto-17ß-estradiol was increased in P-EOSIS not receiving OCPs while 2-hydroxyestradiol and 2-hydroxyestrone were decreased in P-EOSIS receiving OCPs. GI bacterial α-diversity was greater for controls and P-EOSIS that did not receive OCPs. P-EOSIS not utilizing OCPs exhibited a decrease in UG bacterial α-diversity and differences in dominant taxa, while P-EOSIS utilizing OCPs had an increase in UG bacterial α-diversity. P-EOSIS had a strong positive correlation between the GI/UG bacteria species and the concentrations of urinary estrogen and its metabolites. These results indicate an association between microbial dysbiosis and altered urinary estrogens in P-EOSIS, which may impact disease progression.

Urinary oestrogen steroidome as an indicator of the risk of localised prostate cancer progression.

BACKGROUND: Prostate cancer (PCa) is the most common cancer in North American men. Beyond the established contribution of androgens to disease progression, growing evidence suggest that oestrogen-related pathways might also be of clinical importance. The aim of this study was to explore the association of urinary oestrogen levels with clinical outcomes. METHODS: Urine samples from the prospective multi-institutional PROCURE cohort were collected before RP for discovery (n = 259) and validation (n = 253). Urinary total oestrogens (unconjugated + conjugated), including oestrone and oestradiol, their bioactive and inactive catechol and methyl derivatives (n = 15), were measured using mass spectrometry (MS). RESULTS: The median follow-up time for the discovery and replication cohorts was 7.6 and 6.5 years, respectively. Highly significant correlations between urinary oestrogens were observed; however, correlations with circulating oestrogens were modest. Our findings indicate that higher levels of urinary oestriol and 16-ketoestradiol were associated with lower risk of BCR. In contrast, higher levels of 2-methoxyestrone were associated with an increased risk of development of metastasis/deaths. CONCLUSIONS: Our data suggest that urinary levels of oestriol and 16-ketoestradiol metabolites are associated with a more favourable outcome, whereas those of 2-methoxyestrone are associated with an elevated risk of metastasis after RP. Further studies are required to better understand the impact of oestrogens on disease biology and as easily accessible urine-based risk-stratification markers.

Opposite estrogen effects of estrone and 2-hydroxyestrone on MCF-7 sensitivity to the cytotoxic action of cell growth, oxidative stress and inflammation activity.

There are many kinds of estrogens, and endogenous estrogens produce a variety of estrogen metabolites with similar structure but with different physiological effects after metabolism in vivo. Studies have shown that estrone (E1) widely occurs in the environment and animal-derived food. Because of its estrogen effect, E1 can have adverse effects on the human body as an endocrine disruptor. In this study, we found that E1 and 2-hydroxyestrone (2-OH-E1), the hydroxylation metabolite of estrogen, have opposite proliferative effects on breast cancer cells (MCF-7) through cell proliferation experiments and comparison of their effects by molecular docking and detection of ROS, Ca2+, and cell pathway proteins. The effects of 2-methoxyestrone (2-MeO-E1) and 16α-hydroxyestrone (16α-OH-E1) on the biochemical and protein levels of MCF-7 were further studied to compare the effects of metabolic sites and modes on estrogen effects. Hydroxylation of E1 at the C2 site weakened the estrogen effect, down-regulated the expression of the mammalian target of rapamycin (mTOR) and protein kinase B (Akt) pathway proteins, inhibited the proliferation of cancer cells, and enhanced anti-oxidative stress and anti-inflammation. Methoxylation at the C2 position also inhibited the expression of inflammatory and oxidative stress pathway proteins but did not greatly affect the estrogen effects. However, hydroxylation on C16 had no significant effect on the biological effects of estrogen. Therefore, the structural changes of estrogen on C2 are important reasons for the different physiological effects of estrogen and its metabolites. Thus, by regulating the gene Cytochrome P450 1B1(CYP1B1), which affects the hydroxylation metabolism of estrogen, and promoting the hydroxylation of estrone at the C2 position, the estrogen effect of estrone can be effectively reduced, thus reducing the harm its poses in food and the environment.

Markers of Local and Systemic Estrogen Metabolism in Endometriosis.

Estrogen metabolites (EMs) can work independently from their parent hormones. We hypothesize that in endometriosis, estrogen is metabolized preferentially along hormonally active pathways. We recruited 62 women with endometriosis (proven laparoscopically and histologically) and 52 control women (normal findings with laparoscopy) among patients undergoing surgery for pelvic pain and/or infertility during the proliferative phase of the menstrual cycle. Urinary samples were collected preoperatively. Biopsies from eutopic endometrium of control women and women with endometriosis were collected during surgery. EMs in urine and endometrial tissues were extracted and determined using Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS). These included: 2-hydroxyestrone (2OHE1), 16-α hydroxyestrone (16α-OHE1), 2OHE1/16α-OHE1 ratio, 4-hydroxyestrone (4OHE1), 2-hydroxyestradiol (2OHE2), and 4-hydroxyestradiol (4OHE2). Eutopic endometrium of endometriosis patients, as compared to control endometrium, contained significantly higher level of 4OHE1 (0.03 (IQR: 0.03-0.265) versus 0.03 (IQR: 0.03-0.03) µg/g, respectively, P = 0.005), 2-OHE2 (0.241 (IQR: 0.1-0.960) versus 0.1 (IQR: 0.1-0.1) µg/g, respectively, P < 0.001), and 4-OHE2 (0.225 (IQR: 0.22-1.29) versus 0.0.2 (IQR: 0.2-0.2) µg/g, respectively, P < 0.001). Only 2OHE1 showed higher concentration in urine of women with endometriosis than controls (9.9 (IQR: 3.64-14.88) versus 4.5 (IQR: 1.37-17.00) µg/mg creatinine, respectively, P = 0.042). Eutopic endometrium of women with endometriosis metabolizes estrogen preferentially to the biologically active 2OHE2, and potentially genotoxic 4OHE1 and 4OHE2 metabolites. This contributes to further understanding of endometriosis etiology, its link to ovarian cancer, and could help identifying an endometrial biomarker of the disease.

Effect of Interaction between 17ß-Estradiol, 2-Methoxyestradiol and 16α-Hydroxyestrone with Chromium (VI) on Ovary Cancer Line SKOV-3: Preliminary Study.

Ovarian cancer is the leading cause of death from gynecologic malignancies. Some estrogens, as well as xenoestrogens, such as chromium (VI) (Cr(VI)), are indicated as important pathogenic agents. The objective of this study was to evaluate the role of estradiol and some its metabolites upon exposure to the metalloestrogen Cr(VI) in an in vitro model. The changes in cell viability of malignant ovarian cancer cells (SKOV-3 resistant to cisplatin) exposed to 17ß-estradiol (E2) and its two metabolites, 2-methoxyestradiol (2-MeOE2) and 16α-hydroxyestrone (16α-OHE1), upon exposure to potassium chromate (VI) and its interactions were examined. The single and mixed models of action, during short and long times of incubation with estrogens, were applied. The different effects (synergism and antagonism) of estrogens on cell viability in the presence of Cr(VI) was observed. E2 and 16α-OHE1 caused a synergistic effect after exposure to Cr(VI). 2-MeOE2 showed an antagonistic effect on Cr(VI). The examined estrogens could be ranked according to the most protective effect or least toxicity in the order: 2-MeOE2 > E2 > 16α-OHE1. Early pre-incubation (24 h or 7 days) of cells with estrogens caused mostly an antagonistic effect-protective against the toxic action of Cr(VI). The beneficial action of estrogens on the toxic effect of Cr(VI), in the context of the risk of ovarian cancer, seems to be important and further studies are needed.

Effective synthesis of a hexacyclic-steroid derivative from 4-hydroxyestrone.

Several studies have been reported for the preparation of hexacyclic-steroid derivatives; however, some reagents are expensive and require special conditions for handling. In this way, the objective of this study was to synthesize a hexacyclic-steroid derivative from 4-hydroxyestrone. The chemical structure was evaluated through both 1H NMR and 13C NMR spectroscopic analysis. The results showed good performance of the hexacyclic-steroid derivative. In conclusion in this study, an easy method for the preparation of the hexacyclic-steroid derivative is reported.

Endometrial cancer patients have high affinity antibodies for estrogen metabolite-receptor aggregate: A potential biomarker for EC.

AIM: Elevated levels of 16α-hydroxyestrone (16α-OHE1 ) have been described in endometrial cancer (EC) and estrogen receptors (ER) expressed in endometrial tissue, but research on their combined role is lacking. We aimed to investigate the affinity and binding specificity of EC antibodies against the 16α-OHE1 -ERα aggregate in the serum of EC patients. Specificities of EC antibodies were also evaluated according to various clinical characteristics found in these cancer patients. METHODS: The binding specificity and affinity of EC antibodies against 16α-OHE1 -ERα in the serum of 120 EC patients were evaluated by direct binding and competition ELISA and quantitative precipitation titration. Binding of EC antibodies was also determined according to various clinical characteristics in EC patients through competition ELISA. RESULTS: Antibodies from EC patients demonstrated high recognition of 16α-OHE1 -ERα compared to ERα (P < 0.05) or 16α-OHE1 (P < 0.001). The relative affinity of EC IgG was 1.49 × 10-7 M, 1.34 × 10-6 M and 1.13 × 10-6 M for 16α-OHE1 -ERα, ERα and 16α-OHE1 , respectively. Several factors, such as obesity, postmenopausal status, use of hormonal therapy, ER and progesterone receptor (PR) status, low 2-OHE1 /16α-OHE1 ratio, chemotherapy and hypertension, augment the production of antibodies against 16α-OHE1 -ERα in EC patients. CONCLUSION: 16α-OHE1 -ERα is a high-affinity antigen for EC antibodies in the serum of EC patients and might function as a biomarker for this disease. Furthermore, several factors enhanced the production of antibodies against 16α-OHE1 -ERα in the sera of these EC patients.

16α-Hydroxyestrone: Mass Spectrometry-Based Methodologies for the Identification of Covalent Adducts Formed with Blood Proteins.

Elevated levels of the estrone metabolite, 16α-hydroxyestrone (16αOHE1), have been linked with multiple diseases. As an electrophilic reactive metabolite, covalent binding to proteins is thought to constitute one of the possible mechanisms in the onset of deleterious health outcomes associated with 16αOHE1. Whereas mass spectrometry (MS)-based methodologies are currently considered the best suited to monitor the formation of protein covalent adducts, the application of these approaches for the identification of covalent adducts of 16αOHE1 is yet to be provided. In the present study, with the ultimate goal of determining the most adequate methodology for searching for 16αOHE1-derived covalent adducts, we explored multiple liquid chromatography-electrospray ionization tandem high-resolution mass spectrometry (LC-ESI-HRMS/MS)-based approaches to investigate the nature and specific locations of the covalent adducts produced in human hemoglobin (Hb) and human serum albumin (HSA) modified in vitro with 16αOHE1. The application of a "bottom up" proteomics approach, involving the nanoLC-ESI-HRMS/MS analysis of tryptic peptides, allowed the identification of multiple sites of 16αOHE1 adduction in Hb and HSA. As expected, the majority of the adducted peptides occurred in lysine residues following stabilization of the Schiff base formed with 16αOHE1 by reduction or via Heyns rearrangement, yielding the stable α-hydroxyamine and ketoamine adducts, respectively. Noteworthy is the fact that a serine residue was also identified to be covalently modified with 16αOHE1, which to our knowledge constitutes a first-hand report of a keto electrophile as target of hydroxyl-based nucleophilic amino acids. The N-alkyl Edman degradation resulted to be unsuitable for the identification of 16αOHE1adducts formed with the N-terminal valine of Hb, most probably due to stereochemical restraints of the tested derivatizing agents (fluorescein isothiocyanate and phenyl isothiocyanate) on assessing these bulky covalent adducts. Nonetheless, the digestion of adducted proteins to amino acids resulted in the detection of 16αOHE1-derived keto and α-hydroxyamine Lys adducts. The simplicity of this methodology might be beneficial for clinical studies, with the possibility of offering quantitative information with the preparation of synthetic standards of these adducts. The results obtained are crucial not only for the identification and quantification of biomarkers of exposure to 16αOHE1 but also for clarifying the role of protein binding in the onset of diseases associated with elevated levels of this reactive metabolite.

4-Hydroxyestrone, an Endogenous Estrogen Metabolite, Can Strongly Protect Neuronal Cells Against Oxidative Damage.

Earlier studies showed that endogenous estrogens have neuroprotective effect against oxidative damage. The present study seeks to investigate the protective effect of various endogenous estrogen metabolites against oxidative neurotoxicity in vitro and in vivo. Using immortalized mouse hippocampal neuronal cells as an in vitro model, 4-hydroxyestrone, an estrone metabolite with little estrogenic activity, is found to have the strongest neuroprotective effect against oxidative neurotoxicity among 25 endogenous estrogen metabolites tested, and its protective effect is stronger than 17ß-estradiol. Similarly, 4-Hydroxyestrone also exerts a stronger protective effect than 17ß-estradiol against kanic acid-induced hippocampal oxidative damage in rats. Neuroprotection by 4-hydroxyestrone involves increased cytoplasmic translocation of p53 resulting from SIRT1-mediated deacetylation of p53. Analysis of brain microsomal enzymes shows that estrogen 4-hydroxylation is the main metabolic pathway in the central nervous system. Together, these results show that 4-hydroxyestrone is an endogenous neuroestrogen that can strongly protect against oxidative neuronal damage.

The 2-/16α-Hydroxylated Estrogen Ratio-Breast Cancer Risk Hypothesis: Insufficient Evidence for its Support.

During the past 25 years or so a number of studies have been carried out to address the hypothesis that the ratio of 2-hydroxyestrone (2-hydroxy-E1) to 16α-hydroxyestrone (16α-hydroxy-E1) is associated with breast cancer risk. The rationale for this hypothesis is based on data from studies that suggest a tumorigenic and genotoxic effect of 16α-hydroxy-E1 and a protective effect of 2-hydroxy-E1 regarding breast cancer risk. The adverse effect of 16α-hydroxy-E1 has been attributed to its potential to form covalent adducts with macromolecules. Initial studies used radiometric assays and enzyme immunoassays to test the hypothesis. However, concerns about the accuracy of these assays led to the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that is capable of measuring 5 unconjugated and 15 conjugated endogenous estrogens, which include 2- and 16-hydroxylated estrogen metabolites, in serum or urine. The conjugated estrogens are quantified following a deconjugation (hydrolysis) step to remove the sulfate and glucuronide groups. Epidemiologic studies have been using the LC-MS/MS assay to determine whether there is an association between breast cancer risk and the ratio of the sum of the concentrations of metabolites in the 2-hydroxylated estrogen pathway and in the 16-hydroxylated estrogen pathway. However, the validity of the pathways as biomarkers was not evaluated. The 16-hydroxylated estrogen pathway includes estriol, 16-epiestriol, 17-epiestriol and 16-ketoestradiol, in addition to 16α-hydroxy-E1. However, with the exception of 16α-hydroxy-E1, there is no evidence that any of the other estrogens in the pathway have tumorigenic or genotoxic properties, and they do not form covalent adducts with macromolecules. Another deficiency in the epidemiological studies pertains to the accuracy of estrogen metabolite measurements obtained after the hydrolysis step in the LC-MS/MS assays. No validation was performed to demonstrate that a constant efficiency of hydrolysis is found for all the different structural forms of sulfated and glucuronidated conjugates. Other deficiencies in the assays include the need for greater sensitivity so that the very low concentrations of unconjugated 2-hydroxy-E1, 2-hydroxy-E2, and 16α-hydroxy-E1 can be measured in serum. There is also a need to develop assays to measure intact forms of conjugated estrogens in both serum and urine, particularly the sulfates and glucuronides of 2-hydroxylated, 2-methoxylated, and 16α-hydroxylated E1 and E2. This will avoid inaccuracies that stem from hydrolysis procedures. Improvements in LC-MS/MS assay methodology to obtain accurate measurements of unconjugated and conjugated 2-hydroxylated, 2-methoxylated, and 16α-hydroxylated estrogen metabolites are needed. This should provide valuable data for testing the 2-/16α-hydroxylated estrogen-breast cancer risk hypothesis.

Metabolism of 17ß-estradiol by Novosphingobium sp. ES2-1 as probed via HRMS combined with 13C3-labeling.

This study investigated the biodegradation and metabolic mechanisms of 17ß-estradiol (E2) by Novosphingobium sp. ES2-1 isolated from the activated sludge in a domestic sewage treatment plant (STP). It could degrade 97.1% E2 (73.5 µmol/L) in 7 d with a biodegradation half-life of 1.29 d. E2 was initially converted to estrone (E1), then to 4-hydroxyestrone (4-OH-E1), before subsequent monooxygenation reactions cleaved 4-OH-E1 into a metabolite with long-chain ketones structure (metabolite P8). However, when 4-OH-E1 was cleaved through the 4,5-seco pathway, the resulting phenol ring cleavage product could randomly condense with NH3 to yield a pyridine derivative, accompanied by the uncertain loss of a carboxy group at C4 before the condensation. The derivative was further oxidized into the metabolites with both pyridine and long-chain ketones structure (metabolite N5) through a similar formation mechanism as for P8 performed. This research presents several novel metabolites and shows that E2 can be biodegraded into the metabolite with long-chain structure through three optional pathways, thereby reducing E2 contamination.

Obesity alters oestrogen metabolism and contributes to pulmonary arterial hypertension.

Obesity is a common comorbidity for pulmonary arterial hypertension (PAH). Additionally, oestrogen and its metabolites are risk factors for the development of PAH. Visceral adipose tissue (VAT) is a major site of oestrogen production; however, the influence of obesity-induced changes in oestrogen synthesis and metabolism on the development of PAH is unclear. To address this we investigated the effects of inhibiting oestrogen synthesis and metabolism on the development of pulmonary hypertension in male and female obese mice.We depleted endogenous oestrogen in leptin-deficient (ob/ob) mice with the oestrogen inhibitor anastrozole (ANA) and determined the effects on the development of pulmonary hypertension, plasma oestradiol and urinary 16α-hydroxyestrone (16αOHE1). Oestrogen metabolism through cytochrome P450 1B1 (CYP1B1) was inhibited with 2,2',4,6'-tetramethoxystilbene (TMS).ob/ob mice spontaneously develop pulmonary hypertension, pulmonary vascular remodelling and increased reactive oxygen species production in the lung; these effects were attenuated by ANA. Oestradiol levels were decreased in obese male mice; however, VAT CYP1B1 and 16αOHE1 levels were increased. TMS also attenuated pulmonary hypertension in male ob/ob mice. Intra-thoracic fat from ob/ob mice and VAT conditioned media produce 16αOHE1 and can contribute to oxidative stress, effects that are attenuated by both ANA and TMS.Obesity can induce pulmonary hypertension and changes in oestrogen metabolism, resulting in increased production of 16αOHE1 from VAT that contributes to oxidative stress. Oestrogen inhibitors are now in clinical trials for PAH. This study has translational consequences as it suggests that oestrogen inhibitors may be especially beneficial in treating obese individuals with PAH.

Depression enhanced the production of autoantibodies against 16α­hydroxyestrone-estrogen receptor adduct in breast cancer.

Mentally depressed breast cancer (MDBC) patients expressed estrogen receptor (ER) and 16α­hydroxyestrone (16α­OHE1) is directly responsible for causing breast cancer (BC). This study aimed to identify whether depression in breast cancer patients enhanced the production of autoantibodies against 16α­OHE1-ER adduct in breast cancer patients. The antibodies in the serum of 65 breast cancer patients (including 35 MDBC) and 40 control subjects were screened by direct binding, inhibition enzyme-linked immunosorbent assay (ELISA), and quantitative precipitin titration. Competition ELISA was also utilized for the estimation of 16α­OHE1 in the serum of 30 cancer patients. Autoantibodies from MDBC showed strong recognition to 16α­OHE1-ER in comparison to overall breast cancer patients (p < 0.05) and control subjects (p < 0.001). Although breast cancer sera showed high binding to 16α­OHE1-ER in comparison to ER (p < 0.05) or 16α­OHE1 (p < 0.001), the relative affinities of autoantibodies for 16α­OHE1-ER were found to be 1.38 × 10-7 and 1.23 × 10-7 for breast cancer and MDBC patients respectively. No significant difference, either in the level of 16α­OHE1 or 2­hydroxyestrone/16α­OHE1 ratio, was observed in the serum of cancer patients compared with controls, although inflammatory cytokines (IL-6, TNF-α) were significantly high in these patients. Depression in breast cancer patients augments the production of autoantibodies against 16α­OHE1-ER through the generation of inflammatory conditions. Depression in these patients increased the release of pro-inflammatory cytokines that generate more autoantibodies and show strong binding with 16α­OHE1-ER.

16 α-Hydroxyestrone induced adduct generate high affinity autoantibodies in SLE.

PURPOSE: Increased 16 α-hydroxyestrone (16 α-OHE1) and autoantibodies against histone H1 (H1) have been well described in systemic lupus erythematosus (SLE), but the combination effects of 16 α-OHE1 and H1 remains unclear. Here, we tried to assess the affinity and binding specificity of SLE autoantibodies against the 16 α-OHE1-H1 adduct. IgG was induced against this adduct and was also used as immunochemical probe for the estimation of 16 α-OHE1 in the serum of SLE patients. MATERIALS AND METHODS: The affinity and binding specificities of SLE autoantibodies against 16 α-OHE1-H1 were determined by direct binding and inhibition ELISA as well as quantitative precipitation titration in 60 patients and 30 control subjects. RESULTS: Purified SLE autoantibodies showed greater recognition to 16 α-OHE1-H1 over H1 (p < 0.05) or 16 α-OHE1 (p < 0.001). The relative affinity of SLE autoantibodies for 16 α-OHE1-H1, H1 and 16 α-OHE1 was 1.41 × 10-7, 1.31 × 10-6, and 1.03 × 10-6, respectively. The concentration of 16 α-OHE1 in the sera of SLE patients was significantly higher than controls (p < 0.05) as estimated by anti-16 α-OHE1-H1 antibodies. CONCLUSIONS: High affinity of 16 α-OHE1-H1 with SLE autoantibodies might suggest an antigenic role of this adduct in the production of these autoantibodies. The anti-16 α-OHE1-H1 antibody is a good immunochemical probe to measure 16 α-OHE1 in different SLE sera.