Steroid Biochem Research Articles

Production of 11α-Hydroxysteroid Derivatives by Corynebacterium glutamicum Expressing the Rhizopus oryzae Hydroxylating System.

Hydroxylation of steroids has acquired special relevance for the pharmaceutical industry. Particularly, the 11α-hydroxylation of steroids is a process of biotechnological importance currently carried out at industrial scale for the production of contraceptive drugs and glucocorticoids. This process is performed by several fungal species including Rhizopus nigricans, Aspergillus ochraceus, Aspergillus niger, and Rhizopus oryzae that are used to produce by biotransformation hydroxylated steroids for pharmaceutical purposes (Wang et al., J Steroid Biochem Mol Biol 171:254-261, 2017). However, the development of more efficient biotransformation processes is essential since the steroidal derivatives obtained by the in vivo hydroxylation are often a mixture of hydroxylated compounds in different positions of the steroid molecule. This phenomenon is due to the large number of different CYPs contained in the fungal strains.The genes responsible for the 11α-hydroxylase activity in R. oryzae consisting in the cytochrome CYP509C12 and its redox partner, the reductase RoCPR1, have been chemically synthetized forming a synthetic operon named FUN optimized to be expressed in bacteria. To express this operon, we have selected the strain Corynebacterium glutamicum R31 that is a robust and GRAS bacterial strain widely used for industrial purposes. The synthetic operon has been cloned in the pECXK-99E vector, yielding pXKFUN plasmid, and transformed C. glutamicum R31 to generate C. glutamicum R31 (pXKFUN) strain. This strain is not a steroid degrader and can efficiently transport C19 and C21 steroids across the cytoplasmic membrane (García-Fernandez et al. Catalysts 316:1-12, 2017). C. glutamicum can be used as a clean host for steroid biotransformation, because it does not introduce additional undesired side reactions on the steroids, thus reducing the contamination of the final products (Felpeto-Santero et al., Microbiol Biotechnol 12:856-868, 2019). Here we show a proof of concept that C. glutamicum can be used as a suitable chassis to perform steroid biotransformation expressing eukaryotic cytochromes. The protocol below provides detailed information on steroid 11α-hydroxylations by Corynebacterium recombinant strain.

The Importance of Vitamin D in Long-Term Care Residents

The Importance of Vitamin D in Long-Term Care Residents

Simultaneous Determination of Cortisol and Testosterone from Rat Serum by Liquid Chromatography-Tandem Mass Spectrometry

A liquid chromatography-electrospray ionization tandem mass spectrometry in a positive mode (LC-ESI-MS/MS) was developed to determine simultaneously the levels of cortisol and testosterone in rat serum. The MRM (Multiple Reaction Monitoring) was used to optimize ion precursor, fragmentor voltage, ion product, and collision energy. Analytical samples were treated by solid-phase extraction with Bond Elut C18 cartridge. The method was validated according to FDA guidance for bioanalytical method validation. The results indicated that this method showed a wide linearity range (0.1-100 ng/mL for cortisol and 0.025-100 ng/mL for testosterone), good linearity (r2 > 0.999), low limit of quantification (LLOQ values of cortisol and testosterone were 0.1 ng/mL and 0.025 ng/mL, respectively), good recovery (90.4% - 108.0%), suitable repeatability (CV values were in the range of 2.7% - 14.8%), good stability in different conditions. The validated method was applied to quantify cortisol and testosterone levels in several rat blood samples.
 Keywords: LC-ESI-MS/MS, cortisol, testosterone, rat serum, solid-phase extraction.
 References
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Highly Sensitive Simultaneous Measurement of Steroid Hormone in Human Serum by Liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry

Highly Sensitive Simultaneous Measurement of Steroid Hormone in Human Serum by Liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry

Apolipoprotein E polymorphism in Kazakhstani population with Alzheimer’s disease

AbstractBackgroundAdvanced age, apolipoprotein E ε4 (APOE) and gender are well‐known risk factors for Alzheimer's disease (AD). At least one copy of APOE‐ε4 is present in around 60% of AD cases (1). However, there are worldwide variations of APOE‐ε4 carriers by region and by ethnicity (2). There is no available data on distribution of APOE polymorphism among Alzheimer’s patients in Kazakhstan. Thus, we assessed the apolipoprotein E allele and genotype frequencies in patients with Alzheimer’s disease and compared them to normal controls.MethodWe included 53 AD patients and 99 people with normal cognition recruited from city hospital in Astana, Kazakhstan. APOE genotypes were determined by Real Time PCR using TaqMan® SNP Genotyping Assays.ResultThe APOE‐ε4 allele frequencies were found to be 82.7% for ε3, 1.9% for ε2, 41.5% for ε4 in AD group, and 92.9% for ε3, 8.1% for ε2 and 25.2% for ε4 in the control group. Genotype ε4/ε4 was found in 9.4% of AD patients compared to 1% in controls (p<0.005) Genotypic distribution of APOE polymorphism did not differ between gender in study group, however, APOE‐ε4 carrying status was significantly different by ethnic groups (p=0.04), where Kazakhs had 34.3% of APOE‐ε4 carriers, Russian and other ethnic groups 16.6% and 17.6% respectively.ConclusionThis is the first study of APOE polymorphism distribution in AD patients in Kazakhstan. APOE ε4 carrier frequency was similar to other Asian and Central European populations. Our findings are consistent with previous literature on the significance of the subjects' geographical region and ethnic context in the study of APOE genotypes in association with AD. Reference: 1. Riedel BC, Thompson PM, Brinton RD. Age, APOE and sex: Triad of risk of Alzheimer's disease. J Steroid Biochem Mol Biol. 2016 Jun;160:134‐47. doi: 10.1016/j.jsbmb.2016.03.012. Epub 2016 Mar 8. PMID: 26969397; PMCID: PMC4905558. 2. Crean S, Ward A, Mercaldi CJ, Collins JM, Cook MN, Baker NL, Arrighi HM. Apolipoprotein E ε4 prevalence in Alzheimer's disease patients varies across global populations: a systematic literature review and meta‐analysis. Dement Geriatr Cogn Disord. 2011;31(1):20‐30. doi: 10.1159/000321984. Epub 2010 Dec 1. PMID: 21124030.

Trend research of vitamin D receptor: Bibliometric analysis.

Studies on vitamin D receptor (VDR) and its association with multiple disorders are expanding. This bibliometric study aims to find and summarize VDR-related publications, and compare them across various countries, organizations, and journals to demonstrate trends in VDR research. VOSviewer and Excel 2019 were used to classify and summarize Web of Science articles from 1900 to mid-2021. Total records of 8762 articles were analyzed, and maps of co-citations bibliometric keywords co-occurrence were designed. In conclusion, relative research interest and published papers related to VDR were growing in the past 30 years. The United States of America dominates the research regarding VDR. The highest quality of VDR research was achieved by the University of California System, University of Wisconsin System, and Harvard University. J Steroid Biochem Mol Biol, PLoS One, and J Biol Chem are the leading three productive journals on VDR. Various aspects of vitamin D deficiency associated disorders and genetic studies regarding VDR, including single nucleotide polymorphism, gene variants, epigenome, long non-coding ribonucleic acid (lncRNA), and small nucleolar RNA host gene 6 are potentially the recent research hotspot in this field. Moreover, coronavirus disease, polycystic ovary syndrome, non-alcoholic fatty liver disease, gut microbiota, gestational diabetes, systemic sclerosis, and chemoresistance are the trending medical conditions associated with VDR.

Vitamin D and Lumisterol Hydroxyderivatives Can Act on Liver X Receptors (LXRs)

Abstract New pathways of vitamin D3 (D3) activation initiated by CYP11A1 and involving other CYPs have been discovered. At least 15 hydroxyderivatives, including 20(OH)D3 as the major product, are generated by these pathways (1,2) with some being present in human serum, epidermis, and pig adrenals. CYP11A1 can also metabolize 7-dehydrocholesterol to produce 7-dehydropregnenolone, which can be further modified by steroidogenic enzymes generating Δ7-steroids (1,2). Lastly, CYP11A1 and CYP27A1 act on lumisterol (L3) producing at least 9 biologically active derivatives (1,2). Thus, new pathways generating a large number of biologically active secosteroids and lumisterol-derivatives have now been described. These compounds interact with the vitamin D receptor (VDR), retinoic acid receptors (RORs) α and γ, and the aryl hydrocarbon receptor (AhR)(1). These findings challenge dogmas that lumisterol is biologically inactive and that 1,25(OH)2D3 is the only active form of D3 exerting its effects exclusively through interaction with the VDR. In view of the above and since liver X receptors (LXRs) can be activated by oxysterols, we investigated the interactions of novel products of L3 and D3 metabolism with LXRs. Molecular docking, using crystal structures of the ligand binding domains (LBDs) of LXRα and β, revealed high docking scores for L3 and D3 hydroxymetabolites, like those of the natural ligands, predicting good receptor binding. RNA sequencing of murine dermal fibroblasts stimulated with D3-hydroxyderivatives revealed LXR as the second major nuclear receptor signaling pathway for several D3-hydroxyderivatives, including 1,25(OH)2D3. The involvement of LXRs was validated by the induction of several genes downstream of LXR. Furthermore, L3 and D3-hydroxyderivatives activated an LXR-response element (LXRE)-driven reporter in CHO cells and human keratinocytes. For keratinocytes, enhanced expression of LXR target genes was also observed supporting the involvement of LXR. Importantly, L3 and D3 derivatives showed high affinity binding to the LBD of the LXRα and β in LanthaScreen TR-FRET LXRα and β coactivator assays. The majority of metabolites functioned as LXRα/β agonists; however, 1,20,25(OH)3D3, 1,25(OH)2D3, 1,20(OH)2D3 and 25(OH)D3 acted as inverse agonists of LXRα, but as agonists of LXRβ. Molecular dynamics simulations performed for selected compounds, including 1,25(OH)2D3, 1,20(OH)2D3, 25(OH)D3, 20(OH)D3, 20(OH)L3 and 20,22(OH)2L3, showed overlapping and different interactions with LXRs. Identification of D3 and L3 derivatives as ligands for LXRs changes the accepted paradigms on their biological role and mechanism of action. 1. Cell Biochem Biophys. 2020;78(2):165-180. 2. J Steroid Biochem Mol Biol. 2019;186:4-21.

Novel mechanisms of testosterone detoxification in UGT2B17 gene deletion carriers and androgen activation by gut microbiome

Background and objectives Testosterone metabolism by the highly polymorphic UDP-glucuronosyltransferase 2B17 (UGT2B17) is widely recognized, but the metabolic fate of testosterone in the carriers of UGT2B17 gene deletion is not well understood. Similarly, testosterone glucuronide (TG) is prone to secretion into intestinal lumen (1); however, the role of microbiome in activation of testosterone is not studied. Therefore, the aims of this study were: i) to elucidate the alteration in the liver proteome and its effect on steroid metabolism pathways (i.e., metabolic switching) in the UGT2B17 gene deletion, and ii) to assess the TG deconjugation by gut β-glucuronidases. Methods Human liver S9 fraction with gene deletion and high expression (n=3) were used. Untargeted proteomics data was acquired using Easy nano-LC 1200 coupled with the high-resolution QE-HF Orbitrap. The proteomic data were analyzed, processed and pathway mapping was performed using MaxQuant, Perseus and STRING, respectively. In vitro studies in liver S9 fraction of UGT2B17 gene deletion were carried out to investigate testosterone metabolism by aldo-keto reductases (AKRs). The glucuronidation pathway of 5β-dihydrotestosterone (5β-DHT) and 5β-tetrahydrotestosterone (5β-THT) were identified using recombinant UGT2B isoforms. These data were verified using testosterone incubations in human hepatocyte and serum levels of 5β-THTG after oral testosterone dosing (800 mg) in human. Activation mechanism TG to testosterone in the human gut was investigated by incubating TG with recombinant human gut microbial β-glucuronidases and human fecal extracts. Results A total of 1002 liver proteins were quantified in the S9 fraction, of which 14 and 39 proteins were exclusively from the deletion and high-expressor groups, respectively, whereas 949 proteins were common. The following pathways were upregulated in UGT2B17 deletion group: oxidation-reduction, drug metabolic, steroid metabolic, and fatty acid metabolic process. Notable proteins that were upregulated in the gene deletion groups were: AKR1D1, AKR1C4, AKR7DA3, AKR1A1, DHCR7, ADH6, ADH1C, ALDH1A1, ALDH9A1, and ALDH5A1 A deeper molecular pathway analysis revealed that the AKR1D1 and AKR1C4 that metabolize testosterone to inactive 5β-DHT and 5β-THT, respectively (2) (Figure 1) were significantly upregulated in UGT2B17 deletion group. UGT2B7 is primarily responsible for glucuronidation of 5β-THT (Figure 1). Finally, incubation with purified bacterial β-glucuronidases and human fecal extracts confirmed facile but variable deconjugation of TG to testosterone in human gut. Conclusion Novel mechanisms of testosterone metabolic switching in UGT2B17 gene deletion and testosterone activation by gut microbiome have been identified. These findings have a potential to provide deeper insights into the mechanisms of testosterone associated pathophysiological conditions, such obesity, insulin sensitivity, and cancer. References Li, C. Y. et al. J Steroid Biochem Mol Biol (2019). Rižner et al. Steroids (2014).

The Association between Dietary Inflammatory Index and Sex Hormones among Men in the United States.

This study investigated the association between Dietary Inflammatory Index and sex hormones in a large, nationally representative adult male sample. We utilized data from the 2013-2014 and 2015-2016 National Health and Nutrition Examination Survey. Males aged ≥20 years who provided a 24-hour dietary intake history and underwent serum sex hormone testing were included in analysis. Weighted proportions and multivariable analysis controlling for age, race, energy, smoking status, education level, body mass index and time of venipuncture were used to evaluate the associations between Dietary Inflammatory Index and sex hormones. For 4,151 participants, Dietary Inflammatory Index ranged from -5.05 to 5.48. Mean±SD total testosterone was 419.30±176.27 ng/dl. Mean±SD total testosterone was lower among men in the highest tertile compared with men in the lowest tertile group (410.42±171.97 vs 422.71±175.69, p <0.001). A per unit increase in Dietary Inflammatory Index was related to 4.0% (95% CI 0.5-7.6) higher odds of testosterone deficiency. In the fully adjusted multivariable model, males in Dietary Inflammatory Index tertile 3 (the most pro-inflammatory) had 29.6% (3.1-63.0) higher odds of testosterone deficiency than those in tertile 1 (p trend=0.025). Interaction tests revealed no significant effect of body mass index on the association of Dietary Inflammatory Index with testosterone deficiency and all sex hormone parameters. Men adhering to a more pro-inflammatory diet appear to have a higher risk of testosterone deficiency, indicating the important role of diet in male reproductive health.

Reply to Jakovac: “COVID-19 and vitamin D—Is there a link and an opportunity for intervention?”

Reply to Jakovac: “COVID-19 and vitamin D—Is there a link and an opportunity for intervention?”

P0992PHARMACOLOGICAL UPREGULATION OF INTEGRIN-LINKED KINASE (ILK) REDUCES VISCERAL ADIPOSITY IN A METABOLIC SYNDROME MICE MODEL

Abstract Background and Aims Important mechanism associated to the development of Insulin resistance (IR) are dysregulation of lipid adjustment and excessive deposition of extracellular matrix (ECM) in the visceral white adipose tissue (WAT). The transgenic depletion of the ECM-to-cell scaffold intracellular protein Integrin-linked kinase (ILK) modulates IR in WAT [Hatem-Vaquero et al. J Endocrinol. 2017] and body weight gain and adipose malfunction correlates with early downregulation of ILK expression in WAT during a short-term high fat diet (HFD) mice model [Hatem-Vaquero et al. Cell Physiol Biochem 2020]. Thus, we demonstrated that transcriptional regulation of ILK might govern the expansion of adipocytes. Between several transcriptional factors implicated in lipid metabolism, the activation of the transcription factor peroxisome proliferator activated receptor (PPAR) family member PPARβ/δ ameliorates IR. Taking in consideration that it modulates the expression of ILK in other contexts aside metabolism [Di-Poï N et al. J Steroid Biochem Mol Biol. 2003, Zhu B et al. Oncogene. 2014], here we activated PPARβ/δ in vivo and in vitro to upregulate adipose ILK expression and prevent WAT expansion. Method Adult mice were fed with either standard (STD) or HFD diets during 4 weeks and divided into groups treated with PPARβ/δ activator L-165041 (i.p. 2 mg/kg b.w per day) or vehicle for 2 additional weeks with maintained diet challenges [Lim HJ et al. Eur J Pharmacol. 2009]. Body and epidydimal WAT depot (eWAT) weights changes were compared and ILK expression was determined by RT-qPCR. Comparative studies were performed in cultured C3H10t1/2-based adipocytes where ILK expression was deliberately downregulated by the transfection of siRNAs against ILK or in control adipocytes (scramble siRNAs). After cells were treated with L-165041 (1 microM, 24h), we determined the intracellular triglyceride content (by adipo-red dye), the expression of ILK and its downstream substrate AKT (total and active p-AKT in ser473) by RT-qPCR or Western blot. Results HFD increased progressively eWAT and whole body weight gains, which coincide with increase in IR. PPARβ/δ-activated mice under STD or HFD have preventive reduction of weight gains and increased expression of ILK in eWAT. Downregulated ILK expression in culture adipocytes coincide with increased triglyceride content, whereas the activation of PPARβ/δ reduced it at the same time that increased ILK expression and activity. Conclusion We suggest ILK modulation as a novel therapeutic strategy during the obesity establishment. The pharmacological activation of PPARβ/δ increases ILK expression and activity, which correlates with a weight loss in IR-based models in vivo. Although the increased presence of ILK induces the phosphorylation of AKT, in turn one of the main mediators during insulin signalling, we cannot conclude the role of ILK-mediated AKT modulation during the reduction of the adipocyte expansion observed

SUN-LB43 DLK1 Expressing Cells Contribute to the Zonation of the Adrenal Gland

The adrenal cortex is a dynamic organ that undergoes self-renewal and remodeling in response to the demand for steroids. In the mouse it is divided into two concentric layers, the outer zona glomerulosa and the inner zona fasciculata (ZF), that secrete aldosterone and corticosterone, respectively. Cell fate mapping studies have shown that the maintenance of the cortex relies on a pool of stem/progenitor cells located in the capsular and subcapsular compartments. Two interconnected cell populations have been identified, subcapsular undifferentiated cells secreting the morphogen Sonic Hedgehog (Shh) and capsular Gli1+ cells, which can transduce the Shh signal (1); both populations are precursors of steroidogenic cells and newly formed cells migrate in a centripetal fashion to repopulate the gland until they reach the juxtamedullary region where they undergo senescence and apoptosis. Moreover, our lab has shown that the Notch atypical ligand Delta-Like homologue 1 (Dlk1) is expressed in partially undifferentiated cells of the subcapsular region in rat (2)and human (3)adrenals, whilst it is mostly expressed in capsular cells in mice (4,5). To investigate whether Dlk1 expressing cells contribute to the zonation of the adrenal cortex we conducted lineage tracing analyses using a tamoxifen inducible Dlk1CreERT2mouse model carrying the R26tdTom reporter. Pregnant dames were injected with tamoxifen at embryonic day (e) 12.5 and pups were culled at postnatal day (p) 10 and p38. Analysis of tdTomato expression showed that 35% (p10) and 24% (p38) of Steroidogenic Factor 1(Sf1)+cortical cells were tdTomato+, revealing that capsular Dlk1+cells are steroidogenic precursors. On the other hand, postnatal tamoxifen injections (p0) showed tdTomato+/Sf1+ cells only in 1-2% in cortical cells after 24-months chase, suggesting that the contribution of Dlk1+cells to adrenocortical self-renewal is limited postnatally.However, the Dlk1+population could be reactivated in the adult mouse treated with dexamethasone and was shown to contribute to the regeneration of the ZF once dexamethasone treatment was ceased. 1. King P, et al. Shh signaling regulates adrenocortical development and identifies progenitors of steroidogenic lineages. Proc Natl Acad Sci(2009) 106:21185-211902. Guasti L, et al. Dlk1 Up-Regulates Gli1 Expression in Male Rat Adrenal Capsule Cells Through the Activation of β1 Integrin and ERK1/2. Endocrinology(2013) 154:4675-46843. Hadjidemetriou I, et al. DLK1/PREF1 marks a novel cell population in the human adrenal cortex. J Steroid Biochem Mol Biol(2019) 193:1054224. Guasti L, Candy Sze WC, McKay T, Grose R, King PJ. FGF signalling through Fgfr2 isoform IIIb regulates adrenal cortex development. Mol Cell Endocrinol(2013) 371:182-1885. Heikkilä M, et al. Wnt-4 Deficiency Alters Mouse Adrenal Cortex Function, Reducing Aldosterone Production. Endocrinology(2002) 143:4358-4365

GPER1 Signaling Initiates Migration of Female V-SVZ-Derived Cells.

SummaryIn the rodent ventricular-subventricular zone (V-SVZ) neurons are generated throughout life. They migrate along the rostral migratory stream (RMS) into the olfactory bulb before their final differentiation into interneurons and integration into local circuits. Estrogen receptors (ERs) are steroid hormone receptors with important functions in neurogenesis and synaptic plasticity. In this study, we show that the ER GPER1 is expressed in subsets of cells within the V-SVZ of female animals and provide evidence for a potential local estrogen source from aromatase-positive astrocytes surrounding the RMS. Blocking of GPER1 in Matrigel cultures of female animals significantly impairs migration of V-SVZ-derived cells. This outgrowth is accompanied by regulation of phosphorylation of the actin-binding protein cofilin by GPER1 signaling including an involvement of the p21-Ras pathway. Our results point to a prominent role of GPER1 in the initiation of neuronal migration from the V-SVZ to the olfactory bulb.

Abstract P6-04-26: PI3K inhibitors restored the sensitivity to anti-estrogen drugs in endocrine therapy resistant cell lines

Abstract Background The Combination of endocrine therapy with molecular targeted drugs has become one of the standard treatment option for ER positive advanced or metastatic breast cancer. The therapeutic effects of PI3K-mTOR inhibitors, which inhibit intracellular phosphorylation of signaling pathways, have been clarified in clinical trials. However, the effects of these drugs on ER signal pathways in endocrine-resistant breast cancer have not been fully elucidated. We investigated the changes in the expression and functions of ER when endocrine-resistant cell lines, established in our laboratory, were treated with PI3K-mTOR inhibitors Methods Long-term estrogen deprivation-resistant (EDR) cell lines and fulvestrant-resistant cell lines (MFR) were established from MCF-7 cells in our previous studies. These cell lines exhibited different ER expression levels, including high (EDR-1), low (EDR-2), or no expression (MFR). All these cell lines had the same PIK3CA mutations as the parental cell lines. We used buparlisib (BUP, pan-class1 PI3K inhibitor) and alpelisib (ALP, α-specific PI3K inhibitor) as PI3K inhibitors and everolimus (EVE) as an mTOR inhibitor. Tamoxifen and fulvestrant were used in anti-estrogen therapy. In this study, the procedure to examine histone modifications was CHIP-qtPCR analysis. Results EDR-1, -2 and MFR were treated with the PI3K-TOR inhibitors for 24 h. There were no changes in ER expression and ER activity on EDR-1 and MFR before and after the addition of PI3K-mTOR inhibitors. Interestingly, ER expression and ER activity in EDR-2 increased after the addition of PI3K-mTOR inhibitors. In particular, a high re-expression of ER was observed when the PI3K inhibitors were added. Next, in order to evaluate the effects of endocrine therapy on these cell lines Using BUP, ALP and EVE, each drug resistant cell lines were established from EDR-2. When the cell proliferation assay was performed in the established resistant cell line, EDR-2 which showed re-expression of ER on adding the PI3K inhibitors, showed cell growth-inhibitory effects with the addition of anti-estrogen agents. Previously, we reported that epigenetic regulation contributes to the reduction of ER expression in EDR-2. (Tsuboi et al, J Steroid Biochem Mol Biol 2017). Therefore, we investigated the possibility that epigenetic regulation could contribute to the mechanism of ER re-expression when PI3K inhibitors were used in EDR-2. It was revealed that the change of H3K27me3, one of the histonemodifications, correlated with the expression of ER. Conclusion PI3K inhibitor could restore the expression of ER and sensitivity to anti-estrogen drugs in endocrine therapy resistant cell line in which ER expression was lost during acquired resistant period. This restoration might be due to a histone modification (H3K27me3). Citation Format: Emi Tokuda, Shunta Sasaki, Kouki Tsuboi, Masafumi Iida, Toshifumi Niwa, Shigehira Saji, Shin-Ichi Hayashi. PI3K inhibitors restored the sensitivity to anti-estrogen drugs in endocrine therapy resistant cell lines [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-26.

Molecular interactions between sex hormone–binding globulin and nonsteroidal ligands that enhance androgen activity

Molecular interactions between sex hormone–binding globulin and nonsteroidal ligands that enhance androgen activity

Melanin has a Small Inhibitory Effect on Cutaneous Vitamin D Synthesis: A Comparison of Extreme Phenotypes

Epidemiology suggests that melanin inhibits cutaneous vitamin D3 synthesis by UVR. Laboratory investigations assessing the impact of melanin on vitamin D production have produced contradictory results. We determined the effect of melanin on vitamin D3 photosynthesis in healthy young volunteers (n= 102) of Fitzpatrick skin types II-VI (white to black). Participants, irrespective of skin type, were exposed to the same suberythemal UVR dose, to 85% body surface area, using solar simulated UVR or narrowband UVB (311 nm). This was repeated five times with intervals of 3-4 days between UVR exposures. Blood was taken before, during, and after the irradiation and assessed for serum 25-hydroxyvitamin D3 (25[OH]D3) as a marker of vitamin D3 status. Linear UVR dose-dependent increases in 25(OH)D3 were highly significant (P ≤ 7.7 x 10-11). The ratios of regression slopes of the different skin type groups were compared, and only skin type II was significantly steeper than the other groups. Comparisons between extreme skin types II and VI showed melanin inhibition factors of approximately 1.3-1.4, depending on the UVR source. We conclude that the inhibitory effect of melanin on vitamin D3 synthesis is small, compared with erythema, but that this difference may be sufficient to explain the epidemiological data.

OR02-5 Novel Intermediates Are Catalysed By 11βHSD And CYP17A1 In The C11-oxy Backdoor Pathway Leading To The Production Of 11-ketodihydrotestosterone

Adrenal C19 steroids, testosterone and androstenedione, have long been the only androgens considered contributing to clinical conditions characterised by androgen excess. More recently, we showed that adrenal 11β-hydroxyandrostenedione is converted to 11-ketotestosterone and 11-ketodihydrotestosterone (11KDHT), both activating the androgen receptor comparable to dihydrotestosterone (1). The adrenal, however, also produces C11-oxy C21 steroids, 21-deoxycortisol (21dF) and low levels of 11β-hydroxyprogesterone (11OHP4), which are both metabolised to 11KDHT. While 11βHSD2 efficiently converts 11OHP4 and 21dF to 11-ketoprogesterone (11KP4) and 21-deoxycortisone (21dE) respectively, 11OHP4 and 11KP4 are more readily reduced by SRD5A than 21dF and 21dE in the backdoor pathway (2,3). To study the contribution of 11OHP4 and 21dF to the metabolic shunt towards active androgens, 11βHSD and CYP17A1 were expressed in HEK-293 cells and the conversion of novel intermediates (1μM) analysed using UPC2-MS/MS. Once reduced at C3 and the C4-C5 double bond, the 11OHP4 and 21dF intermediates can be further metabolised by 11βHSD2 and CYP17A1 in the C11-oxy backdoor pathway. Conversion by 11βHSD2 yielded alfaxalone (±0.47 μM) and 11-ketoPdiol (±0.75 μM) with 3,11diOH-dihydroprogesterone (3,11diOHDHP4) (±0.52 μM) and 11OHPdiol (±0.24 μM) remaining after 24 hrs. In the conversion of alfaxalone and 11-ketoPdiol by 11βHSD1 most of the substrate was still detected after 24 hrs, ±0.9 μM. Conversion of 1 μM 11OHPdiol and 11-ketoPdiol by CYP17 yielded the lyase products: ±0.18 μM 11β-hydroxyandrosterone (11OHAST) and ±0.25 μM 11ketoandrosterone (11KAST) after 12 hrs. Conversion of 1 μM 3,11diOHDHP4 and alfaxalone yielded the hydroxylase products 11OHPdiol (±0.15 μM) and 11-ketoPdiol (±0.05 μM) and the lyase products, 11OHAST (±0.06 μM) and 11KAST (±0.14 μM). It was only 3,11diOHDHP4 that was further converted to 0.33 μM 11OHPdiol and ±0.16 μM11OHAST after 24hrs. Both adrenal 11OHP4 and 21dF would, via 11OHAST which is also converted to 11KAST by 11βHSD2, readily lead to the production of 11KDHT catalysed by the backdoor steroidogenic enzymes. Data show that the C17 hydroxyl group favours the conversion of intermediates catalysed by 11βHSD2 (75% vs 50 % substrate conversion) with the oxidation reaction by 11βHSD2 being the preferred reaction compared to 11βHSD1. CYP17A1 catalyses both the hydroxylase and 17,21 lyase reactions with the conversion of 3,11diOHDHP4 being the most favourable. Overall, this study highlights the contribution of the C11-oxy C21 steroids to the active androgen pool and relates their physiological relevance to adrenal-linked endocrine diseases characterised by androgen excess. (1) Bloem et al., J. Steroid Biochem. Mol. Biol. 2015 153:80-92 (2) Barnard et al., J. Steroid Biochem. Mol. Biol. 2017 174:86-95 (3) van Rooyen et al., J. Steroid Biochem. Mol. Biol. 178:203-212

Functional deficits in menopause-associated encephalopathy: a behavioural rodent study.

Event Abstract Back to Event Functional deficits in menopause-associated encephalopathy: a behavioural rodent study. Ming-Sum Yu1, Rudi D'Hooge1 and Ann Van Der Jeugd1* 1 KU Leuven, Belgium In this study, 4-months old ovariectomised (OVX) mice (n = 15) and sham control mice of the same age (n = 15) were used. Following surgery and recovery, all mice were subjected to behavioural tests at 6 months to examine the effects of early menopause on behaviour. The tests examined general exploratory activity, anxiety, depression-like behaviours, object recognition memory, social exploration and sociability, and several forms of learning and memory. Results of the open field test showed that, compared to shams, OVX mice did not spend less time in the centre of the arena (Figure 1. A), but were significantly more reluctant to enter the centre (Figure 1. B). In concordance, in the elevated plus maze test, OVX mice spent significantly less time in the open arms of the elevated plus-maze (Figure 1.C) compared to sham mice. When tested for behavioural despair in the forced swim test, OVX mice were more immobile during the first half of the test, reflecting depression-like behaviors in these mice (Figure 1.D). Tested for cognitive functioning, OVX mice showed a significant decrease in novel-object recognition (Figure 2.A). Interestingly, ovariectomy also tended to affect social memory (Figure 2B.), but this needs further investigation. When tested for spatial learning and memory, OVX miced learned the hidden platform position equally well as shams (Figure 2.C). However, on the second probe trial, when the platform was removed, OVX mice spent less time in the target quadrant (Figure 2.D). This lacking preference for the former target in OVX mice was also reflected in the heat maps (Figure 2.E). Finally, when tested for fear memory in the passive avoidance test, OVX mice showed no difference in memory retention for the fearful event 24hrs later when compared to shams (Figure 2.F). Our findings suggest that menopause is associated with anxiety-like behaviour and cognitive problems. They indicate that our mouse model has menopause-associated encephalopathy-like features. We plan to link these first results to neuroplasticity and neurostructural measurements properties. Figure 1 Figure 2 Acknowledgements KU Leuven internal fund to RD, FWO postdoctoral fellowship to AVdJ References (1) Utian WH. Menopause. 2001 Dec;8(6):398–401. (2) Monteleone P et al. Nat Rev Endocrinol. 2018 Apr;14(4):199–215. (3) Weber MT et al. J Steroid Biochem Mol Biol. 2014 Jul;142:90–98. (4) Rocca WA et al. Menopause. 2008 Dec;15(6):1050–1059. (5) Walf AA et al. Pharmacol Biochem Behav. 2004 Jul;78(3):523–529. (6) Lund TD et al. Endocrinology. 2005 Feb;146(2):797–807. (7) de Chaves G et al. Physiol Behav. 2009 Jun 22;97(3-4):420–425. (8) Fratiglioni L et al. Neurology. 1997 Jan;48(1):132–138. (9) Gao S et al. Arch Gen Psychiatry. 1998 Sep;55(9):809–815. (10) Ruitenberg A et al. Neurobiol Aging. 2001 Aug;22(4):575–580. (11) Pike CJ et al. Front Neuroendocrinol. 2009 Jul;30(2):239–258. Keywords: Dementia, Menopause, encephalopathy, Learning and Memory (Neurosciences), Emotional behaviors Conference: 13th National Congress of the Belgian Society for Neuroscience , Brussels, Belgium, 24 May - 24 May, 2019. Presentation Type: Poster presentation Topic: Behavioral/Systems Neuroscience Citation: Yu M, D'Hooge R and Van Der Jeugd A (2019). Functional deficits in menopause-associated encephalopathy: a behavioural rodent study.. Front. Neurosci. Conference Abstract: 13th National Congress of the Belgian Society for Neuroscience . doi: 10.3389/conf.fnins.2019.96.00043 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Apr 2019; Published Online: 27 Sep 2019. * Correspondence: Dr. Ann Van Der Jeugd, KU Leuven, Leuven, 3000, Belgium, ann.vanderjeugd@psy.kuleuven.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Ming-Sum Yu Rudi D'Hooge Ann Van Der Jeugd Google Ming-Sum Yu Rudi D'Hooge Ann Van Der Jeugd Google Scholar Ming-Sum Yu Rudi D'Hooge Ann Van Der Jeugd PubMed Ming-Sum Yu Rudi D'Hooge Ann Van Der Jeugd Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

It's time to Stop Prescribing Ergocalciferol

Vitamin D is important for bone health and, while controversial, may play a role in multiple acute and chronic diseases. As a result, 25-hydroxyvitamin D [25(OH)D] testing is commonly performed, and, when low, supplement recommendations made or prescriptions written to correct hypovitaminosis D. Multiple supplementation options exist including various doses of cholecalciferol (vitamin D3). However, in the United States, hypovitaminosis D treatment often entails prescribing high-dose (50,000 IU) ergocalciferol (vitamin D2) ( 1. Holick M.F. Binkley N. Bischoff-Ferrari H.A. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2011; 96 (et al): 1911-1930 Crossref PubMed Scopus (6809) Google Scholar ). Such treatment occurs despite lack of consensus regarding the optimal 25(OH)D level for health; is it >15 ng/mL, >20 ng/mL, or >30 ng/mL? A major contributor to the lack of clarity regarding what constitutes “low” vitamin D status is inability of routine 25(OH)D assays to provide accurate results. Indeed, despite strides to improve 25(OH)D measurement, spearheaded by the Vitamin D Standardization Program (VDSP), substantial inaccuracy persists in clinical and research laboratories ( 2. Carter G.D. Berry J. Durazo-Arvizu R. Hydroxyvitamin D assays: An historical perspective from DEQAS. J Steroid Biochem Mol Biol. 2018; 177 (et al): 30-35 Crossref PubMed Scopus (62) Google Scholar , 3. Sempos C.T. Betz J.M. Camara J.E. General Steps to Standardize the Laboratory Measurement of Serum Total 25-Hydroxyvitamin D. J AOAC Int. 2017; 100 (et al): 1230-1233 Crossref PubMed Scopus (35) Google Scholar ). Why is this so?

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1: 18O LABELING AND OXYGEN SURROGATE EVIDENCE FOR A ROLE OF A PERFERRYL OXYGEN

Cytochrome P450 (P450) reactions can involve C-C bond cleavage, and several of these are critical in steroid and sterol biosynthesis. The mechanisms of P450s 11A1, 17A1, 19A1, and 51A1 have been controversial, in the context of the role of ferric peroxide (FeO2 (-)) versus perferryl (FeO(3+), compound I) chemistry. We reinvestigated the 17α-hydroxyprogesterone and 17α-hydroxypregnenolone 17α,20-lyase reactions of human P450 17A1 and found incorporation of one (18)O atom (from (18)O2) into acetic acid, consonant with proposals for a ferric peroxide mechanism (Akhtar, M., Lee-Robichaud, P., Akhtar, M. E., and Wright, J. N. (1997) J. Steroid Biochem. Mol. Biol. 61, 127-132; Akhtar, M., Wright, J. N., and Lee-Robichaud, P. (2011) J. Steroid Biochem. Mol. Biol. 125, 2-12). However, the reactions were supported by iodosylbenzene (a precursor of the FeO(3+) species) but not by H2O2 We propose three mechanisms that can involve the FeO(3+) entity and that explain the (18)O label in the acetic acid, two involving the intermediacy of an acetyl radical and one a steroid 17,20-dioxetane. P450 17A1 was found to perform 16-hydroxylation reactions on its 17α-hydroxylated products to yield 16,17α-dihydroxypregnenolone and progesterone, suggesting the presence of an active perferryloxo active species of P450 17A1 when its lyase substrate is bound. The 6β-hydroxylation of 16α,17α-dihydroxyprogesterone and the oxidation of both 16α,17α-dihydroxyprogesterone and 16α,17α-dihydroxypregnenolone to 16-hydroxy lyase products were also observed. We provide evidence for the contribution of a compound I mechanism, although contribution of a ferric peroxide pathway in the 17α,20-lyase reaction cannot be excluded.