scholarly journals Influence of Indomethacin on Steroid Metabolism: Endocrine Disruption and Confounding Effects in Urinary Steroid Profiling of Anti-Doping Analyses

Metabolites ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 463
Author(s):  
Anna Stoll ◽  
Michele Iannone ◽  
Giuseppina De Gregorio ◽  
Francesco Molaioni ◽  
Xavier de la Torre ◽  
...  
Keyword(s):  
Steroid Metabolism ◽  
Control Analysis ◽  
As Doping ◽  
Steroid Profiling ◽  
Steroid Profile ◽  
World Anti Doping Agency ◽  
Androgenic Steroids ◽  
Endogenous Steroid

Anabolic androgenic steroids (AAS) are prohibited as doping substances in sports by the World Anti-Doping Agency. Concentrations and concentration ratios of endogenous AAS (steroid profile markers) in urine samples collected from athletes are used to detect their administration. Certain (non-prohibited) drugs have been shown to influence the steroid profile and thereby sophisticate anti-doping analysis. It was shown in vitro that the non-steroidal anti-inflammatory drug (NSAID) indomethacin inhibits selected steroid-biotransformations catalyzed by the aldo-keto reductase (AKR) 1C3, which plays a key role in the endogenous steroid metabolism. Kinetic parameters for the indomethacin-mediated inhibition of the AKR1C3 catalyzed reduction in etiocholanolone were determined in vitro using two comparing methods. As NSAIDs are very frequently used (not only) by athletes, the inhibitory impact of indomethacin intake on the steroid metabolism was evaluated, and steroid profile alterations were detected in vivo (one male and one female volunteer). Significant differences between samples collected before, during or after the intake of indomethacin for selected steroid profile markers were observed. The presented results are of relevance for the interpretation of results from doping control analysis. Additionally, the administration of NSAIDs should be carefully reconsidered due to their potential as endocrine disruptors.

scholarly journals uPA+/+-SCID Mouse with Humanized Liver as a Model for In Vivo Metabolism of Exogenous Steroids: Methandienone as a Case Study

2009 ◽  
Vol 55 (10) ◽  
pp. 1783-1793 ◽  
Author(s):  
Leen Lootens ◽  
Philip Meuleman ◽  
Oscar J Pozo ◽  
Peter Van Eenoo ◽  
Geert Leroux-Roels ◽  
...  
Keyword(s):  
Animal Model ◽  
Scid Mouse ◽  
Small Animal ◽  
Steroid Metabolism ◽  
Control Analysis ◽  
Chimeric Mouse ◽  
Small Animal Model ◽  
Suitable Alternative

Abstract Background: Adequate detection of designer steroids in the urine of athletes is still a challenge in doping control analysis and requires knowledge of steroid metabolism. In this study we investigated whether uPA+/+-SCID mice carrying functional primary human hepatocytes in their liver would provide a suitable alternative small animal model for the investigation of human steroid metabolism in vivo. Methods: A quantitative method based on liquid chromatography–tandem mass spectrometry (LC-MS/MS) was developed and validated for the urinary detection of 7 known methandienone metabolites. Application of this method to urine samples from humanized mice after methandienone administration allowed for comparison with data from in vivo human samples and with reported methandienone data from in vitro hepatocyte cultures. Results: The LC-MS/MS method validation in mouse and human urine indicated good linearity, precision, and recovery. Using this method we quantified 6 of 7 known human methandienone metabolites in the urine of chimeric mice, whereas in control nonchimeric mice we detected only 2 metabolites. These results correlated very well with methandienone metabolism in humans. In addition, we detected 4 isomers of methandienone metabolites in both human and chimeric mouse urine. One of these isomers has never been reported before. Conclusions: The results of this proof-of-concept study indicate that the human liver–uPA+/+-SCID mouse appears to be a suitable small animal model for the investigation of human-type metabolism of anabolic steroids and possibly also for other types of drugs and medications. .

Influence of Pain Killers on the Urinary Anabolic Steroid Profile

2020 ◽  
Vol 44 (8) ◽  
pp. 871-879 ◽  
Author(s):  
Anna Stoll ◽  
Michele Iannone ◽  
Giuseppina De Gregorio ◽  
Xavier de la Torre ◽  
Francesco Molaioni ◽  
...  
Keyword(s):  
Anabolic Steroids ◽  
Analytical Data ◽  
Urine Samples ◽  
Control Analysis ◽  
Steroid Biotransformation ◽  
Steroid Profile ◽  
Endogenous Steroids ◽  
Antidoping Analysis

Abstract Anabolic androgenic steroids (AAS) are prohibited as performance-enhancing drugs in sports. Among them, testosterone and its precursors are often referred to as “pseudoendogenous” AAS, that is, endogenous steroids that are prohibited when administered exogenously. To detect their misuse, among other methods, the World Anti-Doping Agency-accredited laboratories monitor the steroid profile (concentrations and concentration ratios of endogenous steroids, precursors and metabolites) in urine samples collected from athletes in and out of competition. Alterations in steroid profile markers are used as indicators for misuse of anabolic steroids in sports. Therefore, especially their metabolic pathways with possible interactions are crucial to elucidate. As steroid metabolism is very complex, and many enzymes are involved, certain non-prohibited drugs may influence steroid metabolite excretion. One important group of steroid-metabolizing enzymes is aldo–keto reductases (AKRs). An inhibition of them by non-steroidal anti-inflammatory drugs (NSAIDs), which are neither prohibited nor monitored, but frequently used drugs in sports, was demonstrated in vitro. Thus, this work aims to investigate the influence of NSAID intake on the urinary steroid profile. Kinetic and inhibitory studies were performed using 5α-dihydrotestosterone as substrate. The results obtained from in vitro experiments show that ibuprofen inhibits AKR1C2 and thus influences steroid biotransformation. For in vivo investigations, urine samples prior, during and postadministration of ibuprofen were analyzed using routine methods to monitor the steroid profile. Changes in markers of the steroid profile of volunteers were observed. The combination of in vitro and in vivo results suggests that monitoring of ibuprofen may be useful in doping control analysis. The presented work illustrates the importance to consider co-administration of (non-prohibited) drugs during antidoping analysis. Intake of multiple substances is likely leading to interfering effects. Divergent results in antidoping analysis may therefore be observed and misinterpretation of analytical data may occur. Similar considerations may be appropriate for other fields of forensic applications.

scholarly journals Comparative analysis of the bioenergetics of human adenocarcinoma Caco-2 cell line and postoperative tissue samples from colorectal cancer patients

2018 ◽  
Vol 96 (6) ◽  
pp. 808-817 ◽  
Author(s):  
Lyudmila Ounpuu ◽  
Laura Truu ◽  
Igor Shevchuk ◽  
Vladimir Chekulayev ◽  
Aleksandr Klepinin ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Line ◽  
Respiratory Chain ◽  
Growth Conditions ◽  
Control Analysis ◽  
Respiratory Chain Complexes ◽  
Colon Tissue ◽  
Tissue Samples

The aim of this work was to explore the key bioenergetic properties for mitochondrial respiration in the widely-used Caco-2 cell line and in human colorectal cancer (HCC) postoperational tissue samples. Oxygraphy and metabolic control analysis (MCA) were applied to estimate the function of oxidative phosphorylation in cultured Caco-2 cells and HCC tissue samples. The mitochondria of Caco-2 cells and HCC tissues displayed larger functional activity of respiratory complex (C)II compared with CI, whereas in normal colon tissue an inverse pattern in the ratio of CI to CII activity was observed. MCA showed that the respiration in Caco-2 and HCC tissue cells is regulated by different parts of electron transport chain. In HCC tissues, this control is performed essentially at the level of respiratory chain complexes I–IV, whereas in Caco-2 cells at the level of CIV (cytochrome c oxidase) and the ATP synthasome. The differences we found in the regulation of respiratory chain activity and glycose index could represent an adaptive response to distinct growth conditions; this highlights the importance of proper validation of results obtained from in-vitro models before their extrapolation to the more complex in-vivo systems.

scholarly journals Effects of gonadotrophin in vivo and 2-hydroxyoestradiol-17β in vitro on follicular steroid hormone profile associated with oocyte maturation in the catfish Heteropneustes fossilis

2006 ◽  
Vol 189 (2) ◽  
pp. 341-353 ◽  
Author(s):  
A Mishra ◽  
K P Joy
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Hplc Method ◽  
Heteropneustes Fossilis ◽  
Germinal Vesicle Breakdown ◽  
Steroid Profile ◽  
17 Hydroxyprogesterone ◽  
Envelope Layer

An HPLC method was used to tentatively identify progesterone (P4) and its metabolites (17-hydroxyprogesterone (17-P4) and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)), corticosteroids (cortisol and corticosterone) and testosterone in ovary/follicular preparations of the catfish Heteropneustes fossilis associated with in vivo or in vitro oocyte maturation/ovulation. A single i.p. injection of human chorionic gonadotrophin (100 IU/fish, sampled at 0, 8 and 16 h) induced oocyte maturation and ovulation, which coincided with significant and progressive increases in 17,20β-P, and P4 and 17-P4, the precursors of the former. Both cortisol and corticosterone also increased significantly. Conversely, testosterone decreased significantly and progressively over time. Under in vitro conditions, incubation of post-vitellogenic (intact) follicles or follicular envelope (layer) with 2-hydroxyoestradiol (2-OHE2, 5 μM for 0, 6 and 24 h) elicited a sharp significant increase in 17,20β-P, the increase being higher in the follicular envelope incubate. P4 and 17-P4 also registered significant increases over the time with the peak values at 24 h. Cortisol and corticosterone increased significantly in the intact follicle, but not in the follicular envelope incubate. Testosterone decreased significantly in the intact follicle, but increased significantly (24 h) in the follicular envelope incubate. Coincident with these changes, the percentage of germinal vesicle breakdown (GVBD) increased over the time in the intact follicle incubate (48.9% at 6 h and 79.8% at 24 h). Denuded oocytes on incubation with 2-OHE2 (5 μM) did not produce any significant change in the percentage of GVBD or in the steroid profile. While corticosterone and 17,20β-P were undetected, P4, 17-P4, cortisol and testosterone were detected in low amounts. The results show that the 2-OHE2-induced GVBD response seems to be mediated through the production of 17,20β-P and corticosteroids. It is suggested that hydroxyoestrogens seem to be a component in the gonadotrophin cascade of regulation of oocyte maturation/ovulation in the catfish.

scholarly journals Metabolic evidence for impaired 17α-hydroxylase activity in a kindred bearing the E305G mutation for isolate 17,20-lyase activity

2008 ◽  
Vol 158 (3) ◽  
pp. 385-392 ◽  
Author(s):  
Dov Tiosano ◽  
Carlos Knopf ◽  
Ilana Koren ◽  
Nurit Levanon ◽  
Michaela F Hartmann ◽  
...  
Keyword(s):  
Gas Chromatography Mass Spectrometry ◽  
Enzymatic Reactions ◽  
Hydroxylase Activity ◽  
Lyase Deficiency ◽  
Expression Studies ◽  
Steroid Profiling ◽  
Lyase Activity ◽  
Urinary Steroid

ContextThe CYP17A1 gene encodes many enzymatic reactions including 17α-hydroxylase and 17,20-lyase activities. Mutations that selectively ablate the 17,20-lyase activity, causing isolated 17,20-lyase deficiency, are exceedingly rare and may belong to the rarest of all disorders of steroidogenesis. We have previously reported an E305G mutation in the active site of CYP17A1 that apparently causes isolated 17,20-lyase deficiency. Expression studies suggested intact 17α-hydroxylase activity which was at odds with subnormal tetracosactrin stimulated cortisol in the patients.ObjectivesTo investigate the in vivo activity of the adrenal enzymes, we used the metabolomics approach with urinary steroid profiling by gas chromatography–mass spectrometry.PatientsOf the 11 subjects investigated, 6 patients in the kindred were found to be homozygous, 4 members were asymptomatic heterozygous, and 1 was homozygous for the wild-type allele.ResultsIn the homozygous patients for E305G, both serum and urinary steroids showed a severe lack of androgens (C19-steroids) pointing to the absence of 17,20-lyase activities. Furthermore, precursor/product ratios of urinary steroid metabolites characterizing 17α-hydroxylase activity showed variable decreases in 17α-hydroxylase activities.ConclusionsThe results confirm the complete absence of 17,20-lyase activity in vivo, as in the in vitro expression studies. On the other hand, in vivo 17α-hydroxylase activity was partially impaired. Thus, the in vivo metabolic data seem to be more sensitive than the expression study and suggests that this mutation also impairs 17α-hydroxylase activity.

scholarly journals Inhibitory Effects of Diclofenac on Steroid Glucuronidation in Vivo Do not Affect Hair-Based Doping Tests for Stanozolol

2017 ◽  
Author(s):  
Gergely Zachár ◽  
Nawed I. K. Deshmukh ◽  
Andrea Petróczi ◽  
Andrea D. Székely ◽  
Iltaf Shah ◽  
...  
Keyword(s):  
Steroid Metabolism ◽  
Brown Norway ◽  
Inhibitory Effects ◽  
Spot Urine ◽  
Rat Study ◽  
Weekly Assessment ◽  
Urine And Serum ◽  
Norway Rats

In vitro studies show that diclofenac inhibits enzymatic steroid glucuronidation. This study was designed to investigate the influence of diclofenac on the excretion of stanozolol and 3'-hydroxystanozolol via analyses in hair, blood and urine in vivo in a rat study. Brown Norway rats were administered with stanozolol (weeks 1-3) and diclofenac (weeks 1-6). Weekly assessment of steroid levels in hair was complemented with spot urine and serum tests. Levels of both stanozolol and 3'-hydroxystanozolol steadily increased in hair during stanozolol treatment and decreased post-treatment, but remained readily detectable for 6 weeks. In contrast, compared to control rats, diclofenac significantly reduced urinary excretion of 3’-hydroxystanozolol which was undetectable in most samples. This is the first report of diclofenac altering steroid metabolism in vivo, detrimentally affecting detection in urine, but not in hair which holds considerable advantages over urinalysis for anti-doping tests.

scholarly journals Studies on the solubilized ribonucleic acid polymerase from rat ventral prostate gland

1971 ◽  
Vol 123 (4) ◽  
pp. 619-628 ◽  
Author(s):  
W. I. P. Mainwaring ◽  
F. R. Mangan ◽  
B. M. Peterken
Keyword(s):  
Time Course ◽  
Prostate Gland ◽  
Exchange Chromatography ◽  
Ventral Prostate ◽  
Enzyme Preparations ◽  
Rat Ventral Prostate ◽  
Rapid Stimulation ◽  
Androgenic Steroids

1. By using ultrasonic treatment in media of high ionic strength, the RNA polymerase activities associated with prostatic nuclei and nucleoli can be completely solubilized. Such enzyme preparations are entirely dependent on the provision of added DNA for full activity. 2. The solubilized enzymes from the nucleolar and extranucleolar regions can be separated by ion-exchange chromatography. 3. Based on differences in the optimum DNA templates, pH optima and the effects of ammonium sulphate on the activities in vitro, Mn2+- and Mg2+-specific enzymes are associated with both the nucleolar and extranucleolar regions of prostatic nuclei. 4. Androgenic hormones administered in vivo have a particularly pronounced effect on the activity of Mg2+-dependent enzyme associated with the isolated prostatic nucleolus. 5. Time-course experiments in vivo show that androgens induce a rapid stimulation of the solubilized Mg2+-dependent nucleolar enzyme before a pronounced activation of nucleolar chromatin can be measured. 6. The implications of these findings to the mechanism of action of androgenic steroids are discussed.

Critical Role for DOCK2 in CD8+/TCR− Graft Facilitating Cells Enhancing Engraftment of Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 142-142
Author(s):  
Yujie Wen ◽  
Mary J Elliott ◽  
Yiming Huang ◽  
Deborah R Corbin ◽  
Yoshinori Fukui ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Signaling Pathways ◽  
Microarray Analysis ◽  
Control Analysis ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract Abstract 142 CD8+/TCR− graft facilitating cells (FC) have a potent capability to facilitate relatively small numbers of highly purified hematopoietic stem cells (HSC) in both allogeneic and syngeneic recipients. The mechanisms by which FC promote HSC engraftment have not been fully elucidated. We previously found that FC from non-obese diabetic mice (NOD) were compromised in enhancing engraftment of syngeneic and allogeneic HSC compared with FC from diabetes free congenic NOR mice. We therefore compared gene expression profiling between NOD FC and control NOR FC by microarray analysis. Among 18 most significant differentially expressed genes revealed from 45101 genes by false discovery rate control analysis with a cut-off value at level 0.05, dedicator of cytokinesis 2 (DOCK2) was identified as the gene with the most significant difference (P = 1.07 × 10−7). DOCK2 is a hematopoietic cell-specific member of the Caenorhabditis elegans Ced-5, mammalian DOCK180 and Drosophila melanogaster myoblast city (CDM) family of guanine nucleotide exchange factors. DOCK2 activates the small GTPase Rac and is indispensable for migration of plasmacytoid dendritic cells (pDC). FC is a heterogeneous cell population, with a predominant subpopulation resembling plasmacytoid precursor DC (p-preDC FC). In vitro studies demonstrated that FC increased clonogenicity of HSC and improved the ability of the impaired stroma to support late cobblestone area formation by HSC, which suggests that FC homing to hematopoietic niche as a potential component might be a perquisite for FC to enhance HSC engraftment. Therefore, we hypothesized that signaling pathways controlling cell migration via DOCK2 are critical for FC to enhance HSC engraftment. To test our hypothesis, DOCK2 expression data from microarray analysis was further confirmed using relative quantitative real-time PCR and high content image analysis. The expression level of DOCK2 in FC was significantly lower in NOD mice compared with NOR mice (p < 0.01 vs. NOR FC). Functional phenotypes of DOCK2-deficient FC were determined by Transwell migration assay and colony-forming cell assay in vitro, and by co-transplantation of HSC with FC in vivo as well as enumeration of CellTrack Green labeled FC by flow cytometry in spleen, thymus, and bone marrow of femurs and tibias 18 hours post-transplantation. Deficiency of DOCK2 in FC did not affect the ability of FC in promoting HSC colony formation when the two were cultured together. However, DOCK2-deficient FC were compromised in migration to the α-cheemokine, stromal derived factor-1 (SDF-1) at dose 200 ng/ml (Fig. A, P < 0.01 vs. wild-type FC). Homing of FC to spleen and bone marrow of femurs and tibias was also significantly impaired in DOCK2-deficient FC. Moreover, deficiency of DOCK2 in FC abrogated enhancement of HSC engraftment by FC in the syngeneic and allogeneic in vivo assays (Fig. B, syngeneic model: 500 B6 HSC plus 30K B6 or DOCK2−/−FC into lethally irradiated B6 recipients; Fig. C, allogeneic model: 10K B6 HSC plus 30K B6 or DOCK2−/−FC into lethally irradiated B10.BR recipients, P < 0.05 vs. B6 HSC plus wild-type FC). Taken together, our results indicate that deficiency of DOCK2 in FC leads to the dysfunction in migration, and suggest that the signaling pathways involved in FC migration are crucial for FC to enhance HSC engraftment. Disclosures: Ildstad: Regenerex LLC: Equity Ownership.

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