Insights into the metabolism of CH-PIATA - a novel synthetic cannabinoid featuring an acetamide linker.

The recent change from the popular carboxamide to an acetamide (ATA) linker scaffold in synthetic cannabinoid receptor agonists (SCRAs) can be interpreted as an attempt to circumvent legal regulations, setting new analytical challenges. Metabolites of N-cyclohexyl-2-(1-pentyl-1H-indol-3-yl)acetamide: CH-PIATA, the second ATA type SCRA detected in the EU, were investigated in urine and serum samples by LC-HRMS-MS and LC-MS-MS. Two different in vitro models: a pHLM assay and HepG2-cells as well as an in silico prediction by GLORYx freeware assisted in metabolite formation/identification. CH-PIATA was extensively metabolized, leading to metabolites formed primarily by mono- and dihydroxylation. For urine and serum specimens, monohydroxylation at the indole core or the methylene spacer of the acetamide linker (M1.8), carboxylic acid formation at the N-pentyl side chain (M3.1), and degradation of the latter leading to a tentatively identified N-propionic acid metabolite (M5.1) are suggested as reliable markers for substance intake. The N-propionic acid metabolite could not be confirmed in the in vitro assays as it includes multiple consecutive metabolic reactions. Furthermore, CH-PIATA could be detected as parent substance in blood samples, but not in urine. Both in vitro assays and the in silico tool proved suitable for predicting metabolites of CH-PIATA. Considering effort and costs, pHLM incubations seem to be more effective for metabolite prediction in forensic toxicology. The highlighted phase I metabolites serve as reliable urinary targets for confirming CH-PIATA use. The in silico approach is advantageous when reference material is unavailable.

Elimination profile of low-dose chlortalidone and its detection in hair for doping analysis-Implication for unintentional non-therapeutic exposure.

Chlortalidone (CLT) is a thiazide-type diuretic with high affinity for the erythrocyte carbonic anhydrase. Therapeutically, it is mostly used to treat edema and hypertension due to liver cirrhosis, heart insufficiency, or renal dysfunction. Although diuretics and masking agents are prohibited by the World Anti-Doping Agency (WADA) at all times in sports, substances belonging to this category are constantly detected in athlete samples, according to WADA's annual testing figures. Within this group of structurally diverse compounds, a threshold of 20 ng/mL has been introduced for six substances solely due to their presence as contaminants in other permitted drugs because of pharmaceutical production processes. In a recent presumptive doping case with a low urinary CLT concentration, the question of unintentional doping, for example, by contaminated non-steroidal anti-inflammatory drug intake, arose. To examine this potential scenario, a co-elimination of low-dose CLT and hydrochlorothiazide (HCTA; 20 × 50 µg, 0.2 mg/day each) was conducted on five consecutive days in two volunteers. Urine samples were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Moreover, we examined the incorporation of CLT in scalp hair. HCTA is rapidly excreted renally in comparatively high concentrations. In contrast, the elimination of CLT is considerably slower (terminal elimination half-life extended by a factor of 12) and, consequently, much less concentrated in corresponding urine samples (45 and 53 ng/mL, respectively). Conversely, a higher hair incorporation of chlorthalidone was observed with simultaneous dosing of both. The results suggest that an unintentional intake of sub-therapeutic CLT doses due to contamination might result in an adverse analytical finding.

Endocannabinoids, endocannabinoid-like compounds and cortisone in head hair of health care workers as markers of stress and resilience during the early COVID-19 pandemic.

The pandemic caused by SARS-CoV-2 impacted health systems globally, creating increased workload and mental stress upon health care workers (HCW). During the first pandemic wave (March to May 2020) in southern Germany, we investigated the impact of stress and the resilience to stress in HCW by measuring changes in hair concentrations of endocannabinoids, endocannabinoid-like compounds and cortisone. HCW (n = 178) recruited from multiple occupation and worksites in the LMU-University-Hospital in Munich were interviewed at four interval visits to evaluate mental stress associated with the COVID-19 pandemic. A strand of hair of up to 6 cm in length was sampled once in May 2020, which enabled retrospective individual stress hormone quantifications during that aforementioned time period. Perceived anxiety and impact on mental health were demonstrated to be higher at the beginning of the COVID-19 pandemic and decreased significantly thereafter. Resilience was stable over time, but noted to be lower in women than in men. The concentrations of the endocannabinoid anandamide (AEA) and the structural congeners N-palmitoylethanolamide (PEA), N-oleoylethanolamide (OEA) and N-stearoylethanolamide (SEA) were noted to have decreased significantly over the course of the pandemic. In contrast, the endocannabinoid 2-arachidonoylglycerol (2-AG) levels increased significantly and were found to be higher in nurses, laboratory staff and hospital administration than in physicians. PEA was significantly higher in subjects with a higher resilience but lower in subjects with anxiety. SEA was also noted to be reduced in subjects with anxiety. Nurses had significantly higher cortisone levels than physicians, while female subjects had significant lower cortisone levels than males. Hair samples provided temporal and measurable objective psychophysiological-hormonal information. The hair endocannabinoids/endocannabinoid-like compounds and cortisone correlated to each other and to professions, age and sex quite differentially, relative to specific periods of the COVID-19 pandemic.

Up to the maximum-testosterone dose-dependent effects on anabolic and androgen responsive tissues in orchiectomized rats.

BACKGROUND: Despite the high number of synthetic androgenic-anabolic steroids, testosterone is still misused for doping in amateur and professional sports. However, only few studies investigated the dose-response effects of testosterone beyond its physiological concentrations and in over 90 years of research, no saturation dosage has been experimentally described for exogenous testosterone administration. OBJECTIVES: We want to elucidate the physiological and pathophysiological effects of supra-physiological testosterone application and close this gap in testosterone dose-response data. MATERIALS AND METHODS: Male orchiectomized rats were treated with different testosterone doses ranging from 0.1 to 50 mg/kg body weight for 3 weeks. Several physiological endpoints (e.g., body weight, organ and muscle weight, muscle strength, muscle fiber size) were examined during and after the termination of the treatment with an adjusted Hershberger assay, open-field-test, and (immuno-)histologic. RESULTS: The wet weights of androgen responsive organs (penis, prostate, seminal vesicle) showed a significant increase in a dose-dependent manner. Histological evaluation of the prostate showed a significant higher percentage of KI67 positive prostate nuclei in the highest dosage group and an increasing hyperplasia with increasing testosterone administered. A significant anabolic effect was only observed in Levator ani wet weight, and to minor degree for the cardiac muscle. Regarding other skeletal muscles (Musculus soleus and Musculus gastrognemicus), no significant testosterone effects were observed. We showed a significant increasing dosage-response effect for testosterone in androgen responsive organs with saturation at the two highest concentration of 10 and 50 mg/kg body weight. DISCUSSION AND CONCLUSION: The dose-dependent androgenic effects of testosterone were well observable and the anabolic effects on muscle tissue were visible although to a lesser degree, without the support of aerobic exercise and a protein rich diet. Future studies should investigate a combinatorial effect of testosterone and training. Nevertheless, with the chosen range of applied testosterone, we showed a saturation of testosterone effects in prostate, seminal vesicle, penis, and Levator ani.

Influence of ethanol consumption and food intake on serum concentrations of endogenous steroids.

Steroid biosynthesis and biotransformation are based on a cascade of enzymatic processes being highly sensitive to various external influences. Amongst those, ethanol was shown to affect testosterone metabolism. For doping analyses, athlete steroid profiles comprise seven urinary steroid metabolites, of which relevant ratios are significantly increased following ethanol consumption. This effect is presumably based on the lack of hepatic NAD+-coenzyme as a consequence of ethanol oxidation. Only recently, testosterone (T) and androstenedione (A4) blood profiles have been introduced as additional approach for doping control. However, a potential influence of ethanol intake on testosterone biosynthesis and thus on blood steroid profiles has not been investigated so far. Therefore, steroid concentrations from 10 males and 10 females receiving an ethanol infusion up to a breath alcohol concentration of 0.5 mg/L which was hold as a plateau for two hours were conducted. Blood samples were drawn every 15 min for steroid quantification. An ethanol-dependent T/A4 increase up to 385% resulting from A4 suppression was observed in 14 volunteers. In addition, we observed sporadic A4 increases coinciding with cortisol and ACTH pulses pointing to a meal-induced adrenal stimulation. While testosterone levels in males showed diurnal variation solely, testosterone levels in some females were found to be susceptible to ethanol- and ACTH-dependent perturbations, which is thought to be due to its predominant adrenal synthesis in females. In conclusion, the results of the present study emphasize the importance of blood sampling at a sufficient time interval from food and ethanol intake. This is of interest if T and A4 are used for diagnostics in doping control.

Extracellular Vesicles and Endocannabinoid Signaling in Patients with COVID-19.

Introduction: Endocannabinoids in COVID-19 have immunomodulatory and anti-inflammatory properties but the functional role and the regulation of endocannabinoid signaling in this pandemic disorder is controversial. To exercise their biologic function, endocannabinoids need to travel across the intercellular space and within the blood stream to reach their target cells. How the lipophilic endocannabinoids are transported in the vascular system and how these hydrophobic compounds cross cell membranes is still unclear. Extracellular vesicles (EVs) are released and incorporated by many cell types including immune cells. EVs are small lipid-membrane covered particles and contain RNA, lipids and proteins. They play an important role in intercellular communication by transporting these signaling molecules from their cells of origin to specific target cells. EVs may represent ideal transport vehicles for lipophilic signaling molecules like endocannabinoids and this effect could also be evident in COVID-19. Materials and Methods: We measured the endocannabinoids anandamide, 2-AG, SEA, PEA and OEA in patients with COVID-19 in EVs and plasma. RNA sequencing of microRNAs (miRNAs) derived from EVs (EV-miRNAs) and mRNA transcripts from blood cells was used for the construction of signaling networks reflecting endocannabinoid and miRNA communication by EVs to target immune cells. Results: With the exception of anandamide, endocannabinoid concentrations were significantly enriched in EVs in comparison to plasma and increased with disease severity. No enrichment in EVs was seen for the more hydrophilic steroid hormones cortisol and testosterone. High EV-endocannabinoid concentrations were associated with downregulation of CNR2 (CB2) by upregulated EV-miRNA miR-146a-5p and upregulation of MGLL by downregulated EV-miR-199a-5p and EV-miR-370-5p suggesting counterregulatory effects. In contrast, low EV-levels of anandamide were associated with upregulation of CNR1 by downregulation of EV-miR-30c-5p and miR-26a-5p along with inhibition of FAAH. Immunologically active molecules in immune cells regulated by endocannabinoid signaling included VEGFA, GNAI2, IGF1, BDNF, IGF1R and CREB1 and CCND1 among others. Discussion and Conclusions: EVs carry immunologically functional endocannabinoids in COVID-19 along with miRNAs which may regulate the expression of mRNA transcripts involved in the regulation of endocannabinoid signaling and metabolism. This mechanism could fine-tune and adapt endocannabinoid effects in recipient cells in relationship to the present biological context.

Identification of human in vitro metabolites of the haemoglobin S polymerization inhibitor voxelotor for doping control purposes.

Voxelotor (GBT440) is a haemoglobin S polymerization inhibitor used to treat anaemia in sickle cell disease. Due to an increase of arterial oxygen saturation as well as serum erythropoietin and haemoglobin, the World Anti-Doping Agency included voxelotor in the list of prohibited substances and methods in 2023. The objective of the present study was to identify and characterize metabolites of voxelotor to detect a potential misuse by athletes. The biotransformation was studied in vitro using the human hepatocellular cell line HepG2 and pooled human liver microsomes. The metabolites were analysed using high-performance liquid chromatography (high-resolution) mass spectrometry. In total, three phase I metabolites and six phase II metabolites (resulting from glucuro-conjugation and O-methylation) were formed by the HepG2 cells in a time-dependent manner, and two phase I metabolites were generated by the liver microsomes, among them one also found in the HepG2 incubations. A reduced metabolite and the glucuro-conjugate of a reduced metabolite were the most abundant formed by HepG2 cells. In addition, metabolites resulting from mono-hydroxylation, reduction and O-methylation in different combinations were identified. Voxelotor was also found as glucuro-conjugate with a low abundance. With the spectrometric behaviour of voxelotor and its in vitro metabolites described herein, an implementation in doping control screening and, consequently, a detection of an abuse in an athlete urine sample might be possible.

Effect of HepG2 cell 3D cultivation on the metabolism of the anabolic androgenic steroid metandienone.

The elucidation of the metabolic fate of prohibited substances is crucial for the abuse detection. The human hepatocyte cell line HepG2 can be used to study biotransformation. In order to improve this in vitro model system, we compared the HepG2 spheroid generation using three different techniques: a forced floating, a scaffold-free and a scaffold-based method. We characterized the spheroids with regard to the expression levels of the proliferation marker Mki67, the liver-specific marker albumin and biotransformation enzymes. Moreover, the metandienone metabolite pattern was comparatively analysed by high-performance liquid chromatography mass spectrometry. With all three techniques, HepG2 spheroids were generated showing a degree of differentiation. The forced floating method resulted in very large spheroids (1 mm in diameter) showing signs of necrosis in the centre and a very low metandienone conversion rate. The spheroids formed by the two other techniques were comparable in size with 0.5 mm in diameter on average. Among the three different 3D cultivation methods, the HepG2 spheroids formed on Matrigel® as extracellular matrix were the most promising regarding biotransformation studies on anabolic androgenic steroids. Prospectively, HepG2 spheroids are a promising in vitro model system to study multidrug setups, drug-drug interactions and the biotransformation of other substance classes.

Detection of 18-methyl steroids: Case report on a forensic urine sample and corresponding dietary supplements.

The detection of a putative 18-methyl-19-nortestosterone metabolite in a forensic bodybuilder's urine sample collected as part of a criminal proceeding has triggered a follow-up investigation. Four different dietary supplements in the possession of the suspect were examined with regard to possible precursor steroids. This led to the detection of the declared ingredient methoxydienone, which was confirmed by both, GC-MSMS and LC-HRMSMS. As neither 18-methyl-testosterone, nor 18-methyl-19-nortestosterone were detectable in the supplements, the possibility that the metabolite originates from methoxydienone was investigated. For this purpose, the metabolic fate of methoxydienone was studied in vitro using human HepG2 cells and in vivo by a single oral administration. While the 18-methyl-19-nortestosterone metabolite was not generated by HepG2 cells incubated with methoxydienone, it was observed in the urine samples collected at 2, 6, 10 and 24 h after methoxydienone administration. Moreover, the potential binding of methoxydienone as ligand to the human androgen receptor was modelled in silico in comparison with 18-methylnandrolone, for which androgen receptor activation had been shown in an in vitro approach before. In conclusion, we could ascribe the presence of the 18-methyl-19-nortestosterone metabolite in a forensic urine sample to originate from methoxydienone present in dietary supplements. Methoxydienone was observed to slowly degrade by demethylation of the methoxy substituent in liquid solutions. While no compound-specific intermediates were identified that allowed differentiation from other 18-methyl steroids, the 18-methyl-19-nortestosterone metabolite proved to be a suitable marker for reliable detection in doping analysis.

Agreement of steroid profiles in Athlete Biological Passport residues and corresponding serum samples.

The steroid module of the Athlete Biological Passport (ABP) is based on the analysis of six endogenous steroids in urine samples and a Bayesian statistical approach. However, the urinary steroid concentrations may be affected by confounders like microbial degradation, possible co-administration of diuretics as masking agents, insufficient conjugate hydrolysis or UGT2B17 gene polymorphisms affecting glucuronidation. Therefore, it can be helpful to use other matrices (ABP blood and serum samples) to quantify steroids and thereby support noticeable deviations in the Athlete Biological Passport, for example, abnormally increased urinary testosterone/epitestosterone (T/E) ratios. Aim of the study was to investigate the feasibility to re-use plasma obtained from athlete ABP blood samples for measuring a steroid profile. Therefore, testosterone, androstenedione, cortisol and cortisone were quantified in 36 intra-individual matching ABP blood and serum samples. The steroid levels measured in both matrices showed a high agreement indicating a good stability uninfluenced by storage temperature and duration. Our results pointed out the possibility to expand the athlete ABP blood analysis for steroid profiling.

Application of single hair analysis in a doping case involving amphetamine.

A previously published method for single hair analysis has been applied to a doping case for further clarification. Amphetamine could be detected in multiple micro segments resulting in two distinct concentration peaks in several hairs. The consumption of a contaminated food supplement as possible source for the amphetamine is discussed.

HepG2 as promising cell-based model for biosynthesis of long-term metabolites: Exemplified for metandienone.

In order to detect the abuse of substances in sports, the knowledge of their metabolism is of undisputable importance. As in vivo administration of compounds faces ethical problems and might even not be applicable for nonapproved compounds, cell-based models might be a versatile tool for biotransformation studies. We coincubated HepG2 cells with metandienone and D3 -epitestosterone for 14 days. Phase I and II metabolites were analyzed by high-performance liquid chromatography (HPLC)-tandem mass spectrometry and confirmed by gas chromatography-mass spectrometry (GC-MS). The metandienone metabolites formed by HepG2 cells were comparable with those renally excreted by humans. HepG2 cells also generated the two long-term metabolites 17ß-hydroxymethyl-17α-methyl-18-nor-androst-1,4,13-trien-3-one and 17α-hydroxymethyl-17ß-methyl-18-nor-androst-1,4,13-trien-3-one used in doping analyses, though in an inverse ratio compared with that observed in human urine. In conclusion, we showed that HepG2 cells are suitable as model for the investigation of biotransformation of androgens, especially for the anabolic androgenic steroid metandienone. They further proved to cover phase I and II metabolic pathways, which combined with a prolonged incubation time with metandienone resulted in the generation of its respective long-term metabolites known from in vivo metabolism. Moreover, we showed the usability of D3 -epitestosterone as internal standard for the incubation. The method used herein appears to be suitable and advantageous compared with other models for the investigation of doping-relevant compounds, probably enabling the discovery of candidate metabolites for doping analyses.

Emergence of the less common cannabinoid Δ8 -Tetrahydrocannabinol in a doping sample.

Δ8 -Tetrahydrocannabinol (Δ8 -THC) as isomer of the well-known Δ9 -THC has a similar mode of action, and the potency was estimated to be two thirds compared with Δ9 -THC. Content of Δ8 -THC in plant material is low, but formulations containing Δ8 -THC in high concentrations are gaining popularity. Δ8 -THC is to be regarded as prohibited substance according to the Prohibited List of the World Anti-Doping Agency (WADA). Contradictory results between initial testing procedure and confirmatory quantitation for 11-Nor-9-carboxy-Δ9 -tetrahydrocannabinol (Δ9 -THC-COOH) of a doping control sample gave rise for follow-up testing procedures. After alkaline hydrolysis and liquid-liquid extraction, the sample was analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) using isocratic elution instead of gradient elution, which is used for standard procedure. Isocratic elution resulted in two peaks instead of one using gradient elution. Both peaks showed same fragmentation. Using certified reference materials, one peak could be assigned to Δ9 -THC-COOH and the other one with higher intensity to the less common 11-Nor-9-carboxy-Δ8 -Tetrahydrocannabinol (Δ8 -THC-COOH) in a concentration of approximately 1200 ng/ml. As complementary method, gas chromatography tandem mass spectrometry (GC-MS/MS) can also be used for identification. Here Δ8 - and Δ9 -THC-COOH can be distinguished by chromatography and by fragmentation. Additional investigations of doping control samples containing Δ9 -THC-COOH revealed the simultaneous presence of Δ8 -THC-COOH in low concentrations (0.22-8.91 ng/ml) presumably due to plant origin. Percentage of Δ8 -THC-COOH varies from 0.05 to 2.83%. In vitro experiments using human liver microsomes showed that Δ8 -THC is metabolized in the same way as Δ9 -THC.

Cell culture as a toolbox to generate phase I metabolites for antidoping screening.

The knowledge of the biotransformation of compounds prohibited by the World Anti Doping Agency is of high concern as doping analyses are mostly based on the detection of metabolites instead of the parent compounds abused by athletes. While the self-administration of doping-relevant compounds is from an ethical point of view a rather problematic method to investigate metabolism, the usage of cell culture systems allows for studies on biotransformation in vitro. Five cell culture models with different tissue origin (liver, ovary, skin, kidney, and testis) were comparatively incubated with testosterone and epitestosterone as well as with the synthetic testosterone derivatives 17α-methyltestosterone and 4-chlorotestosterone to investigate the impact of synthetic modifications on phase I metabolic pathways. Cell culture supernatants were analyzed by high-performance liquid chromatography-tandem mass spectrometry. All cell lines possessed the default steroid phase I biotransformation reactions. The highest conversion rate was observed in ovarian (BG-1) and liver cells (HepG2). For BG-1 and skin cells (HaCaT), the 5α-reductase products 5α-dihydrotestosterone (for both) and 5α-androstane-3α/ß,17ß-diol (for BG-1 solely) were found to be prevailing after testosterone incubation. In kidney (COS-1) and HepG2 cells, the 17ß-hydroxysteroid dehydrogenase activity was predominant as supported by the observation that the 17α-OH (epitestosterone) and the methyl group (17α-methyltestosterone) impeded the conversion rate in these cell lines. In conclusion, future work should extend the characterization of the BG-1 and HepG2 cells on phase II metabolic pathways to examine whether they are suitable models for the generation of metabolite reference collections comparable to those obtained by human excretion studies.

Detection of the selective androgen receptor modulator GSK2881078 and metabolites in urine and hair after single oral administration.

Hair and urine concentrations of the nonsteroidal selective androgen receptor modulator GSK2881078 were examined following single oral administration to investigate its hair incorporation and estimate the general suitability of hair testing for selected androgen receptor modulators. Hair segments were collected following a single dose of 1.5 mg GSK2881078 by repeated shaving of scalp hair at Week 0 (blank), Week 1 (representing the pre-application period), Week 3 (ideally focusing the time of incorporation), and Weeks 5 and 9 (post-administration period). The intact compound and various (at least 4) hydroxy-metabolites exhibited similar elimination profiles. The peak urinary concentration (approximately 920 pg/ml) was observed after 8 h and is reduced to the detection limit (2 pg/ml) on Day 42 following administration of 760 µg GSK2881078. Correspondingly, hair concentrations of GSK2881078 (intact compound only) following a single oral dose of 1.5 mg GSK2881078 reached a peak concentration of 1.7 pg/mg in the segments collected 3 weeks post administration, representing the time of ingestion. The concentration rapidly declined to trace amounts of 0.7 (Week 5) and 0.2 pg/mg (Week 9), respectively. In conclusion, measurement of the intact compound GSK2881078 is feasible for both urine and hair analysis. However, concentrations in hair after single oral administration are in the low pg/mg range and can only be detected, if the segments cover the administration period.

Steroid 17-Hydroxyprogesterone in Hair Is a Potential Long-Term Biomarker of Androgen Control in Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency.

INTRODUCTION: To evaluate scalp hair steroid concentrations as a monitoring tool for androgen control and metabolic outcomes in adults with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. METHODS: 17-hydroxyprogesterone (17-OHP), androstenedione, testosterone, cortisol, cortisone, progesterone, prednisolone, and prednisone concentrations were measured in scalp hair by means of LC-MS/MS in 27 women and 15 men with CAH and controls (37 women, 42 men). RESULTS: In CAH men and women, 17-OHP levels in hair showed a significant positive correlation with corresponding levels in serum (ρ = 0.654; p = 0.01; ρ = 0.553, p = 0.003 respectively), while total testosterone levels were only significantly correlated in CAH men (ρ = 0.543; p = 0.036). Androstenedione levels did not show a significant correlation. Receiver-operating characteristic (ROC) curve analysis indicated that a cutoff value of 21.7 pg/mg for 17-OHP in hair provided a sensitivity of 100% and a specificity of 88.9% for identifying men with elevated serum androstenedione. Hair 17-OHP in women showed a poorer performance in terms of identifying those with elevated androstenedione serum levels. However, when applying a cutoff value of 5.5 for the free androgen index as a marker of significant hyperandrogenism in CAH women, 17-OHP >27.6 pg/mg in hair provided a sensitivity of 100% and a specificity of 95.8% (AUC 0.986, 95% CI 0.945-1.000; p < 0.001). Neither hair cortisol nor markers of adrenal androgen control in hair showed significant associations with cardiometabolic outcome or bone health. CONCLUSION: This study shows that scalp hair 17-OHP concentrations may be a promising noninvasive long-term parameter for treatment monitoring in adult patients with CAH.

Stress Related Shift Toward Inflammaging in Cosmonauts After Long-Duration Space Flight.

Space flight exerts a specific conglomerate of stressors on humans that can modulate the immune system. The mechanism remains to be elucidated and the consequences for cosmonauts in the long term are unclear. Most of the current research stems from short-term spaceflights as well as pre- and post-flight analyses due to operational limitations. Immune function of 12 cosmonauts participating in a long-duration (>140 days) spaceflight mission was monitored pre-, post-, and on two time-points in-flight. While the classical markers for stress such as cortisol in saliva where not significantly altered, blood concentrations of the endocannabinoid system (ECS) were found to be highly increased in-flight indicating a biological stress response. Moreover, subjects showed a significant rise in white blood cell counts. Neutrophils, monocytes and B cells increased by 50% whereas NK cells dropped by nearly 60% shortly after landing. Analysis of blood smears showed that lymphocyte percentages, though unchanged pre- and post-flight were elevated in-flight. Functional tests on the ground revealed stable cellular glutathione levels, unaltered baseline and stimulated ROS release in neutrophils but an increased shedding of L-selectin post-flight. In vitro stimulation of whole blood samples with fungal antigen showed a highly amplified TNF and IL-1ß response. Furthermore, a significant reduction in CD4+CD25+CD27low regulatory T cells was observed post-flight but returned to normal levels after one month. Concomitantly, high in-flight levels of regulatory cytokines TGF-ß, IL-10 and IL-1ra dropped rapidly after return to Earth. Finally, we observed a shift in the CD8+ T cell repertoire toward CD8+ memory cells that lasted even one month after return to Earth. Conclusion: Long-duration spaceflight triggered a sustained stress dependent release of endocannabinoids combined with an aberrant immune activation mimicking features of people at risk for inflammation related diseases. These effects persisted in part 30 days after return to Earth. The currently available repertoire of in-flight testing as well as the post-flight observation periods need to be expanded to tackle the underlying mechanism for and consequences of these immune changes in order to develop corresponding mitigation strategies based on a personalized approach for future interplanetary space explorations.

Comparison of the three SARMs RAD-140, GLPG0492 and GSK-2881078 in two different in vitro bioassays, and in an in silico androgen receptor binding assay.

Selective androgen receptor modulators comprise compounds that bind as ligands to the androgen receptor and possess tissue-selective activities. Ideally, they show agonistic properties in anabolic target tissues, while inducing antagonistic or only weak agonistic effects in reproductive organs. Due to their myoanabolic effects, selective androgen receptor modulators are included in the list of prohibited substances and methods of the World Anti-Doping Agency. In the current investigation, the androgenic potential of RAD-140, GSK-2881078 and GLPG0492 was comparably investigated in two different in vitro bioassays. In the yeast androgen screen, the androgenic effects were lower than in the reporter gene assay in prostate carcinoma cells (e.g. for GSK-2881078, the EC50 values were 4.44 × 10-6M in the yeast screen and 3.99 × 10-9M in the prostate cells respectively). For future investigations, it is of importance whether the yeast androgen screen, which has been proven to detect androgenic compounds in urine, can detect an abuse of the selective androgen receptor modulators. Molecular modeling of the binding to the androgen receptor ligand binding domain suggests slight differences in the binding modes of RAD-140, GSK-2881078 and GLPG0492. In conclusion, androgenic activity of the three non-steroidal compounds in the two different in vitro test systems confirmed the results of the in silico modeling of the androgen receptor binding.

In vitro metabolism of selected bioactive compounds of Eurycoma longifolia root extract to identify suitable markers in doping control.

Eurycoma longifolia Jack (Tongkat Ali, Simaroubaceae) is a medicinal plant endemic to South-East Asia. For centuries, different parts of the plant have been used as a natural remedy to treat fever, hypertension, or sexual insufficiency. Today, Eurycoma longifolia preparations are commercially available and advertised to enhance athletic performance and muscle strength. Several studies have demonstrated a testosterone-boosting effect that might be caused by the release of free testosterone from the sex-hormone-binding globulin. To date, many phytochemical constituents of Eurycoma longifolia root extracts have been identified and physiological effects have been examined, while studies on their biotransformation and monitoring are still lacking. Within this study, eurycomalide C, eurycomalactone, 5,6-dehydro-eurycomalactone, longilactone, 14,15ß-dihydroklaieanone, 11-dehydroklaieanone, 9-hydroxycanthin-6-one, and 9-methoxycanthin-6-one isolated from E. longifolia root were incubated with liver microsomes. Respective metabolites were analyzed by liquid chromatography-tandem (high-resolution) mass spectrometry. The compounds were chosen based on their potential androgenic effects (estimated by in vitro assays), their concentrations in plant extracts, and presumptive metabolic pathways. Hydroxylated phase I metabolites were only observed for 5,6-dehydro-eurycomalactone, 11-dehydroklaieanone, 9-hydroxycanthin-6-one, and 9-methoxycanthin-6-one. Moreover, an O-demethylated metabolite of 9-methoxycanthin-6-one was found. Besides, the glucuronide of 9-hydroxycanthin-6-one was detected after in vitro glucuronidation using liver microsomes. The in vitro generated metabolites were comparable to that detected in urine and serum after a single ingestion of either 9-methoxycanthin-6-one or an Eurycoma longifolia root extract. Hence, 9-methoxycanthin-6-one, its glucuronide, and the glucuronide of its O-demethylated biotransformation product are proposed to be the most suitable targets for detection of 9-methoxycanthin-6-one or Tongkat Ali application in urine and serum.

Modulations of Neuroendocrine Stress Responses During Confinement in Antarctica and the Role of Hypobaric Hypoxia.

The Antarctic continent is an environment of extreme conditions. Only few research stations exist that are occupied throughout the year. The German station Neumayer III and the French-Italian Concordia station are such research platforms and human outposts. The seasonal shifts of complete daylight (summer) to complete darkness (winter) as well as massive changes in outside temperatures (down to -80°C at Concordia) during winter result in complete confinement of the crews from the outside world. In addition, the crew at Concordia is subjected to hypobaric hypoxia of ∼650 hPa as the station is situated at high altitude (3,233 m). We studied three expedition crews at Neumayer III (sea level) (n = 16) and two at Concordia (high altitude) (n = 15) to determine the effects of hypobaric hypoxia on hormonal/metabolic stress parameters [endocannabinoids (ECs), catecholamines, and glucocorticoids] and evaluated the psychological stress over a period of 11 months including winter confinement. In the Neumayer III (sea level) crew, EC and n-acylethanolamide (NAE) concentrations increased significantly already at the beginning of the deployment (p < 0.001) whereas catecholamines and cortisol remained unaffected. Over the year, ECs and NAEs stayed elevated and fluctuated before slowly decreasing till the end of the deployment. The classical stress hormones showed small increases in the last third of deployment. By contrast, at Concordia (high altitude), norepinephrine concentrations increased significantly at the beginning (p < 0.001) which was paralleled by low EC levels. Prior to the second half of deployment, norepinephrine declined constantly to end on a low plateau level, whereas then the EC concentrations increased significantly in this second period during the overwintering (p < 0.001). Psychometric data showed no significant changes in the crews at either station. These findings demonstrate that exposition of healthy humans to the physically challenging extreme environment of Antarctica (i) has a distinct modulating effect on stress responses. Additionally, (ii) acute high altitude/hypobaric hypoxia at the beginning seem to trigger catecholamine release that downregulates the EC response. These results (iii) are not associated with psychological stress.