Evaluation of longitudinal steroid profiling with the ADAMS adaptive model for detection of transdermal,intramuscular, and subcutaneous testosterone administration

The steroidal module of the Athlete Biological Passport (ABP) has been used since 2014 for the longitudinal monitoring of urinary testosterone and its metabolites in order to identify samples suspicious for the use of synthetic forms of endogenous anabolic androgenic steroids (EAAS). Samples identified by the module may then be confirmed by isotope ratio mass spectrometry (IRMS) to establish clearly the exogenous origin of testosterone and/or metabolites in the sample. To examine the detection capability of the steroidal ABP model, testosterone administration studies were performed with various doses and three routes of administration – transdermal, intramuscular, and subcutaneous with 15 subjects for each route of administration. Urine samples were collected before, during, and after administration and steroid profiles were analyzed using the steroidal ABP module in ADAMS. A subset of samples from each mode of administration was also analyzed by IRMS. The steroidal ABP module was more sensitive to testosterone use than population‐based thresholds and with high dose administrations there was very good agreement between the IRMS results and samples flagged by the module. However, with low dose administration the ABP module was unable to identify samples where testosterone use was still detectable by IRMS analysis. The testosterone/epitestosterone (T/E) ratio was the most diagnostic parameter for longitudinal monitoring with the exception of low testosterone excretors for whom the 5α‐androstane‐3α, 17β‐diol/epitestosterone (5αAdiol/E) ratio may provide more sensitivity.     

Metabolic studies of 1‐testosterone in horses

1‐Testosterone (17β‐hydroxy‐5α‐androst‐1‐en‐3‐one), a synthetic anabolic steroid, has been described as one of the most effective muscle‐building supplements currently on the market. It has an anabolic potency of 200 as compared to 26 for testosterone. Apart from its abuse in human sports, it can also be a doping agent in racehorses. Metabolic studies on 1‐testosterone have only been reported for human in the early seventies, whereas little is known about its metabolic fate in horses. This paper describes the studies of in vitro and in vivo metabolism of 1‐testosterone in horses, with the aim of identifying the most appropriate target metabolites to be monitored for controlling the misuse or abuse of 1‐testosterone in racehorses. Six in vitro metabolites, namely 5α‐androst‐1‐ene‐3α,17β‐diol (T1a), 5α‐androstane‐3β,17β‐diol (T2), epiandrosterone (T3), 16,17‐dihydroxy‐5α‐androst‐1‐ene‐3‐one (T4 & T5), and 5α‐androst‐1‐ene‐3,17‐dione (T6), were identified. For the in vivo studies, two thoroughbred geldings were each administered orally with 800 mg of 1‐testosterone by stomach tubing. The results revealed that the parent drug and eight metabolites were detected in urine. Besides the four in vitro metabolites (T1a, T2, T3, and T5), four other urinary metabolites, namely 5α‐androst‐1‐ene‐3β,17α‐diol (T1b), 5α‐androst‐1‐ene‐3β,17β‐diol (T1c), 5α‐androstane‐3α,17α‐diol (T7) and 5α‐androstane‐3β,17α‐diol (T8) were identified. This study shows that the detection of 1‐testosterone administration is best achieved by monitoring the parent drug, which could be detected for up to 30 h post‐administration. Copyright © 2012 John Wiley & Sons, Ltd.     

Urinary steroid profile in females – the impact of menstrual cycle and emergency contraceptives

Today's doping tests involving longitudinal monitoring of steroid profiles are difficult in women. Women have more complex hormonal fluctuations than men and commonly take drugs such as hormonal contraceptives that are shown to affect biomarkers used in these doping tests. In this study, we followed six women's urinary steroid profile during one menstrual cycle, including both glucuronides and sulfate conjugated fractions. Additionally, we studied what happens to the steroidal module of the Athlete Biological Passport (ABP) after administration of an emergency contraceptive (levonorgestrel, NorLevo®). The study shows that there are large individual variations in all metabolites included in the ABP and that the administration of emergency contraceptives may lead to suspicious steroid profile findings in the ABP. Urinary epitestosterone concentration increased during the menstrual cycle, leading to a decrease in the testosterone/epitestosterone ratio. The ratios followed in the ABP varied widely throughout the menstrual cycle, the coefficient of variation (CV) ranging from 4 to 99%. There was a 3‐fold decrease in epitestosterone 24 h post administration of the emergency contraceptive pill and androsterone, etiocholanolone, and 5β‐ androstan‐3α,17β‐diol concentrations decreased about 2‐fold. When analyzed with the ABP software, one of the six women had an atypical profile after taking the emergency contraceptive. Furthermore, we could not find any alterations in excretion routes (i.e., if the metabolites are excreted as glucuronide or sulfate conjugates) during the menstrual cycle or after administration of emergency contraceptive, indicating no direct effect on phase II enzymes. Copyright © 2016 John Wiley & Sons, Ltd.     

Detection and metabolic investigations of a novel designer steroid: 3‐chloro‐17α‐methyl‐5α‐androstan‐17β‐ol

In 2012, seized capsules containing white powder were analyzed to show the presence of unknown steroid‐related compounds. Subsequent gas chromatography–mass spectrometry (GC‐MS) and nuclear magnetic resonance (NMR) investigations identified a mixture of 3α‐ and 3β‐ isomers of the novel compound; 3‐chloro‐17α‐methyl‐5α‐androstan‐17β‐ol. Synthesis of authentic reference materials followed by comparison of NMR, GC‐MS and gas chromatography‐tandem mass spectrometry (GC‐MS/MS) data confirmed the finding of a new ‘designer’ steroid. Furthermore, in vitro androgen bioassays showed potent activity highlighting the potential for doping using this steroid. Due to the potential toxicity of the halogenated steroid, in vitro metabolic investigations of 3α‐chloro‐17α‐methyl‐5α‐androstan‐17β‐ol using equine and human S9 liver fractions were performed. For equine, GC‐MS/MS analysis identified the diagnostic 3α‐chloro‐17α‐methyl‐5α‐androstane‐16α,17β‐diol metabolite. For human, the 17α‐methyl‐5α‐androstane‐3α,17β‐diol metabolite was found. Results from these studies were used to verify the ability of GC‐MS/MS precursor‐ion scanning techniques to support untargeted detection strategies for designer steroids in anti‐doping analyses. Copyright © 2015 John Wiley & Sons, Ltd.     

Reference ranges for the urinary steroid profile in a Latin‐American population

The urinary steroid profile has been used in clinical endocrinology for the early detection of enzyme deficiencies. In the field of doping, its evaluation in urine samples is used to diagnose the abuse of substances prohibited in sport. This profile is influenced by sex, age, exercise, diet, and ethnicity, among others; laboratories own reference ranges might compensate for ethnic differences among population and inter‐laboratory biases. This paper shows the reference ranges obtained in the Antidoping Laboratory of Havana for the following steroid profile parameters: ten androgens (testosterone, epitestosterone, androsterone, etiocholanolone, 5α‐androstan‐3α,17β‐diol, 5β‐androstan‐3α,17β‐diol, dehydroepiandrosterone, epiandrosterone, 11β‐hydroxyandrosterone and 11β‐hydroxyetiocholanolone), three estrogens (estradiol, estriol and estrone), two pregnanes (pregnanediol and pregnanetriol) and two corticosteroids (cortisol and tetrahydrocortisol). The urine samples (male: n = 2454 and female: n = 1181) and data obtained are representative of population from Latin‐American countries like Cuba, Venezuela, Mexico, Dominican Republic, Guatemala and Chile. Urine samples were prepared by solid‐phase extraction followed by enzymatic hydrolysis and liquid‐liquid extraction with an organic solvent in basic conditions. Trimethylsilyl derivatives were analyzed by gas chromatography coupled to mass spectrometry. Reference ranges were established for each sex, allowing the determination of abnormal profiles as a first diagnostic tool for the detection of the abuse of androgenic anabolic steroids. The comparison with the Caucasian population confirms that the urinary steroid profile is influenced by ethnicity. Copyright © 2013 John Wiley & Sons, Ltd.     

Multivariate interpretation of the urinary steroid profile and training‐induced modifications. The case study of a Marathon runner

The steroidal module of the athlete biological passport (ABP) introduced by the World Anti‐Doping Agency (WADA) in 2014 includes six endogenous androgenic steroids and five of their concentration ratios, monitored in urine samples collected repeatedly from the same athlete, whose values are interpreted by a Bayesian model on the basis of intra‐individual variability. The same steroid profile, plus dihydrotestosterone (DHT) and DHEA, was determined in 198 urine samples collected from an amateur marathon runner monitored over three months preceding an international competition. Two to three samples were collected each day and subsequently analyzed by a fully validated gas chromatography–mass spectrometry protocol. The objective of the study was to identify the potential effects of physical activity at different intensity levels on the physiological steroid profile of the athlete. The results were interpreted using principal component analysis and Hotelling's T² vs Q residuals plots, and were compared with a profile model based on the samples collected after rest. The urine samples collected after activity of moderate or high intensity, in terms of cardiac frequency and/or distance run, proved to modify the basal steroid profile, with particular enhancement of testosterone, epitestosterone, and 5α‐androstane‐3α,17β‐diol. In contrast, all steroid concentration ratios were apparently not modified by intense exercise. The alteration of steroid profiles seemingly lasted for few hours, as most of the samples collected 6 or more hours after training showed profiles compatible with the “after rest” model. These observations issue a warning about the ABP results obtained immediately post‐competition.     

The Anti‐tumor Effects of Androstene Steroids Exhibit a Strict Structure–Activity Relationship Dependent upon the Orientation of the Hydroxyl Group on Carbon‐17

Androstene steroids are metabolites of dehydroepiandrosterone and exist as androstene‐diols or ‐triols in α‐ and β‐epimeric forms based upon the placement of the hydroxyl groups relative to the plane of the Δ⁵cycloperhydrophenanthrene ring. 5‐Androstene‐3β,17β‐diol (3β,17β‐AED) functions to upregulate immunity and the addition of a third hydroxyl group at C‐7 in the α‐ or β‐orientation (3β,7α,17β‐AET and 3β,7β,17β‐AET, respectively) enhances the immunological activity of the molecule. In contrast, 5‐androstene‐3β,17α‐diol (3β,17α‐AED) possesses potent anti‐tumor activity. We synthesized a new androstene by adding a third hydroxyl group at C‐7 to make 5‐androstene‐3β,7α,17α‐triol (3β,7α,17α‐AET) and compared the anti‐tumor activity of this steroid to the four existing androstenes. The results showed that this modification reduced the activity of 3β,17α‐AED. The ranking of the anti‐tumor activities of these steroids and their IC50 on human glioblastoma and lymphoma cells was: 3β,17α‐AED (～10 μm ) > 3β,7α,17α‐AET (～30 μm ) >> 3β,7α,17β‐AET (～150 μm )> 3β,7β,17β‐AET (not achievable) ≥ 3β,17β‐AED (not achievable). 3β,17α‐AED and 3β,7α,17α‐AET induced autophagy in T98G glioblastoma cells and apoptosis in U937 lymphoma cells. These results indicate that the position of the hydroxyl group on C‐17 dictates the anti‐tumor activity of the androstenes and must be in the α‐configuration, demonstrating a strict structure–activity relationship.     

New oxymesterone metabolites in human by gas chromatography‐tandem mass spectrometry and their application for doping control

Oxymesterone (17α‐methyl‐4, 17β‐dihydroxy‐androst‐4‐ene‐3‐one) is one of the anabolic androgenic steroids (AAS) banned by the World Anti‐Doping Agency (WADA). The biotransformation of oxymesterone is performed in vitro by human heptocytes and human urinary metabolic profiles are investigated after single dose of 20 mg to two adult males as well. Cell cultures and urine samples were hydrolyzed by β‐glucuronidase, extracted, and reacted with N‐Methyl‐N‐trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide (NH₄I), and dithioerythritol. After derivatization, a gas chromatography triple quadruple tandem mass spectrometry (GC‐MS/MS) using full scan and MS/MS modes was applied. The total ion chromatographs of the blank and the positive samples are compared, and 7 new metabolites were found. In addition to the well‐known 17‐epioxymesterone, oxymesterone is metabolized by 4‐ene‐reduction, 3‐keto‐reduction, 11β‐hydroxylation, and 16ξ‐hydroxylation. Based on the behavior of the MS/MS results of product ion and precursor ion modes, a GC‐MS/MS method has been developed monitoring these metabolites. The structures of metabolite 2 and 4 are tentatively identified as 17α‐methyl‐3β, 17β‐dihydroxy‐5α‐androstane‐4‐one and 17α‐methyl‐3α, 4ξ, 17β‐trihydroxy‐5α‐androstane, respectively. Detection of oxymesterone using new metabolites M2 and M4 can extend the detection window up to 4 days since the parent steroid was not detectable. Copyright © 2015 John Wiley & Sons, Ltd.     

Pregnancy greatly affects the steroidal module of the Athlete Biological Passport

Concentrations of urinary steroids are measured in anti‐doping test programs to detect doping with endogenous steroids. These concentrations are combined into ratios and followed over time in the steroidal module of the Athlete Biological Passport (ABP). The most important ratio in the ABP is the testosterone/epitestosterone (T/E) ratio but this ratio is subject to intra‐individual variations, especially large in women, which complicates interpretation. In addition, there are other factors affecting T/E. Pregnancy, for example, is known to affect the urinary excretion rate of epitestosterone and hence the T/E ratio. However, the extent of this variation and how pregnancy affect other ratios has not been fully evaluated. Here we have studied the urinary steroid profile, including 19‐norandrosterone (19‐NA), in 67 pregnant women and compared to postpartum. Epitestosterone was higher and, consequently, the T/E and 5αAdiol/E ratios were lower in the pregnant women. Androsterone/etiocholanolone (A/Etio) and 5αAdiol/5βAdiol, on the other hand, were higher in the first trimester as compared to postpartum (p<0.0001 and p=0.0396, respectively). There was no difference in A/T during pregnancy or after. 19‐NA was present in 90.5% of the urine samples collected from pregnant women. In this study, we have shown that the steroid profile of the ABP is affected by pregnancy, and hence can cause atypical passport findings. These atypical findings would lead to unnecessary confirmation procedures, if the patterns of pregnancy are not recognized by the ABP management units.     

In vivo metabolism of the designer anabolic steroid hemapolin in the thoroughbred horse

Hemapolin (2α,3α‐epithio‐17α‐methyl‐5α‐androstan‐17β‐ol) is a designer steroid that is an ingredient in several “dietary” and “nutritional” supplements available online. As an unusual chemical modification to the steroid A‐ring could allow this compound to pass through antidoping screens undetected, the metabolism of hemapolin was investigated by an in vivo equine drug administration study coupled with GC‐MS analysis. Following administration of synthetically prepared hemapolin to a thoroughbred horse, madol (17α‐methyl‐5α‐androst‐2‐en‐17β‐ol), reduced and dihydroxylated madol (17α‐methyl‐5α‐androstane‐2β,3α,17β‐triol), and the isomeric enone metabolites 17β‐hydroxy‐17α‐methyl‐5α‐androst‐3‐en‐2‐one and 17β‐hydroxy‐17α‐methyl‐5α‐androst‐2‐en‐4‐one, were detected and confirmed in equine urine extracts by comparison with a library of synthetically derived reference materials. A number of additional madol derivatives derived from hydroxylation, dihydroxylation, and trihydroxylation were also detected but not fully identified by this approach. A yeast cell‐based androgen receptor bioassay of available reference materials showed that hemapolin and many of the metabolites identified by this study were potent activators of the equine androgen receptor. This study reveals the metabolites resulting from the equine administration of the androgen hemapolin that can be incorporated into routine GC‐MS antidoping screening and confirmation protocols to detect the illicit use of this agent in equine sports.     

Codeine influences the serum and urinary profile of endogenous androgens but does not interact with the excretion rate of administered testosterone

Today's doping tests involve longitudinal monitoring of urinary steroids including the testosterone glucuronide and epitestosterone glucuronide ratio (T/E) in an Athlete Biological Passport (ABP). The aim of this study was to investigate the possible influence of short‐term use of codeine on the urinary excretion of androgen metabolites included in the steroidal module of the passport prior to and after the co‐administration with testosterone. The study was designed as an open study with the subjects being their own control. Fifteen healthy male volunteers received therapeutic doses of codeine (Kodein Meda) for 6 days. On Day 3, 500 mg or 125 mg of testosterone enanthate (Testoviron®‐Depot) was administered. Spot urine samples were collected for 17 days, and blood samples were collected at baseline, 3, 6, and 14 days after codeine intake. The circulatory concentration of total testosterone decreased significantly by 20% after 3 days' use of codeine (p = 0.0002) and an atypical ABP result was noted in one of the subjects. On the other hand, the concomitant use of codeine and testosterone did not affect the elevated urinary T/E ratio. In 75% of the individuals, the concentration of urinary morphine (a metabolite of codeine) was above the decision limit for morphine. One of the participants displayed a morphine/codeine ratio of 1.7 after codeine treatment, indicative of morphine abuse. In conclusion, our study shows that codeine interferes with the endogenous testosterone concentration. As a result, the urinary steroid profile may lead to atypical findings in the doping test.     

Unambiguous identification and characterization of a long‐term human metabolite of dehydrochloromethyltestosterone

In doping control analysis, the characterization of urinary steroid metabolites is of high interest for a targeted and long‐term detection of prohibited anabolic androgenic steroids (AAS). In this work, the structure of a long‐term metabolite of dehydrochloromethyltestosterone (DHCMT) was elucidated. Altogether, 8 possible metabolites with a 17α‐methyl‐17β‐hydroxymethyl – structures were synthesized and compared to a major DHCMT long‐term metabolite detected in reference urine excretion samples. The confirmed structure of the metabolite was 4α‐chloro‐18‐nor‐17β‐hydroxymethyl‐17α‐methyl‐5α‐androst‐13‐en‐3α‐ol.     

Variability of the urinary and blood steroid profiles in healthy and physically active women with and without oral contraception

The steroidal module of the athlete biological passport (ABP) targets the use of pseudo-endogenous androgenous anabolic steroids in elite sport by monitoring urinary steroid profiles. Urine and blood samples were collected weekly during two consecutive oral contraceptive pill (OCP) cycles in 15 physically active women to investigate the low urinary steroid concentrations and putative confounding effect of OCP. In urine, testosterone (T) and epitestosterone (E) were below the limit of quantification of 1 ng/ml in 62% of the samples. Biomarkers' variability ranged between 31% and 41%, with a significantly lesser variability for ratios (except for T/E [41%]): 20% for androsterone/etiocholanolone (p < 0.001) and 25% for 5α-androstane-3α,17β-diol/5ß-androstane-3α,17β-diol (p < 0.001). In serum, markers' variability (testosterone: 24%, androstenedione: 23%, dihydrotestosterone: 19%, and T/A4: 16%) was significantly lower than in urine (p < 0.001). Urinary A/Etio increased by >18% after the first 2 weeks (p < 0.05) following withdrawal blood loss. In contrast, serum T (0.98 nmol/l during the first week) and T/A4 (0.34 the first week) decreased significantly by more than 25% and 17% (p < 0.05), respectively, in the following weeks. Our results outline steroidal variations during the OCP cycle, highlighting exogenous hormonal preparations as confounder for steroid concentrations in blood. Low steroid levels in urine samples have a clear negative impact on the subsequent interpretation of steroid profile of the ABP. With a greater analytical sensitivity and lesser variability for steroids in healthy active women, serum represents a complementary matrix to urine in the ABP steroidal module.     

Studies on the in vivo metabolism of methylstenbolone and detection of novel long term metabolites for doping control analysis

The anabolic‐androgenic steroid methylstenbolone (MSTEN; 2α,17α‐dimethyl‐17β‐hydroxy‐5α‐androst‐1‐en‐3‐one) is available as a so‐called designer steroid or nutritional supplement. It is occasionally detected in doping control samples, predominantly tested and confirmed as the glucuronic acid conjugate of methylstenbolone. The absence of other meaningful metabolites reported as target analytes for sports drug testing purposes can be explained by the advertised metabolic stability of methylstenbolone. In 2013, a first investigation into the human metabolism of methylstenbolone was published, and two hydroxylated metabolites were identified as potential targets for initial testing procedures in doping controls. These metabolites were not observed in recent doping control samples that yielded adverse analytical findings for methylstenbolone, and in the light of additional data originating from a recent publication on the in vivo metabolism of methylstenbolone in the horse, revisiting the metabolic reactions in humans appeared warranted. Therefore, deuterated methylstenbolone together with hydrogen isotope ratio mass spectrometry (IRMS) in combination with high accuracy/high resolution mass spectrometry were employed. After oral administration of a single dose of 10 mg of doubly labeled methylstenbolone, urine samples were collected for 29 days. Up to 40 different deuterated methylstenbolone metabolites were detected in post‐administration samples, predominantly as glucuronic acid conjugates, and all were investigated regarding their potential to prolong the detection window for doping controls. Besides methylstenbolone excreted glucuronidated, three additional metabolites were still detectable at the end of the study on day 29. The most promising candidates for inclusion into routine sports drug testing methods (2α,17α‐dimethyl‐5α‐androst‐1‐ene‐3β,17β‐diol and 2α,17α‐dimethyl‐5α‐androst‐1‐ene‐3α,17β‐diol) were synthesized and characterized by NMR.     

Monitoring the endogenous steroid profile disruption in urine and blood upon nandrolone administration: An efficient and innovative strategy to screen for nandrolone abuse in entire male horses

Nandrolone (17β‐hydroxy‐4‐estren‐3‐one) is amongst the most misused endogenous steroid hormones in entire male horses. The detection of such a substance is challenging with regard to its endogenous presence. The current international threshold level for nandrolone misuse is based on the urinary concentration ratio of 5α‐estrane‐3β,17α‐diol (EAD) to 5(10)‐estrene‐3β,17α‐diol (EED). This ratio, however, can be influenced by a number of factors due to existing intra‐ and inter‐variability standing, respectively, for the variation occurring in endogenous steroids concentration levels in a single subject and the variation in those same concentration levels observed between different subjects. Targeting an efficient detection of nandrolone misuse in entire male horses, an analytical strategy was set up in order to profile a group of endogenous steroids in nandrolone‐treated and non‐treated equines. Experiment plasma and urine samples were steadily collected over more than three months from a stallion administered with nandrolone laurate (1 mg/kg). Control plasma and urine samples were collected monthly from seven non‐treated stallions over a one‐year period. A large panel of steroids of interest (n = 23) were extracted from equine urine and plasma samples using a C₁₈ cartridge. Following a methanolysis step, liquid‐liquid and solid‐phase extractions purifications were performed before derivatization and analysis on gas chromatography‐tandem mass spectrometry (GC‐MS/MS) for quantification. Statistical processing of the collected data permitted to establish statistical models capable of discriminating control samples from those collected during the three months following administration. Furthermore, these statistical models succeeded in predicting the compliance status of additional samples collected from racing horses. Copyright © 2013 John Wiley & Sons, Ltd.     

Carbon isotope ratio mass spectrometry for detection of endogenous steroid use: A testing strategy

Isotope ratio mass spectrometry (IRMS) testing is performed to determine if an atypical steroid profile is due to administration of an endogenous steroid. Androsterone (Andro) and etiocholanolone (Etio), and/or the androstanediols (5α‐ and 5β‐androstane‐3α,17β‐diol) are typically analyzed by IRMS to determine the ¹³C/¹²C ratio. The ratios of these target compounds are compared to the ¹³C/¹²C ratio of an endogenous reference compound (ERC) such as 5β‐pregnane‐3α,20α‐diol (Pdiol). Concentrations of Andro and Etio are high so ¹³C/¹²C ratios can easily be measured in most urine samples. Despite the potentially improved sensitivity of the androstanediols for detecting the use of some testosterone formulations, additional processing steps are often required that increase labour costs and turnaround times. Since this can be problematic when performing large numbers of IRMS measurements, we established thresholds for Andro and Etio that can be used to determine the need for additional androstanediol testing. Using these criteria, 105 out of 2639 urine samples exceeded the Andro and/or Etio thresholds, with 52 of these samples being positive based on Andro and Etio IRMS testing alone. The remaining 53 urine samples had androstanediol IRMS testing performed and 3 samples were positive based on the androstanediol results. A similar strategy was used to establish a threshold for Pdiol to identify athletes with relatively ¹³C‐depleted values so that an alternative ERC can be used to confirm or establish a true endogenous reference value. Adoption of a similar strategy by other laboratories can significantly reduce IRMS sample processing and analysis times, thereby increasing testing capacity. Copyright © 2013 John Wiley & Sons, Ltd.     

Oestrogenic activity of mimosine on MCF‐7 breast cancer cell line through the ERα‐mediated pathway

Hormone replacement therapy has been a conventional treatment for postmenopausal symptoms in women. However, it has potential risks of breast and endometrial cancers. The aim of this study was to evaluate the oestrogenicity of a plant‐based compound, mimosine, in MCF‐7 cells by in silico model. Cell viability and proliferation, ERα‐SRC1 coactivator activity and expression of specific ERα‐dependent marker TFF1 and PGR genes were evaluated. Binding modes of 17β‐oestradiol and mimosine at the ERα ligand binding domain were compared using docking and molecular dynamics simulation experiments followed by binding interaction free energy calculation with molecular mechanics/Poisson–Boltzmann surface area. Mimosine showed increased cellular viability (64,450 cells/ml) at 0.1 μM with significant cell proliferation (120.5%) compared to 17β‐oestradiol (135.2%). ER antagonist tamoxifen significantly reduced proliferative activity mediated by mimosine (49.9%). Mimosine at 1 μM showed the highest ERα binding activity through increased SRC1 recruitment at 186.9%. It expressed TFF1 (11.1‐fold at 0.1 μM) and PGR (13.9‐fold at 0.01 μM) genes. ERα‐mimosine binding energy was ?49.9 kJ/mol, and it interacted with Thr347, Gly521 and His524 of ERα‐LBD. The results suggested that mimosine has oestrogenic activity.     

Metabolism of 7‐ethoxycoumarin,flavanone and steroids by cytochrome P450 2C9 variants

CYP2C9 is a human microsomal cytochrome P450c (CYP). Much of the variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild‐type CYP2C9 and mutants were coexpressed with NADPH‐cytochrome P450 reductase in Escherichia coli . The hydroxylase activities toward 7‐ethoxycoumarin, flavanone and steroids were examined. Six CYP2C9 variants showed Soret peaks (450 nm) typical of P450 in reduced CO‐difference spectra. CYP2C9.38 had the highest 7‐ethoxycoumarin de‐ethylase activity. All the CYP2C9 variants showed lower flavanone 6‐hydroxylation activities than CYP2C9.1 (the wild‐type). CYP2C9.38 showed higher activities in testosterone 6β‐hydroxylation, progesterone 6β−/16α‐hydroxylation, estrone 11α‐hydroxylation and estradiol 6α‐hydroxylation than CYP2C9.1. CYP2C9.40 showed higher testosterone 17‐oxidase activity than CYP2C9.1; CYP2C9.8 showed higher estrone 16α‐hydroxylase activity and CYP2C9.12 showed higher estrone 11α‐hydroxylase activity. CYP2C9.9 and CYP2C9.10 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.9 and CYP2C9.10 was not changed, but CYP2C9.8, CYP2C9.12 and CYP2C9.40 showed different substrate specificity toward steroids compared with CYP2C9.1; and especially CYP2C9.38 displayed diverse substrate specificities towards 7‐ethoxycoumarin and steroids.     

Metabolic studies of formestane in horses

Formestane (4‐hydroxyandrost‐4‐ene‐3,17‐dione) is an irreversible steroidal aromatase inhibitor with reported abuse in human sports. In 2011, our laboratory identified the presence of formestane in a horse urine sample from an overseas jurisdiction. This was the first reported case of formestane in a racehorse. The metabolism of formestane in humans has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the in vitro and in vivo metabolic studies of formestane in horses, with the objective of identifying the target metabolite with the longest detection time for controlling formestane abuse. In vitro metabolic studies of formestane were performed using homogenized horse liver. Seven in vitro metabolites, namely 4‐hydroxytestosterone (M1), 3β,4α‐dihydroxy‐5β‐androstan‐17‐one (M2a), 3β,4β‐dihydroxy‐5β‐androstan‐17‐one (M2b), 3β,4α‐dihydroxy‐5α‐androstan‐17‐one (M2c), androst‐4‐ene‐3α,4,17β‐triol (M3a), androst‐4‐ene‐3β,4,17β‐triol (M3b), and 5β‐androstane‐3β,4β,17β‐triol (M4) were identified. For the in vivo studies, two thoroughbred geldings were each administered with 800 mg of formestane (32 capsules of Formadex) by stomach tubing. The results revealed that the parent drug and seven metabolites were detected in post‐administration urine. The six in vitro metabolites (M1, M2a, M2b, M2c, M3a, and M3b) identified earlier were all detected in post‐administration urine samples. In addition, 3α,4α‐dihydroxy‐5α‐androstan‐17‐one (M2d), a stereoisomer of M2a/M2b/M2c, was also identified. This study has shown that the detection of formestane administration would be best achieved by monitoring 4‐hydroxytestosterone (M1) in the glucuronide‐conjugated fraction. M1 could be detected for up to 34 h post‐administration. In blood samples, the parent drug could be detected for up to 34 h post administration. Copyright © 2013 John Wiley & Sons, Ltd.     

Longitudinal evaluation of multiple biomarkers for the detection of testosterone gel administration in women with normal menstrual cycle

In women, hormonal fluctuations related to the menstrual cycle may impose a great source of variability for some biomarkers of testosterone (T) administration, which can ultimately disrupt the sensitivity of their longitudinal monitoring. In this study, the sensitivity of the current urinary and haematological markers of the Athlete Biological Passport (ABP), as well as serum steroid biomarkers, was investigated for the monitoring of a 28-day T gel treatment combined with endogenous fluctuation of the menstrual cycle in 14 healthy female subjects. Additionally, the analysis of urinary target compounds was performed on a subset of samples for endogenous/exogenous origin via isotope ratio mass spectrometry (IRMS). In serum, concentrations of T and dihydrotestosterone (DHT) increased significantly during the treatment, whereas in urine matrix the most affected biomarkers were found to be the ratios of testosterone/epitestosterone (T/E) and 5α-androstane-3α,17β-diol/epitestosterone (5αAdiol/E). The detection capability of both urinary biomarkers was heavily influenced by [E], which fluctuated depending on the menstrual cycle, and resulted in low sensitivity of the urinary steroidal ABP module. On the contrary, an alternative approach by the longitudinal monitoring of serum T and DHT concentrations with the newly proposed T/androstenedione ratio showed higher sensitivity. The confirmatory IRMS results demonstrated that less than one third of the tested urine samples fulfilled the criteria for positivity. Results from this study demonstrated that the ‘blood steroid profile’ represents a powerful complementary approach to the ‘urinary module’ and underlines the importance of gathering bundle of evidence to support the scenario of an endogenous prohibited substance administration.