Projets de recherche

Code: 16E12HH 

Gene doping represents a threat to the integrity of sport and the suitable for publication on WADA's website health of athletes. The anti-doping community has been focusing efforts on developing a test for its detection. The current methodology to detect doping genes in an athletes’ blood uses the polymerase chain reaction (PCR) that targets unique sequences in a doping gene, which correspond to exon-exon junctions in the intronless transgene. These so-called real-time PCR assays detect unique sequences in the complementary DNA (cDNA) for human erythropoietin (EPO) and other doping genes such as insulin-like growth factor-1, growth hormone, growth hormone releasing hormone and follistatin. 
As the sequences of cDNA of Epo and other doping genes are known, it is relatively easy to aggravate these tests, which will then result in a false-negative result. Recently, we developed a new gene doping detection assay that will overcome this problem. The test is based on targeted sequencing of doping genes with potential to detect any doping gene in any context with a very high sensitivity. Using an in-house designed next generation sequencing assay, we developed a gene doping detection assay for cDNA of EPO which targets all potential exon-exon junctions of all possible EPO-transcripts. 
We propose to evaluate and further develop a multiplex ‘gene doping detection panel’ which targets genes for, among others, insulin-like growth factor-1, growth hormone, growth hormone releasing hormone and follistatin. The panel allows simultaneous detection of several ‘sport-specific’ genes in one sample, reducing the test’s cost and turn-around-time. This research is crucial in the development of a reliable routine method for detection of gene doping that may be potentially used in all sports. 

Main Findings: 

The main aim of the project was to evaluate and further develop a next generation sequencing-based multiplex ‘gene doping detection panel’. First we developed probes for the detection by sequencing of Erythropoietin, Insulin-like growth factor and Growth Hormone. Second, we evaluated the developed probes in the multiplex sequencing of these genes. Finally, we optimized and fine-tuned the developed method and determined the sensitivity of the developed method.

Our results show that, using the developed probes for next generation sequencing, we were able to simultaneously detect plasmid-derived cDNA copies of Erythropoietin, Insulin-like growth factor and Growth Hormone in a background of genomic DNA with 100% specificity. We were able to detect EPO GH1, GH2, IGF1 and IGF2 cDNA in concentrations below 0.01 percent gDNA at all exon-exon boundaries. For quantification of the amount of cDNA we spiked a GFP plasmid into the samples and found stable numbers of GFP across samples, enabling quantification of gene-doping cDNA levels.

Code: ISF16D21LE 

The administration of recombinant human growth hormone (rhGH) and/or small peptides, i.e. GH releasing factors (GHRF) that stimulate the endogenous production of GH have increased recently as a result of the availability and lack of sensitive tests. Two independent immunoassay methods are currently being employed to detect rhGH doping as well as mass-spectrometric approaches to find GHRFs.  
Our research proposal encompasses projects designed to investigate how the use of rhGH and GHRFs affect the traditional markers as well as putative markers such as miRNA. We will conduct a study in healthy male volunteers that will be given rhGH (Somatropin – two different doses) daily for one week. Some participants will be given two doses of sermorelin (Geref), a GHR peptide, for one week. Urine and serum samples will be collected several times prior to the administration in order to study the different markers longitudinally. Moreover, the steroid profile will be monitored in relation to hrGH/GHRP administration in order to see how the biomarkers of the different ABP modules interact. The use of small GH-releasing peptides is difficult to study in controlled settings since small peptides are not available as traditional drugs. Here we will use samples from patients, both men and women, self-reporting doping with peptides (as well as other doping agents) in order to see which peptides can be identified with the different approaches. 

Main Findings: 

It was found that the GH isoform ratio may detect rhGH intake when 1 and 4IU/day were administered for two weeks to healthy men. Using the biomarker test, none of the participants (n=9) displayed a GH2000 score above the population-based score of 9.98. However, when longitudinally monitored, the GH2000 score and its components IGF-I and P-III-NP, most of the participants showed values outside their individual calculated thresholds (mean four baseline values ±3 SD). Also, the longitudinally testing approach ws studied in four individuals in relation to 5 days GHRH(1-44) administration. It was found that monitoring of IGF-I may be useful for identifying also the intake of GHRH. Additionally, it was investigated if rhGH/GHRH administration exert an impact on ABP biomarkers. A minor increase i RET% and OFF-score after rhGH treatment was found, not resulting in any atypical passport findings. The urinary steroid profile, as well as serum concenctrations of androgens were not affected by rhGH. Moreover, the validity of putative biomarkers was assessed. It was concluded that miRNAs previously associated with GH supplementary treatment were not affected by the rhGH doses given here. Previous findings that fibronectin 1 may be a promising additive protein for detection of rhGH was confirmed.

Longitudinally monitoring of IGF-I and P-III-NP in an endocrine module may be a promising method in the future to increase the chances to detect rhGH/GHRH doping in men. The inclusion of additionaly biomarkers such as fibronectin 1 may increase the effect and/or detection window in some individuals.

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.

Code: ISF16E11FP 

The variable effects of anabolic androgenic steroids (AAS) on increasing skeletal muscle mass and strength has been well documented, as is the misuse of AAS in sport. Recent evidence suggest either a long- or short-term exposure to AAS might have a sustained effect on muscle morphological changes, for example, with increased muscle mass, capillary per fibre, muscle fibre size and myonuclei density, leading to improved performance. A positive correlation between the number of myonuclei and training response
following exposure to AAS in a mouse model seems to suggest a link between the formation of extra myonuclei and the extent of “muscle memory”; an idea that requires further investigation in humans.

Strength training can also increase the number of nuclei in muscle fibres. Adaptations in muscle mass by strength training are significantly enhanced in previously trained individuals despite a prolonged detraining period. Given the persistence of muscle nuclei, the use of AAS combined with training will have a greater impact on muscle hypertrophy than either training or steroid use alone. To detect the long-term effect of AAS and training (even after drugs are no longer detectable in the human system), abnormal changes in
skeletal muscle morphology illustrated by specific gene markers in response to the stimuli exist and will allow these molecular signals to be picked up by modern gene screening methods. In the proposed project, gene expression profiling of skeletal muscle in response to AAS exposure will be carried out using total RNA-seq. The molecular, histological and training response markers will be integrated for the detection of short- and long-term effects of AAS and will be incorporated into the steroid module of the Athlete Biological
Passport for improved validity and reliability.
 

Main Findings:

Fat Free Mass (FFM) of RP2-5, who ceased AAS usage ≤2 weeks prior to visit one with 19-28 weeks between visits, decreased by 3.9-4.7 kg. FFM of RP1, who ceased AAS usage 34 weeks prior to visit one with 28 weeks between visits, decreased by 0.9 kg. Fibre CSA decreased for RP1 and RP2 between visits (7566 vs 6629 µm²; 7854 vs 5677 µm²) whilst myonuclei per fibre remained similar (3.5 vs 3.4; 2.5 vs 2.6). Fibre CSA (7167 vs 7889 µm²) and myonuclei per fibre (2.6 vs 3.3) increased for RP3 between visits. Mean fibre CSA was significantly higher in RT-AS (n=17) (8160 ± 1769 µm²) compared to C (n=5) (6477 ± 1271 µm², p=0.028). There were no significant differences between C, RT (n=15), RT-AS & PREV (n=6) for myonuclei per fibre. Myonuclei per fibre and CSA for all biopsied participants (n=43) was significantly correlated (r=0.8, p<0.001). 
All whole blood samples (n=60) were subjected to RNA-Seq as they had purified total RNA that was of sufficient concentration, purity, and integrity. In comparison with RT participants who ceased AAS exposure <1 week ago (n=10), 1-2 weeks ago (n=5), 10-50 weeks ago (n=4), and >52 weeks ago (n=7), had 612, 464, 173 and 188 genes differentially expressed, respectively. RP2-5 had 33 differentially expressed genes between visits which were mainly associated with the interferon signalling pathway linked to the immune system. RNA-Seq of corresponding muscle samples is currently underway. The finding of comparable myonuclei per fibre numbers despite decrements in fibre CSA post AAS usage is consistent with the “muscle memory” mechanism. RNA-Seq identified 33 genes that could provide novel beneficial biomarkers. 
Further research, particularly the longitudinal monitoring of AAS users post usage is required to confirm these intriguing findings.

Code: 16A11AC 

Urine, as a biological waste material, is an extremely complex fluid counting thousands of components belonging to more than 200 different chemical classes. It is conventionally adopted for investigating the endogenous steroids profile in athletes to prevent doping. Isotopic Ratio Mass Spectrometry (IRMS) is the technique able to distinguish the source of the steroid via carbon isotopic measurements (13C/12C), differentiating the exogenous from the endogenous ones. Reliable IRMS determinations strongly depend on adequate purity of the investigated steroids. This demand is guaranteed by labor-intensive and time consuming preliminary steps (i.e. sample preparation, derivatization, liquid chromatography fractionation).
Multidimensional gas chromatography (MDGC) is a consolidated technique for the separation of complex matrices as well as the investigation of target compounds. In such approach, two columns are arranged in a series (e.g. non-polar stationary phase in the first dimension, followed by a polar stationary phase in the second dimension). The principle is to select the peak of interest in the first dimension and send it (heart-cut) into the second - ideally orthogonal - dimension for further separation.

Main Findings: 

Distinguishing endogenous anabolic steroids from their synthetic copies in urine samples from athletes requires a specific analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS ,IRMS). Such analysis is mandatory to be implemented in a doping control laboratory, since many of the adverse analytical findings involved endogenous steroids whose administration cannot be characterized by other techniques.

In order to obtain suitable and consistent results by IRMS, a laborious and time-consuming sample treatment is required due to both the natural presence of the target analytes and the high comlexity of the matrix, ensuring the convenient purification of the steroids prior to the analysis. Many steps are involved, including one or more preparative high performance liquid chromatography (HPLC) to isolate the steroids from matrix interference. Besides a long time, preparative HPLC necessitates many sample transfer steps, which represents potential losses of the amonut of steroids to be analyzed and possible contribution to the measurement uncertainties.

In order to accelerate the sample preparation and increase the automation of the process, the use of multidimensional gas chromatography (MDGC) prior to IRMS experiments has been investigated as an alternative to preparative HPLC. After preliminary studies already published, a full validation was perfomred in two doping control laboratories accredited by the World Anti-Doping Agency (WADA) to conclude about the advantages in replacing preparative HPLC with MDGC in the routine confirmatory analysis. A well-established instrumental configuration based on two independent GC ovens and one heart-cutting device was used. The first dimension (1D) separation was obtained by a non-polar colum which assured high efficiency and good loading capacity, while the second dimension (2D), based on a mid-polar stationary phase, provided good selectivity. The assembled MDGC set-up was applied for measuring testosterone, 5α- and 5β-androstanediol, androsterone and etiochlanolone as target compounds, and pregnanediol, 11-ketoetiocholanolone and 16-androstenol as endogenous reference compounds. One additional solid phase extraction was included at the end of the conventional sample treatment already implemented in the two laboratories, at which two fractions were obtained to be analyzed, minimizing matrix effects and column overloads. Following WADA regulations, the experiments comprised linearity of the instrument, repeatibility, linear mixing models, method performance assessment and limit quanitification.

Code: 16A19MP

In the fight against doping the laboratories are confronted with an increasing number of substances to screen on. Thus, a comprehensive screening for different classes of substances using dilute-and-inject methods in anti-doping screening is desirable. As lots of xenobiotics are excreted as conjugates a detection of the intact conjugates is performed by this approach. While chemical synthesis of phase-II metabolites works efficiently for compounds 
having only one potential conjugation site, several analogous compounds could not be chemically synthesized effectively, due to their more complex chemical structure. For the synthesis of the phase-II metabolites (glucuronides and sulfates) of these compounds a biotechnological production will be implemented. Fission yeast strains, that enable the biotechnological production of glucuronides and sulfates that cannot be synthesised efficiently via classical chemical synthesis will be generated and used to produce the relevant human conjugates. The produced reference material can be used for method set-up for direct detection. If laboratories still rely on hydrolysis of the conjugates, these reference compounds may
serve as control for hydrolysis efficiency and quality assurance.

As proof of concept the use of the generated fission yeast strains will be demonstrated by generation of salbutamol-sulfate, salbutamol-glucuronide, fenoterol-sulfate and 4-hydroxy-DHEA-sulfate within the project.

Main Findings: 

A complete set of recombinant fission yeast strains each expressing one of the human sulfotransferases hSULT1A1, hSULT1A2, hSULT1A3, hSULT1B1, hSULT1C2, hSULT1C3a, hSULT1C3d, hSULT1C4, hSULT1E1, SULT2A1, hSULT2B1a, hSULT2B1b, SULT4A1, or hSULT6B1, respectively, was successfully generated. For each hSULT two strains were generated, one integrating the sequence in the leu1 gene of the cells, and an additional with a second expression unit in the pREP1-plasmid. Conjugation efficiency of the strains for the sulfonation of one test substrate (known from literature for the respective isoforms) for each SULT isoenzymes was successfully demonstrated. Ten out of twelve enzymes for which substrates are known were already shown to produce the respective sulfoconjugates. Analysis of the two remaining strains is in progress. These results prove that the intracellular production of the cofactor PAPS necessary for the SULT activity in fission yeast is sufficiently high to support metabolite production by whole-cell biotransformation. We also developed a new and convenient SULT activity assay based on the sulfonation of a proluciferin compound and established enzyme bag assays for SULTs. Taken together, we have developed the technology to systematically generate conjugates relevant for doping control by human SULTs.

Code: 16A18IA

The direct detection of phase II metabolites of doping substances suitable for publication on WADA's website and steroids using LC-ESI-MS/(MS) in particular, is a current trend in doping control, offering simplicity, sensitivity and time effectiveness. This approach enables the detection of a great range of phase II metabolites, as the conjugation with glucuronic and/or sulfuric acids clearly improves the ionization efficiency of steroids. Recently, the detection of abundant and/or long term sulfate metabolites has been reported for exogenous AAS using LC-ESI-MS/(MS). However, metabolite characterizations and/or confirmation procedures of sulfate metabolites rely mainly on GC-MS/(MS) methods. This is due to the fact that their analysis in positive ionization mode lacks sensitivity, whereas in negative ionization mode, Collision Induced Dissociation (CID) experiments show dominant product ions that are limited to sulfate moiety and lack structural information. Typically, steroid metabolites have additional functional groups to conjugation site, usually hydroxyls and keto groups, that are prone to derivatization. Chemical derivatization can enhance the ionization efficiency of Phase II metabolites in positive ionization mode, whereas CID product ion spectra are not limited to ions related to conjugated group, alter their fragmentation behavior and hence they can be used as a robust alternative confirmation procedure. This is a novel approach that may facilitate the confirmation of sulfate metabolites, since the laborious deconjugation step will be skipped.  The simultaneous confirmation of intact sulfate and glucuronide metabolites will be examined in specific cases as the proposed methodology permits the confirmation of both sulfo- and gluco-conjugated metabolites possessing specific structural elements like keto groups with a single run. The herein proposed methodology may also used in the future as a complementary tool for the structural elucidation of newly found Phase II metabolites by LC-ESI-MS/(MS). 

Code: 16A01MP

The identification of anabolic androgenic steroids (AAS) is a vital issue in doping control. Due to the performance enhancing properties of AAS, the World Anti-Doping Association (WADA) banned their use but according to the annual report of WADA, steroids are still very popular amongst athletes and are responsible for half of all adverse analytical findings. The search for metabolites with longer detection times remains an important task and the introduction of new long term metabolites for exogenous AAS such as for example stanozolol, methanedione and dehydrochloromethyltestosterone, led to a 4 - 80-fold increase of adverse analytical findings due to the prolonged detection time. 
This project aims at finding new long term metabolites for a number of AAS by application of our newly developed gas chromatography chemical ionization triple quadrupole mass spectrometry (GC-CI-MS/MS) protocol for metabolite detection and identification. Chemical ionization in combination with triple quadruple technology has proven to significantly increase the sensitivity for a wide range of compounds in comparison with electron impact (EI). In addition, GC-CI-MS/MS is characterized by AAS structure correlated fragmentation pathways. The combination of both factors allows the set up of a sensitive MRM method, designed to find previously unknown but expected metabolites by selection of theoretical transitions for expected metabolites. 

Main Findings:

In 2015, a new GC triple quadrupole MS method that used chemical ionization (CI), instead of EI was introduced. This new GC-CI-MS/MS method opened new possibilities in the search for new metabolites as CI is a soft ionization. The correlations between fragmentation behavior and AAS structure could be revealed and fragmentation pathwasy have been postulated. This enabled the search for previously unknown but expected metabolites by selection of their predicted transitions. The aim of the current project was to set up an efficient approach for searching new metabolites by application of these newly dscivered structure depended fragmentation pathways and to find new long term metabolites. The following AAS were selected: drostanolone, metenolone, mesterolone, oxymesterone, formebolone and methyltestosterone.

Novel long-term metabolites for oxymesterone and mesterolone were detected and characterized. This demonstrates that GC-CI-MC/MC is capable of detecting (and characterizing) metabolites that can be missed with other (more frequently) used techniques. For oxymesterone, the metabolite was identified as 18-nor-17β-hydroxymethyl-17α-methyl-4-hydroxy-androst-4,13-dien-3-one. It is primarily excreted as a glucuronide. For mesteroloone, the metabolite was identified as 1α-methyl-5α-androstan-3,6,16-triol-17-one and its sulfate form resulted in a prolonged detection time for mesterolone abuse.

For metenolone, a metabolite, primarily excreted as sulfate, was found to have a slightly improved detection window in comparison with the currently monitored metabolites. Likely, the metabolite is 1β-methyl-5α-androstan-17-one-3ζ-sulphate and for the first time it is documented to provide the longest detection time.

In general, this study illustrates that sulfated steroids are becoming increasingly important for doping control analyses as they allow longer detection times for AAS adn can provide valuable information.