Coupling Complete Blood Count and Steroidomics to Track Low Doses Administration of Recombinant Growth Hormone: An Anti-Doping Perspective

Growth Hormone (GH) under its human recombinant homologue (rhGH), may be abused by athletes to take advantage of its well-known anabolic and lipolytic properties; hence it is prohibited in sports by the World Anti-Doping Agency. Due to the rapid turnover of rhGH, anti-doping screening tests have turned to monitor two endocrine biomarkers (IGF-I and P-III-NP), but unfortunately, they show population-wise variability, limiting the identification rate of rhGH users. Previous studies have evidenced the numerous effects of GH on human physiology, especially in hematopoiesis and steroidogenesis. In this work, aiming to discover novel physiological rhGH biomarkers, we analyzed the complete blood count and the steroidomics profile of healthy, physically active, young males treated either with EPO + rhGH or EPO + placebo. The time-trends of these two physiological routes have been analyzed through geometric trajectory analysis (GTA) and OPLS-DA. Individuals supplemented with micro-doses of rhGH exhibited different leukopoietic and steroidal profiles compared to the control population, suggesting a role of the rhGH in both pathways. In the article, hypotheses on the observed differences are discussed according to the most recent literature and compared to results in animal models. The use of leukopoietic and steroidal biomarkers together with endocrine biomarkers (IGF-1 and P-III-NP) allows to correctly classify over 98% of samples with no false positives, miss-classifying only one single sample (false negative) over a total of 56; a promising result, if compared to the current rhGH detection strategies

Human anogenital distance: an update on fetal smoke-exposure and integration of the perinatal literature on sex differences

study question: Do sex and maternal smoking effects on human fetal anogenital distance (AGD) persist in a larger study and how do these data integrate with the wider literature on perinatal human AGD, especially with respect to sex differences? summary answer: Second trimester sex differences in AGD are broadly consistent with neonatal and infant measures of AGD and maternal cigarette smoking is associated with a temporary increase in male AGD in the absence of changes in circulating testosterone. what is known already: AGD is a biomarker of fetal androgen exposure, a reduced AGD in males being associated with cryptorchidism, hypospadias and reduced penile length. Normative fetal AGD data remain partial and windows of sensitivity of human fetal AGD to disruption are not known. study design, size, duration: The effects of fetal sex and maternal cigarette smoking on the second trimester (11 –21 weeks of gestation) human fetal AGD were studied, along with measurement of testosterone and testicular transcripts associated with apoptosis and proliferation. participants/materials, setting methods: AGD, measured from the centre of the anus to the posterior/caudal root of penis/clitoris (AGDapp) was determined in 56 female and 70 male morphologically normal fetuses. These data were integrated with current literature on perinatal AGD in humans. main results and the role of chance: At 11 – 13 weeks of gestation male fetal AGDapp was 61% (P , 0.001) longer than in females, increasing to 70% at 17 – 21 weeks. This sexual dimorphism was independent of growth characteristics (fetal weight, length, gonad weight). We confirmed that at 14 – 16 weeks of gestation male fetal AGDapp was increased 28% (P , 0.05) by in utero cigarette smoke exposure. Testosterone levels were not affected by smoking. To develop normative data, our findings have been integrated with available data from in vivo ultrasound scans and neonatal studies. Inter-study variations in male/female AGD differences lead to the conclusion that normalization and standardization approaches should be developed to enable confidence in comparing data from different perinatal AGD studies. limitations, reasons for caution: Sex differences, and a smoking-dependent increase in male fetal AGD at 14 – 16 weeks, identified in a preliminary study, were confirmed with a larger number of fetuses. However, human fetal AGD should, be re-assessed once much larger numbers of fetuses have been studied and this should be integrated with more detailed analysis of maternal lifestyle. Direct study of human fetal genital tissues is required for further mechanistic insights

Alternative (backdoor) androgen production and masculinization in the human fetus

Funding: The study was supported by the following grants: Chief Scientist Office (Scottish Executive, CZG/4/742) (PAF and PJOS) (http://www.cso.scot.nhs.uk/funding-2/); NHS Grampian Endowments 08/02 (PAF and PJOS) and 15/1/010 (PAF, PF, US, and PJOS) (https://www.nhsgcharities.com/); the Glasgow Children’s Hospital Research Charity Research Fund, YRSS/PHD/2016/05 (NW, MB, PJOS, and PAF) (http://www.glasgowchildrenshospitalcharity.org/research/glasgow-childrens-hospital-charity-research-fund); the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement number 212885 (PAF) (https://ec.europa.eu/research/fp7/index_en.cfm); Medical Research Council Grants MR/L010011/1 (PAF and PJOS) and MR/K501335/1 (MB, PAF, and PJOS) (https://mrc.ukri.org/); and the Kronprinsessan Lovisas Foundation, “Stiftelsen Gunvor och Josef Anérs,” the “Stiftelsen Jane och Dan Olssons,” and the “Stiftelsen Tornspiran” (KS and OS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Androgenic potential of human fetal adrenals at the end of the first trimester

The onset of steroidogenesis in human fetal adrenal glands (HFA) during the first trimester is poorly investigated. An unresolved question is the capacity of the HFA to produce potent androgen DHT via conventional and/or the backdoor pathway(s) at the end of first trimester, when androgen-responsive organs are developed. Our aim was to explore steroidogenesis and the expression of steroidogenic enzymes and transcription factors in HFA at gestational weeks (GW) 9-12 with focus on their androgenic potential. Steroids in the HFA were analyzed by gas chromatography/mass spectrometry. The expression of steroidogenic enzymes and transcription factors in the HFA at GW9-12 was investigated by qPCR, automated Western blotting and immunohistochemistry. We demonstrated that during GW9-12 HFA produced steroids of the Delta(5), Delta(4) and the backdoor pathways of the biosynthesis of DHT, though the latter was limited to production of 17 alpha-OH-dihydroprogesterone, androsterone and androstanedione without further conversion to DHT. The only androgens identified in the HFA were testosterone and androsterone, a precursor in the biosynthesis of DHT. We also observed higher levels of CYP17A1 but low expression of 3 beta HSD2 at GW11-12 in the HFA. Elevated levels of CYP17A1 were associated with an increased expression of SF-1 and GATA-6. Altogether, our data demonstrate that of those steroids analyzed, the only potent androgen directly produced by the HFA at GW9-12 was testosterone. The onset of steroidogenesis in the HFA is a complex process that is regulated by the coordinated action of related transcription factors

Comparison of water, sediment, and plants for the monitoring of antibiotics: a case study on a river dedicated to fish farming

International audienceOxolinic acid, flumequine, oxytetracycline, and florfenicol are antibiotics commonly used in farming. Because an important percentage of these antibiotics given to fish and cattle ends up, directly or indirectly, in the freshwater environment, suitable tools for the monitoring of these antibiotics are needed. A French river was chosen because of the location of four fish farms and a sewage plant on its main course. First, a passive monitoring program involving water, sediment, and autochthonous bryophytes was performed at 25 sampling sites tested once every three months for one year. Second, an active monitoring method was performed using moss bags for a one-month exposure period, both upstream and downstream of each potential source of antibiotics. Sediment and bryophyte samples, but not water samples, were found to be useful for monitoring environmental contamination by oxolinic acid, flumequine, oxytetracycline, and florfenicol. Sediments and bryophytes also appeared to be complementary media for dating the river's contamination by antibiotics. Data collected by both active and passive monitoring methods confirmed contamination of the river, mainly by flumequine and oxytetracycline, attributable to fish farming but also to terrestrial animal farming and perhaps human pharmaceuticals

Implementation of a semi-automated strategy for the annotation of metabolomic fingerprints generated by liquid chromatography-high resolution mass spectrometry from biological samples

International audienceMetabolomics aims at detecting and semi-quantifying small molecular weight metabolites in biological samples in order to characterise the metabolic changes resulting from one or more given factors and/or to develop models based on diagnostic biomarker candidates. Nevertheless, whatever the objective of a metabolomic study, one critical step consists in the structural identification of mass spectrometric features revealed by statistical analysis and this remains a real challenge. Indeed, this requires both an understanding of the studied biological system, the correct use of various analytical information (retention time, molecular weight experimentally measured, isotopic golden rules, MS/MS fragment pattern interpretation...), or querying online databases. In gas chromatography-electro-ionisation (EI)-mass spectrometry, EI leads to a very reproducible fragmentation allowing establishment of universal EI mass spectra databases (for example, the NIST database - National Institute of Standards and Technology) and thus facilitates the identification step. Unfortunately, the situation is different when working with liquid chromatography-mass spectrometry (LC-MS) since atmospheric pressure ionisation exhibits high inter-instrument variability regarding fragmentation. Therefore, the constitution of LC-MS "in-house'' spectral databases appears relevant in this context. The present study describes the procedure developed and applied to increment 133 and 130 metabolites in databanks dedicated to analyses performed with LC-HRMS in positive and negative electrospray ionisation, and the use of these databanks for annotating quickly untargeted metabolomics fingerprints. This study also describes the optimization of the parameters controlling the automatic processing in order to obtain a fast and reliable annotation of a maximum of organic compounds. This strategy was applied to bovine kidney samples collected from control animals or animals treated with steroid hormones. Thirty-eight compounds were identified successfully in the generated chemical phenotypes, among which five were found to be candidate markers of the administration of these anabolic agents, demonstrating the efficiency of the developed strategy to reveal and confirm metabolite structures according to the high-throughput objective expected from these integrative biological approaches

Population pharmacokinetics/pharmacodynamics modelling of enrofloxacin for the three major trout pathogens Aeromonas salmonicida, Flavobacterium psychrophilum and Yersinia ruckeri

International audienceEnrofloxacin is a fluoroquinolone antimicrobial agent used in freshwater rainbow trout against the main pathogenic bacteria Aeromonas salmonicida, Yersinia ruckeri and Flavobacterium psychrophilum. However, the current “standard” dose (10 mg/kg/day for 10 days) was based only on some old, rather limited experimental data, and needed to be re-assessed. Thus, a pharmacokinetic-pharmacodynamic (PKPD) approach was used by combining a population PK model with new epidemiological data (Minimum Inhibitory Concentrations (MIC)) of the three bacterial species to determine optimal enrofloxacin doses in rainbow trout.Ninety-six rainbow trout (half diploid, half triploid) were randomly assigned to four different groups and received oral (gavage) and then intravenous administration of enrofloxacin at four different doses (range 5–60 mg/kg). Individual blood samples were taken to develop a population PK model.Enrofloxacin should be considered as a long-acting drug in trout due to the observed long plasma half-life (>100 h), which is therefore inadequate with the “standard” dosage based on daily oral administrations. Moreover, the fish ploidy had an impact on the PK of enrofloxacin with a longer persistence of enrofloxacin in triploid individuals, which raises the question of the withdrawal period to apply. The absolute bioavailability of oral enrofloxacin was estimated at ~88%.For F. psychrophilum, the provisional epidemiological cut-off value (CONRI), calculated according to the NRI method, was equal to 0.03 μg/mL. For A. salmonicida and Y. ruckeri, however, no clear bimodal distribution of MIC could be observed, and therefore no relevant CONRI could be obtained.According to our model, a single oral dose of ~5 mg/kg should provide sufficient exposure to treat the wild-type population of F. psychrophilum for 4 days, while complying with the PKPD breakpoints. Then, a maintenance dose of ~2.5 mg/kg could possibly be re-administered every 4 days. The absence of a CONRI did not allow to predict an optimal dose for the two other bacteria. As more than 70% of A. salmonicida isolates in our data set have an enrofloxacin MIC ≥0.25 μg/mL, it seems that enrofloxacin should not be recommended against this bacterium.The PKPD approach allowed us to refine the dosing regimens in rainbow trout, for a more sustainable approach. These new dosing regimens have yet to be clinically confirmed

Development of a molecular recognition based approach for multi-residue extraction of estrogenic endocrine disruptors from biological fluids coupled to liquid chromatography-tandem mass spectrometry measurement

International audienceMulti-residue methods permitting the high-throughput and affordable simultaneous determination of an extended range of endocrine disrupting chemicals (EDCs) with reduced time and cost of analysis is of prime interest in order to characterize a whole set of bioactive compounds. Such a method based on UHPLC-MS/MS measurement and dedicated to 13 estrogenic EDCs was developed and applied to biological matrices. Two molecular recognition-based strategies, either molecular imprinted polymer (MIP) with phenolic template or estrogen receptors (ERα) immobilized on a sorbent, were assessed in terms of recovery and purification efficiency. Both approaches demonstrated their suitability to measure ultra-trace levels of estrogenic EDCs in aqueous samples. Applicability of the MIP procedure to urine and serum samples has also been demonstrated

Integrated metabolomic, lipidomic and steroidomic profiling for revealing signatures and markers of effect induced by bisphenols exposure on human testicular function

International audienceThe Newplast research project (ANR-13-CESA-0012-01) focuses on substitutes and derivatives of bisphenol A (BPA) which are used in the manufacture of polycarbonate and epoxy resins, including food contact materials. Its global objective is to generate data and knowledge as regards (i) their biotransformation and biological impact on the human hepatic and reproductive functions, (ii) their modes of actions at molecular level through ligand-receptor binding / transactivation mechanisms and (iii) human external and internal exposure assessment. The effect of BPA and related substitutes (BPS and BPF) on testicular function was assessed on fetal (FEGA) and adult (TEXAS) models using an integrated metabolomic, lipidomic and steroidomic profiling approach. Three complementary workflows were then combined to generate a unique set of biological signatures as a support for markers of effect discovery. A rationalized sample preparation permitted to fractionate each characterized sample into one polar fraction for RPLC-ESI(+/-)-HRMS metabolomic profiling, and one apolar phase for RPLC-ESI(+/-)-HRMS lipidomic and GC-EI-MS/MS steroidomic profiling. Moreover a comprehensive characterization from both culture medium (exo-metabolome) and testicular tissue (endo-metabolome) was achieved. All together, obtained results give an extended picture of biological disruptions induced by BPA and two main substitutes (BPS and BPF) on human testicular target, including dose-responses and mixture effects. A set of annotated and particularly affected metabolites was revealed from each investigated metabolome sub-component