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.

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

Masculinization of the external genitalia in humans is dependent on formation of 5α-dihydrotestosterone (DHT) through both the canonical androgenic pathway and an alternative (backdoor) pathway. The fetal testes are essential for canonical androgen production, but little is known about the synthesis of backdoor androgens, despite their known critical role in masculinization. In this study, we have measured plasma and tissue levels of endogenous steroids in second trimester human fetuses using multidimensional and high-resolution mass spectrometry. Results show that androsterone is the principal backdoor androgen in the male fetal circulation and that DHT is undetectable (<1 ng/mL), while in female fetuses, there are significantly lower levels of androsterone and testosterone. In the male, intermediates in the backdoor pathway are found primarily in the placenta and fetal liver, with significant androsterone levels also in the fetal adrenal. Backdoor intermediates, including androsterone, are only present at very low levels in the fetal testes. This is consistent with transcript levels of enzymes involved in the alternate pathway (steroid 5α-reductase type 1 [SRD5A1], aldo-keto reductase type 1C2 [AKR1C2], aldo-keto reductase type 1C4 [AKR1C4], cytochrome P450 17A1 [CYP17A1]), as measured by quantitative PCR (qPCR). These data identify androsterone as the predominant backdoor androgen in the human fetus and show that circulating levels are sex dependent, but also that there is little de novo synthesis in the testis. Instead, the data indicate that placental progesterone acts as substrate for synthesis of backdoor androgens, which occurs across several tissues. Masculinization of the human fetus depends, therefore, on testosterone and androsterone synthesis by both the fetal testes and nongonadal tissues, leading to DHT formation at the genital tubercle. Our findings also provide a solid basis to explain why placental insufficiency is associated with disorders of sex development in humans.

The GMO90+ Project: Absence of Evidence for Biologically Meaningful Effects of Genetically Modified Maize-based Diets on Wistar Rats After 6-Months Feeding Comparative Trial.

The GMO90+ project was designed to identify biomarkers of exposure or health effects in Wistar Han RCC rats exposed in their diet to 2 genetically modified plants (GMP) and assess additional information with the use of metabolomic and transcriptomic techniques. Rats were fed for 6-months with 8 maize-based diets at 33% that comprised either MON810 (11% and 33%) or NK603 grains (11% and 33% with or without glyphosate treatment) or their corresponding near-isogenic controls. Extensive chemical and targeted analyses undertaken to assess each diet demonstrated that they could be used for the feeding trial. Rats were necropsied after 3 and 6 months. Based on the Organization for Economic Cooperation and Development test guideline 408, the parameters tested showed a limited number of significant differences in pairwise comparisons, very few concerning GMP versus non-GMP. In such cases, no biological relevance could be established owing to the absence of difference in biologically linked variables, dose-response effects, or clinical disorders. No alteration of the reproduction function and kidney physiology was found. Metabolomics analyses on fluids (blood, urine) were performed after 3, 4.5, and 6 months. Transcriptomics analyses on organs (liver, kidney) were performed after 3 and 6 months. Again, among the significant differences in pairwise comparisons, no GMP effect was observed in contrast to that of maize variety and culture site. Indeed, based on transcriptomic and metabolomic data, we could differentiate MON- to NK-based diets. In conclusion, using this experimental design, no biomarkers of adverse health effect could be attributed to the consumption of GMP diets in comparison with the consumption of their near-isogenic non-GMP controls.

Steroid hormone profiling in human breast adipose tissue using semi-automated purification and highly sensitive determination of estrogens by GC-APCI-MS/MS.

Body mass index is a known breast cancer risk factor due to, among other mechanisms, adipose-derived hormones. We developed a method for steroid hormone profiling in adipose tissue to evaluate healthy tissue around the tumor and define new biomarkers for cancer development. A semi-automated sample preparation method based on gel permeation chromatography and subsequent derivatization with trimethylsilyl (TMS) is presented. Progestagens and androgens were determined by GC-EI-MS/MS (LOQ 0.5 to 10 ng/g lipids). For estrogen measurement, a highly sensitive GC-APCI-MS/MS method was developed to reach the required lower limits of detection (0.05 to 0.1 ng/g lipids in matrix, 100-200 fg on column for pure standards). The combination of the two methods allows the screening of 27 androgens and progestagens and 4 estrogens from a single sample. Good accuracies and repeatabilities were achieved for each compound class at their respective limit of detection. The method was applied to determine steroid hormone profiles in adipose tissue of 51 patients, collected both at proximity and distant to the tumor. Out of the 31 tested steroid hormones, 14 compounds were detected in human samples. Pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone (DHEA), and androstendione accounted together for 80% of the observed steroid hormone profiles, whereas the estrogens accounted for only 1%. These profiles did not differ based on sampling location, except for ß-estradiol; steroid hormone conversions from androgens to estrogens that potentially take place in adipose or tumoral tissue might not be detectable due a factor 100 difference in concentration of for example DHEA and ß-estradiol. Graphical Abstract Schematic overview of the determination of steroid hormones and metabolites in adipose tissue in proximity and distal to the tumor.

Resveratrol inhibits steroidogenesis in human fetal adrenocortical cells at the end of first trimester.

SCOPE: Resveratrol has a diverse array of healthful effects on metabolic parameters in different experimental paradigms but has also potential to inhibit steroidogenesis in rodent adrenals. The aim of the present study was to characterize the effects of resveratrol on human fetal adrenal steroidogenesis at gestational weeks (GW) 9-12. METHODS AND RESULTS: Adrenals from aborted fetuses (GW10-12) were used to prepare primary cultures of human fetal adrenocortical cells (HFAC). HFAC were treated in the presence or absence of ACTH (10 ng/mL) with or without resveratrol (10 µM) for 24 h. The production of steroids by HFAC was analyzed by gas and liquid chromatography coupled to tandem/mass spectrometry. The expression of steroidogenic enzymes at GW 9-12 was quantified by automated Western blotting. We observed that resveratrol significantly suppressed synthesis of dehydroepiandrosterone (DHEA), androstenedione and 11-deoxicortisol by ACTH-activated and unstimulated HFAC, which was associated with inhibition of the activities and expression of cytochromes 17α-hydroxylase/17,20 lyase (CYP17) and 21-hydroxylase (CYP21) in these fetal adrenocortical cells. CONCLUSION: Our in vitro findings on the sensitivity of human fetal adrenal steroidogenesis to resveratrol at GW9-12 suggest that intake of this polyphenol at high doses by women who are at early stages of pregnancy is undesirable.

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 (AGD(app)) 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 AGD(app) 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 AGD(app) 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. WIDER IMPLICATIONS OF THE FINDINGS: Fetal exposure to cigarette smoke chemicals is known to lead to reduced fertility in men and women. Integration of our data into the perinatal human AGD literature shows that more work needs to be done to enable reliable inter-study comparisons. STUDY FUNDING/COMPETING INTERESTS: The study was supported by grants from the Chief Scientist Office (Scottish Executive, CZG/1/109 & CZG/4/742), NHS Grampian Endowments (08/02), the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no 212885 and the Medical Research Council, UK (MR/L010011/1). The authors declare they have no competing interests, be it financial, personal or professional.

Phthalates Exert Multiple Effects on Leydig Cell Steroidogenesis.

Humans are significantly exposed to phthalates via food packaging, cosmetics and medical devices such as tubings and catheters. Testicular Leydig cells (LCs) are suggested to be among the main targets of phthalate toxicity in the body. However, their sensitivity to phthalates is species-dependent. This paper describes the response of the LCs from different species (mouse, rat and human) to phthalate exposure in different experimental paradigms (in vivo, ex vivo and in vitro), with particular focus on mechanisms of phthalate action on LC steroidogenesis. A comprehensive analysis of the impact of phthalate diesters and phthalate monoesters on LCs in different stages of their development is presented and possible mechanisms of phthalates action are discussed. Finally novel, not yet fully elucidated sites of action of phthalate monoesters on the backdoor pathway of 5α-dihydrotestosterone biosynthesis in immature mouse LCs and their effects on steroidogenesis and redox state in adult mouse LCs are reported.

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.

Multi-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.

Recombinant bovine growth hormone identification and the kinetic of elimination in rainbow trout treated by LC-MS/MS.

The efficiency of the administration of recombinant bovine growth hormone (rbGH) to enhance fish growth has been widely reported in the literature. Although its use is probable and has been described in several countries, rbGH is prohibited in the European Union (Council Decisions 1994/936/EC, 1999/879/EC). In this context, an analytical strategy was optimised in order to identify rbGH-treated fish. Currently, one of the most difficult challenges for the detection of rbGH in fish is probably the choice of the matrix and the corresponding available quantity for analysis. Therefore, based on a previous efficient protocol developed for mammalian species, a method was adapted for very limited serum volume (50 µl) and was successfully implemented to analyse serum collected from seven trout treated with rbGH. The detection of rbGH was possible from the very first day after administration and the hormone could easily be identified at least for 1 month with levels in the range 5-10 µg ml(-1). The limits of detection (LODs) estimated around 0.5 µg ml(-1) rbGH in fish serum are far below observed concentrations in incurred samples and therefore attest to the relevance of the developed protocol.

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

Metabolomics 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.

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

Oxolinic 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.