Performance assessment of an equine metabolomics model for screening a range of anabolic agents.

INTRODUCTION: Despite their ban, Anabolic Androgenic Steroids (AAS) are considered as the most important threat for equine doping purposes. In the context of controlling such practices in horse racing, metabolomics has emerged as a promising alternative strategy to study the effect of a substance on metabolism and to discover new relevant biomarkers of effect. Based on the monitoring of 4 metabolomics derived candidate biomarkers in urine, a prediction model to screen for testosterone esters abuse was previously developed. The present work focuses on assessing the robustness of the associated method and define its scope of application. MATERIALS AND METHODS: Several hundred urine samples were selected from 14 different horses of ethically approved administration studies involving various doping agents' (AAS, SARMS, ß-agonists, SAID, NSAID) (328 urine samples). In addition, 553 urine samples from untreated horses of doping control population were included in the study. Samples were characterized with the previously described LC-HRMS/MS method, with the objective of assessing both its biological and analytical robustness. RESULTS: The study concluded that the measurement of the 4 biomarkers involved in the model was fit for purpose. Further, the classification model confirmed its effectiveness in screening for testosterone esters use; and it demonstrated its ability to screen for the misuse of other anabolic agents, allowing the development of a global screening tool dedicated to this class of substances. Finally, the results were compared to a direct screening method targeting anabolic agents demonstrating complementary performances of traditional and omics approaches in the screening of anabolic agents in horses.

Human testis steroidogenesis is inhibited by phthalates.

BACKGROUND: Phthalic acid esters are widely used in the manufacture of plastics. Numerous studies have shown that these phthalates impair testicular testosterone production in the rat. However, the scarce and contradictory data concerning humans have cast doubt over whether these compounds are also anti-androgenic in man. We therefore investigated the direct effects of di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) on organo-cultured adult human testis and a human cell line. METHODS: Adult human testis explants or NCI-H295R adrenocortical human cells were cultured with DEHP or MEHP. The effects of ketoconazole, used as a reference molecule, were also assessed. RESULTS: In both models, DEHP and MEHP significantly inhibited testosterone production. The effects of both phthalates appeared to be specific for steroidogenesis, as INSL3 production by Leydig cells was not altered. Furthermore, the phthalates of interest had no effect on inhibin B production by Sertoli cells or on germ cell apoptosis. As only a small fraction of the phthalates added was found in the testis explants, and as these compounds were found to be metabolized, we estimate that the anti-androgenic effects observed occurred at concentrations of phthalates that are of the same order of magnitude as exposures reported in the literature for men. CONCLUSIONS: We provide the first evidence that DEHP and MEHP can inhibit testosterone production in the adult human testis. This is consistent with recent epidemiological findings of an inverse correlation between exposure to MEHP and testosterone concentrations.

Improvement of estradiol esters monitoring in bovine hair by dansylation and liquid chromatography/tandem mass spectrometry analysis in multiple reaction monitoring and precursor ion scan modes.

RATIONALE: The control of forbidden anabolic practices in cattle in the European Union has become challenging since endogenous compounds such as estradiol derivatives can potentially be used as growth promoters. Due to the great difficulty in establishing a reference threshold value for endogenous steroids, the direct detection of steroid esters in hair is an efficient strategy for the detection of 'natural' steroid abuse in cattle. METHODS: The present study aimed to develop and validate according to the current European standards a specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) analytical strategy to monitor estrogen esters in bovine hair. The analysis was performed by positive ion electrospray ionisation (ESI+) after dansylation. Two acquisition modes were then assessed: single reaction monitoring and precursor ion scanning. RESULTS: The results showed that the introduction of a dansylation step strongly improves the sensitivity of the detection of estradiol-17-esters by LC/(ESI+)-MS/MS. The CCα values are in the range 1-10 ng g(-1) after optimisation, except for estradiol decanoate for which the derivatisation is not efficient. In addition, this LC/MS/MS approach makes it possible to carry out a precursor ion scan to screen for the presence of these estradiol 17-esters in hair samples. CONCLUSIONS: Based on the specific product ions, i.e. m/z 255 in native conditions or m/z 171 after dansylation, this strategy has the advantage of detecting any (un)known estradiol ester and of giving access to the [M + H](+) ion of the suspected ester through only a single analysis.

Thyreostatic drugs, stability in bovine and porcine urine.

Thyreostatic drugs, illegally administrated to livestock for fattening purposes, are banned in the European Union since 1981. For monitoring their illegal use, sensitive and specific analytical methods are required. In this context, the knowledge of the stability in a matrix is of primary importance. This study aimed at evaluating the effects of preservation, number of freeze-thaw cycles, and matrix-related variables on the stability of thyreostatic drugs in the urine of livestock. Finally, the developed conservation approach was applied on incurred urine samples, which displayed traces of the thyreostat thiouracil below the recommended concentration of 10 µg L(-1). The stability study confirmed the negative influence of preservation (8 h) at room temperature and at -70 °C, decreases in concentration of more than 78.0% were observed for all thyreostats, except for 1-methyl-2-mercaptoimidazole and 2-mercaptobenzimidazole. Additionally, investigation of matrix-related variables indicated significant impacts of the presence of copper (p = 0.001) and the pH (p = 0.002). Next, an optimised pre-treatment (pH 1 and 0.1 M ethylenediaminetetraacetic acid disodium salt dehydrate) significantly differing from the original conservation approach (p < 0.05) was developed, which proved capable of delaying the decrease in concentration and improved the detection in time for both spiked as well as incurred urine samples. In the future, it seems highly advisable to apply the developed pre-treatment on incurred urines upon sampling, before thyreostat analysis. Additionally, it is recommendable to limit preservation of urine samples at room temperature, but also in the freezer prior to thyreostat analysis.

Occurrence of priority and emerging organic compounds in fishes from the Rhone River (France).

The main objective of this study was to collect new data on the occurrence, levels of priority and emerging organic compounds in freshwater fish sampled in the Rhone River. The 34 studied contaminants included alkylphenols, bisphenol A, polybromodiphenylethers (PBDE), perfluorinated compounds, hexabromocyclododecanes (HBCD), hexachlorobenzene and hexachlorobutadiene (HCBD). About 50 fish samples (individual specimens or pooled fish) were collected from three sites located upstream and downstream of the Lyon metropolitan area in the Rhone River (France). Four species were caught at each site, namely: the barbel (Barbus barbus), the common bream (Abramis brama), the white bream (Blicca bjoerkna) and the chub (Squalius cephalus). Some contaminants were quantified in all the 32 fish samples analysed: 4-nonylphenol, α-HBCD, the six PBDE congeners (28, 47, 99, 100, 153, 154), perfluorooctanesulfonate (PFOS) and perfluorodecanoic acid. Twenty three of the 32 samples had a concentration of PFOS above the Environmental Quality Standards (EQS) (up to six times higher than the EQS), and all the 32 samples had concentrations of PBDE above the EQS (up to 4,000 times higher, with the sum of six PBDE varying from 4.5 to 182 ng/g dry weight). Clearly, the interest to consider PFOS and HBCD as new priority substances is confirmed. In contrast, the pertinence of a priority status for HCBD, which was never quantified in our study, might have to be reconsidered in the future.

Evidence of complementarity between targeted and non-targeted analysis based on liquid and gas-phase chromatography coupled to mass spectrometry for screening halogenated persistent organic pollutants in environmental matrices.

This study explored the complementarity between targeted (TS) and non-targeted screening (NTS) based on liquid and gas-phase chromatography coupled to (high-resolution) mass spectrometry (LC-/GC-(HR)MS) for the comprehensive characterization of organohalogen fingerprints within a set of Lake Ontario lake trout samples. The concentrations of 86 legacy, emerging and novel halogenated compounds (HCs), were determined through 4 TS approaches involving no less than 6 hyphenated systems. In parallel, an innovative NTS strategy, involving both LC and GC-Q-Orbitrap, was implemented to specifically highlight halogenated signals. Non-targeted HRMS data were processed under the HaloSeeker software based on Cl and Br isotopic ratio and mass defect to extend the screening to unsuspected and unknown HCs. A total of 195 halogenated mass spectral features were characterized in the Lake Ontario lake trout, including well known HCs (PCBs, PBDEs, PBBs, DDT and their degradation products), emerging HCs (novel brominated flame retardants, short-, medium- and long-chain chlorinated paraffins) or suggested molecular formula (mainly polychlorinated ones). Among the 122 HCs highlighted by TS, only 21 were identified by NTS. These results fueled a discussion on the potential and limitations of both approaches, and the current position of NTS within environmental and health monitoring programs.

Metabolomics in chemical risk analysis - A review.

Exposure to chemical hazards is a growing concern in today's society, and it is of utmost interest to know the levels of exposure to chemicals and the risks associated with such exposure in order to implement effective health prevention strategies. Chemical risk analysis represents a complex and laborious task due to the large number of known substances, but also unknown compounds and emerging risks that must be addressed. In this challenging scenario, the study of metabolic perturbations induced by exposure to a given chemical hazard has recently emerged as an interesting alternative approach to apply in chemical risk analysis. Specifically, the biomarkers of effect identified by metabolomics are expected to reveal the adverse effects of chemicals and further link exposure to disease development. In this context, analytical chemistry has become an essential part of the strategy to highlight such biomarkers. The corollary is that the relevance of the discovered biomarkers will largely depend on both the quality of the analytical approaches implemented and the part of the metabolome covered by the analytical technique used. This review focuses on describing significant applications of metabolomics in the field of chemical risk analysis. The different risk assessment steps, including hazard identification, dose-response assessment and exposure assessment, and risk management are addressed through various examples to illustrate that such an approach is fit-for-purpose and meets the expectations and requirements of chemical risk analysis. It can be considered as an innovative tool for predicting the probable occurrence and nature of risks, while addressing the current challenges of chemical risk analysis (e.g. replacement, reduction and refinement (3R) of animal testing, effects of exposure to chemical mixtures at low doses, etc.), and with the aim of responding to chemical exposures concerns in a holistic manner and anticipating human health problems.

Do farming conditions influence brominated flame retardant levels in pig and poultry products?

Brominated flame retardants (BFR) are primarily used as flame retardant additives in insulating materials. These lipophilic compounds can bioaccumulate in animal tissues, leading to human exposure via food ingestion. Although their concentration in food is not yet regulated, several of these products are recognised as persistent organic pollutants; they are thought to act as endocrine disruptors. The present study aimed to characterise the occurrence of two families of BFRs (hexabromocyclododecane (HBCDD) and polybrominated diphenyl ethers (PBDE)) in hen eggs and broiler or pig meat in relation to their rearing environments. Epidemiological studies were carried out on 60 hen egg farms (34 without an open-air range, 26 free-range), 57 broiler farms (27 without an open-air range, 30 free-range) and 42 pig farms without an open-air range in France from 2013 to 2015. For each farm, composite samples from either 12 eggs, five broiler pectoral muscles or three pig tenderloins were obtained. Eight PBDE congeners and three HBCDD stereoisomers were quantified in product fat using gas chromatography-high-resolution mass spectrometry, or high-performance liquid chromatography-tandem mass spectrometry, respectively. The frequencies of PBDE detection were 28% for eggs (median concentration 0.278 ng/g fat), 72% for broiler muscle (0.392 ng/g fat) and 49% for pig muscle (0.403 ng/g fat). At least one HBCDD stereoisomer was detected in 17% of eggs (0.526 ng/g fat), 46% of broiler muscle (0.799 ng/g fat) and 36% of pig muscle (0.616 ng/g fat). Results were similar in concentration to those obtained in French surveillance surveys from 2012 to 2016. Nevertheless, the contamination of free-range eggs and broilers was found to be more frequent than that of conventional ones, suggesting that access to an open-air range could be an additional source of exposure to BFRs for animals. However, the concentration of BFRs in all products remained generally very low. No direct relationship could be established between the occurrence of BFRs in eggs and meat and the characteristics of farm buildings (age, building materials). The potential presence of BFRs in insulating materials is not likely to constitute a significant source of animal exposure as long as the animals do not have direct access to these materials.

Steroidogenic potential of human fetal kidney at early gestational age.

Steroidogenic potential of the human fetal kidney (hFK) at the end of first trimester is poorly investigated. Little is known about the ontogeny of steroidogenic enzymes and activities of steroidogenic pathways in the hFK at early pregnancy. Our aim was to explore steroidogenesis and the expression of steroidogenic enzymes in the hFK at gestational weeks (GW) 9-12. Steroids in the hFK were analyzed by gas chromatography/coupled to tandem mass spectrometry. The expression of steroidogenic enzymes in the hFK at GW 9-12 was investigated by qPCR, automated Western blotting and immunohistochemistry. We observed that the hFK produced substantial amount of steroids of the Δ5 and Δ4 pathways and several steroid precursors in the biosynthesis of DHT via the backdoor pathway but not DHT itself. The levels of steroids and expression of relevant steroidogenic enzymes (e.g., CYP17A1, HSD3B1, HSD3B2, CYP11B1 and AKR1C4) we significantly higher in the hFK at GW11-12 compared to GW9. We also found the expression of sex steroid receptors (e.g., AR, ERα and ERß) in the hFK at GW9-12. No sex-dependent differences in the levels of all identified steroids and expression of steroidogenic enzymes in the hFK from male and female fetuses were found. Altogether, our data indicate that the hFK at early pregnancy is steroidogenic organ with potential to synthesize multiple steroids that may play an important role in the formation and development of this organ in humans.

Quantitative method for conjugated metabolites of bisphenol A and bisphenol S determination in food of animal origin by Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry.

With the objectives of both generating bisphenols (BPs) conjugates occurrence data in food from animal origin but also investigating the origin of associated contamination, the present study deals with the development of an efficient analytical method aiming at monitoring both BPA and BPS conjugated metabolites in food from animal origin. The objective of such monitoring is to determine the origin of BPs contamination (FCM or animal contamination). The targeted compounds were BPA-monoglucuronide (BPA-1G), BPA-diglucuronide (BPA-2G), BPA-monosulfate (BPA-1S), BPA-disulfate (BPA-2S) and BPS-monoglucuronide (BPS-1G). The developed standard operating procedure includes a preliminary solid-liquid extraction step followed by two successive solid phase extraction (SPE) stages, using successively a non-polar phase and a strong cation exchange polymer. Quantification was achieved according to both the isotopic dilution and surrogated quantification methods, using 13C-BPA-1G and BPA-d6-1S as internal standards. Linearity was validated (R2 > 0.99) for each molecule within the concentration range [0-10] µg kg-1. Detection limits ranged from 0.02 µg kg-1 (BPA-1G in muscle, BPA-1S and BPA-2G in liver) to 0.50 µg kg-1 (BPA-2S in muscle). The strategy was then proven on liver samples collected from pregnant ewes subcutaneously exposed to BPA during 105 days, at 50 µg kg-1 per day. BPA-1G, BPA-2G and BPA-1S were detected and quantified at a concentration of 3.81 µg kg-1, 0.80 µg kg-1 and 0.09 µg kg-1, respectively. The analytical method was finally implemented on fifty unpacked food samples from animal origin in which significant free BPA concentrations were previously measured. Since no metabolites of BPA could be measured (

Ontogenesis of human fetal testicular steroidogenesis at early gestational age.

The onset of steroidogenesis in human fetal testes (HFT) during the first trimester is poorly investigated. One important unresolved question is the ontogeny of steroidogenic enzymes and formation of steroidogenic pathways in the HFT at early pregnancy. Our aim was to explore steroidogenesis, the expression of steroidogenic enzymes and their maturation in the HFT at gestational weeks (GW) 8-12. Steroids in the HFT were analyzed by gas chromatography/coupled to tandem mass spectrometry. The expression of steroidogenic enzymes in the HFT at GW8-12 was investigated by qPCR, automated Western blotting and immunohistochemistry. We demonstrated that the HFT at GW8-9 produced low level of testosterone via the Δ4 pathway and progesterone was the major steroid found in the testicular tissue. In contrast, more mature Leydig cells from the HFT at GW11-12 synthesized high levels of androgens via the Δ5 pathway. We also observed a significant upregulation of the expression of StAR, CYP11A1, CYP17A1 and its accessory proteins, P450 oxidoreductase (POR) and cytochrome b5 in the HFT at GW11-12 compared to GW8-9. Altogether, our data suggest that that human fetal Leydig cells differentiate rapidly at the end of the first trimester by acquiring capacity to express high levels of steroidogenic enzymes and switch from the Δ4 to the Δ5 pathways to synthesize high levels of androgens due to maturation of the CYP17-POR-b5 complex.

Development and validation of a method for fipronil residue determination in ovine plasma using 96-well plate solid-phase extraction and gas chromatography-tandem mass spectrometry.

Fipronil, a phenylpyrazole insecticide introduced for pest control on a broad range of crops, undergoes a reinforcement of the regulation within the European Union (2007/52/EC directive) due to its potential effects on environment and human health. In order to assess the plasmatic concentrations of fipronil residues (sulfone, sulfide, fipronil, desulfinyl and amide) in ovine, a methodology based on gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) was developed and validated according to the European standard (2002/657/EC). The proposed method allows a large number of samples to be treated concurrently (n=80) using a reduced sample amounts (0.2 mL), and consents to reach a level of quantification of 0.1 pg microL(-1). The sample preparation consisted of a single solid-phase extraction (SPE) purification on a 96-well plate filled with a styrene-divinyl-benzene phase. Linearity was demonstrated all along the investigated range of concentrations, i.e. from 0.25 to 2000 pg microL(-1), with coefficient of determination (R(2)) from 0.977 to 0.994, depending on target analytes. Calculated decision limit (CCalpha) and detection capability (CCbeta) for fipronil, sulfone and sulphide were in the range 0.05-0.16 and 0.28-0.73 pg microL(-1) respectively.

Comparison between liquid chromatography and supercritical fluid chromatography coupled to mass spectrometry for beta-agonists screening in feeding stuff.

ß-agonistic drugs have been forbidden as growth promoters in rearing animals in Europe since the late 1980s (Dir 96/22/EC). Specific and sensitive analytical methods based on UHPLC-MS/MS allow to monitor a large set of these substances. However, optimal performances are not observed for all the target analytes, especially for those exhibiting the highest polarities. We developed an SFC-MS/MS approach to cover the huge elution window of ß-agonists, from the most polar which are usually eluted in the void volume when using reversed phase chromatography in conventional HPLC to the most apolar ones. The objective was to reach performances in accordance with the European Union recommended level in feeding stuff, i.e. 50 µg kg-1. LC/MS and SFC/MS performances were thoroughly compared in terms of analytical validation data (linearity, selectivity, recovery rates, reproducibility, compounds identification, trueness, decision limit (CCα) and detection capability (CCß)) for 6 ß-agonistic drugs, namely bromobuterol, clenbuterol, isoxsuprine, ractopamine, salbutamol and zilpaterol. As a result, the SFC approach appeared complementary to the LC one because the elution order of compounds was totally different from the one obtained with a classical C18 stationary phase. Moreover, the UPLC-MS/MS approach gave a better response linearity and more accurate values, whereas SFC-MS/MS provided greater data for identification purposes, reproducibility and sensitivity. Both analytical approaches enabled the detection of targeted ß-agonists at a lower concentration than the recommended one (50 µg kg-1).

The human genital tubercle is steroidogenic organ at early pregnancy.

It is generally accepted that androgens produced by fetal Leydig cells (FLC) control proper masculinization of the male external genitalia. Here, we hypothesized that the human genital tubercle (GT) has potential to synthesize androgens independently of FLC at early pregnancy. We observed that human GT of both genders have capacity to synthesize steroids of the Δ4, Δ5 and alternative pathway of DHT synthesis including the androgen itself. The presence of steroids in the GT was associated with the expression of corresponding steroidogenic enzymes. Levels of steroids and the expression of steroidogenic enzymes were similar in the GT from male and female fetuses. In contrast to the GT, the human fetal testis synthesized DHT from testosterone but not via the alternative pathway. Our findings strongly suggest that the human GT at early pregnancy can synthesize DHT via the alternative pathway, which may play an important role in organogenesis of the urethra.

Development of a Cryptosporidium-arsenic multi-risk assessment model for infant formula prepared with tap water in France.

Tap water is used in France to reconstitute powder infant formula, although it is not sterile and possibly contaminated by microbiological and chemical hazards. The present study aims to quantify risks of using tap water in France for the preparation of infant formula, during the first six months of life. Cryptosporidium and arsenic were selected as hazards of greatest concern in microbiology and chemistry, respectively. A probabilistic model was developed using French (when available) and European (alternatively) data. Second order Monte Carlo simulation was used to separate uncertainty and variability of inputs. Outputs were expressed at the individual level as probability of illness and at the population level, using a common metric, the DALY (Disability Adjusted Life Year). Two scenarios of milk preparation were considered: with un-boiled or boiled tap water. Consuming infant formula rehydrated with un-boiled tap water during the first six months of life led to a total of 2250 DALYs per 100,000 infants (90% uncertainty interval [960; 7650]) for Cryptosporidium due to diarrhea, and 1 DALY [0.4; 2] for arsenic due to expected lifetime risk of lung and bladder cancer as a result of early exposure in life. For the entire population, boiling water would suppress the risk from Cryptosporidium. In contrast, the incremental cancer risk was low at the population level but elevated for 5% of the population exposed to high levels of arsenic. A stringent monitoring of tap water supply points should be continued. This multi-risk assessment model could help public health authorities and managers in evaluating both microbiological and chemical safety issues associated with using infant formula prepared with tap water.

Occurence of legacy and novel brominated flame retardants in food and feed in France for the period 2014 to 2016.

Determination of the occurrence levels of legacy and novel BFRs is today required to better understand the trends of BFRs contamination in food consecutive to the EU PBDEs restrictions and to proceed to a recent human food exposure in parallel. Therefore, concentrations of a large set of brominated flame retardants (BFRs) (n = 27) including PBDEs, HBCDDs, TBBPA and novel flame retardants (nBFRs) have been determined in more than 600 food and feed samples collected between 2014 and 2016 in the context of French monitoring plans. Although legacy BFRs had already been studied in France, such a survey constituted the very first determination of nBFRs occurrence in foodstuffs at the national level. The concentration levels measured in fish and fish products were in general higher than in the other food categories. PBDEs were detected in 70% of the samples and were observed as the most abundant congeners (representing 80% of the sum of the monitored BFRs), while α-HBCDD could also be considered as a predominant congener (up to 26% of the sum of the monitored BFRs in fishes). nBFRs concentration levels were most of the time below the LOQ, except PBT, PBBz and HBBz which were more frequently detected at low levels. Also investigated in the study, BRPs exhibited high concentration levels in crustaceous (maximum value > 2700 pg/g ww).

Urinary excretion of 5(10)-estrene-3beta,17alpha-diol and estrone by the female horse: complementary indicators of early pregnancy screened with regard to a putative anabolic doping practice.

Rules of horse racing stipulate that pregnant mares may compete under definite conditions of date, because early pregnant status may be misused for the sake of enhancing physical performance by putative anabolic steroid action. Screening for pregnancy is generally performed by plasma equine gonadotrophin (eCG) immunoassay, which covers the period between Days 40 and 120. In common screening for urinary anabolic steroids performed by gas chromatography-mass spectrometry, inclusion of two complementary criteria, i.e. the evaluation of total conjugates of 5(10)-estrene-3beta,17alpha-diol (EED) and estrone (E1), can easily be performed. Although EED and E1 have no anabolic property per se in the horse, assessing these two markers may be helpful in the period comprised between Days 70 and 250, thereby prolonging the detection period behind that of eCG. Peak values of EED and E1 are then attained, so that visual inspection of chromatographic tracings remains in general sufficient as a diagnostic tool. Comparison of EED and E1 during pregnancy and in an estrus cycle indicates a drastic difference in the attained excretion values, attributable to either the placenta or the ovarian follicle. The identity of EED has been proven by GC-MS(n) in urine and in placental tissue.

Past, present and future of mass spectrometry in the analysis of residues of banned substances in meat-producing animals.

A residue is a trace (microg kg(-1), ng kg(-1)) of a substance, present in a matrix. Banned substances, such as growth promoters, which are abused in animal fattening and where this article is focused on, may be divided into four major groups: thyreostats, anabolics or anabolic steroids, corticosteroids and beta-agonists or repartitioning agents. The combination of chromatographic techniques with mass spectrometry (GC-MS(n), LC-MS(n), etc.) plays a key role in the production of specific results in residue analysis. In this review, the past, present and future of mass spectrometry in this area are discussed in the light of the impact of these substances on human health and the reliable production of analytical results, ready for challenge in a court.

Milk and urine excretion of polycyclic aromatic hydrocarbons and their hydroxylated metabolites after a single oral administration in ruminants.

The aim of this study was to establish the transfer of phenanthrene, pyrene, and benzo[a]pyrene and their major hydroxylated metabolites to milk and to urine after a single oral administration (100 mg per animal of each compound) in 4 lactating goats. Detection and identification of the analytes (native compounds, 1-OH pyrene, 3-OH phenanthrene, 3-OH benzo[a]pyrene) were achieved using gas chromatography-mass spectrometry. Benzo[a]pyrene, phenanthrene, and pyrene were rapidly detected in the plasma stream, whereas 1-OH pyrene and 3-OH phenanthrene appeared later in plasma. These data suggest that pyrene and phenanthrene are progressively metabolized within the organism. Recovery rates of pyrene and phenanthrene in milk over a 24-h period appeared to be very low (0.014 and 0.006%, respectively), whereas the transfer rates of their corresponding metabolites were significantly higher: 0.44% for 1-OH pyrene and 0.073% for 3-OH phenanthrene. Recovery rates in urine were found to be higher (1 to 10 times) than recovery rates in milk. The 1-OH pyrene was found to be the main metabolite in urine as well as in milk. Thus, as has been established for humans, 1-OH pyrene could be considered as a marker of ruminant exposure to polycyclic aromatic hydrocarbons. Because 1-OH pyrene and 3-OH phenanthrene were measured in milk (unlike their corresponding native molecules), metabolites of polycyclic aromatic hydrocarbons should be taken into consideration when evaluating the safety of milk. Benzo[a]pyrene and 3-OH benzo[a]pyrene were (less than 0.005%) transferred to milk and urine in very slight amounts. This very limited transfer rate of both compounds suggests a low risk of exposure by humans to benzo[a]pyrene or its major metabolite from milk or milk products.

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 ∆5, ∆4 and the backdoor pathways of the biosynthesis of DHT, though the latter was limited to production of 17α-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 3ßHSD2 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.