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.

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.

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.

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.

Development and validation of a specific and sensitive gas chromatography tandem mass spectrometry method for the determination of bisphenol A residues in a large set of food items.

BPA-containing products are widely used in foodstuffs packaging as authorized within the European Union (UE no. 10/2011). Therefore, foods and beverages are in contact with BPA which can migrate from food contact material to foodstuffs. An accurate assessment of the exposure of the consumers to BPA is crucial for a non-ambiguous risk characterization. In this context, an efficient analytical method using gas chromatography coupled to tandem mass spectrometry (GC-MS/MS), in the selected reaction monitoring (SRM) mode, was developed for the quantification of BPA in foodstuffs at very low levels (<0.5µgkg(-1)). A standard operating procedure, based on the combination of two successive solid phase extractions (SPE), was developed for various liquid and solid foodstuffs. The use of (13)C12-BPA as internal standard allowed accurate quantification of BPA by isotopic dilution. Control charts based on both blank and certified materials have been implemented to ensure analytical data quality. The developed analytical method has been validated according to in-house validation requirements. R(2) was better than 0.9990 within the range [0-100µgkg(-1)], the trueness was 4.2%. Repeatability and within-laboratory reproducibility ranged from 7.5% to 19.0% and 2.5% to 12.2%, respectively, at 0.5 and 5.0µgkg(-1) depending on the matrices tested for. The detection and quantification limits were 0.03 and 0.10µgkg(-1), respectively. The reporting limit was 0.35µgkg(-1), taking into account the mean of the laboratory background contamination. The global uncertainty was 22.2% at 95% confidence interval.

Determination of residues of oxolinic acid and flumequine in freeze-dried salmon muscle and skin by HPLC with fluorescence detection.

A procedure for the determination of residues of oxolinic acid (OA) and flumequine (FLU) in freeze-dried salmon muscle with attached skin, using reversed-phase high-performance liquid chromatography, is described. OA and FLU were extracted by a solid-liquid extraction procedure: after addition of hydrochloric acid, extraction used successively ethyl acetate, sodium hydroxide and chloroform. Liquid chromatography was performed on a 5 microm PuroSpher RP-18E cartridge using acetonitrile and 0.02 M aqueous orthophosphoric acid solution as mobile phase, with fluorescence detection. The performance of the method was established by spiking tissues with OA and FLU before the freeze-drying step. The method was linear over the concentration range 50-2000 ng/g freeze-dried tissue. Limits of detection and quantitation were 3.2 and 16 ng/g wet weight tissue respectively both for OA and FLU. Mean extraction recoveries of OA and FLU from freeze-dried tissue were 85.5 and 85.2% respectively. The method is suitable as a regulatory one for determination of residues of OA and FLU in freeze-dried salmon tissue.

Determination of warfarin in the yolk and the white of hens' eggs by reversed-phase high-performance liquid chromatography.

A procedure for the determination of warfarin, an anticoagulant rodenticide, in the white and the yolk of hens' eggs, using reversed-phase high-performance liquid chromatography is described. Liquid chromatography was performed on an octadecylsilane cartridge using methanol and ammonium acetate triethylamine buffer as the mobile phase, with UV detection at 281 nm. Samples (5 g) were analysed after liquid-phase extraction using a mixture of acetone and diethyl ether. Linearity, precision and accuracy of the method were determined in the range of 0.5-8.0 microg. Limits of quantitation for warfarin in the white and the yolk were 0.020 and 0.015 microg/g, respectively. Mean recoveries of warfarin from spiked white and yolk samples were 84.6 and 87.4%, respectively. The analytical method was applied to a fourteen-day experimental study conducted in laying hens that had been orally dosed with warfarin.