Screening persistent organic pollutants for effects on testosterone and estrogen synthesis at human-relevant concentrations using H295R cells in 96-well plates.

Many persistent organic pollutants (POPs) are suspected endocrine disruptors and it is important to investigate their effects at low concentrations relevant to human exposure. Here, the OECD test guideline #456 steroidogenesis assay was downscaled to a 96-well microplate format to screen 24 POPs for their effects on viability, and testosterone and estradiol synthesis using the human adrenocortical cell line H295R. The compounds (six polyfluoroalkyl substances, five organochlorine pesticides, ten polychlorinated biphenyls and three polybrominated diphenyl ethers) were tested at human-relevant levels (1 nM to 10 µM). Increased estradiol synthesis, above the OECD guideline threshold of 1.5-fold solvent control, was shown after exposure to 10 µM PCB-156 (153%) and PCB-180 (196%). Interestingly, the base hormone synthesis varied depending on the cell batch. An alternative data analysis using a linear mixed-effects model that include multiple independent experiments and considers batch-dependent variation was therefore applied. This approach revealed small but statistically significant effects on estradiol or testosterone synthesis for 17 compounds. Increased testosterone levels were demonstrated even at 1 nM for PCB-74 (18%), PCB-99 (29%), PCB-118 (16%), PCB-138 (19%), PCB-180 (22%), and PBDE-153 (21%). The MTT assay revealed significant effects on cell viability after exposure to 1 nM of perfluoroundecanoic acid (12%), 3 nM PBDE-153 (9%), and 10 µM of PCB-156 (6%). This shows that some POPs can interfere with endocrine signaling at concentrations found in human blood, highlighting the need for further investigation into the toxicological mechanisms of POPs and their mixtures at low concentrations relevant to human exposure.

Albumin/Mitotane Interaction Affects Drug Activity in Adrenocortical Carcinoma Cells: Smoke and Mirrors on Mitotane Effect with Possible Implications for Patients' Management.

BACKGROUND: Mitotane is the only drug approved for the treatment of adrenocortical carcinoma (ACC). Although it has been used for many years, its mechanism of action remains elusive. H295R cells are, in ACC, an essential tool to evaluate drug mechanisms, although they often lead to conflicting results. METHODS: Using different in vitro biomolecular technologies and biochemical/biophysical experiments, we evaluated how the presence of "confounding factors" in culture media and patient sera could reduce the pharmacological effect of mitotane and its metabolites. RESULTS: We discovered that albumin, the most abundant protein in the blood, was able to bind mitotane. This interaction altered the effect of the drug by blocking its biological activity. This blocking effect was independent of the albumin source or methodology used and altered the assessment of drug sensitivity of the cell lines. CONCLUSIONS: In conclusion, we have for the first time demonstrated that albumin does not only act as an inert drug carrier when mitotane or its metabolites are present. Indeed, our experiments clearly indicated that both albumin and human serum were able to suppress the pharmacological effect of mitotane in vitro. These experiments could represent a first step towards the individualization of mitotane treatment in this rare tumor.

Removal efficiencies and risk assessment of endocrine-disrupting chemicals at two wastewater treatment plants in South China.

Endocrine-disrupting chemicals (EDCs) in the effluent from wastewater treatment plants (WWTPs) are an important pollutant sources of the aquatic system. In this study, the removal efficiencies of eight typical EDCs at two domestic WWTPs in Dongguan City, China, are reported based on instrumental analysis and bioassay results. Bioactivities, including steroidogenesis-disrupting effects, estrogen receptor (ER)-binding activity, and aryl hydrocarbon receptor (AhR)-binding activity were evaluated using the H295R, MVLN, and H4IIE cell bioassays, respectively. The potential environmental risks of these residual EDCs were also evaluated. The results of instrumental analysis showed that nonylphenol was the major chemical type present among the eight tested EDCs. Meanwhile, concentrations of estrogen compounds including estrone, 17ß-estradiol (E2), estriol, 17α-ethinyl estradiol, and diethylstilbestrol were relatively low. The removal rates of all eight EDCs were relatively high. Although the chemical analysis indicated high removal efficiency, the bioassay results showed that steroidogenesis-disrupting effects as well as ER-binding and AhR-binding activities remained, with E2-equivalent values of effluent samples ranging from 0.16 to 0.9 ng·L-1, and 2,3,7,8-tetrachlorodibenzo-p-dioxin-equivalent values ranging from 0.61 to 4.09 ng L-1. Principal component analysis combined with regression analysis suggests that the chemicals analyzed in this study were partly responsible for these ER and AhR activities. Ecological risk assessment of the residual EDCs showed that estrone was the most hazardous chemical among the eight EDCs tested, with a risk quotient of 1.44-5.50. Overall, this study suggests that, despite high apparent removal efficiencies of typical EDCs, their bioactivities and potential ecological risks cannot be ignored.

The influence of TNF-α on the expression profile of key enzymes of steroidogenesis in H295R cells.

INTRODUCTION: Tumor necrosis factor-α (TNF-α) plays an extremely important role in the regulation of hypothalamicpituitary-adrenal axis. It is believed that chronic inflammation is the main cause of cancerogenesis and TNF-α plays a significant role in both of these processes. Unfortunately, the function of TNF-α in human adrenal steroidogenesis has not been explained enough. AIM: To evaluate the changes in transcriptional activity of STAR, CYP11A1, CYP11B1, and CYP11B2 in H295R cell line exposed to TNF-α. MATERIAL AND METHODS: NCI-H295R, human adrenocortical cell line was exposed to human recombinant TNF-α at the concentrations ranging from 0.001 to 10 nM for 3, 12, 24, and 48 h. Cells not exposed to TNF-α were the control of this experiment. RTqPCR assay was used to determine the changes in the expression of genes encoding STAR, CYP11A1, CYP11B1, and CYP11B2. RESULTS: The highest differences between stimulated and non-stimulated cells were observed in the expression of STAR (FC = +2.2; 0.01 nM of TNF-α; 48 h); CYP11A1 (FC = +3.5; 0.1 nM of TNF-α; 24 h); CYP11B1 (FC = +7.0; 10 nM of TNF-α; 48 h); CYP11B2 (FC = +2.5; 10 nM of TNF-α; 48 h). Statistically significant differences (p < 0.05) in the expression were found only for CYP11A1. The interaction effect between genes was also noticed (p < 0.05). CONCLUSIONS: The research showed the impact of TNF-α on the expression of the key genes encoding enzymes involved in adrenal steroidogenesis. Different expression patterns of was observed, depending on time and TNF-α concentration increased synthesis of this pro-inflammatory cytokine may intensify adrenal steroidogenesis.

Interleukin-6 increases adrenal androgen release by regulating the expression of steroidogenic proteins in NCI-H295R cells.

The purpose of this study was to investigate the potential mechanism of interleukin-6 (IL-6) on the stimulation of excessive androgen secretion in human NCI-H295R adrenocortical cells. We performed transcriptome sequencing of cancer and paracancerous tissues obtained from functional adrenal cortical adenomas. The secretion of dehydroepiandrosterone sulfate (DHEAS) in NCI-H295R cells was detected by a chemiluminescence assay. The expression of messenger RNA (mRNA) was detected by real-time polymerase chain reaction and that of protein was detected by western blotting. The expression of secretogranin II (SCG2) and IL-6 were significantly increased in cancer tissues. Upregulation of mRNA and protein levels of AKR1C3, CYP11A, CYP17A1, 3ßHSD, and SULT2A1 was observed after stimulation with IL-6. IL-6 could also increase the expression of StAR mRNA and proteins. Our results suggest that IL-6 can promote androgen secretion by regulating the expression of genes related to androgen pathways.

Four cypermethrin isomers induced stereoselective metabolism in H295R cells.

Cypermethrin (CP) is widely used for controlling agricultural and indoor vermin. Previous studies have reported the stereoselective difference of CP in biological activities. However, little is known about their potential mechanisms between metabolic phenotypes and endocrine-disrupting effects. Herein, nuclear magnetic resonance (NMR)-based metabolomics combining metabolite identification and pathway analysis were applied to evaluate the stereoselective metabolic cdisorders induced by CP isomers in human adrenocortical carcinoma cells (H295R) culture medium. Then, gene expression levels related to disturbed metabolic pathways were assessed to verify according to metabolic phenotypes. Metabolomics profiles showed that [(S)-cyano(3-phenoxyphenyl)methyl](1R,3R)-3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropane-1-carboxylate [(1R,3R,αS)-CP] induced the most significant changes in metabolic phenotypes than did the other stereoisomers. There are 10 differential metabolites (isoleucine, valine, leucine, ethanol, alanine, acetate, aspartate, arginine, lactate, and glucose) as well as two significantly disturbed pathways, including "pyruvate metabolism" and "alanine, aspartate, and glutamate metabolism," that were confirmed in H295R cells culture medium of (1R,3R,αS)-CP compared with other stereoisomers. Polymerase chain reaction (PCR) array also confirmed the results of metabolomics. Our results can help to understand the potential mechanisms between the isomer selectivity in metabolic phenotypes and endocrine-disrupting effects. Data provided here not only lend authenticity to the cautions issued by the scientists and researchers but also offer a solution for the balance between environment and political regulations.

A Targeted Bioinformatics Assessment of Adrenocortical Carcinoma Reveals Prognostic Implications of GABA System Gene Expression.

Adrenocortical carcinoma (ACC) is a rare but deadly cancer for which few treatments exist. Here, we have undertaken a targeted bioinformatics study of The Cancer Genome Atlas (TCGA) ACC dataset focusing on the 30 genes encoding the γ-aminobutyric acid (GABA) system-an under-studied, evolutionarily-conserved system that is an emerging potential player in cancer progression. Our analysis identified a subset of ACC patients whose tumors expressed a distinct GABA system transcriptome. Transcript levels of ABAT (encoding a key GABA shunt enzyme), were upregulated in over 40% of tumors, and this correlated with several favorable clinical outcomes including patient survival; while enrichment and ontology analysis implicated two cancer-related biological pathways involved in metastasis and immune response. The phenotype associated with ABAT upregulation revealed a potential metabolic heterogeneity among ACC tumors associated with enhanced mitochondrial metabolism. Furthermore, many GABAA receptor subunit-encoding transcripts were expressed, including two (GABRB2 and GABRD) prognostic for patient survival. Transcripts encoding GABAB receptor subunits and GABA transporters were also ubiquitously expressed. The GABA system transcriptome of ACC tumors is largely mirrored in the ACC NCI-H295R cell line, suggesting that this cell line may be appropriate for future functional studies investigating the role of the GABA system in ACC cell growth phenotypes and metabolism.

Copper affects steroidogenesis and viability of human adrenocortical carcinoma (NCI-H295R) cell line in vitro.

Copper is an environmental risk factor, which has various effects on reproductive endocrinology. In this study human adrenocortical carcinoma (NCI-H295R) cell line was used as an in vitro biological model to study the effect of copper sulfate (CuSO4.5H2O) on steroidogenesis and cytotoxicity. The cell cultures were exposed to different concentrations (3.90, 62.50, 250, 500, 1000 µM) of CuSO4.5H2O and compared to control group (medium without CuSO4.5H2O). Cell viability was measured by the metabolic activity assay. Quantification of sexual steroid production directly from the medium was performed by ELISA assay. Following 48 h culture of NCI-H295R cell line in the presence of CuSO4.5H2O a dose-dependent depletion of progesterone release was observed even at the lower concentrations of CuSO4.5H2O. The lowest levels of progesterone were detected in groups with the higher doses (≥ 250 µM) of CuSO4.5H2O, which elicited significant cytotoxic action. Testosterone production decreased significantly, and this decline was more prominent in comparison to that of progesterone. The lowest release of testosterone was recorded at 1000 µM of CuSO4.5H2O. The cytotoxic effect of CuSO4.5H2O was evident at all concentrations used in the study. The presented data suggest that copper has detrimental effects on sexual steroid hormones and consecutively on reproductive physiology.

In vitro assessment of corticosteroid effects of eight chiral herbicides.

Information regarding the enantioselective endocrine disruption of chiral herbicides is scarce. This study assessed the disrupting effects of eight typical chiral herbicides on corticosteroids (including glucocorticoids and mineralocorticoids). Enantioselectivity of eight chiral herbicides were evaluated for their agonistic/antagonistic effects on glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) with CHOK1 cell line using reporter gene assay. Their influence on the production of corticosteroids were further investigated in H295R cell line using enzyme-linked immunosorbent assay (ELISA). None of the racemates or enantiomers of eight chiral herbicides exhibited GR or MR agonistic activity at non-cytotoxic concentrations. However, rac-propisochlor and S-imazamox antagonized cortisol-induced transactivation of GR by 21.79% and 38.73% at the concentration of 1.0 × 10-7 M and 1.0 × 10-6 M, respectively, and R-napropamide remarkably attenuated aldosterone-induced MR transactivation by 68.78% at 1.0 × 10-6 M. The secretion of cortisol was significantly restrained after treated with 1.0 × 10-6 M rac-propisochlor and rac-/R-napropamide at the concentration of 1.0 × 10-6 M by 26.49%, 30.10% and 35.27%, respectively, while this glucocorticoid was remarkably induced by 1.0 × 10-5 M rac-diclofop-methyl and its two enantiomers at the concentration of 1.0 × 10-5 M by 75.60%, 100.1% and 68.78%, respectively. Exposure to rac-propisochlor (1.0 × 10-6 M), S-diclofop-methyl (1.0 × 10-5 M) or rac-/S-/R- acetochlor (1.0 × 10-6 M) and rac-/S-/R-lactofen (1.0 × 10-6 M) inhibited the secretion of aldosterone by approximately 40%. Our findings suggested that chiral herbicides disrupted corticosteroid homeostasis in an enantioselective way. Therefore, more comprehensive screening is required to better understand the ecological and health risks of chiral pesticides.

5-oxo-ETE activates migration of H295R adrenocortical cells via MAPK and PKC pathways.

The OXE receptor is a GPCR activated by eicosanoids produced by the action of 5-lipoxygenase. We previously found that this membrane receptor participates in the regulation of cAMP-dependent and -independent steroidogenesis in human H295R adrenocortical carcinoma cells. In this study we analyzed the effects of the OXE receptor physiological activator 5-oxo-ETE on the growth and migration of H259R cells. While 5-oxo-ETE did not affect the growth of H295R cells, overexpression of OXE receptor caused an increase in cell proliferation, which was further increased by 5-oxo-ETE and blocked by 5-lipoxygenase inhibition. 5-oxo-ETE increased the migratory capacity of H295R cells in wound healing assays, but it did not induce the production of metalloproteases MMP-1, MMP-2, MMP-9 and MMP-10. The pro-migratory effect of 5-oxo-ETE was reduced by pharmacological inhibition of the MEK/ERK1/2, p38 and PKC pathways. 5-oxo-ETE caused significant activation of ERK and p38. ERK activation by the eicosanoid was reduced by the "pan" PKC inhibitor GF109203X but not by the classical PKC inhibitor Gö6976, suggesting the involvement of novel PKCs in this effect. Although H295R cells display detectable phosphorylation of Ser299 in PKCδ, a readout for the activation of this novel PKC, treatment with 5-oxo-ETE per se was unable to induce additional PKCδ activation. Our results revealed signaling effectors activated by 5-oxo-ETE in H295R cells and may have significant implications for our understanding of OXE receptor in adrenocortical cell pathophysiology.

Toxic effects of triptolide on adrenal steroidogenesis in H295R cells and female rats.

Triptolide (TP), a major active ingredient of Tripterygium wilfordii, exerts potent immunosuppressive effects in the treatment of rheumatoid arthritis but is not widely used in clinical practice due to its multiorgan toxicity, particularly hepatotoxicity, nephrotoxicity, and reproductive toxicity. An LC-MS/MS approach was employed to explore the endocrine-disrupting effects of TP. The endocrine-disrupting effects of various concentrations (0-100 nM) of TP for 48 hour were firstly investigated using an in vitro model (H295R cell line). It was found that TP did not decrease cell viability. The transcriptional levels of steroidogenic enzymes in H295R cells were assessed by quantificational real-time polymerase chain reaction. The possible adrenal and endocrine effects of oral administration of TP (0, 50, and 500 µg/kg) for 28 days on both normal and collagen-induced arthritis (CIA) rats were also explored. The serum and adrenal tissue hormone levels (corticosterone and progesterone) and adrenal histopathology were analyzed, with the results that TP significantly decreased the level of cortisol in H295R cells and the level of plasma corticosterone in both normal and CIA rats. Histological alterations in adrenal cortex were observed at the dose of 500 µg/kg. Exposure to TP for 48 hour had an obvious inhibitory effect on the messenger RNA transcript levels of HSD3B2, CYP21A2, CYP17A1, and CYP11B1, which is essential for the synthesis of corticosteroids. In a word, TP leads to the disorder of corticosteroid synthesis and secretion, and corticosteroid may be a potential biomarker for the treatment of multiorgan toxicity of TP.

Comparative analysis of endocrine disrupting effects of major phthalates in employed two cell lines (MVLN and H295R) and embryonic zebrafish assay.

Endocrine disruption potentials of phthalates have been widely recognized, but relatively little is known about relative potency of major phthalates. In the present study, six phthalates were chosen, i.e., dimethyl phthalate (DMP), diethyl phthalate (DEP), di(2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DnOP), diisononyl phthalate (DINP) and diisodecyl phthalate (DIDP), and their endocrine disruption effects were evaluated by employing two cell lines and an embryonic zebrafish assay. Binding affinity with estrogen receptors (ERs) and effects on steroidogenesis were evaluated with MVLN and H295R cell assays, respectively. In zebrafish embryos, transcription of genes regulating steroid hormone balance and estrogen receptors was measured. Exposure to DMP, DEP, DEHP, and DnOP significantly increased E2/T ratio in H295R cells. However, no significant binding affinity to ERs was observed in MLVN cells. Exposure to DEHP influenced the expression of vtg1, esr1, and cyp19a1b genes in zebrafish larvae. DMP, DEP, DINP, and DIDP exposure led to significant transcriptional changes even at lower exposure concentrations, suggesting their greater endocrine disruption potency than DEHP in zebrafish. Our findings demonstrate that endocrine disruption upon phthalate exposure varies between in vitro and in vivo assay, and a battery of tests are warranted to understand endocrine disruption potentials of phthalates. Consequences of long-term exposure to phthalates other than DEHP warrant further evaluations.

In vitro and in silico hormonal activity studies of di-(2-ethylhexyl)terephthalate, a di-(2-ethylhexyl)phthalate substitute used in medical devices, and its metabolites.

Plasticizers added to polyvinylchloride used in medical devices can be released into patients' biological fluids. The substitution of di-(2-ethylhexyl)phthalate (DEHP) by alternative plasticizers is essential but their safety must be demonstrated. DEHP, di-(2-ethylhexyl)terephthalate (DEHT) and their metabolites were investigated using level 2 Organization for Economic Co-operation and Development bioassays to screen for in vitro hormonal changes. Differences between the DEHP and DEHT metabolites were observed. Albeit weak, the hormonal activities of DEHT-derived metabolites, e.g., 5-OH metabolite of mono-(ethylhexyl)terephthalate (5-OH-MEHT), were detected and the results of docking experiments performed on estrogen receptor alpha and androgen receptor agreed with the biological results. A co-stimulation of human estrogen receptor alpha and human androgen receptor was also observed. With regard to steroidogenesis, a 16-fold increase in estrogen synthesis was measured with 5-OH-MEHT. Therefore, even if DEHT remains an interesting alternative to DEHP because of its low migration from medical devices, it seems important to verify that multi-exposed patients in neonatal intensive care units do not have urinary levels of oxidized metabolites, in particular 5-OH-MEHT, suggesting a potential endocrine-disrupting effect.

Estrogenic effects of fluorinated diiodine alkanes in MCF-7 cells, H295R cells and zebrafish embryo assays.

Fluorinated diiodine alkanes (FDIAs), important industrial intermediates in the synthesis of various perfluorinated compounds, which are distributed widely in wildlife and humans. Recent studies showed that FDIAs had in vitro estrogenic effects. However, to date, little information is available regarding the in vivo estrogenic effects of FDIAs and the mechanisms are unclear. In this study, a combination of in vitro and in vivo assays was used to investigate the estrogenic effects of FDIAs. We tested the in vitro estrogenic effects and estrogen receptor-related gene expression via MCF-7 cell assay. The hormone level of estradiol and the expression of estrogenic synthesis genes were measured in the H295R cell assay. Finally, the in vivo effects of FDIAs on development and estrogen-related gene expression were assessed in the zebrafish embryos assay. The results demonstrated that FDIAs could exhibit estrogenic activity through inducing cell proliferation (1.6-6.7-fold of the control) and estrogen receptor alpha gene expression (1.07-1.39-fold of the control), altering estradiol production (1.14-1.22-fold of the control) and the major estrogenic synthesis gene expression of CYP19 (1.22-1.31-fold of the control), disrupting the estrogen-related genes (esr1 and cyp19b) levels in zebrafish (1.52-2.99-fold and 2.95-5.00-fold of the control for esr1 and cyp19b, respectively). The current findings indicated the potential estrogenic effects of FDIAs and provided novel information for human risk assessment.

Organotin exposure stimulates steroidogenesis in H295R Cell via cAMP pathway.

Organotin compounds (OTs) are used in a range of industrial products, such as antifouling paints, agricultural pesticides and stabilizers. Owing to potential endocrine-disrupting effects, human exposure to such compounds is a concern. Nevertheless, little is known about the adverse effect of OTs on adrenocortical function in organisms. In this study, the human adrenocortical carcinoma cell (H295R) model was used to investigate effects of OTs on steroidogenesis and potential causes for such endocrine disruption was examined. H295R cells were exposed to several commonly used OTs, including triphenyltin (TPT), tributyltin (TBT), dibutyltin (DBT), and monobutyltin (MBT), and the production level of steroid hormones were quantified. TPT and TBT decreased the production levels of 17ß-estradiol, aldosterone, and cortisol, but increased that of testosterone. Furthermore, the expression levels of ten major steroidogenic genes (HMGR, StAR, CYP11A1, 3ßHSD2, CYP17, CYP19A1, CYP21, CYP11B1, CYP11B2, and 17ßHSD) were examined and both up-regulation of CYP11B2 and down-regulation of StAR, 3ßHSD2, CYP19A1, CYP21 and CYP11B1 by TPT and TBT were observed. Intracellular levels of ATP and cyclic adenosine monophosphate (cAMP) and the activity of adenylate cyclase (AC) decreased in the H295R cells treated with TPT and TBT. No obvious changes in H295R were found with the treatment of DBT and MBT. These results suggest that OTs may stimulate steroidogenesis in vitro via inhibition of cAMP signaling pathway.

Endogenous Purification of NR4A2 (Nurr1) Identified Poly(ADP-Ribose) Polymerase 1 as a Prime Coregulator in Human Adrenocortical H295R Cells.

Aldosterone is synthesized in zona glomerulosa of adrenal cortex in response to angiotensin II. This stimulation transcriptionally induces expression of a series of steroidogenic genes such as HSD3B and CYP11B2 via NR4A (nuclear receptor subfamily 4 group A) nuclear receptors and ATF (activating transcription factor) family transcription factors. Nurr1 belongs to the NR4A family and is regarded as an orphan nuclear receptor. The physiological significance of Nurr1 in aldosterone production in adrenal cortex has been well studied. However, coregulators supporting the Nurr1 function still remain elusive. In this study, we performed RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins), a recently developed endogenous coregulator purification method, in human adrenocortical H295R cells and identified PARP1 as one of the top Nurr1-interacting proteins. Nurr1-PARP1 interaction was verified by co-immunoprecipitation. In addition, both siRNA knockdown of PARP1 and treatment of AG14361, a specific PARP1 inhibitor suppressed the angiotensin II-mediated target gene induction in H295R cells. Furthermore, PARP1 inhibitor also suppressed the aldosterone secretion in response to the angiotensin II. Together, these results suggest PARP1 is a prime coregulator for Nurr1.

Effects of 4-nonylphenol on the steroidogenesis of human adrenocarcinoma cell line (NCI-H295R).

In this study, the human H295R adrenocarcinoma cell line was exposed to different concentrations (0.04, 0.2, 1.0, 2.5 or 5 µg/mL) of nonylphenol (NP) to investigate its impact on the inhibition or induction of the steroid hormones production during 48 h of in vitro culture. The hormone production was measured using ELISA kits. Results of this in vitro study suggest various effect of nonylphenol in relatively low concentrations on the selected steroid hormones production by the human H295R adrenocarcinoma cell line. The inhibiting impact on progesterone and androstenedione production was observed. The amount of progesterone was significantly decreased at 1.0, 2.5 and 5 µg/mL NP. Equally, the androstenedione production significantly decreased at 5 µg/mL NP. On the other hand, the amount of testosterone and 17ß-estradiol was induced after nonylphenol exposition. The significant increase of testosterone level was found out at treatment with 5 µg/mL NP. 17ß-estradiol production significantly increased at the doses of 2.5 and 5 µg/mL NP.

Two simple cleanup methods combined with LC-MS/MS for quantification of steroid hormones in in vivo and in vitro assays.

Measuring both progestagens, androgens, corticosteroids as well as estrogens with a single method makes it possible to investigate the effects of endocrine-disrupting chemicals (EDCs) on the main pathways in the mammalian steroidogenesis. This paper presents two simple methods for the determination of the major steroid hormones in biological matrixes using liquid chromatography tandem mass spectrometry (LC-MS(2)). A novel method was developed for the determination of 14 steroids in the H295R in vitro assay without the need for solid phase extraction (SPE) purification prior to LC-MS(2) analysis. The in vitro assay was validated by exposing H295R cells to prochloraz for inhibiting steroid hormone secretion and by exposing cells to forskolin for inducing steroid hormone secretion. The developed method fulfills the recommendations for the H295R assay suggested by the OECD. Furthermore, a simple off-line SPE methodology was developed for the necessary clean-up of in vivo assays. Samples, such as gonad tissue, plasma and serum, are complex biological matrixes, and the SPE methodology was optimized to remove salts and proteins prior to elution of target analytes. At the same time, lipophilic compounds were retained on the SPE cartridge during elution. This, combined with the multi-steroid LC-MS(2) method, made it possible to determine 10 steroids in male Sprague-Dawley rat gonad tissue. Furthermore, it was possible to quantify 6 steroids in the plasma. In general, the observed concentration of steroid hormones in plasma, testes, and H295R cell medium corresponded well with previous studies. The off-line SPE method was validated using spiked charcoal-stripped serum. Method recovery, accuracy, precision and robustness were all good. Instrument sensitivity was in the range of 55-530 pg/mL (LLOQ).

Endocrine activity of alternatives to BPA found in thermal paper in Switzerland.

Alternatives to bisphenol A (BPA) are more and more used in thermal paper receipts. To get an overview of the situation in Switzerland, 124 thermal paper receipts were collected and analyzed. Whereas BPA was detected in most samples (n=100), some alternatives, namely bisphenol S (BPS), Pergafast® 201 and D-8 have been found in 4, 11 and 9 samples respectively. As no or few data on their endocrine activity are available, these chemicals and bisphenol F (BPF) were tested in vitro using the H295R steroidogenesis assay. 17ß-Estradiol production was induced by BPA and BPF, whereas free testosterone production was inhibited by BPA and BPS. Both non-bisphenol substances did not show significant effects. The binding affinity to 16 proteins and the toxicological potential (TP) were further calculated in silico using VirtualToxLab™. TP values lay between 0.269 and 0.476 and the main target was the estrogen receptor ß (84.4 nM to 1.33 µM). A substitution of BPA by BPF and BPS should be thus considered with caution, since they exhibit almost a similar endocrine activity as BPA. D-8 and Pergafast® 201 could be alternatives to replace BPA, however further analyses are needed to better characterize their effects on the hormonal system.

Endocrine disruptive effects of cadmium on steroidogenesis: human adrenocortical carcinoma cell line NCI-H295R as a cellular model for reproductive toxicity testing.

Cadmium (Cd) is a known endocrine disruptor with the ability to affect the production of hormones involved in the regulation of reproductive processes. In this study human adrenocortical carcinoma cell line NCI-H295R was used as an in vitro biological model to study the effect of cadmium (CdCl2) on steroidogenesis. The cell cultures were exposed to different concentrations of CdCl2 (1.90, 3.90, 7.80, 15.60, 31.20 and 62.50 µM) and compared to control (medium without CdCl2). Cell viability was measured by the metabolic activity (MTT) assay for estimation of mitochondria structural integrity. Quantification of sexual steroid production directly from aliquots of the medium was performed by enzyme linked immunosorbent assay (ELISA). Following 48 h culture of the cells in the presence of CdCl2 a concentration-dependent depletion in progesterone production was observed at the lower concentrations of CdCl2. The lowest amount of progesterone was significantly detected in groups with the higher doses (≥ 31.20 µM) of CdCl2, which elicited significant (P < 0.01) cytotoxic action, too. Cadmium decreased testosterone release in the whole applied range even at the lower concentration of CdCl2. The release of 17ß-estradiol decreased as well, but the decline was less pronounced compared to decrease of progesterone and testosterone. The cytotoxic effect was significantly (P < 0.01) detected at all concentrations of CdCl2 (1.90-62.50 µM) used in the study. However, the cell viability remained relatively high (>75%) up to 7.80 µM of CdCl2 and significantly (P < 0.01) decreased at 15.60 µM and higher concentrations of CdCl2. These results suggest that cadmium has endocrine disruptive effects on sexual steroid synthesis even at very low concentrations.