Early Biological Modulations Resulting from 1-Week Venlafaxine Exposure of Marine Mussels Mytilus galloprovincialis Determined by a Metabolomic Approach.

There is growing evidence of the presence of pharmaceuticals in natural waters and their accumulation in aquatic organisms. While their mode of action on non-target organisms is still not clearly understood, their effects warrant assessment. The present study assessed the metabolome of the Mediterranean mussel (Mytilus galloprovincialis) exposed to a 10 µg/L nominal concentration of the antidepressant venlafaxine (VLF) at 3 time-points (1, 3, and 7 days). Over the exposure period, we observed up- or down-modulations of 113 metabolites, belonging to several metabolisms, e.g., amino acids (phenylalanine, tyrosine, tryptophan, etc.), purine and pyrimidine metabolisms (adenosine, cyclic AMP, thymidine, etc.), and several other metabolites involved in diverse functions. Serotonin showed the same time-course modulation pattern in both male and female mussels, which was consistent with its mode of action in humans, i.e., after a slight decrease on the first day of exposure, its levels increased at day 7 in exposed mussels. We found that the modulation pattern of impacted metabolites was not constant over time and it was gender-specific, as male and female mussels responded differently to VLF exposure.

Metabolomics approach reveals disruption of metabolic pathways in the marine bivalve Mytilus galloprovincialis exposed to a WWTP effluent extract.

Conventional wastewater treatment plants (WWTPs) discharge a highly diverse range of organic contaminants in aquatic environments, including marine waters. The health of marine ecosystems could be threatened by contaminants release. Environmental metabolomics can be helpful to assess the effects of multi-contamination on marine organisms without any a priori information since it is able to provide meaningful information on the biochemical response of organisms to a stress. The aim of the present study was to evaluate the potential of metabolomics to highlight key metabolites disrupted by a WWTP effluent extract exposure and then elucidate the biological effects of such exposure on Mediterranean mussels (Mytilus galloprovincialis). Exposed male mussels showed numerous metabolites altered in response to WWTP effluent exposure. The highlighted metabolites belong mainly to amino acids metabolism (e.g. tyrosine, phenylalanine, leucine, proline, etc.), neurohormones (dopamine and a serotonin metabolite), purine and pyrimidine metabolism (e.g. adenosine, adenine, guanine, uracil etc.), citric acid cycle intermediates (e.g. malate, fumarate), and a component involved in oxidative stress defense (oxidized glutathione). Modulation of these metabolites could reflect the alteration of several biological processes such as energy metabolism, DNA and RNA synthesis, immune system, osmoregulation, byssus formation and reproduction, which may lead to a negative impact of organism fitness. Our study provided further insight into the effects of WWTP effluents on marine organisms.

Urinary excretion of sex steroid hormone metabolites after consumption of cow milk: a randomized crossover intervention trial.

Background: Current cow milk production practices introduce considerable levels of pregnancy hormones into the milk. Humans are exposed to these hormones when cow milk is consumed, and this may explain the observed association between cow milk consumption and several hormone-sensitive cancers. Objectives: The aim of the study was to evaluate whether cow milk consumption is associated with an increase in urinary excretion of sex steroid hormones and their metabolites in humans. Methods: We conducted a randomized crossover intervention feeding experiment. A total of 109 postmenopausal women consumed 1 L of semiskimmed milk (1.5% fat) per day for 4 d and 1 L of whole milk (3.5% fat) per day for 4 d, intersected by 4-d wash-out periods. Sex steroid hormone levels were measured in 24-h urine samples collected at the end of each intervention and wash-out period. Results: Estrogens, androgens, and progesterone were detected in the examined milk samples used for our intervention. Although a very high proportion of the estrogens were conjugated, only small proportions of the androgens and progesterone were conjugated. Milk consumption resulted in a significant increase in urinary estrone (E1) excretion, whereas estradiol (E2), estriol (E3), and 16ketoE2 excretion only increased after semiskimmed milk consumption. Urinary pregnanediol glucuronide excretion was not significantly affected. Conclusion: Cow milk consumption increases urinary excretion of E1 in humans. Ingestion of semiskimmed milk appears also to raise E2, E3, and 16ketoE2 excretion, but future studies need to confirm these associations. This trial was registered at https://www.drks.de as DRKS00003377.

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.

In utero exposure to cigarette smoke dysregulates human fetal ovarian developmental signalling.

STUDY QUESTION: How does maternal cigarette smoking disturb development of the human fetal ovary? SUMMARY ANSWER: Maternal smoking increases fetal estrogen titres and dysregulates several developmental processes in the fetal ovary. WHAT IS KNOWN ALREADY: Exposure to maternal cigarette smoking during gestation reduces human fetal ovarian cell numbers, germ cell proliferation and subsequent adult fecundity. STUDY DESIGN, SIZE, DURATION: The effects of maternal cigarette smoking on the second trimester human fetal ovary, fetal endocrine signalling and fetal chemical burden were studied. A total of 105 fetuses were studied, 56 from mothers who smoked during pregnancy and 49 from those who did not. PARTICIPANTS/MATERIALS, SETTING METHODS: Ovary, liver and plasma samples were collected from electively terminated, normally progressing, second trimester human fetuses. Circulating fetal hormones, levels of 73 fetal ovarian transcripts, protein localization, density of oocytes/primordial follicles and levels of 16 polycyclic aromatic hydrocarbons (PAHs) in the fetal liver were determined. MAIN RESULTS AND THE ROLE OF CHANCE: Circulating fetal estrogen levels were very high and were increased by maternal smoking (ANOVA, P = 0.055-0.004 versus control). Smoke exposure also dysregulated (two-way ANOVA, smoking versus gestation weeks interaction, P = 0.046-0.023) four fetal ovarian genes (cytochrome P450 scc [CYP11A1], NOBOX oogenesis homeobox [NOBOX], activator of apoptosis harakiri [HRK], nuclear receptor subfamily 2, group E, member 1 [NR2E1]), shifted the ovarian Inhibin ßA/inhibin α ratio (NHBA/INHA) transcript ratio in favour of activin (ANOVA, P = 0.049 versus control) and reduced the proportion of dominant-negative estrogen receptor 2 (ERß: ESR2) isoforms in half the exposed fetuses. PAHs, ligands for the aryl hydrocarbon receptor (AHR), were increased nearly 6-fold by maternal smoking (ANOVA, P = 0.011 versus control). A fifth transcript, COUP transcription factor 1 (nuclear receptor subfamily 2, group F, member 1: NR2F1, which contains multiple AHR-binding sites), was both significantly increased (ANOVA, P = 0.026 versus control) and dysregulated by (two-way ANOVA, smoking versus gestation weeks interaction, P = 0.021) maternal smoking. NR2F1 is associated with repression of FSHR expression and smoke-exposed ovaries failed to show the normal increase in FSHR expression during the second trimester. There was a significantly higher number of DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4) VASA-positive (ANOVA, P = 0.016 versus control), but not POU domain, class 1, transcription factor 1 (POU5F1) OCT3/4-positive, oocytes in smoke-exposed fetuses and this matched with a significantly higher number of primordial follicles (ANOVA, P = 0.024 versus control). LIMITATIONS, REASONS FOR CAUTION: The effects of maternal smoking on establishment of the maximum fetal primordial follicle pool cannot be reliably studied in our population since the process is not completed until 28 weeks of gestation and normal fetuses older than 21 weeks of gestation are not available for study. Our data suggest that some fetal ovaries are affected by smoke exposure while others are not, indicating that additional studies, with larger numbers, may show more significant effects. WIDER IMPLICATIONS OF THE FINDINGS: Fetal exposure to chemicals in cigarette smoke is known to lead to reduced fecundity in women. Our study suggests, for the first time, that this occurs via mechanisms involving activation of AHR, disruption of inhibin/activin and estrogen signalling, increased exposure to estrogen and dysregulation of multiple molecular pathways in the exposed human fetal ovary. Our data also suggest that alterations in the ESR2 positive and dominant negative isoforms may be associated with reduced sensitivity of some fetuses to increased estrogens and maternal smoking. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by grants from the Chief Scientist Office (Scottish Executive, CZG/1/109, and CZG/4/742), NHS Grampian Endowments (08/02), the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 212885, a Society for Reproduction & Fertility summer studentship, Medical Research Scotland (research grant 354 FRG) and the Medical Research Council (WBS: U.1276.00.002.00001 and G1100357). The authors declare they have no competing interests, be it financial, personal or professional.

Identification and quantification of 5α-dihydrotestosterone in the teleost fathead minnow (Pimephales promelas) by gas chromatography-tandem mass spectrometry.

The steroid hormone 5α-dihydrotestosterone (DHT) is one of the most physiologically important androgens in male vertebrates, with the exception of teleost fish, in which it is generally assumed that DHT does not play any major physiological role. However, this assumption is challenged by the fact that all the components involved in DHT biosynthesis and action are present and evolutionary conserved in teleost fish. In fact, testosterone (T) is converted into DHT by two isoforms of the enzyme steroid-5-alpha-reductase (5αR), and both 5αRs gene expression and enzymatic activity have been detected in several tissues of different teleost species, which also have an androgen receptor with high binding affinity to DHT. This body of evidence strongly suggest that DHT is synthesised by teleost fish. We investigated this hypothesis using the cyprinid fathead minnow (Pimephales promelas) as the experimental model. The study of the evolutionary and functional conservation of 5αRs in teleost fish was used to support the experimental approach, based on an ultrasensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) method to identify and measure simultaneously T and DHT in fathead minnow biological fluids and tissues. The analyses were performed using plasma samples collected from both male and female adult fish and samples of testicular tissue collected from sexually mature males. Both T and DHT were identified and quantified in all the samples analysed, and in particular, the high concentrations of DHT quantified in the testes suggested that these organs are a likely site of synthesis of DHT in the teleost fathead minnow, as they are in mammals. These results may represent the basis for future studies aimed at elucidating the physiological role, if any, of DHT in teleost fish.

Effects of mono-(2-ethylhexyl) phthalate (MEHP) on chicken germ cells cultured in vitro.

In recent decades, many toxicological tests based on in vivo or in vitro models, mainly from mammalian (rat-mouse) and fish species, were used to assess the risks raised by contact or ingestion of molecules of pharmaceutical, agricultural, or natural origin. But no, or few, in vitro tests using other non-mammalian models such as bird have been explored despite their advantages: the embryonic gonads of birds have a high plasticity of development sensitive to estrogen, and sperm production is nearly two times faster than in rodents. Hence, we have established an in vitro culture of germ cells and somatic cells from chicken post-natal testis, and we have evaluated the sensitivity against the endocrine disruptor compound mono-(2-ethylhexyl) phthalate (MEHP) in comparison to previous studies using rodent and human models. After 96 h of exposure in presence of 10 µM MEHP, chicken seminiferous tubules cultures present a structural alteration, a reduction in cell proliferation and in germ cells population. Apoptosis of germ and somatic cells increases in presence of 1 µM MEHP. Furthermore, MEHP does not affect inhibin B and lactate production by Sertoli cells. These results are in accordance with previous studies using rat, mice, or human culture of testicular cells and in similar range of exposures or even better sensitivity for some "end-points" (biological parameters). In conclusion, the establishment of this postnatal testicular cells culture could be considered as an alternative method to in vivo experiments frequently used for evaluating the impact on the terrestrial wildlife species. This method could be also complementary to mammal model due to the limiting number of animals used and its elevated sensitivity.

Assessment of circulating sex steroid levels in prepubertal and pubertal boys and girls by a novel ultrasensitive gas chromatography-tandem mass spectrometry method.

CONTEXT: Estrogens and androgens play key roles for pubertal onset and sexual maturation. Most currently used immunoassays are not sensitive enough to accurately measure the low circulating levels of sex steroids in children without any signs of puberty. However, this does not exclude that sex steroids have important biological roles in prepubertal children. OBJECTIVES: To accurately determine levels of sex steroid hormones and their metabolites in serum of healthy children before any physical signs of puberty and to evaluate possible sex differences. MAIN OUTCOME MEASURES: Total (unconjugated plus conjugated) serum levels of 17beta-testosterone, 17alpha-testosterone, 5alpha-dihydrotestosterone, 5beta-dihydrotestosterone, androsterone, etiocholanolone, estradiol, and estrone measured by an ultrasensitive method based on gas chromatography-tandem mass spectrometry in samples from 81 healthy schoolchildren (42 boys) without any signs of puberty. For comparison, 48 pubertal children were studied. RESULTS: 17beta-Estradiol levels in prepubertal boys were undetectable or extremely low (median < 3.7 pmol/liter), whereas levels in prepubertal girls were significantly higher (median 9.6 pmol/liter, P < 0.001). Among the older prepubertal children (>8 yr), girls had significantly higher androsterone (4.07 vs. 1.45 nmol/liter, P < 0.05), etiocholanolone (5.45 vs. 1.95 nmol/liter, P < 0.0001), 5alpha-dihydrotestosterone (0.11 vs. <0.10 nmol/liter, P < 0.01), and 17beta-testosterone concentrations (0.69 vs. 0.47 nmol/liter, P < 0.05) compared with similarly aged prepubertal boys. CONCLUSION: Using an accurate and sensitive method, we found significantly higher levels of estrogens as well as androgen metabolites in prepubertal girls compared with age-matched boys. The higher prepubertal sex steroid levels in girls may contribute to their earlier onset of puberty including pubic hair development.

Exposure assessment of prepubertal children to steroid endocrine disruptors. 2. Determination of steroid hormones in milk, egg, and meat samples.

In the present study, the occurrence of the main sex steroid hormones in milk, egg, and meat was evaluated on the basis of a highly specific gas chromatography-tandem mass spectrometry measurement method. Globally, the results indicated that targeted estrogens and androgens occurred at similar levels (concentration levels in the 10-100 ng kg (-1) range) in the analyzed muscle and milk samples. The same compounds occurred at about 10-fold higher concentrations (i.e., in the 100-1000 ng kg (-1) range) in eggs and kidney samples. More precisely, egg and milk appeared as a non-negligible sources of estradiol (i.e., 2.2 +/- 0.8 and 3.1 +/- 2.0 ng day (-1), respectively), whereas testosterone exposure is caused by ingestion of meat and/or egg (i.e., 12.2 +/- 48.2 and 5.2 +/- 2.3 ng day (-1), respectively). The provided exposure data will be further exploited in the scope of a risk assessment study regarding endocrine disruption associated with these molecules.

Exposure assessment of prepubertal children to steroid endocrine disrupters 1. Analytical strategy for estrogens measurement in plasma at ultra-trace level.

Global concern has been raised in recent years over adverse effects that may result from exposure to chemicals that may interfere with the endocrine system. A specific question is related to low-dose effects and long-term exposure consequences, especially for critical populations (foetus, new born, prepubertal children). In this context, we decided to focus our attention on steroid hormones as they are the most potent endocrine disrupters. Our general goal is to investigate whether the steroid intake through food may represent a risk for prepubertal children, from an endocrine disruption point of view, especially with regard to the corresponding endogenous production level in this target population. As a starting point, it was estimated that a (re)-evaluation of the endogenous production of natural estrogens for this population was necessary, on the basis of a very sensitive and specific confirmatory measurement technique (gas chromatography-tandem mass spectrometry or gas chromatography-high resolution mass spectrometry). Thus, a new ultra-sensitive approach for steroid trace measurement in biological samples was developed, which was mainly based on a specific derivatisation (pentafluorobenzyl derivative) and negative chemical ionisation (NCI). Preliminary results obtained by applying this method on plasma samples from healthy prepubertal children demonstrated that estradiol endogenous level in prepubertal children is unsurprisingly very low. Estrone was determined in almost all samples at concentration in the 2-70 ng L(-1) range while 17alpha and 17beta estradiol were quantified in only few samples at concentrations ranging from 2 to 6 ng L(-1). Exogenous contributions of estrogens will therefore constitute a relatively higher proportion of sex hormone activity in the immature child.