Effects of polyethylene microplastics occurrence on estrogens degradation in soil.

Growing focus has been drawn to the continuous detection of high estrogens levels in the soil environment. Additionally, microplastics (MPs) are also of growing concern worldwide, which may affect the environmental behavior of estrogens. However, little is known about effects of MPs occurrence on estrogens degradation in soil. In this study, polyethylene microplastics (PE-MPs) were chosen to examine the influence on six common estrogens (estrone (E1), 17α-estradiol (17α-E2), 17ß-estradiol (17ß-E2), estriol (E3), diethylstilbestrol (DES), and 17α-ethinylestradiol (17α-EE2)) degradation. The results indicated that PE-MPs had little effect on the degradation of E3 and DES, and slightly affected the degradation of 17α-E2, however, significantly inhibited the degradation of E1, 17α-EE2, and 17ß-E2. It was explained that (i) obvious oxidation reaction occurred on the surface of PE-MPs, indicating that PE-MPs might compete with estrogens for oxidation sites, such as redox and biological oxidation; (ii) PE-MPs significantly changed the bacterial community in soil, resulting in a decline in the abundance of some bacterial communities that biodegraded estrogens. Moreover, the rough surface of PE-MPs facilitated the estrogen-degrading bacterial species (especially for E1, E2, and EE2) to adhere, which decreased their reaction to estrogens. These findings are expected to deepen the understanding of the environmental behavior of typical estrogens in the coexisting system of MPs.

Removal of estrogens from primary settled sewage by repeated culture of Selenastrum capricornutum.

Biotransformation and biodegradation of estrogenic compounds by bacteria and even fungi have been reported widely, but the role of microalgae in the elimination of estrogens from municipal wastewater treatment plants and their interaction with other microorganisms in wastewater are not clear. This study reported the feasibility of repeatedly removing a mixture of 17ß-estradiol (E2) and 17α-ethinylestradiol (EE2), each was 100 µg L-1, from primary settled municipal sewage by Selenastrum capricornutum (SC), a ubiquitous microalga, in four exposure cycles, each lasted 7 days, and how they interacted with the microbial consortium in sewage. Mixed estrogen in sewage stimulated the growth of SC, and the indigenous microorganisms in sewage also affected the microalgal growth. The indigenous microorganisms, particularly bacteria, could easily remove E2 (with 99.5% removal), so the role of SC was insignificant. On the contrary, EE2 was difficult to remove by indigenous microorganisms but the removal was significantly enhanced by SC, with almost all spiked EE2 being removed, even at the end of the fourth cycle (with 99.0% removal). These results indicated that SC, together with the indigenous microorganisms in wastewater, could be repeatedly used for simultaneous removal of E2 and EE2 from municipal sewage.

Efficient removal of endocrine disrupting compounds 17 α-ethynyl estradiol and 17 ß-estradiol by Enterobacter sp. strain BHUBP7 and elucidation of the degradation pathway by HRAMS analysis.

Owing to the increased population and their overuse, estrogens are being detected in the environment at alarming levels. They act as endocrine disrupting compounds (EDC's) posing adverse effects on animals and humans. In this study, a strain belonging to Enterobacter sp. strain BHUBP7 was recovered from a Sewage Treatment Plant (STP) situated in Varanasi city, U.P., India, and was capable of metabolizing both 17 α-Ethynylestradiol (EE2) and 17 ß-Estradiol (E2) separately as a sole carbon source. The strain BHUBP7 exhibited high rates of E2 degradation as compared to EE2 degradation. The degradation of E2 (10 mg/L) was 94.3% after four days of incubation, whereas the degradation of EE2 (10 mg/L) under similar conditions was 98% after seven days of incubation. The kinetics of EE2 and E2 degradation fitted well with the first-order reaction rate. FTIR analysis revealed the involvement of functional groups like C = O, C-C, C-OH during the degradation process. The metabolites generated during degradation of EE2 and E2 were identified using HRAMS and a plausible pathway was elucidated. It was observed that metabolism of both E2 and EE2 proceeded with the formation of estrone, which was then hydroxylated to 4-hydroxy estrone, followed by ring opening at the C4-C5 position, and was further metabolized by the 4,5 seco pathway leading to the formation of 3-(7a-methyl-1,5-dioxooctahydro-1H-inden-4-yl) propanoic acid (HIP). It is the first report on the complete pathway of EE2 and E2 degradation in Enterobacter sp. strain BHUBP7. Moreover, the formation of Reactive Oxygen Species (ROS) during the degradation of EE2 and E2 was observed. It was concluded that both hormones elicited the generation of oxidative stress in the bacterium during the degradation process.

Putative adverse outcome pathway development based on physiological responses of female fathead minnows to model estrogen versus androgen receptor agonists.

Several adverse outcome pathways (AOPs) have linked molecular initiating events like aromatase inhibition, androgen receptor (AR) agonism, and estrogen receptor (ER) antagonism to reproductive impairment in adult fish. Estrogen receptor agonists can also cause adverse reproductive effects, however, the early key events (KEs) in an AOP leading to this are mostly unknown. The primary aim of this study was to develop hypotheses regarding the potential mechanisms through which exposure to ER agonists might lead to reproductive impairment in female fish. Mature fathead minnows were exposed to 1 or 10 ng 17α-ethynylestradiol (EE2)/L or 10 or 100 µg bisphenol A (BPA)/L for 14 d. The response to EE2 and BPA was contrasted with the effects of 500 ng/L of 17ß-trenbolone (TRB), an AR agonist, as well as TRB combined with the low and high concentrations of EE2 or BPA tested individually. Exposure to 10 ng EE2/L, 100 µg BPA/L, TRB, or the various mixtures with TRB caused significant decreases in plasma concentrations of 17ß-estradiol. Exposure to TRB alone caused a significant reduction in plasma vitellogenin (VTG), but VTG was unaffected or even increased in females exposed to EE2 or BPA alone or, in most cases, in mixtures with TRB. Over the course of the 14-d exposure, the only treatments that clearly did not affect egg production were 1 ng EE2/L and 10 µg BPA/L. Based on these results and knowledge of hypothalamic-pituitary-gonadal axis function, we hypothesize an AOP whereby decreased production of maturation-inducing steroid leading to impaired oocyte maturation and ovulation, possibly due to negative feedback or direct inhibitory effects of membrane ER activation, could be responsible for causing adverse reproductive impacts in female fish exposed to ER agonists.

Impact of zero-valent iron on nitrifying granular sludge for 17α-ethinylestradiol removal and its mechanism.

Aerobic granulation of nitrifying activated sludge could enhance the removal of 17α-ethinylestradiol (EE2) via abiotic nitration induced by reactive nitrogen species, cometabolism by ammonia-oxidizing bacteria and biodegradation by heterotrophic bacteria. Zero-valent iron (ZVI), a promising and low-cost material, has previously been applied to effectively enhance biological wastewater treatment. The impact and the effect mechanism of ZVI on nitrifying granular sludge (NGS) for EE2 removal was investigated in this study. The results showed that the addition of ZVI achieved better EE2 removal, though ZVI was not conducive to the accumulation of nitrite in NGS which reduced the abiotic transformation of EE2. Moreover, ZVI enriched heterotrophic denitrifying bacteria such as Arenimonas, thus changing the EE2 removal pathway and improving the degradation and mineralization of EE2. In addition, ZVI reduced the emission risk of the greenhouse gas N2O and strengthened the stability of the granules. Metagenomic analysis further revealed that the functional genes related to EE2 mineralization, nitrite oxidation, N2O reduction and quorum sensing in NGS were enriched with ZVI addition. This study provides meaningful guidance for ZVI application in the NGS process to achieve efficient and simultaneous removal of ammonia and emerging contaminants.

Electrochemical degradation of 17α-ethinylestradiol: Transformation products, degradation pathways and in vivo assessment of estrogenic activity.

Conventional water treatment methods are not efficient in eliminating endocrine disrupting compounds (EDCs) in wastewater. Electrochemical Advanced Oxidation Processes (eAOPs) offer a promising alternative, as they electro-generate highly reactive species that oxidize EDCs. However, these processes produce a wide spectrum of transformation products (TPs) with unknown chemical and biological properties. Therefore, a comprehensive chemical and biological evaluation of these remediation technologies is necessary before they can be safely applied in real-life situations. In this study, 17α-ethinylestradiol (EE2), a persistent estrogen, was electrochemically degraded using a boron doped diamond anode with sodium sulfate (Na2SO4) and sodium chloride (NaCl) as supporting electrolytes. Ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was used for the quantification of EE2 and the identification of TPs. Estrogenic activity was assessed using a transgenic medaka fish line. At optimal operating conditions, EE2 removal reached over 99.9% after 120 min and 2 min, using Na2SO4 and NaCl, respectively. The combined EE2 quantification and in vivo estrogenic assessment demonstrated the overall estrogenic activity was consistently reduced with the degradation of EE2, but not completely eradicated. The identification and time monitoring of TPs showed that the radical agents readily oxidized the phenolic A-ring of EE2, leading to the generation of hydroxylated and/or halogenated TPs and ring-opening products. eAOP revealed to be a promising technique for the removal of EE2 from water. However, caution should be exercised with respect to the generation of potentially toxic TPs.

Tributyltin-binding protein type 1 (fish acid glycoprotein) is a potential gatekeeper of ethinylestradiol action in fish.

Tributyltin (TBT)-binding protein type 1 in Japanese medaka (Oryzias latipes) (O.latTBT-bp1) is a fish lipocalin implicated in TBT binding and detoxification. We purified recombinant O.latTBT-bp1 (rO.latTBT-bp1; ca. 30 kDa) by using a baculovirus expression system and His- and Strep-tag chromatography process. Then, we examined O.latTBT-bp1 binding to several endo/exogenous steroid hormones by means of competitive binding assay. The dissociation constants for the binding of rO.latTBT-bp1 to DAUDA and ANS, two fluorescent ligands of lipocalin, were 7.06 and 13.6 µM, respectively. Multiple model validations indicated that a single-binding-site model was the most appropriate for evaluating rO.latTBT-bp1 binding. In the competitive binding assay, testosterone, 11-ketotestosterone, and 17ß-estradiol were each bound by rO.latTBT-bp1; rO.latTBT-bp1 showed the strongest affinity for testosterone (inhibition constant, Ki = 3.47 µM). Endocrine-disrupting chemical (synthetic steroid) also bound to rO.latTBT-bp1; the affinity for ethinylestradiol (Ki = 9.29 µM) was stronger than that for 17ß-estradiol (Ki = 30.0 µM). To determine the function of O.latTBT-bp1, we produced TBT-bp1 knockout medaka (TBT-bp1 KO), which we exposed to ethinylestradiol for 28 days. After exposure, the number of papillary processes in TBT-bp1 KO genotypic male medaka was significantly fewer (3.5), compared to that in wild-type male medaka (22). Thus, TBT-bp1 KO medaka were more sensitive to the anti-androgenic effects of ethinylestradiol than wild-type medaka. These results indicate that O.latTBT-bp1 may bind to steroids and act as a gatekeeper of ethinylestradiol action by regulating the androgen-estrogen balance.

The influence of temperature rise on the metabolic response of Ruditapes philippinarum clams to 17-α-ethinylestradiol.

Untargeted Nuclear Magnetic Resonance metabolomics was employed to study the effects of warming conditions (17-21 °C) and exposure to 17-α-ethinylestradiol (EE2) on the polar metabolome of Ruditapes philippinarum clams, to identify metabolic markers for monitoring/prediction of deviant environmental conditions. Warming alone triggered changes in alanine/aspartate/glutamate, aromatic amino acids, taurine/hypotaurine and homarine/trigonelline pathways, as well as in energy metabolism, suggesting osmoregulatory adaptations and glycolytic/tricarboxylic acid (TCA) cycle activation, possibly accompanied to some extent by gluconeogenesis to preserve glycogen reserves. At 17 °C, the lowest EE2 concentration (5 ng/L) specifically engaged branched-chain and aromatic amino acids to activate the glycolysis/TCA cycle. Notably, a partial metabolic recovery was observed at 25 ng/L, whereas higher EE2 concentrations (125 and 625 ng/L) again induced significant metabolic disturbances. These included enhanced glycogen biosynthesis and increased lipid reserves, sustained by low-level glutathione-based antioxidative mechanisms that seemed active. At 21 °C, response to EE2 was notably weak at low/intermediate concentrations, becoming particularly significant at the highest EE2 concentration (625 ng/L), suggesting higher protection capacity of Ruditapes philippinarum clams under warming conditions. At 625 ng/L, disturbances in alanine/aspartate/glutamate and taurine/hypotaurine metabolisms were observed, with no evidence of enhanced carbohydrate/protein catabolism. This low energy function profile was accompanied by marked antioxidative mechanisms and choline compounds modulation for cell membrane protection/repair. These results help monitor clams´ response to temperature rise and EE2 exposure, paving the way for future effective guidance and prediction of environmental damaging effects.

Effects of 17α-ethinylestradiol on the neuroendocrine gonadotropic system and behavior of European sea bass larvae (Dicentrarchus labrax).

The widespread use of 17α-ethinylestradiol (EE2), and other estrogenic endocrine disruptors, results in a continuous release of estrogenic compounds into aquatic environments. Xenoestrogens may interfere with the neuroendocrine system of aquatic organisms and may produce various adverse effects. The aim of the present study was to expose European sea bass larvae (Dicentrarchus labrax) to EE2 (0.5 and 50 nM) for 8 d and determine the expression levels of brain aromatase (cyp19a1b), gonadotropin-releasing hormones (gnrh1, gnrh2, gnrh3), kisspeptins (kiss1, kiss2) and estrogen receptors (esr1, esr2a, esr2b, gpera, gperb). Growth and behavior of larvae as evidenced by locomotor activity and anxiety-like behaviors were measured 8 d after EE2 treatment and a depuration period of 20 d. Exposure to 0.5 nM EE2 induced a significant increase in cyp19a1b expression levels, while upregulation of gnrh2, kiss1, and cyp19a1b expression was noted after 8 d at 50 nM EE2. Standard length at the end of the exposure phase was significantly lower in larvae exposed to 50 nM EE2 than in control; however, this effect was no longer observed after the depuration phase. The upregulation of gnrh2, kiss1, and cyp19a1b expression levels was found in conjunction with elevation in locomotor activity and anxiety-like behaviors in larvae. Behavioral alterations were still detected at the end of the depuration phase. Evidence indicates that the long-lasting effects of EE2 on behavior might impact normal development and subsequent fitness of exposed fish.

Does dietary exposure to 17α-ethinylestradiol alter biomarkers related with endocrine disruption and oxidative stress in the adult triploid of Danio rerio?

This study was conducted to investigate a comprehensive effect of 17α-ethinylestradiol (EE2) in zebrafish (Danio rerio) with the emphasis on endocrine disruption, oxidative stress and detoxification processes at different levels. Adult male triploid zebrafish were exposed to EE2 administered in feed at two concentrations - 10 and 1000 µg/kg for six weeks. The estrogenic potential of EE2 was evaluated using an analysis of vitellogenin, gene expression focused on reproductive disorders and gonad histological examination. The alterations in antioxidant and detoxification status were assessed using analyses of enzyme activities and changes in transcriptional levels of selected genes. The most significant changes were observed especially in fish exposed to a high concentration of EE2 (i.e., 1000 µg/kg). Such high concentration caused extensive mortality (25 %) mainly in the second half of the experiment followed by a highly significant decrease in the length and body weight. Similarly, highly significant induction of vitellogenin level and vtg1 mRNA expression (about 43,000-fold compared to the control) as well as a significant downregulation of gonad aromatase expression (cyp19a1a) and histological changes in testicular tissue were confirmed in this group. In the group exposed to environmentally relevant concentration of EE2 (i.e., 10 µg/kg), no significant differences in vitellogenin were observed, although all fish were positive in the detection of vitellogenin compared to control, where only 40 % of individuals were positive. In addition, the high concentration of EE2 resulted in significant alterations in most monitored antioxidant and detoxifying enzymes with the exception of catalase, followed by strongly significant upregulation in mRNA expression of gsr, gpx1a, cat and cyp1a genes. Furthermore, a significant decrease in the glutathione reductase activity was recorded in fish exposed to 10 µg EE2/kg. To our knowledge, this is the first study which reports the effects of subchronic per oral exposure to EE2 in adult triploid zebrafish.

17α-ethinylestradiol modulates endothelial function in ovariectomized rat carotid arteries.

17α-ethinylestradiol (EE2), a derivative of 17ß-estradiol (E2), is a potent estrogenic substance that is used as the estrogenic component of oral contraceptives (OCPs). However, women who take OCPs have an increased risk of cardiovascular events. Since few studies have examined EE2 endothelial effects, we explored the effects of EE2 on endothelial function in ovariectomized and isoflavone-free rats. After ovariectomy, 12-week-old female Sprague-Dawley rats were assigned to EE2, E2 or control groups. After 16 weeks, the EE2 and E2 groups were orally administered EE2 (8.3 µg/day) and E2 (12.6 µg/day) for 4 weeks, respectively. At 18 weeks, endothelial denudation of the left common carotid arteries was performed, and they were harvested at 20 weeks. The rats in the EE2 and E2 groups exhibited significantly decreased body weights and significantly increased uterine weights, respectively, but no differences were observed between the EE2 and E2 groups. The EE2 and E2 groups showed significantly enhanced acetylcholine-induced endothelium-dependent relaxation, with apamin plus charybdotoxin inhibiting only the EE2 group. Endothelial nitric oxide (NO) synthase expression was significantly higher in the EE2 group than in the control, but lower than in the E2 group. The intima-to-media ratio of denuded arteries was significantly lower in the E2 group than in the other groups, suggesting that NO decreased in the EE2 group compared to the E2 group. We conclude that EE2 has a weaker ability than E2 to produce NO and, for the first time, we demonstrate the ability of EE2 to enhance the activity of endothelial-derived hyperpolarizing factor.

Toxic effects of a mixture of pharmaceuticals in Mytilus galloprovincialis: The case of 17α-ethinylestradiol and salicylic acid.

The impact of pharmaceuticals on marine invertebrates has been a topic of rising concern, with an increasing number of studies regarding the impacts on bivalves. However, very few investigated the toxicity of mixtures of pharmaceuticals. This knowledge gap was investigated in the present study, where the toxicity of 17α-ethinylestradiol (EE2) and salicylic acid (SA) mixture was evaluated. To this end, Mytilus galloprovincialis mussels were chronically subjected to both pharmaceuticals, acting alone and in combination, and the effects at the cellular level were measured. The Independent Action (IA) model was performed aiming to compare obtained with predicted responses. The integrated biomarker response (IBR) index was used to assess the overall biochemical response given by mussels. The results obtained revealed that the most stressful condition was caused by the combined effect of EE2 and SA, with the highest metabolic capacity, antioxidant (catalase activity) and biotransformation (carboxylesterases activity) activation and cellular damage in organisms exposed to the mixture of both drugs in comparison to responses observed when each drug was acting alone. Predicted responses obtained from the IA model indicate that caution should be paid as frequent deviations to observed responses were found. This study highlights the need for future studies considering the mixture of pollutants, mimicking the actual environmental conditions.

Gene expression and biochemical patterns in the digestive gland of the mussel Mytilus galloprovincialis (Lamarck, 1819) exposed to 17α-ethinylestradiol.

Contaminants of emerging concern (CECs) are a class of chemicals that can spread throughout the environment and may cause adverse biological and ecological effects. While there are many different classes of CECs, one of the most well documented in the aquatic environment are pharmaceutical drugs, such as natural and synthetic estrogens. In particular, the widespread presence of the synthetic estrogen 17 α-Ethinylestradiol (EE2) in water may lead to bioaccumulation in sediment and biota. EE2 is the primary component in contraceptive pills, and is a derivative of the natural hormone estradiol (E2). In this study, the mussel Mytilus galloprovincialis was exposed to EE2 in a semi-static and time-dependent experiment, for a total exposure period of 28 days. Biochemical and transcriptomics analyses were performed on mussel digestive glands after exposure for 14 (T14) and 28 (T28) days. Metabolic and DNA impairments, as well as activation of antioxidant and biotransformation enzymes activation, were detected in T28 exposed mussels. RNA-Seq analysis showed significant differential expression of 160 (T14 compared to controls), 33 (T28 compared to controls) and 79 (T14 compared to T28) genes. Signs of stress after EE2 treatment included up-regulation of gene/proteins involved with immune function, lipid transport, and metabolic and antibacterial properties. This study elucidates the underlying mechanisms of EE2 in a filter feeding organisms to elucidate the effects of this human pharmaceutical on aquatic biota.

Oriented bio-feeding control of the anaerobic biodegradation of ethinyl estradiol.

Up to 95% of hormones are excreted into domestic wastewater with urine or feces, but their macromolecules are difficult to biodegrade. This project studies the treatment of Ethinyl Estradiol (EE2) in swine wastewater in an Upstream Solids Reactor (USR), and explores a new method for oriented bio-feeding to regulate the anaerobic biodegradation process. It was found that the metabolism of lactic acid and propionic acid was accompanied by changes in EE2 content, but lactic acid molecules were not readily bioavailable, so adding propionic acid was more suitable. However, controlling the pH to lower (4.73) and higher (8.73) values inhibited further fermentation of acetic acid and propionic acid, which was not favorable for the removal of EE2. This is simply due to the fact that propionic acid as a carbon source changes the preference of the microbes for consuming EE2. The order of the effect of addition of propionic acid on the removal of EE2 was as follows: P400>P800>P0>P200 (addition of propionic acid). The P400 removal efficiency increased from 60% to 85%. In the metabolism of EE2, after oxidation, hydrolysis, ketosis, hydroxylation and enzymatic action, dienoic acid and oleic acid were generated, and there was no secondary pollution from EE2 metabolites. In conclusion, feeding microorganisms with propionic acid can enhance the anaerobic biodegradation of EE2, providing a new strategy for the anaerobic biodegradation and bioremediation of refractory pollutants.

Transcriptome analysis of changes in M. aeruginosa growth and microcystin production under low concentrations of ethinyl estradiol.

Ethinyl estradiol (EE2) is a synthetic environmental estrogen with considerable estrogenic activity. It has been found to consequently pose a significant threat to the aquatic ecosystem. Harmful algal blooms are a major aquatic ecological issue. However, the relationship between EE2 and cyanobacterial bloom is mainly unknown. In this study, the physiological and molecular responses of Microcystis aeruginosa to EE2 exposure were investigated. A low level of EE2 (0.02 µg/L) significantly enhanced the growth of algal cells (P < 0.05), whereas higher concentrations of EE2 (0.2-200 µg/L) inhibited it. EE2 at doses ranging from 0.02 to 200 µg/L promoted the production of microcystins (MCs), with genes mcyABD playing a key role in the regulation of MC synthesis. The alterations of chlorophyll-a, carotenoid, and phycocyanin contents caused by EE2 showed the same trend as cell growth. At the molecular level, 200 µg/L EE2 significantly down-regulated genes in photosynthetic pigment synthesis, light harvesting, electron transfer, NADPH, and ATP generation. High concentrations of EE2 caused oxidative damage to algal cells on the 4th d. After 12d exposure, although there was no significant change in superoxide dismutase (SOD) content and no damage observed in membrane lipids, genes related to SOD and glutathione were changed. In addition, due to the down-regulation of pckA, PK, gltA, nrtA, pstS, etc., carbon fixation, glycolysis, TCA cycle, nitrogen and phosphorus metabolism were hindered by EE2 (200 µg/L). Gene fabG in fatty acid biosynthesis was significantly up-regulated, promoting energy storage in cells. These findings provide important clues to elucidate the effects and mechanisms of cyanobacterial blooms triggered by EE2 and help to effectively prevent and control cyanobacterial blooms.

Capture-SELEX of DNA Aptamers for Estradiol Specifically and Estrogenic Compounds Collectively.

Estrogenic compounds such as estrone (E1), 17ß-estradiol (E2), and 17α-ethynylestradiol (EE2) are serious environmental contaminants due to their potent biological activities. At least six selections were previously reported to obtain DNA aptamers for E2, highlighting its environmental importance. A careful analysis revealed that the previous aptamers either are too long or do not bind optimally. Herein, a series of new aptamers were obtained from the capture-SELEX method with dissociation constants down to 30 nM as determined by isothermal titration calorimetry (ITC). Two aptamers were converted to structure-switching fluorescent biosensors, which achieved a limit of detection down to 3.3 and 9.1 nM E2, respectively. One aptamer showed similar binding affinities to all the three estrogens, while the other aptamer is more selective for E2. Both aptamers required Mg2+ for binding. The proposed sensors were successfully applied in the determination of E2 in wastewater. Moreover, comparisons were made with previous aptamers based on primary sequence alignment and secondary structures. Among previously reported truncated aptamers, ITC showed binding only in one of them. The newly selected aptamers have the combined advantages of small size and high affinities.

Exposure to 17α-Ethinylestradiol Results in Differential Susceptibility of Largemouth Bass (Micropterus salmoides) to Bacterial Infection.

Disease outbreaks, skin lesions, mortality events, and reproductive abnormalities have been observed in wild populations of centrarchids. The presence of estrogenic endocrine disrupting compounds (EEDCs) has been implicated as a potential causal factor for these effects. The effects of prior EEDC exposure on immune response were examined in juvenile largemouth bass (Micropterus salmoides) exposed to a potent synthetic estrogen (17α-ethinylestradiol, EE2) at a low (EE2Low, 0.87 ng/L) or high (EE2High, 9.08 ng/L) dose for 4 weeks, followed by transfer to clean water and injection with an LD40 dose of the Gram-negative bacteria Edwardsiella piscicida. Unexpectedly, this prior exposure to EE2High significantly increased survivorship at 10 d post-infection compared to solvent control or EE2Low-exposed, infected fish. Both prior exposure and infection with E. piscicida led to significantly reduced hepatic glycogen levels, indicating a stress response resulting in depletion of energy stores. Additionally, pathway analysis for liver and spleen indicated differentially expressed genes associated with immunometabolic processes in the mock-injected EE2High treatment that could underlie the observed protective effect and metabolic shift in EE2High-infected fish. Our results demonstrate that exposure to a model EEDC alters metabolism and immune function in a fish species that is ecologically and economically important in North America.

Activation of natural killer T cells contributes to Th1 bias in the murine liver after 14 d of ethinylestradiol exposure.

BACKGROUND: As the main component of oral contraceptives (OCs), ethinylestradiol (EE) has been widely applied as a model drug to induce murine intrahepatic cholestasis. The clinical counterpart of EE-induced cholestasis includes women who are taking OCs, sex hormone replacement therapy, and susceptible pregnant women. Taking intrahepatic cholestasis of pregnancy (ICP) as an example, ICP consumes the medical system due to its high-risk fetal burden and the impotency of ursodeoxycholic acid in reducing adverse perinatal outcomes. AIM: To explore the mechanisms and therapeutic strategies of EE-induced cholestasis based on the liver immune microenvironment. METHODS: Male C57BL/6J mice or invariant natural killer T (iNKT) cell deficiency (Jα18-/- mice) were administered with EE (10 mg/kg, subcutaneous) for 14 d. RESULTS: Both Th1 and Th2 cytokines produced by NKT cells increased in the liver skewing toward a Th1 bias. The expression of the chemokine/chemokine receptor Cxcr6/Cxcl16, toll-like receptors, Ras/Rad, and PI3K/Bad signaling was upregulated after EE administration. EE also influenced bile acid synthase Cyp7a1, Cyp8b1, and tight junctions ZO-1 and Occludin, which might be associated with EE-induced cholestasis. iNKT cell deficiency (Jα18-/- mice) robustly alleviated cholestatic liver damage and lowered the expression of the abovementioned signaling pathways. CONCLUSION: Hepatic NKT cells play a pathogenic role in EE-induced intrahepatic cholestasis. Our research improves the understanding of intrahepatic cholestasis by revealing the hepatic immune microenvironment and also provides a potential clinical treatment by regulating iNKT cells.

Dietary transference of 17α-ethinylestradiol changes the biochemical and behavioral biomarkers in adult zebrafish (Danio rerio).

The endocrine disruptors (ED), even in low concentration, can change the homeostasis of an organism through the biochemical and physiological pathways; and are gaining more relevance due to their well-reported presence in the natural environment. EDs mainly affect non-target animals, which can bioaccumulate, leading to changes in metabolism. Another problem is due to several organisms that compose the aquatic biota serving as a basis of the food chain and transferring it to higher trophic levels. Here we evaluated the dietary transference of 17α-ethinylestradiol (EE2), in adult zebrafish chronically fed by EE2-bioaccumulated brine shrimp (BS). For this, we evaluated behavioral biomarkers such as the novel tank test (NTT), social preference test (SPT), mirror-induced aggressivity (MIA), and biochemical biomarkers such as acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CTL), and glutathione-S-transferase (GST) activity, cortisol, and lipid peroxidation levels in adult zebrafish. The behavioral effects can be explained by the changed effects on acetylcholinesterase activity as well as in the antioxidant system mainly affected by the high levels of EE2 identified by HPLC shown that had occurred during a dietary transfer for fish. EE2 has a potential pattern for bioaccumulation and dietary transfer in biological tissue and EE2 can affect the behavior of fish. The observed effects could be dangerous to the environment, affecting, other animals and even human health.

Effects of 17α-Ethinylestradiol (EE2) exposure during early life development on the gonadotropic axis ontogenesis of the European sea bass, Dicentrarchus labrax.

Exposure of young organisms to oestrogenic endocrine disrupting chemicals (EDCs) can elicit adverse effects, particularly on the reproductive function. In fish, as in other vertebrates, reproduction is controlled by the neuroendocrine gonadotropic axis, whose components are mainly regulated by sex steroids and may then be targets for EDCs. In the present study, we investigated the effects of a xenoestrogen exposure on the ontogenesis of the gonadotropic axis in European sea bass. After exposure of hatching larvae for 8 days to 17α-ethinylestradiol (EE2) (0.5 nM and 50 nM), gene expression for kisspeptins (kiss1, kiss2), gonadotropin-releasing hormones (gnrh1, gnrh2, gnrh3), gonadotropin beta subunits (lhß and fshß) and brain type aromatase (cyp19a1b) were measured using quantitative real-time PCR. Our results demonstrate that EE2 strongly stimulated the expression of brain type aromatase (cyp19a1b) in sea bass larvae. In addition, EE2 exposure also affected the mRNA levels of kiss1, gnrh1 and gnrh3 by inducing a downregulation of these genes during the early developmental stages, while no effect was seen in gnrh2, lhß and fshß. These results reinforce the idea that the larval development is a sensitive critical period in regard to endocrine disruption and that the gonadotropic axis in the developing sea bass is sensitive to xenoestrogen exposure.