EE2 Group Research Articles

A Proof-of-Concept for a Hypolipidemic Brown Trout Model.

The impacts of hypolipidemic pharmaceuticals on fish lipid metabolism remain unexplored. However, data points to similar effects and mechanisms of action between fish and humans. Therefore, fish may be a strong model for screening hypolipidemic drug candidates and water pollution by lipid-modulating agents. This study aimed to test a new hypolipidemic model assay with juvenile brown trout using atorvastatin (ATV)-a hypolipidemic chemical. We selected 17α-ethinylestradiol (EE2), known to cause hyperlipidemia in fish, to ensure model functionality. Fish received intramuscular injections of 4 μL/g for two weeks under the following experimental conditions: control-C (0.7% NaCl), solvent control-SC (0.7% NaCl, 0.9% ethanol, 0.1% dimethyl sulfoxide), ATV (0.3 μg/g), EE2 (2 μg/g), and a mixture of both compounds-MIX (0.3 μg/g ATV and 2 μg/g EE2). Endpoints included blood lipid biochemistry, hepatic lipid droplet quantification, and liver mRNA expression of lipid-related target genes (related to lipogenesis, lipid transport, and β-oxidation pathways). ATV lowered blood total cholesterol, high-density lipoproteins (HDL), and low-density lipoproteins (LDL) levels, whilst triglycerides and very-low-density lipoproteins (VLDL) were highest under EE2. Hepatic lipid droplet deposition significantly increased in the ATV, EE2, and MIX groups. ATV and MIX caused a significant downregulation of the peroxisome proliferator-activated receptor γ (pparγ) and acetyl Co-A oxidase 3 (acox3). EE2 upregulated acyl-CoA long-chain synthetase 1 (acsl1) and downregulated both fatty acid binding protein 1 (fabp1) and acetyl Co-A oxidase 1-3I (acox1-3I). ATV caused hypolipidemic effects in juvenile brown trout and could even counteract EE2-stimulated hyperlipidemia, reinforcing the potential of fish hypo- and hyperlipidemic models.

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

17α-ethinylestradiol (EE2), a derivative of 17β-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.

The influence of the enriched environment in different periods on neonatal maternal separation-induced visceral pain, anxiousness, and depressive behaviors.

BackgroundNeonatal maternal separation (NMS) is a major early life stress that can induce visceral pain and mental disorders. We have shown that an enriched environment (EE) can alleviate NMS-induced negative effects, but the time window over which EE works is unclear. Therefore, this study aimed to investigate the time window through which EE alleviates NMS-induced visceral pain, anxiousness, and depressive behaviors.MethodsIn this study, we used male C57BL/6J mice. The mice were randomly divided into five groups: control group, NMS group, prepubertal EE group (EE1 group), pubertal EE group (EE2 group), and adult EE group (EE3 group). The visceral pain threshold test (PTT), open field test (OFT), elevated plus maze (EPM), forced swimming test (FST), and sucrose preference test (SPT) were performed in all five groups to assay visceral pain, anxiety-, and depression-like behaviors in mice, respectively. Enzyme-linked immunosorbent assay (ELISA) for corticosterone was performed in all five groups to assess the function of the hypothalamic-pituitary-adrenal (HPA) axis.ResultsThere was no significant change in weight between groups. It was shown that NMS induced visceral pain, anxiety, and depression, and EE1 and EE2 reversed these negative effects, but EE3 had no significant effect. Likewise, EE1 and EE2 reversed the NMS-induced increase of corticosterone, but EE3 did not.ConclusionsAdverse life experiences in early life can lead to visceral pain, anxiety, and depression in adulthood, which can be effectively prevented by EE interventions in prepuberty and puberty.

Selective effects of perinatal estrogen on proliferation and new neurons in hippocampus and piriform cortex of rats at weaning

BackgroundA recent report links heightened prenatal amniotic estrogen levels to an increased risk of autism spectrum disorder (ASD). In this study, we examined the developmental effects of perinatal estrogen treatment on stem cell activity in weaned rats. MethodsSprague-Dawley rats received ethinyl estradiol (EE2, 10 µg/kg/day) or vehicle orally from gestational day 6 until parturition. Offspring were then treated with the same daily dose from postnatal days (PNDs) 1–21. The effects of perinatal estrogen treatment on stem cell activities in the subgranular zone (SGZ) of the hippocampus and the piriform cortex were evaluated in male and female rat pups. ResultsEE2 treatment increased the total Ki67-immunoreactive (Ki67-ir) cell counts in the SGZ of males and females (p < 0.05). However, no treatment or sex differences were detectable in the density of the doublecortin (DCX)-immunoreactive (DCX-ir) deposits in the hippocampus. In the piriform cortex, no treatment or sex differences were detected in Ki67-ir cell counts. However, the EE2 treatment significantly reduced the DCX-ir cell count in male, but not female rats (male EE2 group = 292 ± 22/mm2, male vehicle group = 402 ± 19/mm2, female EE2 group = 342 ± 15/mm2, female vehicle group = 331 ± 9/mm2). ConclusionsPerinatal estrogen treatment increased hippocampal Ki67-ir cell counts in both sexes and selectively reduced DCX-ir cell counts in the piriform cortex of males. These data suggest that exposure to abnormally high levels of estrogens early in life may have an impact on neural cell development. Alterations in development so early in life may have long-term cognitive impact.

Waves of follicle development, growth and degeneration in adult ovary of zebrafish (Danio rerio) on chronic exposure to environmental estrogens in laboratory

Patterns of quantitative production of follicles, their growth, and degeneration in the adult ovary of zebrafish (Danio rerio) in response to long-term (80 days) exposure to environmental estrogens (EE) in the laboratory, were studied. Experimentally naive female D. rerio procured from fish farm were acclimated to the laboratory (natural temperature, 26 ± 1° C, photoperiod, 11.30 L:12.30 D) for two weeks and divided into 10 groups. Each group (n = 20) was housed in a separate glass aquarium containing 10 L of conditioned water (physico-chemical parameters maintained within the permissible range prescribed for zebrafish) along with either 5 ng or 10 ng/L of 17α-ethynylestradiol (EE2) or diethylstilbestrol (DES) or bisphenol A (BPA) or estradiol 17-β (positive control) or water with no chemical (negative control). All experimental fish were fed twice daily on commercial pellets (ad libitum) supplemented with Artemia nauplius, the exposure was semi-static and chemical residues in media samples were determined by ultra-performance liquid chromatography (UPLC). Exposure of fish to estrogens increased (p < 0.05) (i) body mass and gonadosomatic indices (GSI) in E2, EE2 and DES groups (ii) previtellogenic and vitellogenic follicles in E2 and EE2 groups (iii) atretic follicles (AF) in DES and BPA groups compared to controls and (iv) decrease in total oocyte volumes (V = 4/3. π. r3) compared to those of E2 group. These results suggest that the chronic exposure of fish to EE (at environmentally relevant concentrations) has a profound influence on ovarian follicular dynamics and the effects of individual EE are discrete on the ovary.

The Effect of 17α-Ethynilestradiol and GPER1 Activation on Body and Muscle Growth, Muscle Composition and Growth-Related Gene Expression of Gilthead Seabream, Sparus aurata L.

Endocrine-disrupting chemicals include natural and synthetic estrogens, such as 17α-ethynilestradiol (EE2), which can affect reproduction, growth and immunity. Estrogen signalling is mediated by nuclear or membrane estrogen receptors, such as the new G-protein-coupled estrogen receptor 1 (GPER1). The present work studies the effect of EE2 and G1 (an agonist of GPER1) on body and muscle parameters and growth-related genes of 54 two-year-old seabreams. The fish were fed a diet containing EE2 (EE2 group) and G1 (G1 group) for 45 days and then a diet without EE2 or G1 for 122 days. An untreated control group was also studied. At 45 days, the shortest body length was observed in the G1 group, while 79 and 122 days after the cessation of treatments, the shortest body growth was observed in the EE2 group. Hypertrophy of white fibers was higher in the EE2 and G1 groups than it was in the control group, whereas the opposite was the case with respect to hyperplasia. Textural hardness showed a negative correlation with the size of white fibers. At the end of the experiment, all fish analyzed in the EE2 group showed a predominance of the gonadal ovarian area. In addition, the highest expression of the mafbx gene (upregulated in catabolic signals) and mstn2 (myogenesis negative regulator) was found in EE2-exposed fish.

Vaccination of Gilthead Seabream After Continuous Xenoestrogen Oral Exposure Enhances the Gut Endobolome and Immune Status via GPER1.

In fish culture settings, the exogenous input of steroids is a matter of concern. Recently, we unveiled that in the gilthead seabream (Sparus aurata), the G protein-coupled estrogen receptor agonist G-1 (G1) and the endocrine disruptor 17α-ethinylestradiol (EE2) are potent modulators in polyreactive antibody production. However, the integral role of the microbiota upon immunity and antibody processing in response to the effect of EE2 remains largely unexplored. Here, juvenile seabreams continuously exposed for 84 days to oral G1 or EE2 mixed in the fish food were intraperitoneally (i.p.) immune primed on day 42 with the model antigen keyhole limpet hemocyanin (KLH). A critical panel of systemic and mucosal immune markers, serum VTG, and humoral, enzymatic, and bacteriolytic activities were recorded and correlated with gut bacterial metagenomic analysis 1 day post-priming (dpp). Besides, at 15 dpp, animals received a boost to investigate the possible generation of specific anti-KLH antibodies at the systemic and mucosal interphases by the end of the trial. On day 43, EE2 but not G1 induced a significant shift in the serum VTG level of naive fish. Simultaneously, significant changes in some immune enzymatic activities in the serum and gut mucus of the EE2-treated group were recorded. In comparison, the vaccine priming immunization resulted in an attenuated profile of most enzymatic activities in the same group. The gut genes qPCR analysis exhibited a related pattern, only emphasized by a significant shift in the EE2 group’s il1b expression. The gut bacterial microbiome status underwent 16S rRNA dynamic changes in alpha diversity indices, only with the exposure to oral G1, supporting functional alterations on cellular processes, signaling, and lipid metabolism in the microbiota. By the same token, the immunization elevated the relative abundance of Fusobacteria only in the control group, while this phylum was depleted in both the treated groups. Remarkably, the immunization also promoted changes in the bacterial class Betaproteobacteria and the estrogen-associated genus Novosphingobium. Furthermore, systemic and mucosal KLH-specific immunoglobulin (Ig)M and IgT levels in the fully vaccinated fish showed only slight changes 84 days post-estrogenic oral administration. In summary, our results highlight the intrinsic relationship among estrogens, their associated receptors, and immunization in the ubiquitous fish immune regulation and the subtle but significant crosstalk with the gut endobolome.

Effects of α-ethinyl estradiol (EE2) and diethylhexyl phthalate (DEHP) on growth performance, antioxidant status and immune response of juvenile yellow catfish Pelteobagrus fulvidraco

The four experimental groups were carried out to test the response of yellow catfish to EE2 and DEHP: control group was exposed to DMSO; EE2 group was exposed to 1.0 μg/L EE2; DEHP group was exposed to 1.0 mg/L DEHP; mix group was exposed to 1.0 μg/L EE2 and 1.0 mg/L DEHP. The experiment continued for 56 days. Fish survival rate was not different among experimental groups. Fish in DEHP and mix groups had the highest weight gain, and lowest value appeared in control group. The highest hepatosomatic index was found in DEHP and mix groups. Serum alanine transaminase of fish in control group was lower than other groups, but the alkaline phosphatase value was the highest. Serum total anti-oxidation capacity, superoxide dismutase and catalase activities of fish in control group were higher than other groups, but malondialdehyde content is opposite. Respiratory burst and phagocytic indices of fish in EE2 group were the lowest. After 96 h of ammonia stress, the survival rate of fish in mix group was significantly lower than control group. This study indicates that EE2 and DEHP exposure can lead to gain weight of yellow catfish, which is related to liver damage and fat accumulation; EE2 and DEHP exerts its toxic effects by inducing ROS generation, leading to lipid peroxidation and immunosuppression.

Long-term exposure of xenoestrogens with environmental relevant concentrations disrupted spermatogenesis of zebrafish through altering sex hormone balance, stimulating germ cell proliferation, meiosis and enhancing apoptosis

Environmental estrogens are capable of interfering with the spermatogenesis and fertility of fish. However in natural waters, these chemicals are more likely to occur as a combination rather than a single stressor. Whether and how the mixture of xenoestrogens with environmental relevant concentrations may affect fish spermatogenesis remains largely unknown. In this study, male zebrafish adults were administered to 17alpha-ethinylestradiol (EE2) and a mixture of xenoestrogens (Mix (E2, EE2, DES, 4-t-OP, 4-NP and BPA)), with the estrogenic potency equivalent to EE2. After a 60-day exposures, elevated mRNA levels of vitellogenin 1 (vtg1) and estrogen receptor 1 (esr1) in the liver of fish in both treated groups were observed. Moreover, the plasma level of E2 declined significantly in the Mix group and the ratio of 11-KT/E2 was significantly elevated in both treated groups. Consistently, the mRNA level of P450 side-chain cleavage (scc) in the EE2 group and ovarian type aromatase (cyp19a1a) in the Mix group was significantly suppressed. In addition, decreased gonadosomatic index and sperm count in the fish of Mix group were present. Furthermore, increased number of the proliferating germ cells (such as spermatogonia and spermatocytes) was observed in the fish of both groups, suggesting a stimulated germ cell proliferation and meiosis. Accordingly, both exposures significantly up-regulated the mRNA levels of genes in mitosis (cyclinb1) and meiosis (cyp26a1 in EE2 group, aldh1a2, cyp26a1, sycp3 and spo11 in Mix). In addition, decreased number of spermatozoa and increased number of TUNEL-positive signals were present in the testis of fish in the Mix group, indicating an enhanced apoptosis. Further analyses demonstrated the significant elevated expressions of tnfrsf1a and the ratio of tnfrsf1a/tnfrsf1b in the Mix group, suggesting an elevated apoptosis in the testis of fish in the Mix group via extrinsic pathway. The present study greatly extends our understanding of the underlying mechanisms of the reproductive toxicity of xenoestrogens on fish.

Prenatal bisphenol A (BPA) exposure alters the transcriptome of the neonate rat amygdala in a sex-specific manner: a CLARITY-BPA consortium study

Bisphenol A (BPA) is a widely recognized endocrine disruptor prevalent in many household items. Because experimental and epidemiological data suggest links between prenatal BPA exposure and altered affective behaviors in children, even at levels below the current US FDA No Observed Adverse Effect Level (NOAEL) of 5mg/kg body weight (bw)/day, there is concern that early life exposure may alter neurodevelopment. The current study was conducted as part of the CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) program and examined the full amygdalar transcriptome on postnatal day (PND) 1, with the hypothesis that prenatal BPA exposure would alter the expression of genes and pathways fundamental to sex-specific affective behaviors. NCTR Sprague-Dawley dams were gavaged from gestational day 6 until parturition with BPA (2.5, 25, 250, 2500, or 25000μg/kg bw/day), a reference estrogen (0.05 or 0.5μg ethinyl estradiol (EE2)/kg bw/day), or vehicle. PND 1 amygdalae were microdissected and gene expression was assessed with qRT-PCR (all exposure groups) and RNAseq (vehicle, 25 and 250μg BPA, and 0.5μg EE2 groups only). Our results demonstrate that that prenatal BPA exposure can disrupt the transcriptome of the neonate amygdala, at doses below the FDA NOAEL, in a sex-specific manner and indicate that the female amygdala may be more sensitive to BPA exposure during fetal development. We also provide additional evidence that developmental BPA exposure can interfere with estrogen, oxytocin, and vasopressin signaling pathways in the developing brain and alter signaling pathways critical for synaptic organization and transmission.

Transgenerational effects of 17α-ethinyl estradiol on anxiety behavior in the guppy, Poecilia reticulata

Environmental contaminants can cause alterations that can be transgenerationally transmitted to subsequent generations. Estrogens are among those contaminants shown to induce heritable changes that persist over generations in mammals. Results in other vertebrates are few. We have analyzed the effects on anxiety of 17α-ethinyl estradiol (EE2) in the F1 and F2 generations in guppies, Poecilia reticulata, obtained from F0 fish maternally exposed to 0 or 20ng/L EE2 until birth. F0 males and females were bred with fish of the same treatment but different families producing F1 offspring. Behavior in the novel tank test at 6months revealed that males with EE2-exposed parents had significantly longer latency to the upper half of the tank than control males, while no EE2 effects were observed in females. Also in F2, obtained from F1 as above, males in the EE2 group had longer latency time compared to control males, with no differences due to EE2-exposure of F0 observed in females. In the scototaxis (light/dark preference) test, latency to first transition to black compartment and total transitions to black were significantly altered in females due to EE2 exposure of F0 while the total time in black was higher in males with EE2-exposed F0 compared with controls. The increased anxiety in the F2 generation demonstrates a transgenerational anxiety phenotype and shows that non-reproductive behavior can be transgenerationally modified by estrogens in fish.

Environmentally relevant exposure of 17α-ethinylestradiol impairs spawning and reproductive behavior in the brackish medaka Oryzias melastigma

We assessed the effects of 17α-ethinylestradiol (EE2) on spawning and reproductive behavior of the brackish medaka Oryzias melastigma at environmental concentrations. Breeding pairs were exposed to EE2 at either lower concentrations (0, 1, and 10ng/L) or higher concentrations (0, 50, and 100ng/L) for 14days. Swimming performance and reproductive behavior (following, dancing, and copulation) of the males were analyzed at the end of exposure. Additionally, gonad histology was conducted in the males in the higher EE2 experiment. We found that spawning was significantly inhibited in the 50–100ng/L EE2 groups. Swimming performance was not affected in all groups. Dancing and copulation were significantly suppressed in the 50–100ng/L groups. No effect was observed in testis histology in the 50–100ng/L groups. Our data suggests that reproductive behavior of O. melastigma can be utilized as a behavioral endpoint for ecotoxicology studies of endocrine disruptors in estuarine/marine environments.

Chronic exposure to 17α-ethinylestradiol reduces behavioral consistency in male Siamese fighting fish

Both acute and chronic exposure to 17α-ethinylestradiol (EE2), one of the primary active ingredients of birth control pills, decreases courtship and aggression in males of aquatic species. However, how exposure affects behavioral consistency over time is not commonly addressed. To examine the effects of chronic EE2 exposure on behavioral consistency, adult male Siamese fighting fish, Betta splendens, received multiple trials with a dummy male and dummy female presented simultaneously both before and after chronic exposure to a nominal dose (10 ng/l) of EE2. The responses of exposed males were compared before and after exposure and against those of a group of unexposed, control males. Male-directed and female-directed behaviors were reduced after 28 days of EE2 exposure. More importantly, chronic EE2 exposure had dramatic effects on consistent individual differences in female-directed and male-directed behaviors. Repeatability values were markedly lower and level of response was less correlated in the EE2 group, suggesting that exposure disrupts both between-individual variation and behavioral consistency. These results cannot be explained by a temporal effect as they were not found in the control group of unexposed males. This study demonstrates that EE2 exposure may have effects beyond a reduction in overall response. It also demonstrates the importance of studying the effects of chemical exposure on multiple time scales as acute exposure to EE2 affects the consistency of only female-directed behaviors in this species whereas chronic exposure affects consistency of both female-directed and male-directed behavior. Finally, this study suggests that males in this species may differ in their sensitivity, an important area for future research.

Estrogen-like Effects in Male Goldfish Co-exposed to Fluoxetine and 17 Alpha-Ethinylestradiol

The antidepressant fluoxetine (FLX) and the synthetic estrogen, 17 alpha-ethinylestradiol (EE2), are present in municipal sewage discharges. To better understand possible interactions between them, male goldfish were exposed to an ethanol control or to nominal concentrations of FLX (0.54 μg/L) and EE2 (5 ng/L) alone and in combination for 14 days. Real-time reverse-transcription polymerase chain reaction was used to assess effects on hepatic gene expression and liquid chromatography tandem mass spectrometry to analyze the plasma proteome. The results showed an increase in estrogen receptor alpha (esr1) and vitellogenin (vtg) gene expression by 1.9-2.4-fold in the FLX and EE2 groups, but this did not reach statistical significance. In contrast, co-exposure up regulated esr1 and vtg gene expression by 5.5- and 5.3-fold, respectively. Fluoxetine and EE2 alone did not affect estrogen receptor beta (esr2), but the co-exposure down regulated esr2 expression by 50%. There was a significant increase in the number of plasma proteins that were related to endocrine system disorders in the FLX and FLX plus EE2 groups. The level of VTG protein was increased in the plasma from goldfish exposed to EE2, FLX, and FLX plus EE2. Our study demonstrates that low concentrations of FLX and EE2 in a simple mixture produce strong estrogen-like effects in the male goldfish.

Abstract 1390: In utero estrogenic exposures increase tamoxifen resistance in a new pre-clinical model of estrogen receptor positive breast cancer.

Abstract Maternal estrogenic exposures during pregnancy increase breast cancer risk among daughters in humans and in animal studies, possibly through epigenetic mechanisms. In addition, our findings obtained using systems biology tools to analyze mRNA microarray data suggest that in utero estrogenic exposures reduce the efficacy of antiestrogen treatment. Here we tested whether development of tamoxifen (TAM) resistance is pre-programmed in the offspring by maternal exposure to estradiol during pregnancy. Pregnant rats were exposed either to 0 or 0.1 ppm ethinyl estradiol (EE2) during pregnancy, and their offspring were treated with DMBA to induce ER+ mammary tumors. Untreated mammary tumors in the EE2 offspring and in women who subsequently recurred (in Loi dataset), exhibited reduced expression of genes in the ATF6 pathway of unfolded protein response (UPR), including an apoptosis-inducing gene CHOP compared with tumors in the control rats or in women who did not recur. In addition, gene microarray analysis identified SEMA3b (p&amp;lt;.012) as down-regulated and KAL1 (p&amp;lt;.02) as up-regulated in the mammary tumors in the EE2 offspring and in TAM resistant LCC9 human breast cancer cells. SEMA3b is a tumor suppressor, perhaps because it induces apoptosis through Akt-mediated pathway, and is found to be methylated in clinical breast cancer and LCC9 cells. In turn, TGF-β regulated KAL1 gene is overexpressed in breast cancer and suggested to induce cell survival and cancer metastasis. TAM treatment (15 mg/kg TAM via feed) was started when first tumor reached a size of 1.3±0.2 cm in diameter. In rats that exhibited a TAM-resistant tumor, valproic acid (HDAC inhibitor) and hydralazine (DNMT inhibitor) were added as a 2nd line treatment. Control rats were more likely to exhibit complete response to TAM (54%) than in utero EE2 exposed rats (38%), and de novo resistance was also lower in the controls (38%) than in the EE2 group (53%). Acquired resistance (re-growth after a complete response to TAM), characterized by over-expression of UPR gene GRP78, was seen in 23% of the tumors in the control and 42% in the EE2 group (p&amp;lt;0.007). Adding HDACi-DNMTi as a 2nd line of treatment significantly reduced de novo (from 53% to 19%, p&amp;lt;0.001) and acquired TAM resistance (from 42% to 6%, p&amp;lt;0.001) in the EE2 offspring, but did not affect TAM resistance in the controls. Treatment with HDACi-DNMTi also reversed the changes in SEMA3b and KAL1 in the rat tumors and reversed TAM resistance in LCC9 cells. Our findings suggest that in utero estrogenic exposures increase TAM resistance by inducing epigenetic changes leading to down-regulation of specific tumor suppressor genes and up-regulation of pro-survival genes. These changes could be reversed by adding HDACi-DNMTi to TAM treatment. Our results may have important clinical implications for treatment of antiestrogen refractory ER+ breast cancer. Citation Format: Nguyen Nguyen, Anni Warri, Robert Clarke, Katherine Cook, Idalia Cruz, Sonia de Assis, Dominic Kim, Jin Lu, Alan Zwart, Jason Xuan, Leena Hilakivi-Clarke. In utero estrogenic exposures increase tamoxifen resistance in a new pre-clinical model of estrogen receptor positive breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1390. doi:10.1158/1538-7445.AM2013-1390

Developmental treatment with bisphenol A causes few alterations on measures of postweaning activity and learning

Widespread bisphenol A (BPA) exposure necessitates increased knowledge of its potential effects for better risk assessment and regulatory guidance. Here, female Sprague–Dawley rats, reared in low exogenous estrogen environments and bred at adulthood, were gavaged on gestational days 6–21 with vehicle (VEH), 2.5 or 25.0μg/kg/day BPA, or 5.0 or 10.0μg/kg/day ethinyl estradiol (EE2). Offspring were orally treated on postnatal days (PNDs) 1–21 with the same dose their dam received. A naïve control group (NC) was not gavaged. Post-weaning, one offspring/sex/litter (n=11–12/sex/group) was assessed for the typical behaviors measured in developmental neurotoxicology studies. At PND 29, novelty preference was unaffected by treatment; however, relative to the VEH group, males and females of both EE2 groups were more active. VEH males appeared somewhat hypoactive in open field assessments at PNDs 40–42 and, as a result, males of the BPA and EE2 groups were significantly more active. Latency to locate the Barnes maze escape box at PNDs 47–50 was increased in males and females of the 5.0μg/kg/day EE2 group. Relative to other male groups, VEH males exhibited an increased startle response on the first trial block at PND 54 and thus, males of both BPA groups and the 10.0μg/kg/day EE2 group exhibited a significantly decreased startle response. PNDs 43–44 motor coordination and PNDs 75–79 water maze performance were unaffected by treatment. These results indicate few consistent or dose-related effects resulting from developmental treatment with BPA at these doses. Few of these behaviors, however, were sexually dimorphic which may prove more sensitive.

Short-term exposure to an endocrine disruptor affects behavioural consistency in male threespine stickleback

It has been established that exposure to xenooestrogens can have profound effects on reproductive and aggressive behaviours in aquatic organisms. Studies on the effects of 17α-ethinyloestradiol (EE2) on behaviour, however, rarely examine these effects over multiple trials, across time, or across contexts. In addition, it is possible that individuals vary in their sensitivity to EE2 and individuals may differ in the degree to which their behaviour is affected. The study of consistent individual differences in behaviour has grown in popularity in recent years perhaps because of the evolutionary questions this behavioural variation raises. Adult male threespine stickleback ( Gasterosteus aculeatus) from an anadromous population repeatedly received the simultaneous presentation of a dummy male and dummy female conspecific both before and after exposure to a short-term, nominal (15 ng L −1) concentration of 17α-ethinyloestradiol (EE2). These before and after measures were then compared against one another as well as against responses of males in a control group that was not exposed to EE2. Courtship, aggression, and nesting behaviours were all affected by EE2 exposure. In addition, behavioural consistency over trials was markedly reduced after EE2 exposure, as shown by lower repeatability values in the after exposure measures and in weaker correlations between the before and after exposure measures in the EE2 group compared to the control group. This suggests that male threespine stickleback vary in their sensitivity to EE2. This study is one of the first to examine the effects of short-term EE2 exposure on behaviour across trials and has important implications for population-level fitness consequences of xenooestrogens.

Effects of 17β-trenbolone in male eelpout Zoarces viviparus exposed to ethinylestradiol

To evaluate the interaction between 17beta-trenbolone (TB) and 17alpha-ethinylestradiol (EE2), male eelpout, Zoarces viviparus, was exposed for 21 days (April to May 2008) to 5 ng l(-1) EE2 and 5 or 20 ng l(-1) TB, separately or in combination in a flow-through SW system. The effects on hepatosomatic (HSI) and gonadosomatic index (GSI), plasma vitellogenin (Vtg) concentration, gonadal histology, hepatic and testicular Vtg mRNA and estrogen receptor (ERalpha) mRNA expression were investigated. No effects on HSI were observed. A significant decrease was observed in the GSI of all males exposed to EE2 (<0.7%) when compared to controls (1.4%). Histological alterations and immature stages were observed in the testis of all exposed males; however, males exposed to EE2 were the most affected. Increased tubule number and proportionally decreased tubule diameter were observed in the testis of all EE2 groups. No effects in Vtg mRNA expression were observed in the testis; however, a significant decrease in testis ERalpha mRNA was observed in males exposed to 20 ng l(-1) TB. The groups exposed to EE2 showed a significant increase in plasma Vtg (>300-fold), hepatic Vtg mRNA (>450-fold), and ERalpha mRNA (>100-fold) when compared to controls. This study shows that lower concentrations of 17beta-trenbolone are unable to counteract the EE2 estrogenic effects when the exposure is simultaneous.

Advanced fluorescence in situ hybridization to localize and quantify gene expression in Japanese medaka (Oryzias latipes) exposed to endocrine‐disrupting compounds

In an earlier study, we described the development of fluorescence in situ hybridization (FISH) using confocal microscopy to localize and quantify gene expression in fish. Here, we report the results of FISH application to investigate effects of model endocrine-disrupting chemicals (EDCs), 17alpha-ethinylestradiol (EE2) and 17beta-trenbolone (TB), on expressions of EDC-responsive genes in Japanese medaka (Oryzias latipes) at the cellular/tissue level paired with histological observation. Gene expressions of vitellogenin-II (Vit-II), androgen receptor (AR), and cytochrome P450 gonadal aromatase (CYP19a) were determined after exposure to 5, 50, or 500 ng/L of EE2 or 50, 500, or 5,000 ng/L of TB for 7 d. Exposure to the greatest concentration of EE2 or TB significantly reduced fecundity and caused histological alterations in gonads. 17alpha-Ethinylestradiol induced Vit-II expression in both male gonads and liver relative to controls and resulted in greater intensity of hematoxylin staining in hepatocytes, which was significantly correlated with Vit-II induction in liver. When exposed to EE2 at less than 50 ng/L, CYP19a expression associated with early stage oocytes was greater than that in controls. However, at 500 ng/L, this trend was reversed. The greater Vit-II expression in testis from all EE2 groups, and the lesser expression of CYP19a in ovaries from the 500 ng/L group, likely is related to changes in the number of cells in which these genes are predominantly expressed rather than to an increase in expression per cell. 17beta-Trenbolone significantly induced AR expression in ovaries but did not alter AR expression in female liver. It was concluded that FISH combined with histology enables advanced elucidation of molecular effects of chemicals by associating changes in gene expression with certain tissues and/or cell types and allows these changes to be related to histological effects.

Low-dose estrogen treatment suppresses periosteal bone formation in response to mechanical loading

Estrogen and exercise influence cortical bone formation. Both affect bone during growth, but with complex interactions. We hypothesized that estrogen reduces the osteogenic response caused by exercise at the periosteal surface of bone, while it enhances bone formation on the endocortical surface. To test our hypothesis, 16 young (8 weeks old) male Sprague-Dawley rats were randomized into two groups: (1) low-dose 17-α ethynylestradiol treatment+bone loading (EE2) or (2) vehicle-treated+bone loading (vehicle). We applied controlled loading to the right ulna at a peak force of 17 N, 2 min/day, 3 days/week for 5 weeks to simulate exercise. The left nonloaded ulna served as an internal control for loading. Mechanical loading increased cortical area (7.7%) and bone mineral content (8%) in the vehicle-treated group (P<0.05) but only slightly increased cortical area in the EE2 group (P=0.08). Histomorphometry showed 1 week of mechanical loading increased periosteal bone formation rate by 29% in the vehicle group and this response was reduced (P<0.05) to only 15% in the EE2 group. At the endocortical surface, there were no differences in the loading response between the vehicle and EE2-treated groups. We conclude low-dose EE2 suppresses the mechanical loading response on the periosteal surface of long bones, but had no effect on the loading response at the endocortical bone surface in growing male rats.