Vicki L. Marlatt

Auto-regulation of estrogen receptor subtypes and gene expression profiling of 17β-estradiol action in the neuroendocrine axis of male goldfish

Auto-regulation of the three goldfish estrogen receptor (ER) subtypes was examined simultaneously in multiple tissues, in relation to mRNA levels of liver vitellogenin (VTG) and brain transcripts. Male goldfish were implanted with a silastic implant containing either no steroid or 17beta-estradiol (E2) (100 microg/g body mass) for one and seven days. Liver transcript levels of ERalpha were the most highly up-regulated of the ERs, and a parallel induction of liver VTG was observed. In the testes (7d) and telencephalon (7d), E2 induced ERalpha. In the liver (1d) and hypothalamus (7d) ERbeta1 was down-regulated, while ERbeta2 remained unchanged under all conditions. Although aromatase B levels increased in the brain, the majority of candidate genes identified by microarray in the hypothalamus (1d) decreased. These results demonstrate that ER subtypes are differentially regulated by E2, and several brain transcripts decrease upon short-term elevation of circulating E2 levels.

The occurrence of steroidal estrogens in south-eastern Ontario wastewater treatment plants.

We measured steroidal estrogens in wastewater in Ottawa and Cornwall (Ontario, Canada) to determine removal efficiency of these steroids during the treatment process, and whether removal varies during a seasonal cycle. Estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) were found at maximum concentrations in raw sewage (RS), at 104, 66.9 and 5.7 ng L(-1), respectively. For the Ottawa wastewater treatment plant (WWTP), there was sufficient data to show that E1 concentrations in RS correlated with both ambient air temperature and mean daily flow of the WWTP (R(2)=0.792, p=0.003 and R(2)=0.757, p=0.005). E1 removal was correlated with the percent difference in cBOD from RS to FE (final effluent) (R(2)=0.435, p=0.075). However estrogenic potency, as determined by a sensitive in vitro reporter gene assay, did not decrease during the water treatment process, suggesting that many estrogenic chemicals are conserved in FE. E1 and EE2 were found in river water, both upstream and downstream of the WWTPs, and at much lower concentrations than in FE. Our study demonstrates the persistence of steroidal estrogens and estrogenic potency in Ontario WWTP effluents and surface waters, and has uncovered temporal patterns of release that may be used to help predict risks to aquatic organisms in these environments.

Defining Global Neuroendocrine Gene Expression Patterns Associated with Reproductive Seasonality in Fish

Background Many vertebrates, including the goldfish, exhibit seasonal reproductive rhythms, which are a result of interactions between external environmental stimuli and internal endocrine systems in the hypothalamo-pituitary-gonadal axis. While it is long believed that differential expression of neuroendocrine genes contributes to establishing seasonal reproductive rhythms, no systems-level investigation has yet been conducted. Methodology/Principal Findings In the present study, by analyzing multiple female goldfish brain microarray datasets, we have characterized global gene expression patterns for a seasonal cycle. A core set of genes (873 genes) in the hypothalamus were identified to be differentially expressed between May, August and December, which correspond to physiologically distinct stages that are sexually mature (prespawning), sexual regression, and early gonadal redevelopment, respectively. Expression changes of these genes are also shared by another brain region, the telencephalon, as revealed by multivariate analysis. More importantly, by examining one dataset obtained from fish in October who were kept under long-daylength photoperiod (16 h) typical of the springtime breeding season (May), we observed that the expression of identified genes appears regulated by photoperiod, a major factor controlling vertebrate reproductive cyclicity. Gene ontology analysis revealed that hormone genes and genes functionally involved in G-protein coupled receptor signaling pathway and transmission of nerve impulses are significantly enriched in an expression pattern, whose transition is located between prespawning and sexually regressed stages. The existence of seasonal expression patterns was verified for several genes including isotocin, ependymin II, GABAA gamma2 receptor, calmodulin, and aromatase b by independent samplings of goldfish brains from six seasonal time points and real-time PCR assays. Conclusions/Significance Using both theoretical and experimental strategies, we report for the first time global gene expression patterns throughout a breeding season which may account for dynamic neuroendocrine regulation of seasonal reproductive development.

Hepatic protein expression networks associated with masculinization in the female fathead minnow (Pimephales promelas).

Endocrine disruptors that act via the androgen receptor (AR) are less well studied than environmental estrogens, and there is evidence that treatment with AR agonists can result in masculinization of female fish. In this study, female fathead minnows (FHM) were exposed to the model nonaromatizable androgen 5-alpha dihydrotestosterone (DHT) (100 μg/L), the ureic-based herbicide linuron (LIN) (100 μg/L), and a mixture of DHT and LIN (100 μg/L each) to better characterize androgen action in females. LIN was used because of reports that this chemical has an antiandrogenic mode of action in fish. After 21d, DHT and LIN treatments resulted in a significant depression of plasma vitellogenin (Vtg) and DHT and DHT+LIN increased the prevalence of nuptial tubercles in female FHMs indicating masculinization. Using iTRAQ and an LTQ Orbitrap Velos, ∼2000 proteins were identified in the FHM liver and the number of proteins quantified after exposures was >1200. Proteins that significantly and consistently changed in abundance across biological replicates included prostaglandin E synthase 3, programmed cell death 4a, glutathione S transferases, canopy, selenoprotein U, and ribosomal proteins. Subnetwork enrichment analysis identified that interferon and epidermal growth factor signaling were regulated by DHT and LIN, suggesting that these signaling pathways are correlated to depressed plasma vitellogenin. These data provide novel insight into hepatic protein networks that are associated with the process of masculinization in teleosts.

Sex- and tissue-specific effects of waterborne estrogen on estrogen receptor subtypes and E2-mediated gene expression in the reproductive axis of goldfish

This research examined the gene expression profile of three goldfish estrogen receptor (ER) subtypes in multiple tissues in relation to mRNA levels of aromatase B and vitellogenin (VTG) following waterborne estrogen exposures. The protocol consisted of: i) adult male goldfish in late gonadal recrudescence exposed to 1 nM 17beta-estradiol (E2); ii) adult male and female goldfish in early sexual regression exposed to 1 nM E2 for 3, 6, 12 and 24h; and, iii) sexually mature, adult male goldfish exposed to 0.3 nM 17alpha-ethynylestradiol (EE2) for 24h. Liver produced the most consistent response with up-regulation of ERalpha in sexually regressed, mature and recrudescing males and in sexually regressed females. The dose and length of exposure, reproductive state and sex affected the auto-regulation of ERbeta1 by E2. ERbeta2 was not affected in any experiments suggesting it may not be auto-regulated by E2. Aromatase B and VTG gene expression were affected by E2, but also by other experimental conditions. EE2 induced liver ERalpha and VTG mRNA levels indicating that high environmental EE2 levels induce E2-mediated gene expression in a model teleost. These studies reveal a more complicated action of estrogenic compounds that has important implications on estrogenic endocrine disruptors in teleosts.

Lumiestrone is Photochemically Derived from Estrone and may be Released to the Environment without Detection.

Endocrine disrupting chemicals are adversely affecting the reproductive health and metabolic status of aquatic vertebrates. Estrone is often the dominant natural estrogen in urban sewage, yet little is known about its environmental fate and biological effects. Increased use of UV-B radiation for effluent treatments, and exposure of effluents to sunlight in holding ponds led us to examine the effects of environmentally relevant levels of UV-B radiation on the photodegradation potential of estrone. Surprisingly, UV-B-mediated degradation leads to the photoproduction of lumiestrone, a little known 13α-epimer form of estrone. We show for the first time that lumiestrone possesses novel biological activity. In vivo treatment with estrone stimulated estrogen receptor (ER) α mRNA production in the male goldfish liver, whereas lumiestrone was without effect, suggesting a total loss of estrogenicity. In contrast, results from in vitro ER-dependent reporter gene assays indicate that lumiestrone showed relatively higher estrogenic potency with the zebrafish ERβ2 than zfERα, suggesting that it may act through an ERβ-selectivity. Lumiestrone also activated human ERs. Microarray analysis of male goldfish liver following in vivo treatments showed that lumiestrone respectively up- and down-regulated 20 and 69 mRNAs, which was indicative of metabolic upsets and endocrine activities. As a photodegradation product from a common estrogen of both human and farm animal origin, lumiestrone is present in sewage effluent, is produced from estrone upon exposure to natural sunlight and should be considered as a new environmental contaminant.

Triclosan exposure alters postembryonic development in a Pacific tree frog (Pseudacris regilla) Amphibian Metamorphosis Assay (TREEMA)

The Amphibian Metamorphosis Assay (AMA), developed for Xenopus laevis, is designed to identify chemicals that disrupt thyroid hormone (TH)-mediated biological processes. We adapted the AMA for use on an ecologically-relevant North American species, the Pacific tree frog (Pseudacris regilla), and applied molecular endpoints to evaluate the effects of the antibacterial agent, triclosan (TCS). Premetamorphic (Gosner stage 26-28) tadpoles were immersed for 21 days in solvent control, 1.5 μg/L thyroxine (T(4)), 0.3, 3 and 30 μg/L (nominal) TCS, or combined T(4)/TCS treatments. Exposure effects were scored by morphometric (developmental stage, wet weight, and body, snout-vent and hindlimb lengths) and molecular (mRNA abundance using quantitative real time polymerase chain reaction) criteria. T(4) treatment alone accelerated development concomitant with altered levels of TH receptors α and β, proliferating cell nuclear antigen, and gelatinase B mRNAs in the brain and tail. We observed TCS-induced perturbations in all of the molecular and morphological endpoints indicating that TCS exposure disrupts coordination of postembryonic tadpole development. Clear alterations in molecular endpoints were evident at day 2 whereas the earliest morphological effects appeared at day 4 and were most evident at day 21. Although TCS alone (3 and 30 μg/L) was protective against tadpole mortality, this protection was lost in the presence of T(4). The Pacific tree frog is the most sensitive species examined to date displaying disruption of TH-mediated development by a common antimicrobial agent.

The effects of the urea-based herbicide linuron on reproductive endpoints in the fathead minnow (Pimephales promelas).

Linuron is a widely used urea-based herbicide that has anti-androgenic activity in both fish and rodents. To further elucidate the potential mode of action (MOA) of linuron on the vertebrate endocrine system, adult male and female fathead minnows were exposed for 21 days to dechlorinated water, a solvent control, 17β-estradiol (E2; 0.1 μg/L), dihydrotestosterone (DHT; 100 μg/L), linuron (1, 10, 100 μg/L) and one co-treatment of DHT (100 μg/L) and linuron (100 μg/L). There were no effects of linuron on egg hatching, 7 day egg survival, nuptial tubercle formation or gonadal histopathology. Administration of DHT and 1 and 100 μg/L linuron reduced plasma vitellogenin in females, while male plasma vitellogenin were induced after E2 exposure and co-exposure of DHT and linuron. Ovarian mRNA levels were examined for several genes involved in steroidogenesis (e.g. p450scc, cyp19a, star, tspo, hsd17b and hsd11b) and estrogen-mediated responses (esr1, esr2b, esr2a). Only p450scc mRNA was significantly decreased with DHT+linuron co-treatment. Clustering of steroidogenic mRNA transcript expression patterns revealed that patterns for linuron were more similar to E2 compared to DHT. Collectively, this study supports the hypothesis that linuron may not be a pure anti-androgen and may have multiple MOAs that affect vertebrate reproduction.

Interaction of Galaxolide® with the human and trout estrogen receptor-α

Synthetic musks have been detected in sewage effluents, surface waters, and fish tissues where the polycyclic musk compound, HHCB (Galaxolide®) is the dominant compound in those matrices. In the present study, the Galaxolide® formulation was tested in the yeast estrogenicity screening (YES) assay, and also tested in in vitro and in vivo teleost systems to determine whether it interacts with the estrogen receptor as either an agonist or antagonist. In those tests, Galaxolide® did not act as an estrogen agonist, however there was strong evidence of antagonistic activity as Galaxolide® inhibited the estrogenic activity of 17β-estradiol (E2). In the YES assay based on a recombinant strain of yeast containing the human estrogen receptor (i.e. hERα), Galaxolide® inhibited the effects of E2 in a dose-dependent manner (IC50=1.63×10(-5)M). In a luciferase reporter gene assay based on the rainbow trout estrogen receptor (i.e. rtER) transfected into a rainbow trout gonadal (RTG-2) cell line, the IC50 for the antagonistic effect of Galaxolide® was 2.79×10(-9)M. In an in vivo assay based on modulation of vitellogenin in rainbow trout, Galaxolide® i.p. injected into trout at a dose of 3.64mg/kg caused inhibition of E2-induced vitellogenin production. That dose is within the range of concentrations of Galaxolide® that have been detected in tissues of fish from contaminated locations.

Molecular responses to 17β-estradiol in early life stage salmonids.

Environmental estrogens (EE) are ubiquitous in many aquatic environments and biological responses to EEs in early developmental stages of salmonids are poorly understood compared to juvenile and adult stages. Using 17β-estradiol (E2) as a model estrogen, waterborne exposures were conducted on early life stage rainbow trout (Oncorhynchus mykiss; egg, alevin, swim-up fry) and both molecular and physiological endpoints were measured to quantify the effects of E2. To investigate developmental stage-specific effects, laboratory exposures of 1 μg/L E2 were initiated pre-hatching as eyed embryos or post-hatching upon entering the alevin stage. High mortality (∼90%) was observed when E2 exposures were initiated at the eyed embryo stage compared to the alevin stage (∼35% mortality), demonstrating stage-specific sensitivity. Gene expression analyses revealed that vitellogenin was detectable in the liver of swim-up fry, and was highly inducible by 1 μg/L E2 (>200-fold higher levels compared to control animals). Experiments also confirmed the induction of vitellogenin protein levels in protein extracts isolated from head and tail regions of swim-up fry after E2 exposure. These findings suggest that induction of vitellogenin, a well-characterized biomarker for estrogenic exposure, can be informative measured at this early life stage. Several other genes of the reproductive endocrine axis (e.g. estrogen receptors and androgen receptors) exhibited decreased expression levels compared to control animals. In addition, chronic exposure to E2 during the eyed embryo and alevin stages resulted in suppressive effects on growth related genes (growth hormone receptors, insulin-like growth factor 1) as well as premature hatching, suggesting that the somatotropic axis is a key target for E2-mediated developmental and growth disruptions. Combining molecular biomarkers with morphological and physiological changes in early life stage salmonids holds considerable promise for further defining estrogen action during development, and for assessing the impacts of endocrine disrupting chemicals in vivo in teleosts.