DNA methylation and expression of estrogen receptor alpha in fathead minnows exposed to 17α-ethynylestradiol.

The gene expression response thought to underlie the negative apical effects resulting from estrogen exposure have been thoroughly described in fish. Although epigenetics are believed to play a critical role translating environmental exposures into the development of adverse apical effects, they remain poorly characterized in fish species. This study investigated alterations of DNA methylation of estrogen receptor alpha (esr1) in brain and liver tissues from 8 to 10 month old male fathead minnows (Pimephales promelas) after a 2d exposure to either 2.5 ng/L or 10 ng/L 17α-ethynylestradiol (EE2). Changes in the patterns of methylation were evaluated using targeted deep sequencing of bisulfite treated DNA in the 5' region of esr1. Methylation and gene expression were assessed at 2d of exposure and after a 7 and 14d depuration period. After 2d EE2 exposure, males exhibited significant demethylation in the 5' upstream region of esr1 in liver tissue, which was inversely correlated to gene expression. This methylation pattern reflected what was seen in females. No gene body methylation (GBM) was observed for liver of exposed males. Differential methylation was observed for a single upstream CpG site in the liver after the 14d depuration. A less pronounced methylation response was observed in the upstream region in brain tissue, however, several CpGs were necessarily excluded from the analysis. In contrast to the liver, a significant GBM response was observed across the entire gene body, which was sustained until at least 7d post-exposure. No differential expression was observed in the brain, limiting functional interpretation of methylation changes. The identification of EE2-dependent changes in methylation levels strongly suggests the importance of epigenetic mechanisms as a mediator of the organismal response to environmental exposures and the need for further characterization of the epigenome. Further, differential methylation following depuration indicates estrogenic effects persist well after the active exposure, which has implications for the risk posed by repeated exposures..

Assessing the effects of exposure timing on biomarker expression using 17beta-estradiol.

Temporal and spatial variability in estrogenicity has been documented for many treated wastewater effluents with the consequences of this variability on the expression of biomarkers of endocrine disruption being largely unknown. Laboratory exposure studies usually utilize constant exposure concentrations which may produce biological effects that differ from those observed in organisms exposed in natural environments. In this study, we investigated the effects of differential timing of exposures with 17beta-estradiol (E2) on a range of fathead minnow biomarkers to simulate diverse environmentally relevant exposure profiles. Two 21-day, replicate experiments were performed exposing mature male fathead minnows to E2 at time-weighted mean concentrations (similar average exposure to the contaminant during the 21-day exposure period; 17ng E2/L experiment 1; 12ng E2/L experiment 2) comparable to E2 equivalency values (EEQ) reported for several anthropogenically altered environments. A comparable time-weighted mean concentration of E2 was applied to five treatments which varied in the daily application schema: E2 was either applied at a steady rate (ST), in a gradual decreasing concentration (HI), a gradual increasing concentration (LO), applied intermittently (IN), or at a randomly varying concentration (VA). We assessed a range of widely used physiological (vitellogenin mRNA induction and plasma concentrations), anatomical (body and organ indices, secondary sex characteristics, and histopathology), and behavioral (nest holding) biomarkers reported to change following exposure to endocrine active compounds (EACs). All treatments responded with a rise in plasma vitellogenin concentration when compared with the ethanol carrier control. Predicatively, vitellogenin mRNA induction, which tracked closely with plasma vitellogenin concentrations in most treatments was not elevated in the HI treatment, presumably due to the lack of E2 exposure immediately prior to analysis. The ability of treatment male fish to hold nest sites in direct competition with control males was sensitive to E2 exposure and did yield statistically significant differences between treatments and carrier control. Other biological endpoints assessed in this study (organosomatic indices, secondary sex characteristics) varied little between treatments and controls. This study indicates that a broad suite of endpoints is necessary to fully assess the biological consequences of fish exposure to estrogens and that for at least field studies, a combination of vitellogenin mRNA and plasma vitellogenin analysis are most promising in deciphering exposure histories of wild-caught and caged fishes.

Screening California surface waters for estrogenic endocrine disrupting chemicals (EEDC) with a juvenile rainbow trout liver vitellogenin mRNA procedure.

Concern regarding the occurrence of chemicals that disrupt endocrine system functions in aquatic species has heightened over the last 15 years. However, little attention has been given to monitoring for estrogenic endocrine disrupting chemicals (EEDCs) in California's freshwater ecosystems. The objective was to screen surface water samples for estrogenic activity using vitellogenin (Vtg) mRNA quantification in livers of juvenile rainbow trout by real-time reverse transcriptase polymerase chain reaction (Q-RT PCR). Vtg mRNA analysis of livers from fish exposed to 113 ambient water samples collected from surface waters in California's Central Valley and northern area indicated that six samples (5% of total) may have contained EEDCs. The six samples induced marginal, but statistically significant, increases of Vtg mRNA. No ambient water sample evoked Vtg mRNA responses equivalent to those in positive controls (all responses were less than 2% of the positive control response). Thus, EEDC concentrations in these samples were low (at or near the threshold for the procedure) or results may have included false positives. To establish a more definitive assessment of EEDC occurrence, follow-up screening at sites where statistically significant, but weak, estrogenic activity was observed is recommended. Overall, results reveal that a majority of the California surface waters tested were below EEDC detection threshold concentration for the screening procedure utilized.