Regional investigations into the effects of CECs

A molecular framework to identify novel modes of action of endocrine disrupting compounds in shellfish

Concern over human and wildlife health has brought increased attention to a group of emerging environmental contaminants referred to as endocrine disrupting compounds (EDCs). While progress has been made in describing the effects of these compounds, there are still gaps in our understanding of alternative modes of action and physiological effects outside of the reproductive axis, particularly in invertebrates.  One way that EDCs may elicit these changes is through disruptions to normal epigenetic mechanisms.  Epigenetics refers to heritable processes that alter gene activity without manipulating the underlying DNA sequence.  Epigenetic marks, such as DNA methylation, are important regulators of gene expression in both plants and animals.  This research aims to characterize alternative modes of action of EDCs by utilizing molecular tools to examine epigenetic and physiological changes in Pacific oysters (Crassostrea gigas) exposed to the synthetic estrogen, 17α-ethinylestradiol (EE2).

In this experiment, juvenile oysters were exposed to EE2 during gonad maturation.  Sex-ratio and size were evaluated after two months of exposure.  Results of this exposure include a trend toward more females in the EE2 exposed.  In addition, the EE2 exposed females were significantly larger than unexposed females.  To investigate the molecular underpinnings of this phenotype, DNA methylation profiles of control and EE2 exposed females were directly compared using a DNA tiling microarray (MBD-ChIP) in order to test the hypothesis that invertebrate DNA methylation patterns will be altered upon exposure to EDCs.  This analysis revealed a suite of genes that were differentially methylated in response to EE2.  Functional annotations of these genes indicate that a number of biological pathways outside of the reproductive axis are being affected by exposure to EE2. 

This work was performed in the Steven Roberts research lab at the University of Washington.

Contact:

Mackenzie Gavery - University of Washington (mgavery@uw.edu)

Collaborators:

Steven Roberts - SAFS, University of Washington

Innovative approaches to develop biomarkers for emerging contaminants in the Puget Sound

Assessing the impacts of contaminants of emerging concern (CEC) on Puget Sound aquatic resources is complicated by several important data gaps, including: 1) the lack of an understanding of mechanisms of toxicity for this broadly diverse group of contaminants, 2) a poor understanding of occurrence, distribution and environmental fate of CEC in the Puget Sound, and, 3) relevant ecological receptors for inclusion in biomonitoring.  Our laboratories are approaching these data gaps by monitoring for a broad array of CEC in water and fish in Puget Sound locations that receive effluents from wastewater treatment plants, which are known sources for CEC.  We are using biochemical, molecular and physiological biomarkers in an integrated approach to evaluate exposure and effects of CEC in juvenile Chinook salmon (Oncorhynchus tshawytscha) and adult Pacific Staghorn sculpin (Leptocottus armadus).  We are drawing upon the high site fidelity of staghorn sculpin in assessments of CEC-associated reproductive injury, whereas growth parameters are being analyzed in Chinook salmon.  We will undertake a discovery-based biomarker approach using whole genome microarray studies in field sampled Chinook to generate new biomarkers for CEC.  Important CEC identified in field studies will be further evaluated in the laboratory to provide toxicity thresholds, and to strengthen the association between CEC exposure and effects in field studies.  Ultimately, the CEC biomarkers generated from this work can be applied throughout the Puget Sound region to identify sites and sources, and to better inform decision-making associated with the ecological risks of CECs.

This work is being performed in the Evan Gallagher lab at the University of Washington.

Contact:

Evan Gallagher - School of Public Health, University of Washington (evang3@u.washington.edu)

Collaborators:

The pituitary gland as a target of endocrine disrupting compounds in coho salmon, Oncorhynchus kisutch

There is growing evidence that chemicals in the aquatic environment may interfere with reproduction of fish. In vertebrates, the pituitary gland is a central regulator of reproduction, producing the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which regulate gonadal development, sex steroid synthesis and gamete maturation. Despite the pituitary’s central role in regulating reproduction, there are limited data on impacts of contaminants of emerging concern (CECs) on the pituitary gland. Using high-throughput Illumina® sequencing and RNA-Seq we previously found that LH beta subunit (lhb) and FSH beta subunit (fshb) were among the most significantly up- and down-regulated transcripts, respectively, in immature coho salmon exposed to 17α-ethynylestradiol (EE2) for up to six weeks. The aim of this study was to further our understanding of the potential impacts of CECs on gonadotropin synthesis.

To this end, immature coho salmon were exposed to 0, 25 or 500 ng/L fluoxetine (FLX; antidepressant present in wastewater effluent) or 12 ng/L EE2 for up to 6 weeks in flow-through tanks.  Total pituitary RNA was collected and gonadotropin subunit mRNA levels were analyzed by quantitative RT-PCR.  As we previously found, 12 ng/L EE2 significantly up-regulated lhb mRNA levels at 1 and 6 weeks (450-fold and 1200-fold respectively) and down-regulated fshb at 6 weeks (-3-fold).  Six weeks exposure to at least 25 ng/L fluoxetine also decreased fshb transcript levels (-2-fold) but had no effect on levels of lhb.  In conclusion, we have shown that environmentally relevant levels of EE2 and FLX alter lhb and fshb mRNA levels in immature coho salmon.  These results suggest that CECs may disrupt pituitary function and alter gonadotropin synthesis.  Further research is needed to understand the effects of EE2 and FLX on FSH secretion and possible synergistic effects of EE2 and FLX with other chemicals in the environment.

This work is being performed in the Graham Young research lab at the University of Washington.

Contact:

Louisa Harding - University of Washington (lbh4@uw.edu)

Collaborators:

Graham Young - SAFS, University of Washington