ASSESSMENT OF ESTROGENIC AND CYP1A1-INDUCING CHEMICALS IN THE HIGHLY EFFLUENT-DOMINATED ASSABET RIVER

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0231092
• Project Start Date: Oct 1, 2012
• Project End Date: Sep 30, 2016
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003

Performing Department
Veterinary & Animal Science

Non Technical Summary
There is concern over the presence of pollutants in our water supply. Among the pollutants that have elicited the greatest alarm are those that can alter the development and reproduction of aquatic organisms and potentially harm human health. These chemicals, generally referred to as endocrine disrupting compounds (EDCs), enter waterways as runoff from farms, effluent from wastewater treatment and manufacturing plants, and leachate from septic systems. The Assabet River in eastern Massachusetts is a highly impacted waterway with nine towns along the 31 miles of the mainstem. Four municipal wastewater treatment plants; Westborough, Marlborough, Hudson, and Maynard discharge into the Assabet. Seven of the nine Assabet main towns take all or part of their public water supply from groundwater and surface sources in the Assabet Watershed, greatly reducing flow. It's estimated that at certain times of the year as much as 90% of the Assabet River is effluent. Two groups of EDCs frequently detected in effluent are estrogenic and dioxin-like (also called CYP1A1-inducing) compounds. All estrogens, both natural hormones and synthetic compounds can to some extent 'turn on' genes to produce proteins that make cells grow and divide. 17beta-estradiol is a natural estrogen, which is critical for normal growth and development. Levels of 17beta-estradiol vary during development, between the sexes, and, for adult women, throughout the month. Exposure to estrogens, either natural or synthetic, outside of the range needed for normal functioning can disrupt cellular activity and increase the risk of developing disease. A wide range of natural and synthetic compounds share a molecular structure that allows induce the production of the enzyme, cytochrome P4501A1 or CYP1A1. CyP1A1 plays a pivotal role in metabolizing drugs and environmental pollutants, as well as natural hormones and other compounds produced by the body. Overproduction of the enzyme CYP1A1 is associated with increased risk of several diseases. Thus exposure to CYP1A1-inducing compounds can be a serious health risk. Many human-made compounds including polycyclic aromatic hydrocarbons and dioxin-like compounds, bind the aryl hydrocarbon receptor with high affinity. Given the highly-impacted nature of the Assabet River and the ability of many compounds to mimic the actions of estrogenic and CYP1A1-inducing compounds, we have selected to use a biological assay to monitor the Assabet River for the presence of these EDCs. We will use the Japanese medaka, a small fresh water fish, easily maintained in the laboratory to monitor water from the Assabet River. When male Japanese medaka are exposed to estrogenic compounds a gene is turned on that is normally off in males. Measuring mRNA of this gene, vitellogenin, allows us to determine the levels of estrogens in the water. Likewise measuring the extent to which exposure to water from the Assabet River turns on the CYP1A1 gene, allows us to know how much CYP1A1-inducing compounds are in the Assabet. Monitoring the levels and locations of EDCs in the Assabet River can be useful in protecting the aquatic environment.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
112	0399	1150	100%

Knowledge Area
112 - Watershed Protection and Management;

Subject Of Investigation
0399 - Watersheds and river basins, general;

Field Of Science
1150 - Toxicology;

Keywords

estrogens

vitellogenin

cyp1a1

assabet river

medaka

edcs

fish bioassays

Goals / Objectives
1. Monitor the Assabet River and its tributaries for the presence of estrogenic- and CYP1A1-inducing compounds. 2. Identify seasonal changes in the presence of estrogenic- and CYP1A1-inducing compounds. 3. Determine whether storm events result in an increase in the presence of estrogenic- and CYP1A1-inducing compounds. 4. Identify sites that need remediation, e.g. effluent from waste water treatment plants or residential septic systems. 5. Work with colleagues at USEPA regions 5, 8, 10 and the EPA Office of Research and Development (ORD) in Cincinnati to optimize and standardize a larval fathead minnow bioassay for detecting estrogenic compounds in water.

Project Methods
The overall design of this project is to expose laboratory fish to water collected from the Assabet River and its tributaries and to measure changes in vitellogenin and CYP1A1 mRNA as indicators of estrogenic and CYP1A1-inducing compounds. Water will be collected throughout the year to monitor the river at times of both high and low flow. Water also will be collected after severe rain events to capture the effects of additional runoff and combined sewage overflow. Ideally, we would use only larval fathead minnow (FHM) because this bioassay requires the least amount of water and does not include labor intensive dissections. However, this assay is still being optimized and at present the detection limit with larval FHM is higher than that routinely obtained with either adult FHM or adult Japanese medaka. Therefore we will use adult Japanese medaka to test all water samples. In addition we will collaborate with EPA on optimization of the larval FHM bioassay and will test all water samples in both an adult male medaka assay and a larval FHM assay when available. Exposure Assays: Prior to the start of the assay, male fish between 6 and 8 months old will be housed in groups of 15 per tank for 72 hours to acclimate to an all-male environment. Five male fish will be exposed to either 1 L of test water or control water in 5-L plastic tanks with air stones for 72 hours. At 36 hours each tank will receive an 80% water renewal with appropriate test or control water. Positive controls will be 1 nM 17beta-estradiol (E2) for VTG and 1 mg/L beta-naphthoflavone for CYP1A1. General water chemistries will be recorded at time 0, 36 and 72 hours. After 72 hours fish will be sacrificed and livers harvested and stored in Tri Reagent in a -80C freezer until used for mRNA isolation. RNA Isolation, Vitellogenin & CYP1A1 Analyses: Livers will be homogenized in the TissueLyser II, and mRNA isolated using a standard TRI Reagent protocol. RNA quality as indicated by 260/280 ratio will be recorded, and no sample will be used for gene expression analysis that has a ratio outside the range 1.7-2.0 RNA will be diluted to 0.1 ug/ul in RNase-free water and stored at -80C until real time RT-PCR analysis. Vitellogenin, CYP1A1 and the housekeeping gene ribosomal protein L7 (L7) mRNA will be quantified in each liver RNA sample with gene specific primers designed to span introns. Real time RT-PCR reactions will be run in a Roche Lightcycler in capillary format. For each 15 ul total volume reaction, 0.75 ul of sample RNA (at 0.1 ug/ul) will be added to master mix prepared with the Qiagen One-Step RT-PCR kit with SYBR green dye for fluorescent detection and primers. Data Analysis: Vitellogenin and CYP1A1 mRNA level will be normalized to the L7 mRNA level measured in each sample to obtain relative expression levels. Mean vitellogenin and CYP1A1 expression levels will be compared across groups with one-way ANOVAs (with p <0.05).

Progress 10/01/12 to 09/30/16

Outputs
Target Audience:The target audiences include scientists, policy makers and citizens concerned about the environment. Changes/Problems:The overall goal of this project was to further our understanding of human impacts on our nation's water supply and the organisms that live in the water. In addition to working with the EPA to develop standard operating procedures for the vitellogenin expression assay, we worked on two projects investigating 1) human impacts on DNA methylation in bonefish (Albula spp.), and 2) impacts of global warming on DNA methylation in brook trout (Salevelinus fontinalis). For both of these projects, the major contribution of my laboratory was to provide the expertise in DNA methylation analysis. DNA methylation was chosen as the optimal biomarker because it is relatively stable and affected by a range of environmental exposures. Project 1. For the analysis of DNA methylation in bonefish we worked with Professor Andy Danylchuk in the Department of Natural Resources Conservation. We introduced Dr. Danylchuk to a non-lethal method of collecting fish blood on filter paper and we optimized method for extraction of the DNA from the fish blood. Dr. Danylchuk collected blood samples from fish in heavily and less impacted regions of the Florida Keys and the Bahamas. We are near completion of the analysis of DNA methylation in these samples and have begun preparation of a manuscript. Project 2. For the analysis of DNA methylation in brook trout, we provided Dr. Tara Duffy with training and access to a pyrosequencer. Dr. Duffy was a Post Doctorate Fellow working with Dr. Stephen McCormick at the S.O. Conte Anadromous Fish Research Laboratory, USGS in Turners Falls, MA. A manuscript describing the results of this study is under review. What opportunities for training and professional development has the project provided?Training opportunities: During the last year one technician and two undergraduate students received training in molecular biology and statistics as a direct outcome of this project. 1. Elizabeth Punska (technician in the Arcaro lab) was trained in methods of extracting fish DNA from filter paper and examining DNA methylation. Ms. Punska also received training in statistical methods and data preparation. 2. Cassidy Cunningham (Biochemistry major, class of 2016) was trained in methods of DNA extraction and in assay development. Ms. Cunningham assisted Dr. Arcaro and Ms. Punska in developing an assay to estimate DNA methylation in bonefish based on pyrosequencing bisulfite-modified DNA that had been amplified for a repetitive element. 3. Oshiomah Oyageshio (Biochemistry major, class of 2018) received training in basic molecular biology techniques including, extraction and purification of DNA, comparison of methods to evaluate DNA quantity and quality, assessment of DNA methylation using pyrosequencing of fragmented DNA after incubation with methylation specific and tolerant enzymes. Mr. Oyageshio also received training in statistics and study design. Professional development: During the last year two undergraduate students participated in activities related to this project that enhanced their professional development. 1. Cassidy Cunningham presented the results of her research on bonefish and the Massachusetts Undergraduate Research Symposium in April 2016. 2. Oshiomah Oyageshio presented results from his research at a meeting of Dr. Arcaro's colleagues. How have the results been disseminated to communities of interest?During the last year we have taken steps towards disseminating results to communities of interest by submitting two manuscripts for publication (presently under review), and preparing a third manuscript for submission. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? It is well known that human activities are impacting the quality of our water supply by releasing estrogenic compounds into the environment. Exposure to these estrogens has the potential to disrupt reproduction in aquatic animals as well as humans, and may be linked to other health problems. Because environmental estrogens can dispute the function of natural estrogens in the body's endocrine system, they are referred to as endocrine disrupting compounds (EDCs). Complex mixtures of EDCs can enter the environment from wastewater treatments plants, biosolids runoff, and concentrated animal feeding operations, among other human operations. Monitoring and limiting the release of EDCs is a major goal of regulating agencies, however, the complexity of the EDC mixtures makes chemical analysis an unreasonable solution for monitoring environmental inputs. Fish bioassays have been embraced as sensitive and cost effective solution to assessing the estrogenic potential of wastewater and runoff. However, challenges remain because standardized methods of using fish bioassays to quantify estrogenic activity have not been developed. The lack of standardization complicates interpretations among laboratories participating in screening programs. We collaborated with USEPA regions 5, 8 and ORD to standardize methods of assessing the egg yolk precursor protein, vitellogenin, as a biomarker of estrogen exposure in male fish. We assisted in the development of a standard operating procedure (SOP) to evaluate vitellogenin gene (vtg) expression. The SOP was applied to samples collected from studies of male fathead minnows (Pimephales promelas) exposed to 17α-ethinylestradiol (EE2) and minnows exposed to processed wastewater effluent. Variability in gene expression among four laboratories was assessed. Results indicated reasonable consistency among laboratories if LinRegPCR expression analysis was used, with three of four detecting vtg in fish exposed to 5 ng/L EE2 (n = 5). All laboratories significantly detected vtg in male fish exposed to wastewater effluent (n = 15). Finally, the source of high inter-laboratory variability from cDNA to qPCR analyses was determined to be the expression analysis software unique to each real-time quantitative polymerase chain reaction (qPCR) machine. Reanalyzing raw fluorescence data with independent freeware eliminated the inter-laboratory variability. A manuscript describing the results i sunder review.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2017 Citation: MS Title: Tools to Minimize Inter-Laboratory Variability in Vitellogenin Gene Expression Monitoring Programs MS Authors: Denise A. Gordon, Aaron Jastrow, Kasie M. Auger, Elizabeth C. Punska, Kathleen F. Arcaro, Kristen Keteles, Dana Winkelman, David Lattier, Adam Biales, James M. Lazorchak
• Type: Journal Articles Status: Under Review Year Published: 2017 Citation: MS ID#: JEXBIO/2016/155283 MS TITLE: Epigenetic and physiological changes following acclimation to high temperatures in brook trout (Salvelinus fontinalis): evidence for a thermal memory AUTHORS: Tara Duffy, Kathleen Arcaro, and Stephen McCormick

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: The primary target audiences included students at the Univeristy of Massachusetts and students and faculty attening the national meeting of the Society of Environmental Chemistry and Toxicology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Kasie Auger, a graduate student in the Animal Biotechnology Program received her masters degree. Her reserach was based on this NIFA-fundedproject. Dr. Tara Duffy, a postdoctoral fellow at the USGS Anadromous Fish Labortatory at Turnes Falls, MA, conducted part of her research in the Arcaro laboratory using supplies and equipmentobtianed through this NIFA-funded project. Dr. Sangram Parelkar, a postdoctoral fellow in the Polymer Science Department at UMass-Amhers, conducted part of his research in the Arcaro laboratory using supplies and equipmentobtianed through this NIFA-funded project. Dr. Kathleen Arcaro provided guidance and training to Kasie Auger and Drs. Duffy and Parelkar. How have the results been disseminated to communities of interest? Results have been presented at two national meetings and will be presented at another meeting in January 2014. What do you plan to do during the next reporting period to accomplish the goals? We are working with Dr. David Reckhow in Environmental Engineering to collect and analyze water sampels after flood events.

Impacts
What was accomplished under these goals? We have made significant progress on goals 1 and 5. Goal 1. We have tested water mulitple in-streamlocations and effluents and a manuscript is in prepartion. Goal 5. We have worked with the EPA to develop standarized protocls for assesing estrogenic activity; a protocol has been submitted and a manuscript is in preparation.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: A whole-effluent toxicity method for estrogenic wastewater and inter-laboratory comparisons from mesocosm, laboratory, and field exposures. Authors: Adam R. Schwindt, Aaron Jastrow, Denise Gordon, Kathleen Arcaro, Kasie Auger, Kristen Keteles, Dana Winkelman, and James Lazorchak Presentation at the 2013 Annual Meeting of the Society of Environmental Toxicology and Chemistry, Nashville TN
• Type: Theses/Dissertations Status: Other Year Published: 2013 Citation: INVESTIGATING THE ENDOCRINE DISRUPTING POTENTIAL OF THE EFFLUENT-DOMINATED ASSABET RIVER; A Thesis Presented By KASIE M. AUGER; April, 2013. University of Massachusetts
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Poster presentation at the Annual Meeting of The Society for Integrative and Comparative Biology, January 3-7, 2014 at Austin, Texas. DNA methylation in brook trout (Salvelinus fontinalis) following acclimation to thermally stressful environments. Tara A. Duffy, Kathleen Arcaro and Stephen D. McCormick

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: NEW PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
NEW

Publications

• No publications reported this period