Grantee Research Project Results
2000 Progress Report: Detection and Identification of the Toxins from Pfiesteria and Related Harmful Algal Blooms
EPA Grant Number: R826655Title: Detection and Identification of the Toxins from Pfiesteria and Related Harmful Algal Blooms
Investigators: Gawley, Robert E. , Baden, Daniel G.
Institution: University of Miami , University of North Carolina at Greensboro
Current Institution: University of Miami , University of North Carolina at Chapel Hill
EPA Project Officer: Packard, Benjamin H
Project Period: August 1, 1998 through July 31, 2002
Project Period Covered by this Report: August 1, 1999 through July 31, 2000
Project Amount: $353,975
RFA: Ecology and Oceanography of Harmful Algal Blooms (1998) RFA Text | Recipients Lists
Research Category: Water Quality , Nanotechnology , Harmful Algal Blooms , Water , Aquatic Ecosystems
Objective:
This project has three objectives:
- Grow cultures of Pfiesteria and related "Shephard's Crook" dinoflagellates (a not-yet-assigned organism that is present in many Pfiesteria outbreaks), as well as Scripsiellas, Glenodiniums, and Gyrodiniums dinoflagellates. Concentrate the toxins on reversed-phase C-18 HPLC columns by filtration of aquarium water, then elute fractions with organic solvents. Identify toxic fractions by whole-fish bioassay using Tilapia fish fry. Purify the toxins to homogeneity, accumulate enough to allow chemical characterization, and elucidate the toxins' structure using mass spectrometry, nuclear magnetic resonance, infrared spectroscopy, and perhaps x-ray crystallography.
- Use the techniques of combinatorial chemistry to design ligand-selective host(s) that bind toxins from Pfiesteria and related dinoflagellates, while simultaneously effecting a visible signal of such binding in the form of enhanced fluorescence, seeking a host with optimum response to toxin and minimum response to other possible guests. Since the structures of the toxins are not known, this will be accomplished by seeking a correlation of toxicity in toxic fractions with fluorescence signalling of library members on polymer beads. The most highly fluorescent beads could then be produced in bulk for use as a toxin sensor.
- Once hosts that bind the toxin are known, build an affinity column to replace the C-18 column in the aquarium filter, and use this tool to collect the toxin for chemical characterization. This stationary phase could also be used for separating the toxins from other lipophilic components of the toxin extracts. The latter is a prerequisite for work on the biological aspects of these toxins. A large-scale application of this approach might be used in detoxification efforts.
Progress Summary:
Specific Aim 1: Three strains of Pfiesteria piscicida were cultured on Rhodomonas microalgae in Dr. Baden's laboratories in Miami during Year 1. Efforts in Year 2 involved reestablishing these cultures at Dr. Baden's new labs in North Carolina. To the clones that were brought from Miami, we added one that was obtained from the labs of Dr. Karen Steidinger in St Petersburg, Florida, who heads the state's Harmful Algal Bloom task force.
Our goal in this year was two-fold: attempt to use animal blood as a nutrient source for the Pfiesteria cultures in an effort to maintain toxicity, and grow the cultures to larger volumes suitable for extraction. This effort has been met with some frustration on the first point, since the know-how for establishing an entire colony of Pfiesteria piscicida that maintain toxicity to fish has not yet been accomplished. On the other hand, we have enlarged our stocks of the organism and current efforts are focused on inducing and maintaining ichthyotoxicity.
Scientists at the Virginia Institute of Marine Science (VIMS) have established sustainably toxic cultures of a second strain of Pfiesteria, which has yet to be named. For now, it is called Pfiesteria B. We have established a collaboration with these scientists and will begin screening extracts of their strain in November of 2000.
We have established a new bioassay facility in the PIs lab which is equipped with a biosafety cabinet and cell incubator. We have established cell cultures of several epidermal fish cell lines and mammalian neuronal cells, as well as whole fish bioassay. This facility is being used to guide the fractionation of plant extracts to search for bioactive compounds. In Year 3, we will focus on Pfiesteria from the VIMS lab as well as those from Baden's group. In a related project, we have found that extracts of periphyton algal mats from the Everglades show a minimum inhibitory concentration (MIC) of 10 ng/mL against rat B50 neuronal cells. Efforts to isolate the active compound(s) are underway.
Specific Aim 2: As reported last year, we now have all the tools for encoding combinatorial libraries, and have begun work on synthesizing some libraries. We have spent significant amounts of time developing an amino acid that contains an appropriate fluorescent sensor that can be incorporated into the libraries. We did not anticipate some technical problems that were encountered in the synthesis of other possible unnatural amino acid/fluorescent sensors. For example, some chemical intermediates failed to give the expected products when chemical elaboration was attempted, and others produced unwanted byproducts. These efforts taught us some things about the reactivity of our fluorescent sensors, which in the long run, will prove useful to the ultimate goal of incorporation into peptide libraries containing the fluorescent sensor.
Specific Aim 3: This goal is predicated on completion of Aim 2, which is incomplete at this time.
Journal Articles:
No journal articles submitted with this report: View all 9 publications for this projectSupplemental Keywords:
water, watersheds, sediments, marine, estuary, risk assessment, exposure, effects, bioavailability, vulnerability, dose-response, animal, organism, cellular, chemicals, organics, pathogens, environmental chemistry, biology, ecology, analytical, measurement methods, southeast, Atlantic coast, Chesapeake Bay, North Carolina, NC, Virginia, VA., RFA, Scientific Discipline, Water, Geographic Area, Waste, Ecosystem Protection/Environmental Exposure & Risk, Limnology, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Contaminated Sediments, Chemistry, State, Ecological Effects - Environmental Exposure & Risk, Ecological Effects - Human Health, algal blooms, Southeast, Biology, Ecological Indicators, East Coast, ecological exposure, pfiesteria piscicida, North Carolina, agricultural runoff, fish kills, Shephard's Crook, whole-fish bioassay, ecology, suburban watersheds, bloom dynamics, harmful algal blooms, Gyrodiniums, economic assessments, fish lesions, ligand sensitive hosts, Everglades, pfiesteria, toxins, environmental chemistry, dinoflagellate, Glenodriniums, Tilapia, bioassay, fluorescence sensing, Virginia, Florida, ScripsiellasRelevant Websites:
http://www.rsmas.miami.edu/groups/niehs/
http://www.uncwil.edu/cmsr/
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.