Research Project 1: Ciguatera – Dinoflagellate Nutrient Profile and Ecology, Rapid Detection Methods and Human Health

Team

Paul K. Bienfang, Ph.D., Project Leader
Neal A. Palafox, M.D., M.P.H., Co-Investigator
Angel Yanagihara, Ph.D., Co-Investigator
Suzanne DeFelice, Research Technician
Misty Miller, Research Technician
Chelsie Settlemier, Research Technician
Scott Grant, Research Assistant
Darla White, Research Assistant

Fish for Science

Three major aims are to be examined:

1. Examination of the nutrient profile and ecology of Gambierdiscus toxicus in Hawaiian coastal waters;
2. Develop applicable chemical or immunological rapid-detection methods for Ciguatoxin or other polyether toxins in fish;
3. Examination of serum profiles of lipids in Ciguatera fish poisoning and correlate with sera from Chronic Fatigue Syndrome since both diseases have similar symptomologies.

Experiments in Aim 1

This area of study will consist of collection and identification of macroalgae and isolation of G. toxicus.

1. The G. toxicus will be collected and cultured in appropriate sea water medium with added nutrients including: 5 and 6 carbon sugars, vitamins and co-factors under required light and dark conditions.
2. The cultured G. toxicus will be examined for growth, toxicity in mice, effect on sodium channel with neuroblastoma cells (NB-cells) in tissue culture. In addition G. toxicus will be examined for presence of Polyethers immunologically by the membrane immunobead assay (MIA) and the immunofluorescence with MAb-CTX and MAb-CTX-isothiocyanate conjugate respectively.
3. Examine Xylan’s role in G. toxicus growth stimulation and its source and relationship to dinoflagellate proliferation. Macroalgae associated with G. toxicus presences will be examined and extracted with dilute acid. Xylan then precipitated with alcohol and the flocculant precipitate collected by centrifugation. The precipitate is purified further by TLC and Xylan fractions separated determined by color metric assays. Xylan broken down to xylose will be added to G. toxicus culture and growth stimulation assessed.

Experiments in Aim 2

1. Improve specificity and sensitivity of the membrane immunobead assay (MIA) for Ciguatoxin analysis in fish tissue and in sera of Chronic Fatigue Syndrome, Chronic Ciguatera fish poisoning and other disease sera (hepatitis, cancer, etc).
2. Purification of monoclonal antibody to Ciguatoxin (CTX): MAb-CTX will be carried out by Affinity G. chromatography. This step will help to remove non-specific binding proteins in the hybridoma culture medium that may contribute to non-specific reactions.
3. Sensitivity will be examined by use of biotin-avidin complexes in the immunological reactions by the MIA procedure using purified MAb-CTX.
4. Purified Ciguatoxin will be further characterized by NMR, mass spectroscopy and biologically in the sodium channel assay with neuroblastoma cell cultures. It is highly suggestive that Ciguatoxin caused physiological and clinical symptoms via action on sodium channels on nerve, muscle and cardiac cells of the mammalian tissue systems.
5. Ciguatoxin from toxic fish tissue and toxic cultures of G. toxicus will be purified by column chromatography, and thin layer procedures and finally by HLPC.

Experiments in Aim 3

1. Examine lipid profiles with the MAb-CTX in the MIA test of the lipid fractions of sera from Chronic Fatigue Syndrome, Chronic Ciguatera Poisoning and other diseases associated with liver synthetic alterations.
2. Identify each of the lipids and test these lipids in the neuroblastoma assay for their effect on the sodium channel function.
3. Culture human liver cells (available commercially) and study the effects of their lipid synthesis after exposure to ciguatoxin or these synthetic fragments.

It is hoped that these studies will help us to better understand the organism, G. toxicus that produces Ciguatoxin, which initiates Ciguatera fish poisoning in man. It is also hoped to elucidate further knowledge of the mode of action of Ciguatoxin via sodium channels and ultimately, to find drugs or methods to alleviate or neutralize CTX poisoning and indirectly Chronic Fatigue Syndrome.