Grantee Research Project Results
Aquatic Food Web Transfer of Microcystins, a Potent Cyanobacterial Toxin, and Implications for Human HealthEPA Grant Number: F6E11081
Title: Aquatic Food Web Transfer of Microcystins, a Potent Cyanobacterial Toxin, and Implications for Human Health
Investigators: Smith, Juliette L.
Institution: SUNY College of Environmental Science and Forestry
EPA Project Officer: Jones, Brandon
Project Period: September 1, 2006 through September 1, 2008
Project Amount: $110,550
RFA: STAR Graduate Fellowships (2006)
Research Category: Academic Fellowships , Aquatic Ecosystems , Fellowship - Aquatic Biology , Fellowship - Biochemistry
Worldwide, phytoplankton assemblages are switching to communities now dominated by toxin-producing cyanobacteria in response to anthropogenic, accelerated eutrophication. Microcystins (MCs), the most common class of freshwater cyanobacterial toxins, target the liver and hepatopancreas and have led to the death of aquatic and terrestrial animals, including humans. The World Health Organization set a guideline for MC in drinking water at 1 µg L-1 and a tolerable daily intake (TDI) of MC in food at 0.04 µg kg-1 of body weight. In response, numerous studies investigated the concentration of MCs in fish muscle tissue in the field. These studies utilized traditional detection methods that relied on the extraction of MCs from animal tissue via methanol (MeOH). MeOH extraction, however, only releases MCs that are not covalently bound to the target molecule, protein phosphatases, leaving 60 – 90% of the total MC load undetectable. Nonetheless, the values reported were generally accepted to represent the total risk to humans due to the belief that only non-covalently bound MCs were bioavailable.
However, I propose that the covalently bound fraction of MC should be considered when making predictions regarding risk to higher trophic organisms (i.e. piscivores) and humans. I predict that protein phosphatases can be readily digested by proteases in a consumer (e.g., fish or humans), thereby releasing MCs from the diet (e.g., zooplankton or fish fillet). Microcystin, on the other hand, contains a large number of “D” and unique peptide linkages, making the molecule remarkably resistant to trypsin and chymotrypsin protease digestion (G.Boyer, unpubl.). Subsequently MC will regain its toxicity, be absorbed through the GI tract, and eventually accumulate in the consumer.
My objectives are three-fold, (Objective 1) to determine if protease digestion can release and replenish toxicity to three MC variants -LR, -RR and –YR, bound to protein phosphatases PP1 and PP2A, in vitro. Two endopeptidases, trypsin and chymotrypsin, and one exopeptidase, carboxypeptidase, will digest PPases at three pHs, pH 4.0, 7.0 and 9.0, representing optimal pHs for stomach (acidic) or intestinal (alkaline) protease enzyme activity. An in vivo study will then be conducted to answer if covalently bound microcystins in zooplankton are in fact accumulated in fish by comparing mass balances of unbound microcystins (MeOH extraction) and total microcystin load (oxidation extraction, Objective 2). Lastly, fish muscle tissue will be collected weekly from two waterbodies, Oneida Lake, NY and Lake Neahtawanta, NY, to get a measure of total MC load, and therefore possible human exposure, throughout the summer and fall fishing season (Objective 3). Phytoplankton and zooplankton toxicity will also be monitored.Expected Results:
I predict that protease digestion will release microcystins from protein phosphatases PP1 and PP2A. Furthermore, the released microcystins will regain toxicity if the small peptide that is associated with it (originally from the PPases) is not in a position to inhibit microcystin’s covalent binding site. My findings in the laboratory will be compared to an in situ monitoring of microcystins in three trophic levels of the aquatic food web. If my predictions are confirmed then current literature has underestimated the transfer rate of microcystins between trophic levels and the exposure risk to humans.Supplemental Keywords:
Harmful algal blooms, cyanobacterial toxins, microcystins, bioaccumulation,, Health, Scientific Discipline, HUMAN HEALTH, Health Risk Assessment, Exposure, Risk Assessments, Biochemistry, aquatic ecosystem, food chain, human exposure, microcystins, dietary exposure, cyanobacteria