Grantee Research Project Results
2000 Progress Report: Nitric Oxide as an Immune Effector Molecule of Bivalves: Modulation by Environmental Chemicals
EPA Grant Number: R827100Title: Nitric Oxide as an Immune Effector Molecule of Bivalves: Modulation by Environmental Chemicals
Investigators: Anderson, Robert S.
Institution: University of Maryland Center for Environmental Science
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1998 through September 30, 2001
Project Period Covered by this Report: October 1, 1999 through September 30, 2000
Project Amount: $335,064
RFA: Exploratory Research - Environmental Biology (1998) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Human Health , Aquatic Ecosystems
Objective:
The overall goals are to improve our understanding of mechanisms of disease resistance in the Eastern oyster (Crassostrea virginica), and to determine the effects of sublethal exposure to aquatic contaminants on these mechanisms. The hypotheses to be tested are that nitric oxide (NO ) plays a significant role in oyster defense mechanisms, and that exposure to environmental chemicals inhibits NO synthesis, thereby reducing host defenses to disease. Exposure to xenobiotics has been shown to exacerbate progression and severity of important oyster diseases; however, the underlying mechanisms have yet to be identified.Progress Summary:
In previous studies, we have shown that environmental stressors, such as metals, organometals, and hypoxia, increase the intensity and lethality of Perkinsus marinus infection in the oyster Crassostrea virginica. We also showed that this reduction of resistance occurs without concomitant down regulation of production of reactive oxygen intermediates and antimicrobial enzymes, e.g. lysozyme. In this project, we have been investigating the ability of C. virginica hemocytes to produce the antimicrobial agent nitric oxide (NO ), characterizing nitric oxide synthase (NOS), and studying the effects of NO stimulating and inhibiting agents on NOS activity.During Year 1 of this grant, we showed that oyster hemocytes contain NOS, by the use of monoclonal and polyclonal antibodies against murine macrophage inducible form NOS (iNOS) and channel catfish iNOS. Attempts to induce this enzyme by injection of pathogenic bacterial vaccine (Vibrio anguillarum) or endotoxin (LPS) gave mixed results; serum NO levels often rose after treatment, but statistical significance was not established.
Most of year two of this effort was spent attempting to characterize oyster hemocyte NOS and quantify NO production by a series of in vitro experiments. The use of the Griess reagent to measure NO in hemocyte primary culture media was discontinued, even after enzymatic conversion of nitrate to nitrite, because of its lack of sensitivity. A flourescent probe (DAN: 2-3-diaminonaphthalene) was able to show NO in hemocyte media after treatment with nitrate reductase. An apparently constitutive, low-level NO production seems to be only occasionally seen in C. virginica hemocytes. We plan to quantify this by even more sensitive isotopic methods, measuring the conversion of labeled arginine to citruline via adsorption chromatography. However, other Crassostrea species showed consistently higher basal NO production by untreated hemocytes. For example, this was seen with the DAN method for both C. gigas and C. galloprovincialis. A number of agents were tested for their ability to stimulate NO production by the hemocytes; zymosan, phorbal myristate acetate (PMA), calcium ionophores, LPS, formyl peptides, and arachidonic acid. None of these treatments were strong NO inducers (via the DAN reaction) for the three Crassostrea species. However, a species of mussel (Guekensia demissa) which was shown to express constitutive NO , responded to PMA.
Although to date it has been difficult to demonstrate NO directly by the Griess reaction or with the DAN method, there was indirect evidence. When hemocytes are stimulated by zymosan, or other activating agents, they produce a strong chemiluminescent signal (CL) in the presence of luminol; this can be a result of reactive oxygen species (ROS) production and/or peroxynitrite generation. Peroxynitrite is formed by interaction of NO and the ROS, superoxide. Luminol-dependent CL of oyster hemocytes can be inhibited by L-NMMA, a specific NOS inhibitor; this fact implicates NO in the response. It was also noteworthy that L-arginine, a precursor of NO , significantly enhanced the CL response of the hemocytes. In an attempt to better understand the possible coproduction of NO and superoxide by hemocytes, the search for agents that elicit both radicals was initiated. As mentioned, CL is a good probe for oxidizing agents, but it is not entirely specific. Therefore, superoxide dismutase-sensitive nitroblue tetrazolium (NBT) reduction was used as a specific assay for superoxide and the DAN reaction was used to specifically look for NO production by its degradation product, nitrite. Zymosan and PMA triggered NBT reduction, but no DAN response; however, a calcium ionophore and arachidonic acid stimulated activity in both assays.
These results provide additional information on the phenomenon of NO production and control in C. virginica hemocytes. The enzyme responsible for NO generation (nitric oxide synthase) is consistently found in oyster hemocytes, but expression of NO is very low in comparison to that seen in vertebrate macrophages. Several typical NO stimuli are ineffective when applied to the cells in vitro; however, at least two other agents were identified that appear to elicit a low level response. Exposure of the hemocytes in vivo to vaccines of oyster-pathogenic bacteria or bacterial LPS produced little increased NO generation by the cells in preliminary studies. This does not rule out a role for NO in intracellular signaling or other circumstances, several of which will be addressed in the immediate future.
Future Activities:
The studies described above will be completed and published. A standard induction protocol will be established for use in immunotoxicological studies; the effects of sublethal exposure to environmental contaminants on NO responsiveness of oyster hemocytes will be determined. Infection with the protistan parasite Perkinsus marinus down-regulates hemocytic generation of reactive oxygen species; the effects of this pathogen on NO responsiveness will be determined. The NO stimulatory activity of several relevant physiological elicitors will be measured, these include oxidative stress, hypoxia, and contact with yeast (specifically -glucans). Finally, we plan to study the effects of NO modulation by chemical agents on cellular antimicrobial activity and Dermo disease progression and/or lethality.Journal Articles:
No journal articles submitted with this report: View all 2 publications for this projectSupplemental Keywords:
immunology, immunotoxicology, oysters, bivalves, Chesapeake Bay, pathology, metals, organics, toxics, biomarkers of effect, aquaculture., Health, Scientific Discipline, Health Risk Assessment, Epidemiology, Disease & Cumulative Effects, Biochemistry, Biology, Immunology, Pathology, aquatic contaminants, health effects, oyster disease models, antibody-based assays, immune system effects, xenobiotics, bivalves, infectious diseases, bacterial pathogens, disease resistance, harmful environmental agents, biological markers, modulation by environmental chemicals, exposure assessment, immune responseProgress 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.