Grantee Research Project Results
1999 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, 1998 through September 30, 1999
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 understand better 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 exacerbates progression and severity of important oyster diseases; the underlying mechanisms have yet to be identified.Progress Summary:
Initial efforts have been focused on quantifying NO generation by C. virginica hemocytes (blood cells), characterizing inducible nitric oxide synthase (iNOS) in these cells, and studying the effects of classical NO stimulators on iNOS activity.The production of NO by oyster hemocytes was measured in two ways: by quantifying peroxynitrite (a product of NO and superoxide) via luminol-dependent chemiluminescence (CL) and by quantifying NO colorimetrically by the use of the Griess reagent. The cells were previously known to produce superoxide as part of their antimicrobial defense system. The fact that CL products released by activated hemocyte in part depend on NO , was shown by dose-dependent CL inhibition by treatment with competitive enzyme inhibitors specific nitric oxide synthase (NOS). However, the amount of NO produced by oyster hemocytes is low, as compared to that produced under similar conditions by mammalian macrophages. In aqueous conditions, NO is oxidized to both nitrate and nitrite; in our system, nitrate reductase was added to convert nitrate to nitrite, facilitating a positive reading with the nitrite-sensitive Griess reagent. In vitro exposure of hemocytes to bacterial endotoxin (E. coli LPS) seemed to stimulate NO release, but the expected dose-dependency was not seen. The cytokine interferon (IFN ) activates iNOS in mammalian cells, but showed little activity with hemocytes and no synergistic activity with E. coli LPS. It is possible that our current methods lack the required sensitivity to accurately measure small changes in NO production; therefore, a new fluorometric assay is under investigation.
We have demonstrated the presence of iNOS in C. virginica hemocytes with both monoclonal and polyclonal antibodies. Western blots of hemocyte lysates, when probed with a monoclonal antibody (N9-5, donated by Dr. C. Rice of Clemson University) directed against channel catfish macrophage iNOS, were shown to contain cross-reacting protein of approximately 130 kDa. This protein is the same as that to which binds two commercially obtained polyclonal antibodies directed against conserved regions of murine macrophage iNOS. The apparent presence of iNOS in hemocytes from untreated oysters raises interesting questions. Oyster hemolymph, unlike vertebrate blood, is not typically sterile; therefore, constant exposure to low-level stimulation by hemolymph-borne bacteria could result in sustained iNOS expression. In fact, preliminary observations suggest that in vivo administration of Vibrio anguillarum vaccine, which stimulates teleost fish macrophage activity, also will induce iNOS in C. virginica hemocytes.
Future Activities:
The preliminary studies described above will be completed and published. The dose-dependency and kinetics of iNOS induction will be characterized, and a standard induction protocol will be established for use in future immunotoxicological studies. We plan to determine if oyster hemocytes also contain constitutive NOS (cNOS) activity by the use of specific stimuli: phorbol myristate acetate and calcium ionophores. We have shown previously that infection by Perkinsus marinus, an important pathogen of oysters, down-regulates production of antimicrobial oxygen species by oyster hemocytes; the effects of P. marinus on NOS and NO responses by these cells will be studied. Finally, we plan to study the effects of iNOS modulation by environmental chemicals on cellular antimicrobial activity and 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, Chesapeake Bay, pathology, metals, organics, toxic substances 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.