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Final Report: Immunoassay and Serum Markers
EPA Grant Number: R825433C042Subproject: this is subproject number 042 , established and managed by the Center Director under grant R825433
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: EERC - Center for Ecological Health Research (Cal Davis)
Center Director: Rolston, Dennis E.
Title: Immunoassay and Serum Markers
Investigators: Hammock, Bruce
Institution: University of California - Davis
EPA Project Officer: Levinson, Barbara
Project Period: October 1, 1996 through September 30, 2000
RFA: Exploratory Environmental Research Centers (1992)
Research Category: Center for Ecological Health Research , Targeted Research
Description:
Objective:The objective of this research project was to develop and implement immunoassays for monitoring environmental compounds that can contribute to stress in varying components of the ecosystem. When addressing questions of multiple stressors, large numbers of samples for specific compounds are generated. Numerous analytical methods are available, but immunoassays are known to be rapid and inexpensive for large sample loads and utilize simple equipment. Our assays have an additional advantage. We developed these assays to allow simultaneous analysis of varying stress-causing molecules. Compound- and class-selective assays were used to distinguish between the subclasses of compounds through mathematical approaches based on immunoassay arrays. Other efforts included the development of novel immunoassay formats and the evaluation of near infrared technology for immunoassays that could be run faster, with greater sensitivity, and with less expensive equipment. Whenever possible, assays were utilized in conjunction with other program investigations, and technology was transferred to other program laboratories, state laboratories (California Department of Food and Agriculture, Department of Pesticide Regulation), or federal laboratories (U.S. Environmental Protection Agency; National Exposure Research Laboratory, Las Vegas, NV).
Summary/Accomplishments (Outputs/Outcomes):We investigated three potential biomarkers. Releases of enzymes (esterases) from hepatic tissues into the serum were explored as indicators of multiple stresses in vertebrate species. This work was in collaboration with Dr. Barry Wilson's study and focused on neuropathy target esterase. Lipid diols and epoxides, and the enzyme responsible for converting epoxides to diols (soluble epoxide hydrolase [sEH]), were examined as indicators of oxidative stress. This involved the analysis of these oxylipins in urine and other body fluids, as well as an investigation of the toxicity of these lipids to an individual organism and the mechanism of the enzyme.
Because environmental stress on organisms may be decreased by reducing their exposure to toxic environmental chemicals, we virally expressed recombinant peptides designed to disrupt the development of pest insects through viral infection. By applying this virus to crops instead of using chemical pesticides, the environmental impact associated with pest control may be dramatically reduced. Of course, we also are examining the possible deleterious effects of this technology on the ecosystem.
The following activities were accomplished:
• We developed competitive- and sandwich-chelate assays for mercury. This novel assay format uses chelate chemistry instead of antibodies to bind and detect mercury. This format is more robust than the typical immunoassay, while retaining the advantages and flexibility of classical immunoassays for formatting and application to sensors. This assay will be useful to other investigators working on mercury detection.
• We developed an immunoassay and a dioxin-surrogate standard for dioxin analysis. We developed a rapid, simple screening method for dioxin analysis. We also developed a surrogate standard for dioxin. Both of these innovations will allow dioxin analysis to be conducted under less stringent conditions than those required for the classical high resolution gas chromatography-mass spectrometry method, making dioxin analysis available to a wider variety of laboratories.
• We developed compound-, class-, and metabolite-selective assays for the triazine herbicides and pyrethroid insecticides. Development of compound-, class-, and metabolite-selective assays for the major triazine herbicides and pyrethroid insecticides allow for the rapid analysis of these ubiquitous compounds. Academic, commercial, and governmental laboratories can use these tools to provide rapid screening information before classical instrumental analysis.
• We developed class-selective assays for phenyl- and thiophenyl-glucuronides, as well as benzyl and phenyl mercapturates, as potential biomarkers of exposure. Mammalian urinary metabolites, particularly products of secondary conjugation, can be used as indicators of particular compounds and overall oxidative stress. We developed antibodies that can be used in affinity purification procedures to capture all mercapturates or all glucuronides. The individual compounds then can be monitored to develop metabolic profiles of stress.
• We developed a novel, compact disc-based microarray format for monitoring toxic exposure levels in ecological systems. This fast, sensitive assay system has high-throughput capabilities that can potentially increase the efficiency of ecological system monitoring and reduce the costs associated with such work. Ultimately, such a system could result in a biosensor in which multiple analytes can be detected simultaneously. Other investigators will benefit from the speed, ease, and potential developments of our innovation.
• We developed a highly sensitive radioimmunoassay for atrazine and 2,3,7,8-tetrachlorodibenzo-p-dioxin, with a stable label and no radiation hazard or disposal problems. Radioisotope-labeled immunoassays have been used in the past with the advantage of having an intrinsic label, but their use suffered from a perceived radiation hazard, short shelf life of reagents, and radioactive waste disposal problems. In contrast, our highly sensitive assay has a long shelf life, an intrinsic label that avoids handling-recognition problems, and uses levels of radioactivity that are not considered hazardous.
• We developed mathematical approaches to multianalyte analysis using immunoassays with known patterns of cross reactivity. Other investigators now can take advantage of the cross-reactivity patterns of antibodies as a way of conducting multianalyte analysis, not typically considered to be a strength of the immunoassay.
• We developed new substrates, inhibitors, and affinity ligands for carboxylesterase, and applied them to the isolation and purification of neuropathy target esterase. Investigators can use these tools to study other carboxylesterases of interest such as pyrethroid esterase, an enzyme responsible for pyrethroid degradation.
• We found that lipid diols and epoxides can be used as indicators of oxidative stress. We examined lipid diols and epoxides as indicators of oxidative stress and found that not only were lipid epoxides indicators of stress, but several of these materials in both the linoleate series and arachidonic acid series were very toxic to animals. They are, thus, both biomarkers of exposure and effect, and can be used by investigators in further research.
• We cloned and expressed sEH and determined the molecular mechanism for its metabolism of lipid epoxides in animals and plants. We also developed new substrates, inhibitors, and affinity ligands for sEH, and elucidated its role in linoleate and arachidonate metabolism. sEH is known to detoxify xenobiotic epoxides and now has been found to metabolize endogenous lipid epoxides, some of which mediate toxic responses in mammals. Other investigators can build on our work to develop lipid profiles as indicators of mammalian stress.
• We developed recombinant baculoviruses that are more efficient at pest control than the wild-type virus, and may reduce the use of conventional chemical pest control agents. We used a baculovirus system to express recombinant peptides designed to disrupt the development of pest insects. We have studied the interaction of recombinant baculoviruses with the use of traditional pesticides, and it appears that these viruses can dramatically reduce the environmental impact associated with pest control. Our innovation potentially may provide farmers with a safer alternative to chemical insecticides.
Supplemental Keywords:ecosystem, ecosystem protection, environmental exposure and risk, geographic area, international cooperation, water, terrestrial ecosystems, aquatic ecosystem, aquatic ecosystem restoration, aquatic ecosystems and estuarine research, biochemistry, ecological effects, ecological indicators, ecological monitoring, ecology and ecosystems, environmental chemistry, restoration, state, water and watershed, watershed, watershed development, watershed land use, watershed management, watershed modeling, watershed restoration, watershed sustainability, agricultural watershed, exploratory research environmental biology, California, CA, Clear Lake, Lake Tahoe, anthropogenic effects, aquatic habitat, biogeochemical cycling, ecological assessment, ecology assessment models, ecosystem monitoring, ecosystem response, ecosystem stress, environmental stress, environmental stress indicators, fish habitat, hydrologic modeling, hydrology, integrated watershed model, lake ecosystems, lakes, land use, nutrient dynamics, nutrient flux, water management options, water quality, wetlands. , Ecosystem Protection/Environmental Exposure & Risk, Water, INTERNATIONAL COOPERATION, Geographic Area, Scientific Discipline, RFA, ECOSYSTEMS, Ecosystem/Assessment/Indicators, Water & Watershed, exploratory research environmental biology, Restoration, Aquatic Ecosystem Restoration, Aquatic Ecosystems & Estuarine Research, Terrestrial Ecosystems, Ecological Monitoring, Aquatic Ecosystem, Ecological Indicators, Biochemistry, Ecological Effects - Human Health, Watersheds, Environmental Chemistry, Ecological Effects - Environmental Exposure & Risk, Ecosystem Protection, Monitoring/Modeling, Ecology and Ecosystems, Environmental Monitoring, State, biomarkers, runoff, water quality, California (CA), wildlife habitat, watershed modeling, watershed land use, watershed, watershed management, watershed restoration, immunoassay, Clear Lake, ecological risk, ecology assessment models, ecosystem health, Clear Lake , environmental stress indicators, anthropogenic effects, fish habitat, land use, wetlands, accelerator mass spectrometry, watershed development, aquatic habitat, agricultural watershed, aquatic ecosystems, environmental stress, pesticides, lake ecosysyems, watershed sustainablility, ecological assessment, hydrology, water management options, ecosystem stress, serum markers, ecosystem response, ecosystem monitoring, nutrient dynamics
Relevant Websites:
Progress and Final Reports:
1999 Progress Report
2000 Progress Report
Original Abstract
Main Center Abstract and Reports:
R825433 EERC - Center for Ecological Health Research (Cal Davis)
Subprojects under this Center:
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R825433C001 Potential for Long-Term Degradation of Wetland Water Quality Due to Natural Discharge of Polluted Groundwater
R825433C002 Sacramento River Watershed
R825433C003 Endocrine Disruption in Fish and Birds
R825433C004 Biomarkers of Exposure and Deleterious Effect: A Laboratory and Field Investigation
R825433C005 Fish Developmental Toxicity/Recruitment
R825433C006 Resolving Multiple Stressors by Biochemical Indicator Patterns and their Linkages to Adverse Effects on Benthic Invertebrate Patterns
R825433C007 Environmental Chemistry of Bioavailability in Sediments and Water Column
R825433C008 Reproduction of Birds and mammals in a terrestrial-aquatic interface
R825433C009 Modeling Ecosystems Under Combined Stress
R825433C010 Mercury Uptake by Fish
R825433C011 Clear Lake Watershed
R825433C012 The Role of Fishes as Transporters of Mercury
R825433C013 Wetlands Restoration
R825433C014 Wildlife Bioaccumulation and Effects
R825433C015 Microbiology of Mercury Methylation in Sediments
R825433C016 Hg and Fe Biogeochemistry
R825433C017 Water Motions and Material Transport
R825433C018 Economic Impacts of Multiple Stresses
R825433C019 The History of Anthropogenic Effects
R825433C020 Wetland Restoration
R825433C021 Sierra Nevada Watershed Project
R825433C022 Regional Transport of Air Pollutants and Exposure of Sierra Nevada Forests to Ozone
R825433C023 Biomarkers of Ozone Damage to Sierra Nevada Vegetation
R825433C024 Effects of Air Pollution on Water Quality: Emission of MTBE and Other Pollutants From Motorized Watercraft
R825433C025 Regional Movement of Toxics
R825433C026 Effect of Photochemical Reactions in Fog Drops and Aerosol Particles on the Fate of Atmospheric Chemicals in the Central Valley
R825433C027 Source Load Modeling for Sediment in Mountainous Watersheds
R825433C028 Stress of Increased Sediment Loading on Lake and Stream Function
R825433C029 Watershed Response to Natural and Anthropogenic Stress: Lake Tahoe Nutrient Budget
R825433C030 Mercury Distribution and Cycling in Sierra Nevada Waterbodies
R825433C031 Pre-contact Forest Structure
R825433C032 Identification and distribution of pest complexes in relation to late seral/old growth forest structure in the Lake Tahoe watershed
R825433C033 Subalpine Marsh Plant Communities as Early Indicators of Ecosystem Stress
R825433C034 Regional Hydrogeology and Contaminant Transport in a Sierra Nevada Ecosystem
R825433C035 Border Rivers Watershed
R825433C036 Toxicity Studies
R825433C037 Watershed Assessment
R825433C038 Microbiological Processes in Sediments
R825433C039 Analytical and Biomarkers Core
R825433C040 Organic Analysis
R825433C041 Inorganic Analysis
R825433C042 Immunoassay and Serum Markers
R825433C043 Sensitive Biomarkers to Detect Biochemical Changes Indicating Multiple Stresses Including Chemically Induced Stresses
R825433C044 Molecular, Cellular and Animal Biomarkers of Exposure and Effect
R825433C045 Microbial Community Assays
R825433C046 Cumulative and Integrative Biochemical Indicators
R825433C047 Mercury and Iron Biogeochemistry
R825433C048 Transport and Fate Core
R825433C049 Role of Hydrogeologic Processes in Alpine Ecosystem Health
R825433C050 Regional Hydrologic Modeling With Emphasis on Watershed-Scale Environmental Stresses
R825433C051 Development of Pollutant Fate and Transport Models for Use in Terrestrial Ecosystem Exposure Assessment
R825433C052 Pesticide Transport in Subsurface and Surface Water Systems
R825433C053 Currents in Clear Lake
R825433C054 Data Integration and Decision Support Core
R825433C055 Spatial Patterns and Biodiversity
R825433C056 Modeling Transport in Aquatic Systems
R825433C057 Spatial and Temporal Trends in Water Quality
R825433C058 Time Series Analysis and Modeling Ecological Risk
R825433C059 WWW/Outreach
R825433C060 Economic Effects of Multiple Stresses
R825433C061 Effects of Nutrients on Algal Growth
R825433C062 Nutrient Loading
R825433C063 Subalpine Wetlands as Early Indicators of Ecosystem Stress
R825433C064 Chlorinated Hydrocarbons
R825433C065 Sierra Ozone Studies
R825433C066 Assessment of Multiple Stresses on Soil Microbial Communities
R825433C067 Terrestrial - Agriculture
R825433C069 Molecular Epidemiology Core
R825433C070 Serum Markers of Environmental Stress
R825433C071 Development of Sensitive Biomarkers Based on Chemically Induced Changes in Expressions of Oncogenes
R825433C072 Molecular Monitoring of Microbial Populations
R825433C073 Aquatic - Rivers and Estuaries
R825433C074 Border Rivers - Toxicity Studies