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1999 Progress Report: Molecular, Cellular and Animal Biomarkers of Exposure and Effect
EPA Grant Number: R825433C044Subproject: this is subproject number 044 , 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: Molecular, Cellular and Animal Biomarkers of Exposure and Effect
Investigators: Wilson, Barry W.
Institution: University of California - Davis
EPA Project Officer: Levinson, Barbara
Project Period: October 1, 1996 through September 30, 2000
Project Period Covered by this Report: October 1, 1998 through September 30, 1999
RFA: Exploratory Environmental Research Centers (1992)
Research Category: Center for Ecological Health Research , Targeted Research
Description:
Objective:To provide molecular, cellular and animal models to study the actions of habitat and ecosystem disruption on reproductive and neurological functions.
Progress Summary:Molecular methods used in current projects include:
Cholinesterases (ChEs). Studies of the practicality of a new model
of the Test Mate ChE kit at temperatures found in the field were completed by
visiting Brazilian scientist Georgino deOliveira. Unfortunately the results
did not confirm the temperature range in the instructions in which the device
would normalize activities to 25°C for temperatures from 15°C to above
35°C. Unfortunately, direct comparisons showed the values displayed by the
device were not accurate below 20°C limiting its use as a field instrument.
The actual values determined by the machine before correction by an internal
algorithm were not displayed. This year we hope to obtain a machine from the
manufacturer that will show the actual values and work with them to develop
a ChE field device.
ChEs and Neuropathy Target Esterase (NTE). The NTE assay singles out an enzyme of nervous and other tissues implicated in organophosphate induced delayed neuropathy, a disorder brought about by certain organophosphates (OPs) including the pesticides chlorpyrifos and isofenfos. Studies are underway to examine the NTE of mice and its response to neuropathic agents as part of a study of low level exposures of chemical warfare agents, models of pesticides that are present in the environment. We have completed a study of the AChE and NTE activities of mouse and chicken brain homogenates with sarin, an OP known to accidentally have been dispersed to the environment in the Gulf war. The results confirm that sarin inhibits NTE. However, its inhibition of AChE was orders of magnitude greater signifying that the greater risk to a single dose is an acute cholinergic crisis rather than a delayed neuropathy. In other words, the animals would die before they became paralyzed from a single dose. Studies on cells, animals and repeated low exposures continue.
ELISA and Gas Chromatography (GLC). Two heavily used pesticides are the OP diazinon and the pyrethroid esfenvalerate. Diazinon inhibits AChE of insects, man and other animals; esfenvalerate blocks specific ion channels in the nervous system. Major runoff studies of these chemicals during dormant spray season are underway in collaboration with the Cal Fed program and orchard grower groups. This year we participated as the analytical component of the project. GLC methods were validated for the detection of diazinon residues. And a new sensor for esfenvalerate was obtained. A sensitive technique for esfenvalerate is almost finalized due to the efforts of Dr. Oliveira. Samples were stored from runoff studies conducted in collaboration with IPM director Dr. Frank Zalom, hydrologist Wes Wallender and aquatic toxicologist David Hinton. The samples are now being analyzed. (They are also being examined for toxicity by others on the project). Studies will continue into the upcoming spray season.
Hormones. ELISA methods to determine testosterone in the feces of male mice were developed in collaboration with Bill Lasley, providing a non-invasive way to examine the reproductive potential of wild animals. Last year, graduate student Joe Billitti developed and validated an ELISA method for testosterone in the feces of wild mice. This year, we (Billitti, Faulkner and Wilson) finished and reported upon a study of methyl tert butyl ether (MTBE) sponsored by the State of California. The project examined the toxicity of this controversial fuel additive present in the environment. Little acute toxicity was found, but there was a suggestion of testicular damage at high doses. Graduate student Faulkner, together with Bill Lasley, is currently working on an immunoassay to measure testosterone in the droppings of American kestrels, a common raptor species. The first phase of development, characterization of testosterone metabolism in kestrels, is in progress. Last December we injected several birds with radioactive testosterone, and we are currently working to identify the metabolites. The results of HPLC analysis on the samples revealed three peaks, and enzymatic hydrolysis studies have revealed that one of the metabolites is a glucuronide conjugate. Hydrolysis with aryl-sulfatase did not deconjugate the remaining conjugates, indicating that the other HPLC peaks may in fact be non-hydrolyzable or "hard" conjugates. The next steps are additional characterization experiments and mass spectrophometry of the metabolites. In addition, approximately 10% of the injected testosterone was excreted unmetabolized, indicating that it may be possible to use the already existing testosterone immunoassay for detection of parent compound in droppings. Once the development and validation stages are completed the immunoassay will be used to study whether exposure of the kestrel to pesticides during dormant spraying will be detrimental to its reproduction, directly or through exposure of its field mice prey.
Cell Cultures and Development. Highly differentiated cell cultures offer opportunities to detect effects, establish species differences and study metabolic conversions in a cost effective and animal sparing manner. Toxicity differences established in the laboratory on the cells of different species can be checked out in the laboratory and field on the animals themselves.
A. Embryo spinal cord and brain cells and established cell lines (e.g. PC12 cells) provide an alternative to animal studies for the examination of neurological disruptions by putative and known neurotoxins such as pesticides, heavy metals and solvents. Graduate student Ellen Coatney is studying cholinesterases and other proteins of nerve cells exposed to cadmium, organophosphates and combinations of the two. The results suggest toxic interactions occur between the two classes of toxicants. Her current in ovo studies of the developing nervous system explore whether cell death during the development of the spinal cord is affected by OPs and Cd, alone or in combination.
C. Brown fat cells of rodents have been established in culture by physiology graduate student Sean Wilson and his mentor Pamela Pappone to study the role of ATP in their proliferation and electrophysiology. His work has been completed and has resulted in several publications. Such cells may provide an interesting biomarker for migratory species.
D. The action of selenium on development of the Japanese Quail is being studied by Yamamoto, Santolo and Wilson.
Future Activities:Studies of embryo and cellular markers of developmental effects on birds and fish, examination of the impact of dormant sprays on water quality and mammalian and avian wildlife will continue.
Supplemental Keywords:Ecosystem Protection/Environmental Exposure & Risk, Water, Scientific Discipline, Health, RFA, ECOSYSTEMS, Ecosystem/Assessment/Indicators, Endocrine Disruptors - Environmental Exposure & Risk, Water & Watershed, exploratory research environmental biology, Aquatic Ecosystems & Estuarine Research, endocrine disruptors, Terrestrial Ecosystems, Ecological Monitoring, Aquatic Ecosystem, Ecological Indicators, Biochemistry, Watersheds, Environmental Chemistry, Ecological Effects - Environmental Exposure & Risk, Ecosystem Protection, Monitoring/Modeling, Ecology and Ecosystems, Environmental Monitoring, Endocrine Disruptors - Human Health, biomarkers, runoff, water quality, watershed modeling, watershed land use, animal models, endocrine disrupting chemicals, watershed management, watershed restoration, immunoassay, protein kinase, ecological risk, ecology assessment models, ecosystem health, fish, bioindicator, environmental stress indicators, anthropogenic effects, land use, wetlands, models, pesticide exposure, watershed development, molecular markers, aquatic habitat, agricultural watershed, aquatic ecosystems, environmental stress, pesticides, lake ecosysyems, watershed sustainablility, ecological assessment, water management options, agrochemicals, ecosystem stress, ecosystem response
Progress and Final Reports:
Original Abstract
2000 Progress Report
Final Report
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