Grantee Research Project Results

Final Report: Sensitive Biomarkers to Detect Biochemical Changes Indicating Multiple Stresses Including Chemically Induced Stresses

EPA Grant Number: R825433C043
Subproject: this is subproject number 043 , 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: UC Davis Center for Children's Environmental Health and Disease Prevention
Center Director: Van de Water, Judith
Title: Sensitive Biomarkers to Detect Biochemical Changes Indicating Multiple Stresses Including Chemically Induced Stresses
Investigators: Matsumura, Fumio
Institution: University of California - Davis
EPA Project Officer: Packard, Benjamin H
Project Period: October 1, 1996 through September 30, 2000
RFA: Exploratory Environmental Research Centers (1992) RFA Text |  Recipients Lists
Research Category: Center for Ecological Health Research , Targeted Research

Objective:

The objective of this research project was to develop sensitive biomarker technologies that detect multiple stresses on the study organism. We selected four biological systems and investigated which of the stress markers would serve as sensitive and reliable markers of stress.

Summary/Accomplishments (Outputs/Outcomes):

Protein Kinase C (PKC)-Like Proteins as Possible Markers of Stress in Rice Plants

During this research project, we studied the effects of various forms of environmental stress, such as desiccation, high salts, stem and leaf bending, high dry winds, and fungus invasion, to see which markers would serve as the most reliable signs of stress. After several trials, PKC-like proteins were chosen as the best candidate for this purpose.

Antibody Development Against the Most Conserved Stretch of Heat Shock Protein 70s From Various Organisms as a Universal Biomarker of Stress

We have noticed that developing biomarkers for many different species in wildlife is not easy because even in the same group of organisms, their genetic backgrounds often are very different. Our plan is to find a type of stress protein that has not been changed throughout evolution (i.e., those containing a very conserved amino acid sequence). After an extensive homology search, we discovered that heat shock protein 70s from most organisms contains a stretch of amino acid sequence that is identical. This project, therefore, has been designed to concentrate on developing an antibody aimed at this stretch of amino acid sequence that can be used in several organisms to see whether it works as a biomarker of stress.

PKC Isoforms as a Marker of Stress in Relation to the Action of Pesticides as Cancer Promoters in Mouse Hepatocytes

It is now accepted that pesticides cause a distinct type of hyperplasia of hepatocytes in rodents. This type of action of pesticides is likely because of the stress response of hepatocytes. This phenomenon is very consistently observed and, therefore, is regarded as a significant marker of pesticide-induced stress in liver. Pesticide and related lipophilic xenobiotics are known to accumulate particularly in the liver and, therefore, a liver marker of stress could serve as a very sensitive parameter.

Development of an MCF-7 Model as a Screening Method for the Detection of Environmental Endocrine Disruptors, Particularly the Estrogenic Effects of Pesticides

There is no question about the effect of different pesticides that act like estrogen in various organisms, including humans in vivo. Various efforts, however, to develop a suitable screening method to detect their estrogenic actions have failed. The main reason for this problem is that these pesticides show only poor affinities to the estrogen receptor in all binding tests conducted so far. In vivo tests, such as assessing vitellogenin production in live fish, have been successful in detecting their estrogenic properties. The problem is that in vivo tests using wildlife species give very variable results, owing to the heterogeneity of their genetic backgrounds as well as the difficulty of preparing those organisms at precisely the same stage of development before testing. Therefore, it is most desirable to develop an in vitro cell model that would detect the estrogenic potential of these pesticides, although such actions are not because of the agonistic action of the estrogen receptor. After a search, we selected MCF-7 human breast epithelial cancer cells; this cell line is known to express a high titer of estrogen receptor and responds to substances that do not bind directly to the estrogen receptor.

The following activities were accomplished:

• We discovered that PKC-like proteins in rice plants serve as a very sensitive biomarker, responding to even subtle stress. In this study, we were able to show that this protein kinase in young rice plants is altered by winds, drought, fungus infection, lack of sunlight, and high temperature, in addition to herbicides, fungicides, and cupric ions. The response of this enzyme to fungus infection was particularly interesting in view of its relationship to the plant defense signal transduction mechanism that is triggered by invading pathogens. It has been well established that pathogenic fungi give rise to the elicitor response, in which plants produce hydrogen peroxide, phenols, quinones, and other defensive substances. It has not been known, however, that PKC-like proteins are involved in this pathway.

• We developed an antibody against a conserved stretch of amino acids of heat shock proteins, TVPAYFNDSQRQATKDA, which could detect stress responses in two fish, one amphibian, two arthropods, and one plant species through Western blotting. This polyclonal antibody preparation from rabbits was effective in detecting heat shock proteins in all of the species tested. Invariably, heat treatment was found to cause a rise in the titer of heat shock proteinss in all of the organisms tested. Additionally, responses to several different types of environmental stress, including metal, chemical, salt, and changes in pH, were detected depending on the species chosen.

• In the case of mouse hepatocytes, we found that heptachlor epoxide at low concentrations (1 to 100 nM) causes significant downregulation of PKC epsilon without well-known isoforms such as and in cultured Hepa 1 cells from mouse liver hepatoma. Such an action of heptachlor epoxide has not been reported before (Hansen and Matsumura, 2000). Heptachlor epoxide is a very stable pesticidal environmental pollutant and is the most potent congener of technical chlordane. PKC enzymes play many vital roles in most of the organisms studied to date. Our findings indicate that the affected organisms rely on the PKCs to respond and may, in some cases, counteract the toxic stresses to which they are subjected.

• We have developed an MCF-7 cell foci forming assay method. The MCF-7 cell line consists of an estrogen receptor overexpressing human breast epithelial cancer cells. It responds to dichlorodiphenyltrichloroethane, dioxin, hexachlorocyclohexane, and other known disruptors by increasing the number of foci (i.e., pileups of transformed, preneoplastic cells). This method of detection of estrogenic pesticides is an important breakthrough because most of these pesticides do not have high enough affinities to the estrogen receptor, and hence escape detection by most screening for endocrine disruptor assays based on the estrogen receptor binding assays. In addition, we determined the indispensable role of c-Neu tyrosine kinase on the expression of estrogenic actions of chlorinated pesticides. We have found that this kinase is activated by all of the above pollutants at very low concentrations (Hatakeyama and Matsumura, 1999).

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., RFA, Health, Ecosystem Protection/Environmental Exposure & Risk, Scientific Discipline, INTERNATIONAL COOPERATION, Water, ECOSYSTEMS, Aquatic Ecosystem, Aquatic Ecosystems & Estuarine Research, Ecological Indicators, Ecosystem Protection, Watersheds, Environmental Chemistry, Ecosystem/Assessment/Indicators, Monitoring/Modeling, Endocrine Disruptors - Human Health, Ecological Monitoring, endocrine disruptors, Ecological Effects - Environmental Exposure & Risk, Biochemistry, Endocrine Disruptors - Environmental Exposure & Risk, Water & Watershed, Ecology and Ecosystems, Environmental Monitoring, watershed management, nutrient dynamics, lake ecosysyems, ecosystem health, accelerator mass spectrometry, protein kinase, watershed land use, watershed modeling, water management options, water quality, pesticides, ecological assessment, bioindicator, pesticide exposure, watershed restoration, anthropogenic effects, fish habitat, hydrology, ecosystem monitoring, ecosystem response, ecosystem stress, environmental stress, endocrine disrupting chemicals, integrated watershed model, immunoassay, wetlands, land use, ecology assessment models, environmental stress indicators, watershed development, ecological risk, aquatic ecosystems, aquatic habitat, agricultural watershed, agrochemicals, Clear Lake, biomarkers, watershed sustainablility

Relevant Websites:

http://ice.ucdavis.edu/cehr/ Exit

Progress and Final Reports:

Original Abstract

1997

1998

1999 Progress Report

Main Center Abstract and Reports:

R825433    UC Davis Center for Children's Environmental Health and Disease Prevention

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