Grantee Research Project Results
2002 Progress Report: FRIENDS Analytical Toxicology Core Facility
EPA Grant Number: R829390C003Subproject: this is subproject number 003 , established and managed by the Center Director under grant R829390
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Water Innovation Network for Sustainable Small Systems
Center Director: Reckhow, David A.
Title: FRIENDS Analytical Toxicology Core Facility
Investigators: Kostyniak, Paul J. , Olson, James , Fitzpatrick, Richard
Current Investigators: Kostyniak, Paul J.
Institution: The State University of New York at Buffalo
EPA Project Officer: Aja, Hayley
Project Period: October 17, 2001 through October 16, 2002
Project Period Covered by this Report: October 17, 2001 through October 16, 2002
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2001) RFA Text | Recipients Lists
Research Category: Human Health , Children's Health
Objective:
The objective of the Analytical Toxicology Core Facility (ATCF) is to provide analytical support to both the epidemiological and animal research projects of the FRIENDS Children's Environmental Health Center. The ATCF support involves the development of new analytical techniques, analyses of samples and analyses of data. All procedures are controlled by a strict QC/QA program. The techniques available Include GC with EC, FID, PID and Hall detection, HPLC with diode array, fluorescence and electrochemical detection, GC-MS, AAS, mercury determinations, high temperatures GPC for absolute molecular weight and molecular weight distribution determinations. This laboratory has extensive experience determining specific congeners of PCBs and pesticides and heavy metals at ppb levels in biological samples, and has participated in nationwide proficiency programs for PCBs, pesticides and mercury. The ATCF also performs a reporter gene bioassay for the quantification of total dioxin (TCDD) toxic equivalents (TEQs) in Hepa 1c1c7, mouse hepatoma cells, which are stably transfected with the reporter plasmid p2Dluc containing 2 copies of the DRED consensus sequence.
Progress Summary:
Considerable effort was devoted to the preparation of the appropriate PCB congener mix to be used for the animal studies throughout the five year project. PCB congener profiles were obtained for 15 subjects consuming Fox River fish. There was considerable variation in absolute levels of total PCBs but a similar congener profile. Women with an extensive history of breast feeding tended to have lower PCB levels particularly of the higher chlorinated congeners which tend to bioaccumulate. PCB congener profiles for human subjects and PCB congener profiles Fox River walleye and white bass, were compared. As might be expected, fish PCB congener profiles did not correspond directly with that found in the subjects. Fish tended to have a congener profile with a more concentrated Arochlor 1242 like pattern in which there was a more predominant emphasis on lower chlorinated congeners than in the subjects. It was decided that for animal studies, which are intended to mimic human exposures, we would prepare an animal dosing mixture, which mimicked Fox River fish PCB profiles. We constructed various theoretical mixtures of PCBs from published congener contents of various Aroclors. The Aroclor mix, which predicted a profile most similar to that found in the Fox River fish was a mixture containing 35% Aroclor1242, 35% Aroclor 1248, 15 % Aroclor 1254 and 15% Aroclor 1260. The mixture was prepared and is currently undergoing confirmation analysis by GC/ECD, to verify its similarity with the congener profiles in Fox River Fish.
In an attempt to assess other potential vectors of PCB exposure in the study population, imported foodstuffs primarily from Tailand were obtained. These foods are obtained from specialty food stores in Green Bay and are used on a regular basis by the study population. The foodstuffs for PCB analysis include seven of the most popular brands of fish sauce, ten varieties of pickled and canned fish, four varieties of crab and shrimp paste, and shrimp crackers. Methods of extraction are being modified for each of these sample matrices to enable PCB congener and pesticide determination, in addition to assessment of total dioxin like activity by the reporter gene assay.
The reporter gene bioassay was developed as an economical, rapid bioassay that can screen environmental and biological specimens for complex mixtures of compounds that elicit dioxin-like activity. Dioxin-like compounds include including polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) with chlorine on at least the 2,3,7,8 positions and the coplanar (non-ortho) and mono-ortho substituted PCBs that have biological and toxicological activities similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent HAH. The reporter gene bioassay system was developed based on the premise that dioxin-like compounds initially bind to the AhR and subsequently that the ligand-activated AhR binds to dioxin response elements on DNA. A murine hepatoma cell line (hepa 1c 1c7) was stably transfected with the p2Dluc plasmid containing two DREs upstream from the firefly luciferase gene. A 96-well cell culture plate format has been optimized for this cell-based reporter gene bioassay system to assess AhR-responsive luciferase activity of dioxin-like chemicals, individually and in complex, real world mixtures. The reporter gene bioassay is exceptionally sensitive, detecting levels of TCDD as low as 1 pM (0.06 pg/200 µl/well. The only remaining factor to be optimized for the reporter gene bioassay is the range of the absolute quantity of extracted lipid that from a given specimen that can be added to the cell culture medium. We are currently optimizing extraction and analysis methods for the foodstuffs described above Results will be expressed as ppt (part per trillion) of dioxin TEQs in each foodstuff on a wet weight and lipid weight basis.
Future Activities:
The ATCF will provide analytical support for the routine measurement of PCB congeners, pesticides and methymercury in human and animal samples generated in the various Center projects. The ATCF will also continue to improve the congener specific PCB and pesticide analytical methods to include the most prevalent PCB metabolites. We will also expand the assay to include relevant polybrominateddiphenylethers, which are found in Great Lakes fish and have been implicated in neurodevelopment.
Supplemental Keywords:
PCBs, Methymercury, dioxin-like, TEQ, PBDE., RFA, Scientific Discipline, Health, ENVIRONMENTAL MANAGEMENT, Geographic Area, POLLUTANTS/TOXICS, Midwest, Toxicology, Health Risk Assessment, Epidemiology, Chemistry, Chemicals, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Disease & Cumulative Effects, Ecological Risk Assessment, Molecular Biology/Genetics, Children's Health, genetic susceptability, Biology, Risk Assessment, human data, neurotoxic, behavioral assessment, PCBs, pesticides, animal model, electrochemical detection, children, neurotoxicity, motor development, behavioral deficits, methylmercury, PCB, cognitive development, human exposure, Wisconsin (WI), animal studies, reproductive health, exposure assessment, biomedical research, heavy metalsProgress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R829390 Water Innovation Network for Sustainable Small Systems Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R829390C001 Neurobehavioral Effects of PCBs and Methylmercury in Rats
R829390C002 Perinatal PCB Exposure and Neuropsychological/Auditory Function
R829390C003 FRIENDS Analytical Toxicology Core Facility
R829390C004 Developmental Effects of PCBs and Methylmercury
The 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.