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Grantee Research Project Results

1997 Progress Report: Development of a Bioassay for AhR-mediated Toxicity to Rainbow Trout

EPA Grant Number: R825371
Title: Development of a Bioassay for AhR-mediated Toxicity to Rainbow Trout
Investigators: Giesy, John P.
Current Investigators: Giesy, John P. , Richter, Catherine A. , Blankenship, Alan L. , Villeneuve, Daniel L.
Institution: Michigan State University
EPA Project Officer: Hahn, Intaek
Project Period: December 15, 1996 through December 14, 1998
Project Period Covered by this Report: December 15, 1996 through December 14, 1997
Project Amount: $304,771
RFA: Exploratory Research - Environmental Biology (1996) RFA Text |  Recipients Lists
Research Category: Biology/Life Sciences , Aquatic Ecosystems

Objective:

The objective of the research was to develop and characterize a novel in vitro bioassay which utilizes rainbow trout cells stably transfected with a luciferase reporter gene under control of dioxin responsive enhancers (DRE) to provide an integrative measure of fish- specific potency of aryl hydrocarbon receptor (AhR)-active agents to which cells are exposed.

Progress Summary:

We have developed a stable recombinant rainbow trout cell line, RLT 2.0. Those cells have been used to develop a bioanalytical system which we are currently characterizing. This report details recent progress in adapting the RLT 2.0 bioassay to a 96-well microplate, and optimizing the assay for high throughput and efficiency. It also provides RLT 2.0-derived potency estimates for several dioxin and furan congeners, not tested previously, and describes the results of a sensitivity analysis used to estimate the range of uncertainty associated with application of those estimates.

The RLT 2.0 bioassay was successfully adapted to and optimized for a 96-well microplate format. RLT 2.0 cells seeded into clear bottomed 96-well microplates are exposed to samples for 72-hr. Analysis is performed using a microplate reading luminometer. Use of a glow-type luciferase assay reagent and cycle mode of luminometer operation, allows plates to be scanned in approximately 2 min, compared to the 40 min scan time required by the previous method which used a flash-type reagent. The bioassay procedure was further streamlined by substituting visual inspection of test wells for a cell viability assay prior to luciferase assay and using microplates with transparent bottoms (which are covered by a reflective sticker for luciferase assay). These modifications eliminated washing and lysate transfer steps which represented potential steps where additional variability and/or error could be introduced. In vitro protein assay performed after luciferase assay, provides a secondary quantitative measure of potential differences in cell number and thus viability per well. Overall, the 96-well RLT 2.0 bioassay developed greatly increased assay efficiency and throughput without significant loss of sensitivity.

Dioxin and furan congeners including 2,3,7,8-TCDF, 1,2,3,7,8-PCDF, 1,2,3,4,7,8- HxCDF, and 1,2,3,4,7,8-HxCDD were analyzed using the 96-well RLT 2.0 and their relative potencies were estimated. EC-50s estimates for the congeners tested ranged from 100 to 2000 pM (in-well), while potencies expressed relative to a 2,3,7,8-TCDD standard ranged from 0.917 for 1,2,3,4,7,8-HxCDF to 0.208 for 1,2,3,7,8-PCDF. RLT 2.0-derived relative potencies were similar to those based on other rainbow trout specific and mammalian bioassays.

Bioassay derived relative potencies are often applied to calculate theoretical toxic (or TCDD) equivalents (TEQ) based on instrumental analyses. Thus, a sensitivity analysis was conducted to estimate the range of uncertainty associated with TEQ estimates based on RLT 2.0-derived relative potencies. Results suggest that RLT 2.0-TEQ estimates should be applied with a ten-fold (? 5x) uncertainty factor. Within this degree of uncertainty, the RLT 2.0 bioassay showed no definitive biases or inaccuracies relative to similar mammalian- or fish-specific in vitro bioassays.

Future Activities:

Fish and mammals have been reported to have different sensitivity to mono- and certain non- ortho PCB congeners. The current hypothesis is that the RLT 2.0 bioassay should respond differently to samples containing these compounds than mammalian bioassay systems. In order to test this hypothesis, future experiments will characterize RLT 2.0 responses to laboratory prepared and environmental samples containing mixtures of PCB congeners and compare them to results of a mammalian based in vitro luciferase assay.


Journal Articles on this Report : 2 Displayed | Download in RIS Format

Publications Views
Other project views: All 8 publications 4 publications in selected types All 3 journal articles
Publications
Type Citation Project Document Sources
Journal Article Richter CA, Tieber VL, Denison MS, Giesy JP. An in vitro rainbow trout cell bioassay for aryl hydrocarbon receptor-mediated toxins. Environmental Toxicology and Chemistry 1997;16(3):543-550. R825371 (1997)
R825371 (Final)
not available
Journal Article Villeneuve DL, Richter CA, Blankenship AL, Giesy JP. Rainbow trout cell bioassay-derived relative potencies for halogenated aromatic hydrocarbons: Comparison and sensitivity analysis. Environmental Toxicology and Chemistry 1999, Volume: 18 , Number: 5 (MAY) , Page: 879-888. R825371 (1997)
R825371 (Final)
not available

Supplemental Keywords:

aquatic toxicology, fish, cell, bioassay, dioxin, PCB., RFA, Scientific Discipline, Toxics, Ecosystem Protection/Environmental Exposure & Risk, Ecology, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Chemical Mixtures - Environmental Exposure & Risk, Environmental Chemistry, pesticides, Chemistry, Ecological Effects - Environmental Exposure & Risk, Ecological Effects - Human Health, Biology, Ecological Indicators, bioindicator, complex mixtures, dioxin, chemical contaminants, pulp mill effluents, PCB, polychlorinated biphenyls, bioassay, biochemical measurements, hydrocarbons, fish , immune systems, luciferase, reproductive health, dioxin exposure

Progress and Final Reports:

Original Abstract
  • Final Report
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    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.

    Project Research Results

    • Final Report
    • Original Abstract
    8 publications for this project
    3 journal articles for this project

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