Grantee Research Project Results
Final Report: Development of a Bioassay for AhR-mediated Toxicity to Rainbow Trout
EPA Grant Number: R825371Title: Development of a Bioassay for AhR-mediated Toxicity to Rainbow Trout
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 Amount: $304,771
RFA: Exploratory Research - Environmental Biology (1996) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Aquatic Ecosystems
Objective:
RLT 2.0 cells are rainbow trout hepatoma cells stably transfected with a luciferase reporter gene under control of dioxin responsive elements (DREs). Binding of the aryl hydrocarbon receptor (AhR)-ligand complex to the DREs results in an upregulation of luciferase transcription, which upon addition of the substrate, luciferin, catalyzes a light-producing reaction. The luminescent end-point can be measured with a luminometer to provide a sensitive measure of AhR-mediated gene expression potency. Greater sensitivity, selectivity, and dynamic range have been cited as potential advantages of using luciferase-transfected cell lines rather than cytochrome P4501A1 (CYP1A1) expression in wild-type cells as an end-point.The purpose of this study was to adapt the previously developed RLT 2.0 assay to a 96-well plate format and increase assay efficiency without significant loss of sensitivity or resolution. Such modifications should increase the utility of the assay for screening and bioassay-directed fractionation applications. In addition, a previously reported list of RLT 2.0-derived relative potencies (RPs) was expanded to aid mass-balance evaluations. New congeners tested included several mono- and di-ortho PCB congeners, 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzofuran (PeCDF), and 1,2,3,4,7,8-hexachlorodibenzofuran (HxCDF). RLT 2.0-derived RPs were compared to both fish-specific and mammalian RPs and toxic equivalency factors (TEFs) commonly used in risk assessment and characterization of environmental samples. A sensitivity analysis was performed to characterize the uncertainty associated with application of RLT 2.0-derived values. Results presented should help guide the use of RLT 2.0-derived potency to characterize environmental samples and evaluate aquatic exposure to halogenated aromatic hydrocarbons (HAHs).
Summary/Accomplishments (Outputs/Outcomes):
Overall the results of this study contribute to ongoing efforts to develop in vitro models as an alternative to whole animal testing in environmental toxicology research. To be viable alternatives to whole animal tests, in vitro bioassays must be rapid and cost-effective, yet sensitive. Furthermore, they must be calibrated to whole animal responses if they are to provide useful information for environmental risk assessment. The uncertainties associated with application of these models also must be addressed. This study was designed to address some of these issues for the RLT 2.0 in vitro bioassay as a possible alternative to whole fish testing for characterizing exposure to dioxin-like HAHs in environmental matrices and potential risks associated with such exposures.Two methods for performing the RLT 2.0 bioassay, using a 96-well plate format, were developed that generated more variable results than those using original methods. Instrument limitations made it impossible to determine whether the difference in variability was due to the assay method or changes in the stability of the cell line and its response to HAHs. The 96-well plate methods provided a substantial increase in assay throughput and efficiency. Using a 96-well plate method with a glow-type reagent, it is possible to analyze up to 50 plates per instrument in a single 8-hour day, which would generally allow for screening of approximately 400 samples, or characterization of a full dose-response curves for 90 samples per day. This increased throughput was achieved without loss of sensitivity or resolving power. Overall, assuming differences in the assay method were the cause of the greater variability, the original RLT 2.0 assay method remains effective for precise determinations of relative potency, but the 96-well plate methods are more suitable for rapid screening and characterization of numerous samples.
RLT 2.0 bioassay using a 96-well plate format with a glow-type reagent was used to characterize the potency of a number of HAHs relative to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a highly potent, prototypical AhR agonist. RLT 2.0-based RP estimates were compared to rainbow-trout-specific RPs based on other end-points in vitro and in vivo (Table 1). RLT 2.0-derived RPs were well correlated with early life-stage mortality (ELSM)-based RPs, but poorly correlated with in vivo and in vitro biomarker-based RPs in rainbow trout. The lack of strong correlation between the various sets of rainbow-trout specific RPs suggest that within the range of uncertainty associated with these assays, there is no consensus rank order of potency for the dioxins, furans, and nonortho PCBs among rainbow-trout-specific assays. Overall, the RLT 2.0 bioassay appeared to be a reasonable method for screening samples for potential to cause dioxin-like biological responses in rainbow trout. RLT 2.0 RPs may slightly over or underestimate the potency of individual congeners, but no definitive biases or inaccuracies were apparent based on comparison to other rainbow trout RPs.
Table 1. Comparison of RLT 2.0 bioassay-derived relative potencies (RPs) to rainbow-trout-specific RPs based on other in vitro and in vivo end-points and World Health Organization (WHO) toxic equivalency factors (TEFs) for fish. A correlation matrix is presented. Correlations were calculated using only those congeners for which point estimates of RP were available for both columns being compared.
Compound | RLT 2.0 | In vitro EROD a |
In vivo EROD b |
In vivo ELSM c |
WHO TEF d |
2,3,7,8 TCDD | 1.0 | 1.0 | 1.0 | 1.0 | 1 |
1,2,3,7,8 PeCDD | 0.225e | 2.6 | 1.8 | 0.73 | 1 |
1,2,3,4,7,8 HxCDD | 0.762f | 1.1 | 0.4 | 0.319 | 0.5 |
2,3,7,8 TCDF | 0.235f | 0.2 | 0.5 | 0.028 | 0.05 |
1,2,3,7,8 PeCDF | 0.208f | 0.2 | 0.4 | 0.034 | 0.05 |
2,3,4,7,8 PeCDF | 0.296e | 1.9 | 2.0 | 0.359 | 0.5 |
1,2,3,4,7,8 HxCDF | 0.917f | 1.1 | 0.4 | 0.280 | 0.1 |
PCB 77 (3,3',4,4') | 5.95 x 10-3 e* | ? | ? | 0.00016 | 0.0001 |
PCB 105 (2,3,3',4,4') | <5 x 10-5 f | ? | ? | <7x10-5 | <5x10-6 |
PCB 118 (2,3'4,4',5) | <6 x 10-3 f | ? | ? | <7x10-5 | <5x10-6 |
PCB 126 (3,3',4,4',5) | 6.28 x 10-3 e | ? | ? | 0.005 | 0.005 |
| |||||
Correlation Matrix | R2 | R2 | R2 | R2 | R2 |
RLT 2.0 | 1.0 | -0.072 | -0.34 | 0.61 | 0.45 |
In vitro EROD | ? | 1.0 | 0.83 | 0.58 | 0.70 |
In vivo EROD | ? | ? | 1.0 | 0.49 | 0.63 |
In vivo ELSM | ? | ? | ? | 1.0 | 0.95 |
b Parrot et al. 1995
c ELSM = early life stage mortality; Walker and Peterson 1991
d Van den Berg et al. 1999
e Richter et al. 1997
f this study
* EC-50 estimates based on incomplete dose-response curves
RLT 2.0 RPs were similar to RPs generated using a mammalian in vitro bioassay (Table 2). Previous studies reported differences in the RPs of mono-ortho polychlorinated biphenyls between fish and mammals. An inability to generate point estimates of RP for the mono- and di-ortho PCBs tested in this study, makes unclear whether the RLT 2.0 cell line is a better model for predicting the in vivo potency of these compounds to fish.
Table 2. Comparison of RLT 2.0 bioassay derived relative potencies (RPs) to RPs based on in vitro bioassay with H4IIE-rat liver cells (recombinant [luc] and wildtype [wt]) and international toxic equivalency factors (TEFs).
Compound | RLT 2.0 RP |
H4IIE-luc b RP |
H4IIE-wta RP |
International TEFs a |
2,3,7,8 TCDD | 1.0 | 1.0 | 1.0 | 1.0 |
1,2,3,7,8 PeCDD | 0.225c | 0.79 | 0.420 | 0.5 |
1,2,3,4,7,8 HxCDD | 0.762d | ? | .0830 | 0.1 |
2,3,7,8 TCDF | 0.235d | ? | 0.200 | 0.1 |
1,2,3,7,8 PeCDF | 0.208d | ? | 0.200 | 0.05 |
2,3,4,7,8 PeCDF | 0.296c | 0.69 | 1.40 | 0.5 |
1,2,3,4,7,8 HxCDF | 0.917d | ? | 0.020 | 0.1 |
PCB 77 (3,3',4,4') | 5.95 x 10-3 c* | 0.00071 | 1.8 x 10-5 | 5 x 10-4 |
PCB 105 (2,3,3',4,4') | < 5 x 10-5 d | <1 x 10-6 | 8 x 10-6 | 1 x 10-4 |
PCB 118 (2,3,4',4',5) | < 6 x 10-3 d | <1 x 10-6 | 3.5 x 10-7 | 1 x 10-4 |
PCB 126 (3,3',4,4',5) | 6.28 x 10-3 c | 0.017 | 2.2 x 10-2 | 0.1 |
PCB 156 (2,3,3',4,4',5) | < 3.6 x 10-2 d | ? | 5.5 x 10-5 | 5 x 10-4 |
PCB 169 (3,3',4,4',5,5') | < 5 x 10-3 d | 0.00055 | 4.7 x 10-4 | 1 x 10-2 |
b Sanderson et al. 1996
c Richter et al. 1997
d this study
* EC-50 estimates based on incomplete dose response curves
A common use for RPs and TEFs is in deriving a single value to characterize the biological potency of a sample based on instrumental analysis, which is generally calculated by multiplying the concentration of each compound detected by its RP or TEF and summing the total for all compounds. The value attained is known as a TCDD equivalent (TEQ). TEQs can be used to characterize potential biological potency based on instrumental analysis alone, or they can be applied in conjunction with bioassay. TEQs calculated for risk-assessment purposes tend to be based on TEFs. TEQs used in a mass balance context, in conjunction with bioassay, tend to be based on assay-specific RPs. A comparison of RP-TEQs with bioassay-derived TCDD-equivalents (TCDD-EQs) provides a method to evaluate mass balance and possible interactions between compounds. In the case of an unknown sample, if bioassay-derived TCDD-EQs are approximately equal to a calculated TEQ, one can assume that the active compounds in the sample have been detected and justified. Large differences between TCDD-EQs and TEQs suggest either nonadditive interactions between compounds or failure to detect and explain all active compounds in the sample. Bioassay-directed fractionation and further instrumental analysis can be applied to elucidate which of the above circumstances apply.
A sensitivity analysis was conducted to determine the uncertainty contributed to the assessment of complex environmental mixtures of HAHs by uncertainties in RPs determined for individual congeners and by differences in RPs between in vitro and in vivo responses and among species. The sensitivity analysis was conducted by calculating a set of TEQs from representative sets of RPs and TEFs. Instrumental analyses reported for samples of three species of fish (carp, walleye, and alewife) collected from Saginaw Bay, MI, were used. Congener concentrations and relative distributions in these samples should be representative of fish, from a variety of trophic levels, exposed to HAHs in situ.
The sensitivity analysis was used to determine which RLT 2.0 RPs are most likely to contribute to the uncertainty in RLT 2.0-derived TEQs. A frequency distribution of TEQs was generated for each species and a sensitivity analysis was performed to determine the relative contribution of each RP to the total variation in the TEQ estimate. Values within the TEQ distributions for carp, walleye, and alewife varied up to fifteen-fold. Within the 95-percent confidence range, carp and alewife TEQs varied up to six-fold and walleye TEQs varied up to eight-fold. This suggests that a ten-fold uncertainty factor (? 5 fold) is probably appropriate for TEQs derived from the current set of RLT 2.0 RPs.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 8 publications | 4 publications in selected types | All 3 journal articles |
---|
Type | Citation | ||
---|---|---|---|
|
Khim JS, Villeneuve DL, Kannan K, Lee KT, Snyder SA, Koh CH, Giesy JP. Alkylphenols, polycyclic aromatic hydrocarbons, and organochlorines in sediment from Lake Shihwa, Korea: instrumental and bioanalytical characterization. Environ Toxicol Chem 1999;18(11):2424-2432. |
R825371 (Final) |
not available |
|
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 |
|
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:
RLT 2.0, cell, in vitro bioassay, relative potency, TEF, rainbow trout, fish, PCBs, PCDDs, PCDFs, dioxins, furans, TEQ, TCDD-EQ, environmental toxicology, aquatic toxicology, risk assessment, reporter gene, luciferase, HAHs, sensitivity analysis, Monte-Carlo., 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 exposureProgress and Final Reports:
Original AbstractThe 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.