Endocrine Active Chemical Screening Tests Optimized to Improve Precision, Accuracy, and Timeliness
Scientist examining cells under a microscope
Scientists optimized existing endocrine active chemical screening tests to improve their precision, accuracy, and ability to screen more samples in a shorter time-frame. The optimization was done to more rapidly obtain results from the bioassays so that research on the risks of endocrine active chemical exposure can proceed more rapidly.
Major challenges arise when prioritizing the tens of thousands of potential endocrine active chemicals in production today. This information is needed to determine if chemical exposures pose a risk for adverse effects on humans and other organisms. Screening tests offer a way to prioritize risk in a scientifically defensible, consistent and cost efficient manner.
Existing screening tests that use luciferase-reporter-gene expression linked to DNA response elements have been an effective method to screen samples for endocrine activity. However, the cost and time required to screen large numbers of chemicals or samples becomes unfeasible because the test is limited to 96-well plates. In addition, current testing methods are challenged because some endocrine active chemicals are toxic to cells used in the assay, which can interfere with the test and lead to biased and inconsistent results.
Therefore, scientists at the U.S. Geological Survey (USGS) cell bioassay laboratory at Columbia Environmental Research Center focused on optimizing existing screening methods to increase the number of samples that can be screened in a shorter time-frame while also improving the precision and accuracy of the tests by co-testing for sample toxicity. USGS scientists optimized two existing assays that traditionally used 96-well plates:
- the chemically-activated luciferase expression (CALUX) humanbreast VM7Luc4E2 cell bioassay for estrogens/anti-estrogen like chemicals, and
- a CALUX-type human breast MDA-kb2 cell bioassay for androgens/anti-androgen like chemicals and glucocorticoids/anti-glucocorticoid like chemicals in 384-well plates.
The optimized method was capable of producing consistent and precise results for the 384-well plate compared to the existing method that utilizes a 96-well plate. This four-fold increase in analytical capability can assist scientists to more rapidly determine exposure risks in a scientifically defensible and cost-effective manner. This is important so that a distinction can be made between perceived and actual risks of exposure to endocrine active chemicals.
The optimization of this method is part of a larger effort in the USGS Environmental Health programs to build rigorous, sustainable, and timely core analytical capabilities that can be used to prioritize ongoing research to understand contaminant effects on exposed organisms.
The USGS Contaminant Biology Program funded this study.
References
Brennan, J.C., and Tillitt, D.E., 2018, Development of a dual luciferase activity and fluorescamine protein assay adapted to a 384 micro-well plate format--Reducing variability in human luciferase transactivation cell lines aimed at endocrine active substances: Toxicology in Vitro, v. 47, no. Supplement C, p. 18-25, doi:10.1016/j.tiv.2017.10.030.
Brennan, J.C., 2017, Development of a dual luciferase-fluorescamine assay adapted to a 384 micro-well plate format-data: U.S. Geological Survey Data Release.
Below are other science projects associated with this Activities and Expertise.
Endocrine Disrupting Compounds in the Chesapeake Bay Watershed Science Team
Scientists optimized existing endocrine active chemical screening tests to improve their precision, accuracy, and ability to screen more samples in a shorter time-frame. The optimization was done to more rapidly obtain results from the bioassays so that research on the risks of endocrine active chemical exposure can proceed more rapidly.
Major challenges arise when prioritizing the tens of thousands of potential endocrine active chemicals in production today. This information is needed to determine if chemical exposures pose a risk for adverse effects on humans and other organisms. Screening tests offer a way to prioritize risk in a scientifically defensible, consistent and cost efficient manner.
Existing screening tests that use luciferase-reporter-gene expression linked to DNA response elements have been an effective method to screen samples for endocrine activity. However, the cost and time required to screen large numbers of chemicals or samples becomes unfeasible because the test is limited to 96-well plates. In addition, current testing methods are challenged because some endocrine active chemicals are toxic to cells used in the assay, which can interfere with the test and lead to biased and inconsistent results.
Therefore, scientists at the U.S. Geological Survey (USGS) cell bioassay laboratory at Columbia Environmental Research Center focused on optimizing existing screening methods to increase the number of samples that can be screened in a shorter time-frame while also improving the precision and accuracy of the tests by co-testing for sample toxicity. USGS scientists optimized two existing assays that traditionally used 96-well plates:
- the chemically-activated luciferase expression (CALUX) humanbreast VM7Luc4E2 cell bioassay for estrogens/anti-estrogen like chemicals, and
- a CALUX-type human breast MDA-kb2 cell bioassay for androgens/anti-androgen like chemicals and glucocorticoids/anti-glucocorticoid like chemicals in 384-well plates.
The optimized method was capable of producing consistent and precise results for the 384-well plate compared to the existing method that utilizes a 96-well plate. This four-fold increase in analytical capability can assist scientists to more rapidly determine exposure risks in a scientifically defensible and cost-effective manner. This is important so that a distinction can be made between perceived and actual risks of exposure to endocrine active chemicals.
The optimization of this method is part of a larger effort in the USGS Environmental Health programs to build rigorous, sustainable, and timely core analytical capabilities that can be used to prioritize ongoing research to understand contaminant effects on exposed organisms.
The USGS Contaminant Biology Program funded this study.
References
Brennan, J.C., and Tillitt, D.E., 2018, Development of a dual luciferase activity and fluorescamine protein assay adapted to a 384 micro-well plate format--Reducing variability in human luciferase transactivation cell lines aimed at endocrine active substances: Toxicology in Vitro, v. 47, no. Supplement C, p. 18-25, doi:10.1016/j.tiv.2017.10.030.
Brennan, J.C., 2017, Development of a dual luciferase-fluorescamine assay adapted to a 384 micro-well plate format-data: U.S. Geological Survey Data Release.
Below are other science projects associated with this Activities and Expertise.