Abstract:
Substantial public health concerns exist over the potential endocrine
disrupting capabilities of a wide variety of untested or under-tested natural
and industrial chemicals. It is clear that the development of accurate, high-throughput, and inexpensive testing regimens will be key to mitigating public concern. Here we report on the development of a novel screening assay for estrogenic activity that utilizes an autonomously bioluminescent human cell line to provide direct bioavailability data. To construct this cell line, estrogen-responsive human breast carcinoma cells (T-47D) were genetically
engineered to express the full bacterial bioluminescence gene cassette
(luxCDABEfrp), generating an autonomously bioluminescent cell line
(T-47D/Lux) capable of maintaining bioluminescent output independent of
substrate addition. Bioluminescence emitted from T-47D/Lux cells was
correlated tightly (R2 > 0.99) to the number of cells present in a population,
permitting the use of light production dynamics as an indicator of cell
proliferation. Additionally, the substrate-free nature of the lux system allowed
for continuous, near real-time monitoring of the same cell population
throughout exposure to the tested compounds. A significant change in
bioluminescent production (p < 0.05) compared with unexposed control was
observed 3 days after exposure to concentrations of 17β-estradiol (E2) as
low as 1 pM. The EC50 for E2 in this assay was determined to be
approximately 10 pM. These results are similar to those obtained using a
traditional cell proliferation assay, but offer the advantage that data
acquisition can be performed in a fully automated fashion since the need for
sample destruction or substrate addition is removed, making it an ideal
candidate for high-throughput analysis.
Tingting Xu1,, Dan Close2, Sarah Price1, and Gary Sayler1,21, Joint Institute for Biological Sciences, The University of Tennessee, Knoxville, TN 2490 BioTech Inc., Knoxville, TN
