Screening the ToxCast Chemical Libraries for Binding to Transthyretin

Transthyretin
(TTR) is one of the serum binding proteins
responsible
for transport of thyroid hormones (TH) to target tissue and for maintaining
the balance of available TH. Chemical binding to TTR and subsequent
displacement of TH has been identified as an end point in screening
chemicals for potential disruption of the thyroid system. To address
the lack of data regarding chemicals binding to TTR, we optimized
an in vitro assay utilizing the fluorescent probe
8-anilino-1-napthalenesulfonic acid (ANSA) and the human protein TTR
to screen over 1500 chemicals from the U.S. EPA’s ToxCast ph1_v2,
ph2, and e1k libraries utilizing a tiered approach. Testing of a single
high concentration (target 100 μM) resulted in 888 chemicals
with 20% or greater activity based on displacement of ANSA from TTR.
Of these, 282 chemicals had activity of 85% or greater and were further
tested in 12-point concentration–response with target concentrations
ranging from 0.015 to 100 μM. An EC50 was obtained for 276 of
these 301 chemicals. To date, this is the largest set of chemicals
screened for binding to TTR. Utilization of this assay is a significant
contribution toward expanding the suite of in vitro assays used to identify chemicals with the potential to disrupt
thyroid hormone homeostasis.