High-Throughput Effect-Directed Analysis Using Downscaled in Vitro Reporter Gene Assays To Identify Endocrine Disruptors in Surface Water

Effect-directed analysis (EDA) is
a commonly used approach for
effect-based identification of endocrine disruptive chemicals in complex
(environmental) mixtures. However, for routine toxicity assessment
of, for example, water samples, current EDA approaches are considered
time-consuming and laborious. We achieved faster EDA and identification
by downscaling of sensitive cell-based hormone reporter gene assays
and increasing fractionation resolution to allow testing of smaller
fractions with reduced complexity. The high-resolution EDA approach
is demonstrated by analysis of four environmental passive sampler
extracts. Downscaling of the assays to a 384-well format allowed analysis
of 64 fractions in triplicate (or 192 fractions without technical
replicates) without affecting sensitivity compared to the standard
96-well format. Through a parallel exposure method, agonistic and
antagonistic androgen and estrogen receptor activity could be measured
in a single experiment following a single fractionation. From 16 selected
candidate compounds, identified through nontargeted analysis, 13 could
be confirmed chemically and 10 were found to be biologically active,
of which the most potent nonsteroidal estrogens were identified as
oxybenzone and piperine. The increased fractionation resolution and
the higher throughput that downscaling provides allow for future application
in routine high-resolution screening of large numbers of samples in
order to accelerate identification of (emerging) endocrine disruptors.