Combination of Metabolomics with Cellular Assays Reveals New Biomarkers and Mechanistic Insights on Xenoestrogenic Exposures in MCF‑7 Cells

The
disruptive potential of xenoestrogens like bisphenol A (BPA) lies
in their 17β-estradiol (E2)-like binding to estrogen receptors
(ERs) followed by concomitant modulation of ER target gene expression.
Unsurprisingly, most endocrine testing systems focus on the quantification
of canonical transcripts or ER-sensitive reporters. However, only
little information is available about the corresponding metabolomic
changes in vitro. This knowledge gap becomes particularly
relevant in the context of potential mixture effects, for example,
as a consequence of coexposure to potentially estrogenically active
pollutants (e.g., Cd2+). Such effects are often difficult
to dissect with molecular tools, especially with regard to potential
physiological relevance. Metabolomic biomarkers are well-suited to
address this latter aspect as they provide a comprehensive readout
of whole-cell physiology. Applying a targeted metabolomics approach
(FIA-MS/MS), this study looked for biomarkers indicative of xenoestrogenic
exposure in MCF-7 cells. Cells were treated with E2 and BPA in the
presence or absence of Cd2+. Statistical analysis revealed
a total of 11 amino acids and phospholipids to be related to the compound’s
estrogenic potency. Co-exposure to Cd2+ modulated the estrogenic
profile. However, the corresponding changes were found to be moderate
with cellular assays such as the E-screen failing to record any Cd2+-specific estrogenic effects. Overall, metabolomics analysis
identified proline as the most prominent estrogenic biomarker. Its
increase could clearly be related to estrogenic exposure and concomitant
ERα-mediated induction of proliferation. Involvement of the
latter was confirmed by siRNA-mediated knockdown studies as well as
by receptor inhibition. Further, the underlying signaling was also
found to involve the oncoprotein MYC. Taken together, this study provides
insights into the underlying mechanisms of xenoestrogenic effects
and exemplify the strength of the complementary use of metabolomics
and cellular and molecular assays.