Identification of xenoestrogens in food additives by an integrated in silico and in vitro approach

Abstract

In the search for xenoestrogens within food additives, we have analyzed the Joint FAO-WHO expert committee database, containing 1500 compounds, using an integrated in silico and in vitro approach. This analysis identified 31 potential estrogen receptor alpha ligands that were reduced to 13 upon applying a stringent filter based on ligand volume and binding mode. Among the 13 potential xenoestrogens, four were already known to exhibit an estrogenic activity, and the other nine were assayed in vitro, determining the binding affinity to the receptor and biological effects. Propyl gallate was found to act as an antagonist, and 4-hexylresorcinol was found to act as a potent transactivator; both ligands were active at nanomolar concentrations, as predicted by the in silico analysis. Some caution should be issued for the use of propyl gallate and 4-hexylresorcinol as food additives.

Figure 1

Ribbon and surface representation of ERα in complex with the agonist diethylstilbestrol (a) (PDB code: 3ERD), closed conformation, and with a dihydrobenzoxathiin SERM (b) (PDB code: 1XPC), open conformation. H12 (red) exhibits different orientations in the two complexes.

Figure 2

Overlay of the ERα binding site complexed with 17β-estradiol (PDB code: 1ERE, yellow), 4-hydroxytamoxifen (PDB code: 3ERT, blue), and raloxifene (PDB code: 1ERR, light-blue). Only the ligands and the key interacting residues are represented in capped sticks. Glu353 and Arg394, which interact with the estradiol A ring, show a very similar behavior in the different complexes. On the contrary, His524, which interacts with the estradiol D ring, displays different orientations in different complexes, reflecting the higher degree of freedom of ligand molecules in this portion of the binding pocket. The side chain of Asp351 shows two distinct orientations, depending on the presence (SERMs) or absence (agonist) of a bulky side chain able to interact with this residue.

Figure 3

Correlation between the experimental pK_i and the HINT-calculated pK_i for the 57 ERα—ligand complexes.

Figure 4

Binding titrations for propyl gallate (a) and 4-hexylresorcinol (c) and transactivation assays for propyl gallate (b), 4-hexylresorcinol (d), and 17β-estradiol (e). The antagonist activity of propyl gallate was tested in a transactivation assay (f). Each experiment was carried out in triplicate, according to the procedure reported in the Experimental Procedures.

Figure 5

Predicted binding modes for the 13 food additives identified as potential ERα ligands by virtual screening. (a) Propyl 4-hydroxybenzoate and butyl 4-hydroxybenzoate, (b) butyl hydroxyanisole, (c) delphinidin, (d) 4-hexylresorcinol, (e) malvidin, (f) peonidin, (g) propyl gallate, (h) octyl gallate, (i) nordihydroguaiaretic acid, (j) capsaicin, (k) curcumin, and (l) erythrosine.