Studying the role of estrogen receptor alpha in the developmental toxicity of diethylstilbestrol using alternative testing strategies

Diethylstilbestrol (DES) is a synthetic estrogen that has been used between the 1940s and 1970s by pregnant women to prevent miscarriages and premature delivery by stimulating the synthesis of estrogen and progesterone in the placenta. However, use of DES appeared to cause a wide range of adverse effects, such as clear cell vaginal adenocarcinoma in the daughters of women who took the drug, and developmental and reproductive toxicity. These adverse effects have often been attributed to the functional estrogen receptor alpha (ERα), since it has been reported that ERα is needed to induce DES-mediated adverse developmental and reproductive effects in neonates. The question has been raised why DES behaves differently from the endogenous ERα agonist 17β-estradiol (E2), even though the molecular dimensions and binding orientations of DES and E2 to the ERα are almost identical. The research described in this thesis aimed to investigate the possible differences in the estrogenicity and developmental toxicity between DES and E2, using different in vitro and in silico approaches, focussing on the potential role of possible differences in ERα-mediated effects in the underlying mode of action. Accordingly, first the effect of DES and E2 on ERα-mediated reporter gene expression, ERα-mediated T47D breast cancer cell proliferation, and ERα-coregulator interactions and gene expression in T47D cells were evaluated. In addition, the effects of DES and E2 in two alternative developmental toxicity assays (the ES-D3 cell differentiation assay of the embryonic stem cell test (EST) and the zebrafish embyotoxicity test (ZET)) and the potential role of ERα in these effects were evaluated. Finally, possible dose-dependent differences in internal dose levels of DES and E2 were evaluated with help of PBK modelling, in order to elucidate to what extent possible differences in kinetics could play a role in differential in vivo effects of DES and E2. Altogether, the data show that two estrogens E2 and DES differ in their biological effects related to development in a subtle but significant way. At the cellular level, DES and E2 show high similarities in the molecular pathways that relate to ERα-mediated effects with small significant differences that may contribute to the developmental toxicity in part via potential epigenetic effects of DES. The in vitro developmental toxicity assays EST and ZET can discriminate DES from E2 in terms of developmental toxicity, but at the same time do not capture the full mode of action underlying DES-induced developmental toxicity. Finally, it was shown that in addition to the subtle differences in toxicodynamics, substantial differences in internal concentrations (endogenous E2 concentrations compared to predicted DES concentrations in women that took DES as medication), add to the differential in vivo effects of E2 and DES