Aziza Hussein

Diethylstilbestrol (DES) is a synthetic hormone that was first prescribed in the period of 1938 to 1971 for pregnant women to prevent miscarriage and premature delivery, and to women in general for treatment of menstrual problems and cancer. However, from later studies it was concluded that these claimed beneficial effects were not observed, and even adverse effects were reported, such as clear cell carcinoma, and reproductive and developmental toxicity. Although increased levels of the endogenous hormone 17β-estradiol (E2) have been reported to increase the chances on developing cancer, such as breast cancer, the typical adverse effects observed for DES were not reported to the same extent for E2, although DES is structurally similar to E2 and it is suggested that DES acts by mimicking the effects of E2. The aim of this thesis was to investigate the differences in 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 changes in the underlying mode of action.

Chapter 1 provides background information introducing the topic. In addition, the adverse effects related to DES-exposure and the known mechanisms of action of DES are described. The knowledge gaps and principal aim of the thesis are defined and the testing strategies to be used are explained and a general outline of the thesis is provided.

Chapter 2 investigated the possible differences between DES and E2 in induction of ERα-mediated cellular effects, including ERα-mediated reporter gene expression in the U2OS CALUX assay, ERα-mediated cell proliferation, and ERα-mediated-coregulator interactions and gene expression in cells of the T47D breast cancer cell line. The results obtained indicate that DES and E2 activate ERα-mediated reporter gene transcription and T47D cell proliferation in a similar way. However, minor but significant differences between DES- and E2-induced binding of the ERα to coregulator motifs and in transcriptomic signatures were observed. These differences, including especially E2-induced binding of the ERα with several co-repressor motifs, DES-induced downregulation of genes involved in histone deacetylation and DNA methylation, and upregulation of CYP26A1 and CYP26B1, may play a role in the differential in vivo effects reported for DES and E2. Thus, coregulator binding and transcriptomic signatures could discriminate DES from E2.

In Chapter 3 an alternative testing strategy to quantitatively predict the in vivo developmental toxicity of DES was evaluated. To this end a physiologically based kinetic (PBK) model was defined that was subsequently used to translate concentration-response data for the in vitro developmental toxicity of DES, obtained in the ES-D3 cell differentiation assay, into predicted in vivo dose-response data for developmental toxicity. Previous studies showed that the PBK modeling-facilitated reverse dosimetry approach is a useful approach to quantitatively predict the developmental toxicity of several developmental toxins. The results obtained in this chapter show that the PBK model adequately predicted DES blood concentrations in rats. Furthermore, the study revealed that DES tested positive in the ES-D3 differentiation assay and that DES-induced inhibition of the ES-D3 cell differentiation could be counteracted by the ERα antagonist fulvestrant, indicating that the in vitro ES-D3 cell differentiation assay was able to capture the role of ERα reported in the mode of action underlying the developmental toxicity of DES in vivo. Finally, the in vitro data were combined with the PBK model to predict a dose-response curve for the in vivo developmental toxicity of DES, and the results clearly showed that this combination did not adequately predict the in vivo developmental toxicity of DES in a quantitative way. Thus, it was concluded that although the EST qualifies DES as a developmental toxin and detects the role of ERα in this process, the ES-D3 cell differentiation assay of the EST apparently does not adequately capture the full mode of action underlying DES-induced developmental toxicity in vivo. This may in part be related to the fact that the ES-D3 cell differentiation assay lacks the complex biological system and the metabolic capacity of an intact organism and/or that the assay may not reflect all modes of action possibly underlying developmental toxicity, including for example epigenetic effects, reported to play an important role in DES-mediated developmental effects.

Because of these potential limitations of the EST, Chapter 4 assessed the developmental toxicity of DES compared to E2 in the zebrafish embryotoxicity test (ZET). In addition, it was investigated whether the role of the ERα in DES-mediated developmental toxicity could also be demonstrated in the ZET. To this end, the in vitro embryotoxicity of DES and E2 was quantified in the ZET in the absence and presence of the ERα antagonist fulvestrant. Results obtained in the ZET showed that DES induced growth retardation, cumulative mortality and malformations in zebrafish embryos, while E2 showed only growth retardation and cumulative mortality with a lower potency compared to DES. Additionally, DES induced pericardial edema formation in zebrafish embryos, which was not observed in E2-exposed zebrafish embryos. This effect could be counteracted by co-exposure to fulvestrant, indicating that the ZET was able to capture the role of ERα in the mode of action underlying this developmental toxicity effect of DES in zebrafish. Overall, it is concluded that the ZET differentiates between E2 and DES with respect to their developmental toxicity, while confirming the role of ERα in the specific developmental toxicity effects found for DES. Furthermore, like the EST, also the ZET appeared unable to capture the relatively high in vivo potency of DES as a developmental toxicant.

Finally, in Chapter 5 of the thesis it was investigated to what extent differences in kinetics and internal dose levels may add to the potential in vivo differences in effects of E2 vs DES on development. It was hypothesised that part of the in vivo differences may originate from differences in the internal dose levels of these two estrogens during pregnancy and/or DES treatment. To enable quantification of dose-dependent internal dose levels, physiologically based kinetic (PBK) models for E2 and DES in pregnant women were defined. The models predicted the kinetics of DES and E2 in pregnant women to be comparable. Therapeutic doses of DES as given to pregnant women were predicted to result in blood levels that are 3 to 4 orders of magnitude higher than endogenous E2 blood levels. It is concluded that the PBK models developed enable quantification of dose-dependent plasma concentrations of DES and E2 in pregnant women and reveal that differences in effects of DES and E2 on development may at least in part be due to differences in internal exposure levels.

In Chapter 6 an overview and discussion of the results obtained is provided. The chapter also presents remaining data gaps and future perspectives. Altogether, it is concluded that the 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.