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

Assessment of the in vitro developmental toxicity of diethylstilbestrol and estradiol in the zebrafish embryotoxicity test

The present study investigated the developmental toxicity of diethylstilbestrol (DES) in the zebrafish embryotoxicity test (ZET). This was done to investigate whether the ZET would better capture the developmental toxicity of DES than the embryonic stem cells test (EST) that was previously shown to underpredict the DES-induced developmental toxicity as compared to in vivo data, potentially because the EST does not capture late events in the developmental process. The ZET results showed DES-induced growth retardation, cumulative mortality and dysmorphisms (i.e. induction of pericardial edema) in zebrafish embryos while the endogenous ERα agonist 17β-estradiol (E2) showed only growth retardation and cumulative mortality with lower potency compared to DES. Furthermore, the DES-induced pericardial edema formation in zebrafish embryos could be counteracted by co-exposure with ERα antagonist fulvestrant, indicating that the ZET captures the role of ERα in the mode of action underlying the developmental toxicity of DES. Altogether, it is concluded that the ZET differentiates DES from E2 with respect to their developmental toxicity effects, while confirming the role of ERα in mediating the developmental toxicity of DES. Furthermore, comparison to in vivo data revealed that, like the EST, in a quantitative way also the ZET did not capture the relatively high in vivo potency of DES as a developmental toxicant.</p

Apoptotic Potential of Glucomoringin Isothiocyanate (GMG-ITC) Isolated from <i>Moringa oleifera</i> Lam Seeds on Human Prostate Cancer Cells (PC-3)

Inhibition of several protein pathways involved in cancer cell regulation is a necessary key in the discovery of cancer chemotherapy. Moringa oleifera Lam is often used in traditional medicine for the treatment of various illnesses. The plant contains glucomoringin isothiocyanate (GMG-ITC) with therapeutic potential against various cancer cells. Therefore, GMG-ITC was evaluated for its cytotoxicity against the PC-3 prostate cancer cell line and its potential to induce apoptosis. GMG-ITC inhibited cell proliferation in the PC-3 cell line with IC50 value 3.5 µg/mL. Morphological changes as a result of GMG-ITC-induced apoptosis showed chromatin condensation, nuclear fragmentation, and membrane blebbing. Additionally, Annexin V assay showed proportion of cells in early and late apoptosis upon exposure to GMG-ITC in a time-dependent manner. Moreover, GMG-ITC induced a time-dependent G2/M phase arrest, with reduction of 39.1% in the PC-3 cell line. GMG-ITC also activates apoptotic genes including caspase, tumor suppressor gene (p53), Akt/MAPK, and Bax of the proapoptotic Bcl family. Early apoptosis proteins (JNK, Bad, Bcl2, and p53) were significantly upregulated upon GMG-ITC treatment. It is concluded that apoptosis induction was observed in PC-3 cells treated with GMG-ITC. These phenomena suggest that GMG-ITC from M. oleifera seeds could be useful as a future cytotoxic agent against prostate cancer

The in vivo developmental toxicity of diethylstilbestrol (DES) in rat evaluated by an alternative testing strategy

In the present study, we evaluated an alternative testing strategy to quantitatively predict the in vivo developmental toxicity of the synthetic hormone diethylstilbestrol (DES). 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. The previous studies showed that the PBK model-facilitated reverse dosimetry approach is a useful approach to quantitatively predict the developmental toxicity of several developmental toxins. The results obtained in the present study show that the PBK model adequately predicted DES blood concentrations in rats. Further studies 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 estrogen receptor alpha (ERα) antagonist fulvestrant, indicating that the in vitro ES-D3 cell differentiation assay was able to mimic the role of ERα reported in the mode of action underlying the developmental toxicity of DES in vivo. In spite of this, combining these in vitro data with the PBK model did not adequately predict the in vivo developmental toxicity of DES in a quantitative way. It is 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 processes underlying DES-induced developmental toxicity in vivo.</p

The in vivo developmental toxicity of diethylstilbestrol (DES) in rat evaluated by an alternative testing strategy

In the present study, we evaluated an alternative testing strategy to quantitatively predict the in vivo developmental toxicity of the synthetic hormone diethylstilbestrol (DES). 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. The previous studies showed that the PBK model-facilitated reverse dosimetry approach is a useful approach to quantitatively predict the developmental toxicity of several developmental toxins. The results obtained in the present study show that the PBK model adequately predicted DES blood concentrations in rats. Further studies 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 estrogen receptor alpha (ERα) antagonist fulvestrant, indicating that the in vitro ES-D3 cell differentiation assay was able to mimic the role of ERα reported in the mode of action underlying the developmental toxicity of DES in vivo. In spite of this, combining these in vitro data with the PBK model did not adequately predict the in vivo developmental toxicity of DES in a quantitative way. It is 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 processes underlying DES-induced developmental toxicity in vivo.</p