The prostate cancer drug enzalutamide shortens anogenital distance in male rat offspring by blocking the androgen receptor

Abstract Background: Enzalutamide is a non-steroidal anti-androgen drug used to treat prostate cancer. It is a potent androgen receptor (AR) antagonist, with an in vitro Lowest Observed Effect Concentration (LOEC) of 0.05 μM. In this study, we wanted to assess its utility as a model compound for future mechanistic studies aimed at delineating mechanism-of-action of anti-androgenic effects in the developing fetus. Methods: Enzalutamide in vitro activity was tested using an Androgen receptor reporter assay (AR-EcoScreenTM) and a steroidogenesis assay (H295R assay). For in vivo characterization, pregnant Sprague-Dawley rats were exposed to 10 mg/kg bw/day enzalutamide from gestational day 7-21. At gestational day 21, enzalutamide exposure concentrations were measured both in amniotic fluids and fetal plasma, alongside Anogenital distance (AGD). Fetal testes were collected and for testosterone measurements and gene expression profiling. Results: Enzalutamide was a strong AR antagonist in vitro and we also observed disrupted androgen synthesis in the H295R steroidogenic assay with a LOEC of 3.1 μM. In utero exposure resulted in about 20% shorter anogenital distance (AGD) in male fetuses., as well as signs of dysregulated expression of the steroidogenic genes Star, Cyp11a1 and Cyp17a1 in the fetal testes at gestational day 21. Intra-testicular testosterone levels were unaffected. Conclusions: Based on these observations, together with in vitro LOECs and the fetal plasma levels of enzalutamide, we propose that the effect on male AGD was caused by AR antagonism rather than suppressed androgen synthesis. Due to the characteristic mechanism of action of enzalutamide, we suggest to use it as a new model compound in research on anti-androgenic environmental chemicals.