Steroidogenesis in vitro: towards relevant models for endocrine disruptor screening

Abstract

Starting our search for in vitro alternative methods to screen for steroidogenesis toxicity, we focused on the effects of (suggested) endocrine disrupting compounds (EDCs) on cytochrome P450 17 (CYP17) enzyme activity. CYP17 is responsible for conversion of progestagens to dehydroepiandrosterone (DHEA), the main precursor for androgens and estrogens, important for (fetal) sexual functioning and development. We developed a new assay to screen for effects on CYP17 activity using porcine adrenal cortex microsomes (PACMs) and compared this with the well-known H295R steroidogenesis assay. Also, effects of selected compounds on CYP19 (aromatase) activity in the H295R assay as well as human placental microsomes were assessed. 28 (suggested) EDCs were screened. The PACM assay showed to be an effective, rapid tool for screening of direct interaction of EDCs with CYP17. Furthermore, we discovered that CYP17 activity is regulated differently from CYP19. The addition of a specific and validated CYP17 assay to an EDC screening battery would be of added value on top of the existing steroidogenesis assays that mainly consider effects on the CYP19 enzyme. We further investigated a specific class of suggested EDCs: the conazole fungicide, some of which have been shown to affect male fertility parameters in vitro and in vivo. Ten conazole fungicides were selected and tested for potential in vitro (anti-)androgenic effects using the murine MA-10 cell line as well as an androgen receptor (AR) activity reporter assay. Exposure of MA-10 cells to six of these conazoles resulted in a decrease in basal testosterone (T) secretion. Nine of the tested conazole fungicides showed an inhibition of T-induced AR activation. Thus, several of the tested conazole fungicides showed distinct in vitro anti-androgenic effects in the performed assays. These effects are a result of two mechanisms, i.e. inhibition of T secretion and AR antagonism, suggesting that these compounds are potential testicular toxicants in vivo. Subsequently, the effects of two bisphenol analogues, two perfluorinated compounds, and two phthalates on ABC efflux transporters in the blood-testis barrier (BTB) were studied. Bisphenol A (BPA), tetrabromobisphenol A (TBBPA), perfluorooctanoic acid (PFOA), as well as perfluorooctane-sulfonic acid (PFOS) inhibited breast cancer resistance protein (BCRP) activity. BPA and PFOA showed to be substrates for BCRP. TBBPA, PFOA, and PFOS also showed inhibition of multidrug resistance proteins 1 and 4 (MRP1/4) and P-glycoprotein. In MA-10 cells, BPA and TBBPA concentration-dependently increased T secretion. For TBBPA, this could be blocked by inhibition of MRPs, generating the hypothesis that transporter-mediated efflux of T precursors is inhibited by TBBPA resulting in a higher availability of precursors for T production. This research indicates that, in EDC risk assessment, ABC efflux transporters present in the BTB are of toxicological and clinical relevance for testicular toxicity and should be taken into account. Next, effects of these several (structural) bisphenol analogues on AR and glucocorticoid receptor (GR) activity modulation were tested, as well as testicular steroidogenesis in MA-10 cells. BPA, bisphenol F (BPF), and TBBPA, but not bisphenol S (BPS) showed differential inhibition of AR as well as GR activity. BPF and BPS exposure of MA-10 cells mainly resulted in higher levels of progestagens that are formed at the beginning of the steroidogenic pathway, whereas TBBPA shows a distinct profile more directed towards the endpoint of testicular steroidogenesis, i.e. T and other androgens production. Furthermore, full steroid profiling of the in vitro MA-10 cell model shows the presence of a more fetal-like, testicular-specific steroidogenesis pathway. This so-called “backdoor pathway” renders the MA-10 assay added value on top of the H295R steroidogenesis assay for tissue-specific questions related to EDC screening.