Tissue viability and steroid enzyme expression is maintained following treatment with steroidogenic inhibitors
Examination of the cultured tissue fragments was used to determine whether the investigated concentrations of the selected inhibitors affected HFA tissue morphology and viability. Overall, no apparent changes in tissue morphology were observed in the inhibitor-treated samples compared with vehicle controls (Fig. 2a). Likewise, in cultured tissue fragments treated with Abiraterone acetate, Osilodrostat, or Efavirenz proliferating cells (BrdU+) were present, while no or few apoptotic (cPARP+) cells were observed which was similar to vehicle controls. Treatment with a 10-fold higher dose of Abiraterone acetate and Osilodrostat also did not affect proliferation and apoptosis (based on BrdU+ and cPARP+ cells) compared to vehicle controls (data not shown). This together suggests that the effects of the selected inhibitors on adrenal steroidogenesis were not due to cytotoxicity. The steroidogenic enzymes expected to be inhibited by the selected inhibitors were all expressed in the ex vivo cultured tissue samples after the 14 days ex vivo culture period (Fig. 2b). Since no apparent difference in expression pattern or level compared to vehicle controls was observed, effects of the examined inhibitors are suggested to be antagonistic rather than affecting the expression levels of the steroidogenic enzymes.
Effects of Abiraterone acetate treatment on human fetal adrenal steroidogenesis
Treatment with Abiraterone acetate (1 μM) affected the secretion of all investigated steroids. Specifically, Abiraterone acetate caused decreased androgen levels, a small rise in glucocorticoid levels, and a considerable increase in the level of some steroidogenic intermediates under basal conditions (Fig. 3a). The inhibitory effect on HFA androgen biosynthesis was evident from reduced levels of testosterone (2.2-fold decrease, p < 0.05) and androstenedione (4.9-fold decrease, p < 0.01) and a tendency towards decreased DHEAS levels although this was not statistically significant. Abiraterone acetate treatment increased the levels of most measured glucocorticoids, including cortisone (2.0-fold, p < 0.05) and cortisol (2.4-fold, p < 0.05), and further resulted in a substantial increase in the secretion of the steroidogenic intermediates progesterone (11-fold, p < 0.001), corticosterone (11-fold, p < 0.001), and 17-hydroxyprogesterone (1.6-fold, p < 0.05). Treatment with Abiraterone acetate (1 μM) under ACTH-stimulated conditions affected the secretion of all measured steroids compared with effects of ACTH-stimulation alone, including DHEAS and 11-deoxycortisol that were not altered after Abiraterone acetate treatment under basal conditions (Fig. 3a). In particular, the inhibitory effect on adrenal androgen biosynthesis was more pronounced under ACTH-stimulated conditions with reduced biosynthesis of testosterone (47-fold decrease, p < 0.0001), androstenedione (47-fold decrease, p < 0.0001), and DHEAS (3.0-fold decrease, p < 0.01) when compared with ACTH-stimulation alone. Also, the effects of Abiraterone acetate under stimulated conditions caused elevated levels of cortisone (6.1-fold, p < 0.001) and cortisol (1.6-fold, p < 0.05), while the glucocorticoid intermediates 11-deoxycortisol (3.5-fold decrease, p < 0.05) and 17-hydroxyprogesterone (1.7-fold decrease, p < 0.01) levels were reduced under ACTH-stimulated conditions. Finally, the levels of progesterone (14-fold, p < 0.0001) and corticosterone (13-fold, p < 0.0001) increased following Abiraterone acetate treatment under stimulated conditions.
The inhibitory effect of Abiraterone acetate under basal and ACTH-stimulated conditions was further evident from the CYP17A1 product/substrate ratios (Fig. 3b). Thus, the 17α-hydroxylase product/substrate ratio (17-hydroxyprogesterone/progesterone) decreased 6.8-fold (p < 0.01) under basal conditions and 24-fold (p < 0.0001) under ACTH-stimulated conditions. Accordingly, the 17,20-lyase product/substrate ratio (androstenedione/17-hydroxyprogesterone) decreased 7.8-fold (p < 0.001) under basal conditions and 27-fold (p < 0.0001) under ACTH-stimulated conditions. A potent androgen inhibition was also evident from the ratios reflecting the combined CYP17A1 activity after Abiraterone acetate treatment (Fig. 3c). The androstenedione/corticosterone ratio decreased 55-fold (p < 0.001) under basal conditions and 608-fold (p < 0.001) under ACTH-stimulated conditions and the androstenedione/cortisol ratio decreased 12-fold (p < 0.0001) under basal conditions and 76-fold (p < 0.0001) under ACTH-stimulated conditions.
The observed increase in HFA glucocorticoid levels upon treatment with Abiraterone acetate (1 μM) led us to speculate whether this could be the result of incomplete CYP17A1 inhibition. Therefore, the effects of a 10-fold higher concentration of Abiraterone acetate were examined under basal conditions in HFA tissue cultured ex vivo for 14 days (Additional file 2: Figure S1). Treatment with Abiraterone acetate (10 μM) reduced the levels of testosterone (3.4-fold decrease, p < 0.001), androstenedione (30-fold decrease, p < 0.0001), and DHEAS (24-fold decrease, p < 0.0001) in accordance with the observations from treatment with the lower concentration of Abiraterone acetate (1 μM). However, the 10 μM dose further decreased androstenedione levels 5.9-fold (p < 0.0001) and DHEAS levels 10-fold (p < 0.0001) compared with treatment effects of the 1 μM dose. Interestingly, the 10-fold higher concentration of Abiraterone acetate shifted the effect from an increase (at 1 μM) to a decrease (at 10 μM) in the secretion of glucocorticoids, including reduced levels of cortisone (3.7-fold decrease, p < 0.0001), cortisol (2.6-fold decrease, p < 0.01), and the intermediate 11-deoxycortisol (8.0-fold decrease, p < 0.0001), compared with vehicle control. Thus, the increased Abiraterone acetate concentration resulted in a 7.2-fold decrease in cortisone levels (p < 0.0001), 6.2-fold decrease in cortisol levels (p < 0.0001), and an 11-fold decrease in 11-deoxycortisol levels (p < .0001) compared with treatment effects of 1 μM Abiraterone acetate. Abiraterone acetate treatment (10 μM) also increased the levels of the steroidogenic intermediate progesterone (68-fold, p < 0.0001) and corticosterone (15-fold, p < 0.0001) causing an additional 3.2-fold increase (p < 0.0001) in progesterone levels compared with treatment effects following the 1 μM dose.
Effects of Osilodrostat treatment on human fetal adrenal steroidogenesis
Treatment with Osilodrostat (1 μM) altered the secretion of the investigated steroidogenic intermediates and androgens under basal conditions in ex vivo cultured HFA tissue (Fig. 4a). Thus, treatment with Osilodrostat affected HFA androgen secretion causing an increase in testosterone (9.5-fold, p < 0.001) and androstenedione (10.3-fold, p < 0.0001), while DHEAS levels were unaffected. Although no effects were detected on the secretion of cortisone and cortisol, Osilodrostat treatment did result in increased levels of glucocorticoid intermediates upstream of CYP11B1 activity. This increase in the levels of intermediates included 17-hydroxyprogesterone (3.8-fold, p < 0.001) and the 11β-hydroxylase substrate 11-deoxycortisol (50-fold, p < 0.0001). Additionally, treatment with Osilodrostat reduced corticosterone levels (2.9-fold decrease, p < 0.001) and increased progesterone levels (2.1-fold, p < 0.01) under basal conditions. ACTH-stimulation prevented the treatment-induced increase in adrenal androgens and glucocorticoid intermediates levels that was observed following treatment with Osilodrostat (1 μM) under basal conditions (Fig. 4a). Thus, no effects of Osilodrostat treatment were observed on adrenal androgen levels under stimulated conditions compared with ACTH-stimulation alone, while the increase in 11β-hydroxylase substrate 11-deoxycortisol (2.5-fold, p< 0.001) was less pronounced than the response under basal conditions. Following ACTH-stimulation, only effects on corticosterone (2.1-fold decrease, p < 0.01) and progesterone (1.6-fold increase, p < 0.05) levels were of similar level as the effects of Osilodrostat treatment under basal conditions.
The effect of Osilodrostat treatment on CYP11B1/2 product/substrate ratios showed effective inhibition under both basal and ACTH-stimulated conditions (Fig. 4b). The corticosterone/progesterone ratio decreased 6.1-fold (p < 0.0001) under basal and 3.4-fold (p< 0.001) under ACTH-stimulated conditions. Additionally, the cortisol/11-deoxycortisol ratio decreased 31-fold (p < 0.0001) under basal conditions and 2.1-fold (p < 0.05) under ACTH-stimulated conditions. The effect on steroidogenic intermediates and glucocorticoid levels under basal conditions was also clear from the ratios reflecting the combined CYP11B1/2 activity (Fig. 4c) with increased androstenedione/corticosterone (29-fold, p < 0.0001) and androstenedione/cortisol ratios (6.3-fold p < 0.001) under basal conditions, while no effects on these ratios were observed under ACTH-stimulated conditions (Fig. 4c).
Treatment with Osilodrostat (1 μM) did not lead to a decrease in secretion of cortisol in ex vivo cultured HFA tissues as has previously been reported in patient studies [25]. Since Osilodrostat is known to be a more potent inhibitor of aldosterone synthase activity than 11β-hydroxylase activity [26], we speculated whether the effect of Osilodrostat on glucocorticoid secretion could be dose-dependent. Therefore, the effects of a 10-fold higher concentration of Osilodrostat were examined under basal conditions in HFA tissue cultured ex vivo for 14 days (Additional file 3: Figure S2). Treatment with Osilodrostat resulted in a dose-dependent inhibition of HFA glucocorticoid secretion. Interestingly, the higher concentration of Osilodrostat (10 μM) caused a reduction in the levels of both cortisone (1.5-fold decrease, p < 0.05) and cortisol (3.6-fold decrease, p < 0.01) compared with vehicle controls, and a 5.4-fold decrease in cortisol levels (p < 0.0001) compared with treatment effects of 1 μM Osilodrostat. Treatment with Osilodrostat (10 μM) also increased the levels of steroidogenic intermediates upstream of 11β-hydroxylase activity (11-deoxycortisol 31-fold, p < 0.0001; and 17-hydroxyprogesterone 2.7-fold p < 0.0001) in accordance with the observations from treatment with the lower concentration of Osilodrostat (1 μM). The increase in progesterone (5.3-fold, p < 0.0001) and reduction in corticosterone levels (25-fold decrease, p < 0.0001) compared with vehicle controls were 2.5-fold higher for progesterone (p < 0.001) and 9.1-fold lower for corticosterone (p < 0.0001) compared with treatment effects of 1 μM Osilodrostat. Treatment with Osilodrostat (10 μM) increased the levels of testosterone (3.4-fold, p < 0.01), androstenedione (3.8-fold, p < 0.0001), and reduced the levels of DHEAS (2.2-fold decrease, p < 0.01) compared with vehicle controls. This corresponded to a 2.6-fold decrease of testosterone (p < 0.05) and 2.3-fold decrease in androstenedione (p < 0.001) levels compared with treatment effects of 1 μM Osilodrostat.
Effects of Efavirenz treatment on human fetal adrenal steroidogenesis
Treatment with Efavirenz (10 μM) in ex vivo cultured HFA tissue only affected the secretion of testosterone (4.3-fold decrease, p < 0.05) under basal conditions, with no statistically significant effects observed on glucocorticoid and steroidogenic intermediate levels (Fig. 5a). However, under ACTH-stimulated conditions, treatment with Efavirenz inhibited the biosynthesis of both adrenal androgens, glucocorticoids and steroidogenic intermediates when compared with ACTH-stimulation alone (Fig. 5a). Under stimulated conditions, Efavirenz reduced the levels of testosterone (20-fold decrease, p < 0.001) and androstenedione (20-fold decrease, p < 0.0001) as well as the levels of cortisol (4.4-fold decrease, p < 0.01), 11-deoxycortisol (16-fold decrease, p < 0.0001) and 17-hydroxyprogesterone (5.0-fold decrease, p < 0.001).
The unspecific inhibitory effect of Efavirenz observed under ACTH-stimulated conditions was further evident from the product/substrate ratios reflecting the activity of CYP21A2 and CYP17A1. The corticosterone/progesterone ratio reflecting CYP21A2 and CYP11B1/2 activity was unaffected by Efavirenz under both basal and stimulated conditions (Fig. 5b), while the 11-deoxycortisol/17-hydroxyprogesterone ratio reflecting decreased CYP21A2 activity (3.1-fold p < 0.001) under ACTH-stimulated conditions (Fig. 5b). Also, CYP17A1 product/substrate ratio was unaffected by Efavirenz under basal conditions (Fig. 5c), while under ACTH-stimulated conditions both the 17-hydroxyprogesterone/progesterone (6.0-fold, p < 0.01) and androstenedione/17-hydroxyprogesterone (3.9-fold, p< 0.001) ratios were decreased (Fig. 5c). The overall inhibitory effect of Efavirenz on adrenal steroidogenic enzymes was further evident from the 8.5-fold decrease (p < 0.001) in the androstenedione/corticosterone ratio and a 4.4-fold decrease (p < 0.05) in the androstenedione/cortisol ratio under stimulated conditions (Fig. 5d). Thus, treatment with Efavirenz under stimulated conditions appears to inhibit androgen rather than corticosteroid biosynthesis suggesting unspecific inhibition of several steroidogenic enzymes rather than specific CYP21A2 inhibition.