Is the unique benzodiazepine structure interacting with CYP enzymes to affect steroid synthesis in vitro?

The aim of this project was to investigate the endocrine disrupting effects of three γ-aminobutyric acid type A receptor (GABAAR) agonists, diazepam (DZ), oxazepam (OX) and alprazolam (AL) using the steroidogenic in vitro H295R cell line assay, a recombinant CYP17A1 assay, qPCR analysis and computational modelling. Similar effects for DZ and OX on the steroidogenesis were observed in the H295R experiment at therapeutically relevant concentrations. Progestagens and corticosteroids were increased up to 10 fold and androgens were decreased indicating CYP17A1 lyase inhibition. For DZ the inhibition on both the hydroxylase and lyase was confirmed by the recombinant CYP17A1 assay, whereas OX did not appear to directly affect the recombinant CYP17A1 enzyme. Androgens were decreased when exposing the H295R cells to AL, indicating a CYP17A1 lyase inhibition. However, this was not confirmed by the recombinant CYP17A1 assay but a down-regulation in gene expression was observed for StAR and CYP17A1. The present study showed that the three investigated benzodiazepines (BZDs) are rather potent endocrine disruptors in vitro, exerting endocrine effects close the therapeutic Cmax. Both direct and indirect effects on steroidogenesis were observed, but molecular modelling indicated no direct interactions between the heme group in the steroidogenic CYP enzymes and the unique diazepin structure. In contrast, physicochemical properties such as high log P, structure and molecular weight similar to that of steroids appeared to influence the endocrine disrupting abilities of the investigated pharmaceuticals in vitro. Docking of the three BZDs in CYP17A1 and CYP21A2 confirmed that shape complementarity and hydrophobic effects seem to determine the binding modes.

Oral anti-diabetic drugs as endocrine disruptors in vitro - No evidence for additive effects in binary mixtures.

Diabetes is one of the World's most concerning health problems and millions of patients are using anti-diabetic drugs (ADDs) in order to control blood glucose. The in vitro H295R steroidogenesis assay was implemented to investigate endocrine effects of three ADDs, metformin (MET), glimepiride (GLIM), sitagliptin (SIT) and the cholesterol-lowering drug simvastatin (SIM) individually and in three binary mixtures. Steroid hormones were analyzed using LC-MS/MS. Mixture effects were assessed by applying the Concentration Addition (CA) model. All tested drugs and binary mixtures interrupted the H295R steroidogenesis with different potency. The effects of MET:GLIM on the steroidogenesis were overall similar to the steroidogenic profile of GLIM, however effects were less pronounced. The binary mixture of MET:SIT showed overall minor effects on steroid production and only at very high concentrations. The SIM:SIT mixture showed inhibition downstream from cholesterol, which was attributed to the effects of SIM. The CA model partly predicted the effect of MET:SIT on some steroids but significantly overestimated the effects of MET:GLIM and SIM:SIT. Thus, the applicability of the CA model was limited and cocktail effects appeared to be intermediate responses of individual drugs, rather than additive. The complexity of dynamic pathways such as steroidogenesis appears to significantly reduce the use of the CA model. In conclusion, more dynamic models are needed to predict mixture effects in complex systems.

Microcystins and Microcystis aeruginosa PCC7806 extracts modulate steroidogenesis differentially in the human H295R adrenal model.

The aim of this study was to investigate the potential interference of cyanobacterial metabolites, in particular microcystins (MCs), with steroid hormone biosynthesis. Steroid hormones control many fundamental processes in an organism, thus alteration of their tissue concentrations may affect normal homeostasis. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate the modulation of 14 hormones involved in the adrenal steroid biosynthesis pathway using forskolin-treated H295R cells, following exposure with either microcystin-LR (MC-LR) alone, a mixture made up of MC-LR together with eight other MCs and nodularin-R (NOD-R), or extracts from the MC-LR-producing Microcystis aeruginosa PCC7806 strain or its MC-deficient mutant PCC7806mcyB-. Production of 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) was increased in the presence of MC-LR in a dose-dependent manner, indicating an inhibitory effect on 3ß-hydroxysteroid dehydrogenase (3ß-HSD). This effect was not observed following exposure with a MCs/NOD-R mixture, and thus the effect of MC-LR on 3ß-HSD appears to be stronger than for other congeners. Exposure to extracts from both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB- had an opposite effect on 3ß-HSD, i.e. concentrations of pregnenolone, 17-hydroxypregnenolone and DHEA were significantly decreased, showing that there are other cyanobacterial metabolites that outcompete the effect of MC-LR, and possibly result instead in net-induction. Another finding was a possible concentration-dependent inhibition of CYP21A2 or CYP11ß1, which catalyse oxidation reactions leading to cortisol and cortisone, by MC-LR and the MCs/NOD-R mixture. However, both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB- extracts had an opposite effect resulting in a substantial increase in cortisol levels. Our results suggest that MCs can modulate steroidogenesis, but the net effect of the M. aeruginosa metabolome on steroidogenesis is different from that of pure MC-LR and independent of MC production.