Extended steroid profiling in H295R cells provides deeper insight into chemical-induced disturbances of steroidogenesis: Exemplified by prochloraz and anabolic steroids.

Human adrenocortical H295R cells have been validated by the OECD Test Guideline 456 to detect chemicals disrupting testosterone and 17ß-estradiol (estradiol) biosynthesis. This study evaluated a novel approach to detect disturbances of steroidogenesis in H295R cells, exemplified by prochloraz and five anabolic steroids. Steroid profiles were assessed by an untargeted LC-MS-based method, providing a relative quantification of 57 steroids annotated according to their accurate masses and retention times. Such a panel of steroids included several mineralocorticoids, glucocorticoids, progestins and adrenal androgens. The coverage of a high number of metabolites in this extended steroid profiling facilitated grouping of chemicals with similar effects and detecting subtler differences between chemicals. It allowed, for example, distinguishing between the effects of turinabol and oxymetholone, supposed to act similarly in a previous characterization including only nine adrenal steroids. Furthermore, the results revealed that product/substrate ratios can provide superior information on altered enzyme activities compared to individual metabolite levels. For example, the 17α-hydroxypregnenolone/pregnenolone ratio was found to be a more sensitive marker for detecting 17α-hydroxylase inhibition by prochloraz than the corresponding individual steroids. These results illustrate that chemical grouping and calculation of product/substrate ratios can provide valuable information on mode-of-action and help prioritizing further experimental work.

Profiling of anabolic androgenic steroids and selective androgen receptor modulators for interference with adrenal steroidogenesis.

Anabolic-androgenic steroids (AAS) are testosterone derivatives developed for steroid-replacement and treatment of debilitating conditions. They are widely used by athletes in elite sports and bodybuilding due to their muscle-building and performance-enhancing properties. Excessive AAS use is associated with cardiovascular diseases, mood changes, endocrine and metabolic disorders; however, the underlying mechanisms remain unknown. Selective androgen receptor modulators (SARMs) aim to reduce adverse androgenic effects, while maximizing anabolic effects. This study assessed potential steroidogenic disturbances of 19 AAS and 3 SARMs in human adrenocortical carcinoma H295R cells, comparing basal and forskolin-activated states by mass spectrometry-based quantification of nine major adrenal steroids. Mesterolone, mestanolone and methenolone increased mineralocorticoid but decreased adrenal androgen production, indicating CYP17A1 dysfunction. Cell-free activity assays failed to detect direct CYP17A1 inhibition, supported by molecular modeling. The mRNA expression levels of 3ß-HSD2, CYP17A1, CYP21A2, CYP11B1 and CYP11B2 were unaffected, suggesting indirect inhibition involving post-translational modification and/or impaired protein stability. Clostebol and oxymetholone decreased corticosteroid but increased dehydroepiandrosterone biosynthesis in H295R cells, suggesting CYP21A2 inhibition, sustained by molecular modeling. These AAS did not affect the expression of key steroidogenic genes. None of the SARMs tested interfered with steroidogenesis. The chosen approach allowed the grouping of AAS according to their steroidogenic-disrupting effects and provided initial mechanistic information. Mesterolone, mestanolone and methenolone potentially promote hypertension and cardiovascular diseases via excessive mineralocorticoid biosynthesis. Clostebol and oxymetholone might cause metabolic disturbances by suppressing corticosteroid production, resulting in adrenal hyperplasia. The non-steroidal SARMs exhibit an improved safety profile and represent a preferred therapeutic option.

Identification of the fungicide epoxiconazole by virtual screening and biological assessment as inhibitor of human 11ß-hydroxylase and aldosterone synthase.

Humans are constantly exposed to a multitude of environmental chemicals that may disturb endocrine functions. It is crucial to identify such chemicals and uncover their mode-of-action to avoid adverse health effects. 11ß-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) catalyze the formation of cortisol and aldosterone, respectively, in the adrenal cortex. Disruption of their synthesis by exogenous chemicals can contribute to cardio-metabolic diseases, chronic kidney disease, osteoporosis, and immune-related disorders. This study applied in silico screening and in vitro evaluation for the discovery of xenobiotics inhibiting CYP11B1 and CYP11B2. Several databases comprising environmentally relevant pollutants, chemicals in body care products, food additives and drugs were virtually screened using CYP11B1 and CYP11B2 pharmacophore models. A first round of biological testing used hamster cells overexpressing human CYP11B1 or CYP11B2 to analyze 25 selected virtual hits. Three compounds inhibited CYP11B1 and CYP11B2 with IC50 values below 3 µM. The most potent inhibitor was epoxiconazole (IC50 value of 623 nM for CYP11B1 and 113 nM for CYP11B2, respectively); flurprimidol and ancymidol were moderate inhibitors. In a second round, these three compounds were tested in human adrenal H295R cells endogenously expressing CYP11B1 and CYP11B2, confirming the potent inhibition by epoxiconazole and the more moderate effects by flurprimidol and ancymidol. Thus, the in silico screening, prioritization of chemicals for initial biological tests and use of H295R cells to provide initial mechanistic information is a promising strategy to identify potential endocrine disruptors inhibiting corticosteroid synthesis. A critical assessment of human exposure levels and in vivo evaluation of potential corticosteroid disrupting effects by epoxiconazole is required.

Steroid profiling in H295R cells to identify chemicals potentially disrupting the production of adrenal steroids.

The validated OECD test guideline 456 based on human adrenal H295R cells promotes measurement of testosterone and estradiol production as read-out to identify potential endocrine disrupting chemicals. This study aimed to establish optimal conditions for using H295R cells to detect chemicals interfering with the production of key adrenal steroids. H295R cells' supernatants were characterized by liquid chromatography-mass spectrometry (LC-MS)-based steroid profiling, and the influence of experimental conditions including time and serum content was assessed. Steroid profiles were determined before and after incubation with reference compounds and chemicals to be tested for potential disruption of adrenal steroidogenesis. The H295R cells cultivated according to the OECD test guideline produced progestins, glucocorticoids, mineralocorticoids and adrenal androgens but only very low amounts of testosterone. However, testosterone contained in Nu-serum was metabolized during the 48h incubation. Thus, inclusion of positive and negative controls and a steroid profile of the complete medium prior to the experiment (t=0h) was necessary to characterize H295R cells' steroid production and indicate alterations caused by exposure to chemicals. Among the tested chemicals, octyl methoxycinnamate and acetyl tributylcitrate resembled the corticosteroid induction pattern of the positive control torcetrapib. Gene expression analysis revealed that octyl methoxycinnamate and acetyl tributylcitrate enhanced CYP11B2 expression, although less pronounced than torcetrapib. Further experiments need to assess the toxicological relevance of octyl methoxycinnamate- and acetyl tributylcitrate-induced corticosteroid production. In conclusion, the extended profiling and appropriate controls allow detecting chemicals that act on steroidogenesis and provide initial mechanistic evidence for prioritizing chemicals for further investigations.

Site-Specific Polymer Conjugation Stabilizes Therapeutic Enzymes in the Gastrointestinal Tract.

The site-specific conjugation of polymers to multiple engineered cysteine residues of a prolyl endopeptidase leads to its stabilization in the gastrointestinal tract of rats, without compromising the activity relative to the native enzyme. The importance of polymer attachment sites is investigated, as well as the significance of polymer structure.