Incorporation of 14C-cholesterol in human adrenal corticocarcinoma H295R cell line and online-radiodetection of produced 14C-steroid hormone metabolites

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Graphical abstractHighlightsRadiolabeled steroidogenic pathway allows for an improved mechanistic understanding.14C-cholesterol in H295R human cell line produce radiolabeled steroid metabolites.HPLC-UV-Flow Scintillation Analyzer method was developed to monitor metabolites.New steroid metabolites confirmed in this in-vitro assay (OECD guideline 456).Study indicates new metabolites of corticosteroids, androgens or estrogens.This study demonstrates the addition of 14C-cholesterol to the human cell line H295R will in-situ form radiolabeled steroid hormones allowing for new mechanistic and metabolic insights. The aim of the present study was to in-situ radiolabel steroid hormones from cell line-incorporated 14C-cholesterol using the OECD guideline 456, H295R steroidogenesis in-vitro assay. Radiodetection of the steroid metabolites of the steroidogenic pathway allows for an improved understanding of the various enzymatic mechanisms involved without necessarily being dependent on quantification. Generated radiolabeled steroids were analyzed using HPLC hyphenated with a Flow Scintillation Analyzer (FSA). H295R cells were incubated with radiolabeled cholesterol and cell media were collected and prepared by solid phase extraction and analyzed with HPLC-FSA. For successful radiolabeling of the steroids in the steroidogenesis of H295R cells, radioactive cholesterol may potentially only need to be added just before the cells are incubated for 72 h in well plates. Based on the obtained HPLC-FSA chromatograms, and confirmation of the observations by studies in the literature, a qualitative time profile for the production of steroid hormones was estimated. Multiple radiolabeled steroid hormones were identified by means of analytical standards and UV (ultraviolet) co-chromatography, though the elucidation of multiple metabolites remains unresolved. Although online radiodetection proved to suffer from suboptimal sensitivity, the concept of radiolabeling the steroidogenesis in H295R cells with 14C-cholesterol and detecting the radiolabeled steroid hormones online was proved and may assist in further toxicological studies.

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