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Incorporation of (14)C-cholesterol in human adrenal corticocarcinoma H295R cell line and online-radiodetection of produced (14)C-steroid hormone metabolites
England.
Kristianstad University, School of Education and Environment, Avdelningen för Naturvetenskap. Kristianstad University, Plattformen för molekylär analys.ORCID iD: 0000-0003-2283-2927
Danmark.
Danmark.
2017 (English)In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 145, p. 569-575Article in journal (Refereed) Published
Abstract [en]

This study demonstrates the addition of (14)C-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 (14)C-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 72h 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 (14)C-cholesterol and detecting the radiolabeled steroid hormones online was proved and may assist in further toxicological studies.

Place, publisher, year, edition, pages
2017. Vol. 145, p. 569-575
Keywords [en]
Flow scintillation analysis, NCI-H295R, OECD guideline 456, Online radiodection, Radiolabeling, Steroidogenesis
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:hkr:diva-17079DOI: 10.1016/j.jpba.2017.06.058PubMedID: 28777968OAI: oai:DiVA.org:hkr-17079DiVA, id: diva2:1133592
Available from: 2017-08-16 Created: 2017-08-16 Last updated: 2017-08-16Bibliographically approved

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