title: Corticosteroid production in H295R cells during exposure to 3 endocrine disrupters analyzed with LC-MS/MS.
abstract: The adrenocortical human cell line H295R is a valuable tool for screening endocrine disrupting compounds. In general, previous research focus has been on the production of the 2 sex steroids, 17β-estradiol and testosterone, and less attention has been paid to other important steroid end points in the steroidogenesis with a wide range of physiological functions, such as the glucocorticoids (corticosterone and cortisol). A newly developed and validated solid phase extraction (SPE) liquid chromatography-mass spectroscopy (LC-MS/MS) method was used to measure the production of cortisol and corticosterone in the H295R cell line. The method was applied by studying the effects of 2 model endocrine disrupters, ketoconazole and prochloraz, the pharmaceutical budesonide, and the inducer forskolin on the steroid production in this cell line. Dose-response curves were obtained for the correlation between hormone concentrations and the concentration of the individual disruptors. Exposing cells to ketoconazole resulted in a decrease in cortisol and corticosterone concentrations in a dose-dependent manner with EC₅₀ values of 0.24 and 0.40 μmol/L, respectively. The same applied for cells exposed to prochloraz with EC₅₀ values of 0.06 and 0.09 μmol/L for cortisol and corticosterone, respectively. Budesonide also inhibited glucocorticoid secretion. The EC₅₀ value for cortisol was 19.50 μmol/L, whereas the EC₅₀ value for corticosterone was 71.42 μmol/L. Forskolin induced the secretion of both cortisol (EC₅₀ = 4.09 μmol/L) and corticosterone (EC₅₀ = 0.28 μmol/L). The results obtained demonstrated the validity of the method. Based on these findings, quality criteria for the production of these steroids in this cell line were suggested.
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2100, Denmark. bjarne.styrishave@sund.ku.dk
authors.forename: Christina S
authors.initials: CS
authors.lastname: Winther
authors.forename: Frederik K
authors.initials: FK
authors.lastname: Nielsen
authors.forename: Martin
authors.initials: M
authors.lastname: Hansen
authors.forename: Bjarne
authors.initials: B
authors.lastname: Styrishave
mesh_terms: D018268:Adrenocortical Carcinoma / Q000378:metabolism*; D019819:Budesonide / Q000494:pharmacology; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D005576:Colforsin / Q000494:pharmacology; D052244:Endocrine Disruptors / Q000494:pharmacology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007654:Ketoconazole / Q000494:pharmacology; D053719:Tandem Mass Spectrometry
chemical_list: D052244:Endocrine Disruptors; D007093:Imidazoles; D005576:Colforsin; D019819:Budesonide; C045362:prochloraz; D007654:Ketoconazole
keywords: H295R; corticosterone; cortisol; endocrine disruption; steroidogenesis