title: H295R cells as a model for steroidogenic disruption: a broader perspective using simultaneous chemical analysis of 7 key steroid hormones.
abstract: The effects of three model endocrine disruptors, prochloraz, ketoconazole and genistein on steroidogenesis were tested in the adrenocortical H295R cell line to demonstrate that a broader mechanistic understanding can be achieved in one assay by applying chemical analysis to the H295R assay. Seven key steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, estrone and 17β-estradiol) were analyzed using a novel and thoroughly validated GC-MS/MS method. In addition to the simultaneous quantification of 7 steroid hormones, the present method also negates the potential problems of cross-reactivity that can be encountered in some immunoassays. Although all 3 test compounds decrease the concentrations of the main sex steroids, the chemicals exerted different effects upstream in the pathway. Exposure to prochloraz resulted in increased hormone levels upstream of steroid 17 alpha-hydroxylase/17,20 lyase (P450c17) and decreases downstream. Ketoconazole inhibited the entire pathway, while exposure to genistein resulted in increased hormone levels upstream of 3-β-hydroxysteroid dehydrogenase (3β-HSD) and decreases downstream. The results demonstrate that chemical analysis combined with the H295R cell assay is an useful tool for studying the mechanisms by which endocrine disruptors interfere with the steroidogenic pathway.
authors.affiliation: Section of Toxicology and Environmental Chemistry, Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark. fkn@farma.ku.dk
authors.forename: Frederik Knud
authors.initials: FK
authors.lastname: Nielsen
authors.forename: Cecilie Hurup
authors.initials: CH
authors.lastname: Hansen
authors.forename: Jennifer Anna
authors.initials: JA
authors.lastname: Fey
authors.forename: Martin
authors.initials: M
authors.lastname: Hansen
authors.forename: Naja Wessel
authors.initials: NW
authors.lastname: Jacobsen
authors.forename: Bent
authors.initials: B
authors.lastname: Halling-Sørensen
authors.forename: Erland
authors.initials: E
authors.lastname: Björklund
authors.forename: Bjarne
authors.initials: B
authors.lastname: Styrishave
mesh_terms: D001681:Biological Assay / Q000379:methods; D045744:Cell Line, Tumor; D002849:Chromatography, Gas; D052244:Endocrine Disruptors / Q000494:pharmacology*; D019833:Genistein / Q000494:pharmacology*; D012739:Gonadal Steroid Hormones / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D007654:Ketoconazole / Q000494:pharmacology*; D013256:Steroids / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D052244:Endocrine Disruptors; D012739:Gonadal Steroid Hormones; D007093:Imidazoles; D013256:Steroids; C045362:prochloraz; D019833:Genistein; D007654:Ketoconazole