title: Steroid profiling in H295R cells to identify chemicals potentially disrupting the production of adrenal steroids.
abstract: 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.
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland; Swiss Centre for Applied Human Toxicology (SCAHT), Universities of Basel and Geneva, Basel, Switzerland.
authors.forename: Petra
authors.initials: P
authors.lastname: Strajhar
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva and University of Lausanne, Pavillon des Isotopes 20, Boulevard d'Yvoy, 1211 Geneva, Switzerland; Swiss Centre for Applied Human Toxicology (SCAHT), Universities of Basel and Geneva, Basel, Switzerland.
authors.forename: David
authors.initials: D
authors.lastname: Tonoli
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva and University of Lausanne, Pavillon des Isotopes 20, Boulevard d'Yvoy, 1211 Geneva, Switzerland; Swiss Centre for Applied Human Toxicology (SCAHT), Universities of Basel and Geneva, Basel, Switzerland.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Jeanneret
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
authors.forename: Raphaella M
authors.initials: RM
authors.lastname: Imhof
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Malagnino
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland; Swiss Centre for Applied Human Toxicology (SCAHT), Universities of Basel and Geneva, Basel, Switzerland.
authors.forename: Melanie
authors.initials: M
authors.lastname: Patt
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
authors.forename: Denise V
authors.initials: DV
authors.lastname: Kratschmar
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva and University of Lausanne, Pavillon des Isotopes 20, Boulevard d'Yvoy, 1211 Geneva, Switzerland.
authors.forename: Julien
authors.initials: J
authors.lastname: Boccard
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva and University of Lausanne, Pavillon des Isotopes 20, Boulevard d'Yvoy, 1211 Geneva, Switzerland; Swiss Centre for Applied Human Toxicology (SCAHT), Universities of Basel and Geneva, Basel, Switzerland.
authors.forename: Serge
authors.initials: S
authors.lastname: Rudaz
authors.affiliation: Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland; Swiss Centre for Applied Human Toxicology (SCAHT), Universities of Basel and Geneva, Basel, Switzerland. Electronic address: alex.odermatt@unibas.ch.
authors.forename: Alex
authors.initials: A
authors.lastname: Odermatt
mesh_terms: D000311:Adrenal Glands / Q000166:cytology / Q000187:drug effects*; D045744:Cell Line, Tumor; D002934:Cinnamates / Q000633:toxicity; D002951:Citrates / Q000633:toxicity; D005576:Colforsin / Q000494:pharmacology; D052244:Endocrine Disruptors / Q000633:toxicity*; D023381:Endpoint Determination; D004958:Estradiol / Q000378:metabolism; D005786:Gene Expression Regulation; D017408:Guidelines as Topic; D006801:Humans; D011804:Quinolines / Q000633:toxicity; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D013256:Steroids / Q000378:metabolism*; D013739:Testosterone / Q000378:metabolism; D018675:Toxicity Tests
chemical_list: D002934:Cinnamates; D002951:Citrates; D052244:Endocrine Disruptors; D011804:Quinolines; D012333:RNA, Messenger; D013256:Steroids; C014953:2-acetyltributylcitrate; D005576:Colforsin; D013739:Testosterone; C483909:torcetrapib; D004958:Estradiol; C118580:octylmethoxycinnamate
keywords: Adrenal toxicity; Endocrine disrupting chemical; H295R; Profiling; Steroid