Mixture Effects of 3 Mechanistically Different Steroidogenic Disruptors (Prochloraz, Genistein, and Ketoconazole) in the H295R Cell Assay.
Mixture effects of 3 model endocrine disruptors, prochloraz, ketoconazole, and genistein, on steroidogenesis were tested in the adrenocortical H295R cell line. Seven key steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, estrone, and 17β-estradiol) were analyzed using gas chromatography and tandem mass spectrometry (GC-MS/MS) to investigate the effects throughout the steroidogenic pathway. Current modeling approaches often rely on models assuming compounds acting independently and that the individual effects in some way can be summarized to predict a mixture effect. In H295R cells with an intact steroidogenic pathway, such assumptions may not be feasible. The purpose of this study was therefore to evaluate whether effects of a mixture with differing modes of action followed or deviated from additivity (concentration addition) and whether the H295R cell line was suitable for evaluating mixture toxicity of endocrine disruptors with different modes of action. The compounds were chosen because they interfere with steroidogenesis in different ways. They all individually decrease the concentrations of the main sex steroids downstream but exert different effects upstream in the steroidogenic pathway. Throughout the study, we observed lowest observed effect concentrations of mixtures at levels 2 to 10 times higher than the predicted EC(50), strongly indicating antagonistic effects. The results demonstrate that chemical analysis combined with the H295R cell assay is a useful tool also for studying how mixtures of endocrine disruptors with differing modes of action interfere with the steroidogenic pathway and that existing models like concentration addition are insufficient in such cases. Furthermore, for end points where compounds exert opposite effects, no relevant models are available.
Toxicology Laboratory, Analytical Biosciences, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark frederik.nielsen@gmail.com.
Frederik Knud
FK
Nielsen
Toxicology Laboratory, Analytical Biosciences, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
Cecilie Hurup
CH
Hansen
Toxicology Laboratory, Analytical Biosciences, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
Jennifer Anna
JA
Fey
Department of Integrative Biology, University of California, Berkeley, CA 94720 and Department of Civil and Environmental Engineering, University of California, Berkeley, CA 94720, USA.
Martin
M
Hansen
Toxicology Laboratory, Analytical Biosciences, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
Bent
B
Halling-Sørensen
School of Education and Environment, Division of Natural Sciences, Kristianstad University, SE-291 88 Kristianstad, Sweden.
Erland
E
Björklund
Toxicology Laboratory, Analytical Biosciences, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
Bjarne
B
Styrishave
D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004347:Drug Interactions; D052244:Endocrine Disruptors / Q000633:toxicity*; D019833:Genistein / Q000633:toxicity*; D006801:Humans; D007093:Imidazoles / Q000633:toxicity*; D007654:Ketoconazole / Q000633:toxicity*; D013256:Steroids / Q000096:biosynthesis*
D052244:Endocrine Disruptors; D007093:Imidazoles; D013256:Steroids; C045362:prochloraz; D019833:Genistein; D007654:Ketoconazole
GC-MS/MS; antagonism; concentration addition; endocrine disruption; steroidogenesis