Mixture Effects of 3 Mechanistically Different Steroidogenic Disruptors (Prochloraz, Genistein, and Ketoconazole) in the H295R Cell Assay

Frederik Knud Nielsen; Cecilie Hurup Hansen; Jennifer Anna Fey; Martin Hansen; Bent Halling-Sørensen; Erland Björklund; Bjarne Styrishave

doi:10.1177/1091581815599375

Mixture Effects of 3 Mechanistically Different Steroidogenic Disruptors (Prochloraz, Genistein, and Ketoconazole) in the H295R Cell Assay

Frederik Knud Nielsen, Cecilie Hurup Hansen, Jennifer Anna Fey, Martin Hansen, Bent Halling-Sørensen, Erland Björklund, Bjarne Styrishave

5 Citations (Scopus)

Abstract

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 EC₅₀, 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.

Original language	English
Journal	International Journal of Toxicology
Volume	34
Issue number	6
Pages (from-to)	534-42
Number of pages	9
ISSN	1091-5818
DOIs	https://doi.org/10.1177/1091581815599375
Publication status	Published - Nov 2015

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Mixture Effects of 3 Mechanistically Different Steroidogenic Disruptors (Prochloraz, Genistein, and Ketoconazole) in the H295R Cell Assay. / Nielsen, Frederik Knud; Hansen, Cecilie Hurup; Fey, Jennifer Anna et al.

In: International Journal of Toxicology, Vol. 34, No. 6, 11.2015, p. 534-42.

Research output: Contribution to journal › Journal article › Research › peer-review

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title = "Mixture Effects of 3 Mechanistically Different Steroidogenic Disruptors (Prochloraz, Genistein, and Ketoconazole) in the H295R Cell Assay",

abstract = "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 EC50, 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.",

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AU - Fey, Jennifer Anna

AU - Hansen, Martin

AU - Halling-Sørensen, Bent

AU - Björklund, Erland

AU - Styrishave, Bjarne

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N2 - 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 EC50, 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.

AB - 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 EC50, 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.

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Mixture Effects of 3 Mechanistically Different Steroidogenic Disruptors (Prochloraz, Genistein, and Ketoconazole) in the H295R Cell Assay

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