Mixture effects of imidazole fungicides on cortisol and aldosterone secretion in human adrenocortical H295R cells

Exposure to chemicals commonly occurs in the form of mixtures. Methods and models are required to analyze and predict the effect of mixtures in order to improve risk assessment. The steroidogenesis and hormone production of the adrenal gland is a sensitive target for endocrine-disrupting chemicals including imidazoles. Here, we exposed human adrenocortical H295R cells to the individual imidazole fungicides prochloraz, ketoconazole, imazalil and their mixtures and analyzed the effects on secretion of cortisol and aldosterone and the effects on steroidogenic gene expression. The individual imidazole fungicides prochloraz, ketoconazole and imazalil and their mixtures inhibited cortisol secretion in a similar monotonic dose-response pattern with an EC50 value of approximately 0.1 mu M. Aldosterone secretion, in contrast, displayed a biphasic dose-response, with low-dose stimulation of up to maximum twofold and high-dose inhibition. Biphasic dose-responses were found following prochloraz and ketoconazole exposure and their mixtures, but not following imazalil exposure. The inhibition of cortisol secretion was equally well predicted with the concentration addition (CA) and independent action (IA) models, while the biphasic aldosterone response was partially predicted by a modified CA model and not predicted well by a modified IA model. Changes in expression levels of steroidogenic genes could not conclusively explain the different effects on the two hormone endpoints or the different specificities of the imidazoles. We conclude that single imidazoles and mixtures have specific effects on adrenal hormone secretion. These effects can only partly be predicted using current models and need to be further analyzed in terms of in vivo relevance and human risk assessment. (C) 2010 Elsevier Ireland Ltd. All rights reserved.