Erik Ullerås

A biphasic effect of the fungicide prochloraz on aldosterone, but not cortisol, secretion in human adrenal H295R cells - underlying mechanisms.

The widely used imidazole fungicide prochloraz displays anti-androgenic effects partly via inhibition of testicular steroidogenesis and testosterone secretion. Adrenal steroidogenesis and hormone secretion may also be a target of this endocrine disruptor. Herein, we demonstrate a dose-dependent inhibition of cortisol secretion and a biphasic effect on aldosterone secretion, with a 2-fold stimulation at low concentrations and a strong inhibition at high concentrations, following prochloraz treatment (0-10 microM) of human adrenocortical H295R cells. Analysis of the dose-dependent effects of prochloraz on the secretion of steroidogenic intermediates suggested that the observed effects on cortisol and aldosterone secretion might be mediated by inhibition of the steroidogenic steps catalysed by CYP17A1 and CYP21A2. The inhibition of CYP17A1 was reflected on the level of expression of steroidogenic genes as analysed by quantitative RT-PCR. In addition, analysis of enzyme activity showed a dose-dependent inhibitory effect of prochloraz on the activity of CYP17A1 and CYP21A2, but not CYP11B1. We have demonstrated specific effects of prochloraz on adrenal steroidogenic pathways and hormone secretion via inhibition of steroidogenic CYP enzymes. The disruption of adrenal hormone secretion may result in altered endocrine homeostasis and affect human health.

Secretion of cortisol and aldosterone as a vulnerable target for adrenal endocrine disruption - screening of 30 selected chemicals in the human H295R cell model.

The adrenal gland is a vulnerable target for toxic insult. Disruption of adrenal steroidogenesis and hormone secretion may cause serious effects on human health. A human in vitro model is needed to predict effects, and elucidate mechanisms of endocrine disruption and adrenal toxicity. The human adrenocortical cell line H295R has been used to screen for effects on sex hormones. Here, we have analyzed the effect of 30 potential endocrine disrupting chemicals on the secretion of cortisol and aldosterone from the H295R cells, using specific ELISA assays. The effect of chemicals was analyzed for basal and forskolin‐ or angiotensin II‐stimulated hormone secretion. The chemicals were tested at the highest concentration where they displayed no evident unspecific cytotoxicity. Quantitative and qualitative differences in effects on hormone secretion were demonstrated for the various chemicals. A subset of the chemicals displayed different effects on cortisol and aldosterone secretion, and in some cases the effects were different between basal and stimulated hormone secretion. Aminoglutethimide, prochloraz, ketoconazole, 6‐hydroxyflavone, imazalil and etomidate had the most marked inhibitory effects on cortisol (with or without forskolin) and ketoconazole, 6‐hydroxyflavone, imazalil and etomidate had the most marked effects on aldosterone (with or without angiotensin II). The results are discussed in terms of known effects, structural similarity and possible mechanisms. We have shown that adrenal steroidogenesis is a vulnerable target for toxic insult and that the H295R assay is a useful in vitro model for screening purposes. Copyright

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 EC(50) value of approximately 0.1 microM. 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.

Mixture effects of dietary flavonoids on steroid hormone synthesis in the human adrenocortical H295R cell line.

Humans are exposed to a mixture of dietary flavonoids with a variety of potential beneficial and harmful effects. Flavonoids are endocrine disruptors, acting both at receptor level and by interfering with steroid hormone synthesis. Due to a high dietary intake and the potential to cause mixture effects, assessment of combined exposure of flavonoids is required. We have studied effects on cortisol, aldosterone, testosterone and oestradiol secretion of the individual isoflavones daidzein and genistein, the flavone apigenin and the mixture of the three flavonoids in human adrenocortical H295R cells. The most vulnerable targets of the flavonoids were the secretion of cortisol and testosterone, which were inhibited by daidzein and genistein with IC50 values below 1 μM. An equimolar mixture of the flavonoids caused inhibition of cortisol, aldosterone and testosterone secretion in an additive manner. The observed mixture effect was described well by both concentration addition (CA) and independent action (IA) prediction models. Both prediction models underestimated the effect on oestradiol secretion. We conclude that the three flavonoids exhibit specific effects on steroid hormone secretion. A mixture of the flavonoids caused additive effects emphasizing the need to assess flavonoids together as a group. The prediction models are valuable tools for mixture assessment.

Mast Cell Survival and Mediator Secretion in Response to Hypoxia

Tissue hypoxia is a consequence of decreased oxygen levels in different inflammatory conditions, many associated with mast cell activation. However, the effect of hypoxia on mast cell functions is not well established. Here, we have investigated the effect of hypoxia per se on human mast cell survival, mediator secretion, and reactivity. Human cord blood derived mast cells were subjected to three different culturing conditions: culture and stimulation in normoxia (21% O2); culture and stimulation in hypoxia (1% O2); or 24 hour culture in hypoxia followed by stimulation in normoxia. Hypoxia, per se, did not induce mast cell degranulation, but we observed an increased secretion of IL-6, where autocrine produced IL-6 promoted mast cell survival. Hypoxia did not have any effect on A23187 induced degranulation or secretion of cytokines. In contrast, cytokine secretion after LPS or CD30 treatment was attenuated, but not inhibited, in hypoxia compared to normoxia. Our data suggests that mast cell survival, degranulation and cytokine release are sustained under hypoxia. This may be of importance for host defence where mast cells in a hypoxic tissue can react to intruders, but also in chronic inflammations where mast cell reactivity is not inhibited by the inflammatory associated hypoxia.

Developmental toxicity of albendazole and its three main metabolites in zebrafish embryos.

Albendazole (ABZ) is used as an anthelmintic drug in humans and animals. ABZ has been shown to cause developmental toxicity in experimental animals, however it is not clear if this is caused by the parent compound or a metabolite. Zebrafish embryos were exposed from 1 to 144hpf (hours post fertilization) to investigate the developmental toxicity of ABZ, the first metabolite albendazole sulphoxide and the subsequent metabolites albendazole sulphone (ABZSO(2)) and albendazole-2-aminosulphone (ABZSO(2)NH(2)). The results showed that ABZ caused malformations of head and tail and embryonic lethality from 0.3μM. In contrast, the metabolites did not display developmental toxicity at any tested concentration. Dechorionation did not influence the developmental toxic potential of ABZ and ABZSO, indicating that bioavailability was not a limiting factor. Chemical analysis showed that at sublethal concentrations, most of ABZ was metabolized to ABZSO. The results demonstrate that in zebrafish embryos ABZ rather than ABZSO displays developmental toxicity.

Effects of cadmium on calcium transporter SPCA, calcium homeostasis and β-casein expression in the murine mammary epithelium.

Maternal cadmium (Cd) exposure during lactation causes neurobehavioral effects in the suckling offspring as well as involution like disturbances in the mammary glands of rodents. The aim of the present study was to examine Cd-induced effects in secreting mammary epithelial cells in relation to calcium (Ca) transport and β-casein expression. Reduced protein expression of secretory pathway Ca-ATPase (SPCA) was revealed in the mammary glands of lactating mice exposed to Cd during peak lactation. In concordance, SPCA gene expression was down regulated and total intracellular Ca levels reduced in murine mammary epithelial HC11 cells treated with Cd for 72 h. Cd reduced β-casein gene expression in a concentration dependent manner in the HC11 cells. Our findings on Cd-induced reduction of Ca levels, SPCA and β-casein expression in the mammary epithelium resemble the effects observed in the mammary glands as a result of forced weaning. In conclusion, maternal Cd exposure during lactation may disturb Ca regulation and decrease the levels of β-casein in milk with potential nutritional and developmental implications for the breast-fed newborn.