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Integrated approach to explore the mechanisms of aromatase inhibition and recovery on fathead minnow ovaries

ABSTRACT: Aromatase, a member of the cytochrome P450 superfamily, is a key enzyme in estrogen synthesis and is responsible for the aromatization of androgens into estrogens. Here, we used an integrated approach to better understand the effects of aromatase inhibition as well as the mechanisms involved in the adaptation and recovery process. We exposed female fathead minnows (Pimephales promelas) to 30 μg/L of a model aromatase inhibitor, fadrozole, during 8 days (exposure phase). Fish were then held in clean water for 8 more days (recovery phase). Samples were collected at 1, 2, 4, and 8 days of both the exposure and the recovery phase. We used transcriptomics, metabolomics, and network inference to understand changes and infer connections at the transcript and metabolite level in the ovary. Other apical endpoints such as plasma estradiol, testosterone, and vitellogenin levels were also measured. An integrated analysis of the data revealed changes in gene expression consistent with increased testosterone. Metabolites such as glycogen and taurine were strongly correlated with increased testosterone levels. Comparison of in vivo and ex vivo data suggested the accumulation of steroidogenic enzymes, including aromatase, as a mechanism to compensate for aromatase inhibition.

ORGANISM(S): Pimephales promelas

PROVIDER: GSE51961 | GEO | 2013/11/01

SECONDARY ACCESSION(S): PRJNA225936

REPOSITORIES: GEO

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Project description:Cytochrome P450 aromatase is an essential enzyme for vertebrates. It is responsible for the conversion of androgens to estrogens in the brain and gonadal tissues. Aromatase converts testosterone into estradiol and therefore participates in the regulation of many processes which are controlled by estrogens such as development and fertility. Teleosts have been characterized as having exceptionally high levels of brain estrogen biosynthesis when compared to the brains of the other vertebrates. Little is known about the effects of estrogens on brain function in teleosts. The main objective of this study was to assess the effects of fadrozole, a powerful aromatase inhibitor, on gene expression in the zebrafish brain. To achieve this, male zebrafish were exposed to fadrozole for 10 days. This exposure causes a decrease in estrogens along with an increase in androgens. Plasma testosterone levels showed a 3-fold increase in response to the exposure. In the telencephalon, 235 genes were identified by Affymetrix GeneChip analysis as being differentially regulated. Real-time RT-PCR was used to validate the data obtained from the microarrays. This technique was also used to evaluate the response in the hypothalamus, which appears to follow similar transcriptional regulation. Gene ontology analysis of the microarray data revealed common biological processes and molecular functions such as sensory perception and detection of light stimulus, homeobox and transcription factor complexes. This study has identified genes which are directly and/or indirectly controlled by estrogens and androgens which helped to demonstrate the pronounced effects of endocrine disrupting chemicals on gene expression in the brain of teleosts. Collectively, the results provide a better understanding of the effects of aromatase inhibitors on gene expression and also shed light on the underlying effects of sex hormone variation and their importance in the brain of teleosts. Adult male zebrafish were exposed to fadrozole (200 ug/L) for 10 days. Telencephalons were pooled; approximately 30 per array. 3 treated samples, 3 control samples

Project description:The fathead minnow (Pimephales promelas) is a small fish species widely used for ecotoxicology research and regulatory testing in North America. This study used a 2000 gene oligonucleotide microarray to evaluate the effects of the aromatase inhibitor, fadrozole, on gene expression in the liver and brain tissue of exposed females. Reproductive measures, plasma vitellogenin, and gene expression data for the brain isoform of aromatase (CYP19B), vitellogenin precursors, and transferrin all provided evidence supporting the efficacy of the fadrozole exposure. Unsupervised analysis of the microarray results identified 20 genes in brain and 41 in liver as significantly up-regulated and 7 genes in brain and around 45 in liver as significantly down-regulated. Differentially expressed genes were associated with a broad spectrum of biological functions, many with no obvious relationship to aromatase inhibition. However, in brain, fadrozole exposure elicited significant up-regulation of several genes involved in the cholesterol synthesis, suggesting it as one potentially impacted pathway. Gene ontology-based analysis of expression changes in liver suggested overall down-regulation of protein biosynthesis. While real-time PCR analyses supported some of the microarray responses, others could not be verified. Overall, results of this study provide a foundation for developing novel hypotheses regarding the system-wide effects of fadrozole, and other chemical stressors with similar modes of action, on fish biology. Keywords: chemical stress response --Fadrozole exposure-- Treatments: 0, 60 ug fadrozole/L Replication: 4 replicate tanks per treatment, 2 replicate pairs (1 male, one female per pair) per tank for a total of 8 male, 8 female per treatment. Route of exposure: Waterborne, continuous flow through, without the use of carrier solvents, flow rate approx. 45 ml/min. Tanks: 20 L tanks containing 10 L of exposure water Fadrozole (purity â¥ 99%; Dr. H. Cooper Eckhardt, Summit, NJ, USA). Fish: Adult (ca. 6 month old) male (4.65Â±0.93 g) and female (1.90Â±0.44 g) Conditions: (25Â°C, 16:8 light:dark photoperiod, fed adult brine shrimp twice daily) Duration: 7 d (Â± 4 h) Sampling: Fish humanely euthanized in tricaine methanesulfonate (Finquel; Argent, Redmond, WA, USA). Liver, and brain samples transferred directly to pre-weighed vials of RNAlaterÂ® (Sigma, St. Louis, MO). Plasma samples were stored frozen at -80Â°C. Average water quality characteristics (Â±SD): temperature 25.5Â±0.1 Â°C; pH 7.21Â±0.04; dissolved oxygen 5.74Â±0.52 mg/L. --Microarray analysis-- n=3 microarrays per treatment and tissue Experimental samples: total RNA pooled from n=2 fish from the same treatment and replicate (except for pooled sample BC3 which included fish from two different replicate tanks). Reference sample: pooled liver, brain, and gonad samples from adult male and female fathead minnows Microarray design: Two color, reference design Hybridization: Experimental samples Cy5, reference sample Cy3

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Integrated approach to explore the mechanisms of aromatase inhibition and recovery on fathead minnow ovaries

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