Adverse effects of BDE-47 on in vivo developmental parameters, thyroid hormones, and expression of hypothalamus-pituitary-thyroid (HPT) axis genes in larvae of the self-fertilizing fish Kryptolebias marmoratus.

2,2',4,4'-tetrabromodiphenylether (BDE-47) is known to have the potential to disrupt the thyroid endocrine system in fishes due to its structural similarity to the thyroid hormones triiodothyronine (T3) and thyroxine (T4). However, the effects of BDE-47 on thyroid function in fishes remain unclear. In this study, abnormal development (e.g. deformity, hemorrhaging) and an imbalance in thyroid hormone (TH) homeostasis was shown in the early developmental stages of the mangrove killifish Kryptolebias marmoratus in response to BDE-47 exposure. To examine the thyroid endocrinal effect of BDE-47 exposure in mangrove killifish K. marmoratus larvae, transcript levels of genes involved in TH homeostasis and hypothalamus-pituitary-thyroid (HPT) axis-related genes were measured. The expression of thyroid hormone metabolism-related genes (e.g. deiodinases, UGT1ab) and HPT axis-related genes was up-regulated and there were significant changes in TH levels (P < 0.05) in response to BDE-47 exposure. This study provides insights into the regulation of TH homeostasis at the transcriptional level and provides a better understanding on the potential impacts of BDE-47 on the thyroid endocrine system of fishes.

Custom microarray construction and analysis for determining potential biomarkers of subchronic androgen exposure in the Eastern Mosquitofish (Gambusia holbrooki).

BACKGROUND: The eastern mosquitofish (Gambusia holbrooki) has the potential to become a bioindicator organism of endocrine disrupting chemicals (EDCs) due to its androgen-driven secondary sexual characteristics. However, the lack of molecular information on G. holbrooki hinders its use as a bioindicator coupled with biomarker data. While traditional gene-by-gene approaches provide insight for biomarker development, a holistic analysis would provide more rapid and expansive determination of potential biomarkers. The objective of this study was to develop and utilize a mosquitofish microarray to determine potential biomarkers of subchronic androgen exposure. To achieve this objective, two specific aims were developed: 1) Sequence a G. holbrooki cDNA library, and 2) Use microarray analysis to determine genes that are differentially regulated by subchronic androgen exposure in hepatic tissues of 17ß-trenbolone (TB) exposed adult female G. holbrooki. RESULTS: A normalized library of multiple organs of male and female G. holbrooki was prepared and sequenced by the Illumina GA IIx and Roche 454 XLR70. Over 30,000 genes with e-value ≤ 10⁻4 were annotated and 14,758 of these genes were selected for inclusion on the microarray. Hepatic microarray analysis of adult female G. holbrooki exposed to the vehicle control or 1 µg/L of TB (a potent anabolic androgen) revealed 229 genes upregulated and 279 downregulated by TB (one-way ANOVA, p < 0.05, FDR α = 0.05, fold change > 1.5 and < -1.5). Fifteen gene ontology biological processes were enriched by TB exposure (Fisher's Exact Test, p < 0.05). The expression levels of 17ß-hydroxysteroid dehydrogenase 3 and zona pellucida glycoprotein 2 were validated by quantitative polymerase chain reaction (qPCR) (Student's t-test, p < 0.05). CONCLUSIONS: Coupling microarray data with phenotypic changes driven by androgen exposure in mosquitofish is key for developing this organism into a bioindicator for EDCs. Future studies using this array will enhance knowledge of the biology and toxicological response of this species. This work provides a foundation of molecular knowledge and tools that can be used to delve further into understanding the biology of G. holbrooki and how this organism can be used as a bioindicator organism for endocrine disrupting pollutants in the environment.

The ribosomal protein L19 mRNA is induced by copper exposure in the swordtail fish, Xiphophorus helleri.

Teleosts are useful vertebrate model species for understanding copper toxicity due to the dual entry route for copper intake via the gills and intestine. In this present study, we utilized the differential display reverse transcription-polymerase chain reaction to isolate potential novel hepatic genes induced by sublethal copper exposure in the freshwater swordtail fish, Xiphophorus helleri. Full length cloning of a cDNA fragment induced by copper exposure to 1 µg/ml during 24 h resulted in the positive identification of a hepatic ribosomal protein L19 (RPL19) gene. Further characterization of this gene revealed that its transcriptional expression was dependent on dosage and time of copper exposure. This study describes for the first time the involvement of RPL19 in copper toxicity, probably as a result of increase in ribosome synthesis rate to support activities such as cellular protein translation, transcriptional activation and mRNA stabilization during sublethal copper exposure.

Endocrine disrupting chemicals (bisphenol A, 4-nonylphenol, 4-tert-octylphenol) modulate expression of two distinct cytochrome P450 aromatase genes differently in gender types of the hermaphroditic fish Rivulus marmoratus.

To understand the effect of endocrine-disrupting chemicals (EDCs) on cytochrome P450 aromatase (rm-cyp19) gene expression between gender types in the hermaphroditic fish Rivulus marmoratus, we cloned two distinct rm-cyp19 genes using RT-PCR with degenerative primers, obtained full-length cDNAs using 5'- and 3'-RACE-PCR methods, and completely sequenced them. The brain aromatase (rm-cyp19b) cDNA consisted of 2,124 bp including the open reading frame (ORF), which encoded a putative protein of 505 amino acids. The ovarian aromatase (rm-cyp19a) cDNA consisted of 2,075 bp, including the ORF encoding a putative protein of 516 amino acids. Expression patterns of rm-cyp19b and rm-cyp19a mRNAs were investigated in embryos of different developmental stages and in seven different tissues of adult fish. The rm-cyp19b gene in hermaphrodite and secondary male R. marmoratus was predominantly expressed in the brain, while the rm-cyp19a gene was expressed gender-specifically in the gonad. The expression of rm-cyp19b mRNA increased from stage 1 (2 d post fertilization) to stage 4 (12 d post fertilization) in a developmental stage-dependent manner but steeply decreased in the hatching stage. Compared to the rm-cyp19b gene, the abundance of ovarian aromatase rm-cyp19a transcripts was very low, and its expression was first detected at stage 3 and then decreased gradually to the hatching stage. Alteration of rm-cyp19b and rm-cyp19a gene expression was further analyzed in the brain and gonad by real-time RT-PCR 96 h after EDC exposure in hermaphrodites and secondary males. The brain aromatase rm-cyp19b gene was up-regulated in the brain after 4-nonylphenol (4-NP)-exposure, while the ovarian aromatase rm-cyp19a gene was significantly down-regulated in the gonad. In 300 microg/L 4-tert octylphenol (4-tert-OP), or 600 microg/L bisphenol A-exposed brain and gonad, both rm-cyp19b and rm-cyp19a genes were up-regulated. In the case of secondary males, the rm-cyp19b gene was highly expressed in the 4-NP-exposed brain, while expression of the rm-cyp19a gene was not detected in the gonad. These results indicate that the expression of rm-cyp19a and rm-cyp19b genes is differently modulated according to estrogenic compounds and gender type of R. marmoratus.