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ABSTRACT: Exposure to estrogens during the period of sexual differentiation is known to adversely affect the development of testes in African clawed frogs (Xenopus laevis), but little is known about the molecular changes that coincide with the development of altered phenotypes. Therefore, the transcriptome-level effects of exposure to 17α-ethynylestradiol (EE2) during sexual differentiation of X. laevis were evaluated by use of Illumina sequencing coupled with RNA-Seq expression analysis. Overall, a number of processes were affected by 17α-ethynylestradiol, including steroid biosynthesis, thyroid hormone signaling and metabolism, testicular development, and spermatogenesis. Some of the altered pathways, such as thyroid hormone signaling and testicular development, could be linked with biological effects on metamorphosis and gonadal phenotypes, respectively, that were observed in frogs that were exposed to 17α-ethynylestradiol throughout metamorphosis and the early post-metamorphic period. Thus, early changes at the transcriptome-level may have been predictive of pathologies that did not manifest until later in development. To validate the quantitative capacity of RNA-Seq, a subset of transcripts identified to have altered abundances in individuals exposed to 17α-ethynylestradiol was also evaluated by use of quantitative polymerase chain reaction (qPCR). While small sample sizes (n=3) limited the ability to draw conclusions pertaining to differences in qPCR-derived abundances of transcripts between control and exposed tadpoles, there was a significant relationship (r2=0.78) between fold-changes for RNA-Seq and qPCR.
Environmental Science & Technology 04/2013; · 4.80 Impact Factor
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ABSTRACT: Populations of amphibians are in decline in certain locations around the world, and the possible contribution of environmental contaminants, including estrogenic compounds, to these declines is of potential concern. In the current study, responses of the wood frog (Lithobates sylvaticus) to exposure to 17α-ethynylestradiol (EE2), the synthetic estrogen used in oral contraceptives, during the larval period were characterized. Exposure of L. sylvaticus to 1.08, 9.55, or 80.9μg EE2/L had no effects on survival, growth, or metamorphic endpoints monitored in the current study. However, there were significant effects of exposure to EE2 on phenotypic sex ratios. In general, lesser proportions of L. sylvaticus developed as phenotypic males and greater proportions developed as phenotypic females or with mixed sex phenotypes at all concentrations of EE2 tested. Utilizing the data collected in the current study, the EC(50) for complete feminization of L. sylvaticus was determined to be 7.7μg EE2/L, and the EC(50) for partial feminization was determined to be 2.3μg EE2/L. In addition, after chronic exposure, abundances of transcripts of vitellogenin A2, high density lipoprotein binding protein, and 7-dehydrocholesterol reductase were 1.8-280-fold greater in livers from L. sylvaticus exposed to EE2 compared to controls. Overall, there were significant effects of exposure to all concentrations of EE2 tested, the least of which was within about 2-fold of estrogen equivalent concentrations previously measured in the environment.
Aquatic toxicology (Amsterdam, Netherlands) 10/2012; 126C:42-51. · 3.12 Impact Factor
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ABSTRACT: Several studies have shown that exposure of amphibians, including the African clawed frog (Xenopus laevis), to potent estrogens at critical times during development results in feminization and/or demasculinization. However, genotyping of X. laevis has only recently become possible, so studies performed in the past were rarely able to make explicit linkages between genetic and phenotypic sex. Therefore, to further characterize this relationship, X. laevis tadpoles were exposed during development to 0.09, 0.84, or 8.81μg/L 17α-ethynylestradiol (EE2), which is the estrogen analog commonly used in oral contraceptives. Exposure to all concentrations of EE2 tested resulted in significant delays in time to metamorphosis. Genotyping showed that genetic sex ratios were similar among treatments. However, morphological evaluation revealed that a significant number of individuals with a male genotype displayed mixed sex and abnormal phenotypes. Additionally, both genetic males and females exposed to EE2 exhibited greater presence of vitellogenin protein relative to the respective controls. Since estrogens function downstream of the initial molecular signals of sexual differentiation, it is likely that genetic male animals received mixed endogenous male and exogenous female signals that caused disordered sexual development. The production of vitellogenin was probably temporally separated and independent from primary effects on sexual differentiation, and might have contributed to delays in metamorphosis observed in individuals exposed to EE2.
Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 06/2012; 156(3-4):202-10. · 2.62 Impact Factor
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ABSTRACT: Populations of sturgeon (Acipenseridae) are declining in many places in the world because of several potential factors, including overharvesting, habitat alteration, and pollution. In North America, populations of the white sturgeon (Acipenser transmontanus) have been experiencing poor annual recruitment in major river systems for more than three decades. Metal pollution has been hypothesized as a potential contributing factor to the poor recruitment in some of the water bodies. In general, little is known about the toxicity of metals such as Cu, Cd, and Zn to white sturgeon and their potential influence on survival of embryos and juveniles. The present study was conducted to establish baseline toxicity data for the subchronic exposure of early life stages of white sturgeon to Cu, Cd, and Zn that can be used in metal-related risk assessments. Embryos, larvae, and fry were exposed to increasing concentrations of dissolved Cu, Cd, or Zn for 66 d using laboratory-based flow-through exposure systems. Hatching success was greater than 79% for all controls, and no significant differences were observed among treatment groups or between treatments and controls. Chronic lethal concentrations at which 20% mortality occurred (LC20s) for Cd (1.5 µg/L), Cu (5.5 µg/L), and Zn (112 µg/L) obtained for white sturgeon in the present study were comparable to those of sensitive salmonid species. Based on LC20 values for 19 or 58 d posthatch white sturgeon, the United States national ambient water quality criteria and the Canadian water quality guidelines for the protection of aquatic life that have been established for Cd, Cu, and Zn protect white sturgeon early life stages.
Environmental Toxicology and Chemistry 07/2011; 30(11):2497-505. · 2.81 Impact Factor
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ABSTRACT: Effects of two model imidazole-type fungicides, prochloraz (PCZ) and ketoconazole (KTC), on the hypothalamic-pituitary-gonadal (HPG) axis of the Japanese medaka (Oryzias latipe) [corrected] were examined by use of real time PCR (RT-PCR) array. Fourteen-week-old Japanese medaka were exposed for seven days to concentrations of PCZ or KTC from 3.0 to 300 microg/L Exposure [corrected] to KTC or PCZ caused significant reduction of fecundity of Japanese medaka and down-regulated expression of estrogen receptor (ER)-alpha and egg precursors in livers of males and females. However, PCZ was more potent than KTC both in modulating transcription and causing lesser fecundity. Exposure to nominal 30 microg PCZ/L resulted in 50% less fecundity and significant down-regulation of vitellogenin II expression, but KTC did not cause such effects at this concentration. Exposure to PCZ caused a compensatory upregulation in cytochrome P450 c17alphahydroxylase, 17,20-lyase (CYP17) and aromatase (CYP19) expression in the ovary, while KTC did not. Furthermore, the ecologically relevant end point, fecundity was log-log related to mRNA level of six genes in livers of females.
Environmental Science and Technology 10/2008; 42(17):6762-9. · 5.23 Impact Factor
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ABSTRACT: Both the anabolic androgen 17beta-trenbolone (TRB) and the aromatase inhibitor fadrozole (FAD) can cause decreased plasma concentrations of estrogen (E2) and reduce fecundity of fish. However, the underlying mechanisms and the molecular pathways involved are largely unknown. The present study was designed to assess time-dependent effects of FAD and TRB on the transcriptional responses of the hypothalamic-pituitary-gonadal (HPG) axis of Japanese medaka (Oryzias latipes). Fourteen-week-old Japanese medaka were exposed to 50 microg FAD/L or 2 microg TRB/L in a 7-d static renewal test, and the expression profiles of 36 HPG axis genes were measured by means of a medaka HPG real-time reverse-transcription polymerase chain reaction array after 8 h, 32 h, or 7 d of exposure. Exposure to TRB or FAD caused lesser fecundity of Japanese medaka and down-regulated transcription of vitellogenin and choriogenin (CHG) gene expression in the liver of females. Exposure to FAD for 8 h resulted in an 8-fold and 71-fold down-regulation of expression of estrogen receptor alpha and choriogenin L (CHG L), respectively, in female liver. 17beta-Trenbolone caused similar down-regulation of these genes, but the effects were not observed until 32 h of exposure. These results support the hypothesis that FAD reduces plasma E2 more quickly by inhibiting aromatase enzyme activity than does TRB, which inhibits the production of the E2 precursor testosterone. Exposure to FAD and TRB resulted in rapid (after 8 h) down-regulation of luteinizing hormone receptor and low-density-lipoprotein receptor in the testis to compensate for excessive androgen levels. Overall, the molecular responses observed in the present study differentiate the mechanisms of the reduced fecundity by TRB and FAD.
Environmental Toxicology and Chemistry 09/2008; 27(12):2504-11. · 2.81 Impact Factor
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Xiaowei Zhang,
Markus Hecker,
June-Woo Park, Amber R Tompsett,
John Newsted,
Kei Nakayama,
Paul D Jones,
Doris Au,
Richard Kong,
Rudolf S S Wu,
John P Giesy
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ABSTRACT: This paper describes the development and validation of a PCR array for studying chemical-induced effects on gene expression of selected endocrine pathways along the hypothalamic-pituitary-gonadal (HPG) axis of the small, oviparous fish, the Japanese medaka (Oryzias latipes). The Japanese medaka HPG-PCR array combines the quantitative performance of SYBR Green-based real-time PCR with the multiple gene profiling capabilities of a microarray to examine expression profiles of 36 genes associated with endocrine pathways in brain, liver and gonad. The performance of the Japanese medaka HPG-PCR array was evaluated by examining effects of two model compounds, the synthetic estrogen, 17alpha-ethinylestradiol (EE2) and the anabolic androgen, 17beta-trenbolone (TRB) on the HPG axis of the Japanese medaka. Four-month-old medaka was exposed to three concentrations of EE2 (5, 50, 500 ng/L) or TRB (50, 500, 5000 ng/L) for 7d in a static renewal exposure system. A pathway-based approach was implemented to analyze and visualize concentration-dependent mRNA expression in the HPG axis of Japanese medaka. The compensatory response to EE2 exposure included the down-regulation of male brain GnRH RI and testicular CYP17. The down-regulation of AR-alpha expression in brain of EE2-exposed males was associated with suppression of male sexual behavior. Compensatory responses to TRB in the female HPG axis included up-regulation of brain GnRH RII and ovary steroidogenic CYP19A. Overall, the results suggested that the Japanese medaka HPG-PCR array has potential not only as a screening tool of potential endocrine-disrupting chemicals but also in elucidating mechanisms of action.
Aquatic Toxicology 08/2008; 88(3):173-82. · 3.76 Impact Factor
