Browsing by Author "Fu, Keng-Yen"

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Application of a yeast estrogen screen in non-biomarker species Varicorhinus barbatulus fish with two estrogen receptor subtypes to assess xenoestrogens
(2007-06-01)
Fu, Keng-Yen
;
Chen, Chung-Yuan
;
Chang, Whei-meih
;
環境工程研究所
;
Institute of Environmental Engineering
Xenoestrogens can interfere with normal estrogen signaling by competitively binding to the estrogen receptor (ER) and activating transcription of target genes. In this study, we cloned the estrogen receptor alpha (vbER alpha) and beta 2 (vbER beta 2) genes from liver of the indigenous Taiwanese cyprinid fish Varicorhinus barbatulus and tested the direct impact of several xenoestrogens on these ERs. Transcriptional activity of xenoestrogens was measured by the enzymatic activity of estrogen responsive element (ERE)-containing beta-galactosidase in a yeast reporter system. The xenoestrogens tested were phenol derivatives, DDT-related substances, phthalic acid esters, and polychlorinated biphenyls, with 17 beta-estradiol (E2) as a subjective standard. The phenol derivatives [4-nonylphenol (4-NP), 4-t-octylphenol (4-t-OP) and bisphenol A (BPA)] exhibited significant dose-dependent responses in both ligand potency and ligand efficiency. Consistent with yeast assays using human or rainbow trout ERs, we observed a general subtype preference in that vbER alpha displayed higher relative potencies and efficiencies than vbER beta 2, although our assays induced a stronger response for xenoestrogens than did human or trout ERs. Whereas 4-NP and 4-t-OP have similar EC50 values relative to E2 for both ER subtypes, the strong estrogenic response of BPA markedly differentiates vbER alpha from vbER beta 2, suggesting possible species-specific BPA sensitivity. We report that the ameliorative yeast tool is readily applicable for indigenous wildlife studies of the bio-toxic influence of xenoestrogens with wildlife-specific estrogen receptors. (C) 2006 Elsevier Ltd. All rights reserved.
Molecular cloning and tissue distribution of three estrogen receptors from the cyprinid fish Varicorhinus barbatulus
(2008-02-01)
Fu, Keng-Yen
;
Chen, Chung-Yuan
;
Lin, Chi-Tsai
;
Chang, Whei-Meih
;
環境工程研究所
We present molecular cloning and tissue expression analysis of three estrogen receptor (ER) subtypes, vbER alpha, vbER beta 1 and vbER beta 2, from liver of the cyprinid fish Varicorhinus barbatulus through reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The sequence alignment and phylogenetic analysis reconfirmed the evolutionary relationship of V. barbatulus within the family Cypriniformes. Directional constraints for subtype-specific substitution of critical amino acids were observed in the E2 binding region. For amino acid substitution, vbER beta exhibited a M517L change in the ligand-dependent transactivation region. The tissue distributions were investigated using RT-PCR with subtype-distinguishable primers. Both vbER alpha and vbER beta 1 were most highly expressed in liver, while vbER beta 2 was higher in intestine. Here we demonstrate that the identification and cloning of ER subtypes using PCR is feasible in wildlife in that the temporal and spatial observations are consistent with those from phylogeny analysis and crystal structural investigation by others.