Nobuaki Tominaga

Fluorotelomer alcohols induce hepatic vitellogenin through activation of the estrogen receptor in male medaka (Oryzias latipes)

Here we report on the in vivo estrogenic effects of two fluorotelomer alcohols, such as 1H,1H,2H,2H-perfluorooctan-1-ol (6:2 FTOH) and 1H,1H,2H,2H-perfluorodecan-1-ol (8:2 FTOH), in male medaka (Oryzias latipes). An in vitro yeast two-hybrid assay indicated a significant, dose-dependent interaction between medaka estrogen receptor alpha (ERalpha) and coactivator TIF2 upon treatment with 6:2 FTOH, 8:2 FTOH or 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-nonadecafluoro-1-decanol (NFDH). The relative ranks of tested chemicals on the estrogenic effects for medaka ERalpha descended in the order of estradiol-17beta (100)>>6:2 FTOH (0.16)>NFDH (0.016)>8:2 FTOH (0.0044). In contrast, no interaction with the ERalpha was observed upon treatment with perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorododecanoic acid (PFDA) or perfluoroundecanoic acid (PFUnDA). Expression analysis of hepatic vitellogenin (VTG) protein showed estrogenic potentials with, 6:2 FTOH and 8:2 FTOH, indicative of the induction of VTG synthesis in the livers of male medaka. We also investigated mRNA expression levels of two ER subtypes (ERalpha and beta) and two VTGs (VTG I and VTG II) in the livers of male medaka following exposure to FTOHs. Quantitative real-time polymerase chain reaction analyses revealed that hepatic ERalpha, VTG I, and VTG II mRNA responded rapidly to FTOHs such as 6:2 FTOH and 8:2 FTOH after 8-h exposure, whereas no effects of these compounds on ERbeta mRNA transcription were observed. These results from both in vitro and in vivo assays strongly suggest that certain FTOHs, such as 6:2 FTOH and 8:2 FTOH, induce hepatic VTG through activation of ERalpha in male medaka.

In vivo and in silico analyses of estrogenic potential of bisphenol analogs in medaka (Oryzias latipes) and common carp (Cyprinus carpio).

Various studies have demonstrated the estrogenic effect of bisphenol A (BPA), a member of bisphenol analogs (BPs), in in vitro and in vivo assays. However, limited data are available on the estrogenic potentials and risks of other BPs in aquatic organisms. In addition, the estrogenic effect of chemicals is known to have species-specific responses in teleost fish. The objective of this study was to evaluate the potential estrogenic effects of BPs on the medaka (Oryzias latipes) and common carp (Cyprinus carpio) using in vivo and in silico assays. Our quantitative real-time PCR analyses revealed that the expression levels of several hepatic estrogen-responsive biomarker genes in male medaka responded to various types and concentrations of BPs in a dose-response manner. The order of in vivo estrogenic potencies of BPs was as follows: BPC≈BPAF>BPB>BPA⋙BPP. To further investigate the interaction potential of BPs with medaka estrogen receptor α (ERα) in silico, a three-dimensional model of the ERα ligand-binding domain (LBD) was built and docking simulations were performed. The docking simulation analysis revealed that BPC interaction potential for medaka ERα LBD was the most potent, followed by BPAF and BPA. Comparing this with carp ERα LBD revealed that the interaction potentials of these BPs to medaka ERα LBD were more stable than to carp ERα LBD. Furthermore, we identified key amino acid residues in medaka ERα LBD that interacted with BPC (Glu356, Arg397, and Cys533), BPAF (Thr350 and Glu356), and BPA (Glu356 and Met424), and found some differences in these key amino acid residues between medaka and carp ERα LBDs. These results of in vivo and in silico analyses showed potential estrogenic effects of BPs in teleost fish, and they also indicated that the differences in interaction potentials and key amino acid residues between medaka and carp ERα LBDs may be due to the differences between the species and estrogenic potencies of the selected BPs.

A Novel Material Incorporation Technique for Medaka (Oryzias latipes) Eggs Using Nanosecond Pulsed Electric Fields

The effects of nanosecond pulsed electric fields (nsPEFs) on medaka eggs were examined. Although embryogenesis was not affected by nsPEF treatment alone, significant harmful effects were observed when the eggs were treated with nsPEFs in the presence of cycloheximide in the outer solution. Nanosecond PEF treatment affected the permeability of both the egg envelope (chorion) and the cell membrane, which resulted in intracellular incorporation of cycloheximide.

Graphical Gaussian Modeling for Gene Association Structures Based on Expression Deviation Patterns Induced by Various Chemical Stimuli

Activity patterns of metabolic subnetworks, each of which can be regarded as a biological function module, were focused on in order to clarify biological meanings of observed deviation patterns of gene expressions induced by various chemical stimuli. We tried to infer association structures of genes by applying the multivariate statistical method called graphical Gaussian modeling to the gene expression data in a subnetwork-wise manner. It can be expected that the obtained graphical models will provide reasonable relationships between gene expressions and macroscopic biological functions. In this study, the gene expression patterns in nematodes under various conditions (stresses by chemicals such as heavy metals and endocrine disrupters) were observed using DNA microarrays. The graphical models for metabolic subnetworks were obtained from these expression data. The obtained models (independence graph) represent gene association structures of cooperativities of genes. We compared each independence graph with a corresponding metabolic subnetwork. Then we obtained a pattern that is a set of characteristic values for these graphs, and found that the pattern of heavy metals differs considerably from that of endocrine disrupters. This implies that a set of characteristic values of the graphs can representative a macroscopic biological meaning.

Induction of the estrogen‐responsive genes encoding choriogenin H and L in the liver of male medaka (Oryzias latipes) upon exposure to estrogen receptor subtype‐selective ligands

Choriogenin (Chg) H and L are estrogen‐induced chorion precursors. We measured the induction of ChgH and ChgL mRNA in the livers of male medaka fish treated with Orthoester‐2k, a selective ligand for estrogen receptor (ER) α, and 2‐(4‐hydroxyphenyl)‐5‐hydroxy‐1,3‐benzoxazole (HPHB), a selective ligand of ERβ. Although both ChgH and ChgL mRNA were induced by treatment with Orthoester‐2k or HPHB separately, their combination induced much greater expression of each Chg. ChgH expression correlated more closely with Orthoester‐2k dosage when combined with a small fixed dose of HPHB (1 μm), whereas ChgL mRNA expression was more responsive to HPHB dose when combined with a fixed dose of Orthoester‐2k (2.8 nm). Moreover, upon long‐term treatment with Orthoester‐2k, ChgH mRNA and ERα mRNA expression showed similar patterns with peak expression between days 6 and 10. These results imply that ERβ primarily regulates ChgL mRNA expression and ERα action primarily regulates ChgH mRNA expression. Thus, it is necessary to develop screening methods for fish ER subtype‐specific ligands. Copyright

Endocrine-disrupting potentials of equine estrogens equilin, equilenin, and their metabolites, in the medaka Oryzias latipes: in silico and DNA microarray studies

Although several previous studies have demonstrated the presence of equine estrogens in the aquatic environment, limited data are currently available on the endocrine‐disrupting potentials in fish and the risks they pose to aquatic organisms. To investigate the interactions of major equine estrogens equilin (Eq) and equilenin (Eqn), as well as their metabolites 17α‐dihydroequilin, 17β‐dihydroequilin, 17α‐dihydroequilenin and 17β‐dihydroequilenin, with the estrogen receptor α (ERα) of medaka (Oryzias latipes), a three‐dimensional model of the ligand‐binding domain (LBD) of ERα was built in silico, and docking simulations were performed. The docking simulation analysis indicated that the interaction of 17β‐dihydroequilenin with the ERα LBD is the most potent, followed by those of 17α‐dihydroequilin and 17β‐dihydroequilin, whereas those of Eq and Eqn were least potent. We further analyzed gene expression profiles in the livers of male medaka exposed to Eq and Eqn. A DNA microarray representing 6000 genes revealed that 24‐h exposure to Eq and Eqn (100 ng/L) upregulated the expression of 6 and 34 genes in the livers of males, respectively. Genes upregulated by Eq included the estrogenic biomarker genes vitellogenins and choriogenins, suggesting the estrogenic potential of Eq. In contrast, Eqn exposure upregulated several cancer‐related genes, such as mediator complex subunit 16 and RAS oncogene family members, suggesting a carcinogenic potential for Eqn. These results suggest that equine estrogens may have not only endocrine‐disrupting potentials via the ERα signaling pathway but also carcinogenic potency in male medaka. Copyright

Effects of lithium on growth, maturation, reproduction and gene expression in the nematode Caenorhabditis elegans

Lithium (Li) has been widely used to treat bipolar disorder, and industrial use of Li has been increasing; thus, environmental pollution and ecological impacts of Li have become a concern. This study was conducted to clarify the potential biological effects of LiCl and Li2CO3 on a nematode, Caenorhabditis elegans as a model system for evaluating soil contaminated with Li. Exposure of C. elegans to LiCl and Li2CO3 decreased growth/maturation and reproduction. The lowest observed effect concentrations for growth, maturation and reproduction were 1250, 313 and 10 000 µm, respectively, for LiCl and 750, 750 and 3000 µm, respectively, for Li2CO3. We also investigated the physiological function of LiCl and LiCO3 in C. elegans using DNA microarray analysis as an eco‐toxicogenomic approach. Among approximately 300 unique genes, including metabolic genes, the exposure to 78 µm LiCl downregulated the expression of 36 cytochrome P450, 16 ABC transporter, 10 glutathione S‐transferase, 16 lipid metabolism and two vitellogenin genes. On the other hand, exposure to 375 µm Li2CO3 downregulated the expression of 11 cytochrome P450, 13 ABC transporter, 13 lipid metabolism and one vitellogenin genes. No gene was upregulated by LiCl or Li2CO3. These results suggest that LiCl and Li2CO3 potentially affect the biological and physiological function in C. elegans associated with alteration of the gene expression such as metabolic genes. Our data also provide experimental support for the utility of toxicogenomics by integrating gene expression profiling into a toxicological study of an environmentally important organism such as C. elegans. Copyright

C. elegans as a tool for environmental toxicology

We describe the utility of the free-living soil nematode Caenorhabditis elegans (C. elegans) as a model organism for toxicity tests. Dose response data were observed on larvae C. elegans exposed to xenobiotic chemicals, CdCl2 and Bisphenol-A (BPA), under liquid toxicity test system. The end points measured were 5, 8, 24 hr mortality. The LD50 value was low level or the same level in C. elegans, although a longer duration was needed in yeast toxicity test. For mortality, CdCl2 was more toxic than BPA. In addition to measurement of mortality after chemical exposure, we analyzed the effects of xenobiotic chemicals at the molecular level in C. elegans. In order to identify genes that are differentially expressed as a consequence of toxic stress due to CdCl2 or BPA, we used the cDNA microarray system to compare mRNA expression patterns in C. elegans. Using cDNA microarray technique, we found that a large number of target genes positively and negatively regulated by each chemicals in C. elegans. Chemicals affected gene expression for a diverse range of cellular functions and the differential gene expression pattern was observed between different chemical exposures. These data suggest that C. elegans is a suitable test organism for chemical bioassay of environmental xenobiotic chemicals.

A study of material incorporation for medaka (Oryzias latipes) EGGS by various voltage pulses

The purpose of this study is to investigate the effects of pulsed electric fields on fertilized eggs and to establish a technique of incorporating materials into them without damage to inside tissues, embryo-cell and yolk. Medaka (Oryzias latipes) egg was used, since its growth can be observed easily with a microscope because of its transparency of outer-shell. In addition, it is easy to handle and breed them. Then a chemical substance called cycloheximide, which is a protein synthesis inhibitor, was selected as the first material to be incorporated. If it is succeeded to incorporate into egg, embryogenesis will be obstructed or delayed directly.

Nanosecond pulsed electric field incorporation technique to predict molecular mechanisms of teratogenicity and developmental toxicity of estradiol-17β on medaka embryos: DEVELOPMENTAL EFFECTS OF E2 ON MEDAKA EMBRYOS BY NSPEF INCORPORATION

Herein, we propose using a nanosecond pulsed electric field (nsPEF) technique to assess teratogenicity and embryonic developmental toxicity of estradiol‐17β (E2) and predict the molecular mechanisms of teratogenicity and embryonic developmental defects caused by E2 on medaka (Oryzias latipes). The 5 hour post‐fertilization embryos were exposed to co‐treatment with 10 μm E2 and nsPEF for 2 hours and then continuously cultured under non‐E2 and nsPEF conditions until hatching. Results documented that the time to hatching of embryos was significantly delayed in comparison to the control group and that typical abnormal embryo development, such as the delay of blood vessel formation, was observed. For DNA microarray analysis, 6 day post‐fertilization embryos that had been continuously cultured under the non‐E2 and nsPEF condition after 2 hour co‐treatments were used. DNA microarray analysis identified 542 upregulated genes and one downregulated gene in the 6 day post‐fertilization embryos. Furthermore, bioinformatic analyses using differentially expressed genes revealed that E2 exposure affected various gene ontology terms, such as response to hormone stimulus. The network analysis also documented that the estrogen receptor α in the mitogen‐activated protein kinase signaling pathway may be involved in regulating several transcription factors, such as FOX, AKT1 and epidermal growth factor receptor. These results suggest that our nsPEF technique is a powerful tool for assessing teratogenicity and embryonic developmental toxicity of E2 and predict their molecular mechanisms in medaka embryos.