Potential mechanisms underlying embryonic developmental toxicity caused by benzo[a]pyrene in Japanese medaka (Oryzias latipes).

Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), are widely distributed in air, water, and sediments; however, limited data are available regarding their potential adverse effects on the early life stages of fish. In this study, we evaluated the embryonic teratogenicity and developmental toxicity of BaP in Japanese medaka (Oryzias latipes) using a nanosecond pulsed electric field (nsPEF) technique and predicted their molecular mechanisms via transcriptome analysis. The gas chromatography/mass spectrometry analyses revealed that the BaP was efficiently incorporated into the embryos by nsPEF treatment. The embryos incorporating BaP presented typical teratogenic and developmental effects, such as cardiovascular abnormalities, developmental abnormalities, and curvature of backbone. DNA microarray analysis revealed several unique upregulated genes, such as those involved in cardiovascular diseases, various cellular processes, and neural development. Furthermore, the gene set enrichment and network analyses found several genes and hub proteins involved in the developmental effects of BaP on the embryos. These findings suggest a potential mechanism of teratogenicity and developmental toxicity caused by exposure to BaP. The nsPEF and transcriptome analyses in combination can be effective for evaluating the potential effects of chemical substances on medaka embryos.

Rapid point-of-care testing for epidermal growth factor receptor gene mutations in patients with lung cancer using cell-free DNA from cytology specimen supernatants.

Epidermal growth factor receptor (EGFR) mutations are associated with responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). Our previous study revealed a rapid point-of-care system for detecting EGFR mutations. This system analyzes cell pellets from cytology specimens using droplet-polymerase chain reaction (d-PCR), and has a reaction time of 10 min. The present study aimed to validate the performance of the EGFR d-PCR assay using cell-free DNA (cfDNA) from supernatants obtained from cytology specimens. Assay results from cfDNA supernatant analyses were compared with those from cell pellets for 90 patients who were clinically diagnosed with, or suspected of having, lung cancer (80 bronchial lavage fluid samples, nine pleural effusion samples and one spinal fluid sample). EGFR mutations were identified in 12 and 15 cases using cfDNA supernatants and cell pellets, respectively. The concordance rates between cfDNA-supernatant and cell­pellet assay results were 96.7% [kappa coefficient (K)=0.87], 98.9% (K=0.94), 98.9% (K=0.79) and 98.9% (K=0.79) for total EGFR mutations, L858R, E746_A750del and T790M, respectively. All 15 patients with EGFR mutation-positive results, as determined by EGFR d-PCR assay using cfDNA supernatants or cell pellets, also displayed positive results by conventional EGFR assays using tumor tissue or cytology specimens. Notably, EGFR mutations were even detected in five cfDNA supernatants for which the cytological diagnoses of the corresponding cell pellets were 'suspicious for malignancy', 'atypical' or 'negative for malignancy.' In conclusion, this rapid point-of-care system may be considered a promising novel screening method that may enable patients with NSCLC to receive EGFR-TKI therapy more rapidly, whilst also reserving cell pellets for additional morphological and molecular analyses.

Nanosecond pulsed electric field incorporation technique to predict molecular mechanisms of teratogenicity and developmental toxicity of estradiol-17ß on medaka embryos.

Herein, we propose using a nanosecond pulsed electric field (nsPEF) technique to assess teratogenicity and embryonic developmental toxicity of estradiol-17ß (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.

A novel, rapid point-of-care test for lung cancer patients to detect epidermal growth factor receptor gene mutations by using real-time droplet-PCR and fresh liquid cytology specimens.

Epidermal growth factor receptor gene (EGFR) mutations are associated with response to tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC). We developed a novel, rapid EGFR mutation assay using a real-time droplet-polymerase chain reaction machine (EGFR d-PCR assay). The purpose of this study was to validate the performance of the EGFR d-PCR assay using fresh liquid cytology specimens. We analyzed three major EGFR mutations (L858R in exon 21, E746_A750del in exon 19 and T790M in exon 20) in 80 fresh liquid cytology specimens of adenocarcinoma (ADC) or NSCLC-not otherwise specified (NOS) via the EGFR d-PCR assay and conventional real-time PCR assay using the therascreen® EGFR RGQ PCR kit (Therascreen assay). In addition, we performed sensitivity assays using cell lines with EGFR mutations. The EGFR d-PCR assay detected 16 L858Rs, 8 E746_A750dels and 1 T790M mutation and the Therascreen assay detected 16 L858Rs, 11 deletions in exon 19 and 1 T790M mutation. The results were concordant between the two assays. The reaction time of the EGFR d-PCR assay was 8 min and 10 sec, but that of the Therascreen assay was 1 h and 45 min. Sensitivity, as assessed by the detection limit of the EGFR d-PCR assay was 0.5, 0.05 and 0.5% for L858R, E746_A750del and T790M, respectively. The EGFR d-PCR assay markedly reduced the detection time of major EGFR mutations with high sensitivity compared with the conventional Therascreen assay and is expected to expedite EGFR-TKI therapy for lung cancer patients, especially those in advanced stages.

A Rapid and Automated Device for Purifying Nucleic Acids.

We have developed a rapid, automated nucleic acid purification device in a single cartridge containing silica-coated magnetic beads. We succeeded in extracting the matrix protein gene of influenza A virus from pharyngeal swab samples within 3 min. The device will be widely applicable to detect a specific gene from the various samples for clinical diagnosis and genetic research.

A novel automated device for rapid nucleic acid extraction utilizing a zigzag motion of magnetic silica beads.

We report a novel automated device for nucleic acid extraction, which consists of a mechanical control system and a disposable cassette. The cassette is composed of a bottle, a capillary tube, and a chamber. After sample injection in the bottle, the sample is lysed, and nucleic acids are adsorbed on the surface of magnetic silica beads. These magnetic beads are transported and are vibrated through the washing reagents in the capillary tube under the control of the mechanical control system, and thus, the nucleic acid is purified without centrifugation. The purified nucleic acid is automatically extracted in 3 min for the polymerase chain reaction (PCR). The nucleic acid extraction is dependent on the transport speed and the vibration frequency of the magnetic beads, and optimizing these two parameters provided better PCR efficiency than the conventional manual procedure. There was no difference between the detection limits of our novel device and that of the conventional manual procedure. We have already developed the droplet-PCR machine, which can amplify and detect specific nucleic acids rapidly and automatically. Connecting the droplet-PCR machine to our novel automated extraction device enables PCR analysis within 15 min, and this system can be made available as a point-of-care testing in clinics as well as general hospitals.

Evaluation of saliva as diagnostic materials for influenza virus infection by PCR-based assays.

BACKGROUND: Immunochromatographic antigen tests have been widely used for detection of influenza virus; however its low sensitivity restricts the use of clinical materials other than nasopharyngeal swabs. Saliva is obtained non-invasively and has utility for diagnosis of influenza. Polymerase chain reaction (PCR) is not typically used for rapid testing because it is time consuming. We evaluated the utility of saliva as diagnostic materials for influenza virus infection by PCR-based assays. METHODS: Nasopharyngeal swabs and saliva were simultaneously collected from 144 patients and investigated by reverse transcription-quantitative PCR (RT-qPCR) and droplet-RT-PCR. RESULTS: Overall concordance of results from nasopharyngeal swabs and saliva were 95.8%. Influenza gene was detectable in less than 12min in saliva by the droplet-RT-PCR. Saliva as well as nasopharyngeal swabs contained more than 1×10(2) copies/µl of the influenza gene. About half of the patients provided positive results in nasopharyngeal swabs and saliva within 24h from the onset of the symptoms. CONCLUSION: The study demonstrates that saliva can be used as an alternative specimen source to nasopharyngeal swabs. When rapid PCR assay including RNA extraction to be full-automation in a miniaturized machine, point-of-care test based on PCR may be realized using saliva without restriction of materials.

Rapid diagnosis of acute promyelocytic leukemia with the PML-RARA fusion gene using a combination of droplet-reverse transcription-polymerase chain reaction and instant-quality fluorescence in situ hybridization.

BACKGROUND: Acute promyelocytic leukemia (APL) with the PML-RARA fusion gene can be effectively cured using molecular-targeted therapies, which require both detection and quantification of the PML-RARA fusion gene. Here, we developed a rapid assay for identifying and measuring the PML-RARA fusion gene in patients with APL using droplet-reverse transcription-polymerase chain reaction (droplet-RT-PCR) and instant quality-fluorescence in situ hybridization (IQ-FISH). METHODS: RNA for droplet-RT-PCR and fixed-cell suspensions for IQ-FISH were prepared from five patients with APL and three controls. We evaluated the amplification efficiency and reaction time with droplet-RT-PCR and signal clarity and hybridization time with IQ-FISH. RESULTS: The reaction using droplet-RT-PCR was completed in 26min. The PML-RARA fusion gene was detected in all samples from the five patients. IQ-FISH yielded clear signals after 1h of hybridization. There were no significant differences in signal clarity or positive signal ratios between IQ-FISH and conventional FISH. CONCLUSIONS: Simultaneous droplet-RT-PCR and IQ-FISH, in addition to morphological examination of blood smears, can be used to diagnose patients as having APL within 4h based on molecular/cytogenetic results. Rapid diagnosis can allow effective therapies to be started promptly.

Development of a rapid and sensitive one-step reverse transcription-nested polymerase chain reaction in a single tube using the droplet-polymerase chain reaction machine.

BACKGROUND: Reverse transcription (RT)-nested polymerase chain reaction (PCR) is a time-consuming procedure because it has several handling steps and is associated with the risk of cross-contamination during each step. Therefore, a rapid and sensitive one-step RT-nested PCR was developed that could be performed in a single tube using a droplet-PCR machine. METHODS: The K562 BCR-ABL mRNA-positive cell line as well as bone marrow aspirates from 5 patients with chronic myelogenous leukemia (CML) and 5 controls without CML were used. We evaluated one-step RT-nested PCR using the droplet-PCR machine. RESULTS: One-step RT-nested PCR performed in a single tube using the droplet-PCR machine enabled the detection of BCR-ABL mRNA within 40min, which was 10(3)-fold superior to conventional RT nested PCR using three steps in separate tubes. The sensitivity of the one-step RT-nested PCR was 0.001%, with sample reactivity comparable to that of the conventional assay. CONCLUSIONS: One-step RT-nested PCR was developed using the droplet-PCR machine, which enabled all reactions to be performed in a single tube accurately and rapidly and with high sensitivity. This one-step RT-nested PCR may be applicable to a wide spectrum of genetic tests in clinical laboratories.

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.

Rapid single nucleotide polymorphism based method for hematopoietic chimerism analysis and monitoring using high-speed droplet allele-specific PCR and allele-specific quantitative PCR.

BACKGROUND: Chimerism analysis is important for the evaluation of engraftment and predicting relapse following hematopoietic stem cell transplantation (HSCT). We developed a chimerism analysis for single nucleotide polymorphisms (SNPs), including rapid screening of the discriminable donor/recipient alleles using droplet allele-specific PCR (droplet-AS-PCR) pre-HSCT and quantitation of recipient DNA using AS-quantitative PCR (AS-qPCR) following HSCT. METHODS: SNP genotyping of 20 donor/recipient pairs via droplet-AS-PCR and the evaluation of the informativity of 5 SNP markers for chimerism analysis were performed. Samples from six follow-up patients were analyzed to assess the chimerism via AS-qPCR. These results were compared with that determined by short tandem repeat PCR (STR-PCR). RESULTS: Droplet-AS-PCR could determine genotypes within 8min. The total informativity using all 5 loci was 95% (19/20). AS-qPCR provided the percentage of recipient DNA in all 6 follow-up patients without influence of the stutter peak or the amplification efficacy, which affected the STR-PCR results. CONCLUSION: The droplet-AS-PCR had an advantage over STR-PCR in terms of rapidity and simplicity for screening before HSCT. Furthermore, AS-qPCR had better accuracy than STR-PCR for quantification of recipient DNA following HSCT. The present chimerism assay compensates for the disadvantages of STR-PCR and is readily performable in clinical laboratories.

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 ERß. 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 ERß 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.

Novel high-speed droplet-allele specific-polymerase chain reaction: application in the rapid genotyping of single nucleotide polymorphisms.

BACKGROUND: Single nucleotide alterations such as single nucleotide polymorphisms (SNP) and single nucleotide mutations are associated with responses to drugs and predisposition to several diseases, and they contribute to the pathogenesis of malignancies. We developed a rapid genotyping assay based on the allele-specific polymerase chain reaction (AS-PCR) with our droplet-PCR machine (droplet-AS-PCR). METHODS: Using 8 SNP loci, we evaluated the specificity and sensitivity of droplet-AS-PCR. Buccal cells were pretreated with proteinase K and subjected directly to the droplet-AS-PCR without DNA extraction. The genotypes determined using the droplet-AS-PCR were then compared with those obtained by direct sequencing. RESULTS: Specific PCR amplifications for the 8 SNP loci were detected, and the detection limit of the droplet-AS-PCR was found to be 0.1-5.0% by dilution experiments. Droplet-AS-PCR provided specific amplification when using buccal cells, and all the genotypes determined within 9 min were consistent with those obtained by direct sequencing. CONCLUSIONS: Our novel droplet-AS-PCR assay enabled high-speed amplification retaining specificity and sensitivity and provided ultra-rapid genotyping. Crude samples such as buccal cells were available for the droplet-AS-PCR assay, resulting in the reduction of the total analysis time. Droplet-AS-PCR may therefore be useful for genotyping or the detection of single nucleotide alterations.

Rapid detection of PML-RARA fusion gene by novel high-speed droplet-reverse transcriptase-polymerase chain reaction: possibility for molecular diagnosis without lagging behind the morphological analyses.

BACKGROUND: Acute promyelocytic leukemia (APL) is an aggressive disease requiring prompt diagnosis and treatment. Rapid detection of the PML-RARA fusion gene provides the molecular basis for a highly effective therapy with all-trans retinoic acid. We developed a rapid assay by novel droplet-reverse transcriptase-polymerase chain reaction (droplet-RT-PCR) for the detection of the PML-RARA fusion gene in APL patients. METHODS: RNA was extracted from 7 samples obtained from 5 APL patients with the PML-RARA fusion gene confirmed by nested RT-PCR and fluorescence in situ hybridization. Using these 7 samples, we evaluated the reaction time and amplification efficiency of the droplet-RT-PCR. RESULTS: Using the droplet-RT-PCR, we could detect the PML-RARA fusion gene in all 7 samples. The reaction time for 50 cycles of droplet-RT-PCR was 27 min. The amplification by the droplet-RT-PCR assay was considered positive for the PML-RARA fusion gene in less than 22 min, at the point when the fluorescence exceeded the threshold level. CONCLUSIONS: Our novel droplet-RT-PCR assay is specific for the detection of the PML-RARA fusion gene and has a markedly reduced reaction time. Thus, the novel droplet-RT-PCR assay contributes to the rapid diagnosis of APL without lagging behind the morphological assessment.

A novel high-speed droplet-polymerase chain reaction can detect human influenza virus in less than 30 min.

BACKGROUND: The polymerase chain reaction (PCR) has been widely used for diagnosis of infection. Rapid detection of influenza virus is useful for therapeutic decisions. We attempted to develop a novel assay by real-time droplet-PCR machine for influenza virus. METHODS: RNA extracted from nasal swabs or primary swabs pretreated only were used for PCR analyses. We evaluated reaction time, amplification efficiency, sensitivity, and specificity of the novel droplet-PCR. RESULTS: The reaction time of the novel droplet-PCR was 28 min, whereas that of PCR using the conventional PCR machine was 80 min. The standard curve constructed from the amplification plots by the novel droplet-PCR was: y=-3.6x+42.9; that by PCR using the conventional PCR machine was: y=-3.5x+37.8. The sensitivity and specificity of the novel droplet-PCR were 86.7% and 91.7% for the influenza A and 100.0% and 100.0% for the influenza B, respectively. The novel droplet-PCR provided the specific amplification when using primary swabs without RNA extraction. CONCLUSIONS: Our novel droplet-PCR markedly reduced the reaction time while showing same reactivity as that by PCR using the conventional PCR machine. Thus, the novel droplet-PCR assay can be used as a rapid assay for detection of influenza virus.

(17α,20E)-17,20-[(1-methoxyethylidene)bis(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11) is a partial agonist of the androgen receptor.

A novel steroid compound, (17α,20E)-17,20-[(1-methoxyethylidene)bis(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11), was found to be a partial agonist of the androgen receptor (AR) in an androgen responsive element (ARE)-luciferase reporter assay. YK11 accelerates nuclear translocation of AR. Furthermore, YK11 does not induce amino/carboxyl-terminal (N/C) interaction and prevents 5-α-dihydrotestosterone (DHT)-mediated N/C interaction. Thus, YK11 activates AR without causing N/C interaction, which may in turn be responsible for the partially agonistic nature of YK11 observed in the ARE-luciferase reporter system. YK11 acts as a gene-selective agonist of AR in MDA-MB 453 cells. The effect of YK11 on gene expression relative to that of androgen agonist varies depending on the gene context. YK11 activated the reporter gene by inducing the translocation of the AR into the nuclear compartment, where its amino-terminal domain (NTD) functions as a constitutive activator of AR target genes. Our results suggest that YK11 might act as selective androgen receptor modulator (SARM).

Biochemical and molecular characterization of senescence-related cysteine protease-cystatin complex from spinach leaf.

Cysteine proteases (CPs) with N-succinyl-Leu-Tyr-4-methylcoumaryl-7-amide (Suc-LY-MCA) cleavage activity were investigated in green and senescent leaves of spinach. The enzyme activity was separated into two major and several faint minor peaks by hydrophobic chromatography. These peaks were conventionally designated as CP1, CP2 and CP3, according to their order of elution. From the analyses of molecular mass, subunit structure, amino acid sequences and cDNA cloning, CP2 was a monomer complex (SoCP-CPI) (51 kDa) composed of a 41-kDa core protein, SoCP (Spinacia oleracea cysteine protease), and 14-kDa cystatin, a cysteine protease inhibitor (CPI), while CP3 was a trimer complex (SoCP-CPI)(3) (151 kDa) of the same subunits as SoCP-CPI and showed a wider range of specificity toward natural substrates than SoCP-CPI. Trimer (SoCP-CPI)(3) was irreversibly formed from monomers through association. The results of reverse transcription-polymerase chain reaction (RT-PCR) revealed that mRNAs of CPI and SoCP are hardly expressed in green leaves, but they are coordinately expressed in senescent leaves, suggesting that these proteases involve in senescence. Purified recombinant CPI had strong inhibitory activity against trimer SoCP, (SoCP)(3) , which had a cystatin deleted with K(i) value of 1.33 × 10(-9) M. After treatment of the enzyme with a succinate buffer (pH 5) at the most active pH of the enzyme, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and activity analyses showed that cystatin was released from both monomer SoCP-CPI and trimer (SoCP-CPI)(3) complexes with a concomitant activation. Thus, the removal of a cystatin is necessary to activate the enzyme activity.

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.

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.

Short-term effects of endocrine-disrupting chemicals on the expression of estrogen-responsive genes in male medaka (Oryzias latipes).

To evaluate the estrogenic activities of selected estrogenic compounds such as estradiol-17beta (E2), nonylphenol (NP), 4-(1-adamantyl)phenol (AdP), bisphenol A (BPA), BPA metabolite 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) and 4,4'-dihydroxy-alpha-methylstilbene (DHMS) in the shortest possible time, we investigated the expression of estrogen-responsive genes such as vitellogenin I, vitellogenin II and alpha-type estrogen receptor genes in the liver of male medaka (Oryzias latipes) using reverse transcription-polymerase chain reaction (RT-PCR) techniques. These estrogen-responsive genes responded rapidly to selected estrogenic compounds after 8 h exposure, and the expression of hepatic vitellogenin II and estrogen receptor alpha mRNA was found to be more responsive than that of vitellogenin I mRNA. As a result, the relative estrogenic potencies of tested chemicals descended in the order of E2 (100)>MBP (0.38)>AdP (0.25)>DHMS (0.05)>NP (0.02)>BPA (0.001). Moreover, this preliminary study indicates that AdP and DHMS should be considered as candidate estrogenic compounds with the potential to induce hepatic estrogen-responsive genes in male medaka. These results suggest that vitellogenin I, vitellogenin II and estrogen receptor alpha gene expression patterns alter in male medaka treated with selected estrogenic compounds, and that these genes may be useful molecular biomarkers for screening estrogenic endocrine-disrupting chemicals in the shortest possible time.