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Objectives In patients with RA, baricitinib not only improves arthritis symptom severity, but also patients’ neuropsychological symptoms, such as depression and fatigue. However, the cellular mechanisms through which baricitinib can affect neural activity is unexplored. While the blood–brain barrier (BBB) permeability of this drug remains unclear, Janus kinase inhibitors (JAKi) might reach the area postrema, which is a unique brain region with a weak BBB function. Our recent study demonstrated microglial activation during experimental arthritis in the area postrema. Therefore, we sought to assess the effect of baricitinib on microglia in the area postrema using the CIA mouse model. Methods Microglia number and morphology in the area postrema were assessed by immunostaining for ionized calcium-binding adaptor molecule-1 (Iba-1). Data were collected on post-immunization day 35 (early phase) and 84 (late phase), and compared between baricitinib- and vehicle-treated mice. The effect on signal transducers and activators of transcription (STAT3) in the area postrema was also immunohistochemically examined. Behavioural outcomes were assessed by examining feeding behaviours and sucrose preference tests. Results In the early phase, activated microglial levels in the area postrema were decreased by baricitinib, accompanied by the inhibition of phosphorylated-STAT3 and recovery of food intake and sucrose preference. On the other hand, baricitinib did not affect microglial morphology in the late phase. Conclusion Our results demonstrate that baricitinib can affect brain cells, specifically microglia, in the brain region with a weak BBB and mitigate aberrant behaviours during autoimmune arthritis, pointing to the potential therapeutic effect of JAKi on brain pathologies underpinning RA.

Cetaceans accumulate high levels of environmental pollutants, yet their toxicological studies have been difficult due to technical and ethical issues. It is essential to identify and fill the curre...

Equine estrogens (EQs) are steroidal hormones isolated from the urine of pregnant mares and are used in the formulation of human medications. This study initially investigated the embryonic developmental toxicity of equilin (Eq) and equilenin (Eqn) in medaka (Oryzias latipes). Malformations were observed in embryos exposed to nominal concentrations of 1 and 10 mg/L of Eq and Eqn. Delayed hatching was observed at 1 mg/L of Eq. To further investigate the molecular mechanism of developmental toxicity caused by Eq and Eqn, transcriptome and bioinformatics analyses were performed. Among 2016 and 3855 total differentially expressed genes (DEGs), 1117 DEGs overlapped between Eq. (55.4 % of total DEGs) and Eq. (29.0 % of total DEGs). Gene ontology indicated effects in terms related to blood circulation and cell junctions. Pathway analyses using DEGs revealed that both Eq and Eqn treatments at 10 mg/L affected various KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, such as neuroactive ligand-receptor interaction, mitogen-activated protein kinase signaling, retinol metabolism, and cytokine-cytokine receptor interaction. These results suggest that the disruption of these KEGG pathways is involved in the developmental toxicity of EQs in medaka embryos.

This study aimed to establish zebrafish-based in vivo and in silico assay systems to evaluate the antiandrogenic potential of environmental chemicals. Zebrafish embryos were exposed to 17α-methyltestosterone (TES) alone or coexposed to TES and representative antiandrogens including flutamide, p,p'-DDE, vinclozolin, fenitrothion, and linuron. We assessed the transcript expression of the androgen-responsive gene sulfotransferase family 2, cytosolic sulfotransferase 3 (sult2st3). The expression of sult2st3 was significantly induced by TES in the later stages of embryonic development. However, the TES-induced expression of sult2st3 was inhibited by flutamide in a concentration-dependent manner (IC

The present study evaluated the binding potencies (equilibrium dissociation constant: KD) of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) with the constitutive androstane receptor (CAR)_ligand binding domain (LBD) of the Baikal seal (bsCAR_LBD) and mouse (mCAR_LBD) using a surface plasmon resonance (SPR) biosensor. The binding affinities of individual congeners with mCAR_LBD tended to be higher than those with bsCAR_LBD but the differences were within the same order of magnitude. Notably, PBDE congeners showed higher binding affinities for both CAR_LBDs than PCB congeners. In silico docking simulations demonstrated that PBDEs had more non-covalent interactions with specific amino acid residues in both CAR_LBDs than PCBs, supporting the results of their binding affinities. Binding affinity comparisons among congeners revealed the structural requirements for higher binding; mono or di ortho-, tri meta-, and di para‑chlorine substitutions for PCBs, and di or tri ortho-, mono meta-, and di para‑bromine substitutions for PBDEs. The binding potencies of these congeners unlikely accounted for their previously reported CAR-mediated transactivation potencies, implying that their transactivation is regulated in a ligand-dependent, but a distinct manner from ligand binding. Risk assessment analysis showed that the KD values of individual PCB and PBDE congeners were 1-4 orders of magnitude higher than their respective hepatic concentrations in wild Baikal seal population.

Juvenile hormone (JH) are a family of multifunctional hormones regulating larval development, molting, metamorphosis, reproduction, and phenotypic plasticity in arthropods. Based on its importance in arthropod life histories, many insect growth regulators (IGRs) mimicking JH have been designed to control harmful insects in agriculture and aquaculture. These JH analogs (JHAs) may also pose hazards to nontarget species by causing unexpected endocrine-disrupting (ED) effects such as molting and metamorphosis defects, larval lethality, and disruption of the sexual identity. This critical review summarizes the current knowledge of the JH-mediated effects in the freshwater cladoceran crustaceans such as Daphnia species on JHA-triggered endocrine disruptive outputs to establish a systematic understanding of JHA effects. Based on the current knowledge, adverse outcome pathways (AOPs) addressing the JHA-mediated ED effects in cladoceran leading to male offspring production and subsequent population decline were developed. The weight of evidence (WoE) of AOPs was assessed according to established guidelines. The review and AOP development aim to present the current scientific understanding of the JH pathway and provide a robust reference for the development of tiered testing strategies and new risk assessment approaches for JHAs in future ecotoxicological research and regulatory processes.

Whales accumulate high levels of environmental pollutants. Exposure to polychlorinated biphenyls (PCBs) and their metabolites (OH-PCBs) could be linked to abnormal behavior, which may lead to mass stranding of marine mammals. Whales may thus suffer from adverse effects such as neuronal dysfunction, yet testing the neurotoxicity of these compounds has never been feasible for these species. This study established neurons chemically reprogrammed from fibroblasts of mass stranded melon-headed whales (Peponocephala electra) and used them for in vitro neurotoxicity assays. Exposure to 4-hydroxy-2',3,5,5'-tetrachlorobiphenyl (4'OH-CB72), a metabolite of PCBs, caused apoptosis in the reprogrammed neurons. Transcriptome analysis of 4'OH-CB72-treated whale neurons showed altered expressions of genes associated with oxidative phosphorylation, chromatin degradation, axonal transport, and neurodegenerative diseases. These results suggest that 4'OH-CB72 exposure may induce neurodegeneration through disrupted apoptotic processes. A comparison of the results with human reprogrammed neurons revealed the specific effects on the whale neurons. Our noninvasive approach using fibroblast-derived neurons is useful for hazard and risk assessments of neurotoxicity in whales.

Juvenile hormone (JH) is an important endocrine factor regulating many biological activities in arthropods. In daphnids, methoprene-tolerant (Met) belongs to a basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family protein which has recently been confirmed as a JH receptor and can bind and be activated by JHs and JH agonists. Although the activation of the JH signaling pathway causes many physiological effects, the molecular basis for the structural feature and ligand binding properties of Daphnia Met are not fully understood. To study the ligand preference in terms of structural features of Daphnia Met, we built in silico homology models of the PAS-B domain of Daphnia Mets from cladoceran crustaceans, Daphnia pulex and D. magna. Structural comparison of two Daphnia Met PAS-B domain models revealed that the volume in the main cavity of D. magna Met was larger than that of D. pulex Met. Compared with insect Met, Daphnia Met had a less hydrophobic cavity due to polar residues in the core-binding site. Molecular docking simulations of JH and its analogs with Daphnia Met indicated that the interaction energies were correlated with each of the experimental values of in vivo JH activities based on male induction and in vitro Met-mediated transactivation potencies. Furthermore, in silico site-directed mutagenesis supported experimental findings that Thr292 in D. pulex Met and Thr296 in D. magna Met substitution to valine contribute to JH selectivity and differential species response. This study demonstrates that in silico simulations of Daphnia Met and its ligands may be a tool for predicting the ligand profile and cross species sensitivity.

This study assessed the transcription levels of estrogen-responsive genes, such as vitellogenins (Vtg1 and Vtg2), choriogenins (ChgL, ChgH, and ChgHm), cytochrome P450 aromatase (CYP19a1b), and ER subtypes (ERα, ERβ1, and ERβ2), in 7 days-post-fertilization (dpf) embryos and 9 and 12 dpf larvae of medaka (Oryzias latipes) exposed to estrogenic endocrine-disrupting chemicals (EDCs). The5 h-post-fertilization embryos were exposed to EDCs such as 17β-estradiol (E2), p-n-nonylphenol (NP), and bisphenol A (BPA). In E2 (0.10-222 nM)-treated 7 dpf embryos and 9 or 12 dpf larvae, ChgL, ChgH, and ChgHm expression was up-regulated in a concentration-dependent manner. By contrast, interestingly, Vtg1 and Vtg2 expression was not induced in E2-treated 7 dpf embryos but was significantly induced in 9 and 12 dpf larvae, suggesting a developmental-stage-specific regulatory mechanism underlying Vtg expression. The maximum concentrations of NP (0.09-1.5 μM) and BPA (1.8-30 μM) up-regulated Chg expression in 9 or 12 dpf larvae, and the relative estrogenic potencies (REPs) of E2, NP, and BPA were 1, 2.1 × 10

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.

To assess the effect of exposure to persistent organic pollutants (POPs) on the estrogen receptor (ER) signaling pathway in Baikal seals (Pusa sibirica), we investigated the molecular characterizations and functions of two Baikal seal ER (bsER) isoforms, bsERα and bsERβ. The bsERα and bsERβ cDNA clones isolated have an open reading frame of 595 and 530 amino acid residues, respectively. The tissue distribution analyses of bsER mRNAs showed that bsERα transcripts were primarily found in the ovary and uterus, and bsERβ in the muscle in wild Baikal seals. The immunofluorescence staining assay showed that 17β-estradiol (E2) treatment promoted the nuclear translocation of in vitro-expressed bsERα. Transient transfection of bsERα in U2OS cells enhanced the transcription of pS2, an ER target gene of E2. We then measured bsER-mediated transactivation potencies of POPs in an in vitro reporter gene assay system, in which a bsERα or bsERβ expression vector was transfected into COS-1 cells. For comparison, transactivation potencies of POPs on mouse ERs (mERα and mERβ) were also evaluated in the same manner. Results showed significant dose-dependent responses of bsERs and mERs when treated with p,p’-dichlorodiphenyltrichloroethane (p,p’-DDT), and p,p’-dichlorodiphenyldichloroethylene (p,p’-DDE). bsERs and mERs showed no response when exposed to polychlorinated biphenyls (PCBs) or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Comparison of the dose-response curves of DDTs across species (bsERs vs. mERs) showed that bsERα had a response similar to mERα, but bsERβ was less sensitive than mERβ. Comparing the lowest observable effective concentrations of p,p′-DDT (2.8 μM) and p,p′-DDE (10 μM) for in vitro bsERα-mediated transactivation with their hepatic concentrations in wild Baikal seals indicated that some individuals accumulated these compounds at levels comparable to the effective concentrations, suggesting the potential disruption of the bsERα signaling pathway in the wild population by these compounds. Co-transfection experiments with bsER and the aryl hydrocarbon receptor (AHR) suggested that high accumulation of estrogenic compounds exerts a synergistic effect with dioxin-like congeners on ER signaling through AHR activation in the wild seal population.

Equine estrogens (EEs) are widely used in hormone replacement therapy pharmaceuticals for postmenopausal women. Previous studies have shown that EEs occur in the aquatic environment; however, the potential estrogenicity and risk of EEs in aquatic organisms, including fish, have yet to be studied in detail. Therefore, we evaluated the estrogenic potential of major EEs, namely equilin (Eq), 17α-dihydroequilin (17α-Eq), 17β-dihydroequilin (17β-Eq), equilenin (Eqn), 17α-dihydroequilenin (17α-Eqn), and 17β-dihydroequilenin (17β-Eqn), on medaka (Oryzias latipes) using in vivo and in silico assays. Quantitative real-time RT-PCR analyses revealed that expression levels of choriogenin L (ChgL) and choriogenin H (ChgH) in medaka embryos responded to various types and concentrations of EEs in a concentration-dependent manner, whereas transcription levels of vitellogenin 1 were not significantly affected by any of the EEs in the concentration range tested. The order of the in vivo estrogenic potencies of EEs was as follows: 17β-Eq > Eq > 17β-Eqn > Eqn > 17α-Eqn > 17α-Eq. Additionally, the 50% effective concentrations (EC50) of 17β-Eq was lower than that of 17β-estradiol. We also investigated the interaction potential of EEs with medaka estrogen receptor (ER) subtypes in silico using a three-dimensional model of the ligand-binding domain (LBD) for each ER and docking simulations. All six EEs were found to interact with the LBDs of ERα, ERβ1, and ERβ2. The order of the in silico interaction potentials of EEs with each ER LBD was as follows: 17β-Eq > 17α-Eq > Eq > 17β-Eqn > 17α-Eqn > Eqn. Furthermore, we identified the key amino acids that interact with EEs in each ER LBD; our findings suggest that amino acids and/or their hydrogen bonding may be responsible for the ligand-specific interactions with each ER. This study is the first to comprehensively analyze the estrogenic potential of EEs in medaka both in vivo and in silico.

Nonylphenol (NP) has been classified as an endocrine-disrupting chemical. In this study, we conducted mysid DNA microarray analysis with which has 2240 oligo DNA probes to observe differential gene expressions in mysid crustacean (Americamysis bahia) exposed to 1, 3, 10 and 30 μg/l of NP for 14 days. As a result, we found 31, 27, 39 and 68 genes were differentially expressed in the respective concentrations. Among these genes, the expressions of five particular genes were regulated in a similar manner at all concentrations of the NP exposure. So, we focused on one gene encoding cuticle protein, and another encoding cuticular protein analogous to peritrophins 1-H precursor. These genes were down-regulated by NP exposure in a dose-dependent manner, and it suggested that they were related in a reduction of the number of molting in mysids. Thus, they might become useful molecular biomarker candidates to evaluate molting inhibition in mysids.

Aryl hydrocarbon receptor (AHR) is well conserved from invertebrates to vertebrates, and it mediates the toxic effects of exogenous ligands, including dioxins. Recent studies reported that AHRs activated by endogenous ligands play critical roles in mammalian physiological homeostasis. Avian species possess at least two AHR isoforms (AHR1 and AHR2), which exhibit species- and isoform-specific transactivation potencies to exogenous ligands, whereas mammals possess a single AHR. To delineate the profiles and roles of endogenous ligands for avian AHR isoforms, we investigated in vitro transactivation potencies of avian AHRs (AHR1 and AHR2 from the jungle crow, Corvus macrorhynchos; common cormorant, Phalacrocorax carbo; and black-footed albatross, Phoebastria nigripes) treated with the endogenous tryptophan metabolites 6-formylindolo [3,2-b] carbazole (FICZ), l-kynurenine (l-Kyn), kynurenic acid (KYNA), and indoxyl sulfate (IS). Furthermore, we analyzed the binding mode of these ligands to each avian AHR isoform by in silico docking simulations. The EC50 of FICZ (0.009-0.032nM) was similar regardless of the species or isoform of AHR. The estimated in silico binding mode of FICZ to AHRs was well conserved in both isoforms. The transactivation potencies of avian AHRs to other tryptophan metabolites were 10(5)-10(7) fold lower than those for FICZ, and EC50 values varied in a species- and isoform-specific manner. This was consistent with poor conservation of the binding mode of l-Kyn, KYNA, and IS predicted in in silico docking simulations. Our results suggest that in avian species, FICZ is the most potent endogenous AHR ligand, and that AHR1 and AHR2 are physiologically functional.

In this study, we assessed the binding affinities of perfluoroalkyl substances (PFASs), including perfluoroalkyl carboxylates (PFCAs) and perfluoroalkyl sulfonates (PFSAs), to the ligand-binding domains (LBDs) of Baikal seal ( Pusa sibirica; bs) and human (h) peroxisome proliferator-activated receptor alpha (PPARα). An in vitro competitive binding assay showed that six PFCAs and two PFSAs could bind to recombinant bs and hPPARα LBD proteins in a dose-dependent manner. The relative binding affinities (RBAs) of PFASs to bsPPARα were as follows: PFOS > PFDA > PFNA > PFUnDA > PFOA > PFHxS > PFHpA > PFHxA. The RBAs to bsPPARα showed a significant positive correlation with those to hPPARα. In silico PPARα homology modeling predicted that there were two ligand-binding pockets (LBPs) in the bsPPARα and hPPARα LBDs. Structure-activity relationship analyses suggested that the binding potencies of PFASs to PPARα might depend on LBP binding cavity volume, hydrogen bond interactions, the number of perfluorinated carbons, and the hydrophobicity of PFASs. Interspecies comparison of the in vitro binding affinities revealed that bsPPARα had higher preference for PFASs with long carbon chains than hPPARα. The in silico docking simulations suggested that the first LBP of bsPPARα had higher affinities than that of hPPARα; however, the second LBP of bsPPARα had lower affinities than that of hPPARα. To our knowledge, this is the first evidence showing interspecies differences in the binding of PFASs to PPARαs and their structure-activity relationships.

The aim of this study was to understand the cytochrome P450 (CYP)-dependent metabolic pathway and potency of polychlorinated biphenyls (PCBs) in the Baikal seal (Pusa sibirica). In vitro metabolism of 62 PCB congener mixtures was investigated by using liver microsomes of this species. A decreased ratio of over 20% was observed for CB3, CB4, CB8, CB15, CB19, CB22, CB37, CB54, CB77, and CB105, suggesting the preferential metabolism of low-chlorinated PCBs by CYPs. The highly activated metabolic pathways in Baikal seals that were predicted from the decreased PCBs and detected hydroxylated PCBs (OH-PCBs) were CB22 to 4'OH-CB20 and CB77 to 4'OH-CB79. The total amount of OH-PCBs detected as identified and unidentified congeners accounted for only a 3.8 ± 1.7 mol % of loaded PCBs, indicating many unknown PCB metabolic pathways. To explore factors involved in CYP-dependent PCB metabolism, we examined the relationships among the structural and physicochemical properties of PCBs, the in silico PCB-CYP docking parameters, and the in vitro PCB decreased ratios by principal component analysis. Statistical analysis showed that the decreased PCB ratio was at least partly accounted for by the substituted chlorine number of PCBs and the distance from the Cl-unsubstituted carbon of docked PCBs to the heme Fe in CYP2A and 2B.

This study aimed to elucidate the catalytic function of cytochrome P450 (CYP) 1 enzymes in aquatic mammals. Alkoxyresorufin O-dealkylation (AROD) activities including methoxy- (MROD), ethoxy- (EROD), pentoxy- (PROD), and benzyloxyresorufin O-dealkylation (BROD), and 2- and 4-hydroxylation activities of 17β-estradiol (E2) were measured by using yeast-expressed Baikal seal (Pusa sibirica) CYP1A1, 1A2, and 1B1 proteins. Heterologous protein expression of the Baikal seal CYP1s (bsCYP1s) in yeast microsomes was confirmed by reduced CO-difference spectra and immunoblotting. Heterologously expressed human CYP1 enzyme (hCYP1) activities were simultaneously measured and compared with those of bsCYP1 isozymes. Recombinant bsCYP1A1 protein showed the highest Vmax of EROD, followed by MROD, PROD, and BROD, similar to that of hCYP1A1. Vmax/Km ratios of all AROD activities catalyzed by bsCYP1A1 were lower than those catalyzed by hCYP1A1, suggesting less potential for AROD by bsCYP1A1. Enzymatic assays for bsCYP1A2 showed no or minimal AROD activities, while hCYP1A2 displayed MROD and EROD activities. bsCYP1B1 showed an AROD profile (EROD>BROD>MROD>>PROD) similar to that of hCYP1B1; however, Vmax/Km ratios of all AROD activities by bsCYP1B1 were higher. Yeast microsomes containing bsCYP1A1 and 1B1 and hCYP1A1, 1A2, and 1B1 metabolized E2 to 2-OHE2 and 4-OHE2, whereas bsCYP1A2 showed no such activity. Comparison of 4- and 2-hydroxylations of E2 by CYP1As suggests that bsCYP1A1, hCYP1A1, and 1A2 preferentially catalyze 2- rather than 4-hydroxylation. As for CYP1B1, the Vmax/Km ratios suggest that both Baikal seal and human CYPs catalyze 4- rather than 2-hydroxylation. Interspecies comparison showed that bsCYP1B1 has higher metabolic potencies for both E2 hydroxylations than does hCYP1B1, whereas the activity of bsCYP1A1 was lower than that of hCYP1A1. Messenger RNA expression levels of bsCYP1s in the liver of Baikal seals indicated that bsCYP1A1 and 1A2 enzymes contributed to 16.2% and 83.7% of total CYP1s, respectively; bsCYP1B1 accounted for only 0.06%. Addition of anti-human CYP1A1 antibody in seal liver microsomes suppressed EROD activity more than did anti-human CYP1A2 antibody. Therefore, EROD may be catalyzed by hepatic bsCYP1A1 but not bsCYP1A2, consistent with the results of yeast-expressed bsCYP1A1 and 1A2. In silico substrate-docking models of bsCYP1s suggested that the defect in bsCYP1A2 enzymatic activities may be accounted for by the Pro substitution of highly conserved Thr in the I-helix, which is involved in formation of a hydrogen bond with the hydroperoxy intermediate on the heme. This Thr-Pro substitution is evolutionarily conserved across aquatic mammals and could explain their lower metabolic potential for persistent organic pollutants.

Nuclear hyperpolarization is a phenomenon that can be used to improve the sensitivity of magnetic resonance molecular sensors. However, such sensors typically suffer from short hyperpolarization lifetime. Herein we report that [15N, D14]trimethylphenylammonium (TMPA) has a remarkably long spin–lattice relaxation time (1128 s, 14.1 T, 30 °C, D2O) on its 15N nuclei and achieves a long retention of the hyperpolarized state. [15N, D14]TMPA-based hyperpolarized sensor for carboxylesterase allowed the highly sensitive analysis of enzymatic reaction by 15N NMR for over 40 min in phophate-buffered saline (H2O, pH 7.4, 37 °C).

To evaluate species- and isoform-specific responses to dioxins and related compounds (DRCs) via aryl hydrocarbon receptor (AHR) in the red seabream ( Pagrus major ), we constructed a reporter gene assay system. Each expression plasmid of red seabream AHR1 (rsAHR1) and AHR2 (rsAHR2) together with a reporter plasmid containing red seabream CYP1A 5'-flanking region were transfected into COS-7 cells. The cells were treated with graded concentrations of seven DRC congeners including 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 1,2,3,4,7,8-HxCDD, 2,3,7,8-TCDF, 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDF, and PCB126. Both rsAHR1 and rsAHR2 exhibited dose-dependent responses for all the tested congeners. The rsAHR isoform-specific TCDD induction equivalency factors (rsAHR1- and rsAHR2-IEFs) were calculated on the basis of 2,3,7,8-TCDD relative potency derived from the dose-response of each congener. The rsAHR1-IEFs of PeCDD, HxCDD, TCDF, PeCDF, and HxCDF were estimated as 0.17, 0.29, 2.5, 1.5, and 0.27, respectively. For PCB126, no rsAHR1-IEF was given because of less than 10% 2,3,7,8-TCDD maximum response. The rsAHR2-IEFs of PeCDD, HxCDD, TCDF, PeCDF, HxCDF, and PCB126 were estimated as 0.38, 0.13, 1.5, 0.93, 0.20, and 0.0085, respectively. The rsAHR1/2-IEF profiles were different from WHO toxic equivalency factors for fish. In silico docking simulations supported that both rsAHRs have potentials to bind to these congeners. These results suggest that dioxin toxicities may be mediated by both rsAHRs in red seabreams.

BACKGROUND AND PURPOSE: It has not been clarified whether the postoperative resolution of vertebral deformities achieved by KP are superior to those achieved by VP. We compared the outcomes of KP and VP, taking into account the presence of vertebral mobility, to determine whether the balloon inflation in KP may contribute to postoperative resolution of the vertebral deformities in patients with VCFs. MATERIALS AND METHODS: The subjects had 34 vertebral bodies treated by VP, and 43 vertebral bodies treated by KP. In all patients, preoperative dynamic imaging was performed to assess the presence of vertebral mobility. First, the vertebral height restoration and kyphotic change were compared between the VP and KP groups. Then, the patients were further divided into 2 groups on the basis of the presence of vertebral mobility, and they were compared within and between the VP and KP groups in relation to the presence of vertebral mobility. RESULTS: Overall, no significant differences were observed in either the vertebral height restoration or kyphotic change between the VP and KP groups (P > .20). Preoperative dynamic imaging identified 19 vertebral bodies each with vertebral mobility in the VP (56%) and KP groups (44%) (P = .21). Within the VP and KP groups, the vertebral height restoration and kyphotic changes were significantly better in patients with vertebral mobility than in those without (P < .01). There were no significant differences between the 2 treatment groups after adjustment for the presence of vertebral mobility (P > .30). CONCLUSIONS: In both the VP and KP groups, the vertebral height restoration and kyphotic change largely depended on the preoperative vertebral mobility. The use of the balloon itself contributed little to resolution of the vertebral deformities.

Percutaneous vertebral body perforation is a new technique for treating painful vertebral compression fractures (VCFs). We prospectively evaluated this treatment in 45 consecutive patients with 63 VCFs treated at our hospital between September and December 2009. In all patients, long-term conservative treatment had failed to achieve pain relief, resulting in reduced changes in activities of daily living (ADL). We evaluated visual analogue scale (VAS) scores for pain before and after vertebral body perforation as well as in the chronic phase, changes in ADL, whether recipients would recommend this treatment, and complications. The mean VAS score improved from 6.9 ± 1.9 before surgery to 3.6 ± 2.9 immediately after surgery, and to 2.5 ± 2.1 at 3 months. ADL was improved in 27 patients, and walking became possible in 16 of 24 patients who had been unable to walk. Nearly two-thirds of patients would strongly recommend this treatment to others. The analgesic effect of vertebral body perforation was significantly lower in patients with than in those without vertebral pseudarthrosis before surgery. Only 1 patient suffered a new fracture during the 3-month period after surgery, but there were no perioperative complications. Vertebral body perforation for painful VCFs can achieve early improvement of ADL due to the analgesic effect in carefully selected patients and is a safe treatment with a low frequency of complications.

Dioxins cause various toxic effects through the aryl hydrocarbon receptor (AHR) in vertebrates, with dramatic species and strain differences in susceptibility. Although inbred mouse strains C3H/HeJ-lpr/lpr (C3H/lpr) and MRL/MpJ-lpr/lpr (MRL/lpr) are known as dioxin-sensitive and dioxin-resistant mice, respectively, the molecular mechanism underlying this difference remains unclear. The difference in the hepatic proteome of the two mouse strains treated with vehicle or 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) was investigated by a proteomic approach of two-dimensional electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF). To confirm the strain-difference in response to TBDD treatment, cytochrome P450 (CYP) 1A1 and 1A2 protein levels were measured in both strains. A dose of 10 µg/kg body weight of TBDD induced hepatic CYP1A1 and CYP1A2 expression in both strains, but the expression levels of both CYP1A proteins were higher in C3H/lpr mice than in MRL/lpr mice, supporting that C3H/lpr mice are more sensitive to dioxins than MRL/lpr mice. Proteins that were more induced or suppressed by TBDD treatment in C3H/lpr mice were successfully identified by 2-DE and MALDI-TOF/TOF, including proteins responsible for AHR activation through production of endogenous ligands such as aspartate aminotransferase, indolethylamine N-methyltransferase, and aldehyde dehydrogenases, as well as proteins reducing oxidative stress, such as superoxide dismutase and peroxiredoxins. Taken together, our results provide insights into the molecular mechanism underlying the high dioxin susceptibility of the C3H/lpr strain, in which AHR activation by TBDD is more prompted by the production of endogenous ligands, but the adaptation to oxidative stress is also acquired.

The aryl hydrocarbon receptor (AHR) mediates dioxin toxicities. Several studies have suggested that two amino acid residues corresponding to the 324th and 380th positions in the ligand binding domain (LBD) of the chicken AHR1 (Ile_Ser as high sensitivity, Ile_Ala as moderate sensitivity, and Val_Ala as low sensitivity), could be an important factor determining dioxin sensitivity in avian species. Here, we analyzed the association between ecological factors and AHR1 LBD genotypes of 113 avian species. Cluster analyses showed that 2 major clusters and sub-clusters of the cluster 3 were associated with specific AHR1 genotypes depending on the food, habitat, and migration of the animal. The majority of the species with Ile_Ala type were the Passeriformes, which are omnivorous or herbivorous feeders in the terrestrial environment. The species with Val_Ala type was primarily composed of raptors and waterbirds, which have been exposed to naturally occurring dioxins. An in vitro reporter gene assay revealed that the sensitivity to a natural dioxin, 1,3,7-tribromodibenzo-p-dioxin was in the order of Ile_Ser > Ile_Ala > Val_Ala. These results suggest that ecological factors related to the exposure of natural dioxins contribute to natural selection of the avian AHR1 genotype, which consequently leads to different sensitivity to man-made dioxins.

A long-term station blackout accident of a BWR is analysed using TRAC-BF1, and the calculated results are compared with the observed data at the unit 2 reactor of the Fukushima Daiichi nuclear power plant. The RCIC is assumed to be actuated, and the reactor pressure and the liquid level in the core are found to be stable and the dry well pressure increases gradually. The effects of leakage and heat release from the containment are discussed. The reactor pressure increases to the SRV set point and the liquid level and the dry well pressure decrease after the termination of RCIC. The calculated results are in good agreement with the observed data, and the thermal–hydraulic transient of unit 2 reactor is shown to be simulated well.

An unprecedented earthquake and tsunami struck the Fukushima Dai-ichi Nuclear Power Plants on 11 March 2011. Although extensive efforts have been continuing on investigations into the causes and consequences of the accident, and the Japanese Government has presented a comprehensive report on the accident in the IAEA Ministerial Conference held in June 2011, there is still much to be clarified on what happened during the accident and why. This article aims at identifying what should be clarified further about the progression of the accident at Units 1–3 through the review and analysis of information released from Tokyo Electric Power Company and government authorities. It also discusses the safety issues raised by the accident based on the insights gained, in order to contribute to establishing a new framework that pursues continuous improvement toward the highest standards of safety that can reasonably be achieved.

The present study demonstrates the seasonal variations of concentrations of pharmaceutical and personal care products(PPCPs) in influents and effluents at two sewage treatment plants (STPs) and the surface water of Mankyung River, South Korea. PPCPs were measured using a high performance liquid chromatograph coupled with a tandem mass spectrometer(LC-MS/MS). In the two STPs surveyed, all of the 13 targeted PPCPs were detected at concentrations ranging from ND (not detected) to 13700 ng/L in the influents, and ND to 1940 ng/L in the effluents, with total PPCP concentrations being higher in winter than in other seasons in both STP influents and effluents. While the removal efficiencies of disopyramide, ibuprofen, triclosan, erythromycin, atenolol and indomethacin of the STPs were relatively high (average 60-90%), those of clarithromycin, carbamazepine, fluconazole, propranolol and ifenprodil were extremely low (average 0-30%).The prevalent contaminants in the river water in all seasons were carbamazepine (ND to 595 ng/L), followed by atenolol (ND-690 ng/L), ibuprofen (ND-414 ng/L), mefenamic acid (ND-326 ng/L), levofloxacin (ND-380 ng/L), clarithromycin(ND-443 ng/L), fluconazole (ND-111 ng/L), erythromycin (ND-152 ng/L), propranolol (ND-268 ng/L), ifenprodil (ND-38.5 ng/L), indomethacin (ND-58.2 ng/L), triclosan (ND-46.1 ng/L) and disopyramide (ND). Concentrations of PPCPs in surface river water collected from five sample sites trended to be higher in November than in March, May and August. It was proposed that the reasons for the observed seasonal variations in PPCP concentrations may be increased consumption by the inhabitants of the Mankyung River basin and the decreased river flow during this period leading to increased relative concentrations of these compounds in water. In addition, PPCP concentrations were greater in the surface water samples collected in the vicinity of STPs, so STPs act as a point source of PPCP pollution.

We investigated the effects of environmentally relevant concentrations of nonylphenol (NP) on survival, growth, sexual development and molting of the mysid Americamysis bahia (Crustacea: Mysidacea), including assay of 20-hydroxyecdysone (20E) levels. Seven-day-old mysids were exposed to NP concentrations of 0.3, 1, 3, 10 and 30 microg/L for 14 days. Among growth-related traits we assayed, an effect on body length was the most prominent, with the effects observed at concentrations as low as 1 microg/L NP. In addition, the total number of molting exposed to NP for 14 days was significantly decreased in the treatment groups at 10 or 30 microg/L relative to the controls. These results clearly indicate that subchronic exposure to environmentally relevant concentrations of NP affects growth of mysids. Furthermore, we investigated the effect of NP on production of 20E in female mysids during the molting period. Production of 20E in pre-molting mysids exposed to 30 microg/L of NP was lower than those in control groups, and exposure of female mysids to NP disrupted molting cycles. Taken together, the results suggest that environmentally relevant concentrations of NP can disrupt growth of A. bahia via inhibition of 20E levels and an associated delay in molting.

This study demonstrates the effects of synthetic polycyclic musks such as Galaxolide (HHCB), Tonalide (AHTN), Traseolide (ATII), Celestolide (ADBI), Phantolide (AHMI) and Cashmeran (DPMI), both on the early life stage and on gene expression in the livers of male medaka (Oryzias latipes). The toxicity ranking (the 96-h median lethal concentration) of the chemicals tested on 24-h-old medaka larvae descended in the order HHCB (0.95 mg/L)=ATII (0.95 mg/L)AHTN (1.0 mg/L)AHMI (1.2 mg/L)ADBI (2.0 mg/L)DPMI (12 mg/L), indicating high acute toxicity of these compounds on the early life stages of medaka. Expression analysis of hepatic vitellogenin (VTG) protein showed potential estrogenic effects upon the addition of AHTN and HHCB, indicative of the induction of VTG synthesis in the livers of male medaka. We also investigated mRNA expression levels of two estrogen receptor (ER) subtypes (ERalpha and beta), two VTGs (VTG I and II), pregnane X receptor (PXR), and two cytochromes P450 (CYP) 3As (CYP3A38 and 3A40) in the livers of male medaka treated with AHTN and HHCB at 5, 50 and 500 microg/L. Quantitative real-time PCR analyses revealed that hepatic ERalpha, VTG I, VTG II, and CYP3A40 mRNA responded to 500 microg/L of AHTN and/or HHCB after 3 days exposure, whereas no effects of these compounds on ERbeta, PXR, and CYP3A38 mRNA transcription were observed. These results suggest that certain polycyclic musks, including AHTN and HHCB, induce the expression levels of hepatic ERalpha and VTG mRNA/protein and modulate expression levels of CYP3A40 mRNA in the livers of male medaka.

Fucoxanthin exhibits a preventative function with degenerative diseases such as cancer and metabolic syndrome without side effects. Therefore, commercial-scale preparation of fucoxanthin is needed, but it has been very difficult to find the required resources to do so. The aim of this study is to develop a preparation method of fucoxanthin using waste parts of cultured kombu (Laminalia japonica). Around 79,000 t of cultured kombu is discarded in Japan during thinning out and forming processes, which includes a high amount of fucoxanthin (21.3-17.8 mg/100 g fresh weight). Waste parts of kombu were examined to obtain better quality fucoxanthin. Heating increased fucoxanthin recovery, and additional washing with tap water reduced the salt content of the fucoxanthin extract. Cutting waste parts of kombu into 5-mm wide strips made extraction easier without the leakage of fucoxanthin during handling. After freezing and transportation to the extraction factory, kombu showed the best recovery of fucoxanthin and the lowest content of salt following two extractions with 3 volumes of absolute ethanol. To remove chlorophylls the extract was subjected to silica gel column chromatography. Finally, 1490 g fucoxanthin was obtained from 10 t of waste parts of kombu and the recovery ratio was 82%. The fucoxanthin obtained was stable and reduced by only 2% in 6 months storage at 4°C. Thus, waste parts of cultured kombu are a good bioresource for fucoxanthin extraction.

To clarify the degradation pathway of acrinol by light, isolation and identifi cation of acrinol degradation products (ANDP) were attempted. ANDP2, on eo ft he ANDPs, was isolated by extraction with methanol from cloths dampened with acrinol solution, and purified by column chromatography on Diaion HP-10, Sephadex LH-20 and G25. Th es tructural elucidation of ANDP-2 was examined by infrared, 1 H-NMR, 13 C-NMR and Fast atom bombardment mass (FAB-MS) spectra studies. From the spectroscopic data, the structure of ANDP-2 was determined to be 4-amino-6-ethoxyl-2,3dicarboxyquinoline, that was found to be a novel degradation product of acrinol by light. The solution of ANDP-2 has fluorescence with a blue color, though that of acrinol has fluorescence with green. ANDP-2 did not show growth inhibition at a concentration of 100µg/ml against Gram-positive, -negative bacteria, yeast and fungi.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces a broad spectrum of toxic effects including craniofacial malformation and neural damage in fish embryos. These effects are mainly mediated by the aryl hydrocarbon receptor (AHR). However, the mode of action between TCDD-induced AHR activation and adverse outcomes is not yet understood. To provide a comprehensive picture of the AHR signaling pathway in fish embryos exposed to TCDD, red seabream (Pagrus major) embryos were treated with graded concentrations of TCDD (0.3-37nM) in seawater, or with a mixture of TCDD and 500nM CH223191, an AHR-specific antagonist. The transcriptome of red seabream embryos was analyzed using a custom-made microarray with 6000 probes specifically prepared for this species. A Jonckheere-Terpstra test was performed to screen for genes that demonstrated altered mRNA expression levels following TCDD exposure. The signals of 1217 genes (as human homologs) were significantly altered in a TCDD concentration-dependent manner (q-value

The objective of this study was to investigate the reproductive success (fertility and fecundity) of medaka (Oryzias latipes) exposed to 4-nonylphenol (4-NP) in water and the viability of their F(1) offspring. In addition, we measured the bioconcentration of 4-NP in eggs. After a 21-d exposure to 100microg/l 4-NP, medaka showed reduced egg production and fertility. Hepatic vitellogenin levels were increased significantly in males treated with 10, 50 and 100microg/l of 4-NP. In the F(1) generations, the hatchability and time to hatching of embryos in the 100microg/l treatment group were adversely affected, and 2-7microg 4-NP/g egg was found in spawned eggs (the bioconcentration factor: 30-100). These results indicate that (1) 4-NP adversely affected the reproduction (fecundity and fertility) of adult medaka, (2) 4-NP accumulated in eggs through maternal transfer and (3) these levels of 4-NP were associated with adverse effects in the F(1) offspring.

In a recent study, it was reported that 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), a metabolite of bisphenol A (BPA; 2,2-bis(4-hydroxyphenyl)propane), showed estrogenic activity in several in vitro assays, and the estrogenic activity of MBP was higher than that of BPA. In this study, we have investigated the early life stage toxicity and estrogenic effect of MBP on medaka (Oryzias latipes). The 96-h median lethal concentration value of MBP and BPA with 24-h-old larvae was estimated to be 1,640 and 13,900 microg/l, respectively. The hatchability of fertilized eggs exposed to MBP and BPA over 14 days was significantly decreased at doses of 2,500 microg/l and 12,500 microg/l, respectively. Moreover, to compare the potency of estrogenic activity in vivo, male medaka were exposed to various concentrations of MBP and BPA for 21 days. The lowest-observed-effect concentrations of MBP and BPA for hepatic vitellogenin induction in male medaka were estimated to be 4.1 and 1,000 microg/l, respectively. These results suggest that MBP has high toxicity for early life stages of the medaka, and that the estrogenic activity of MBP was about 250-fold higher than that of BPA to male medaka.

We investigated the effects of nonylphenol (NP) and triclosan (TCS) on production of vitellogenin (Vg), testosterone (T), and hepatic cytochrome P450 1A and 2B activities in male South African clawed frogs (Xenopus laevis). In a 14-d waterborne exposure test, no significant differences in the level of plasma Vg synthesis in male frogs were observed among the control, 10, 50, and 100 microg/l NP and 20, 100, and 200 microg/l TCS treatment groups. Intraperitoneal injection of male frogs with 2, 20, and 200 microg/g body weight NP resulted in no significant differences in plasma Vg levels among the control and all treatment groups. However, the levels of plasma Vg in all TCS treatment groups (intraperitoneal injection of 4, 40, and 400 microg/g body weight) were lower than that in the solvent control group, and male frogs injected with high doses of NP or TCS had lower T levels than the control group. No significant differences in hepatic cytochrome P450 1A and 2B activities were observed among the all treatment groups. Male frogs injected with 20 microg/g body weight of estradiol-17beta had significantly higher plasma Vg levels than the control group. These results suggest that profiles of plasma Vg and T production in male Xenopus laevis could be useful biomarkers for detecting hormonally active agents.

Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) is widely used as antibacterial agent in various industrial products, such as textile goods, soap, shampoo, liquid toothpaste and cosmetics, and often detected in wastewater effluent. In this study, the effects of TCS on the early life stages and reproduction of medaka (Oryzias latipes) were investigated. The 96-h median lethal concentration value of TCS for 24-h-old larvae was 602 microg/l. The hatchability and time to hatching in fertilized eggs exposed to 313 microg/l TCS for 14 days were significantly decreased and delayed, respectively. An assessment of the effects of a TCS 21-day exposure period on the reproduction of paired medaka showed no significant differences in the number of eggs produced and fertility among the control and 20, 100 and 200 microg/l TCS treatment groups. However, concentrations of hepatic vitellogenin were increased significantly in males treated with TCS at 20 and 100 microg/l. In the F(1) generations, although the hatching of embryos in the 20 microg/l treatment showed adverse effects, there was no dose-response relationship between hatchability and TCS treatment levels. These results suggest that TCS has high toxicity on the early life stages of medaka, and that the metabolite of TCS may be a weak estrogenic compound with the potential to induce vitellogenin in male medaka but with no adverse effect on reproductive success and offspring.

The acute toxicity of endocrine disrupters in two crustaceans, Americamysis bahia (A. bahia) and Daphnia magna (D. magna), was investigated and the toxicological responses compared. Bisphenol A had the lowest toxicity to D. magna, the 48 hr median lethal concentrations (LC50) values was 12.8 mg/l. However, the toxic sensitivity of A. bahia to bisphenol A was approximately 10-fold higher than for D. magna (48 hr LC50; 1.34 mg/l). The 48 hr LC50 of estradiol-17β was 2.97 and 1.69 mg/l in D. magna and A. bahia, respectively. Nonylphenol had the highest lethal toxicity against both A. bahia (48 hr LC50; 0.051 mg/l) and D. magna (48 hr LC50; 0.18 mg/l). However, ecdysteroids, and ecdysteroidal activity insecticides, such as tebufenozide, and juvenile hormone analog, had no toxic effects against either A. bahia or D. magna at the concentrations tested in this study. These results suggest that A. bahia is a more suitable toxicity test organism for endocrine disrupters than D. magna because of the higher sensitivity of A. bahia to the toxicological effect.

Effectiveness of debris cooling by water that fills a gap between the debris and the lower head wall was estimated through steady calculations in reactor scale. In those calculations, the maximum coolable debris depth was assessed as a function of gap width with combination of correlations for critical heat flux and turbulent natural convection of a volumetrically heated pool. The results indicated that the gap with a width of 1 to 2 mm was capable of cooling the debris under the conditions of the TMI-2 accident, and that a significantly larger gap width was needed to retain a larger amount of debris within the lower plenum. Transient models on gap growth and water penetration into the gap were developed and incorporated into CAMP code along with turbulent natural convection model developed by Yin, Nagano and Tsuji, categorized in low Reynolds number type two-equation model. The validation of the turbulent model was made with the UCLA experiment on natural convection of a volumetrically heated pool. It wa...

The aryl hydrocarbon receptor (AHR) mediates toxic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other dioxin-like compounds (DLCs). Avian species possess multiple AHR isoforms (AHR1, AHR1β, and AHR2) that exhibit species- and isoform-specific responses to ligands. To account for the ligand preference in terms of the structural features of avian AHRs, we generated in silico homology models of the ligand-binding domain of avian AHRs based on holo human HIF-2α (PDB entry 3H7W ). Molecular docking simulations of TCDD and other DLCs with avian AHR1s and AHR2s using ASEDock indicated that the interaction energy increased with the number of substituted chlorine atoms in congeners, supporting AHR transactivation potencies and World Health Organization TCDD toxic equivalency factors of congeners. The potential interaction energies of an endogenous AHR ligand, 6-formylindolo [3,2-b] carbazole (FICZ) to avian AHRs were lower than those of TCDD, which was supported by a greater potency of FICZ for in vitro AHR-mediated transactivation than TCDD. The molecular dynamics simulation revealed that mean square displacements in Ile324 and Ser380 of TCDD-bound AHR1 of the chicken, the most sensitive species to TCDD, were smaller than those in other avian AHR1s, suggesting that the dynamic stability of these amino acid residues contribute to TCDD preference. For avian AHR2, the corresponding residues (Val/Ser or Val/Ala type) were not responsible for differential TCDD sensitivity. Application of the three-dimensional reference interaction site model showed that the stabilization of TCDD binding to avian AHRs may be due to the solvation effect depending on the characteristics of two amino acids corresponding to Ile324 and Ser380 in chicken AHR1. This study demonstrates that in silico simulations of AHRs and ligands could be used to predict isoform-, ligand-, and species-specific interactions.

Objective The postoperative time course of redundant nerve roots (RNRs) in patients with lumbar spinal canal stenosis (LSCS) is currently unknown. The purpose of this study is to investigate the relationship between postoperative morphologic changes in detected RNRs and the clinical outcome of patients with LSCS. Methods A total of 33 symptomatic patients with LSCS who demonstrated RNRs on magnetic resonance imaging (MRI) were treated with decompressive laminectomy alone. On the basis of the MRI scans obtained 7 days after surgery, patients were stratified into two groups: group 1 included patients with resolution of RNRs and group 2 included patients with persistent RNRs. Comparative parameters were examined between the two groups of patients. Results We found that 24 of the 33 patients showed resolution of RNRs and 9 showed persistent RNRs. Although there was no difference in the Japanese Orthopedic Association score between the two groups before treatment, group 1 showed a greater recovery of Japanese Orthopedic Association score 1 month postoperatively. MRI demonstrated that the cross-sectional area of the preoperative dural sac at the stenotic lesion was smaller in group 2 than in group 1; however, there was no difference in cross-sectional area of the postoperative dural sac between the two groups. Within 12 months, there was no evidence of RNRs in six of the nine cases in group 2. Conclusion Although most patients with LSCS show postoperative resolution of RNRs detected on MRI, some show persistent RNRs postoperatively. The functional outcome of these patients remains poor even if sufficient expansion of the dural sac is achieved postoperatively.