Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

Major histocompatibility complex (MHC) II genes are key factors in presenting antigens derived from extracellular pathogens to CD4(+) T cells. These genes are known to be associated with disease resistance in livestock, including cultured fish species. Genome-wide linkage analysis of RSIV-resistant red sea bream has revealed that MHC II beta is linked to the RSIV-resistant trait and was suggested as a probable candidate gene responsible for the resistant trait. In the present study, diversity of the MHC II beta gene in wild and farmed red sea bream populations was evaluated by analysis of an intronic minisatellite locus, PmaMHC2b. Haplotypes of the beta I domain of MHC II beta were identified by cloning, and linkage of minisatellite polymorphisms with haplotype was confirmed in farmed populations. Large numbers of alleles were observed in the wild population (30), while the domesticated populations showed significantly lower values (13). Thirteen haplotypes of MHC II beta were observed; most haplotypes possessed the unique PmaMHC2b allele, but some were present in more than one PmaMHC2b allele. Each haplotype possessed a distinct amino acid sequence with no synonymous mutations. Among 16 amino acid positions involved in peptide-binding grooves, four were monomorphic and 12 polymorphic. Several positions differed in the RSIV-resistant and RSIV-susceptible phenotypes: five in hydrophilic/hydrophobic character and five in polarity. These results provide insight applicable to broodstock management and contribute to understanding RSIV resistance.

DOI： 10.1016/j.aquaculture.2022.738038

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

A combined treatment of heavy oil (HO) exposure and virus infection induces increased mortality in Japanese flounder (Paralichthys olivaceus). In this study, we addressed how HO exposure affects the immune system, especially antiviral activities, in Japanese flounder. The fish were infected with viral hemorrhagic septicemia virus (VHSV), followed by exposure to HO. We analyzed virus titers in the heart and mRNA expression in the kidney of surviving fish. The virus titers in fish exposed to heavy oil were higher than the threshold for onset. The results suggest that HO exposure may allow the replication of VHSV, leading to higher mortality in the co-treated group. Gene-expression profiling demonstrated that the expression of antiviral-activity-related genes, such as those for interferon and apoptosis induction, were lower in the co-treated group than in the group with VHSV infection only. These results helped explain the high virus titers in fish treated with both stressors. Thus, interferon production in the virus-infected cells and apoptosis induction by natural killer cells worked normally in the VHSV-infected fish without HO exposure, but these antiviral activities were slightly suppressed by HO exposure, possibly leading to extensive viral replication in the host cells and the occurrence of VHS.

DOI： 10.1016/j.fsi.2022.03.046

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.11403/jset.25.1

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Language：English   Publishing type：Research paper (scientific journal)  

Commercial mixtures of decabromodiphenyl ether (deca-BDE), a brominated flame retardant, contain not only polybrominated diphenyl ethers (PBDEs, mainly BDE-209) as the main component but also dioxin-like compounds (DLCs) such as polybrominated dibenzofurans (PBDFs). Deca-BDE handling facilities (DHFs) and sewage treatment plants receiving effluent from DHFs are point sources of DLC and flame retardant (FR) pollution. Here, we examined their emission in Japan. For DHF effluents, DLCs detected by the dioxin-responsive chemically activated luciferase expression (DR-CALUX) assay were 1.3-890 pg TCDD-EQ/L (median 46 pg TCDD-EQ/L), while PBDEs and other FRs were <2.0-110,000 ng/L (610 ng/L) and 150-4,800,000 ng/L (41,000 ng/L). Risk quotients based on predicted no-effect concentrations suggested that DLCs, decabromodiphenyl ethane (DBDPE), tris(2,3-dibromopropyl) isocyanurate (TDBP-TAZTO), and bisphenol A bis(diphenyl phosphate) (BPA-BDPP) present significant risks for aquatic organisms. The concentrations of PBDFs, which are impurities in deca-BDE, were expected to decrease with the inclusion of deca-BDE in the Stockholm Convention list of persistent organic pollutants (May 2017). However, DLCs other than PBDFs and alternative FRs such as DBDPE, TDBP-TAZTO, and BPA-BDPP are likely still discharged. Additional findings indicate that strong (e.g., DLCs, DBDPE, and BPA-BDPP), but not weak (e.g., TDBP-TAZTO), hydrophobic compounds are sufficiently removed by current wastewater treatment processes in Japan.

DOI： 10.1021/acs.est.0c06359

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Authorship：Corresponding author   Language：English   Publisher：The Japanese Society of Environmental Toxicology  

<p>In order to develop an optimal method for the investigation of relative potencies of dioxin-like compounds using Japanese medaka, the present study conducted five independent early-life stage toxicity tests using 2,3,7,8-tetrachlorodibenzo-<i>p</i>-dioxin (TCDD) as the positive control. First, both short- and long-term effects of TCDD were examined to determine sensitive and highly reproducible endpoints. Then, the outcome and threshold of TCDD toxicities under different experimental conditions, e.g., exposure timing and duration, were compared to develop a cost-effective method. Finally, we decided to observe yolk sac/pericardial edema, hatching failure, and mortality of medaka embryos/larvae within a 28-d experimental period, after 6-h exposure to TCDD at 0-d post fertilization. We obtained the LC₅₀ values of TCDD at 28-d post fertilization based on its concentration in water (6.84 ng/L) or in egg (753 pg/g-egg), and the LC₅₀ values were comparable with those reported in the literature. Thus, in our future studies, relative toxic potencies of dioxin-like compounds will be tested using the method established in this study.</p>

DOI： 10.11403/jset.24.1

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The arylhydrocarbon receptor (AhR) is an important signaling pathway in the immune system of mammals. In addition to its physiological functions, the receptor mediates the immunotoxic actions of a diverse range of environmental contaminants that bind to and activate the AhR, including planar halogenated aromatic hydrocarbons (PHAHs or dioxin-like compounds) and polynuclear aromatic hydrocarbons (PAHs). AhR-binding xenobiotics are immunotoxic not only to mammals but to teleost fish as well. To date, however, it is unknown if the AhR pathway is active in the immune system of fish and thus may act as molecular initiating event in the immunotoxicity of AhR-binding xenobiotics to fish. The present study aims to examine the presence of functional AhR signaling in immune cells of rainbow trout (Oncorhynchus mykiss). Focus is given to the toxicologically relevant AhR2 clade. By means of RT-qPCR and in situ hybdridization, we show that immune cells of rainbow trout express ahr 2α and ahr 2β mRNA; this applies for immune cells isolated from the head kidney and from the peripheral blood. Furthermore, we show that in vivo as well as in vitro exposure to the AhR ligand, benzo(a)pyrene (BaP), causes upregulation of the AhR-regulated gene, cytochrome p4501a, in rainbow trout immune cells, and that this induction is inhibited by co-treatment with an AhR antagonist. Taken together, these findings provide evidence that functional AhR signaling exists in the immune cells of the teleost species, rainbow trout.

DOI： 10.3390/ijms21176323

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DOI： 10.1016/j.aquaculture.2019.03.039

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Gobiida is a basal subseries of percomorphs in teleost fishes, holding a useful position for comparisons with other orders of Percomorpha as well as other cohort of teleosts. Here, we describe a telencephalic atlas of a Gobiida species Rhinogobius flumineus (Mizuno, Memoirs of the College of Science, University of Kyoto, Series B: Biology, 1960; 27, 3), based on cytoarchitectural observations, combined with analyses of the distribution patterns of neurochemical markers and transcription factors. The telencephalon of R. flumineus shows a number of features distinct from those of other teleosts. Among others, the followings were of special note. (a) The lateral part of dorsal telencephalon (Dl), which is known as a visual center in other teleosts, is composed of as many as seven regions, some of which are conspicuous, circumscribed by cell plates. These subdivisions of the Dl can be differentiated clearly by differential soma size and color with Nissl-staining, and distribution patterns of neural markers. (b) Cell populations continuous with the ventral region of dorsal part of ventral telencephalon (vVd) exhibit extensive dimension. Especially, portion 1 of the central part of ventral telencephalon appears to represent a cell population laterally translocated from the vVd, forming a large cluster of small cells that penetrate deep into the central part of dorsal telencephalon. (c) The magnocellular subdivision of dorsal part of dorsal telencephalon (Ddmg) contains not only large cells but also vglut2a-positive clusters of small cells that cover a wide range of the caudal Ddmg. Such clusters of small cells have not been observed in the Ddmg of other teleosts.

DOI： 10.1002/cne.24547

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Language：Japanese   Publisher：The Japan Institute of Marine Engineering  

<p>The effects of chemicals in sediments in coastal areas on environmental organisms are almost unknown, although a number of investigations on the distribution of pollutants in these sediments were performed, often resulting in the detection of chemical residues with high concentrations. In this study, toxicities of sediments collected from 11 locations in Hiroshima Bay, Japan, was assessed using embryos of Java medaka, <i>Oryzias javanicus</i>. The embryos were placed on sediments containing a little pore water without adding overlying water. The results showed that as high as 37% of the embryos exposed to sediments collected near Hatsukaichi City died. Additionally, the mortality was 33% at a site distant slightly from an industrial area in Otake City. Delayed hatching was observed at six locations. Thus, the certain effects of exposure to sediments collected from the bay in the embryos were observed, and the authors concluded that these effects are most likely to have been caused by chemicals in the sediments. </p>

DOI： 10.5988/jime.54.860

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The infection caused by a kinetoplastid flagellate, Azumiobodo hoyamushi, in an ascidian, Halocynthia roretzi, results in softening of the tunic, and finally death. This disease is usually recognized using palpation of the softening tunic, and A. hoyamushi infection is detectable using microscopy or PCR amplification of specific gene fragments. The present study is the first quantitative evaluation of the symptoms of soft tunic syndrome by measuring the amount of bending (bending) and the peak force required to pierce the tunic (force). There was a strong correlation between bending and force. Correlation analyses among other parameters (ascidian total weight, tunic thickness, and tunic water content) indicated that larger ascidians had harder and thicker tunics with a higher water content. As compared to the tunic of healthy individuals, softened tunic was thinner and had lower water content. Infected tunics thus possibly lose water and become softer and thinner. Mechanisms for maintaining the appropriate water level content may be crucial for preventing tunic softening.

DOI： 10.2108/zs180072

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The complement system plays an important role in immune regulation and acts as the first line of defense against any pathogenic attack. To comprehend the red sea bream (Pagrus major) immune response, three complement genes, namely, pmC1r, pmMASP and pmC3, belonging to the classical, lectin and alternative complement cascade, respectively, were identified and characterized. pmC1r, pmMASP, and pmC3 were comprised of 2535, 3352, and 5735 base mRNA which encodes 732, 1029 and 1677 aa putative proteins, respectively. Phylogenetically, all the three studied genes clustered with their corresponding homologous clade. Tissue distribution and cellular localization data demonstrated a very high prevalence of all the three genes in the liver. Both bacterial and viral infection resulted in significant transcriptional alterations in all three genes in the liver with respect to their vehicle control counterparts. Specifically, bacterial challenge affected the pmMASP and pmC3 expression, while the viral infection resulted in pmC1r and pmC3 mRNA activation. Altogether, our data demonstrate the ability of pmC1r, pmMASP and pmC3 in bringing about an immune response against any pathogenic encroachment, and thus activating, not only one, but all the three complement pathways, in red sea bream.

DOI： 10.1016/j.fsi.2018.08.027

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer New York LLC  

We previously reported a body color deformity in juvenile red sea bream, which shows transparency in the juvenile stage because of delayed chromatophore development compared with normal individuals, and this finding suggested a genetic cause based on parentage assessments. To conduct marker-assisted selection to eliminate broodstock inheriting the causative gene, developing DNA markers associated with the phenotype was needed. We first conducted SNP mining based on AFLP analysis using bulked-DNA from normal and transparent individuals. One SNP was identified from a transparent-specific AFLP fragment, which significantly associated with transparent individuals. Two alleles (A/G) were observed in this locus, and the genotype G/G was dominantly observed in the transparent groups (97.1%) collected from several production lots produced from different broodstock populations. A few normal individuals inherited the G/G genotype (5.0%), but the A/A and A/G genotypes were dominantly observed in the normal groups. The homologs region of the SNP was searched using a medaka genome database, and intron 12 of the Nell2a gene (located on chromosome 6 of the medaka genome) was highly matched. We also mapped the red sea bream Nell2a gene on the previously developed linkage maps, and this gene was mapped on a male linkage group, LG4-M. The newly found SNP was useful in eliminating broodstock possessing the causative gene of the body color transparency observed in juvenile stage of red sea bream.

DOI： 10.1007/s10126-018-9810-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ireland Ltd  

The ciliate Miamiensis avidus causes scuticociliatosis in Japanese flounder Paralichthys olivaceus. We previously reported three serotypes of this ciliate distinguishable by serotype-specific antigenic polypeptides (serotype I, 30kDa; serotype II, 38kDa; serotype III, 34kDa). In this study, we determined the localization site of the serotype-specific polypeptides in the ciliate and determined the genes encoding the polypeptides, using the isolates IyoI (serotype I), Nakajima (serotype II), and Mie0301 (serotype III). SDS-PAGE and immunoblot analysis of cilia, membrane proteins, and cytoskeletal elements of the ciliates revealed that the polypeptides were abundant in the former two. Scanning electron microscopy of ciliates immobilized by homologous antiserum showed morphological changes in the cilia. These evidences suggested that the polypeptides were ciliary membrane immobilization antigens. The ciliary genes identified showed low identity scores-<51.5% between serotypes. To differentiate the serotypes, we designed serotype-specific PCR primer sets based on the DNA sequences. The PCR-based serotyping results were completely consistent with conventional serotyping methods (immobilization assay and immunoblot analysis). Twenty of 21 isolates were classified as either serotype I or II, and one isolate was undistinguishable. The combination of species-specific PCR previously reported and three serotype-specific PCR could be useful for identifying, serotyping, and surveillance for occurrences of new serotypes of M. avidus.

DOI： 10.1016/j.parint.2017.10.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Fish Pathology  

Miamiensis avidus is the causative agent of scuticociliatosis in various marine fish species. The virulence factors of the parasite have not been identified, so far. In this study, we examined M. avidus extracellular proteinases (ECPs) as potential virulence factors, using culture supernatants as an ECPs source. We investigated the substrate specificity of ECPs using artificial peptides, and the cytotoxicity of the ECPs was examined using CHSE-214 cells. To elucidate the role of ECPs in ciliate growth, M. avidus was cultured on CHSE-214 cells in the presence of proteinase inhibitors. We detected proteinase activities from the supernatant of M. avidus. Viable CHSE-214 cells decreased significantly in number, when incubated in a medium supplemented with the culture supernatant of M. avidus. The growth of ciliates on CHSE-214 cells was delayed in the presence of PMSF (serine proteinase inhibitor) and E-64 (cysteine proteinase inhibitor). These results suggested that the culture supernatant contained ECPs showing cytotoxicity, and the proteinases facilitated nutrient uptake by the ciliates. Thus, ECPs may be responsible for virulence factors of M. avidus.

DOI： 10.3147/jsfp.53.1

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Language：Japanese   Publisher：Japan Society of Material Cycles and Waste Management  

Brominated dioxins such as polybrominated dibenzo-<i>p</i>-dioxins (PBDDs), polybrominated dibenzofurans (PBDFs), and coplanar polybrominated biphenyls (Co-PBBs) are considered to have toxicity profiles (toxic, persistent and bioaccumulation) similar to those of the chlorinated analogues which have been prohibited as persistent organic pollutants (POPs) under the Stockholm Convention on POPs. Therefore, a joint World Health Organization (WHO) and United Nations Environment Programme (UNEP) expert panel has recommended the use of WHO toxicity equivalency factor for risk management of brominated dioxins. The Ministry of the Environment Japan (MOEJ) continues to investigate its actual emission of brominated dioxins such as PBDD/Fs. These have a close relationship with the lifecycle of decaboromodiphenyl ether (decaBDE), especially through manufacturing, recycling and incinerating. This is because PBDD/Fs are well known to be an impurity contained in decaBDE and in unintentionally generated compounds through photo and thermal degradation of brominated flame retardants (BFRs), including decaBDE. Since 2017, decaBDE has been listed in the Stockholm Convention and marked for elimination. Because of this, emission changes for brominated dioxins resulting from the shift to alternatives for decaBDE should be investigated from now on. As the WHO and UNEP expert panel pointed out, toxicity studies with brominated dioxins in fish and mammalian models are also needed for their appropriate risk management.

DOI： 10.3985/mcwmr.29.470

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Red sea bream iridoviral disease (RSIVD) is a major viral disease in red sea bream farming in Japan. Previously, we identified one candidate male individual of red sea bream that was significantly associated with convalescent individuals after RSIVD. The purpose of this study is to identify the quantitative trait loci (QTL) linked to the RSIVD-resistant trait for future marker-assisted selection (MAS). Two test families were developed using the candidate male in 2014 (Fam-2014) and 2015 (Fam-2015). These test families were challenged with RSIV, and phenotypes were evaluated. Then, de novo genome sequences of red sea bream were obtained through next-generation sequencing, and microsatellite markers were searched and selected for linkage map construction. One immune-related gene, MHC class IIβ, was also used for linkage map construction. Of the microsatellite markers searched, 148 and 197 were mapped on 23 and 27 linkage groups in the female and male linkage maps, respectively, covering approximately 65% of genomes in both sexes. One QTL linked to an RSIVD-resistant trait was found in linkage group 2 of the candidate male in Fam-2014, and the phenotypic variance of the QTL was 31.1%. The QTL was closely linked to MHC class IIβ. Moreover, the QTL observed in Fam-2014 was also significantly linked to an RSIVD-resistant trait in the candidate male of Fam-2015. Our results suggest that the RSIVD-resistant trait in the candidate male was controlled by one major QTL closely linked to the MHC class IIβ gene and could be useful for MAS of red sea bream.

DOI： 10.1007/s10126-017-9779-z

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

To develop a test for assessing the immunomodulatory effects of chemical pollutants on fish, we evaluated the effects of dexamethasone on the natural host-pathogen interaction between common carp (Cyprinus carpio) and Aeromonas salmonicida. Carp were exposed to 1mgL-1 dexamethasone for the entire experimental period. One week after the exposure test started, the exposed fish, as well as unexposed fish, were bath-infected with A. salmonicida. One hundred percent mortality was observed in bacteria-infected fish exposed to dexamethasone, whereas no infection-associated mortality was observed in infected fish in the absence of dexamethasone exposure. In a separate experiment, dexamethasone exposure significantly suppressed hemolytic complement activity in bacteria-infected fish. These results clearly indicate that exposure to a high concentration of dexamethasone suppressed the carp immune system and caused subsequent mortality. Thus, this proposed test method is likely to be useful for evaluating the immunomodulatory effects of chemicals in fish.

DOI： 10.1016/j.marpolbul.2016.12.060

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We investigated the effect of combined exposure to nodavirus infection and TBT on medaka (Oryzias latipes). Medaka larvae were infected by immersion in medium containing nodavirus at titers of 102.5, 103.5, or 104.5 TCID50/mL. Infected fish then were exposed to TBT at 0, 0.17, 0.52, 1.6, or 4.7μg/L. Of the 12 groups exposed to both stressors, the mortalities of 6 (102.5 TCID50/mL+0.52, 1.6, or 4.7μg/L, 103.5 TCID50/mL+4.7μg/L and 104.5 TCID50/mL+1.6 or 4.7μg/L) were significantly higher than that of each TBT control. Specifically, mortality was 46±5.5% in the group exposed to both 102.5 TCID50/mL virus and 0.52μg/L TBT, which represent the lowest observed effective dose and concentration, respectively, among the 6 groups with increased mortalities. Our results suggest that combined exposure to both stressors suppresses antiviral mechanisms in the fish, thus increasing mortality.

DOI： 10.1016/j.marpolbul.2017.02.020

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Pyrene, a member of the polycyclic aromatic hydrocarbons (PAHs), contributes to abnormality in the size of the brain and the swimming behavior of pufferfish (Takifugu niphobles) larvae. We hypothesized that the aryl hydrocarbon receptor (AHR) may mediate pyrene-induced toxic effects because AHR is assumed to be a candidate for the downstream target of PAHs in many cases. To identify the contribution of AHR on developing pufferfish, we performed exposure experiments using β-naphthoflavone, an agonist of AHR. We found that the toxic effects of pyrene and β-naphthoflavone in pufferfish larvae are fundamentally different. Pyrene specifically induced problems in the developing midbrain and in swimming behavior, while β-naphthoflavone affected the heartbeat rate and the size of the yolk. These results suggest that the behavioral and morphological abnormality caused by pyrene exposure is mediated by an AHR-independent pathway. Alternatively, defects caused by pyrene may be attributed to the inhibition of the FGF signal.

DOI： 10.1016/j.marpolbul.2017.02.058

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Linear alkylbenzene sulfonate (LAS) is an anionic surfactant commonly used in cleaning agents such as laundry detergents. Trace amounts of LAS are released into environmental waters after processing in wastewater treatment plants after the use of this chemical. Acute toxicity of LAS has been well-studied using various organisms, and its effects are particularly well known in fish. LAS damages fish gill morphology and induces mucous excretion from these organs. LAS also causes hematological changes. These observations suggest that LAS might induce hypoxic conditions in fish. However, the connections between hypoxia and hematological changes at the cellular and molecular levels remain unknown. Common carp were exposed to LAS at concentrations of 625, 1250, and 2500 μg/L for 96 h. A total of 9-10 fish were sampled at the end of the exposure period for each concentration. For hematological analysis, carp blood was sampled from the caudal vein. Gill tissue was used for real-time PCR analysis to evaluate transcriptional changes of hypoxia-induced genes. The number of normal red blood cells and the number of immature red blood cells were significantly decreased and increased, respectively, in fish exposed to 2500 µg/L LAS. The hypoxic marker genes hypoxia inducible factor 1α, myoglobin 1, and erythropoietin 2 were upregulated in these fish. Our results suggest that LAS decreases erythrocyte numbers and induces hypoxic conditions. In addition, LAS-exposed fish increase production of immature erythrocytes and upregulate myoglobin expression in gills to improve oxygen transport and absorption. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 122-130, 2017.

DOI： 10.1002/tox.22217

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Benzo[c]phenanthrene (BcP) is a highly toxic polycyclic aromatic hydrocarbon (PAHs) found throughout the environment. In fish, it is metabolized to 3-hydroxybenzo[c]phenanthrene (3-OHBcP). In the present study, we observed the effects of 1nM 3-OHBcP on the development and gene expression of Japanese medaka (Oryzias latipes) embryos. Embryos were nanoinjected with the chemical after fertilization. Survival, developmental stage, and heart rate of the embryos were observed, and gene expression differences were quantified by messenger RNA sequencing (mRNA-Seq). The exposure to 1nM 3-OHBcP accelerated the development of medaka embryos on the 1st, 4th, and 6th days post fertilization (dpf), and increased heart rates significantly on the 5th dpf. Physical development differences of exposed medaka embryos were consistent with the gene expression profiles of the mRNA-Seq results for the 3rd dpf, which show that the expression of 780 genes differed significantly between the solvent control and 1nM 3-OHBcP exposure groups. The obvious expression changes in the exposure group were found for genes involved in organ formation (eye, muscle, heart), energy supply (ATPase and ATP synthase), and stress-response (heat shock protein genes). The acceleration of development and increased heart rate, which were consistent with the changes in mRNA expression, suggested that 3-OHBcP affects the development of medaka embryos. The observation on the developmental stages and heart beat, in ovo-nanoinjection and mRNA-Seq may be efficient tools to evaluate the effects of chemicals on embryos.

DOI： 10.1016/j.aquatox.2016.11.020

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The present study evaluated the environmental risks to common carp (Cyprinus carpio) posed by glucocorticoids present in sewage treatment plant (STP) effluent. To gather information on the seasonal variations in glucocorticoid concentration, the authors sampled the effluent of a Japanese STP every other week for 12 mo. Six of 9 selected glucocorticoids were detected in the effluent, with clobetasol propionate and betamethasone 17-valerate detected at the highest concentrations and frequencies. The present study's results indicated that effluent glucocorticoid concentration may depend on water temperature, which is closely related to the removal efficiency of the STP or to seasonal variations in the public's use of glucocorticoids. In a separate experiment, to clarify whether glucocorticoids in environmental water increase susceptibility to bacterial infection in fish, the authors examined the responses to bacterial infection (Aeromonas veronii) of common carp exposed to clobetasol propionate. Clobetasol propionate exposure did not affect bacterial infection-associated mortality. In fish infected with A. veronii but not exposed to clobetasol propionate, head kidney weight and number of leukocytes in the head kidney were significantly increased (p < 0.05), whereas these effects were not observed in infected fish exposed to clobetasol. This suggests that clobetasol propionate alleviated bacterial infection-associated inflammation. Together, these results indicate that susceptibility to bacterial infection in common carp is not affected by exposure to glucocorticoids at environmentally relevant concentrations.

DOI： 10.1002/etc.3136

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：INTER-RESEARCH  

Soft tunic syndrome is a fatal disease in the edible ascidian Halocynthia roretzi, causing serious damage to ascidian aquaculture in Korea and Japan. In diseased individuals, the tunic, an integumentary extracellular matrix of ascidians, softens and eventually tears. This is an infectious disease caused by the kinetoplastid flagellate Azumiobodo hoyamushi. However, the mechanism of tunic softening remains unknown. Because cellulose fibrils are the main component of the tunic, we compared the contents and structures of cellulose in healthy and diseased tunics by means of biochemical quantification and X-ray diffractometry. Unexpectedly, the cellulose contents and structures of cellulose microfibrils were almost the same regardless of the presence or absence of the disease. Therefore, it is unlikely that thinning of the microfibrils occurred in the softened tunic, because digestion should have resulted in decreases in crystallinity index and crystallite size. Moreover, cellulase was not detected in pure cultures of A. hoyamushi in biochemical and expressed sequence tag analyses. These results indicate that cellulose degradation does not occur in the softened tunic.

DOI： 10.3354/dao02913

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

Glucocorticoids (GCs) are widely used as anti-inflammatory drugs. Our previous study demonstrated that several GCs such as cortisol and dexamethasone (Dex) were frequently detected in effluents collected from Japanese sewage treatment plants (STPs) in 2012. In this study, we used the GC-Responsive Chemical-Activated LUciferase gene eXpression (GR-CALUX) assay to elucidate GC receptor (GR) agonistic activities of ten pure synthetic GCs and selected STP effluents in Japan for assessment of the risks associated with the presence of GR agonists. The tested GCs demonstrated dose-dependent agonistic effects in the GR-CALUX assay and their EC50 values were calculated for estimation of relative potencies (REPs) compared to Dex. The GR agonistic potency was in the rank of: clobetasol propionate > clobetasone butyrate > betamethasone 17-valerate > difluprednate > betamethasone 17,21-dipropionate > Dex > betamethasone > 6α-methylprednisolone > prednisolone > cortisol. The GR agonistic activity in STP effluents as measured in Dex-equivalent (Dex-EQ) activities ranged from < 3.0-78 ng L(-1) (median: 29 ng L(-1), n = 50). To evaluate the contribution of the target GCs, theoretical Dex-EQs were calculated by multiplying the concentrations of each GC by its respective REP. Our calculation of Dex-EQ contribution for individual GR agonists indicated that the well-known GCs cortisol and Dex should not be given priority for subsequent in vivo testing, monitoring and removal experiments, but rather the highly potent synthetic GCs clobetasol propionate and betamethasone 17-valerate (REP = 28 and 3.1) as well as other unidentified compounds are important GR agonists in STP effluents in Japan.

DOI： 10.1016/j.scitotenv.2015.05.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Berlin Heidelberg  

A sensitive and comprehensive analytical method for glucocorticoids (GCs) in water samples was developed and applied to effluent of sewage treatment plants (STPs). In the present study, totally 10 natural and synthetic GCs, including cortisol, betamethasone valerate, clobetasol propionate, clobetasone butyrate, difluprednate, betamethasone, dexamethasone, betamethasone dipropionate, methylprednisolone, and prednisolone, were targeted. Analytes were extracted and concentrated using an OASIS HLB solid phase extraction cartridge. Chromatographic separation and quantification were achieved using an ultrahigh performance liquid chromatograph coupled with a tandem mass spectrometer (UHPLC-MS/MS). Method detection limits were 0.05 to 0.89 ng/L, which were 1-2 orders of magnitude more sensitive than in the previous reports. Cortisol was detected in more than half of (27 out of 50) analyzed effluent samples at concentrations in the range of ND-1.36 ng/L, indicating continuous discharge of natural GC via STP effluent. On the other hand, dexamethasone + betamethasone, prednisolone, betamethasone valerate, and clobetasol propionate were detected in 25, 8, 20, and 9 samples among 50 effluent samples, respectively, suggesting not extreme but significant administration of synthetic GCs.

DOI： 10.1007/s11356-015-4626-0

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The dinoflagellate Karenia mikimotoi forms blooms in the coastal waters of temperate regions and occasionally causes massive fish and invertebrate mortality. This study aimed to elucidate the toxic effect of K. mikimotoi on marine organisms by using the genomics approach; RNA-sequence libraries were constructed, and data were analyzed to identify toxin-related genes. Next-generation sequencing produced 153,406 transcript contigs from the axenic culture of K. mikimotoi. BLASTX analysis against all assembled contigs revealed that 208 contigs were polyketide synthase (PKS) sequences. Thus, K. mikimotoi was thought to have several genes encoding PKS metabolites and to likely produce toxin-like polyketide molecules. Of all the sequences, approximately 30 encoded eight PKS genes, which were remarkably similar to those of Karenia brevis. Our phylogenetic analyses showed that these genes belonged to a new group of PKS type-I genes. Phylogenetic and active domain analyses showed that the amino acid sequence of four among eight Karenia PKS genes was not similar to any of the reported PKS genes. These PKS genes might possibly be associated with the synthesis of polyketide toxins produced by Karenia species. Further, a homology search revealed 10 contigs that were similar to a toxin gene responsible for the synthesis of saxitoxin (sxtA) in the toxic dinoflagellate Alexandrium fundyense. These contigs encoded A1-A3 domains of sxtA genes. Thus, this study identified some transcripts in K. mikimotoi that might be associated with several putative toxin-related genes. The findings of this study might help understand the mechanism of toxicity of K. mikimotoi and other dinoflagellates.

DOI： 10.1371/journal.pone.0142731

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Hepatic concentrations of persistent organochlorines (OCs) were determined in the common minke whale (Balaenoptera acutorostrata) from the North Pacific. To investigate the effects of OCs on the transcriptome in the minke whale, the present study constructed a hepatic oligo array of this species where 985 unique oligonucleotides were spotted and further analyzed the relationship between the OC levels and gene expression profiles of liver tissues. The stepwise multiple linear regression analysis identified 32 genes that correlated with hepatic OC levels. The mRNA expression levels of seven cytochrome P450 (CYP) genes, CYP1A1, 1A2, 2C78, 2E1, 3A72, 4A35, and 4V6 showed no clear correlations with the concentration of each OC, suggesting that the accumulated OCs in the liver did not reach levels that could alter CYP expression. Among the genes screened by the custom oligo array analysis, hepatic mRNA expression levels of 16 genes were further measured using quantitative real-time reverse transcription polymerase chain reaction. The mRNA levels of vitamin D-binding protein (DBP) were negatively correlated with non-ortho coplanar polychlorinated biphenyl (PCB) levels. Androgen receptor-associated coregulator 70 (ARA70) expression levels showed a significant positive correlation with concentrations of non-ortho coplanar PCB169. These correlations suggest that coplanar PCB-reduced DBP expression could suppress vitamin D receptor-mediated signaling cascades in peripheral tissues. Alternatively, the suppression of vitamin D receptor signaling cascade could be enhanced through competition with the androgen receptor signaling pathway for ARA70. In addition, a negative correlation between kynureninase and PCB169 levels was also observed, which suggest an enhanced accumulation of an endogenous aryl hydrocarbon receptor agonist, kynurenine in the minke whale population. Further studies are necessary to translate the changes in the transcriptome to toxicological outcomes including the disruption of the nervous and immune systems.

DOI： 10.1016/j.ecoenv.2014.06.028

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Spills of heavy oil (HO) have an adverse effect on marine life. We have demonstrated previously that exposure to HO by fertilized eggs of the pufferfish (Takifugu rubripes) induces neural disruption and behavioral abnormality in early-hatched larvae. Here, two kinds of polycyclic aromatic hydrocarbons, pyrene and phenanthrene, were selected to examine their toxic effects on larval behavior of another pufferfish species (T. niphobles). Larvae exposed to pyrene or phenanthrene exhibited no abnormalities in morphology. However, those exposed to pyrene but not phenanthrene swam in an uncoordinated manner, although their swimming distance and speed were normal. The optic tectum, a part of the midbrain, of pyrene-exposed larvae did not grow to full size. Thus, these findings are indicated that pyrene might be a contributor to the behavioral and neuro-developmental toxicity, although there is no indication that it is the only compound participating in the toxicity of the heavy oil mixture.

DOI： 10.1016/j.marpolbul.2014.04.022

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Uptake and biological effects of synthetic glucocorticoids (GCs) were analyzed using common carp (Cyprinus carpio). Fish were exposed to clobetasol propionate (CP) or clobetasone butyrate (CB) individually or in mixture at 1 μg L(-1) for 21 days. Bioconcentration factor (BCF) of CB was calculated as 100, and BCF of CP was less than 16. No effects were found in fish erythrocyte and leukocyte numbers and serum glucose levels after exposure to the selected GCs. On the other hand, serum concentrations of free amino acids significantly increased in GC-exposed groups. Thus, exposures to synthetic GCs at relatively low concentrations seemed to cause enhancement of protein degradation and subsequent increase of serum free amino acids without a corresponding increase in serum glucose levels, an effect which might be related to partial induction of gluconeogenesis by GC.

DOI： 10.1016/j.marpolbul.2014.01.042

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Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease (LCD). In this study, we investigated the mechanisms of lymphocystis cell (LCC) formation from the viewpoint of gene expression changes in the infected fish. LCC occurrence and virus titers in the experimentally infected Japanese flounder, Paralichthys olivaceus were monitored by visual confirmation and real-time PCR, respectively. The gene expression changes in the fish fin were investigated by microarray experiments. LCCs firstly appeared in the fish at 21 days post infection (dpi). LCD incidence increased with time and reached 92.9% at 62 dpi. LCDV genome was firstly detected from dorsal fins at 14 dpi, and the relative amount of the genome gradually-increased until 56 dpi. Since the occurrence of LCC was approximately synchronized with increasing of the virus genome, virus replication might play important roles for LCC formation. The microarray detected a few gene expression changes until 28 dpi. However, the number of expression changed genes dramatically increased between 28 and 42 dpi in which LCCs formation was active. From the microarray data analyses, apoptosis and cell division related genes were down-regulated, whereas cell fusion and collagen related genes were up-regulated at 42 dpi. Together with the observation of morphological changes of LCCs in previous reports, it is suggested that the following steps are involved in LCC formation: the virus infected cells were (1) inhibited apoptotic death and (2) cell division before enlargement, (3) hypertrophied by cell fusion, and (4) surrounded by a hyaline capsule associated with the alteration of collagen fibers.

DOI： 10.1016/j.fsi.2014.03.028

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

PURPOSE: The immunotoxicities of oil and its components on fish immunities have been investigated, but there is little literature on the recovery of the fish from the immune suppression. Therefore, the recovery of Japanese flounder Paralichthys olivaceus from an immunosuppressive effect due to heavy oil (HO) exposure was investigated in this study. METHODS: Fish were exposed to HO at a concentration of 0.385 g/L for 2 days, while control fish received no exposure. Seven fish were sampled at 0, 3, 7, and 14 days post-exposure. The respiratory rate was measured everyday as an indicator of the acute effect of HO exposure. Fish serum was collected and used for antibacterial activity assay against Edwardsiella tarda. Expression changes of respiratory and immune-related genes were evaluated by real-time PCR. RESULTS AND DISCUSSION: The respiratory rate was significantly increased in the HO-exposed group until 4 days post-exposure. A respiratory-related gene, β-hemoglobin, was also significantly downregulated in the spleen both at 0 and 7 days post-exposure and kidney at 3 days post-exposure in HO-exposed fish. Immunotoxicity, including suppression of antibacterial activities and downregulation of the IgM gene, was observed in HO-exposed fish until 3 days post-exposure, but not after that time. From these results, we conclude that the fish likely return to normal status around 1 week.

DOI： 10.1007/s11356-012-0737-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Heavy oil (HO) pollution is one of the most important environmental issues globally. However, little is known about the immunotoxicity of HO in fish. We therefore investigated the effects of HO exposure on immunocompetence and expression of immune-related genes in Japanese flounder, Paralichthys olivaceus. To test immunocompetency, serum collected from the fish was mixed with Edwardsiella tarda, plated, and the resultant numbers of bacterial colonies were counted. Plates with serum from HO-exposed fish (5 d postexposure [dpe]) had significantly higher numbers of colonies than those of the untreated control group, suggesting that HO exposure suppresses immunocompetency. Downregulation of the immunoglobulin light chain (IgM) gene in HO-exposed fish at 5 dpe was detected by real-time polymerase chain reaction. These results suggest that IgM-mediated immunity is suppressed by HO exposure. We measured polycyclic aromatic hydrocarbon (PAH) concentrations in the liver of the fish. Low molecular weight PAHs were found to be taken up at high concentrations in fish liver; therefore, they are likely the cause of immune suppression in the fish.

DOI： 10.1002/etc.1743

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We have previously indicated that accumulation of chlorinated dioxins and related compounds (DRCs) induced cytochrome P450 (CYP) 1A1, 1A2 and 1B1 isozymes in the liver of wild Baikal seals (Pusa sibirica). Here we attempt to assess the potential effects of DRCs triggered by the induction of these CYP1 isozymes in this species, using an integrative approach, combining gene expression monitoring and biochemical assays. To screen genes that may potentially respond to the exposure of DRCs, we constructed a custom cDNA oligo array that can target mRNAs in Baikal seals, and monitored hepatic mRNA expression levels in the wild population. Correlation analyses between the hepatic total 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs) and mRNA levels supported our previous findings that high accumulation of DRCs induces the transcription of CYP1A1, CYP1A2 and CYP1B1 genes. In addition, our integrative assessment indicated that the chronic exposure to DRCs may alter the hepatic transcript levels of genes related to oxidative stress, Fe ion homeostasis, and inflammatory responses. The expression levels of CYP1A2 showed significant positive correlations with levels of malondialdehyde, a biomarker of lipid peroxidation, and of etheno-dA, a DNA adduct, suggesting that the lipid peroxidation may be enhanced through the production of reactive oxygen species (ROS) triggered by CYP1A2 induction. Moreover, there was a positive correlation between heme oxygenase activities and malondialdehyde levels, suggesting the prompted heme degradation by ROS. Fetuin-A levels, which are suppressed by inflammation, showed a significant negative correlation with TEQ levels, and hepcidin levels, which are conversely increased by inflammation, had significant positive correlations with malondialdehyde and etheno-dA levels, implying the progression of inflammation by DRC-induced oxidative stress. Taken together, we propose here that wild Baikal seals may suffer from effects of chronic exposure to DRCs on the induction of CYP1 isozymes, followed by increased oxidative stress, heme degradation and inflammation.

DOI： 10.1016/j.aquatox.2011.05.007

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PURPOSE: We evaluate malathion toxicity to Japanese medaka (Oryzias latipes) juveniles by using a mass spectrometry combined with gas chromatography (GC/MS) metabolomics approach. METHODS: Medaka were exposed to low (L) and high (H) concentrations (nominally 20 and 2,000 μg/L, respectively) of water-borne malathion. Metabolites were extracted from the fish, derivatized, and analyzed by GC/MS. Identified metabolites were subjected to one-way analysis of variance and principal component analysis (PCA). We examined the variations in the amounts of the metabolites during the exposure period. RESULTS AND DISCUSSION: At 24 h, control, L, and H groups were separated along PC1, suggesting that the effects of malathion depended on exposure concentration. The PCA results at 96 h suggest that the metabolite profiles variations of the L and H groups differed, and thus that the effects of malathion in groups differed. At 24 h, the amounts of amino acids in both exposed groups were lower than the control group amounts, perhaps owing to accelerated protein synthesis. At 96 h, the amounts of almost all the amino acids increased in the L group but decreased in the H group relative to the control group amounts, suggesting the proteolysis occurred in the L group while protein synthesis continued in the H group, that the high malathion exposure affected the fish. In addition, at 96 h, gluconeogenesis may have been induced in the L group but not in H group. CONCLUSIONS: Malathion exposure may have altered the balance between protein synthesis and degradation and induced gluconeogenesis in medaka. Our results suggest that metabolomics will be useful for comprehensive evaluation of toxicity.

DOI： 10.1007/s11356-012-0834-z

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BACKGROUND AND OBJECTIVE: Spills of heavy oil (HO) over the oceans have been proven to have an adverse effect on marine life. It has been hypothesized that exposure of early larvae of sinking eggs to HO leads largely to normal morphology, whereas abnormal organization of the developing neural scaffold is likely to be found. HO-induced disruption of the nervous system, which controls animal behavior, may in turn cause abnormalities in the swimming behavior of hatched larvae. To clarify the toxicological effects of HO, we performed exposure experiments and morphological and behavioral analyses in pufferfish (Takifugu rubripes) larvae. EXPERIMENTAL PROCEDURE: Fertilized eggs of pufferfish were exposed to 50 mg/L of HO for 8 days and transferred to fresh seawater before hatching. The hatched larvae were observed for their swimming behavior, morphological appearance, and construction of muscles and nervous system. RESULTS AND DISCUSSION: In HO-exposed larvae, we did not detect any anomaly of body morphology. However, they showed an abnormal swimming pattern and disorganized midbrain, a higher center controlling movement. Our results suggest that HO-exposed fishes suffer developmental disorder of the brain that triggers an abnormal swimming behavior and that HO may be selectively toxic to the brain and cause physical disability throughout the life span of these fishes.

DOI： 10.1007/s11356-012-0833-0

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The full-length cDNA sequence of tributyltin-binding protein type 1 in Japanese medaka (Oryzias latipes) (Olat.TBT-bp1) was determined by means of rapid amplification of cDNA ends (RACE) of liver tissue. Analysis of the structure of the gene encoding Olat.TBT-bp1 revealed that the exonintron organization of this gene corresponds to that of the genes encoding lipocalin superfamily proteins, suggesting that Olat.TBT-bp1 can be categorized as a member of the lipocalin superfamily, which may play an important role in transportation, detoxification, and excretion of xenobiotic compounds. Reverse transcription - PCR revealed that Olat.TBT-bp1 was expressed mainly in the liver, and upregulation of its expression was detected 1, 2, and 4 weeks post hatching. Relative expression of the Olat.TBT-bp1 gene was significantly downregulated, compared with that in the solvent control, by exposure to tributyltin at 0.01 mg/l or triclosan at 1.7 mg/l. Further studies on Olat.TBT-bp1 expression in conjunction with other biochemical and physiological toxicities in response to chemical exposures are needed to increase our understanding and information of TBT-bps mechanisms and as molecular biomarkers of chemical exposures. The role of Olat.TBT-bp1 in xenobiotic detoxification and/or excretion needs more investigations.

DOI： 10.2108/zsj.28.281

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In this study, individual Japanese flounder were intraperitoneally injected with 2 μg tributyltin-d27 (TBT-d27) fish⁻¹. Blood samples were collected on day 7 after injection. TBT-binding protein types 1 and 2 (TBT-bp1, -bp2) in the blood serum were quantified by western blotting analysis. As a result, the concentration of TBT-bp2 in TBT-d27 treated group increased to 220% of that in the solvent control, whereas the TBT-bp1 concentration decreased to 65% of that in the solvent control. Additionally, a positive relationship between the concentrations of TBT-bp2 and TBT was observed in blood sera of wild and cultured flounder. We suggest that TBT-bp2 is produced in response to TBT exposure and may play an important role in fish physiology.

DOI： 10.1016/j.marpolbul.2010.12.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Heavy oil (HO) on the sea surface penetrates into fish eggs and prevents the normal morphogenesis. To identify the toxicological effects of HO in the context of the egg types, we performed exposure experiments using floating eggs and sinking eggs. In the course of development, HO-exposed embryos of floating eggs showed abnormal morphology, whereas early larva of the sinking eggs had almost normal morphology. However, the developing peripheral nervous system of sinking eggs showed abnormal projections. These findings suggest that HO exposed fishes have problems in the developing neurons, although they have no morphological malformations. Through these observations, we conclude that HO is strongly toxic to floating eggs in the morphogenesis, and also affect the neuron development in both floating and sinking eggs.

DOI： 10.1016/j.marpolbul.2011.04.018

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The relationship between chemical exposure and disease outbreak in fish has not been fully defined due to the limitations of experimental systems (model fish and pathogens). Therefore, we constructed a system using the Japanese flounder, Paralichthys olivaceus, and viral haemorrhagic septicemia virus (VHSV), and evaluated it by heavy oil (HO) exposure. The fish were exposed to HO at 0.3, 0.03, 0.003, and 0 g/L following VHSV infection at doses of 10(2.5) or 10(3.5) tissue culture infectious dose (TCID)50/fish. As a result, groups given the dual stressors showed more than 90% mortality. Although VHSV infection at 10(2.5) and 10(3.5) TCID50/fish without HO exposure also induced high mortality, at 68.8% and 81.3%, respectively, HO exposure induced faster and higher mortality in the virus carrier fish, indicating that chemical stressors raise the risk of disease outbreak in fish. The experimental system established in this study could be useful for chemical risk assessment.

DOI： 10.1016/j.marpolbul.2011.01.020

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Contamination status of brominated flame retardants (BFRs) and persistent organic pollutants (POPs) in blubber of finless porpoises (Neophocaena phocaenoides) stranded along the coasts of Seto Inland Sea and Omura Bay in Japan were investigated. Levels of PCBs, DDTs and CHLs were significantly higher than those of HCHs, HCB, PBDEs and HBCDs. Concentrations of PBDEs and HBCDs, as well as organochlorine compounds in males increased with body length (p<0.05). Among 14 PBDE congeners analyzed, BDE-47 was the predominant, which is similar to those generally reported in biota. PBDEs, HBCDs and PCBs showed no obvious temporal trend in concentrations during the study period, suggesting continuous environmental release of these chemicals. On the other hand, levels of DDT, CHLs and HCHs have decreased. Concentrations of PCBs in liver trematode infected individuals were significantly higher than those in not infected individuals, implying there could be a relationship between contaminant levels and parasitic infection.

DOI： 10.1016/j.marpolbul.2011.01.014

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The effects of polychlorinated biphenyls (PCBs) on liver function and their differences between sexes were analyzed in Japanese medaka (Oryzias latipes) exposed to PCB126 or Kanechlor-400 (KC-400) using microarray. PCB exposure induced vitellogenin 1 expression in female medaka while suppressing choriogenin genes, which suggests that the effects of PCBs on estrogen-responsive genes do not occur directly through an estrogen receptor-mediated pathway. Reduction of androgen receptor alpha expression was also observed, and the gene expression pattern in PCB-exposed males changed to become more similar to that of females. Furthermore, changes in glycolysis-related genes indicate that PCB exposure might enhance glucose production via gluconeogenesis in the liver of medaka. Taken together, our results suggest that PCBs disrupt the endocrine system, especially androgen function, and may have the potential to cause demasculinizing effects. Additionally, induction of gluconeogenesis might be a response to maintain glucose levels consumed as a result of PCB exposures.

DOI： 10.1016/j.marpolbul.2011.01.015

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It has been well known that oil spills cause serious problems in the aquatic organisms. In particular, some species of teleosts, which develop on the sea surface thought to be affected by heavy oil (HO). During the embryogenesis, the nervous system is constructed. Therefore, it is important to study the toxicological effects of HO on the developing neurons. We exposed HO to eggs of Japanese flounder (Paralichthys olivaceus) and investigated the neural disorder. In larvae exposed by HO at the concentration of 8.75 mg/L, the facial and lateral line nerves partially entered into the incorrect region and the bundle was defasciculated. Furthermore, in the HO-exposed larvae, Sema3A, a kind of axon guidance molecule, was broadly expressed in second pharyngeal arch, a target region of facial nerve. Taken together, we suggested the possibility that the abnormal expression of Sema3A affected by HO exposure causes disruption of facial nerve scaffolding.

DOI： 10.1016/j.marpolbul.2011.01.022

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Here we propose a novel microarray data analysis method applicable to evaluation of the chemical effects on wild animals. First, we analyzed correlations between log-transformed hepatic 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalent (TEQ) levels and probe signals detected in wild cormorant liver to screen contaminant-responsive genes. Second, principal component analysis (PCA) was conducted using the screened probes. Third, these probes were divided into two groups based on our PCA result. Finally, we calculated Euclidian distance of signals, which is equivalent to variance of gene expressions, in each probe set, and analyzed the relationship between log-transformed hepatic TEQ levels and Euclidian distances. A probe set whereby the calculated Euclidian distance was positively correlated with TEQ levels, could indicate genes that were directly affected by dioxins or other persistent organic pollutants (POPs), hence they can be used as biomarkers. By contrast, there were a number of probes whereby the Euclidian distance was negatively correlated with TEQ levels. In the latter probe group, the smaller Euclidian distances in highly contaminated individuals could point to changes in physiological activities of wild cormorants. Therefore, our microarray data analysis method will provide new insights into POPs-responsive genes in field-collected samples for toxicogenomics studies.

DOI： 10.1016/j.scitotenv.2010.08.065

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Organotins (OTs) are globally ubiquitous contaminants. Contamination of OTs, particularly butyltins (BTs), has been suspected to cause immunosuppressive effects leading to subsequent infectious diseases or opportunistic infection by pathogens in marine mammals. In the present study, we collected individuals from five different populations of finless porpoises (Neophocaena phocaenoides) from coastal areas of Japan (Seto Inland Sea, Ise Bay, Omura Bay, and Pacific coast) and Hong Kong, and measured OT concentrations in their livers. In addition, infection status of lung nematodes and liver trematodes was also observed to analyze the relationship between OT contamination and parasitic infection in finless porpoises. Hepatic OT concentrations were highest in the Hong Kong population, while levels in the Japanese populations were equivalent to those of other cetaceans from different locations. OT levels in Japan have been decreasing in the past few decades, while OT contamination has shown little improvement in Hong Kong. Observation of parasite infection in Seto Inland Sea porpoises revealed that 16 and 11 out of 22 individuals were infected by lung nematodes and liver trematodes, respectively. Additionally, a significant association between BT levels and parasitic infection status of lung nematodes was detected. Thus, the present study suggests that BTs could be a factor affecting parasitic infection, especially the presence or severity of lung nematodes in finless porpoises. Since chemical exposure may alter the susceptibility of organisms to infectious diseases, the interaction of chemical contamination with infectious diseases needs to be investigated in greater depth to understand the risk of population decline due to these factors in marine mammals.

DOI： 10.1016/j.scitotenv.2009.08.043

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer Japan  

In recent years, discharges of anthropogenic chemicals to the environment have been increasing in association with industrial development. These chemicals and their degradation products are released to the environment, discharged into water, and may ultimately contaminate aquatic organisms. Polychlorinated biphenyls (PCBs) and tributyltin (TBT) are particularly ubiquitous pollutants. © 2009 Springer Japan.

DOI： 10.1007/978-4-431-85709-9_11

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DOI： 10.1111/j.1365-2761.2009.01089.x

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Authorship：Lead author,　Corresponding author   Language：Japanese  

DOI： 10.11403/jset.11.75

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Contamination levels of perfluorinated compounds (PFCs), including perfluorooctanesulfonate (PFOS), perfluorononanoic acid (PFNA), perfluorooctane sulfonamide (PFOSA), perfluorohexanesulfonate (PFHS), and perfluorooctanoic acid (PFOA), were determined in the livers of wild common cormorants (Phalacrocorax carbo) from Lake Biwa, Japan. Potential effects of PFCs alone and combined effects by complex mixture of PFCs and dioxins and related compounds (DRCs) were also assessed by gene expression profiling using a cormorant oligo array. Perfluorooctanesulfonate, PFNA, and PFOSA were detected in almost all liver samples analyzed, while concentrations of PFHS and PFOA were below the limit of quantification. The microarray data analyses revealed that hepatic PFC levels were correlated with the expression of 74 genes. Real-time reverse-transcript polymerase chain reaction data demonstrated that PFOS concentration was positively correlated with mRNA levels of glutathione peroxidase 1 and glutathione S-transferase alpha 3 and negatively correlated with levels of heat shock 70-kDa protein 8 and tumor rejection antigen 1 mRNAs. These results suggest the induction of antioxidant enzymes in response to oxidative stress caused by PFCs and the suppression of molecular chaperones, leading to reduction in protein stability. Moreover, multiple regression analyses identified seven significant models in which certain genes showed expression levels altered by accumulation of PFCs and DRCs. The regression models explained associations with cytochrome P450 1A mRNA and protein expression levels, and its catalytic activity, ethoxyresorufin-O-deethylase of both PFNA and the 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalent levels. Thus, the regression models suggested the potential of PFCs to enhance toxicities of DRCs. Since mixture toxicity is an urgent issue, further study is required to understand the effects of mixtures of PFCs and DRCs in wild cormorants.

DOI： 10.1897/07-614.1

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This paper describes the development and validation of a PCR array for studying chemical-induced effects on gene expression of selected endocrine pathways along the hypothalamic-pituitary-gonadal (HPG) axis of the small, oviparous fish, the Japanese medaka (Oryzias latipes). The Japanese medaka HPG-PCR array combines the quantitative performance of SYBR Green-based real-time PCR with the multiple gene profiling capabilities of a microarray to examine expression profiles of 36 genes associated with endocrine pathways in brain, liver and gonad. The performance of the Japanese medaka HPG-PCR array was evaluated by examining effects of two model compounds, the synthetic estrogen, 17alpha-ethinylestradiol (EE2) and the anabolic androgen, 17beta-trenbolone (TRB) on the HPG axis of the Japanese medaka. Four-month-old medaka was exposed to three concentrations of EE2 (5, 50, 500 ng/L) or TRB (50, 500, 5000 ng/L) for 7d in a static renewal exposure system. A pathway-based approach was implemented to analyze and visualize concentration-dependent mRNA expression in the HPG axis of Japanese medaka. The compensatory response to EE2 exposure included the down-regulation of male brain GnRH RI and testicular CYP17. The down-regulation of AR-alpha expression in brain of EE2-exposed males was associated with suppression of male sexual behavior. Compensatory responses to TRB in the female HPG axis included up-regulation of brain GnRH RII and ovary steroidogenic CYP19A. Overall, the results suggested that the Japanese medaka HPG-PCR array has potential not only as a screening tool of potential endocrine-disrupting chemicals but also in elucidating mechanisms of action.

DOI： 10.1016/j.aquatox.2008.04.009

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Language：Japanese  

DOI： 10.15017/9849

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Polychlorinated biphenyls (PCBs) are known as neurotoxic chemicals and possibly alter animal behavior. We previously reported that PCB-exposure induced abnormal schooling behavior in Japanese medaka (Oryzias latipes). This abnormal behavior might be caused by the functional alteration of central or terminal nervous system. To understand the mechanism(s) of behavioral change by PCB-exposure, we analyzed the gene expression profiles in the brain of medaka exposed to 3,3',4,4',5-pentachlorobiphenyl (PCB126) or a PCB mixture (Kanechlor-400: KC-400) using a cDNA microarray that we constructed. Twelve FLF-II strain medaka (six individuals per treatment) were dietary exposed to PCB126 (0.01 microg/g b.w./day) or KC-400 (1 microg/g b.w./day) for three weeks. For the control, six fish were fed a control diet. After the exposure period, fish were dissected, and the brain samples were collected. The samples from control fish were pooled and used as a common reference in the microarray experiment. Microarray data were normalized by the LOWESS method, and we screened the genes whose expression levels were altered more than 1.5-fold. Gene expression profiling showed 97 down-regulated and 379 up-regulated genes in the brain of medaka exposed to PCB126. KC-400 exposure suppressed 15 genes and induced 266 genes in medaka brain. Among these genes, the expression levels of 7 and 188 genes were commonly down- or up-regulated, respectively in both treatment groups. On the other hand, 31 gene expressions were significantly different between PCB126 and KC-400 treatment groups, and three out of 31 genes were received opposite effects. In addition, the microarray data showed that thyroid hormone-responsive genes were up-regulated by PCB-exposure, which may imply that PCBs or their metabolites mimic thyroid hormone effects in the brain of PCB-exposed medaka.

DOI： 10.1016/j.marpolbul.2008.02.020

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Heavy oil contamination is one of the most important environmental issues. Toxicities of polycyclic aromatic hydrocarbons (PAHs), including immune toxicities, are well characterized, however, the immune toxic effects of heavy oil, as a complex mixture of PAHs, have not been investigated. In the present study, we selected Japanese flounder (Paralichthys olivaceus) as a model organism, and observed alteration of immune function by the exposure to heavy oil. To analyze the expression profiles of immune system-related genes, we selected 309 cDNAs from our flounder EST library, and spotted them on a glass slide. Using this cDNA array, alteration of gene expression profiles was analyzed in the kidneys of flounders exposed to heavy oil. Six Japanese flounders (mean body weight: 197 g) were acclimated to laboratory conditions at 19-20 degrees C. Three fish were exposed to heavy oil C (bunker C) at a concentration of 3.8 g/L for 3 days, and the others were kept in seawater without heavy oil and used as the control. After the exposure period, the fish were transferred into control seawater and maintained for 4 days, and then they were dissected and their kidneys were removed. Total RNA was extracted from the kidney samples to use in gene expression analyses. The microarray detected alteration of immune system-related genes in the kidneys of heavy oil-exposed flounders, including down-regulation of immunoglobulin light chain, CD45, major histocompatibility complex class II antigens and macrophage colony-stimulating factor precursor, and up-regulation of interleukin-8 and lysozyme. These results suggest that pathogen resistance may be weakened in heavy oil-exposed fish, causing a subsequent bacterial infection, and then proinflammatory genes may be induced as a defensive response against the infection. Additionally, we found candidate genes for use as biomarkers of heavy oil exposure, such as N-myc downstream regulated gene 1 and heat shock cognate 71 kDa proteins.

DOI： 10.1016/j.marpolbul.2008.02.021

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

As basic research for the effect of heavy oil on the fish immune system, in this study, the number of leukocyte was counted in Japanese flounder Paralichthys olivaceus, after exposure to heavy oil at a concentration of 30 g/8L for 3 days. To compare the numbers of bacteria in the skin mucus between oil-exposed and control fish, viable bacteria were enumerated by counting colony forming unit (CFU). Compared with 5.79+/-1.88 x 10(7)leukocytes/mL in the controls, the exposed fish demonstrated higher counts, averaging 1.45+/-0.45 x 10(8)cells/mL. The bacterial numbers of control fish were 4.27+/-3.68 x 10(4)CFU/g, whereas they were 4.58+/-1.63 x 10(5)CFU/g in the exposed fish. The results suggest that immune suppression of the fish occurred due to heavy oil stressor, and bacteria could invade in the mucus, resulting in the increasing leukocyte number to prevent infectious disease.

DOI： 10.1016/j.marpolbul.2008.01.024

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

It is well known that heavy oil (HO) on the sea surface causes serious problems in the aquatic environment. In particular, some species of teleosts which develop on the sea surface are thought to be affected by the HO which flows out from tankers or coastal industry. However, the toxicological effects of HO are not fully understood. We performed exposure experiments using the Pleuronectiformean fish, spotted halibut (Verasper variegatus), which is an important fishery resource in Japan. In course of the development, HO-exposed embryos showed remarkable delay in developmental processes including somite formation. We further observed abnormal development of the head morphology. Notably, treated embryos had relatively small eyes and craniofacial structures. These findings strongly suggest that HO seriously affects the cell proliferation and differentiation of the embryo. In addition, HO-exposed embryos showed abnormal neuronal development. We also performed the exposure in the larval stage. Treatment of post-hatching larvae with HO resulted in significantly greater mortality compared with controls. Through these observations, we finally conclude that HO is strongly toxic to halibut in their early life stages.

DOI： 10.1016/j.marpolbul.2008.02.043

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

We measured the concentrations of monoamines in the brain of Japanese medaka, Oryzias latipes, exposed to tributyltin (TBT). Fish were exposed to 0, 1, 5, 25, or 125 microg g(-1) of TBT via the diet for 21 days. After the administration period, six males and six females in each treatment group were dissected and their brains were collected. The following monoamines were analyzed: dopamine (DA), norepinephrine (NE), epinephrine (E), and 5-hydroxytryptamine (5-HT). The metabolites of DA, 3,4-dihydroxyphenylacetic acid and homovanilic acid, and the metabolite of 5-HT, 5-hydroxyindolacetic acid were also analyzed. The concentration of DA in the brain of male medaka and the concentrations of 5-HT and NE in the female brains were significantly decreased by exposure to 125 microg TBT g(-1). The concentrations of 5-HT and NE in males and of DA in females were slightly decreased by 125 micrg g(-1) of TBT, although the differences were not statistically significant. The present study demonstrates that TBT alters monoamine concentrations in the brain of medaka.

DOI： 10.1002/tox.20233

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

We examined the effect of tributyltin (TBT) on reproduction in the Japanese whiting, Sillago japonica. Mature fish were placed in indoor 500-L polyethylene tanks (five males and three females per tank) with a flow-through system and received dietary exposure to tributyltin oxide at concentrations of 2, 20, or 200 microg/g for 30 days during the active spawning period. Eggs spawned from the fish were collected daily, and the floating egg rate, larval deformity, hatchability, and viable hatch were monitored. TBT concentration in eggs of 200-microg/g group ranged from 85.0 to 159.6 ng/g in the evaluation period (days 5-30). In this period, the floating egg rate (83.2%), viable hatchability (82.2%), and total number of viable larvae (422,000 larvae per 100 g of female) were all significantly decreased in the 200-microg/g group compared with the control group (93.0%, 91.9%, and 709,000 larvae, respectively). The rate of deformity (2.6%) in the 200-microg/g group was about three times that in the control group (0.8%), although this difference was not significant. From these results, the lowest observed effect concentration of TBT in eggs on reproduction in Japanese whiting was estimated to be less than 159.6 ng/g-eggs.

DOI： 10.1016/j.marenvres.2006.04.030

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

To establish a monitoring system for gene expression profiles related to chemical contamination in wild common cormorants (Phalacrocorax carbo), the present study constructed an oligo array designed from expressed sequence tag (EST) sequences of the cormorant liver, where 1061 unique oligonucleotides were spotted. Common cormorants were collected from Lake Biwa, Japan in May 2001 and 2002. With the use of this oligo array, gene expression profiles in the liver of individual specimens were evaluated. To determine the expression patterns of genes altered by environmental contaminants, relationships between concentrations of persistent organochlorines including polychlorinated dibenzo-p-dioxins, furans, polychlorinated biphenyls, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane and its metabolites (DDTs), hexachlorocyclohexane isomers (HCHs), chlordane compounds (CHLs), butyltins, and bisphenol A (BPA) and expression levels of each gene in the cormorant liver were examined using stepwise multiple regression analysis. The reliability of data obtained by the oligo array was further confirmed by quantifying the expression levels of certain genes using real-time RT-PCR. The 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalent (TEQ) level was positively correlated with both cytochrome P4501A4 and 1A5 gene expression. In addition, the mRNA level of an antioxidant enzyme, Cu/Zn superoxide dismutase, was negatively correlated with hepatic total TEQ. Other antioxidant enzymes, glutathione peroxidase 3 and glutathione S-transferase class mu, were negatively correlated with HCHs and BPA levels, respectively. The mRNA expression level of a nonenzymatic antioxidant, haptoglobin, was negatively but not significantly correlated with CHLs. These results led to a hypothesis that wild cormorant population may suffer from oxidative stress due to chemically induced formation of reactive oxygen species and subsequent reduction of antioxidant resistance. Thus, the cormorant oligo array may be a useful monitoring tool to identify specific gene expression profiles altered by various environmental contaminants. Although further research is required to clarify a definitive cause-and-effect relationship, the current study provides valuable information on contaminant-responsive genes to predict potential effects on wildlife in a real environment.

DOI： 10.1021/es051386m

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SETAC  

The effects of polychlorinated biphenyls (PCBs; Kanechlor 400) on the schooling behavior of Japanese medaka (Oryzias latipes) were tested after feeding with various concentrations of PCBs (0, 1, 5, 25, and 125 microg/g). To test schooling, three PCB-exposed medaka and three untreated fish were placed in one chamber, and their swimming trajectories were recorded. Swimming velocity, turning angle, and nearest-neighbor distance (NND) were analyzed individually, and the polarization and expanse of each schooling group were analyzed. Fractal dimension analysis was performed for trajectory, swimming velocity, and turning angle. Six behaviors (school formed by six swimming medaka, school formed by six stationary medaka, school formed by three to five swimming medaka, school formed by three to five stationary medaka, swimming without schooling, and stationary without schooling) were quantified with time. Behavioral parameters were the same for PCB-exposed and unexposed medaka in the same schooling group. Swimming velocity decreased in a dose-dependent manner, and NND in the group exposed to 25 microg/g of PCBs was significantly longer (126%) than that in the controls. The fractal dimensions of trajectory and turning angle increased significantly in the highest PCB-exposure group, indicating that schools containing PCB-exposed individuals frequently changed direction. The PCBs shortened the time of school formed by six swimming fish, which was 23.7% of the control value in the group exposed to 25 microg/g of PCBs. The frequency of collisions between individuals was significantly greater in the highest-exposure group (31.3) than in the controls (13.3). Thus, PCB exposure influences the behavior of unexposed fish in the same school, consequently affecting schooling behavior.

DOI： 10.1897/04-518R2.1

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

The present study examined the effects of tributyltin (TBT), polychlorinated biphenyls (PCBs), and mixtures of both chemicals on reproduction in Japanese medaka, Oryzias latipes. For 21 d we gave groups of medaka freeze-dried brine shrimp flakes contaminated with a mixture of either 0, 1, 5, or 25 microg TBT g(-1) plus 0 or 25 .micro PCBs g(-1). We measured the fecundity and fertility of the parent fish and assessed the deformity, hatchability, time-to-hatching, and swim-up failure rate of the next generation. Fertilization success in the third week of the administration period was significantly decreased by administration of 25 microg TBT g(-1) (77%) compared with the control group (87%). Both TBT and PCBs were transferred maternally into the eggs of the next generation, causing early life-stage toxicity. Administration of 1 microg TBT g (-1) was not toxic to embryological development, but abnormal eye development (i.e., small eyes or no eyes) occurred when TBT at the same concentration was mixed with PCBs (6.4%). Administration of TBT alone significantly decreased hatchability and increased swim-up failure, and administration of PCBs alone significantly increased time-to-hatching. Statistical analysis by two-way analysis of variance detected an interaction between TBT and PCBs in these three parameters. TBT induces abnormal development of the eyes, reduced hatchability, and increased swim-up failure, whereas PCBs delay time-to-hatching. Administration of mixtures of TBT and PCBs has more adverse effects on the developmental stage of medaka than does that of each chemical alone.

DOI： 10.1897/04-157r.1

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

We evaluated the effects of tributyltin (TBT), polychlorinated biphenyls (PCBs), and a mixture of TBT and PCBs on reproduction, gonadal histology, and sexual behavior in Japanese medaka (Oryzias latipes). We administered TBT (1 microg g(-1) body weight [b.w.] daily), PCBs (1 microg g(-1) b.w. daily), or both to medaka for 3 weeks. We assessed reproductive success during week 3 and the sexual behavior of male medaka after the exposure period. Fertilization success was significantly decreased in groups that received TBT. The profiles of fertility developed a bimodal distribution in the TBT and TBT+PCBs groups, and the numbers of males that performed "following" and "dancing" were significantly decreased in the TBT+PCBs group. The frequency of "dancing" also decreased after treatment with TBT, whereas fish that received PCBs only showed no difference in sexual behavior. Testes that had oocytes showed normal spermatogenesis, even though two of the five males treated with TBT+PCBs had some indication of testis-ova, which did not occur in any other group. Our results indicate that TBT, but not PCBs, affects sexual behavior and reproduction in medaka.

DOI： 10.1016/j.chemosphere.2003.11.050

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：KYUSHU UNIV, FACULTY AGRICULTURAL PUBLICATIONS  

We examined the general behavior of male Japanese medaka, Oryzias latipes, after exposed to tributyltin (TBT), polychlorinated biphenyls (PCBs), or a mixture of these chemicals at a concentration of 1 mug/g body weight daily for 3 weeks. We analyzed swimming velocity as an indicator of acute toxicity and counted the frequencies of straight swimming and swimming in circles. We also calculated the entropy of the positions of the fish within the experimental chamber. Neither TBT nor PCBs nor their mixture affected the swimming velocity, suggesting that there were no acute toxic effects. However, PCBs exposure increased the frequencies of both straight and circular swimming, suggesting hyperactivity. TBT increased the positional entropy value. Both TBT and PCBs affect the general behavior of mature male medaka, although exposure to the 2 chemicals together has no significant additive behavioral effect.

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Language：English  

DOI： 10.11403/jset.7.123

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Language：English   Publishing type：Research paper (scientific journal)  

The distribution of tributyltin (TBT) and its metabolites, dibutyltin (DBT) and monobutyltin (MBT), was examined in the liver, brain and fat tissues in a two-generation reproductive toxicity study of tributyltin chloride (TBTCl) in rats using dietary supplementation at concentrations of 5, 25 and 125 ppm. In the liver, irrespective of TBTCl dietary concentration, gender or generation, the highest concentration of metabolite was consistently MBT, followed by DBT, and then TBT. In contrast, TBT was consistently present at the highest concentration in the brain, nearly always followed by DBT and MBT. In fat tissues, the concentrations of the three butyltin compounds showed similar relationships to those observed in the brain, although the concentrations were much lower. In the liver, the concentration of TBT was higher in females, and those of DBT and MBT were higher in males. Factorial ANOVA also suggested the effect of gender on the concentrations of the three butyltin compounds in the liver. The results of this study suggest tissue-dependent distribution of TBT, DBT and MBT and gender-dependent distribution of the three metabolites in the liver of rats.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Mating pairs of Japanese medaka (Oryzias latipes) received 17beta-estradiol (E2; dose, 3 and 30 microg/g body weight daily) in their diet and E2-free diet (control) for two weeks. The effect on reproductive success was assessed in the mating pairs, and sexual and nonsexual behaviors in the male were quantified. The fecundities of the E2-treated groups were reduced significantly (p < 0.05) compared with those of control, and the sexual behaviors (following, dancing, floating, and crossing) of male fish in response to prostaglandin F2alpha (PG)-injected E2-unexposed females were suppressed dramatically. While the fertility of spawned eggs and the frequencies of the nonsexual behaviors of resting and swimming in a straight line performed by male were unchanged in E2-exposed groups. Therefore, sexual behavior in male may be linked with reproductive success in mating pair of madaka exposed to E2. This assay for sexual behavior using PG may provide a useful tool for assessing the effect of endocrine-disrupting chemicals.

DOI： 10.1016/S0045-6535(02)00494-0

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：AMERICAN SOCIETY TESTING AND MATERIALS  

After fertilization, blue crab eggs are embedded in a "sponge" which is attached to the female abdomen during embryo development. Embryos after 9 stages in the egg sac hatch into a swimming zoea stage (stage 10). We have developed a bioassay where embryo development is monitored in culture plates with and without toxicants in the water. Toxicant effects are based on determining the percentage of embryos which hatch to toes. Hatching EC50 (toxicant concentration at which 50% of the embryos fail to hatch) for a number of pesticides, organometallics and metals were determined. The test takes from 2 to 6 days depending on the embryo stage selected for the study. In addition to embryo development effects the prevalence of DNA single-strand breaks in individual embryo cells were determined using the single cell gel electrophoresis method (Comet assay). A good correlation between DNA strand breakage and embryo defects was found after exposure to genotoxic contaminants. Thus, the bioassay linking DNA damage to embryo hatching effects is rapid, sensitive and mechanistically relevant.

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Language：Japanese   Publisher：日本微生物生態学会  

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Language：Japanese   Publisher：Kyushu University  

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Other Link： http://hdl.handle.net/2324/9849

Language：English   Publisher：International Coastal Research Center, Ocean Research Institute, the University of Tokyo  

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Other Link： http://hdl.handle.net/2261/20990

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DOI： 10.11403/jset.9.81

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER CHEMICAL SOC  

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Language：Japanese   Publisher：環境ホルモン学会(日本内分泌撹乱化学物質学会)  

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DOI： 10.11403/jset.7.61

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DOI： 10.11457/swsj1965.53.224

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DOI： 10.11457/swsj1965.53.224

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