Kazuhiko Mochida

Development of enzyme-linked immunosorbent assays for two forms of vitellogenin in Japanese common goby (Acanthogobius flavimanus).

Two vitellogenins (Vgs) were detected in serum from estradiol-17beta (E(2))-injected Japanese common goby (Acanthogobius flavimanus). Vitellogenins with molecular masses of 530 kDa (Vg-530) and 320 kDa (Vg-320) were purified, and used to raise specific antisera in rabbits. Sandwich enzyme-linked immunosorbent assays (ELISAs) for Vg-530 and Vg-320 were developed using the antisera and the isolated Vgs. The sensitivity ranges of these ELISAs were 1.25-160 ng/ml for Vg-530 and 0.26-66 ng/ml for Vg-320, and very low cross-reactivity was found with the alternate Vg in each assay. Treatment of male gobys with E(2) by injection and immersion induced both Vgs in sera in a dose-dependent manner. The mean concentrations of the Vgs increased from 10 ng/L E(2) exposure for three weeks. Serum concentrations of the two Vgs in field-collected maturing females increased in accordance with increment of E(2) level and ovarian development, and the mean concentrations of Vg-530 were higher than those of Vg-320 in maturing female. These results indicate that the sandwich ELISAs for Vg-530 and Vg-320 developed in the present study is useful as an assay system for surveys of estrogenic activity in coastal areas of Japan.

Acute toxicity of pyrithione antifouling biocides and joint toxicity with copper to red sea bream (Pagrus major) and toy shrimp (Heptacarpus futilirostris)

We evaluated the median lethal concentrations (LC50s) of the pyrithione (PT) antifoulants copper pyrithione (CuPT) and zinc pyrithione (ZnPT) to a teleost, red sea bream (Pagrus major), and a crustacean, toy shrimp (Heptacarpusfutilirostris). The 96-h LC50 values of CuPT and ZnPT, on the basis of actual concentrations, were 9.3 and 98.2 R.g/L, respectively, for red sea bream and 2.5 and 120 microg/L, respectively, for toy shrimp. Histological observations revealed that the secondary lamellae of the gill filaments of the experimental fish were heavily damaged after exposure to the PTs, suggesting that fatal hypoxemia was one cause of death. Because CuPT and ZnPT are usually used in combination with Cu, we also estimated the joint toxicities of the PTs with Cu using the LC50 values of the PTs and those of Cu (84.4 and 113 microg/L for red sea bream and toy shrimp, respectively). The results suggested that the joint toxicity of the ZnPT and Cu mixture is more than the additive toxicities of CuPT and Cu, especially in toy shrimp. The enhancement of toxicity in the mixture was inferred to be caused by conversion of ZnPT to the more toxic CuPT in the presence of Cu.

Salmonid olfactory system-specific protein (N24) exhibits glutathione S-transferase class pi-like structure.

Abstract: A salmonid olfactory system‐specific protein (N24) that has been identified in lacustrine sockeye salmon (Oncorhynchus nerka) was characterized by biochemical and molecular biological techniques. N24 is a homodimer, and the intact molecular mass is estimated as ∼43.3 kDa by gel filtration. Furthermore, N24 was located only in the cytosolic fraction of the olfactory tissues as determined by subcellular fractionation. cDNA encoding the lacustrine sockeye salmon N24 was isolated and sequenced. This cDNA contained a coding region encoding 216 amino acid residues and the molecular mass of this protein is calculated to be 242,224.77. The protein and nucleotide sequencing demonstrates the existence of a remarkable homology between N24 and glutathione S‐transferase (GST; EC 2.5.1.18) class pi enzymes. Northern analysis showed that N24 mRNA with a length of 950 bases is expressed in lacustrine sockeye salmon olfactory epithelium. Olfactory receptor cells showed strong hybridization signals for N24 mRNA in the olfactory epithelium. N24 demonstrated glutathione binding activity in affinity‐purified GST column experiments. The present study describes for the first time cDNA cloning of GST in fish olfactory epithelium.

A high molecular weight glycoprotein in seminal plasma is a sperm immobilizing factor in the teleost Nile tilapia, Oreochromis niloticus.

Sperm that have acquired potential for motility are kept immotile in seminal plasma in the teleost, Nile tilapia. In order to investigate the mechanism of immobilization, several experiments were performed using a previously characterized monoclonal antibody (TAT‐30) against a molecular weight (Mr) = 120 000 protein that is secreted by Sertoli cells and epithelial cells of the sperm duct, and is also bound to the head of the spermatozoon. First, we assessed sperm motility in the seminal plasma protein fraction (SPP), and demonstrated that the sperm motility is inhibited by SPP in a concentration‐dependent manner. Furthermore, sperm motility was recovered if SPP was pretreated with TAT‐30, suggesting that the TAT‐30 antigen is one of the components of the sperm immobilizing factor. Calibration by gel filtration followed by sodium dodecylsulfate‐ polyacrylamide gel electrophoresis (SDS‐PAGE) and western blotting with TAT‐30 demonstrated that the sperm immobilizing factor was more than Mr = 1 000 000 in seminal plasma, suggesting that it is a homopolymer of the Mr = 120 000 – TAT‐30 positive protein. Additionally, lectin blot analysis showed that the TAT‐30 antigen was reactive with Lens culinarin agglutinin (LCA) and Conavalia ensiformis agglutinin (ConA), indicating that it is a glycoprotein. Immunohistochemical studies showed that the TAT‐30 antigen was localized specifically on the heads of spermatozoa and on the apical surface, lysosomes and rough endoplasmic reticulum of Sertoli cells.

Early life‐stage toxicity test for copper pyrithione and induction of skeletal anomaly in a teleost, the mummichog (Fundulus heteroclitus)

We used a teleost fish, the mummichog (Fundulus heteroclitus), to conduct early life-stage toxicity testing for copper pyrithione (CuPT). Fertilized mummichog eggs were exposed to CuPT at various concentrations for 50 d under continuous flow-through conditions. Hatchability, survival, growth, and morphologic abnormalities were measured. Hatchability did not differ significantly between any experimental group and control groups. Survival and growth were significantly reduced at 50 d in the groups exposed to 2 or 4 microg/L CuPT. During the test, morphologic abnormalities, such as vertebral deformity and formation of inflammatory masses in the lateral muscles, occurred in fish exposed to CuPT. Light and electron microscopic studies indicated that muscle dysfunction played a role in the vertebral deformity and revealed that the inflammatory mass was composed mainly of macrophages and necrotic myocytes. We consider that macrophages infiltrated and phagocytized necrotic cells, thus forming the inflammatory mass. In addition, acetylcholinesterase activity was markedly decreased in the 2- and 4-microg/L exposure groups, suggesting the skeletal deformity was due to mechanisms similar to those proposed for organophosphorous pesticide exposure.

Molecular Cloning of Two Estrogen Receptors Expressed in the Testis of the Japanese Common Goby, Acanthogobius flavimanus

Abstract We isolated cDNA clones of two estrogen receptors (ER1 and ER2) from testis of the Japanese common goby (Acanthogobius flavimanus). The cDNAs of ER1 contained 3,796 nucleotides, including an open reading frame encoding 564 amino acids (Mr: 61.9 kDa); the cDNA for ER2 was 3,274 nucleotides long, with an open reading frame of 567 amino acids (Mr: 63.5 kDa). The deduced aminoacid sequences of ER1 and ER2 each had a characteristic ER structure consisting of five domains (A/B, DNA-binding, D, ligand-binding, and F) and were homologous to ERα and ERβ of other vertebrates. We therefore named ER1 and ER2 as goby ERα (gERα) and goby ERβ (gERβ), respectively. The DNA- and ligand-binding domains in each gER showed high similarity to the corresponding domains of other vertebrates. Analysis of the distribution of gERα and gERβ mRNAs in tissue by reverse transcription–polymerase chain reaction (RT-PCR) analysis revealed that gERα was expressed at high levels in brain and testis, and gERβ was expressed most strongly in testis. In situ hybridization showed that the mRNA of each gER was expressed mainly in the Sertoli cells of goby testis.

Use of species sensitivity distributions to predict no-effect concentrations of an antifouling biocide, pyridine triphenylborane, for marine organisms

We used species sensitivity distributions (SSDs) and a Bayesian statistical model to carry out a primary risk assessment for pyridine triphenylborane (PTPB) in Hiroshima Bay, Japan. We used SSDs derived from toxicity values, such as EC₅₀ and LC₅₀, obtained from this study and previous work to calculate hazardous concentrations that should protect 95% and 99% of species (HC₅ and HC₁) and demonstrated that the medians of the HC₅ and HC₁ were 0.78 and 0.17 μg/L, respectively. We also used liquid chromatography/mass spectrometry to investigate the occurrence of PTPB in seawater from several coastal sites of Hiroshima Bay and detected PTPB at concentrations of 4.8-21 pg/L. Comparison of environmental concentrations to the HC values suggests that the current ecological risk posed by PTPB in Hiroshima Bay is low. This is the first report of the detection of PTPB in the natural marine environment.

Induction of apoptosis in testis of the marine teleost mummichog Fundulus heteroclitus after in vivo exposure to the antifouling biocide 4,5-dichloro-2-n-octyl-3(2H)-isothiazolone (Sea-Nine 211).

4,5-dichloro-2-n-octyl-3(2H)-isothiazolone (Sea-Nine 211) has been widely used as an effective antifouling biocide. However, little is known about its reproductive toxicity in fish. Here we investigated testicular toxicity in a marine teleost, the mummichog Fundulus heteroclitus, after exposure to Sea-Nine 211 for 28 d. Although Sea-Nine 211 exposure did not affect germ cell proliferation in testis, terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labeling revealed that the number of apoptotic spermatocytes was increased in the 1.0- and 3.0-μg L(-1) groups, and significant differences emerged between the 1.0-μg L(-1) group and control groups. Immunohistochemistry showed that the numbers of cysts expressing caspases 2, 3, 6, and 8 (apoptosis-associated proteins) were significantly increased in the 1.0-μg L(-1) group, whereas the signal intensity of an anti-apoptotic protein Bcl-xL was reduced in a dose-dependent manner. Moreover, the number of cysts positive for neuronal nitric oxide synthase was twofold higher in the 1.0-μg L(-1) group than in the control groups. These results suggest that long-term exposure to Sea-Nine 211 induces apoptosis in the testicular germ cells of mummichogs via a caspase-dependent pathway and that oxidative stress via nitric oxide synthesized by neuronal nitric oxide synthase is involved in this induction.

Spatial analysis of 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (Sea-Nine 211) concentrations and probabilistic risk to marine organisms in Hiroshima Bay, Japan.

We analyzed the spatial distribution of an antifouling biocide, 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (Sea-Nine 211) in the surface water and sediments of Hiroshima Bay, Japan to determine the extent of contamination by this biocide. A quantitative estimate of the environmental concentration distribution (ECD) and species sensitivity distributions (SSDs) for marine organisms were derived by using a Bayesian statistical model to carry out a probabilistic ecological risk analysis, such as calculation of the expected potentially affected fraction (EPAF). The spatial distribution analysis supported the notion that Sea-Nine 211 is used mainly for treatment of ship hulls in Japan. The calculated EPAF suggests that approximately up to a maximum of 0.45% of marine species are influenced by the toxicity of Sea-Nine 211 in Hiroshima Bay. In addition, estimation of the ecological risk with a conventional risk quotient method indicated that the risk was a cause for concern in Hiroshima Bay.

Primary risk assessment of dimethyldithiocarbamate, a dithiocarbamate fungicide metabolite, based on their probabilistic concentrations in a coastal environment

The primary ecological risk of dimethyldithiocarbamate (DMDC), a dithiocarbamate fungicide (DTC) metabolite, was evaluated based on their probabilistic environmental concentration distributions (ECDs) in the coastal environment, Hiroshima Bay, Japan. And their behavior and temporal trends was further considered. This is the first report of the identification of DMDC from environmental seawater and sediment samples. DMDC concentrations in bottom seawater were substantially higher than those in surface seawater, which are associated with the leachability from sediments in bottom seawaters, and with photodegradation in surface seawaters. Furthermore, seasonal risks are dominated by higher concentrations from April to June, indicating temporal variation in the risk to exposed species. Hierarchical Bayesian analysis offered DMDC ECD medians and range (5th to 95th percentiles) of 0.85 ng L(-1) (0.029, 22), 12 ng L(-1) (3.2, 48) and 110 ng kg dry(-1) (9.5, 1200) in surface seawater, bottom seawater and sediment, respectively. Considering that DMDC and DTCs have similar toxicological potential to aquatic organisms, the occurrence of the compound in water is likely to be of biological relevance. In summary, this work provides the first demonstration that the ecological risk of DMDC and its derived DTCs in Hiroshima Bay is relatively high, and that DTCs should be a high priority for future research on marine contamination, especially in bottom seawaters.