Thiobencarb herbicide reduces growth, photosynthetic activity, and amount of Rieske iron-sulfur protein in the diatom Thalassiosira pseudonana.

We investigated the effects of the herbicide thiobencarb on the growth, photosynthetic activity, and expression profile of photosynthesis-related proteins in the marine diatom Thalassiosira pseudonana. Growth rate was suppressed by 50% at a thiobencarb concentration of 1.26 mg/L. Growth and photosystem II activity (Fv /Fm ratio) were drastically decreased at 5 mg/L, at which the expression levels of 13 proteins increased significantly and those of 11 proteins decreased significantly. Among these proteins, the level of the Rieske iron-sulfur protein was decreased to less than half of the control level. This protein is an essential component of the cytochrome b6 f complex in the photosynthetic electron transport chain. Although the mechanism by which thiobencarb decreased the Rieske iron-sulfur protein level is not clear, these results suggest that growth was inhibited by interruption of the photosynthetic electron transport chain by thiobencarb.

Growth-phase dependent variation in photosynthetic activity and cellular protein expression profile in the harmful raphidophyte Chattonella antiqua.

This study investigated temporal variations in the potential maximum quantum yield of photosystem II (F(v)/F(m) ratio) and growth-phase dependent cellular protein expressions of Chattonella antiqua under laboratory conditions. Despite the culture conditions, significant positive correlations between the F(v)/F(m) ratio and daily growth rate were observed. Threshold F(v)/F(m) ratios associated with positive cell growth were calculated to be >0.44, >0.44, and >0.37, and those associated with active cell growth (growth rate >0.5 div. d(-1)) were >0.58, >0.60, and >0.49 under control culture, low nutrient and intense light conditions, respectively. Proteome profiles obtained by two-dimensional gel electrophoresis (2-DE) indicated that 42 protein spots were differentially expressed at various growth phases of C. antiqua, which indicates changes in cellular physiological status throughout the growth cycle, and suggests that oxygen evolving enhancer 1 and 2-cysteine peroxiredoxin play roles in maintaining the positive growth of C. antiqua.

Molecular cloning, sequencing, and gene expression analysis of tributyltin-binding protein type 1 in Japanese medaka fish, Oryzias latipes.

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.

The effects of methyltestosterone on the sexual development and reproduction of adult medaka (Oryzias latipes).

We examined the effects of methyltestosterone (MT) on the reproduction of medaka (Oryzias latipes). Six mating pairs of medaka were exposed for 3 weeks to MT at the measured concentrations of 22.5, 46.8, 88.1, 188, and 380 ng/L. The fecundity and fertility of paired medaka were significantly decreased in the 46.8, 88.1, 188, and 380 ng/L MT groups compared with those of controls. The gonadosomatic indices of female fish exposed to >or=46.8 ng/L were increased significantly. Gonadal histology showed degeneration of oocytes in the ovaries of fish in all MT treatment groups. Hepatic vitellogenin concentrations were significantly decreased in female fish treated with MT at 188 and 380 ng/L, and the hatchability and survival rate of the offspring were decreased in all MT treatment groups. These results clearly demonstrate that MT at >or=46.8 ng/L inhibited gonadal development and adversely affected the reproduction of medaka.

Alteration of gene expression profiles in the brain of Japanese medaka (Oryzias latipes) exposed to KC-400 or PCB126.

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.

Purification and characterization of tributyltin-binding protein type 2 from plasma of Japanese flounder, Paralichthys olivaceus.

We used gel filtration chromatography, anion-exchange chromatography and polyacrylamide gel electrophoresis to purify tributyltin-binding protein type 2 (TBT-bp 2) from plasma of Japanese flounder (Paralichthys olivaceus) injected intraperitoneally with TBT (5.0 mg/kg body weight). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular mass of TBT-bp 2 was approximately 48 kDa, and isoelectric focusing-polyacrylamide gel electrophoresis indicated that the isoelectric point was approximately 3.0. TBT-bp 2 contained 40% N-glycan. The complete cDNA nucleotide sequence and the genome sequence of TBT-bp 2 were determined by means of rapid amplification of cDNA ends of liver tissue of Japanese flounder and a genome-walking technique, respectively. The 216 amino acid sequence of TBT-bp 2 showed 47% identity to the sequences of puffer fish (Takifugu pardalis) saxitoxin- and tetrodotoxin-binding protein but only 27% similarity to the sequence of TBT-bp 1. Analysis of the motif sequence of the amino acid sequence and the structure of the gene encoding TBT-bp 2 suggested that this protein belongs to the lipocalin superfamily.

Effect of tributyltin on veliger larvae of the Manila clam, Ruditapes philippinarum.

We investigated the effects of waterborne and maternal exposure to tributyltin (TBT) on veliger larvae of the Manila clam, Ruditapes philippinarum. In a waterborne exposure test, veliger larvae (D-larvae stage: 24h after fertilization) were exposed to TBT at measured concentrations of <0.01 (control), 0.055, 0.130, 0.340, and 0.600microg/l for 13d. The percentage of normal veliger larvae (the ratio of normal veliger larvae to all larvae) decreased significantly in all TBT treatment groups compared with that in the control group. In a maternal exposure test, 100 clams were exposed to TBT at measured concentrations of <0.01 (control), 0.061, and 0.310microg/l at 20-22 degrees C for 3 weeks, and the percentage of normal veliger larvae assessed for 13d. No maternal effects on veliger larvae from TBT were observed in TBT treatment groups as compared with the control group. These results demonstrate that waterborne TBT affects Manila clam veliger larvae, and indicates that TBT may have reduced Manila clam populations by preventing the development and survival of veliger larvae.

Effects of tributyltin on the energy metabolism of pen shell (Atrina pectinata japonica).

We examined the effects of tributyltin (TBT) on aerobic and anaerobic energy metabolism of pen shell (Atrina pectinata japonica). We exposed pen shells to TBT at nominal concentrations of 0 (control) and 1.0microg/l for 72h under aerobic condition. At the end of the exposure, half of the pen shells in each treatment were wrapped in plastic wrap to simulate exposure to hypoxia and held at 25 degrees C for another 12h. The concentrations of the products of energy metabolism, namely lactate, pyruvate, fumarate and succinate, in adductor muscle were measured. The exposure to TBT under aerobic condition significantly elevated lactate, pyruvate and fumarate concentrations (p<0.001). After subsequent exposure to anaerobic condition, the mean concentration of succinate in the TBT treatment group was 64% of that in the control group, but there were no significant differences. Our results suggest that the energy metabolism of pen shell is disrupted by exposure to TBT.

Tributyltin causes abnormal development in embryos of medaka, Oryzias latipes.

We examined the effects of tributyltin (TBT) on embryonic development, hatching success and sexual differentiation in Japanese medaka (Oryzias latipes). Embryos (within 8h after fertilization) were exposed to TBT in ovo via nanoinjection at concentrations of 0 (control), 0.16, 0.80, 3.96, 19.2 and 82.1 ng/egg. Embryonic survival, development and hatching were observed. Hatched fry were reared until 60 days when they sexually matured, and sexual differentiation was also examined by accordance of genetic and phenotypic sex, based on existence of DMY (a male determining gene in medaka) and secondary sex characteristics. As results, TBT caused a concentration-dependent mortality and impaired the embryonic development. However, no masculinization was detected at 60 dph medaka adults. Lowest observed effective concentration for inducing abnormal embryonic development was estimated to 0.16 ng/egg (ca. 160 ng/g egg).

Interaction patterns and toxicities of binary and ternary pesticide mixtures to Daphnia magna estimated by an accelerated failure time model.

Organisms in natural environments are often exposed to a broad variety of chemicals, and the multi-chemical mixtures exposure may produce significant toxic effects, even though the individual chemicals are present at concentrations below their no-observed-effect concentrations. This study represents the first attempt that uses the accelerated failure time (AFT) model to quantify the interaction and toxicity of multi-chemical mixtures in environmental toxicology. We firstly conducted the acute immobilization tests with Daphnia magna exposed to mixtures of diazinon (DZN), fenitrothion (MEP); and thiobencarb (TB) in single, binary, and ternary formulations, and then fitted the results to the AFT model. The 48-h EC50 (concentration required to immobilize 50% of the daphnids at 48h) values for each pesticide obtained from the AFT model are within a factor of 2 of the corresponding values calculated from the single pesticide exposure tests, indicating the methodology is able to provide credible toxicity values. The AFT model revealed either significant synergistic (DZN and MEP; DZN and TB) or antagonistic (MEP and TB) interactions in binary mixtures, while the interaction pattern of ternary mixture depended on both the concentration levels and concentration ratios of pesticides. With a factor of 2, the AFT model accurately estimated the toxicities for 78% of binary mixture formulations that exhibited significant synergistic effects, and the toxicities for all the ternary formulations. Our results showed that the AFT model can provide a simple and efficient way to quantify the interactions between pesticides and to assess the toxicity of their mixtures. This ability may greatly facilitate the ecotoxicological risk assessment of exposure to multi-chemical mixtures.

Effects of tributyltin maternal and/or waterborne exposure on the embryonic development of the Manila clam, Ruditapes philippinarum.

We examined the effect of tributyltin (TBT) on embryonic development of the Manila clam, Ruditapes philippinarum. In a maternal exposure test, 100 clams were exposed to TBT at measured concentrations of <0.01 (control), 0.061, 0.310, or 0.350 microg/l at 20-22 degrees C for 3 weeks, and the embryo developmental success (the ratio of normal D-larvae to all larvae) was measured. There was a significant negative correlation between embryo developmental success and TBT concentration in the female Manila clams (p < 0.001). These results indicated that TBT accumulated in the female clam decreased embryo developmental success. In a waterborne exposure test, fertilized eggs (4 h after fertilization) were exposed to TBT at measured concentrations of <0.01 (control), 0.062, 0.140, 0.320, or 0.640 microg/l for 23 h. Embryo developmental success was also significantly decreased in all TBT treatment groups compared with that in the control group. TBT accumulated in female adults and waterborne TBT clearly inhibit reproductive success of the clam.

Anti-androgen flutamide affects gonadal development and reproduction in medaka (Oryzias latipes).

The effects of an anti-androgenic chemical on the reproduction of medaka (Oryzias latipes) were examined. Six mating pairs of medaka were exposed to flutamide (FLU) at measured concentrations of 0.101, 0.202, 0.397, 0.787, or 1.56 mg/L for 3 weeks. During the exposure period, one female died in the 1.56-mg/L treatment group, but no lesions or pathological abnormalities were observed. The fecundity and fertility of paired medaka were significantly decreased at 1.56 mg/L compared with those of the controls. Histological examination showed intersex gonads (testis-ova) in males exposed to FLU at 0.202, 0.397, and 0.787 mg/L. However, concentrations of hepatic vitellogenin (Vtg) in both sexes were not statistically different from those in the controls. These results demonstrate that FLU affected gonadal development in male medaka and reproductive capacity in paired medaka. This is the first report of testis-ova in male medaka exposed to FLU.

Effect of tributyltin on reproduction in Japanese whiting, Sillago japonica.

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.

Effects of polychlorinated biphenyls on the schooling behavior of Japanese medaka (Oryzias latipes).

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.

Quantitative bio-imaging analysis for evaluation of sexual differentiation in germ cells of olvas-GFP/ST-II YI medaka (Oryzias latipes) nanoinjected in ovo with ethinylestradiol.

We examined the effects on sexual differentiation of nanoinjecting ethinylestradiol (EE2) into embryos of olvas-GFP/ST-II YI medaka (Oryzias latipes). This strain contains the green fluorescent protein (GFP) gene fused to the regulatory region of the medaka vasa gene, and germ-cell-specific expression of GFP can be visualized in living (transparent) individuals. The number of germ cells in untreated genotypic females (XX) was approximately 10-fold that in untreated genotypic males (XY) at 10 d posthatch (dph). Germ cell proliferation was prevented in XX females that developed from embryos nanoinjected with 0.5, 2.5, or 5.0 ng of EE2. Some 10-dph XY males from embryos injected with 0.5 ng or more of EE2 showed a larger fluorescent area and more germ cells than those of pooled control groups. Males and females from embryos injected with 5.0 ng of EE2 had no significant difference in germ cell number or fluorescent area. Thus, EE2 injection into embryos caused abnormal gonadal development in both sexes. Observations of external secondary sex characteristics and histological examination of adult gonads showed complete sex reversal in some males after 0.5-, 2.5-, and 5.0-ng treatments but no changes in XX females after any treatment. Thus, quantitative bio-imaging can aid in evaluating the effects of endocrine-disrupting chemicals on fish within 10 dph.

Early-life-stage toxicity in offspring from exposed parent medaka, Oryzias latipes, to mixtures of tributyltin and polychlorinated biphenyls.

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.

Photosensitizing hemolytic toxin in Heterocapsa circularisquama, a newly identified harmful red tide dinoflagellate.

Red tides of Heterocapsa circularisquama (H. circularisquama), recently identified as a novel species of dinoflagellate, have frequently caused mass mortality of several species of bivalves in Japan, while no harmful effects of this flagellate on fish have been reported so far. We found that the cell-free ethanol extract prepared from H. circularisquama caused hemolysis of rabbit erythrocytes and demonstrated cytotoxic effects in HeLa cells and on the microzooplankton rotifer (B. plicatilis) in a dose- and time-dependent manner. Interestingly, the hemolytic activity and cytotoxic effects of the extract were completely dependent on the presence of light. When the experiments were conducted in the dark, no hemolysis was observed even at very high concentration of the extract. These results suggest that H. circularisquama has photosensitizing hemolytic toxin which can be easily extracted into ethanol. This may be the first report documenting the occurrence of photosensitizing hemolytic toxin in marine phytoplankton species.

Production of superoxide anion and hydrogen peroxide by the red tide dinoflagellate Karenia mikimotoi.

We found that the red tide dinoflagellate Karenia mikimotoi (Gymnodinium mikimotoi) generates reactive oxygen species (ROS). In chemiluminescence analysis using an O2- specific probe, a slightly lower but significant level of O2- was detected in a K. mikimotoi cell suspension as compared to one containing Chattonella marina, a well-known ROS-producing red tide phytoplankton. Interestingly, the chemiluminescence response pattern caused by K. mikimotoi was different from that of C. marina, although both responses were almost completely abolished in the presence of superoxide dismutase (SOD). By fluorescence spectrophotometric analysis, H2O2 was also detected in the K. mikimotoi cell suspension, but the level of H2O2 was approximately 10% of that in the C. marina suspension based on the cell number. No significant increase in O2- generation by K. mikimotoi was observed in the presence of lectins such as concanavalin A (Con A) and wheat germ agglutinin (WGA) or fish mucus substances prepared from the skin and gills of yellowtail, whereas C. marina generated an increased level of O2- in response to these stimuli. Furthermore, O2- production in C. marina, but not in K. mikimotoi, was inhibited by treatment with proteinase K.

Suppression of sexual behavior in male Japanese medaka (Oryzias latipes) exposed to 17beta-estradiol.

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.

Effect of 17beta-estradiol on the reproduction of Japanese medaka (Oryzias latipes).

Estrogenic compounds such as 17beta-estradiol (E2) and its analogs are present in the aquatic environment and can adversely affect the reproductive systems of aquatic organisms. Although E2 has been detected at a maximum concentration of 64 ng/l in effluents of sewage treatment works (STWs), few reports address the associated effects on reproduction in fish. Therefore, we exposed adult medaka (Oryzias latipes) to mean measured E2 concentrations of 29.3, 55.7, 116, 227, and 463 ng/l for 21 d and assessed the effects on the egg number and fertility of paired medaka during the exposure period. In addition, we determined the hepatic vitellogenin (Vtg) concentration and histologically assessed the gonads of these fish. The number of egg produced and fertility of the paired medaka exposed to 463 ng/l E2 were significantly less compared with those of the control fish. Males in all treatment groups had developed testis-ova. Males treated with E2 concentrations = 55.7 ng/l contained relating great concentrations of hepatic Vtg. Therefore, although only the greatest E2 concentration tested in our study affected fecundity and fertility, effects of E2 were observed on induction of Vtg and testis-ova in male medaka exposed to environmentally relevant concentrations of E2.