Seiichi Uno

1H NMR‐based metabolomics approach to assess toxicity of bunker a heavy oil to freshwater carp, Cyprinus carpio

Using a 1H NMR metabolomics approach, the effects of dietary exposure of bunker A heavy oil (0.01, 0.1, 1, and 5% in diet) on freshwater carp, Cyprinus carpio, were examined. Statistical analysis by PCA score plots showed that the amount of metabolites in exposure groups 0.1, 1, and 5% differed from those in the control group. Although no discernible effects on metabolites were noted in the 0.1% exposure group as well as in the lowest concentration (0.01%) group, several metabolites such as amino acid (e.g., leucine, isoleucine, valine, glutamine, histidine, proline, and methionine), 3‐D‐hydroxybutyrate, and glycerol were elevated, while another metabolite such as formate was reduced in 1 and 5% groups. These changes in the metabolites associated with the tri‐carboxylic‐acid (TCA) cycle suggest that oil exposure resulted in the disturbance of the TCA cycle in the liver of the carp. Isobutyrate, a marker of anoxia, was also increased in 1 and 5% exposures groups and was directly related to low hemoglobin concentrations leading to reduced oxygen transport by blood. In addition, significant elevation of creatinine in the plasma of carps exposed to 5% heavy oil suggests disturbance in kidney function. Thus, metabolomics approach can detect toxic effects of hazardous pollutants on fish.

Comparative Study of Bioconcentration and EROD Activity Induction in the Japanese Flounder, Red Sea Bream, and Java Medaka Exposed to Polycyclic Aromatic Hydrocarbons

Japanese flounder (Paralichthys olivaceus), red sea bream (Pagrus major), and Java medaka (Oryzias javanicus) were exposed to water borne polycyclic aromatic hydrocarbons (PAHs) for 10 days to compare PAH bioconcentration and P450 enzyme induction by ethoxyresorufin‐O‐deethylase (EROD) activity for use in oil spill biomonitoring in Asian waters. Target exposure concentration for phenanthrene, pyrene, and chrysene were 30 μg/L each, while benzo[a]pyrene was 3.0 μg/L. Phenanthrene and pyrene were accumulated in the flounder and red sea bream; chrysene was found only in the livers of the red sea bream, while Java medaka accumulated the high molecular weight benzo[a]pyrene along with the other PAHs. Total PAH concentrations increased with duration of exposure in the red sea bream from 184 ± 37 ng/g wet weight (w.w.) in day 2 to 572 ± 72 ng/g (w.w.) in day 10; It, however, decreased in the other two species. Among the three fish species, Java medaka had the highest initial total PAH concentration of 388 ± 62 ng/g (w.w.); this was, however, reduced to the lowest final concentration of 52.3 ± 3 ng/g (w.w.). It also had the highest EROD activity of 4.2 ± 2.8 n mol/min/mg protein compared to the lowest of 0.11 ± 0.03 n mol/min/mg protein in the Japanese flounder. Java medaka with high EROD activity induction and bioaccumulation of all PAHs will be suitable for PAH biomonitoring in Asian waters. Due to its high PAH bioconcentration red sea bream is also recommended for consideration for biomonitoring and PAH chronic toxicity tests.

Assessment of pesticide residues in freshwater areas affected by rice paddy effluents in southern Japan.

Pesticide residues in five freshwater areas that are directly affected by rice paddy effluents in southern Japan were measured to determine their maximum concentrations and temporal variations. Water samples were collected every week during the 2005 rice planting season in Kagoshima Prefecture and stations were established in Amori River, Sudo River, Nagaida River (that drains into the bigger Kotsuki River), rice paddy drainage canal, and wastewater reservoir (that collects effluents from rice paddy fields). Of the 14 target pesticides examined, a total of 11 were detected in all stations. Mefenacet, fenobucarb, and flutolanil were the three pesticides with the highest maximum concentrations and were also detected frequently. Analysis of temporal variations of pesticides showed that herbicides had relatively higher concentrations in the earlier stages of the rice planting season, while insecticides and fungicides had relatively higher concentrations at the later stages. There was no significant difference among stations with regards to the temporal patterns of the top three pesticides. The calculated toxic units were less than 1 in all stations, implying low or negligible environmental risk of pesticides detected to freshwater organisms.

Measurement of urinary zearalenone concentrations for monitoring natural feed contamination in cattle herds: On-farm trials1

The aims of the present study were to investigate the efficacy of measuring bovine urinary zearalenone (ZEN) concentrations by using a commercially available ELISA method in cattle kept under different feeding conditions to monitor the natural contamination of feeds at the farm level, and to investigate the effects of supplementation of a mycotoxin adsorbent (MA) product in the feed based on urinary ZEN concentration. First, Japanese Black cattle herds kept for breeding (4 herds) and fattening (4 herds) purposes were provided with similar feeding conditions. Then, urinary samples from 5 cows in each herd were collected and analyzed. Second, dairy cows from 1 herd fed with total mixed rations (TMR) were selected. After thorough mixing of the MA (40 g/d) with TMR, the supplemented TMR was fed according to the following schedule: with MA for 2 wk, without MA for 3 wk; then with MA for 2 wk and without MA for 6 wk. Urine samples were collected from cows (n = 6 to 7) and examined before and after each interval. Zearalenone concentrations were measured by the ELISA and liquid chromatography-tandem mass spectrometry methods. The concentration of ZEN and its metabolites was expressed after creatinine (Crea) correction [ZEN or metabolites (pg/mL)/Crea (mg/dL); pg/mg of Crea]. In the first experiment, the urinary concentrations of ZEN and its metabolites were variable in all herds, and significant differences were observed between herds. In 1 fattening herd, in particular, urinary ZEN concentrations were greater (P < 0.001) than in the other 3 herds. This might reflect significant natural ZEN contamination of the feed at the farm level. In Exp. 2, urinary ZEN concentrations displayed peculiar trends after supplementation with MA. After 2 wk of supplementation, a significant decrease of ZEN (P < 0.05) was observed. Zearalenone concentrations remained at a reduced amount during 3 wk without MA supplementation and 2 wk with MA supplementation. When MA was not added to the feed for the next 6 wk, the concentrations increased to the original quantity. These findings indicate the usefulness of measuring concentrations of urinary ZEN and its metabolites not only for monitoring the natural ZEN contamination of cattle feed at the farm level but also for in vivo evaluation of MA function after supplementing feeds with MA.

Detection of zearalenone and its metabolites in naturally contaminated follicular fluids by using LC/MS/MS and in vitro effects of zearalenone on oocyte maturation in cattle

Zearalenone (Zen) and its metabolites are estrogenic and may be important factors involved in reproductive disorders in domestic animals. We aimed to (1) simultaneously detect Zen and its metabolites in bovine follicular fluids (FFs) by liquid chromatography-tandem mass spectrometry and (2) examine the in vitro effects of Zen on bovine oocytes. Zen and its metabolites were detected in 6 of 32 normal follicles and 7 of 20 cystic follicles. Bovine oocytes were cultured in a maturation media containing various Zen concentrations (0 [control], 1, 10, 100, and 1000microg/L), fertilized, and cultured further. Maturation rates decreased dose-dependently. Further, maturation of 62 (50%) of 124 oocytes examined in the 1000-microg/L group was arrested in metaphase I, without affecting the fertilization rate. Blastocyst-formation rates did not significantly differ among the groups. Zen and its metabolites were detectable in bovine FFs. High Zen concentration may adversely affect meiotic competence but not the fertilization and development rates.

Java Medaka : A Proposed New Marine Test Fish for Ecotoxicology

Java medaka (Oryzias javanicus), which is distributed in estuarine waters of East to Southeast Asia, is proposed here for consideration as a new marine test fish. Semi‐static acute toxicity tests were conducted at 26°C for 1 week, 2 weeks, and 1 month old Java‐medaka exposed to 4‐chlorophenol and zinc, and 2 weeks olds exposed to potassium dichromate (hexavalent chromium) and sodium hypochlorite (residue chlorine). Ninety‐six hours LC50s of 4‐chlorophenol, zinc, hexavalent chromium, and residue chlorine were from 3.0 to 4.1, from 12.4 to 14.7, 7.4, and 0.05 mg/L, respectively. There were no large differences in sensitivity for 4‐chlorophenol and zinc among the three growth stages. Compared with other fish species, these LC50s were relatively low suggesting that Java medaka has relatively high sensitivity to chemicals. This fish is quite easy to keep in the laboratory, with low mortalities at early stages and it takes about 3 months to mature from hatching, suggesting that this fish can be used not only for short‐term toxicity tests but also for early life stage toxicity tests. From these results, Java‐medaka is highly recommended as a marine test fish for ecotoxicity tests.

Distributions of polycyclic aromatic hydrocarbons and alkylated polycyclic aromatic hydrocarbons in Osaka Bay, Japan

Contaminations in sediments by polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs were investigated at 44 sites in Osaka Bay, Japan. Concentrations of total PAHs and alkylated PAHs were in the range 6.40-7800 ng/g dry weights and 13.7-1700 ng/g dry weights, respectively. The PAH concentrations tended to be higher along the shoreline in the vicinities of big ports, industrialized areas, and densely populated regions such as the cities of Osaka and Kobe. The major sources appeared to be pyrogenic or both pyrogenic and petrogenic at most of the sites. PAH concentrations were remarkably high at a site near Kobe, where the concentrations of dibenzo(a,h)anthracene and benzo(g,h,i)perylene exceeded the effects-range-medium concentration and eight PAHs were above the corresponding effects-range-low concentrations. Those PAHs may have been derived from the great fire associated with the large earthquake in 1995.

Histopathological alterations in gill, liver and kidney of common carp exposed to chlorpyrifos

Histopathological alterations in gill, liver and kidney of common carp, Cyprinus carpio, intoxicated with sub-lethal concentrations of chlorpyrifos (O,O,-diethyl-O-3,5,6-trichloro-2-pyridyl phosphorothioate) pesticide (1 and 100 μg/L) for a period of 14 days were analyzed under light microscope. Gill exhibited hyperplasia and hypertrophy of gill epithelium, blood congestion, dilation of marginal channel, epithelial lifting, lamellar fusion, lamellar disorganization, lamellar aneurysm, rupture of the lamellar epithelium, rupture of pillar cells and necrosis. Alterations in hepatocytes were more pronounced, including nuclear and cellular hypertrophy, cellular atrophy, irregular contour of cells and nucleus, cytoplasmic vacuolation, cytoplasmic and nuclear degeneration, cellular rupture, pyknotic nucleus, necrosis and melanomacrophages aggregations. Histopathological lesions in kidney were cellular and nuclear hypertrophy, narrowing of tubular lumen, cytoplasmic vacuolation, hyaline droplet degeneration, nuclear degeneration, occlusion of tubular lumen, tubular regeneration, dilation of glomerular capillaries, degeneration of glomerulus and hemorrhage in Bowmans space. The most significant conclusion drawn from this study was that with the increased concentration and duration the toxicosis of chlorpyrifos would be enhanced as shown through the analysis of mean assessment value (MAV) and degree of tissue changes (DTC) also.

Bioaccumulation of nitroarenes in bivalves at Osaka Bay, Japan

This paper reports for the first time the detection and occurrence of nitroarenes (NPAHs) in marine organisms. Mussels and oysters collected from Osaka Bay, Japan, had total NPAHs concentrations that ranged from 2380 to 24,688 pg/g dry and 2672 to 25,961 pg/g dry, respectively. Relatively higher concentrations were detected in sampling sites located near the central district and suburbs of Osaka City implying that the most probable sources of NPAHs in the two bivalves are exhaust gases and smokes emitted by automobiles and industrial plants. Bivalves had relatively higher residues of 1-nitronaphthalene, 2-nitronaphthalene, 3-nitrophenanthrene, and 9-nitrophenanthrenes. Residues of 2-nitrofluorene, 1-nitropyrene, 4-nitropyrenes, and 6-nitrochrysene were much lower compared to nitronaphthalenes and nitrophenanthrenes. Inter-species differences was only observed for 2-nitronaphthalene with oysters exhibiting significantly higher residues than mussels.

Effect of heavy oil exposure on antibacterial activity and expression of immune‐related genes in Japanese flounder Paralichthys olivaceus

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.