Development of a Medium-term Animal Model Using gpt Delta Rats to Evaluate Chemical Carcinogenicity and Genotoxicity

In this study, the potential for development of an animal model (GPG46) capable of rapidly detecting chemical carcinogenicity and the underlying mechanisms of action were examined in gpt delta rats using a reporter gene assay to detect mutations and a medium-term rat liver bioassay to detect tumor promotion. The tentative protocol for the GPG46 model was developed based on the results of dose-response exposure to diethylnitrosamine (DEN) and treatment with phenobarbital over time following DEN administration. Briefly, gpt delta rats were exposed to various chemicals for 4 weeks, followed by a partial hepatectomy (PH) to collect samples for an in vivo mutation assay. The mutant frequencies (MFs) of the reporter genes were examined as an indication of tumor initiation. A single intraperitoneal (ip) injection of 10 mg/kg DEN was administered to rats 18 h after the PH to initiate hepatocytes. Tumor-promoting activity was evaluated based on the development of glutathione S-transferase placental form (GST-P)-positive foci at week 10. The genotoxic carcinogens 2-acetylaminofluorene (2-AAF), 2-amino-3-methylimidazo [4,5-f] quinolone (IQ) and safrole (SF), the non-genotoxic carcinogens piperonyl butoxide (PBO) and phenytoin (PHE), the non-carcinogen acetaminophen (APAP) and the genotoxic non-hepatocarcinogen aristolochic acid (AA) were tested to validate the GPG46 model. The validation results indicate that the GPG46 model could be a powerful tool in understanding chemical carcinogenesis and provide valuable information regarding human risk hazards.     

Habitat management of little terns in Japan’s highly developed landscape

The little tern (Sterna albifrons) is a species of shorebird that nests in colonies on sandy beaches and riverbanks with little vegetation cover. In Japan, the natural breeding habitats of the terns have decreased drastically, and the species is listed under the category “vulnerable” in the Red List of the Government of Japan. To reduce the effects of habitat loss on the terns, conservationists are attempting to create and manage artificial colony sites in highly developed landscapes, such as reclaimed lands located in Tokyo Bay, central Japan. We present the factors that contribute to breeding success for this species and the habitat characteristics related to nest-site selection in the artificial colony sites along Tokyo Bay. Our data show that around 3 ha of a building rooftop without vegetation cover could sustain more than 2000 little tern nests. We found a strong positive relationship between colony size and hatching success, observing that a colony size of more than 100 lowered predation rates of eggs. On sites built upon white crushed-concrete, nest densities were high and predation rates of eggs were low. We also found that feeding rates were affected by foraging habitats, of which the best types were the shoreline of sandy beaches and mud flats. The 40 km dispersal range of the breeding terns, inferred from distribution data, should be considered when establishing a network of the multiple colony sites.     

Fractionation of metal complexes with dissolved organic matter in a rhizosphere soil solution of a humus-rich Andosol using size exclusion chromatography with inductively coupled plasma-mass spectrometry

To better understand the behavior of metals in soil–plant systems, their physicochemical forms in rhizosphere soil should be elucidated. The dissolved organic matter (DOM) in a soil solution influences the mobility and bioavailability of metals in soil. The present study examined the effects of plant growth on DOM–metal complexes in a rhizosphere soil solution using size exclusion chromatography combined with an inductively coupled plasma-mass spectrometer system (SEC–ICP-MS) and an ultrafiltration technique. Humus-rich volcanic ash soil from the surface of an agricultural field was used in a pot cultivation experiment. Brassica rapa nothovar. was cultivated in a pot in which rhizosphere soil (R) was separated by a nylon net screen from non-rhizosphere soil (NR). Soil solutions were collected using a high-speed centrifugation method 3 weeks after sowing and analyzed using SEC–ICP-MS. Some peaks of DOM with a high molecular size were detected in the ultraviolet-absorbing chromatograph (280 nm) of the soil solution samples. Their concentrations were much higher in the R solution than in the NR solution. Metals including Al, Fe, Cu, Zn, Pb, Y, La and U were detected at the ultraviolet peak positions of the DOM. The ultrafiltration experiment showed that the size distributions of the organic materials to which the metals were combined differed between the R and NR soil solutions. These results suggest that plant growth enhanced the dissolution of metals adsorbed with organic matter from the solid phase in rhizosphere soil.     

Development of EST-SSR markers and construction of a linkage map in faba bean (Vicia faba)

To develop a high density linkage map in faba bean, a total of 1,363 FBES (Faba bean expressed sequence tag [EST]-derived simple sequence repeat [SSR]) markers were designed based on 5,090 non-redundant ESTs developed in this study. A total of 109 plants of a ‘Nubaria 2’ × ‘Misr 3’ F₂ mapping population were used for map construction. Because the parents were not pure homozygous lines, the 109 F₂ plants were divided into three subpopulations according to the original F₁ plants. Linkage groups (LGs) generated in each subpopulation were integrated by commonly mapped markers. The integrated ‘Nubaria 2’ × ‘Misr 3’ map consisted of six LGs, representing a total length of 684.7 cM, with 552 loci. Of the mapped loci, 47% were generated from multi-loci diagnostic (MLD) markers. Alignment of homologous sequence pairs along each linkage group revealed obvious syntenic relationships between LGs in faba bean and the genomes of two model legumes, Lotus japonicus and Medicago truncatula. In a polymorphic analysis with ten Egyptian faba bean varieties, 78.9% (384/487) of the FBES markers showed polymorphisms. Along with the EST-SSR markers, the dense map developed in this study is expected to accelerate marker assisted breeding in faba bean.     

A method for estimating radioactive cesium concentrations in cattle blood using urine samples

In the region contaminated by the Fukushima nuclear accident, radioactive contamination of live cattle should be checked before slaughter. In this study, we establish a precise method for estimating radioactive cesium concentrations in cattle blood using urine samples. Blood and urine samples were collected from a total of 71 cattle on two farms in the ‘difficult‐to‐return zone’. Urine ¹³⁷Cs, specific gravity, electrical conductivity, pH , sodium, potassium, calcium, and creatinine were measured and various estimation methods for blood ¹³⁷Cs were tested. The average error rate of the estimation was 54.2% without correction. Correcting for urine creatinine, specific gravity, electrical conductivity, or potassium improved the precision of the estimation. Correcting for specific gravity using the following formula gave the most precise estimate (average error rate = 16.9%): [blood ¹³⁷Cs] = [urinary ¹³⁷Cs]/([specific gravity] ? 1)/329. Urine samples are faster to measure than blood samples because urine can be obtained in larger quantities and has a higher ¹³⁷Cs concentration than blood. These advantages of urine and the estimation precision demonstrated in our study, indicate that estimation of blood ¹³⁷Cs using urine samples is a practical means of monitoring radioactive contamination in live cattle.     

Suppressing effect of neocarrabiose 4-O-sulfate on thermal and freeze denaturation of myosin subfragment-1 and myofibrils

Suppressive effects of neocarrabiose 4-O-sulfate on the thermal and freeze denaturation of myosin subfragment-1 (S-1) and on myofibrils were investigated. The compound strongly suppressed the thermal denaturation of S-1. Its suppressive effect was greater than that of sorbitol and similar to that of maltose. However, it tended to accelerate the denaturation of myofibrils, suggesting a loss of protection by F-actin upon its addition. The compound suppressed the freeze denaturation of myofibrils and S-1. The effect was similar to that of sorbitol or maltose, and completely different from that of Na-sulfate. The compound solubilized myofibrils at concentrations similar to KCl. Therefore, it was concluded that neocarrabiose 4-O-sulfate behaved as an ionic salt in the thermal treatment process, whereas it behaved as a sugar in the frozen storage process.     

Immunoreactive changes in pituitary FSH and LH cells during seasonal reproductive and spawning cycles of female chub mackerel <Emphasis Type="Italic">Scomber japonicus</Emphasis>

The physiological functions of pituitary gonadotropins (GtHs) are well established in higher vertebrates, whereas those in teleosts are still poorly understood. To describe the role of GtHs during gonadal development of female chub mackerel Scomber japonicus, changes in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells were investigated immunohistochemically during the seasonal reproductive and spawning cycles. FSH and LH cells were identified in the different cell types of the proximal pars distalis (PPD); FSH cells were located in the central PPD, whereas LH cells were localized along the border of the pars intermedia. To examine changes in FSH and LH cells, the percentage of FSH or LH cell-occupying area in the PPD was evaluated and represented as FSHβ-immunoreactive (ir) or LHβ-ir levels, respectively. FSHβ-ir levels increased significantly from immature to the completion of vitellogenesis, whereas LHβ-ir levels were maintained at high levels from early vitellogenesis to post-spawning. During the spawning cycle, which consisted of four stages from just after spawning to the next oocyte maturation, both FSHβ-ir and LHβ-ir levels showed no significant changes among different stages; however, LHβ-ir levels remained relatively high, and FSHβ-ir levels were constantly low. These results suggest that both FSH and LH may be involved in vitellogenesis and LH may act at final oocyte maturation in female chub mackerel, although the role of FSH during the spawning cycle is still unclear.     

Age validation,growth and annual reproductive cycle of chub mackerel <Emphasis Type="Italic">Scomber japonicus</Emphasis> off the waters of northern Kyushu and in the East China Sea

The age and growth of chub mackerel Scomber japonicus collected from the East China Sea and the northern waters off Kyushu between June 2000 and June 2001 were determined by observing the otolith surface after dipping it in xylene. The translucent and opaque zones on the otolith surface were identified, and the number of translucent zones was counted. Monthly changes in the frequency of fish with translucent zones on the otolith margin, and in the marginal increments, indicated that the translucent zones were formed between April and June. The seasonal pattern of annulus formation on the otolith became clear by observing the otoliths of fish with known ages, and the otolith formation in wild fish was consistent with that of fish with known ages. The mean gonadosomatic index of male and female fish was high from March to May, and spawning females were observed from mid-March to mid-May. The estimated ages were 1–5 years for males and 1–6 years for females. The von Bertalanffy growth parameters did not significantly differ between male and female. The model was obtained as FL _t=406×{1−exp[−0.372×(t+1.68)]     

Isolation and identification of DPPH radical scavenging compounds in Kurosu (Japanese unpolished rice vinegar)

Dihydroferulic acid (DFA) and dihydrosinapic acid (DSA) were isolated from Kurosu (unpolished rice vinegar) as the major constituents responsible for Kurosu's radical scavenging activity. The levels of antioxidative activity of DFA and DSA in DPPH radical scavenging were higher than those of their respective structurally related compounds, ferulic acid and sinapic acid. The concentrations of DFA and DSA were low in common rice vinegar (polished rice vinegar), suggesting that Kurosu is more advantageous than rice vinegars as an antioxidative food item. As the concentrations of DFA and DSA were low in unpolished rice, too, these acids are thought to be produced in Kurosu through the process of the fermentation from ferulic acid and sinapic acid, respectively.     

Development of a sandwich enzyme-linked immunosorbent assay (ELISA) to quantify γ-glutamyl-carboxylated clotting factor IX and assess redox susceptibility of anticoagulant chemicals

Anticoagulant chemicals (ACCs) such as warfarin are widely used in medical applications as well as for their rodenticide properties. Their efficacy is greatly influenced by polymorphisms in the gene encoding vitamin K epoxide reductase (VKOR). Evaluation of the activity of ACCs toward VKOR variants is essential to determine their proper use. Presently, this is achieved by co-expressing VKOR of Rattus Norvegicus and human clotting factor IX in cultured cells and measuring inhibition of vitamin K-dependent gamma-glutamyl carboxylation of factor IX (glaFIX) activity. However, glaFIX has only been quantified using indirect methods like blood coagulation assays. We have developed a sandwich enzyme-linked immunosorbent assay using a glaFIX-specific antibody to quantify glaFIX and used this to analyze inhibition of VKOR activity by warfarin.