Testing mosses exposed in bags as biointerceptors of airborne radiocaesium after the Fukushima Dai-ichi Nuclear Power Station accident.

Eight years after the Fukushima nuclear accident, mosses exposed in bags were used to investigate their ability to accumulate radiocaesium and therefore to act as biointerceptors of 134Cs and 137Cs in the evacuated area of the Fukushima territory. Bags were filled with 3 widely studied moss species (Sphagnum palustre, Hypnum cupressiforme, and Hypnum plumaeforme) and exposed for 3, 6 or 9 weeks at 5 former residential sites within the Fukushima area and, for comparison, at three background sites located 700 km away. The radiocaesium activity concentrations found in moss bags were evaluated as function of exposure time, site conditions and moss species. In the Fukushima area, the moss bags accumulated 137Cs at all exposure sites and in all exposure periods, with S. palustre having the highest 137Cs accumulation ability. The 137Cs activity concentrations (from 28 to 4700 Bq kg-1) measured in moss bags increased with the exposure time and were consistent with the decontamination status of each exposure site, highlighting the big potential of moss bags to discriminate among exposure sites. Time dependency of 137Cs activity concentrations measured in mosses allowed the calculation of location-specific and species-specific factors, which can be used to predict radiocaesium accumulation trends in future biomonitoring surveys performed in the same area with the same experimental design. Autoradiography and electron microscopy analyses of the moss surfaces revealed a prevalence of soil-derived particulate form of radiocaesium, suggesting the use of moss bags as warning sensors of resuspended particles potentially harmful for local residents.

Spinal cord involvement by atrophy and associations with disability are different between multiple sclerosis and neuromyelitis optica spectrum disorder.

BACKGROUND AND PURPOSE: The cervical and thoracic cross-sectional spinal cord area (CS-SCA) in multiple sclerosis (MS) correlates with disability, whilst such a correlation remains to be established in neuromyelitis optica spectrum disorder (NMOSD). Our aim was to clarify differences between MS and NMOSD in spinal cord segments where CS-SCA is associated with disability. METHODS: The CS-SCA at C2/C3, C3/C4, T8/T9 and T9/T10 vertebral disc levels was measured in 140 MS patients (111 with relapsing-remitting MS and 29 with progressive MS) and 42 NMOSD patients with anti-aquaporin-4 immunoglobulin G. Disability was evaluated by Expanded Disability Status Scale (EDSS) scores. Multivariate associations between CS-SCA and disability were assessed by stepwise forward multiple linear regression. RESULTS: Thoracic CS-SCA was significantly smaller in NMOSD patients than in MS patients even after adjusting for age, sex and disease duration (P = 0.002 at T8/T9), whilst there was no difference in cervical CS-SCA between the two diseases. Cervical and thoracic CS-SCA had a negative correlation with EDSS scores in MS patients (P < 0.0001 at C3/C4 and P = 0.0002 at T8/T9) whereas only thoracic CS-SCA correlated with EDSS scores in NMOSD patients (P = 0.0006 at T8/T9). By multiple regression analyses, predictive factors for disability in MS were smaller cervical CS-SCA, progressive course, older age and a higher number of relapses, whilst those in NMOSD were smaller thoracic CS-SCA and older age. CONCLUSIONS: Thoracic CS-SCA is a useful predictive marker for disability in patients with NMOSD whilst cervical CS-SCA is associated with disability in patients with MS.

Suppression of streptozotocin-induced type-1 diabetes in mice by radon inhalation.

We examined the protective effect of radon inhalation on streptozotocin (STZ)-induced type-1 diabetes in mice. Mice inhaled radon at concentrations of 1000, 2500, and 5500 Bq/m3 for 24 hours before STZ administration. STZ administration induced characteristics of type-1 diabetes such as hyperglycemia and hypoinsulinemia; however, radon inhalation at doses of 1000 and 5500 Bq/m3 significantly suppressed the elevation of blood glucose in diabetic mice. Serum insulin was significantly higher in mice pre-treated with radon at a dose of 1000 Bq/m3 than in mice treated with a sham. In addition, superoxide dismutase activities and total glutathione contents were significantly higher and lipid peroxide was significantly lower in mice pre-treated with radon at doses of 1000 and 5500 Bq/m3 than in mice treated with a sham. These results were consistent with the result that radon inhalation at 1000 and 5500 Bq/m3 suppressed hyperglycemia. These findings suggested that radon inhalation suppressed STZ-induced type-1 diabetes through the enhancement of antioxidative functions in the pancreas.

Radioactivity of 210Pb in Japanese cigarettes and radiation dose from smoking inhalation.

It is well known that cigarette tobaccos contain naturally occurring radioactive nuclides such as (210)Pb and (210)Po. In many countries, the radioactivity of tobaccos has been measured to estimate the effective dose from smoking inhalation. The present study covered 24 cigarette brands including the top 20 of sales in Japan between April 2008 and March 2009. The activity concentrations of (210)Pb were measured by gamma-ray spectrometry, and then those of its progeny ((210)Po) were evaluated assuming the radioactive equilibrium between the two nuclides. Their concentrations were in the range of 2-14 mBq cigarette(-1) with an arithmetic mean of 8±3 mBq cigarette(-1). The annual committed effective doses were also calculated, based on the scenario that a smoker consumes 20 cigarettes a day. The average doses from (210)Pb and (210)Po inhalations were 22±9 and 68±27 µSv y(-1), respectively.

Straw preservation effects of Arundo donax L. on its allelopathic activity to toxic and bloom-forming Microcystis aeruginosa.

In this study, straw preservation effects of Arundo donax L. on its allelopathic activity to toxic and bloom-forming Microcystis aeruginosa were investigated. The aquatic extracts of fresh straw significantly inhibited the growth of M. aeruginosa. After 7 d of cultivation, the percentage inhibitions (Pls) of all treated groups were above 60% and the maximum almost reached 100%. However, when the straw was used after two-month preservation, its allelopathic activities decreased significantly with all PIs lower than 60%. To unclose the substance differences between fresh and long-time preserved straws, the contents of dissolved organic carbon (DOC) and ultraviolet-visible and fluorescence characteristics were analyzed. The results show that long-time preserved straw had lower DOC and UV254 values, weaker fluorescence intensities, and fluorescent substance loss. Further, the allelopathic fractions were isolated from the aquatic extracts of the straws by solvent extraction. The potential allelochemicals were analyzed by gas chromatography-mass spectrometry (GC-MS). The strong antialgal fractions of both fresh and long-time preserved straws were found to have various potential allelochemicals, including esters, ketones, alkaloids and phenolic acids. Their number was decreased from 21 to 9 after two-month preservation. The characteristics analysis of potential allelochemicals hinted that fresh A. donax might have more highly-effective allelochemicals than long-time preserved one.

Elution and adsorptive concentration of Japanese cedar (Cryptomeria japonica) pollen allergen in environmental water.

Elution of Japanese cedar pollen allergens (Cry j I and others) from pollen grains and its adsorptive concentration onto hydrophobic and hydrophilic surfaces were investigated using the surface plasmon resonance technique. Results showed that the allergen elution was obviously enhanced when the ion concentration was higher than that within the human body, indicating that the pollen tend to release its allergen in environmental water having a high ion concentration. However, higher adsorption capacity was observed on hydrophobic surface than hydrophilic surface. These results indicate that water puddles on roadsides beside heavy traffic including large amounts of ion compounds and hydrophobic diesel exhaust particles (DEPs) are a pollen allergen-DEP complex generator. DEPs are easily absorbed into the living body; therefore these mechanisms may be responsible for causing the highest incidence of pollinosis among residents living alongside roads with heavy automobile traffic.

Enhanced in vitro maturation of subcultivated fetal human hepatocytes in three dimensional culture using poly-L-lactic acid scaffolds in the presence of oncostatin M.

Fetal human liver cell fractions, which contain large numbers of hepatocyte progenitors, have high proliferation potential in vitro. To create an engineered liver tissue equivalent of a clinically significant size, however, repeated subcultivation and functional maturation are necessary in vitro. A commercially available human fetal liver cell fraction that was cultivated for some time in vitro has been reported to lose liver specific functions almost completely. We therefore investigated the effects of oncostatin M (OSM) and hepatocyte growth factor (HGF) in long-term three-dimensional (3D) culture using macroporous poly-L-lactic acid (PLLA) scaffolds on the restoration of such liver-specific functions of the fraction. 3D culture using PLLA scaffolds with OSM remarkably enhanced the albumin production and cytochrome P450 1A1/2 capacity with the culture time. HGF alone had no preferable effect on these functions even in 3D culture. Alpha-fetoprotein production was consistently suppressed in the 3D culture compared with that in monolayers. This suppression was not observed in the same types of culture of hepatocarcinoma Hep G2 cells. Despite these favorable observations on the 3D culture with OSM, the final attained functional levels at the 5th week were still over ten-times lower than those of Hep G2 cells when standardized with a cellular DNA amount. Although further improvement is needed for the complete functional restoration and maturation in vitro, these results demonstrate that a combination of 3D culture using PLLA scaffolds and OSM offers promising culture conditions for in vitro maturation of human hepatocyte progenitors.

Catalytic ozonation of an organophosphorus pesticide using microporous silicate and its effect on total toxicity reduction.

Catalytic ozonation is promising as one of the advanced oxidation processes because of its effective use of ozone and its improved treatability of organic compounds through radical reactions. In this article, we investigated the feasibility of microporous silicates as a potential catalyst for a catalytic ozonation process. Organophosphorus insecticide, dichlorvos (DDVP), was employed as a model chemical for assessing conventional ozonation and catalytic ozonation, because its oxidative intermediate is toxic and is not degraded by direct ozonation. It was found that ozone was well adsorbed and simultaneously decomposed in microporous silicates, resulting in the production of possible radical species. In the presence of microporous silicates, radical reactions by decomposed ozone were evidenced by phosphate ion release that shows a degradation of a toxic intermediate of DDVP. Accordingly, cytotoxicity was successfully decreased. In a continuous treatment process combining a conventional ozonation vessel and a microporous silicate column for an effective use of residual ozone, enhanced degradation of DDVP was demonstrated by a decrease of DOC, an increase of the PO4(3-) concentration and reduction in the cytotoxicity. This new treatment mechanism is likely to be promising as an advanced water treatment process particularly when we think about better toxicity reduction of wastewaters.

Preservation of human cell bioassay device for the on-site evaluation of environmental waters.

We have already developed a novel disposable bioassay device based on the low-density lipoprotein (LDL) uptaking activity of human hepatoblastoma Hep G2 cells in our previous work. However, this device is not readily applicable to evaluate river water toxicity on-site because it cannot be preserved for more than one week. In this work, we developed the method for preservation of the device to enable it to be preserved for at least one month. The device can be supplied to individual environmental sites without any facilities for cell culture. We can evaluate river water toxicity by 2 hours of exposure after thawing. Therefore, this kind of device could be a promising tool for daily water quality management.

Cultured human-cell-based bioassay for environmental risk management.

Among bioassays for evaluating various impacts of chemicals on humans and ecosystems, those based on cultured mammalian-cells can best predict acute lethal toxicity to humans. We expect them to be employed in the future in environmental risk management alongside mutagenicity tests and endocrine-disrupting activity tests. We recently developed a disposable bioassay device that immobilizes human hepatocarcinoma cells in a small micropipette tip. This enables very quick (within 2 h) evaluation of acute lethal toxicity to humans. For bioassay-based environmental management, 2 promising approaches have been demonstrated by the US-EPA: toxicity identification evaluation (TIE) and toxicity reduction evaluation (TRE). The Japanese Ministry of Environment has been supporting a multi-center validation project, aimed at assembling a bioassay database. To make full use of these resources, we present a numerical model that describes contribution of individual chemical to observed toxicity. This will allow the selection of the most effective countermeasure to reduce the toxicity. Bioassay-based environmental risk management works retrospectively, whereas impact assessment using substance flow models and toxicity databases works prospective. We expect that these 2 approaches will exchange information, act complementarily, and work effectively in keeping our environment healthy in the 21 st century.

A modelling approach to global nitrate leaching caused by anthropogenic fertilisation.

Water quality associated with nitrate (NO3-) leaching from agricultural soils is an important environmental issue. This paper describes a new modelling approach to quantitatively evaluate the effect of the use of fertilisers on global nitrate leaching. A global process-based simulation model was previously developed for the nitrogen cycle in terrestrial ecosystems, in which soil inorganic nitrogen in the form of ammonium (NH4+) and NO3- was considered. After introducing data on world fertiliser consumption (FAO, 1995) into the steady-state model, the extent of disturbance to the nitrogen cycle caused by fertilisation was calculated. Although fertilisation resulted in an annual increase in net primary production (NPP, represented as carbon) of 18 Gt year-1, NO3- leaching and gaseous losses of nitrogen oxides and ammonia were also accelerated. Most regions with heavy fertiliser application (over 100 kg ha-1 year-1) showed a high annual leaching load equal to or more than 20 kg ha-1 year-1. About 2.5% of the land area occupied by terrestrial ecosystems suffered a serious leaching load of more than 30 kg ha-1 year-1. Of the total amount of applied fertilisers (138 Tg year-1) 19% was lost to NO3- leaching, 8% to gaseous ammonia, and 3% to gaseous nitrogen oxides. The rest was assumed to be fixed in the ecosystems through vegetation uptake.

A mathematical model of a shallow and eutrophic lake (the Keszthely Basin, Lake Balaton) and simulation of restorative manipulations.

Concern about the overall management of lakes has been growing, and a lake ecological model provides the guidelines necessary for such management. In this study, an ecological model describing the ecosystem of the Keszthely Basin, Lake Balaton, Hungary, one of the typical shallow and eutrophic lakes, was proposed. This model includes three types of zooplankton and two types of fish as well as two types of algae and nutrients. Parameters concerning the algae and fish were estimated based on observations in the basin between 1991 and 1995. The other parameters and the structure of the model were determined by our previous study. The parameters of the model were calibrated with the Monte Carlo technique, and its predictability was confirmed. The effects on the basin's ecosystem of three restorative manipulations, namely a biomanipulation, reduction of loading phosphorus, and dredging the sediment, were assessed by simulation studies using the proposed model. The simulation results indicated that a biomanipulation that removed 90% of the bream should suppress the growth of algae temporarily through bottom-up regulation; however, this effect seemed to not be perpetuated in this basin. The reduction of loading phosphorus seemed to be the most effective means to suppress algal growth, while dredging of sediment seemed to be the most desirable restoration method from the standpoint of the overall management of the lake, because it was expected to accelerate the growth of fish population as well as to suppress algal growth. Furthermore, the algal growth suppression mechanism of the dredging was discussed on the basis of the model calculations.

Biological efficiency and nutritional value of Pleurotus ostreatus cultivated on spent beer grain.

Unpretreated spent beer grains were successfully used as a basic substrate material for the cultivation of Pleurotus ostreatus. The effects of spent grain types, additives, substrate moisture content, and substrate packing density on the yield and nutrition of fruit bodies were investigated. The cultivation results showed that few fruit bodies were formed on spent grain alone; however, a significantly high biological efficiency (19.1%) was obtained with the addition of wheat bran to (45%). The chemical analysis of fruit bodies indicated that P. ostreatus cultivated on spent grain substrate had a higher nutritional value than those grown on other reported types of substrates. The total amino acid content in the fruit bodies was 347.5 mg/g dry matter, and the crude protein content was as high as 53.3% on a dry weight basis. It was also found that the cultivation of P. ostreatus increased the crude protein content, while it decreased the ratio of lignin to cellulose, of the spent grain substrate.

Development of a rapid and sensitive bioassay device using human cells immobilized in macroporous microcarriers for the on-site evaluation of environmental waters.

We developed a novel disposable bioassay device based on the fluorescein isothiocyanate-labelled low-density lipoprotein-uptake activity of human hepatoblastoma Hep G2 cells. The cells were cultured in porous microcarriers at a high cell density and packed in a filter tip that has a hydrophobic membrane. Upon evaluation of water samples, the culture medium was decanted by pipetting it down with a micropipet, and the samples were then introduced to the cell-immobilizing part of the tip only by pipetting them up after mixing them with x10 concentrated culture medium. The new device enabled us to detect almost the same toxicity levels of river water within 2 h of exposure as those detected by a conventional 48-h cell-survival assay. This is the first bioassay device for the rapid on-site evaluation of environmental waters using cultured human cells, and therefore promising for water-quality management based on risk to humans.

Rapid and sensitive neurotoxicity test based on the morphological changes of PC12 cells with simple computer-assisted image analysis.

In order to develop a rapid and sensitive bioassay for the screening of chemicals with possible neurotoxicity, a computer-assisted simple image-analysis system was developed to quantify small changes in the specific morphology of the cultured pheochromocytoma cell line, PC12. This cell forms a neuron-like microfibril network (neurites) in response to a nerve growth factor (NGF) stimulation in vitro. Dichrolvos (DDVP) and methylmercury chloride (MMC) were employed as model neurotoxicants. In DDVP treatment, there was no large difference in the ED50s (effective dose that reduces the morphological index by 50%) among the toxicities determined from various morphological indices, but they were significantly lower than those observed by whole-cell-area-based toxicity assay using the hepatoblastoma cell line, Hep G2. In contrast, in MMC treatment, neurite-length-based toxicity was observed as early as 2 h, and at 48 h this was lower by over three orders of magnitude compared with whole-cell-area-based one (2.06 x 10(-7) mM vs. 6.42 x 10(-4) mM). These results demonstrate that the developed bioassay using image analysis of nerve-tissue-derived cell morphology allows us to screen possible neurotoxic chemicals very rapidly with highly enhanced sensitivity, particularly for some chemicals that preferentially act on nerve fibers.

Preservation of microplate-attached human hepatoma cells and their use in cytotoxicity tests.

We investigated the feasibility of hypothermic- orcryogenically-preserved human hepatoma Hep G2 cell preculturedin 96-well plates in cytotoxicity testings. First, we observedthat microplates precoated with both collagen (CN) and pronectin (PN) showed significantly improved living cell adhesion (71.0 +/- 5.5%) after 48 hr of cryopreservation with 10%-DMSO containing culture medium, whereas non-coated surfaces gave very low living cell adhesion (33.5 +/- 2.1%). Hypothermic preservation was most suitable for short-term storage, and cryogenic preservation at -20 degrees C allowed cells to be used within a week of the storage period. Only cryopreservation in a deep freezer (-85 degrees C) gave satisfactory results in much longer period of storage. Second, we evaluated the cytotoxicity of ten chemicals during 48 hr of exposure using hypothermically - (4 degrees C for 2 days) or cryogenically - (-85 degrees C for 7 days) preserved cells cultured inCN/PN-precoated microplates in comparison with results fromfreshly inoculated cells. Although almost the same LD(50)values were obtained, LD(10) values of relatively hydrophilic chemicals obtained with cryopreserved cell were significantly lowered. These results shown that CN/PN-precoating is effective in keeping cells attached even in recultivation of preserved cells and that the toxicities of relatively hydrophilic chemicals tend to be overestimated when we use preserved cells in that manner.

Quantitative comparison of rat hepatocyte functions in two improved culture systems with or without rat liver epithelial cell line.

We quantitatively evaluated two recently-developed novel techniques for hepatocyte cultivation in a dish level; that is, spheroid culture and membrane-supported collagen (CN) gel sandwich culture, in terms of cellular maintenance, albumin secretion and 7-ethoxycoumarin (7EC) metabolism to 7-hydroxycoumarin (7HC) as a marker for cytochrome P450 IA1 activity in the presence and absence of rat liver epithelial cell line (RLEC) during one month of culture, together with conventional coculture with RLEC in CN-coated dishes as a control. RLEC prevented spheroid loss caused by its detachment from the culture dishes often occurring in pure culture. CN-gel sandwich by itself improved remarkably hepatocyte maintenance when compared with CN-gel free systems, thereby resulting in enhancement of overall functional expressions as compared with CN-gel free systems. RLEC in CN-gel sandwhich, however, reduced cellular sustainment probably due to its suppression of hepatocyte growth. Although there were no significant differences in albumin secretion per cell among the five cultures examined, CN-gel sandwich expressed markedly higher 7EC metabolizing activity per cell, where RLEC presence had a preferable influence. Consequently, membrane-supported CN-gel sandwich was the most superior technique for hepatocyte cultivation from the standpont of both cellular maintenance and its functional expressions per cell.

Long-term culture of primary rat hepatocytes with high albumin secretion using membrane-supported collagen sandwich.

Primary cultured rat hepatocytes in a membrane-supported collagen sandwich maintained their normal cell morphology and high level of albumin secretion for over 56 days. It was found that the existence of an upper layer of collagen gel is crucial for long-term culture and that the transference of cellular nutrients between the culture media and hepatocytes from both the upper and the lower sides of gel layers promotes albumin secretion. These facts suggest that the membrane-supported collagen sandwich mimics well the in vivo environment of hepatocytes. This method has great potential for the long-term culture of primary cells.

Protein separation using membrane-encapsulated soluble ligand conjugates.

A new approach for isolating and recovering biological macromolecules using membrane-encapsulated soluble ligand conjugates was investigated. Membrane-encapsulated solid adsorbents have been successfully developed and employed in our laboratory to isolate and purify proteins and enzymes directly from culture broths. This new concept also makes it possible to use soluble ligand conjugates instead of solid adsorbents inside membrane capsules. In this work, model membrane-encapsulated soluble and insoluble ligands comprising Blue Dextran and Blue Sepharose entrapped within calcium alginate membranes were studied to compare adsorption characteristics such as capacities and rates. Experimental results suggest that membrane-encapsulated soluble ligands may be expected to result in higher overall adsorption capacity compared to membrane-encapsulated solid adsorbents with comparable adsorption rates.

A new isolation and purification method for staphylococcal protein A using membrane encapsulated rabbit IgG-agarose.

A new isolation and purification method for bioproducts using membrane-encapsulated affinity adsorbents was investigated. The new method involves encapsulation of affinity adsorbents, batch adsorption of the bioproduct from whole fermentation broth and rapid batch desorption after dissolution of the capsule membranes. Recovery of protein A from Staphylococcus aureus was used as the model experimental system. Affinity adsorbents such as rabbit IgG-agarose were successfully encapsulated within calcium alginate membranes and used directly to recover protein A from whole cell homogenate containing a number of macromolecular contaminants as well as suspended solids. Both high yield and high purity of protein A were recovered by this method in comparison with various previously reported methods.