Koji Matsuno

Placental transfer of melamine and its effects on rat dams and fetuses.

In 2008, an epidemic of cases of renal failure among Chinese infants, due to melamine contamination of milk, raised international concern. Thus, numerous studies on the metabolism of melamine were broadly undertaken. However, little is known about placental transfer of melamine. In this study, the possibility of placental transfer of melamine and its effects on fetuses and pregnant dams were determined. Melamine was respectively administered at 0, 40 and 400mg/kg body weight by daily gavage from gestation day (GD) 13 to GD 20 to control (C), low melamine (LM) and high melamine (HM) groups of pregnant female F344 rats. Rats were sacrificed 30min after the last gavage. Melamine was not detected in any of the control and placental samples, or in amniotic fluid from the LM group. Plasma and fetal melamine concentrations in the HM group were significantly higher than in the LM group (P<0.01). Liver enzyme determination revealed no differences among the three groups. However, plasma creatinine, plasma uric acid and blood urea nitrogen concentrations in dams were significantly increased by melamine (P<0.05). These results show that ingested melamine affects renal function in dams and dose-dependently passes the placental barrier to reach the fetus.

Inhibition of proliferation by agricultural plant extracts in seven human adult T-cell leukaemia (ATL)-related cell lines

Adult T-cell leukaemia (ATL) is caused by human T-cell leukaemia virus type I (HTLV-I) infection and is resistant to conventional chemotherapy. We evaluated the inhibitory effects of agricultural plants on the proliferation of seven ATL-related human leukaemia cells, using three ATL cell lines (ED, Su9T01 and S1T), two human T-cell lines transformed by HTLV-I infection (HUT-102 and MT-2) and two HTLV-I-negative human T-cell acute lymphoblastic leukaemia cell lines (Jurkat and MOLT-4). A total of 52 samples of 80% ethanol extracts obtained from 30 types of agricultural plants were examined. On the basis of IC50 values, we selected samples with greater activity than genistein, which was used as a positive control. The highest inhibitory effect was observed with extracts from leaves of Vaccinium virgatum Aiton (blueberry) on four cell lines (ED, Su9T01, HUT-102 and Jurkat); seeds of Momordica charantia L. (bitter gourd) exhibited the second highest activity. The bitter gourd seeds suppressed the proliferation of three cell lines (Su9T01, HUT-102 and Jurkat). The extracts from edible parts of Ipomea batatas LAM. (sweet potato), edible parts of Colocasia esculenta (L.) Schott (taro), skin of taro and seeds of Prunus mume Sieb. et Zucc. (mume) showed markedly greater inhibitory effects on Su9T01 than genistein. These findings suggest that ATL-preventative bioactive compounds may exist in these agricultural plants, which are considered to be functional foods.

Efficacy of Brazilian Propolis against Herpes Simplex Virus Type 1 Infection in Mice and Their Modes of Antiherpetic Efficacies

Ethanol extracts (AF-06, 07, and 08, 10 mg/kg) of Brazilian propolis were administered orally to cutaneously herpes simplex virus type 1 (HSV-1)-infected mice three times daily on days 0 to 6 after infection to evaluate their efficacies against HSV-1 infection and significantly limited development of herpetic skin lesions. AF-07 and 08 significantly reduced virus titers in brain and/or skin on day 4 without toxicity, but AF-08 had no anti-HSV-1 activity in vitro. AF-06 and 08 significantly enhanced delayed-type hypersensitivity (DTH) to inactivated HSV-1 antigen in infected mice. Oral AF-08-administration significantly augmented interferon (IFN)-γ production by HSV-1 antigen from splenocytes of HSV-1-infected mice, while direct exposure of splenocytes of infected mice to AF-06 significantly elevated IFN-γ production in vitro. Thus, AF-08 might have components that are active in vivo even after oral administration and those of AF-06 might be active only in vitro. Because DTH is a major host defense for intradermal HSV-1 infection, augmentation of DTH response by AF-06 or 08, directly or indirectly, respectively, may contribute to their efficacies against HSV-1 infection. In addition, AF-06 and 07 possibly contain anti-HSV-1 components contributing to their efficacies. Such biological activities of Brazilian propolis may be useful to analyze its pharmacological actions.

8-Hydroxydeoxyguanosine generated in the earthworm Eisenia fetida grown in metal-containing soil

Heavy metal pollution of soil causes biological problems, such as mutagenicity to living organisms, including human beings. However, few methods have been developed to assess metal mutagenicity in soil. To avoid metal mutagenicity, an adequate bio-monitoring method is required. In the present study, to determine if the analysis of oxidative DNA damage generated in the earthworm is a useful bio-monitoring method for soil mutagenicity, the accumulation of 8-hydroxydeoxyguanosine (8-OH-dG), a major form of oxidative DNA damage, in Eisenia fetida (Savigny, 1826) treated with cadmium chloride (CdCl2) or nickel chloride (NiCl2) was analyzed. E. fetida was treated with Cd (10 or 200 microg/g soil) or Ni (10 or 200 microg/g soil) for 1, 2, and 3 weeks or 3 months. After metal exposure, the metal concentration in E. fetida was analyzed by atomic absorption spectrometry and the 8-OH-dG accumulated in E. fetida was analyzed by HPLC analyses and immunohistochemistry. Atomic absorption spectrometry revealed that Cd, but not Ni, accumulated within E. fetida. The 8-OH-dG levels in the DNA of E. fetida treated with Cd for 3 months were significantly higher than those in control E. fetida. Moreover, immunohistochemical analyses revealed that positive signals for 8-OH-dG accumulation in seminal vesicles were detected only in E. fetida treated with 10 microg of Cd for 3 months. Although some points remain unresolved, a bio-monitoring system analyzing the DNA damage generated in the earthworm might be useful for the assessment of the mutagenicity of soil contaminated with various heavy metals, such as Cd.

Activation of Cellular Immunity in Herpes Simplex Virus Type 1-Infected Mice by the Oral Administration of Aqueous Extract of Moringa oleifera Lam. Leaves.

Moringa oleifera Lam. is used as a nutritive vegetable and spice. Its ethanol extract has been previously shown to be significantly effective in alleviating herpetic skin lesions in mice. In this study, we evaluated the alleviation by the aqueous extract (AqMOL) and assessed the mode of its anti‐herpetic action in a murine cutaneous herpes simplex virus type 1 (HSV‐1) infection model. AqMOL (300 mg/kg) was administered orally to HSV‐1‐infected mice three times daily on days 0 to 5 after infection. AqMOL significantly limited the development of herpetic skin lesions and reduced virus titers in the brain on day 4 without toxicity. Delayed‐type hypersensitivity (DTH) reaction to inactivated HSV‐1 antigen was significantly stronger in infected mice administered AqMOL and AqMOL augmented interferon (IFN)‐γ production by HSV‐1 antigen from splenocytes of HSV‐1‐infected mice at 4 days post‐infection. AqMOL administration was effective in elevating the ratio of CD11b+ and CD49b+ subpopulations of splenocytes in infected mice. As DTH is a major host defense mechanism for intradermal HSV infection, augmentation of the DTH response by AqMOL may contribute to their efficacies against HSV‐1 infection. These results provided an important insights into the mechanism by which AqMOL activates cellular immunity. Copyright

Establishment of a Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics Method and Its Application to Lichen Profiling

Direct-injection electron ionization-mass spectrometry (DI-EI-MS) is a multivariate analysis method useful for characterizing biological materials. We demonstrated the use of DI-EI-MS for metabolic profiling using several closely related lichen species: Cladonia krempelhuberi, C. gracilis, C. pseudogymnopoda, and C. ramulosa. The methodology involves conversion of total ion chromatograms to integrated chromatograms and assessment of reproducibility. The qualitative DI-EI-MS method was used to profile the major and/or minor constituents in extracts of lichen samples. It was possible to distinguish each lichen sample by altering the electron energy in DI-EI-MS and examining the resulting data using one-way analysis of variance. Previously undetectable peaks, which are easy to fragment could be revealed by varying the electron energy. Our results suggest that metabolic profiling using DI-EI-MS would be useful for discriminating between subgroups within the same species. This is the first study to report the use of DI-EI-MS in a metabolomics application.

Comparison of Cultivars and Seasonal Variation in Blueberry (Vaccinium Species) Leaf Extract on Adult T-Cell Leukemia Cell Line Growth Suppression

The inhibitory effects of blueberry leaves on the proliferation of adult T-cell leukemia (ATL) cell lines have previously been reported. A comparison of blueberry leaf extracts from different cultivars and seasonal variation were investigated regarding their effects on ATL cell line proliferation. The inhibitory effects of 80% ethanol leaf extracts from different blueberry cultivars collected from April to December in 2006 or 2008 were evaluated using two ATL cell lines. The bioactivities of leaf extracts of rabbit-eye blueberry (Vaccinium virgatum Aiton; RB species), southern highbush blueberry (V. spp.; SB species), northern highbush blueberry (V. corymbosum L.; NB species), and wild blueberry (V. bracteatum Thunb.; WB species) were compared. Of these, leaves of the RB species collected in December showed a significantly stronger inhibitory effect in both cell lines than the SB, NB, or WB species. These results suggest elevated biosynthesis of ATL-preventative bioactive compounds in the leaves of the RB species before the defoliation season.

Identification of a Bioactive Compound against Adult T-cell Leukaemia from Bitter Gourd Seeds

In our previous report, an 80% ethanol bitter gourd seed extract (BGSE) was found to suppress proliferation of adult T-cell leukemia (ATL) cell lines. The present study aimed to identify the bioactive compounds from BGSE specific against ATL. From the result of an HPLC-MS analysis, α-eleostearic acid (α-ESA) was present in BGSE at 0.68% ± 0.0022% (±SD, n = 5). In the cell proliferation test, α-ESA potently suppressed proliferation of two ATL cell lines (ED and Su9T01; IC50 = 8.9 and 29.3 µM, respectively) more than several other octadecanoic acids. However, α-ESA moderately inhibited phytohemagglutinin-activated human peripheral blood mononuclear cells (PBMC; IC50 = 31.0 µM). These results suggest that BGSE-derived α-ESA has potential as a functional food constituent because of its activity against ATL, particularly against ED cells. Moreover, α-ESA might be effective for the prevention of moderate adverse effects of ATL on normal T cells.