Naohide Kinae

Intestinal absorption of luteolin and luteolin 7-O-β-glucoside in rats and humans

In this study, we investigated the intestinal absorption of luteolin and luteolin 7‐O‐β‐glucoside in rats by HPLC. The absorption analysis using rat everted small intestine demonstrated that luteolin was converted to glucuronides during passing through the intestinal mucosa and that luteolin 7‐O‐β‐glucoside was absorbed after hydrolysis to luteolin. Free luteolin, its conjugates and methylated conjugates were present in rat plasma after dosing. This suggests that some luteolin can escape the intestinal conjugation and the hepatic sulfation/methylation. LC/MS analysis showed that the main conjugate which circulates in the blood was a monoglucuronide of the unchanged aglycone. Luteolin in propyleneglycol was absorbed more rapidly than that in 0.5% carboxymethyl cellulose. The plasma concentration of luteolin and its conjugates reached the highest level 15 min and 30 min after dosing with luteolin in propyleneglycol, respectively. HPLC analysis also allowed us to demonstrate the presence of free luteolin and its monoglucuronide in human serum after ingestion of luteolin.

Radioprotective effects of antioxidative plant flavonoids in mice

Radioprotective effects of tea infusions and plant flavonoids were investigated by using the micronucleus test for anticlastogenic activity and the thiobarbituric acid assay for antioxidative activity. A single gastric intubation of rooibos tea (Aspalathus linearis) infusion at 1 ml per mouse 2 h prior to gama-ray irradiation (1.5 Gy) reduced the frequency of micronucleated reticulocytes (MNRETs). After the fractionation of rooibos tea infusion, the flavonoid fraction was found to be most anticlastogenic and antioxidative. From this fraction, luteolin was isolated as an effective component. Then, anticlastogenic effects of 12 flavonoids containing luteolin and their antioxidative activities against lipid peroxidation by Fentons reagent were examined. A good correlation (r=0.717) was observed between both activities. Luteolin showed the most effective potency. A gastric intubation of luteolin (10 micromoles/kg) 2 h prior to gamma-ray irradiation (6 Gy) suppressed lipid peroxidation in mouse bone marrow and spleen and a trend of protective effect of luteolin against the decrease of endogenous ascorbic acid in mouse bone marrow after gamma-ray irradiation (3 Gy) was observed. These results suggest that plant flavonoids, which show antioxidative potency in vitro, work as antioxidants in vivo and their radioprotective effects may be attributed to their scavenging potency towards free radicals such as hydroxyl radicals. Therefore, the flavonoids contained in tea, vegetables and fruits seem to be important as antioxidants in the human diet.

Development of genotoxicity assay systems that use aquatic organisms.

Our aim is to develop and evaluate monitoring systems that use aquatic organisms to assess the genotoxicity of water in the field and in the laboratory. In a field study, we have shown that the micronucleus assay is applicable to freshwater and marine fishes and that gill cells are more sensitive than hematopoietic cells to micronucleus-inducing agents. Gill cells from Carassius sp. (Funa) and Zacco platypus (Oikawa) collected upstream on the Tomio River (Nara, Japan), tended to have lower micronucleus frequencies than gill cells from fish collected at the midstream of the river. Leiognathus nuchalis (Hiiragi) and Ditrema temmincki (Umitanago), small marine fishes collected periodically at Mochimune Harbor (Shizuoka, Japan), showed seasonal differences in the frequencies of micronucleated gill cells and erythrocytes; they were highest in summer. For laboratory studies, we developed a method for analyzing chromosomal aberrations and micronuclei using Rhodeus ocellatus ocellatus (rose bitterling) embryos. One day after artificial insemination (gastrula stage), we observed structural chromosomal aberrations and micronuclei in the cells of embryos grown in water containing trichloroethylene. Although more work is needed to fully assess their sensitivity, these assays show promise as a means of detecting environmental genotoxins.

Detection of DNA lesions induced by chemical mutagens using the single-cell gel electrophoresis (comet) assay. 2. Relationship between DNA migration and alkaline condition.

The alkaline condition is an important factor for the alkaline single-cell gel electrophoresis (SCG) assay to detect the genotoxic effects of chemicals. In order to understand the relationship between DNA migration and alkaline condition, the effect of 13 model chemical mutagens with different modes of action was evaluated with the alkaline SCG assay under two different alkaline conditions (pH 12.1 and 12.6). CHO cells were sampled just after treatment for 1 h. The X-ray mimetic mutagen BLM increased DNA migration at pH 12.1 and 12.6 and the results were the same at both pH values. Six alkylating mutagens MNU, ENU, MNNG, ENNG, MMS, and EMS and one base adduct inducer 4-NQO induced a dose-dependent response only at pH 12.6. Two DNA crosslinking agents, MMC and DDP, and AMD had negative results. MMC and DDP, however, reduced the positive response of BLM, suggesting that DNA crosslinks could be detected. These results demonstrated that the alkaline condition was important factor for the alkaline SCG assay to detect the genotoxic effects of chemicals.

Protective effect of flavonoids on doxorubicin-induced cardiotoxicity

We have examined the effect of alpha G-Rutin and luteolin on doxorubicin (DOX) toxicity in mice. In the heart, the lipid peroxide level, increased to 1.5 times of the normal level induced by DOX, decreased to the normal level after treatment with alpha G-Rutin or luteolin (i.p.). Glutathione peroxidase (GSHpx) activity, decreased to 73% of normal activity after DOX treatment, was shown to recover by the combined flavonoids. The lipid peroxide level in bone marrow cells increased to 5.9 times of the normal level by DOX treatment, whereas this level in the extra bone marrow cells did not change by treatment with DOX. The combination of alpha G-Rutin and luteolin with DOX significantly inhibited the DOX induced-increment of the lipid peroxide level in bone marrow cells. Flavonoids have also reduced the effect of DOX toxicity by oral administration. It is suggested that it is possible to reduce DOX toxicity by the intake of food including flavonoids. In NADPH-dependent lipid peroxidation, alpha G-Rutin and luteolin showed concentration-dependent inhibition. Therefore, we considered that the reduction effect of DOX toxicity by flavonoids was caused by antioxidative action and other effect of the flavonoids.

Genotoxicity of nano/microparticles in in vitro micronuclei, in vivo comet and mutation assay systems

BackgroundRecently, manufactured nano/microparticles such as fullerenes (C60), carbon black (CB) and ceramic fiber are being widely used because of their desirable properties in industrial, medical and cosmetic fields. However, there are few data on these particles in mammalian mutagenesis and carcinogenesis. To examine genotoxic effects by C60, CB and kaolin, an in vitro micronuclei (MN) test was conducted with human lung cancer cell line, A549 cells. In addition, DNA damage and mutations were analyzed by in vivo assay systems using male C57BL/6J or gpt delta transgenic mice which were intratracheally instilled with single or multiple doses of 0.2 mg per animal of particles.ResultsIn in vitro genotoxic analysis, increased MN frequencies were observed in A549 cells treated with C60, CB and kaolin in a dose-dependent manner. These three nano/microparticles also induced DNA damage in the lungs of C57BL/6J mice measured by comet assay. Moreover, single or multiple instillations of C60 and kaolin, increased either or both of gpt and Spi- mutant frequencies in the lungs of gpt delta transgenic mice. Mutation spectra analysis showed transversions were predominant, and more than 60% of the base substitutions occurred at G:C base pairs in the gpt genes. The G:C to C:G transversion was commonly increased by these particle instillations.ConclusionManufactured nano/microparticles, CB, C60 and kaolin, were shown to be genotoxic in in vitro and in vivo assay systems.

Studies on the toxicity of pulp and paper mill effluents—I. Mutagenicity of the sediment samples derived from Kraft paper mills

Sediment samples collected at three different coastal points (Shinguu, Ooigawa and Tagonoura) were extracted with diethylether and then methanol. Each extract was separated into five fractions by high-speed liquid chromatography and they were submitted to mutagenic assay using B. subtilis and S. typhimurium and to GC-MS analysis. The ether fraction of sediment sample from Shinguu showed DNA damaging potency on B. subtilis and exhibited mutagenic effect on S. typhimurium TA 98, TA 100 and TA 1537. 2,4,6-Trichlorophenol, 3,4,5,6-tetrachloroguaiacol, dehydroabietic acid, pyrene and fluoranthene were mutagenic substances among 28 substances identified and tested.

Depressive symptoms are independently correlated with lipid peroxidation in a female population: Comparison with vitamins and carotenoids

OBJECTIVE Lipid peroxidation (LPO) is involved in oxidative tissue injuries. The present investigation examined the association between LPO and psychological depressive symptoms. METHODS A cross-sectional study was conducted on 66 female volunteers aged 38-70. Lipid peroxides (LOOH) in serum were evaluated by hemoglobin-methylene blue (Hb-MB) method; additionally, serum antioxidants were also detected. To assess depressive symptoms, the Center for Epidemiologic Studies Depression (CES-D) Scale and a subscale in the 28-item General Health Questionnaire (GHQ) were applied. RESULTS LOOH concentration displayed a significant positive correlation with CES-D and GHQ depression scores. Multiple regression analysis was performed in which LOOH concentration served as a dependent variable and CES-D scores and antioxidants as independent variables. Consequently, CES-D scores demonstrated significant positive correlation with LOOH. CONCLUSIONS The positive relationship between depressive symptoms and LPO in a female population may support the hypothesis that LPO may affect depressive symptoms.

Detection of in vivo genotoxicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) by the alkaline single cell gel electrophoresis (Comet) assay in multiple mouse organs

We tested the genotoxicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) in the mouse in 6 organs (liver, lung, kidney, brain, spleen, and bone marrow) and in the mucosa of stomach, jejunum, ileum, colon, and bladder using the alkaline single-cell gel electrophoresis (SCG) (Comet) assay modified by us. Mice were sacrificed 1, 3, 6, and 24 h after oral administration of the mutagen at 100 mg/kg. MX yielded statistically significant DNA damage in the liver, kidney, lung, and brain and in all the mucosa samples. While DNA damage persisted in the gastrointestinal and urinary tract for 6-24 h after a single oral dosing, it peaked in the liver at 1 h and returned to almost the control level at 3 h. Our present results suggest that MX is genotoxic for various mouse organs, but not for the hematopoietic system, and that the alkaline SCG assay with a homogenization technique can be used to predict genotoxicity in the gastrointestinal and urinary tracts.

The clastogen-suppressing effects of green tea, Po-lei tea and Rooibos tea in CHO cells and mice.

The suppressing effects of crude extracts of three kinds of tea-green tea (GT) from Japan, Po-lei tea (PT) from China, and Rooibos tea (RT) from South Africa-on the induction of chromosome aberrations in cultured CHO cells and mice were studied. When CHO cells were exposed to each tea extract in the presence of rat liver microsomal enzymes (S9 mix) together with benzo[a]pyrene (B(a)P) or mitomycin C (MMC), a decrease in the frequency of chromosome aberrations was observed. PT and RT, but not GT, also suppressed the induction of chromosome aberrations by MMC in the absence of S9 mix. When cells were treated with tea extract after B(a)P or MMC treatment, RT suppressed the induction of chromosome aberrations in the presence and absence of S9 mix whereas GT and PT showed suppressing effects only in the presence of S9 mix. These data suggest that catechines, well-known antimutagens in tea samples, might account for the inhibitory effect in the case of GT and PT. Since RT contains few catechines, several unknown antimutagenic components could be responsible for its effect. The antimutagenic effects of tea extracts at concentration levels consumed by humans were examined in mice using micronucleus induction with B(a)P or MMC. When mice received oral gavage of 0.2% GT, 0.1% PT, and 0.1% RT at 1.0 ml/mouse 6 h before intraperitoneal injection of MMC, a decrease in the frequency of micronuclei was observed. The induction of micronuclei by B(a)P was suppressed by oral dosage of GT, PT and RT at 1.0 ml/mouse/day for 28 days. This was not due to a delay in the maturation of micronucleated reticulocytes. In conclusion, intake of tea might suppress the mutagenic activity of certain potent mutagens in human beings.