In vivo examination of the genotoxicity of the urban air and surface soil pollutant, 3,6-dinitrobenzo[e]pyrene, with intraperitoneal and intratracheal administration.

3,6-Dinitrobenzo[e]pyrene (3,6-DNBeP) was identified as a new potent mutagen toward Salmonella strains in surface soil and airborne particles. Because data of in vivo examination of the genotoxicity of 3,6-DNBeP are limited, micronucleus test was performed in peripheral blood and bone marrow, and comet assay in the lungs of mice treated with 3,6-DNBeP. In male ICR mice intraperitoneally (i.p.) injected with 3,6-DNBeP, the frequency of micronuclated polychromatic erythrocytes (MNPCEs) was increased in the peripheral blood and bone marrow after 24 h in a dose-dependent manner. Compared to controls, the highest dose of 3,6-DNBeP (40 mg/kg B.W.) induced 7.3- and 8.7-fold increases of MNPCE frequency in the peripheral blood and bone marrow, respectively. Furthermore, when 3,6-DNBeP was intratracheally (i.t.) instilled to male ICR mice, 3,6-DNBeP at the highest dose of 0.1 mg/kg body exhibited 3.1-fold increase of DNA tail moment in the lungs at 3 h after the instillation compared to controls. The values of DNA tail moment at 9 and 24 h after the instillation were increased up to 3.5 and 4.2-fold, respectively. These data indicate that 3,6-DNBeP is genotoxic to mammalians in in vivo and suggest that 3,6-DNBeP may be a carcinogenic compound present in the human environment.

Associations of depressive symptoms with serum proportions of palmitic and arachidonic acids, and α-tocopherol effects among male population--a preliminary study.

BACKGROUND & AIMS: Recent years, inflammation and oxidative stress have been addressed in relation to interactions between fatty acid (FA) and depression. To study the associations between FAs and depressive symptoms in men, serum FA proportion was compared with perceived depression. We also measured α-tocopherol (a-Toc) levels to investigate the associations with FA functions. METHODS: A cross-sectional study was performed on 113 male workers recruited from a software development company in Japan. Depressive symptoms were assessed according to the 20-item Center for Epidemiologic Studies Depression (CES-D) scale. Twenty-four FAs in the serum from the peripheral blood were examined. RESULTS: CES-D scores were significantly positively correlated with the serum percentage of palmitic acid (PA), while they were negatively correlated with arachidonic acid (AA). The CES-D scores were not correlated with the serum ratio of docosahexaenoic acid or eicosapentaenoic acid (n-3 poly-unsaturated FAs (PUFAs)). CES-D scores were significantly negatively correlated with a-Toc/PA ratio (correlation: adjusting for possible confounders). CONCLUSIONS: Although no associations were found between depressive symptoms and n-3 PUFA proportions in male population, depressive symptoms were positively correlated with PA percentages and negatively correlated with AA percentages. PA may increase neural vulnerability and AA may decrease the severity of depression. Moreover, a-Toc may have protective effects against depressive symptoms.

Change in mutagenic activity of genistein after a nitrite treatment.

This study examined the mutagenic activity of genistein after a nitrite treatment under acidic conditions. Nitrite-treated genistein exhibited mutagenic activity toward Salmonella typhimurium strains TA 100 and TA 98 with or without S9 mix. Nitrite-treated genistein was demonstrated by electron spin resonance to generate radicals. An instrumental analysis showed 3'-nitro-genistein to have been formed in the reaction mixture. However, 3'-nitro-genistein did not exhibit mutagenic activity toward the S. typhimurium strains, suggesting that other mutagens might also have been formed in the reaction mixture. The clastogenic properties of nitrite-treated genistein and 3'-nitro-genistein were examined by a micronucleus test with male ICR mice. Nitrite-treated genistein and 3'-nitro-genistein showed a significantly higher frequency of micronucleated reticulocytes in mice than in the control group. These results suggest that a daily oral intake of genistein and nitrite through foodstuffs might induce the formation of various mutagenic compounds in the body.

Acrylamide genotoxicity in young versus adult gpt delta male rats.

The recent discovery that the potent carcinogen acrylamide (AA) is present in a variety of fried and baked foods raises health concerns, particularly for children, because AA is relatively high in child-favoured foods such as potato chips and French fries. To compare the susceptibility to AA-induced genotoxicity of young versus adult animals, we treated 3- and 11-week-old male gpt delta transgenic F344 rats with 0, 20, 40 or 80 p.p.m. AA via drinking water for 4 weeks and then examined genotoxicity in the bone marrow, liver and testis. We also analysed the level of N7-(2-carbamoyl-2-hydroxyethyl)-guanine (N7-GA-Gua), the major DNA adduct induced by AA, in the liver, testis and mammary gland. At 40 and 80 p.p.m., both age groups yield similar results in the comet assay in liver; but at 80 p.p.m., the bone marrow micronucleus frequency and the gpt-mutant frequency in testis increased significantly only in the young rats, and N7-GA-Gua adducts in the testis was significantly higher in the young rats. These results imply that young rats are more susceptible than adult rats to AA-induced testicular genotoxicity.

Genotoxicity of acrylamide in vitro: Acrylamide is not metabolically activated in standard in vitro systems.

The recent finding that acrylamide (AA), a genotoxic rodent carcinogen, is formed during the frying or baking of a variety of foods raises human health concerns. AA is known to be metabolized by cytochrome P450 2E1 (CYP2E1) to glycidamide (GA), which is responsible for AA's in vivo genotoxicity and probable carcinogenicity. In in-vitro mammalian cell tests, however, AA genotoxicity is not enhanced by rat liver S9 or a human liver microsomal fraction. In an attempt to demonstrate the in vitro expression of AA genotoxicity, we employed Salmonella strains and human cell lines that overexpress human CYP2E1. In the umu test, however, AA was not genotoxic in the CYP2E1-expressing Salmonella strain or its parental strain. Moreover, a transgenic human lymphoblastoid cell line overexpressing CYP2E1 (h2E1v2) and its parental cell line (AHH-1) both showed equally weak cytotoxic and genotoxic responses to high (>1 mM) AA concentrations. The DNA adduct N7-GA-Gua, which is detected in liver following AA treatment in vivo, was not substantially formed in the in vitro system. These results indicate that AA was not metabolically activated to GA in vitro. Thus, AA is not relevantly genotoxic in vitro, although its in vivo genotoxicity was clearly demonstrated.

Induction effect of coadministration of soybean isoflavones and sodium nitrite on DNA damage in mouse stomach.

We have already found that nitrite-treated isoflavones exhibit genotoxic activities toward Salmonella typhimurium TA 100 and 98 strains (submitted: nitrite-treated genistein). However, we have not demonstrated genotoxic activity induced by simultaneous treatment with isoflavones and NaNO(2)in vivo. In the present study, we examined whether coadministration of isoflavones (such as daidzein and genistein) and NaNO(2) induces DNA damage in the stomach of ICR male mice. Mice were coadministered with isoflavones (1mg/kg body weight) and NaNO(2) (10mg/kg body weight), and dissected to collect tissues at 1, 3, and 6h after administration. We used comet assay combined with repair enzyme formamidopyrimidine-N-glycosylase (FPG) to detect FPG-sensitive sites. An HPLC-ECD system was employed to determine 8-oxo-2'-deoxyguanosine (8-oxodG) in the stomach. In addition, we observed leukocyte infiltration by histopathological investigation, and measured total superoxide dismutase (SOD) in the stomach. We confirmed that oxidative DNA damage in the stomach was significantly increased by coadministration. Total SOD activities were also significantly stimulated by coadministration. However, the induction of inflammation in the stomach was not found. These data suggest that coadministration of isoflavones and NaNO(2) can cause DNA damage in the stomach because of the formation of radicals.

Leaf extract of Wasabia japonica relieved oxidative stress induced by Helicobacter pylori infection and stress loading in Mongolian gerbils.

Infection with Helicobacter pylori (H. pylori) can induce gastric disorders, and though its presence cannot explain disease pathogenesis and does not have associations with other factors, it is well known that H. pylori infection causes stomach inflammation following oxidative stress. We examined the suppressive effects of a leaf extract of Wasabia japonica on H. pylori infection and on stress loading in Mongolian gerbils. Following oral administration of wasabi extract of 50 and 200 mg/kg B.W./d for 10 d, the animals were exposed to restraint stress for 90 and 270 min. As for the results, the level of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in the stomach and oxidative DNA damage in peripheral erythrocytes at 270 min significantly increased. That elevation was significantly suppressed by the addition of the leaf extract. We concluded that the simultaneous loading of H. pylori infection and physical stress loading might induce oxidative DNA damage additively, while a leaf extract attenuated this DNA damage in the stomach as well as the peripheral erythrocytes.

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

BACKGROUND: Recently, 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. RESULTS: In 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. CONCLUSION: Manufactured nano/microparticles, CB, C60 and kaolin, were shown to be genotoxic in in vitro and in vivo assay systems.

Effects of chronic ingestion of catechin-rich green tea on hepatic gene expression of gluconeogenic enzymes in rats.

Normal rats were given catechin-rich green tea as drinking fluid and the effects on hepatic gene expression were examined. The results of DNA microarray analysis and quantitative real-time reverse transcription-polymerase chain reaction indicated the down-regulated expression of genes for glucose-6-phosphatase (G6Pase) and fatty acid synthase, and the up-regulated expression of peroxisome proliferator activated receptor alpha in the rats given green tea for 4 weeks as compared with the water-given animals. One may expect anti-diabetic activity by catechin-rich green tea through its chronic down-regulatory effect on G6Pase expression.

Genotoxicity and estrogenic activity of 3,3'-dinitrobisphenol A in goldfish.

3,3'-Dinitrobisphenol A (dinitro-BPA) is formed in a mixture of bisphenol A (BPA) and nitrite under acidic conditions. It shows genotoxicity in male ICR mice on a micronucleus test, but its estrogenic activity has not been examined in vivo. We examined its estrogenic activity using goldfish (Carassius auratus) by measuring plasma levels of vitellogenin (VTG) by the ELISA method. Expression of VTG didn't increase in the plasma of goldfish intraperitoneal injected with dinitro-BPA at a dose of 10 mg/kg of body weight. We also examined the genotoxicity of dinitro-BPA by single-cell gel electrophoresis (comet assay) and a micronucleus test using goldfish. The DNA tail moment of blood cells increased after intraperitoneal injection of dinitro-BPA. Dinitro-BPA at the same dose significantly increased micronucleus frequency in gills of goldfish. On the other hand, BPA did not significantly increase the frequency of micronucleated cells. In conclusion, we found that dinitro-BPA did not show estrogenic activity, but had genotoxic potency stronger than that of BPA.

Application of a new bioassay technique using goldfish for assessment of water toxicity.

There are a variety of chemicals in aquatic environment, so it is important to assess the toxicity. The biomarkers such as induction of DNA damage, micronuclei, vitellogenin, and hepatic P450 in fish are known to be effective for monitoring genotoxic and/or estrogenic chemicals. However, there is little study to use these biomarkers in same fish. Goldfish (Carassius auratus) is widely used and is suitable in size to collect blood or organs. In this study, validity of multiple-biomarkers in goldfish was checked using standard chemicals and applied in the river water. Ho River, which flows through the textile dyeing factory in Shizuoka Prefecture, Japan, was reported to show genotoxicity toward Salmonella typhimurium TA98 and YG1024. When the goldfish were exposed to Ho River, DNA damage, estrogenic activity, and CYP1A induction were observed. Through the study, it was assumed that not only mutagens/carcinogens but also endocrine disrupting chemicals and poly aromatic hydrocarbons were present in Ho River. Therefore, chemical identification should be required. We could evaluate both genotoxicity and estrogenic activity simultaneously, so goldfish might be a good experimental model for estimation of chemical contamination levels in aquatic environment.

Functional analyses of neutrophil-like differentiated cell lines under a hyperglycemic condition.

Diabetic patients are prone to severe bacterial infections. The functional alterations of neutrophils by hyperglycemia are thought to be partially responsible for such infections. In this study, we investigated the functional changes of neutrophil-like differentiated cell lines (dHL-60, dTHP-1, and dNB-4) by treatment with 5.5 mM, 11 mM, or 35 mM of glucose. In dHL-60 cells, the incubation with high glucose (35 mM) resulted in the enhancement of cell aggregation, the suppression of cellular fragility, the induction of reactive-oxygen species (ROS) production by phorbol myristate acetate (PMA) stimulation, and the impairment of phagocytosis. In dTHP-1 cells, the treatment with higher glucose generated the suppression of cellular fragility and extremely impaired phagocytosis (by 35 mM), and induced ROS production due to PMA stimulation (by 11 mM). Furthermore, the higher glucose exposure to dNB-4 cells enlarged intracellular vacuoles (by 35 mM) and induced ROS production due to PMA stimulation (by 11 mM). Since the ROS generation of those cells was enhanced only after PMA stimulation under the higher glucose conditions, glucose may have a priming effect rather than a triggering effect. These extraordinary sensitivities caused by the higher glucose treatments may reflect the dysfunction or overactivation of neutrophils.

Responses of ultra-weak chemiluminescence and secretory IgA in saliva to the induction of angry and depressive moods.

Oxidative damage to tissues and cells contributes to disease processes. We used ultra-weak chemiluminescence (uwCL) as an indicator of oxidative activity to examine the effects of psychological challenges on oxidative responses. We also examined the association of underlying psychological characteristics with oxidative and immune responses. Eighteen healthy men and women with a mean age of 24.1 were recruited. Anger and depressive symptoms were evaluated using the State-Trait Anger Expression Inventory and the Center for Epidemiological Studies Depression Scale, respectively. Following a baseline period, participants were required to complete two separate speech tasks where they were asked to recall life events that made them feel angry (AT) or depressed (DT). The tasks were separated by a 30-min recovery period and the order was randomized between participants using a counterbalanced design. Saliva was sampled and assayed for uwCL and secretory immunoglobulin A (sIgA). The level of uwCL was significantly increased in response to both tasks (p<.05), whereas sIgA concentrations decreased significantly in response to DT (p<.05). At 30 min after each task, uwCL values were positively related to anger-in (p<.005), anger expression (p<.05) and trait anger (p<.05) post-AT, and sIgA concentrations were positively related to anger-out (p<.05) post-AT and -DT, after controlling for covariates. The present study suggests that induction of angry and depressive moods can increase oxidative activity and transiently weaken immunity indicated by salivary sIgA concentrations. In addition, anger personality traits may modify these responses.

Comparison of peroxidase response to mental arithmetic stress in saliva of smokers and non-smokers.

Saliva is the first body fluid to encounter exogenous materials or gases such as cigarette smoke (CS). The aim of this study was to examine whether smoking affects oral peroxidase (OPO) reactivity to mental stress. The subjects were 39 non-smokers and 10 smokers. In the experiment, the Kraepelin psychodiagnostic test as a psychological stressor and saliva was sampled 30 min before, just before, immediately after, and 30 min after the beginning of the test. OPO reactivity to the test between smokers and non-smokers was measured in addition to uric acid concentration, flow rate, IgA, thiocyanate (SCN-) concentration, amylase activity as a salivary stress marker, and ultra-weak chemiluminescence (UCL) level, which is indicative of salivary antioxidative and antibacterial abilities. Moreover, we studied the effect of smoking on the response of salivary peroxidase (SPO) and myeloperoxidase (MPO) activity to mental stress, respectively. The results showed that the IgA concentration, amylase activity, SCN(- concentration, and UCL level are higher in the non-smoking group than smoking group and the IgA concentration and UCL level increased in the non-smokers significantly just after the Kraepelin test. The levels of SCN-) were higher in smokers than in non-smokers and OPO activity was greater in the non-smoking group in all sessions. Furthermore, only the non-smokers had significantly increased MPO activity just after the test. MPO may play a crucial role in the response to acute psychological stress besides inflammation, and CS suppresses this response significantly.

Food and health longevity.

Japan has the longest life expectancy in the world. It was an average 85.59 years for women and 78.64 years for men and 77.2 years for women and 71.9 years for men in average health expectancy in 2004. As the diet becomes more westernized, the number of patients with diabetic, cardiovascular, and cerebrovascular diseases due to the metabolic syndrome is increasing. In 2002, our university was selected as a COE21st century participant by the Ministry of Education, Culture, Sports, Science and Technology. Our program is entitled "Center of Excellence for Evolutionary Human Health Sciences". The aim of this program is to obtain and provide significant information for maintaining human health through research/education on food and medicine. The combined use of specialized foods and medicines will enhance the effectiveness and decrease the adverse effects of medicines. Safety evaluation methods will be developed for the combined use, thus establishing a novel academic field for human health sciences. We call this "borderless sciences of food and medicine" (yaku-shoku dogen).

The anti-fibrotic effect of green tea with a high catechin content in the galactosamine-injured rat liver.

Previously, we reported that the oral administration of green tea rich in catechins restored levels of several biomarkers increasing in galactosamine-treated rats to nearly control values. These biomarkers included serum transaminase activities, serum concentrations of tumor necrosis factor-alpha and interleukin 1-beta, and the hepatic mRNA expression of these inflammatory cytokines. In the present study, we examined possible anti-fibrotic effects of green tea in galactosamine-induced hepatitis. The results of the reverse transcription and polymerase chain reaction indicated that the increase in gene expression of the alpha1 chain of collagen type 1 and transforming growth factor beta-1 in the injured liver 24 h post-injection of galactosamine was suppressed by the administration of green tea. Masson's trichrome staining demonstrated that the extent of fibrogenesis after 14 days was greater in the galactosamine-injured livers not treated with green tea than the treated ones. These results suggest that the drinking of green tea with a high catechin content may help to prevent and/or attenuate the development of fibrosis in hepatitis.

Evaluation of mutagenic activities of leachates in landfill sites by micronucleus test and comet assay using goldfish.

To develop a simple system for monitoring the presence of mutagens/carcinogens in the leachates from landfill sites, we used a micronucleus test and a single cell gel electrophoresis (comet) assay originally developed for mice and rats on goldfish (Carassius auratus). The goldfish were exposed for 9 days to the leachate with chemical and biological treatment (treated leachate) or without treatment (raw leachate). The goldfish exposed to several samples died because of the high concentrations of NaCl or ammonium ion (NH4+). In the comet assay using peripheral erythrocytes, the raw leachates showed higher mutagenic activity than the treated leachates. In the micronucleus test, it was difficult to detect the micronuclei in peripheral erythrocytes. On the other hand, the frequency of micronuclei was high in gill cells of goldfish exposed to the raw leachates compared to the treated leachates. A combination of the two bioassays was shown to be useful to evaluate the mutagenic activity of the leachates. We also propose a new scoring method for determination of water quality by using acute toxicity and mutagenic activity.

Renoprotective effects of tea catechin in streptozotocin- induced diabetic rats.

Tea catechins, a class of flavonoids, are suggested to have biological effects, possibly mediated through their antioxidative properties. Recent data indicated that tea catechins suppressed proliferative changes in glomeruli and inhibited the development of glomerulosclerosis in partially nephrectomized rats. We thus sought to determine whether tea catechins may protect against renal dysfunction in streptozotocin-induced diabetic rats. Four groups of male Sprague-Dawley rats (n=11-15 per group), with and without streptozotocin-induced diabetes, were treated with and without catechins (5 mg/day) administered in the drinking water for 12 weeks. At the end of the treatment period, 24-hour urinary albumin excretion rate (AER), serum lipid peroxides as thiobarbituric acid reactive substrates (TBARS) and blood pressure were measured. Renal glomerular volume and interstitial fibrosis were assessed morphologically. Albuminuria developed progressively in untreated diabetic rats, resulting in a mean AER of 559+/-124 (mean+/-SE) versus 63+/-7 microg/day/100 g body weight in non-diabetic rats at 12 weeks (P<0.001). Catechin treatment significantly reduced AER to 287+/-56 microg/day/100 g body weight in diabetic rats (P=0.017 versus untreated diabetic rats). Increased interstitial fibrosis in the kidney, observed in untreated diabetic rats, was completely normalized with catechin treatment. Serum levels of TBARS and blood pressure were comparable among the four groups. In conclusion, administration of tea catechin retards the progression of functional and morphological changes in the kidney of streptozotocin-induced diabetic rats.

Utilization study of stems and leaves of Tienchi Ginseng. I. Anti-hypertensive effect of stems and leaves of Tienchi Ginseng on stroke-prone spontaneously hypertensive rat (SHRSP).

Tienchi ginseng tea was prepared from stems and leaves of Tienchi ginseng which is a special product in China. The increase in systolic blood pressure (SBP) was significantly inhibited by the consumption of a 4% this tea solution as drinking water from the prehypertensive stage (6 weeks of age) in male stroke-prone spontaneously hypertensive rats (SHRSP). A similar intake from the hypertensive stage also showed an anti-hypertensive effect. In contrast, a similar intake had no effect on SBP of normotensive Wistar Kyoto rats. We found that the rhizome of Tienchi ginseng contained two types of saponin: 20(s)-protopanaxadiol (PPD) such as ginsenosides Rb(1) and Rd having a hypotensive effect, and 20(s)-protopanaxatriol (PPT) such as ginsenosides Rg(1) and Re having a hypertensive effect. In contrast, the tea sample contained PPD and gamma-aminobutyric acid (GABA), but no PPT. These results suggest that drinking this tea infusion would be useful for controlling hypertension.

Changes in the mutagenic and estrogenic activities of 17beta-estradiol after treatment with nitrite.

We determined the changes in the mutagenic and estrogenic activities of 17beta-estradiol after a nitrite treatment. Nitrite-treated 17beta-estradiol showed mutagenic activities toward Salmonella typhimurium strains TA 100 and TA 98. We confirmed that nitrite-treated 17beta-estradiol generated radicals from the results of an analysis of electron spin resonance. By applying an instrumental analysis, we identified 2-nitro-17beta-estradiol to have been formed in the reaction mixture. 2-Nitro-17beta-estradiol did not exhibit mutagenic activities toward Salmonella typhimurium strains, suggesting that other mutagens might have been formed in the reaction mixture. The clastogenic properties of nitrite-treated 17beta-estradiol and 2-nitro-17beta-estradiol were analyzed by a micronucleus test with male ICR mice. Nitrite-treated 17beta-estradiol and 2-nitro-17beta-estradiol induced a significantly higher frequency of micronucleated reticulocytes in mice. The estrogenic activity of 2-nitro-17beta-estradiol was found to be lower than that of 17beta-estradiol. These data suggest that a daily oral intake of 17beta-estradiol and nitrite might induce the formation of mutagenic compounds in our body.