Placental tissue of greenhouse muskmelon (Cucumis melo L.) contains more gamma-aminobutyric acid with antioxidant capacity than the fleshed pulp.

Our previous study revealed that gamma-aminobutyric acid (GABA) in Earl's muskmelon is more concentrated in the inner than the outer parts of the fruit. Here, the GABA and antioxidant capacity of the placental tissue of muskmelon, which is considered waste, were evaluated for possible use as a source of bioactive compounds. The concentrations of GABA and related substances in the placental tissue were significantly higher than in the fleshed pulp, whereas glutamic acid and sugar levels were significantly lower. The two sites showed no difference in GAD activity. Furthermore, the placental site showed high antioxidant capacities based on 2,2-diphenyl-1-picrylhydrazyl and oxygen radical absorbance capacity for hydrophilic compounds assays compared with the fleshed pulp, because of the higher levels of total phenolic and L-ascorbic acids. Therefore, the placental tissue of muskmelons may be useful for developing functional foods, which would also reduce the amount of residues during muskmelon processing.

Differential GABA concentration gradients are present in the edible parts of greenhouse melon (Cucumis melo L.) during all four seasonal croppings.

Food-derived gamma-aminobutyric acid (GABA) exhibits health-promoting benefits, and melon contain high GABA concentrations. Greenhouse melons (Cucumis melo L. "Earl's Favorite") cultivated in Japan have identical or more edible parts than cultivars in other countries, however GABA distribution and the effects of seasonal variations are unclear. Thus, the present study aimed to evaluate GABA concentration gradients in four seasonal melons and how glutamic acid (Glu) influences the establishment of these gradients. GABA concentration was significantly lower near the exocarp than in the peduncle, equator, and remnant style regions in most seasons. Glu and GABA concentrations showed similar trends and were significantly correlated near the remnant style. No significant differences in GABA and Glu concentration were detected at concyclic sites across horizontal sections. These data indicate that GABA and Glu concentration differs substantially along a vertical melon section, but less so along a horizontal section, among sampling regions, sites, and cropping season.

Usefulness of combined in vivo skin comet assay and in vivo skin micronucleus test.

We have already found that the in vivo skin comet assay is useful for the evaluation of primary DNA damage induced by genotoxic chemicals in epidermal skin cells. The aim of the present study was to evaluate the sensitivity and specificity of the combined in vivo skin comet assay and in vivo skin micronucleus (MN) test using the same animal to explore the usefulness of the new test method. The combined alkaline comet assay and MN test was carried out with three chemicals: 4-nitroquinoline-1-oxide (4NQO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo[a]pyrene (B[a]P). In the first experiment, we compared DNA- and chromosome-damaging effects of 3 [72, 24 and 3 hours (h) before sacrifice] and 4 applications (72, 48, 24 and 3h before sacrifice) of 4NQO, which induces dermal irritancy. The animals were euthanized and their skin was sampled for the combination test. As a result, the 4-application method was able to detect both DNA- and chromosome-damaging potential with a lower concentration; therefore, in the second experiment, MNNG and B[a]P were topically applied four times, respectively. The animals were euthanized, and then their skins were sampled for combination tests. In the alkaline comet assay, significant differences in the percent of DNA (%DNA) in the tail were observed in epidermal skin cells treated with MNNG and B[a]P. In the MN test, an increased frequency of MN cells (%MN) cells was observed by treatment with MNNG; however, there were no significant increases. In contrast, significant differences in %MN were observed by treatment with B[a]P. From these results, we conclude that the combined in vivo skin comet assay and in vivo MN test was useful because it can detect different genotoxicity with the same sampling time and reduce the number of animals used.

Use of the in vivo skin comet assay to evaluate the DNA-damaging potential of chemicals applied to the skin.

The aim of the present study was to evaluate both sensitivity and specificity of an in vivo skin comet assay using chemically treated, hairless mouse dorsal skin as a model. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.0125-0.2%), 4-nitroquinoline-1-oxide (4NQO, 0.01-0.25%), mitomycin C (MMC, 0.0125-0.05%), benzo[a]pyrene (B[a]P, 0.25-2%), and 7,12-dimethylbenz[a]anthracene (DMBA, 0.25-1%) were each applied once to the dorsal skin of hairless male mice; after 3h, epidermal skin cells were isolated, and the alkaline comet assay was performed. The assay was performed after 24h for only the B[a]P and DMBA. Furthermore, B[a]P and DMBA were evaluated by alkaline comet assay using liver cells after both 3 and 24h. The mean percent of DNA (%DNA) in tail in the 0.05-0.2% MNNG and 0.1-0.25% 4NQO treatment groups was markedly higher than in the control group at 3h post-application. Although the mean %DNA values in the tail in the B[a]P and DMBA groups were the same as the controls at 3h post-application, the 2% B[a]P and 1% DMBA groups showed significantly higher values versus controls 24h after application. No significant increases in the mean %DNA in the tail were observed in the MMC group. No clear increases in %DNA in the tail were observed in the B[a]P and DMBA groups at 3 or 24h after application in the liver. These results suggest that the in vivo skin comet assay is able to accurately identify DNA-damaging potential with a skin-specific response and is a useful method to detect the DNA-damaging potential of genotoxic chemicals on the skin.

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.

Practical long-term storage of strawberries in refrigerated containers at ice temperature.

This study investigated the effect of storage temperature in the presence or absence of film packaging on the Benihoppe and Kirapika varieties of Japanese strawberries stored for 28 days at 0°C and 3°C. The study was conducted in a 20-foot reefer container for practicality. Storage at 0°C suppressed decay and reduction in sugars and organic acids more efficiently than that at 3°C. Softening of fruit hardness was also suppressed depending on the variety. The reduction in sugars and organic acids did not affect strawberry palatability. Along with low temperature, long-term storage of strawberries also requires the use of film packaging, which prevents drying. Without film packaging, storage at both 0°C and 3°C decreased fresh weight significantly, resulting in loss of commercial value. In contrast, storage in film packaging decreased weight reduction to <5%, even after 28 days cold storage.

Effects of distribution of sugars and organic acids on the taste of strawberries.

The concentrations of sugars and organic acids as well as the total soluble solid (TSS) in different parts of the strawberry fruit were characterized. The data were used to create simulated fruit juice jellies, in order to clarify how the sugar and organic acid levels affect the taste. Such an approach eliminates the influence of external factors such as size, color, and texture when using real fruits in sensory evaluations. Further, the use of a jelly allowed us to simulate the concentration differences between various parts of the fruit. In the strawberry fruit, the sugar content is higher in the apex than in the peduncle; however, the level of organic acids is the same throughout. It was revealed that the sweetness and sourness in the apex and peduncle could be sufficiently recognized by humans as tastes. Also, a layered jelly sample replicating the sugar and acid distribution in real strawberry was perceived as less sweet and more sour, compared to a homogeneous one with the same overall composition. The likely reason is that the sourness in the peduncle is accentuated by the low TSS level, which decreases the TSS/total organic acid ratio that affects the sweetness/sour perceptions. Based on these results, factors for the appropriate sensory evaluation of fresh fruits in general were considered. Specifically, the distribution of sugars and organic acids in the fruit should be analyzed first, and bite-sized parts with concentrations close to the average provide the most accurate evaluation results.

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.

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.

In vivo comet assay of acrylonitrile, 9-aminoacridine hydrochloride monohydrate and ethanol in rats.

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay, we examined the ability of acrylonitrile, 9-aminoacridine hydrochloride monohydrate (9-AA), and ethanol to induce DNA damage in the liver and glandular stomach of male rats. Acrylonitrile is a genotoxic carcinogen, 9-AA is a genotoxic non-carcinogen, and ethanol is a non-genotoxic carcinogen. Positive results were obtained in the liver cells of male rats treated with known genotoxic compounds, acrylonitrile and 9-AA.

Ovarian dysfunction, obesity and pituitary tumors in female mice following neonatal exposure to low-dose diethylstilbestrol.

In a previous study, we found that early life exposure to low-dose diethylstilbestrol (DES) induced early onset of spontaneous abnormalities in estrus cycle and shortened survival in female Sprague-Dawley rats. In order to confirm the repeatability of the previous study, neonates of C57BL/6J mice were orally administered DES at doses of 0.005, 0.05, 0.5 and 5 µg/kg/day, and the aging of their reproductive function was observed. As a result, delayed toxicity on ovarian function was found in females treated with 0.5 µg/kg/day of DES. Concomitantly, the females in the 0.05 µg/kg/day of DES, or greater, groups, had increased body weights and, in the 0.5 µg/kg/day of DES, or greater, groups, had developed pituitary tumors, which were causal factors in their accelerated mortality. Thus, we found that early life exposure to low-dose DES induced early onset of spontaneous abnormalities in estrus cycle not only in female rats but also in female mice.

Genotoxic potential and in vitro tumour-promoting potential of 2-dodecylcyclobutanone and 2-tetradecylcyclobutanone, two radiolytic products of fatty acids.

The DNA-damaging and tumour-promoting effects of two 2-alkylcyclobutanones (2-ACBs), which are found in irradiated fat-containing foods, were investigated by use of the comet assay and in an azoxymethane (AOM)-induced colon-carcinogenesis study in rats, respectively. We conducted genotoxicity tests of 2-dodecylcyclobutanone (2-dDCB) and 2-tetradecylcyclobutanone (2-tDCB) according to the test guidelines for chemicals or drugs. In addition, a cell-transformation assay with Bhas 42 cells was performed to investigate their promoting potential in vitro. The Salmonella typhimurium mutagenicity assay (Ames test), conducted with five tester strains, revealed that neither 2-dDCB nor 2-tDCB possessed mutagenic activity. Moreover, both in the in vitro chromosomal aberration test on CHL/IU cells and the in vivo bone-marrow micronucleus test where mice were given 2-dDCB and 2-tDCB (orally, up to 2000 mg/kg bw/day), we did not detect any clastogenic effects. Furthermore, DNA strand-breaks were not detected in the in vitro comet assay with CHL/IU cells, and DNA adducts derived from 2-dDCB and 2-tDCB were not detected in the colon tissues of the mice used for the micronucleus tests, in rats from a repeated dose 90-day oral toxicity test (0.03% 2-tDCB in the diet), or in rats from the AOM-induced carcinogenesis study (0.025% 2-tDCB in the diet). An in vitro tumour-promotion assay with Bhas 42 cells revealed that the number of transformed foci increased significantly following treatment of cells in the stationary phase with 2-dDCB or 2-tDCB for 10 days. Our results indicate that neither 2-dDCB nor 2-tDCB were genotoxic chemicals. However, they exhibited promoting activity, at least in vitro, when Bhas 42 cells were continuously exposed to these chemicals at toxic doses.

Evaluation of effect during cell isolation process in alkaline comet assay using epidermal skin cells.

The aim of the present study was to evaluate the effect of the cell isolation process in the alkaline comet assay using epidermal skin cells. When we explored the cell isolation method for the alkaline comet assay using the 3-dimensional (3D) human epidermal skin model, we found that DNA damage and cytotoxicity were induced during the cell isolation process. In particular, trypsin 5 min treatment with ethylene diamine tetraacetic acid (EDTA) showed about 5 times %DNA in the tail value compared to without EDTA treatment. In general, EDTA is commonly used for cell isolation, but it is known to induce genotoxicity due to secondary effects. We therefore evaluated the effect of EDTA and pH in the alkaline comet assay on a monolayer culture of rat keratinocytes. As a result, there was a significant increase of %DNA in tail values by treatment with 0.1 w/v% EDTA for 60 min; however, there was no difference in the %DNA in tail values between 0.1 w/v% EDTA/PBS(-) (pH 6.8) and 0.1 w/v% EDTA/PBS(-) (pH 7.4). These data imply that there is a need to control the EDTA conditions for cell isolation in the epidermal skin cells.

Assessment of technical protocols for novel embryonic stem cell tests with molecular markers (Hand1- and Cmya1-ESTs): a preliminary cross-laboratory performance analysis.

The Hand1- and Cmya1-ESTs are novel short-term tests for embryotoxic chemicals using genetically engineering mouse ES cells for luciferase reporter gene assays. These ESTs allow convenient determination of differentiation toxicity and cell viability in a short duration with high throughput 96-well microplates for prediction of embryotoxicity of chemicals. To assess the Hand1-EST technical protocol, we firstly compared reporter gene assay and cytotoxicity test data for a representative compound (hydroxyurea) from four different laboratories with tests carried out under the same experimental conditions. Extensive investigations of the Hand1- and Cmya1-ESTs were then performed to explore reproducibility by comparing a set of 6 well-known test chemicals, including hydroxyurea, across the laboratories. The results gave good correspondence in all four laboratories, indicating that transferability, intra-laboratory variability and inter-laboratory variability of the present technical protocols of the ESTs were sufficient to conduct further validation studies.

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.

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.