Identification and antioxidant activity of flavonoid metabolites in plasma and urine of eriocitrin-treated rats.

Eriocitrin, a flavonoid glycoside present in lemon fruit, is metabolized in vivo to a series of eriodictyol, methylated eriodictyol, 3,4-dihydroxyhydrocinnamic acid, and their conjugates. Plasma antioxidant activity increased following oral administration of aqueous eriocitrin solutions to rats. Eriocitrin metabolites were found in plasma and renal excreted urine through HPLC and LC-MS analyses. Eriocitrin was not detected in plasma and urine, but eriodictyol, homoeriodictyol, and hesperetin in their conjugated forms were detected in plasma of 4.0 h following administration of eriocitrin. In urine for 24 h, both nonconjugates and conjugates of these metabolites were detected. 3,4-Dihydroxyhydrocinnamic acid, which is metabolized from eriodictyol by intestinal bacteria, was detected in slight amounts with each form in 4.0-h plasma and 24-h urine. Eriocitrin was suggested to be metabolized by intestinal bacteria, and then eriodictyol and 3,4-dihydroxyhydrocinnamic of its metabolite were absorbed. Following administration of eriocitrin, plasma exhibited an elevated resistance effect to lipid peroxidation. Eriocitrin metabolites functioning as antioxidant agents are discussed.

Functional properties of wasabi and horseradish.

Wasabi (Wasabi japonica) and horseradish (Cholearia arnoracia) are used as spices of daily foodstuffs. Allylisothiocyanate (AIT) is a potent component in both plants and occurs by grating them. It is well known that AIT shows inhibitory effect on the growth of food poisoning bacteria and fungi. In this work, several functional properties of roots and leaves from wasabi and horseradish were examined in vitro. Each sample showed peroxidase activity. They also exhibited antioxidative and superoxide scavenging potency. Antimutagenic activity was observed toward 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline [MeIQx], a well-known mutagen/carcinogen in broiled fish and meat. They also decreased His+ revertant colonies of 3-chloro-4-dichloromethyl-5-hydroxy-2(5H)-furanone (MX) in the Ames test, a strong mutagen and carcinogen in chlorine disinfected tap water. Isolation of antimutagenic components in wasabi root was done. Three components including (-)-(R)-7-methylsulfinylheptyl isothiocyanate were identified. These data show that wasabi and horseradish might be potent functional foods for keeping human health.

Metabolic fate of luteolin and its functional activity at focal site.

Luteolin has been shown to possess potent antioxidant and anti-inflammatory/anti-allergic activities. In order to evaluate a chemopreventive role of luteolin in inflammatory responses involved in the pathogenesis of atherosclerosis and cancer etc., the metabolic fate of luteolin in rats and humans was investigated by HPLC analysis, and its effect on cell surface expression of adhesion molecules in human umbilical vein endothelial cells(HUVECs) was examined by ELISA. Luteolin monoglucuronide, which was a main metabolite, and free luteolin were detected in rat plasma and human serum. Luteolin monoglucuronide was hydrolyzed to free luteolin by beta-glucuronidase released from neutrophils stimulated with lonomycin and Cytocharasine B. Luteolin suppressed the TNF-alpha induced ICAM-1 expression significantly. Among nine flavonoids (40 microM) examined, chrysin, apigenine, quercetin and galangin also demonstrated suppressive effct on it. These results suggest the posssibility that deconjugation of luteolin monoglucuronide occurs and that free luteolin showed functional acyivities such as suppression of TNF-alpha induced ICAM- 1 expression at inflammation site.

Promoting effects of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone on rat glandular stomach carcinogenesis initiated with N-methyl-N'-nitro-N-nitrosoguanidine.

The modifying effects of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a mutagenic by-product in chlorinated water, on the development of glandular stomach cancers were investigated in Wistar rats. A total of 120 males, 6 weeks of age, were divided into six groups. After initiation with 100 ppm N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) solution and 5% NaCl diet for 8 weeks, 30 rats each in groups 1-3 were given MX in the drinking water at concentrations of 30, 10, or 0 ppm for the following 57 weeks. Ten animals each in groups 4-6 were administered the MX without prior carcinogen exposure. There were no statistical significant differences in final body weights between the groups. The incidences and multiplicities of adenocarcinomas in the glandular stomachs were significantly higher (P < 0.05) in the initiated 30 ppm MX group than those in the MNNG/NaCl group. The incidences of atypical hyperplasias in the glandular stomachs were also significantly increased (P < 0.05 or 0.01) by the MX treatments. With their multiplicity, the effects were clearly dose dependent. Interestingly, the 30 ppm MX alone itself induced atypical hyperplasias in the pylorus, although the incidences and severity were low. Moreover, MX showed a tendency to enhance the development of intrahepatic cholangiocellular tumors and thyroid follicular cell tumors in the MNNG-treated animals. The results of the present study thus indicate that MX exerts promoting effects when given during the postinitiation phase of two-stage glandular stomach carcinogenesis in rats.

3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX): toxicological properties and risk assessment in drinking water.

MX (3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone), one of the byproducts formed during the chlorine disinfection process of drinking water, shows strong mutagenic activity for Salmonella strains in the Ames test. In several countries, the contribution of MX to the total mutagenicity of drinking water is estimated to range from 7% to 67%. To assess the risk of MX for human health, we summarized the toxicological properties of MX and estimated the tolerable daily intake (TDI) or tolerable concentration in drinking water. MX is genotoxic in cultured mammalian cells and causes in vivo DNA damage in several tissues. MX is carcinogenic for rodents in addition to possessing skin and gastric promotion activities. From these toxicological profiles of MX, we estimated the virtual safety dose (VSD) for genotoxic action as 5 ng/kg/d and the TDI for non-genotoxic action of MX as 40 ng/kg/d. We assumed a tolerable MX concentration of 150 ng/L in drinking water. Because of the uncertainty about human genotoxicity, however, and the lack of information on reproductive or developmental toxicity, the estimated tolerable dose level may be provisional.

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.

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) sensitizes mammalian cells to UV radiation by causing the S-phase arrest, not by inhibiting the repair of DNA damage as observed in Escherichia coli.

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) is known to be a mutagen and carcinogen isolated from the charred parts of cooked foods. We found previously that Trp-P-1 enhanced UV-induced lethality and mutation frequency in Escherichia coli by inhibiting the repair of UV-induced DNA damage. In the present study, we investigated whether Trp-P-1 also potentiated UV-induced lethality by inhibiting the repair of UV-induced DNA damage in cultured mammalian cells. As a result, Trp-P-1 enhanced UV-induced lethality in a concentration-dependent manner in human and Chinese hamster cells. However, Trp-P-1 was unable to inhibit the repair of the two major photolesions (cyclobutane pyrimidine dimers and (6-4)photoproducts) from the genomic DNA, as determined using monoclonal antibodies specific for each type of lesion. On the other hand, Trp-P-1, with or without UV irradiation, efficiently suppressed DNA synthesis and arrested cells in S phase in concentration- and time-dependent manners, as measured by pulse-labelling with 3H-thymidine and flow cytometry. Thus, the present results suggest that Trp-P-1 potentiates UV-induced lethality in cultured mammalian cells by causing the S-phase arrest, not by inhibiting the repair of UV-induced DNA damage as observed in Escherichia coli.

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.

Food-derived heterocyclic amines potentiate the mutagenicity of a drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX).

We investigated the enhancing effect of heterocyclic amines on base-substitution mutations with 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (MeIQ). We compared the mutagenicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in the presence and absence of the heterocyclic amines in E. coli WP2 (trpE) and in excision repair-deficient strains WP2s (uvrA, trpE) and ZA500 (uvrA, rfa, trpE). Since the assay was performed without microsomal metabolic activation, Trp-P-1 and MeIQ alone were not mutagenic. In WP2, trp+ reversions induced by MX were greatly potentiated by Trp-P-1 and slightly potentiated by MeIQ. Mutation enhancement was not observed in strains WP2s and ZA500, suggesting that a functional DNA excision repair system is necessary for the combined action of MX and heterocyclic amines. Our finding implies that the combined effect of mutagens as well as the effect of individual mutagens, should be considered in risk evaluation.

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.

Protection by alpha G-rutin, a water-soluble antioxidant flavonoid, against renal damage in mice treated with ferric nitrilotriacetate.

The protective effect of alpha G-Rutin against ferric nitrilotriacetate (Fe-NTA)-induced renal damage was studied in male ICR mice. Fe-NTA induces renal lipid peroxidation, leading to a high incidence of renal cell carcinoma in rodents. Administration of alpha G-Rutin (50 mumol as rutin/kg) by gastric intubation 30 min after i.p. injection of Fe-NTA (7 mg Fe/kg) most effectively suppressed renal lipid peroxidation. Repeated i.p. injection of Fe-NTA (2 mg Fe/kg/day for the first 3 days and 3 mg Fe /kg/day for 12 days, 5 days a week) causes subacute nephrotoxicity as revealed by induction of karyomegalic cells in renal proximal tubules. A protective effect was observed in mice given alpha G-Rutin 30 min after each Fe-NTA treatment. To elucidate the mechanism of protection by alpha G-Rutin, the pharmacokinetics and hydroxyl radical-scavenging effect of alpha G-Rutin were investigated by HPLC analysis and by electron spin resonance (ESR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), respectively. When mice were given alpha G-Rutin (50 mumol as rutin/kg) by gastric intubation, rapid absorption into the circulation was observed. The plasma concentration of alpha G-Rutin reached the highest level 30 min after oral administration and then decreased to the control level within 60 min, alpha G-Rutin inhibited the formation of DMPO-OH in a concentration-dependent manner. Further, chelating activity of alpha G-Rutin to ferric ions was shown by spectrophotometric analysis. These results suggest that absorbed alpha G-Rutin works as an antioxidant in vivo either by scavenging reactive oxygen species or by chelating ferric ions and this serves to prevent oxidative renal damage in mice treated with Fe-NTA.

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) inhibits the binding activity of T4 endonuclease V to UV-damaged DNA.

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) is a mutagen/carcinogen derived from cooked foods which enhances the induction of mutations and chromosome aberrations by UV without microsomal activation. These co-mutagenic effects are considered to arise from inhibition of DNA excision repair at the incision step. However, the inhibition mechanism has not been clarified. In this study we show, using agarose gel electrophoresis, that Trp-P-1 inhibits incision by T4 endonuclease V, which cleaves DNA at the site of cyclobutane dimers. Trp-P-1 also inhibits the binding of this enzyme to UV-damaged DNA in a gel shift assay. In addition, the results of DNA unwinding assay with topoisomerase I suggest that Trp-P-1 intercalates into DNA molecules. The known intercalators ethidium bromide and acriflavine demonstrate similar effects in these experiments. However, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which showed no co-mutagenic effects in our previous study, does not demonstrate such effects. These results suggest that Trp-P-1 changes DNA conformation by intercalation, causing inhibition of binding of repair enzymes to UV-damaged DNA, and this in turn leads to inhibition of DNA excision repair and to co-mutagenic effects.

Radioprotective effect of antioxidative flavonoids in gamma-ray irradiated mice.

The anticlastogenic effect of 12 structurally different flavonoids was investigated in whole body gamma-ray irradiated mice. Each flavonoid was administered to ICR male mice by a single gastric intubation (5 mumol/kg) 6 h before gamma-ray irradiation (1.5 Gy) and the frequency of micronucleated reticulocytes (MNRETs) in peripheral blood was determined. In order to elucidate the mechanism of the anticlastogenic effect of these flavonoids, their antioxidative activities were examined by the thiobarbituric acid method using methyl linoleate and Fenton's reagent (Fe2+/H2O2). Of the 12 flavonoids, luteolin had the most marked effect on reducing the frequencies of MNRETs and also inhibiting lipid peroxidation. However, quercetin tetramethylether, which has methoxy groups instead of hydroxyl groups at the 3,7,3',4'-positions, and phloretin with an open C-ring showed the least anticlastogenic and antioxidative activity. A good correlation (r = 0.717, P < 0.01) was observed between the anticlastogenic activity and the antioxidative activity of the 12 flavonoids. These results suggest that the radioprotective effect of flavonoids in mice may be attributed to the hydroxyl radical scavenging potency in a direct or an endogenous enzyme mediated manner.

Enhancing effects of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) on cell proliferation and lipid peroxidation in the rat gastric mucosa.

The influence of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone, a mutagen in chlorinated water, on cell proliferation and lipid peroxidation in the glandular stomach mucosa was investigated in male 4-week-old Wistar rats. Animals were given 50 p.p.m., 25 p.p.m., 12.5 p.p.m., 6.25 p.p.m. or 0 p.p.m. of MX solution in their drinking water for 5 weeks. At the end of this period, cell proliferation in the mucosal epithelia of the gastric fundus was increased in a dose-dependent manner up to 25 p.p.m., at which dose the induction was statistically significant as compared with the control value (P < 0.05). The MX treatment was also associated with increased lipid peroxidation levels in the gastric mucosa as well as in the urine, with loose dose-dependence, although not at 50 p.p.m. Mucosal lipid peroxidation was significantly increased in animals given 25 p.p.m. as compared with controls (P < 0.05). Similarly, the levels of urinary lipid peroxidation were significantly higher in rats given 25 p.p.m. or 12.5 p.p.m. than in the controls (P < 0.05). Histopathologically, gastric erosion was noted in rats receiving 25 p.p.m. or more of MX. There were no statistical differences between groups for serum biochemical data. The results thus suggest that MX may exert a gastric tumor-promoting action in rats, even at low doses which do not give rise to toxic effects, because of the clear dose-response relationship evident at low levels.

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) inhibits the removal of both cyclobutane dimers and (6-4) photoproducts from the DNA of ultraviolet-irradiated E. coli.

Heterocyclic amines have been isolated from cooked foods and found to be mutagens and carcinogens. Among them, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) were also found to enhance UV-induced mutation frequencies in Escherichia coli at the concentrations where they were neither toxic nor mutagenic by themselves. Using an immunological method recently developed to detect UV-induced DNA damage, we investigated the inhibitory effect of Trp-P-1 on the removal of both cyclobutane dimers and (6-4)photoproducts from the DNA of UV-irradiated E.coli. Cells repaired 60% of the initial cyclobutane dimers within 30 min and 75% at 120 min after UV-irradiation. Furthermore, the same cells repaired 90% of the initial (6-4)photoproducts within 30 min. On the other hand, Trp-P-1 clearly showed inhibition of repair of both photolesions in a concentration-dependent manner. The levels of repair inhibition by Trp-P-1 were almost the same between cyclobutane dimers and (6-4)photoproducts. These results suggested that the enhancing effect of Trp-P-1 on UV-induced mutagenesis in E.coli stemmed from the inhibition of the removal of photolesions from the DNA.

A possible correlation between environmental chemicals and pigment cell neoplasia in fish.

A croaker (Nibea mitsukurii) has a high incidence of the skin melanoma, chromatophoroma, in a Pacific coastal area in Japan. A sea catfish (Plotosus anguillaris) bearing skin melanosis is also found in the same area. For elucidation of a correlation between these pigment cell neoplasms of the skin and environmental contaminants, an epidemiological survey was conducted to determine the distribution and prevalence of tumor-bearing fish. Based upon observations of a high prevalence of skin neoplasms near the discharge point for kraft pulp mills, experiments were conducted to determine the neoplastic induction efficiency of the effluent on the croaker and sea catfish species. Isolation and identification of mutagens in effluent extracts were carried out using the Ames test, followed by mass spectral analysis of mutagenic fractions. The effluent induced a chromatophoroma on one croaker of the 100 tested, and it induced pigment cell hyperplasia on 70 to 100% of the sea catfish. These skin neoplasms were grossly similar to those observed in the field. Five chloroacetones were identified from the Ames-positive fractions of the effluent, and tetrachlorocyclopentene-1,3-dione and two alpha-dicarbonyl compounds were also detected as mutagens. The above experiments indicate that the mutagenic contaminants found in kraft mill effluent may play an important role in the induction of skin neoplastic disease in fish.

Environment: peculiar pigment cell neoplasm in fish.

Chromatophoroma in the croaker (Nibea mitsukurii) showed a unique geographic distribution. The contribution of environmental chemicals to the cause of chromatophoroma in the feral croaker is considered likely on the basis of the following results in our studies. 1) Chromatophoroma was induced in tank-reared N. mitsukurii by administration of certain kinds of known carcinogens such as 7,12-dimethyl-benz(a)anthracene, N-methyl-N'-nitro-N-nitrosoguanidine, and nifurpirinol. 2) Local accumulation of pigment-cell hyperplasia in the catfish (Protosus anguillaris) showed similar tendencies to those of chromatophoroma in N. mitsukurii. 3) Removal of contaminated sediment from the harbor and the river appeared to reduce the incidence from 47% in 1973-1983 to about 20% in 1985-1987. 4) Waste water from a factory located at the station where the incidence of the neoplasm was the highest contained mutagenic substances such as chloroacetones and glyoxals [5]. Exposure of catfish to the waste water induced pigment-cell hyperplasia on the skin.

Detection of 2-amino-3-methylimidazo[4,5-f]quinoline in cigarette smoke condensate.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), which is mutagenic and carcinogenic, was found to be present in cigarette smoke condensate by liquid chromatography with an electrochemical detector and a photodiode-array detector. The amount of IQ was estimated to be 0.26 ng per cigarette.

Identification and quantification of 2-amino-3,4,8-trimethylimidazo- [4,5-f]quinoxaline (4,8-DiMeIQx) in beef extract.

2-Amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) was found in bacteriological-grade beef extract by liquid chromatographies with electrochemical detection and a photodiode array detector. 4,8-DiMeIQx was estimated to be present at a level of 10.0 ng per g of beef extract, and to account for 9% of the total mutagenicity of the extract.

Quantification of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in beef extracts by liquid chromatography with electrochemical detection (LCEC).

A simple and sensitive method was developed for quantification of mutagenic/carcinogenic aminoimidazoquinoline and aminoimidazoquinoxaline compounds in heated materials. Samples were partially purified by blue-cotton treatment, 0.1 N HCl-methylene dichloride partition and separation in a SEP-PAK silica cartridge. The recoveries of aminoimidazoquinoline and aminoimidazoquinoxaline compounds at the step of partial purification were estimated by spiking with 14C-labeled compounds. The compounds in partially purified materials were analyzed by liquid chromatography with electrochemical detection using a combination of two columns of octadecyl silane and cation exchange. Bacteriological-grade beef extract was found to contain 41.6 and 58.7 ng/g of 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), respectively. MeIQx was also detected at a level of 3.1 ng per g in food-grade beef extract.