Deglucuronidation of a flavonoid, luteolin monoglucuronide, during inflammation.

In this study, we investigated whether luteolin monoglucuronide was converted to free aglycone during inflammation using human neutrophils stimulated with ionomycin/cytochalasin B and rats treated with lipopolysaccharide (LPS). beta-Glucuronidase activity was assayed using 4-methylumbelliferyl-glucuronide and methanol extracts of rat plasma containing luteolin monoglucuronide. The released 4-methylumbelliferone, a fluorescent molecule, was quantified by fluorometry. Deglucuronidation of luteolin monoglucuronide was examined by high-performance liquid chromatography (HPLC) analysis. HPLC analyses showed that the supernatants obtained from neutrophils stimulated with ionomycin/cytochalasin B hydrolyzed luteolin monoglucuronide to free luteolin. beta-Glucuronidase activity in human serum from patients on hemodialysis increased significantly compared with that from healthy volunteers. The beta-glucuronidase activity in rat plasma increased after i.v. injection of LPS. The ratio of luteolin to luteolin monoglucuronide in plasma of LPS-treated rats also increased. These results suggest that during inflammation beta-glucuronidase is released from stimulated neutrophils or certain injured cells and then deglucuronidation of flavonoids occurs.

Oxidative DNA damage induced by high glucose and its suppression in human umbilical vein endothelial cells.

In order to investigate the mechanism of the production of oxidative DNA damage by hyperglycemia, we measured formamidopyrimidine N-glycosylase (FPG)-sensitive sites by the comet assay in human umbilical vein endothelial cells (HUVECs) cultured under various conditions including high glucose. Mean values of FPG-sensitive sites were higher in HUVECs cultured for 5 days in high glucose (45 mM) compared with normal glucose (5mM) medium (P<0.001). FPG-sensitive sites increased in a time-dependent manner under high glucose treatment (3 days: P<0.05, 5 days: P<0.001), whereas L-glucose, which is taken up poorly into the cells, gave a slight increase in FPG-sensitive sites (P<0.05). Flow cytometric analysis using 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) showed that incubation with L-glucose produced more reactive oxygen species than incubation with D-glucose. However, these increases were slight (1.22- and 1.12-folds, respectively). Incubation of HUVECs with aminoguanidine (100 microM) or pyridoxamine (1mM), which are inhibitors of glycation, decreased the levels of FPG-sensitive sites (P<0.001). However, these inhibitors did not suppress the intracellular generation of reactive oxygen species induced by high glucose. These results indicate that FPG-sensitive sites induced by high glucose are not due to intracellular reactive oxygen species. In order to clarify what caused the induction of FPG-sensitive sites, we investigated the effect of glyoxal and 3-deoxyglucosone (3-DG) on the induction of FPG-sensitive sites and the intracellular production of reactive oxygen species in HUVECs. Glyoxal and 3-DG at a concentration of 100 microg/ml induced FPG-sensitive sites (P<0.001, P<0.01, respectively). In contrast, glyoxal did not generate reactive oxygen species inside HUVECs. The results shown in this study suggest that glyoxal formed intracellularly or extracellularly during high glucose treatment might induce FPG-sensitive sites by a mechanism not involving reactive oxygen species.

Dietary intakes of flavonols, flavones and isoflavones by Japanese women and the inverse correlation between quercetin intake and plasma LDL cholesterol concentration.

The intake of flavonols, flavones and isoflavones by Japanese women was calculated from our food-phytochemical composition table. The relationship between intake of these phytochemicals and various anthropometric and blood chemistry data was analyzed in a cross-sectional study. The subjects were 115 women volunteers, aged 29-78 y, living in the northern part of Japan. Each subject completed a 3-d dietary record and received a health check up, including urine and blood sampling for biochemical analysis. Total mean intakes of flavonoids (sum of flavonols and flavones) and isoflavones were 16.7 and 47.2 mg/d, respectively. The major source of flavonoids was onions (45.9%) and that of isoflavones was tofu (37.0%). Total intake of isoflavones exceeded that of other dietary antioxidants, such as flavonoids, carotenoids (3.5 mg/d) and vitamin E (8.2 mg/d), and was approximately one half of the vitamin C intake (109 mg/d). The total intake of flavonoids was inversely correlated with the plasma total cholesterol concentration (TC) (r = -0.236, P: < 0.05) and plasma LDL cholesterol concentration (LDL-C) (r = -0.220, P: < 0.05), after the adjustment for age, body mass index and total energy intake. As a single component, quercetin was inversely correlated with both TC (r = -0.261, P: < 0.01) and LDL-C (r = -0. 263, P: < 0.01). Among Japanese, flavonoid and isoflavone intake is the main component among nonnutrient phytochemicals with antioxidant potential in the diet. These results suggest that a high consumption of both flavonoids and isoflavones by Japanese women may contribute to their low incidence of coronary heart disease compared with women in other countries.

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.

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

In this study, we investigated the intestinal absorption of luteolin and luteolin 7-O-beta-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-beta-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.

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.

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.

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 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 Fenton's 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.

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.