Quantification method of DNA adducts at the practical sensitivity was eagerly awaited in several study area such as mutagenesis, carcinogenesis, aging, DNA repair and molecular epidemiology. Recent development of liquid chromatography electrospray tandem mass spectrometry (LC/ESI/MS/MS) enables us to detect 25 fg of adducted deoxynucleoside, which is corresponding to a few DNA adducts in 109 bases. This means that LC/ESI/MS/MS analysis of DNA adducts reaches to the practical sensitivity.
To evaluate the aneugenic effects of carbendazim (MBC) and griseofulvin (GF) on meiosis, we cultured mouse oocytes, allowing them to mature in vitro in the presence of MBC and GF. The incidence of hyperploidy was significantly increased in oocytes cultured with 0.6 μg/mL or higher concentration of MBC, while the incidence of diploidy was significantly increased in oocytes cultured with 1 or 3 μg/mL of GF. To investigate the stage-specific effects of these chemicals during meiotic progression, the oocytes were exposed to chemicals for the initial 7 h or final 8 h of the culture. Both MBC and GF were found to effectively induce numerical aberration during the final half of the culture. Thus, in vitro maturation of mouse oocytes is a useful model for the detection of chemically induced aneuploidy and for the detection of stage-specific effects of chemicals during meiosis.
Swimming pool water may accumulate chlorinated organic compounds because of continuous chlorination and contamination with organic compounds in the form of sweat, urine, epithelial cells, hair, etc. Some of these chlorinated compounds are suspected as carcinogens or mutagens. In this paper, we assayed the mutagenicity of 5 samples of swimming pool water in Kitakyushu-city. The samples were concentrated with adsorbent (CSP800) and the mutagenic activity was assayed on Salmonella typhimurium TA100 and TA98 strains with or without S9 mix. The water samples were also analyzed for volatile organic compounds and some factors prescribed by the conventional water quality surveillance protocol. The samples tested showed TA100 mutagenicity of 2,100 to 13,030 rev./L without S9 mix. High concentration of ammonia was detected in one of the samples, but not chloroform. The users of the facility, where the sample was obtained, had complained of eye irritation. In addition to ammonia, chloramine formed from ammonia and chlorine is known to be irritative to eyes and to give unpleasant odor in swimming pool water. Therefore, we examined the level of chloroform, residual chlorine, and the mutagenicity when ammonia was added to chlorine-treated humic acid as a model for generation of chlorinated organic compound. Chloroform level and the mutagenicity were decreased, while residual chlorine level was increased during chloramine formation.
Hepatic cytochrome P-450 (CYP) 1A1, 51 and 53kDa proteins and constitutive CYP1A2 (3.8-fold) and 2B2 (4.0-fold), but not CYP2B1, 2E1 and 3A2, were induced by i.g. treatment with 85 mg/kg 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) eight times over 11 days in female SD rats. Feeding 400 p.p.m. α-naphthyl isothiocyanate (ANIT) in a high fat diet for 3 weeks induced three CYP1A proteins but not CYP1A2; this enhancement was much lower compared to that seen with PhIP. PhIP treatment and subsequent use of liver S9 fraction in S.typhimurium TA98 mutation assay showed 2.8-12.9-fold elevation of mutagenicity by PhIP, four other heterocyclic amines (HCAs) and benzo[a]pyrene (BP), whereas no significant alterations in the TA98 mutagenic activities of these chemicals were produced with S9 prepared after gavage with ANIT. The combination of PhIP and ANIT markedly decreased three liver CYP1A proteins except CYP1A1 and CYP2B2, and the liver S9 from this combination showed reduced mutagenic activities of HCAs and BP. PhIP and ANIT enhanced UDP-glucuronyltransferase (UDPGT) activity towards 4-nitrophenol 2.2- and 1.6- fold, respectively, and the combined administration showed much higher induction (4.7-fold). These results clearly indicate that chemoprevention by ANIT of PhIP-induced rat mammary carcinogenesis can be attributed to a dual action mechanism; a decrease in metabolic activation of PhIP by hepatic CYP1A2 and an increase in the detoxification by UDPGT but not by CYP1A1. On the other hand, no significant alterations in the hepatic levels of these enzymes and activities were produced by s.c. treatment with 0.5 mg/kg N-nitrosomethylbenzylamine (NMBA) three times per week for 5 weeks, feeding of —0.2% curcumin in the diet for 6 weeks, or both, in male F344 rats. In contrast, gavage of curcumin decreased the constitutively detected CYP2B1 and 2E1 in the esophagus, and a single dose of 270 mg/kg curcumin caused 40% decreases (P<0.01) in both CYP species. This suggests that suppression by curcumin of NMBA-induced esophageal carcinogenesis can be attributed to a decrease in esophageal activation of NMBA during the initiation phase, but not attributed to metabolic activation and inactivation via glucuronidation in the liver. Nevertheless, these are the first demonstrations that a HCA can induce hepatic CYP2B2, that an isothiocyanate can suppress metabolic activation by hepatic CYP1A and 2B, and that curcumin shows a suppressive effect on esophagus CYP2B and 2E1.
Dietary factors are estimated to account for approximately 30% of cancer deaths in the United States. According to a WHO/FAO report in 2003 based on a review of published epidemiological studies, convincing evidence linking diet-related factors and cancer is available for the following associations: physical activity (colorectal cancer), overweight/obesity (cancers of esophagus <adenocarcinoma>, colorectum, breast <postmenopausal>, endometrium and kidney), alcohol (cancers of oral cavity, pharynx, esophagus, liver and breast), aflatoxin (liver) and Chinese-style salted fish (nasopharyngeal cancer). Fruits and vegetables (cancers of oral cavity, esophagus, stomach and colorectum) and physical activity (breast cancer) probably reduce the risk, while preserved and red meat (colorectal cancer), salt-preserved foods and high salt intake (stomach cancer) and very hot drinks and foods (cancers of oral cavity, pharynx and esophagus) probably increase the risk. Since these evidences are mainly based on epidemiological studies in Western countries, more evidence from studies in Japan is essential to establish appropriate recommendations for reducing the risk of developing cancer among Japanese. Recent information from the Japan Public Health Centerbased prospective study (JPHC Study), on-going population-based cohort study in 11 areas nationwide, is providing evidence for the association between dietary factors and the risk of gastric and breast cancers.
Recent studies on the bioavailability, pharmacokinetics and metabolism of flavonoids which are common dietary components of vegetables, fruits, wine, and tea have largely focused on evaluating their in vivo functional activities and their roles in the chemoprevention of diseases, including cancer, atherosclerosis, and others. A number of studies have reported that the absorbed flavonoids are primarily metabolized to the glucuronide and/or sulfate conjugates in vivo, which then circulate in the blood and are released into the bile and urine for excretion. It remains unknown, however, whether these conjugates still retain biological functions in vivo as their aglycones do. Notably, a glucuronided metabolite of quercetin was earlier reported to possess antioxidant activity and to prevent vascular smooth muscle cell hypertrophy induced by angiotensin II. We thus set out to study the effects of quercetin conjugates on TNF-α-induced expression of intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVECs) by using an ELISA detection method. We found that quercetin-3-glucuronide and quercetin-3-sulfate failed to show suppressive effects. Moreover, quercetin-3-glucuronide showed no antiproliferative effects in MCF-7 mammary tumor cells. Accordingly, these results suggest that deconjugation is required for a quercetin glucuronide to exert its effects. We found that β-glucuronidase released from neutrophils and certain injured cells can hydrolyze glucuronidated flavonoids to free aglycones when inflammation occurs. In addition, we also found that incubation of endothelial cells with cytokines such as IL-1α resulted in an increased permeability to glucuronides. Therefore, it is suggested that conjugated flavonoids may pass through the vascular monolayers and are then converted to free aglycones by the hydrolyzing enzymes released from inflammatory cells or tissue cells to exert their biological activity at the inflammation site. Hence, it is our belief that the conjugated flavonoids in circulation may still play an important role as chemopreventive agents at the early stage of an inflammatory disease process.
Co-intake of grapefruit juice with drugs results in a substantial increase in oral drug bioavailability. In contrast, DNA damage in target organ induced by a food-derived carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was reduced in rats by grapefruit juice intake. Aflatoxin B1-induced DNA damage was also suppressed in rats treated with grapefruit juice and an ethyl acetate extract of grapefruit juice. A significant decrease in hepatic CYP3A content, but not in CYP1A, CYP2C, glutathione S-transferase and microsomal epoxide hydrolase contents was observed in rats after grapefruit juice intake. No significant differences in the portal blood and liver concentrations of aflatoxin B1, nor in blood concentration of PhIP, were observed between control rats and rats ingesting grapefruit juice. Thus, grapefruit juice intake causes suppression of carcinogen-induced DNA damage at least in part through decreased metabolic activation in rat liver.
An important group of compounds that have a chemopreventive property is organosulfur compounds such as isothiocyanates. In the present study, we clarified the molecular mechanism underlying the relationship between benzyl isothiocyanate (BITC)-induced cell cycle arrest and apoptosis. The exposure of the cultured cells to BITC resulted in the inhibition of the G2/M progression that coincided with apoptosis induction. An experiment using phase-specific synchronized cells demonstrated that the G2/M phase-arrested cells are more sensitive to undergoing apoptotic stimulation by BITC than the cells in other phases. We identified phosphorylated Bcl-2 as a key molecule linking the p38 MAPK-dependent cell cycle arrest with JNK activation by BITC. We also found that BITC induced a cytotoxic effect more preferentially in the proliferating normal human colon epithelial cells than the quiescent cells. Moreover, down-regulation of p53 resulted in the enhancement of susceptibility to undergoing apoptotic stimulation by BITC. These findings suggested that p53 might play a negative regulating role in BITC-induced apoptosis. In conclusion, the results from this study provided biological evidence that BITC has a potential to induce apoptosis selectively in p53-mutated proliferating pre-cancerous cells.
Heirloom vegetables in Kyoto, that have preserved their seeds by a traditional cultivation, have a more savory taste than conventional vegetables. The bioantimutagenic activity in such vegetables has been examined using the UV-induced mutation assay of Escherichia coli B/r WP2 and human fibroblasts. We report three major findings. Firstly, extracts from heirloom Kamo-nasu eggplant, Shishigatani-kabocha pumpkin and Katsura-uri pickling melon revealed stronger bioantimutagenicity than extracts from the counterpart conventional vegetables. Secondly, seven strains of the heirloom daikon in Kyoto were found to have a higher potency of antimutagenicity than conventional daikon. The active bioantimutagen principle was identified to be 4-(methylthio)-3-butenyl isothiocyanate, which causes the pungency in daikon. Thirdly, the aqueous fraction in Fushimi-togarashi sweet pepper facilitated the repair of UV-induced cyclobutane pyrimidine dimer in human fibroblasts.
We are daily exposed to many kinds of chemicals through air, drinking water and foodstuffs. Some of these chemicals show mutagenic and carcinogenic activities. N-nitrosamines are potential candidates to induce human carcinogenesis, as these compounds are carcinogenic in animal studies and because they are formed by reaction of secondary amines with nitrite in the stomach environment. In Japan, stomach cancer is the most common malignancy. Therefore it is an important task to know how to inhibit carcinogenesis induced by N-nitrosamines. In this study, we examined if green tea acts as an inhibitor of the formation of N-nitrosamines. Furthermore, we determined the catechin concentration in a cup of green tea prepared from tea found in 13 countries, and examined the relationship between such concentrations and the incidence of cancer in several organs. Green tea leaves were brewed 8 times in hot water. Sodium nitrite and morpholine were added to each extract and adjusted to pH 3.0. After incubation at 37°C for 30 min, the concentration of formed N-nitrosomorpholine was determined by HPLC with a UV detector. The first and second green tea extracts inhibited the formation of N-nitrosomorpholine, while subsequent extracts accelerated its formation. These results suggested that the formation of nitrosamines in stomach may be reduced by green tea brews and that repeated brewing results in diminished inhibition of nitrosamine formation. We also demonstrated that the intake of catechins in tea producing areas is higher than that in non-producing areas. We observed a significant inverse correlation between standardized mortality ratio (SMR) of stomach cancer in males and the catechin concentration in green tea extracts. From these data, it is evident that green tea might be a potential chemopreventive agent to inhibit the formation of N-nitrosamine.