Chemo-preventive effects and antitumorigenic mechanisms of beer and nonalcoholic beer toward 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) - induced lung tumorigenesis in A/J mice.

We investigated the chemopreventive effects of beer, nonalcoholic beers (NABs), and beer-components (glycine betaine (GB)) on NNK-induced lung tumorigenesis in A/J mice, and the possible mechanisms underlying the antitumorigenic effects of beer, NABs, and beer-components. Beer, NABs, and GB reduced NNK-induced lung tumorigenesis. We investigated the antimutagenicity of beer, NABs and beer-components (GB and pseudouridine (PU)) toward the mutagenicity of 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Beer, NABs, and beer components were antimutagenic toward MNNG and NNK in the Ames test using S. typhimurium TA1535. In contrast, MNNG and NNK mutagenicity detected in S. typhimurium YG7108, a strain lacking O6-methylguanine DNA methyltransferases (ogtST and adaST) did not decrease in the presence of beer, NABs, or beer components, suggesting that they may mediate its antimutagenic effect by enhancing DNA damage repair. Phosphorylation of Akt and STAT3, with or without epidermal growth factor stimulation, in lung epithelial-like A549 cells were significantly decreased following beer, NABs, GB and PU. They targeted both the initiation and growth/progression steps of carcinogenesis, specifically via antimutagenesis, stimulation of alkyl DNA-adduct repair, and suppression of Akt- and STAT3- mediated growth signaling. GB and PU may contribute, in part, to the biological effects of beer and NABs via the suppression of Akt and STAT3 phosphorylation.

Chemopreventive effects and anti-tumorigenic mechanisms of Actinidia arguta, known as sarunashi in Japan toward 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)- induced lung tumorigenesis in a/J mouse.

BACKGROUND: Previously, we reported the inhibitory effect of Actinidia arguta juice, known as sarunashi juice (sar-j) in Japan, on mutagenesis, inflammation, and mouse skin tumorigenesis. The components of A. arguta responsible for the anti-mutagenic effects were identified to be water-soluble, heat-labile phenolic compounds. We proposed isoquercetin (isoQ) as a candidate anticarcinogenic component. In this study, we sought to investigate the chemopreventive effects of A. arguta juice and isoQ on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice, and identify the possible mechanisms underlying the anti-tumorigenic effects of A. arguta. RESULTS: The number of tumor nodules per mouse lung in the group injected with NNK and administered A. arguta juice orally was significantly lower than that in the group injected with NNK only. Oral administration of isoQ also reduced the number of nodules in the mouse lungs. As expected, the mutagenicity of NNK and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) detected using S. typhimurium TA1535 decreased in the presence of sar-j. However, NNK and MNNG mutagenicity detected using S. typhimurium YG7108, a strain lacking the O6-methylguanine DNA methyltransferases (ogtST and adaST) did not decrease in the presence of sar-j suggesting that sar-j may mediate its antimutagenic effect by enhancing the DNA damage repair by ogtST and adaST. Phosphorylation of Akt, with or without epidermal growth factor stimulation, in A549 cells was significantly decreased following sar-j and isoQ treatment, indicating that components in sar-j including isoQ suppressed the PI3K/AKT signaling pathways. CONCLUSIONS: Sar-j and isoQ reduced NNK-induced lung tumorigenesis. Sar-j targets both the initiation and growth/progression steps during carcinogenesis, specifically via anti-mutagenesis, stimulation of alkyl DNA adduct repair, and suppression of Akt-mediated growth signaling. IsoQ might contribute in part to the biological effects of sar-j via suppression of Akt phosphorylation, but it may not be the main active ingredient.

Antimutagenic, anti-inflammatory, and antioxidative activities of the juice of Vitis ficifolia var. Ganebu, a woody vine in the grape family, known as Ryukyu-ganebu in Japan.

BACKGROUND: Mutation, inflammation, and oxidative damage including lipid-peroxidation are factors involved in the development of cancer. We investigated the antimutagenic, in vivo and in vitro anti-inflammatory, and antioxidative effects of the juice of Vitis ficifolia var. ganebu (known as Ryukyu-ganebu in Japan) harvested in Kuchinoshima island (hereafter, the juice is referred to as ganebu-K) in comparison with the juice of Vitis coignetiae (crimson glory vine, known as yamabudo in Japan; hereafter, the juice is referred to as yamabudo) which we found antimutagenic and anti-inflammatory effects. RESULTS: Ganebu-K inhibited the mutagenic activity of several carcinogens, MeIQx, IQ, Trp-P-2(NHOH), and MNNG, model compounds of tumor initiation. Using S. typhimurium YG7108, a strain lacking O6-methylguanine DNA methyltransferases, ganebu-K showed no significant inhibition of the mutagenicity of MNNG. Thus, DNA repair of O6-methylguanine produced by MNNG might be an antimutagenic target of the components in ganebu-K. Topical application of ganebu-K to the dorsal sides of mice resulted in potent suppression of acute edema induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Ganebu-K, but not yamabudo, exhibited significant inhibition of the induction of prostaglandin E2 (PGE2) induced by TPA. Components contained in ganebu-K, but not in yamabudo, might be responsible for the inhibition of the induction of PGE2. Ganebu-K inhibited in vivo lipid peroxidation and decreased the level of glutamic oxaloacetic transaminase induced by CCL4 treatment. CONCLUSIONS: These results suggest that the active components in ganebu-K juice are not the same as those in yamabudo, and the components in ganebu-K are attractive candidates as chemopreventive agents.

Chemopreventive effects and anti-tumorigenic mechanisms of 2,6-dimethoxy-1,4-benzoquinone, a constituent of Vitis coignetiae Pulliat (crimson glory vine, known as yamabudo in Japan), toward 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice.

Previously, we isolated and identified anti-mutagenic and anti-inflammatory components from Vitis coignetiae (crimson glory vine, known as yamabudo in Japan) as 2,6-dimethoxy-1,4-benzoquinone (DBQ), fertaric acid and caftaric acid. We also reported that the oral intake of a partially purified fraction from yamabudo juice (yamabudo-fr) or DBQ affords significant protection against two-stage skin carcinogenesis in mice. In this study, we found that oral intake of yamabudo-fr or DBQ affords significant protection against a tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse model of lung tumorigenesis. Furthermore, we investigated the anti-tumorigenic mechanisms of yamabudo juice and DBQ. NNK is known to be a DNA-methylating and alkylating agent; thus, we investigated the anti-tumorigenic mechanisms of yamabudo juice and DBQ in relation to DNA methylation. Pretreatment with yamabudo-fr or DBQ dose-dependently decreased formation of O6-methylguanine and N7-methylguanine in DNA of the A549 human lung epithelial-like cell line treated with a methylating agent, 1-methyl-3-nitro-1-nitrosoguanidine. Yamabudo juice and DBQ inhibited the mutagenicity of NNK in the Ames test using Salmonella typhimurium TA1535 but not S. typhimurium YG7108, an alkylguanine DNA alkyltransferase-deficient strain (same as TA1535 but Δadast::Kmr, Δogtst::Cmr). Yamabudo juice and DBQ might accelerate the repair of DNA damage caused by NNK and reduce DNA damage to cells. We also investigated the effects of yamabudo juice and DBQ on signaling pathways in A549 cells. With or without epidermal growth factor stimulation, phosphorylation of Erk1/2, Akt and Stat3 in A549 cells was significantly decreased in the presence of yamabudo juice or DBQ, indicating that yamabudo juice and DBQ suppressed PI3K/AKT, MAPK/ERK and JAK/STAT3 signaling pathways. These results suggest that both initiation and growth/progression steps in carcinogenesis, especially anti-oxidant effects, stimulation of repair of alkyl DNA adducts and suppressed growth signaling pathways are potential anti-tumorigenic targets of yamabudo juice and DBQ in NNK-induced lung tumorigenesis.

2,6-Dimethoxy-1,4-benzoquinone, isolation and identification of anti-carcinogenic, anti-mutagenic and anti-inflammatory component from the juice of Vitis coignetiae.

Previously we demonstrated the anti-tumorigenic, anti-mutagenic and anti-inflammatory effects of the juice of Vitis coignetiae (yamabudo), and identified caftaric acid as an anti-mutagenic component from the juice. In the present study, we investigated the isolation of anti-inflammatory components in yamabudo juice supposing that the anti-inflammatory components in yamabudo are also responsible for the anti-tumorigenic activity. The suppressing effect on nitric oxide production in mouse leukemic monocyte with LPS was used as a separation marker. Three components comprising 2,6-dimethoxy-1,4-benzoquinone (DBQ), fertaric acid and caftaric acid were isolated and identified from the juice of V. coignetiae as anti-inflammatory ingredients. Inhibitory effects were found of DBQ on the mutagenicity of dimethylbenzo[a]anthracene, aflatoxin B1, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the Ames test. Topical application of DBA significantly inhibited TPA-induced edema of mouse ears. The anti-tumorigenic effect of DBQ on the promotion and initiation stages of mouse skin tumorigenesis was investigated, and topical administration of DBQ on the promotion stage significantly decreased tumor development in mice skin. DBQ is a potential candidate for the chemopreventive effect of V. coignetiae.

Increase of somatic cell mutations in oxidative damage-sensitive drosophila.

BACKGROUND: Oxidative damage is an important genotoxic source for almost all organisms. To efficiently detect mutations induced by oxidative damage, we previously developed a urate-null Drosophila strain. Using this Drosophila strain, we showed the mutagenic activity of environmental cigarette smoke (ECS) and the herbicide paraquat, which are known to produce reactive oxygen species (ROS). In the present study, we examined the mutagenic activities of carcinogenic mutagens that are considered to cause mutations by adduct formation, alkylation, or crosslinking of cellular DNA in the oxidative damage-sensitive Drosophila to evaluate how the oxidative damage induced by these mutagens is involved in causing mutations. In addition, we evaluated whether these oxidative damage-sensitive flies may be useful for mutation assays. METHODS: We performed the wing-spot test in oxidative damage-sensitive Drosophila (urate-null strains) to examine the mutagenicity of 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx), mitomycin C (MMC), 4-nitroquinoline N-oxide (4NQO), N-nitrosodimethyl-amine (NDMA), and N-nitrosodiethylamine (NDEA). We also observed the mutagenicity of X-ray irradiation as a control in which mutations should be mainly caused by oxidative damage. RESULTS: As expected, the mutagenic activity of X-ray irradiation was higher in the urate-null Drosophila than in the wild-type Drosophila. The mutagenic activities of the tested compounds were also higher in the urate-null Drosophila than in the wild-type Drosophila. In experiments using another urate-null strain, the mutagenicity of N-nitrosodialkylamines was also higher in the urate-null flies than in the wild-type ones. CONCLUSIONS: The tested compounds in this study were more mutagenic in urate-null Drosophila than in wild-type Drosophila. It was supposed that ROS were generated and that the ROS might be involved in mutagenesis. The present results support the notion that in addition to causing DNA lesions via adduct formation, alkylation, or DNA crosslinking, these mutagens also cause mutations via ROS-induced DNA damage. As such, urate-null Drosophila appear to be useful for detecting the mutagenic activity of various mutagens, especially those that produce reactive oxygen. If the mutation rate increases on a mutation assay using urate-null Drosophila, it might suggest that the mutagen generates ROS, and that the produced ROS is involved in causing mutations.

Inhibitory effect of Actinidia arguta on mutagenesis, inflammation and two-stage mouse skin tumorigenesis.

BACKGROUND: Actinidia arguta, known as sarunashi in Japan, is a vine tree native to east-Asia, including Japan, that produces small fruit rich in anthocyanins, catechins, vitamin C, chlorophyll, beta-carotene and other polyphenols. RESULTS: Our study revealed the inhibitory effect of the juice of A. arguta (arguta-juice) toward the mutagenicity of food-derived carcinogens and polycyclic aromatic hydrocarbons using the Ames test, and antioxidant activity of arguta-juice as determined using a free radical scavenging assay. The formation of DNA adducts in liver of mice fed 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) decreased significantly following administration of arguta-juice. The preventive effect of arguta-juice on the induction of inflammation of mouse ear by 12-O-tetradecanoylphorbol-13-acetate (TPA) was revealed. The anti-carcinogenic effect of a topically applied partially purified fraction of A. arguta was revealed on skin tumorigenesis in mice induced by treatment with 7,12-dimethylbenz(a)anthracene and TPA. In an effort to reveal the mechanisms for antimutagenicity of arguta-juice, effects on the enzymes that metabolize xenobiotics were examined. Combined effects comprising i) inhibition of the metabolic activation of mutagens with phase I enzymes, but ii) no prevention on the activity of phase II detoxification enzyme, UGT, were observed. We also investigated the characterization and partial purification of the antimutagenic components in A. arguta, which suggested that the components in A. arguta responsible for the antimutagenicity were water-soluble, heat-labile phenolic compounds. CONCLUSIONS: These results suggested that components in A. arguta are attractive candidates for potential use as chemopreventive agents.

Somatic-cell mutation induced by short exposures to cigarette smoke in urate-null, oxidative stress-sensitive Drosophila.

We previously reported that a urate-null strain of Drosophila is hypersensitive to cigarette smoke (CS), and we suggested that CS induces oxidative stress in Drosophila because uric acid is a potent antioxidant. Although the carcinogenic risk of CS exposure is widely recognized; documentation of in vivo genotoxic activity of environmental CS, especially gaseous-phase CS, remains inconclusive. To date, somatic-cell mutations in Drosophila resulting from exposure to CS have not been detected via the somatic mutation and recombination test (wing spot test) with wild-type flies, a widely used Drosophila assay for the detection of somatic-cell mutation; moreover, genotoxicity has not been documented via a DNA repair test that involves DNA repair-deficient Drosophila. In this study, we used a new Drosophila strain (y v ma-l; mwh) to examine the mutagenicity induced by gaseous-phase CS; these flies are urate-null due to a mutation in ma-l, and they are heterozygous for multiple wing hair (mwh), a mutation that functions as a marker for somatic-cell mutation. In an assay with this newly developed strain, a superoxide anion-producing weed-killer, paraquat, exhibited significant mutagenicity; in contrast, paraquat was hardly mutagenic with a wild-type strain. Drosophila larvae were exposed to CS for 2, 4 or 6h, and then kept at 25°C on instant medium until adulthood. After eclosion, mutant spots, which consisted of mutant hairs on wings, were scored. The number of mutant spots increased significantly in an exposure time-dependent manner in the urate-null females (ma-l (-/-)), but not in the urate-positive females (ma-l (+/-)). In this study, we showed that short-term exposure to CS was mutagenic in this in vivo system. In addition, we obtained suggestive data regarding reactive oxygen species production in larva after CS exposure using the fluorescence probe H2DCFDA. These results suggest that oxidative damage, which might be countered by uric acid, was partly responsible for induction of somatic cell mutations in Drosophila larvae exposed to CS.

Mutagenicity and clastogenicity of extracts of Helicobacter pylori detected by the Ames test and in the micronucleus test using human lymphoblastoid cells.

Epidemiological studies have demonstrated a close association between infection with Helicobacter pylori (H.pylori) and the development of gastric carcinoma. Chronic H.pylori infection increases the frequency of mutation in gastric epithelial cells. However, the mechanism by which infection of H.pylori leads to mutation in gastric epithelial cells is unclear. We suspected that components in H.pylori may be related to the mutagenic response associated with DNA alkylation, and could be detected with the Ames test using a more sensitive strain for alkylating agents. Our investigation revealed that an extract of H.pylori was mutagenic in the Ames test with Salmonella typhimurium YG7108, which is deficient in the DNA repair of O(6)-methylguanine. The extract of H.pylori may contain methylating or alkylating agents, which might induce O (6)-alkylguanine in DNA. Mutagenicity of the alkylating agents N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-N-nitrosoguanidine in the Ames test with S.typhimurium TA1535 was enhanced significantly in the presence of the extract of H.pylori. The tested extracts of H.pylori resulted in a significant induction of micronuclei in human-derived lymphoblastoid cells. Heat instability and dialysis resistance of the extracts of H.pylori suggest that the mutagenic component in the extracts of H.pylori is a heat-unstable large molecule or a heat-labile small molecule strongly attached or adsorbed to a large molecule. Proteins in the extracts of H.pylori were subsequently fractionated using ammonium sulphate precipitation. However, all fractions expressed enhancing effects toward MNU mutagenicity. These results suggest the mutagenic component is a small molecule that is absorbed into proteins in the extract of H.pylori, which resist dialysis. Continuous and chronic exposure of gastric epithelial cells to the alkylative mutagenic component from H.pylori chronically infected in the stomach might be a causal factor in the gastric carcinogenesis associated with H.pylori.

Somatic cell mutations caused by 365 nm LED-UVA due to DNA double-strand breaks through oxidative damage.

Evidence is accumulating indicating that UVA (320-400 nm ultraviolet light) plays an important role in photo-carcinogenesis. UVA is thought to produce reactive oxygen species in irradiated cells through photo-activation of inherent photosensitizers, and was recently reported to cause DNA double-strand breaks (DSBs) in exposed cells. We have investigated the involvement of UVA in mutations and DNA damage in somatic cells using Drosophila melanogaster larvae. Using the Okazaki Large Spectrograph, we previously observed that longer wavelength UVA (>330 nm) was more mutagenic in post-replication repair-deficient D. melanogaster (mei-41) than in the nucleotide excision repair-deficient strain (mei-9). LED-light has recently been developed as a high-dose-rate UVA source. LED-UVA light (365 nm) was also more mutagenic in mei-41 than in mei-9. The mei-41 gene was shown to be an orthologue of the human ATR gene, which is involved in the repair of DSBs through phosphorylation of histone H2AX. In order to estimate the extent to which oxidative damage contributes to mutation, we established a new D. melanogaster strain (urate-null mutant) that is sensitive to oxidative damage and has a marker to detect somatic cell mutations. When somatic cell mutations were examined using this strain, LED-UVA was mutagenic in the urate-null strain at doses that were non-mutagenic in the urate-positive strain. In an effort to investigate the generation of DSBs, we examined the presence of phosphorylated histone H2AvD (H2AX D. melanogaster homologue). At high doses of LED-UVA (>800 kJ m(-2)), levels of phosphorylated H2AvD (γ-H2AvD) increased significantly in the urate-null strain. Moreover, the level of γ-H2AvD increased in the excision repair-deficient strain but not in the ATR-deficient strain following UVA-irradiation. These results supported the notion that the generation of γ-H2AvD was mediated by the function of the mei-41 gene. It was reported that ATR functions on DSB repair in D. melanogaster. Taken together, we propose a possible pathway for UVA-induced mutation, whereby DNA double-strand breaks resulting from oxidative stress might be responsible for UVA-induced mutation in somatic cells of D. melanogaster larvae.

Chemopreventive effects of the juice of Vitis coignetiae Pulliat on two-stage mouse skin carcinogenesis.

Our study revealed the inhibitory effect of Vitis coignetiae Pulliat, known as Yamabudo in Japan, at the stages of multi-step carcinogenesis. The juice of Vitis coignetiae (Y-grape juice) was antimutagenic toward dimethylbenzo[a]anthracene (DMBA), aflatoxin B1, and benzo[a]pyrene in the Ames test. The Y-grape juice was also antigenotoxic in the micronucleus test using HepG2 cells toward DMBA and aflatoxin B1. Topical and oral administration of the Y-grape juice to mice inhibited the induction of inflammation of 12-O-tetradecanoylphorbol-13-acetate (TPA). Topical and oral administration of the Y-grape juice significantly decreased the incidence and mean number of tumors in mice skin with the 2-stage tumorigenesis protocol. To elucidate the mechanisms underlying the antiinflammatory and antitumor promotion activity of the Y-grape juice, the effect of Y-grape juice on cyclooxygenase-2 (COX-2) activity in mouse ear treated with TPA was studied. Both topical and oral application of the Y-grape juice inhibited the TPA-induced increase in COX-2 activity. Caftaric acid, isolated and identified from the Y-grape juice, was antimutagenic toward DMBA and prevented TPA-induced inflammation in mice, suggesting caftaric acid participates in chemopreventive effect/activities of Y-grape juice.

Anti-genotoxic activity of Vitis coignetiae Pulliat towards heterocyclic amines and isolation and identification of caftaric acid as an antimutagenic component from the juice.

Our study demonstrated that the formation of DNA adducts in liver, lungs, colon and kidneys of mice given a carcinogenic heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), in the diet significantly decreased following the administration of the juice of Vitis coignetiae, purple berries from a vine tree. The juice of V. coignetiae significantly inhibited the clastogenicity and mutagenicity of heterocyclic amines in the micronucleus assay and the Ames test, and was an effective inhibitor of the activities of phase I enzymes (cytochrome P450 1A1 and cytochrome P450 1A2) and enhancer of the activities of phase II enzymes (uridine 5'-diphospho-glucuronosyltransferase and glutathione S-transferase). We investigated the purification and isolation of an active compound in the juice of V. coignetiae using antimutagenicity as a separation marker. Caftaric acid, a polyphenolic compound, was identified as a component responsible for antimutagenicity in the juice of V. coignetiae towards the carcinogenic heterocyclic amine 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). This is the first report of antimutagenicity of caftaric acid. Caftaric acid was reported as an inhibitor of the protein-protein interactions mediated by the Src-family kinases. The impact of the juice of V. coignetiae and its constituents on tumor initiation and promotion thus warrants further study.

Photomicronucleus assay of phototoxic and pseudophotoclastogenic chemicals in human keratinocyte NCTC2544 cells.

Photochemical genotoxicity was evaluated in human keratinocyte NCTC2544 cells. The cells were pre-treated with photogenotoxic or pseudophotoclastogenic chemicals and irradiated with a solar-simulator for 50min at a total UV dose of 5J/cm(2) or placed in the dark for the same period. After washing, the cells were cultured for 1.5-2 cell cycles with fresh culture medium. At the end of culturing, slide specimens were prepared and examined for micronucleus formation. 8-Methoxypsoralen, a photogenotoxic chemical, strongly induced micronucleated cells with UV irradiation but not under non-irradiation conditions. Therefore, NCTC2544 cells were subjected to further investigation to evaluate the possible photogenotoxicity of other chemicals. 6-Methylcoumarin, 3,3',4',5-tetrachlorosalicylanilide and protoporphyrin IX disodium salt, which are all known phototoxic substances, induced micronucleated cells with irradiation but not in the non-irradiation state. These phototoxic substances were confirmed to be photogenotoxic. Tetrabenzoporphine and 5-aminolevulinic acid, which are used for photodynamic therapy, showed phototoxicity. However, these chemicals did not induce micronucleated cells in the irradiated or non-irradiated state, suggesting a lack of photogenotoxicity. Among 3 pseudophotoclastogenic chemicals having no light absorbance at 290-700nm, neither cycloheximide nor disulfoton induced micronucleated cells with or without irradiation; zinc oxide induced micronucleated cells with irradiation and, to a lesser extent, without irradiation. Based on the results of the photogenotoxicity assays of these 9 chemicals, NCTC2544 cells are considered to be a suitable test system to evaluate the photogenotoxic potential of chemicals.

A possible photosensitizer: Tobacco-specific nitrosamine, 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), induced mutations, DNA strand breaks and oxidative and methylative damage with UVA.

We discovered the directly acting mutagenicity of the tobacco-specific nitrosamine, 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), with UVA light (320-400nm) in Ames bacteria and phage M13mp2 in the absence of metabolic activation. We have investigated the spectrum of mutations caused by UVA-activated NNK. The majority (57%) of induced sequence changes were comprised of GC to CG, GC to TA and GC to AT. This suggested that modification of guanine residues was responsible for these mutations. Hence, we explored the formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) and O(6)-methylguanine (O(6)meG) in the DNA. When calf thymus DNA was treated with NNK and UVA, the amount of 8-oxodG/dG and O(6)meG/G in the DNA increased up to 20-fold and 100-fold, respectively, compared with the untreated control. DNA strand breaks were observed following NNK and UVA treatment, and the strand breaks were suppressed in the presence of scavengers for oxygen and NO radical. The formation of NO was also observed in NNK solutions irradiated with UVA. We analyzed the photodynamic spectrum of mutation induction, 8-oxodG formation and NO formation using monochromatic radiation. The patterns of the action spectra were comparable to the absorption spectrum of NNK. We conclude that NNK may act as a photosensitizer in response to UVA to produce NO and other oxidative and alkylative intermediates following the formation of 8-oxodG and O(6)meG in DNA, which may lead to mutations and DNA strand breaks.

Inhibitory effects of beer on mutation in the Ames test and DNA adduct formation in mouse organs induced by 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

An evaluation of the antigenotoxic potential of beer components against carcinogens contained in the human diet, namely heterocyclic amines (HCAs) including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was determined. The protective mechanism involved was also investigated. Beer samples were found to inhibit the mutagenicity of HCAs in the Ames test. Beer solution, consisting of a freeze-dried and dissolved sample, given as drink-water significantly reduced the formation of PhIP-DNA adducts in mouse colon and lung compared to control mice fed with PhIP in the absence of beer solution. Furthermore, beer solution added in the diet as a food additive mimic significantly reduced the amount of DNA adducts present in the liver and lung of mice fed with PhIP. In an effort to investigate the mechanism responsible for the observed protective effect, the effect of beer solutions on HCA metabolizing enzymes was investigated. Beer solutions inhibited the activity of CYP1A1 and CYP1A2, as determined from deethylation and demethylation assays using 7-ethoxy- and 7-methoxyresolufin, respectively. Considering the overall suppression of PhIP genotoxicity by beer, this study confirmed that beer components can interfere with the enzyme activity involved in the metabolism of HCAs and subsequently suppress the observed genotoxicity. The results of this study showed that beer components act in a protective capacity against the genotoxic effects of heterocyclic amines in vivo.

Inhibition of PhIP-induced mammary carcinogenesis in female rats by ingestion of freeze-dried beer.

This study evaluated the modulating effect of non-alcoholic constituents of beer on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis. Female Sprague-Dawley (SD) rats at 6 weeks of age were divided into four groups (n=26-30) and fed either a high fat diet or high fat diets containing 1, 2 or 4% freeze-dried beer (FD beer). One week after the start of feeding, rats received PhIP at a dose of 85 mg/kg by gavage four times weekly for 2 weeks. There were no differences in the body weights or diet intakes of rats between the control and the experimental groups. Weekly observation of palpable tumors indicated that tumor incidence and tumor multiplicity in the 2 and 4% FD beer groups were lower than in the control group throughout the experiment. Neoplastic lesions were pathologically examined at the end of the 22-weeks experiment. Tumor development was inhibited by FD beer intake in a dose-dependent manner. Tumor incidence (38.5%) and tumor multiplicity (0.8+/-0.4) for the group fed with a diet containing 4% FD were significantly reduced as compared with the control group (73.3% and 1.8+/-0.7). Supplementation with FD beer for 3 weeks together with the PhIP treatments resulted in increased liver GST activity, decreased liver CYP1A2 activity and a decrease in the number of DNA adducts in the mammary tissue, though these values were not significant. In conclusion, our results suggest that intake of FD beer may reduce the risk of carcinogenesis caused by heterocyclic amines.

Evaluation of photo-mutagenicity and photo-cytotoxicity of food coloring agents.

Pigments extracted from natural products are widely used for food coloration in Japan. An investigation concerning the photo-mutagenicity and photo-carcinogenicity of frequently used colorants in Japan was performed. Colorants examined were from Laccifer lacca (lac-color), Coccus cacti (cochineal-color), Carthamus tinctorius (carthamus yellow), Gardenia augusta (gardenia yellow and gardenia blue), Monascus anka and Monascus purpureus (monascus red), the skin of Vitis vinifera and Vitis labrusca (grape-skin color), Tamarindus indica (tamarind brown) and Beta vulgaris (beet red). No significant increase in bacterial mutation was found when Salmonella typhimurium TA98, TA100 and TA102 were simultaneously treated with colorants and subjected to UVA irradiation for 30 min. When colorant solutions were subjected to UVA irradiation for 4 h, irradiated solutions containing lac-color became slightly mutagenic toward S.typhimurium TA98 without metabolic activation. A decrease in cell survival resulted when WTK-1 cells were subjected to UVA irradiation for 60 min in the presence of purpurin at 1 mg/ml. Delayed cytotoxicity was also observed following 24 h incubation in fresh medium of samples that were subjected to UVA irradiation for 60 min in the presence of colorant (carthamus yellow, grape-skin color, gardenia blue, cochineal-color, monascus red or purpurin).

Inhibitory effects of heterocyclic amine-induced DNA adduct formation in mouse liver and lungs by beer.

An evaluation of the in vivo antigenotoxic potential of beer components on heterocyclic amines including 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 3-amino-1-methyl-5H-pyrido[4.3-b]indole (Trp-P-2) was determined with particular focus on the target organs of tumorigenesis, and the protective mechanisms involved were investigated. Beer-solution, consisting of a freeze-dried and dissolved sample, given as drinking water, reduced the formation of MeIQx-DNA adducts in mouse liver and lungs. Beer-solution added in the diet as a mimic of food additives also significantly reduced the amount of DNA adducts present in the liver, lung, and kidney DNA of mice fed with MeIQx compared to control mice fed with MeIQx in the absence of beer-solution. The amount of adducts present in the liver of mice with single or continuous administration of Trp-P-2 was significantly reduced when beer-solution was given as part of the diet compared to control mice given Trp-P-2 without beer-solution. Protective effects were observed both with lager- and stout-type samples. In an effort to investigate the mechanisms responsible for the observed protective effects, the effects of beer-solution on metabolizing enzymes for heterocyclic amines were examined. Beer-solutions inhibited the metabolic activation of Trp-P-2 to Trp-P-2(NHOH), as demonstrated by HPLC analysis. Considering the overall suppression of the genotoxicity of MeIQx and Trp-P-2 by beer, we have shown that beer components can inhibit the metabolic activation of heterocyclic amines and subsequently suppress the observed genotoxicity. The results of this study show that beer components are protective against the genotoxic effects of heterocyclic amines on target organs associated with tumorigenesis in vivo.

Inhibitory effects of beer on heterocyclic amine-induced mutagenesis and PhIP-induced aberrant crypt foci in rat colon.

Anti-mutagenic and anti-carcinogenic effects of beer on heterocyclic amine (HCA)-induced carcinogenesis were studied in vitro and in vivo. Four commercial beers (two pilsner-type, black, and stout) showed inhibitory effects against five HCAs, 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ), in the Ames assay using Salmonella typhimurium TA98 in the presence of rat S9 mix. The inhibitory effects of dark-colored beers (stout and black beer) were greater than those of pilsner-type beers. Dark-colored beers suppressed CYP1A2 activity in a dose-dependent manner, suggesting that inhibition of HCA activation is partly responsible for their strong anti-mutagenic effects. Anti-mutagenic effects were also observed when the pooled human S9 mix or activated IQ was used in the assay. The micronucleus test using Chinese hamster lung CHL/IU cells showed that the addition of freeze-dried samples of pilsner-type and stout beer to the culture medium significantly reduced the number of cells with micronuclei induced with PhIP or Trp-P-2. Single-cell gel electrophoresis assay (comet assay) revealed that oral ingestion of pilsner-type and stout beers for 1 week significantly inhibited DNA damage in the liver cells of male ICR mice exposed to MeIQx (13 mg/kg, i.p.). A decrease in the formation of DNA adducts was also observed using a 32P-postlabeling method. Male Fischer 344 rats orally received PhIP (75 mg/kg, five times a week for 2 weeks) and aberrant crypt foci (ACF) formation in the colon was analyzed after 5 weeks. The number of ACF was significantly reduced in rats fed a diet containing freeze-dried beer. These results suggest that beer inhibits the genotoxic effects of HCAs and may reduce the risk of carcinogenesis caused by food borne carcinogens.

Inhibitory effects of (-)-epigallocatechin gallate on the mutation, DNA strand cleavage, and DNA adduct formation by heterocyclic amines.

Green tea is known to be a potential chemopreventive agent against cancer. In this study, we investigated the inhibitory activities of tea extracts, and in particular the polyphenolic component (-)-epigallocatechin gallate (EGCG), against heterocyclic amine-induced genotoxicity. The tea extracts displayed inhibition of 2-hydroxyamino-6-methyldipyrido[1,2-a,3',2'-d]imidazole (Glu-P-1(NHOH))-induced mutagenicity. This inhibition can be accounted for by the presence of EGCG in the extracts. The mutagenic effect of Glu-P-1(NHOH), which induces single-strand cleavage in supercoiled circular DNA under neutral conditions, was inhibited by EGCG. Using the Drosophila repair test, a test for gross DNA damage, and DNA adduct detection by (32)P-postlabeling, we showed that EGCG prevented 2-amino-3,8-dimethylimidazo[4,5-f]quinoline-induced DNA damage and adduct formation in insect DNA. EGCG was found to accelerate the degradation of Glu-P-1(NHOH) in vitro. This observation suggested that the inhibition by EGCG is associated with an accelerated degradation of metabolically activated heterocyclic amines.