Homologous recombination contributes to the repair of acetaldehyde-induced DNA damage.

Acetaldehyde, a chemical that can cause DNA damage and contribute to cancer, is prevalently present in our environment, e.g. in alcohol, tobacco, and food. Although aldehyde potentially promotes crosslinking reactions among biological substances including DNA, RNA, and protein, it remains unclear what types of DNA damage are caused by acetaldehyde and how they are repaired. In this study, we explored mechanisms involved in the repair of acetaldehyde-induced DNA damage by examining the cellular sensitivity to acetaldehyde in the collection of human TK6 mutant deficient in each genome maintenance system. Among the mutants, mismatch repair mutants did not show hypersensitivity to acetaldehyde, while mutants deficient in base and nucleotide excision repair pathways or homologous recombination (HR) exhibited higher sensitivity to acetaldehyde than did wild-type cells. We found that acetaldehyde-induced RAD51 foci representing HR intermediates were prolonged in HR-deficient cells. These results indicate a pivotal role of HR in the repair of acetaldehyde-induced DNA damage. These results suggest that acetaldehyde causes complex DNA damages that require various types of repair pathways. Mutants deficient in the removal of protein adducts from DNA ends such as TDP1-/- and TDP2-/- cells exhibited hypersensitivity to acetaldehyde. Strikingly, the double mutant deficient in both TDP1 and RAD54 showed similar sensitivity to each single mutant. This epistatic relationship between TDP1-/- and RAD54-/- suggests that the protein-DNA adducts generated by acetaldehyde need to be removed for efficient repair by HR. Our study would help understand the molecular mechanism of the genotoxic and mutagenic effects of acetaldehyde.

Administration of Apple Polyphenol Supplements for Skin Conditions in Healthy Women: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

This clinical study was performed to evaluate the effects of continuous apple polyphenol (AP) administration on facial skin conditions and pigmentation induced by ultraviolet (UV) irradiation in healthy women participants. Participants (n = 65, age 20-39 years) were randomized to receive tablets containing AP (300 or 600 mg/day) or placebo in a double-blinded, placebo-controlled clinical trial. Continuous administration of AP for 12 weeks significantly prevented UV irradiation induced skin pigmentation (erythema value, melanin value, L value), although a dose-dependent relationship was not clearly observed. In contrast, no significant differences were detected between the groups with regard to water content and trans-epidermal water loss. Our study demonstrated that APs and their major active compounds, procyanidins, have several health benefits. Here, we report that continuous administration of AP for 12 weeks alleviated UV irradiation induced skin pigmentation, when compared with placebo, in healthy women.

Apple- and Hop-Polyphenols Inhibit Porphyromonas gingivalis-Mediated Precursor of Matrix Metalloproteinase-9 Activation and Invasion of Oral Squamous Cell Carcinoma Cells.

BACKGROUND: Recent epidemiologic studies have revealed a significant association between periodontitis and oral squamous cell carcinoma (OSCC). Furthermore, periodontitis is markedly associated with orodigestive cancer mortality, whereas Porphyromonas gingivalis (Pg) infection has been identified as a specific and potentially independent microbial factor related to increased risk of orodigestive cancer death. The authors previously reported that Pg induced the precursor form of matrix metalloproteinase-9 (proMMP-9) production via proteinase-activated receptor (PAR)-related pathways, after which proMMP-9 was activated by gingipains to enhance cellular invasion of SAS cells. In the present study, effects of selected polyphenols as inhibitors of cellular invasion caused by Pg gingipains in SAS cells are examined. METHODS: OSCC cells were infected with Pg strains including gingipain mutants. To evaluate effects of inhibitors: 1) apple polyphenol (AP); 2) hop bract polyphenol (HBP); 3) high-molecular-weight fractions of HBP (HMW-HBP); 4) low-molecular-weight fractions of HBP (LMW-HBP); 5) epigallocatechin gallate (EGCg); 6) KYT-1 (Arg-gingipain inhibitor); and KYT-36 (Lys-gingipain inhibitor) in combination are used. PAR2 and PAR4 mRNA expressions are examined using real-time reverse transcription polymerase chain reaction, and signaling pathways are evaluated by western blotting analysis. RESULTS: KYT-1/KYT-36, AP, HBP, and HMW-HBP significantly inhibited PAR2 and PAR4 mRNA expressions, proMMP-9 activation, and cellular invasion. Furthermore, AP, HBP, and HMW-HBP reduced activation of heat shock protein 27 and Ets1 and nuclear translocation of nuclear factor-kappa B, whereas EGCg and LMW-HBP did not. CONCLUSION: These results suggest that AP, HBP, HMW-HBP are potent inhibitors of proMMP-9 activation and cellular invasion mediated with Pg in OSCC cells.

Alanine-fortified tomatoes relieve the acute alcohol-induced adverse effects in healthy men: a randomized cross-over study.

BACKGROUND: Little is known about the effects of dietary components on the regulation of the gastric emptying rate of alcohol and its impact on alcohol metabolism. We recently found that the crude water-insoluble dietary fibers from several types of botanical foods maintained aqueous ethanol solutions. Additionally, the ethanol-absorbing ability of the dietary fibers correlated with the inhibition of the blood ethanol elevation by delaying gastric emptying. Moreover, we found that the synergism between tomatoes and alanine to reduce the absorption of alcohol in rats was attributable to the effect of alanine on precipitates, such as the crude water-insoluble dietary fibers of tomatoes. In the present study, we assess whether an alanine-fortified tomato (AFT) is effective in relieving acute alcohol-induced adverse effects by lowering the alcohol action in healthy human volunteers following the ingestion of alcohol with a meal. METHODS: Twenty healthy males ingested the AFT or sugar as the control, with 1.2 g/kg of alcohol and a micronutrient-fortified meal in a randomized cross-over study. Breath alcohol concentrations were temporally measured, and blood and urine samples were obtained during the trial. The study protocol was repeated with the AFT and sugar groups reversed 4 weeks later. RESULTS: Various analyses were performed using the data from the 15 subjects. The breath alcohol concentrations significantly decreased when AFT was ingested. A decrease in the urinary pH was also noted following the ingestion of AFT. Moreover, the sum of seven sedative scores as subjective sensation after alcohol ingestion was significantly reduced by AFT the next morning. CONCLUSIONS: Our study demonstrates that the simultaneous ingestion of AFT under the consumption of excess alcohol and a micronutrient-fortified meal relieved the acute alcohol-induced adverse effects in male volunteers. These results are consistent with the effectiveness observed in rats as previously reported.

Alanine with the Precipitate of Tomato Juice Administered to Rats Enhances the Reduction in Blood Ethanol Levels.

Delay in gastric emptying (GE) lowers the blood ethanol concentration (BEC) after alcohol administration. We previously demonstrated that water-insoluble fractions, mainly comprising dietary fiber derived from many types of botanical foods, possessed the ability to absorb ethanol-containing aqueous solutions. Furthermore, there was a significant correlation between the absorption of ethanol and lowering of BEC because of delay in GE. Here we identified dietary nutrients that synergize with the water-insoluble fraction of tomatoes to lower BEC in rats. Consequently, unlike tomato juice without alanine, tomato juice with 5.0% alanine decreased BEC depending on the delay in GE and mediated the ethanol-induced decrease in the spontaneous motor activity (an indicator of drunkenness). Our findings indicate that the synergism between tomato juice and alanine to reduce the absorption of ethanol was attributable to the effect of alanine on precipitates such as the water-insoluble fraction of tomatoes.

Oral administration of apple condensed tannins delays rheumatoid arthritis development in mice via downregulation of T helper 17 (Th17) cell responses.

Apples are known to contain high concentrations of phenolic compounds such as condensed tannins. Consumption of condensed tannins has been reported to reduce the risk of many types of chronic diseases including allergies. However, their therapeutic effectiveness and potential in treating autoimmune disease remain controversial. Here, the effect of oral administration of apple condensed tannins (ACT) prepared from apples (Malus pumila cv. Fuji) on bovine type II collagen (CII)-induced arthritis in DBA1/J mice, a well-established murine model of human rheumatoid arthritis (RA), was evaluated. As compared to the control (without ACT administration) group, RA development was delayed and a significant reduction in the RA clinical score was observed in the ACT-administered group. Using cultured splenocytes isolated from CII-immunized mice, ACT-administration was shown to decrease the CII-induced increases in IL-17 expression and production in vitro. We propose that downregulation of T helper (Th) 17 cells is responsible for the ACT-induced RA suppression.

Cloning and characterization of a novel O-methyltransferase from Flammulina velutipes that catalyzes methylation of pyrocatechol and pyrogallol structures in polyphenols.

A novel O-methyltransferase gene was isolated from Flammulina velutipes. The isolated full-length cDNA was composed of a 690-nucleotide open reading frame encoding 230 amino acids. A database search revealed that the deduced amino acid sequence was similar to those of other O-methyltransferases; the highest identity was only 61.8% with Laccaria bicolor. The recombinant enzyme was expressed by Escherichia coli. BL21 (DE3) was assessed for its ability to methylate (-)-epigallocatechin-3-O-gallate (EGCG). LC-TOF-MS and NMR revealed that the enzyme produced five kinds of O-methylated EGCGs: (-)-epigallocatechin-3-O-(3-O-methyl)gallate, (-)-epigallocatechin-3-O-(4-O-methyl)gallate, (-)-epigallocatechin-3-O-(3,4-O-dimethyl)gallate, (-)-epigallocatechin-3-O-(3,5-O-dimethyl)gallate, and (-)-4'-O-methylepigallocatechin-3-O-(3,5-O-dimethyl)gallate. The substrate specificity of the enzyme for 20 kinds of polyphenols was assessed using the crude recombinant enzyme of O-methyltransferase. This enzyme introduced methyl group(s) into polyphenols with pyrocatechol and pyrogallol structures.

Metabolic stability and inhibitory effect of O-methylated theaflavins on H2O2-induced oxidative damage in human HepG2 cells.

Seven new O-methylated theaflavins (TFs) were synthesized by using O-methyltransferase from an edible mushroom. Using TFs and O-methylated TFs, metabolic stability in pooled human liver S9 fractions and inhibitory effect on H(2)O(2)-induced oxidative damage in human HepG2 cells were investigated. In O-methylation of theaflavin 3'-O-gallate (TF3'G), metabolic stability was potentiated by an increase in the number of introduced methyl groups. O-methylation of TF3,3'G did not affect metabolic stability, which was likely because of a remaining 3-O-galloyl group. The inhibitory effect on oxidative damage was assessed by measuring the viability of H(2)O(2)-damaged HepG2 cells treated with TFs and O-methylated TFs. TF3,3'G and O-methylated TFs increased cell viabilities significantly compared with DMSO, which was the compound vehicle (p < 0.05), and improved to approximately 100%. Only TF3'G did not significantly increase cell viability. It was suggested that the inhibitory effect on H(2)O(2)-induced oxidative damage was potentiated by O-methylation or O-galloylation of TFs.

Purification and characterization of a novel O-methyltransferase from Flammulina velutipes.

An enzyme catalyzing the methylation of phenolic hydroxyl groups in polyphenols was identified from mycelial cultures of edible mushrooms to synthesize O-methylated polyphenols. Enzyme activity was measured to assess whether methyl groups were introduced into (-)-epigallocatechin-3-O-gallate (EGCG) using SAM as a methyl donor, and (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3″Me), (-)-epigallocatechin-3-O-(4-O-methyl)-gallate (EGCG4″Me), and (-)-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate (EGCG3″,5″diMe) peaks were detected using crude enzyme preparations from mycelial cultures of Flammulina velutipes. The enzyme was purified using chromatographic and two-dimensional electrophoresis. The purified enzyme was subsequently analyzed on the basis of the partial amino acid sequence using LC-MS/MS. Partial amino acid sequencing identified the 17 and 12 amino acid sequences, VLEVGTLGGYSTTWLAR and TGGIIIVDNVVR. In database searches, these sequences showed high identity with O-methyltransferases from other mushroom species and completely matched 11 of 17 and 9 of 12 amino acids from five other mushroom O-methyltransferases.

Cardiac electrophysiological alterations in heart/muscle-specific manganese-superoxide dismutase-deficient mice: prevention by a dietary antioxidant polyphenol.

Cardiac electrophysiological alterations induced by chronic exposure to reactive oxygen species and protective effects of dietary antioxidant have not been thoroughly examined. We recorded surface electrocardiograms (ECG) and evaluated cellular electrophysiological abnormalities in enzymatically-dissociated left ventricular (LV) myocytes in heart/muscle-specific manganese-superoxide dismutase-deficient (H/M-Sod2(-/-)) mice, which exhibit dilated cardiomyopathy due to increased oxidative stress. We also investigated the influences of intake of apple polyphenols (AP) containing mainly procyanidins with potent antioxidant activity. The QRS and QT intervals of ECG recorded in H/M-Sod2(-/-) mice were prolonged. The effective refractory period in the LV myocardium of H/M-Sod2(-/-) mice was prolonged, and susceptibility to ventricular tachycardia or fibrillation induced by rapid ventricular pacing was increased. Action potential duration in H/M-Sod2(-/-) LV myocytes was prolonged, and automaticity was enhanced. The density of the inwardly rectifier K(+) current (I K1) was decreased in the LV cells of H/M-Sod2(-/-) mice. The AP intake partially improved these electrophysiological alterations and extended the lifespan in H/M-Sod2(-/-) mice. Thus, chronic exposure of the heart to oxidative stress produces a variety of electrophysiological abnormalities, increased susceptibility to ventricular arrhythmias, and action potential changes associated with the reduced density of I K1. Dietary intake of antioxidant nutrients may prevent oxidative stress-induced electrophysiological disturbances.

Comprehensive separation and structural analyses of polyphenols and related compounds from bracts of hops (Humulus lupulus L.).

A novel sequential chromatographic technique was applied to the comprehensive separation of polyphenols and related compounds from a hop bract extract. Over 100 types of constituents were effectively isolated from only 25 g of extract in high yields by high-speed countercurrent chromatography followed by hydrophilic interaction chromatography and reversed-phase high performance liquid chromatography. Among the materials isolated, the structures of 39 compounds were elucidated on the basis of their spectroscopic data including electrospray ionization time-of-flight mass spectrometry and one-dimensional/two-dimensional nuclear magnetic resonance. Three new compounds, 1 known compound identified for the first time in plants, and 20 known compounds that have not been reported in hops, were found. The hop bract extract also contained an abundance of highly oligomeric proanthocyanidins, which consisted of B-type procyanidin structures.

Prevention of allergic disease development and symptoms by food factors.

A fundamental means of allergic disease prevention, via the use of functional food factors, is desirable. A number of studies on the role of functional food factors in preventing allergic diseases have been reported. In this review, the preventive effects of polyphenols, carotenoids, polysaccharides, and non-digestible oligosaccharides on allergic diseases are discussed.

O-methylated theaflavins suppress the intracellular accumulation of triglycerides from terminally differentiated human visceral adipocytes.

A known O-methylated theaflavin, theaflavin 3-O-(3-O-methyl)gallate (3MeTF3G), and the new theaflavin 3-O-(3,5-di-O-methyl)gallate (3,5diMeTF3G) were synthesized via the O-methylation of theaflavin 3-O-gallate (TF3G). Both 3MeTF3G and 3,5diMeTF3G are more stable than TF3G at pH 7.5 in the order 3,5diMeTF3G > 3MeTF3G > TF3G. The inhibitory effects of these compounds on the intracellular accumulation of triglycerides from terminally differentiated human visceral adipocytes were investigated. Compound 3MeTF3G exhibited an inhibitory effect similar to that of TF3G at 3 µM and a slightly lower effect than that of TF3G at 10 µM. The result suggested that the degradants and oxidatively polymerized products of TF3G may also have inhibitory effects. For cells treated with 3,5diMeTF3G at 3 and 10 µM, intracellular triglyceride accumulation was dose dependent and significantly lower compared with that for other compounds. It was suggested that the higher effect of 3,5diMeTF3G was due to its higher stability and likely improved absorption owing to di-O-methylation.

Characterization and activity of anthocyanins in Zijuan tea (Camellia sinensis var. kitamura).

Zijuan tea is a new cultivar produced in Yunnan province of China. Unlike most tea cultivars, Zijuan tea is anthocyanin-rich. The composition and antioxidant activities of anthocyanins of Zijuan tea were studied for the first time in this paper. Anthocyanins were extracted with acidified methanol and quantified as 707 ± 28 µg/g of dry weight (cyanidin-3-O-ß-D-glucoside equivalent) by high-performance liquid chromatography (HPLC) analysis. Four anthocyanins were successfully identified after Amberlite XAD-7HP adsorption column chromatography and octadecyl silane (ODS) flash chromatography. Among the four, delphinidin-3-O-ß-D-galactoside (1) and cyanidin-3-O-ß-D-galactoside (2) were confirmed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) and HPLC. Delphinidin-3-O-ß-D-(6-(E)-p-coumaroyl) galactopyranoside (3) and cyanidin-3-O-ß-D-(6-(E)-p-coumaroyl) galactopyranoside (4) were characterized by the high-resolution time-of-flight-mass spectrometry (HRTOF-MS) and nuclear magnetic resonance (NMR) spectrometry. The antioxidant activities of compounds 3 and 4, which composed approximately 75% of the total anthocyanins content in HPLC analysis, were evaluated by DPPH and FRAP assays. Results showed that both had higher antioxidant activities than commercial antioxidants butylated hydroxytoluene (BHT) used as one of controls in these assays.

Ninety-day dietary toxicity study of apple polyphenol extracts in Crl: CD (SD) rats.

To examine the safety of Dietary Applephenon® (AP) in feed, Crl: CD (SD) rats of each sex were divided into four groups and given diets containing AP at 0%, 1.25%, 2.5%, or 5.0% for 90 days. All rats survived and toxic changes were not observed throughout the study. Body weight and food efficiency in the 5.0% AP group of both sexes were significantly decreased compared with that in controls. These changes were considered to be caused by the physiological effects of AP (including the inhibitory effects on pancreatic lipase activity). Slight hypertrophy in acinar cells in the parotid and submandibular glands appeared in the 2.5% and 5.0% groups. These were suggested not to be toxicological but physiologic adaptive responses to oral stimuli by the lower pH of AP-containing diets. In conclusion, dietary AP in feed, up to a maximum level of 5.0% for 90 days, given to rats did not induce toxicological effects.

Apple polyphenols suppress antigen presentation of ovalbumin by THP-1-derived dendritic cells.

Apple polyphenol extract (AP) and procyanidin contained in AP were investigated for their immunomodulatory effects using THP-1-derived human dendritic cells (TDDCs). The expression levels of HLA-DR (MHC class II) and CD86 (costimulatory molecule) were measured as an indicator of antigen presentation in TDDCs. A significant decrease in HLA-DR expression was observed in the AP and fractionated procyanidin-treated cells in the presence of ovalbumin (OVA), but no effect on CD86 expression was observed. The uptake of OVA was not inhibited by AP treatment, and the gene expression of membrane-associated RING-CH ubiquitin E3 ligase, MARCH1, was up-regulated by AP treatment. It can therefore be presumed that AP suppresses HLA-DR expression via the ubiquitin-proteasome pathway. Furthermore, the up-regulation of IL-12 and TNF-α was found in the procyanidin trimers-treated cells in the presence of OVA. These results suggest that apple polyphenols would be an effective factor for the development of immunomodulatory agents with suppressive effects of antigen presentation.

Isolation and structure elucidation of tetrameric procyanidins from unripe apples (Malus pumila cv. Fuji) by NMR spectroscopy.

Procyanidins are plant secondary metabolites widely consumed and known to have various physiological functions, but their bioavailability and mechanism of action are still unclear especially for larger oligomers. One of the reasons is scarce information about the detailed structure of oligomeric procyanidins. As for apple, structures of procyanidin components larger than trimers are scarcely known. In this study, 11 tetrameric procyanidins including two known compounds were isolated from unripe apples (Malus pumila cv. Fuji) and identified by NMR spectroscopic analysis and phloroglucinol degradation. As a result, the detailed structural diversity of tetrameric procyanidins in apple was established.

A procyanidin trimer, C1, promotes NO production in rat aortic endothelial cells via both hyperpolarization and PI3K/Akt pathways.

Procyanidins, which are condensed catechins, have been elucidated as absorbable polyphenols, but their health-benefits remain unclear. The aim of this study was, thus, to clarify the efficacy and mechanism of each procyanidin oligomer in NO activation in rat aortic endothelial cells (RAECs). Treatment of RAECs with 50µM procyanidin C1 (4ß→8 trimer) resulted in a time- and dose-dependent hyperpolarization using the membrane potential-sensitive probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol, while no effect was observed for (-)-epicatechin (a monomer) and procyanidin B2 (4ß→8 dimer). The C1-induced hyperpolarization was inhibited by iberiotoxin, a specific inhibitor of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel, as well as 2-aminoethyl diphenylborinate (2-APB), a store-operated Ca(2+) entry inhibitor. Procyanidin C1 caused a significant increase in NO production from RAECs via phosphorylation of both eNOS and Akt, and the effect was completely inhibited by N(G)-monomethyl-l-arginine or combined treatment with iberiotoxin and the phosphatidylinositol 3-kinase (PI3K) specific inhibitor, wortmannin, as well as combined treatment with 2-APB and wortmannin. Taken together, these findings provide critical evidence that procyanidin C1, but not B2, has potential to induce NO production in RAECs via both Ca(2+)-dependent BK(Ca) channel-mediated hyperpolarization and Ca(2+)-independent PI3K/Akt pathways.

Apple procyanidins induce hyperpolarization of rat aorta endothelial cells via activation of K+ channels.

Apple procyanidins (AP), one of the polyphenol-rich compounds, showed an endothelial-dependent vasorelaxation in rat aorta, but the mechanisms of beneficial effects are still unclear. The present study was designed to clarify the potential role of AP in rat aorta endothelial cells (RAECs). The treatment of RAECs with AP (1-10 µg/ml) resulted in a dose-dependent hyperpolarization with a maximum effect at 10 µg/ml, and for this reason, AP (10 µg/ml) was used in all the following experiments. AP-induced hyperpolarization was significantly inhibited by pretreatment of nonspecific K(+) inhibitor, tetraethyl ammonium chloride or specific K(+) channel inhibitors, iberiotoxin, glibenclamide, 4-aminopyridine and BaCl(2), as well as by high KCl or Ca(2+)-free solution. AP-induced hyperpolarization was also proved using 64-channel multielectrode dish system that can monitor a direct and real-time change of membrane potential. Furthermore, AP treatment caused a significant increase of nitric oxide (NO) production and cyclic guanosine monophosphate levels via endothelial NO synthase messenger RNA expression. The NO production was inhibited by N(G)-monoethyl-l-arginine or Ca(2+)-free solution and was completely abolished by their combination. Also, AP inhibited endothelial proliferation, while the effect was significantly abolished by N(G)-monoethyl-l-arginine or tetraethyl ammonium chloride. These findings suggest that AP induces both hyperpolarization of RAECs via multiple activation of K(+) channels and activation of NO/cyclic guanosine monophosphate pathway via increasing NO production or is responsible for antiangiogenic effect. Diminishment of hyperpolarization as well as NO production of AP in Ca(2+)-free solution implicated that AP would play a crucial role in promoting Ca(2+) influx into endothelial cells so as to promote both actions.

Apple Procyanidins Suppress Amyloid ß-Protein Aggregation.

Procyanidins (PCs) are major components of the apple polyphenols (APs). We previously reported that treatment with PC extended the mean lifespan of Caenorhabditis elegans (Sunagawa et al., 2011). In order to estimate the neuroprotective effects of PC, we investigated the antiaggregative activity of PC on amyloid ß-protein (Aß) aggregation, which is a pathological hallmark of Alzheimer's disease. We herein report that PC significantly suppressed Aß42 aggregation and dissociated Aß42 aggregates in a dose-dependent manner, indicating that PC is a potent suppressor of Aß aggregation. Furthermore, PC significantly inhibited Aß42 neurotoxicity and stimulated proliferation in PC-12 cells. These results suggested that the PC and AP acted as neuroprotective factors against toxic Aß aggregates.