Cytotoxic and cytoprotective effects of tryptamine-4,5-dione on neuronal cells: a double-edged sword.

Serotonin (5-hydroxytryptamine) is a putative substrate for myeloperoxidase, which may convert it into the reactive quinone tryptamine-4,5-dione (TD). In this study, we found that the viability of human SH-SY5Y neuroblastoma cells treated with 25 µM TD was increased to approximately 117%. On the other hand, the cell viability was significantly decreased by exposure to TD (150-200 µM), with an increase in intracellular reactive oxygen species (ROS). Interestingly, pre-treatment of SH-SY5Y cells with 100 µM TD prevented cell death and suppressed intracellular ROS generation evoked by the addition of hydrogen peroxide (H2O2). Expression of the phase-II antioxidant enzyme NAD(P)H: quinone oxidoreductase 1 and haem oxygenase 1 were upregulated by TD at a concentration of 50-100 µM. Nuclear factor erythroid 2-related factor 2 (Nrf2), the regulator of these enzyme, was translocated from the cytosol to the nucleus by 100 µM TD. In summary, moderate concentrations of TD may increase the self-defence capacity of neuronal cells against oxidative stress.

Phytoestrogenic activity of blackcurrant (Ribes nigrum) anthocyanins is mediated through estrogen receptor alpha.

SCOPE: Blackcurrants (Ribes nigrum L., Grossulariaceae) contain high amounts of anthocyanin polyphenols, which have antioxidant and anti-carcinogenic health benefits. This study analyzed the potential phytoestrogenic effects of blackcurrant extract (BCE) in breast cancer (MCF-7) and human endometrial cancer (Ishikawa) cell lines that over-express estrogen receptor alpha (ERα), as well as in immature female rats. METHODS AND RESULTS: Microarray analysis and Ingenuity® Pathway Analysis showed that BCE activated the ERα pathway, whereas quantitative-PCR confirmed that BCE and four types of anthocyanins up-regulated genes downstream of ERα. BCE (0.1-1.0 µg/mL) and anthocyanins (0.1-10 µM) induced MCF-7 cell proliferation; however, this effect was blocked by ER antagonist fulvestrant. Flow cytometry showed that anthocyanins reduced and increased the number of MCF-7 cells in the G0/G1 and G2/M phases, respectively. Anthocyanins stimulated ERα transcriptional activity in human ERα reporter assays and induced alkaline phosphatase activity in Ishikawa cells. Competition assays and in silico analysis indicated that anthocyanins bind to ERα. Finally, BCE focally induced stratification of columnar epithelial cells in the rat uterus and increased cytoplasmic mucin levels in these cells. CONCLUSION: These results suggest that blackcurrant anthocyanins act as phytoestrogens in vitro and in vivo.

Protective effects of raw and cooked blackcurrant extract on DNA damage induced by hydrogen peroxide in human lymphoblastoid cells.

CONTEXT: Blackcurrant (Ribes nigrum L.) is a classical fruit that has long been used to make juice, liqueur and sometimes medicines in Europe. The beneficial effects of blackcurrant, which are inhibition of lipopolysaccharide-stimulated inflammatory, anticarcinogenesis and other health effects, have been reported. OBJECTIVE: Previously, we reported the antimutagenic activities of blackcurrant using a yeast gene mutation assay. In this study, we investigated whether this antimutagenicity of blackcurrant was confirmed in human cells. MATERIALS AND METHODS: We prepared four types of aqueous blackcurrant extracts (BCE) from mature and premature with or without heat treatment by microwave. Antioxidant activities of BCE were measured by the DPPH radical scavenger assay. In the DPPH radical scavenger assay, the maximum concentration of BCE was 1.6 mg/reaction. We investigated the antigenotoxic activities of BCE by the comet assay and micronucleus test using the human lymphoblastoid cell line TK6. In the comet assay, TK6 was treated with 300 µM H2O2 without or with BCE at concentrations of 0.5, 1.0, 2.0 and 3.0 mg/mL. In the micronucleus test, TK6 was treated with 1 mg/mL BCE without or with H2O2. RESULTS: All BCEs exhibited more than 90% of inhibition rates of DPPH radicals at the maximum concentration of BCE. DNA damage and micronuclei induced by H2O2 significantly decreased in the each BCE-treated condition. CONCLUSION: The results suggest that BCE treatment can reduce the genomic instability induced by H2O2 in human cells. We consider that these antigenotoxic effects are related to polyphenols, l-ascorbic acid and other antioxidant compounds.

Role of lipid peroxide in the neurodegenerative disorders.

Nervous system controls all the organs in the living like a symphony. In this chapter, the mechanism of neuronal death in aged is discussed in relation to oxidative stress. Polyunsaturated fatty acid (PUFA) is known to be rich in the membranous component of the neurons and plays an important role in maintaining the neuronal functions. Recent reports revealed that oxidation of omega-3 and omega-6 PUFAs, such as docosahexaenoic acid (DHA) and arachidonic acid (ARA), are potent antioxidant but simultaneously, their oxidation products are potentially toxic. In this chapter, the existence of early oxidation products of PUFA is examined in the samples from neurodegenerative disorders and the cellular model. Accumulation of proteins with abnormal conformation is suggested to induce neuronal death by disturbance of proteolysis and mitochondrial function. The role of lipid peroxide and lipid-derived aldehyde adduct proteins is discussed in relation to brain ageing and age-related neurodegeneration.

Plasma metabolites of dietary flavonoids after combination meal consumption with onion and tofu in humans.

SCOPE: The effect of food combination on metabolic profile in postprandial plasma has hardly been reported. We investigated the absorption and metabolism of quercetin and soy isoflavones in humans after combination meal consumption. METHODS AND RESULTS: Five healthy volunteers ingested sautéed onion and tofu, and the plasma metabolites of quercetin and isoflavones were analyzed. Quercetin and genistein were incubated with human intestinal Caco-2 cells and human hepatoma HepG2 cells to further analyze the influence of simultaneous supply to the small intestine and the liver. Glucuronosyl conjugates of quercetin and methylated quercetin were the major plasma metabolites in the case of onion intake. Plasma metabolites with the single serving of tofu were both glucuronide and sulfate metabolites of isoflavones. Interestingly, quercetin sulfate was only detected after the combined intake of sautéed onion and tofu, accompanied with a decrease in sulfated isoflavones. Besides, quercetin was shown as the preferential substance for phase II enzymes over genistein in both Caco-2 and HepG2 cells. CONCLUSION: These results indicate that, when flavonoids and isoflavonoids were ingested together, the metabolic conversions in the small intestine and/or the liver could be altered, resulting in the variation of the postprandial profiles of the plasma metabolites.

Identification of proteoglycan from salmon nasal cartilage.

There has been no structural information about the core protein of salmon nasal cartilage proteoglycan although its physiological activities have been investigated. Internal amino acid sequencing using nano-LC/MS/MS revealed that the salmon proteoglycan was aggrecan. Primer walk sequencing based on the amino acid information determined that the salmon aggrecan cDNA is comprised of 4207bp nucleotides predicted to encode 1324 amino acids with a molecular mass of 143,276. It exhibited significant similarities to predicted pufferfish aggrecan, zebrafish similar to aggrecan, zebrafish aggrecan, bovine aggrecan and human aggrecan isoform 2 precursor; whose amino acid identities were 56%, 55%, 49%, 31% and 30%, respectively. Salmon cartilage aggrecan had globular domains G1, G2 and G3 as in mammalian aggrecans. Neither the putative keratan sulfate attachment domain enriched with serine, glutamic acid and proline, nor the putative chondroitin sulfate attachment domain with repeating amino acid sequence containing serine-glycine, found in mammalian aggrecans were observed in salmon, however, random serine-glycine (or glycine-serine) sequences predicted to the sugar chain attachment sites were observed. Based on cDNA analysis and amino acid analysis after ß-elimination, the ratio of serine attached to sugar chains was calculated to be approximately 37.7% of total serine, that is, 46 of 123 serine residues.

Continuous intake of polyphenolic compounds containing cocoa powder reduces LDL oxidative susceptibility and has beneficial effects on plasma HDL-cholesterol concentrations in humans.

BACKGROUND: Cocoa powder is rich in polyphenols such as catechins and procyanidins and has been shown in various models to inhibit LDL oxidation and atherogenesis. OBJECTIVE: We examined whether long-term intake of cocoa powder alters plasma lipid profiles in normocholesterolemic and mildly hypercholesterolemic human subjects. DESIGN: Twenty-five subjects were randomly assigned to ingest either 12 g sugar/d (control group) or 26 g cocoa powder and 12 g sugar/d (cocoa group) for 12 wk. Blood samples were collected before the study and 12 wk after intake of the test drinks. Plasma lipids, LDL oxidative susceptibility, and urinary oxidative stress markers were measured. RESULTS: At 12 wk, we measured a 9% prolongation from baseline levels in the lag time of LDL oxidation in the cocoa group. This prolongation in the cocoa group was significantly greater than the reduction measured in the control group (-13%). A significantly greater increase in plasma HDL cholesterol (24%) was observed in the cocoa group than in the control group (5%). A negative correlation was observed between plasma concentrations of HDL cholesterol and oxidized LDL. At 12 wk, there was a 24% reduction in dityrosine from baseline concentrations in the cocoa group. This reduction in the cocoa group was significantly greater than the reduction in the control group (-1%). CONCLUSION: It is possible that increases in HDL-cholesterol concentrations may contribute to the suppression of LDL oxidation and that polyphenolic substances derived from cocoa powder may contribute to an elevation in HDL cholesterol.

Effects of volatile constituents of rosemary extract on lung inflammation induced by diesel exhaust particles.

Epidemiological and experimental studies have implicated that diesel exhaust particles are involved in increases in morbidity and mortality from lung diseases. Recently, we have demonstrated that rosmarinic acid, a polyphenolic liquid component in perilla, inhibits lung inflammation induced by diesel exhaust particles in vivo, partly through its antioxidative property. We have also shown the antioxidative activities of volatile constituents of rosemary extract, the gaseous component in perilla, in vitro. The purpose of this study was to evaluate the effects of intratracheal administration of volatile rosemary extract on lung inflammation induced by diesel exhaust particles. ICR mice were treated with intratracheal administration of volatile rosemary extract before intratracheal exposure to diesel exhaust particles. Twenty-four hr later, diesel exhaust particles exposure elicited lung inflammation characterized by the infiltration of neutrophils and eosinophils, which was confirmed by cellular profile of bronchoalveolar lavage fluid and histological examination. Diesel exhaust particles enhanced the protein expressions of interleukin-1beta, macrophage inflammatory protein-1alpha, macrophage chemoattractant protein-1, and keratinocyte chemoattractant in the lung. Pretreatment with rosemary extract significantly inhibited the diesel exhaust particles-induced lung inflammation. Rosemary extract treatment also suppressed the diesel exhaust particles-enhanced lung expression of macrophage inflammatory protein-1alpha, macrophage chemoattractant protein-1, and keratinocyte chemoattractant. These results suggest that intratracheal administration of rosemary extract can prevent lung inflammation induced by diesel exhaust particles. The preventive effect is mediated, at least partly, through the inhibition of the enhanced lung expressions of macrophage inflammatory protein-1alpha, macrophage chemoattractant protein-1, and keratinocyte chemoattractants.

Effects of volatile constituents of a rosemary extract on allergic airway inflammation related to house dust mite allergen in mice.

Perilla leaf extract is known to have anti-inflammatory properties. Recently, we have demonstrated that rosmarinic acid, a polyphenolic liquid component in perilla, inhibits the allergic airway inflammation induced by house dust mites (HDMs) in vivo. The purpose of this study was to evaluate the effects of intratracheal (i.t.) exposure to volatile constituents of a rosemary extract (VR), gaseous components in perilla, on a murine model of allergic asthma induced by HDM. C3H/HeN mice were treated 7 times weekly with i.t. exposure. The HDM allergen challenge elicited a pulmonary eosinophilic inflammation accompanied by an increase in the lung expression of interleukin (IL)-5, IL-13, and eotaxin. VR inhibited increases in the number of eosinophils, neutrophils, and mononuclear cells around the airways and those in the bronchoalveolar lavage fluid. VR exposure also significantly suppressed the expression of IL-13 enhanced by HDM allergen. These results suggest that i.t. exposure to VR can, at least partially, prevent allergic airway inflammation induced by HDM. The preventive effect is associated with inhibition of the enhanced local expression of IL-13.

Protective role of antioxidative food factors in oxidative stress caused by hyperglycemia.

Hyperglycemia causes the autoxidation of glucose, glycation of proteins, and the activation of polyol metabolism. These changes accelerate generation of reactive oxygen species (ROS) and increases in oxidative chemical modification of lipids, DNA, and proteins in various tissues. Oxidative stress may play an important role in the development of complications in diabetes such as lens cataracts, nephropathy, and neuropathy. Glycation reactions, especially Maillard reactions, occur in vivo as well as in vitro and are associated with the chronic complications of diabetes mellitus and aging and age-related diseases by increases in oxidative chemical modification of lipids, DNA, and proteins. In particular, long-lived proteins such as lens crystallines, collagens, and hemoglobin may react with reducing sugars to form advanced glycation end products (AGEs). Recently, we found a novel type of AGE, named MRX, and we found that MRX is a good biomarker for detecting oxidative stress produced during Maillard reaction. We also examined in detail the role of lipid peroxidation reaction in hyperglycemia and found that hexanoyl modification formed by the reaction of oxidized lipids and proteins must be important for oxidative stress. Detailed analyses of the formation mechanism of hexanoyl lysine (HEL) moiety in proteins were conducted, and excretion of HEL into urine was quantified by using LC/MS/MS. Macrophages and neutrophils play an important role in oxidative stress during hyperglycemia, and we determined that oxidatively modified tyrosines are a good biomarker for formation of oxidative stress at an early stage. Immunochemical analyses by application of monoclonal antibodies specific to lipid hydroperoxide-modified proteins produced by polyunsaturated fatty acids including docosahexaenoic acid (DHA) in oxidative stress caused by hyperglycemia were conducted, and the relationship between glycation and lipid peroxidation reactions both by chemical and immunochemical approaches are discussed. Recently, we put much more focus on dietary antioxidants for prevention of diabetic complications. Curcuminoids, the main yellow pigments in Curcuma longa (turmeric), have been used widely and for a long time in the treatment of sprain and inflammation in indigenous medicine. Curcumin is the main component of turmeric, and two minor components are also present as the curcuminoids. Curcuminoids possess antioxidant activity. Protective effects of curcumin (U1) and one of its major metabolites, tetrahydrocurcumin (THU1), have been examined for development of diabetic cataract in 25% galactose-fed SD rats. Through detailed examination of protective mechanisms of THU1, it was found that THU1 showed that scavenger ROS not only formed during hyperglycemia, but also induced antioxidative enzymes including detoxification enzymes such as glutathine S-transferase. THU1 also showed significant increase of glutathione concentration in the cultured rat lens. Glutathione (gamma-glutamylcysteinyl glycine [GSH]) is thought to be an important factor in cellular function and defense against oxidative stress, and we found that dietary GSH suppresses oxidative stress in vivo in prevention of diabetic complications such as diabetic nephropathy and neuropathy.

Histochemical features of stress-induced aggregates in alpha-synuclein overexpressing cells.

alpha-Synuclein is a major component of intracytoplasmic inclusions including Lewy bodies (LB), Lewy neurites (LN) and glial cytoplasmic inclusions, and plays a key role in neurodegenerative processes in Parkinson's disease (PD) and other synucleinopathies. Although the molecular mechanisms of the disease process still remain to be elucidated, recent studies have suggested that an interaction between reactive oxygen species (ROS) and alpha-synuclein may be closely associated with the initiation and/or the progression of synucleinopathies. In this study, we established human dopaminergic SH-SY5Y cell lines overexpressing wild-type or mutant alpha-synuclein and exposed them to various ROS generators. After the exposure to ROS, alpha-synuclein aggregates were formed in the cytoplasm of these cells, and these were immunopositive for ubiquitin, nitrotyrosine and dityrosine, and positive for thioflavin S staining. Thus, the obtained cytoplasmic aggregates shared many features with inclusion bodies in synucleinopathies. The gamma-tubulin and molecular chaperones coexisted as well, suggesting that the aggregate formation is associated with the intracellular transport along microtubules and may reflect protective responses against neuronal insults. This cellular model not only will be informative for our understanding of the pathophysiological process in synucleinopathies, but also can be applied to the screening of neuroprotective molecules with therapeutic potential.

Bactericidal effects of konjac fluid on several food-poisoning bacteria.

In this study, the bactericidal effects of Japanese alkaline foods on food-poisoning bacteria were evaluated. Konjac is an alkaline food soaked in calcinated calcium (the pH of konjac fluid ranges from 11.42 to 12.53). Konjac fluids completely inactivated Escherichia coli, enterohemorrhagic E. coli O157:H7 and E. coil O26:H9, Salmonella Enteritidis, Vibrio parahemolyticus. and Staphylococcus aureus. The initial level of 6 log CFU/ml dramatically decreased after incubation with konjac fluid, and no viable gram-negative bacterium cells could be detected within 1 to 2 days and no viable S. aureus cells could be detected within 3 to 5 days. On the other hand, treatment with konjac fluid was also effective in reducing levels of spore-forming bacteria (Bacillus subtilis, Bacillus cereus, Clostridium perfringens, and Clostridium botulinum type E and type A). At least a 4-log reduction of spore-forming bacteria was obtained in konjac fluid within 7 to 14 days. Vegetative cells were more susceptible to konjac fluid than spores were. When the initial cell count was 6 log CFU/ml, a few surviving spores remained for 60 to 90 days, but no spores could be detected after 120 days. When the initial count of spore-forming bacteria was 3 to 4 log CFU/ml, the cells considered vegetative were completely inactivated within I to 3 days. Repeated treatment with konjac fluid caused complete inactivation of spores in less than 1 to 3 days. Our studies indicate that konjac fluid, which has a long history of use in food, will control food-poisoning bacterial contamination during the production or preservation of konjac and other foods and has a preventive effect on bacteria that can cause severe disease at uniquely low levels.

Rosmarinic acid inhibits lung injury induced by diesel exhaust particles.

Epidemiological and experimental studies have suggested that diesel exhaust particles (DEP) may be involved in recent increases in lung diseases. DEP has been shown to generate reactive oxygen species. Intratracheal instillation of DEP induces lung inflammation and edema in mice. Rosmarinic acid is a naturally occurring polyphenol with antioxidative and anti-inflammatory activities. We investigated the effects of rosmarinic acid on lung injury induced by intratracheal administration of DEP (500 microg/body) in mice. Oral supplementation with administration of rosmarinic acid (2 mg/body for 3 d) inhibited DEP-induced lung injury, which was characterized by neutrophil sequestration and interstitial edema. DEP enhanced the lung expression of keratinocyte chemoattractant (KC), interleukin-1beta, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1alpha, which was inhibited by treatment with rosmarinic acid. DEP enhanced expression of iNOS mRNA and formation of nitrotyrosine and 8-OHdG in the lung, which was also inhibited by rosmarinic acid. These results suggest that rosmarinic acid inhibits DEP-induced lung injury by the reduction of proinflammatory molecule expression. Antioxidative activities of rosmarinic acid may also contribute to its protective effects.