Salivary hydrogen peroxide produced by holding or chewing green tea in the oral cavity

Tea (Camellia sinensis) catechins have been studied for disease prevention. These compounds undergo oxidation and produce H₂O₂. We have previously shown that holding tea solution or chewing tea leaves generates high salivary catechin levels. Herein, we examined the generation of H₂O₂ in the oral cavity by green tea solution or leaves. Human volunteers holding green tea solution (0.1-0.6%) developed salivary H₂O₂ with C_max = 2.9-9.6 μM and AUC_0 → ∞ = 8.5-285.3 μM min. Chewing 2 g green tea leaves produced higher levels of H₂O₂ (C_max = 31.2 μM, AUC_0 → ∞ = 1290.9 μM min). Salivary H₂O₂ correlated with catechin levels and with predicted levels of H₂O₂ (C_{max(expected)} = 36 μM vs C_{max(determined)} = 31.2 μM). Salivary H₂O₂ and catechin concentrations were similar to those that are biologically active in vitro. Catechin-generated H₂O₂ may, therefore, have a role in disease prevention by green tea.     

Salivary hydrogen peroxide produced by holding or chewing green tea in the oral cavity

Tea (Camellia sinensis) catechins have been studied for disease prevention. These compounds undergo oxidation and produce H₂O₂. We have previously shown that holding tea solution or chewing tea leaves generates high salivary catechin levels. Herein, we examined the generation of H₂O₂ in the oral cavity by green tea solution or leaves. Human volunteers holding green tea solution (0.1–0.6%) developed salivary H₂O₂ with C_max = 2.9–9.6 μM and AUC_{0 → ∞} = 8.5–285.3 μM min. Chewing 2 g green tea leaves produced higher levels of H₂O₂ (C_max = 31.2 μM, AUC_{0 → ∞} = 1290.9 μM min). Salivary H₂O₂ correlated with catechin levels and with predicted levels of H₂O₂ (C_{max(expected)} = 36 μM vs C_{max(determined)} = 31.2 μM). Salivary H₂O₂ and catechin concentrations were similar to those that are biologically active in vitro. Catechin-generated H₂O₂ may, therefore, have a role in disease prevention by green tea.     

Contribution of catalase to hydrogen peroxide resistance in Enterococcus faecalis

Enterococcus faecalis exhibits high resistance to oxidative stress. Several enzymes are responsible for this trait. The role of alkyl hydroperoxide reductase (Ahp), thiol peroxidase (Tpx), and NADH peroxidase (Npr) in oxidative stress defense was recently characterized. Enterococcus faecalis, in contrast to many other streptococci, contains a catalase (KatA), but this enzyme can only be formed when the bacterium is supplied with heme. We have used this heme dependency of catalase activity and mutants deficient in KatA and Npr to investigate the role of the catalase in resistance against exogenous and endogenous hydrogen peroxide stress. The results demonstrate that in the presence of environmental heme catalase contributes to the protection against toxic effects of hydrogen peroxide.     

The mechanism of pH-dependent hydrogen peroxide cytotoxicity in vitro

The present paper is concerned with the influence of hydrogen ion concentration and composition of the medium on clonogenic survival of epithelial cells exposed to hydrogen peroxide in vitro. The survival of cells incubated with H2O2 in phosphate-buffered saline at pH 6.5 was 1 x 10(-2) and increased abruptly to 9 x 10(-2) at pH 7.0. The pH dependence of the cytocidal effect was particularly conspicuous when Eagle's minimum essential medium (SFMEM) was used for cell exposure to H2O2: the survival was characterized by exponential pH dependence and varied from 1 x 10(-1) to 9 x 10(-1) for pH 6.5 and 7.5, respectively, with a superimposed sharp peak value of 9 x 10(-1) at pH 7.0. The enhanced pH dependence of the H2O2 cytotoxicity in SFMEM was found to result from the additive action of glucose and histidine present in this medium. Glucose alone protected the cells with the efficiency decreasing with increasing hydrogen ion concentration. Histidine was responsible for the intermediate maximum in the pH-dependent survival spectrum. In addition, the changes in cell survival were accompanied by pH-dependent release of GSSG from the exposed cells. The GSSG efflux was inhibited by glucose in the medium. The influence of glucose on both the pattern of cell survival and the associated GSSG release indicate that the glutathione peroxidase activity supported by the pentose phosphate pathway is crucial in cell protection against extracellular H2O2 toxicity.     

Involvement of hydrogen peroxide in chromosomal aberrations induced by green tea catechins in vitro and implications for risk assessment

Catechins, which are polyphenol compounds found in abundance in green tea, have elicited high interest due to their beneficial effects on health. Catechins have also been demonstrated to induce chromosomal aberrations in vitro, although no clastogenicity was confirmed in studies in vivo. We investigated the mechanism of catechin-induced chromosomal aberrations in CHL/IU cells. Addition of catalase suppressed chromosomal aberrations, indicating involvement of hydrogen peroxide (H2O2). We confirmed that substantial amounts of H2O2 are generated when catechins are incubated under in vitro culture conditions, whereas, interestingly, extremely low amounts of H2O2 were detected when catechins were incubated at the same concentration in water. Generation of H2O2 increased steeply above pH 6, indicating that pH is a key factor in determining how much H2O2 is generated via catechins in vitro. Our assessment indicates that humans have practically non-existent exposure to H2O2 when catechins are ingested in a beverage. Polyphenols, including catechins, are known to act as antioxidants due to their reducing potential. However, under in vitro culture conditions, catechins are thought to act primarily as pro-oxidants by reducing ambient or dissolved oxygen to form H2O2. Based on the above observations, we conclude that in vitro culture conditions as currently employed are inappropriate to address genotoxicity concerns regarding polyphenols, including catechins.     

Role of lipophilicity and hydrogen peroxide formation in the cytotoxicity of flavonols.

We compared the lipophilicity and toxicity of the four flavonols, galangin, kaempferol, quercetin and myricetin, which respectively have no, one, two and three hydroxyl groups on the B-ring. The lipophilicity was in the order of myricetin < quercetin < kaempferol < galangin. The cytotoxicity determined by a colony-formation assay with Chinese hamster lung fibroblast V79 cells was in the order of quercetin < kaempferol < galangin < myricetin. Apart from myricetin, the order of lipophilicity was the same as that of cytotoxicity, implying that the cytotoxicity was attributable to the lipophilicity. The cytotoxicity of myricetin was attributable to the hydrogen peroxide formed by autoxidation.     

Reduction in tumor necrosis factor binding and cytotoxicity by hydrogen peroxide

The regulatory effect of H2O2 on both the cytotoxic activity and the specific binding of TNF-alpha was studied by using TNF-alpha-sensitized murine L929 cells. When these cells were exposed simultaneously to TNF-alpha and H2O2 (100 to 500 microM), the cytotoxic activity of TNF-alpha was inhibited by up to 66.6%. This inhibition was also effective when the cells were pretreated by H2O2, but not when TNF-alpha alone was preexposed to H2O2. These data suggest that H2O2 altered the cell sensitivity to TNF-alpha, without modifying the activity of the TNF-alpha molecule. Maximum loss of cell sensitivity to TNF-alpha occurred after 30-min preexposure to 500 microM H2O2. Complete restoration of TNF-alpha sensitivity was obtained within 12 h after H2O2 removal. It required protein synthesis as demonstrated by the suppressive effect of actinomycin D. The inhibitory effect of H2O2 was suppressed by catalase, but was unaffected by the scavengers of hydroxyl radical and hypochlorous acid, suggesting that H2O2 but not one of its metabolites was responsible for this inhibition. H2O2 inhibitory effect did not implicate any change in prostaglandin production or in PKC activity. In contrast, H2O2 effect was associated with an about 50% loss of the density of cell membrane 125I-TNF-alpha receptors (2949 vs 5620 binding sites per cell), without change in their affinity (3.9 vs 3.4 nM). Moreover H2O2 did not affect the rate of degradation of TNF-alpha, and only slightly increased the degree of internalization of 125I-TNF-alpha receptors. These findings indicate that H2O2 can down-regulate the cellular response to TNF-alpha, possibly by reducing the TNF-alpha-binding capacity.     

Endogenous defenses against the cytotoxicity of hydrogen peroxide in cultured rat hepatocytes

The catalase activity of cultured rat hepatocytes was inhibited by 90% pretreatment with 20 mM aminotriazole without effect on the activities of glutathione peroxidase or glutathione reductase, or on the viability of the cells over the subsequent 24 h. Glutathione reductase was inhibited by 85% by pretreatment with 300 microM 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) without effect on glutathione peroxidase, catalase, or on viability. Both pretreatments sensitized the hepatocytes to the cytotoxicity of H2O2 generated either by glucose oxidase (0.05-0.5 units/ml) or by the autoxidation of the one-electron-reduced state of menadione (50-250 microM). Aminotriazole pretreatment had no effect on the GSH content of the hepatocytes. BCNU reduced GSH levels by 50%. Depletion of GSH levels to less than 20% of control by treatment with diethyl maleate, however, did not sensitize the cells to either glucose oxidase or menadione, indicating that the effect of BCNU is related to inhibition of the GSH-GSSG redox cycle rather than to the depletion of GSH. With glucose oxidase, most of the cell killing in hepatocytes pretreated with either aminotriazole or BCNU occurred between 1 and 3 h. The antioxidant diphenylphenylenediamine (DPPD) had no effect on viability at 3 h. Catalase added to the culture medium 1 h after the addition of glucose oxidase prevented the cell killing measured at 3 h. The sulfhydryl reagents dithiothreitol (200 microM), N-acetyl-L-cysteine (4 mM), and alpha-mercaptopropionyl-L-glycine (2.5 mM) prevented the cell killing with exogenous H2O2 in hepatocytes sensitized by the inhibition of catalase or glutathione reductase. With menadione, there was no killing of nonpretreated hepatocytes at 1 h, and DPPD did not prevent the cell death after 3 h. Aminotriazole pretreatment enhanced the cell killing at 3 h but not at 1 h, and DPPD was not protective. Catalase added to the medium at 1 h inhibited the cell death measured at 3 h. In contrast, menadione killed hepatocytes pretreated with BCNU within 1 h. DPPD prevented cell death at 1 h, and there was evidence of lipid peroxidation in the accumulation of malondialdehyde in the culture medium. Catalase added with menadione did not prevent the cell killing at 1 h but did prevent it at 3 h. These data indicate that catalase and the GSH-GSSG cycle are active in the defense of hepatocytes against the toxicity of H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of diethyldithiocarbamate and endogenous polyamine content on cellular responses to hydrogen peroxide cytotoxicity.

In exponential-phase Chinese-hamster cells, 0.1 mM-diethyldithiocarbamate (DDC) afforded greater than 1 log survival protection to cultures treated before and during exposure to 1 mM-H2O2. Both DDC and H2O2 treatment stimulated the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, within 4 h of exposure. DDC, and to a lesser degree H2O2, also stimulated the activity of spermidine N1-acetyltransferase (SAT), the rate-limiting enzyme in polyamine catabolism. The increase in SAT activity, after exposure to DDC or another stress (heat shock), was inhibited in cells depleted of putrescine and spermidine by alpha-difluoromethylornithine (DFMO), the enzyme-activated suicide inhibitor of ODC. Pretreatment with DFMO or heat shock also induced resistance to H2O2 cytotoxicity. Since SAT activity is low in resting cells, yet stimulation of enzyme activity depends on endogenous spermidine pools, these results suggest that the expression of SAT activity occurs by a mechanism involving a stress-dependent displacement of spermidine into a new intracellular compartment. The stimulation of ODC and SAT activities does not appear to be a necessary component of the mechanism by which DDC protects cells from H2O2 cytotoxicity, although spermidine displacement may be a common facet of the cellular response to stress.     

The protective role of polyphenols in cytotoxicity of hydrogen peroxide.

A variety of synthetic and natural polyphenols protect mammalian cells from hydrogen peroxide (H2O2). Cytotoxicity of H2O2 on Chinese hamster V79 cells was assessed with a colony formation assay, and several polyphenols prevented the decrease in the number of colonies caused by H2O2. A study of the structure-activity relationship revealed that affinity of the polyphenols for the cell membrane and the presence of an ortho-dihydroxy moiety in their structure proved essential to this protection.     

Differential cytotoxicity of daunomycin in tumour cells is related to glutathione-dependent hydrogen peroxide metabolism

Addition of 0.5mm-daunomycin, a quinone anti-cancer drug, causes severe inhibition of respiration in Ehrlich ascites cells, whereas Yoshida ascites cells were almost as resistant as rat hepatocytes. An inverse relationship appears to exist in the two types of tumour cells (which are both catalase-deficient) between the extent of cellular damage brought about by intracellular formation of superoxide anion occurring on reaction with O₂ of the drug free radical and the efficiency of the glutathione-mediated H₂O₂-detoxifying system.     

Antiangiogenic properties of natural polyphenols from red wine and green tea

Epidemiological studies have indicated that regular consumption of red wine and green tea is associated with a reduced risk of coronary heart disease and tumor progression. The development of tumors and of atherosclerosis lesions to advanced plaques, which are prone to rupture, is accelerated by the formation of new blood vessels. These new blood vessels provide oxygen and nutrients to neighboring cells. Therefore, recent studies have examined whether red wine polyphenolic compounds (RWPCs) and green tea polyphenols (GTPs) have antiangiogenic properties. In vitro investigations have indicated that RWPCs and GTPs are able to inhibit several key events of the angiogenic process such as proliferation and migration of endothelial cells and vascular smooth muscle cells and the expression of two major proangiogenic factors, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2, by both redox-sensitive and redox-insensitive mechanisms. Antiangiogenic properties of polyphenols have also been observed in the chick embryo chorioallantoic membrane since the local application of RWPCs and GTPs strongly inhibited the formation of new blood vessels. Moreover, intake of resveratrol or green tea has been shown to reduce corneal neovascularization induced by proangiogenic factors such as VEGF and fibroblast growth factor in mice. The ability of RWPCs and GTPs to prevent the formation of new blood vessels contributes, at least in part, to explain their beneficial effect on coronary heart disease and cancer. This review focuses on the antiangiogenic properties of natural polyphenols and examines underlying mechanisms.     

The role of hydrogen peroxide in the in vitro cytotoxicity of 3-hydroxykynurenine

Previous studies have indicated that the generation of H₂O₂ may be a key step in the mechanism mediating the in vitro cytotoxicity of 3-hydroxykynurenine (3HK). An exposure protocol resulting in a delayed toxicity was utilized in order to further examine the role of H₂O₂ in the in vitro toxicity of 3HK in a neural hybrid cell line. 3HK-induced cell lysis was significantly attenuated by administration of catalase after termination of 3HK exposure and was abolished when intracellular peroxidase activity was elevated by pretreatment of cultures with horseradish peroxidase. In addition, a dose-dependent attenuation of 3HK toxicity was observed when cultures were exposed to 3HK in the presence of the iron chelator, desferrioxamine (DFO). Pretreatment with DFO also resulted in a significant attenuation of 3HK toxicity. These data suggest a direct role for H₂O₂ and metal ions in the cytotoxic action of 3HK and indicate that cell lysis results from the intracellular accumulation of toxic levels of H₂O₂.     

Ilex paraguariensis extracts are potent inhibitors of nitrosative stress: a comparative study with green tea and wines using a protein nitration model and mammalian cell cytotoxicity

Due to the increasing importance of nitrosative stress in pathology and the efficacy displayed by flavonoids in cancelling the effects of peroxynitrite, we decided to conduct a comparative study of three commonly used beverages with the highest polyphenol contents and proven antioxidant properties: mate (Ilex paraguariensis); green tea (Camelia sinensis) extracts and white and red wines of the main varietals. We directly evaluated and compared the extracts and wines as protein nitration inhibitors using 3-nitrotyrosine as a biomarker, we studied the extracts as protectors from OONO-induced cytotoxicity in two mammalian cell lines. Both green tea and mate extracts have a high polyphenol content, in the case of Ip, its higher concentration and higher free radical quenching activity on the DPPH assay may be mainly due to the sui generis extraction procedure. When BSA was incubated in the presence of SIN-1, a time and dose dependent nitration of the protein is clearly shown. Co-incubation of BSA with Ip, green tea or red wines led to a dose dependent inhibition of the effect. Ip displayed the highest inhibitory activity, followed by red wines and the green tea. Dilutions as low as 1/1500 produced more than 80% inhibition of albumin nitration. When we studied peroxynitrite-induced cytotoxicity in murine RAW 264.7 macrophages and 31EG4 mammary cells., we found a potent, dose-dependent protective effect that was Ilex paraguariensis > red wines > green tea. Taken together, our results indicate that when the herbal preparations studied here are prepared the way they are usually drunk, Ip displays the highest inhibition of protein nitration, and the highest promotion of cell survival, whereas green tea or red wines display significant but lesser effects at the same concentrations. Further studies aiming at isolation of the active principles and assessment of their bioavailability are warranted.     

Prehemolytic effects of hydrogen peroxide and t-butylhydroperoxide on selected red cell properties

To provide further understanding of how oxidative damage affects red cell membrane function, the effects of low levels of two different types of oxidants on selected red cell properties have been studied. Hydrogen peroxide (H2O2), an example of a water soluble oxidant, and t-butylhydroperoxide (tBHP), a hydrophobic hydroperoxide, were compared with respect to their effects on membrane permeability, membrane mechanical properties and binding of autologous serum antibodies to the cell surface. Whereas H2O2 treatment resulted in a dose-dependent increase in membrane permeability to potassium that was evident after one hour of oxidant exposure, cells treated with tBHP at doses up to 5 mumol/ml cells showed no immediate change in cation permeability. H2O2 also caused a marked decrease in membrane deformability, whereas tBHP-treated cells showed minimal loss of deformability. However, tBHP treatment did result in a dose-dependent increase in the susceptibility of the membrane to fragmentation under high shear stress. With exclusion of treated samples that bound excess rabbit anti-spectrin antibody, indicating exposure of intracellular components, neither agent promoted the binding of autologous serum antibody in amounts comparable to that found in vivo on high density or some pathologic red cells. Taken together, the results suggest that tBHP and H2O2 cause damage to human red cells by distinct oxidative mechanisms which do not lead directly to substantive generation of binding sites for autologous serum antibodies.     

Role of hydrogen peroxide in the cytotoxicity of the xanthine/xanthine oxidase system.

1. The survival of mammalian epithelial cells exposed in vitro to the xanthine/xanthine oxidase system in phosphate-buffered saline (PBS) or serum-containing medium (SCMEM) was investigated. 2. The cytotoxic effect observed depended on the composition of the medium in which the enzymic reaction was carried out; a surviving fraction of 5 x 10(-5) was found for cells exposed in PBS and 5.2 x 10(-1) for those in SCMEM. 3. The cytotoxic product(s) formed by the xanthine/xanthine oxidase system was relatively stable in PBS; survival of cells incubated after completion of the enzymic reaction was always less than that found for cells exposed during the reaction in the same system. 4. Superoxide dismutase or mannitol present during the enzymic reaction did not inhibit the cytotoxic effect. 5. NaN3 (a single-oxygen quencher and a catalase inhibitor) added to the system in SCMEM caused a reduction in survival to the level observed for cells exposed to the enzymic reaction in PBS. 6. Catalase completely protected cells, but no protection was observed when both catalase and NaN3 were present in the reaction mixture. 7. A similar cytotoxic effect was produced when cells were treated with H2O2 alone. 8. The rate of H2O2 decomposition in medium was accelerated by the presence of serum, but this was completely inhibited by NaN3. 9. It is concluded that H2O2 is the major cytotoxic product formed by the xanthine/xanthine oxidase system.     

Polyphenols from green tea prevent antineuritogenic action of Nogo‐A via 67‐kDa laminin receptor and hydrogen peroxide

Axonal regeneration after injury to the CNS is hampered by myelin‐derived inhibitors, such as Nogo‐A. Natural products, such as green tea, which are neuroprotective and safe for long‐term therapy, would complement ongoing various pharmacological approaches. In this study, using nerve growth factor‐differentiated neuronal‐like Neuroscreen‐1 cells, we show that extremely low concentrations of unfractionated green tea polyphenol mixture (GTPP) and its active ingredient, epigallocatechin‐3‐gallate (EGCG), prevent both the neurite outgrowth‐inhibiting activity and growth cone‐collapsing activity of Nogo‐66 (C‐terminal domain of Nogo‐A). Furthermore, a synergistic interaction was observed among GTPP constituents. This preventive effect was dependent on 67‐kDa laminin receptor (67LR) to which EGCG binds with high affinity. The antioxidants N‐acetylcysteine and cell‐permeable catalase abolished this preventive effect of GTPP and EGCG, suggesting the involvement of sublethal levels of H₂O₂ in this process. Accordingly, exogenous sublethal concentrations of H₂O₂, added as a bolus dose (5 μM) or more effectively through a steady‐state generation (1–2 μM), mimicked GTPP in counteracting the action of Nogo‐66. Exogenous H₂O₂ mediated this action by bypassing the requirement of 67LR. Taken together, these results show for the first time that GTPP and EGCG, acting through 67LR and elevating intracellular sublethal levels of H₂O₂, inhibit the antineuritogenic action of Nogo‐A.

     

The origin of red cell fluorescence caused by hydrogen peroxide treatment

Fluorescence in red cells following hydrogen peroxide treatment has been attributed to lipid peroxidation of the membrane. The putative relationship between lipid peroxidation and fluorescence was questioned by the finding that BHT and alpha-tocopherol, which are thought to inhibit lipid peroxidation, do not inhibit the fluorescence detected by flow cytometry. Furthermore, lipid peroxidation induced in red cells by the Fe(III)-ADP-ascorbate system did not produce fluorescence. These results require an alternative explanation for the hydrogen peroxide-induced fluorescence. A role for reduced hemoglobin is indicated by the inhibition of fluorescence by pretreatment of cells with CO that binds strongly to ferrohemoglobin and nitrite that oxidizes ferrohemoglobin. Our earlier studies have shown the formation of fluorescent heme degradation products during the reaction of purified hemoglobin with hydrogen peroxide, which was also inhibited by CO and nitrite pretreatment. The fluorescence produced in red cells after the addition of hydrogen peroxide can, therefore, be attributed to fluorescent heme degradation products.     

Detection of hydrogen peroxide production in the isolated rat lung using Amplex red

Abstract

The objectives of this study were to develop a robust protocol to measure the rate of hydrogen peroxide (H₂O₂) production in isolated perfused rat lungs, as an index of oxidative stress, and to determine the cellular sources of the measured H₂O₂ using the extracellular probe Amplex red (AR). AR was added to the recirculating perfusate in an isolated perfused rat lung. AR’s highly fluorescent oxidation product resorufin was measured in the perfusate. Experiments were carried out without and with rotenone (complex I inhibitor), thenoyltrifluoroacetone (complex II inhibitor), antimycin A (complex III inhibitor), potassium cyanide (complex IV inhibitor), or diohenylene iodonium (inhibitor of flavin-containing enzymes, e.g. NAD(P)H oxidase or NOX) added to the perfusate. We also evaluated the effect of acute changes in oxygen (O₂) concentration of ventilation gas on lung rate of H₂O₂ release into the perfusate. Baseline lung rate of H₂O₂ release was 8.45?±?0.31 (SEM) nmol/min/g dry wt. Inhibiting mitochondrial complex II reduced this rate by 76%, and inhibiting flavin-containing enzymes reduced it by another 23%. Inhibiting complex I had a small (13%) effect on the rate, whereas inhibiting complex III had no effect. Inhibiting complex IV increased this rate by 310%. Increasing %O₂ in the ventilation gas mixture from 15 to 95% had a small (27%) effect on this rate, and this O₂-dependent increase was mostly nonmitochondrial. Results suggest complex II as a potentially important source and/or regulator of mitochondrial H₂O₂, and that most of acute hyperoxia-enhanced lung rate of H₂O₂ release is from nonmitochondrial rather than mitochondrial sources.