Studies on the mutagenic activity of ascorbic acid in vitro and in vivo

In vitro data are presented to show that ascorbic acid does not have intrinsic mutagenicity towards strain TA100 of S. typhimurium if deionized water is used to prepare the incubation medium. The addition of Cu2+ ions to the bacterial medium that contains ascorbic acid, or the use of tap water and ascorbic acid alone, causes a mutagenic and cytotoxic response that is blocked by EDTA. Additional in vitro data demonstrate that hydrogen peroxide is mutagenic to S. typhimurium strain TA100 and it is suggested that ascorbic acid may be mutagenic and cytotoxic through the generation of hydrogen peroxide. In vivo studies using a sensitive intrahepatic host-mediated mutagenicity assay indicate that ascorbic acid is not genotoxic in guinea pigs even when the dietary intake of vitamin C is above the level required for tissue saturation (5000 mg/kg body weight/day).     

Disinfection of model indicator organisms in a drinking water pilot plant by using PEROXONE

PEROXONE is an advanced oxidation process generated by combining ozone and hydrogen peroxide. This process stimulates the production of hydroxyl radicals, which have been shown to be superior to ozone for the destruction of some organic contaminants. In this study, pilot-scale experiments were conducted to evaluate the microbicidal effectiveness of PEROXONE and ozone against three model indicator groups. Escherichia coli and MS2 coliphage were seeded into the influent to the preozonation contactors of a pilot plant simulating conventional water treatment and were exposed to four ozone dosages (0.5, 1.0, 2.0, and 4.0 mg/liter), four hydrogen peroxide/ozone (H2O2/O3) weight ratios (0, 0.3, 0.5, and 0.8), and four contact times (4, 5, 12, and 16 min) in two source waters--Colorado River water and state project water--of different quality. The removal of heterotrophic plate count bacteria was also monitored. Results of the study indicated that the microbicidal activity of PEROXONE was greatly affected by the applied ozone dose, H2O2/O3 ratio, contact time, source water quality, and type of microorganism tested. At contact times of 5 min or less, ozone alone was a more potent bactericide than PEROXONE at all H2O2/O3 ratios tested. However, this decrease in the bactericidal potency of PEROXONE was dramatic only as the H2O2/O3 ratio was increased from 0.5 to 0.8. The fact that the bactericidal activity of PEROXONE generally decreased with increasing H2O2/O3 ratios was thought to be related to the lower ozone residuals produced. The viricidal activity of PEROXONE and ozone was comparable at all of the H2O2/O3 ratios. Heterotrophic plate count bacteria were the most resistant group of organisms. Greater inactivation of E. coli and MS2 was observed in Colorado River water than in state project water and appeared to result from differences in the turbidity and alkalinity of the two waters. Regardless of source water, greater than 4.5 log10 of E. coli and MS2 was inactivated at an applied ozone dosage of 2.0 mg/liter (and a 4-min contact time) when the H2O2/O3 ratio was less than or equal to 0.5. Comparative disinfection experiments indicated that free chlorine was the most potent bactericidal agent, followed (in descending order of effectiveness) by ozone, PEROXONE, and chloramines. These results indicate that the PEROXONE process must be optimized for each source water to achieve microbicidal effectiveness.     

Disinfection of model indicator organisms in a drinking water pilot plant by using PEROXONE.

PEROXONE is an advanced oxidation process generated by combining ozone and hydrogen peroxide. This process stimulates the production of hydroxyl radicals, which have been shown to be superior to ozone for the destruction of some organic contaminants. In this study, pilot-scale experiments were conducted to evaluate the microbicidal effectiveness of PEROXONE and ozone against three model indicator groups. Escherichia coli and MS2 coliphage were seeded into the influent to the preozonation contactors of a pilot plant simulating conventional water treatment and were exposed to four ozone dosages (0.5, 1.0, 2.0, and 4.0 mg/liter), four hydrogen peroxide/ozone (H2O2/O3) weight ratios (0, 0.3, 0.5, and 0.8), and four contact times (4, 5, 12, and 16 min) in two source waters--Colorado River water and state project water--of different quality. The removal of heterotrophic plate count bacteria was also monitored. Results of the study indicated that the microbicidal activity of PEROXONE was greatly affected by the applied ozone dose, H2O2/O3 ratio, contact time, source water quality, and type of microorganism tested. At contact times of 5 min or less, ozone alone was a more potent bactericide than PEROXONE at all H2O2/O3 ratios tested. However, this decrease in the bactericidal potency of PEROXONE was dramatic only as the H2O2/O3 ratio was increased from 0.5 to 0.8. The fact that the bactericidal activity of PEROXONE generally decreased with increasing H2O2/O3 ratios was thought to be related to the lower ozone residuals produced. The viricidal activity of PEROXONE and ozone was comparable at all of the H2O2/O3 ratios. Heterotrophic plate count bacteria were the most resistant group of organisms. Greater inactivation of E. coli and MS2 was observed in Colorado River water than in state project water and appeared to result from differences in the turbidity and alkalinity of the two waters. Regardless of source water, greater than 4.5 log10 of E. coli and MS2 was inactivated at an applied ozone dosage of 2.0 mg/liter (and a 4-min contact time) when the H2O2/O3 ratio was less than or equal to 0.5. Comparative disinfection experiments indicated that free chlorine was the most potent bactericidal agent, followed (in descending order of effectiveness) by ozone, PEROXONE, and chloramines. These results indicate that the PEROXONE process must be optimized for each source water to achieve microbicidal effectiveness.     

Factors Influencing the Effectiveness of Swimming Pool Bactericides

Techniques for culturing, harvesting, and testing bacteria to evaluate bactericidal chemicals for swimming pools are described. Concentrations of 25, 50, and 100 mg of the chlorine stabilizer cyanuric acid per liter increased the time required for a 99% kill of Streptococcus faecalis by 0.5 mg of chlorine per liter at pH 7.4 and 20 C from less than 0.25 min without cyanuric acid to 4, 6, and 12 min, respectively. The effect of concentrations of ammonia nitrogen in the range found in swimming pools on the rate of kill of 0.5 mg of chlorine per liter and of chlorine plus cyanuric acid was tested. At concentrations of ammonia nitrogen greater than 0.05 mg per liter, faster rates of kill of S. faecalis were obtained with 100 mg of cyanuric acid per liter plus 0.5 mg of chlorine per liter than with 0.5 mg of chlorine per liter alone. When water samples from four swimming pools with low ammonia levels were used as test media, 0.5 mg of added chlorine per liter killed 99.9% of the added S. faecalis in less than 2 min, but water from a pool with a large number of children required 60 to 180 min of treatment.     

Preparation of dehydro-L-ascorbic acid dimer by air oxidation of L-ascorbic acid in the presence of catalytic amounts of copper(II) acetate and pyridine

The catalytic system Cu(AcO)2-pyridine 1:4 mol% in methanol, slowly catalyses the air oxidation of ascorbic acid to the 2-methyl hemi-ketal of dehydroascorbic acid 5, and hydrogen peroxide. However, with Cu(AcO)2-pyridine 3:4 mol% the air oxidation is quite fast and no hydrogen peroxide is present at the end of the reaction. Removal of the catalyst and refluxing the foamy 5 in MeCN gives the oxidized, dimeric, dehydroascorbic acid in very good yields (approximately 70%) contaminated by approximately 1-2% MeCN.     

Iron prevents ascorbic acid (vitamin C) induced hydrogen peroxide accumulation in copper contaminated drinking water

Ascorbic acid (vitamin C) induced hydrogen peroxide (H₂O₂) formation was measured in household drinking water and metal supplemented Milli-Q water by using the FOX assay. Here we show that ascorbic acid readily induces H₂O₂ formation in Cu(II) supplemented Milli-Q water and poorly buffered household drinking water. In contrast to Cu(II), iron was not capable to support ascorbic acid induced H₂O₂ formation during acidic conditions (pH: 3.5-5). In 12 out of the 48 drinking water samples incubated with 2 mM ascorbic acid, the H₂O₂ concentration exceeded 400 μM. However, when trace amounts of Fe(III) (0.2 mg/l) was present during incubation, the ascorbic acid/Cu(II)-induced H₂O₂ accumulation was totally blocked. Of the other common divalent or trivalent metal ions tested, that are normally present in drinking water (calcium, magnesium, zinc, cobalt, manganese or aluminum), only calcium and magnesium displayed a modest inhibitory activity on the ascorbic acid/Cu(II)-induced H₂O₂ formation. Oxalic acid, one of the degradation products from ascorbic acid, was confirmed to actively participate in the iron induced degradation of H₂O₂. Ascorbic acid/Cu(II)-induced H₂O₂ formation during acidic conditions, as demonstrated here in poorly buffered drinking water, could be of importance in host defense against bacterial infections. In addition, our findings might explain the mechanism for the protective effect of iron against vitamin C induced cell toxicity.     

Prevention of oxidative DNA degradation by copper-binding peptides

Free divalent ions of copper (Cu) are capable of generating radical species such as hydroxyl radicals in the presence of hydrogen peroxide or ascorbic acid through Harbor-Weiss-like reactions under physiological conditions. It has been reported that radical-mediated damage to DNA molecules in animal cells leads to programmed cell death. Hence it is important to seek for methods to prevent Cu-mediated DNA damage. In this study we identified on effect of Cu binding of short peptides (chiefly Gly-Gly-His tripeptide) in the prevention of DNA degradation caused by Cu-mediated reactions in the presence of hydrogen peroxide and of ascorbic acid.     

Urea hydrogen peroxide reduces the numbers of lactobacilli, nourishes yeast, and leaves no residues in the ethanol fermentation

Urea hydrogen peroxide (UHP) at a concentration of 30 to 32 mmol/liter reduced the numbers of five Lactobacillus spp. (Lactobacillus plantarum, L. paracasei, Lactobacillus sp. strain 3, L. rhamnosus, and L. fermentum) from approximately 10(7) to approximately 10(2) CFU/ml in a 2-h preincubation at 30 degrees C of normal-gravity wheat mash at approximately 21 g of dissolved solids per ml containing normal levels of suspended grain particles. Fermentation was completed 36 h after inoculation of Saccharomyces cerevisiae in the presence of UHP, even when wheat mash was deliberately contaminated (infected) with L. paracasei at approximately 10(7) CFU/ml. There were no significant differences in the maximum ethanol produced between treatments when urea hydrogen peroxide was used to kill the bacteria and controls (in which no bacteria were added). However, the presence of L. paracasei at approximately 10(7) CFU/ml without added agent resulted in a 5.84% reduction in the maximum ethanol produced compared to the control. The bactericidal activity of UHP is greatly affected by the presence of particulate matter. In fact, only 2 mmol of urea hydrogen peroxide per liter was required for disinfection when mashes had little or no particulate matter present. No significant differences were observed in the decomposition of hydrogen peroxide in normal-gravity wheat mash at 30 degrees C whether the bactericidal agent was added as H(2)O(2) or as urea hydrogen peroxide. NADH peroxidase activity (involved in degrading H(2)O(2)) increased significantly (P = 0.05) in the presence of 0.75 mM hydrogen peroxide (sublethal level) in all five strains of lactobacilli tested but did not persist in cells regrown in the absence of H(2)O(2). H(2)O(2)-resistant mutants were not expected or found when lethal levels of H(2)O(2) or UHP were used. Contaminating lactobacilli can be effectively managed by UHP, a compound which when used at ca. 30 mmol/liter happens to provide near-optimum levels of assimilable nitrogen and oxygen that aid in vigorous fermentation performance by yeast.     

Real-time detection of L-ascorbic acid and hydrogen peroxide in crude food samples employing a reversed sequential differential measuring technique of the SIRE-technology based biosensor.

Detection of the common electrochemical interferents, ascorbic acid and hydrogen peroxide, using a SIRE (Sensors based on Injection of the Recognition Element) technology based biosensor in reverse mode operation is reported. The differential measuring principle employed in the SIRE biosensor during operation in reverse mode is such that the sample is measured first in the presence of enzyme (yielding matrix signal only), and then measured again in the absence of enzyme (yielding signal from matrix+analyte). Subtraction of the signal obtained in the presence of enzyme from the signal obtained in the absence of enzyme gives a specific signal for the analyte only and correlates directly to its concentration in solution. The linear range for the determination of ascorbic acid and hydrogen peroxide was 0-3 mM and 0-2 mM, respectively, with an enzyme concentration of 25 U ascorbate oxidase/ml and 1000 U catalase/ml. The reproducibility was 5% for ascorbic acid (R.S.D. n=15) and 10% for hydrogen peroxide (R.S.D. n=18). The cost per measurement was 0.28 USD for ascorbic acid analysis and 0.0008 USD for hydrogen peroxide analysis. The degradation of ascorbic acid in cereal was followed in real-time, as was the stabilization of low pH on the degradation process.     

Effects of citrinin on iron-redox cycle

The ability of the mycotoxin citrinin to act as an inhibitor of iron-induced lipoperoxidation of biological membranes prompted us to determine whether it could act as an iron chelating agent, interfering with iron redox reactions or acting as a free radical scavenger. The addition of Fe3+ to citrinin rapidly produced a chromogen, indicating the formation of citrinin-Fe3+ complexes. An EPR study confirms that citrinin acts as a ligand of Fe3+, the complexation depending on the [Fe3+]:[citrinin] ratios. Effects of citrinin on the iron redox cycle were evaluated by oxygen consumption or the o-phenanthroline test. No effect on EDTA-Fe2+-->EDTA-Fe3+ oxidation was observed in the presence of citrinin, but the mycotoxin inhibited, in a dose-dependent manner, the oxidation of Fe2+ to Fe3+ by hydrogen peroxide. Reducing agents such as ascorbic acid and DTT reduced the Fe3+-citrinin complex, but DTT did not cause reduction of Fe3+-EDTA, indicating that the redox potentials of Fe3+-citrinin and Fe3+-EDTA are not the same. The Fe2+ formed from the reduction of Fe3+-citrinin by reducing agents was not rapidly reoxidized to Fe3+ by atmospheric oxygen. Citrinin has no radical scavenger ability as demonstrated by the absence of DPPH reduction. However, a reaction between citrinin and hydrogen peroxide was observed by UV spectrum changes of citrinin after incubation with hydrogen peroxide. It was also observed that citrinin did not induce direct or reductive mobilization of iron from ferritin. These results indicate that the protective effect on iron-induced lipid peroxidation by citrinin occurs due to the formation of a redox inactive Fe3+-citrinin complex, as well as from the reaction of citrinin and hydrogen peroxide.     

Iron prevents ascorbic acid (vitamin C) induced hydrogen peroxide accumulation in copper contaminated drinking water

Ascorbic acid (vitamin C) induced hydrogen peroxide (H₂O₂) formation was measured in household drinking water and metal supplemented Milli-Q water by using the FOX assay. Here we show that ascorbic acid readily induces H₂O₂ formation in Cu(II) supplemented Milli-Q water and poorly buffered household drinking water. In contrast to Cu(II), iron was not capable to support ascorbic acid induced H₂O₂ formation during acidic conditions (pH: 3.5–5). In 12 out of the 48 drinking water samples incubated with 2 mM ascorbic acid, the H₂O₂ concentration exceeded 400 μM. However, when trace amounts of Fe(III) (0.2 mg/l) was present during incubation, the ascorbic acid/Cu(II)-induced H₂O₂ accumulation was totally blocked. Of the other common divalent or trivalent metal ions tested, that are normally present in drinking water (calcium, magnesium, zinc, cobalt, manganese or aluminum), only calcium and magnesium displayed a modest inhibitory activity on the ascorbic acid/Cu(II)-induced H₂O₂ formation. Oxalic acid, one of the degradation products from ascorbic acid, was confirmed to actively participate in the iron induced degradation of H₂O₂. Ascorbic acid/Cu(II)-induced H₂O₂ formation during acidic conditions, as demonstrated here in poorly buffered drinking water, could be of importance in host defense against bacterial infections. In addition, our findings might explain the mechanism for the protective effect of iron against vitamin C induced cell toxicity.     

Bactericidal effect of hydrogen peroxide on Salmonella typhimurium in liquid whole egg

The effect of hydrogen peroxide on Salmonella typhimurium in whole egg was evaluated. The bactericidal effects observed on the test organism at 5 degrees and 20 degrees C were found to be similar. There was a 99% kill in the presence of 0.5% and 1.0% H2O2. Addition of the test organism and H2O2 after pre-heating the egg material at 40 degrees C for 15 min caused a rapid kill which was 10,000-fold greater than that produced by H2O2 alone.     

多种小麦种子储藏蛋白的电泳分析

采用不同的提取液,对１０个小麦品种的非酶功能性种子储藏蛋白进行提取,分别进行梯度凝胶电泳分析。电泳依据提取液的不同,分别采用酸性或碱性系统。对酸性凝胶催化系统,采用Ａｐ－Ｖｃ－ＦｅＳＯ４系统代替Ｈ２Ｏ２－Ｖｃ－ＦｅＳＯ４系统,克服了酸性凝胶的不足,提高了凝胶的性质性能并使之容易操作。应用新的催化系统配制的酸性梯度胶,提高了分辨率。并初步尝试以酸性系统分析种子谷蛋白,获得了成功,经过对不同提取液蛋白     

Copper(II) ethylenediaminetetraacetate complex does activate hydrogen peroxide in the presence of biological reductants.

The reaction of CuII (edta) (edta: ethylenediaminetetraacetic acid) with hydrogen peroxide (H2O2) was studied in the pH range of 6.0 to 8.0. CuII (edta) did not react with H2O2 in the all pH range examined in the absence of biological reductants. CuII (edta), however, could react with H2O2 in the presence of biological reductants such as ascorbic acid, cysteine and NADH to give thibarbituric acid (TBA) reactive substance, regardless of the pH. From these results, it is concluded that CuII (edta) cannot be bound to H2O2 and that the change of the redox potential of Cu2+ ion on ligating with edta may cause CuII (edta) to be unable to oxidize H2O2.     

Oxalic acid is available as a natural antioxidant in some systems

Oxalic acid is found in a wide variety of plants. This study showed that oxalic acid suppressed in vitro lipid peroxidation in a concentration-dependent manner. Furthermore, oxalic acid reduced the rate of ascorbic acid oxidation in the presence of hydrogen peroxide and Cu(2+). These results suggest that oxalic acid is available as a natural antioxidant.     

Redox reactions of copper complexes formed with different beta-amyloid peptides and their neuropathological [correction of neuropathalogical] relevance

The binding stoichiometry between Cu(II) and the full-length beta-amyloid Abeta(1-42) and the oxidation state of copper in the resultant complex were determined by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) and cyclic voltammetry. The same approach was extended to the copper complexes of Abeta(1-16) and Abeta(1-28). A stoichiometric ratio of 1:1 was directly observed, and the oxidation state of copper was deduced to be 2+ for all of the complexes, and residues tyrosine-10 and methionine-35 are not oxidized in the Abeta(1-42)-Cu(II) complex. The stoichiometric ratio remains the same in the presence of more than a 10-fold excess of Cu(II). Redox potentials of the sole tyrosine residue and the Cu(II) center were determined to be ca. 0.75 and 0.08 V vs Ag/AgCl [or 0.95 and 0.28 V vs normal hydrogen electrode (NHE)], respectively. More importantly, for the first time, the Abeta-Cu(I) complex has been generated electrochemically and was found to catalyze the reduction of oxygen to produce hydrogen peroxide. The voltammetric behaviors of the three Abeta segments suggest that diffusion of oxygen to the metal center can be affected by the length and hydrophobicity of the Abeta peptide. The determination and assignment of the redox potentials clarify some misconceptions in the redox reactions involving Abeta and provide new insight into the possible roles of redox metal ions in the Alzheimer's disease (AD) pathogenesis. In cellular environments, the reduction potential of the Abeta-Cu(II) complex is sufficiently high to react with antioxidants (e.g., ascorbic acid) and cellular redox buffers (e.g., glutathione), and the Abeta-Cu(I) complex produced could subsequently reduce oxygen to form hydrogen peroxide via a catalytic cycle. Using voltammetry, the Abeta-Cu(II) complex formed in solution was found to be readily reduced by ascorbic acid. Hydrogen peroxide produced, in addition to its role in damaging DNA, protein, and lipid molecules, can also be involved in the further consumption of antioxidants, causing their depletion in neurons and eventually damaging the neuronal defense system. Another possibility is that Abeta-Cu(II) could react with species involved in the cascade of electron transfer events of mitochondria and might potentially sidetrack the electron transfer processes in the respiratory chain, leading to mitochondrial dysfunction.     

Determination of catalase-like activity in plants based on the amperometric monitoring of hydrogen peroxide consumption using a carbon paste electrode modified with ruthenium(IV) Oxide

A carbon paste electrode containing ruthenium(IV) oxide as a modifier was tested as an effective hydrogen peroxide amperometric sensor in bulk measurements (hydrodynamic amperometry). Factors that influence its overall analytical perform ance, such as pH and the applied potential, were examined. The RuO2-modified electrode displayed high sensitivity towards hydrogen peroxide, with detection limits as low as 0.02 mm at pH 7.4 and 0.007 mM at pH 9.0. The method was applied for monitoring the decomposition of hydrogen peroxide (by catalase) in phosphate buffer of pH 7.4. The relative response of the electrode towards ascorbic acid was assessed and it was found that the selectivity of the RuO2-modified electrode towards hydrogen peroxide over ascorbic acid could be significantly improved by electro-polymerizing m-phenylenediamine on its surface prior to measurements. The RuO2-modified electrode was used for the kinetic (fixed time) determination of catalase activity in the range of 4-40 U/mL (detection limit 1.2 U/mL). The method was applied to the determination of catalase-like activity in various plant materials (recov-ery ranged from 93 to 101%, detection limit 480 U/100 g).     

Protective effect of seminal plasma proteins on the degradation of ascorbic acid

The objectives of this study were to determine ascorbic acid stability and its effect on antiproteinase activity of seminal plasma in the presence of an oxidant. Effect of seminal plasma, and additives: glutathione, albumin, hydrogen peroxide and Tris buffer, on ascorbic acid degradation was investigated by UV absorbance. Antiproteinase against trypsin amidase activity was measured spectrophotometrically using N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as substrate. Ascorbic acid was destroyed much more rapidly with the addition of hydrogen peroxide than in Tris buffer at pH 8.2 alone. Seminal plasma protected ascorbic acid more efficiently than glutathione and albumin alone. The protective effect of seminal plasma on ascorbic acid degradation may closely relate to the function of ascorbic acid in reproductive system of scurvy-prone animals including teleost fish. Within the range of 1–8 mM concentrations, ascorbic acid had a pro-oxidant action on seminal plasma antiproteinase activityin vitro when they were incubated with hydrogen peroxide.Abbreviations AA Ascorbic acid - BAPNA N-benzoyl-DL-arginine-p-nitroanilide - DMSO dimethyl sulfoxide - GSH glutathione - H₂O₂ hydrogen peroxide     

Inactivation of gerbil-cultured Giardia lamblia cysts by free chlorine.

Giardia lamblia cysts were harvested from Mongolian gerbils and exposed to free chlorine in buffered water at pH 5, 7, and 9 at 15 degrees C. The contact times required to obtain a 2-log reduction in cyst survival (i.e., a 99% kill) were interpolated from survival curves generated at fixed concentrations of chlorine in the range of 0.25 to about 16 mg/liter. Concentration-time (C.t') products for 99% inactivation ranged from about 120 to nearly 1,500 mg.min/liter. These values are higher than those reported previously for free chlorine using G. lamblia cysts from infected humans. The cysts isolated from gerbils, as with other Giardia cysts, were unusually sensitive to chlorine in alkaline solutions.     

The effect of hydrogen peroxide on spores of Clostridium bifermentans.

The effect of hydrogen peroxide on the germination, colony formation and structure of spores of Clostridium bifermentans was examined. Treatment with 0.35 M-hydrogen peroxide increased the germination rate at 25 degrees C but increasing the temperature or concentration of hydrogen peroxide decreased both the germination rate and colony formation. The presence of Cu2+ increased the lethal effect of hydrogen peroxide on colony formation as much as 3000-fold. Pre-incubation of spores with Cu2+ before treatment with hydrogen peroxide produced a similar increase, but this could be eliminated by washing the spores with dilute spores--apparently from the coat--and treatment with dithiothreitol, which also removes spore-coat protein, increased the lethal effect of hydrogen peroxide 500-fold, suggesting that spore-coat protein has a protective effect against hydrogen peroxide.