The effects of oxaloacetate on hydrogen peroxide generation from ascorbate and epigallocatechin gallate in cell culture media: potential for altering cell metabolism.

Several phenolic compounds as well as ascorbate can oxidise in certain cell culture media (especially Dulbecco's modified Eagle's medium (DMEM)) to generate hydrogen peroxide. Addition of oxaloacetate decreased the levels of H(2)O(2) detected and the oxaloacetate was depleted. Oxaloacetate was approximately as effective as pyruvate in decreasing H(2)O(2) levels and more effective than α-ketoglutarate. Our data raise important issues to consider when interpreting the behaviour and metabolism of cells in culture (which are both altered by the oxidative stress of cell culture) and their apparent response to addition of autooxidisable compounds such as ascorbate and epigallocatechin gallate.

Oral zinc supplementation does not improve oxidative stress or vascular function in patients with type 2 diabetes with normal zinc levels.

OBJECTIVE: There is considerable controversy about what constitutes optimal zinc intakes in patients with type 2 diabetes mellitus. Several studies suggest that higher zinc intakes improve vascular function and decrease oxidative damage. We aimed to assess the effects of zinc supplementation using a range of reliable biomarkers of oxidative damage and vascular function in patients with type 2 diabetes. METHODS: Forty male type 2 diabetic patients were supplemented either with 240 mg/day of zinc as zinc gluconate (n=20) or with placebo (n=20) for 3 months. Blood and spot urine samples were taken at baseline, days 3 and 7, months 1, 2 and 3 during supplementation and 1 month after cessation. Serum zinc, reliable biomarkers of oxidative damage (F(2)-isoprostanes, neuroprostanes, cholesterol oxidation products, allantoin) as well as hydroxyeicosatetraenoic acid products and vascular-related indices (augmentation index, pulse wave velocity and aortic pressure) were measured. RESULTS: Despite significantly higher levels of serum zinc in the treatment group, markers of oxidative damage, levels of hydroxyeicosatetraenoic acid products and vascular indices were unchanged by zinc supplementation during the four-month study period. CONCLUSION: Improving the zinc status in patients with type 2 diabetes with normal zinc levels did not have any impact on oxidative damage and vascular function, and such supplementation may not be generally beneficial in these individuals.

Artefacts in cell culture: α-Ketoglutarate can scavenge hydrogen peroxide generated by ascorbate and epigallocatechin gallate in cell culture media.

Ascorbate and several phenolic compounds readily oxidise in cell culture media to generate hydrogen peroxide. However, addition of α-ketoglutarate, which is known to be released by several cell types, decreased the levels of H(2)O(2), and the α-ketoglutarate was depleted and converted to succinate. These observations could account for previous reports of the protective effects of α-ketoglutarate in promoting the growth of cells in culture, and may contribute to explaining some of the variability in the literature in reported rates of H(2)O(2) production from autoxidisable compounds in cell culture systems.

Instability of, and generation of hydrogen peroxide by, phenolic compounds in cell culture media.

Many papers in the literature have described complex effects of flavonoids and other polyphenols on cells in culture. In this paper we show that hydroxytyrosol, delphinidin chloride and rosmarinic acid are unstable in three commonly-used cell culture media (Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 (RPMI) and Minimal Essential Medium Eagle (MEM)) and undergo rapid oxidation to generate H2O2. This may have confounded some previous studies on the cellular effects of these compounds. By contrast, apigenin, curcumin, hesperetin, naringenin, resveratrol and tyrosol did not generate significant H2O2 levels in these media. Nevertheless, curcumin and, to a lesser extent, resveratrol (but not tyrosol) were also unstable in DMEM, so the absence of detectable H2O2 production by a compound in cell culture media should not be equated to stability of that compound. Compound instability and generation of H2O2 must be taken into account in interpreting effects of phenolic compounds on cells in culture.

Artefacts in cell culture: pyruvate as a scavenger of hydrogen peroxide generated by ascorbate or epigallocatechin gallate in cell culture media.

Ascorbate and several phenolic compounds readily oxidise in cell culture media to generate hydrogen peroxide. However, media containing pyruvate showed much less H(2)O(2) production, apparently because pyruvate can scavenge H(2)O(2) in the medium. Researchers must be aware that compounds under test can sometimes readily oxidise in cell culture media, that this might not be detected by measurement of H(2)O(2) if the media contain pyruvate, and that pyruvate can be substantially depleted in the media as a result.

Limited antioxidant effect after consumption of a single dose of tomato sauce by young males, despite a rise in plasma lycopene.

This study investigated the effect of a single dose of tomato sauce on healthy male volunteers in a randomized crossover study. Healthy male subjects (n = 10) were enrolled. Placebo (rice and olive oil) or tomato (tomato sauce, rice and olive oil) meals were provided to the volunteers. Blood and urine samples were taken before consumption of meal (0 h) and 2, 4, 6, 24 and 48 h after meal. Consumption of tomato sauce increased plasma lycopene level by 5-22%, with a maximum level at 24 h (p<0.01) after the meal. Levels of plasma F(2)-isoprostanes, hydroxyeicosatetraenoic acid products, allantoin and urinary 8-hydroxy-2'-deoxyguanosine did not change after either meal, but urinary F(2)-isoprostanes (p<0.05) significantly decreased at 48 h compared to 0 h after the tomato sauce meal. This study showed that a single dose of tomato sauce meal had only a limited antioxidant effect in vivo.

Different patterns of oxidized lipid products in plasma and urine of dengue fever, stroke, and Parkinson's disease patients: cautions in the use of biomarkers of oxidative stress.

Many products of lipid oxidation have been associated with human diseases. These include F2-isoprostanes (F2-IsoPs), hydroxyeicosatetraenoic acid products (HETEs), and cholesterol oxidation products (COPs). Here we present measurements of F2-IsoPs, HETEs, COPs, and arachidonate in single plasma samples of patients with acute (dengue fever and ischemic stroke) and chronic (Parkinson's) diseases, and in age-matched study controls. Urine samples were collected for F2-IsoPs analysis. Our analysis demonstrated elevated F2-IsoPs levels in ischemic stroke, HETEs in Parkinson's disease, dengue fever, and ischemic stroke, and COPs in Parkinson's disease and dengue fever patients, as compared with those in age-matched study controls. Strong but complex correlations were observed between levels of certain oxidized lipid products and age. The relations between various oxidized lipids and dengue fever, stroke, and Parkinson's disease are discussed in relation to the selection and application of biomarkers of oxidative lipid damage, in particular the need for corrections for age and lipid levels.

Different cytotoxic and clastogenic effects of epigallocatechin gallate in various cell-culture media due to variable rates of its oxidation in the culture medium.

Positive genotoxicity results are often observed using mammalian cells in culture with agents that are not in vivo genotoxins. We here illustrate one possible explanation: interaction of test chemicals with the cell-culture media used. We find that the toxicity and clastogenicity of epigallocatechin gallate (EGCG) to Chinese Hamster ovary (CHO) cells is affected by the culture medium used and appears largely or entirely due to variable rates of formation of hydrogen peroxide (H(2)O(2)) by chemical reactions of EGCG with the culture media. Catalase decreased EGCG toxicity substantially. Of seven different types of commonly used media evaluated, F-10 and F-12 nutrient mixtures were the least prone to produce this artefact. Although it generated H(2)O(2) in the culture media, ascorbate was not toxic to CHO cells because the H(2)O(2) levels achieved were insufficient to kill these cells. Thus, the culture medium, the cell type and the presence or absence of catalase (e.g. its variable amounts in S9 fractions) must be taken into account in in vitro genotoxicity testing.

Cautions in the use of biomarkers of oxidative damage; the vascular and antioxidant effects of dark soy sauce in humans.

Dark soy sauce (DSS) is a powerful antioxidant in vitro. We investigated whether this effect could occur in vivo and improve vascular function. Healthy human subjects were given DSS or placebo meals in a randomized, crossover study. Blood and urine were sampled before and 1, 2, 3, and 4h after the meal for F(2)-isoprostanes (total, free, and esterified) and 8OHdG measurements. Blood pressure, vascular augmentation index (AIx), and heart rate (HR) were also measured. Plasma total F(2)-isoprostanes significantly decreased 3h after placebo and the decrease was greater for DSS. Plasma free and esterified F(2)-isoprostanes were also significantly decreased after DSS. Both placebo and DSS meals increased urinary F(2)-isoprostanes at 1h but not thereafter, and lowered urinary 8OHdG levels, DBP and AIx, and increased HR. We conclude that DSS decreases lipid peroxidation in vivo. However, oxidative damage biomarkers changed after the placebo meal, a phenomenon to consider when designing interventional studies.

Establishing biomarkers of oxidative stress: the measurement of hydrogen peroxide in human urine.

Hydrogen peroxide (H(2)O(2)) can be detected in freshly-voided human urine from healthy subjects and has been proposed as a "biomarker" of oxidative stress. This paper summarizes our studies to examine the extent to which urinary H(2)O(2) measurement fulfils the criteria for the "ideal biomarker". Levels of H(2)O(2), standardised for creatinine, varied widely between subjects. In most subjects, levels also varied considerably when measurements were made at different times and on different days. A reproducible increase in urinary H(2)O(2) was detected in all subjects examined after drinking coffee, a beverage rich in H(2)O(2). By contrast, green tea decreased urinary H(2)O(2) levels. We conclude that the H(2)O(2) in coffee is not excreted into urine. Instead, hydroxyhydroquinone from coffee is absorbed, excreted and oxidises in urine to produce H(2)O(2). No other confounders of urinary H(2)O(2) have been identified to date. Work is underway to compare H(2)O(2) levels with variations in other biomarkers of oxidative damage, to test the possibility that there are daily or other periodic variations in oxidative damage rates.

Contribution of hydrogen peroxide to the cytotoxicity of green tea and red wines.

Green tea and red wine are claimed to have health benefits because of their high content of polyphenolic compounds, but they have also been reported as mutagenic in some test systems. In this paper, we show that a commonly used cell culture medium, Dulbecco's modified Eagle's medium (DMEM), catalyses oxidation of green tea and red wines to generate H(2)O(2). The level of H(2)O(2) produced from green tea accounted for all of the cytotoxic effects of this beverage on PCl2 cells. By contrast, H(2)O(2) was only responsible for part of the cytotoxicity of the red wines examined. Our data illustrate the danger of extrapolating from cell culture studies to predict the effects of complex beverages in vivo.

Factors affecting the ascorbate- and phenolic-dependent generation of hydrogen peroxide in Dulbecco's Modified Eagles Medium.

Ascorbate and several polyphenolic compounds have been reported to undergo oxidation in cell culture media to generate hydrogen peroxide (H2O2), but the mechanism underlying this has not been established. We therefore investigated the parameters affecting H2O2 production. H2O2 generation from ascorbate, gallic acid and other phenolic compounds in Dulbecco's Modified Eagles' Medium (DMEM) at 37 degrees C under 95% air - 5% CO2 was not significantly inhibited by high (5-10 mM) concentration of EGTA, o-phenanthroline or desferriox-amine, but partial inhibition by EDTA and diethylene-triaminepentaacetic acid (DTPA) was observed. Incubation of DMEM alone at 37 degrees C led to an upward drift of pH, even under an atmosphere of 95% air - 5% CO2. Prevention of this pH rise by increasing the concentration of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes) buffer lowered the levels of H2O2 generated by ascorbate and phenolic compounds, but there was still substantial H2O2 generated at pH 7.4. Mixtures of ascorbate and phenolic compounds led to less H2O2 generation than would be expected from the rates observed with ascorbate or phenolic compounds alone. Ascorbate prevented the loss of gallic acid incubated in DMEM. The role of metal ions and other constituents of the culture medium in promoting H2O2 generation is discussed.

The cytotoxicity of dopamine may be an artefact of cell culture.

Administration of L-DOPA is commonly used to treat Parkinson's disease, yet controversy continues as to whether the dopamine arising from it aggravates neuronal loss. Several authors have reported cytotoxic effects of L-DOPA and dopamine on cultured cells, but others have not. In this report using the rat pheochromocytoma cell line PC12 and the M14 human melanoma cell line we show that dopamine-mediated cell death is not specific for neuronal cells. Moreover, our data show that both L-DOPA and dopamine interact with commonly used cell culture media, undergoing oxidation to generate hydrogen peroxide and dopamine semiquinones/quinones. Catalase and reduced glutathione could protect against cytotoxicity. These results suggest that caution needs to be employed when using cell culture studies to predict effects of L-DOPA and/or dopamine in vivo because of the extracellular generation of reactive species in the culture media.