The rate of cellular hydrogen peroxide removal shows dependency on GSH: mathematical insight into in vivo H2O2 and GPx concentrations.

Although its concentration is generally not known, glutathione peroxidase-1 (GPx-1) is a key enzyme in the removal of hydrogen peroxide (H2O2) in biological systems. Extrapolating from kinetic results obtained in vitro using dilute, homogenous buffered solutions, it is generally accepted that the rate of elimination of H2O2 in vivo by GPx is independent of glutathione concentration (GSH). To examine this doctrine, a mathematical analysis of a kinetic model for the removal of H2O2 by GPx was undertaken to determine how the reaction species (H2O2, GSH, and GPx-1) influence the rate of removal of H2O2. Using both the traditional kinetic rate law approximation (classical model) and the generalized kinetic expression, the results show that the rate of removal of H2O2 increases with initial GPx(r), as expected, but is a function of both GPx(r) and GSH when the initial GPx(r) is less than H2O2. This simulation is supported by the biological observations of Li et al. Using genetically altered human glioma cells in in vitro cell culture and in an in vivo tumour model, they inferred that the rate of removal of H2O2 was a direct function of GPx activity x GSH (effective GPx activity). The predicted cellular average GPx(r) and H2O2 for their study are approximately GPx(r) < or =1 microm and H2O2 approximately 5 microm based on available rate constants and an estimation of GSH. It was also found that results from the accepted kinetic rate law approximation significantly deviated from those obtained from the more generalized model in many cases that may be of physiological importance.

Manganese superoxide dismutase suppresses hypoxic induction of hypoxia-inducible factor-1alpha and vascular endothelial growth factor.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that governs cellular responses to reduced O2 availability by mediating crucial homeostatic processes. HIF-1 is composed of an HIF-1alpha subunit and an HIF-1beta subunit. HIF-1alpha is degraded following enzyme-dependent hydroxylation of prolines of HIF-1alpha in the presence of molecular oxygen, Fe2+, alpha-ketoglutarate, and ascorbate. These cofactors contribute to the redox environment of cells. The antioxidant enzyme manganese superoxide dismutase (MnSOD) also modulates the cellular redox environment. Here we show that MnSOD suppressed hypoxic accumulation of HIF-1alpha protein in human breast carcinoma MCF-7 cells. This suppression was biphasic depending on MnSOD activity. At low levels of MnSOD activity, HIF-1alpha protein accumulated under hypoxic conditions. At moderate levels of MnSOD activity (two- to six-fold increase compared to parent cells), these accumulations were blocked. However, at higher levels of MnSOD activity (>6-fold increase), accumulation of HIF-1alpha protein was again observed. This biphasic modulation was observed under both 1 and 4% O2. Coexpression of mitochondrial hydrogen peroxide-removing proteins prevented the accumulation of HIF-1alpha protein in cells with high levels of MnSOD; this effect demonstrates that the restabilization of HIF-1alpha observed in high MnSOD overexpressors is probably due to hydrogen peroxide, most likely produced from MnSOD. Hypoxic induction of vascular endothelial growth factor (VEGF) protein was also suppressed by elevated MnSOD activity and its levels reflected HIF-1alpha protein levels. These observations demonstrated that HIF-1alpha accumulation and VEGF expression could be modulated by the antioxidant enzyme MnSOD.

Nitric oxide as a cellular antioxidant: a little goes a long way.

Nitric oxide (NO*) is an effective chain-breaking antioxidant in free radical-mediated lipid oxidation (LPO). It reacts rapidly with peroxyl radicals as a sacrificial chain-terminating antioxidant. The goal of this work was to determine the minimum threshold concentration of NO* required to inhibit Fe2+ -induced cellular lipid peroxidation. Using oxygen consumption as a measure of LPO, we simultaneously measured nitric oxide and oxygen concentrations with NO* and O2 electrodes. Ferrous iron and dioxygen were used to initiate LPO in docosahexaenoic acid-enriched HL-60 and U937 cells. Bolus addition of NO* (1.5 microM) inhibited LPO when the NO* concentration was greater than 50 nM. Similarly, using (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate as a NO* donor we found that an average steady-state NO* concentration of at least 72 +/- 9 nM was required to blunt LPO. As long as the concentration of NO* was above 13 +/- 8 nM the inhibition was sustained. Once the concentration of NO* fell below this value, the rate of lipid oxidation accelerated as measured by the rate of oxygen consumption. Our model suggests that a continuous production of NO* that would yield a steady-state concentration of only 10-20 nM is capable of inhibiting Fe2+ -induced LPO.

Ascorbate enhances the toxicity of the photodynamic action of Verteporfin in HL-60 cells.

As a reducing agent, ascorbate serves as an antioxidant. However, its reducing function can in some settings initiate an oxidation cascade, i.e., seem to be a "pro-oxidant." This dichotomy also seems to hold when ascorbate is present during photosensitization. Ascorbate can react with singlet oxygen, producing hydrogen peroxide. Thus, if ascorbate is present during photosensitization the formation of highly diffusible hydrogen peroxide could enhance the toxicity of the photodynamic action. On the other hand, ascorbate could decrease toxicity by converting highly reactive singlet oxygen to less reactive hydrogen peroxide, which can be removed via peroxide-removing systems such as glutathione and catalase. To test the influence of ascorbate on photodynamic treatment we incubated leukemia cells (HL-60 and U937) with ascorbate and a photosensitizer (Verteporfin; VP) and examined ascorbic acid monoanion uptake, levels of glutathione, changes in membrane permeability, cell growth, and toxicity. Accumulation of VP was similar in each cell line. Under our experimental conditions, HL-60 cells were found to accumulate less ascorbate and have lower levels of intracellular GSH compared to U937 cells. Without added ascorbate, HL-60 cells were more sensitive to VP and light treatment than U937 cells. When cells were exposed to VP and light, ascorbate acted as an antioxidant in U937 cells, whereas it was a pro-oxidant for HL-60 cells. One possible mechanism to explain these observations is that HL-60 cells express myeloperoxidase activity, whereas in U937 cells it is below the detection limit. Inhibition of myeloperoxidase activity with 4-aminobenzoic acid hydrazide (4-ABAH) had minimal influence on the phototoxicity of VP in HL-60 cells in the absence of ascorbate. However, 4-ABAH decreased the toxicity of ascorbate on HL-60 cells during VP photosensitization, but had no affect on ascorbate toxicity in U937 cells. These data demonstrate that ascorbate increases hydrogen peroxide production by VP and light. This hydrogen peroxide activates myeloperoxidase, producing toxic oxidants. These observations suggest that in some settings, ascorbate may enhance the toxicity of photodynamic action.

A new paradigm: manganese superoxide dismutase influences the production of H2O2 in cells and thereby their biological state.

The principal source of hydrogen peroxide in mitochondria is thought to be from the dismutation of superoxide via the enzyme manganese superoxide dismutase (MnSOD). However, the nature of the effect of SOD on the cellular production of H(2)O(2) is not widely appreciated. The current paradigm is that the presence of SOD results in a lower level of H(2)O(2) because it would prevent the non-enzymatic reactions of superoxide that form H(2)O(2). The goal of this work was to: a) demonstrate that SOD can increase the flux of H(2)O(2), and b) use kinetic modelling to determine what kinetic and thermodynamic conditions result in SOD increasing the flux of H(2)O(2). We examined two biological sources of superoxide production (xanthine oxidase and coenzyme Q semiquinone, CoQ(*-) that have different thermodynamic and kinetic properties. We found that SOD could change the rate of formation of H(2)O(2) in cases where equilibrium-specific reactions form superoxide with an equilibrium constant (K) less than 1. An example is the formation of superoxide in the electron transport chain (ETC) of the mitochondria by the reaction of ubisemiquinone radical with dioxygen. We measured the rate of release of H(2)O(2) into culture medium from cells with differing levels of MnSOD. We found that the higher the level of SOD, the greater the rate of accumulation of H(2)O(2). Results with kinetic modelling were consistent with this observation; the steady-state level of H(2)O(2) increases if K<1, for example CoQ(*-)+O(2)-->CoQ+O(2)(*-). However, when K>1, e.g. xanthine oxidase forming O(2)(*-), SOD does not affect the steady state-level of H(2)O(2). Thus, the current paradigm that SOD will lower the flux of H(2)O(2) does not hold for the ETC. These observations indicate that MnSOD contributes to the flux of H(2)O(2) in cells and thereby is involved in establishing the cellular redox environment and thus the biological state of the cell.

Detection of lipid radicals using EPR.

Cells oxidize molecules to generate energy and to make the materials to build and support the structures and functions needed for life. However, unwanted oxidations can damage these same structures and impair function. Lipids (the lipids in membranes and lipoproteins) are targets of unwanted oxidations. The primary mechanism of these oxidations is free radical-mediated chain reactions. Here we provide an overview of how electron paramagnetic resonance (EPR) can be used to detect the free radicals formed during lipid peroxidation. Although direct detection of lipid-derived radicals has been accomplished, the approach is not feasible for detecting these radicals in cells. Spin trapping with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 5,5-dimethyl-pyrroline-1-oxide has provided the most information on cellular lipid peroxidation. We present some considerations for successful detection of lipid radicals by EPR.

Phospholipid hydroperoxide glutathione peroxidase induces a delay in G1 of the cell cycle.

Phospholipid hydroperoxide glutathione peroxidase (PhGPx) is an antioxidant enzyme that reduces cellular phospholipid hydroperoxides (PLOOHs) to alcohols. Cellular peroxide tone has been implicated in cell growth and differentiation. By reducing the PLOOH level in the cell membrane, PhGPx regulates the peroxide tone and thereby might be involved in cell growth. We hypothesized that overexpression of PhGPx in human breast cancer cells would decrease their growth rate. We stably transfected MCF-7 cells (Wt) with L-PhGPx and measured cell doubling time, plating efficiency, and cell cycle phase transit times. P-4 cells (8-fold increase in PhGPx activity) showed a 2-fold increase in doubling time; doubling time increased directly with PhGPx activity (r = 0.95). The higher the PhGPx activity, the lower the plating efficiency (r = -0.86). The profile of other antioxidant enzymes was unchanged. Overexpression of PhGPx lowered the steady-state level of PLOOH (by > 60%). Results from bromodeoxyuridine pulse-chase experiments and flow cytometry indicate that PhGPx induced a delay in MCF-7 proliferation that was primarily due to a slower progression from G1 to S. These results support the hypothesis that PhGPx plays a regulatory role in the progression of MCF-7 cells from G1 to S possibly by regulating the steady-state levels of PLOOH. These data suggest that PhGPx can lower the peroxide tone, which might change the cellular redox environment resulting in a delay in G1 transit. Thus, PhGPx could be an important factor in cell growth.

Overexpression of manganese superoxide dismutase promotes the survival of prostate cancer cells exposed to hyperthermia.

It has been hypothesized that exposure of cells to hyperthermia results in an increased flux of reactive oxygen species (ROS), primarily superoxide anion radicals, and that increasing antioxidant enzyme levels will result in protection of cells from the toxicity of these ROS. In this study, the prostate cancer cell line, PC-3, and its manganese superoxide dismutase (MnSOD)-overexpressing clones were subjected to hyperthermia (43 degrees C, 1 h). Increased expression of MnSOD increased the mitochondrial membrane potential (MMP). Hyperthermic exposure of PC-3 cells resulted in increased ROS production, as determined by aconitase inactivation, lipid peroxidation, and H2O2 formation with a reduction in cell survival. In contrast, PC-3 cells overexpressing MnSOD had less ROS production, less lipid peroxidation, and greater cell survival compared to PC-3 Wt cells. Since MnSOD removes superoxide, these results suggest that superoxide free radical or its reaction products are responsible for part of the cytotoxicity associated with hyperthermia and that MnSOD can reduce cellular injury and thereby enhance heat tolerance.

A simple and sensitive assay for ascorbate using a plate reader.

We have developed a rapid, inexpensive, and reliable assay for the determination of ascorbate using a plate reader. In this assay, ascorbic acid is oxidized to dehydroascorbic acid using Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy) and then reacted with o-phenylenediamine to form the condensation product, 3-(dihydroxyethyl)furo[3,4-b]quinoxaline-1-one. The rate of appearance of this product is monitored over time using fluorescence. With this method, it is possible to analyze 96 wells in less than 10min. This permits the analysis of 20 samples with a full set of standards and blanks, all in triplicate. The assay is robust for a variety of samples, including orange juice, swine plasma, dog plasma, and cultured cells. To demonstrate the usefulness of the assay for the rapid determination of experimental parameters, we investigated the uptake of ascorbate and two different ascorbate derivatives in U937 cells. We found similar plateau levels of intracellular ascorbate at 24h for ascorbate and ascorbate phosphate. However, the intracellular accumulation of ascorbate via the phosphate ester had an initial rate that was three to five times slower than that via the palmitate ester. Only lower concentrations of the palmitate ester could be examined because the ethanol needed as solvent decreased cell viability; it behaved similarly to the other two compounds at lower concentrations. To come to these conclusions, only nine plates needed to be analyzed to provide us with the end result after only 7h of analysis. This clearly demonstrates the strength of the plate reader assay, which allows the analysis of large-sample sets in a fraction of the time required for the methods that are most commonly used today. The assay is quick, is very economical, and provides results with uncertainties on the order of only 5%.

Comparing beta-carotene, vitamin E and nitric oxide as membrane antioxidants.

Singlet oxygen initiates lipid peroxidation via a nonfree radical mechanism by reacting directly with unsaturated lipids to form lipid hydroperoxides (LOOHs). These LOOHs can initiate free radical chain reactions leading to membrane leakage and cell death. Here we compare the ability and mechanism by which three small-molecule membrane antioxidants (beta-carotene, alpha-tocopherol and nitric oxide) inhibit lipid peroxidation in membranes. We demonstrate that beta-carotene provides protection against singlet oxygen-mediated lipid peroxidation, but does not slow free radical-mediated lipid peroxidation. Alpha-Tocopherol does not protect cells from singlet oxygen, but does inhibit free radical formation in cell membranes. Nitric oxide provides no direct protection against singlet oxygen exposure, but is an exceptional chain-breaking antioxidant as evident from its ability to blunt oxygen consumption during free radical-mediated lipid peroxidation. These three small-molecule antioxidants appear to have complementary mechanisms for the protection of cell membranes from detrimental oxidations.