The generation of hydroxyl radicals following superoxide production by neutrophil NADPH oxidase

Stimulated neutrophils generate superoxide and hydroxyl radicals. A membrane-bound NADPH oxidase, inactive in the resting state, is responsible for superoxide production. The production of hydroxyl radicals is through a secondary reaction. A Fenton-catalysed Haber—Weiss reaction is proposed. Transferrin was used as the catalyst in this investigation.     

Manganese complexes and the generation and scavenging of hydroxyl free radicals

In a wide variety of biological systems non-enzyme complexes of the metals copper (Cu) and iron (Fe) have been shown to enhance oxygen radical damage by increasing the production of an oxidative species generally believed to be the hydroxyl free radical (.OH) via "Fenton" and possibly "Haber-Weiss" type reactions. However, the behavior of the chemically and biologically similar transition metal manganese (Mn) with .OH is unknown. Unlike Fe and Cu, inorganic complexes of Mn are known to exist in high concentrations in certain cells. Three different oxygen free radical generating systems and four .OH detection methods were used to investigate the activity of biologically relevant inorganic Mn complexes. These complexes were compared to compounds reported to scavenge and generate .OH. The direct and indirect effects of Mn on the .OH flux were compared by attempting to distinguish the effects of hydrogen peroxide (H2O2), superoxide (O2-), and .OH through the use of selective scavengers and generators. Mn-EDTA and biologically relevant Mn-pyrophosphates and polyphosphates, in contrast to Fe-EDTA, do not generate .OH in these systems. The results suggest that Mn in various forms does, indeed, inhibit oxy-radical damage mediated by .OH, but only if the .OH production is dependent on the presence of O2- or H2O2. Thus, with .OH, as with O2- and H2O2, Mn complexes appear to behave in a fundamentally different fashion from Cu and Fe.     

Electron paramagnetic resonance highlights that the oxygen effect contributes to the radiosensitizing effect of paclitaxel

Background

Paclitaxel (PTX) is a potent anti-cancer chemotherapeutic agent and is widely used in the treatments of solid tumors, particularly of the breast and ovaries. An effective and safe micellar formulation of PTX was used to administer higher doses of PTX than Taxol® (the current commercialized drug). We hypothesize that PTX-loaded micelles (M-PTX) may enhance tumor radiosensitivity by increasing the tumor oxygenation (pO₂). Our goals were (i) to evaluate the contribution of the “oxygen effect” to the radiosensitizing effect of PTX; (ii) to demonstrate the therapeutic relevance of the combination of M-PTX and irradiation and (iii) to investigate the underlying mechanisms of the observed oxygen effect.

Methodology and Principal Findings

We used (PEG-p-(CL-co-TMC)) polymeric micelles to solubilize PTX. pO₂ was measured on TLT tumor-bearing mice treated with M-PTX (80 mg/kg) using electron paramagnetic resonance (EPR) oximetry. The regrowth delay following 10 Gy irradiation 24 h after M-PTX treatment was measured. The tumor perfusion was assessed by the patent blue staining. The oxygen consumption rate and the apoptosis were evaluated by EPR oximetry and the TUNEL assay, respectively. EPR oximetry experiments showed that M-PTX dramatically increases the pO₂ 24 h post treatment. Regrowth delay assays demonstrated a synergy between M-PTX and irradiation. M-PTX increased the tumor blood flow while cells treated with M-PTX consumed less oxygen and presented more apoptosis.

Conclusions

M-PTX improved the tumor oxygenation which leads to synergy between this treatment and irradiation. This increased pO₂ can be explained both by an increased blood flow and an inhibition of O₂ consumption.     

The effect of pH on the conversion of superoxide to hydroxyl free radicals

The conversion of superoxide (O-.2) to the hydroxyl (HO.) free radical by superoxide-driven Fenton reactions was measured by the formation of hydroxylated derivatives from benzoate. Among a range of catalysts required for the conversion, the Fe3+EDTA complex was the most effective. The effect of superoxide dismutase and catalase indicated that O-.2 and H2O2 were essential reactants, while the formation of authentic HO. was confirmed by the inhibiting capacities of formate, t-butanol, and mannitol. The conversion of O-.2 to HO. was tested over a broad pH range, and was found to be highest at pH 4.8 whether Fe3+EDTA or free Fe3+ were used as the catalysts. When Fe3+EDTA was used at the optimum pH, every HO. produced required 3.7 O-.2 radicals, close to the theoretical limit of one HO. from every three O-.2 radicals generated.     

An electron paramagnetic resonance study of free radicals in cells

An electron paramagnetic resonance study was performed on cell lines of the following strains: HeLa, 37RC, L, FLC, NRK/RSV, 3T3/SV40.Unsynchronized and synchronized HeLa cells were studied with particular attention paid to the relation between growth and free radical concentration. Free radical levels were shown to be a function of the growth stage and different phases of the cell cycle.     

Electron spin resonance evidence for the formation of free radicals in plants exposed to ozone

Electron spin resonance (ESR) spectroscopy was used to demonstrate that free radicals are formed in O₃-fumigated plant leaves prior to the formation of visible leaf injury. ESR signals with a g-value of 2.0037 to 2.0043, were observed in pea ( Pisum sativum L. cv. Feltham first) and bean ( Phaseolus vulgaris L. cv. Pinto) plants that had been fumigated for 4 h with 70–300 nl l⁻¹ of ozone after they had been treated with the spin-trap N- t -butyl-α-phenylnitrone (PBN). The size of the ESR signals increased with the concentration of ozone used but the nature of the trapped radicals could not be identified. However, further experiments using an inhibitor of ethylene biosynthesis, arninoethoxyvinyl glycine (AVG), showed that the reaction between ozone and ethylene is the cause for ozone toxicity.     

Oxygen toxicity in Streptococcus sanguis. The relative importance of superoxide and hydroxyl radicals

Streptococcus sanguis, whose growth appears to be independent of the availability of iron, makes no hemes, contains neither catalase nor peroxidase, and can accumulate millimolar concentration levels of H2O2 during aerobic growth. It possesses a single manganese-containing superoxide dismutase whose concentration can be varied over a 50-100-fold range by manipulating the availability of oxygen during growth. Cell extracts contain a soluble NADH-plumbagin diaphorase which mediates O2- production in vitro and presumably also in vivo. Plumbagin increased oxygen consumption by S. sanguis and imposed an oxygen-dependent toxicity. Cells grown aerobically and containing elevated levels of superoxide dismutase were resistant to this toxicity. Dimethyl sulfoxide, which was shown to permeate S. sanguis freely, was used as an indicating scavenger of OH. An in vitro enzymic source of O2- plus H2O2 generated formaldehyde from dimethyl sulfoxide, an indication of OH. production. Either superoxide dismutase or catalase inhibited this OH. production and iron salts augmented it. Intact, aerobic cells of S. sanguis also gave evidence of OH. production, in the presence of plumbagin, but all of it appeared to be generated outside the cells. In addition, 0.5 M dimethyl sulfoxide did not diminish the oxygen-dependent toxicity of plumbagin. We conclude that, in S. sanguis, O2- can exert a toxic effect independent of the production of OH..     

An electron paramagnetic resonance study of free radicals in cells.

An electron paramagnetic resonance study was performed on cell lines of the following strains: HeLa, 37RC, L, FLC, NRK/RSV, 3T3/SV40. Unsynchronized and synchronized HeLa cells were studied with particular attention paid to the relation between growth and free radical concentration. Free radical levels were shown to be a function of the growth stage and different phases of the cell cycle.     

Electron paramagnetic resonance study of the generation of reactive oxygen species catalysed by transition metals and quinoid redox cycling by inhalable ambient particulate matter

A range of epidemiological studies in the 1990s showed that exposure to ambient particulate matter (PM) is associated with adverse health effects in the respiratory system and increased morbidity and mortality rates. Oxidative stress has emerged as a pivotal mechanism that underlies the toxic pulmonary effects of PM. A key question from a variety of studies was whether the adverse health effects of PM are mediated by the carbonaceous particles of their reactive chemical compounds adsorbed into the particles. Experimental evidence showed that PM contains redox-active transition metals, redox cycling quinoids and polycyclic aromatic hydrocarbons (PAHs) which act synergistically to produce reactive oxygen species (ROS). Fine PM has the ability to penetrate deep into the respiratory tree where it overcomes the antioxidant defences in the fluid lining of the lungs by the oxidative action of ROS. From a previous study [Valavanidis A, Salika A, Theodoropoulou A. Generation of hydroxyl radicals by urban suspended particulate air matter. The role of iron ions. Atmospher Environ 2000; 34 : 2379-2386], we established that ferrous ions in PM play an important role in the generation of hydroxyl radicals in the presence of hydrogen peroxide (H2O2). In the present study, we investigated the synergistic effect of transition metals and persistent quinoid and semiquinone radicals for the generation of ROS without the presence of H2O2. We experimented with airborne particulate matter, such as TSPs (total suspended particulates), fresh automobile exhaust particles (diesel, DEP and gasoline, GEP) and fresh wood smoke soot. Using electron paramagnetic resonance (EPR), we examined the quantities of persistent free radicals, characteristic of a mixture of quinoid radicals with different structures and a carbonaceous core of carbon-centred radicals. We extracted, separated and analysed the quinoid compounds by EPR at alkaline solution (pH 9.5) and by TLC. Also, we studied the direct production of superoxide anion and the damaging hydroxyl radical in aqueous and in DMSO suspensions of PM without H2O2. From these results, it is suggested that the cytotoxic and carcinogenic potential of PM can be partly the result of redox cycling of persistent quinoid radicals, which generate large amounts of ROS. In the second phase, the water-soluble fraction of PM elicits DNA damage via reactive transition metal-dependent formation of hydroxyl radicals, implicating an important role for hydrogen peroxide. Together, these data indicate the importance of mechanisms involving redox cycling of quinones and Fenton-type reactions by transition metals in the generation of ROS. These results are supported by recent studies indicating cytotoxic effects, especially mitochondrial damage, by PM extracts and differential mechanisms of cell killing by redox cycling quinones.     

ESR evidence for superoxide, hydroxyl radicals and singlet oxygen produced from hydrogen peroxide and nickel(II) complex of glycylglycyl-L-histidine

ESR studies using spin traps, 5,5-dimethylpyrroline-N-oxide and alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone, revealed that hydroxyl radical adducts are produced by the decomposition of hydrogen peroxide in the presence of nickel(II) oligopeptides. Order of catalytic activities of nickel(II) oligopeptides used in the production of hydroxyl radical adducts was tetraglycine greater than pentaglycine greater than triglycine greater than GlyGly, GlyHis. Ni(II) GlyGlyHis plus hydrogen peroxide produced superoxide in addition to hydroxyl radical adduct. Trapping experiments with 2,2,6,6-tetramethyl-4-piperidone suggested that singlet oxygen was generated by the reaction of hydrogen peroxide with Ni(II) GlyGlyHis, but not in the case of tetraglycine, pentaglycine, triglycine, GlyGly or GlyHis.     

Investigation of heavy-ion-induced sucrose radicals by electron paramagnetic resonance

The production of sucrose radicals with heavy-ion irradiation was investigated by an EPR (electron paramagnetic resonance) spectroscopic method. We examined the correlation between the production of sucrose radicals and the ion species, as well as LET (linear energy transfer). The spectral pattern obtained was the same for various ion species, including helium, carbon, neon, argon and iron ions. Quantitative EPR analyses showed that the production of sucrose radicals depended on both the ion species and the LET for the same dose of 50 Gy. The spin yield obtained showed a logarithmic correlation with the LET. In addition, the EPR response had a linear relationship with dose in the dose range of 5-60 Gy. Thus the present EPR results show that sucrose can be used to monitor the ionizing particle based on the radical yield.     

Electron spin resonance study of free radicals generated from retinyl- and ionyl-derivatives

Free radicals generated from alpha- and beta-ionyl bromides gave well resolved ESR spectra, but retinyl bromide and chloride gave only broad signals. Delocalised radicals were also spectroscopically observed on hydrogen abstraction from alpha-ionane, alpha-ionyltrimethylsilylether and buten-3-ynyl-2,6,6-trimethyl-2-cyclohexene. Retinyl and beta-ionyl radicals, derived from the corresponding xanthates, were successfully spin trapped with nitrosodurene. The results suggested that the secondary sites C(7) and C(9) were the most reactive in the beta-ionyl radical and that the secondary sites C(7) and C(11) and probably the primary site C(15) were the most reactive in the retinyl radical.