Biomedical subjects

J E Biaglow

Publications and source records attributed to J E Biaglow.

At least 19 recordsLinked to original sources

PubMed2001

Redox cycling by motexafin gadolinium enhances cellular response to ionizing radiation by forming reactive oxygen species.

PURPOSE: To examine the mechanism of radiation enhancement by motexafin gadolinium (Gd-Tex) in vitro. METHODS AND MATERIALS: Oxidation of ascorbate and NADPH by Gd-Tex was evaluated in a neutral buffer. Growth inhibition of human uterine cancer cell line MES-SA was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye. Clonogenic assays were used to measure radiation response in MES-SA, A549 human lung carcinoma, E89, a CHO cell line variant deficient in glucose-6-phosphate dehydrogenase activity, and murine lymphoma cell lines LYAR and LYAS. RESULTS: Gd-Tex catalyzed the oxidation of NADPH and ascorbate under aerobic conditions, forming hydrogen peroxide. Decreased viability was observed in MES-SA cells incubated with Gd-Tex in media containing NADPH or ascorbate. Gd-Tex and ascorbate increased fluorescence in dichlorofluorescin acetate-treated cultures. Synergistic effects on the aerobic radiation response in MES-SA and A549 were seen using Gd-Tex in combination with L-buthionine-(S,R)-sulfoximine (BSO). Incubation with Gd-Tex in the presence of ascorbate increased the aerobic radiation response of E89 and the apoptosis-sensitive B-cell line (LYAS). CONCLUSIONS: Gd-Tex sensitizes cells to ionizing radiation by increasing oxidative stress as a consequence of futile redox cycling. Optimization of the concentration of ascorbate (or other reducing species) may be required when evaluating Gd-Tex activity in vitro.

Animals↗

PubMed2001

Absence of Crabtree effect in human melanoma cells adapted to growth at low pH: reversal by respiratory inhibitors.

Because many tumors are acidic and hypoxic relative to normal tissues, glycolysis and oxygen consumption were investigated in early-passage human melanoma cells adapted to growth at pH 6.7. In the absence of glucose, the basal rate of oxygen consumption in low pH-adapted cells was 75% of that in cells grown at pH 7.3. The rate of lactic acid production in low pH-adapted cells was increased 4-fold by exposure to 16.7 mM glucose compared with a 10-fold increase in cells grown at pH 7.3. Furthermore, in low pH-adapted cells the rate of oxygen consumption was stimulated by the addition of glucose in contrast to the inhibition of oxygen consumption by elevated glucose in cells grown at pH 7.3 (i.e., the Crabtree effect). Both low pH-adapted cells and cells grown at pH 7.3 exposed to glucose plus 0.35 mM meta-iodo-benzylguanidine (MIBG), an inhibitor of mitochondrial respiration, had oxygen consumption reduced by approximately 60% and lactic acid production increased by approximately 65% relative to glucose alone. Although adaptation to growth at low pH was associated with a loss of the Crabtree effect and a higher ratio of oxygen consumption to lactic acid production, the rate of glycolysis was the same in both growth conditions in the presence of 0.1 mM dinitrophenol, an uncoupler of ATP synthesis. This indicates that the glycolytic capacity of low pH-adapted cells remains unchanged. Therefore, tumor acute acidification and oxygenation can be achieved by exposure to hyperglycemia combined with MIBG to improve therapeutic response.

3-Iodobenzylguanidine↗

PubMed2000

G6PD deficient cells and the bioreduction of disulfides: effects of DHEA, GSH depletion and phenylarsine oxide.

We used Glucose 6 phosphate dehydrogenase (G6PD) minus cells (89 cells) and G6PD containing cells (K1) to understand the mechanisms of bioreduction of disulfide and the redox regulation of protein and non protein thiols in mammalian cells. The 89 cells reduce hydroxyethyldisulfide (HEDS) to mercaptoethanol (ME) at a slower rate than K1 cells. HEDS reduction results in loss of nonprotein thiols (NPSH) and a decrease in protein thiols (PSH) in 89 cells. The effects are less dramatic with K1 cells. However, the loss of NPSH and PSH in K1 cells are increased in the absence of glucose. Glutathione-depletion with L-BSO partially blocks HEDS reduction in K1 and 89 cells. Treatment with the vicinal thiol reagent phenyl arsenic oxide (PAO) blocks reduction of HEDS in both cells. Surprisingly, dehydroepiandrosterone (DHEA), a known inhibitor of G6PD, inhibits the growth and blocks the reduction of HEDS both in 89 and K1 cells suggesting that its mechanism for inhibition of growth is not G6PD related.

Animals↗

PubMed2000

A method for measuring disulfide reduction by cultured mammalian cells: relative contributions of glutathione-dependent and glutathione-independent mechanisms.

A method is described for measuring bioreduction of hydroxyethyl disulfide (HEDS) or alpha-lipoate by human A549 lung, MCF7 mammary, and DU145 prostate carcinomas as well as rodent tumor cells in vitro. Reduction of HEDS or alpha-lipoate was measured by removing aliquots of the glucose-containing media and measuring the reduced thiol with DTNB (Ellman's reagent). Addition of DTNB to cells followed by disulfide addition directly measures the formation of newly reduced thiol. A549 cells exhibit the highest capacity to reduce alpha-lipoate, while Q7 rat hepatoma cells show the highest rate of HEDS reduction. Millimolar quantities of reduced thiol are produced for both substrates. Oxidized dithiothreitol and cystamine were reduced to a lesser degree. DTNB, glutathione disulfide, and cystine were only marginally reduced by the cell cultures. Glucose-6-phosphate deficient CHO cells (E89) do not reduce alpha-lipoate and reduce HEDS at a much slower rate compared to wild-type CHO-K1 cells. Depletion of glutathione prevents the reduction of HEDS. The depletion of glutathione inhibited reduction of alpha-lipoate by 25% and HEDS by 50% in A549 cells, while GSH depletion did not inhibit alpha-lipoate reduction in Q7 cells but completely blocked HEDS reduction. These data suggest that the relative participation of the thioltransferase (glutaredoxin) and thioredoxin systems in overall cellular disulfide reduction is cell line specific. The effects of various inhibitors of the thiol-disulfide oxidoreductase enzymes (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), arsenite, and phenylarsine oxide) support this conclusion.

Animals↗

PubMed2000

Apoptosis induced by dithiothreitol in HL-60 cells shows early activation of caspase 3 and is independent of mitochondria.

Previous studies have shown that under certain conditions some thiol-containing compounds can cause apoptosis in a number of different cell lines. Herein, we investigated the apoptotic pathways in HL-60 cells triggered by dithiothreitol (DTT), used as a model thiol compound, and tested the hypothesis that thiols cause apoptosis via production of hydrogen peroxide (H2O2) during thiol oxidation. The results show that, unlike H2O2, DTT does not induce apoptosis via a mitochondrial pathway. This is demonstrated by the absence of early cytochrome c release from mitochondria into the cytosol, the lack of mitochondrial membrane depolarization at early times, and the minor role of caspase 9 in DTT-induced apoptosis. The first caspase activity detectable in DTT-treated cells is caspase 3, which is increased significantly 1 - 2 h after the start of DTT treatment. This was shown by following the cleavage of both a natural substrate, DFF-45/ICAD, and a synthetic fluorescent substrate, z-DEVD-AFC. Cleavage of substrates of caspases 2 and 8, known as initiator caspases, does not start until 3 - 4 h after DTT exposure, well after caspase 3 has become active and at a time when apoptosis is in late stages, as shown by the occurrence of DNA fragmentation to oligonucleosomal-sized pieces. Although oxidizing DTT can produce H2O2, data presented here indicate that DTT-induced apoptosis is not mediated by production of H2O2 and occurs via a novel pathway that involves activation of caspase 3 at early stages, prior to activation of the common 'initiator' caspases 2, 8 and 9.

Amino Acid Chloromethyl Ketones↗

PubMed2000

Oxidation of cellular thiols by hydroxyethyldisulphide inhibits DNA double-strand-break rejoining in G6PD deficient mammalian cells.

PURPOSE: We investigated the effect of protein- and non protein-thiol oxidation on DNA double-strand-break (DSB) rejoining after irradiation and its relevance in the survival of CHO cells. MATERIALS AND METHODS: We used mutant cells null for glucose 6 phosphate dehydrogenase (G6PD) activity since reducing equivalents, required for reduction of oxidized thiols, are typically generated through G6PD regulated production of NADPH. Cellular thiols were oxidized by pre-incubating the cells with hydroxyethyldisulphide (HEDS), the oxidized form of mercaptoethanol (ME). The concentrations of the intracellular and extracellular non-protein thiols (NPSH), glutathione, cysteine and mercaptoethanol were quantitated by HPLC. Protein thiols (PSH) were estimated using Ellman's reagent. Cell survival was determined by clonogenic assay. The induction and rejoining of DSB in cells was quantitated by Pulse Field Gel Electrophoresis after exposure to ionizing radiation. RESULTS: Much lower bioreduction of HEDS was found in the G6PD deficient mutants (E89) than in the wild-type cells (K1). A 1 h treatment of E89 cells with HEDS produced almost complete depletion of non-protein thiol (NPSH) and a 26% decrease in protein thiols. Only minor changes were found under similar conditions with K1 cells. When exposed to gamma radiation in the presence of HEDS, the G6PD null mutants exhibited a higher cell killing and decreased rate and extent of rejoining of DSB than were observed in K1 cells. Moreover, when the G6PD deficient cells were transfected with the gene encoding wild-type G6PD (A1A), they recovered close to wild-type cellular thiol status, cell survival and DSB rejoining. CONCLUSIONS: These results suggest that a functioning oxidative pentose phosphate pathway is required for DSB rejoining in cells exposed to a mild thiol oxidant.

Animals↗

PubMed1999

Mechanism of copper-catalyzed autoxidation of cysteine.

The kinetics of copper-catalyzed autoxidation of cysteine and its derivatives were investigated using oxygen consumption, spectroscopy and hydroxyl radical detection by fluorescence of a coumarin probe. The process has complex two-phase kinetics. During the first phase a stoichiometric amount of oxygen (0.25 moles per mole of thiol) is consumed without production of hydroxyl radicals. In the second reaction phase excess oxygen is consumed in a hydrogen peroxide-mediated process with significant *OH production. The reaction rate in the second phase is decreased for cysteine derivatives with a free aminogroup and increased for compounds with a modified aminogroup. The kinetic data suggest the catalytic action of copper in the form of a cysteine complex. The reaction mechanism consists of two simultaneous reactions (superoxide-dependent and peroxide-dependent) in the first phase, and peroxide-dependent in the second phase. The second reaction phase begins after oxidation of free thiol. This consists of a Fenton-type reaction between cuprous-cysteinyl complex and following oxidation of cysteinyl radical to sulfonate with the consumption of excessive oxygen and significant production of hydroxyl radicals.

Catalase↗

PubMed1998

The measurement of bioreductive capacity of tumor cells using methylene blue.

PURPOSE: Methylene blue (MB) can be used as an intracellular electron acceptor. The purpose of this study was to demonstrate the usefulness of MB for the determination of total bioreductive capacity of cell suspensions. METHODS AND MATERIALS: We measured oxygen consumption by Clark electrode and pentose cycle activity by release of 14CO2 from 1-14C-glucose. RESULTS: Methylene blue catalyzes the reaction of intracellular reductants NADPH, NADH, and reduced glutathione (GSH) with oxygen, causing the production of hydrogen peroxide. The reaction rate correlates with the negative charge of molecule (NADPH(-4) > NADH(-2) > GSH(-1)), suggesting that reaction with positively charged oxidized MB is the limiting step of the reaction. In a cellular system MB causes the electron flow from cellular endogenous substrates to oxygen. It is activated by the disruption of the NADP+/NADPH ratio due to several processes. These are direct oxidation of NADPH and GSH, the GSH peroxidase catalyzed reaction of GSH with H2O2, followed by NADPH oxidation by oxidized glutathione (GSSG). This results in increased cellular oxygen consumption and stimulation of the oxidative limb of pentose cycle (PC) in the presence of MB. The cellular effect of MB differs from other electron accepting drugs. Diamide and tert-butylhydroperoxide act as direct oxidants, while MB is an electron carrier to oxygen. Accordingly, MB shows the highest effect on PC activation and oxygen consumption. CONCLUSIONS: Our results indicate that MB may be used for the determination of the total bioreductive capacity of the cells, measured by oxygen consumption and PC activation.

Carbon Dioxide↗

PubMed1998

MIBG inhibits respiration: potential for radio- and hyperthermic sensitization.

INTRODUCTION: Meta-iodobenzylguanidine (MIBG) in its 131I-labeled form is clinically used as a tumor-targeted radiopharmaceutical in the diagnosis and treatment of adrenergic tumors. This well established drug may have additional clinical applications as a radiosensitizer or hyperthermic agent, ie., MIBG reportedly inhibits mitochondrial respiration in vitro. The mechanism for MIBG inhibition of cellular oxygen consumption is uncertain. Moreover, MIBG reportedly stimulates glycolysis both in vitro and in vivo. Our studies show the effect of MIBG on 9L glioma oxygen consumption and redox status with tumors cells in vitro and in vivo. MATERIALS AND METHODS: The effects on electron transfer were determined by following oxygen consumption with a Clark oxygen electrode. Fluorescence measurements were used to determine effects of MIBG on intracellular electron acceptors, NADPH and flavoproteins, in vitro and in vivo. 31P-NMR was used to determine alterations in tumor cell pH in vivo. RESULTS: Our results show the inhibition of oxygen utilization with MIBG for cell suspensions in vitro. The same results were demonstrated for tumor cell suspensions rapidly isolated from tumors grown in rats. Moreover, NAD(P)H and flavoprotein (Fp) fluorescence changes were observed to rapidly occur following MIBG addition in vitro. Changes in intracellular pH measured with 31P-NMR, in vivo, precede the changes in fluorescence of NAD(P)H and Fp obtained with frozen sections of tumor. CONCLUSIONS: We conclude that 31P-NMR measurements and fluorescence changes, following MIBG injection, can be used as criterion for selecting the proper time to treat tumors with ionizing radiation or hyperthermia.

3-Iodobenzylguanidine↗

PubMed1998

Mechanism of copper-catalyzed oxidation of glutathione.

The mechanism of copper-catalyzed glutathione oxidation was investigated using oxygen consumption, thiol depletion, spectroscopy and hydroxyl radical detection. The mechanism of oxidation has kinetics which appear biphasic. During the first reaction phase a stoichiometric amount of oxygen is consumed (1 mole oxygen per 4 moles thiol) with minimal .OH production. In the second reaction phase, additional (excess) oxygen is consumed at an increased rate and with significant hydrogen peroxide and .OH production. The kinetic and spectroscopic data suggest that copper forms a catalytic complex with glutathione (1 mole copper per 2 moles glutathione). Our proposed reaction mechanism assumes two parallel processes (superoxide-dependent and peroxide-dependent) for the first reaction phase and superoxide-independent for the second phase. Our current results indicate that glutathione, usually considered as an antioxidant, can act as prooxidant at physiological conditions and therefore can participate in cellular radical damage.

Copper↗

PubMed1998

Autoxidation of ferrous ion complexes: a method for the generation of hydroxyl radicals.

The kinetics of the production of hydroxyl radicals during the autoxidation of ferrous ion complexes at pH 7.4 was investigated using the fluorescent probe coumarin-3-carboxylic acid. Polyphosphates (tri- and tetrapolyphosphate and their adenosine derivatives), citrate, and acetic derivatives of ethyleneamine ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), triethylenetetraminehexaacetic acid (TTHA), ethylenediamine-(N,N')-diacetic acid (EDDA) and nitrilotriacetic acid (NTA) were used as iron chelators. Production of hydroxyl radical in these chemical systems was compared with that by radiation to determine the equivalent doses of radiation that produced equal amounts of .OH. The amount of .OH formed during ferrous ion autoxidation is determined by the concentration of the complex, its structure and the radical scavenging by the chelator molecule. Production of .OH for homologous ethylenamine acetates increases with increased complex stability: NTA < EDDA < TTHA < EDTA < DTPA. The radiation dose equivalence for 0.1 mM complexes increased from 5 Gy for NTA to 25 Gy for DTPA. The radiation dose equivalence for polyphosphates was 15 Gy for tripolyphosphate and 32 Gy for tetrapolyphosphate. The dose equivalences for adenosine phosphates are lower, 5 Gy for ATP and 10 Gy for adenosine tetraphosphate, due to intramolecular .OH scavenging. The rate of generation of .OH shows an inverse correlation with the charge of the ferrous ion complex, varying from 2 cGy/s for DTPA to 1.2 Gy/s for EDTA. The data presented indicate the usefulness of autoxidation of ferrous ion complexes for generation of .OH in chemical systems. The ability to control the amount and the rate of production of .OH may prove useful for examining the cytotoxic effects of .OH generated in biological systems.

Ferrous Compounds↗

PubMed1997

Effect of oncogene transformation of rat embryo cells on cellular oxygen consumption and glycolysis.

We found an unique effect of oncogene transfections on rat embryo cell (REF) respiration, glycolysis and radiation response. Radioresistance, defined as an increase in Do, increases for REF cells transfected with v-myc or H-ras oncogenes. The combination of both oncogenes confers the maximal radioresistance. Our work shows inhibition of oxygen uptake when cells are transfected with v-myc or H-ras alone. However, oxygen uptake increases when cells are transfected simultaneously with v-myc + H-ras (3.7,2.1,2.8). A higher oxygen consumption results from increased utilization of pyruvate via the Kreb's cycle. Succinate stimulates cellular oxygen consumption. The maximum stimulation of oxygen consumption by succinate occurred with v-myc + H-ras transfected cells. The glycolysis of the transfected cells is also altered by the oncogenes. Our glycolytic measurements indicate the H-ras oncogene causes the largest stimulation of glycolysis. Our data shows that transfection with oncogenes has a major effect on cellular glycolysis, oxidative metabolism as well as the subsequent radiation response.

Animals↗

PubMed1997

Quantitation of hydroxyl radicals produced by radiation and copper-linked oxidation of ascorbate by 2-deoxy-D-ribose method.

We have established controlled conditions for studying the reaction of chemically and radiolytically produced hydroxyl radical (.OH) with 2-deoxy-D-ribose (2-DR). Ascorbate (ASC) or dithiothreitol (DTT) and cuprous or cupric ions were used to generate the OH-radical. The OH-radical was detected using the classical method of measuring the amount of thiobarbituric acid reactive products (TBARP) formed by .OH-mediated 2-DR degradation, but using sensitive fluorescent detection of the TBARP production to quantify the OH-radical. All experiments were performed with adequate O(2) concentrations. The copper reaction with ASC consumes O(2) in a manner that is strongly dependent on copper concentration, and less dependent on ascorbate concentration. For an independent check of the Cu2+ catalyzed ASC oxidation kinetics, the decay of ASC absorbency at 265 nm, as well as the increase of H(2)O(2) absorbency at approximately 240 nm, were also monitored. These spectral changes agree well with the O(2) consumption data. TBARP production from 2-DR incubated with a Cu2+-ASC mixture or gamma-irradiated were also compared. gamma-Irradiation of 2-DR solutions shows a dose and 2-DR concentration dependent increase of TBARP generation. Other electron donors, such as DTT, are more complicated in their mechanism of OH-radical production. Incubation of 2-DR with Cu2+-DTT mixtures shows a delay (approximately 50 min) before OH-radical generation is detected. Our results suggest that the Cu2+-ASC reaction can be used to mimic the effects of ionizing radiation with respect to OH-radical generation. The good reproducibility and relative simplicity of the 2-DR method with fluorescence detection indicates its usefulness for the quantitation of the OH-radical generated radiolytically or chemically in carefully controlled model systems.

Ascorbic Acid↗

PubMed1997

The importance of sodium pyruvate in assessing damage produced by hydrogen peroxide.

Instability of hydrogen peroxide solutions was noted during the experimental exposure of human cells in culture to hydrogen peroxide in experiments designed to study the production and repair of DNA single-strand breaks. A hydrogen peroxide concentrate was diluted into culture medium, which was then added to experimental cell cultures at various times, with all cultures assessed for DNA damage at 2 h. Only cells treated by the first addition had observable DNA damage. This result was unexpected since these cells had had the maximum repair time. It was determined that the hydrogen peroxide had been eliminated by the culture medium. To determine the mechanism of this elimination, 200 microM hydrogen peroxide was added to various cell culture components, and the solutions were assayed for hydrogen peroxide after 1 h at 37 degrees C. Although most components (except the balanced salts) showed some hydrogen peroxide degradation, it was found that sodium pyruvate was most effective, by a wide margin, in eliminating hydrogen peroxide and its toxic effects. This was confirmed by addition of pyruvate to balanced salt solutions or buffers, and observing the same elimination of hydrogen peroxide. We subsequently found a few earlier reports describing the decarboxylation reaction between hydrogen peroxide and pyruvate, but no kinetic measurements have been published and there seems to be no general appreciation for the very high efficiency of this reaction. The present work presents a preliminary assessment of the importance of pyruvate in the study of hydrogen peroxide and other reactive oxygen species in mammalian cell culture.

Azides↗

PubMed1997

Effect of purine nucleoside phosphates on OH-radical generation by reaction of Fe2+ with oxygen.

The influence of various purine nucleotides, nucleosides and nucleoside phosphates on the generation of OH-radicals by the reaction of Fe2+ with oxygen was investigated. Coumarin-3-carboxylic acid was used as a fluorescent detector of OH.. Nucleoside triphosphates caused the enhancement of OH. production due to chelation of ferrous ion by the phosphate moiety. About 30% of produced OH. are intramolecularly scavenged by the nucleoside moiety of the chelator molecule. Nucleoside diphosphates cause a slight enhancement of OH. yield. Nucleotides, nucleosides and nucleoside monophosphates decrease the OH. production. Rate constants of reaction between OH. and nucleoside derivatives were determined from the competitive scavenging of OH radicals, produced by oxidation of Fe(2+)-EDTA complex. Derivatives of guanosine and xanthine are more efficient scavengers in comparison to adenine and inosine. Phosphate groups do not affect the constant of reaction of nucleoside with OH.. Our results suggest that the yield of OH. in the presence of the nucleotide derivatives is determined by chelation of ferrous with polyphosphates and preferential OH. scavenging by the organic portion of molecule. We propose that the generation of active oxygen intermediates in the reaction between nucleoside triphosphate complexes of iron and molecular oxygen is involved in iron-related cellular injury.

Coumarins↗

PubMed1997

Mechanism of production of hydroxyl radicals in the copper-catalyzed oxidation of dithiothreitol.

We have undertaken detailed studies of the mechanisms involved in the production of OH radicals during the copper-catalyzed oxidation of dithiothreitol (DTT). Most of these studies were conducted in real time, detecting .OH based on its reaction with coumarin-3-carboxylic acid to produce the fluorescent derivative 7-hydroxycoumarin-3-carboxylic acid (7-OHCCA). Studies of the kinetics of oxidizing DTT show that production of .OH occurs in two stages: an initial lag period during which there is little production of 7-OHCCA, and a second reaction phase during which there is more rapid generation of .OH. The duration of the initial reaction period depends on the concentrations of both DTT and Cu2+. During this initial stage, oxygen consumption is high, although an increasing concentration of Cu2+ decreases the oxygen consumption. The rate of production of .OH during the second phase of the reaction depends on the concentration of Cu2+, but little oxygen is consumed. A mechanism is proposed whereby a Cu2+-DTT complex is formed and catalyzes oxidation of free DTT via formation of an oxygen-containing intermediate. The second phase of the reaction begins after complete oxidation of free DTT and involves production of O2.- and H2O2 followed by generation of .OH via reduction of H2O2 by cuprous ion.

Copper↗

PubMed1997

The generation of hydroxyl radicals in the reaction of molecular oxygen with polyphosphate complexes of ferrous ion.

The reaction of Fe2+ with molecular oxygen (autoxidation) was investigated in 20 mM phosphate buffer (pH 7.4) at 37 degrees C using a fluorescent OH probe, coumarin-3-carboxylic acid. The autoxidation of unchelated Fe2+ produces OH radicals. Polyphosphatic chelators (pyrophosphate and tri- and tetrapoly phosphate) enhanced the generation of radicals. This effect was explained by an alteration of the reaction mechanism. The two-electron reduction of the oxygen molecule and the generation of hydrogen peroxide intermediates are the major reactions during Fe2+ autoxidation. The polyphosphatic complexes of ferrous ion reduce molecular oxygen and reactive oxygen intermediates by a one-electron mechanism. The chelation of ferrous ion increases the generation of the superoxide radical and production of OH during ferrous ion autoxidation and in the Fenton reaction. The results consider the ferrous ion-polyphosphate system as a convenient model for the generation of hydroxyl radical in biological systems.

Ferrous Compounds↗

PubMed1997

Coumarin-3-carboxylic acid as a detector for hydroxyl radicals generated chemically and by gamma radiation.

Coumarin-3-carboxylic acid (3-CCA) was used as a detector for hydroxyl radicals (.OH) in aqueous solution. The .OH was generated by gamma irradiation or chemically by the Cu2+-mediated oxidation of ascorbic acid (ASC). The excitation and emission spectra of 3-CCA, hydroxylated either chemically or by gamma irradiation, were nearly identical to those of an authentic 7-hydroxycoumarin-3-carboxylic acid (7-OHCCA). The pH-titration curves for the fluorescence at 450 nm (excitation at 395 nm) of 3-CCA, hydroxylated either chemically or by gamma radiation, were also identical to those of authentic 7-OHCCA (pK = 7.4). Time-resolved measurements of the fluorescence decays of radiation- or chemically hydroxylated 3-CCA, as well as those of 7-OHCCA, indicate a monoexponential fit. The fluorescence lifetime for the product of 3-CCA hydroxylation was identical to that of 7-OHCCA (approximately 4 ns). These data, together with analysis of end products by high-performance liquid chromatography, show that the major fluorescent product formed by radiation-induced or chemical hydroxylation of 3-CCA is 7-OHCCA. Fluorescence detection of 3-CCA hydroxylation allows real-time measurement of the kinetics of .OH generation. The kinetics of 3-CCA hydroxylation by gamma radiation is linear, although the kinetics of 3-CCA hydroxylation by the Cu2+-ASC reaction shows a sigmoid shape. The initial (slow) step of 3-CCA hydroxylation is sensitive to Cu2+, but the steeper (fast) step is sensitive to ASC. Analysis of the kinetics of 3-CCA hydroxylation shows a diffusion-controlled reaction with a rate constant 5.0 +/- 1.0 x 10(9) M(-1) s(-1). The scavenging of .OH by 3-CCA was approximately 14% for chemical generation with Cu2+-ASC and approximately 50% for gamma-radiation-produced .OH. The yield of 7-OHCCA under the same radiation conditions was approximately 4.4% and increased linearly with radiation dose. The 3-CCA method of detection of .OH is quantitative, sensitive, specific and therefore accurate. It has an excellent potential for use in biological systems.

Chromatography, High Pressure Liquid↗