Determination of hydroxylated aromatic compounds produced via superoxide-dependent formation of hydroxyl radicals by liquid chromatography/electrochemistry

Hydroxyl radicals may be formed in a xanthine oxidase/hypoxanthine system, where the superoxide anion radical O-.2 and H2O2 are produced. The superoxide-dependent production of the OH. radicals may be monitored by determining the amount of hydroxylated aromatic compounds formed in such a system. Liquid chromatography/electrochemistry is a powerful tool for the determination of hydroxylated aromatic compounds. A technique is presented in which aniline and phenol are hydroxylated in xanthine oxidase/hypoxanthine incubations. No sample derivatization is needed for the determinations which can be accomplished by direct injection of the incubation mixture. Detection limits for 1,2- and 1,4-hydroxylated compounds are in the picomole range.     

Mutagenicity of 80 chemicals in Escherichia coli tester strains IC203, deficient in OxyR, and its oxyR(+) parent WP2 uvrA/pKM101: detection of 31 oxidative mutagens

Strain IC203, deficient in OxyR, and its oxyR(+) parent WP2 uvrA/pKM101 (denoted IC188) are the basis of a new bacterial reversion assay, the WP2 Mutoxitest, which has been used in the evaluation of 80 chemicals for oxidative mutagenicity. The following 31 oxidative mutagens were recognized by their greater mutagenic response in IC203 than in IC188: (1) peroxides: hydrogen peroxide (HP), t-butyl hydroperoxide (BOOH) and cumene hydroperoxide (COOH); (2) benzoquinones (BQ): 2-methyl-1,4-BQ, 2,6-dimethyl-1,4-BQ and 2,3, 5,6-tetramethyl-1,4-BQ; (3) naphthoquinones (NQ): 1,4-NQ, 2-methyl-1, 4-NQ and 2-hydroxy-1,4-NQ; (4) phenol derivatives: catechol, hydroquinone, pyrogallol, 1,2,4-benzenetriol, t-butylhydroquinone, gallic acid and 4-aminophenol; (5) catecholamines: DL- and L-dopa, DL- and L-epinephrine, dopamine and L-norepinephrine; (6) thiols: L-cysteine methyl ester, L-cysteine ethyl ester, L-penicillamine and dithiothreitol; (7) diverse: 3,4-dihydroxyphenylacetic acid, hypoxanthine and xanthine, both in the presence of xanthine oxidase, L-ascorbic acid plus copper (II) and phenazine methosulfate. Among these oxidative mutagens, 25 were found to be uniquely positive in IC203. With the exception of BOOH and COOH, mutagenesis by all oxidative mutagens was inhibited by catalase present in rat liver S9, indicating that it is mediated by HP generation, probably in autoxidation reactions. These catalase-sensitive oxidative mutagens were poor inducers of mutations derived from 8-oxoguanine lesions, whereas such mutations were efficiently induced by organic hydroperoxides. The results support the usefulness of incorporating IC203 in the bacterial battery for testing of chemicals. The well-characterized oxidative mutagens available with the use of the WP2 Mutoxitest may serve as a reference in studies on the genotoxicity of oxidative stress.     

The pathophysiology of superoxide: roles in inflammation and ischemia

The superoxide radical plays major roles in the neutrophil-medicated acute inflammatory response and in postischemic tissue injury, although the sources and actions of the radical are quite different in these two pathological states. While neutrophils produce superoxide for the primary purpose of aiding in the killing of ingested microbes, a second useful function has evolved. The superoxide released from actively phagocytosing neutrophils serves to attract more neutrophils by reacting with, and activating, a latent chemotactic factor present in plasma. Superoxide dismutase, by preventing the activation of this superoxide-dependent chemotactic factor, exerts potent anti-inflammatory action. During ischemia, energy-starved tissues catabolize ATP to hypoxanthine. Calcium transients in these cells appear to activate a calmodulin regulated protease which attacks the enzyme xanthine dehydrogenase, converting it to a xanthine oxidase capable of superoxide generation. When the tissue is reperfused and reoxygenated, all the necessary components are present (xanthine oxidase, hypoxanthine, and oxygen) to produce a burst of superoxide which results in extensive tissue damage. Ischemic tissues are protected by superoxide dismutase or allupurinol, an inhibitor of xanthine oxidase.     

Antioxidants inhibit the effect of vitamin C on oxygen radical-induced sister-chromatid exchanges

The mechanism of vitamin C-induced sister-chromatid exchanges in cultured mammalian cells was studied. Chinese hamster ovary cells, when exposed to an enzymatic oxygen radical-generating system (xanthine oxidase plus hypoxanthine), develop increased numbers of sister-chromatid exchanges. Inclusion of ascorbate (greater than or equal to 0.1 mM) in these incubations resulted in an augmentation of this effect. Superoxide dismutase (100 microliter/ml) and catalase (220 microliter/ml) caused a significant reduction in the number of sister-chromatid exchanges induced by xanthine oxidase, hypoxanthine and vitamin C. Their heat-inactivated counterparts had no effect. These results confirm that vitamin C (greater than or equal to 0.1 mM) potentiates the genetic toxicity of oxygen radicals and that this effect is mediated by toxic oxygen intermediates.     

Reaction products of 1-naphthol with reactive oxygen species prevent NADPH oxidase activation in activated human neutrophils, but leave phagocytosis intact

Activation of human neutrophils with opsonized particles in the presence of a nontoxic dose of 1-naphthol resulted in inhibition of superoxide anion production but not of the phagocytotic activity of the cells. In this study we have investigated the mechanism of action of 1-naphthol. The inhibition is not at the level of cellular activation since the FMLP-induced rise of intracellular free calcium was unaffected. Our results show that the (metabolic) activation of 1-naphthol to 1,4-naphthoquinone by reaction with H2O2 from the oxidative burst is a necessary event for the inhibition to occur. The study provides evidence that by its reactivity with essential thiol groups 1,4-naphthoquinone (1,4-NQ) prevents the assembly of a functional NADPH-oxidase in the neutrophil membrane.     

Effect of silybin on phorbol myristate actetate-induced protein kinase C translocation, NADPH oxidase activity and apoptosis in human neutrophils.

Mechanism of the action of silybin (1) and its derivatives (2-4), possessing different lipid solubility in PMA-stimulated neutrophils was evaluated. Silybin (1) inhibited the calcium, phosphatidylserine- and diacylglycerol-dependent protein kinase C translocation and the NADPH oxidase activity in PMA-stimulated neutrophils and resulted in decreased apoptosis. Furthermore, silybin (1) inhibited xanthine oxidase activity and hem-mediated oxidative degradation of low-density lipoprotein, as well. Its derivatives (2-4), possessing different lipid-solubility, affected all the studied parameters. The lipid solubility of silybin (1) was enhanced by methylation (5'7'4'trimethylsilybin: 2), whereas a decrease in lipid-solubility by acetylation of compound 2 (5',7,'4"-trimethylsilybin-acetate: 3) or all the hydroxyl groups of silybin (peracetyl-silybin: 4) attenuated the antioxidant capacity by decreasing the inhibition in PKC translocation and NADPH oxidase activation. All the derivatives of silybin (2-4) showed no inhibition in cell free systems; e.g. did not alter the xanthine oxidase activity and the hem-mediated oxidative degradation of LDL. In conclusion, the antioxidant activity of (1) might be due to its ability to inhibit PKC translocation and NADPH oxidase activation in PMA-stimulated neutrophils. The increase of lipid solubility of silybin (1) supports its penetration through cell membrane and enhances its inhibitory effects. This structural modification of (1) might have pharmacological consequences.     

Ferritin and superoxide-dependent lipid peroxidation

Ferritin was found to promote the peroxidation of phospholipid liposomes, as evidenced by malondialdehyde formation, when incubated with xanthine oxidase, xanthine, and ADP. Activity was inhibited by superoxide dismutase but markedly stimulated by the addition of catalase. Xanthine oxidase-dependent iron release from ferritin, measured spectrophotometrically using the ferrous iron chelator 2,2'-dipyridyl, was also inhibited by superoxide dismutase, suggesting that superoxide can mediate the reductive release of iron from ferritin. Potassium superoxide in crown ether also promoted superoxide dismutase-inhibitable release of iron from ferritin. Catalase had little effect on the rate of iron release from ferritin; thus hydrogen peroxide appears to inhibit lipid peroxidation by preventing the formation of an initiating species rather than by inhibiting iron release from ferritin. EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide was used to observe free radical production in this system. Addition of ferritin to the xanthine oxidase system resulted in loss of the superoxide spin trap adduct suggesting an interaction between superoxide and ferritin. The resultant spectrum was that of a hydroxyl radical spin trap adduct which was abolished by the addition of catalase. These data suggest that ferritin may function in vivo as a source of iron for promotion of superoxide-dependent lipid peroxidation. Stimulation of lipid peroxidation but inhibition of hydroxyl radical formation by catalase suggests that, in this system, initiation is not via an iron-catalyzed Haber-Weiss reaction.     

Acceleration of adhesion of cancer cells and neutrophils to endothelial cells in the absence of de novo protein synthesis: possible implication for involvement of hydroxyl radicals

The adhesion of colon cancer cells (colo201) and neutrophils to endothelial cells which had been briefly exposed to either hypoxanthine/xanthine oxidase, or hydrogen peroxide, or peroxynitrite was analyzed in the absence of de novo protein synthesis. Such treatments accelerated the adhesions of both colo201 cells and neutrophils to endothelial cells. These effects were blocked by SOD/catalase or EDTA. The results provided evidence that hydroxyl radicals affect the cell surface of endothelial cells and accelerate cell adhesion.     

Reactive oxygen metabolite-induced toxicity to cultured bovine endothelial cells: Status of cellular iron in mediating injury

We aimed to determine the status of iron in mediating oxidant-induced damage to cultured bovine aortic endothelial cells. Chromium-51-labeled cells were exposed to reaction mixtures of xanthine oxidase/hypoxanthine and glucose oxidase/glucose; these produce superoxide and hydrogen peroxide, or hydrogen peroxide, respectively. Xanthine oxidase caused a dose dependent increase of ⁵¹Cr release. Damage was prevented by allopurinol, oxypurinol, and extracellular catalase, but not by superoxide dismutase. Prevention of xanthine oxidase-in-duced damage by catalase was blocked by an inhibitor of catalase, aminotriazole. Glucose oxidase also caused a dose-dependent increase of ⁵¹Ci release. Glucose oxidase-induced injury, which was catalase-inhibitable, was not prevented by extracellular superoxide dismutase. Both addition of and pretreatment with deferoxamine (a chelator of Fe³⁺) prevented glucose oxidase-induced injury. The presence of phenanthroline (a chelator of divalent Fe²⁺) prevented glucose oxidase-induced ⁵¹Cr release, whereas pretreatment with the agent did not. Apotransferrin (a membrane impermeable iron binding protein) failed to influence damage. Neither deferoxamine nor phenanthroline influenced cellular antioxidant defenses, or inhibited lysis by non-oxidant toxic agents. Treatment with allopurinol and oxypurinol, which inhibited cellular xanthine oxidase, failed to prevent glucose oxidase injury. We conclude that (1) among the oxygen species extracellularly generated by xanthine oxidase/hypoxanthine, hydrogen peroxide induces damage via a reaction on cellular iron; (2) deferoxamine and phenanthroline protect cells by chelating Fe³⁺ and Fe²⁺, respectively; and (3) reduction of cellular stored iron (Fe³⁺) to Fe²⁺ may be a prerequisite for mediation of oxidantinduced injury, but this occurs independently of extracellular superoxide or cellular xanthine oxidase-derived superoxide. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

The oxidation of tetrachloro-1,4-hydroquinone by microsomes and purified cytochrome P-450b. Implications for covalent binding to protein and involvement of reactive oxygen species

The enzymatic oxidation of tetrachloro-1,4-hydroquinone (1,4-TCHQ), resulting in covalent binding to protein of tetrachloro-1,4-benzoquinone (1,4-TCBQ), was investigated, with special attention to the involvement of cytochrome P-450 and reactive oxygen species. 1,4-TCBQ itself reacted very rapidly and extensively with protein (58% of the 10 nmol added to 2 mg of protein, in a 5-min incubation). Ascorbic acid and glutathione prevented covalent binding of 1,4-TCBQ to protein, both when added directly and when formed from 1,4-TCHQ by microsomes. In microsomal incubations as well as in a reconstituted system containing purified cytochrome P-450b, 1,4-TCHQ oxidation and subsequent protein binding was shown to be completely dependent on NADPH. The reaction was to a large extent, but not completely, dependent on oxygen (83% decrease in binding under anaerobic conditions). Inhibition of cytochrome P-450 by metyrapone, which is also known to block the P-450-mediated formation of reactive oxygen species, gave a 80% decrease in binding, while the addition of superoxide dismutase prevented 75% of the covalent binding, almost the same amount as found in anerobic incubations. A large part of the conversion of 1,4-TCHQ to 1,4-TCBQ is apparently not catalyzed by cytochrome P-450 itself, but is mediated by superoxide anion formed by this enzyme. The involvement of this radical anion is also demonstrated by microsomal incubations without NADPH but including the xantine/xantine oxidase superoxide anion generating system. These incubations resulted in a 1.6-fold binding as compared to the binding in incubations with NADPH but without xantine/xantine oxidase. 1,4-TCHQ was shown to stimulate the oxidase activity of microsomal cytochrome P-450. It is thus not unlikely that 1,4-TCHQ enhances its own microsomal oxidation.     

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O2.- and H2O2 during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-beta-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O2 consumption, only slightly but substantially inhibited in a competitive manner the O2.- -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new spectrophotometric assay for enzymes of purine metabolism. I. Determination of xanthine oxidase activity.

A new method for the determination of xanthine oxidase activity with xanthine or hypoxanthine is described. The hydrogen peroxide produced by the oxidation of the substrates is reduced by catalase in the presence of high concentrations of ethanol. The acetaldehyde formed is further oxidized by aldehyde dehydrogenase NAD or NADP-dependent. The reduction rate of the coenzymes were measured at 334 nm and utilized as indicators for the xanthine oxidase. The sensitivity of the method with xanthine as substrate can be doubled by the addition of uricase, which oxidizes uric acid to allantoin.     

On radical production by PMA-stimulated neutrophils as monitored by luminol-amplified chemiluminescence.

The means by which neutrophils within the body ward off infectious and neoplastic processes by the activation of molecular oxygen, as well as how such mechanisms dysfunction, is the subject of extensive ongoing research. Most previous studies of neutrophil activation indicate that there is a transient production of reactive oxygen species. Luminol-amplified chemiluminescence surveillance of O2-. and H2O2 supported these general findings. Yet, recent studies showed that production of reactive oxygen species by PMA-stimulated neutrophils is not transient but persistent; however, luminol-dependent methods do not corroborate such findings. The kinetics of O2-. production by human neutrophils were studied using luminol-amplified chemiluminescence (CL), spin trapping combined with electron spin resonance detection, and ferricytochrome c reduction. The effects of pH and O2 level on luminol-amplified CL were determined using hypoxanthine/xanthine oxidase to produce O2-. and H2O2 in cell-free systems. As we have found by electron spin resonance and ferricytochrome c reduction, stimulated neutrophils continued to generate O2-. for several hours, yet when luminol-amplified CL was used to continuously follow radical production, CL was shortly lost. Similar loss of CL was observed with continuous enzymatic formation of O2-. and H2O2. The failure of the CL assay to report O2-. and H2O2 formation results from some luminol reaction product which interferes with the light reaction. Our results show that the cells are operative for long periods indicating that cell exposure to prolonged O2-. fluxes does not terminate radical production, and even when pH, [O2], and reagents are optimized, the use of luminol-amplified CL is not a valid assay for continuous monitoring of O2-. and H2O2 generated by either stimulated neutrophils or in cell-free systems.     

Effect of oxygen-derived reactive species on cartilage proteoglycan-hyaluronate aggregates

Proteoglycan-hyaluronate aggregates were incubated with oxygen-derived reactive species generated enzymatically by the action of xanthine oxidase upon hypoxanthine. Analysis of the products of the incubation by caesium sulphate zonal sedimentation revealed that degradation of aggregate had occurred. This effect was reversed by inclusion of superoxide dismutase, catalase or diethylenetriaminepentaacetic acid in the incubations suggesting that hydroxyl radicals were the active species. Separate analysis by gel filtration chromatography on Sepharose CL-2B of proteoglycan monomer subjected to a similar treatment indicated that the molecule is minimally degraded. These results are discussed with reference to the well established degradation of hyaluronate by oxygen-derived reactive species.     

Identification of Hypoxanthine Transport and Xanthine Oxidase Activity in Brain Capillaries

Microvessel segments were isolated from rat brain and used for studies of hypoxanthine transport and metabolism. Compared to an homogenate of cerebral cortex, the isolated microvessels were 3.7-fold enriched in xanthine oxidase. Incubation of the isolated microvessels with labeled hypoxanthine resulted in its rapid uptake followed by the slower accumulation of hypoxanthine metabolites including xanthine and uric acid. The intracellular accumulation of these metabolites was inhibited by the xanthine oxidase inhibitor allopurinol. Hypoxanthine transport into isolated capillaries was inhibited by adenine but not by representative pyrimidines or nucleosides. Similar results were obtained when blood to brain transport of hypoxanthine in vivo was measured using the intracarotid bolus injection technique. Thus, hypoxanthine is transported into brain capillaries by a transport system shared with adenine. Once inside the cell, hypoxanthine can be metabolized to xanthine and uric acid by xanthine oxidase. Since this reaction leads to the release of oxygen radicals, it is suggested that brain capillaries may be susceptible to free radical mediated damage. This would be most likely to occur in conditions where the brain hypoxanthine concentration is increased as following ischemia.     

Role of extracellular superoxide in neutrophil activation: interactions between xanthine oxidase and TLR4 induce proinflammatory cytokine production

Reactive oxygen species (ROS) contribute to neutrophil activation and the development of acute inflammatory processes in which neutrophils play a central role. However, there is only limited information concerning the mechanisms through which extracellular ROS, and particularly cell membrane-impermeable species, such as superoxide, enhance the proinflammatory properties of neutrophils. To address this issue, neutrophils were exposed to superoxide generating combinations of xanthine oxidase and hypoxanthine or lumazine. Extracellular superoxide generation induced nuclear translocation of nuclear factor-kappaB (NF-kappaB) and increased neutrophil production of the NF-kappaB-dependent cytokines tumor necrosis factor-alpha (TNF-alpha) and macrophage inhibitory protein-2 (MIP-2). In contrast, there were no changes in TNF-alpha or MIP-2 expression when neutrophils lacking Toll-like receptor-4 (TLR4) were exposed to extracellular superoxide. Immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer (FRET) studies demonstrated association between TLR4 and xanthine oxidase. Exposure of neutrophils to heparin attenuated binding of xanthine oxidase to the cell surface as well as interactions with TLR4. Heparin also decreased xanthine oxidase-induced nuclear translocation of NF-kappaB as well as production of proinflammatory cytokines. These results demonstrate that extracellular superoxide has proinflammatory effects on neutrophils, predominantly acting through an TLR4-dependent mechanism that enhances nuclear translocation of NF-kappaB and increases expression of NF-kappaB-dependent cytokines.     

Structure prerequisite for antioxidant activity of silybin in different biochemical systems in vitro.

Structural analogues (flavanone: 2-4 and flavone: 5 and 6, respectively) of silybin (1a) were synthesized and tested for inhibitory activity on O(2)(-) release and PKC translocation in PMA-stimulated neutrophils as well as xanthine oxidase activity in order to identify the molecular structures responsible for the antioxidant property of silybin. Concerning the prevention of hem-mediated oxidative modification of LDL by silybin, the hydroxyl radical scavenging activity of its structural analogues was also determined. We demonstrated that the basic skeleton of 1a (4) is responsible for its inhibitory activity on O(2)(-) release in PMA-stimulated neutrophils via inhibition of PKC translocation, since introduction of a double bound and hydroxyl groups at C-5 and C-7 position (5 and 6) did not result in further increase in inhibition of O(2)(-) release. It has been shown that the presence of the phenolic hydroxyl group at C-5 and C-7 of 1a is essential for the inhibition of xanthine oxidase activity. Moreover, introduction of a double bond into the C-ring of 2 and 3, resulting in flavone derivatives (5 and 6), markedly enhanced the antioxidant effect in all the tested systems. Finally, silybin (1a) and its flavon derivatives (5 and 6) directly scavenged hydroxyl radicals as well. On the basis of these results it might be concluded that different moiety of silybin is responsible for inhibition of overproduction of O(2)(-) in stimulated neutrophils, xanthine oxidase activity, and for prevention of hem-mediated oxidative modification of LDL.     

A prodigiosin analogue inactivates NADPH oxidase in macrophage cells by inhibiting assembly of p47phox and Rac

Prodigiosins are natural red pigments that have multi-biological activities. Recently, we discovered a marine bacterial strain, which produces a red pigment. Extensive two-dimensional nuclear magnetic resonance and mass spectrometry analysis showed that the pigment is a prodigiosin analogue (PG-L-1). Here, we investigated the effect of PG-L-1 on NADPH oxidase activity in macrophage cells. PG-L-1 significantly inhibited superoxide anion (O(2)(-)) production by phorbol 12-myristate 13-acetate (PMA)-stimulated RAW 264.7 cells, a mouse macrophage cell line. The ED(50) value was estimated to be approximately 0.3 microM. PG-L-1 had no direct scavenging effect on O(2)(-) generated by hypoxanthine/xanthine oxidase system in electron spin resonance-spin trapping determinations, suggesting that this compound directly acts on the O(2)(-) production system, NADPH oxidase, in macrophage cells. We further investigated the effect of PG-L-1 on the behaviour of the cytosolic components of the NADPH oxidase, p67(phox), p47(phox), p40(phox), Rac and protein kinase C (PKC), in PMA-stimulated RAW 264.7 cells. Although PG-L-1 showed no effect on the activation of PKC, the immunoblotting analysis using specific antibodies showed that PG-L-1 strongly inhibits the association of p47(phox) and Rac in the plasma membrane of PMA-stimulated RAW 264.7 cells. These results suggest that PG-L-1 inactivates NADPH oxidase through the inhibition of the binding of p47(phox) and Rac to the membrane components of NADPH oxidase.     

Purpurogallin--a natural and effective hepatoprotector in vitro and in vivo.

Purpurogallin is a plant phenol that is sometimes added as an oxidation retardant to fats-oils or to certain fuels or lubricants. However, it was unknown if purpurogallin is cytoprotective. Here we examined this issue, both in isolated hepatocytes and in vivo. From 0.5 to 2.0 mM, purpurogallin prolongs survival of rat hepatocytes substantially against oxyradicals generated with xanthine oxidase and hypoxanthine. The protection was dose dependent and surpassed that given by such antioxidants as ascorbate, mannitol, superoxide dismutase, catalase, and Trolox, when each was examined at or near its optimal concentration in the same system. When 1.5, 3, and 6 mumol of purpurogallin in saline were infused into rats with postischemic livers shortly before reperfusion, the mean hepatic salvages were 42, 76, and 86%, respectively. Such salvage effects would rank purpurogallin highly among the hepatoprotectors known. Over the range of 31 to 500 microM, purpurogallin inhibited the rate of O2 consumption in the xanthine oxidase reaction by approximately 90%, which was 2- to several-fold higher than the inhibition elicited by allopurinol over the same concentrations. Thus, purpurogallin is an effective natural hepatoprotector that may operate partly or principally as an inhibitor of xanthine oxidase.     

Hyperthermia enhances the cytotoxic effects of reactive oxygen species to Chinese hamster cells and bovine endothelial cells in vitro.

Hyperthermia is under intensive investigation as a treatment for tumors both alone and in combination with other therapeutic agents. Hyperthermia has a profound effect on the function and structural integrity of tumor microvasculature; this has often been cited as a reason for its effectiveness in treatment of tumors. To test the role of hyperthermia in cytotoxic effects of active oxygen species, Chinese hamster, V79, and bovine endothelial cells were treated by the active oxygens, O not equal to 2 and H2O2, generated from the hypoxanthine/purine and xanthine oxidase reactions. It was found that cytotoxicity to V79 cells depends on the concentrations of purine and xanthine oxidase. A high level of cytotoxicity may be initiated in hyperthermia-treated tumors because high xanthine oxidase activity is known to be associated with tumors and endothelial cells, and degradation processes produce high concentrations of xanthine oxidase substrates in tumors. Since the cytotoxic effect can be reduced by the xanthine oxidase inhibitor, allopurinol, and the H2O2 removal enzyme, catalase, the cytotoxic effect in this experimental system is dependent on xanthine oxidase and H2O2. Adding erythrocytes at the same time as purine and xanthine oxidase could also prevent the cytotoxicity. Elevated temperatures stimulated the reaction of purine and xanthine oxidase and resulted in an increased cytotoxic effect. A similar effect is observed in growth inhibition and colony formation in endothelial cells without adding xanthine oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)