Radical-generating System Research Articles

Endogenous vasoactive substances and oxygen-derived free radicals in pulpal haemodynamics

An adequate blood supply to the dental pulp is essential to the health of the tooth. A recent concept is that repeated stimulation of sensitive teeth may induce pulpal changes; this could occur through induction of neurogenic inflammation and alteration of pulpal blood flow. One possibility is that production of oxygen-derived free radicals at sites of inflammation contributes to alterations in local blood flow. The first target of free radicals, generated in several pathological processes, is the vascular system (essentially the endothelium). Although the exact mechanism by which free radicals induce changes in vascular conductance is still uncertain, they may act directly on vascular smooth muscle or modify vascular tone by interacting with the production and/or biological activity of endogenous vasoactive mediators. Recent data indicate that the oxygen-derived, free radical-generating system can decrease pulpal blood flow in the dog via endothelial dysfunction when applied locally in deep dentinal cavities. In addition to the part played by oxygen-derived free radicals, the measurement of pulpal blood flow and the effects of endogenous vasoactive substances on flow are discussed.

Effect of pyruvate on oxidant injury to isolated and cellular DNA

Drawing upon the capacity of pyruvate to detoxify H2O2, we demonstrate that pyruvate (i) protects against H2O2-dependent, hydroxyl radical-mediated degradation of isolated DNA; (ii) reduces the amount of 8-hydroxy-2-deoxyguanosine detected following oxidative injury to isolated DNA and (iii) diminishes the amounts of detectable hydroxyl radical generated by a H2O2-dependent system. Compared to mannitol, pyruvate protects weakly against oxidative degradation of DNA induced by a H2O2-independent, hydroxyl radical-generating system. The protective effects of pyruvate against H2O2-instigated DNA damage were also evinced in cells in culture exposed to H2O2. In contrast to its protective effects against H2O2-dependent injury to DNA, pyruvate failed to offer convincing protection to another intracellular, H2O2-vulnerable target, glyceraldehyde-3-phosphate dehydrogenase. The protection conferred by pyruvate to intracellular H2O2-vulnerable targets is thus influenced by the nature of the target exposed to H2O2. Pyruvate was markedly protective in a model of cytotoxicity induced by the concomitant depletion of cellular glutathione and inhibition of catalase activity; pyruvate can thus function as an intracellular antioxidant and in this latter model, no evidence of DNA damage was observed. Pyruvate, in contrast to catalase, is a potent protector against cytotoxicity induced by organic peroxides, a finding that cannot be explained by the scavenging of organic peroxides, differences in glutathione content or attenuation in oxidative injury to DNA. We conclude that while DNA damage is a key pathogenetic event in oxidative stress induced by H2O2, such nuclear changes may not universally subserve a critical role in models of H2O2-dependent cell death. We also conclude that the antioxidant capabilities of pyruvate extend beyond scavenging of H2O2 to include potent protection against cytotoxicity induced by organic peroxides.

NADPH-, NADH- and cumene hydroperoxide-dependent metabolism of benzo[a]pyrene by pyloric caeca microsomes of the sea star Asterias rubens L. (Echinodermata: Asteroidea).

1. Benzo[a]pyrene (BaP) metabolism was studied in microsomes of the pyloric caeca (main digestive tissue and site of P450) of the echinoderm sea star (starfish) Asterias rubens. 2. NADPH-dependent metabolism of BaP produced phenols (36% of total metabolism), quinones (19%), dihydrodiols (25%) and putative protein adducts (20%). 3. NADH-dependent rates of BaP metabolism were approximately twice those found for NADPH-dependent metabolism, and metabolite formation was shifted towards dihydrodiols and quinones. 4. Cumene hydroperoxide (CHP)-dependent rates of BaP metabolism were also higher than NADPH-dependent rates by a factor of six for quinone and putative protein adduct production, and by a factor of four for phenol and dihydrodiol production. 5. Microsomal rates of BaP metabolism in BaP-exposed sea stars appeared to be elevated more in the case of NADPH-dependent than for CHP-dependent metabolism (respectively, increases of 130 and 41%), indicating the induction of forms of P450 preferentially catalysing NADPH-dependent metabolism. 6. 1,1,1-Trichloropropene-2,3-oxide (TCPO) inhibited dihydrodiol formation from both NADPH- and CHP-dependent BaP metabolism, indicating the involvement of epoxide hydratase in BaP metabolism. 7. Incubations of pyloric caeca microsomes with BaP and a superoxide anion radical-generating system (xanthine/xanthine oxidase) produced putative protein adducts but no free metabolites.

Oxygen radical-induced neurotoxicity in spinal cord neuron cultures.

The neurotoxic effects of oxygen radicals on spinal cord neuron cultures derived from fetal mouse have been studied. Oxygen radicals, superoxide radical and hydrogen peroxide, were generated by adding xanthine oxidase and hypoxanthine in the culture medium. Exposure of neurons to this oxygen radical-generating system resulted in a significant cell death and decrease of choline acetyltransferase (ChAT) activity in a time-dependent manner in spinal cord neuron cultures. The decrease in cell viability and ChAT enzyme activity induced by the oxygen radicals was blocked by scavengers such as superoxide dismutase (SOD), catalase, and tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a metal chelator. Antagonists of the N-methyl-D-aspartate (NMDA) receptor, including MK801 (a noncompetitive NMDA antagonist), D-2-amino-5-phosphonovaleric acid (APV) (a competitive NMDA antagonist), and 7-chlorokynurenic acid (an antagonist at the glycine site associated with the NMDA receptor), similarly blocked oxygen radical-induced decrease in cell viability and ChAT activity in spinal cord neuron cultures. These results indicate that both oxygen radicals and excitotoxic amino acids were involved in the oxidant-initiated neurotoxicity of spinal cord neurons.

Effect of reactive oxygen species on lysosomal membrane integrity. A study on a lysosomal fraction.

Using a lysosome-enriched "light mitochondrial" fraction of a rat liver homogenate, the effects of the reactive oxygen species hydrogen peroxide, superoxide- and hydroxyl radicals were determined. Alterations in the intralysosomal pH and the release of a lysosomal marker enzyme, N-acetyl-glucosaminidase, were used as indicators of changes in the lysosomal membrane integrity. Lipid peroxidation of the fraction was assayed by TBARS measurement. Neither superoxide radicals, generated by hypoxanthine/xanthine oxidase, nor a bolus dose of hydrogen peroxide (0.5-1.5 mM) induced any lysosomal damage. If, however, Fe(III)ADP was included in the superoxide radical-generating system, lysosomal membrane damage was detected, both as an increase in lysosomal pH and as a release of N-acetyl-glucosaminidase, but only after a lag phase of about 7 min. Lipid peroxidation, on the other hand, proceeded gradually. Lysosomes treated with hydrogen peroxide displayed similar dose-dependent alterations, albeit only if both Fe(III)ADP and the reducing amino acid cysteine were added. In the latter system, however, alterations of the lysosomal membrane stability occurred more rapidly, showing a lag phase of only 2 min. Lipid peroxidation, which proceeded faster and displayed no lag phase, levelled out within 10 min. The results indicate that neither superoxide radicals nor hydrogen peroxide are by themselves damaging to lysosomes. Available catalytically active iron in Fe(II) form, however, allows reactions yielding powerful oxidative species--probably hydroxyl radicals formed via Fenton reactions--to take place inducing peroxidation of the lysosomal membranes resulting in dissipation of the proton-gradient and leakage of their enzyme contents.

2-Oxo-histidine as a novel biological marker for oxidatively modified proteins

We report a promising finding that oxidative modification of proteins by free radicals could be monitored by the formation of oxidized histidine that is detectable by reverse-phase HPLC with electrochemical detection (HPLC-ECD). When the N-protected histidine derivative ( N-benzoyl-histidine) was exposed to a free radical-generating system (copper/ascorbate), a number of products were detected by HPLC-ECD and the main product among them was found to be identical to N-benzoyl-2-oxo-histidine. The acid hydrolysis of N-benzoyl-2-oxo-histidine provided a single product (2-oxo-histidine) that was detected sensitively by HPLC-ECD. Thus 2-oxo-histidine was indeed generated as the main product in the oxidatively modified proteins by free radicals. Taken together, 2-oxo-histidine may be a useful biological marker for assessing protein modifications under oxidative stress.

Amyloidogenicity of rodent and human βA4 sequences

Previously we have shown that aggregation of the C-terminal 100 residues (A4CT) of the βA4 amyloid protein precursor (APP) and also of βA4 itself depends on the presence of metal-catalyzed oxidation systems [T. Dyrks et al. (1988) EMBO J. 7, 949-957]. We showed that aggregation of the amyloidogenic peptides induced by radical generation systems requires amino acid oxidation and protein cross-linking. Here we report that aggregation of A4CT and βA4 induced by radical generation systems involves oxidation of histidine, tyrosine and methionine residues. The roden βA4 sequence lacking the single tyrosine and one of the three histidine residues of human βA4 and a βA4 variant in which the tyrosine and the three histidine residues were replaced showed a reduced tendency for aggregation. Thus our results may explain why βA4 amyloid deposits could so far not been detected in the rodent brain.

Electron paramagnetic resonance study of radicals derived from 2- and 3-fluoropropene

Using the TiCl3–H2O2, –NH2OH, −Na2S2O8, and −H2O2/Na2SO3, continuous-flow, radical-generating methods at 25 °C, we have successfully characterized by electron paramagnetic resonance spectroscopy radicals formed by the reaction of each of the substrates 2- and 3-fluoropropene with the radicals •OH, •NH3+, •OSO3−, and •SO3−, respectively. With 2-and 3-fluoropropene, respectively, the radicals identified were of general formula CH2(X)ĊFCH3 and CH2(X)ĊHCH2F where -X is the added group, except that treatment of 3-fluoropropene with the TiCl3–H2O2 reaction system also yielded •CH2CH(X)CH2F. The results obtained are discussed in terms of the effect of the presence of the fluorine substituent on both the apparent reactivity of the substrate molecules towards the radical-generating systems and the spectroscopic properties of the radicals observed.

Biologic Effects of Parenteral Bisulfite on Human Vascular Tissue

Antioxidant properties of bisulfite are used to stabilize parenteral amino acid solutions. After reports of adverse reactions to dietary sulfites, we evaluated whether the infusion of bisulfite had biologic effects on human vascular tissue. Because an endothelial oxidative injury can affect mediators of vasoreactivity, vascular pressure and prostaglandin production were studied in an intact human vein model infused with clinically relevant amino acid solutions differing only by their metabisulfite content (0 versus 300 mg/L). The amino acid solution containing bisulfite presented higher venous pressure (p < 0.01) and prostaglandin production (6-keto-prostaglandin F1 alpha, p < 0.01; and prostaglandin E2, p < 0.05). A hydroxyl radical-generating system added to the solutions did not modify the pressure readings, but it resulted in an overall decrease in prostacyclin production (p < 0.05). Despite this known inhibitory effect on oxidative challenge on prostaglandin I2 production, prostaglandins remained higher in the presence of bisulfite. The results suggest that the effect of bisulfite takes place before prostaglandin H2 synthesis in the eicosanoid cascade, whereas the oxidative challenge affects specifically the synthesis of prostaglandin I2, after prostaglandin H2, indicating that there is no interaction between bisulfite and the hydroxyl generating system. Bisulfite has local vascular effects on endothelial mediators, separate from its antioxidant properties.

Antioxidant action of dietary restriction in the aging process.

Dietary restriction is widely recognized to be the most effective means of intervening in the aging process in laboratory animals. Recent studies have produced substantial evidence that indicates this nutritional manipulation can modulate many aspects of free radical metabolism, including free radical generation, lipid peroxidation, DNA damage and cytosolic defense systems. Some of our laboratory's recent work is summarized in this presentation.

Reaction of cyclopenta[c,d]pyrene-3,4-epoxide with DNA and deoxynucleotides.

Cyclopenta[c,d]pyrene (CPP) is a widespread polycyclic aromatic hydrocarbon with potent mutagenic and carcinogenic activity. The trans isomer of 3,4-dihydro-3,4-dihydroxy-cyclopenta[c,d]pyrene has been shown to be the major metabolic product of CPP in rat, mouse or human microsomal systems, as well as in peroxyl radical-generating systems, indicating the preferential formation of its obligatory precursor, CPP-3,4-epoxide. The direct mutagenicity of CPP-3,4-epoxide, the inactivity of 3,4-dihydro-CPP and the DNA adduct forming capacity of CPP in vivo has prompted analysis of the DNA adducts produced by CPP-3,4-epoxide to provide information pertaining to: (i) the role this postulated major ultimate mutagenic metabolite may play in the formation of DNA adducts in vivo; (ii) the base selectivity of CPP-3,4-epoxide DNA adducts; and (iii) the role of CPP-3,4-epoxide in the mutagenicity/carcinogenicity of CPP. CPP-3,4-epoxide was reacted with calf thymus DNA, dGp, dAp, dTp, dCp, poly dG-dC, poly dA-dT and poly dG. Adducts were analyzed by the butanol-enhanced version of 32P-postlabeling. Four major and at least three minor adducts formed with DNA in vitro, which were further analyzed for their base selectivity. A similar spectrum of adducts was exhibited by dGp, poly dG-dC and poly dG. dCp, dTp, and dAp formed one, two, and four adducts respectively. The relative binding in adducts per 10(7) nucleotides was in the following descending order: dGp (6000), poly dG-dC (5800), dTp (5300), dAp (4800), calf thymus DNA (3800), poly dA-dT (2300), poly dG (2600) and dCp (20). Adducts derived from either dGp, poly dG-dC or poly dG co-migrated with the DNA adducts in three solvent systems, indicating that CPP-3,4-epoxide forms DNA adducts almost exclusively with deoxyguanosine.

Modification of contractile proteins by oxygen free radicals in rat heart.

This study was undertaken to investigate the effects of oxygen free radicals on myofibrillar creatine kinase activity. Isolated rat heart myofibrils were incubated with xanthine+xanthine oxidase (a superoxide anion radical-generating system) or hydrogen peroxide and assayed for creatine kinase activity. To clarify the involvement of changes in sulfhydryl groups in causing alterations in myofibrillar creatine kinase activity, 1) effects of N-ethylmaleimide (sulfhydryl groups reagent) on myofibrillar creatine kinase activity, 2) effects of oxygen free radicals on myofibrillar sulfhydryl groups content, and 3) protective effects of dithiothreitol (sulfhydryl groups-reducing agent) on the changes in myofibrillar creatine kinase activity due to oxygen free radicals were also studied. Xanthine+xanthine oxidase inhibited creatine kinase activity both in a time- and a concentration-dependent manner. Superoxide dismutase (SOD) showed a protective effect on the depression in creatine kinase activity caused by xanthine+xanthine oxidase. Hydrogen peroxide inhibited creatine kinase activity in a concentration-dependent manner; this inhibition was prevented by the addition of catalase. N-ethylmaleimide reduced creatine kinase activity in a dose-dependent manner. The content of myofibrillar sulfhydryl groups was decreased by xanthine+xanthine oxidase; this reduction was prevented by SOD. Furthermore, the depression in myofibrillar creatine kinase activity by xanthine+xanthine oxidase was protected by the addition of dithiothreitol. Oxygen free radicals may inhibit myofibrillar creatine kinase activity by modifying sulfhydryl groups in the enzyme protein. The reduction of myofibrillar creatine kinase activity may lead to a disturbance of energy utilization in the heart and may contribute to cardiac dysfunction due to oxygen free radicals.

The effect of L-carnitine and acetyl-L-carnitine on the disappearance of DNA single-strand breaks in human peripheral blood lymphocytes.

DNA single-strand breaks (SSBs) and their disappearance during repair incubation were determined by alkaline filter elution in freshly isolated human peripheral blood lymphocytes (PBLs) after in vitro treatment with either the oxygen radical-generating system of xanthine oxidase (XOD) plus hypoxanthine (HYP) or the alkylating agent N-ethyl-N'-nitrosourea (ENU). The elution curves obtained with DNA from PBLs treated with XOD/HYP were markedly nonlinear, possibly as a result of a nonrandom induction of SSBs along the DNA strands. The disappearance of XOD/HYP-induced SSBs during the initial repair period was quite slow; only 20 +/- 7% (n = 6) of the induced SSBs had disappeared after a 2 1/2 h repair incubation. However, by 24 h the elution curves obtained with DNA from treated PBLs were indistinguishable from those obtained with DNA from nontreated control cells, indicating complete repair. Treatment of PBLs with ENU resulted in linear elution curves. Approximately 50% of the total amount of ENU-induced SSBs had disappeared within 1 h in PBLs from most donors; the additional SSBs were found to be persistent (Beorrigter, M.E.T.I., Mullaart, E., Berends, F., and Vijg, J. (1991) Induction and disappearance of DNA strand breaks and/or alkali-labile sites in human lymphocytes exposed to N-ethyl-N'-nitrosoureas. Carcinogenesis, 12, 77-82). Preincubation of PBLs with 5 mM L-carnitine, a trans-mitochondrial carrier of acetyl and long-chain acyl groups, or 5 mM acetyl-L-Carnitine, resulted in a more rapid disappearance of XOD/HYP-induced SSBs (48 +/- 23% and 48 +/- 30% respectively). Preincubation of PBLs with different doses of L-carnitine, before exposure to 0.5 mM ENU, increased SSB disappearance dependent on the dose and donor PBLs. In conclusion, these studies suggest that treatment with L-carnitine accelerates the disappearance of SSBs induced by oxygen radicals and alkylating agents.

Induction of glutathione peroxidase by reactive oxygen in the yeast Hansenula mrakii

The yeast Hansenula mrakii IFO 0895 induces glutathione peroxidase when exposed to exogenous lipid hydroperoxides (Inoue, Y. et al., Agric. Biol. Chem., 54, 3289–3293, 1990). When protoplasts of H. mrakii were treated in a hydroxyl radical- and superoxide radical-generating systems; i.e., H 2 O 2 + Fe 2+, and xanthine + xanthine oxidase, respectively, glutathione peroxidase was also induced. This suggested that the induction of glutathione peroxidase is one of the mechanisms in H. mrakii for adaptation to various oxidative stresses.

Ca 2+-induced, phospholipase-independent injury during reoxygenation of anoxic mitochondria

Reoxygenation of rat-liver mitochondria after anoxic incubation induced release of matrix proteins. As assessed by release of a matrix enzyme, it was proportional to the rate of H 2O 2 production. The release was not observed with low concentrations of extramitochondrial free Ca 2+, indicating a Ca 2+-dependent pathway. Phospholipase A 2 was not involved in the reoxygenation injury, because non-esterified fatty acids did not increase on reoxygenation even when re-acylation was inhibited and because inhibitors of phospholipase A 2 had little effect on enzyme release. Cyclosporin A, ATP, ADP and inhibitors of pyridine nucleotide oxidation had a protective effect, strongly suggesting involvement of so-called Ca 2+-dependent permeability transition. Ca 2+ was also released from reoxygenated mitochondria and inhibition of reuptake of released Ca 2+ attenuated the enzyme release. Similar releases of aspartate aminotransferase and Ca 2+ were observed with mitochondria in an oxygen radical-generating system, hypoxanthine and xanthine oxidase. In this system, lecithin-cardiolipin liposomes also released entrapped Ca 2+ without disruption of the membrane. From these results, we conclude that during reoxygenation, Ca 2+ release and subsequent reuptake induced permeability transition of mitochondria, resulting in reoxygenation injury.

Neuroprotective effects of carvedilol, a new antihypertensive agent, in cultured rat cerebellar neurons and in gerbil global brain ischemia.

Free radical generation mediates part of the ischemic neuronal damage caused by the excitatory amino acid glutamate. Carvedilol, a novel multiple-action antihypertensive agent, has been shown to scavenge free radicals and inhibit lipid peroxidation in swine heart and rat brain homogenates. Therefore, we studied the neuroprotective effect of carvedilol on cultured cerebellar neurons and on CA1 hippocampal neurons of gerbils exposed to brain ischemia. Neuroprotective mechanisms were studied using an in vitro ischemia model of cultured rat cerebellar granule cell neurons exposed to either glutamate or oxygen free radical-generating systems. Prevention of lipid peroxidation by carvedilol was studied by measuring the formation of thiobarbituric acid-reactive substance. Gerbil CA1 neuron survival was examined by direct neuronal count 7 days after 6 minutes of global ischemia with reperfusion. Carvedilol protected cultured neurons in a dose-dependent manner against glutamate-mediated excitotoxicity (inhibitory concentration [IC50] = 1.1 microM) as well as against a 20-minute oxidative challenge (IC50 = 5 microM). The IC50 against the oxidative challenge was lowered to 1.3 microM by growing neurons for 24 hours in the presence of carvedilol. At 10 microM carvedilol inhibited lipid peroxidation 50% and 73% (n = 4, p < 0.001) in neurons exposed to two different free radical-generating systems. Neuroprotection of 52% (n = 22, p = 0.009 versus vehicle) of gerbil CA1 hippocampal neurons was achieved by pretreatment and posttreatment with subcutaneous injection of 3 mg/kg carvedilol twice a day for 4 and 3 days, respectively. Carvedilol provided neuroprotection in both in vitro and in vivo models of neuroinjury, where oxygen radicals are likely to play an important role. Therefore, carvedilol may reduce the risk of cerebral ischemia and stroke by virtue of both its antihypertensive action and its antioxidative properties.

Adenosine A1 receptor activation attenuates cardiac injury produced by hydrogen peroxide.

Adenosine has been shown to protect the ischemic and reperfused myocardium. To examine whether the protective effect of the nucleoside is mediated by modulation of oxidative stress, isolated rat hearts were perfused for 30 minutes with 100 microM H2O2 or an exogenous free radical-generating system consisting of purine (3.06 mM) and xanthine oxidase (10 units/l) in the presence or absence of drugs acting on adenosine A1 or A2 receptors. H2O2 alone produced a greater than 90% loss in contractility concomitant with a threefold elevation in resting tension, although these effects occurred in the absence of ultrastructural damage. Two A1 receptor agonists N6-cyclopentyladenosine (CPA, 1 microM) and R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA, 1 microM) significantly attenuated the cardiodepressant effects of H2O2 and depressed the elevation in resting tension; however, only the effect of CPA was found to be significant with regard to the latter parameter. A similar concentration of S(+)-N6-(2-phenylisopropyl)adenosine (S-PIA), a markedly less potent A1 receptor agonist, was found to be without beneficial effect. However, a significant protective effect against both the reduction in contractility and the elevation in resting tension was seen with a 10-fold elevation in the concentration of S-PIA (10 microM). The protective effects on functional parameters were associated with preservation of high-energy phosphate and adenine nucleotide contents after 30 minutes of H2O2 treatment. The salutary effects of all drugs were reversed in the presence of the A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (0.5 microM). An A2 receptor agonist 2-[p-(carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine, termed CGS 21680 (1 microM), failed to alter the cardiac response to H2O2 with regard to all parameters studied. Neither a 50% reduction in external CaCl2 concentration nor treatment with 10 microM DL-propranolol exerted salutary effects against H2O2-induced dysfunction. None of the A1 receptor agonists modulated the response to purine plus xanthine oxidase. Our results demonstrate a selective protective effect of adenosine A1 receptor activation against the cardiac toxicity of H2O2 and provide, at least in part, a basis for the cardioprotective actions of adenosine and its analogues.

C-reactive protein selectively enhances the intracellular generation of reactive oxygen products by IgG-stimulated monocytes and neutrophils.

The acute phase protein, C-reactive protein (CRP), when heat-aggregated (Agg-CRP), potentiates immunoglobulin G (IgG) Fc receptor-mediated luminol-enhanced chemiluminescence (CL) in human monocytes and neutrophils. Luminol-CL is a sensitive measure of phagocyte respiratory burst activity; however, the nature of oxidative products contributing to the light emission and their site of generation remain incompletely defined. To more precisely describe the oxidative burst of monocytes and neutrophils to Agg-CRP, superoxide anion release was measured by cytochrome c reduction. In addition, the extracellular release of hydrogen peroxide was distinguished from hydrogen peroxide generation using a phenol red oxidation assay. Finally, a flow cytometric determination of dichlorofluorescein (DCFH) oxidation was employed as an index of intracellular peroxide production. Although Agg-CRP alone did not stimulate hydrogen peroxide generation by either monocytes or neutrophils, it significantly enhanced hydrogen peroxide generation in response to heat-aggregated IgG (Agg-IgG). In contrast, Agg-CRP did not enhance the extracellular release of either hydrogen peroxide or superoxide anion from Agg-IgG-stimulated cells. The capacity of Agg-CRP to enhance selectively intracellular oxidative product generation was confirmed when measuring DCFH oxidation in Agg-IgG-stimulated cells. To evaluate whether this selective enhancement of intracellular oxidative events could be attributed, at least in part, to a scavenging effect of Agg-CRP, a cell-free oxygen radical-generating system was employed. Agg-CRP did not significantly diminish the lucigenin-amplified CL response induced by the xanthine/xanthine oxidase reaction. These results indicate that although Agg-CRP enhances the intracellular generation of reactive oxygen intermediates by monocytes and neutrophils, extracellular release of those products is not influenced by cell interaction with Agg-CRP. It is tempting to speculate that CRP can selectively boost the microbicidal activities of monocytes and neutrophils within an inflammatory site by amplifying the intracellular generation of reactive oxygen products without increasing damage to surrounding normal tissues.

Depression of cardiac sarcolemmal phospholipase D activity by oxidant-induced thiol modification.

Myocardial phospholipase D (PLD) is primarily localized at the sarcolemmal level and selectively hydrolyzes phosphatidylcholine to form phosphatidic acid as part of the signal transduction mechanisms for regulating Ca2+ movements in the heart. Since the myocardial cell damage induced by oxidative stress is associated with abnormalities in Ca2+ homeostasis and thiol status, we examined the thiol group dependence and the effects of oxidant species on this enzyme. Sarcolemmal membranes isolated from rat heart were exposed to several types of thiol group modifiers. Alkylation with N-ethylmaleimide or methyl methanethiosulfonate, mercaptide formation with p-chloromercuriphenylsulfonic acid, and thiol-disulfide exchange with 5,5'-dithio-bis(2-nitrobenzoate) depressed sarcolemmal PLD activity; in all cases the depression was prevented by dithiothreitol. At different concentrations of N-ethylmaleimide the PLD depression correlated well (r = 0.98) with the decrease in total thiol group content of the membrane. The enzyme activity was not affected by xanthine-xanthine oxidase, a superoxide anion-generating system, but was depressed by hydrogen peroxide (H2O2) in a concentration-dependent manner. This inhibitory effect was prevented by catalase as well as by dithiothreitol, but not by D-mannitol. The effect of a hydroxyl radical-generating system (Fenton reaction) could not be assessed because of an interfering direct inhibition by Fe2+. Dithiothreitol was also able to restore PLD activity in H2O2-pretreated membranes and to prevent a severe deactivation of the enzyme by hypochlorous acid (HOCI). Protection by glutathione and inhibition by its oxidized form were also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amyloidogenicity of beta A4 and beta A4-bearing amyloid protein precursor fragments by metal-catalyzed oxidation.

Previously we have shown that the COOH-terminal 100 residues (A4CT) of the amyloid protein precursor (APP), which carry the sequence of the amyloid beta A4 protein of Alzheimer's disease at N-terminal position, form highly insoluble aggregates if expressed in the rabbit reticulocyte lysate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Dyrks, T., Weidemann, A., Multhaup, G., Salbaum, J.M., Lemaire, H.-G., Kang, J., Müller-Hill, B., Masters, C. L., and Beyreuther, K. (1988) EMBO J. 7, 949-957). Here we report that aggregation of this COOH-terminal APP fragment A4CT and also of beta A4 itself depends on additional factors. In contrast to the reticulocyte expression system, expression of A4CT and beta A4 in the wheat germ expression system resulted in only monomeric forms. We have identified the factors which are capable of transforming both soluble A4CT and beta A4 into insoluble and aggregating molecules. Monomeric A4CT or beta A4 expressed in the wheat germ lysate could be transformed into aggregating molecules by the addition of metal-catalyzed oxidation systems. The addition of radical scavengers such as ascorbic acid, trolox, and amino acids prevented the aggregation process induced by the radical initiators. Thus, the aggregation of amyloidogenic APP fragments if analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis requires amino acid oxidation and protein cross-linking induced by radical generation systems.