Reaction kinetics and targeting to cellular glutathione S-transferase of the glutathione peroxidase mimetic PhSeZnCl and its D,L-polylactide microparticle formulation.

Catalytic properties and cellular effects of the glutathione peroxidase (GPx)-mimetic compound PhSeZnCl or its d,l-lactide polymer microencapsulation form (M-PhSeZnCl) were investigated and compared with the prototypical Se-organic compounds ebselen and diselenide (PhSe)2. PhSeZnCl was confirmed to catalyze the ping-pong reaction of GPx with higher Vmax than ebselen and (PhSe)2, but the catalytic efficiency calculated for the cosubstrates glutathione (GSH) and H2O2, and particularly the high reactivity against thiols (lowest KM for GSH in the series of test molecules), suggested poor biological applicability of PhSeZnCl as a GPx mimetic. Cytotoxicity of PhSeZnCl was demonstrated in various cancer cell lines via increased reactive oxygen species (ROS) generation, depletion of intracellular thiols, and induction of apoptosis. Experiments carried out in GSH S-transferase P (GSTP)-overexpressing K562 human erythroleukemia cells and in GSTP1-1-knockout murine embryonic fibroblasts (MEFs) demonstrated that this cytosolic enzyme represents a preferential target of the redox disturbances produced by this Se-compound with a key role in controlling H2O2 generation and the perturbation of stress/survival kinase signaling. Microencapsulation was adopted as a strategy to control the thiol reactivity and oxidative stress effects of PhSeZnCl, then assessing applications alternative to anticancer. The uptake of this "depowered" GPx-mimetic formulation, which occurred through an endocytosis-like mechanism, resulted in a marked reduction of cytotoxicity. In MCF-7 cells transfected with different allelic variants of GSTP, M-PhSeZnCl lowered the burst of cellular ROS induced by the exposure to extracellular H2O2, and the extent of this effect changed between the GSTP variants. Microencapsulation is a straightforward strategy to mitigate the toxicity of thiol-reactive Se-organic drugs that enhanced the antioxidant and cellular protective effects of PhSeZnCl. A mechanistic linkage of these effects with the expression pattern and signaling properties of GSTP . This has overcome the GPx-mimetic paradigm proposed for Se-organic drugs with a more pragmatic concept of GSTP signaling modulators.

Peroxiredoxin VI oxidation in cerebrospinal fluid correlates with traumatic brain injury outcome.

Traumatic brain injury (TBI) patients would benefit from the identification of reliable biomarkers to predict outcomes and treatment strategies. In our study, cerebrospinal fluid (CSF) from patients with severe TBI was evaluated for oxidant stress-mediated damage progression after hospital admission and subsequent ventriculostomy placement. Interestingly, substantial levels of peroxiredoxin VI (Prdx6), a major antioxidant enzyme normally found in astrocytes, were detected in CSF from control and TBI patients and were not associated with blood contamination. Functionally, Prdx6 and its associated binding partner glutathione S-transferase Pi (GSTP1-1, also detected in CSF) act in tandem to detoxify lipid peroxidation damage to membranes. We found Prdx6 was fully active in CSF of control patients but becomes significantly inactivated (oxidized) in TBI. Furthermore, significant and progressive oxidation of "buried" protein thiols in CSF of TBI patients (compared to those of nontrauma controls) was detected over a 24-h period after hospital admission, with increased oxidation correlating with severity of trauma. Conversely, recovery of Prdx6 activity after 24h indicated more favorable patient outcome. Not only is this the first report of an extracellular form of Prdx6 but also the first report of its detection at a substantial level in CSF. Taken together, our data suggest a meaningful correlation between TBI-initiated oxidation of Prdx6, its specific phospholipid hydroperoxide peroxidase activity, and severity of trauma outcome. Consequently, we propose that Prdx6 redox status detection has the potential to be a biomarker for TBI outcome and a future indicator of therapeutic efficacy.

Role for Prdx1 as a specific sensor in redox-regulated senescence in breast cancer.

Recent studies suggest that Peroxiredoxin 1 (Prdx1), in addition to its known H2O2-scavenging function, mediates cell signaling through redox-specific protein-protein interactions. Our data illustrate how Prdx1 specifically coordinates p38MAPK-induced signaling through regulating p38MAPKα phosphatases in an H2O2 dose-dependent manner. MAPK phosphatases (MKP-1 and/or MKP-5), which are known to dephosphorylate and deactivate the senescence-inducing MAPK p38α, belong to a group of redox-sensitive phosphatases (protein tyrosine phosphatases) characterized by a low pKa cysteine in their active sites. We found that Prdx1 bound to both MKP-1 and MKP-5, but dissociated from MKP-1 when the Prdx1 peroxidatic cysteine Cys52 was over-oxidized to sulfonic acid, which in turn resulted in MKP-1 oxidation-induced oligomerization and inactivity toward p38MAPKα. Conversely, over-oxidation of Prdx1-Cys52 was enhancing in the Prdx1:MKP-5 complex with increasing amounts of H2O2 concentrations and correlated with a protection from oxidation-induced oligomerization and inactivation of MKP-5 so that activation toward p38MAPK was maintained. Further examination of this Prdx1-specific mechanism in a model of reactive oxygen species-induced senescence of human breast epithelial cells revealed the specific activation of MKP-5, resulting in decreased p38MAPKα activity. Taken together, our data suggest that Prdx1 orchestrates redox signaling in an H2O2 dose-dependent manner through the oxidation status of its peroxidatic cysteine Cys52.

Allelic variants of glutathione S-transferase P1-1 differentially mediate the peroxidase function of peroxiredoxin VI and alter membrane lipid peroxidation.

The dual-functioning antioxidant enzyme peroxiredoxin VI (Prdx6) detoxifies lipid peroxides particularly in biological membranes, and its peroxidase function is activated by glutathione S-transferase Pi (GSTP). The GSTP gene is polymorphic in humans, with the wild-type GSTP1-1A (Ile105, Ala114) and three variants: GSTP1-1B (Ile105Val, Ala114), GSTP1-1C (Ile105Val, Ala114Val), and GSTP1-1D (Ile105, Ala114Val). The focus of this study was to determine the influence of these polymorphisms on Prdx6 peroxidase function. Using extracellular generation of OH radicals and fluorescence (DPPP dye) detection, we found a fast (~300 s) onset of lipid peroxidation in membranes of MCF-7 cells transfected with a catalytically inactive Y7F mutant of GSTP1-1 and either GSTP1-1B or GSTP1-1D. However, this effect was not detected in cells expressing either GSTP1-1A or GSTP1-1C. Imaging of DPPP-labeled MCF-7 cells showed fluorescence localized in the plasma membrane, but intensity was substantially diminished in the GSTP1-1A- and GSTP1-1C-expressing cells. Moreover, in the Y7F mutant of GSTP1-1A-, GSTP1-1B-, and GST1-1D-expressing cells ()OH generation resulted (after 36 h) in plasma membrane-permeability-related cell death, whereas GSTP1-1A- and GSTP1-1C-expressing cells had significantly better survival. We used FRET analyses to measure in vitro binding of purified GSTP1-1 allelic variant proteins to purified recombinant Prdx6. The affinities for Prdx6 binding to GSH-loaded GSTP1-1's either mirrored their observed peroxidase activities (using phospholipid hydroperoxide as a substrate), GSTP1-1A>GSTP1-1C (K(D)=51.0 vs 57.0 nM), or corresponded to inactivation, GSTP1-1B (GSTP1-1D) (K(D)=101.0 (94.0) nM). In silico modeling of the GSTP1-1-Prdx6 heterodimer revealed that the sites of GSTP1-1 polymorphism (Ile105 and Ala114) are in close proximity to the binding interface. Thus, there is a hierarchy of effectiveness for polymorphic variants of GSTP1-1 to regulate Prdx6 peroxidase function, a feature that may influence human population susceptibilities to oxidant stress.

Preclinical pharmacokinetic analysis of NOV-002, a glutathione disulfide mimetic.

NOV-002 is a glutathione disulfide (GSSG) mimetic that is the subject of clinical investigation in oncology indications. GSSG is reduced by glutathione reductase (GR) to form glutathione (GSH), thereby maintaining redox homeostasis. The purpose of the study was to report the pharmacokinetic properties of NOV-002 and evaluate the effect that NOV-002 elicits in redox homeostasis. The pharmacokinetic analysis and tissue distribution of NOV-002 and GSH was evaluated in mice following a dose of 250 mg/kg, i.p. The redox potential and total protein thiol status was calculated. Here we show that NOV-002 is a substrate for GR and that GSH is a primary metabolite. Non-linear pharmacokinetic modeling predicted that the estimated absorption and elimination rate constants correspond to a half-life of approximately 13 min with an AUC of 1.18 µgh/mL, a C(max) of 2.16 µg/ml and a volume of distribution of 42.61 L/kg. In addition, measurement of the redox potential and total protein thiol status indicated the generation of a transient oxidative signal in the plasma compartment after administration of NOV-002. These results indicate that NOV-002 exerts kinetic and dynamic effects in mice consistent with the GSSG component as the active pharmacological constituent of the drug. A longer-lasting decrease in total plasma free thiol content was also seen, suggesting that the oxidative effect of the GSSG from NOV-002 was impacting redox homeostasis.

Activation of the antioxidant enzyme 1-CYS peroxiredoxin requires glutathionylation mediated by heterodimerization with pi GST.

1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, can protect cells against membrane oxidation through glutathione (GSH)-dependent reduction of phospholipid hydroperoxides to corresponding alcohols. However, purified native or recombinant enzyme in vitro generally lacks GSH peroxidase (GPx) activity because of oxidation of its single conserved cysteine. Reduction of the resultant oxidized cysteine is difficult because of its protected location within the homodimer formed by the oxidized protein monomers. Partial purification of 1-cysPrx from bovine lung revealed the presence of pi GST in an active preparation, while purification to homogeneity yielded enzyme that inactivated with time. We show that heterodimerization of 1-cysPrx with GSH-saturated pi GST results in glutathionylation of the oxidized cysteine in 1-cysPrx followed by subsequent spontaneous reduction of the mixed disulfide and restoration of enzymatic activity. Maximum activation of 1-cysPrx occurred with a 1:1 molar ratio of GSH-saturated pi GST and a 2:1 molar ratio of GSH to 1-cysPrx. Liposome-mediated delivery of oxidized recombinant enzyme into NCI-H441 cells that lack 1-cysPrx but express pi GST resulted in 1-cysPrx activation, whereas activation in MCF7 cells required co-delivery of pi GST. Our data indicate a physiological mechanism for glutathionylation of the oxidized catalytic cysteine of 1-cysPrx by its heterodimerization with pi GST followed by its GSH-mediated reduction and enzyme activation.

Roles for platelet-activating factor and *NO-derived oxidants causing neutrophil adherence after CO poisoning.

Studies were conducted with rats to investigate whether platelet activating factor (PAF) and nitric oxide (*NO)-derived oxidants played roles in the initial adherence of neutrophils to vasculature in the brain after carbon monoxide (CO) poisoning. Before CO poisoning, rats were treated with the competitive PAF receptor antagonist WEB-2170 or with the peroxynitrite scavenger selenomethionine. Both agents caused significantly lower concentrations of myeloperoxidase in the brain after poisoning, indicating fewer sequestered neutrophils. Similarly, both agents reduced the concentration of nitrotyrosine, indicating less oxidative stress due to *NO-derived oxidants. There were no alterations in whole brain homogenate PAF concentration measured by immunoassay and bioassay, nor were there changes in phosphatidylcholine concentration. Immunohistochemical imaging showed PAF to be more heavily localized within perivascular zones after CO poisoning. Neutrophils colocalized with both PAF and nitrotyrosine in brains of rats killed immediately after CO poisoning. We conclude that qualitative changes in brain PAF are responsible for neutrophil adherence immediately after CO poisoning and that activated neutrophils trigger the initial rise in brain nitrotyrosine. Persistent PAF-mediated neutrophil adherence required production of *NO-derived oxidants because when oxidants were scavenged, neutrophil adherence was not maintained.

Oxidative burst and NO generation as initial response to ischemia in flow-adapted endothelial cells.

Shear stress modulates endothelial physiology, yet the effect(s) of flow cessation is poorly understood. The initial metabolic responses of flow-adapted bovine pulmonary artery endothelial cells to the abrupt cessation of flow (simulated ischemia) was evaluated using a perfusion chamber designed for continuous spectroscopy. Plasma membrane potential, production of reactive O2 species (ROS), and intracellular Ca(2+) and nitric oxide (NO) levels were measured with fluorescent probes. Within 15 s after flow cessation, flow-adapted cells, but not cells cultured under static conditions, showed plasma membrane depolarization and an oxidative burst with generation of ROS that was inhibited by diphenyleneiodonium. EGTA-inhibitable elevation of intracellular Ca(2+) and NO were observed at approximately 30 and 60 s after flow cessation, respectively. NO generation was decreased in the presence of inhibitors of NO synthase and calmodulin. Thus flow-adapted endothelial cells sense the altered hemodynamics associated with flow cessation and respond by plasma membrane depolarization, activation of NADPH oxidase, Ca(2+) influx, and activation of Ca(2+)/calmodulin-dependent NO synthase. This signaling response is unrelated to cellular anoxia.

Phospholipid hydroperoxides are substrates for non-selenium glutathione peroxidase.

This study investigated phospholipid hydroperoxides as substrates for non-selenium GSH peroxidase (NSGPx), an enzyme also called 1-Cys peroxiredoxin. Recombinant human NSGPx expressed in Escherichia coli from a human cDNA clone (HA0683) showed GSH peroxidase activity with sn-2-linolenoyl- or sn-2-arachidonoyl-phosphatidylcholine hydroperoxides as substrate; NADPH or thioredoxin could not substitute for GSH. Activity did not saturate with GSH, and kinetics were compatible with a ping-pong mechanism; kinetic constants (mM(-1) min(-1)) were k(1) = 1-3 x 10(5) and k(2) = 4-11 x 10(4). In the presence of 0.36 mM GSH, apparent K(m) was 120-130 microM and apparent V(max) was 1.5-1.6 micromol/min/mg of protein. Assays with H(2)O(2) and organic hydroperoxides as substrate indicated activity similar to that with phospholipid hydroperoxides. Maximal enzymatic activity was at pH 7-8. Activity with phospholipid hydroperoxide substrate was inhibited noncompetitively by mercaptosuccinate with K(i) 4 miroM. The enzyme had no GSH S-transferase activity. Bovine cDNA encoding NSGPx, isolated from a lung expression library using a polymerase chain reaction probe, showed >95% similarity to previously published human, rat, and mouse sequences and does not contain the TGA stop codon, which is translated as selenocysteine in selenium-containing peroxidases. The molecular mass of bovine NSGPx deduced from the cDNA is 25,047 Da. These results identify a new GSH peroxidase that is not a selenoenzyme and can reduce phospholipid hydroperoxides. Thus, this enzyme may be an important component of cellular antioxidant defense systems.

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.

Radiation-sensitive tyrosine phosphorylation of cellular proteins: sensitive to changes in GSH content induced by pretreatment with N-acetyl-L-cysteine or L-buthionine-S,R-sulfoximine.

PURPOSE: At relatively high concentrations, ie., > 20 mM, N-acetyl-L-cysteine (NAC) scavenges reactive oxygen species produced by ionizing radiation in aqueous solution. Therefore, the ability of NAC to block signal transduction reactions in vivo, has lead to the suggestion that ROS are necessary for the normal propagation of these signals. In this paper we investigate the mechanism by which NAC alters signal transduction in whole cells. RESULTS: Exposing CHO-K1 cells to ionizing radiation results in elevated pp59fyn kinase activity. Moreover, we observe changes in the phosphotyrosine content of multiple cellular proteins, including one prominent phosphotyrosyl protein with a Mr of 85 kDa. Both the radiation-induced changes in pp59fyn kinase activity and the changes in phosphotyrosine content of pp85 were not affected by exposing K1 cells to NAC during the time of irradiation, suggesting that ROS generated extracellularly are not involved in the radiation-induced changes observed in phosphotyrosyl proteins. We also demonstrate that the cell membrane is an effective barrier against negatively charged NAC. Therefore, it seems unlikely that NAC's ability to block signal transduction reactions is related to scavenging of ROS intracellularly. Chronic exposure, ie., 1 h, to 20 mM NAC lead to a twofold elevation in GSH levels and resulted in a 17% decrease in the phosphotyrosine content of pp85 after exposure to 10 Gy. Moreover, pretreatment with L-buthionine-S,R-sulfoximine (BSO) decreased GSH levels and resulted in elevated phosphotyrosine levels in pp85 isolated from irradiated CHO-K1 cells. CONCLUSIONS: Since many signaling molecules contain redox sensitive cysteine residues that regulate enzyme activity, we suggest that the effects of NAC on radiation-induced signal transduction are due to its ability to alter the intracellular reducing environment, and not related to direct scavenging of ROS.

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.

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.

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.

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.

The importance of choice of anaesthetics in studying radiation effects in the 9L rat glioma.

In the present study we demonstrate that the glycolysis of the tumour 9L glioma, in vivo, may be manipulated with ketamine/xylazine combinations of anaesthetics. Xylazine alone or in combination with ketamine causes hyperglycaemia which is enhanced by glucose injections. Intracellular tumour pH is acidified when glucose is administered with ketamine/xylazine. However, the combination of inorganic phosphate and insulin with ketamine/xylazine and glucose caused an alkaline shift in the tumour pH as measured by 31P NMR. The anaesthetic combination of ketamine/acepromazine did not produce alterations in blood glucose or in tumour pH status as detected by 31P NMR spectroscopy. These results demonstrate dramatic effects of ketamine/xylazine on the acidification or alkalinisation of the cells of 9L glioma. These altered metabolic states are of potential therapeutic importance. The choice of xylazine alone would be useful for chemotherapy and hyperthermia modalities, both known to be dependent upon glucose metabolism and resultant acidification.

Role of guanosine triphosphate in ferric ion-linked Fenton chemistry.

We measured the production of reactive hydroxyl radical (OH.) by Fe2+ itself or complexed with nucleotide triphosphates or tripolyphosphate (TPP). Coumarin-3-carboxylic acid (3-CCA) reacts with the OH. produced by Fe2+, Fe3+ or Cu2+ plus ascorbate and with various iron complexes. We measured in real time the increased fluorescence of 3-CCA after hydroxylation to 7-hydroxy-coumarin-3-carboxylic acid (7-OHCCA). Phosphate-buffered solutions do not affect the yield of Fe(2+)-linked OH. as do other organic buffer solutions. Our results show that guanosine triphosphate enhances the Fe(2+)-linked production of OH.. We also tested inosine triphosphate, adenosine triphosphate and xanthine triphosphate for their capacity to produce OH. with Fe2+. Inosine triphosphate is the most effective nucleotide in the production of OH.. However, the Fe(2+)-mediated yield of OH. is greater in the presence of TPP compared to the nucleotide triphosphates. Organic buffers as well as the purine and ribose portion of nucleotides compete for OH. and decrease the yield of fluorescent 7-OHCCA. We also decreased the yield of OH. by adding guanosine to the Fe2+/TPP-generating system. Adenosine, ribose and deoxyribose also react with Fe(2+)-generated OH.. The decreased yield of 7-OHCCA occurs because the ribose and purine part of the molecule reacts with OH.. The maximal production of reactive OH., compared to all nucleotides and phosphates tested, occurs with a ratio of 2 TPP/Fe2+ complex. In conclusion, the real-time measurement of the production of fluorescent 7-OHCCA provides a convenient means for measuring chemically generated OH.. The TPP/Fe(2+)-generating mixture, in the presence of 3-CCA, can be used to study the scavenging ability of other competing molecules.

Benzoyl peroxide acts as a promoter of radiation induced malignant transformation in vitro.

The experiments reported here demonstrate that benzoyl peroxide (BP) can promote radiation induced transformation in vitro. BP is shown to be capable of generating free radicals, determined by the kinetics of hydroxylation as measured by fluorescence of coumarin-3-carboxylic acid. Although the mechanisms involved in the BP enhancement of radiation transformation are unknown, we hypothesize that lipid peroxidation produced by benzoyl radicals in the vicinity of membrane associated unsaturated lipids could contribute to the promotion of transformation in vitro.

The molecular organization of the influenza virus surface. Studies using photoreactive and fluorescent labeled phospholipid probes.

The membrane structures of remantadin-sensitive and remantadin-resistant influenza virus strains were studied using a photoreactive fatty acid as well as analogues of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, carrying a fluorescent or photoreactive reporter group at the end of one of the aliphatic chains. The results obtained demonstrated for the first time that the phospholipids of the viral membrane form lateral domains differing by the fluidity of their hydrocarbon chains and, probably, by the head-group composition of the lipids. The hemagglutinin small subunit (HA2) was shown to protrude into the apolar region of the phospholipid bilayer, whereas the M1 protein makes contact only with the inner surface. In the remantadin-sensitive virions the heavy hemagglutinin chain (HA1) appears not to be in contact with the lipid bilayer, whereas in the remantadin-resistant strain HA1 has a hydrophobic segment that proved to be inserted into the bilayer.

Lipid-specific fluorescent probes in studies of biological membranes.

Lipid-specific fluorescent probes are natural lipids carrying an apolar fluorophore in one of the hydrocarbon chains. Since such probes retain the head groups and resemble the molecular shape of native membrane lipids, they largely mimic the behaviour of their natural prototypes in biological membranes. Information provided by the lipid-specific probes is more differentiated and easier to interpret than that obtained from non-lipid probes. The principles of design of lipid-specific probes are formulated and the relative advantages and disadvantages of various fluorophores are discussed. In order to reduce ambiguities caused by perturbation of the probe environment, it is proposed to use, in a comparative manner, two or more lipid-specific probes resembling each other in all aspects except the polar head groups (the 'two probes' concept). Two types of fluorophores, the anthrylvinyl group and the perylenoyl group, were found to be well suited for the synthesis of lipid-specific probes. Use of both types of probes 'in tandem' opens new possibilities for studying lipid-protein and lipid-lipid interactions in biological membranes. The anthrylvinyl- and perylenoyl-labeled lipids were applied in studies of serum lipoproteins and erythrocyte membranes. A new highly sensitive ligand-receptor binding assay and a new approach to biological signal amplifying based on the use of lipid-specific probes are described.