Mechanism of ultrasound enhanced porphyrin cytotoxicity. Part I: A search for free radical effects

Intense ultrasound beams may have the potential to treat malignant tumours when combined with sensitizers, often called sonodynamic agents. Some of these agents, e.g., the porphyrins, are currently used for photodynamic therapy. However, the experimental evidence for ultrasound activation of sensitizers is inconsistent. This paper attempts to discover whether they yield of free radicals such as .OH and .H, which are produced by transient cavitation, could explain the killing of Chinese hamster ovary (CHO) cells in vitro with and without sonodynamic agents. CHO cells were irradiated with ultrasound beams in phosphate-buffered saline or in growth medium, and the immediate cell lysis and loss of cell colony forming ability were measured. Under our specific conditions, in which the standing wave patterns were minimized, a general correlation was observed between the transient cavitation, free radical production, and cytotoxicity. However, the yield of free radicals was much too small to explain the cell killing observed. We conclude that cytotoxicity is not linked to attack from free radicals formed outside the cells. In our experiments, immediate cell lysis is closely linked to the transient cavitation, which is known to produce shear forces that disrupt cellular membranes. We hypothesize that the loss of cell colony forming ability is also linked to damage of cellular membranes.     

The fate of the oxidizing tyrosyl radical in the presence of glutathione and ascorbate. Implications for the radical sink hypothesis

Cellular systems contain as much as millimolar concentrations of both ascorbate and GSH, although the GSH concentration is often 10-fold that of ascorbate. It has been proposed that GSH and superoxide dismutase (SOD) act in a concerted effort to eliminate biologically generated radicals. The tyrosyl radical (Tyr.) generated by horseradish peroxidase in the presence of hydrogen peroxide can react with GSH to form the glutathione thiyl radical (GS.). GS. can react with the glutathione anion (GS-) to form the disulfide radical anion (GSSG-). This highly reactive disulfide radical anion will reduce molecular oxygen, forming superoxide and glutathione disulfide (GSSG). In a concerted effort, SOD will catalyze the dismutation of superoxide, resulting in the elimination of the radical. The physiological relevance of this GSH/SOD concerted effort is questionable. In a tyrosyl radical-generating system containing ascorbate (100 microM) and GSH (8 mM), the ascorbate nearly eliminated oxygen consumption and diminished GS. formation. In the presence of ascorbate, the tyrosyl radical will oxidize ascorbate to form the ascorbate radical. When measuring the ascorbate radical directly using fast-flow electron spin resonance, only minor changes in the ascorbate radical electron spin resonance signal intensity occurred in the presence of GSH. These results indicate that in the presence of physiological concentrations of ascorbate and GSH, GSH is not involved in the detoxification pathway of oxidizing free radicals formed by peroxidases.     

The structure of free radical metabolites detected by EPR spin trapping and mass spectroscopy from halocarbons in rat liver microsomes

Electron impact (EI) tandem mass spectrometry (MS/MS) combined with EPR spin trapping was used to detect and identify the free radical metabolites of various halocarbons in rat liver microsomal dispersions. EPR spectra of the spin adducts of radical metabolites derived from fluorine-containing halocarbons display fluorine hyperfine splitting, which can be used as proof for the identification of this kind of halocarbon-derived free radical spin adduct. For halocarbons without fluorine atoms, MS/MS was found to be a very useful and simple method for the detection and identification of the structures of halocarbon-derived spin adducts from radical metabolites. The molecular ions from spin adducts of these halocarbon-derived free radical intermediates were observed for the first time by scanning the precursor ion spectrum of m/z 57. These assignments were further confirmed by the use of perdeuterated tert-butyl PBN which provides the precursor ion spectrum of m/z 66.     

Vibrational spectrum associated with the reduction of tyrosyl radical D* in photosystem II: a comparative biochemical and kinetic study

Photosystem II (PSII) contains a redox-active tyrosine, D. Difference FT-IR spectroscopy can be used to obtain structural information about this species, which is a neutral radical, D*, in the photooxidized form. Previously, we have used isotopic labeling, site-directed mutagenesis, and kinetics to assign a vibrational line at 1477 cm-1 to D*; these studies were performed on highly resolved PSII preparations at pH 7.5 ?Kim et al. (1998) Biochim. Biophys. Acta 1364, 337-360; publisher's correction, Biochim. Biophys. Acta 1366, 330-354?. Here, we use kinetics to assign vibrational features to tyrosyl radical, D*, in PSII membranes. EPR and fluorescence controls identify a time regime in which D* decay occurs independently of redox changes involving the PSII quinone acceptors. Difference FT-IR spectra, acquired over this time regime, exhibit decreases in the amplitude of a 1477 cm-1 line; quantitative comparison with EPR transients supports the assignment to D*. Conditions, requiring the use of phosphate/formate, have been described for observation of a dissimilar FT-IR spectrum, which has been assigned to tyrosyl radical D*; this spectrum lacks a 1477 cm-1 line ?Hienerwadel et al. (1997) Biochemistry 36, 14712-14723?. Under these conditions, we have observed (1) an acceleration in the rate of D* decay and a decrease in D* yield attributable to the presence of formate, (2) a proportional decrease in the amplitude of FT-IR spectra acquired over the time regime in which D* decays, (3) frequency shifts in the D* - D FT-IR spectrum, (4) large-scale structural changes, as assessed by the amide I line shape, and (5) contributions to the FT-IR spectrum from the phosphate/formate buffer in the absence of PSII. We conclude that changes in the FT-IR spectrum, observed in the presence of phosphate/formate, are caused by alterations in the environment of D* and by direct phosphate/formate contributions to the spectrum.     

Tree-ring formation during vacuum arc remelting of INCONEL 718: Part I. Experimental investigation

In the production of turbine discs, the final wrought structure is critically dependent on aspects of the ingot grain structure produced by vacuum arc remelting (VAR) prior to cogging and forging. Variations in the as-cast grain structure in the nickel-based superalloy INCONEL 718 were investigated, focusing upon regions where strings of equiaxed grains interrupt a predominately columnar-dendritic structure. These features, termed “tree rings,” form concentric circles that can be observed visually on etched transverse sections of the VAR ingot. These structures are of interest because they correlate with perturbations in control of the process and have also been associated with the occurrence of defects. This article describes an experimental study of these tree rings, which both characterizes them and investigates possible mechanisms for their formation. Fluctuations in the macroscopic heat, mass, and momentum transfer, which can increase the grain nucleation at the mushy zone front (similar to a columnar-to-equiaxed transition), were considered to be the most likely of the mechanisms hypothesized. In a second article, a multiscale mathematical model is developed to quantify these concepts and to determine whether they account for the features observed.     

Photosensitization with anticancer agents. 17. EPR studies of photodynamic action of hypericin: formation of semiquinone radical and activated oxygen species on illumination

The newly synthesized 14-alkoxymetopon derivatives, 14-methoxymetopon, 14-ethoxymetopon, 14-methoxy-5-methyl-morphinone, exhibit high affinity for the naloxone binding sites in rat brain. A substantial decrease in affinity was observed, in the presence of NaCl indicating a high degree of agonist activity. All three 14-alkoxymetopon derivatives displayed high affinity for [3H][D-Ala2,(Me)Phe4,Gly-ol5]enkephalin ([3H]DAMGO) binding sites, much less potency toward delta sites and were the least effective at kappa sites. Isolated tissue studies using the guinea pig ileum preparation confirmed their high agonist potency. Following administration the new compounds produced naloxone reversible antinociceptive effects and were 130-300 times more potent than morphine in the acetic acid induced abdominal constriction model in the mouse, and the hot plate and tail flick tests in the rat. The compounds also produced dose-dependent muscle rigidity, and potentiated barbiturate-induced narcosis. The in vivo apparent pA2 values for naloxone against 14-ethoxymetopon and morphine were similar in analgesia, suggesting an interaction with the same (mu) receptor site. The dependence liability of 14-alkoxymetopon derivatives in the withdrawal jumping test was less pronounced than that of morphine in either rats or mice, similar to tolerance to the their analgesic action. It is concluded that the 14-alkoxymetopon derivatives studied are selective and potent agonists at mu opioid receptors, with reduced dependence liability.     

Radiation-induced radical formation in beta-glycerophosphate single crystals studied by electron paramagnetic resonance and electron nuclear double resonance spectroscopy: formation of an allylic-type radical

The purpose of this study was to better understand students' perceptions of and preferences for inclusion or pull-out service delivery models. Thirty-two students with and without learning disabilities who had participated in both models during the past 2 or 3 years were interviewed individually. Key questions assessed their perceptions of which model was most conducive to academic learning and which was most likely to yield social benefits, and the reasons for their beliefs. Results indicated that students' views varied. Overall, more children identified pull-out as the model of choice, but many children were confident that inclusion was meeting their academic and social needs. We interpret the results of this study as providing support for maintaining a continuum of service delivery options and for considering the placement of each child individually, based on his or her unique needs.     

Isolation and characterization of maturation inhibiting compound in bovine follicular fluid

The protein fraction which is responsible for the inhibition of maturation of bovine oocytes in vitro was isolated from cow follicular fluid by means of column chromatography on a Sephadex G-200 and a Sepharose 4B, both in 0.1 M ammonium acetate, pH 6.7. The molecular weight of the maturation inhibiting protein fraction is approximately 60 kDa. At a concentration of 2.0 mg/mL in cultivation medium, 100% of the oocytes were arrested at the germinal vesicle stage. At a concentration of 0.25 mg/mL, the protein fraction still had some meiosis inhibiting effects, but 56% of the oocytes were capable of maturing to the metaphase of the second meiosis (MII). Without compact cumulus the inhibiting fraction had no meiosis retarding effect on the oocytes. Cow follicular fluid also exhibited this inhibitory effect on oocyte maturation in vitro. However, the follicular fluid from follicles of 2.5-5.0 mm diameter showed higher meiosis inhibiting effects than the follicular fluid from follicles of 5-10 mm diameter.     

Cysteinyl and substrate radical formation in active site mutant E441Q of Escherichia coli class I ribonucleotide reductase

All classes of ribonucleotide reductase are proposed to have a common reaction mechanism involving a transient cysteine thiyl radical that initiates catalysis by abstracting the 3'-hydrogen atom of the substrate nucleotide. In the class Ia ribonucleotide reductase system of Escherichia coli, we recently trapped two kinetically coupled transient radicals in a reaction involving the engineered E441Q R1 protein, wild-type R2 protein, and substrate (Persson, A. L., Eriksson, M., Katterle, B., P?tsch, S., Sahlin, M., and Sj?berg, B.-M. (1997) J. Biol. Chem. 272, 31533-31541). Using isotopically labeled R1 protein or substrate, we now demonstrate that the early radical intermediate is a cysteinyl radical, possibly in weak magnetic interaction with the diiron site of protein R2, and that the second radical intermediate is a carbon-centered substrate radical with hyperfine coupling to two almost identical protons. This is the first report of a cysteinyl free radical in ribonucleotide reductase that is a kinetically coupled precursor of an identified substrate radical. We suggest that the cysteinyl radical is localized to the active site residue, Cys439, which is conserved in all classes of ribonucleotide reductase, and which, in the three-dimensional structure of protein R1, is positioned to abstract the 3'-hydrogen atom of the substrate. We also suggest that the substrate radical is localized to the 3'-position of the ribose moiety, the first substrate radical intermediate in the postulated reaction mechanism.     

Separation of the two double-chain bovine intermediates of the proinsulin-insulin conversion I. Chemical, immunochemical, circular dichroism, and biological characterization

The intermediates of the proinsulin-insulin conversion were separated by cation exchange. The circular dichroism spectra of the intermediates showed less alpha-helix than insulin and proinsulin. It is suggested that the C-peptide interacts with the section of alpha-helix contained between residues 2 and 8 in the A-chain of the insulin moieties and unwinds the alpha-helix. The in vivo activities of the intermediates were found to be in the order of 50% relative to insulin. In the fat cell assay, the A-chain-substituted form is weaker (9%) than the B-chain-substituted form (19%). The C-peptide segments of the two forms reacted with C-peptide-specific antibodies as fully as the free C-peptide, on a molar basis. In contrast, the insulin segments were hindered from reacting with insulin-specific antibodies as fully as the insulin.     

Dioxygen inactivation of pyruvate formate-lyase: EPR evidence for the formation of protein-based sulfinyl and peroxyl radicals

We here report EPR studies that provide evidence for radical intermediates generated from the glycyl radical of activated pyruvate formate-lyase (PFL) during the process of oxygen-dependent enzyme inactivation, radical quenching, and protein fragmentation. Upon exposure of active PFL to air, a long-lived radical intermediate was generated, which exhibits an EPR spectrum assigned to a sulfinyl radical (RSO*). The EPR spectrum of a sulfinyl radical was also generated from the activated C418A mutant of PFL, indicating that Cys 418 is not the site of sulfinyl radical formation. Exposure of the activated C419A mutant or C418AC419A double mutant to air on the other hand, resulted in a new EPR spectrum that we assign to the alpha-carbon peroxyl radical (ROO*) of the active-site glycine, G734. These findings suggest that C419 is the site of sulfinyl radical formation and that replacement of this cysteine with alanine results in the accumulation of the carbon peroxyl radical. The results also support the proposal that the peroxyl radical and the sulfinyl radical are intermediates in the oxygen-dependent inactivation and cleavage of the protein. Moreover, these observations are consistent with the hypothesis that C419 and G734 are in close proximity in the activated enzyme and may participate in a glycyl/thiyl radical equilibrium. A mechanism that accounts for the formation of the radical intermediates is proposed.     

Degradation of hyaluronic acid, poly- and monosaccharides, and model compounds by hypochlorite: evidence for radical intermediates and fragmentation

Degradation of hyaluronic acid by oxidants such as HO. and HOCl/CIO- is believed to be important in the progression of rheumatoid arthritis. While reaction of hyaluronic acid with HO. has been investigated extensively, reaction with HOCl/ClO- is less well defined. Thus, little is known about the site(s) of HOCl/ClO- attack, the intermediates formed, or the mechanism(s) of polymer degradation. In this study reaction of HOCl/ClO- with amides, sugars, polysaccharides, and hyaluronic acid has been monitored by UV-visible (220-340 nm) and EPR spectroscopy. UV-visible experiments have shown that HOCl/ClO- reacts preferentially with N-acetyl groups. This reaction is believed to give rise to transient chloramide (R-NCl-C(O)-R') species, which decompose rapidly to give radicals via either homolysis (to produce N. and Cl.) or heterolysis (one-electron reduction, to give N. and Cl.) of the N--C bond. The nature of the radicals formed has been investigated by EPR spin trapping. Reaction of HOCl/ClO- with hyaluronic acid, chondroitin sulphates A and C, N-acetyl sugars, and amides gave novel, carbon-centered, spin adducts, the formation of which is consistent with selective initial attack at the N-acetyl group. Thus, reaction with hyaluronic acid and chondroitin sulphate A, appears to be localized at the N-acetylglucosamine sugar rings. These carbon-centered radicals are suggested to arise from rapid rearrangement of initial nitrogen-centered radicals, formed from the N-acetyl chloramide, by reactions analogous to those observed with alkoxyl radicals. The detection of increasing yields of low-molecular-weight radical adducts from hyaluronic acid and chondroitin sulphate A with increasing HOCl/ClO-concentrations suggests that formation of the initial nitrogen-centered species on the N-acetylglucosamine rings, and the carbon-centered radicals derived from them, brings about polymer fragmentation.     

An investigation of the formation of cytotoxic, genotoxic, protein-reactive and stable metabolites from naphthalene by human liver microsomes

Chemically reactive epoxide metabolites have been implicated in various forms of drug and chemical toxicity. Naphthalene, which is metabolized to a 1,2-epoxide, has been used as a model compound in this study in order to investigate the effects of perturbation of detoxication mechanisms on the in vitro toxicity of epoxides in the presence of human liver microsomes. Naphthalene (100 microM) was metabolized to cytotoxic, protein-reactive and stable, but not genotoxic, metabolites by human liver microsomes. The metabolism-dependent cytotoxicity and covalent binding to protein of naphthalene were significantly higher in the presence of phenobarbitone-induced mouse liver microsomes than with human liver microsomes. The ratio of trans-1,2-dihydrodiol to 1-naphthol was 8.6 and 0.4 with the human and the induced mouse microsomes, respectively. The metabolism-dependent toxicity of naphthalene toward human peripheral mononuclear leucocytes was not affected by the glutathione transferase mu status of the co-incubated cells. Trichloropropene oxide (TCPO; 30 microM), an epoxide hydrolase inhibitor, increased the human liver microsomal-dependent cytotoxicity (19.6 +/- 0.9% vs 28.7 +/- 1.0%; P = 0.02) and covalent binding to protein (1.4 +/- 0.3% vs 2.8 +/- 0.2%; P = 0.03) of naphthalene (100 microM), and reversed the 1,2-dihydrodiol to 1-naphthol ratio from 6.6 (without TCPO) to 2.6, 0.6 and 0.1 at TCPO concentrations of 30, 100 and 500 microM, respectively. Increasing the human liver microsomal protein concentration reduced the cytotoxicity of naphthalene, while increasing its covalent binding to protein and the formation of the 1,2-dihydrodiol metabolite. Co-incubation with glutathione (5 mM) reduced the cytotoxicity and covalent binding to protein of naphthalene by 68 and 64%, respectively. Covalent binding to protein was also inhibited by gestodene, while stable metabolite formation was reduced by gestodene (250 microM) and enoxacin (250 microM). The study demonstrates that human liver cytochrome P450 enzymes metabolize naphthalene to a cytotoxic and protein-reactive, but not genotoxic, metabolite which is probably an epoxide. This is rapidly detoxified by microsomal epoxide hydrolase, the efficiency of which can be readily determined by measurement of the ratio of the stable metabolites, naphthalene 1,2-dihydrodiol and 1-naphthol.     

Characterization of high- and low-molecular weight zinc-dependent acid phosphatases in bovine liver

We have purified two forms of Zn2+-dependent acid phosphatase (Zn2+-APase) from bovine liver, both of which require Zn2+ to hydrolyze the substrate p-nitrophenyl phosphate in an acidic environment. The apparent molecular weights of these two forms of Zn2+-APase were estimated to be about 100,000 and 62,000 by gel filtration, and about 44,000 and 31,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, respectively. The low-molecular weight (LMW) Zn2+-APase catalyzed the hydrolysis of myo-inositol-1-phosphate in the presence of 3 mM Mg2+ at physiological pH, but the high-molecular weight (HMW) enzyme did not. The LMW-Zn2+-APase of bovine liver was recognized by polyclonal antibodies developed against the Zn2+-APase of bovine brain, but the HMW-Zn2+-APase was not.     

Modulation of quantum yield of primary radical pair formation in photosystem II by site-directed mutagenesis affecting radical cations and anions

Pigment-protein interactions play a significant role in determining the properties of photosynthetic complexes. Site-directed mutants of Synechocystis PCC 6803 have been prepared which modify the redox potential of the primary radical pair anion and cation. In one set of mutants, the environment of P680, the primary electron donor of Photosystem II, has been modified by altering the residue at D1-His198. It has been proposed that this residue is an axial ligand to the magnesium cation. In the other set, the D1-Gln130 residue, which is thought to interact with the C9-keto group of the pheophytin electron acceptor, has been changed. The effect of these mutations is to alter the free energy of the primary radical pair state, which causes a change in the equilibrium between excited singlet states and radical pair states. We show that the free energy of the primary radical pair can be increased or decreased by modifications at either the D1-His198 or the D1-Gln130 sites. This is demonstrated by using three independent measures of quantum yield and equilibrium constant, which exhibit a quantitative correlation. These data also indicate the presence of a fast nonradiative decay pathway that competes with primary charge separation. These results emphasize the sensitivity of the primary processes of PS II to small changes in the free energy of the primary radical pair.     

Hypoglycemia, hypoxia, and ischemia in a corticostriatal slice preparation: electrophysiologic changes and ascorbyl radical formation

Experimental and clinical data suggest that oxygen and/or glucose deprivation alters electrical transmission in the brain and generates free radicals, which may mediate neuronal death. We have analyzed the effects of oxygen and/or glucose deprivation on both excitatory transmission, by measuring field potential amplitude, and free radical production, by using electron spin resonance (ESR) spectroscopy, in a corticostriatal slice preparation. Combined oxygen and/or glucose deprivation (ischemia) lasting 10 to 20 minutes induced a long-term depression of field potential amplitude. The ascorbyl radical could only be detected in brain slices during the reperfusion-phase after 30 minutes of ischemia. It appeared in the early minutes after the washout of ischemic medium and remained stable throughout the reperfusion phase. This radical was never detected in the external medium. Ischemia induced only a slight, but progressive, release of lactate dehydrogenase (LDH) into the external medium during the reperfusion phase. In contrast, exposure of slices to hypoxia or hypoglycemia alone resulted in transient depression of field potential amplitude, and no generation of ascorbyl radicals was observed on reperfusion. We propose that the long-lasting loss of electrical signals is the early sign of neuronal damage during ischemia. On the other hand, ascorbyl radical formation may be considered an indicator of neuronal injury after prolonged energy deprivation.     

Relationship between age of blastocyst formation and interferon-tau secretion by in vitro-derived bovine embryos

The quantitative analysis of a three-dimensional (3-D) intracoronary ultrasound (ICUS) image data set permits a more comprehensive assessment of coronary arterial segments. The 3-D image sets are generally acquired during continuous motorized pullbacks. However, the cyclic changes of vascular dimensions and the cyclic spatial displacement of the ICUS transducer relative to the vessel wall can result in characteristic image artifacts, which may limit the applicability of quantitative automated analysis systems. This limitation may be overcome by an ECG-gated image acquisition. In the present study we acquired in vivo (1) nongated and (2) ECG-gated 3-D ICUS image sets of 15 human atherosclerotic coronary arteries and performed a computer-assisted contour detection of the lumen and total vessel boundaries. Total vessel and lumen volumes measured significantly larger in the nongated versus ECG-gated end-diastolic image sets (753+/-307 mm3 vs. 705+/-305 mm3; 411+/-154 mm3 vs. 388+/-165 mm3, both: P < 0.05). Both end-diastolic and systolic measurements were available in nine arteries, showing a larger total vessel and lumen volume at systole (664+/-221 mm3 vs. 686+/-227 mm3, P=0.03; 384+/-164 mm3 vs. 393+/-170 mm3, P=0.08). The differences observed may be of particular interest for volumetric ICUS studies, addressing presumably small differences in vessel or lumen dimensions.     

Distribution of annexins I, II, and IV in bovine mammary gland

Annexins belong to a family of proteins that are characterized by their ability to bind phospholipids in a Ca(2+)-dependent manner that is thought to be involved in a variety of biological processes. The present study determined the localization of annexins in subcellular fractions, nuclei in particular, of cow mammary gland by immunoblot analysis using monoclonal antibodies to annexins I, II, IV, and VI. The analysis revealed that annexins I, II, and IV were present in cytosol, but VI was not. Annexins I and IV were found in the nuclear fraction, but annexin II was only faintly present. Annexin VI was also undetectable in this fraction. Cytosolic annexin I had a molecular mass of 36 kDa. The 36-kDa annexin I was also found in the nuclear fraction. A 38-kDa annexin I was additionally detected in nuclei. The cytosolic and nuclear 36-kDa annexin I and the nuclear 38-kDa annexin I showed different isoelectric points, as revealed by two-dimensional PAGE. Annexin IV from cytosolic and nuclear fractions had similar molecular masses and isoelectric points.