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Biomedical subjects

A W Girotti

Publications and source records attributed to A W Girotti.

At least 73 records · Page 4Linked to original sources
PubMed1987

Lipid peroxidation in erythrocyte membranes: cholesterol product analysis in photosensitized and xanthine oxidase-catalyzed reactions.

The effects of singlet oxygen- and oxygen radical-induced lipid peroxidation on cell membrane integrity were compared, using the human erythrocyte ghost as a model system. Resealed ghosts underwent lipid peroxidation and lysis (release of trapped glucose-6-P) when irradiated in the presence of uroporphyrin (UP) or when incubated with xanthine (X), xanthine oxidase (XO) and iron. The UP-sensitized process was inhibited by azide but not by phenolic antioxidants, consistent with singlet oxygen (nonradical) involvement. This was confirmed by showing that the predominant photoproduct of membrane cholesterol was the 5 alpha-hydroperoxide. Total hydroperoxide (LOOH) content in UP-photooxidized ghosts increased linearly during the prelytic lag and throughout the period of rapid lysis. Unlike the photoreaction, X/XO/iron-dependent peroxidation and lysis was inhibited by catalase, superoxide dismutase and phenolic antioxidants, indicating O2-/H2O2 intermediacy and a free radical mechanism. Correspondingly, only radical reaction products of cholesterol were formed, notably the 7 alpha-, 7 beta-hydroperoxide pair. Membrane lysis had a distinct lag as in photooxidation; however, the LOOH profile was more complex, with an initial lag followed by a sharp increase and then slow decline. X/XO/iron-induced lysis commenced when LOOH levels were 2-3 times higher than in photosensitized lysis, suggesting that the pathways of membrane lesion formation are different in the two systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Ascorbic Acid↗
PubMed1987

Singlet oxygen intermediacy in the photodynamic action of membrane-bound hematoporphyrin derivative.

The cell-damaging photochemistry of hematoporphyrin derivative (HPD) has been investigated using isolated erythrocyte membranes as a test system. Irradiation of membranes in the presence of the tumor-localizing fraction of HPD resulted in formation of singlet molecular oxygen (1O2) as measured by the phosphorescence at 1268 nm. The authentic product of 1O2 attack on cholesterol, 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide, was identified in this system. Relatively insignificant amounts of free radical-derived hydroperoxides were detected. These results suggest that 1O2 plays a major role in the HPD-sensitized photokilling of tumor cells in vivo.

Cholesterol↗
PubMed1987

Photodynamic action of merocyanine 540 on artificial and natural cell membranes: involvement of singlet molecular oxygen.

The photochemistry of merocyanine 540 (MC 540), a sensitizing dye that binds preferentially to leukemia and electrically excitable cells, has been investigated. MC 540-mediated photooxidation of histidine, arachidonate, and unsaturated phospholipid vesicles was assessed by spin label oximetry and shown to involve type II (singlet oxygen) chemistry. The dye was also shown to be a potent sensitizer of lipid peroxidation in a natural cell membrane, the erythrocyte ghost. Inhibition by azide, stimulation by 2H2O, and identification of the cholesterol product 5 alpha-cholest-6-ene-3 beta,5-diol in this system, all were consistent with singlet oxygen intermediacy. Finally, MC 540 was found to be considerably more phototoxic to K-562 leukemia cells in 2H2O than in H2O. We conclude that singlet oxygen plays a major role in the phototherapeutic effects of this dye.

Cell Line↗
PubMed1986

Inhibition of cell membrane lipid peroxidation by cadmium- and zinc-metallothioneins.

The effects of all-zinc metallothionein (Zn-metallothionein) and predominantly cadmium metallothionein (Cd/Zn-metallothionein) on free radical lipid peroxidation have been investigated, using erythrocyte ghosts as the test system. When treated with xanthine and xanthine oxidase, Zn-metallothionein and Cd/Zn-metallothionein underwent thiolate group oxidation and metal ion release that was catalase-inhibitable, but superoxide dismutase-non-inhibitable. Similar treatment in the presence of ghosts and added Fe(III) resulted in metallothionein oxidation that was significantly inhibited by superoxide dismutase. Ghosts incubated with xanthine/xanthine oxidase/Fe(III) underwent H2O2- and O2--dependent lipid peroxidation, as measured by thiobarbituric acid reactivity. Neither type of metallothionein had any effect on xanthine oxidase activity, but both strongly inhibited lipid peroxidation when added to the membranes concurrently with xanthine/xanthine oxidase/iron. This inhibition was far greater and more sustained than that caused by dithiothreitol at a concentration equivalent to that of metallothionein thiolate. Significant protection was also afforded when ghosts plus Cd/Zn-metallothionein or Zn-metallothionein were preincubated with H2O2 and Fe(III), and then subjected to vigorous peroxidation by the addition of xanthine and xanthine oxidase. These results could be mimicked by using Cd(II) or Zn(II) alone. Previous studies suggested that Zn(II) inhibits xanthine/xanthine oxidase/iron-driven lipid peroxidation in ghosts by interfering with iron binding and redox cycling. Therefore, the primary determinant of metallothionein protection appears to be metal release and subsequent uptake by the membranes. These results have important implications concerning the antioxidant role of metallothionein, a protein known to be induced by various prooxidant conditions.

Catalase↗
PubMed1986

Xanthine oxidase-catalyzed crosslinking of cell membrane proteins.

Isolated erythrocyte membranes exposed to protease-free xanthine oxidase plus xanthine and ferric iron undergo lipid peroxidation and protein crosslinking (appearance of high molecular weight aggregates on sodium dodecyl sulfate (SDS) gel electrophoresis). Spectrin is more susceptible to crosslinking than the other polypeptides. Thiol-reducible bonds (disulfides) as well as nonreducible bonds are generated, the former type relatively rapidly (detected within 10-20 min) and the latter type more slowly (usually detected after 1 h). Reducible crosslinking is inhibited by catalase, but not by superoxide dismutase, desferrioxamine, butylated hydroxyltoluene, and mannitol; whereas nonreducible crosslinking, like free radical lipid peroxidation, is inhibited by all of these agents except mannitol. Zinc(II) also inhibits lipid peroxidation, but stimulates disulfide bond formation to the virtual exclusion of all other crosslinking. Our results indicate that disulfide formation is dependent on H2O2, but not O2- or iron. However, O2-, H2O2, and iron are all required for lipid peroxidation and nondisulfide crosslinking, suggesting the intermediacy of OH generated via the iron-catalyzed Haber-Weiss reaction. The possible role of malonaldehyde (MDA, a by-product of lipid peroxidation) in the latter type of crosslinking was examined. Solubilized samples of xanthine/xanthine oxidase-treated membranes showed a strong visible fluorescence (emission maximum 450 nm; excitation 390 nm). This resembled the fluorescence of membranes treated with authentic MDA, which forms conjugated imine linkages between amino groups. Fluorescence scanning of SDS gels from MDA-treated membranes showed a strong signal coincident with crosslinked proteins and also one in the low molecular weight, nonprotein region, suggestive of aminolipid conjugates. Similar scanning on xanthine/xanthine oxidase-reacted membranes indicated that all fluorescence is associated with the lipid fraction. Thus, nonreducible protein crosslinks in this system do not appear to be of the MDA-derived, Schiff base type.

Catalysis↗
PubMed1985

Phototoxic effects of hematoporphyrin derivative and its chromatographic fractions on hormone-producing human malignant trophoblast cells in vitro.

The in vitro phototoxicity of HPD on malignant cells relative to normal cells has been examined. Two human malignant cell lines were studied: the BeWo line of choriocarcinoma cells, which secrete the tumor marker human chorionic gonadotropin (hCG) and its alpha-subunit; and the CaSki line of human cervical carcinoma cells, which secrete hCG and its beta-subunit. Trophoblast-derived, hCG-secreting cells from human amniotic fluid were used as normal controls. In all experiments with HPD plus light, a close correlation was found 24 h after light between cell number and RIA-detectable marker concentration in the medium. Phototoxicity was greater when HPD was introduced in serum-free rather than serum-containing medium. No toxicity was observed in light and dark controls. Cells in Leighton tubes were incubated 1 h with HPD (25 micrograms/ml) in serum-free medium, then rinsed and incubated with medium containing 10% serum. At 2 and 3 days after contact with HPD, flasks were exposed to cool white fluorescent light (1 mW/cm2) for 5 min. Viable cell counts taken 1 day after the light dose indicated that HPD is significantly more phototoxic to BeWo than to CaSki cells; and that both malignant cell types are more photosensitive than amniotic fluid cells, presumably because the latter retain HPD less effectively. In another aspect of this work BeWo cells were used as a model system for comparing the phototoxic effects of the fast (F) and slow (S) eluting fractions of HPD obtained by Bio-Gel P-10 chromatography. Cells in light-shielded tubes were sensitized by incubating with porphyrins for 20 h in media containing 10% calf serum. At 2, 3, or 4 days after removal of porphyrin, with daily replacement of serum-containing medium, flasks were irradiated (see above), and then incubated in the dark for 2 to 4 additional days. Daily culture fluids were analyzed for hormone levels (hCG alpha), and cell counts were performed 2 or 3 days after the light dose. HPD-S (25 micrograms/ml) had no effect on either hormone secretion or cell viability in any of the flasks, whether exposed to light or not. HPD-F at low concentrations (0.25 or 2.5 micrograms/ml) had no detectable effect on cell count or hormone secretion in irradiated flasks. At 10 micrograms/ml, HPD-F was innocuous in dark controls, but caused a large decrease in cell count and hormone output in irradiated flasks.(ABSTRACT TRUNCATED AT 400 WORDS)

Amniotic Fluid↗
PubMed1985

Lipid photooxidation in erythrocyte ghosts: sensitization of the membranes toward ascorbate- and superoxide-induced peroxidation and lysis.

The damaging effects of ascorbate (AH-) and superoxide (O-2) on resealed erythrocyte ghosts containing predetermined levels of lipid hydroperoxides (LOOHs) have been studied. Continuous blue light irradiation of membranes in the presence of protoporphyrin resulted in steadily increasing LOOH levels and enhanced release of a trapped marker, glucose 6-phosphate (G6P), after a 3- to 4-h lag. Neither superoxide dismutase (SOD) nor catalase inhibited these effects, ruling out O-2 and H2O2 as reactive intermediates. A 1-h light dose produced partially photoperoxidized ghosts, which, in the dark at 37 degrees C, released G6P no faster than unirradiated controls (approximately 7%/h). When xanthine oxidase plus xanthine (XO/X) was introduced as a source of O-2 and H2O2, the irradiated membranes lysed rapidly (t1/2 approximately 2 h). EDTA or SOD inhibited the reaction, whereas catalase had little or no effect. Unirradiated ghosts were not lysed by XO/X unless the system was supplemented with Fe(III), in which case total protection was afforded by SOD or catalase. In all experiments there was an excellent correlation between postirradiation lipid peroxidation (thiobarbituric acid reactivity) and G6P release. Similar observations were made with AH-. For example, dark incubation of photooxidized ghosts in the presence of 0.5 mM AH- resulted in rapid lysis (t1/2 approximately 1 h), which was stimulated approximately twofold by 50 microM Fe(III) and was inhibited by EDTA. By comparison, unirradiated ghosts showed no net peroxidation or lysis after 3 h exposure to Fe(III)/AH-. Neither SOD nor catalase protected against AH--stimulated damage. AH--promoted lipid peroxidation was inhibited by butylated hydroxytoluene, a lipophilic antioxidant, but was unaffected by 2,5-dimethylfuran or ethanol, singlet oxygen, and hydroxyl radical traps, respectively. These results suggest that a mechanism exists by which photogenerated LOOHs undergo redox metal-mediated reduction to alkoxy radicals (LO.), which trigger a burst of membrane-disrupting lipid peroxidation.

Ascorbic Acid↗
PubMed1985

Mechanisms of lipid peroxidation.

This article provides an overview of how peroxidation of unsaturated lipids takes place and how it can be measured. Several different aspects of free-radical-mediated lipid peroxidation are discussed, including: the catalytic role of chelated iron and other redox metal ions; induction by reducing agents such as superoxide, ascorbate, and xenobiotic free radicals; suppression by antioxidant chemicals and enzymes; and how peroxidation that depends on pre-existing hydroperoxides (lipid hydroperoxide-dependent initiation of lipid peroxidation) can be distinguished from that which does not (lipid hydroperoxide-independent initiation of lipid peroxidation). Attention is also given to non-radical, singlet oxygen-driven peroxidation and how this can be resolved from radical-driven processes.

Animals↗
PubMed1985

Inhibitory effect of zinc(II) on free radical lipid peroxidation in erythrocyte membranes.

New evidence in support of zinc's role as a membrane antioxidant is presented. Human erythrocyte membranes in buffered saline underwent catalase- and superoxide dismutase-inhibitable lipid peroxidation when incubated with xanthine, xanthine oxidase, and Fe(III). Free radical mediated peroxidation was measured in terms of thiobarbituric acid reactivity and iodometric determination of lipid hydroperoxides. Whereas Ca(II) had relatively little effect on lipid peroxidation, Zn(II) strongly inhibited the reaction and suppressed peroxidation-dependent lysis of resealed membranes. Inhibition of lipid peroxidation was essentially complete in the presence of 0.1 mM Zn(II), a concentration equivalent to that of added Fe(III). By contrast, Zn(II) had no effect on rose bengal-photosensitized lipid peroxidation, a predominantly nonradical, singlet oxygen-driven process. Zinc(II) also interfered with xanthine/xanthine oxidase/iron-induced peroxidation of Triton X-100-dispersed membranes, but had no effect if EDTA was present. Trivial reasons for inhibition, for example, inactivation of xanthine oxidase or complex formation with O2-, were ruled out by showing that the rate of reduction of cytochrome c by xanthine/xanthine oxidase is not affected by Zn(II). We speculate that Zn(II) acts by interfering with the redox cycling of iron, possibly by competing with the latter for membrane binding sites.

Calcium↗
PubMed1984

Damaging effects of oxygen radicals on resealed erythrocyte ghosts.

Resealed ghosts of human erythrocytes are sensitive to oxidative damage induced by xanthine oxidase acting on xanthine in the presence of iron. Damage was assessed in terms of lipid peroxidation and increased permeation of trapped markers, Na+ and glucose-6-P. Key findings are as follows. (a) Marker efflux from xanthine/xanthine oxidase/iron-treated ghosts accelerated after a lag, Na+ emerging far ahead of glucose-6-P. (b) Both effluxes and lipid peroxidation were stimulated by Fe(III) in a dose-dependent fashion and inhibited by chelating agents. (c) The antioxidant butylated hydroxytoluene effectively halted lipid peroxidation and net glucose-6-P efflux, but slowed Na+ efflux only partially. (d) Lipid peroxidation and marker release could be completely inhibited by superoxide dismutase or catalase, indicating that O2- and H2O2 are both required, possibly as precursors of OH. via the iron-catalyzed Haber-Weiss reaction (O2- + H2O2 leads to OH- + OH. + O2). (e) OH. scavengers, e.g. ethanol, mannitol, choline, had no protective effect against marker efflux and lipid peroxidation. Yet these agents did intercept OH. in the bulk medium, since they inhibited the degradation of 2-deoxyribose added as an extramembranous OH. probe. It is proposed that OH. produced on the membrane at iron binding sites reacts so rapidly with target molecules that scavengers cannot compete. (f) Desferrioxamine abolished all effects, including net egress of Na+. EDTA, while totally inhibitory toward lipid peroxidation and glucose-6-P release, diminished Na+ release partially, changing it to first order, approximately 3-fold faster than background. The latter response was totally inhibited by catalase, but only marginally by superoxide dismutase. This and other evidence suggests that different forms of membrane damage are responsible for enhanced permeation of the two markers; although glucose-6-P depends on lipid peroxidation, Na+ does not, certainly when EDTA is present.

Allopurinol↗
PubMed1984

Superoxide and hydrogen peroxide-dependent lipid peroxidation in intact and triton-dispersed erythrocyte membranes.

Isolated erythrocyte membranes incubated with xanthine, xanthine oxidase, and Fe(III) underwent lipid peroxidation, as indicated by the thiobarbituric acid reaction and iodometric determination of hydroperoxides. In detergent-free medium (phosphate buffered saline) peroxidation was inhibited by superoxide dismutase, catalase, and EDTA; but was promoted by OH. scavangers, eg. mannitol. Generation of OH. in the system via iron-catalyzed reduction of H2O2 by O-2 was demonstrated by EPR spectrometry using spin trapping. In membranes treated with Triton X-100 lipid peroxidation was stimulated by EDTA and suppressed by OH. traps. This and other evidence suggests that OH. in the medium was an effective initiator of lipid peroxidation in detergent-dispersed membranes, but not in intact membranes.

Catalase↗
PubMed1983

Photodynamic action of protoporphyrin on resealed erythrocyte membranes: mechanisms of release of trapped markers.

Photoactivation of protoporphyrin IX (PP) bound to resealed human erythrocyte (RBC) ghosts results in membrane damage which is manifested by the release of trapped markers Na+ and glucose-6-phosphate (G6P). Efflux of Na+ was rapid, continuous, and virtually complete before the onset of G6P efflux. The sugar phosphate emerged abruptly after a long lag. The antioxidant butylated hydroxytoluene (BHT) had no effect on the permeation of Na+, but greatly suppressed that of G6P. These results suggest that the markers are emitted via different mechanisms. For G6P, disruption of the bilayer by free radical lipid peroxidation appears to be necessary, inasmuch as BHT inhibited peroxidation as measured by thiobarbituric acid reactivity and appearance of phospholipid and cholesterol hydroperoxides on thin layer chromatograms. It is deduced that non-lipid damage is sufficient for Na+ release. This effect is manifested at low light intensities and low PP concentrations. Protein regulators of passive cation permeability may be the primary targets in this case. When sensitive sulfhydryl groups on these proteins were blocked with p-chloromercuri-benzene-sulfonate, Na+ leaked out rapidly, but G6P was unaffected, thereby mimicking the early stages of membrane photodamage.

Cell Membrane Permeability↗
PubMed1982

Lysis of resealed erythrocyte ghosts by photoactivated tetrapyrroles: estimation of photolesion dimensions.

1. Resealed erythrocyte ghosts containing Na+ and glucose-6-P (G6P) as markers of membrane integrity were used as a model system for probing the damaging effects of photoactivated tetrapyrroles on cell membranes. 2. Continuous blue-light irradiation of bilirubin (BR)-sensitized and protoporphyrin (PP)-sensitized ghosts made them progressively more permeable to Na+, the cation emerging well ahead of G6P. 3. G6P efflux occurred abruptly after a lag period and resembled an all-or-none process. 4. These and other results suggest that a relatively subtle structural modification (possibly in some crucial protein(s) is sufficient for Na+ release, whereas gross disruption of the bilayer (probably by free-radical lipid peroxidation) is necessary for G6P release. 5. The dimensions of the G6P-releasing photolesions were estimated by density floatation centrifugation, using saccharides of increasing molecular size. Both BR and PP produced pores greater than 11 A but less than 42 A in diam., which is considerable smaller than the size range estimated in hypotonically lysed ghosts.

Cell Membrane Permeability↗
PubMed1980

Photosensitized cross-linking of erythrocyte membrane proteins. Evidence against participation of amino groups in the reaction.

Exposure of human erythrocyte ghosts (pH 8, 10 degrees C) to visible light in the presence of the photosensitizer, methylene blue, results in a relatively rapid loss of spectrin (bands 1 and 2 on sodium dodecyl sulfate gel electropherograms) and the appearance of high molecular weight cross-linked derivatives. Isolated spectrin also undergoes photosensitized cross-linking, indicating that the reaction is not lipid-dependent. Extensive cross-linking was neither reversed by dithiothreitol nor prevented by prior blocking of SH groups with N-ethylmaleimide, suggesting that cysteine residues are not crucial bridging sites. The possible requirement for NH2 groups, as suggested by previous model studies (Dubbelman, T.M.A.R., de Goeij, A.F.P.M. and van Steveninck, J. (1978) Biochim. Biophys. Acta 511, 141--151), was tested. Succinylation of spectrin protected against cross-linking, but this effect is attributed to the disruption of quaternary structure, as deduced from sedimentation measurements. However, virtually complete blocking of NH2 groups by amidination perturbed overall structure relatively little, and had no effect on cross-linking. Moreover, exogenous amines such as ethylamine, added in large excess to spectrin prior to irradiation, did not interfere with cross-link formation. These results suggest that NH2 groups are not involved in the reaction.

Cross-Linking Reagents↗