Gary J. Bachowski

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.

PHOTOPEROXIDATION OF CHOLESTEROL IN HOMOGENEOUS SOLUTION, ISOLATED MEMBRANES, AND CELLS: COMPARISON OF THE 5α‐ AND 6β‐HYDROPEROXIDES AS INDICATORS OF SINGLET OXYGEN INTERMEDIACY

Abstract— Singlet oxygen (1O2) can react with cholesterol (Ch) to give three possible ene‐addition hydroperoxides: 3β‐hydroxy‐5α‐cholest‐6‐ene‐5‐hydroperoxide (5α‐OOH), 3β‐hydroxycholest‐4‐ene‐6α‐hydroperoxide (6α‐OOH), and 3β‐hydroxycholest‐4‐ene‐6β‐hydroperoxide (6β‐OOH). The rates of dye‐sensitized photogeneration and also the fates of 5α‐OOH and 6β‐OOH in membrane bilayers have been studied and compared. Irradiation of unilamellar [14C]Ch/phospholipid vesicles in the presence of aluminum phthalocyanine tetrasulfonate or merocyanine 540 resulted in formation of 5α‐OOH and 6β‐OOH, as determined by high performance liquid chromatography with radiochemical or electrochemical detection. The initial rate of 6β‐OOH formation was 335% that of 5α‐OOH in a variety of liposomal systems. However, after a lag, 5α‐OOH invariably decayed via allylic rearrangement to 7α‐OOH (also known to be a free radical product), whereas 6β‐OOH accumulated in unabated fashion until Ch depletion became limiting. Photooxidation of Ch in an isolated natural membrane (erythrocyte ghost) or in L1210 leukemia cells gave similar results. When the reaction was carried out in pyridine or methanol, the rate of 6β‐OOH formation relative to 5α‐OOH was reduced by approximately half, with essentially no isomerization of the latter to 7α‐OOH. These results suggest that (i) environmental factors in a lipid bilayer somehow make photogeneration of 6β‐OOH more favorable than in homogeneous solution; and (ii) due to its relative stability, 6β‐OOH may be a more reliable probe of 1O2 intermediacy than 5α‐OOH.

Chromatographic separation and electrochemical determination of cholesterol hydroperoxides generated by photodynamic action

Reverse-phase HPLC with electrochemical detection (HPLC-EC) was used to separate and quantitate photochemically generated cholesterol hydroperoxides. The EC measurements were performed in the reduction mode under anaerobic conditions. When cholesterol-containing liposomes were irradiated in the presence of a phthalocyanine dye, at least four major oxidation products of cholesterol were detected by HPLC-EC:5 alpha-hydroperoxide (5 alpha-OOH), 6 beta-hydroperoxide (6 beta-OOH), 7 alpha-hydroperoxide (7 alpha-OOH), and 7 beta-hydroperoxide (7 beta-OOH). The detection limit for each compound was found to be approximately 25 pmol. Product identification was based on matching HPLC and TLC behavior of standards and on physical indicators (melting points and NMR chemical shifts). The cholesterol hydroperoxides were barely separated from EC-silent diol derivatives, which could be detected by 210 nm absorbance after reduction of the hydroperoxides with triphenylphosphine. Irradiation of a dye-sensitized natural membrane, the human erythrocyte ghost, also resulted in formation of 5 alpha-OOH, 6-OOH, and 7-OOH, as evidenced by HPLC-EC. Under the chromatographic conditions used, these species were well separated not only from one another but also from a family of at least six phospholipid hydroperoxides. These results illustrate the strengths of HPLC-EC as a relatively convenient, sensitive, and selective means of analyzing cholesterol hydroperoxides in biological samples.

PHOTOSENSITIZED LIPID PEROXIDATION AND ENZYME INACTIVATION BY MEMBRANE‐BOUND MEROCYANINE 540: REACTION MECHANISMS IN THE ABSENCE AND PRESENCE OF ASCORBATE*

The lipophilic photosensitizing dye merocyanine 540 (MC540) is being studied intensively as an antitumor and antiviral agent. Since plasma membranes are believed to be the principal cellular targets of MC540‐mediated photodamage, we have studied membrane damage in a well characterized test system, the human erythrocyte ghost. When irradiated with white light, MC540‐sensitized ghosts accumulated lipid hydroperoxides (LOOHs derived from phospholipids and cholesterol) at a rate dependent on initial dye concentration. Neither desferrioxamine nor butylated hydroxytoluene inhibited LOOH formation, suggesting that Type I (iron‐mediated free radical) chemistry is not important. By contrast, azide inhibited the reaction in a dose‐dependent fashion, implicating a Type II (singlet oxygen, 1O2) mechanism. Stern‐Volmer analysis of the data gave a 1O2 quenching constant ∼50 times lower than that determined for an extramembranous target, lactate dehydrogenase (the latter value agreeing with literature values). This suggests that 1O2 reacts primarily at its membrane sites of origin and that azide has limited access to these sites. Using [14C]cholesterol‐labeled membranes and HPLC with radiodetection, we identified 3β‐hydroxy‐5α‐cholest‐6‐ene‐5‐hydroperoxide as the major cholesterol photoproduct, thereby confirming 1O2 intermediacy. Irradiation of MC540‐sensitized membranes in the presence of added iron and ascorbate resulted in a large burst of lipid peroxidation, as shown by thiobarbituric acid reactivity and appearance of 7‐hydroperoxycholesterol and 7‐hydroxycholesterol as major oxidation products. Amplification of MC540‐initiated lipid peroxidation by iron/ ascorbate (attributed to light‐independent reduction of nascent photoperoxides, with ensuing free radical chain reactions) could prove useful in augmenting MC540s phototherapeutic effects.

Ascorbate-enhanced lipid peroxidation in photooxidized cell membranes: Cholesterol product analysis as a probe of reaction mechanism

Cholesterol was used as an in situ probe for studying mechanisms of lipid peroxidation in isolated erythrocyte membranes subjected to different prooxidant conditions. The membranes were labeled with [14C]cholesterol by exchange with prelabeled unilamellar liposomes and photosensitized with hematoporphyrin derivative. Irradiation with a dose of blue light resulted in thiobarbituric acid-detectable lipid peroxidation that was increased markedly by subsequent dark incubation with 0.5–1.0 mM ascorbate (AH−). Ascorbate-stimulated lipid peroxidation was inhibited by EDTA, desferrioxamine (DOX) and butylated hydroxytoluene (BHT), suggesting that the process is free radical in nature and catalyzed by membrane-bound iron. Thin layer chromatography and radiometric scanning of extracted lipids from photooxidized membranes revealed that the major oxidation product of cholesterol was the 5α-hydroperoxide (5α-OOH), a singlet oxygen adduct. Post-irradiation treatment with AH−/Fe(III) resulted in an almost-total disappearance of 5α-OOH and the preponderance of free radical oxidation products, e.g. 7-ketocholesterol, the epimeric 7α-/7β-hydroperoxides (7α-/7β-OOH) and their respective alcohols (7α-/7β-OH). EDTA, DOX and BHT inhibited the formation of these products, while catalase and superoxide dismutase had no effect. These results are consistent with a mechanism involving 1-electron reduction of photogenerated hydroperoxides to oxyl radical, which trigger bursts of free radical lipid peroxidation. Though generated in this system, partially reduced oxygen species, viz. superoxide, hydrogen peroxide and hydroxyl radical, appear to be relatively unimportant in the autoxidation process.

Photodynamic action of merocyanine 540 on erythrocyte membranes: structural perturbation of lipid and protein constituents

erocyanine 540 (MC540) is a membrane-directed photosensitizing dye with antileukemic and antiviral properties. In this study, biophysical and biochemical techniques have been used to examine MC540-sensitized photooxidative damage in the lipid and protein compartments of a test membrane, the human erythrocyte ghost. Irradiation of MC540-sensitized ghosts with white light resulted in oxidative damage to proteins, as manifested by (i) loss of sulfhydryl groups; (ii) intermolecular cross-linking of major polypeptides; and (iii) loss of Mg(2+)-ATPase and Na+,K(+)-ATPase activities. Photooxidation also produced a rapid and progressive increase in general protein motion, as measured by electron paramagnetic resonance spectrometry (EPR) with the sulfhydryl spin label MAL-6. In addition to these effects, ghosts exposed to MC540 and light underwent lipid peroxidation. EPR with two lipophilic spin probes, 5-doxylstearate and 16-doxylstearate, showed that lipid peroxidation is accompanied by a progressive decrease in bilayer fluidity (motional freedom). At a given dye concentration, structural perturbations of proteins were detected at much lower light fluences than those of lipids. When photoreactions were carried out in the presence of ascorbate and iron, there was a strong stimulation of lipid peroxidation (attributed to free radical chain reactions), with a concomitant greater decrease in lipid mobility. Thus, the deleterious effects of photoperoxidation on lipid structure and motional freedom were greatly exacerbated by ascorbate and iron. Membrane damage similar to that described here may play a role in the phototherapeutic activity of MC540.

CHARACTERIZATION OF LIPID HYDROPEROXIDES GENERATED BY PHOTODYNAMIC TREATMENT OF LEUKEMIA CELLS

A new technique, high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop (HPLC-EC), has been used for analyzing lipid hydroperoxide (LOOH) formation in photooxidatively stressed L1210 leukemia cells. Highly specific and sensitive for peroxides (detection limits <0.5 pmol for cholesterol hydroperoxides and <50 pmol for phospholipid hydroperoxides), this approach allows different classes of LOOH to be separated and determined in minimally damaged cells. L1210 cells in serum-containing growth medium were irradiated in the presence of merocyanine 540 (MC540), a lipophilic photosensitizing dye. Lipid extracts from cells exposed to a light fluence of 0.11 J/cm2 (which reduced clonally assessed survival by 30%) showed 12–15 well-defined peaks in HPLC-EC. None of these peaks was observed when cells were irradiated without MC540 or when dye/light-treated samples were reduced with triphenylphosphine prior to analysis. Three peaks of relatively low retention time (<12 min) were assigned to the following species by virtue of comigration with authentic standards: 3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide (5α-OOH), 3β-hydroxycholest-4-ene-6β-hydroperoxide (6β-OOH), and 3β-hydroxycholest-5-ene-7α/7β-hydroperoxide (7α/7β-OOH). Formation of 5α-OOH and 6β-OOH (singlet oxygen adducts) was confirmed by subjecting [14C]cholesterol-labeled cells to relatively high levels of photooxidation and analyzing extracted lipids by HPLC with radiochemical detection. Material represented in a major peak at 18–22 min on HPLC-EC was isolated in relatively large amounts by semipreparative HPLC and shown to contain phospholipid hydroperoxides (predominantly phosphatidylcholine species, PCOOH) according to the following criteria: (i) decay of 18–22 min peak during Ca2+/phospholipase A2 treatment, with reciprocal appearance of fatty acid hydroperoxides; (ii) reduction of peroxide during treatment with reduced glutathione and phospholipid hydroperoxide glutathione peroxidase, but not glutathione peroxidase; and (iii) comigration with PCOOH standards in thin-layer chromatography. HPLC-EC analysis revealed quantifiable amounts ofPCOOH and ChOOH at a light fluence that clonally inactivated <10% of the cells, which allows for the possibility that photoperoxidative damage plays a causal role in cell killing.

Phthalocyanine-sensitized lipid peroxidation in cell membranes: Use of cholesterol and azide as probes of primary photochemistry

Various phthalacyanine (Pc) derivatives of phototherapeutic interest have been shown to be efficient type II (singlet oxygen, 1O2) sensitizers in aqueous and non-aqueous solutions. However, primary Pc photochemistry in biological environments, e.g. cell membranes, has not been studied in a definitive manner. To address this question, we used endogenous cholesterol in the erythrocyte ghost as a mechanistic reporter lipid Membranes sensitized with chloroaluminum Pc tetrasulfonate (AlPcS) and exposed to white light at 10 degrees C underwent lipid peroxidation, as indicated by the accumulation of hydroperoxides and thiobarbituric acid reactivity. Specific analysis of cholesterol photo-products by thin layer chromatography and high performance liquid chromatography revealed the presence of 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), with much smaller amounts of 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide (7 alpha-OOH) and 5 alpha-cholest-6-en-3 beta, 5-diol and cholest-5-en-3 beta, 7 alpha-diol (5 alpha-OH and 7 alpha-OH). Identification of 5 alpha-OOH as a major photoproduct provides unambiguous evidence for large scale 1O2 intermediacy. Azide inhibited lipid peroxidation in a dose-dependent fashion, providing additional support for a type II mechanism. However, the 1O2 quenching constant from Stern-Volmer analysis was approximately 50 times lower than that determined for a non-membrane probe, lactate dehydrogenase. The latter value agreed with literature values. A probable explanation is that membrane-bound dye generates most of the 1O2 involved in lipid peroxidation. Although azide can intercept any 1O2 escaping into (or formed in) the medium, it has limited access to 1O2 generated on the membrane and reacting (or being quenched) near its site of origin.

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 5α-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 reactions products of cholesterol were formed, notably the 7α-, 7β-hydroperoxide pair. Membranes 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. In low concentrations, ascorbate exacerbated the damaging effects of photoperoxidation, switching the reaction from primarily singled oxygen- to oxygen radical-dependence, as indicated by cholesterol product analysis.

PORPHYRIN‐SENSITIZED PHOTOREACTIONS IN THE PRESENCE OF ASCORBATE: OXIDATION OF CELL MEMBRANE LIPIDS AND HYDROXYL RADICAL TRAPS

Abstract— Photooxidation reactions in ascorbate (AH)‐containing erythrocyte membrane suspensions have been studied in broad perspective by simultaneously monitoring lipid peroxidation in the membrane compartment and formation of hydrogen peroxide (H2O2) and hydroxyl radical (OH) in the aqueous compartment. Non‐bound uroporphyrin (UP) and membrane‐bound protoporphyrin (PP) were used as sensitizers. Photoreduction of UP to the radical anion (UP‐) was detected by electron spin resonance when UP/AH/membrane mixtures were irradiated anaerobically. Aerobic irradiation resulted in a strong AH‐‐stimulation of lipid peroxidation, H2O2 formation, and OH‐ generation (detected with 2‐deoxyribose (DOR) and the spin trap 5,5‐dimethyl‐l‐pyrroline‐N‐oxide). Use of diagnostic agents (e.g. catalase, desferrioxamine, mannitol) revealed that OH‐ is involved in light‐stimulated DOR oxidation, but not in lipid peroxidation. Similar irradiation in the presence of PP resulted in far greater lipid peroxidation than observed with UP, but less DOR oxidation, and insignificant accumulation of H2O2. This suggests that photoreduction of membrane‐bound PP is less efficient, possibly due to hindered access of AH‐.