Lipid Peroxidation-Dependent Chemiluminescence from the

We also thank Dr. Thérèse Wilson, Dr. Rafael Radi, and Dr. Bruce Freeman for a ... Niu, Q., and Mendenhall, G. D. (1990) Structural effects on the y...
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Chem. Res. Toxicol. 1997, 10, 1090-1096

Lipid Peroxidation-Dependent Chemiluminescence from the Cyclization of Alkylperoxyl Radicals to Dioxetane Radical Intermediates Graham S. Timmins,§ Rosemeire E. dos Santos,‡ Adrian C. Whitwood,§ Luiz H. Catalani,‡ Paolo Di Mascio,† Bruce C. Gilbert,§ and Etelvino J. H. Bechara*,† Department of Chemistry, University of York, York, U.K., and Departamento de Bioquı´mica and de Quı´mica Fundamental, Instituto de Quı´mica, Universidade de Sa˜ o Paulo, CP 26077, 05599-970 Sa˜ o Paulo, Brazil Received May 7, 1997X

This work reveals a novel mechanism for triplet carbonyl formation (and hence chemiluminescence) during lipid peroxidation, whose chemiluminescence has been attributed to both triplet carbonyls and singlet oxygen. As a model for polyunsaturated fatty acid hydroperoxides, we have synthesized 3-hydroperoxy-2,3-dimethyl-1-butene by photooxygenation of tetramethylethylene. One-electron oxidation of this hydroperoxide with heme proteins and peroxynitrite to the corresponding alkylperoxyl radical results in chemiluminescence, both direct and 9,10dibromoanthracene-2-sulfonate-sensitized, the latter attributed to the formation of triplet acetone. It is postulated that triplet acetone results from the cyclization of the alkylperoxyl radical to a dioxetane radical intermediate followed by its thermolysis. This is supported by EPR spin-trapping experiments in which discrimination between carbon-centered radicals derived from the alkyloxyl and alkylperoxyl radicals is achieved through the use of one-electron oxidants and reductants, e.g., FeII and TiIII.

Introduction The importance of the peroxidation of polyunsaturated fatty acids (PUFA)1 and their reactive products in aging and disease processes linked to oxidative stress has been well established by many groups. The chemiluminescence associated with lipid peroxidation has found widespread use in the sensitive detection of oxidative stress both in vitro and in vivo (1-4). It is known that at least two excited species are involved in this chemiluminescence: dimol red emission from singlet oxygen (λmax at 634 and 703 nm) and blue-green phosphorescence from triplet carbonyls (λmax at 450-550 nm). Singlet oxygen can be produced both by the quenching of triplet carbonyls by ground-state oxygen and by the annihilation of alkylperoxyl radicals (Russell reaction) (2-5). Indeed, the Russell reaction has been shown to form singlet oxygen in high yields (ca. 10%) (6). There remains, however, some debate as to the mechanisms of formation of the triplet carbonyl species. It has been proposed that the Russell reaction of alkylperoxyl radicals might also be responsible for triplet carbonyl formation in lipid peroxidation (Scheme 1a) (24), but the fact that the corresponding yield of triplet ketones is less than 0.01% (7), and that this is a * Address correspondence to this author. Tel: 55-11-8183869. Fax: 55-11-8155579. E-mail: [email protected]. § University of York. † Departamento de Bioquı´mica, Universidade de Sa ˜ o Paulo. ‡ Departamento de Quı´mica Fundamental, Universidade de Sa ˜o Paulo. X Abstract published in Advance ACS Abstracts, September 15, 1997. 1 Abbreviations: polyunsaturated fatty acid (PUFA), tetramethylethylene (TME), 3-hydroperoxy-2,3-dimethyl-1-butene (TMEOOH), 3,5dibromo-4-nitrosobenzenesulfonic acid (DBNBS), 5,5-dimethyl-1pyrroline N-oxide (DMPO), sodium 9,10-dibromoanthracene-2-sulfonate (DBAS), sodium 9,10-diphenylanthracene-2-sulfonate (DPAS), horseradish peroxidase (HRP).

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Scheme 1. Potential Sources of Triplet Ketones in Lipid Peroxidation: (a) Russell Reaction, (b) Alkyloxyl Radical Dismutation, and (c) Dioxetane Thermolysis

bimolecular reaction (less favored for short-lived intermediates such as radicals in biological systems, where only low steady-state radical concentrations are expected due to the many possible reactions with other substrates available at high concentrations in these systems), might make this pathway less likely. Similarly, the disproportionation of alkoxyl radicals (Scheme 1b), although producing up to a 10% yield of triplet carbonyls (8), would be even more disfavored than alkylperoxyl species, as alkoxyl radicals are much more reactive than alkylperoxyl radicals toward biological substrates and may also undergo intramolecular rearrangements (such as β-scission and 1,2 H-shifts) (9). Because the thermolysis of dioxetanes can produce high yields of triplet carbonyls (10-60%) (10), it has been proposed that the cycloaddition of singlet oxygen to PUFA, producing such dioxetanes, could provide a plausible mechanism for chemiluminescence (Scheme 1c) (1-4). However, it has been established that reaction of singlet oxygen with linoleic acid results almost exclusively in the formation of hydroperoxides and 1,4-endoperoxides, not the 1,2-cycloaddition products, dioxetanes (11). Nevertheless, the involvement of dioxetane in lipid peroxidation is worth pursuing as a plausible alternative explanation for the observed chemiluminescence. © 1997 American Chemical Society

Dioxetane from Peroxyl Radical Cyclization Scheme 2. Working Hypothesis for Dioxetane Formation in Lipid Peroxidation (left) and in One-Electron Oxidation of a Model Hydroperoxide (TMEOOH) (right)

Dioxetane intermediates are hypothesized here to be formed by the cyclization of alkylperoxyl radicals formed either during lipid peroxidation or by the one-electron oxidation of PUFA-derived hydroperoxides, PUFAOOH (Scheme 2). Herein, studies were pursued to determine whether such reactions and dioxetane chemiluminescence occur. The hydroperoxide formed by photooxidation of tetramethylethylene (TME), 3-hydroperoxy-2,3-dimethyl1-butene (TMEOOH), was used as a model for the reactions of PUFAOOH. Its reactions with a range of one-electron oxidants and reductants have been studied by (i) electron paramagnetic resonance spectroscopy (EPR, also known as electron spin resonance, ESR) following spin-trapping of free radical intermediates, (ii) chemiluminescence spectrophotometry to identify and quantify triplet carbonyl generation, and (iii) product analysis by thin layer chromatography (TLC) and gas chromatography (GC) to provide estimates of the contributions of the possible reaction pathways. The oneelectron oxidants studied have included CeIV, as well as heme proteins, and peroxynitrite (OONO-), which are both thought to play an important role in many diseases associated with oxidative stress. Heme proteins (such as horseradish peroxidase, HRP) can produce alkylperoxyl radicals through two sequential one-electron oxidations of hydroperoxide by compounds I and II (12), while peroxynitrite (or its conjugate peroxynitrous acid, HOONO), formed by the reaction of nitric oxide with superoxide, reacts with the hydroperoxide either directly, by formation of a hydroxyl radical-like species, or through a CO2-derived intermediate (13-15). The spin-traps used were 5,5-dimethyl-1-pyrroline N-oxide (DMPO) which forms stable adducts with a wide range of species (such as carbon-centered, alkoxyl, and alkylperoxyl radicals) and 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS), a nitroso trap that can provide greater information due to additional splitting(s) from the added radical (16, 17).

Materials and Methods All reagents used were commercial samples of the highest available purity from Sigma Ltd. (except Chelex, which was obtained from BioRad) and used as supplied, except DBNBS (17), TMEOOH (11), and peroxynitrite (18) which were synthe-

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1091 sized as previously described. DMPO was purified by treatment with activated charcoal. All buffers used in the EPR experiments were treated with Chelex to remove transition metal contaminants; otherwise, water deionized with Millipore MilliQ equipment (Bedford, MA) was used. When using alkaline peroxynitrite solutions, the prereaction mixture was titrated with HCl to produce the required pH after its addition. Peroxynitrite was quantified by spectrophotometry at 302 nm ( ) 1670 M-1 cm-1) (12). Sodium 9,10-dibromoanthracene-2-sulfonate (DBAS) and sodium 9,10-diphenylanthracene-2-sulfonate (DPAS) were synthesized by the procedure of Catalani et al. (DBAS at 405 nm ) 7750 M-1 cm-1 and DBAS at 397 nm ) 8050 M-1 cm-1) (19). Caution: Distillation of peroxides can result in explosion. All preparations should be of small quantities and the distillation performed carefully in suitably armored/shielded fume hoods. If not thoroughly acquainted with such procedures, then other purification techniques, such as column chromatography, must be used. TMEEOH Preparation. TMEOOH was prepared by photooxygenation of TME by a modification of the procedure used by Di Mascio et al. to synthesize tetraethylethylene-derived peroxide (11). TME (2 g, 24 mmol) was dissolved in 30 mL of CH2Cl2 containing a few crystals of tetraphenylporphine ( 102) (10). Moreover, 10 µM HRP could be replaced by 10 µM hematin or cytochrome c without significant changes in the maximum intensity of chemiluminescence elicited by 10 mM TMEOOH. Hemoglobin and myoglobin were less efficient as oxidants (not shown), in agreement with the well-known reactivity of hematin and heme proteins with hydroperoxides to yield peroxyl radicals. Although the chemiluminescent data provide strong evidence for the formation of a triplet ketone, this species is not unambiguously identified in these experiments as the triplet acetone resulting from thermolysis of dioxetane or its radical. Product Analysis. The products of the reaction of TMEOOH with HRP at different concentrations were studied using TLC and GC. The yield of acetone was of particular interest as this species is only expected to be produced from the dioxetane intermediate III, formed by the cyclization of TMEOO• radical (II). The yield of acetone (retention time 0.52 min) when 10 mM TMEOOH was treated with 1 µM HRP was 18%. This yield dropped to only 6% with 10 µM HRP [reaction conducted in 50 mM phosphate buffer (pH 7.4) for 10 min]. TLC analysis of 2,4-dinitrophenylhydrazine products of this spent reaction mixture contained a hydrazone that cochromatographed with a standard prepared from acetone (Rf ) 0.35), supporting the GC analysis. Polarographic studies showed that the reaction does not consume oxygen, and nitrogen-purging did not affect the DBAS-sensitized chemiluminescence intensity from the standard reaction mixture. That HRP reacted with TMEOOH was confirmed by the observed shift and intensification (13%) of the native HRP Soret band at 403 nm ( ) 1.02 × 105 M-1 cm-1) to 420 nm, the λmax of HRP compound II ( ) 1.05 × 105 M-1 cm-1; data not shown) (21). It can be seen that there is a strong codependence upon HRP concentration (and hence “steady-state concentration” of TMEOO• radical) of the yield of the different RH2C• species, the yield of triplet acetone, and the yield of acetone in the reaction mixture. Altogether, these data provide strong evidence for the formation and role in chemiluminescence of the dioxetanyl radical III formed by cyclization of the TMEOO• radical (Scheme 3).

Timmins et al.

Since TMEOOH may provide a simple model for studying PUFA peroxidation (Scheme 2), it is postulated that the chemiluminescence observed upon lipid peroxidation may also arise from cyclization-derived dioxetane intermediates. Here the alkylperoxyl radical precursor might be formed either by reaction of carbon-centered PUFA radicals with oxygen or via oxidation of PUFAOOH to PUFAOO• radical species by heme or heme proteins. It is also apparent that the dioxetane-forming pathway provides a mechanism for the amplification of lipid peroxidation, since it will produce both a free radical species (either directly or by hydrogen abstraction by the dioxetane radical III) and potentially high levels of triplet carbonyl (yield of up to 60%) (10), which itself has a reactivity similar to an alkoxyl radical and is able to further abstract hydrogen from PUFA thus propagating PUFA peroxidation (22-24). Reactions of OONO-/HOONO with TMEOOH. Since study of the reactions of TMEOOH has indicated that this system can provide useful information as to the types and relative rates of radical species formed by oxidants and reductants, we have used this system to study the reactivity of peroxynitrite (an important biological oxidant) with hydroperoxides, to determine how it might be involved in lipid peroxidation. Peroxynitrite and its conjugate peroxynitrous acid (HOONO, pKa 6.8) are formed by the reaction of nitric oxide and superoxide (k2 ) 6.9 × 109 M -1 s-1) and are important sources of oxidative stress in vivo, capable of reacting with nucleic acids, proteins, and PUFA to exert deleterious effects (13-15, 25-28). It is thought that nitric oxide can exert an antioxidant activity (in the absence of superoxide) and that the ratio of nitric oxide to peroxynitrite modulates the damaging or protective effects of nitric oxide (29). The term OONO-/HOONO will be used for the mixture of peroxynitrite and peroxynitrous acid found between pH 5 and 9, with the specific term only being used when the reactive species is critical. Upon protonation of peroxynitrite, peroxynitrous acid is formed which decomposes rapidly (k ) 0.6 s-1) to yield nitrate (in ca. 70% yield) and a species with hydroxyl radical-like reactivity (in ca. 30% yield, thought to be responsible for much of the activity of OONO-/HOONO) although its identity remains uncertain; ground-state OONO-/HOONO itself can also oxidize some substrates such as cytochromes, thiols, and ascorbate (13, 14, 25). It has also recently been shown that peroxynitrite can react via a CO2-derived radical species (15). Although much evidence suggests that peroxynitrous acid and its hydroxyl radical-like decomposition product are responsible for the reactivity of OONO-/HOONO, it remains unclear which of the species in OONO-/HOONO can be reactive and whether peroxynitrite itself initiates lipid peroxidation. Therefore the reactions of these species with TMEOOH were studied. Upon the reaction of OONO-/HOONO acid with TMEOOH in the presence of DBNBS (pH 7.4) a spectrum (Figure 4) was obtained, showing the presence of radical adducts that were assigned as in the case of HRP (i.e., methyl X, two primary carbon-centered radicals III/VIII and XIII, and tertiary carbon-centered radical XII), indicating that OONO-/HOONO can one-electron oxidize TMEOOH to TMEOO• radical (II). No signals were observed in the absence of any one component of the reacting system. DMPO was not used as a radical trap here because of its known reactivity with OONO-/ HOONO to yield stable adducts (26).

Dioxetane from Peroxyl Radical Cyclization

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1095

Figure 4. EPR spectrum of TMEOOH-derived radical adducts to DBNBS obtained upon one-electron oxidation by peroxynitrite. Reaction mixture was 20 mM TMEOOH, 10 mM DBNBS, and 1 mM peroxynitrite in 100 mM phosphate buffer (pH 7.4) at 20 °C. Spectrometer settings as in Figure 1B.

Figure 6. Chemiluminescence spectrum of the TMEOOH/ peroxynitrite system. Reaction conditions: 2.2 mM TMEOOH and 2.3 mM ONOO- in aqueous medium at 25 °C. See Materials and Methods.

Figure 5. Ratio of the DBNBS adducts of [III and/or VIII] to [XIII] generated by peroxynitrite oxidation of TMEOOH concentration vs the reaction pH. Reaction conditions: 50 mM TMEOOH, 20 mM DBNBS, and 1 mM peroxynitrite in 100 mM phosphate buffer (pH 7.4) at 20 °C. Spectrometer settings as in Figure 1C.

When the pH of the reaction mixture was varied (using NaH2PO4/Na2HPO4, pKa 6.8), a dependence of the ratio of radical adducts III/VIII to XIII upon pH could be determined (Figure 5), analogous to the dependence upon [HRP] in Figure 2. It can be seen that there is a strong correspondence between the relative ratio of adducts of III and/or VIII to XIII and the calculated percentage of OONO-/HOONO present in the peroxynitrous acid form. Since this indicates that the concentration of the alkylperoxyl radical II (and hence rate of reaction with TMEOOH) is lower at acidic pH, then the peroxynitrite itself must be capable of oxidizing TMEOOH to II and at a more rapid rate than the decomposition of peroxynitrous acid. Since at physiological pH (near 7.4 for most tissues) 80% of OONO-/HOONO will be in the anion form, it is possible that the oxidation of low endogenous levels of PUFAOOH to PUFAOO• radical by OONO-/ HOONO is responsible for the metal-independent induction of lipid peroxidation by OONO-, and this would be a consequence of OONO- and not HOONO. Chemiluminescence studies with TMEOOH were also conducted with OONO-/HOONO as the oxidizing species. A 0.68 mL aliquot of 113 mM peroxynitrite was added to 8.0 mL of 10 mM TMEOOH in aqueous medium. The observed chemiluminescence intensity integrated over the whole spectrum (2.5 × 105 cps) was at least 2 orders of magnitude more intense than that in the presence of HRP, even in the absence of fluorescers like DBAS. The

chemiluminescence spectrum arising from the treatment of TMEOOH with OONO-/HOONO has a maximum at about 450 nm (Figure 6), as expected for acetone phosphorescence (30). The maximum direct chemiluminescence intensity from the reaction of 1 mM TMEOOH and 1 mM peroxynitrite was 6.0 × 104 cps, and it was 1.2 × 105 cps in the presence of 100 µM DBAS. In both cases, chemiluminescence emission decayed exponentially to basal levels within 5 min. Upon addition of 0.3 mM sorbate DBAS-sensitized reaction mixtures, chemiluminescence was quenched by 50%. Thus it can be assumed that the one-electron oxidation of TMEOOH by peroxynitrite to yield the dioxetane radical III follows the same oxidation mechanism as proposed for HRP. Noteworthy in this respect is the possibility that direct oxygentransfer from TMEOOH to peroxynitrite may also generate singlet oxygen as shown previously with hydrogen peroxide as acceptor (27).

Conclusions It has been shown that one-electron oxidation of TMEOOH results in the formation of a variety of radical species, chemiluminescence, and acetone formation. The observed codependence of the nature of the radical intermediates, the yield of chemiluminescence, and the yield of acetone upon HRP concentration, taken together with the nature of the chemiluminescence, provide strong evidence for a pathway constituting a route to triplet acetone chemiluminescence via cyclization of alkylperoxyl radical to dioxetane species. With TMEOOH serving as a model for PUFA peroxidation, it is concluded that cyclization of PUFAOO• radical to chemiluminescent dioxetane species may occur in concert with the Russell reaction. This mechanism may thus be an important route to the lipid peroxidation-associated chemiluminescence both in vivo and in vitro, rather than the cycloaddition of singlet oxygen to form dioxetanes (already disproved by recent studies) (11). Moreover, we demonstrate that heme proteins such as plant peroxidases and peroxynitrite will act as one-electron oxidants of PUFA hydroperoxides. It has also been shown that the peroxynitrite anion may directly induce lipid peroxidation without the involvement of peroxynitrous acid-derived species.

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Acknowledgment. The authors are grateful to the Fundac¸ a˜o de Amparo Åa Pesquisa do Estado de Sa˜o Paulo (FAPESP), the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), the Programa de Apoio ao Desenvolvimento Cientı´fico e Tecnolo´gico (PADCT), the EPSRC and Association for International Cancer Research (provision of EPR facilities), the Coordenac¸ a˜o de Aperfeic¸ oamento de Pessoal de Nı´vel Superior (CAPES), the British Council for supporting a collaboration between the Universities of York and Sa˜o Paulo, the Yorkshire Cancer Research Campaign (fellowship to G.S.T.), and the John Simon Guggenheim Memorial Foundation (fellowship to E.J.H.B.). We thank Mr. Sı´lvio T. Sasaki for helping with the preparation of peroxynitrite, Mr. De´cio F. Lima for helping to perform the GC analyses, and Dr. Iseli L. Nantes for advice in the TMEOOH/heme protein studies. We also thank Dr. The´re`se Wilson, Dr. Rafael Radi, and Dr. Bruce Freeman for a critical reading of this manuscript.

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