Interest of photochemical methods for induction of lipid peroxidation

Biochimie (1994) 76, 355-368

355

© Soci6t6 frangaise de biochimie et biologic mol6culaire / Elsevier, Pads

Review

Interest of photochemical methods for induction of lipid peroxidation N Paillous, S Fery-Forgues Laboratoh'e des IMRCP, URA 470 au CNRS, UniversiM Paul Sabatiet; 118, route de Narbonne, 31062 Toulouse Cedex, France

(Received 30 April 1993; accepted 24 June 1993)

Summary w Lipid peroxidation, which plays a part in a wide variety of biological processes, is an integral process in the oxidation of unsaturated fatty acids v/a a radical chain reaction. Among the various species which may induce this reaction in vivo, reactive forms of oxygen such as peroxide anion, the hydroxyl radical and singlet oxygen are of cardinal importance. These species may be generated enzymatically, chemically or by various radiochemical and photochemical reactions. We present here the advantages of photochemical induction of peroxidation, and we describe the principles of the reactions, the photosensitizers that can be employed to generate the various reactive species of oxygen, and the techniques, direct (ESR) or indirect (specific traps), used to detect the reactive species. Photosensitization can induce the formation of a whole gamut of products of lipid peroxidation: conjugated dienes, aldehydes, hydroperoxides, etc. The relative proportions of the various hydroperoxides of fatty acids or cholesterol depend on the nature of the reactive species involved. Utilization of photochemical reactions is an effective and clean way of inducing peroxidation, allowing fine control of both initiation and orientation.

photosensitization / UV / superoxide anion / singlet oxygen / free radical / hydroperoxide

Introduction The act of respiration, which effectively optimizes the energy balance of the organism, is a characteristic of evolved species. Unfortunately, aerobic life is not without risk to the organism as it exposes vital biological components to powerful oxidative processes. None of the main classes of compounds found in biological systems elude oxidative processes [1], which may degrade substances as diverse as sugars, nucleic acids, proteins, free amino acids and lipids. Since the first report of de Saussure in 1820, peroxidation of lipids [2] has been of particular concern to biologists, organic chemists and nutritionists. Since fats are turned rancid by oxidation, this process is a prime concem in the oil, margarine, rubber, plastic and paint industries. Recent work has shown that peroxidation of unsaturated lipids in vivo may be responsible for pathological processes such as d~ag-induced photo~oxicity, atherosclerosis and even aging. In cells, oxidation underlies effects such as the enhanced permeability to ions, loss of membrane fluidity, cross-linking of aminolipids and polypeptides, inact.i.vafion of enzymes and membrane receptors, release of lysosomal enzymes and breakage of nucleic acid chains. On a

more positive note, it should be borne in mind that peroxidation is involved in such fundamental processes as phagocytosis and the biosynthesis of prostaglandins. This type of reaction can be profitably induced and investigated using photochemical methods as the products of peroxidation obtained photochemically are analogous to those obtained by autoxidation or other routes of induction. We describe here how photochemical control of the nature and quantity of species involved can help gain insight into the mechanisms and kinetics of peroxidation reactions.

General mechanisms of lipid peroxidation Lipid peroxidation comprises the oxidative degradation of cholesterol, phospholipids and unsaturated fatty acids [3-10]. It is a radical chain reaction leading to the formation of intermediate hydroperoxides (LOOH). The reaction can be schematized considering the auto-oxidation of the polyunsaturated fatty acid, linolenic acid. The reaction is initiated by removal of a weakly bound allylic proton, either by an excited species of oxygen, or by a photosensitizer in the triplet

356 INITIATION

(a)

(',.H)

H

,

3

C

~

(CH2)6COOH

HaC~lCH'zlsCOOH

(L')

HaC~(GH2}sCOOH

PROPAGATION

(b)

DNA. The radicals may also combine in dimerization and dismutation reactions (fig lc). Peroxidation reactions arising in the course of this chain reaction can give rise to a wide variety of secondary products [7, 11]. Depending on the particular conditions, different products resulting from mono-, di- and tri-oxidations are formed, which may subsequently undergo rearrangements. The decomposition of these oxidation products leads to a myriad of carbonyl (aldehydes, ketones) compounds, aliphatic hydrocarbons, furans, etc. At all stages, intermediates are formed, which may damage cellular structures and functions [12-15] t~espite the presence of protective detoxification systems within the organism. Initiation thus appears to be the key phase in lipid peroxidation, and we will focus on this aspect of the process in the following discussion. Induction processes - Active species

"~,- L O O ° ~

~

LH

TERMINATION

(c)

21."

~

LL

~

LOOL + 02

L" + LOO"

~

LOOL

RH + L"

~

R'+ LH

~

R'+ LOOH

2 LOO"

RH + L O O "

Fig 1. a. Initiation reactions, b. Propagation reactions. c. Termination reactions.

excited state. The alkyl radical L • produced forms a mesomeric allylic system, which is stabilized by resonance. It is readily oxidized further (fig I a). Capture of a molecule of oxygen produces a peroxyl radical LO0", which may in turn r,:r~,:,,le a hydrogen atom from another polyunsaturate6 fatty acid giving rise to a chain reaction. As the addition of oxygen depends essentially on diffusion, oxidation will spread as long as lipids and oxygen are present (fig lb). In fact, the propagation reaction competes with various termination reactions stemming from removal of hydrogen atoms from the surrounding medium by alkyi and peroxyl radicals. In biological systems, these hydrogen atoms may be on non-lipid substrates such as anti-oxidants, amino acid residues, proteins or

A variety of endogenous factors may induce lipid peroxidation in the organism. In fact, oxygen itself is not particularly toxic, as it has relatively low intrinsic reactivity in the fundamental state. However, highly reactive activated forms are produced within the organism via enzyme reactions involving electron transfers

(fig 2). These reduced intermediates of oxygen, such as superoxide anion (02"-) and the hydroxyl radical (HO') have been widely studied, and are the subject of several reviews [16--18]. All these radical species formed in the organism in situ may initiate peroxidation. Superoxide anion

A large number of biological oxidations whether spontaneous or enzymatic (mitochondrial or microsomal electron transport chains, amino acid oxidases, NADPH oxidases in phagocytes) generate superoxide ion 02"- [ 19, 20]. The role of 02"- in the peroxidation of lipids presents rather a conundrum as from a purely chemical viewpoint it is incapable of removing an allylic hydrogen atom from an unsaturated fatty acid. Furthermore, 02"- is the predominant form at physio-

+e 02

~ Oa ;"

+e +2H+ = H202

e + H+ +e +H+ = OH" =

I

H20

I 02 + 4 e + 4 H÷

~

,2 H20

Fig 2. Redox reactions involved in the radical reactions of oxygen.

357 logical pH, and only the acid form HO 2" is sufficiently reactive to initiate lipid peroxidation. This active form is only found in appreciable amounts in media at acidic pH as in the vacuoles of phagocytes. In addition, the amount of 02"- in the organism is regulated by an enzyme, superoxide dismutase (SOD), which is found in nearly all aerobic organisms. This enzyme catalyzes the dismutation of 02"- into two less reactive species, molecular oxygen in the fundamental state and hydrogen peroxide: SOD 202"- + 2H + ---->H202 + 302 It was long thought that superoxide anion plays little part in lipid peroxidation, although its nucleophilicity has been shown to confer toxic properties [21, 22]. In general its action is now assumed to result from the formation of more reactive species in the presence of traces of iron [23].

The hydroxyl radical Superoxide anion may give rise, via the Haber-Weiss reaction, to the hydroxyl radical, which is incriminated in many destructive processes [24, 25]. This reaction is catalyzed by iron (Fenton reagent) or by other transition metals found in the cellular environment: 02"- + Fe 3+ --->02 + Fe 2+ H202 + ge 2+ + H + --~ HO" + ge 3+ + H20 The OH" radical is particularly destructive in the biological environment as there are no detoxification systems for this species. In aqueous solution, HO" has a radius of diffusion of around 20 A and a life time of around 2 ns or less in the presence of potentially oxidizable substrates. This species can thus only induce peroxidation if it is generated in the neighborhood of the particular lipid. Hydroxyl radicals may also be formed by reaction of hydrogen peroxide with a semiquinone: semiquinone" + H202 + H + ---->quinone + HO" + H20 although there is as yet no evidence for the induction of peroxidation via this mechanism in vivo [26, 27]. Traces of iron may also react with oxygen in the fundamental state to produce perferryl (Fe3+O2"-) or ferryl ions (FeO2+), which have both been shown to be involved in various reactions, the simplest example being that of the oxidation of olefins [28].

Singlet oxygen It has been suggested by numerous workers that superoxide anion may be a precursor of singlet oxygen via a spontaneous dismutation:

202"-+ 2H + --->H202 + IO2 However, Foote et al [29] in a study of trapping with cholesterol demonstrated that singlet oxygen is not a major product of dismutation of 02"-. It has also been suggested that singlet oxygen may derive from a process involving both superoxide anion and the hydroxyl radical [30]: 02"- + HO" + H + ~ 102 + H20 The probability of occurrence of this reaction is limited by the high reactivity of the hydroxyl ion which tends to react with the nearest biological substrate. In cells, singlet oxygen is produced enzymat~cally in neutrophils activated by myeloperoxidase and lactoperoxidase in the presence of a halogen [31]. Lipid peroxidation does appear to produce singlet oxygen, as the reaction involving free radicals is accompanied by weak chemiluminescence attributed to the decay of 10 2. Singlet oxygen is thought to be generated by disproportionation of peroxyl radicals in a Russell reaction. In this reaction, two peroxyl radicals react together to form a metastable tetroxide which decomposes into an alcohol, ketone and 02: 2 LOO" --> (LOO)2 --> LO + LOH + IO2 (or 302) Singlet oxygen is a good oxidant [32, 33], giving rise to hydroperoxide via a concerted mechanism: OOH

I

-CHE-CH=CH- + IO2 --> -CH=CH-CHwhich does not lead to a chain reaction. Singlet oxygen can react effectively with oxidizable substrates as its diffusion radius is around 200 A in water (1000 A in lipid membranes), which is of the same order of magnitude as the distance between organelles within cells. It has a sufficiently long life time in water (4.4 las) [34] to allow it to diffuse over such distances, and studies in micellar media and vesicles [35] have shown that it has an even longer life time in such condensed media. Although singlet oxygen may be responsible for damage to lipids [36, 37], it is thought to be less involved in lipid peroxidation than radical species. In fact, IO2 is 1000-fold less reactive than a competing radical towards olefins (fig 3).

Photochemical models of induction processes Principle tn order to gain more understanding of the mechan,sms of action of activated species within the orga-

358 I02

R.CH=CH-CH2-R' Radical R-CH---CH-~I-R'=

02

RCH-CIt=CII-R' I (3Oil [ 302

R-~lt-Ctl---Ctl-R'

OOH

I R-CH=CH-CH-R Fig 3. Reactivities of singlet oxygen and alkyl radicals with an olefin. nism, these species can be generated in the laboratory. A variety of methods are available involving enzymatic, photochemical, radiolytic and, most commonly, chemical reactions [38]. Peroxidation can be stimulated chemically by a wide range of compounds, including salts of iron and other metals (heavy or transition), organic hydroperoxides, halogenated hydrocarbons (CCI4, CHCI3), ethanol, ozone, nitrogen dioxide, compounds with effects on the redox cycle (Paraquat, adriamycin, mitomycin, menadione, aromatic nitro compounds, etc), hydrazine, numerous pesticides, antibiotics and even cigarette smoke. Chemical induction has two main drawbacks: the production of the relevant species is not readily quantified, and the action of the species involved cannot be controlled over a short enough time scale to enable kinetic determinations. Furthermore, the exact effect of an inducer at the cellular level is far from clear. We will concentrate here on photochemical induction [2], which is both a clean and readily implemented method to generate a high concentration of activated species in a brief time interval. Direct irradiation For the sake of simplicity we will consider the effect of UV alone in the absence of photosensitizer. Induction of a chemical reaction by UV radiation requires that the substrate absorbs at the particular wavelength used. Unsaturated lipids (non-conjugated) do not absorb in the UV-visible region. It has been shown that irradiation of lipoproteins [39] and liposomes of phosphatidylcholine [40] at 365 nm (UVA) has no effect on the lipids themselves. Paradoxically, many workers report on lipid peroxidation induced by exposure to radiation at these wavelengths [41-46]. UVB can initiate formation of malonaldehyde from various unsaturated fatty acids in aqueous solution [42, 4 7 -

52]. Irradiation with UV in the absence of photosensitizer is now widely used to obtain peroxidized lipids [42, 53--61], and generally speaking there is a linear increase in peroxidation with increasing dose of irradiation. The effect of radiation at these wavelengths is not well understood, but it may stem from traces of peroxidation products from autoxidation reactions. Such species absorb slightly in the UV region. This can account for the slower peroxidation of fresh liposomes of phosphatidylcholine than 2--4-day old liposomes, which may have undergone some autoxidation [62]. The intervention of trace amounts of endogenous photosensitizers cannot be ruled out [39], particularly in biological media where they may well be responsible for the effects observed after irradiation of cells or cell homogenates. Irradiation with UVC is relatively ineffective [42, 43]. It tends to give rise to superoxide anion [63], and may induce other reactions [62] not involving malonaldehyde [42]. Photosensitized irradiation When a compound does not absorb directly at the wavelength used, as is the case for lipids in the UVvisible region, excitation can occur via the intervention of a photosensitizer. Systems such as LDL, membranes or whole cells contain numerous substances that may act as a photosensitizer. This compound, which absorbs at the irradiating wavelength, may intervene at various stages: 1) It may transfer its energy to a substrate:

3S +LH --> S +3LH 3S represents the triplet state of the photosensitizer. From a purely photochemical viewpoint, for transfer of energy to occur, the energy of the excited state of the photosensitizer must be higher than that of the substrate. In the above example, this transfer is not likely as the energy of the triplet state is particularly elevated. 2) It may also transfer its energy to molecular oxygen generating singlet oxygen:

3S 4- 302 -"> IO 2 4" IS which then reacts with the phospholipid. Nearly all aromatic compounds can generate singlet oxygen. The best photosensitizers of this type are compounds whose energy of the excited state is close to that of singlet oxygen. 3) The photosensitizer may exchange an electron or hydrogen atom with the substrate. In the case of a lipid LH and for most photosensitizers, removal of hydrogen is thermodynamically more favorable than stripping of an electron. Thus: 3S +LH --> S'H + L"

359 Hydrogen may be removed from other compounds in the medium creating other organic radicals, which may in turn attack the lipid. Intramolecular hydrogen transfer may also occur. 4) An electron may be transferred between the excited photosensitizer and oxygen, giving rise to superoxide anion: *S + 02 --> S "+ + 02"In common with the previous cases, the superoxide anion may lead to HO" or H202 via the action of superoxide dismutase in a Haber-Weiss reaction. Photosensitization can thus produce ~inglet oxygen, superoxide anion, HO" radicals and alkyl radicals.

Nature of photosensitizers Depending on the nature of the photosensitizer, a variety of activated species that may initiate peroxidation can be produced after irradiation in the visible or UV regions.

Generation of superoxide ion and hydroxyl radical Photochemical systems are particularly specific, and a variety of systems are available for production of superoxide and ferryl ions~ or hydroxyl radicals. Photolysis of H202 at wavelengths below 380 nm gives rise to abundant OH" radicals. Judicious use of catalysts can steer the reaction towards other oxygenated radicals [64, 65]. In the absence of hydrogen peroxide, other photosensitizers can be v~sed to generate radical species. For example, FMN [66] in combination with reducing agents generates an irradiation-dependent quantity of superoxide radicals. Systems such as anthraquinone-2-sulfonate in DMSO generate large amounts of 02"-. In aqueous solution, 02"- is rapidly converted into OH" radicals [67]. The hydroxyl radical may also be produced directly by photolysis of Nhydroxy-2-pyridinethione [68, 69], or prepared from hydroperoxides produced by photooxygenation of the corresponding phthalimides [70]. 02"- may also be generated by irradiation of various dyes. Addition of ferric ions to the system induces production of OH" [71 ], ferryl and perferryl ions. A similar mechanism is thought to account for the photoperoxidation of lipids by merocyanin 540 [72]. These methods are those of conventional photochemistry, and their value lies in the fact that specific radicals may be produced in high yield. Induction is strictly dependent on the dose of irradiation and stops immediately once the irradiation is extinguished. All these methods should be profitably employed in studies on lipid peroxidation. Generation of singlet oxygen The oxidation by singlet oxygen is a type II photosensitization reaction, in contrast to the type I or radi-

cal photosensitized oxidation reactions. A large number of sensitizers have been employed to initiate lipid peroxidation. They include dyes [71, 73-83] such as acridine orange, methylene blue, erythrosin, nile blue A, azur blue, toluidine blue, rose bengal as well as the derivatives employed in photodynamic therapy such as porphyrins [40, 73, 84-87] phtha!c~cyanins [88-91] and merocyanin [90, 92-95]. Other useful sensitizers include drugs which have given rise to photoallergic or phototoxic reactions such as amiodarone [96], afloqualone [97], chlorpromazine [98], psoralen [45, 85], quinolones [99], griseofulvin [ 100] or molecules of biological origin such as flavins [ 101 ], tryptophan [39, 102] and chlorophyll [57, 78, 79, 103, 104], which all interact with light. Aromatic compounds such as thiopben [105], dimethylanthracene [35] and hydroxytoluene [106] have also been employed. These compounds, which may also be incorporated in the biological environment, interact with irradiation to produce singlet oxygen. The characteristics of their triplet states and the quantum yields for production of singlet oxygen are known. The photooxidation yield can thus be readily compared with the quantum yield of production of singlet oxygen or intersystem crossing. Photooxidation of all lipid systems may be profitably investigated using toO2generated via photosensitization. This can help gain more understanding on both the effect of organization on photooxidation [88, 90], and the influence of the localization of the photosensitizer in the organized phase [35, 96]. It can also give rise to regio- and stereospecific oxidations. An interesting example is the oxidation of linoleic acid using porphyrin as photosensitizer in a sandwich arrangement in cyclodextrin, which mimics the activity of lipoxygenase [ 107]. It should be pointed out, however, that certain sensitizers lack specificity and may generate other activated species of oxygen. This is the case for merocyanin 540, which apart from singlet oxygen, produces large amounts of hydroxyl ions [72, 95]. Depending on the nature of the reaction medium, type II reactions may also occur. For example, the photooxidation of methyl linoleate sensitized by riboflavin has been shown to take place via a radical mechanism in aqueous medium [81], whereas in acetonitrile singlet oxygen is involved [101]. In the case of the peroxidation of phenyl linoleate in methanol, there appears to be an equilibrium between the type I and II reactions [73]. The rate of type II processes depends mainly on the concentration of oxygen in solution, which is around 0.9-1.2 10-2 M in organic solvents saturated with oxygen [108] and 2.6 10-4 M in water saturated with air. Generally speaking, mechanisms involving IO2 are favored in oxygen-enriched media

360 such as organic solvents, while radical mechanisms are favored in media containing low concentrations of oxygen and high levels of reducing agents. The latter media are found in cells [71 ]. Certain photosensitizers may also give rise to electron transfer reactions. One technique worthy of mention is that of heterogeneous photosensitization. It has the advantage of producing pure singlet oxygen with an absence of other activated species. In this case, singlet oxygen is produced by irradiation of rose bengal in a flow chamber and then bubbled into a lipid suspension [109, 110]. The concentration of IOz can thus be kept constant in the reaction medium, allowing kinetic studies [ 1101.

Generation of alkyl radicals Via excited carbonyls. Numerous compounds after excitation can remove a hydrogen atom from a substrate giving rise to an alkyl radical [ 111]. The best known examples of such sensitizers are carbonyl derivatives such as flavins [811, benzophenones [112, 113], acetone [I 14], etc. The action of these ketones on lipids has been extensively studied by laser photolysis, and the rate of removal of secondary, allylic or doubly ailylic hydrogens has been determined [115]. To enhance selectivity, benzophenone may be incorporated in various fatty acids. Its localization in the linoleate/SDS system is now known. This has enabled study of the influence of molecular organization on the behavior of the sensitizer as well as on the reactivity of excited species in general [ 116]. Since lipid peroxidation leads to production of excited carbonyl species, the action of these ketones can mimic reactions occurring in vivo. It has been shown for example that peroxidation may give rise to excited carbonyls via a Russell reaction involving two peroxyl radicals: LOO" + LOO" --> LO* + LOH + 02 Other processes, such as the cleavage of a dioxetane formed by reaction of singlet oxygen on unsaturated fatty acids:

I I

I

I

-C-C- --->-C-O* + -C=O

I I O-O or disproportionation of alkoxyl radicals: LO" + LO" --->LOH + LO* represent further sources of excited carbonyls. In biological media, these reactions are catalyzed by enzymes of the peroxidase type, which thus constitute a protective microenvironment. The lower the number of deactivating collisions the greater will be the production of excited carbonyls. However, the

excited carbonyl may transfer its energy to appropriate acceptors such as flavins, which in turn give rise to addition or radical chain reactions.

By other radicals. Besides excited carbonyls and the activated forms of oxygen, other radicals (ter-BuO', SO:-, CCI3OO', CF3CHCIOO', etc) may generate alkyl radicals by stripping hydrogen atoms from lipid molecules. Most of these radicals can be generated photochemically and used to study lipid peroxidation [117]. Examples are the nitrenes produced from a photolabile precursor [40], and phenoxyl and methyl ions generated by the photosensitizer FMN in the presence of appropriate substrates. The reactivity and selectivity of ter-BuO" radicals has also been determined by flash photolysis [I 18] and photolysis coupled with ESR. Although outside the scope of this discussion, free radicals are commonly generated by pulsed radiolysis. Mention can, however, be made of studies on dienyl [119], ter-butoxyl [120], sulfite [121] and thiyl RS" [122-124] radicals. Thiyl radicals are of interest as they mimic the reactions of cysteinyl radicals with linoleic, linolenic and arachidonic acids. The rates of reaction and recombination have been determined for all these reactions.

Propagation reaction As illustrated in figure 1, alkyl radicals react mainly with oxygen forming peroxyl radicals L" + 02 --->LOO'. A chain reaction may then take place: LOO" + L'H -> LOOH + L". The reaction products LOOH, referred to as lipid monohydroperoxides, decompose spontaneously on heating. However, the presence of metallic catalysts such as hemoproteins markedly enhances decomposition, thereby initiating other chain reactions: LOOH + Fe 3+ ---->LOO" + H ÷ + Fe2+ LOO" + LH ---->LOOH + L" etc. Peroxyl radicals LOO', which may undergo various intramolecular rearrangements, are thought to be the main agents of chain propagation [125-127]. The recombination of two peroxyl radicals requires a highly peroxidized medium, wh~.ch is rarely encoun~ tered in normal physiological conditions. Chemical and photochemical process may thus interact synergistically. The presence of ferrous iron may also induce formation of alkoxyl radicals:

361 LOOH + Fe2+ --~ LO" + HO- + Fe 3+. The LO" radicals are probably highly unstable in polar solvents, and react rapidly with biological substrates such as lipids, nucleic acids and nucleosides. LO" + LH -~ LOH + L"

[ 137]. The position of the radical center on deuterated fatty acids has thus been defined. It should be borne in mind that radicals of similar structure derived from fatty acids are not always distinguishable as the spin-labelled products tend to have the same hyperfine structure. In this case, high performance liquid chromatography may be profitably employed to characterize the adducts [ 138].

UV photolysis may also induce production of these radicals by homolytic cleavage, which has been exploited in several investigations [ 128, 129]

Emission spectroscopy of singlet oxygen

Study of activated species formed by photosensitization

Since singlet oxygen emits in the infra-red at 1269 nm, spectroscopy is of particular value for detecting this species, especially in biological systems [ 139].

Depending on the nature of the photosensitizer, one or more of the above-mentioned activated species may be formed. In order to make sure that a particular species is involved it must either be detected directly by a physicochemical method or be trapped and then detected.

Identification of radicals by ESR Radicals may be detected directly either by flash photolysis or ESR, although the latter technique is most commonly employed for detection of radicals in biological systems [ 130, 131 ]. ESR can detect radicals in concentrations down to 10-8 M, although higher concentrations may be required to resolve the hyperfine components of the spectra and obtain structural information on the radical. This direct detection method is hampered by the fact that these radicals tend to be produced in relatively small amounts and are also highly reactive. Rapid freezing and freezedrying of continuous flow systems have been used to get round this problem, but artefacts are not uncommon. ESR is most effective when associated with photochemical methods [117], which generate large numbers of radicals. For example, the reaction with antioxidants of peroxyl radicals generated by UV irradiation has been studied directly, enabling determination of kinetics and activation energies [ 132]. Spin-labelling can be used to trap highly reactive radicals by converting them into more stable adducts that can be detected with greater ease. The choice of spin-traps for oxygenated radicals has been reviewed recently [133]. Certain traps are quite specific. For example the hydroxyl radical may be trapped by 2, 4, 6-trimethoxyphenyl tert-butyl nitrone without any interference of other oxygenated radicals in the medium [ 134, 135]. Spin-traps can also help discriminate between peroxyl radicals and radicals centered on the carbon [ 136], and they have also been used to characterize alkoxyl and peroxyl radicals produced during photolytic decomposition of hydroperoxides

Specific traps One of the simplest methods of finding out whether a radical or excited species is involved in a reaction is to test the action of specific inhibitors of the species. For radical species of oxygen in biological systems, such traps form part of the natural defenses of the organism [140]. Photoperoxidation slowed by superoxide dismutase, glutathione peroxidase or catalase is indicative of the intervention of 02"- and.H20. "~s reaction intermediates. However, SOD used alone can also enhance lipid peroxidation by producing H202 which can generate in the presence of metal traces hydroxyl radicals much more efficient than the superoxide anion in inducing lipid peroxidation. HO" scavengers such as ethanol, isopropanol, mannitol and deoxyribose are expected to protect lipid against peroxidation if HO" is participating in the damage process. HO" may also react with DMSO to form the stable methane sulfinic acid, which may be assayed colorimetrically [ 141, 142]. When HO" results from 02"- by an Haber-Weiss reaction, chelating agents such as transferrin, ferritin, desferrioxamine, DTPA and EDTA may inhibit the production of HO" by sequestrating iron atoms. Secondary defense systems which are highly specific for LO" and LOO" radicals may also be employed to inhibit these species in investigations on reaction mechanisms. Among antioxidants, tocopherol, a natural component of vitamin E, is a good example: L" +TH2 --->LH+TH" LOO" + IH2 --->LOOH + TH" Other quinonoids of biological origin (ubiquinone, catechin, etc) and synthesis inhibitors (butyl hydroxytoluene, butyl gallate, promethazine, diphenyl-p-phenylenediamine, etc) may be employed in vitro, although in vivo they tend to be less effective than vitamin E as they are metabolized more rapidly.

362 '02 quenchers such as sodium azide, dimethylfuran, DABCO, histidine and [;-carotene may decrease lipid damages due to this species. For example, azides inhibit the photoperoxidation of cell membranes induced by the sensitizers HPD and merocyanin 540 [27, 93]. The reactivity of all these quenchers is related to their accessibility to the reactive site. Hydrophilic inhibitors, for example, are not effective traps for species formed inside membranes. Exploitation of a medium effect may profitably be combined with the use of scavengers. Singlet oxygen for example has a 14-fold longer life time in D20 (55 ps) than in H20 (4.4 ps). A faster reaction in D20 is indicative of the involvement of toO2[34]. However, the reve,,'=~ situation does not necessarily rule out the involvement of singlet oxygen, which may be generated within a hydrophobic medium, such as that of a lipid bilayer, effectively precluding exchange with 1)20. Many quenchers are not really specific. For example, over a range of 1 mM to 10 mM, quenchers of singlet oxygen such as azides and histidine react with ~O2, deactivate the excited triplet states of the photosensitizers [ 143] and chelate iron [ 111 ]. Furthermore, some of these inhibitors may also react with HO" radicals [25]: sodium azide for example, deactivates HO" 10 times faster than mannitol does [144]. In the same manner, both tocopherol and hydroxytoluene not only react with HO', but also react rapidly with singlet oxygen. As a result, effects of quenchers on the peroxidation rates are not necessarily unequivocal in the conclus|ons they bring. Complementary experiments such as the measurement of reaction rate constants or the characterization of the products of photodegradation, are often necessary to establish the nature of the species involved in the photoinduced peroxidation process.

detected by a variety of methods. These methods have been extensively reviewed [2, 4, 7, 28, 145-147], but none are specific to photochemical systems. It should be borne in mind that chemical peroxidation proceeds essentially via a radical mechanism, whereas photochemical peroxidation may involve several types of activated species. In this case, the mechanism may be identified from the formation of characteristic peroxidation products.

HydroperoMdes offatty acids Polyunsaturated fatty acids give rise to different peroxidation products depending on whether singlet oxygen or a radical reaction is involved [28]. Singlet oxygen attacks either side of a double bond indiscri-

CH3-CHt-CH--CH-CHa-CH=CH-CH2-CH--CH-(CHa) TCOOH HO" " ~

p"

CH3-CH2-CH=CH-CH-CH=CH-CH2-CH--CH-(CH2) rCOOH Alkyl radical

GH3-CH2-CH-CH~K~H-CH---CH-CH2-CH--CH-(CH2) TCOOH 02 - 1

A/kj4ra0~ca/ (Conjugateddiene)

CHa-CH2.CH.CH---CH.CHffiCH-CHa-CH_-CH-(CH2)TCOOH 6-0" RH ~ Peroxylradicai. Radical~i~n reaction ~ R" CH3-CH2-.CH-CH=CH-CH--CH-CH2-CHffiCH-(CH2) TCOOH ~oH

F 2.

• ~j,

Peroxidation products As mentioned above, lipid peroxidation generally !eads to a sequence of complex reactions, and there are only a few examples in which all products have been analyzed. Identification of the main families of .products formed during the first stages of peroxidation is an achievable goal. They include: i)hydrope~oxides; ii) conjugated dienes (with other functional groups on the chains); iii)aldehydes derived from cleavage of the hydroperoxides; and iv) hydrocarbons. Pentane and ethane from decomposition of peroxidized fatty acids in whole organisms or isolated organs may also be detectable (fig 4). Whether generated chemically, photochemically or enzymatically, the products of peroxidation can be

Unolenicacid

H=O

,

~ -~ Fe~-

Hydroperoxide

CH3-CH2-CH-CH=CH-CH=CH-CH2-CH=CH-(CHa)TCOOH O" + OH" Ch,~H2

+

~

Alkoxyiradical

H%

o=,C-CH=CH-CHffiCH.CH2-CH=CH.(CH2)TCOOH Aldehyde

CHa-CH3 Ethane

Fig 4. Products of radical pcroxidation of linolenic acid.

363

CI-~(CI-12)a~ (CH~BCOOH e'., H +

CHa(CH2)3"

V-V=v

(CH2)sCOOH

CI-~(CH2)a~ 00"

Ii 9-OOH, 13-OOH I

~

(CH2)sCOOH

l~0-OOHI 02

C1-13(C1-~2)a~ (CH2)sCOOH "OO C I - l a ( C H 2 ) 3 ~ (CH2)6COOH

OOH CHdCH~a~

~

OOH C h ( C H 2 ) a ~ (CH2)BCOOH

HOO C I - 1 3 ( C H 2 ) a ~ (CH~BCOOH

LH L"

(CH~6e-.,OOH

HOO

113-OOH! CH3(CH2)3~(CH2)eCOOH I ,OOHI Fig 5. Dependence of peroxidation products of linoleic acid on mode of induction.

I 12-OOH I

364 minately. Thus from iinoleate one obtains equivalent amounts of peroxides oxidized in positions 9, 10, 12, 13, 14 and 15, whereas radical peroxidation only involves the end positions of a conjugated diene (fig 5). For linolenate (18:3), the oxidized derivatives in positions l0 and 15, and for arachidonate (20:4), the 6 and 16 derivatives are thus clues to the action of singlet oxygen (cffig 5). Assuming that singlet oxygen is uniformly distributed, its conaibution relative to that of radicals can be calculated. A recent study [73] has shown that methylene blue, erythrosin, or hematoporphyrin peroxidize esters of iinoleate via a type II mechanism in methanol, whereas riboflavins induce a combination of type I and type II mechanisms. The secondary products of peroxidation will also depend on the nature of the processes involved [ 11 ]. For linoleate, the cyclic peroxides derived from the 10 and 12 hydroperoxides are readily separated by

HOO CH3(CH2)a~

-H"

1

HPLC, and characterize the action of singlet oxygen (fig 6).

Hydroperoxides of cholesterol In common with the polyunsaturated fatty acids, cholest~:rol gives rise to characteristic peroxidation produc~ depending on the type of peroxidation [89, 90, 148-154] (fig 7). Purely radical mechanisms do not give rise to the 5tx-OOH derivative, which is characteristic of oxidation by singlet oxygen. However, 5(x-OOH may derive from an allylic rearrangement of the 7a-OOH and 7[3OOH derivatives [150, 155]. This tends to occur more readily in solution in chloroform or methanol than in membranes. This possibility may be resolved by determining the 5a-OOH/7-OOH ratio in photooxidized membranes in the presence and absence of D20. OOH

(CH2)sCOOH

1lo-ooH!

CH3(C1"~3~

1

-H" O

CH3(CH2)a~

(CH2)sCOOH

1

O-O CH3(CH2)3~~~j (CH2)6COOH

H"~ O 2 HOO O--O CH3(CH2)3V~~ (CH2)sCOOH Fig6.1,3-cyclizatioof n the10-and12-hydropcroxides oflinoleicacid.

(CH2)6COOH 12-OOH]

0

•

CH3(CH2)a~

(CH2)6COOH

1

O-O CH3(CHa)a/~V~j(cHd6CooH H" " ~ 0 2 0-0 OOH CH3(CH2)a/k~V,,[~ (CH2)sCOOH

365

hv / sens HO

02

HO OOH

HO

:

15

i

'

-OOH l

HO

oo.I Fig 7. Dependence of peroxidation products of cholesterol on mode of induction. An increased ratio in D20 is indicative of competition between type I and type II reactions. Leaving aside this obstacle, cholesterol is found in significant amounts in natural membranes (40% of erythrocyte membrane lipids) and can be detected with greater ease than the multitude of polyunsaturated fatty acids with similar structures [134, 135]. It has recently been employed as a probe in situ [27, 28]. One disadvantage nonetheless is that the quantum yield is low, often requiring the use of 14C-labelled cholesterol [92].

Conclusion We have seen how lipid peroxidation can be studied using photochemical methods. They are both clean and efficient, producing similar organic radicals to those formed chemically. Furthermore these radicaJs are only generated during exposure to the radiation. This is particularly convenient as the progress of the reaction and the nature of the products formed can be readily controlled. These methods could be improved by development of more efficient and more selective photosensitizers. Many questions remain unanswered about the processes of initiation and mechanisms of protection against peroxidation. Of particular interest is the

transformation of certain hydroperoxides into potent physiological mediators, which are now known to initiate and control whole sequences of biochemical reactions in vivo [156-158].

References 1 Sies H (1985) Oxidative Stress. Academic Press, London 2 Kappas H (1985) Lipid peroxidation: mechanisms, analysis, enzymology and biological relevance, ht: Oxidative sn'ess (Sies H, ed) Academic Press. London, 273-310 3 Frankel EN (1988) Hydropero~idation of unsaturated fatty esters. Basic Life Sci 49, 265-282 4 Girotti AW (1985) Mechanism of lipid peroxidation. J Free Radicals Biol Med I. 87-95 5 Gutteridge JMC, Halliwell B (1990) The measurement and mechanism of lipid peroxidation in biological system. Trends Biochem Sci 15, 129-135 6 Hsieh RJ, Kinsella JE (1989) Oxidation of polyunsaturated fatty acids: mechanisms, products, and inhibition with emphasis on fish. Adv Food Nutr Res 33, 233-341 7 Kagan VE (1988) Lipid Peroxidatio,t in Biomemhranes. CRC Press, Boca RatonFlorida 8 Mead JF ( i 976) Free radical mechanisms of lipid damage and consequences for cellular membranes. In: Free Radicals b~ Biology (Pryor WA, ed) Academic Press, New York, 51-68 9 Porter NA (1984) Chemistry of lipid peroxidation. Methods En-vmol 105, 273-282 10 Kappus H (1991) Lipid peroxidation: mechanism and biological relevance. hi: Free Radicals Food Addit (Aruoma OI, Halliwell B, eds) Taylor & Francis, London, 59-75 i I Frankel EN (1987) Secondary products of lipid oxidation. Chem Phys Lipids 44, 73-85

366 12 Bors W. Erben-Russ Mo Michel C. Saran M (1990) Radical mechanisms in fatty acid and lipid pcroxidalion. In: Free Radicals, Lipoproteins, and Membrane Lipids (Crastes de Paulet A. Douste-Blazy L. Paoletti R. eds) NATO ASi Series. New York. Plenum Press 13 KikugavJa K, Beppu M (1987) Involvement of lipid oxidation products in the formation of fluorescent and cross-linked proteins. Chem Phys Lipids 44, 277-296 14 Reed GA (1987) Co-oxidation of xenobiotics: lipid peroxyl derivatives as mediators of metabolism. Chem Phys Lipids 44, 127-148 15 Vaca CE. Wilhelm J. Harms-Ringdahl M (1988) Interaction of lipid peroxidation products with DNA. A review. Murat ICes 195, 137-149 16 Ferra_dini C (1986) Esl~ces activ6es radicalaires de i'oxyg~ne. Biochimie 68. 779--785 17 Fridovitch I (1976) Oxygen Radicals. Hydrogen Peroxide. and Oxygen Toxicity, In: Free Radicals in Biology (pryor WA. ed) Academic Press. NewYork I. 239-277 18 Asmus KD (1988) Formation and properties of oxygen radicals. NATO AS! Ser, Ser A. 146, 37-54 19 Deby C. Goutier R (1990) New perspectives on the biochemistry of superoxide anion and the efficiency of superoxide dismutases. Biochem Pharmaco139. 399-405 20 Fridovitch [ (1986) Biological effects of the superoxide radical. Awl: Biothem Biophys 247. I-I I 21 Dietz R, Fomo AES, Larcombe PE, Peover ME (1970) Nucleophilic reactions of electrogenerated superoxide ion. J Chem Soc B. 8 ! 6--820 22 Niehaus WG (1978) A proposed role of superoxide anion as a biological nucleophile in the deesterification of phospholipids. Bioorg Chem 7. 77-84 23 Minotti G, Aust SD (1987) The role of iron in the initiation of lipid peroxidation. Chem Phys Lipids 44. 191-208 24 Czapski G (1971) Radiation chemistry of oxygenated aqueous solutions. Annu Rev Phys Chem 22. 171-208 25 Dorfman LM. Adams GE (1973) Reactivity of the hydroxyl radical in aqueous solutions. Nat Stand Ref Data Ser Nat Bur Stand 46.1-59 26 Borg DC. Schaich DM (1974) Cytotoxicity from coupled redox cycling of autoxidizing xenobiotics and metals. Israel J Chem 24, 38-53 27 Girotti AW. Thomas JP (1984) Superoxide and hydrogen peroxide-dependent lipid peroxidation in intact and triton-dispersed erythrocyte membrane. Biochem Biophys Res Commun 118. 474--480 28 Girotti AW (1990) Photodynamic lipid peroxidation in biological systems. Photochem Photabiol 5 !. 497-509 29 Foote CS, Shook FC, Abakerli RA (1980) Chemistr) of superoxide ion. 4. Singlet oxygen is not a major product of dismutation J Am Chem Soc 102, 2503-2504 30 Ameson RM (1970) Substrate-indnced chemiluminescence of xanthine oxidase and aldehyde oxidase. Arch Biochem Biophys 136, 352-360 31 Kanofsky JR (1984) Singlet oxygen production by lactoperoxidase: halide dependence and quantitation of yield. J Photochem 25, 105-113 32 Foote CS (1982) Light. oxygen, and toxicity. In: Pathology of oxygen (Autor AR ed) Academic Press. New York. 21--44 33 Gorman AA (1992) The bimolecular reactivity of singlet molecular oxygen. Adv Photoch,,m 17.217-274 34 Rodgers MAJ, Snowden PT (1982) Lifetime of O,('Ag) in liquid water as determined by time-resolved infrared luminescence measurements. J Am Chem Soc 1(}4,5541-5543 35 Dearden SJ (1986) Kinetics of O4'Ag) photo-oxidation reactions in egg-yolk lecithin vesicles. J Chem Soc Faraday Trans ! 82, 1627-1635 36 Valenzeno DP (1987) Photomodification of biological membranes with emphasis on singlet oxygen mechanisms. Photochem Photobiol 46, 147-156 37 Rawls HR, Van Santen PJ (1970) A possible role for singlet oxygen in the initiation of fatty acid autoxidation. J Am Oil Chem Soc 47, 12 !-!25 38 Kappus H (1987) A survey of chemicals inducing lipid peroxidation in biological systems. Chem Phys Lipids 45,105-115 39 Salmon S, Mazi~re JC, Santus R, Morli~re P, Bouchemal N (1990) UVBInduced photoperoxidation of lipids of human low and high density lipoproreins. A possible role of tryptophan residues. Photochem Photobhff 52, 541-545 40 Szebeni J. Tollin G (1988) Some relationships between ultraviolet light and hemeprotein-induced peroxidative lipid breakdown in liposomes, as reflected by fluorescence changes: the effect of negative surface charge. Photochem Photobio147, 475-479

41 Bose B. Agarwal S. Chatterjee SN (1989) UV-A induced lipid peroxidation in liposomai membrane. Radiat Environ Biophys 28, 59-65 42 Dennis KJ, Shibamoto T (1989) Production of malonaldehyde from squalene. a major skin surface lipid, during UV-irradiation. Photochem Photabiol 49,711-716 43 Bathia R, Viswanathan PN, Kakkar P (1991) Liposomes as a model for studying the effect of UV radiations on biomembranes. J Microencapsulation 8, 349-357 44 Bose B, Agarwal S, Chatterjee SN (I 990) Membrane lipid peroxidation by UV-A: mechanism and implications. Biotechnol Appl Biochem 12, 557561 45 Matsuo 1. Yoshino K, Ohkido M (1983) Mechanism of skin surface lipid peroxidation. Curr Prohl Dermatol ! !. 135-143 46 Chatterjee SN. Agarwal S. Bose B (1990) An approach towards understanding the genesis of sunlight-induced skin cancer. Indian J Biochem Biophys 27, 254--263 47 Brona E, Petit E, Beljan-Leymarie M, Huynh S, Nouvelot A (1989) Specific susceptibility of docosahexaenoic acid and eicosapentaenoic acid to peroxidation in aqueous solution. Lipids 24, 970-~75 48 Chatterjee SN. Agarwal S (1983) Lipid peroxidation by ultraviolet light and high energy and particles from a cyclotron. Radiat Environ Biophys 21, 275-280 49 Punnonen K, Jansen CT (1989) UVB irradiation induces changes in the distribution and release of arachidonic acid. J Invest Dermatol 88, 61 ! 614 50 Schieberle P. Trebert Y. Firl J, Grosch W (1988) Photolysis of unsaturated fatty acid hydroperoxides. 4. Fatty acid products from the aerobic decomposition of methyl 13(S)-hydroperoxy-9(Z), ! ! (E)-octadecadienoate dissolved in cyclohexane. Chem Phys Lipids 48, 281-288 5 ! Niyati-Shirkhodaee F, Shibamoto T (1992) In vitro determination of toxic aldehydes formed from the skin lipid, triolein, upon ultraviolet irradiation: formaldehyde and acrolein. J Toxicol Cutaneous Ocul Toxicol I I, 285-292 52 Yeo HCH. Shibamoto T (1992) Formation of formaldehyde and malonalde hyde by photooxidation of squalene. Lipids 27, 50-53 53 Agarwal S, Ghosh A. Chatterjee SN (1987) Spontaneous release of malondialdehyde from ultraviolet light exposed liposomal membranes. Z Natarforsch C Biosci 42, 585-588 54 Sugiyama M, Kajiyama K, Hidaka T, Kumano S, Ogura R (1984) Lipid peroxidation and radical formation in methyl iinoleate following ultraviolet light exposure. J Dermatol ! !, 455--459 55 Baker N, Wilson L (1974) UItraviolet-autoxidized linolenic acid in high yield for cancer study. Lipids 9, 346-352 56 Seligman ML, Mitmura J° Shera N, Demopoulos HB (1979) Corticosteroid (methylprednisolone) modulation of photoperoxidation by ultraviolet light in liposomes. Photochem Photobio129, 549-558 57 Khan NA, Lundberg WO, Holman RT (1954) Displacement analysis of lipides. IX. Products of the oxidation of methyl linoleate. J Am Chem Soc 76, 1779-1784 58 N~gre-Salvayre A, Paillous N, Dousset N, Bascoul J, Salvayre R (1992) Wavelength dependence of photoinduced peroxidation and cytotoxicity of human low density lipoproteins. Photochem Photohio155. 197-204 59 Stepien K, Porebska-Budny M. Hoilek AM, Wilczok T (1992) The inhibiting effect of catecholamine-melanins on UV-induced lecithin peroxidation. J Photochem Photobiol B 15, 223-23 ! 60 Kowalska E, Naglik T (1990) Interactions of ferrous ions with peroxidized lecithin-liposomes. Acta Biochim Po137, 85-88 61 Pelle E, Maes D, Padulo GA, Kim EK, Smith WP (1990) An in vitro model to test relative antioxidant potential: ultraviolet-induced lipid peroxidation in liposomes. Arch Biochem Biophys 283, 234-240 62 Islomov AI, Azizova OA, Odinokova GG, Roschchupkin DI, Remizov AN, Vladimirov YA 0979) Free radicals formed during UV-irradiation of oxidized phosphatidylcholine. BioJizika 24, 403-407 63 Yamashoji S, Yoshida H, Kajimoto G (1979) Photooxidation of linoleic acid by ultraviolet light and effect of superoxide anion quencher. Agric Biol Chem 43, 1249-1254 64 Lunak S, Sedlak P (1992) Photoinitiated reactions of hydrogen peroxide in the liquid phase. J Photochem Phoiobioi A 68, 1-33 65 Zepp RG, Faust BC, Hoigne J (1992) Hydroxyl radical formation in aqueous reactions (pH 3-8) of iron (It) with hydrogen peroxide: the photo-Fenton reaction. Environ Sci Techno126, 313-3 i 9

367 66 Proila TA, Mehlom RJ (1990) A photochemical system for generating free radicals: superoxide, phenoxyi, ferryl and methyl. Free Radical Res Commun 9, 135-146 67 Lang K, Wagnerova DM, Stopka P, Damerean W (1992) Reduction of dioxygen to superoxide photosensitized by anthraquinone-2-suifonate. J Photochem Photobiol A 67, 187-195 68 Boivin V, Crepon E, Zard SZ (1990) N-Hydroxy-2-pyridinethione: a mild and convenient source of hydroxyl radicals. Tetrahedron Lett 31, 6869--6872 69 Hess KM, Dix TA (1992) Evaluation of N-hydroxy-2-thiopyridone as a nonmetal dependent source of the hydroxyl radical (HO') in aqueous systems. Anal Biochem 206, 309-314 70 Matsugo S, Takayama M, Matsuura T, Saito I (1989) Design of a new photochemical hydroxyl radical generator and its DNA cleaving activity. Photomed Photohiol ! 1, 9-13 71 Martin JP, Burch P (1990) Production of oxygen radicals by photosensitization. Methods Enzymol 186, 635-645 72 Sarna T, Pilas B (1991) The role of free radicals in photosensitized oxidation reactions induced by merocyanine 540. J Photochem Photobiol 58, 339347 73 Chacon JN, Jamieson GR. Sinclair RS (1987) Dye-sensitized photooxidation of the methyl and phenyl esters of oleic and iinoleic acids. Chem Phys Lipids 43, 81-99 74 Chacon JN, McLearie J, Sinclair RS (1988) Singlet oxygen yields and radical contributions in the dye-sensitised photo-oxidation in methanol of esters of polyunsaturated fatty acids (oleic, linoleic, linolenic and arachidonic). Photochera Photobio147, 647--656 75 Terao J, Matsushita q (1977) Products formed by photosensitized oxidation of unsaturated fatty a~:id esters. J Am Oil Chem Soc 54, 234-238 76 Krinsky NI, Andersen SM (1973) Protective action of carotenoid pigments against photodynamic :lamage to liposomes. Photochem Photobiol 18, 403--408 77 Dearden SJ, Hunter IF, Phil:~J (1985) Fatty acid analysis as a function of photooxidation in egg yolk lecithin vesicles. Photochem Photobiol 41. 213-215 78 Rawls HR, van Santen PJ (1968) Singlet oxygen and the initiation of fatty acid autoxidation. Tetrahedron Letter 14, 1675-1678 79 Frankel EN, Neff WE, Bessler TR (1979) Analysis of autoxidized fats by gas chromatography-mass spectrometry: V. Photosensitized oxidation. Lipids 14, 961-967 80 Matsushita S, Terao J, Yamauchi R (1978) Photosensitized oxidation of unsaturated fatty acid esters and the quenching effects of tocopherols on singlet oxygen. In: Tocopherol Oaygen Biomembr Proc Int Symp 1977, 23-39 81 Chan HWS (1977) Photo-sensitized oxidation of unsaturated fatty acid methyl esters. The identification of different pathways. J Am Oil Chem Soc 54, 100-104 82 Thomas M.I, Pryor WA (1980) Singlet oxygen oxidation of methyl linoleate: isolation and characterization of the sodium borohydride-reduced products. Lipids 15,544-548 83 Porter NA, Logan J, Kontoyiannidou V (1979) Preparation and purification of arachidonic acid hydroperoxides of biological importance. J Org Chem 44, 3177-3181 84 Candide C, Reyflmann JP, Santus R, Mazi~re JC, Morli~re P, Goldstein S (1988) Modification of epsilon-amino group of tysines, cholesterol oxidation and oxidized lipid-apoprotein cross~!ink~f6~a:ion by po~hyrin-photosensitized oxidation of human low density lipoprotems. Photochem Photobio148, 137-146 85 Carraro C, Pathak MA (1988) Studies on the ~lature of in vitro and in vivo phot~.ensitization reactions by psoralens ~ d porphyrins. J Invest Dermatol 90, 267-275 86 Ohtani B, Nishida M, Nishimoto St, Kagiya T (1986) Photooxygenation of methyl linoleate sensitized by porphyrins and dyes in acetonitrile solution and aqueous emulsion systems. Photochem Phoobiol 44, 725-732 87 Thomas JP, Girotti AW (1989) Reactivity of photochemically-generated lipid hydroperoxides in cell membranes with glutathione peroxidase. Photochem Photobio149, 153-156 88 Nonell S, Braslavsky SE, Schaffner K (1990) Quantum yield of production of singlet molecular oxygen in aqueous dispersions of small unilamellar lipid vesicles. A time-resolved near-IR phosphorescence study. Photochem ~hotobio151, 551-556

89 Bachowsk:: GJ, Ben-Hur E, Girotti AW (1991) Phtalocyanine-sensitized lipid peroxidation in cell membranes: use of cholesterol and azide as probes of primary photochemistry. J Photochem Photobiol B 9, 307-32 I 90 Kory*,owski W, Bachowski G J, Girotti AW (I 992) Photoperoxidation of cholesterol in homogeneous solution, isolated membranes, and cells: comparison of the 5a-and 6~-hydroperoxides as indicators of singlet oxygen intermediacy. Photochem Photobio156, !-8 9! Agarwal R, Zaidi SIA, Athar M, Bickers DR, Mukhtar H (1992) Photodynamic effects of chloroaluminium phthalocyanine tetrasulfonate are mediated by singlet oxygen: in vivo and in vitro studies utilizing hepatic microsomes as a model membrane source. Arch Biochem Bioph.w 294, 30-37 92 Bachowski GJ, Thomas JP, Girotti AW (1988) Ascorbate-enhanced lipid peroxidation in photooxidized cell membranes: cholesterol product analysis as a probe of reaction mechanism. Lipids 23,580-586 93 Kalyana.-aman B, Feix JB, Sieber F, Thomas JP, Girotti AW (1987) Photodynamic action of merocyanine 540 on artificial and natural cell membranes: involvement of singlet molecular oxygen. Proc Natl Acad Sci USA 84, 2999-3003 94 Bachowski G J, Pintar TJ, Girotli AW (1991) Photosensitized lipi:t peroxidation and enzyme inactivation by membrane-bound merocyanine 540: reaction mechanisms in the absence and presence of ascorbate. Photochem Phoyobio153, 481--491 95 Feix JB, Kalyanaraman B (1991) Production of singlet oxygen-derived hydroxyl radical adducts during merocyanine-540-mediated photosensitization: analysis by ESR-spin trapping and HPLC with radiochemical detection. Arch Bioehem Biophys 291,43-5 ! 96 Sautereau AM, Tournaire C, Suares M, Tocanne JF, Paillous N (1992) Biochem Pharmaco143, 2559-2566 97 Hayakawa K, Matsuo I (1989) Afloqualone phototoxicity - photosensitized lipid peroxidation potency and its biological significance. Tokai J Exp Clin Med 14, 15-24 98 Testylier G, Clement G, Leterrier F (1984) Photochemical interactions of chlorpromazine with phospholipids and cholesterol in artificial and natural membranes. Photochem Photobio139, 277-280 99 Cardenas D. AM, Cortes MP, Fernandez BE, Pena W (1992) Lipid peroxidation and loss of potassium from red blood cells produced by phototoxic quinolones. Toxicology 72, 145-151 100 Matsuo I, Inukai N, Fujita H, Ohkido M (1990) Possible involvement of oxidation of lipids in inducing griseofulvin photosensitivity. Photodermatol Photoimmunol Photomed 7, 213-217 101 Fukuzumi S, Tanii K, Tanaka T (1989) Flavin-sensitized photooxidation of unsaturated fatty acids. J Chem Soc Perkin Trans 2 12, 2103-2108 102 Kochevar IE, Yoon M (1983) Photosensitization of methyl linolealc oxidaiion by tryptophan in peptides. Photochem Photobio137, 279-285 103 Clements AH, Van den Engh RH, Frost DJ, Hoogenhout K (i973) Participation of singlet oxygen in photosensitized oxidation of 1,4-dienoic systems and photooxidation of soybean oil. J Am Oil Chem Soc 50, 325-330 104 Cobern D, Hobbs JS, Lucas RA, M ~kenzi¢ DJ (1966) Location of hydroperoxyde groups in monohydroperoxydes formed by chlorophyll-photosensitized oxi~at.;on of unsaturated esters. J Chem Soc C 21, 1897-1902 105 Sasaki M. Tokiwa K, Hiroyuki M, Sakata 1", Fujita H (i990) Peroxidation of model lipids in the presence of thiophene derivatives with UV-A light. Photomed Photobiol 12, 177-178 106 Logani MK (1984) tert-Butylated-hydroxytoluene-sensitized photooxi&ttion of fatty acids: inhibition by 1,4-diazabicyclo[2.2.2]octane and role of singlet oxygen. J Photochem 25, 275-284 107 Kuroda Y, Sera T, Ogoshi H (1991) Regioselectivities and stereoselectivities of singlet oxygen generated by cyclodextrin-sandwiche~.~'~o~hyrin sensitization. Lipoxygenase-like activity. J Am Chem Soc I 13, 2793-2794 108 Wilhelm E, Battino R (1973) Thermodynamic functions of the solubilities of gases in l':quids at 25°C. Chem Rev 73, 1-9 109 Eisenberg WC, Taylor K, Grossweiner LI (1984J Oxidatiofi o|" egg phospbatidyicholine liposomes by singlet oxygen generated in the gas phase. Photothem Photobiol 40, 55-58 110 Eisenberg WC, Anand J, Wang S, Stevenson RJ (1992) Oxidation of phosphatidylcholine membranes by singlet oxygen generated in the gas phase. Photochem Photobio156, 44 !--445 111 Halliwell B, Gutteridge JMC (1985) Interaction of amiodarone with model membranes and amiodarone-photoinduced peroxidation of lipids. Free Radicals ht Biology. Clarendon Press, Oxford .~,

368 !i2 Barclay LRC, Baskin KA. Locke SJ. Vinqvist MR (1989) Absolute rate constants for lipid peroxidation and inhibition in model biomembranes. Can J Chem 67, i 366-1369 113 Barclay LRC. Baskin KA. Dakin KA. Lock SJ, Vinqvist MR (1990)The antioxi "dant activities of phenolic antioxidants in free radical peroxidation of phospholipid membranes. Can J Chem 68, 2258-2269 114 Indig GL, Campa A, Bechara FJH. Cilento G (1988) Conjugated diene formation promoted by triplet acetone acting upon arachidonic acid. Photothem Photobio148. 719-723 115 Markovic DZ, Patterson LK (1989) Radical processes in lipids. Selectivity of hydrogen abstraction from lipids by benzophenone triplet. Photochem Photobio149, 531-535 116 Markovic DZ. Durand T. Patterson LK (1990) Hydrogen abstraction from iipids by triplet states of derivatized benzopbenone photosensitizers. Photothem Photohiol 5 I. 389-394 117 Bascetta E. Gunstone FD. Walton JC (1983) An ESR study of fatty acids and esters. I. Hydrogen abstraction from olefinic and acetylenic long chain esters. J Chem Soc Perkin Trans 2 603-613 118 Small RD. Scaiano JC. Patterson LK (1979) Radical processes in lipids. A la,~er photolysis study of tert-butoxy radical reactivity towards fatty acids. Pi,lott~'hem Photobio129, 49-5 ! 119 Heijman MGJ. Nauta H. Levine YK {1985) A pulse radiolysis study of the dienyl radical in oxygen-free linoleate solutions: time and iinoleate concentr:~tion dependence. Radiat Phys Chem 26. 73-82 120 Erben-Russ M. Bars W. Michel C, Saran M (1986) Reaction of polyunsaturated fatty acids with the ter-butoxyl radical in aqueous solution. Superoxide Superoxide dismutase Chem Biol Med Proc int Conf4th, 1985, 35--37 121 Erben-Russ M. Bars W. Winter R. Saran M (1986) The reaction of sulfite anion radical (SO~) with polyunsaturated fatty acids. Radiat Phys Chem 27, 419-424 122 Schoeneich C. Asmus KD, Dillinger U. Van Bruchhausen F (1989) Thiyl radical attack on polyun~turated fatty acids: a possible route to lipid peroxidaiion. Bi~whem Biophys Res Commun 16 !. i 13-120 123 Schocneich C, Dillinger U. Von Bmchhausen F. Asmus KD (1992) Oxidation of polyunsaturated fatty acids and lipids through thiyl and sulfonyl radicals: reaction kinetics, and influence of oxygen and structure of thiyl radicals. Arch Biochem Biophys 292. 456-467 124 Schoeneich C, Bonifacic M. Diilinger U. Asmus KD (1990) Hydrogen abstraction by thiyl radicals from activated carbon-hydrogen bonds of alcohols. ethers and polyunsaturated fatty acids. NATO ASI Ser Ser A, 197, 367376 125 Willson RL (1985) Organic peroxy free radicals as ultimate agents in o~ygen toxicity. In: Oxidatire stress (Sies H. ed) Academic Press, Londo~ ~!-7~ 126 f.~ardner. HW (1989) Oxygen radical chemistry of polyunsaturated |a;ty acids. Free Radical Bioi Med 7.65-86 127 Bars W. Erben-Russ. M. Michel C. Saran M (1990) Radical mechanisms in fatty acid and lipid peroxidation, NATO AS! Ser Ser A |89, !-~|6 128 Bars W. Michel C, Saran M (1984) inhibition of the bleaching of the carotenoid crocin. A rapid test for quantifying antioxidant activity. Biochim Biophys Acta 796. 312-319 129 Neff WE, Frankel EN (1984) Photosensitized oxidaLion of methyl linoleate monohydropero~tidcs: hydropcroxy cyclic peroxides, dihydroperoxides and hydroperoxy-bis-cyclic peroxides. Lipids 19, 952-957 130 Davies MJ (1987) Applicatior~s of electron spin resonance spectroscopy to the identification of radicals produced during lipid peroxidation. Chem Phys LiFids 44. 149-173 131 Kalyanaraman B, Sivamjah K (1984) The electron spin resonance study of free radicals formed during the arachidonic acid cascade and cooxidalion of xenobiotics by proslaglandin synthase. In: Free Radicals in Biology 6. (pryor WA. ed) Academic Press, Orlando. Florida, 149-198 132 Zhu J. Johnson WJ, Seviila CL Herrington JW, Sevilla MD (1990) An electron spin resonance study of the reactions of lipid peroxyl radicals with antioxidants. J Phys Chem 94. 7 ! 85-7190 .,pp~ng 133 Buettner GR. Mason RP (1990) ~,,ln ~n" _.,,, • methods for detecting supertou, oxide and hydroxyl free radicais in ritro and in riv,. Meili,ds t~tt.),tttclt .......... , ,o,~ 127-133 134 Janzen EG. DuBose CM. Kotake Y (1990) 2,4,6-Trimethoxyphenyl tertbutyl nitrone, (MO),PBN, as a detector of hydroxyl radicals in the presence of peroxides, superoxide and peroxyl radicals. Tetrahedron Lett 31, 7395-7398

135 Korytowski Wo Bachowski G, Girotti AW (1991) Chromatographic separation and electrochemical determination of cholesterol hydroperoxides generated by hydrodynamic action. Anal Biochem 197, 149-!56 136 Zhang JC. Zhao BL. Guo YJ, Xin WJ (1991) Evidenco for L- against LOO" being spin-trapped by 4-POBN during the reaction of iron (2+)-induced lipid peroxidation. Appl Magn Reson 2, 52 !-53 i 137 Davies MJ, Slater TF (1986) Studies on the photolytic breakdown of hydroperoxides and peroxidized fatty acids by using electron spin resonance spectroscopy. Biochem J 240, 789-795 ! 38 Sugata R. lwahashi H, lshii T, Kido R (1989) Separation of polyunsaturated fatty acid radicals by high-performance liquid chromatography with electron spin resonance and ultraviolet detection. J Chromatogr 487, 9--16 139 Gorman AA, Rodgers MAJ (1992) Current perspectives of singlet oxygen detection in biological environment. J Photochem Photobiol B 14, 159176 140 Bars W. Saran M.. Lengfeider E. Michel C. Fuchs C, Frenzel, C (1978) Detection of oxygen radicals in biological reactions. Photochem Photobiol 28. 629-638 141 Popham PL. Novacky A ( 1991 ) Use of dimethyi sulfoxide to detect hydroxyl radical during bacteria-induced hypersensitive reaction. Plant Physiol 96, 1157-1160 142 Babbs CF. Steiner MG (1990) Detection and quantitation of hydroxyl radical using dimethyl sulfoxide as molecular probe. Methods Enzymol 186, 137-147 143 Foote CS (1979) Quenching of singlet oxygen. In: Singlet oxygen (Wasserman HH, Murray RW, eds) Academic Press. New York, 139--171 144 Farhataziz, Ross AB (1977) Selected specific rates of reactions of transients from water in aqueous solution, ilL Hydroxyl radicals and perhydroxyl radical and their radicals ions. Nail Stand Ref Data Ser Natl Bur Stand 59, I-II3 145 Duthie GG (! 99 l) Measuring oxidation and antioxidant status ha viva. Chem Ind 2, 42--44 146 Piretti MV, pagliuca G (1989) Systematic isolation and identification of membrane lipid oxidation products. Free Radical Bioi Med 7, 219-221 147 Cheeseman KH (1990) Methods of measuring lipid peroxidation in biological systems: an overview. NATO ASI Ser Ser A 189. 143-152 148 Roy TA. Field FH. Lin YY, Smith LL (1979) Hydroxyl ion negative chemical ionization mass spectra of steroids. Anal Chem 5 i. 272-278 149 Scbenck GO, Gollnick K, Neumueller OA (1957) Zur photosensibilisieren Autoxydation der Steroide. Darstellung van Steroid-Hydroperoxyden mittels phototoxischer Photosensibilisatoren. Anal Chem 603.46-59 150 Smith LL. Teng Jl. Kulig MJ, Hill FL (1973) Sterol metabolism XXIil. Cholesterol oxidation by radical-induced processes. J Org Chem 38, 17631765 151 Kulig M, Smith LL (1973) Steroi metabolism. XXV. Cholesterol oxidation by singlet molecular oxygen. J Org Chem 38, 3639-3042 152 Smith LL. Teng II, Kulig MJ. Hill FL (1973) Steroid metabolism XXiII. Cholesterol oxidation by radical-induced processes. J Org Chem 38, 17631765 153 Suwa K. Kimura T, Schaap AP (1977) Reactivity of singlet molecular oxygen with cholesterol in a phospholipid matrix. A model for oxidative damage in membranes. Biochem Biophys Res Commun 75,785-792 154 Girotti AW (1992) Photosensitized oxidation of cholesterol in biological systems: reaction pathways, cytotoxie effects and defense mechanisms. J Photochem Photobiol B 13. 105-118 155 Smith LL (198 l) Cholesterol autoxidation. Plenum Press, New York. 674 p 156 Lands WEM. Kulmacz RJ, Marshall PJ (1984) Lipid peroxide actions in the regulation of prostaglandin biosynthesis. In: Free Radicals ha Biology 6 (Pryor WA, ed) Academic Press, Orlando, Florida, 39-61 157 Marnett LJ (1984) Hydroperoxide-dependent oxidations during prostaglandin biosynthesis, in: Free Radicals in Biology 6 (Pryor WA, ed) Academic Press. Orlando, Florida, 63-94 158 Ran GHR, Gerrard JM, Eaton JW, White IG (1978) Arachidonic acid peroxidation, prostaglandin synthesis and platelet function, Photochem Photobio128, 845-850