New trends in photobiology

.I. Photochem.

Photobiol. B: BioZ., 13 (1992)

105

105-118

Invited Review (New Trends in Photobiology)

Photosensitized oxidation of cholesterol in biological systems: reaction pathways, cytotoxic effects and defense mechanisms Albert

W. Girotti

Department

(Received

of Biochemistry,

December

Medical

College of Wmconsin, Milwaukee,

15, 1991; accepted

December

WI 53226

(USA)

28, 1991)

Abstract Cholesterol resembles other unsaturated lipids in being susceptible to peroxidative degradation when exposed to a sensitizing agent, exciting light of suitable wavelength and molecular oxygen. Selected hydroperoxides of cholesterol can be used as relatively convenient and reliable indicators of primary photochemical mechanisms, allowing a distinction to be made between free radical-mediated and singlet oxygen-mediated reactions. When generated in cell membranes, hydroperoxides of cholesterol and other lipids can have deleterious effects on membrane structure and function. Such damage may be exacerbated if these photoproducts undergo one-electron reduction to oxylrradicals which in turn initiate chain peroxidation reactions. Cells can resist these effects by using a membrane-based glutathione peroxidase to catalyze the two-electron reduction and detoxification of lipid hydroperoxides. Recent advances in our understanding of cholesterol photo-oxidation from the standpoints of (a) mechanistic information, (b) cytotoxicity and (c) cytoprotection are discussed in this article.

Keywords:

oxygen,

Photosensitization, lipid peroxidation, free radicals, cytoprotection.

cholesterol

hydroperoxides,

singlet

1. Introduction

Cholesterol (3/3-hydroxy-cholest-5ene) is a neutral lipid found most abundantly in plasma membranes of eukaryotic cells, in plasma lipoproteins and in bile. Lie all unsaturated lipids, cholesterol is subject to oxidative modification, some aspects of which are physiologically important (e.g. cytochrome P450-catalyzed hydroxylation in the synthesis of steroid hormones, bile salts and vitamin D), whereas others can be pathological (e.g. enzymatic and non-enzymatic peroxidation). The latter aspect will be reviewed in this article, with special attention given to dye- and pigment-sensitized photoperoxidation of cholesterol in membrane systems. Photo-oxidation of cholesterol and other cell membrane lipids in surface tissues may conceivably play a role in various skin phototoxicities, photoaging or photocarcinogenesis [l-3]. Cholesterol may also be

Elsevier Sequoia

106 an important lipid target in many of the therapeutic applications of photosensitization, e.g. the eradication of neoplastic cells or pathogenic viruses by photodynamic therapy (PDT). In addition to the physiological and biomedical implications of cholesterol photo-oxidation, identification of discrete photoproducts can provide valuable information about reaction mechanisms [2]. This article focuses on (a) the use of cholesterol as an endogenous mechanistic probe, (b) the cytotoxicity of cholesterol hydroperoxides and (c) the detoxification or repair pathways.

2. Mechanisms

of cholesterol

photo-oxidation

2.1. Type I vs. type II reactions All photodynamic oxidations, including those involving cholesterol, fall into two mechanistic categories: type I (involving interaction of triplet sensitizer with substrate) and type II (involving interaction of triplet sensitizer with 02) [4]. Both commence with the absorption of a photon by the sensitizer (S), elevating it to a singlet excited state (%), some fraction of which undergoes intersystem crossing to a relatively longlived triplet state (3S) (Fig. 1). The reaction then proceeds in the type I or type II direction, depending on (among other things) the relative concentrations of molecular oxygen and reducing substrate that 3S encounters. In a type I reaction, ?3 abstracts a hydrogen or electron from a reductant (depicted in Fig. 1 as an unsaturated lipid (LH) which could be cholesterol). LH oxidation typically occurs via hydrogen abstraction, with the formation of the lipid alkyl radical (L’) and sensitizer radical (SH), deprotonation of which gives the sensitizer radical anion (s’-). This intermediate has been identified in several different porphyrinsensitized reaction systems [5-71. Of special interest here is the study involving hematoporphyrin-sensitized photo-oxidation of cholesterol and various other unsaturated lipids [5], which provided convincing electron spin resonance (ESR) evidence for the formation of s’- and L’ under anaerobic conditions. Although photoproducts were not characterized, the most prominent cholesterol radical generated in this system was probably the carbon-centered species at C-7 [8]. Rapid reaction of L’ with ground LH . LOO'

O2 --L H202 -

OH'

L' + H20

LH LOOH Fig, 1. Type I and type II pathways of photodynamic lipid such as cholesterol.

lipid peroxidation.

LJ-I denotes an oxidizable

107

state Oz propagates free radical lipid peroxidation via peroxyl radical intermediates (LOO’), with ultimate formation of lipid hydroperoxides (LOOHs) (Fig. 1). Concomitantly, autoxidation of s’- regenerates S and gives rise to superoxide (Oz-), which can undergo disproportionation or reduction to hydrogen peroxide (H,O& The latter may react with 02- via the iron-catalyzed Haber-Weiss cycle [2, 91, generating hydroxyl radical (OH’), a powerful, but relatively indiscriminate, oxidant that can initiate lipid peroxidation by hydrogen abstraction (Fig. 1). Ensuing free radical reactions can radiate from initiation sites until terminated by antioxidants or by depletion of O2 or oxidizable lipid. In membrane systems, OH-mediated photoperoxidation of cholesterol and other lipids is most likely to occur in a “site-specific” manner, i.e. at or near specific ironbinding sites [lo, 111. It is apparent that two different reaction routes are possible in type I photoperoxidation, one in which ?S activates lipids directly, and the other in which s’- (generated by hydrogen or electron transfer from LH or some other reductant) activates oxygen. Although the initiation steps differ in these reactions, the propagation steps are mechanistically similar (Fig. 1) and resemble those of ordinary autoxidation [2, 91. In type II photo-oxidation, 3S transfers its energy to ground state oxygen t30,), elevating it to the ‘A9 singlet excited state (Fig. 1). (In rare instances, electron transfer from 3S to 302 can occur, with formation of O,-.) Singlet molecular oxygen (‘0,) can react directly with cholesterol and other unsaturated lipids (ene addition) to give allylic hydroperoxides [12, 131. All atoms of the hydroperoxyl group derive from ‘02 and the target lipid, with no involvement of free radical intermediates. By contrast, in type I photoperoxidation both oxygen atoms of the hydroperoxyl group derive from 302, with the possible exception of any ‘02 that might be generated via Russell-type disproportionation of type I peroxyl radicals [14]. No photochemical examples of ‘02 arising in this fashion have been reported; however, the weak chemiluminescence that often accompanies non-photochemical autoxidation is typically attributed to ‘02 [15, 161. From the discussion thus ‘far, it is clear that type II lipid peroxidation is much less complex mechanistically than type I. In the case of cholesterol, the type II process also generates far fewer LOOH products (see Section 2.3). The relative importance of type I and type II reactions in a membrane bilayer will depend on factors such as (a) the ability of the sensitizer to interact with the membrane, (b) the rate constant for the reaction of 3S with 302 VS. that of 3S with LH and (c) the relative concentrations of 302 and LH that 3S encounters if membrane bound. Since a membrane-bound sensitizer would be in relatively close proximity to LH, this would be expected to favor type I chemistry. However, since the concentration of oxygen in the membrane tends to be higher than in solution [9], it is difficult to predict how 3S might partition without detailed information about its local environment. As discussed in Section 2.3, however, such information can be readily obtained by using cholesterol as an “internal” reporter. Suffice it to say that, by this approach, most membrane-based photoperoxidation reactions studied up to now have been shown to be predominantly mediated by ‘Oz

PI2.2. Reduction of photoperoxides It is important to note that in type I or type II peroxidation, LOOHs can be end-products or intermediates, depending on the reaction conditions [2, 91. In simple cell-free systems lacking reducing agents, photoperoxides tend to accumulate in direct proportion to increasing light dose [17]. However, in cells LOOHs may undergo (a) one-electron reduction reactions, which exacerbate potentially lethal peroxidative dam-

108

age, or (b) two-electron reduction reactions, which prevent, or at least minimize, peroxidative damage (Fig. 2). In the one-electron pathway, a reducing agent such as Oz- or ascorbate typically drives the iron-catalyzed reduction of nascent LOOHs to lipid oxyl radicals (Lo’), which either directly or indirectly [18] initiate rounds of free radical peroxidation by hydrogen abstraction. Enhanced formation of free radical intermediates (Lo’, LOO) or carbonyl by-products in these reactions may result in cytotoxicity which far exceeds that due to photoperoxidation alone [19-211. Alternatively, LOOHs may undergo two-electron reductive metabolism mediated by glutathione(GSH)dependent peroxidases of transferases [2]. Oxidative stress arises when toxic oneelectron reduction of photoperoxides outpaces two-electron detoxification, resulting in aggravated cell damage (see Section 4).

2.3. Use of cholesterol as a mechanistic probe Photoperoxidation of cholesterol and other membrane lipids can be “diagnosed” for ‘02 or free radical intermediacy in a variety of ways, each with certain advantages and disadvantages. An approach that has been widely used is to determine whether the reaction is inhibited by well-established ‘02 scavengers (e.g. azide, histidine, /?carotene) or by free radical scavengers (e.g. mannitol for OH’, butylated hydroxytoluene for LOO’ or Lo’). Unfortunately, many of these agents lack absolute specificity (e.g. azide intercepts both IO2 and OH). Many, including azide and mannitol, are too polar to interact with membranes, and others (e.g. butylated hydroxytoluene) may interact, but in doing so may perturb membrane structure. Approaches based on the stimulation of a reaction have also been used. For example, D20 increases the lifetime of IO2 by about 15-fold and on this basis would be expected to accelerate ‘Oz.-mediated reactions [22]. However, in order to work, this strategy requires (among other things) that D20 be exchanged efficiently for HZ0 in the reaction system. Since photoactive sites on membranes may be Hz0 poor, lack of a DzO effect may be wrongly interpreted as non-involvement of ‘Oz [13]. A more rigorous alternative to these approaches is based on product screening, i.e. identification of species that are uniquely characteristic of either ‘02 or free radical intermediacy. The biological substrate that has been exploited to greatest advantage in this regard is cholesterol. Although phospholipid polyunsaturated fatty acyl groups One-electron Pathway

Two-electron Pathway

le- (

LOOH ,

\

: I 8 I

OH-+LO'

2e-, 2H+ LOH + H20

LH LOOH

LOH

I!

Fig. 2. One-electron

x

VS. two-electron

reduction of photochemically

generated

lipid hydroperoxides.

109

have also been considered as possible reaction probes, cholesterol is clearly superior for the following reasons: (a) it exists naturally in membranes as a single molecular species, making product determination less complicated; (b) during sample preparation, potentially artifactual hydrolysis steps are unnecessary with cholesterol; (c) it can be easily exchange-radiolabeled in membranes and cells, pe~itting highly sensitive and specific product analysis. Cholesterol products generated by photo-oxidation and by a variety of other pro-oxidant conditions (e.g. exposure to ionizing radiation, ozone or oxidative enzymes) have been well characterized in simple systems such as organic solvents, micelles and liposomal membranes [8,23-291. In free radical-mediated reactions, the epimeric pair, 3P-hydroxycholest-5-ene-7ahydroperoxide (7cu-OOH) and 3@-hydroxycholest-5-ene-7P-hydroperoxide (7/?-OOH), are generally the most prominent products (Fig. 3), followed by lesser amounts of the dihydroxy derivatives cholest-5ene-3/3,7tr-diol(7~OH) and cholest-5-ene-3~,7~-diol (7@OH), and 3~-hydro~cholest5-ene-7-one (7-one). Small amounts of epimeric 5,depoxides and side-chain oxidation products (20-, 24- and 25OOH) may also be observed, together with trace levels of various other species [8]. The free radical-derived hydroperoxides arise via direct attack of an oxidant on cholesterol (initiation) and/or via propagation reactions (see Fig. 1). In ‘02-mediated reactions, the major product uniquely indicative of this oxidant is 3/3-hydroxy-Sat-cholest-6-ene-5-hydroperoxide (SLY-OOH) (Fig. 3). The only other products known to be generated by ‘02 attack on cholesterol (albeit in much lower yields than 5a-OOH) are 3~-hydro~cholest-4-ene-6~-hydropero~de (6a-OOH) and 3/3-hydro~cholest-4-ene-6~-hydroperoxide (6/3-OOH). Although ‘02 does not produce 7~ or 7/3-OOH, this pair can arise via allylic rearrangement of 5~00H, which is especially pronounced in non-polar solvents [30,31]. On the other hand, no 5a-OOH is generated in reactions that are purely free radical in nature [25]. It should be pointed out that, compared with other biological acceptors (e.g. certain amino acids), choiesterol reacts with ‘02 at a low rate (k = 7 X lo4 MI s-’ in pyridine [23, 321) which usually translates into low product yields in photoreactions carried out under physiologically relevant conditions. Problems of detection and quantitation have been alleviated for the most part by the availabili~ of radiolabeled cholesterol of high specific activity and also by the development of ultrasensitive new detectors for chromatographic analysis ]331* Sensitizers of the flavin, ketone or quinone family are believed to be intrinsically more disposed towards free radical photochemist~ than ~rphyrin, thiazine or xanthene sensitizers [34-361. In at least one instance this has been clearly confirmed by photooxidizing cholesterol. Thus UVA (320-400 nm) irradiation of menadione-sensitized cholesterol in methanol solution produced type I products exclusively, mainly 7~ and 7&00H, which were identified by thin Iayer chromatography (TLC) subsequent to borohydride reduction [36]. Proof of ‘Oz intermediacy by cholesterol product analysis has been cited more frequently in the literature, beginning with the classic studies of Schenck and collaborators. Schenck et al. [23] and, later, Kulig and Smith [24] showed that exposure of hematopo~hyrin-sensit~ed cholesterol in pyridine solution to broadband visible light resulted in high yields of .5jcu-OOH, with relatively little (l%-2%) 6ar/6@00H. It was noted that rearrangement of 5cr-OOH to 7~00H (and thence epimerization to 7/3-OOH) occurs much more slowly in pyridine than in chloroform or hexane 123, 241. More recent studies have shown that 5a-OOH is quite resistant to rearrangement in membrane bilayers containing unsaturated phospholipids, but loses this property during lipid extraction [20, 371. Any conversion of SC+OOH to 7&& OOH during a photoreaction and/or sample preparation may cause confusion in assigning mechanisms. This point is considered further in the next section.

110

Ch

1

Type

Type II Oo*)

PO*) \

/

c8 H17

HO dP

HO

70(iOOH 78-00 H

3 OOH H

: ’ iOH SK-OOH

R2 H OOH

e

Ho ’

veH17

;,

% R2

6a-OOH VOOH

Rl OOH

R2 H

H

OOH

Fig. 3. Cholesterol hydroperoxides generated by type I and type II photochemistry. Peroxides are typically derived from ground state oxygen (302) in type I reactions and from singlet excited oxygen (‘0,) in type II reactions. In the former case, only the major photoproducts are represented, namely 7~ and 7@00H.

3. Photogeneration

of cholesterol

hydroperoxides

in membranes

3.2. Model systems That cholesterol can serve as a specific and sensitive probe of type I vs. type II photochemistry in membrane systems was first demonstrated by Suwa et al. [26, 271. Using hematoporphyrin-sensitized [14C]-cholesterol in unilamellar phospholipid vesicles (liposomes), these workers determined (a) that 5a-OOH was the most abundant photoproduct, establishing ‘02 as the major reactive intermediate, and (b) that the yield of SWOOH increased with temperature in parallel with the phase transition of

111

phospholipid, suggesting that membrane fluidity influences the reactivity of ‘02 with cholesterol. Relatively recent studies in our laboratory have shown that cholesterol can also serve as an effective reporter in a natural membrane model, the human erythrocyte ghost. Using TLC and high performance liquid chromatography (HPLC) for separating and analyzing photoproducts (in some instances [r4C]-labeled species), we found that several different sensitizers, including protoporphyrin IX, uroporphyrin I, purified hematoporphyrin derivative (HPD-A), merocyanine 540 (MC540) and aluminum phthalocyanine tetrasulfonate (AlPcS), generate 5a-OOH preferentially in ghost membranes [17, 20, 21, 3S-401. In most cases, smaller amounts of 7cu/7@00H were also observed; almost invariably, these were attributed to Sa-OOH rearrangement rather than authentic type I photochemistry [31]. Screening of cholesterol products has also provided valuable insights into interactions of photoperoxides with one-electron donors in cells (see section 2.2 and Fig. 2). For example, when HPD-A-sensitized ghosts were irradiated in the presence of ascorbate (AH-) and supplemental iron (Fe3+), there was a large burst of thiobarbituric acid (TBA) reactivity, indicative of free radical peroxidation [20]. Concomitantly, SWOOH was replaced by 7a/7P-OOH, 7cJ7P-OH and 7-one in the product population, consistent with switching from a ‘02dominated reaction to a free radical-dominated process. Our evidence indicated that the 7-oxysterols were produced by reduction of type II hydroperoxides (including 5~ OOH and phospholipid species) rather than by AH--driven type I photochemistry [2]. This is one of the first reported examples of how cholesterol can be used to monitor mechanistic changes under different reaction conditions. In more recent work, the kinetics of formation of cholesterol photoperoxides (5~ OOH, 6-OOH and 7-OOH) in various membrane systems have been measured and compared for the first time [41]. A newly developed analytical approach, HPLC with electrochemical (EC) detection [33], was used for highly sensitive and specific monitoring of cholesterol hydroperoxides. Irradiation of AlPcS- or MC540-sensitized cholesterol in phospholipid vesicles or erythrocyte ghosts resulted in SOY-OOH and 6P-OOH formation, the initial rate of the latter being approximately 30%-35% that of the former. (Though also detected, ~CZ-OOH accumulated much more slowly than 6pOOH.) By comparison, the photogeneration rate of 6P-OOH in pyridine or methanol was only lo%-15% that of 5~00H, resulting in relatively poor yields of the 6-isomer in these solvents, as had been observed previously [23, 241. Unlike 6@00H, which accumulated in apparent linear fashion during irradiation, 5~00H reached a peak yield of 5%-lo%, and then decayed via rearrangement to 7~00H [41]. After prolonged photo-oxidation periods, virtually all of the Sa-OOH appeared as 7a/7P-OOH. The factors responsible for the 5~00H threshold and for the high yield of 6P-OOH in membranes vs. homogeneous solution are not yet clear. On the basis of its relative stability and reasonably good yields (by modern detection standards) in photo-oxidized membranes, 6P-OOH is proposed as a more reliable indicator of IO2 intermediacy and ‘0, reaction kinetics than 5~00H. 3.2. Cellular systems When generated in cells, species such as i02, oxyradicals or lipid-derived radicals may have extremely short lifetimes due to their rapid quenching by a profusion of cellular acceptors. This often precludes direct determination of these species by spectroscopic means [42]. In the case of photochemically generated ‘02, this problem has been partially overcome by the development of supersensitive detectors which allow measurement of the weak luminescence of ‘02 at 1270 nm [43]. In addition to being quite esoteric, this approach, like many others based on light emission, is subject

112

to artifacts. Moreover, the physical detection of ‘02 escaping from a site of origin in a cell does not necessarily imply that ‘02 reacts with vital targets such as unsaturated lipids. From this standpoint, the advantages of using cholesterol as an in situ chemical probe are quite clear. We have recently adapted this approach to living cells [41]. Murine leukemia L1210 cells were exchange-radiolabeled with [‘4C]-cholesterol and subjected to AlPcS-sensitized photo-oxidation at 10 “C. HPLC with radiochemical detection revealed that both 5c~-OOH and 6/3-OOH were produced in this system, the latter at approximately one-third the initial rate of the former (1% h-’ US. 2.7% h-‘), as observed in the model membrane systems (see above). Identification and quantitation of these species provide not only the first unequivocal chemical evidence for the photogeneration of ‘02 in a cell, but also for the reaction of ‘02 with an important cellular target. It should be pointed out, however, that in order to generate detectable levels of 5a-OOH and 6P-OOH, it was necessary to use light doses that were far in excess of normal killing doses [41]. However, there is no reason not to believe that ‘02 was also generated during the early stages of irradiation and that it produced lethal damage.

4. Cytotoxicity

of cholesterol

hydroperoxides

Cholesterol hydroperoxides generated by autoxidation or by sensitized photooxidation have been shown to be cytotoxic and/or mutagenic [8, 44, 451. Cytotoxicity may reflect the fact that cholesterol hydroperoxides, like other LOOHs, physically perturb membrane structure and function. On the other hand, these species may undergo partial reduction to free radicals, which trigger peroxidative damage that affects membranes and other structures (see Fig. 2). An example of cell damage mediated specifically by photoperoxidized cholesterol was reported several years ago by Lamola et al. [44]. They irradiated cholesterol in the presence of protoporphyrin and incorporated the major photoproduct (designated as Scr-OOH) into phospholipid vesicles. When incubated with these vesicles, human erythrocytes underwent an initial decrease in osmotic fragility, followed by a large increase in fragility and then lysis (hemoglobin release). These effects were shown to be dependent on hydroperoxide uptake by the cells, which presumably occurred via an exchange process. Lamola et al. [44] also showed that 5a-OOH is generated in isolated erythrocyte membranes during porphyrin-sensitized photo-oxidation. This suggested that 5a-OOH may be an important molecular mediator of some of the phototoxic effects (e.g. cutaneous lesions) associated with certain porphyrias. Geiger et al. [46] have recently reported that photoperoxidized cholesterol can also cause lethal injury to cultured cells. By contrast, peroxidized egg phosphatidylcholine was found to be relatively innocuous. In this study, unilamellar liposomes composed of cholesterol, dimyristoyl phosphatidylcholine and dicetylphosphate (the last two constituents being non-oxidizable) were sensitized with rose bengal and then irradiated for different periods to give preparations of increasing cholesterol hydroperoxide content, as determined iodometrically. When given to murine L1210 cells in increasing peroxide (constant lipid) doses, the liposomes were found to be cytotoxic, as determined by clonogenic assay (LC,, = 75 PM peroxide). Cholesterol hydroperoxide was mainly in the form of ~cY/~P-OOH (due to 5c~-OOH rearrangement) when incubated with the cells. Control liposomes containing the diol analog 7/3-OH instead of 7a/7&00H were found to be minimally toxic (LDsO > 250 PM), ruling out the possibility that enhanced cell killing was a non-specific oxysterol effect. Cells pretreated with desferrioxamine or butylated hydroxytoluene were much less sensitive to

113

photo-oxidized cholesterol than non-treated cells, implying that cytodamage was mediated by iron and free radicals. A mechanism based on intracellular reduction of cholesterol hydroperoxides to noxious free radical intermediates is consistent with these findings (see Section 2.2). 5. Metabolic

detoxification

of cholesterol

hydroperoxides

As is true for other oxidative damage in cells, photoperoxidation of cholesterol and other lipids can be prevented, or at least minimized, by a variety of enzymatic and non-enzymatic defense systems [47]. Prominent among the enzymatic systems are (a) superoxide dismutase and catalase, which scavenge Oz- and HzOz respectively, (b) glutathione (GSH)-dependent selenoperoxidases, which reduce and detoxify H,Oz and a variety of organoperoxides, including lipid-derived species, and (c) non-selenoGSH-S-transferases, which can also reduce organoperoxides. The reaction catalyzed by the selenoperoxidases and peroxidatic transferases is shown in eqn. (1) (refer also to Fig. 2) ROOH + 2GSH -

ROH + GSSG + H,O

NADPH + H+ + GSSG -

(I)

NADP + + 2GSH

(2) where ROOH denotes a hydroperoxide and ROH its alcohol derivative. Equation (2) depicts the natural regeneration of GSH catalyzed by GSSG-reductase, which uses NADPH as the two-electron donor. Two selenoperoxidases are known to exist: (i) classical GSH-peroxidase (GPX), a tetramer located in cytosol and mitochondrial matrix [48, 491; and (ii) the more recently discovered phospholipid hydroperoxide GSH-peroxidase (PHGPX), a much less abundant monomer located in both cytosol and membranes [50]. Both enzymes can act on relatively polar substrates such as H202 and fatty acid hydroperoxides. However, only PHGPX is capable of acting directly on phospholipid hydroperoxides in membrane bilayers [50,51]; GPX, and also the peroxidatic transferases, require the prior hydrolytic release of the sn-2 fatty acyl hydroperoxide moiety, as catalyzed by phospholipase A2 (PLAz), for example [52, 531. It has been proposed that detoxification of membrane-associated peroxides is one of the major physiological functions of PHGPX, whereas detoxification of free peroxides in the cytosol and mitochondrial matrix is mediated primarily by GPX [51]. 5.1. Studies on isolated membrane systems Most of the above information on GPX and PHGPX reactivities was acquired using artificial membranes (liposomes) containing oxidizable phospholipids, but not cholesterol [50, 52, 531. More recent evidence has indicated that photochemically generated phospholipid hydroperoxides in a natural cell membrane, the erythrocyte ghost (60 wt.% phospholipid; 25 wt.% cholesterol), must also be hydrolyzed by PI& before peroxide groups are recognized by GPX [54, 551. However, the cholesterol hydroperoxides were found to be totally resistant to GPX, even after extraction from the membrane and solubilization in dilute alcohol. Furthermore, incubation of photoperoxidized ghosts with AH- and iron resulted in a burst of TBA-detectable (free radical) lipid peroxidation that was only partially inhibited by prior PLAJGPX treatment. Since the inhibition was partial, whereas the reduction of mobilized fatty acid hydroperoxides was total, it was apparent that cholesterol-derived oxyl radicals (see Fig. 2) were the primary chain initiators in the AH--driven reaction. How potentially lethal cholesterol hydroperoxides might be detoxified in cells was an open question at that point [54]. Subsequent studies with PHGPX provided some insights. Most important

114

was the observation that all photochemically generated hydroperoxides in ghost membranes (cholesterol- as well as phospholipid-derived) were completely reduced in situ during incubation with GSH/PHGPX [56, 571. In agreement with previous results [54, 551, the LOOHs were inert to GSH/GPX alone, and were reduced by only 60%-65% after treating with GSH/GPX subsequent to PLA2. PHGPX treatment of photo-oxidized ghosts rendered them completely insensitive to AH- and iron, clearly demonstrating the protective effectiveness of PHGPX compared with GPX. 5.2. Studies on cellular systems As a follow-up to our findings in cell-free systems (preceding section), we asked the following question: do cells use selenoperoxidases to defend themselves against peroxide stress, specifically that induced by photochemically generated cholesterol hydroperoxides? Since GPX and PHGPX have no known specific inhibitors, selenium manipulation is the only viable option for modulating these enzymes in an effort to elucidate their protective roles. A relatively straightforward approach for depleting selenium in cultured cells is to limit the concentration of serum in the growth media. Using 1% serum, Geiger et al. [46] were able to grow selenium-deficient L1210 cells (designated Se(-)) that expressed less than 5% of the GPX or PHGPX activity of selenium-supplemented controls (designated Se( + )) or selenium-repleted Se( - ) cells (designated Se( -/+)). Clonogenic survival assays indicated that Se(-) cells were much more sensitive to exogenous cholesterol hydroperoxides presented in liposomal form (see Section 4) than Se( +) or Se( - /+) cells (LC,, = 10 PM vs. LC,, = 75 PM). The involvement of GSH in cytoprotection (as a selenoperoxidase substrate and possibly also as a direct free radical interceptor) was established by showing that Se(+) cells were more sensitive to peroxides (mainly 7a/7@-OOH) after treating with buthionine sulfoximine, an inhibitor of GSH biosynthesis [58]. Preliminary evidence [59] has indicated that Se( - ) cells metabolize cholesterol hydroperoxides to diols at a lower initial rate than Se( +) cells, consistent with the fact that the latter are more peroxide resistant. If protection depended on metabolic detoxification of cholesterol hydroperoxides as they entered the cells, then PHGPX will be uniquely involved, since GPX does not react with cholesterol hydroperoxides. PHGPX would have been involved at a secondary level if any oxidation of cellular phospholipids or cholesterol had occurred (see Fig. 2). On the other hand, if toxic fatty acid hydroperoxides had been liberated during secondary oxidation, GPX might also have contributed to cytoprotection, making the overall process quite complex [46]. Related studies have provided new insights into how tumor cells cope with photo-oxidative stress during PDT treatment [60]. In these studies, Se( - ) L1210 cells were found to be much less resistant to HPD-Asensitized photoperoxidation (endogenous formation of cholesterol and phospholipid hydroperoxides) and also to photokilling than Se( + ) controls, implying a strong selenoperoxidase contribution to cytoprotection. Among the many unanswered questions in this area (applying to photochemical as well as non-photochemical systems) is the relative importance of PHGPX and GPX in cytoprotection, whether it be against exogenous or endogenous hydroperoxides. Also unclear is how cells repair or refashion membranes in which various LOOH compounds have been enzymatically reduced and/ or excised [61]. 6. Concluding

remarks

Cholesterol is an important molecular target of biological photodynamic action, particularly in plasma membranes, where it typically comprises 40-45 mol.% of the

115

total lipid. Together with unsaturated phospholipids, cholesterol is converted to hydroperoxide photoproducts, which may act as physical perturbants of membrane structure and function or as nuclei for free radical lipid peroxidation, which exacerbates membrane damage. From the mechanistic perspective, membrane-associated cholesterol can be used as an unequivocal probe of singlet oxygen-mediated ZX. free radical-mediated photochemistry. Recent advances in the high resolution separation and detection of peroxide photoproducts have made this approach feasible for complex cellular systems. This should allow reaction pathways for new sensitizers of biomedical interest (e.g. PDT dyes) to be probed under physiologically relevant conditions. Cholesterol hydroperoxides have been shown to be cytotoxic on the one hand and subject to metabolic detoxification on the other. Our understanding of this particular aspect is still rather fragmentary, not only for peroxides of cholesterol (photochemically generated or otherwise), but also for those of other lipids.

Acknowledgments

The author is grateful to G. J. Bachowski, P. G. Geiger, W. Korytowski and J. P. Thomas for their valuable contributions to some of the work described in this article. He also wishes to thank L. L. Smith and J. I. Teng for helpful discussions about HPLC analysis of cholesterol oxidation productions. The generous support of the National Science Foundation and the National Cancer Institute of the United States Public Health Service is also acknowledged. References

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for free radical induction in unsaturated

fatty acids and for singlet oxygen production,

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