photo-oxidation of cells generates long-lived intracellular protein peroxides

Original Contribution

PHOTO-OXIDATION OF CELLS GENERATES LONG-LIVEDINTRACELLULAR PROTEIN PEROXIDES

ADAM WRIGHT, CLARE L. HAWKINS, and MICHAEL J. DAVIES

Free Radical Group, Heart Research Institute, Sydney, New South Wales, Australia

(Received 8 July 2002;Revised 6 November 2002;Accepted 15 November 2002)

Abstract—Singlet oxygen is generated by several cellular, enzymatic, and chemical reactions as well as by exposureto UV or visible light in the presence of a sensitizer. Consequently, this oxidant has been proposed to be a damagingagent many pathologies. Proteins are major targets for singlet oxygen as a result of their abundance and high rateconstants for reaction. In this study, we show that illumination of viable rose bengal-loaded THP-1 (human monocyte-like) cells with visible light gives rise to intracellular protein-derived peroxides. The peroxide yield increases withillumination time, requires the presence of rose bengal, is enhanced in D2O, and is decreased by azide, consistent withthe mediation of singlet oxygen. The concentration of peroxides detected, which is not affected by glucose or ascorbateloading of the cells, corresponds to about 1.5 nmoles peroxide per 106 cells, or 10 nmoles/mg cell protein, and accountfor up to approximately 15% of the O2 consumed by the cells. Similar peroxides have been detected on isolated cellularproteins exposed to light in the presence of rose bengal and oxygen. After cessation of illumination, cellular proteinperoxide levels decrease with t1/2 about 4 h at37°C. Decomposition of protein peroxides formed within cells, or onisolated cellular proteins, by metal ions gives rise to radicals as detected by EPR spin trapping. These studiesdemonstrate that exposure of intact cells to visible light in the presence of a sensitizer leads to novel long-lived, butreactive, intracellular protein peroxides via singlet oxygen-mediated reactions. © 2003 Elsevier Science Inc.

Keywords—Protein oxidation, Singlet oxygen, Peroxides, Photo-oxidation, Free radicals

INTRODUCTION

Singlet oxygen (molecular oxygen in its1�g state;1O2) isknown to be produced in several heme protein (e.g.,reactions involving myelo-, lacto-, chloro-, and eosino-phil peroxidases) and lipoxygenase-catalyzed reactions[1–3]. 1O2 generation has also been detected with stim-ulated cell types including neutrophils, eosinophils, andmacrophages [4–6]. UV exposure can also generate1O2,as can visible light in the presence of suitable (exogenousor endogenous) sensitizers [7].1O2 reacts with a broadspectrum of biological molecules including DNA, cho-lesterol, lipids, amino acids, and proteins [8], and it hasbeen postulated that1O2-mediated damage plays a role inthe development of human pathologies including cata-ract, sunburn, and some skin cancers, as well as in theaging process [9].

Previous studies have elucidated some of the productsarising from the reaction of1O2 with cholesterol, lipids,and DNA (reviewed in [10]). With each of these targets,reactive peroxides (endoperoxides or hydroperoxides)are generated as key intermediates. Although convincingevidence has been presented for1O2-mediated oxidationof cholesterol, lipids, and DNA in cells [11–13], andcholesterol oxidation products have been detected inintact photo-oxidized rat skin [14], it is clear that proteinsare also major intracellular targets for1O2 due to theirabundance and fast rates of reaction [15–17].

Amino acid residues within proteins can react with1O2 by two mechanisms. Under physiological conditions,physical quenching is only a major reaction pathway forTrp, although this amino acid also undergoes chemicalreaction (rate constants of about 2–5� 107 and 3� 107

dm3 mol�1 s�1 for physical and chemical reactions,respectively [18]). All other amino acids are oxidized by1O2 via chemical reaction. Of the normal physiologicalamino acids, only five are appreciably oxidized by1O2.These include: His (k about 3.2� 107 dm3 mol�1 s�1 at

Address correspondence to: Dr. Michael J. Davies, Heart ResearchInstitute, 145 Missenden Road, Camperdown, Sydney, NSW 2050,Australia; Tel:�61 (2) 9550-3560; Fax:�61 (2) 9550-3302; E-Mail:[email protected].

Free Radical Biology & Medicine, Vol. 34, No. 6, pp. 637–647, 2003Copyright © 2003 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/03/$–see front matter

doi:10.1016/S0891-5849(02)01361-8

637

pH 7.1, though this value is markedly pH dependent);Trp (k 3 � 107 dm3 mol�1 s�1); Tyr (k 0.8 � 107 dm3

mol�1 s�1); Met (k 1.6 � 107 dm3 mol�1 s�1); and Cys(k 8.9 � 106 dm3 mol�1 s�1) [16]. All other side-chainsreact with k � 0.7 � 107 dm3 mol�1 s�1 [16]. At highpH values, where there is a significant concentration ofthe unprotonated (neutral) form, Arg and Lys also un-dergo photo-oxidation [19].

The chemical consequences of the reaction of 1O2

with proteins have been partially elucidated. These in-clude oxidation of side-chains, backbone fragmentation(though this appears to be only a minor process withmost proteins), dimerization or aggregation, unfolding orconformational changes, and alterations in cellular han-dling and turnover (reviewed in [7,17,20,21]). Cellularresponses to such alterations include the induction ofvarious genes, including those for heme oxygenase [22],mitogen-activated kinases [23], matrix metalloprotein-ase-1 [24], collagenases [25], NF-�B [26], and AP-2[27], and the occurrence of apoptosis [28,29]. The anti-apoptotic protein Bcl-2 has been reported to be particu-larly susceptible to 1O2 damage, with its loss promotingthe induction of apoptosis in MCF-7 cells [30].

Previous studies have shown that oxidation by 1O2 ofTrp, His, and Tyr residues on isolated peptides andproteins can result in the formation of semistable perox-ides. The structures of some of these peroxides have beendetermined [31–34]. Decomposition of such peroxideshas been demonstrated to yield further reactive species,including oxygen- and carbon-centered radicals [34,35],and result in protein cross-linking and enzyme inhibition[34–36]. These reactive species may, therefore, play arole in the observed biological consequences of 1O2

generation within cells.To examine the potential role of 1O2-mediated protein

peroxide in cellular damage, we have investigated theoxidation of cellular proteins to peroxidic species by 1O2,the stability of such peroxides, and their decompositionto radicals.

MATERIALS AND METHODS

Materials

All water used was filtered through a four-stage MilliQ system (Millipore-Waters, Milford, MA, USA)equipped with a 0.2 �m pore size final filter. All chem-icals were of the highest grade commercially availableand used as received with the exception of DMPO, whichwas purified before use by treatment with activated char-coal. Protein concentrations were determined using theBCA assay (Pierce, Rockford, IL, USA) using a standardcurve prepared using BSA. Solutions of Fe2�-EDTA(1:1 molar ratio) were prepared by the addition of solid

FeSO4 to a solution of deoxygenated (bubbling withO2-free nitrogen) EDTA in water. This solution wascontinually deoxygenated during use to prevent autoxi-dation.

Cell culture

THP-1 cells, a human monocyte-like cell line, werecultured in RPMI-1640 medium (CSL, North Ryde,NSW, Australia) with 10% fetal calf serum (Life Tech-nologies, Mulgrave, VIC, Australia), 2 mM L-glutamine(Trace Scientific, Melbourne, Victoria, Australia), 100IU ml�1 penicillin, and 100 �g ml�1 streptomycin (Sig-ma Chemical Co., St. Louis, MO, USA), at 37°C, underan atmosphere of 5% CO2. Cells were prepared forexperiments by washing twice with HBSS (SigmaChemical Co.). They were then incubated with HBSSand 5 �M rose bengal for 30 min at 37°C in 5% CO2, inthe absence of light. Non-rose bengal-containing controlexperiments were treated with HBSS alone. The cellswere then washed twice with HBSS, to remove extracel-lular rose bengal, and resuspended in HBSS at a concen-tration of 4 � 106 cells ml�1. When stated, cells wereresuspended at an identical concentration in phosphate-buffered saline (PBS), PBS containing 5 mM glucose,HBSS made with D2O in place of H2O, and HBSScontaining 5 mM sodium azide. Loading of cells withascorbate was achieved by incubation of 4 � 106 cellsml�1 for 30 min with 100 �M dehydroascorbic acid, asdescribed previously [37]. Subsequently, the cells werepelleted, washed twice with normal HBSS to removeexcess dehydroascorbate, and then resuspended as out-lined above. Ascorbate concentrations were measured byHPLC as described below. This treatment gave intracel-lular ascorbate concentrations of 10 nmol mg cell pro-tein�1 when loading was performed in the presence ofrose bengal, and 13 nmol mg cell protein�1 in the ab-sence of rose bengal. Ascorbate levels in nonloaded cellswere below the sensitivity limit of the detection system(� about 1 pmol mg cell protein�1).

Intracellular photochemical 1O2 generation andassessment of peroxide yields

Illumination of cells was performed in 6-well plateswith 2 ml of cell suspension per well. The light sourceused was a commercially available 40 W tungsten fila-ment lamp (Osram, Pennant Hills, NSW, Australia), fil-tered through a 345 nm cut-off filter, which was posi-tioned 5 cm distant from the cell suspensions. Illuminationwas carried out at 4°C for periods of up to 20 min.Peroxide levels were then assayed using a modified FOXassay [38]. After the cessation of photolysis, cellularproteins were precipitated from 1.5 ml of cell suspensionwith cold TCA (10% final concentration). Pellets were

638 A. WRIGHT et al.

washed with 1 ml cold 10% TCA and resuspended in 900�l of 25 mM H2SO4. Next, 50 �l of a stock solutioncontaining 5 mM FeSO4 and 2 mM xylenol orange (in 25mM H2SO4) were added to the samples, which were thenvortex mixed, and, subsequently, incubated in the dark atroom temperature for 30 min. The samples were centri-fuged to remove cellular debris and their absorbance at560 nm was measured. Peroxide concentrations weredetermined by comparison to a standard curve preparedusing H2O2.

Photo-oxidation of isolated cellular proteins anddetermination of peroxide yields

Bulk cellular proteins were precipitated with TCA(5% w/v), washed (5% TCA), and then resuspended in 6M urea at a concentration of about 1.5 mg ml�1. The pHof the solution was subsequently adjusted to 7.4 usingNaOH. Rose bengal (10 �M) was then added to theprotein samples and illuminated at 4°C using a KodakS-AV 2050 slide projector (250 W bulb; Eastman Kodak,Rochester, NY, USA) filtered through a 345 nm cut-offfilter, with constant aeration for purified proteins as de-scribed previously [34,35]. Peroxides formation wasquantified using a modified FOX assay [38]; 50 �l of 5mM FeSO4 and 2 mM xylenol orange (in 25 mMH2SO4) were added to the samples, which were thenincubated in the dark at room temperature for 30 min.The absorbance of the samples at 560 nm was subse-quently measured and compared to a standard curveprepared using H2O2. Previous studies have shownthat this assay gives peroxide values that are similarbut slightly higher (by � 30%) than those detectedusing an iodometric method (A. Wright, C. L.Hawkins, M. J. Davies, unpublished data).

Measurement of cell viability

Cell viability was estimated by measuring the releaseof DNA into the medium, as a percentage of total DNAin the same number of intact cells after lysis with TritonX-100, using ethidium bromide [39]. After the requiredphotolysis period, an aliquot of cell suspension was takenand diluted to 2 � 106 cells ml�1 with HBSS. The cellswere then pelleted by centrifugation at 800 � g for 5min. The supernatant was transferred to a fresh tube andthe cell pellet lysed by the addition of 1 ml of 1% TritonX-100 (Sigma Chemical Co.). Then, 10 �l of 2.5 mMethidium bromide was added to both supernatant andlysate samples and the fluorescence measured using �ex

360 nm and �em 580 nm. Appropriate blanks (HBSS orTriton) were subtracted and the cell death was calculatedusing the following equation: 100 � ({(lysate � blankfluorescence)/[(sample � blank fluorescence) � (lysate� blank fluorescence)]} � 100).

Oxygen consumption experiments

Oxygen consumption was measured using a Clark-typeoxygen electrode (Rank Brothers Ltd., Cambridgeshire, En-gland), with concentrations recorded manually every 30 s.Cell suspensions (4 � 106 cells ml�1 in HBSS, 5.5 ml totalvolume) were stirred slowly and allowed to equilibrate withair at room temperature (�22°C) and atmospheric pressure.When steady state readings were obtained, the vessel wassealed and the cells illuminated using a 40 W tungstenfilament bulb at a distance of 5 cm through the water-jacketof the cell. The electrode was calibrated by setting themaximum value to that obtained with fresh normoxic bufferand the minimum value to that obtained after thoroughdeoxygenation of the buffer sample with Ar, followed bythe addition of a few grains of fresh sodium dithionite. Thereadings were converted to concentrations using the knownconcentration of dissolved O2 in air-saturated HBSS [40].

EPR spectroscopy

EPR spectra were recorded at room temperature usinga Bruker EMX X-band spectrometer (Bruker, Karlsruhe,Germany) equipped with 100 kHz modulation and acylindrical ER4103 TM cavity. Samples were containedin a flattened aqueous sample cell. Spectral accumula-tions were initiated within 2 min of sample preparationunless stated otherwise. Hyperfine couplings were mea-sured directly from the field scan and confirmed byspectral simulation using the WINSIM program. Spec-trometer parameters were as follows: gain, 1 � 106;modulation amplitude, 0.05 mT; time constant, 81 ms;scan time, 42 s; center field, 348 mT; field scan, 8 mT;microwave power, 31.7 mW; resolution, 1024 points;and frequency, 9.76 GHz, with 4 scans accumulated.

Measurement of cellular ascorbate levels

Cell suspensions (0.5 ml) were pelleted by centrifuga-tion (1000 � g, 5 min) and the pellet was extracted with 0.5ml of ice-cold methanol containing EDTA (1 mM). Theextracts were centrifuged at 16,000 � g for 2 min and thesupernatants collected. Ascorbate levels in the supernatantswere determined using a reverse-phase ODS HPLC column(Supelco, Bellefonte, PA, USA; 25 � 0.46 cm, 5 �mparticle size) with electrochemical detection. The mobilephase employed was 40 mM sodium acetate, 7.5% (v/v)methanol, 0.25% (v/v) dodecyl-triethylammonium phos-phate, and 0.45 mM EDTA, at pH 4.75 [41].

RESULTS

Viability of THP-1 cells during photo-oxidation

In preliminary studies, experimental conditions wereestablished under which minimal loss of cell viability

639Photo-oxidation of cells yields protein peroxides

was detected during illumination with visible light in thepresence of rose bengal. Under the conditions employed,and with illumination times of �20 min, greater than85% of the cells remained viable, as assessed usingethidium bromide binding to released DNA (Fig. 1b).LDH release and Trypan blue dye exclusion assays werefound to be unsuitable for such measurements as photo-oxidation can result in direct inactivation of LDH anddye bleaching (data not shown). The minor losses of cellviability detected were found to be independent of lightexposure and the presence of rose bengal, and not sta-tistically significant (two-way ANOVA with Tukey’sposthoc testing). Similar cell viabilities were observedwhen experiments were performed in HBSS made withD2O, with PBS in place of HBSS, in the presence of 5mM glucose, after ascorbate loading, and in the presence

of 5 mM N3�. None of these treatments resulted in

statistically significant differences compared to controls(one-way ANOVA).

Formation of protein peroxides in photo-oxidized cells

Rose bengal-loaded THP-1 cells (4 � 106 cells ml�1)were illuminated with visible light in the presence of O2

for up to 20 min. Immediately after the cessation ofphotolysis, the cells were treated with TCA to precipitatecellular proteins, and the resultant pellets were washedextensively. The resuspended material tested positive forperoxides, with the concentration of this material in-creasing with increasing photolysis time (Fig. 1). Negli-gible levels of protein-bound peroxides were detected incontrol cells (no rose bengal loading and no illumina-

Fig. 1. Formation of protein-bound peroxides (a and c) and levels of cell viability (b and d) during illumination of THP-1 cells. (a andb) Cells were preloaded with (●) and without (■ ) rose bengal (5 �M in HBSS) for 30 min in the absence of light as described inMaterials and Methods. Cells (4 � 106 cells ml�1) were resuspended in HBSS before photolysis and assay for (a) peroxide formationor (b) cell viability, as described in Materials and Methods. (c and d) Cells were loaded with rose bengal as in (a) except that they wereresuspended in (�) HBSS, (E) HBSS made with D2O in place of H2O, or (‚) HBSS containing 5 mM sodium azide. Samples werethen photolysed and assayed for (c) peroxide formation or (d) cell viability. The variation in absolute peroxide levels and cell viabilitybetween (a) and (c) and between (b) and (d) reflect experiments carried out with different batches of cells with marginally differentcell numbers in the 6-well plates. Data are means (of four to eight determinations) � SD.


tion), in cells that had been loaded with rose bengal andthen kept in the dark, or in cells that were exposed tolight in the absence of rose bengal (Fig. 1). As theperoxide assays were performed on washed precipitates,H2O2 generated during the photo-oxidation process isunlikely to contribute significantly to the peroxide levels.Washing of the precipitates with methanol and hexane(see Materials and Methods) to remove contaminatinglipids before assay did not significantly alter the peroxideconcentrations (7.3 � 0.1 �M before methanol/hexanewashing, 7.0 � 1.0 �M after washing; n � 8 determi-nations, p � 0.48, two-tailed t-test). The precipitatedmaterial is believed to consist primarily of proteins; thus,these peroxides are referred to hereafter as protein-boundperoxides.

The yield of protein-bound peroxides detected inthese cell-derived precipitates was markedly enhancedwhen the cells were resuspended in buffer made withD2O in place of H2O, and the yield was dramaticallydecreased when the buffer contained 5 mM sodium azide(Fig. 1). These data are consistent with the observedperoxides being generated via 1O2-mediated reactions.

Effect of glucose and ascorbate levels on proteinperoxide formation in photo-oxidized cells

Illumination of rose bengal-loaded cells for 20 min inPBS, or PBS containing 5 mM glucose, did not result inany significant differences in peroxide level (data notshown; p � 0.95, two-tailed t-test) when the cells wereexamined immediately after the cessation of illumina-tion. The peroxide levels detected were not significantlydifferent than those detected when HBSS was employedin place of PBS (data not shown).

As many cultured cells are ascorbate deficient [37], andthis material reacts rapidly with 1O2 [16], analogous exper-iments were carried out with cells that had been preloadedwith ascorbate by prior incubation with dehydroascorbate.HPLC analysis demonstrated efficient enhancement of cel-lular ascorbate levels using this methodology (about 10nmol mg cell protein�1 for loaded cells, � 1 pmol mg cellprotein�1 for control cells). TCA-precipitable peroxide lev-els detected on illumination of such ascorbate-replete andcontrol cells, after rose bengal loading, were found to beidentical within experimental error (data not shown; p �0.35, two-tailed t-test).

Effect of cellular integrity on protein peroxideformation

The formation of protein-bound (TCA-precipitable)peroxides, in both intact cells and cells that had beenlysed by multiple freeze-thaw cycles prior to illumina-tion, was examined to investigate the effect of cellularintegrity on peroxide levels. Identical cell numbers and

incubation volumes were employed in each case. Theyields of peroxides detected in both systems were iden-tical, within experimental error, when short illuminationtimes were employed (Fig. 2); however, at longer illu-mination times higher yields of peroxides were detectedfrom the lysed cells. This is consistent with the presenceof factors that prevent the accumulation of high yields ofprotein-bound peroxides in intact, but not lysed, cells.

Stability of protein peroxides within cells

The stability of protein-bound peroxides within cells wasinvestigated by assessing the levels of these materialswithin cells at various periods after the cessation of illumi-nation (as described above), with the cells kept in HBSS at37°C in the absence of light during the postphotolysisperiod. A statistically significant decrease in protein perox-ide concentrations was observed on incubation after thecessation of illumination, with the half-lives of these per-oxides being about 4 h (Fig. 3). Longer incubation periodswere not examined. No effect on either the rate or extent ofloss of protein-bound peroxides was observed with cellsthat were incubated in the presence of 5 mM glucose orloaded with ascorbate (as described above), compared tocontrol cells (data not shown).

Formation of radicals on decomposition of cellularprotein peroxides

Treatment of illuminated, rose bengal-loaded cells,which had been exposed previously to visible light for 20min and contained about 10 �M protein peroxides with

Fig. 2. Formation of protein-bound peroxides on illumination of intact(●, ■ ) and lysed (�, Œ) THP-1 cells in the presence (●, �) andabsence (Œ, ■ ) of rose bengal. Intact cells were prepared, illuminated,and peroxide levels assayed as described in Fig. 1. THP-1 cell lysateswere prepared as described in Materials and Methods, illuminated, andperoxide levels assayed as described in Fig. 1.


Fe2�-EDTA (91 �M) in the presence of the spin trapMNP (9.1 mM), resulted only in the detection of weakradical adduct signals by EPR. This is likely to be due tothe intracellular nature of the protein peroxides and theextracellular location of the Fe2�-EDTA complex. Incontrast, when similar protein peroxide-containing cellswere lysed by repeated freeze-thaw cycles before expo-sure to Fe2�-EDTA and MNP (concentrations as above),poorly resolved EPR signals were detected (Fig. 4). Thebroad nature of the observed spectra are consistent withthe presence of a large, slowly tumbling radical adduct.These signals were not observed in the absence of eitheradded Fe2�-EDTA or spin trap. Furthermore, these sig-nals were not detected with lysates prepared from cellsthat had been illuminated for an identical period in theabsence of rose bengal, from cells that had been loadedwith rose bengal but not exposed to light, or lysates fromcontrol cells (Fig. 4). These signals have been assignedto multiple protein-derived carbon-centered radical ad-ducts generated as a result of the decomposition of theprotein-bound peroxides by the added Fe2�-EDTA, andreaction of these species with the spin trap.

Formation and decomposition of peroxides formed onisolated cellular proteins

Isolation of bulk cellular proteins (see Materials andMethods), resuspension in 6 M urea, and subsequentillumination with visible light in the presence of rosebengal (10 �M) and O2 resulted in the formation ofprotein-bound peroxides, as determined by the FOX as-say, with about 130 �M peroxides detected after 60 minphotolysis. This concentration of peroxides is higher thanthose detected with intact cells as a result of the morepowerful light source and the longer illumination timesemployed. Incubation of these protein peroxides (about90 �M peroxide) with Fe2�-EDTA (176 �M) in thepresence of MNP (11.8 mM) gave intense EPR signals(Fig. 5a). These signals were not observed when anycomponent of the reaction mixture was omitted. Thesesignals have been assigned to (at least) two proteinperoxide-derived, carbon-centered spin adducts [hyper-fine coupling constants for radical 1: a(N) 1.54, a(H) 0.16mT; for radical 2: a(N) 1.67, a(H) 0.15 mT], togetherwith features from tert-BuNHO• (a(N) 1.44, a(H) 1.44mT) and di-tert-butyl nitroxide (a(N) 1.70 mT), arisingfrom reduction and decomposition of the spin trap, re-spectively. These coupling constants were confirmed by

Fig. 3. Effect of the incubation of cells at 37°C after the cessation ofillumination on the cellular content of protein-bound peroxides. Cellswere prepared and loaded with rose bengal as described in Fig. 1, thenilluminated for 20 min. After the cessation of illumination (t � 0), thecells were reincubated at 37°C, in the absence of light, under a 5% CO2

atmosphere for the indicated period; the levels of protein peroxideswere subsequently assessed, as described in Fig. 1. Cells loaded withrose bengal and exposed to light (●) and cells loaded with rose bengaland incubated in the dark (■ ) are shown. Data from cells exposed tolight in the absence of rose bengal and nonloaded cells incubated in thedark have been omitted for clarity; these experiments yielded data thatwere not statistically different from the loaded cells incubated in thedark. Values are means (n � 8 determinations) � SEM. *Indicatesstatistically significant difference from controls (two-way ANOVAwith Tukey’s posthoc testing).

Fig. 4. EPR spectra observed on decomposition of intracellular proteinperoxides by metal ions in the presence of the spin trap MNP. Cellswere prepared as described in Fig. 1. Immediately after the cessation ofillumination (or incubation in the dark for controls), the cells werelysed and the lysates (about 10 �M peroxide) incubated with Fe2�/EDTA (91 �M, 1:1 complex) in the presence of MNP (9.1 mM) andexamined by EPR spectroscopy. (a) Cells loaded with rose bengal andsubsequently illuminated. (b) Cells loaded with rose bengal and incu-bated in the dark. (c) Nonloaded cells exposed to light. (d) Nonloadedcells incubated in the dark. Signals assigned to multiple protein-derivedcarbon-centered radicals with the exception of features marked ●,which are assigned to di-tert-butyl-nitroxide. Spectrometer parameterswere as follows: gain, 1 � 106; modulation amplitude, 0.05 mT; timeconstant, 81 ms; scan time, 42 s; center field, 348 mT; field scan, 8 mT;power, 31.7 mW, with four scans accumulated.


computer simulation (Fig. 5b). To confirm that H2O2,and HO• that may arise from this species, is not involvedin the generation of these adducts, control experimentswere carried out where H2O2 (900 �M) was added toisolated, nonilluminated cell proteins in the presence ofFe2�-EDTA and MNP (concentrations as above). Suchexperiments did not result in the detection of any protein-derived adducts.

Quantification of oxygen consumption and peroxideformation

The extent of conversion of molecular O2 to protein-bound peroxides was examined using an oxygen elec-trode, with the THP-1 cells (4 � 106 cells ml�1) illumi-nated within the electrode chamber at 22°C. Illuminationof the cells under these conditions resulted in the forma-tion of peroxides, as indicated by the FOX assay, and thedepletion of dissolved O2 in a parallel, time-dependentmanner (Fig. 6). Control experiments using illuminated,nonloaded cells showed negligible levels of protein per-oxides and low levels of O2 consumption. At short illu-

mination times, a significant proportion of the O2 con-sumed was converted into protein peroxides (� 14% atthe earliest time points, see Fig. 6). These values mayunderestimate the total flux of protein peroxides due tothe thermal decay of the protein-bound peroxides at thetemperature employed.

DISCUSSION

Previous studies on radical-mediated oxidation of iso-lated peptides and proteins, in the presence of O2, haveprovided evidence for the formation protein-derived per-oxides [42–44]. More recently, reports have appeared onthe detection of similar protein-bound peroxides in se-rum [45], mouse myeloma cells exposed to � irradiation(i.e., HO•) [45], and U937 cells exposed to a thermalsource of peroxyl radicals [46]. In both cell studies, theextent of cell viability at the end of the exposure time isdifficult to ascertain or was not reported. It is, therefore,possible that the peroxides detected may include contri-butions from materials released from lysed cells.

We have demonstrated that the formation of proteinperoxides is not confined to radical-mediated reactions,as exposure of isolated peptides and proteins to 1O2,generated by visible light in the presence of the sensitizerrose bengal, leads to high yields of peptide- and protein-bound peroxides [34–36]. A major role for radicals in theformation of these previously characterized peroxideshas been discounted, even though rose bengal can gen-

Fig. 5. (a) EPR spectrum observed on the decomposition of proteinperoxides formed on isolated cell proteins exposed to metal ions in thepresence of the spin trap MNP. Isolated cell protein peroxides (about 90�M) were treated with Fe2�/EDTA (176 �M, 1:1 complex) in thepresence of MNP (11.8 mM), then examined by EPR spectroscopy.Spectrometer parameters were as follows: gain, 1 � 106; modulationamplitude, 0.05 mT; time constant, 81 ms; scan time, 42 s; center field,348 mT; field scan, 8 mT; power, 31.7 mW, with four scans accumu-lated. Lines marked ● are assigned to di-tert-butyl nitroxide and thosemarked ■ to tert-BuNHO. (b) Computer simulation of the experimentalspectrum in (a) using the parameters reported in the text.

Fig. 6. Oxygen consumption (�) and protein peroxide formation (●) inrose bengal-loaded THP-1 cells exposed to visible light. Cells wereprepared as described in Fig. 1, placed in a Clark-type oxygen electrodeat 22°C, then illuminated as described in Materials and Methods. At theindicated times, aliquots were removed and protein peroxides assayedas described in Fig. 1. Oxygen electrode readings were converted toconcentrations as described in Materials and Methods. Values markedon the plot indicate the percentage of oxygen (mean � SEM from threeseparate experiments) converted to protein-bound peroxides at eachtime point.


erate radicals on photolysis with short wavelength UVlight [47], on the basis that well-characterized radical-mediated products such as di-tyrosine and 3,4-dihy-droxyphenylalanine (DOPA) were not detected in theseexperiments [34]. The nature of some of these 1O2-generated peroxides have been characterized in detail[31,34].

Evidence has been presented for the decomposition ofboth radical- and 1O2-generated protein peroxides toreactive radicals in the presence of metal-ion complexes[34,35,48,49]. It has been demonstrated that these spe-cies can induce damage to a range of cellular targetsincluding other proteins and enzymes [36,50], lipids (Da-vies et al., unpublished), and DNA [51–53] by bothradical-mediated and nonradical reactions.

In this study, it has been shown, for the first time, thatexposure of rose bengal-loaded cells to low levels ofvisible light yields peroxides that can be precipitatedwith the protein precipitant TCA. Experiments usingwashed precipitates confirm that this peroxidic materialis not H2O2 formed during the photo-oxidation, thoughevidence has been presented for the formation of thisspecies [36,54]. Determination of the peroxide levels inTCA precipitates that had been delipidated with metha-nol/hexane confirmed that this material is not lipid de-rived. Identical results were obtained with bulk cellularproteins that were isolated from the same cell type, priorto photo-oxidation. These peroxides are, therefore, as-signed to protein-bound species generated on cell-de-rived proteins. Under the conditions employed, the for-mation of these peroxides did not result in any significantdecrease in cell viability compared to controls.

The yield of these materials increased with increasingillumination time over the range examined, with onlylow levels of peroxides detected in control cells. Thesedata are consistent with the generation of peroxides re-quiring both the sensitizer and light. The increased yieldof peroxides detected in the presence of D2O, and thedecreased levels formed in the presence of azide, areconsistent with, but do not categorically prove, that theseperoxides are generated via 1O2-mediated reactions. Thedecreased yield of peroxides detected in the presence ofazide might also arise from other effects of this ion, suchas its action as a metabolic inhibitor. Nevertheless, theseresults are in accord with previous studies of isolatedproteins and peptides [34,35].

The negligible levels of protein-derived peroxidesdetected in cells that had been illuminated but not loadedwith rose bengal suggests that, under the conditionsemployed, endogenous cellular materials (e.g., porphy-rins, flavins; [55]) are not efficient sensitizers of peroxideformation, possibly as a result of their low concentration.The absence of peroxides in nonilluminated rose bengal-

loaded cells confirmed that the observed peroxides donot arise via “dark” reactions of rose bengal.

Examination of the rate and extent of O2 consumptionand peroxide formation has shown that, particularly atshort illumination times, there is efficient conversion ofO2 into protein peroxides, with this accounting for up to14 � 5% of the O2 consumed. This is likely to be anunderestimated amount, as it is unclear whether all theprotein peroxides formed are measured by the FOXassay and no account has been taken of peroxide decay toother materials during the illumination process or theperiod required for sample preparation and analysis.These data are consistent with previous studies that haveshown that proteins are major intracellular targets[16,17].

The peroxide levels detected on proteins isolated fromthese photo-oxidized cells are considerably higher thanthose that have been detected on proteins from cellsexposed to either � radiation or peroxyl radicals (c.f. Fig.1a, and values of 1–1.5 �M reported for somewhathigher cell numbers in [45,46]). This may reflect a num-ber of different factors including the type of cells em-ployed, a more efficient generation of peroxides by 1O2,differences in the lifetime of these materials (c.f. previ-ous studies that were carried out at 37°C, where suchperoxides have shorter lifetimes [44]), or differences inthe manner in which cells handle and detoxify suchperoxides. In particular, the rose bengal system used inthe current study appears to generate protein peroxideswith greater efficiency than the thermal decomposition ofazo compounds (AAPH) employed previously [46].Thus, the current data suggest that about eight moleculesof O2 are consumed for each protein peroxide moleculegenerated, whereas about 200 peroxyl radicals (and,hence, about 200 molecules of O2) are required whenAAPH is the oxidant [46]. Furthermore, the oxygenconsumption data in the current study have not beencorrected for consumption arising from cellular respira-tion.

The absence or presence of glucose in the cell me-dium (cells illuminated in PBS, compared with PBScontaining 5 mM glucose), which would be expected tomaintain reducing equivalents within the cell during il-lumination, had no effect on the peroxide levels detected.This suggests that peroxide generation is either not lim-ited by the maintenance of reductive capacity within thecell or that any glucose-dependent repair process is rap-idly inactivated. Further studies are required to elucidatewhich of these factors is most important.

Ascorbate reacts rapidly with 1O2, with k about 107

M�1 s�1 [16], and can also reduce both radiation-gen-erated [46,56] and 1O2-mediated protein peroxides (P.Morgan, R.T. Dean, and M. J. Davies, unpublished data).However, the similar yields of protein-derived peroxides


observed in ascorbate-loaded and control cells indicatesthat ascorbate was unable to either scavenge the 1O2 thatgenerates these peroxides or reduce the protein-derivedperoxides once formed. The inability of ascorbate todirectly scavenge 1O2 may be due to the binding of rosebengal to cellular proteins or its presence within mem-brane structures [15], which might result in 1O2 beingproduced in close proximity to its target. However, it isalso possible that the level of ascorbate loading achievedis insufficient to compete successfully with the reactionof 1O2 with the high concentration of cellular proteins.This is consistent with the data obtained with � radiationand peroxyl radicals, where intracellular antioxidantswere unable to prevent intracellular protein peroxidegeneration [45,46].

The increased level of protein-bound peroxides de-tected with lysed cells, compared to intact cells at longillumination times (c.f. Fig. 2), suggests that cellularintegrity can have a limiting effect on the accumulationof protein peroxides. This may be due to the loss ordisruption of enzymatic removal/repair processes (or thereducing equivalents required for such systems) ratherthan low molecular weight scavengers. It has alreadybeen established that 1O2-generated protein peroxidescan inhibit key intracellular enzymes such as glyceralde-hyde-3-phosphate dehydrogenase and glutathione reduc-tase [36], which maintain reducing equivalents within thecell, and the caspase family [57]. The loss of peroxidesduring the postillumination phase may be due to suchreactions.

Taking a value of 9 �M for the peroxide level de-tected after 20 min illumination (c.f. Fig. 1a), the currentdata correspond to a peroxide concentration of about 1.5nmoles per 106 cells, or 10 nmoles/mg cell protein. Thesevalues are much lower than those reported in a previousstudy using AAPH as the oxidant (770 nmoles perox-ide/mg cell protein [46], though it appears that this valueis a typographical error and should be pmoles perox-ide/mg protein; J. Gebicki, personal communication).The value obtained in the current work corresponds, if anaverage molecular mass of 50,000 is taken for cellularproteins, to 10 nmoles per 20 nmoles protein, or 0.5peroxide groups per protein molecule.

The absolute levels of peroxide groups detected areslightly higher, but of the same order of magnitude, asanother generic marker (protein carbonyls) of oxidativedamage in cells. Thus, aged cultured human fibroblastcells have been reported to accumulate 2–4 nmoles pro-tein carbonyls/mg cell protein, and up to 8 nmoles pro-tein carbonyls have been detected in diseased brain tissue([58–60], reviewed in [61,62]). The levels of a numberof more specific markers of protein oxidation are con-siderably lower, being typically of the order of a few

pmoles per mg protein even in heavily diseased tissue[61,62].

Previous studies have shown that amino acid- andprotein-derived peroxides are labile at elevated temper-atures, on exposure to UV light, and in the presence ofcertain metal ions. Such decomposition can yield alco-hols and carbonyl species, as well as reactive radicals[34,35,48,49,52,53,56]. Weak EPR signals, which couldnot be definitively assigned, were observed on incubationof cells containing protein peroxides in the presence ofspin traps after the cessation of illumination. These sig-nals are probably of poor intensity as a result of the lowlevel of peroxides present and a slow rate of radicalgeneration. The addition of Fe2�-EDTA, which is knownto stimulate radical formation from isolated peptide andprotein peroxides [34,35,48,49,52,53], did not signifi-cantly enhance the radical adduct concentration, proba-bly because this membrane-impermeant complex can notgain access to the intracellular peroxides. In contrast,when Fe2�-EDTA was added to cells that had beenphoto-oxidized and subsequently lysed, intense EPR sig-nals were detected and have been assigned to carbon-centered protein-derived species.

Similar signals were detected with cellular proteinsthat were subject to photo-oxidation after isolation, andthen incubated with Fe2�-EDTA in the presence ofMNP. In this case, further information on the nature ofthese adducts has been obtained as a result of the moreisotropic nature of the spectra. This is ascribed to thepresence of urea, which unfolds proteins and give rise tomore rapid molecular motion. These signals are assignedto side chain-derived, carbon-centered radicals on thebasis of their coupling constants and comparison withdata for other protein peroxide-derived radicals[34,35,48,49,52,53]. The exact nature of these speciescan not be determined, though these signals are similar tothose detected on decomposition of 1O2-mediated perox-ides present on BSA, which have been assigned to Tyr-and His-derived radicals [34,35].

In conclusion, it has been demonstrated that photo-oxidation of rose bengal-loaded cells leads to the forma-tion of protein peroxides. Both light and a sensitizer arerequired for peroxide formation, and the yield of perox-ides is enhanced by D2O and inhibited by azide, provid-ing strong evidence that the reactions leading to the thesespecies are mediated by 1O2. These peroxides have arelatively long biological half-life at physiological tem-peratures, due to their poor removal by cellular enzy-matic reactions (P. Morgan, R. T. Dean, and M. J.Davies, unpublished data; c.f. data on related radiation-generated peroxides [63]). The slow removal of thesespecies may allow such peroxides to undergo furtherradical or nonradical reactions that may be deleterious tocell function. It has been shown already that such per-


oxides can inhibit key cellular enzymes such as thecaspase family [36,57] and induce DNA damage [51–53]. Such processes may prevent the induction of apo-ptosis and allow the inappropriate accumulation of cellscontaining damaged DNA.

Acknowledgements — The authors thank the Australian ResearchCouncil and the Wellcome Trust for financial support. A. W. acknowl-edges the receipt of an Australian Postgraduate Award administeredthrough the University of Sydney. The authors are grateful to ProfessorRoger Dean for helpful discussions.

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ABBREVIATIONS

AAPH—2,2'-azobis(amidinopropane) dihydrochlorideDMPO—5,5-dimethyl-1-pyrroline N-oxideHBSS—Hank’s balanced salt solutionMNP—2-methyl-2-nitrosopropane1O2—singlet oxygen (molecular oxygen in its 1�g state)PBN—N-tert-butyl-�-phenylnitronePBS—phosphate-buffered salineTCA—trichloroacetic acid


photo-oxidation of cells generates long-lived intracellular protein peroxides

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