Light-stimulated formation of hydrogen peroxide and hydroxyl radical in the presence of uroporphyrin and ascorbate

Free RadicalBiology& Medicine, Vol.5, pp. 3-6, 1988

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Original Contribution I

LIGHT-STIMULATED FORMATION OF HYDROGEN PEROXIDE AND HYDROXYL RADICAL IN THE PRESENCE OF UROPORPHYRIN AND ASCORBATE

GARY J. BACHOWSKI and ALBERT W. GIROTTI* Department of Biochemistry,Medical College of Wisconsin, Milwaukee, WI 53226 (Received 2 June 1987;Revised 5 August 1987)

Abstract--Blue light irradiation of 2-deoxyribose (DOR) in the presence of uroporphyrin I (UP), ascorbate (All-), trace iron, and phosphate buffer resulted in a strong stimulation of hydroxyl radical (OH')-dependent oxidation of DOR. Photostimulated generation of H202 was monitored after removal of residual AH- (i) by ascorbate oxidase treatment, or (ii) by anion exchange on mini-columns of DEAE-Sephadex. Irradiation of the above mixture produced a strong burst of 1-1202 which was intensified by desferrioxamine and suppressed by catalase or EDTA. The mechanism suggested by these observations is one in which photoreduction of UP to the radical anion initiates the formation of H202, which gives rise to OH" via Fenton chemistry. This is the first known investigation of H202 fluxes in a Type I (free radical) photoreaction involving AH- as the electron donor. Keywords--Photosensitization, Photoreduction, Porphyrins, Ascorbate, Hydrogen peroxide, Hydroxyl radical

INTRODUCTION

Certain dyes and pigments that are capable of sensitizing photodynamic processes are being studied intensively in connection with their cytotoxic effects as well as therapeutic potential, t'2 A photodynamic reaction requires a sensitizing agent, exciting light (typically in the visible range), molecular oxygen, and an oxidizable substrate. Photodynamic reactions fall into two major categories: (i) Type I involves electron or hydrogen transfer from a substrate to triplet excited sensitizer, with generation of free radicals and other reactive intermediates, some of which may be derived from 02, e.g. superoxide (O2-), hydrogen peroxide (H202), and hydroxyl radical (OH'); (ii) Type H involves energy transfer from triplet sensitizer to 02, with formation of singlet molecular oxygen (IO2). Activated oxygen species such as these have been implicated in membrane lipid peroxidation, DNA modification, and other types of cell damage. 3 Detection and quantitation of these species is often difficult because of their instability and the many sources of interference. For example, most existing methods for the determination of H202 are subject to interference by *Correspondenceshould be addressed to: Albert W. Girotti, Ph.D., BiochemistryDepartment, Medical College of Wisconsin, Milwaukee, WI 53226.

reductants such as thiols or ascorbate. Therefore, although H202 has been shown to be an intermediate in ascorbate-driven, Type I photoreactions by virtue of catalase inhibition, 4,s its actual formation and decay during the course of irradiation has not been studied. We have now accomplished this in a test system containing uroporphyrin, ascorbate, and deoxyribose as photosensitizer, electron donor, and hydroxyl radical trap, respectively. Newly devised procedures for rapidly removing residual ascorbate prior to peroxide analysis are described.

MATERIALS AND METHODS

Ascorbate oxidase, Cu/Zn-superoxide dismutase, catalase(thymol-free),horseradishperoxidase,2-deoxyribose, 4-aminoantipyrine, and DEAE-Sephadex A25-120 were obtained from Sigma Chemical Co. (St. Louis, MO). Uroporphyrin I, desferrioxamine, and sodium ascorbate were provided by Porphyrin Products (Logan, UT), Ciba-Geigy (Suffern, NY), and BDH Chemicals (Poole, England), respectively. All other chemicals were of the highest purity available, and all solutions were prepared with deionized, glass-distilled water. Reactions were carried out at 37"C in thermostatted Stirrer Bath vessels (Yellow Springs Instruments), as

4

G . J . BACHOWSKI and A. W. GIROTTI

described previously. 6 Stock A H - t in H20 was prepared immediately before use and added as the final component in reaction mixtures. Solutions were irradiated with filtered blue light 6 at an incident fluence rate of - 9 mW/cm 2. Other details are provided in the figure and table captions. Hydrogen peroxide was determined according to Frew et al.,7 using the peroxidase/phenol/aminoantipyrine system. In this assay, H202 oxidatively couples with aminoantipyrine and phenol to give a quinoneimine chromogen which absorbs maximally at 505 nm. Calculations were based on an extinction coefficient (¢) of 6,400 M-~cm -~ at 505 nm, which was determined with spectrophotometrically standardized H202 [e = 43.5 M - l c m -~ at 240 nm (Ref. 8)]. As little as 10/zM A H - caused a measurable interference in the assay (e.g. 10% decrease in A505 with 60/~M H202). Therefore, residual A H - in reaction samples was removed by either of the following procedures: A. Ascorbate oxidase treatment. Ascorbate oxidase catalyzes the oxidation of AH- to A (dehydroascorbate), with accompanying reduction of 02 t o H 2 0 . 9 Samples were mixed with 10 pM Dox (which stabilized H202 by inactivating iron), incubated with ascorbate oxidase (2 units/ml) for 2 - 3 min at 25°C, and then assayed immediately for H202. (Prolonged preincubation with the enzyme, > 5 rain, was avoided, as it resulted in some loss of peroxide.) This treatment effectively depleted A H - at starting concentrations up to 2 mM and generated no detectable H202 on its own. Trial runs involving 0.5 mM A H - and known concentrations of H202 (75-450 pM) gave excellent results (Table 1). B. Anion exchange on DEAE-Sephadex. Stock resin prepared by consecutive washings with 10 mM EDTA, 0.2 N NaOH, 1 M NaC1, and PBS was poured into 0.5 ml syringe columns. Before use, the columns (in holding tubes) were damp-dried by spinning in a table-top centrifuge. Samples (0.2 ml) from reaction mixtures were loaded onto the columns, washed through by centrifugation using 0.75 ml of H20 containing 10 pM Dox, and then assayed. When the procedure was carefully standardized, eluent volume was constant and recovered H202 approached 100% (Table 1). The columns were regenerated by flushing with 2 M NaCI, H20, and PBS.

All manipulations of Methods A and B were done under minimal room illumination when UP was present. Deoxyribose oxidation products were determined by "?The abbreviations used are: A H - , ascorbate; UP, uroporphyrin I; D O R , 2-deoxyribose; E D T A , ethylenediaminetetraacetic acid; Dox, d e s f e r r i o x a m i n e ; PBS, 25 m M s o d i u m p h o s p h a t e / 1 2 5 m M NaCI (pH

7.4).

Table 1. Recovery of Hydrogen Peroxide from Ascorbate-Containing Mixtures: Comparison of DEAE-Sephadex and Ascorbate Oxidase Treatments * H202 (Micromolar) Recovered (after A H - ) Expt. No.

Initial (before A H - )b

1 2 3 4 5

76 ± 1 156 ± 2 ND d 346 ± 14 456 ± 2

DEAE_Sephadex o 86 ± 177 ± 300± 362 ± 453 ±

13 14 32 11 11

Ascorbate Oxidase c 70 148 265 338 428

± ± ± ± ±

2 1 8 3 3

aA stock solution of H202 (determined spectrally at 240 nm) was diluted to the following concentrations: 75/aM, 150/aM, 305/aM, 338/aM, and 450/aM in H20 containing 20/aM Dox (expts. 1, 2, 3, 4, and 5, respectively). Hydrogen peroxide was determined before and after the addition of 0.5 mM AH- to each mixture. Samples were withdrawn within 5 min. after the addtion of AH , subjected to DEAE-Sephadex chromatugraphy or ascorbate oxidase treatment, and tested for recovered H202 by peroxidase assay. Residual AH- in chromatographed or oxidase-treated samples was < 10 /aM. All manipulations were carried out at 25°C. bMean ~ deviation of values from two determinations. CMean --- SD of at least five determinations, dND, not determined.

thiobarbituric acid assay, as described previously. 1° Absorbance values of thiobarbituric acid adducts were measured at 532 nm. Ascorbate was determined spectrophotometrically by measuring the reduction of Fe(III) [ o-phenanthroline]2, using 0.18 mM FeC13 and 1.3 mM o-phenanthroline in 0.1 N acetate buffer, pH 4.5. I~ The extinction coefficient, based on the 2-electron oxidation of ascorbate, was determined to be 23,000 M-~cm -1 at 515 nm. RESULTS

Evidence for photosensitized generation of H202 by UP in the presence of AH- is shown in Table 2. Note Table 2. Light-Stimulated Generation of Hydrogen Peroxide in the Presence of Uroporphyrin and Ascorbate: Peroxide Determination Before and After Removal of Ascorbate a H202 (Micromolar)

Treatment None DEAE-Sephadex Aseorbate oxidase (1 unit/ml) Ascorbate oxidase (3 units/ml)

Dark Control b

Irradiated Mixture c

0 9 ± 8

0 102 -+ 14

2 - 1

103 ± 2

2 --- 0

102 ± 3

aA stock mixture containing 1 mM A l l - , 10 ,uM UP, and 10/aM Dox at 37°C was either incubated in the dark (Dark Control) or irradiated for 10 rain (Irradiated Mixture), after which samples were either assayed directly or subsequent to anion exchange or ascorbate oxidase treatment. Residual A l l - in the irradiated sample assay with no treatment was approximately 0.15 mM, while that in the treated samples was <10/aM. Values shown represent H202 in the reaction mixture. bMean --+-deviation of values from two determinations on one experiment. CMean --- SD of four determinations on duplicate experiments.

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Fig I. Time course of hydrogenperoxidegenerationand deoxyribosedegradation. The standardreactionmixture (O) contained l0 mM DOR, l0 gM UE 5 gM FeCI3, and 1 mM AH- in PBS at 37°C. Other additions were as follows: 20/zM EDTA (A); 20 gM Dox (V); 10 p.g/ml superoxide dismutase ([-]); 10 ug/ml catalase (<>); and 10 mM mannitol (+). The control (×) lacked AH-. (A) Oxidationof DOR in the dark. (B) Light-stimulatedoxidationof DOR (total reaction minus dark background). (C) Light-stimulatedgenerationof H202. Residual AH- in samples to be analyzedfor H202was removedwith ascorbate oxidase prior to analysis. Points with error bars are means -+ deviation of values from duplicate experiments. that after 10 min of irradiation under the specified conditions, - 1 0 0 pM H202 was measured in the medium, with excellent agreement between the two types of sample treatment. No H202 could be detected if residual A H - ( - 0 . 1 5 mM in this experiment) was not removed, illustrating the extreme magnitude of A H interference. Relatively little, if any, H202 accumulated in the nonirradiated control; values shown are near the detection limits of the assay. The time course of H202 generation in a reaction system containing UP, A H - , trace Fe(III), and DOR as the oxidizable substrate is depicted in Figure 1. The dark control (Fig, 1A) showed significant oxidation of DOR, which was dependent on A H - , but not UP. In agreement with earlier observations, 1° this reaction was stimulated by EDTA and strongly inhibited by Dox or catalase. Superoxide dismutase had no effect, whereas the OH" scavenger mannitol (equimolar with DOR) caused a sizeable inhibition. These observations are consistent with a reaction mechanism involving iron-catalyzed, AH--driven reduction of 02 to OH', the ultimate oxidant of DOR. In this mechanism, OH" would arise via Fenton chemistry, i.e. reduction of H202by Fe(II). As shown in Figure 1B, irradiation produced a large stimulation of DOR oxidation; note the 5-fold increase in initial rate relative to that of the basal reaction (Fig. 1A). An increment was observed in the absence of added iron, but was much smaller and more variable. Like the light-independent reaction, light-stimulated oxidation of DOR was further enhanced by EDTA,

inhibited by Dox, catalase, or mannitol, and unaffected by superoxide dismutase. An irradiated control containing UP, but lacking A H - , showed no reaction, indicating that the reductant was absolutely necessary for OH" formation. Hydrogen peroxide profiles for this system are shown in Figure 1C. Continuous irradiation resulted in a large burst of H202, which reached an apparent steady state level of - 9 0 / z M at 15-20 min. If any H202 was present in the light control (minus AH- ) or dark control (plus A H - ) over this time period, its concentration was too low to be measured. EDTA caused a large decrease in the steady level of H202 relative to that of the standard reaction mixture, whereas Dox had the opposite effect, allowing almost three times as much peroxide to accumulate after 20 min. These results are consistent with the known effects of these chelators on iron's redox properties. 12 Thus, by facilitating Fe(III) reduction by A H - , EDTA would enhance OH" formation and suppress H202levels, as actually observed. Inactivation of Fe(III) by Dox would be expected to inhibit OH" formation and elevate H202 levels, as also observed. (Similar results have been reported for xanthine oxidase-catalyzed formation of H202.13) Mannitol had no effect on photogenerated H202 (Figure 1C), whereas catalase, not surprisingly, abolished it. Although superoxide dismutase neither stimulated nor inhibited DOR oxidation, it caused a small elevation in H202 throughout the illumination. Thus, while reduction of 02- to H202 was probably favored by the high concentration of A H - , 14 some for-

6

G . J . BACHOWSKIand A. W. GIROTTI

mation via catalyzed dismutation of 02- appeared possible.

applicable to other prooxidant systems in which H202 is a crucial intermediate.

DISCUSSION

Acknowledgments--The authors are grateful to J. P. Thomas for helpful discussions. This work was supported by NSF grant DCB8501894.

The following reaction scheme may at least partially account for H202 and OH" formation in the system we describe. AH- + Fe(III) O2 + Fe(II)

~ A: + Fe(II) + H ÷ ~ 02- + Fe(III)

AH- + 02- + H + H20 2 +

Fe(II)

UP + hv 3Up + AHUP ~ + 02

(1) (2)

~ H202 + A ~-

(3)

~ OH" + OH- + Fe(III)

(4)

) IUp

> 3Up

~ UP: + A ~ + H + ~ UP + 02-

(5)

(6) (7)

In the absence of light, iron-catalyzed autoxidation of AH- produces 02- (Eqs. 1 and 2), H202 (Eq. 3), and OH" (Eq. 4, the Fenton reaction). DOR oxidation is inhibited by catalase, indicating that H202 is an intermediate in the formation of OH" under these conditions. However, H202 was barely detectable (Fig. I C), evidently because of its relatively slow rate of generation (Eq. 3) and rapid depletion (Eq. 4). The much higher levels of H202 attained when the UP-containing reaction mixtures were irradiated is attributed primarily to Type I photochemistry (Eqs. 5-7). In these reactions UP (acting as a catalytic photosensitizer) is converted sequentially to IUP, 3UP, and U P : , its excited singlet, excited triplet, and radical anion forms, respectively. The porphyrin radical, a strong reducing agent formed by electron transfer from AH- (Eq. 6), reacts rapidly with O2 to give 02- (Eq. 7). Subsequent reduction of 02- by AH- (Eq. 3) and possibly by ascorbate radical, A : (not shown) provides a large burst of H202 which stimulates OH" formation and DOR oxidation. (Any competing deactivation of 3 U p by 02, cf. Eq. 6, would give rise to LO2, but this is unreactive with DOR.) There is ample evidence for the photogeneration of porphyrin radical anions. ~5-~7In the specific case of UP and A H - , we recently demonstrated the existence of UP : , using ESR under anaerobic conditions. 18Our evidence for OH" formation under aerobic conditions is entirely consistent with the spin trapping data reported previously for other photosensitizers. 4,5,19 Although many different assays for H202 exist, 7 most are subject to interference by reducing agents, either through destruction of chromogen/fluorogen indicators or generation of false signals. By eliminating AH- interference in our analyses, we have been able to probe the dynamic state of H202 in the reaction system described. Similar approaches could well be

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