Intracellular generation of electronically excited states. Polymorphonuclear leukocytes challenged with a precursor of triplet acetone

Biochimica et Biophysica Acta 881 (1986) 337-342

337

Elsevier BBA 22302

Intracellular generation of electronically e x c i t e d states. P o l y m o r p h o n u c l e a r i e u k o c y t e s challenged with a precursor of triplet a c e t o n e A n a L u c i a T.O. N a s c i m e n t o a, Luis M a r c o s d a F o n s e c a b, I g u a t e m y L. B r u n e t t i and Giuseppe Cilento a

a

~' Instituto de Quimica, Universidade de Silo Paulo, C.P. 20. 780, Silo Paulo, SP, and b Faculdade de Ci~ncias Farmac~uticas, UNESP, Araraquara, SP (Brazil)

(Received October 1st, 1985)

Key words: Myeloperoxidase; Enol oxidation; Triplet acetone; (Polymorphonuclear leukocyte)

When the enoi of isobutanal is added to polymorphonuclear cells, it undergoes an intraceUular, myeloperoxidase-catalyzed aerobic oxidation with the formation of triplet acetone. The latter induces considerable damage if the enol concentration exceeds 2 mM. Cells which do not have myeloperoxidase are not damaged.

Introduction

Materials and Methods

The horseradish peroxidase-catalyzed aerobic oxidation of isobutanal in phosphate buffer leads to the formation of triplet acetone [1]. As reported in the preceding paper [2], the true substrate is the enol form of isobutanal. With the enol, the reaction also occurs in other buffers. The reaction further requires a peracid or hydrogen peroxide [3]. Despite the presence of oxygen, acetone phosphorescence is observed [1,2]. Polymorphonuclear leukocytes are rich in myeloperoxidase (5% of dry wt.) [4]. It is known that when myeloperoxidase acts upon a halide factor in the presence of H202, electronically excited singlet oxygen (1AglO2) is formed [5-8]. Given the capability of myeloperoxidase to catalyze the generation of electronic energy (see also Ref. 9), it was of considerable interest to verify whether polymorphonuclear cells challenged with either isobutanal or an enolic precursor of isobutanal would produce triplet acetone.

All chemicals were analytical-grade reagents. Glycogen (type II), catalase and glucose oxidase were from Sigma Chemical Co. Stock solutions of the trimethylsilyl enol ether of isobutanal (0.26 M) and of isobutanal [2] (1 : 5; v/v) were prepared in ethanol and used within hours. The sodium salt of anthracene-2-sulfonic acid was prepared concomitantly with the dibromo derivative [2].

Preparation of polymorphonuclear leukocytes. Female Wistar rats (Rattus norvegicus) weighing 300-400 g were used throughout. Peritoneal exudates were obtained by injection of 0.5% glycogen in saline. After 12 h [10], the animals were killed with diethyl ether, and the cells were collected by washing the peritoneal cavity with Dulbecco's phosphate-buffered saline (pH 7.3). After centrifugation (700 X g for 10 min), contaminating erythrocytes were removed (when necessary) by hypotonic lysis with cold deionized water for a few seconds. The cells were immediately collected by centrifugation, washed twice, and resuspended in Dulbecco's buffer (pH 7.3) to give a final concentration of 107 cells/ml.

0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

338 This procedure routinely produced cell populations with 98% viability, as assessed by Trypan blue exclusion, consisting of up to 96% polymorphonuclear leukocytes (approx. 90% neutrophils). Differential cell counts were performed on smears prepared from each sample using Leishman's stain. The cell suspensions were maintained throughout at 0°C. Unless otherwise stated, the standard reaction mixture contained 2 mM trimethylsilyl enol ether, 0.13 M ethanol and 5.106 cells/ml in 0.1 M phosphate buffer (pH 7.3) containing 0.02% glucose. The final volume was 2.6 ml and the temperature was 37 + I°C. Equipment for measuring 02 uptake and light emission is described in the preceding paper [2]. Results and Discussion

Addition of 1-6 mM enol to polymorphonuclear cells leads to a very fast oxygen consumption (Fig. 1) accompanied by light emission (Fig. 2). The emission intensity can be increased by addition of H202, the increase being small at enol concentrations of 1-2 raM, and substantial (almost 2-fold) at high enol concentrations (not shown). Presumably the rate of 02 uptake is also increased; however, such an increase could not be detected by our equipment because the reaction in the absence of H202 is already too fast. Neither reaeration nor addition of H202 regenerates emission; however, addition of further enol leads again to light emission which is even more intense (not shown). This indicates that under the experimental conditions used, the substrate is the limiting reagent for the reaction. Indeed, upon formation in situ from the trimethylsilyl precursor, the enol undergoes fast conversion to the aldehyde, which was found to be essentially inactive. Thus, 02 uptake was barely detectable and the emission was only twice the background level when isobutanal was added to the cells, even at a high concentration (84 mM) in a medium containing phosphate buffer (0.1 M) which catalyzes the enol ~ keto conversion [3]. The negative result with isobutanal can be rationalized on the basis of the very low equilibrium constant (1.28.10 -4) [11] for enolization. In another experiment, isobutanal was added at the end of the reaction with trimethylsilyl enol

precurosr. Again, the results were negative even when H202 was added to the system. Taken together, these results suggest that: (i) if isobutanal penetrates into the cells, it is not appreciably enolized; (ii) the addition of the enol precursor to the cells does not result in the release of myeloperoxidase. The latter inference is of particular interest since activation of polymorphonuclear leukocytes by certain stimuli can cause the release of myeloperoxidase into the extracellular space [12,13]. Interestingly, when the cells were sonicated, the emission induced by the enol increased by one order of magnitude. The effect of H202 merits additional comment. The quantity of endogenous H202 [14] is probably sufficient to carry out the intracellular oxidative processes when the enol concentration is limiting. The fact that the second addition of enol leads to a stronger emission is consistent with the burst of oxidative metabolism which accompanies stimulation of leukocytes and releases, among other species, H202 [12-15]. The enol also undergoes a

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Fig. 1. Oxygen consumption by polymorphonuclearcells following addition of the trimethylsilylenol ether of isobutanal. e, 1 mM enol ether; zx, 2 raM; O, 4 mM; A, 6 raM. Fig. 2. Temporal behavior of the chemiluminescent emission from polymorphonuclear cells elicited by the trimethylsilyl enol ether of isobutanal, e, 1 mM enol ether; O, 1.5 raM; zx, 2 mM; O, 4 mM; A, 6 mM.

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Fig. 3. The effect of incubating polymorphonuclear leukocytes with 1 mM N 3- (O) and of heating these cells at 100°C for 30 min (D) upon the emission elicited by the enol ether (ix). Fig. 4. The effect of incubating polymorphonuclear leukocytes with 1 mM N -3 (O) and of heating these cells at 100°C for 30 rain (El) upon oxygen uptake following addition of the enol ether (zx).

non-chemiluminescent oxidation (see below) and it is likely that the latter reaction stimulates the formation of H202. The H202 effect suggests that the enol undergoes a peroxidase-oxidase-catalyzed oxidation in the cells. Since the most likely candidate for the enzyme involved is myeloperoxidase, the cells were incubated with 1 mM N f , a very efficient inhibitor of this enzyme [15-20]. Fig. 3 shows that the emission is dramatically quenched. Although oxygen consumption still occurred to some extent (Fig. 4), this may reflect competitive oxidation of the enol by other non-luminescent pathways. That the oxidative process leading to the observed emission is enzymatic is conclusively shown by the total inhibition of 02 uptake (Fig. 4) and emission (Fig. 3) upon heating the cells in a water-bath [16,21]. Additional experiments were performed to obtain further evidence that the enol is oxidized intracellularly. It is known that, upon incubation, polymorphonuclear leukocytes lose some myelo-

peroxidase; the amount increases with time, with values as high as 2.7% of the total enzyme having been reported [22]. In our system, essentially the same emission was observed when the enol precursor was added to polymorphonuclear leukocytes which were freshly prepared or several hours old. Myeloperoxidase is stored in the azurophilic granules. However, when neutrophils are stimulated, this enzyme is released [5,21]. Hence, it is possible that the reaction with the enol occurs in the cytoplasm. Enol precursor concentrations above 2 mM produce significant damage to the cells. This may be the reason for the much stronger emission at 4 mM enol (fig. 2). Pertinent results and data are collated in Table I. It must be pointed out that even with 6 mM enol the cell integrity was preserved, the number of cells being not significantly altered. An important question is whether the damage to the cells induced by high concentrations of the enol precursor is connected with the myeloperoxidase-catalyzed aerobic oxidation. Consequently, the enol was added under anaerobic conditions, achieved by addition of glucose (0.3%), glucose oxidase and catalase. Just after the cells had been collected, there were more viable cells compared to controls which received the enol under aerobic conditions; however, cellular death started immediately upon aeration. This observation strongly indicates that damage is induced by the myeloperoxidase-catalyzed oxidation of the enol. Full confirmation was provided by the fol-

TABLE I RESULTS PERTAINING TO THE OXIDATION OF THE ENOL OF ISOBUTANAL BY P O L Y M O R P H O N U C L E A R LEUKOCYTES (5.106 CELLS/ml) Trimethylsilyl enol ether (mm)

Maximal intensity (103 counts/5 s)

Viable cells (%)

1.0 1.5 2.0 2.0 a 4.0 4.0 a 4.0

10 17 27 38 70 126 84

95 95 85 85 45 30 5

a H202 (8"10 ~ M) added.

340

lowing observation in the presence of 5.5 mM enol: although polymorphonuclear leukocytes were totally damaged, mononuclear cells (such as

lymphocytes) remained completely intact (Fig. 5). Lymphocytes do not have myeloperoxi.dase and there is no reason to expect that the enol does not penetrate into these cells. The destruction seen in Fig. 5 results from the preparation of the smear and is ultimately due to the damaged membrane seen with Trypan blue exclusion.

Nature of the excited species formed By analogy to the horseradish peroxidase-catalyzed reaction [1-3,9], the excited species generated in the myeloperoxidase-catalyzed process should be triplet acetone. For conclusive evidence, we resorted to the use of the 9,10-dibromoanthracene-2-sulfonate ion and the parent nonhalogenated analogue as probes. Triplet acetone generated both chemically (dioxetane decomposition) [23,24] and enzymically [1] elicites strong emission from the 9,10-dibromoanathracene-2-

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Fig. 6. The effect of adding 12 /LM 9,10-dibromoanthracene-2sulfonate (O) and 12/~M anthracene-2-sulfonate ( . . . . . . ) upon the emission elicited by addition of the enol ether to polymorphonuclear cells (zx). Also shown ( O ) is the 9,10-dibromoanthracene-2-sulfonate enhanced emission observed with cells which had been previously exposed to the enhancer for 15 min. centrifuged and resuspended.

Fig. 5. Representative photographs of Leishman's stain of polymorphonuclear leukocytes initially (A), after exposure to 2 mm enol of isobutanal (B), and after exposure to 5.5 mM enol (C). The intact cell (lower-right corner in C) is a lymphocyte. Original magnification x 1000. Lymphocytes were consistently undamaged.

Fig. 7. The emission spectrum ( ) of the 9,10-dibromoanthracene-2-sulfonate (12 ffM) -sensitized emission resulting upon addition of the enol ether to polymorphonuclear leukocytes. The curve ( . . . . . . ) denotes the emission spectrum of the optically excited 9,10-dibromoanthracene-2-sulfonate in the spent mixture. For the optically excited spectrum the slit was 2 nm, whereas in the case of the enzymic system the slit was 20 nm. ~ .... 383 nm.

341

sulfonate ion, while the anthracene-2-sulfonate ion has little or no effect. This behavior was reproduced with the enol/polymorphonuclear leukoc y t e / O 2 system, even in the absence of H202 (Fig. 6). If the cells were first sonicated, the 9,10-dibromoanthracene-2-sulfonate ion emission was strong enough to be observed with the darkadapted eye and the spectrum could be recorded on conventional equipment (Fig. 7). Neither of these probes had any effect whatsoever upon the rate of 02 uptake. Clearly, it was of considerable interest to verify whether the 9,10-dibromoanthracene-2-sulfonate ion is taken up by the cells. For this reason, the system was incubated with this probe for 15 min and the cells were centrifuged, collected and resuspended. Strong emission could be seen upon addition of the enol precursor (Fig. 7). These results show conclusively that triplet acetone can transfer energy intracellularly to the 9,10-dibromoanthracene-2-sulfonate ion. An extra bonus of this experiment is that there was less cellular death and improved cellular integrity in the presence of this acceptor, suggesting competition between the acceptor and cell constituents involved in quenching of triplet acetone, this latter quenching interaction presumably giving rise to the deleterious effects. The following scheme summarizes our results:

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The observation that myeloperoxidase induces the formation of electronically excited triplet carbonyl during catalysis of the aerobic oxidation of the enol is of considerable interest, especially since the reaction appears to occur intracellularly. This intracellular generation of a triplet carbonyl compound can lead to cell damage and death at high substrate concentrations. The considerable protection observed in the presence of the 9,10-dibromoanthracene-2-sulfonate ion indicates that this electronic energy acceptor binds to sites close to the loci where triplet acetone is generated. This is quite consistent with the fact that transfer of electronic energy occurs. Such a favorable situation is not entirely surprising, since polymorphonuclear leukocytes are very rich in myeloperoxidase. In conclusion, given the appropriate substrate, these cells are very efficient in generating electronically excited species. More broadly, the in situ generation of electronically excited species by adding appropriate substrates to appropriate cells opens new horizons in toxicology and possibly in chemotherapy.

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Concluding remarks

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H3C ~ C=O * --~ > C = O + hvp H3C H3C 3H3C > C---O * + cellular constituents ~ quenching (and damage) H3C 3H3C

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H3C

> C=O + 9,10-dibromoanthracene-2-sulfonate 1 H3C

1 9,10-dibromoanthracene-2-sulfonate * ---*9,10-dibromoanthracene-2-sulfonate+ huf

342

Acknowledgements The authors wish to express their deep gratitude to Professor Frank H. Quina for advice and for a critical reading of the manuscript and to Mrs. Primavera B. Garcia for valuable advice in the experimental work. This work benefited from grants from FINEP (Rio de Janeiro), FAPESP (S~o Paulo), CNPq (Brasilia) and from the Volkswagen Foundation (Hannover).

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