Journal of Photochemistry

and Photobiology,

B: Biology, 5 (1990)

495

495 - 503

XeCl LASER ACTION AT MEDIUM FLUENCES ON BIOLOGICAL TISSUES: FLUORESCENCE STUDY AND SIMULATION WITH A CHEMICAL SOLUTION J. P. BERTHIERT

and E. RAYNAL

Laboratoire de Physique des Lasers, Universite’ Paris XIII, 93430

Villetaneuse (Fmnce)

S. KIMEL Department

of Chemistry, Technion-Zsrael Institute of Technology,

Haifa 32000

(Israel)

S. AVRILLIER Laboratoire de Physique des Lasers, Universite' Paris XIII, 93430

Villetaneuse (France)

J. P. OLLIVIER Departement

de Cardiologie, H8pital du Val de Grace, 75005 Paris (France)

(Received June 29, 1989; accepted October 15, 1989)

Keywords. XeCl excimer laser, rat heart fluorescence, respiratory chain, NADH, FAD, cytochrome c.

Abbreviations APD ablative photodecomposition ATP adenosine triphosphate cytochrome c reductase Cyt FAD flavin adenine dinucleotide NAD nicotinamide adenine dinucleotide PBS phosphate-buffered saline summary

A rat heart, isolated and perfused, was irradiated with a XeCl excimer laser at 308 run. The evolution of the fluorescence spectrum was measured. For an incident energy E > 4 kJ m-* per pulse the fluorescence changed with time in a complex and spectrally non-uniform way. The proposed interpretation is that the radiation acts on the cellular respiratory chain. Buffered solutions of NADH, cytochrome c and FAD, which play a role in the respiratory chain, were irradiated in order to simulate the in uiuo findings. The conclusion of this study is that XeCl radiation introduces a modification in the functioning of the respiratory chain : it accelerates electron transfer, but this quickly leads to an interruption of the respiratory chain. *Author to whom correspondence loll-1344/90/$3.50

should be addressed. 0 EIsevier Sequoia/Printed

in The Netherlands

496

1. Introduction The study of the interaction of laser radiation with biological tissues is of general interest since one or more of the unique characteristics of the laser can be used: high dose rate, high spatial resolution, high wavelength selectivity and high temporal resolution (with pulsed lasers). In previous studies [ 1, 21 we have used a specially designed spectrofluorometer to investigate the fluorescence of intact cardiac tissue excited by nitrogen laser radiation at 337 nm. The in uiuo fluorescence evolution is related to the ratio [NADH]/[NAD+] [3 - 51, thus enabling us to monitor, in real time, changes in respiratory events in cardiac cells. In this study we report the action of XeCl excimer (308 nm) laser pulses on the perfused heart. It is well known that the XeCl laser is capable of causing ablative photodecomposition (APD) [ 61 when the energy density exceeds a certain threshold, e.g. E > 14 kJ m-’ per pulse for arterial tissue [ 71. So as to monitor non-ablative interactions only we limited the incident fluence to E < 7.5 kJ mm2 per pulse, i.e. well below APD threshold levels. It is fortunate that physical-chemical processes related to the respiratory chain, which are generated by XeCl laser radiation, can be monitored by the fluorescence induced by the same laser. We have recorded the fluorescence emission spectrum (and its evolution) of the isolated and perfused rat heart. The analysis of the spectrum yields novel cellular and molecular information about the action of XeCl laser radiation on intact cardiac tissue. The interaction of pulsed lasers, in the 300 - 315 nm spectral region, with tissue is of great interest for various laser applications in medicine, particularly in angioplasty. This is because strong interactions, such as APD, can be obtained with less deleterious side effects than when irradiating at shorter wavelengths [ 81. 2. Materials and methods 2.1. Experimental arrangement A schematic diagram of the experimental set-up is given in Fig. 1. The excimer laser (Sopra, model 1250 E2, Bois-Colombes, France) emitted pulses of 0.3 J and 20 ns duration at a repetition rate of 1 Hz at 308 nm. The fluorescence emission passed through a 308 nm blocking filter (F) and was collected by a Jarell-Ash monochromator ($ m) equipped with a grating (1200 lines mm-‘) blazed at 300 nm; the effective spectral slit width was 2 nm. Fluorescence signals were measured with a Hamamatsu R 928 photomultiplier (PM), and reference signals from the XeCl laser were measured with a fast silicon photodiode (PD). An HP 54 111 D dual-beam digitizing oscilloscope (Hewlett-Packard, Santa Clara, CA) sampled both signals at 1 ns intervals. The oscilloscope was controlled by an HP 9310 computer, connected to a plotter. The fluorescence of the perfused rat heart was measured by a backward detection method using an optical fibre (diameter, 1 mm) to excite the tissue

497 r-------7 I

/Id I

or

I

Cuvette

I

I

L-------l ~-____--------_

+LZG-.>ConputeF)>...FI Fig. 1. Schematic diagram of experimental set-up (see text).

and to collect the fluorescence emission. The rat heart was electrically stimulated at 3 Hz with a pulse generator (Gen) and was perfused with an oxygenated medium at 37 “C, buffered at pH 7.4. It is important to note that the penetration depth of 308 nm radiation in the tissue is rather small, about 0.05 mm [7]. In addition, we measured (in a cuvette) the in vitro fluorescence of premixed solutions of selected chemical compounds in a PBS buffer at pH 7.4, which simulated the observed temporal evolution of the in vivo fluorescence at different wavelengths. All chemicals were obtained from Sigma Chemicals (Paris) and were used as received. Cytochrome c reductase was used for the simulation study rather than cytochrome c because it contains components which fluoresce at 380 nm (the same as the tissue). 2.2. Methods The PM and PD signals were measured simultaneously. For each laser pulse the fluorescence signal at a given wavelength was sampled at 1 ns intervals for 20 ns, and its maximum value was stored in the computer. Similarly, the laser energy profile was sampled by the PD at 1 ns intervals during the pulse, and its maximum value was also stored. The computer calculated, in real time, the average value of the PM readings for eight pulses, then the average value of the PD readings for two pulses and, finally, the ratio between these averages. In this procedure one value for the ratio (the normalized fluorescence at a given wavelength F,) was obtained every 10 s. This procedure was continued for 10 min to obtain Fh(t), the temporal evolution of the normalized fluorescence. Measurements were performed for six wavelength settings of the monochromator (at 350, 390, 420, 450, 480 and 520 nm), chosen for their relevance in a mechanistic interpretation of the in viuo fluorescence data, as will be discussed below. Figure 2 illustrates a typical result. For each curve, Fh(t), a different site on the rat heart was selected. Similarly, fresh solutions in the cuvette were prepared for each in vitro measurement of FA(t). The measurements on perfused hearts were

498

...

480

_,-------_

0

2

I

520

,.\_._I’._._

-__Z___-.--._

3

4

5

t/mln

Fig. 2. Evolution of the fluorescence of the perfused rat heart, PA(~), at six wavelengths (nm); E = 6 kJ mm2 per pulse.

repeated with six different rats. Absolute values of individual measurements F(h) varied to some extent from animal to animal. However, the temporal evolution at a given wavelength Fh( t), normalized to the value Fh( 0) at t = 0, was identical for all animals to within 5%.

3. Results and discussion 3.1. Fluorescence evolution From Fig. 2, where the fluorescence evolution, FL(t), is plotted for six selected wavelengths, we constructed an orthogonal representation: the fluorescence spectrum at a given time, F,(X). Figure 3 shows such a six-point spectrum at four different times: t = 0, t = 1 min, t = 2 min and t = 5 (or 10) min; the data were not corrected for the spectral sensitivity of the spectrometer or detector. In Fig. 3(A), obtained with an incident energy density E = 4

350

400

450

500

350

400

450

500

Wavdength/nm

Fig. 3. Fluorescence spectrum, F&), pulse, t = 0 (...), t = 10 min (-* * -); ),t-2min(----),t=5min(---). (-

of the perfused rat heart: (A) E = 4 kJ me2 per (B) E = 6 kJ me2 per pulse, t = 0 (. . .), t = 1 min

499

kJ mm2 per pulse, little temporal evolution is observed. In contrast, in Fig. 3 (B), obtained with E = 6 kJ mm2 per pulse, a significant evolution of F(h) us. time is observed : (i) a monotonic decrease in F( 350) ; (ii) a 40% decrease in F(480) in the first minute followed by a gradual increase; (iii) a similar evolution of F(520) but less pronounced than F(480). With nitrogen laser excitation we have shown that the fluorescence emission spectrum is practically that of pure NADH [2]. In the present study, using medium-fluence 308 nm excimer laser excitation, a complex spectrum (F(h)) is found. This is already the case at t = 0 and, moreover, F(h) is seen to evolve with time. In view of our previous findings [2], the analysis of the present data for F,(h) suggests that XeCl laser radiation introduces modifications in the functioning of the respiratory chain of cardiac cells. This is probably not the only light-induced action, but it is the one accessible through measurement of F,(h), as has been reported previously by Chance and coworkers [3, 41 for tissue and by Kohen et al. [ 51 for cells. The respiratory chain consists of a succession of oxidation-reduction reactions [9]. The hypothesis that XeCl laser radiation acts on the respiratory chain was tested by using the metabolic inhibitor rotenone, which is known to block the chain at the NADH site (Fig. 4). On addition of 1 mM rotenone to the perfusion fluid, the NADH fluorescence no longer decreases (Fig. 5), which indeed proves that the respiratory chain can be activated by 308 nm radiation. In order to elucidate the chemical processes occurring in the chain we carried out spectral measurements of the critical components in the chain (Fig. 4) which fluoresce on direct excitation at 308 nm: Cyt shows maximum fluorescence around 360 nm (Fig. 6, curve A) and NADH has a maximum around 464 nm (Fig. 6, curve B). In addition, FAD in mixtures can be excited indirectly, by absorption of the Cyt and NADH fluorescence at 360 and 460 nm, to give broad-band FAD fluorescence around 515 nm. The evolution of the rat heart fluorescence F@) (Fig. 3) suggests the sequential participation of these three compounds. It should be noted that the pure compounds Cyt and NADH do not show fluorescence evolution. In fact, a superposition of the curves 6A and 6B resembles the fluorescence curve F&) (Fig. 3(B)) of the perfused heart at t = 0 (Fig. 6, curve C). As

NAD 4

FAD

coa I

ATP-ADP

ATP -AW

SITE

I

Cytb

CytdIII)

Cyta

H$I

4 ATP -Mp

SITE II

-Fig. 4. Simplified respiratory chain processes.

SITE

III

I 0

1

3

2

4

5

t/mln

Fig. 5. (A) Fluorescence evolution, Fedt), of the perfused rat heart;(B) min after addition of 1 mM rotenone to the perfusing fluid.

350

400

450

500

same as (A) 25

550

Wavelength/m

Fig. 6. Fluorescence

spectrum, F(X):(A)

Cyt in PBS; (B) NADH in PBS;(C)

rat heart (at

t = 0).

noted above FAD quenches the fluorescence of Cyt and NADH and, in turn, emits at 515 nm. In the three-component system NADH + Cyt + FAD the strong absorbance of FAD (between 370 and 450 nm) causes a red shift (Ax = 475 - 465 nm = 10 nm) of the NADH fluorescence, as is indicated in Fig. 6 (compare curves B and C). 3.2. Simulation We studied the evolution of the fluorescence spectra of solutions containing the three model compounds. The stoichiometric proportions of these compounds involved in the cellular respiratory chain are [NADH] : [FAD] : [Cyt] = 1:1:2 [lo]. However, in cells, where respiratory chain proteins are located in the mitochondrial membranes, values of effective concentrations may be different from the stoichiometric proportions. Thus, we adjusted the relative concentrations of the three model compounds to [NADH]: [FAD] : [Cyt] = 1:0.15 :1.25 and obtained, empirically, a fluorescence signal

501

350

400

450

500

550

Wavetength/nm

Fig. 7. Evolution of fluorescence spectrum Ft(h) of a solution NADH (17.0 PM), FAD (2.7 /.LM)and Cyt (21.5 /JM) in PBS at pH 7.4.

containing

(at t = 1 min) which looked similar to that of the rat heart (at t = 0). The time difference, At = 1 min, is due to the fact that at t = 0 Cyt in solution is in the oxidized form, whereas in the in uiuo situation Cyt is partially in the reduced form. A certain time is required before the composition of the solution attains the in vim equilibrium value. The results are plotted in Fig. 7. NADH reduces FAD as is apparent from the decrease in F,(460). In turn, photogenerated FADHz reduces Cyt( Fe”‘), and Cyt( Fen) reduces ambient oxygen present in the solution [ 111. Since NADH is consumed, the amount of radiative energy that can be transferred to FAD diminishes; consequently, the fluorescence intensity of FAD at 515 nm also decreases, thus causing a gradual decrease with time of the red shift Ah of the NADH emission. Without irradiation, the evolution in the three-component system is qualitatively the same but occurs at a much slower rate. Thus, XeCl laser radiation seems to act as a photocatalyst of the dark reaction. The solution spectra F,(X) in Fig. 7 can be compared with those of the perfused rat heart (Fig. 3(B)). The spectra are similar during the second minute, in the spectral region X > 450 nm. However, there are a number of differences: (i) in the solution the evolution of F(480) is about twice as fast as in the perfused heart; (ii) in the region around 350 nm the heart tissue fluoresces more strongly than the solution; (iii) for the heart tissue, after 1 min, F,(480) increases, whereas for the solution F,(480) continues to decrease. This suggests that the three-component model considered thus far is oversimplified. Elements in the respiratory chain have been left out so that compounds with high redox. potential interact directly with those of low redox potential. Also, in heart tissue the three components are located in the membrane and are not in direct contact with each other. Therefore, the model must be generalized to include membrane interactions and this point will be dealt with in a future publication. In an attempt to explain these differences we varied the mode of irradiation. The heart tissue was irradiated with bursts of NP consecutive laser pulses, at a repetition rate of 1 Hz; bursts were separated by dark periods,

502

012345

L13 tInIn

5 Number

7

of bursts

9 (Nb)

Fig. 8. Evolution of fluorescence signal at 480 nm following excitation of a perfused rat heart with bursts of NP laser pulses (6 kJ mm2 per pulse; repetition rate, 1 Hz) followed by dark periods, td: (A) us. elapsed time; (B) number of bursts N, (when not indicated otherwise N, = IO). td = 10 s y’--); td=30 s (...); td=l min ( -); td’2 min (-‘-); td=5 min (-“-);td=Imin,Np=5(---);td=lmin,Np=20(-----).

td. Fluorescence measurements were made at 480 nm and the results are presented in Fig. 8(A), plotted as a function of elapsed time, and in Fig. 8 (B), plotted as a function of the number of bursts, N,,. After the first burst F,s, decreases, whereas the second burst reverses this trend and causes an increase in F,,. This result is nearly independent of the duration of the dark periods, td (Fig. 8(B)). Intuitively, this result may be explained by assuming that the first burst catalyses the reaction and accelerates processes in the respiratory chain, whereas the second burst interrupts the chain by causing irreversible changes in the tissue or proteins. It is possible that, on the cellular level, the first burst alters the mitochondrial membranes. Indeed, damaged mitochondria are known to transfer electrons at a more rapid rate than intact mitochondria [ 121. In accordance with this interpretation it is not surprising that with Np = 5 the tissue destruction is slower and with Np = 20 the destruction is faster than with N,, = 10 (Fig. 8).

4. Conclusions We have shown that XeCl laser radiation initiates chemical reactions in cardiac tissue. Some of these reactions seem to occur in the cellular respiratory chain where the radiation appears to accelerate electron transfer. This action begins at a radiation dose as low as E = 4 kJ rnp2 per pulse. It is possible that light-induced acceleration occurs in an uncoupled mode, i.e. without concomitant ATP production [12]. However, evidence exists that light can induce coupled reactions leading to enhanced ATP production [ 131. In order to clarify this point it would be interesting to repeat our measurements with single cells and mitochondria [14] and to study the correla-

503

tion between light-induced variations in mitochondrial

changes in the respiratory chain and localized activity reported for myocardial cells in culture

c151. References 1 G. Renault, E. Raynal, M. Sinet, J. P. Berthier, B. Godard and J. Cornillault, A laser fluorimeter for direct cardiac metabolism investigation, Opt. Laser Technot., 6 (1982) 143 - 148. 2 G. Renault, E. Raynal, M. Sinet, M. MuffatJoly, J. M. Vallois, J. P. Berthier, J. Cornillault and B. Godard, Cardiac metabolism monitoring with fiber optic laser fluorimeter, Am. Heart J., 108 (1984) 428 - 429. 3 B. Chance, J. R. Williamson, D. Jamieson and B. Schoener, Properties and kinetics of reduced pyridine nucleotide fluorescence of the isolated and in vivo rat heart, Biochem. Z., 341 (1965) 357 - 377. 4 R. Scholz, R. G. Thurman, J. R. Williamson, B. Chance and T. Bucher, Flavin and pyridine nucleotide oxidation-reduction changes in perfused rat liver. I. Anoxia and subcellular localization of fluorescent flavoproteins, J. Biol. Chem., 244 (1969) 2317 - 2324. 5 E. Kohen, B. Thorell, C. Kohen and J. M. Salmon, Studies on metabolic events in localized compartments of the living cell by rapid microspectrofluorometry, Adu. Biol. Med. Phys., 15 (1974) 271 - 297. 6 R. Srinivasan, Ablation of polymers and biological tissue by ultraviolet lasers, Science, 234 (1986) 559 - 565. I D. L. Singleton, G. Parakevopoulous, R. S. Taylor and L. A. J. Higginson, Excimer laser angioplasty: tissue ablation arterial response and fiber optic delivery, IEEE J. Quantum Electron., 23 (1987) 1772 - 1787. 8 C. M. Colella, P. Bogani, G. Agati and F. Fusi, Genetic effects of UV-B: mutagenicity of 308 nm light in Chinese hamster V79 cells, Photochem. Photobiol., 43 (1986) 437 - 442. 9 R. V. Bensasson, E. J. Land and T. G. Truscott, Flash photolysis and pulse radiolysis, Contributions to ihe Chemistry of Biology and Medicine, Pergamon Press, Oxford, 1983, pp. 135 - 163. 10 A. L. Lehninger, Principles of Biochemistry, Worth Publishers, New York, 6th edn., 1972, Chapt. 17. 11 A. Ferri, D. Patti, P. Chiozzi and M. Cattozzo, Photoexcitation of the methionineiron bond in iron(II1) cytochrome c: bimolecular reaction with NADH, J. Photothem. Photobiol. B, 2 (1988) 341 - 353. 12 D. E. Metzler, Biochemistry, Academic Press, New York, 1982, Chapt. 10. 13 R. Hilf, R. S. Murant, U. Narayanan and S. L. Gibson, Relationship of mitochondrial function and cellular adenosine triphosphate levels to hematoporphyrin derivativeinduced photosensitization in R3230AC mammary tumors, Cancer Res., 46 (1986) 211 - 217. 14 C. Salet, S. Passarella and E. Quagliariello, Effects of selective irradiation on mammalian mitochondria, Photochem. Photobiol., 45 (1987) 433 - 438. 15 A. Siemens, R. Walter, L.-H. Liaw and M. W. Berns, Laser stimulated fluorescence of submicrometer regions within single mitochondria of rhodamine-treated myocardial cells in culture, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 466 - 470.

XeCl laser action at medium fluences on biological tissues: fluorescence study and simulation with a chemical solution.

A rat heart, isolated and perfused, was irradiated with a XeCl excimer laser at 308 nm. The evolution of the fluorescence spectrum was measured. For a...
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