Phorochembrry und Photobiology Vol. 53, No. 4, pp. 549-553. 1991 Printed in Great Britain. All rights rcservcd
0031-8655/91 $03.00 +O.W Copyright @ 1991 Pergamon Prcss plc
RESEARCH NOTE
THE PHOTODEGRADATION OF PORPHYRINS IN CELLS CAN BE USED TO ESTIMATE THE LIFETIME OF SINGLET OXYGEN JOHAN MOAN*and KRISTIAN BERG Institute for Cancer Research, 0310 Montebello, Oslo 3, Norway (Received 25 June 1990; accepted 3 October 1990)
Abstract-NHIK 3025 cells were incubated with Photofrin I1 (PII) and/or tetra (3hydroxypheny1)porphyrin (3THPP) and exposed to light at either 400 or 420 nm, i.e. at the wavelengths of the maxima of the fluorescence excitation spectra of the two dyes. The kinetics of the photodegradation of the dyes were studied. When present separately in the cells the two dyes are photodegraded with a similar quantum yield. 3THPP is degraded 2-6 times more efficiently by light quanta absorbed by the fluorescent fraction of 3THPP than by light quanta absorbed by the fluorescent fraction of PI1 present in the same cells. The distance diffused by the reactive intermediate, supposedly causing the photodegradation was estimated to be on the order of 0.01-0.02 pm, which mainly lo2, corresponds to a lifetime of 0.01-0.04 ps of the intermediate in the cells. PI1 has binding sites at proteins in the cells as shown by an energy transfer band in the fluorescence excitation spectrum at 290 nm. During light exposure this band decays faster than the Soret band of PI1 under the present conditions. Photoproducts (lo2etc.) generated at one binding site contribute significantly in the destruction of remote binding sites.
INTRODUCTION
Porphyrins are degraded or modified by light (Cox et af.,1982; Krieg and Whitten, 1984). Considerable interest has been focused on such photodegradation (often called photobleaching), since it can be taken advantage of in photodynamic therapy of cancer (Moan, 1986; Dougherty, 1987, Mang et af.,1987). In fact, the concept of photobleaching has been successfully applied in human clinical trials (Mang et af., 1990). The mechanisms of photodegradation are different for porphyrins in solution and for porphyrins in biological systems (Krieg and Whitten, 1984). In the latter case the rate constants are significantly larger and different products are formed. To a first approximation the photodegradation follows first order kinetics and the rate constants are independent of the initial porphyrin concentration, both in cells and in tissues (Mang er af., 1987). This indicates that an excited porphyrin molecule is degraded either by products formed by itself or by direct interaction with its surrounding. However, this is a crude approximation since the kinetics deviate from first order after extended (but still clinically relevant) exposure times. Furthermore, the rate constants for degradation of Photofrin I1 (PII)? are *To whom correspondence should be addressed. tAbbreviations: HPLC, high performance liquid chroma-
tography; MEM, minimal essential medium, NCS, newborn calf serum, PII, Photofrin 11; PBS, Dulbeccos phosphate buffered saline; 3THPP, tetra (3hydroxyphenyl) porphyrin. PAP 53:4-I
larger when cells are exposed t o light in a D20buffer than when they are exposed in a H 2 0 buffer, indicating that '02-production plays a role (Moan et af., 1988). In order to elucidate the photobleaching mechanisms further we have incubated cells with two different porphyrins, Photofrin I1 (PII) and tetra (3hydroxylphenyl) porphyrin (3THPP). which have different fluorescence excitation and emission spectra. Thus, even when they are present in the same cell they can be excited and monitored with some selectivity. Both are negatively charged lipophilic dyes which, according to fluorescence microscopic studies (data not shown), localize almost identically in the cells, presumably in membrane structures. In such a system it is possible to study the degradation of one dye caused by excitation of the other dye, and to estimate the distance travelled by reactive intermediates before deactivation. MATERIALS AND METHODS
Chemicals. Photofrin I1 was obtained from Photomedica, Raritan, NJ. The drug was stored frozen in small vials for up to 8 months before being used in the present experiments. It was thoroughly checked by HPLC, and by cell survival experiments that the drug did not change in its characteristics during the time of storage. The procedures of HPLC and cell survival experiments are described elsewhere (Moan et a / . , 1982; Moan and Sommer, 1983). The 3THPP was obtained from Porphyrin Products, Logan, UT. Stock solutions (0.1 mg/mL) were prepared in 0.05 M NaOH as earlier described (Moan et al., 1987). Cell cultivation. Cells of the line NHIK 3025 (derived
549
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JOHANMOANand KRlsnAN BERG
from a human carcinoma in situ) were cultivated in MEM containing 10% NCS. For fluorescence experiments 1W cells were incubated in 25 cm2 Falcon tissue culture dishes. Six hours later the medium was changed to MEM with 3% NCS with either 25 pg PII/mL and/or 1 pg 3THPPhL. These concentrations were chosen on the basis of pilot experiments, in view of optimal analysis of the fluorescence spectra of samples containing both drugs. At these concentrations the photosensitivity and the fluorescence yield were about a factor 2 larger for cells with PI1 than for cells with 3THPP, when exposed to equal fluence rates at the optimal wavelengths (400 nm for PI1 and 420 nm for 3THPP). After an incubation period of 18 h with the dyes the cells were washed five times in icecold PBS, brought into suspension by trypsinization, washed once more with PBS and finally suspended in PBS at a concentration of approx. loh cellshl-as determined by Biirker chamber counting. Irradiation of the samples. The light source was a 900 W Osram high pressure xenon lamp fitted to a Bausch & Lomb grating monochromator. The bandwidth of the light was 15 nm as measured with a second monochromator (Jarrel Ash) with narrow slits (Ah = 1 nm) and a UDT 1la detector (United Detector Technology, Santa Monica, CA). This detector is calibrated and was also used to determine the fluence rates at the position of the cells: 30 mW/cm2 at both wavelengths (400 and 420 nm). The samples were irradiated in a fluorescence cuvette (3 x 3 x 20 mm) and were gently stirred during the light exposure. Fluorescence measurements. Fluorescence spectra were recorded by means of a Perkin-Elmer LS 5 spectrofluorimeter equipped with a Hamamatsu R928 red sensitive photomultiplier tube. A 580 nm cut-off filter was used to remove scattered light from the light entering the emission slit, which was usually set to give AX = 5 nm. The distortion of the spectra resulting from such slit widths were taken into consideration in the evaluation of the data but found to be of minor importance. Using a microcuvette with a cross section of only 3 x 3 mm, inner filter effects played no significant role.
ml
Loo
m
Wavelength hm)
RESULTS AND DISCUSSION
Figure 1. Fluorescence excitation spectra of PI1 and 3THPP in NHIK 3025 cells 18 h incubation in MEM with 3% NCS containing 25 pm PII/mL and/or 2 pg/mL 3THPP. The dotted line in the spectrum of PI1 shows the spectrum in methanol in the region 250-329 nm.
We have earlier shown that the fluorescence quantum yields of PI1 and 3THPP in NHIK 3025 cells are similar to within 30% (Moan et al., 1987). Therefore, and since it is difficult to record the absorption spectra of dyes in cells with accuracy, we decided to base our calculations on the fluorescence measurements. In fact, it is more relevant to use fluorescence spectra than absorption spectra for investigations of this type, since the aggregated fraction of the dyes in the cells is both non-fluorescent and photochemically inactive. This has been shown by means of absorption, fluorescence and action spectroscopy for both dyes considered in the present work (Western and Moan, 1988; Moan and Sommer, 1984). Figure 1 shows the fluorescence excitation spectra of cells with PII, 3THPP and with the mixture of the two dyes. There is a peak in the spectra at about 290 nm, which, in the case of PII, is entirely due to energy transfer from proteins containing aromatic amino acids to porphyrin molecules (Moan et al., 1988). Thus, using the known shape of the excitation spectra of PI1 in liposomes or lipids with properties
comparable to those of cell membranes but without proteins, it is possible to separate the energy transfer peak from the direct excitation of PI1 as indicated by the dotted line on the spectrum for PI1 (Fig. 1, Moan et al., 1988). In the case of 3THPP such a separation was not attempted, since this dye has a peak in its own excitation spectrum in this wavelength region due to the phenyl rings. One can easily separate the spectra of samples containing both dyes (Fig. l ) , both manually and by means of a simple computer program. To a first approximation and for exposure times shorter than 6 min, the decay kinetics of the Soret band is of first order for both dyes (Figs. 2 and 3). The same is true for the energy transfer band at 290 nm (Fig. 2). However, the rate constant for the decay of the energy transfer band is larger than that for the decay of the Soret band. Thus, the decay of the energy transfer band is due to at least two factors: the photodegradation of PI1 and the destruction of binding sites at proteins. Since the rate constant for the decay of the energy transfer
Research Note
Figure 2. The decay of the Soret band of PI1 in NHIK 3025 cells monitored with the excitation and emission wavelengths set at 400 and 625 nm, respectively; and of the energy transfer band (Acxc = 290 nm, Acm = 625 nm), the contribution from direct excitation being subtracted. The wavelength of the exposure light was 400 nm.
55 1
6 min) as indicated by the regression lines drawn in Fig. 3. Table 1 shows that the quantum yield of photodegradation of PI1 by light quanta absorbed by PI1 itself is comparable to the corresponding yield for 3THPP or slightly lower. However, the relative value of the quantum yield of photodegradation of 3THPP caused by quanta absorbed by 3THPP is 3-6 times larger than the relative value for photodegradation of the dye caused by quanta absorbed by PII. (Only the fluorescent and photosensitizing fraction of the dyes are considered). Similarly, the relative value of the quantum yield of photodegradation of PI1 caused by light absorption in PI1 is more than twice as large as the relative value for photodegradation caused by light absorption in 3THPP. This is in agreement with our earlier conclusion that the photodegradation of porphyrins in cells is predominantly a first order process (Mang et al., 1987). Therefore, the present results indicate that a photoproduct, like lo2,generated by the absorption of a quantum of light in a porphyrin molecule causes damage mainly to that molecule and not to other porphyrin molecules in the vicinity. The average intracellular concentrations of 3THPP can be estimated to 40 p M under the present conditions, and that of PI1 can be estimated to 80 p M of porphyrin rings (mol. wt 600) (Moan ef al., 1987). In these estimations it is assumed that the amount of dye in the nucleus is negligible (Moan er al., 1989). The nucleus constitutes about 30% of the volume of these cells (Moan and Boye, 1981). Thus, spheres of radius 0.02 pm located randomly in the cytoplasm contain on the average 1 fluorescent molecule of 3THPP. It is, of course, a very crude approximation that the dye molecules are homogeneously distributed in the cytoplasm, but it may serve in a first approximative calculation of the distance travelled by the reactive intermediates generated by the photoexcitation of the dye molecules. If we assume that the membranes constitute about 10% of the cytoplasmic volume (White et al., 1978) and that the present lipophilic dyes are localized mainly in membrane structures, their local concentrations are a factor of 10 larger than estimated above and the radius of a sphere containing on average 1 fluorescent molecule of 3THPP is about 0.01 pm. Singlet oxygen (lo2) is almost certainly a reactive intermediate generated under the present conditions (Moan et al., 1987). The lifetime rA of lo2under the present conditions can be estimated by use of the formula 6 = ( 6 0 T~)”’, where 6 is the distance diffused by lo2 before it is quenched and D is the diffusion coefficient of ‘ 0 2 . If we assume that D = 1.4 x cm2 s-l (Moan and Boye, 1981) and 6 is within the range 0.01-0.02 p , rA is within the range 0.01-0.04 ps. Such a short lifetime is consistent with the lack of a D 2 0 effect in many photosensitized processes in which lo2is very likely to be involved. Using the
-
band is dependent on the concentration of PI1 in the cells, the binding sites at proteins can be degraded by photoproducts generated remotely from these binding sites (Moan ef al., 1988). From Fig. 3 rate constants for the first part of the decay of the Soret band of the dyes can be determined. In the calculations we have taken into account only the two first exposure times (i.e. 3 and
Figure 3. Decay curves for the Soret bands of PI1 and 3THPP separately or simultaneouslypresent in NHIK 3025 cells exposed to light at either 400 or 420 nm. Incubation conditions, see legend of Fig. 1.
JOHAN MOANand KRISTIAN BERG
552
Table l(a). Photodegradation of 3THPP ~
Concentrations WmL)
3THPP 1 1 1 1
PI1 0 0 25 25
~~~~~~
hCxp
(nm)
A(3THPP)
A(P1I)
k(min-')
400 420 400 420
0.2 1.0 0.2 I .0
0 0 2 2
0.014 0.10 0.07 0. 14 ~
@(3THPP33THPP) Q(3THPP.PII)
0.07 0.10 0.022 0.016
~~~~~
Table l(b). Photodegradation of PI1 Concentrations ()lLg/mL) PI1 25 25 25 25
3THPP 0 0 1 1
Lip (nm) 400
420 400 420
A(PI1)
A(3THPP)
2.0 2.0 2.0 2.0
0
n
0.2 1 .0 ~~
k(min-')
0. 13 0.11 0.13 0.11 ~~~
@(PII,PII)
(D(P11.3THPP)
0.M5 0.055
< 0.03' ~~
Aexp is the wavelength of the photodegrading light. A(PI1) and A(3THPP) are relative numbers of light quanta absorbed by fluorescing and photoactive molecules of PI1 and 3THPP in the samples, approximated in relative values by the convolution integrals of the fluorescence excitation spectra and the spectra of the photodegrading light at 400 and 420 nm rcspcctively, with AA = 15 nm. In this approximation it is assumed that the fluorescence quantum yield of the fluorescing molecules of 3THPP in the cells is similar to that of fluorescing molecules of PII, which is suggested by our earlier work (Moan et al., 1988). @(3THPP, 3THPP) is the yield of degradation of 3THPP resulting from absorption of light by the fluorescent fraction of 3THPP. Correspondingly. @(3THPP. PII) is the yield of degradation of 3THPP resulting from light absorption by thc tluorcsccnt fraction of PI1 in the cells. @(PII, PII) and @(PII, 3THPP) are defined analoguously. The yields arc given in relative values. "0.03 is the upper limit of @(PII, 3THPP) since the maximal error in the relative valucs of k (thc rate constants for photodegradation of the dyes as estimated by the fluorescence experiments) is 30% in ihc present data as estimated from two parallel experimental series.
value T,, = 0.04 ps water will account for only 1% of the quenching of '02in a cell and no D 2 0 effect can be expected. This is consistent with experiments indicating that T A < 0.6 ps in cells (Firey et al., 1988). Thus, the time resolution of experiments intended to determine in cells directly would have to be improved by more than a factor of 10 from what can be achieved at present. It should be noted that detection of the 1272 nm lo2phosphorescence is probably the only way to prove that is generated in a cell, since scavenger experiments can always be questioned because of inhomogeneity of the dye- and scavenger molecules and reactions of most scavengers with other possible intermediates. The possibility that scavenger molecules and sensitizer molecules are present in different micro-compartments of the cells should always be considered, even in the case that both types of molecules have a similar lipophilicity. Since most efficient scavenger molecules are only weakly fluorescent, it is difficult to study their interacellular localization pattern. The use of two different lipophilic, fluorescent and photosensitizing dyes may give valuable information about the processes of photosensitization in cells as indicated by the present results. The conclusion that the main reactive intermediated diffuse of the order of 0.01-0.02 pm in the cells is in agreement with our observation
that photoexcitation of a porphyrin present at or outside the cell wall of an Escherichia cofi cell, whose thickness is approx. 0.03 pm, does not result in any photodamage to DNA in the bacteria (Boye and Moan, 1980) and that uroporphyrin, which produces with a high quantum yield (0.7-0.8) when photoexcited (Blum and Grossweiner, 1985) is inefficient in sensitizing cells when present in the medium outside the cells during light exposure (Nelson et al., 1986; Madslien, K., unpublished data). Acknowledgement-The authors want to express their thanks to professor Claude Rimington, F.R.S. for valuable comments and advice during the preparation of the manuscript. REFERENCES
Blum, A. and L. I. Grossweiner (1985) Singlet oxygen generation by hematoporphyrin IX.uroporphyrin I and hematoporphyrin derivative at 546 nm in phosphate buffer and in the presence of egg phosphatidylcholine liposomes. Phorochem. Photobiol. 41, 27-32. Cox, G. C., C. Bobillier and D. G. Whitten (1982) Photooxidation and singlet oxygen sensitization by protoporphyrin IX and its photooxidation products. Phorochem, Phorobiol. 36, 401-407. Dougherty, T. J. (1987) Photosensitizers: Therapy and detection of malignant tumors. Photochem. Photobiol. 45, 879-889.
Research Note Fiery, P. A , , T. W. Jones, G. Jori and M. A. J. Rodgers (1988) Photoexcitation of zinc phthalocyanine in mouse myeloma cells: the observation of triplet states but not of singlet oxygen. Photochem. Photobiol. 48, 357-360. Krieg, M. and D. Whitten (1984) Self-sensitized photooxidation of protoporphyrin IX and related porphyrins in erythrocyte ghosts and microemulsions: A novel photo-oxidation pathway involving singlet oxygen. J . Photochem. 25, 235-252. Mang, T. S., T. J. Dougherty, W. R. Potter, D. G. Boyle, S. Sommer and J. Moan (1987) Photobleaching of porphyrins used in photodynamic therapy and implications for therapy. Photochem. Photobiol. 45, 501-506. Mang, T. S., B. D. Wilson and S. Kahn (1990) An evaluation of the role of photobleaching concepts of Photofrin I1 in photodynamic therapy to human clinical trials. Photochem. Photobiol. 51s. 72s. Moan, J. (1986) Effect of bleaching of porphyrin sensitizers during photodynamic therapy. Cancer Lett. 33, 45-53.
Moan, J., K. Berg, E. Kvam, A. Western, Z. Malik, A. Ruck and H. Schnekenburger, (1989). Intracellular localization of photosensitizers. In Photosensitizing Compounds: Their Chemistry, Biology and Clinical Use. (Edited by S . Hernet), pp. 95-111. Ciba Foundation Symp. No. 146. Wiley, New York. Moan, J. and E. Boye (1981) Photodynamic effect on DNA and cell survival of human cells sensitized by hematoporphyrin. Photobiochem. Photobiophys. 2 , 301-307.
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Moan, J., T. Christensen and S. Sommer (1982) The main photosensitizing components of hematoporphyrin derivative. Cancer Lett. 15, 161-166. Moan, J., 0.Peng, J. F. Evensen, K. Berg, A. Western and C. Rimington (1987) Photosensitizing efficiencies, tumor- and cellular uptake of different photosensitizing drugs relevant for photodynamic therapy of cancer. Photochem. Phorobiol. 46, 713-721. Moan, J., C. Rimington and Z. Malik (1988) Photoinduced degradation and modification of Photofrin I1 in cells in vitro. Photochem. Photobiol. 47, 363-367. Moan, J. and S . Sommer (1983) Uptake of the components of hematoporphyrin derivative by cells and tumors. Cancer Lett. 21, 167-174. Moan, J. and S . Sommer (1984) Action spectra for hematoporphyrin derivative and photofrin I1 with respect to sensitization of cells in vitro to photoinactivation. Photochem. Photobiol. 40,631-634. Nelson, J. S., C. H. C. Sun and M. W. Berm (1988) Study of the in vivo and in virro photosensitizing capabilities of Uroporphyrin I compared to Photofrin 11. Lasers Surg. Med. 6 , 131-136. Western, A. and J. Moan (1988) Action spectra for photoinactivation of cells in the presence of tetra (3hydrooyphenyl) porphyrin, chlorine e,, and aluminium phthalocyanine tetrasulfonate. In Light in Biology and Medicine (Edited by R. H. Douglas, J. Moan and F. Dall'Acqua), vol. 1, pp. 5689. Plenum, New York. White, A. P. Handler, E. L. Smith, R. L. Hill and 1. R. Lehman (1978). Principles of Biochemistry, p 301. McGraw-Hill Kogakusha.
0031-8655/91 $03.00 +O.W Copyright @ 1991 Pergamon Prcss plc
RESEARCH NOTE
THE PHOTODEGRADATION OF PORPHYRINS IN CELLS CAN BE USED TO ESTIMATE THE LIFETIME OF SINGLET OXYGEN JOHAN MOAN*and KRISTIAN BERG Institute for Cancer Research, 0310 Montebello, Oslo 3, Norway (Received 25 June 1990; accepted 3 October 1990)
Abstract-NHIK 3025 cells were incubated with Photofrin I1 (PII) and/or tetra (3hydroxypheny1)porphyrin (3THPP) and exposed to light at either 400 or 420 nm, i.e. at the wavelengths of the maxima of the fluorescence excitation spectra of the two dyes. The kinetics of the photodegradation of the dyes were studied. When present separately in the cells the two dyes are photodegraded with a similar quantum yield. 3THPP is degraded 2-6 times more efficiently by light quanta absorbed by the fluorescent fraction of 3THPP than by light quanta absorbed by the fluorescent fraction of PI1 present in the same cells. The distance diffused by the reactive intermediate, supposedly causing the photodegradation was estimated to be on the order of 0.01-0.02 pm, which mainly lo2, corresponds to a lifetime of 0.01-0.04 ps of the intermediate in the cells. PI1 has binding sites at proteins in the cells as shown by an energy transfer band in the fluorescence excitation spectrum at 290 nm. During light exposure this band decays faster than the Soret band of PI1 under the present conditions. Photoproducts (lo2etc.) generated at one binding site contribute significantly in the destruction of remote binding sites.
INTRODUCTION
Porphyrins are degraded or modified by light (Cox et af.,1982; Krieg and Whitten, 1984). Considerable interest has been focused on such photodegradation (often called photobleaching), since it can be taken advantage of in photodynamic therapy of cancer (Moan, 1986; Dougherty, 1987, Mang et af.,1987). In fact, the concept of photobleaching has been successfully applied in human clinical trials (Mang et af., 1990). The mechanisms of photodegradation are different for porphyrins in solution and for porphyrins in biological systems (Krieg and Whitten, 1984). In the latter case the rate constants are significantly larger and different products are formed. To a first approximation the photodegradation follows first order kinetics and the rate constants are independent of the initial porphyrin concentration, both in cells and in tissues (Mang er af., 1987). This indicates that an excited porphyrin molecule is degraded either by products formed by itself or by direct interaction with its surrounding. However, this is a crude approximation since the kinetics deviate from first order after extended (but still clinically relevant) exposure times. Furthermore, the rate constants for degradation of Photofrin I1 (PII)? are *To whom correspondence should be addressed. tAbbreviations: HPLC, high performance liquid chroma-
tography; MEM, minimal essential medium, NCS, newborn calf serum, PII, Photofrin 11; PBS, Dulbeccos phosphate buffered saline; 3THPP, tetra (3hydroxyphenyl) porphyrin. PAP 53:4-I
larger when cells are exposed t o light in a D20buffer than when they are exposed in a H 2 0 buffer, indicating that '02-production plays a role (Moan et af., 1988). In order to elucidate the photobleaching mechanisms further we have incubated cells with two different porphyrins, Photofrin I1 (PII) and tetra (3hydroxylphenyl) porphyrin (3THPP). which have different fluorescence excitation and emission spectra. Thus, even when they are present in the same cell they can be excited and monitored with some selectivity. Both are negatively charged lipophilic dyes which, according to fluorescence microscopic studies (data not shown), localize almost identically in the cells, presumably in membrane structures. In such a system it is possible to study the degradation of one dye caused by excitation of the other dye, and to estimate the distance travelled by reactive intermediates before deactivation. MATERIALS AND METHODS
Chemicals. Photofrin I1 was obtained from Photomedica, Raritan, NJ. The drug was stored frozen in small vials for up to 8 months before being used in the present experiments. It was thoroughly checked by HPLC, and by cell survival experiments that the drug did not change in its characteristics during the time of storage. The procedures of HPLC and cell survival experiments are described elsewhere (Moan et a / . , 1982; Moan and Sommer, 1983). The 3THPP was obtained from Porphyrin Products, Logan, UT. Stock solutions (0.1 mg/mL) were prepared in 0.05 M NaOH as earlier described (Moan et al., 1987). Cell cultivation. Cells of the line NHIK 3025 (derived
549
550
JOHANMOANand KRlsnAN BERG
from a human carcinoma in situ) were cultivated in MEM containing 10% NCS. For fluorescence experiments 1W cells were incubated in 25 cm2 Falcon tissue culture dishes. Six hours later the medium was changed to MEM with 3% NCS with either 25 pg PII/mL and/or 1 pg 3THPPhL. These concentrations were chosen on the basis of pilot experiments, in view of optimal analysis of the fluorescence spectra of samples containing both drugs. At these concentrations the photosensitivity and the fluorescence yield were about a factor 2 larger for cells with PI1 than for cells with 3THPP, when exposed to equal fluence rates at the optimal wavelengths (400 nm for PI1 and 420 nm for 3THPP). After an incubation period of 18 h with the dyes the cells were washed five times in icecold PBS, brought into suspension by trypsinization, washed once more with PBS and finally suspended in PBS at a concentration of approx. loh cellshl-as determined by Biirker chamber counting. Irradiation of the samples. The light source was a 900 W Osram high pressure xenon lamp fitted to a Bausch & Lomb grating monochromator. The bandwidth of the light was 15 nm as measured with a second monochromator (Jarrel Ash) with narrow slits (Ah = 1 nm) and a UDT 1la detector (United Detector Technology, Santa Monica, CA). This detector is calibrated and was also used to determine the fluence rates at the position of the cells: 30 mW/cm2 at both wavelengths (400 and 420 nm). The samples were irradiated in a fluorescence cuvette (3 x 3 x 20 mm) and were gently stirred during the light exposure. Fluorescence measurements. Fluorescence spectra were recorded by means of a Perkin-Elmer LS 5 spectrofluorimeter equipped with a Hamamatsu R928 red sensitive photomultiplier tube. A 580 nm cut-off filter was used to remove scattered light from the light entering the emission slit, which was usually set to give AX = 5 nm. The distortion of the spectra resulting from such slit widths were taken into consideration in the evaluation of the data but found to be of minor importance. Using a microcuvette with a cross section of only 3 x 3 mm, inner filter effects played no significant role.
ml
Loo
m
Wavelength hm)
RESULTS AND DISCUSSION
Figure 1. Fluorescence excitation spectra of PI1 and 3THPP in NHIK 3025 cells 18 h incubation in MEM with 3% NCS containing 25 pm PII/mL and/or 2 pg/mL 3THPP. The dotted line in the spectrum of PI1 shows the spectrum in methanol in the region 250-329 nm.
We have earlier shown that the fluorescence quantum yields of PI1 and 3THPP in NHIK 3025 cells are similar to within 30% (Moan et al., 1987). Therefore, and since it is difficult to record the absorption spectra of dyes in cells with accuracy, we decided to base our calculations on the fluorescence measurements. In fact, it is more relevant to use fluorescence spectra than absorption spectra for investigations of this type, since the aggregated fraction of the dyes in the cells is both non-fluorescent and photochemically inactive. This has been shown by means of absorption, fluorescence and action spectroscopy for both dyes considered in the present work (Western and Moan, 1988; Moan and Sommer, 1984). Figure 1 shows the fluorescence excitation spectra of cells with PII, 3THPP and with the mixture of the two dyes. There is a peak in the spectra at about 290 nm, which, in the case of PII, is entirely due to energy transfer from proteins containing aromatic amino acids to porphyrin molecules (Moan et al., 1988). Thus, using the known shape of the excitation spectra of PI1 in liposomes or lipids with properties
comparable to those of cell membranes but without proteins, it is possible to separate the energy transfer peak from the direct excitation of PI1 as indicated by the dotted line on the spectrum for PI1 (Fig. 1, Moan et al., 1988). In the case of 3THPP such a separation was not attempted, since this dye has a peak in its own excitation spectrum in this wavelength region due to the phenyl rings. One can easily separate the spectra of samples containing both dyes (Fig. l ) , both manually and by means of a simple computer program. To a first approximation and for exposure times shorter than 6 min, the decay kinetics of the Soret band is of first order for both dyes (Figs. 2 and 3). The same is true for the energy transfer band at 290 nm (Fig. 2). However, the rate constant for the decay of the energy transfer band is larger than that for the decay of the Soret band. Thus, the decay of the energy transfer band is due to at least two factors: the photodegradation of PI1 and the destruction of binding sites at proteins. Since the rate constant for the decay of the energy transfer
Research Note
Figure 2. The decay of the Soret band of PI1 in NHIK 3025 cells monitored with the excitation and emission wavelengths set at 400 and 625 nm, respectively; and of the energy transfer band (Acxc = 290 nm, Acm = 625 nm), the contribution from direct excitation being subtracted. The wavelength of the exposure light was 400 nm.
55 1
6 min) as indicated by the regression lines drawn in Fig. 3. Table 1 shows that the quantum yield of photodegradation of PI1 by light quanta absorbed by PI1 itself is comparable to the corresponding yield for 3THPP or slightly lower. However, the relative value of the quantum yield of photodegradation of 3THPP caused by quanta absorbed by 3THPP is 3-6 times larger than the relative value for photodegradation of the dye caused by quanta absorbed by PII. (Only the fluorescent and photosensitizing fraction of the dyes are considered). Similarly, the relative value of the quantum yield of photodegradation of PI1 caused by light absorption in PI1 is more than twice as large as the relative value for photodegradation caused by light absorption in 3THPP. This is in agreement with our earlier conclusion that the photodegradation of porphyrins in cells is predominantly a first order process (Mang et al., 1987). Therefore, the present results indicate that a photoproduct, like lo2,generated by the absorption of a quantum of light in a porphyrin molecule causes damage mainly to that molecule and not to other porphyrin molecules in the vicinity. The average intracellular concentrations of 3THPP can be estimated to 40 p M under the present conditions, and that of PI1 can be estimated to 80 p M of porphyrin rings (mol. wt 600) (Moan ef al., 1987). In these estimations it is assumed that the amount of dye in the nucleus is negligible (Moan er al., 1989). The nucleus constitutes about 30% of the volume of these cells (Moan and Boye, 1981). Thus, spheres of radius 0.02 pm located randomly in the cytoplasm contain on the average 1 fluorescent molecule of 3THPP. It is, of course, a very crude approximation that the dye molecules are homogeneously distributed in the cytoplasm, but it may serve in a first approximative calculation of the distance travelled by the reactive intermediates generated by the photoexcitation of the dye molecules. If we assume that the membranes constitute about 10% of the cytoplasmic volume (White et al., 1978) and that the present lipophilic dyes are localized mainly in membrane structures, their local concentrations are a factor of 10 larger than estimated above and the radius of a sphere containing on average 1 fluorescent molecule of 3THPP is about 0.01 pm. Singlet oxygen (lo2) is almost certainly a reactive intermediate generated under the present conditions (Moan et al., 1987). The lifetime rA of lo2under the present conditions can be estimated by use of the formula 6 = ( 6 0 T~)”’, where 6 is the distance diffused by lo2 before it is quenched and D is the diffusion coefficient of ‘ 0 2 . If we assume that D = 1.4 x cm2 s-l (Moan and Boye, 1981) and 6 is within the range 0.01-0.02 p , rA is within the range 0.01-0.04 ps. Such a short lifetime is consistent with the lack of a D 2 0 effect in many photosensitized processes in which lo2is very likely to be involved. Using the
-
band is dependent on the concentration of PI1 in the cells, the binding sites at proteins can be degraded by photoproducts generated remotely from these binding sites (Moan ef al., 1988). From Fig. 3 rate constants for the first part of the decay of the Soret band of the dyes can be determined. In the calculations we have taken into account only the two first exposure times (i.e. 3 and
Figure 3. Decay curves for the Soret bands of PI1 and 3THPP separately or simultaneouslypresent in NHIK 3025 cells exposed to light at either 400 or 420 nm. Incubation conditions, see legend of Fig. 1.
JOHAN MOANand KRISTIAN BERG
552
Table l(a). Photodegradation of 3THPP ~
Concentrations WmL)
3THPP 1 1 1 1
PI1 0 0 25 25
~~~~~~
hCxp
(nm)
A(3THPP)
A(P1I)
k(min-')
400 420 400 420
0.2 1.0 0.2 I .0
0 0 2 2
0.014 0.10 0.07 0. 14 ~
@(3THPP33THPP) Q(3THPP.PII)
0.07 0.10 0.022 0.016
~~~~~
Table l(b). Photodegradation of PI1 Concentrations ()lLg/mL) PI1 25 25 25 25
3THPP 0 0 1 1
Lip (nm) 400
420 400 420
A(PI1)
A(3THPP)
2.0 2.0 2.0 2.0
0
n
0.2 1 .0 ~~
k(min-')
0. 13 0.11 0.13 0.11 ~~~
@(PII,PII)
(D(P11.3THPP)
0.M5 0.055
< 0.03' ~~
Aexp is the wavelength of the photodegrading light. A(PI1) and A(3THPP) are relative numbers of light quanta absorbed by fluorescing and photoactive molecules of PI1 and 3THPP in the samples, approximated in relative values by the convolution integrals of the fluorescence excitation spectra and the spectra of the photodegrading light at 400 and 420 nm rcspcctively, with AA = 15 nm. In this approximation it is assumed that the fluorescence quantum yield of the fluorescing molecules of 3THPP in the cells is similar to that of fluorescing molecules of PII, which is suggested by our earlier work (Moan et al., 1988). @(3THPP, 3THPP) is the yield of degradation of 3THPP resulting from absorption of light by the fluorescent fraction of 3THPP. Correspondingly. @(3THPP. PII) is the yield of degradation of 3THPP resulting from light absorption by thc tluorcsccnt fraction of PI1 in the cells. @(PII, PII) and @(PII, 3THPP) are defined analoguously. The yields arc given in relative values. "0.03 is the upper limit of @(PII, 3THPP) since the maximal error in the relative valucs of k (thc rate constants for photodegradation of the dyes as estimated by the fluorescence experiments) is 30% in ihc present data as estimated from two parallel experimental series.
value T,, = 0.04 ps water will account for only 1% of the quenching of '02in a cell and no D 2 0 effect can be expected. This is consistent with experiments indicating that T A < 0.6 ps in cells (Firey et al., 1988). Thus, the time resolution of experiments intended to determine in cells directly would have to be improved by more than a factor of 10 from what can be achieved at present. It should be noted that detection of the 1272 nm lo2phosphorescence is probably the only way to prove that is generated in a cell, since scavenger experiments can always be questioned because of inhomogeneity of the dye- and scavenger molecules and reactions of most scavengers with other possible intermediates. The possibility that scavenger molecules and sensitizer molecules are present in different micro-compartments of the cells should always be considered, even in the case that both types of molecules have a similar lipophilicity. Since most efficient scavenger molecules are only weakly fluorescent, it is difficult to study their interacellular localization pattern. The use of two different lipophilic, fluorescent and photosensitizing dyes may give valuable information about the processes of photosensitization in cells as indicated by the present results. The conclusion that the main reactive intermediated diffuse of the order of 0.01-0.02 pm in the cells is in agreement with our observation
that photoexcitation of a porphyrin present at or outside the cell wall of an Escherichia cofi cell, whose thickness is approx. 0.03 pm, does not result in any photodamage to DNA in the bacteria (Boye and Moan, 1980) and that uroporphyrin, which produces with a high quantum yield (0.7-0.8) when photoexcited (Blum and Grossweiner, 1985) is inefficient in sensitizing cells when present in the medium outside the cells during light exposure (Nelson et al., 1986; Madslien, K., unpublished data). Acknowledgement-The authors want to express their thanks to professor Claude Rimington, F.R.S. for valuable comments and advice during the preparation of the manuscript. REFERENCES
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