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Photohemolytic activity of lichen metabolites
37
J. Photochem. Photobiol. B: Biol., 21 (1993) 3740
Photohemolytic M.E. Hidalgo+,
activity of lichen metabolites
E. Fernfindez
and W. Quilhot
Chemistry and Pharmacy School, Faculty of Medicine, University of Valparatko, Vakamiko (Chile)
E.A. Lissi Chem&y
Department, Faculty of Science, University of Santiago of Chile, Santiago (Chile)
(Received November 5, 1992; accepted June 10, 1993)
Abstract Irradiation of pannarin, l’-chloropannarin and atranorin with 366 nm light leads to significant hemolysis in a red cell suspension. However, their mechanism of action is different. Hemolysis induced by pannarin and l’chloropannarin increases in the presence of oxygen, whereas hemolysis induced by atranorin is higher in nitrogenpurged solutions. The effect of free radical scavengers, and the lack of effect of Dr.0 in the medium, suggest that the hemolysis induced by pannarin and 1’-chloropannarin is not mediated by ‘O> Roth the hemolytic and photohemolytic activities of the depsidones, particularly 1’-chloropannarin, increase when the temperature increases from 21 to 37 “C.
Key wom!s: Photohemolytic
activity; Lichen metabolite;
1. Introduction
It has been reported that phenolic compounds present in lichens produce dermal alterations. In photosensitive subjects, lichens produce contact and photocontact dermatitis due to the presence of allergens, such as depsides, depsidones and usnic acid derivatives [l, 21. The hemolytic capacity of lichen metabolites, mediated by damage to the red cell membrane constituents, has been attributed to the production of singlet oxygen [3]. Protein damage and/or lipid peroxidation of the membrane constituents could contribute to the photodamage induced by the lichen compounds [4]. Photoirradiation of pannarin, l’-chloropannarin and atranorin with 366 nm light leads to their decomposition [5]. However, while the photodecomposition of pannarin and l’-chloropannarin is reduced by oxygen, suggesting competition between primary photoprocesses and oxygen quenching, significant photodecomposition of atranorin takes place only in the presence of oxygen [5]. These results suggest that the photochemistry of the chlorinated compounds could be very different from the photochemistry of compounds not bearing +Autbor to wbom correspondence
loll-1344/93/$6.00
should be addressed.
Irradiation;
Red cell suspension
chlorine atoms, e.g. atranorin. For the latter compound, the photohemolytic capacity has been related to the production of singlet oxygen [3]. This work aims to evaluate the photohemolytic capacity of chlorine-atom-bearing lichen compounds, and to compare their behavior with that of atranorin.
2. Materials
and methods
Deuterium oxide (Uvasol), anhydrous D( +)glucose for biochemical purposes, L( + )-sodium ascorbate, sodium chloride and sodium phosphates (Merck) were of the highest purity available and were employed as received. Atranorin, pannarin and 1’-chloropannarin were obtained and purified according to the reported procedure 16, 71. Red blood cells of healthy adult donors were used. Shortly after collection, the heparinized blood was centrifuged at 200% and the plasma and huffy coat discarded. The remaining red cells were washed three times with an isotonic solution (0.15 M NaCl on 0.01 M sodium phosphate (PBS), pH 7.4). The red cells were resuspended to approximately 2% v/v, kept at 6 “C and used in the next 72 h. 0 1993 - Elsevier Sequoia. All rights reserved
38
ME. Hidalgo et al. I Photohemolytic activity of lichen metabolites
The percentage of hemolysis was determined immediately after irradiation by measuring the hemoglobin liberated in the medium from solutions containing 0.4% red cells. The measurements were carried out spectrophotometrically after centrifugation at 2000g. Measurements were carried out at 540 and 630 nm using a Beckman 35 instrument, and the percentages of hemoglobin and methemoglobin were evaluated according to the procedures given by Khan and Fleischaker [8] and Trotta et al. [9] respectively. Photohemolysis was promoted by irradiation from a medium pressure mercury lamp (Hannovia) fitted with a glass filter to isolate 366 mn radiation. The E values of the compounds at this wavelength are 3250, 3820 and 3702 M-’ cm-’ for pannarin, l’-chloropannarin and atranorin respectively. The employed intensity corresponds to 21.3 J m-* s-l. The experiments were performed at 20 and 37 “C. The concentrations of the lichen compounds employed correspond to their solubility in PBS: 12.5 ,uM for atranorin and 7.8 PM for pannarin and 1 ‘-chloropannarin.
3. Experimental
results
The three compounds considered present negligible hemolytic capacity at 20 “C (data not shown). Incubation of the red cell suspension with pannarin produces only about 0.8% hemolysis after 180 min, and the extent of hemolysis is even smaller for the other compounds. Increasing the temperature to 37 “C produces a noticeable increase in hemolysis (Fig. 1). The effect is most important for l’chloropannarin, whose incorporation leads to 61% hemolysis after 55 min of incubation. No significant
0
0
40
80
120
160
hemolysis was observed in the absence of additives for incubation times up to 180 min. Figure 2 shows the extent of aerobic photohemolysis at 20 “C for pannarin and l’-chloropannarin. These data show a significant degree of hemolysis by both compounds. However, no (less than 2%) hemolysis was detected in the irradiation of atranorin under similar conditions. The different behavior of atranorin can be explained in terms of the different photodecomposition mechanisms reported for chlorinated and non-chlorinated lichen metabolites [5]. Increasing the temperature increases the degree of hemolysis, particularly for l’-chloropannarin, whose irradiation at 37 “C leads to almost quantitative lysis after 45 min (Fig. 2). Conversely, only 41% hemolysis is observed after 180 min of irradiation in the presence of atranorin at 37 “C. The photohemolysis induced by pannarin and l’-chloropannarin at 20 “C is promoted by oxygen. Removing the oxygen by nitrogen bubbling reduces the photohemolysis percentages measured after 180 min to less than 5%. In order to evaluate if the process is mediated by singlet oxygen, experiments were carried out employing PBS prepared in D20. The results obtained (data not shown) were identical to those observed employing PBS prepared in H,O. Although interpretation of this type of experiment in biological samples is not straightforward due to the short lifetime of the intracellularly generated singlet oxygen [lo], the results favor a mechanism which does not involve singlet oxygen as the main damaging agent. This conclusion is further supported by the results 100
10
2
Time (min)
Fig. 1. Dark hemolysis induced by incubation at 37 “C: 0, l’chloropannarin (7.8 PM); f, pannarin (7.8 PM); 0, atranorin (12.5 /AM).
Time (mid
Fig. 2. Photohemolysis as a function of irradiation time: 0, l’chloropannarin, 37 “C; +, I’-chloropannarin, 21 “C, 0, pannarin, 21 “C; A, pannarin, 37 “C.
ME. Hidalgo et al. I Photohemolyric activiv of lichen metabolites TABLE 1. Percentage of irradiation
of photohemolysis
inhibition after 180 min
Additive
Photosensitizer
Inhibition W)
1’-Chloropannarin Pannarin
Glucose (5.5 mM) Ascorbate (0.5 mM) Glucose (5.5 mM) Ascorbate (0.5 mh4)
40
80
99 93 73 60
M
120
160
i
Timetmin)
Fig. 3. Percentage of methemoglobin production as a function of irradiation time in the presence of l’-chloropannarin (0) (scale right) or pannarin (0) (scale left).
obtained in the presence of additives that can reduce the damage induced by free radicals. The data in Table 1 show that the addition of glucose or ascorbate protects the red cell from photohemolysis induced by pannarin and l’-chloropannarin. Photohemolysis is accompanied by methemoglobin production, and the time profiles of both processes are similar (Figs. 2 and 3). At long irradiation times (180 min), l’-chloropannarin produced more methemoglobin than pannarin in aerated solutions. Furthermore, in similar conditions, atranorin, which does not produce significant photohemolysis, does not lead to measurable hemoglobin oxidation. However, in anaerobic conditions, only atranorin produces significant (8 f 1%) methemoglobin.
39
does it take place almost exclusively at a single level [ll]. The results obtained in this work indicate that both pannarin and l’chloropannarin have significant hemolytic capacity at 37 “C, and that this effect is notably increased by irradiation. An oxygen-dependent photohemolytic capacity of pannarin and l’-chloropannarin is also observed at lower (21 “C) temperatures where dark hemolysis is negligible. The time profile of the hemolysis is compatible with that expected for a colloid osmotic photohemolysis [4]. The lack of a deuterium oxide effect and the protective effect of ascorbic acid and glucose indicate that the damage is mediated by oxygenated radicals and not by singlet oxygen. This conclusion is contrary to that reached for the photohemolytic mechanism of other lichen compounds [3]. The free-radical-mediated toxicity of pannarin and l’-chloropannarin may be due to a bimolecular process between the excited molecules and red cell components (a type I photo-oxidative process) and/or to secondary reactions of radicals produced in unimolecular processes of the excited molecules. However, it should be noted that the effect of oxygen observed in this work is contrary to that reported for the photoconsumption of the parent compounds [5], since the quantum yield of the latter process decreases in the presence of oxygen, most probably due to competitive triplet quenching. In order to explain the present results, it can be assumed that the primary photoprocess decreases in the presence of oxygen, but this is necessary to lead to significant photodamage, probably produced by radicals and/or chain reactions, such as that involved in lipid peroxidation. In addition, it has been shown that the damage to proteins mediated by free radicals notably increases in the presence of oxygen [ 121. We cannot at present offer a mechanistic interpretation of the strong effect of temperature on the hemolytic and photohemolytic activity observed for the depsidones considered. However, strong increases in hemolytic rates with temperature (including physiological) are not unusual [13].
4. Discussion The red cell is a very simple system for the evaluation of the toxic potential of xenobiotics, mostly due to its reduced enzymatic activity and the presence of only one (the cytosolic) membrane. However, the toxicological action can take place at the level of lipids (lipid peroxidation), membrane proteins and/or hemoglobin, and only exceptionally
Acknowledgments
This work was supported by a grant from FONDECYT, proyecto 92-426 and DICYT Universidad de Valparaiso, proyecto UV 12/91.
40
M.E. Hidalgo et al. 1 Photohemolytic activity of lichen metabolites
References 1 P. Thune, Contact allergy due to lichens in patients with a history of photosensitivity, Contact Dermatitk, 3 (1977) 267-272. 2 P. Thune, Y. Solberg, N. McFadden, F. Staerfelt and M. Sandberg, Perfume allergy due to oak moss and other lichens, Contact DermaUk, 8 (1982) 396-400. 3 G. Wennersten, Photodynamic reactions induced by compounds derived from lichens, Acta Derm-Venereol., 59 (1979) 197-200. 4 J.P. Pooler, The kinetics of colloid osmotic hemolysis. II. Photohemolysis, Biochim. Biophys. Acta, 812 (1985) 199-205. 5 M.E. Hidalgo, E. Fernandez, W. Quilhot and E. Lissi, Solubilization and photophysical and photochemical behaviour of depsides and depsidones in water and Brij-35 solutions at different pHs, J. Photochem. Photobiol. A: Chem., 67 (1992) 245-254. 6 W. Quilhot, B. Didyk, V. Gambaro and J.A. Garbarino, Studies on Chilean Lichens. VI. Depsidones from Erioderma Chilense, J. Nat. Prod., 46 (1983) 942-943. 7 M. Piovano, M.J. Garrido, V. Gambaro, J.A. Garbarino and W. Quilhot, Studies on Chilean Lichens. VIII. Depsidones from Psoroma species, J. Nat Prod., 48 (1985) 854-855.
8 G. Khan and B.I. Fleischaker, Red blood cell hemolysis by photosensitizing compounds, J. Invest. Dermatol., 50 (1971) 85-90. 9 R. Trotta, S.G. Sullivan and A. Stem, Lipid peroxidation and haemoglobin degradation in red blood cells exposed to I-butyl hydroperoxide, Biochem. J., 212 (1983) 759-772. 10 A. Baker and J.R. Kanofsky, Of singlet oxygen by biomolecules from L1210 leukemia cells, Photochem. PhorobioZ., 55 (1992) 523-528; D.P. Valenzeno, Photomodification of biological membranes with emphasis on singlet oxygen mechanisms, Phorochem. Photobiol., 46 (1987) 147-160; E.A. Lissi, M.V. Encinas, E. Lemp and M.A. Rubio, Singlet oxygen biomolecular processes. Solvent and compartmentalization effects, Chem. Rev., 93 (1993) 699-723. 11 D.E. Moore, Photosensitization by drugs, J. Pharm. Sci., 66 (1977) 1282-1284. 12 E.A. Lissi and N. Clavero, Inactivation of lysozyme by alkylpyroxyl radicals, Free Radical Res. Commun., 10 (1990) 177-184. 13 LB. Zavodnik, T.P. Piletskaia and 1.1. Stepuro, Influence of temperature on the lysis of human erythrocytes by palmitic acid, Biophysics, 36 (1991) 1064-1068; I.V. Yamaikina and Y.A. Chernitskii, Thermohemolysis of erythrocytes in the temperature range including physiologic (autohemolysis), Biophysics, 36 (6) (1991) 1059-1063.
J. Photochem. Photobiol. B: Biol., 21 (1993) 3740
Photohemolytic M.E. Hidalgo+,
activity of lichen metabolites
E. Fernfindez
and W. Quilhot
Chemistry and Pharmacy School, Faculty of Medicine, University of Valparatko, Vakamiko (Chile)
E.A. Lissi Chem&y
Department, Faculty of Science, University of Santiago of Chile, Santiago (Chile)
(Received November 5, 1992; accepted June 10, 1993)
Abstract Irradiation of pannarin, l’-chloropannarin and atranorin with 366 nm light leads to significant hemolysis in a red cell suspension. However, their mechanism of action is different. Hemolysis induced by pannarin and l’chloropannarin increases in the presence of oxygen, whereas hemolysis induced by atranorin is higher in nitrogenpurged solutions. The effect of free radical scavengers, and the lack of effect of Dr.0 in the medium, suggest that the hemolysis induced by pannarin and 1’-chloropannarin is not mediated by ‘O> Roth the hemolytic and photohemolytic activities of the depsidones, particularly 1’-chloropannarin, increase when the temperature increases from 21 to 37 “C.
Key wom!s: Photohemolytic
activity; Lichen metabolite;
1. Introduction
It has been reported that phenolic compounds present in lichens produce dermal alterations. In photosensitive subjects, lichens produce contact and photocontact dermatitis due to the presence of allergens, such as depsides, depsidones and usnic acid derivatives [l, 21. The hemolytic capacity of lichen metabolites, mediated by damage to the red cell membrane constituents, has been attributed to the production of singlet oxygen [3]. Protein damage and/or lipid peroxidation of the membrane constituents could contribute to the photodamage induced by the lichen compounds [4]. Photoirradiation of pannarin, l’-chloropannarin and atranorin with 366 nm light leads to their decomposition [5]. However, while the photodecomposition of pannarin and l’-chloropannarin is reduced by oxygen, suggesting competition between primary photoprocesses and oxygen quenching, significant photodecomposition of atranorin takes place only in the presence of oxygen [5]. These results suggest that the photochemistry of the chlorinated compounds could be very different from the photochemistry of compounds not bearing +Autbor to wbom correspondence
loll-1344/93/$6.00
should be addressed.
Irradiation;
Red cell suspension
chlorine atoms, e.g. atranorin. For the latter compound, the photohemolytic capacity has been related to the production of singlet oxygen [3]. This work aims to evaluate the photohemolytic capacity of chlorine-atom-bearing lichen compounds, and to compare their behavior with that of atranorin.
2. Materials
and methods
Deuterium oxide (Uvasol), anhydrous D( +)glucose for biochemical purposes, L( + )-sodium ascorbate, sodium chloride and sodium phosphates (Merck) were of the highest purity available and were employed as received. Atranorin, pannarin and 1’-chloropannarin were obtained and purified according to the reported procedure 16, 71. Red blood cells of healthy adult donors were used. Shortly after collection, the heparinized blood was centrifuged at 200% and the plasma and huffy coat discarded. The remaining red cells were washed three times with an isotonic solution (0.15 M NaCl on 0.01 M sodium phosphate (PBS), pH 7.4). The red cells were resuspended to approximately 2% v/v, kept at 6 “C and used in the next 72 h. 0 1993 - Elsevier Sequoia. All rights reserved
38
ME. Hidalgo et al. I Photohemolytic activity of lichen metabolites
The percentage of hemolysis was determined immediately after irradiation by measuring the hemoglobin liberated in the medium from solutions containing 0.4% red cells. The measurements were carried out spectrophotometrically after centrifugation at 2000g. Measurements were carried out at 540 and 630 nm using a Beckman 35 instrument, and the percentages of hemoglobin and methemoglobin were evaluated according to the procedures given by Khan and Fleischaker [8] and Trotta et al. [9] respectively. Photohemolysis was promoted by irradiation from a medium pressure mercury lamp (Hannovia) fitted with a glass filter to isolate 366 mn radiation. The E values of the compounds at this wavelength are 3250, 3820 and 3702 M-’ cm-’ for pannarin, l’-chloropannarin and atranorin respectively. The employed intensity corresponds to 21.3 J m-* s-l. The experiments were performed at 20 and 37 “C. The concentrations of the lichen compounds employed correspond to their solubility in PBS: 12.5 ,uM for atranorin and 7.8 PM for pannarin and 1 ‘-chloropannarin.
3. Experimental
results
The three compounds considered present negligible hemolytic capacity at 20 “C (data not shown). Incubation of the red cell suspension with pannarin produces only about 0.8% hemolysis after 180 min, and the extent of hemolysis is even smaller for the other compounds. Increasing the temperature to 37 “C produces a noticeable increase in hemolysis (Fig. 1). The effect is most important for l’chloropannarin, whose incorporation leads to 61% hemolysis after 55 min of incubation. No significant
0
0
40
80
120
160
hemolysis was observed in the absence of additives for incubation times up to 180 min. Figure 2 shows the extent of aerobic photohemolysis at 20 “C for pannarin and l’-chloropannarin. These data show a significant degree of hemolysis by both compounds. However, no (less than 2%) hemolysis was detected in the irradiation of atranorin under similar conditions. The different behavior of atranorin can be explained in terms of the different photodecomposition mechanisms reported for chlorinated and non-chlorinated lichen metabolites [5]. Increasing the temperature increases the degree of hemolysis, particularly for l’-chloropannarin, whose irradiation at 37 “C leads to almost quantitative lysis after 45 min (Fig. 2). Conversely, only 41% hemolysis is observed after 180 min of irradiation in the presence of atranorin at 37 “C. The photohemolysis induced by pannarin and l’-chloropannarin at 20 “C is promoted by oxygen. Removing the oxygen by nitrogen bubbling reduces the photohemolysis percentages measured after 180 min to less than 5%. In order to evaluate if the process is mediated by singlet oxygen, experiments were carried out employing PBS prepared in D20. The results obtained (data not shown) were identical to those observed employing PBS prepared in H,O. Although interpretation of this type of experiment in biological samples is not straightforward due to the short lifetime of the intracellularly generated singlet oxygen [lo], the results favor a mechanism which does not involve singlet oxygen as the main damaging agent. This conclusion is further supported by the results 100
10
2
Time (min)
Fig. 1. Dark hemolysis induced by incubation at 37 “C: 0, l’chloropannarin (7.8 PM); f, pannarin (7.8 PM); 0, atranorin (12.5 /AM).
Time (mid
Fig. 2. Photohemolysis as a function of irradiation time: 0, l’chloropannarin, 37 “C; +, I’-chloropannarin, 21 “C, 0, pannarin, 21 “C; A, pannarin, 37 “C.
ME. Hidalgo et al. I Photohemolyric activiv of lichen metabolites TABLE 1. Percentage of irradiation
of photohemolysis
inhibition after 180 min
Additive
Photosensitizer
Inhibition W)
1’-Chloropannarin Pannarin
Glucose (5.5 mM) Ascorbate (0.5 mM) Glucose (5.5 mM) Ascorbate (0.5 mh4)
40
80
99 93 73 60
M
120
160
i
Timetmin)
Fig. 3. Percentage of methemoglobin production as a function of irradiation time in the presence of l’-chloropannarin (0) (scale right) or pannarin (0) (scale left).
obtained in the presence of additives that can reduce the damage induced by free radicals. The data in Table 1 show that the addition of glucose or ascorbate protects the red cell from photohemolysis induced by pannarin and l’-chloropannarin. Photohemolysis is accompanied by methemoglobin production, and the time profiles of both processes are similar (Figs. 2 and 3). At long irradiation times (180 min), l’-chloropannarin produced more methemoglobin than pannarin in aerated solutions. Furthermore, in similar conditions, atranorin, which does not produce significant photohemolysis, does not lead to measurable hemoglobin oxidation. However, in anaerobic conditions, only atranorin produces significant (8 f 1%) methemoglobin.
39
does it take place almost exclusively at a single level [ll]. The results obtained in this work indicate that both pannarin and l’chloropannarin have significant hemolytic capacity at 37 “C, and that this effect is notably increased by irradiation. An oxygen-dependent photohemolytic capacity of pannarin and l’-chloropannarin is also observed at lower (21 “C) temperatures where dark hemolysis is negligible. The time profile of the hemolysis is compatible with that expected for a colloid osmotic photohemolysis [4]. The lack of a deuterium oxide effect and the protective effect of ascorbic acid and glucose indicate that the damage is mediated by oxygenated radicals and not by singlet oxygen. This conclusion is contrary to that reached for the photohemolytic mechanism of other lichen compounds [3]. The free-radical-mediated toxicity of pannarin and l’-chloropannarin may be due to a bimolecular process between the excited molecules and red cell components (a type I photo-oxidative process) and/or to secondary reactions of radicals produced in unimolecular processes of the excited molecules. However, it should be noted that the effect of oxygen observed in this work is contrary to that reported for the photoconsumption of the parent compounds [5], since the quantum yield of the latter process decreases in the presence of oxygen, most probably due to competitive triplet quenching. In order to explain the present results, it can be assumed that the primary photoprocess decreases in the presence of oxygen, but this is necessary to lead to significant photodamage, probably produced by radicals and/or chain reactions, such as that involved in lipid peroxidation. In addition, it has been shown that the damage to proteins mediated by free radicals notably increases in the presence of oxygen [ 121. We cannot at present offer a mechanistic interpretation of the strong effect of temperature on the hemolytic and photohemolytic activity observed for the depsidones considered. However, strong increases in hemolytic rates with temperature (including physiological) are not unusual [13].
4. Discussion The red cell is a very simple system for the evaluation of the toxic potential of xenobiotics, mostly due to its reduced enzymatic activity and the presence of only one (the cytosolic) membrane. However, the toxicological action can take place at the level of lipids (lipid peroxidation), membrane proteins and/or hemoglobin, and only exceptionally
Acknowledgments
This work was supported by a grant from FONDECYT, proyecto 92-426 and DICYT Universidad de Valparaiso, proyecto UV 12/91.
40
M.E. Hidalgo et al. 1 Photohemolytic activity of lichen metabolites
References 1 P. Thune, Contact allergy due to lichens in patients with a history of photosensitivity, Contact Dermatitk, 3 (1977) 267-272. 2 P. Thune, Y. Solberg, N. McFadden, F. Staerfelt and M. Sandberg, Perfume allergy due to oak moss and other lichens, Contact DermaUk, 8 (1982) 396-400. 3 G. Wennersten, Photodynamic reactions induced by compounds derived from lichens, Acta Derm-Venereol., 59 (1979) 197-200. 4 J.P. Pooler, The kinetics of colloid osmotic hemolysis. II. Photohemolysis, Biochim. Biophys. Acta, 812 (1985) 199-205. 5 M.E. Hidalgo, E. Fernandez, W. Quilhot and E. Lissi, Solubilization and photophysical and photochemical behaviour of depsides and depsidones in water and Brij-35 solutions at different pHs, J. Photochem. Photobiol. A: Chem., 67 (1992) 245-254. 6 W. Quilhot, B. Didyk, V. Gambaro and J.A. Garbarino, Studies on Chilean Lichens. VI. Depsidones from Erioderma Chilense, J. Nat. Prod., 46 (1983) 942-943. 7 M. Piovano, M.J. Garrido, V. Gambaro, J.A. Garbarino and W. Quilhot, Studies on Chilean Lichens. VIII. Depsidones from Psoroma species, J. Nat Prod., 48 (1985) 854-855.
8 G. Khan and B.I. Fleischaker, Red blood cell hemolysis by photosensitizing compounds, J. Invest. Dermatol., 50 (1971) 85-90. 9 R. Trotta, S.G. Sullivan and A. Stem, Lipid peroxidation and haemoglobin degradation in red blood cells exposed to I-butyl hydroperoxide, Biochem. J., 212 (1983) 759-772. 10 A. Baker and J.R. Kanofsky, Of singlet oxygen by biomolecules from L1210 leukemia cells, Photochem. PhorobioZ., 55 (1992) 523-528; D.P. Valenzeno, Photomodification of biological membranes with emphasis on singlet oxygen mechanisms, Phorochem. Photobiol., 46 (1987) 147-160; E.A. Lissi, M.V. Encinas, E. Lemp and M.A. Rubio, Singlet oxygen biomolecular processes. Solvent and compartmentalization effects, Chem. Rev., 93 (1993) 699-723. 11 D.E. Moore, Photosensitization by drugs, J. Pharm. Sci., 66 (1977) 1282-1284. 12 E.A. Lissi and N. Clavero, Inactivation of lysozyme by alkylpyroxyl radicals, Free Radical Res. Commun., 10 (1990) 177-184. 13 LB. Zavodnik, T.P. Piletskaia and 1.1. Stepuro, Influence of temperature on the lysis of human erythrocytes by palmitic acid, Biophysics, 36 (1991) 1064-1068; I.V. Yamaikina and Y.A. Chernitskii, Thermohemolysis of erythrocytes in the temperature range including physiologic (autohemolysis), Biophysics, 36 (6) (1991) 1059-1063.