Journal of Photochemistry
and Photobiology,
B: Biology, 4 (1990) 335 - 342
News and Views
Mutagenic and genotoxic properties of singlet oxygen JACQUES PIETTE Experimental (Belgium)
Physics, Institute of Physics B5, University of LiGge, B-4000 LiGge
335
336
NEWS AND VIEWS
Because of its action at the DNA base level and maybe at the level of the DNA backbone, singlet oxygen should be considered as a DNA damaging agent when it is generated in the vicinity of DNA, and its effect on genetic information is worth studying in detail. The appearance of porphyrin derivatives and phthalocyanines as nontoxic drugs that have a high potential for use in photomedical applications has also focused interest on the genotoxic effects of singlet oxygen [lo]. Although these photosensitizers tend to accumulate in the membrane fractions, singlet oxygen seems to be one of the activated species generated upon visible light irradiation of these drugs. The singlet oxygen may diffuse to the nucleus and cause alterations of the genetic material [ll]. But we should keep in mind that genotoxicity could also be due to an oxidation product, arising from an initial reaction between singlet oxygen and membrane phospholipids, which is capable of subsequently reacting with the nucleic acids. 2. Mutagenic properties Evidence for the mutagenic effects of singlet oxygen has been provided for a wide variety of organisms ranging from yeast [12] and bacteria [13] to bacteriophages [ 14, 151. This important information has been deduced from experiments performed with different photosensitizers having strong affinities for nucleic acids and localized in the nucleus. Although these photosensitized reactions can generate other oxidizing species, the involvement of singlet oxygen in genetic changes has been demonstrated by the effectiveness of the reaction, which can be decreased by adding azide or enhanced by using a deuterated medium [16]. In 1983, Midden and Wang [17] described the separated-surface-sensitizer technique which permitted the exposure of biological material to pure singlet oxygen. The’ device prevents contact between the photosensitizer and the target, and thus avoids effects due to direct reaction either with the excited photosensitizer or with other oxygen radical species having a short lifetime. Decuyper-Debergh et al. [18] have also used the separated-surface-sensitizer to study the mutagenic consequences of damages induced by singlet oxygen in a double-stranded viral DNA suspended in deuterated water. These authors used a simple forward mutational system capable of detecting all classes of mutational events. The transfection of the oxidized DNA into bacteria leads to a 16-fold increase in the mutation frequency. This mutagenicity turns out to be largely due to single-nucleotide substitutions, frameshift events or double mutations, and the single mutations which occur predominently are G:C to T:A transversions. Moreover, the spectrum of mutations detected among the phages surviving singlet oxygen is totally different from those appearing spontaneously. From these experiments it can be concluded that guanine oxidation products mediated by singlet oxygen constitute premutational lesions if they are not repaired. However, it is not possible to discriminate between the various oxidation products as to which is the more likely major premutational lesion. The generation of singlet oxygen close to DNA can lead to
NEWS AND VIEWS
331
oxidation of guanine residues, and the unrepaired lesions can constitute premutational events leading mainly to G + T transversions. When generated outside bacteria, singlet oxygen is unable to promote mutagenicity in 26 S. typhimurium histidine auxotrophic strains killed to 35% survival [19]. These strains included a variety of base pair substitution or frameshift target sequences for reversion, including targets responsive to oxidative damage and those rich in GC base pairs. This work indicates that singlet oxygen generated outside bacteria does not react significantly with the bacterial chromosome in ways leading to DNA sequence changes or damages that interfere with DNA replication or SOS induction. Moreover, it seems that singlet oxygen is unable to generate oxidation products in the bacterial membrane which can secondarily promote lesion at the level of the genetic material. Photosensitizing agents such as hematoprophyrin derivatives and phthalocyanine are located in the cell membranes and are known to produce mainly singlet oxygen. It has been found that these photosensitizing reactions are poorly mutagenic in various loci such as the X-linked hypoxanthine phosphoribosyltransferase [20] and Na+/K+ ATPase [21]. Since mutants harboring multilocus lesions, such as those induced by activated oxygen species [22] are recovered with a low level at both of these loci, the problem has been re-examined at the thymidine kinase locus of mouse L5178Y cells heterozygous or hemizygous for the thymidine kinase gene on chromosome 11 [23]. Surprisingly, treatment with phthalocyanine plus light is clearly mutagenic at the heterozygous thymidine kinase locus in the strain LY-Sl. However, it remains to be shown what renders this strain mutable and whether or not singlet oxygen is implicated in the phenomenon. 3. Cy totoxic and genotoxic properties Using pure singlet oxygen allowed its potent cytotoxicity to be demonstrated when it is generated outside bacteria [ 24, 251, or a primary culture of human lymphocytes [26] or ciliated respiratory epithelium of hamster trachea [27]. Numerous works based on the use of various photosensitizers generating singlet oxygen confirm that this activated oxygen species attacks cellular components critical for cell survival. For instance, lesions in membranes, mitochondria, or other limiting essential substances or organelles could be responsible for the cytotoxicity. DNA lesions do not appear to be involved in the cytotoxic process. Hematoporphyrin and visible light can induce genotoxic response in human NHIK 3025 cells observed as sister chromatid exchanges and singlestranded breaks visualized by an alkali labilization [ 281. The frequencies of these events are about five times lower than those observed after X-ray irradiation, and in both cases the single-stranded breaks are practically completely repaired within 15 min. The frequencies of sister chromatid exchanges per chromosome have been followed after treatment of Allium cepa meristematic cells with red light and either hematoporphyrin or mesotetra(4-pyridyl)porphine [29]. In the case of both photosensitized reactions,
338
NEWS AND VIEWS
the frequency of the chromatid exchanges is increased significantly the values associated with the photoreaction mediated by the cationic porphyrin are higher than those recorded with hematoporphyrin. The generation of pure singlet oxygen outside human lymphocytes cultivated in vitro also increases the frequency of the sister chromatid exchanges per cell [26]. In these two last cases, the values turn out to be statistically different from that for the control but rather low in comparison with those published in the literature concerning other well-known genotoxic agents [ 301. Although nothing is known about the lesions responsible for the increase in the sister chromatid exchange frequency, the more likely hypothesis is that singlet oxygen reacts with lipids of the cellular membrane to form hydroperoxides which in turn can decompose to give several types of secondary lipid free radicals [31]. These radicals could then participate in secondary reactions with chromatin as target. To conclude this short review, we should point out that singlet oxygen can oxidize DNA. The unrepaired guanine oxidation products constitute premutational lesions. When it is generated outside either the nucleus or the cell, the mutagenic and the genotoxic actions of singlet oxygen drop drastically, being abolished in the case of bacteria. Jacques Piette is senior research associate from the Belgian National Fund for Scientific Research (Brussels, Belgium). We thank Prof. A. Van de Vorst for his comments on the manuscript. 1 J. L. Marx, Oxygen free radicals linked to many diseases, Science, 235 (1985) 529 531. 2 P. B. Merkel, R. Nilson and D. R. Kearns, Radiationless decay of singlet molecular oxygen in solution. An experimental and theoretical study of electronic to vibrational energy transfer, J. Am. Chem. Sot., 94 (1972) 7244 - 7253. 3 M. A. J. Rodgers, Activated oxygen. In R. V. Bensasson, G. Jori, E. J. Land and T. G. Truscott (eds.), Primary Photoprocesses in Biology and Medicine, Plenum, New York, 1985, pp. 181- 195. 4 P. C. C. Lee and M. A. J. Rodgers, Laser flash photokinetic studies of rose bengal sensitized photodynamic interactions of nucleotides and DNA, Photochem. Photobiol., 45 (1987) 79 - 86. 5 J. Cadet, M. Berger, C. Decarroz, J. R. Wagner, J. E. Van Lieu, Y. M. Ginot and P. Vigny, Photosensitized reactions of nucleic acids, Biochimie, 68 (1986) 813 - 834. 6 A. W. R. Niewint, J. M. Aubry, F. Arwert, H. Kortbeek, S. Herzberg and H. Joenje, Inability of chemically generated singlet oxygen to break the DNA backbone. Free Rad. Res. Commun., 1 (1985) 1 - 9. 7 M. V. M. Lafleur, A. W. M. Nieuwint, J. M. Aubry, H. Kortbeek, F. Arwert and H. Joenje, DNA damage by chemically generated singlet oxygen, Free Rad. Res. Comm., 2 (1987) 343 - 350. 8 P. Di Mascio, H. Wefers, H.-P. Do-Thi, M. V. M. Lafleur and H. Sies, Singlet molecular oxygen causes loss of biological activity in plasmid and bacteriophage DNA and induces single-stranded breaks, Biochim. Biophys. Acta, 1007 (1989) 151 - 157. 9 E. R. Blazek, J. G. Peak and M. J. Peak, Singlet oxygen induces frank strand breaks as well as alkali- and piperidine-labile sites in supercoiled plasmid DNA, Photochem. Photobiol., 49 (1989) 607 - 613. 10 T. J. Dougherty, Photosensitizers: therapy and detection of malignant tumors, Photothem. Photobio!., 45 (1987) 879 - 889.
NEWS AND VIEWS
339
11 A. Nye, G. Rosen, E. Gabrielson, J. Keana and V. Prabhu, Diffusion of singlet oxygen into human bronchial epithelial cells, Biochim. Biophys. Acta, 928 (1987) 1. 12 K. Kobayashi and T. Ito, Further in vivo studies on the participation of singlet oxygen in the photodynamic inactivation and induction of genetic changes in Saccharomyces cerevisiae, Photochem. Photobiol., 23 (1976) 21 - 28. 13 B. Gutter, W. T. Spech and H. S. Rosenkranz, A study of the photoinduced mutagenicity of methylene blue, Mutat. Res., 44 (1977) 177 - 182. 14 J. Piette, C. M. Calberg-Bacq and A. Van de Vorst, Photodynamic effect of proflavine on @X174 bacteriophage, its DNA replicative form and its isolated single-stranded DNA: inactivation, mutagenesis and repair, Mol. Gen. Genet., 167 (1978) 95 - 103. 15 N. Houba-Herin, C. M. Calberg-Bacq, J. Piette and A. Van de Vorst, Mechanisms for dye-mediated photodynamic action: singlet oxygen production, deoxyguanosine oxidation and phage inactivating efficiencies, Photochem. Photobiol., 36 (1982) 297 306. 16 T. Ito and K. Kobayashi, A survey of in vivo photodynamic activity of xanthenes, thiazines and acridines in yeast cells, Photochem. Photobiol., 26 (1977) 581 - 588. 17 W. R. Midden and S. Y. Wang, Singlet oxygen generation for solution kinetics: clean and simple, J. Am. Chem. Sot., 105 (1983) 4129 - 4135. 18 D. Decuyper-Debergh, J. Piette and A. Van de Vorst, Singlet oxygen-induced mutations in Ml3 Zac Z phage DNA, EMBO J., 6 (1987) 3155 - 3161. 19 T. A. Dahl, W. R. Midden and P. E. Hartman, Pure exogenous singlet oxygen: nonmutagenicity in bacteria, Mutat. Res., 201 (1988) 127 - 136. 20 C. J. Gomer, N. Rucker, A. Banerjee and W. F. Benedict, Comparison of mutagenicity and induction of sister chromatid exchange in Chinese hamster cells exposed to hematoporphyrin derivative photoradiation, ionizing radiation, or ultraviolet radiation, Cancer Res., 43 (1983) 2622 - 2627. 21 E. Ben-Hur, T. Fujihara, F. Suzuki and M. M. Eklind, Genetic toxicology of the photosensitization of Chinese hamster cells by phthalocyanines, Photochem. Photobiol., 45 (1987) 227 - 230. 22 A. W. Hsie, L. Recio, D. S. Katz, C. Q. Lee, M. Wagner and R. L. Schenley, Evidence for reactive oxygen species inducing mutations in mamalian cells, Proc. Natl. Acad. Sci. USA, 83 (1986) 9616 - 9620. 23 H. H. Evans, R. M. Rerko, J. Mencl, M. E. Clay, A. R. Antunez and N. L. Oleinick, Cytotoxicity and mutagenic effects of the photodynamic action of chloroaluminium phthalocyanine and visible light in L5178Y cells, Photochem. Photobiol., 49 (1989) 43 - 47. 24 S. A. Bezman, P. A. Burtis, T. P. J. Izod and M. A. Thayer, Photodynamic inactivation of E. coli by rose bengal immobilized on polystyrene beads, Photochem. Photobiol., 28 (1978) 325 - 329. 25 T. A. Dahl, W. R. Midden and P. E. Hartman, Pure singlet oxygen cytotoxicity for bacteria, Photochem. Photobiol., 46 (1987) 345 - 352. 26 D. Decuyper-Debergh, J. Piette, C. Laurent and A. Van de Vorst, Cytotoxicity and genotoxic effects on extracellular generated singlet oxygen in human lymphocytes in vitro, Mutat. Res., 225 (1989) 11 - 14. 27 L. J. Schiff, W. C. Eisenberg, J. Dziuba, K. Taylor and S. J. Moore, Cytotoxic effects of singlet oxygen, Environ. Health Persp., 76 (1987) 199 - 203. 28 J. Moan, H. Waksvik and T. Christensen, DNA single-stranded breaks and sister chromatid exchanges induced by treatment with hematoporphyrin and light or by Xrays in human NHIK 3025 cells, Cancer Res., 40 (1980) 2915 - 2918. 29 M. J. Hazen, A. Villaneuva and J. C. Stockert, Induction of sister chromatid exchanges in Allium cepa meristematic cells exposed to meso-tetra (4-pyridyl) porphine and hematoporphyrin photoradiation, Phoiochem. Photobiol., 46 (1987) 469 - 476. 30 C. Laurent, SCE increases after an accidental acute inhalation exposure to Et0 and recovery to normal after two years, Mutat. Res., 204 (1988) 711 - 717. 31 C. E. Vaca, J. Wihelm and M. Harms-Ringdahl, Interaction of lipid peroxidation products with DNA. A review, Mutat. Res., 195 (1988) 137 - 149.
and Photobiology,
B: Biology, 4 (1990) 335 - 342
News and Views
Mutagenic and genotoxic properties of singlet oxygen JACQUES PIETTE Experimental (Belgium)
Physics, Institute of Physics B5, University of LiGge, B-4000 LiGge
335
336
NEWS AND VIEWS
Because of its action at the DNA base level and maybe at the level of the DNA backbone, singlet oxygen should be considered as a DNA damaging agent when it is generated in the vicinity of DNA, and its effect on genetic information is worth studying in detail. The appearance of porphyrin derivatives and phthalocyanines as nontoxic drugs that have a high potential for use in photomedical applications has also focused interest on the genotoxic effects of singlet oxygen [lo]. Although these photosensitizers tend to accumulate in the membrane fractions, singlet oxygen seems to be one of the activated species generated upon visible light irradiation of these drugs. The singlet oxygen may diffuse to the nucleus and cause alterations of the genetic material [ll]. But we should keep in mind that genotoxicity could also be due to an oxidation product, arising from an initial reaction between singlet oxygen and membrane phospholipids, which is capable of subsequently reacting with the nucleic acids. 2. Mutagenic properties Evidence for the mutagenic effects of singlet oxygen has been provided for a wide variety of organisms ranging from yeast [12] and bacteria [13] to bacteriophages [ 14, 151. This important information has been deduced from experiments performed with different photosensitizers having strong affinities for nucleic acids and localized in the nucleus. Although these photosensitized reactions can generate other oxidizing species, the involvement of singlet oxygen in genetic changes has been demonstrated by the effectiveness of the reaction, which can be decreased by adding azide or enhanced by using a deuterated medium [16]. In 1983, Midden and Wang [17] described the separated-surface-sensitizer technique which permitted the exposure of biological material to pure singlet oxygen. The’ device prevents contact between the photosensitizer and the target, and thus avoids effects due to direct reaction either with the excited photosensitizer or with other oxygen radical species having a short lifetime. Decuyper-Debergh et al. [18] have also used the separated-surface-sensitizer to study the mutagenic consequences of damages induced by singlet oxygen in a double-stranded viral DNA suspended in deuterated water. These authors used a simple forward mutational system capable of detecting all classes of mutational events. The transfection of the oxidized DNA into bacteria leads to a 16-fold increase in the mutation frequency. This mutagenicity turns out to be largely due to single-nucleotide substitutions, frameshift events or double mutations, and the single mutations which occur predominently are G:C to T:A transversions. Moreover, the spectrum of mutations detected among the phages surviving singlet oxygen is totally different from those appearing spontaneously. From these experiments it can be concluded that guanine oxidation products mediated by singlet oxygen constitute premutational lesions if they are not repaired. However, it is not possible to discriminate between the various oxidation products as to which is the more likely major premutational lesion. The generation of singlet oxygen close to DNA can lead to
NEWS AND VIEWS
331
oxidation of guanine residues, and the unrepaired lesions can constitute premutational events leading mainly to G + T transversions. When generated outside bacteria, singlet oxygen is unable to promote mutagenicity in 26 S. typhimurium histidine auxotrophic strains killed to 35% survival [19]. These strains included a variety of base pair substitution or frameshift target sequences for reversion, including targets responsive to oxidative damage and those rich in GC base pairs. This work indicates that singlet oxygen generated outside bacteria does not react significantly with the bacterial chromosome in ways leading to DNA sequence changes or damages that interfere with DNA replication or SOS induction. Moreover, it seems that singlet oxygen is unable to generate oxidation products in the bacterial membrane which can secondarily promote lesion at the level of the genetic material. Photosensitizing agents such as hematoprophyrin derivatives and phthalocyanine are located in the cell membranes and are known to produce mainly singlet oxygen. It has been found that these photosensitizing reactions are poorly mutagenic in various loci such as the X-linked hypoxanthine phosphoribosyltransferase [20] and Na+/K+ ATPase [21]. Since mutants harboring multilocus lesions, such as those induced by activated oxygen species [22] are recovered with a low level at both of these loci, the problem has been re-examined at the thymidine kinase locus of mouse L5178Y cells heterozygous or hemizygous for the thymidine kinase gene on chromosome 11 [23]. Surprisingly, treatment with phthalocyanine plus light is clearly mutagenic at the heterozygous thymidine kinase locus in the strain LY-Sl. However, it remains to be shown what renders this strain mutable and whether or not singlet oxygen is implicated in the phenomenon. 3. Cy totoxic and genotoxic properties Using pure singlet oxygen allowed its potent cytotoxicity to be demonstrated when it is generated outside bacteria [ 24, 251, or a primary culture of human lymphocytes [26] or ciliated respiratory epithelium of hamster trachea [27]. Numerous works based on the use of various photosensitizers generating singlet oxygen confirm that this activated oxygen species attacks cellular components critical for cell survival. For instance, lesions in membranes, mitochondria, or other limiting essential substances or organelles could be responsible for the cytotoxicity. DNA lesions do not appear to be involved in the cytotoxic process. Hematoporphyrin and visible light can induce genotoxic response in human NHIK 3025 cells observed as sister chromatid exchanges and singlestranded breaks visualized by an alkali labilization [ 281. The frequencies of these events are about five times lower than those observed after X-ray irradiation, and in both cases the single-stranded breaks are practically completely repaired within 15 min. The frequencies of sister chromatid exchanges per chromosome have been followed after treatment of Allium cepa meristematic cells with red light and either hematoporphyrin or mesotetra(4-pyridyl)porphine [29]. In the case of both photosensitized reactions,
338
NEWS AND VIEWS
the frequency of the chromatid exchanges is increased significantly the values associated with the photoreaction mediated by the cationic porphyrin are higher than those recorded with hematoporphyrin. The generation of pure singlet oxygen outside human lymphocytes cultivated in vitro also increases the frequency of the sister chromatid exchanges per cell [26]. In these two last cases, the values turn out to be statistically different from that for the control but rather low in comparison with those published in the literature concerning other well-known genotoxic agents [ 301. Although nothing is known about the lesions responsible for the increase in the sister chromatid exchange frequency, the more likely hypothesis is that singlet oxygen reacts with lipids of the cellular membrane to form hydroperoxides which in turn can decompose to give several types of secondary lipid free radicals [31]. These radicals could then participate in secondary reactions with chromatin as target. To conclude this short review, we should point out that singlet oxygen can oxidize DNA. The unrepaired guanine oxidation products constitute premutational lesions. When it is generated outside either the nucleus or the cell, the mutagenic and the genotoxic actions of singlet oxygen drop drastically, being abolished in the case of bacteria. Jacques Piette is senior research associate from the Belgian National Fund for Scientific Research (Brussels, Belgium). We thank Prof. A. Van de Vorst for his comments on the manuscript. 1 J. L. Marx, Oxygen free radicals linked to many diseases, Science, 235 (1985) 529 531. 2 P. B. Merkel, R. Nilson and D. R. Kearns, Radiationless decay of singlet molecular oxygen in solution. An experimental and theoretical study of electronic to vibrational energy transfer, J. Am. Chem. Sot., 94 (1972) 7244 - 7253. 3 M. A. J. Rodgers, Activated oxygen. In R. V. Bensasson, G. Jori, E. J. Land and T. G. Truscott (eds.), Primary Photoprocesses in Biology and Medicine, Plenum, New York, 1985, pp. 181- 195. 4 P. C. C. Lee and M. A. J. Rodgers, Laser flash photokinetic studies of rose bengal sensitized photodynamic interactions of nucleotides and DNA, Photochem. Photobiol., 45 (1987) 79 - 86. 5 J. Cadet, M. Berger, C. Decarroz, J. R. Wagner, J. E. Van Lieu, Y. M. Ginot and P. Vigny, Photosensitized reactions of nucleic acids, Biochimie, 68 (1986) 813 - 834. 6 A. W. R. Niewint, J. M. Aubry, F. Arwert, H. Kortbeek, S. Herzberg and H. Joenje, Inability of chemically generated singlet oxygen to break the DNA backbone. Free Rad. Res. Commun., 1 (1985) 1 - 9. 7 M. V. M. Lafleur, A. W. M. Nieuwint, J. M. Aubry, H. Kortbeek, F. Arwert and H. Joenje, DNA damage by chemically generated singlet oxygen, Free Rad. Res. Comm., 2 (1987) 343 - 350. 8 P. Di Mascio, H. Wefers, H.-P. Do-Thi, M. V. M. Lafleur and H. Sies, Singlet molecular oxygen causes loss of biological activity in plasmid and bacteriophage DNA and induces single-stranded breaks, Biochim. Biophys. Acta, 1007 (1989) 151 - 157. 9 E. R. Blazek, J. G. Peak and M. J. Peak, Singlet oxygen induces frank strand breaks as well as alkali- and piperidine-labile sites in supercoiled plasmid DNA, Photochem. Photobiol., 49 (1989) 607 - 613. 10 T. J. Dougherty, Photosensitizers: therapy and detection of malignant tumors, Photothem. Photobio!., 45 (1987) 879 - 889.
NEWS AND VIEWS
339
11 A. Nye, G. Rosen, E. Gabrielson, J. Keana and V. Prabhu, Diffusion of singlet oxygen into human bronchial epithelial cells, Biochim. Biophys. Acta, 928 (1987) 1. 12 K. Kobayashi and T. Ito, Further in vivo studies on the participation of singlet oxygen in the photodynamic inactivation and induction of genetic changes in Saccharomyces cerevisiae, Photochem. Photobiol., 23 (1976) 21 - 28. 13 B. Gutter, W. T. Spech and H. S. Rosenkranz, A study of the photoinduced mutagenicity of methylene blue, Mutat. Res., 44 (1977) 177 - 182. 14 J. Piette, C. M. Calberg-Bacq and A. Van de Vorst, Photodynamic effect of proflavine on @X174 bacteriophage, its DNA replicative form and its isolated single-stranded DNA: inactivation, mutagenesis and repair, Mol. Gen. Genet., 167 (1978) 95 - 103. 15 N. Houba-Herin, C. M. Calberg-Bacq, J. Piette and A. Van de Vorst, Mechanisms for dye-mediated photodynamic action: singlet oxygen production, deoxyguanosine oxidation and phage inactivating efficiencies, Photochem. Photobiol., 36 (1982) 297 306. 16 T. Ito and K. Kobayashi, A survey of in vivo photodynamic activity of xanthenes, thiazines and acridines in yeast cells, Photochem. Photobiol., 26 (1977) 581 - 588. 17 W. R. Midden and S. Y. Wang, Singlet oxygen generation for solution kinetics: clean and simple, J. Am. Chem. Sot., 105 (1983) 4129 - 4135. 18 D. Decuyper-Debergh, J. Piette and A. Van de Vorst, Singlet oxygen-induced mutations in Ml3 Zac Z phage DNA, EMBO J., 6 (1987) 3155 - 3161. 19 T. A. Dahl, W. R. Midden and P. E. Hartman, Pure exogenous singlet oxygen: nonmutagenicity in bacteria, Mutat. Res., 201 (1988) 127 - 136. 20 C. J. Gomer, N. Rucker, A. Banerjee and W. F. Benedict, Comparison of mutagenicity and induction of sister chromatid exchange in Chinese hamster cells exposed to hematoporphyrin derivative photoradiation, ionizing radiation, or ultraviolet radiation, Cancer Res., 43 (1983) 2622 - 2627. 21 E. Ben-Hur, T. Fujihara, F. Suzuki and M. M. Eklind, Genetic toxicology of the photosensitization of Chinese hamster cells by phthalocyanines, Photochem. Photobiol., 45 (1987) 227 - 230. 22 A. W. Hsie, L. Recio, D. S. Katz, C. Q. Lee, M. Wagner and R. L. Schenley, Evidence for reactive oxygen species inducing mutations in mamalian cells, Proc. Natl. Acad. Sci. USA, 83 (1986) 9616 - 9620. 23 H. H. Evans, R. M. Rerko, J. Mencl, M. E. Clay, A. R. Antunez and N. L. Oleinick, Cytotoxicity and mutagenic effects of the photodynamic action of chloroaluminium phthalocyanine and visible light in L5178Y cells, Photochem. Photobiol., 49 (1989) 43 - 47. 24 S. A. Bezman, P. A. Burtis, T. P. J. Izod and M. A. Thayer, Photodynamic inactivation of E. coli by rose bengal immobilized on polystyrene beads, Photochem. Photobiol., 28 (1978) 325 - 329. 25 T. A. Dahl, W. R. Midden and P. E. Hartman, Pure singlet oxygen cytotoxicity for bacteria, Photochem. Photobiol., 46 (1987) 345 - 352. 26 D. Decuyper-Debergh, J. Piette, C. Laurent and A. Van de Vorst, Cytotoxicity and genotoxic effects on extracellular generated singlet oxygen in human lymphocytes in vitro, Mutat. Res., 225 (1989) 11 - 14. 27 L. J. Schiff, W. C. Eisenberg, J. Dziuba, K. Taylor and S. J. Moore, Cytotoxic effects of singlet oxygen, Environ. Health Persp., 76 (1987) 199 - 203. 28 J. Moan, H. Waksvik and T. Christensen, DNA single-stranded breaks and sister chromatid exchanges induced by treatment with hematoporphyrin and light or by Xrays in human NHIK 3025 cells, Cancer Res., 40 (1980) 2915 - 2918. 29 M. J. Hazen, A. Villaneuva and J. C. Stockert, Induction of sister chromatid exchanges in Allium cepa meristematic cells exposed to meso-tetra (4-pyridyl) porphine and hematoporphyrin photoradiation, Phoiochem. Photobiol., 46 (1987) 469 - 476. 30 C. Laurent, SCE increases after an accidental acute inhalation exposure to Et0 and recovery to normal after two years, Mutat. Res., 204 (1988) 711 - 717. 31 C. E. Vaca, J. Wihelm and M. Harms-Ringdahl, Interaction of lipid peroxidation products with DNA. A review, Mutat. Res., 195 (1988) 137 - 149.
