Mutation Research, 275 (1992) 367-375 © 1992 Elsevier Science Publishers B.V. All rights reserved 0921-8734/92/$05.00
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MUTAGI 0253
Singlet oxygen induced DNA damage H e l m u t Sies a and Carlos F.M. M e n c k b lnstitut far Physiologische Chemic I, Uni~'ersitiitDiisseldo~ Diisseldo~ Germany and t, Departamento de Biologia, Instituto de Biociencias, Unit,ersidadede Sao Paulo, Sao Paulo 05499, Brazil {Received 8 March 1992) (Revision received 18 May 1992) (Accepted 27 May 1992)
Keywords: Singlet oxygen; Endoperoxides; DNA damage; Shuttle vectors
Summary Singlet oxygen generated by photoexcitation and by ehemiexcitation selectively reacts with the guanine moiety in nueleosides (kq + k r about 5 × 10 6 M - I s - i ) and in DNA. The oxidation products include 8-oxo-7.hydro.deoxyguanosine (8-oxodG; also called 8-hydroxydeoxyguanosine) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Singlet oxygen also causes alkali-labile sites and single-strand breaks in DNA. The biological consequences include a loss of transforming activity as studied with plasmids and bacteriophage DNA, and mutagenicity and genotoxicity. Employing shuttle vectors, it was shown that double-stranded vectors carrying singlet oxygen induced lesions seem to be processed in mammalian cells by DNA repair mechanisms efficient in preserving the biological activity of the plasmid but highly mutagenic in mammalian cells. Biological protection against singlet oxygen is afforded by quenchers, notably carotenoids and tocopherols. Major repair occurs by excision of the oxidized deoxyguanosine moieties by the Fpg protein, preventing mismatch of 8-oxodG with dA, which would generate G: C to T: A transversions.
Electronically excited molecular oxygen (singlet oxygen, ~O2) can be generated by photocheraical reactions through transfer of excitation energy to ground-state oxygen (30 2) from a suitable excited triplet-state sensitizer (photoexeitation). Since the discovery of singlet oxygen by Kautsky and de Bruijn (1931), the type I1 photooxygenation reactions have been intensely investigated (Goilniek, 1968; Foote, 1990). However, singlet oxygen can also be produced by dark reactions (chemiexcitation), e.g., by the decomposition of
Correspondence: ProL Dr. H. Sies, lnstitut ffir Physiologische Chemie I, Moorenstrasse 5, W-4000-Diisseidorf, German;,.
endoperoxides and several other types of reaction (Wasserman and Murray, 1979; Adan. and Cilento, 1982). In biological systems, these dark reactions occur in lipid peroxidation and by enzyme reactions such as those catalyzed by lactoperoxidase, lipoxygenase and chloroperoxidase (for reviews, see Cadenas and Sies, 1984; Kanofsky, 1989). Singlet oxygen is also produced during photooxidation of a variety of biological compounds and xenobiotics (Krinsky, 1977; Krasnovsky, 1991). It is relatively long-lived, with half-times in the range of 4-50/~s, so that diffusion of singlet oxygen is possible within a radius estimated to be in the range of 100 A (Schnuriger and Bourdon, 1968; Moan, 1990).
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Reactions of singlet oxygen Reactions of singlet oxygen are physical and/or chemical (Kasha and Khan, 1970; Krasnovsky, 1979). The physical reactivlty is characterized by photoemissive decay, which can occur in two modes, termed monomol and dimol reactions, characterized by the emission at 1268 nm, and at 634 nm and 703 nm, respectively; this photoemission is used for the detection of singlet oxygen, employing liquid nitrogen-cooled germanium photodiode detectors for the 1268 nm infrared emission and red-sensitive photomultipliers cooled to about -25°C for the dimol emission, also called ultraweak chemiluminescence. The chemical reactions are manifold, including 1,2, 1,3 and 1,4 additions to olefins, forming dioxetanes, allylic hydroperoxides ('ene reaction'), and endoperoxides, respectively, as well as oxidation of sulfides or phenols to form sulfoxides or hydroperoxydienones, for example (Wasserman and Murray, 1979; Frimer, 1985; Aubry, 1991). This chemical reactivity is the basis of biological damage inflicted by ringlet oxygen.
Singlet oxygen reactivity with nucleosides and DNA
Guanine oxidation products This topic has recently been reviewed by Piette (1991). Photosensitization causes damage to deO
oxyguanosine almost exclusively, either as free nucleoside or in DNA (Simon and van Vunakis, 1962; Gutter et al, 1977; Friedman and Brown, 1978; Piette and Moore, 1982; Piette et al., 1984; Kawanishi et al., 1986). Model experiments by Cadet et al. (1983,1986) using Y,5'-di-O-acetyl2'-deoxyguanosine showed that three main products arose from the reaction with singlet oxygen, namely the two diastereomers of the 4,8-dihydro4-hydroxy-8-oxo-deoxyguanosine derivative, and the cyanuric acid derivative. It is assumed that the former arise from a 1,4-cycloaddition leading to an unstable endoperoxide (Ravanat et al., 1991). The cyanuric acid derivative is likely to be formed via a transient enamine as a substrate for a second addition of a ringlet oxygen molecule (Matsuura et al., 1972). Boiteux et al. (1992) have detected the formation of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) as a product from deoxyguanosine residues in calf thymus DNA using photosensitization with visible light and methylene blue. 8-Hydroxydeoxyguanosine, better called 8-oxo7-hydrodeoxyguanosine (abbreviated 8-oxodG), was described by Floyd et al. (1989) as a product of deoxyguanosine oxidation by ringlet oxygen in DNA, employing methylene blue and light. This was extended to other photosonsitizers, e.g., thiazinc dyes (Floyd ¢t al. 1990), It should he mentioned that the generation of singlet oxygen is not the only reaction occurring, e.g., with methylene
0
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blue only 55%, so that other (type !) reactions might influence the damage pattern. Recently, Devasagayam et al. (1991b) demonstrated that a dark reaction, i.e., the generation of singlet oxygen by thermodissociation of an endoperoxide (DiMascio and Sies, 1989), also results in the formation of 8-oxoG or 8-oxodG. When deoxyadenosine was exposed to singlet oxygen, there was no formation of the corresponding 8-oxo product (Devasagayam et al., 1991b). However, radiation-chemically generated hydroxyl radicals give rise to the corresponding 8-oxo products from both deoxyguanosine and deoxyadenosine (Cadet and Berger, 1985; Dizdaroglu, 1985; Steenken, 1989). It thus appears that the formation of the 4-hydroxy-8-oxoguanosine derivative is a selective initial product generated from singlet oxygen via an unstable endoperoxide (Ravanat et al., 1991). As discussed by Devasagayam et al. (1991b), such a type of intermediate may be on the pathway of 8-oxoG or 8-oxodG formation. For the endoperoxide to give the 8-oxopurine, two reducing equivalents have to be supplied (Fig. 1). Boiteux et ai. (1992) suggested that the formation of FapyGua and 8-oxodG might be explained by an initial electron transfer from the guanine ring to singlet oxygen, resulting in the generation of a guanine radical cation and the superoxide radical anion. Further reactions of the guanine radical cation may then result in the for,nation of a C-8 OH adduct radical of guanine. T~7 C-8 OH adduct radicals of purines are ktiown ~o produce formamidopyrimidines upon ring-opening followed by one-electron reduction, and 8-oxopufines are formed upon one-electron oxidation (for review, see Steenken, 1989).
Quenching of photoemission The quenching of monomol photoemission of singlet oxygen by guanosine occurs with the overall singlet oxygen quenching rate constant, kq + k r, of 6.2 × 106 M-Is -1, where kqis the physical quenching rate constant and k, is the chemical reaction rate constant. The value for the overall quenching rate constant for deoxyguanosine is 5.2× 106 M - i s -t (Devasagayam et al., 1991b). This value is almost identical with that (5.3 x 106 M - i s -1) measured for 2'-deoxyguanosine-5'-
monophosphate by use of time-resolved singlet oxygen emission decay techniques (Lee and Rodgers, 1987). Single-strand break formation by singlet oxygen
Singlet oxygen was shown to lead to singlestrand breaks in plasmid DNA (Wefers et al., 1987; DiMascio et al., 1989b; Blazek et al., 1989; Devasagayam et al., 1991a) and in bacteriophage DNA (DiMascio et al., 1989b). In these studies, several techniques for the generation of singlet oxygen were employed, including the thermal dissociation of an endoperoxide, as mentioned above, as well as the use of microwave discharge and the diffusion from immobilized rose bengal on a cover slip. Earlier work by Nieuwint et al. (1985, 1987) and Lafleur et al. (1987) resulted in no single-strand breaks; in these experiments, much lower concentrations of singlet oxygen available to the DNA were employed. That singlet oxygen mediates the single-strand breaks is supported by the enhancement of the effect caused by solvent deuteration, which is known to increase the lifetime of singlet oxygen, and by a quenching effect caused by the addition of sodium azide (Wefers et al., 1987; DiMascio et al., 1989b; Blazck et al., 1989; Devasagayam et al., 1991a). Some biologically occurring thiols, glutathione, cysteamine and cysteine, enhanced the single-strand breaks in plasmid pBR322 DNA; lipoate (thioctic acid) protected (Devasagayam et al., 1991,~,,b). Epe et al. (1988) examined the singlet oxygen induced DNA damage profile in PM2 DNA and found it to be different from that induced by hydroxyl radicals, as did Lafleur et al. (1987), with single-strand breaks making up only less than 5% of the total DNA damage. That single-strand breaks are relatively rare events as compared to, e.g., 8-oxodG formation is known from the work by Schneider et al. (1990), who calculated that single-strand nicking was approx. 17 times less than 8-oxodG formation, using methylene blue plus light. The mechanism of singlet oxygen induced strand break formation is unknown. It is possible that 8-oxodG and strand breaks are derived from a common precursor, possibly the initial endoper-
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oxide mentioned above (Fig. 1). This is in agreement with the observations by Devasagayam et al. (1991b) that (i) the time courses of the two effects are similar, and (ii) that the single-strand breaks occur selectively at guanine residues. The latter was determined using a plasmid containing a fragment of the mouse metallothionein I promoter and a novel end-labeling technique; no major differences (hot spots) were observed between individual guanines in these experiments (Devasagayam et al., 1991b). A major type of singlet oxygen induced DNA damage is the generation of alkali-labile sites (ca. 30% of all the damage), as studied by Nieuwint et al. (1985) and Lafleur et al. (1987). In comparison, strand-breaks are formed only at a frequency of 2-5% (DiMascio et al., 1989b; Epe et al., 1988; Joenje et al., 1991).
TABLE 1 COMPARISON OF LOSS OF TRANSFORMING ACTIVITY AND LOSS OF SUPERCOILED DNA UPON EXPO. SURE OF pBR322 TO SINGLET OXYGEN
Control tO 2 tO 2, D20 tO 2, plus NaN3 (20 raM) IO 2, plus methionine (10 raM) t o 2, plus methionine sulfone (10 raM)
Transforming activity a (%)
Supercoiled DNA b (%)
100 32.1 ±4.6 14.1 ± 1.5
91,9 :i:0.7 49,1 ± 1.8 30,25:2.2
84.3 ± 3.8
86.2 ± 2.6
77.5±1,6
69.1+1.7
58.3 ± 6,1
45.8 ± 3.1
DNA solutions were exposed for 20 min to a gas stream containing tO z generated by microwave discharge. a Data from Wefers et aL (1987). b Dala from Di Mascio et aL (1989b).
Biological consequences
Loss of transforming activity in experiments with plasmid pBR322 DNA (Wefers et al., 1987) and with phage $X174 DNA (Lafleur et al., 1987; DiMascio et ai., 1989b), it was observed that the transforming activity in E. coli was lost, with the single-stranded form of the phage DNA being more sensitive than the double-stranded form. This indicates that nonrepaired guanine oxidation products and/or singlestrand breaks have the capacity to inhibit plasmid or phage DNA replication in competent bacteria. Loss of transforming capacity essentially parallels single-strand break formation (Table 1; Wefers et ai., 1987).
Mutagenicity and genotoxicity The cytotoxicity as well as mutagenicity and genotoxicity exhibited by singlet oxygen have been studied in recent years, also with regard to photodynamic therapy; this will not be discussed here. The most recent surveys on the genotoxicity of singlet oxygen have been given by Epe (1991) and by Piette (1991). An enhancement of mutagenesis was detected in rodent cells exposed to photosensitized rose bengal (Gruener and Lockwood, 1979) and phthalocyanines (Ben-Hur et al., 1987). DNA sin-
gle-strand breaks and sister-chromatid exchanges were shown to be induced in human cells treated with hematoporphyrin and light (Moan et ai., 1980). A dose-dependent increase of sister-chromatid exchanges was observed after treatment of human lymphocytes with extracellularly generated singlet oxygen (Decuyper-Debergh ¢t al., 1989), employing the surface-separated method of Midden and Wang (1983). The most frequent mutations detected in M13 iacZ phage DNA were O : C to T : A transversions; no significant mutagenicity was induced at T: A base pairs (Decuyper-Debergh et al., 1987). The potential mutagenie effect of 8-oxodG suspected from experiments by Kuchino et al. (1987) was recently established (Wood et ai., 1990; Moriya et al., 1991; Shibutani et aL, 1991). 8-oxodO also base-pairs with dA in DNA, since the oxidized base is pref¢rably present in its syn form (Kouchakdiian et al., 1991). Thus, 8-oxodG contributes to spontaneous mutagenesis by favoring the G : C to T: A transversion, Using human fibroblasts, TyrreU and Pidoux (1989) showed that cell killing by UVA (334 and 365 nm) and near-visible (405 nm) radiations may involve singlet oxygen. The implication of singlet oxygen in these effects was based mainly on an enhancement of the effects by deuterium oxide
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and on the diminution of the effects by singlet oxygen quenchers such as azide. However, there is no evidence that the genetic alterations which were observed are directly resultant from the interaction between singlet oxygen and the chromosomes. It is also possible that singlet oxygen first reacts with cell membrane components leading to lipid peroxidation, the products of which in turn might react with the DNA (Vaca et al., 1988). Shuttle vector experiments In order to detect the direct consequences of singlet oxygen induced DNA damage in mammalian cells, our own recent work was performed using shuttle vectors. These are hybrid plasmids containing DNA sequences derived from pBR322 and from a mammalian virus, for replication both
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Fig. 3. Mutagenesis per single strand break in ss (o) and ds (o) SV40-based shuttle vectors. Data are from Fig. 2.
in bacteria and in eukaryotic cells; hence the term shuttle vector. The plasmids used were SV40-based vectors derived from those described before in detail (Menck et al., 1989). In these experiments, the vectors were treated in vitro as naked DNA with the thermolabile endoperoxide generating singlet oxygen (DiMascio and Sies, 1989). The damaging action of singlet oxygen on the shuttle vectors themselves results in single-strand breaks in both dsDNA (DiMascio et ai., 1990a) and ssDNA (Ribeiro et al., 1992b). Moreover, double-strand breaks were also detectable in dsDNA after exposure to high doses of singlet origen (DiMascio et aL, 1990a). The vectors replicated in monkey COS7 cells, and the results revealed that there is a dose-dependent increase in the mutation frequency on the supF gene in the singlet oxygen treated ds or ss plasmids (Fig. 2A). The higher susceptibility of ssDNA may be due to increased accessibility of singlet oxygen to its main target, the guanine residues, which are potentially less readily reachable in the bases arranged in the double helix. Likewise, the mutation frequency is higher for ssDNA than for dsDNA (Fig. 2B). However, as shown in Fig. 3, this higher mutagenicity cannot be attributed solely to the greater number of lesions. In fact, when the mutagenesis data are plotted against the number of breaks, the figure shows exactly the inverse, that is, more mutations per break are detected in dsDNA than in ssDNA.
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One explanation could be that premutagenic lesions other than breaks, i.e., 8-oxodG or FapyGua, may be more abundant in dsDNA. As mentioned above, 8-oxodG was found to occur 17-fold more frequently than single-strand breaks (Schneider et ai., 1990). Ribeiro et al. (1992a) observed that in vitro DNA polymerase blocking sites also exceed the number of breaks in singlet oxygen treated ssDNA by 20-fold. A ~cond explanation for the higher mutability of damage in dsDNA is that the singlet oxygen induced lesions may be processed in mammalian cells differently in dsDNA and ssDNA. For ssDNA, the absence of a template makes the repair of damage more difficult, so that the lesions must be bypassed during replication. Lesions not bypassed would lead to vector inactivation. Therefore, the results shown in Fig. 3 indicate that the singlet oxygen induced lesions in dsDNA are processed by error-prone repair in mammalian cells. This repair is unable to act on ssDNA vectors. Such conclusions are consistent with the much higher level of inactivation of damaged vectors transfected in the ss structure in relation to dsDNA (Ribeiro et al., 1992b). Moreover, the DNA sequence of those singlet oxygen induced mutants revealed that part of them contain multiple base changes (Oliveira et al., unpublished results), a type of mutation which can be due to error-prone DNA polymerization during excision repair (Seidman ct al., 1987). Thus, the ds vectors carrying singlet oxygen induced lesions seem to be processed by DNA repair mechanisms efficient in preserving the biological activity of the plasmid but highly mutagenic in mammalian cells.
Protection and repair mechanisms As is obvious from this discussion, defense against singlet oxygen induced mutagenesis and genotoxic lesions is multiple. In terms of protection, the prevention of the reaction of singlet oxygen with guanosines in DNA by quenching is of major importance. A number of biological compounds have been shown to quench singlet oxygen, notably the carotenoids (Foote and Denny, 1968; Krasnovsky, 1979). The capability of canthaxanthin, a carotenoid not serving as provitamin A, in protecting against chemically and physically induced neoplastic transformation just
as p-carotene does, has sparked recent interest in the biological properties of carotenoids not linked to their functions as precursor of vitamin A (lung et al., 1988; Bertram et al., 1991). Lycopene is the most efficient biological singlet oxygen quencher (DiMascio et al., 1989a; Corm et al., ~991). Also, tocopherols and some biological thiols are singlet oxygen quenchers (Kaiser et al., 1990; Devasagayam et al., 1991c; DiMascio et al., 1990b). Whether these reactivities are related to the biological protection of DNA in cells and tissues is being studied in several laboratories. The repair of DNA containing the oxidized guanosine residues occurs by excision. The excised product, 8-oxodG, can be detected in urine, as an indicator of in vivo oxidative DNA damage (Shigenaga and Ames, 1990; Shigenaga et al., 1991; Park et al., 1992). The enzyme catalyzing excision of these purine lesions in E. coil is Fpg protein (formamidopyrimidine-DNA glycosylase), described by Boiteux et al. (1990, 1992). An enzyme which has a DNA glycosylase activity similar to Fpg protein exists in mammalian cells (Margisson and Pegg, 1981) and has been partially purified (Breimer, 1984; Laval et al., 1990). Kasai et al. (1986) have detected 8-oxodG in DNA from mammalian cells irradiated with ,/-rays. They also found that the amount of this lesion decreases with time after irradiation, suggesting the presence of repair enzymes acting on 8-oxodGs in these cells. More recently, 8-oxodG was measured in various organs from rats of different ages (Fraga et al., 1990). The amount of this DNA lesion was found to increase with age in liver, kidney and intestine, but remained unchanged in brain and testes. These authors have also shown the excretion of 8-oxodG in the urine of these animals, as a possible consequence of its repair from DNA by nuclease activity. The amount excreted in the urine decreased with age. Thus, the age-dependent accumulation of 8oxodO residues in DNA may be related to the. loss of DNA repair activity or to an increase in the rate of oxidative DNA damage.
Acknowledgements We gratefully acknowledge the fruitful cooperation and helpful discussion of our colleagues
373
and coworkers as cited in the references. Our studies were. supported by the National Foundation for Cancer Research, Bet.hesda, by the Jung-Stiftung fiir Medizin, Hamburg, and by CNPO and FAPESP, Brazil. References Adam, W. and Cilento, G. (1982) Chemical and Biological Generation of Electronically Excited States. Academic Press, New York. Aubry, J.M. (1991) New chemical sources of singlet oxygen. In: C. Vigo-Pelfrey (Ed.), Membrane Lipid Peroxidation II. CRC Press, Boca Raton, FL, pp. 65-102. Ben-Hut, E., Fujihara, T., Suzuki, F. and Elkind, M.M. (1987) Genetic toxicology of the photosensitization of Chinese hamster cells by phthalocyanines. Photochem. Photobiol. 45, 227-230. Bertram, J.S., Pung, A., Churley, M., Kappock, T.J., Wiikins, L.R. and Cooney, R.V. (1991) Diverse carotenoids protect against chemically induced neoplastic transformation. Carcinogenesis 12, 671-678. Blazek, E.R., Peak, J.G. and Peak, M.J. (1989) Singlet oxygen induces frank strand breaks as well as alkali- and piperidine-labile sites in supercoiled plasmid DNA. Photochem. Photobiol. 49, 607-613. Boiteux, S., O'Connor, T.R., Lederer, F., Gouyette, A. and Laval, J. (1990) Homogeneous Escherichia coli Fpg protein. A DNA glycosylase which excises imidazole ringopened purines and nicks DNA at apurinic/apyrimidinic sites. J, Biol. Chem. 265, 3916-3922. Boiteux, S., Gajewski, E., Laval, J. and Dizdarogln, M. (1992) Substrate specificity of the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase): Excision of purine lesions in DNA produced by ionizing radiation or photosensitization, Biochemistry 31, 106-110. Breimer, L.H. (1984) Enzymatic excision from ~-irradiated polynueleotides of adenine residues whose imidazole ring has been ruptured. Nucleic Acids Res. 12, 6359-6367. Cadenas, E. and Sies, H. (1984) low-level chemiluminescence as an indicator of singlet molecular oxygen in biological systems. Meth. Enzymol. 105, 221-231. Cadet, J. and Berger, M. (1985) Radiation-induced decomposition of the purine bases with DNA and related model compounds. Int. J. Radiat. Biol. 47, 127-143. Cadet, J., Decarroz, C., Wang, S.Y. and Midden, W.R. (1983) Mechanisms and prod,.~cts of photosensitized degradation of nucleic acids and related model compounds. Israel J. Chem. 23, 420-429. Cadet, J., Berger, M., Decarroz, C., Wagner, J.R., Van Lier, J.E., Ginto, Y.M. and Vig-y, P. (1986) Photosensitized reactions of nucleic acids. Biochimie 68, 813-834. Corm, P.F., Schalch, W. and Truscott, T.G. (1991) The singlet oxygen and carotenoid interaction. J. Photochem. Photobiol. B, Biol. 11, 41-47. Davidson, R.S., Goodwin, D. and Pratt, J.E. (1987) Problems
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Murphy, M.E. and Sies, H. (1990) Visible-range low-level chemiluminescence in biological systems. Meth. Enxymol. 186,595-610. Nieuwint, A.W.R., Aubry, J.M., Arwert, F., Kortbeek, H., Herzberg, S. and Joenje, H. (1985) Inability of chemically generated singlet oxygen to break the DNA backbone. Free Radical Res. Commun. 1, 1-9. Nieuwint, A.W.R., Aubry, J.M., Arwerl, F., Kortbeek, H., Herzberg, S. and Joenje, H. (1987) DNA damage by chemically generated singlet oxygen. Fret: Radical Res. Commun. 2, 343-350. Park, E.-M., Shigenaga, M.K., Degan, P., Korn, T.S., Kitzler, J.W., Wehr, C.M., Kolachana, P. and Ames, B.N. (1992) The assay of excised oxidized DNA lesions: Isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with monoclonal antibody columns. Proc. Natl. Acad. Sci. USA 89, 3375-3379. Piette, J. (1991) Biological consequences associated with DNA oxidation mediated by singlet oxygen. J. Photochem. Photobiol. B, Biol. 11,241-260. Piette, J. and Moore, P.D. (1982) DNA synthesis on phiXI74 template damaged by proflavine and light treatment. Photochem. Photobiol. 35, 705-708. Piette, J., Calberg-Bacq, C.M., Lopez, M. and Van de Vorst, A. (1984) Termination of DNA synthesis on "proflavine and light" treated phiX174 single-stranded DNA. Biochim. Biophys. Acta, 781,257-264. Pung, A., Rundhaug, J.E., Yoshizawa, C.N. and Bertram, J.S. (1988) ~-Carotene and canthaxanthin inhibit chemicallyand physically-induced neoplastic transformation in 10TI/2 cells. Carcinogenesis 9, 1533-1539. Ravanat,J.-L., Berger, M., Buchko, G.W., Benard, J.-F., van Lier, J. and Cadet, J. (1991) Photooxidation sensibilis6e de la desoxy.2'-guanosine par des phthalocyanines et naphthulocyanines. D~termination de I'importance des m~canismes de type I e t de type II. J. Chim. Phys. 88, 1069-1076. Ribeiro, D.T., Bourre, F., Sarasin, A., Di Mascio, P. and Menck, C.F.M. (1992a) DNA synthesis blocking lesions induced by singlet oxygen are targeted to deoxyguanosines. Nucleic Acids Res. 20, 2465-2469. Ribeiro, D.T., Madzak, C., Sarasin, A,, Di Mascio, P., Sies, H. and Menck, C.F.M. (1992b) Singlet oxygen induced DNA damage and mutagenicity in a single-stranded SV40-based shuttle vector. Photochem. Photobiol. 55, 39-45. Schneider, J.E., Price, S., Maidt, L., Gutteridge, J.M.C. and Floyd, R.A. (1990) Methylen~ blue plus light mediates
8-hydroxy-2'-deoxyguanosine formation in DNA preferentially over strand breakage. Nucleic Acids Res. 18, 631635. Schnuriger, B. and Bourdon, J. (1968) Photosensitized oxidation through stearate monomolecular films. Photochem. Photobiol. 8, 361-368. Seidman, M.M., Bredberg, A., Seetharam, S. and Kraemer, K.H. (1987) Multiple point mutations in a shuttle vector propagated in human cells: evidence for an error-prone DNA polymerasc activity. Prec. Natl. Acad. Sci. USA 84, 4944 -4948. Shibutani, S., Takeshita, M. and Grollman, A. (1991) Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature 349. 431-433. Shigenaga, M.K. and Ames, B.N. (1991) Assays for 8-hydroxy2'-deoxyguanosine: a biomarker of in rive oxidative DNA damage. Free Radical Biol. Med. 10, 211-216. Shigenaga, M.K., Park, J.W., Cundy, K.C., Gimeno, C.J. and Ames, B.N. (1990) In rive oxidative DNA damage: measurement of 8-hydroxy-2'-deoxyguanosine in DNA and urine by high-performance liquid chromatography with electrochemical detection. Meth. Enzymol. 186, 521-530. Simon, M.I. and Van Vunakis, H. (1962) The photodynamic reaction of methylene blue with deoxyribonucleic acid. J. Mol. Biol. 4, 488-499. Steenken, S. (1989) Purine bases, nucleosides and nucleotides: Aqueous solution redox chemistry and transformation reactions of their radical cations and e - and OH adducts. Chem. Rev. 89, 503-520. Tyrrell, R.M. and Pidoux, M. (1989) Singlet oxygen involvement in the inactivation of cultured human fibroblasts by UVA (334 nm, 365 nm) and near-visible (405 nm) radiations. Photochem. Photobiol. 49, 407-412. Vaca, C.E., Wihelm, L and Harms-Ringdahl. M. (1988) Interaction of lipid peroxidation products with DNA. A review. Mutation Res. 195, 137-48. Wasserman, H.H. and Murray, R.W. (Eds.) (1979) Singlet Oxygen. Academic Press, New York. Wefers, H., Schulte~Frohlinde, D. and Sies. H. (1987) Loss of transforming activity of plasmid DNA (pBR3221 in E. coil caused by singlel molecular oxygen. FEBS Lctt. 211, 49-52. Wood, M.L,, Dizdar~.glu, M., Gajewski, A. and Essigmann, J.M. (1990) Mechanistic studies of ionizing radiation and oxidative mutagenesis, genetic effects of a single 8-hydroxyguanine (7-hydro-8-oxoguanine) residue ,n,~erted at a unique site in a viral genome. Biochemistry 29, 7024-7032.
367
MUTAGI 0253
Singlet oxygen induced DNA damage H e l m u t Sies a and Carlos F.M. M e n c k b lnstitut far Physiologische Chemic I, Uni~'ersitiitDiisseldo~ Diisseldo~ Germany and t, Departamento de Biologia, Instituto de Biociencias, Unit,ersidadede Sao Paulo, Sao Paulo 05499, Brazil {Received 8 March 1992) (Revision received 18 May 1992) (Accepted 27 May 1992)
Keywords: Singlet oxygen; Endoperoxides; DNA damage; Shuttle vectors
Summary Singlet oxygen generated by photoexcitation and by ehemiexcitation selectively reacts with the guanine moiety in nueleosides (kq + k r about 5 × 10 6 M - I s - i ) and in DNA. The oxidation products include 8-oxo-7.hydro.deoxyguanosine (8-oxodG; also called 8-hydroxydeoxyguanosine) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Singlet oxygen also causes alkali-labile sites and single-strand breaks in DNA. The biological consequences include a loss of transforming activity as studied with plasmids and bacteriophage DNA, and mutagenicity and genotoxicity. Employing shuttle vectors, it was shown that double-stranded vectors carrying singlet oxygen induced lesions seem to be processed in mammalian cells by DNA repair mechanisms efficient in preserving the biological activity of the plasmid but highly mutagenic in mammalian cells. Biological protection against singlet oxygen is afforded by quenchers, notably carotenoids and tocopherols. Major repair occurs by excision of the oxidized deoxyguanosine moieties by the Fpg protein, preventing mismatch of 8-oxodG with dA, which would generate G: C to T: A transversions.
Electronically excited molecular oxygen (singlet oxygen, ~O2) can be generated by photocheraical reactions through transfer of excitation energy to ground-state oxygen (30 2) from a suitable excited triplet-state sensitizer (photoexeitation). Since the discovery of singlet oxygen by Kautsky and de Bruijn (1931), the type I1 photooxygenation reactions have been intensely investigated (Goilniek, 1968; Foote, 1990). However, singlet oxygen can also be produced by dark reactions (chemiexcitation), e.g., by the decomposition of
Correspondence: ProL Dr. H. Sies, lnstitut ffir Physiologische Chemie I, Moorenstrasse 5, W-4000-Diisseidorf, German;,.
endoperoxides and several other types of reaction (Wasserman and Murray, 1979; Adan. and Cilento, 1982). In biological systems, these dark reactions occur in lipid peroxidation and by enzyme reactions such as those catalyzed by lactoperoxidase, lipoxygenase and chloroperoxidase (for reviews, see Cadenas and Sies, 1984; Kanofsky, 1989). Singlet oxygen is also produced during photooxidation of a variety of biological compounds and xenobiotics (Krinsky, 1977; Krasnovsky, 1991). It is relatively long-lived, with half-times in the range of 4-50/~s, so that diffusion of singlet oxygen is possible within a radius estimated to be in the range of 100 A (Schnuriger and Bourdon, 1968; Moan, 1990).
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Reactions of singlet oxygen Reactions of singlet oxygen are physical and/or chemical (Kasha and Khan, 1970; Krasnovsky, 1979). The physical reactivlty is characterized by photoemissive decay, which can occur in two modes, termed monomol and dimol reactions, characterized by the emission at 1268 nm, and at 634 nm and 703 nm, respectively; this photoemission is used for the detection of singlet oxygen, employing liquid nitrogen-cooled germanium photodiode detectors for the 1268 nm infrared emission and red-sensitive photomultipliers cooled to about -25°C for the dimol emission, also called ultraweak chemiluminescence. The chemical reactions are manifold, including 1,2, 1,3 and 1,4 additions to olefins, forming dioxetanes, allylic hydroperoxides ('ene reaction'), and endoperoxides, respectively, as well as oxidation of sulfides or phenols to form sulfoxides or hydroperoxydienones, for example (Wasserman and Murray, 1979; Frimer, 1985; Aubry, 1991). This chemical reactivity is the basis of biological damage inflicted by ringlet oxygen.
Singlet oxygen reactivity with nucleosides and DNA
Guanine oxidation products This topic has recently been reviewed by Piette (1991). Photosensitization causes damage to deO
oxyguanosine almost exclusively, either as free nucleoside or in DNA (Simon and van Vunakis, 1962; Gutter et al, 1977; Friedman and Brown, 1978; Piette and Moore, 1982; Piette et al., 1984; Kawanishi et al., 1986). Model experiments by Cadet et al. (1983,1986) using Y,5'-di-O-acetyl2'-deoxyguanosine showed that three main products arose from the reaction with singlet oxygen, namely the two diastereomers of the 4,8-dihydro4-hydroxy-8-oxo-deoxyguanosine derivative, and the cyanuric acid derivative. It is assumed that the former arise from a 1,4-cycloaddition leading to an unstable endoperoxide (Ravanat et al., 1991). The cyanuric acid derivative is likely to be formed via a transient enamine as a substrate for a second addition of a ringlet oxygen molecule (Matsuura et al., 1972). Boiteux et al. (1992) have detected the formation of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) as a product from deoxyguanosine residues in calf thymus DNA using photosensitization with visible light and methylene blue. 8-Hydroxydeoxyguanosine, better called 8-oxo7-hydrodeoxyguanosine (abbreviated 8-oxodG), was described by Floyd et al. (1989) as a product of deoxyguanosine oxidation by ringlet oxygen in DNA, employing methylene blue and light. This was extended to other photosonsitizers, e.g., thiazinc dyes (Floyd ¢t al. 1990), It should he mentioned that the generation of singlet oxygen is not the only reaction occurring, e.g., with methylene
0
m
HN~I"~N-~(dlribose
0 +2[H]
OH \\(d)ribose
:_
H2N'~NO~oHNX~Hibdose )r -H20
H
IN.o
H2N H ~ N ~ I ~ \ ( d ) ribose Fig. 1. A pathwayfrom the initial guanineendoperoxideto 8-oxoG,From Ravanatet al. (1991)and Devasagayamet aL (1991b). Note that Boiteaxet al. (1992)haveproposedan alternativepathway,via electrontransfergeneratingG+ andO~" (seetext).
369
blue only 55%, so that other (type !) reactions might influence the damage pattern. Recently, Devasagayam et al. (1991b) demonstrated that a dark reaction, i.e., the generation of singlet oxygen by thermodissociation of an endoperoxide (DiMascio and Sies, 1989), also results in the formation of 8-oxoG or 8-oxodG. When deoxyadenosine was exposed to singlet oxygen, there was no formation of the corresponding 8-oxo product (Devasagayam et al., 1991b). However, radiation-chemically generated hydroxyl radicals give rise to the corresponding 8-oxo products from both deoxyguanosine and deoxyadenosine (Cadet and Berger, 1985; Dizdaroglu, 1985; Steenken, 1989). It thus appears that the formation of the 4-hydroxy-8-oxoguanosine derivative is a selective initial product generated from singlet oxygen via an unstable endoperoxide (Ravanat et al., 1991). As discussed by Devasagayam et al. (1991b), such a type of intermediate may be on the pathway of 8-oxoG or 8-oxodG formation. For the endoperoxide to give the 8-oxopurine, two reducing equivalents have to be supplied (Fig. 1). Boiteux et ai. (1992) suggested that the formation of FapyGua and 8-oxodG might be explained by an initial electron transfer from the guanine ring to singlet oxygen, resulting in the generation of a guanine radical cation and the superoxide radical anion. Further reactions of the guanine radical cation may then result in the for,nation of a C-8 OH adduct radical of guanine. T~7 C-8 OH adduct radicals of purines are ktiown ~o produce formamidopyrimidines upon ring-opening followed by one-electron reduction, and 8-oxopufines are formed upon one-electron oxidation (for review, see Steenken, 1989).
Quenching of photoemission The quenching of monomol photoemission of singlet oxygen by guanosine occurs with the overall singlet oxygen quenching rate constant, kq + k r, of 6.2 × 106 M-Is -1, where kqis the physical quenching rate constant and k, is the chemical reaction rate constant. The value for the overall quenching rate constant for deoxyguanosine is 5.2× 106 M - i s -t (Devasagayam et al., 1991b). This value is almost identical with that (5.3 x 106 M - i s -1) measured for 2'-deoxyguanosine-5'-
monophosphate by use of time-resolved singlet oxygen emission decay techniques (Lee and Rodgers, 1987). Single-strand break formation by singlet oxygen
Singlet oxygen was shown to lead to singlestrand breaks in plasmid DNA (Wefers et al., 1987; DiMascio et al., 1989b; Blazek et al., 1989; Devasagayam et al., 1991a) and in bacteriophage DNA (DiMascio et al., 1989b). In these studies, several techniques for the generation of singlet oxygen were employed, including the thermal dissociation of an endoperoxide, as mentioned above, as well as the use of microwave discharge and the diffusion from immobilized rose bengal on a cover slip. Earlier work by Nieuwint et al. (1985, 1987) and Lafleur et al. (1987) resulted in no single-strand breaks; in these experiments, much lower concentrations of singlet oxygen available to the DNA were employed. That singlet oxygen mediates the single-strand breaks is supported by the enhancement of the effect caused by solvent deuteration, which is known to increase the lifetime of singlet oxygen, and by a quenching effect caused by the addition of sodium azide (Wefers et al., 1987; DiMascio et al., 1989b; Blazck et al., 1989; Devasagayam et al., 1991a). Some biologically occurring thiols, glutathione, cysteamine and cysteine, enhanced the single-strand breaks in plasmid pBR322 DNA; lipoate (thioctic acid) protected (Devasagayam et al., 1991,~,,b). Epe et al. (1988) examined the singlet oxygen induced DNA damage profile in PM2 DNA and found it to be different from that induced by hydroxyl radicals, as did Lafleur et al. (1987), with single-strand breaks making up only less than 5% of the total DNA damage. That single-strand breaks are relatively rare events as compared to, e.g., 8-oxodG formation is known from the work by Schneider et al. (1990), who calculated that single-strand nicking was approx. 17 times less than 8-oxodG formation, using methylene blue plus light. The mechanism of singlet oxygen induced strand break formation is unknown. It is possible that 8-oxodG and strand breaks are derived from a common precursor, possibly the initial endoper-
370
oxide mentioned above (Fig. 1). This is in agreement with the observations by Devasagayam et al. (1991b) that (i) the time courses of the two effects are similar, and (ii) that the single-strand breaks occur selectively at guanine residues. The latter was determined using a plasmid containing a fragment of the mouse metallothionein I promoter and a novel end-labeling technique; no major differences (hot spots) were observed between individual guanines in these experiments (Devasagayam et al., 1991b). A major type of singlet oxygen induced DNA damage is the generation of alkali-labile sites (ca. 30% of all the damage), as studied by Nieuwint et al. (1985) and Lafleur et al. (1987). In comparison, strand-breaks are formed only at a frequency of 2-5% (DiMascio et al., 1989b; Epe et al., 1988; Joenje et al., 1991).
TABLE 1 COMPARISON OF LOSS OF TRANSFORMING ACTIVITY AND LOSS OF SUPERCOILED DNA UPON EXPO. SURE OF pBR322 TO SINGLET OXYGEN
Control tO 2 tO 2, D20 tO 2, plus NaN3 (20 raM) IO 2, plus methionine (10 raM) t o 2, plus methionine sulfone (10 raM)
Transforming activity a (%)
Supercoiled DNA b (%)
100 32.1 ±4.6 14.1 ± 1.5
91,9 :i:0.7 49,1 ± 1.8 30,25:2.2
84.3 ± 3.8
86.2 ± 2.6
77.5±1,6
69.1+1.7
58.3 ± 6,1
45.8 ± 3.1
DNA solutions were exposed for 20 min to a gas stream containing tO z generated by microwave discharge. a Data from Wefers et aL (1987). b Dala from Di Mascio et aL (1989b).
Biological consequences
Loss of transforming activity in experiments with plasmid pBR322 DNA (Wefers et al., 1987) and with phage $X174 DNA (Lafleur et al., 1987; DiMascio et ai., 1989b), it was observed that the transforming activity in E. coli was lost, with the single-stranded form of the phage DNA being more sensitive than the double-stranded form. This indicates that nonrepaired guanine oxidation products and/or singlestrand breaks have the capacity to inhibit plasmid or phage DNA replication in competent bacteria. Loss of transforming capacity essentially parallels single-strand break formation (Table 1; Wefers et ai., 1987).
Mutagenicity and genotoxicity The cytotoxicity as well as mutagenicity and genotoxicity exhibited by singlet oxygen have been studied in recent years, also with regard to photodynamic therapy; this will not be discussed here. The most recent surveys on the genotoxicity of singlet oxygen have been given by Epe (1991) and by Piette (1991). An enhancement of mutagenesis was detected in rodent cells exposed to photosensitized rose bengal (Gruener and Lockwood, 1979) and phthalocyanines (Ben-Hur et al., 1987). DNA sin-
gle-strand breaks and sister-chromatid exchanges were shown to be induced in human cells treated with hematoporphyrin and light (Moan et ai., 1980). A dose-dependent increase of sister-chromatid exchanges was observed after treatment of human lymphocytes with extracellularly generated singlet oxygen (Decuyper-Debergh ¢t al., 1989), employing the surface-separated method of Midden and Wang (1983). The most frequent mutations detected in M13 iacZ phage DNA were O : C to T : A transversions; no significant mutagenicity was induced at T: A base pairs (Decuyper-Debergh et al., 1987). The potential mutagenie effect of 8-oxodG suspected from experiments by Kuchino et al. (1987) was recently established (Wood et ai., 1990; Moriya et al., 1991; Shibutani et aL, 1991). 8-oxodO also base-pairs with dA in DNA, since the oxidized base is pref¢rably present in its syn form (Kouchakdiian et al., 1991). Thus, 8-oxodG contributes to spontaneous mutagenesis by favoring the G : C to T: A transversion, Using human fibroblasts, TyrreU and Pidoux (1989) showed that cell killing by UVA (334 and 365 nm) and near-visible (405 nm) radiations may involve singlet oxygen. The implication of singlet oxygen in these effects was based mainly on an enhancement of the effects by deuterium oxide
371
and on the diminution of the effects by singlet oxygen quenchers such as azide. However, there is no evidence that the genetic alterations which were observed are directly resultant from the interaction between singlet oxygen and the chromosomes. It is also possible that singlet oxygen first reacts with cell membrane components leading to lipid peroxidation, the products of which in turn might react with the DNA (Vaca et al., 1988). Shuttle vector experiments In order to detect the direct consequences of singlet oxygen induced DNA damage in mammalian cells, our own recent work was performed using shuttle vectors. These are hybrid plasmids containing DNA sequences derived from pBR322 and from a mammalian virus, for replication both
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Fig. 2. Induction of DNA single-strand breaks (A) and mutagenesis (13) in ss (0) and ds (e) DNA after NDPO 2 treatment. The DNA samples were treated with the indicated concentrations of NDPO 2.
20-
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Fig. 3. Mutagenesis per single strand break in ss (o) and ds (o) SV40-based shuttle vectors. Data are from Fig. 2.
in bacteria and in eukaryotic cells; hence the term shuttle vector. The plasmids used were SV40-based vectors derived from those described before in detail (Menck et al., 1989). In these experiments, the vectors were treated in vitro as naked DNA with the thermolabile endoperoxide generating singlet oxygen (DiMascio and Sies, 1989). The damaging action of singlet oxygen on the shuttle vectors themselves results in single-strand breaks in both dsDNA (DiMascio et ai., 1990a) and ssDNA (Ribeiro et al., 1992b). Moreover, double-strand breaks were also detectable in dsDNA after exposure to high doses of singlet origen (DiMascio et aL, 1990a). The vectors replicated in monkey COS7 cells, and the results revealed that there is a dose-dependent increase in the mutation frequency on the supF gene in the singlet oxygen treated ds or ss plasmids (Fig. 2A). The higher susceptibility of ssDNA may be due to increased accessibility of singlet oxygen to its main target, the guanine residues, which are potentially less readily reachable in the bases arranged in the double helix. Likewise, the mutation frequency is higher for ssDNA than for dsDNA (Fig. 2B). However, as shown in Fig. 3, this higher mutagenicity cannot be attributed solely to the greater number of lesions. In fact, when the mutagenesis data are plotted against the number of breaks, the figure shows exactly the inverse, that is, more mutations per break are detected in dsDNA than in ssDNA.
372
One explanation could be that premutagenic lesions other than breaks, i.e., 8-oxodG or FapyGua, may be more abundant in dsDNA. As mentioned above, 8-oxodG was found to occur 17-fold more frequently than single-strand breaks (Schneider et ai., 1990). Ribeiro et al. (1992a) observed that in vitro DNA polymerase blocking sites also exceed the number of breaks in singlet oxygen treated ssDNA by 20-fold. A ~cond explanation for the higher mutability of damage in dsDNA is that the singlet oxygen induced lesions may be processed in mammalian cells differently in dsDNA and ssDNA. For ssDNA, the absence of a template makes the repair of damage more difficult, so that the lesions must be bypassed during replication. Lesions not bypassed would lead to vector inactivation. Therefore, the results shown in Fig. 3 indicate that the singlet oxygen induced lesions in dsDNA are processed by error-prone repair in mammalian cells. This repair is unable to act on ssDNA vectors. Such conclusions are consistent with the much higher level of inactivation of damaged vectors transfected in the ss structure in relation to dsDNA (Ribeiro et al., 1992b). Moreover, the DNA sequence of those singlet oxygen induced mutants revealed that part of them contain multiple base changes (Oliveira et al., unpublished results), a type of mutation which can be due to error-prone DNA polymerization during excision repair (Seidman ct al., 1987). Thus, the ds vectors carrying singlet oxygen induced lesions seem to be processed by DNA repair mechanisms efficient in preserving the biological activity of the plasmid but highly mutagenic in mammalian cells.
Protection and repair mechanisms As is obvious from this discussion, defense against singlet oxygen induced mutagenesis and genotoxic lesions is multiple. In terms of protection, the prevention of the reaction of singlet oxygen with guanosines in DNA by quenching is of major importance. A number of biological compounds have been shown to quench singlet oxygen, notably the carotenoids (Foote and Denny, 1968; Krasnovsky, 1979). The capability of canthaxanthin, a carotenoid not serving as provitamin A, in protecting against chemically and physically induced neoplastic transformation just
as p-carotene does, has sparked recent interest in the biological properties of carotenoids not linked to their functions as precursor of vitamin A (lung et al., 1988; Bertram et al., 1991). Lycopene is the most efficient biological singlet oxygen quencher (DiMascio et al., 1989a; Corm et al., ~991). Also, tocopherols and some biological thiols are singlet oxygen quenchers (Kaiser et al., 1990; Devasagayam et al., 1991c; DiMascio et al., 1990b). Whether these reactivities are related to the biological protection of DNA in cells and tissues is being studied in several laboratories. The repair of DNA containing the oxidized guanosine residues occurs by excision. The excised product, 8-oxodG, can be detected in urine, as an indicator of in vivo oxidative DNA damage (Shigenaga and Ames, 1990; Shigenaga et al., 1991; Park et al., 1992). The enzyme catalyzing excision of these purine lesions in E. coil is Fpg protein (formamidopyrimidine-DNA glycosylase), described by Boiteux et al. (1990, 1992). An enzyme which has a DNA glycosylase activity similar to Fpg protein exists in mammalian cells (Margisson and Pegg, 1981) and has been partially purified (Breimer, 1984; Laval et al., 1990). Kasai et al. (1986) have detected 8-oxodG in DNA from mammalian cells irradiated with ,/-rays. They also found that the amount of this lesion decreases with time after irradiation, suggesting the presence of repair enzymes acting on 8-oxodGs in these cells. More recently, 8-oxodG was measured in various organs from rats of different ages (Fraga et al., 1990). The amount of this DNA lesion was found to increase with age in liver, kidney and intestine, but remained unchanged in brain and testes. These authors have also shown the excretion of 8-oxodG in the urine of these animals, as a possible consequence of its repair from DNA by nuclease activity. The amount excreted in the urine decreased with age. Thus, the age-dependent accumulation of 8oxodO residues in DNA may be related to the. loss of DNA repair activity or to an increase in the rate of oxidative DNA damage.
Acknowledgements We gratefully acknowledge the fruitful cooperation and helpful discussion of our colleagues
373
and coworkers as cited in the references. Our studies were. supported by the National Foundation for Cancer Research, Bet.hesda, by the Jung-Stiftung fiir Medizin, Hamburg, and by CNPO and FAPESP, Brazil. References Adam, W. and Cilento, G. (1982) Chemical and Biological Generation of Electronically Excited States. Academic Press, New York. Aubry, J.M. (1991) New chemical sources of singlet oxygen. In: C. Vigo-Pelfrey (Ed.), Membrane Lipid Peroxidation II. CRC Press, Boca Raton, FL, pp. 65-102. Ben-Hut, E., Fujihara, T., Suzuki, F. and Elkind, M.M. (1987) Genetic toxicology of the photosensitization of Chinese hamster cells by phthalocyanines. Photochem. Photobiol. 45, 227-230. Bertram, J.S., Pung, A., Churley, M., Kappock, T.J., Wiikins, L.R. and Cooney, R.V. (1991) Diverse carotenoids protect against chemically induced neoplastic transformation. Carcinogenesis 12, 671-678. Blazek, E.R., Peak, J.G. and Peak, M.J. (1989) Singlet oxygen induces frank strand breaks as well as alkali- and piperidine-labile sites in supercoiled plasmid DNA. Photochem. Photobiol. 49, 607-613. Boiteux, S., O'Connor, T.R., Lederer, F., Gouyette, A. and Laval, J. (1990) Homogeneous Escherichia coli Fpg protein. A DNA glycosylase which excises imidazole ringopened purines and nicks DNA at apurinic/apyrimidinic sites. J, Biol. Chem. 265, 3916-3922. Boiteux, S., Gajewski, E., Laval, J. and Dizdarogln, M. (1992) Substrate specificity of the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase): Excision of purine lesions in DNA produced by ionizing radiation or photosensitization, Biochemistry 31, 106-110. Breimer, L.H. (1984) Enzymatic excision from ~-irradiated polynueleotides of adenine residues whose imidazole ring has been ruptured. Nucleic Acids Res. 12, 6359-6367. Cadenas, E. and Sies, H. (1984) low-level chemiluminescence as an indicator of singlet molecular oxygen in biological systems. Meth. Enzymol. 105, 221-231. Cadet, J. and Berger, M. (1985) Radiation-induced decomposition of the purine bases with DNA and related model compounds. Int. J. Radiat. Biol. 47, 127-143. Cadet, J., Decarroz, C., Wang, S.Y. and Midden, W.R. (1983) Mechanisms and prod,.~cts of photosensitized degradation of nucleic acids and related model compounds. Israel J. Chem. 23, 420-429. Cadet, J., Berger, M., Decarroz, C., Wagner, J.R., Van Lier, J.E., Ginto, Y.M. and Vig-y, P. (1986) Photosensitized reactions of nucleic acids. Biochimie 68, 813-834. Corm, P.F., Schalch, W. and Truscott, T.G. (1991) The singlet oxygen and carotenoid interaction. J. Photochem. Photobiol. B, Biol. 11, 41-47. Davidson, R.S., Goodwin, D. and Pratt, J.E. (1987) Problems
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