Photosensitization of Guanine-Specific DNA Damage by 2

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Chem. Res. Toxicol. 1999, 12, 38-45

Photosensitization of Guanine-Specific DNA Damage by 2-Phenylbenzimidazole and the Sunscreen Agent 2-Phenylbenzimidazole-5-sulfonic Acid Clarke Stevenson and R. Jeremy H. Davies* School of Biology and Biochemistry, Medical Biology Centre, Queen’s University, Belfast BT9 7BL, Northern Ireland Received July 7, 1998

Gel sequencing experiments with end-labeled synthetic oligodeoxyribonucleotides have established that 2-phenylbenzimidazole (PBZ) and the common sunscreen constituent 2-phenylbenzimidazole-5-sulfonic acid (PBSA) function as efficient photosensitizers of DNA damage when they are exposed to UV-B (290-320 nm) radiation or natural sunlight. Although neither compound binds specifically to DNA, both are active at sub-millimolar concentrations and induce the formation of piperidine-labile cleavage sites that map almost exclusively to the positions of guanine residues. The pattern of attack on single-stranded DNA, where all guanines are modified to a similar extent, is typical of photooxidation by singlet oxygen. The involvement of singlet oxygen is consistent with the effect of quenchers and scavengers on the reaction, and is supported by the demonstration that UV-B irradiation of 2′-deoxyguanosine with PBSA in oxygenated solution generates the diagnostic compound 4,8-dihydro-4-hydroxy-8-oxo-2′-deoxyguanosine in comparatively high yield. In contrast, the main photoinduced cleavage sites in double-helical DNA are located at the 5′-guanines of GG and (to a lesser degree) GA doublets. This characteristic behavior implies that electron transfer from DNA to the photoexcited sensitizer is the predominant mechanism in this conformation. A similar dichotomy of reactivity toward denatured and native DNA has been reported for riboflavin and certain pterin derivatives which resemble PBZ and PBSA in not binding tightly to DNA. The photosensitizing properties of PBSA could possibly detract from its fitness as a sunscreen agent.

Introduction Numerous proprietary sunscreen preparations are currently available for protecting human skin from sunburn and the longer-term consequences of exposure to sunlight, such as actinic aging and carcinogenesis (1). Their active ingredients include organic compounds which absorb strongly at UV-B1 (290-320 nm) and/or UV-A (320-400 nm) wavelengths and thereby attenuate the harmful high-energy photons in sunlight. However, this beneficial shielding effect can be compromised if the photoexcited sunscreen agents are prone to decomposition or react chemically with cellular components. DNA molecules, in particular, are susceptible to genotoxic damage caused by the independent light absorption of endogenous or xenobiotic compounds that act as photosensitizers (2-7). Several sunscreen active ingredients (8) have been found to sensitize the production of singlet molecular oxygen (1O2), including p-aminobenzoic acid which can also form photoadducts with thymine and thymidine (9). The related compound Padimate-O [(2ethylhexyl)-4-(dimethylamino)benzoate] has been shown (10) to induce mutagenic lesions in DNA via a free radical mechanism. * To whom correspondence should be addressed. Telephone: 00-441232-272102. Fax: 00-44-1232-236505. E-mail: [email protected]. 1 Abbreviations: DABCO, 1,4-diazabicyclo[2.2.2]octane; dGuo, 2′deoxyguanosine; 1O2, singlet molecular oxygen; 4-OH-8-oxodGuo, 4,8dihydro-4-hydroxy-8-oxo-2′-deoxyguanosine; PBSA, 2-phenylbenzimidazole-5-sulfonic acid; PBZ, 2-phenylbenzimidazole; PN, perinaphthenone; UV-A, 320-400 nm radiation; UV-B, 290-320 nm radiation.

Here, we report that 2-phenylbenzimidazole-5-sulfonic acid (PBSA), which is widely used as a UV-B filter in sunscreen formulations and cosmetics (11), behaves as an efficient photosensitizer of guanine base damage in DNA. This property is also exhibited by the parent compound 2-phenylbenzimidazole (PBZ) and may therefore extend to other PBZ derivatives, some of which possess antileukemic activity (12) or act as topoisomerase I poisons (13). Our experimental evidence indicates that (as frequently occurs) PBSA is capable of sensitizing photooxidation by both type I and type II mechanistic pathways. Type I processes involve electron or hydrogen atom transfer between excited state sensitizer molecules and the substrate, whereas type II processes are initiated by triplet energy transfer from the sensitizer to molecular oxygen, thus generating highly reactive 1O2 (14). A wide variety of photosensitizers are known that will damage DNA when they are excited at UV or visible wavelengths (2, 5). Their action can be investigated very effectively by means of gel sequencing protocols (15-17) with end-labeled DNA molecules. Although DNA segments obtained by digestion with restriction endonucleases are routinely used for this purpose, shorter synthetic oligodeoxyribonucleotides offer some advantages because they can be designed to incorporate specific sequence contexts and to avoid the possibility of partial renaturation with single-stranded molecules. It is now well-established (14) that photosensitization of DNA, under aerobic conditions, leads predominantly to oxidative modifications of the guanine bases; these lesions can

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Photosensitization of DNA by Phenylbenzimidazoles

be detected as alkali-labile chain cleavage sites when the DNA is subsequently heated with piperidine. Their locations can be mapped at nucleotide resolution by comparing the electrophoretic mobility of the cleavage fragments with those in a reference chemical sequencing ladder (18). This approach has revealed distinct patterns of photodamage associated with type I and type II processes. In agreement with theoretical predictions (19) that the 5′-guanines of GG doublets are the most readily oxidized nucleobases in native DNA, some type I photosensitizers selectively induce damage at these sites with little modification of other base residues (20-23). Oneelectron oxidation of the reactive guanines [which may occur by hole migration (24, 25)] leads, via the guanine radical cation, to alkali-labile end products, including 7,8dihydro-8-oxo-2′-deoxyguanosine (20, 23, 25, 26) and an oxazolone derivative (26). By contrast, 1O2 (characteristic of type II photosensitization) modifies guanine bases fairly uniformly in all sequence contexts (27), but the rate of attack is much greater for denatured than for native DNA (28, 29). Again, 8-oxo-7,8-dihydro-2′-deoxyguanosine is a major product (6, 30), but it can be oxidized further to the deoxyriboside of cyanuric acid (31). In this study, we show that PBSA and PBZ act as photosensitizers toward DNA when they are irradiated either exclusively with UV-B or with natural sunlight. The distribution of alkali-labile lesions observed in target oligonucleotides suggests that the mechanism of photooxidation is predominantly type I in duplex DNA and type II in single-stranded DNA.

Experimental Section Materials. 2-Phenylbenzimidazole (Aldrich, 97% pure) and 2-phenylbenzimidazole-5-sulfonic acid (Janssen Chimica, 98% pure) were decolorized with charcoal and recrystallized from aqueous ethanol before use; perinaphthenone ([1H]phenalen-1one) from Aldrich (97% pure) was recrystallized from butan-2ol. After purification, all three compounds eluted as single sharp peaks on HPLC with detection at 214 nm. [γ-32P]ATP, [R-32P]ddATP, and terminal deoxynucleotidyl transferase (calf thymus) were supplied by Amersham International, and T4 polynucleotide kinase was supplied by New England Biolabs. Reagents for oligonucleotide synthesis were obtained from Applied Biosystems/Perkin-Elmer; all other reagents and enzymes were from Sigma. Target Oligonucleotides. The oligodeoxyribonucleotides used in this study were assembled from nucleoside β-cyanoethyl N,N-diisopropylaminophosphoramidites on an Applied Biosystems 380B DNA synthesizer. Specific reference is made to the following oligomers: 34-mer, 5′-CATATGACGGCATTCGTTCACGGGTTACTGCAAA-3′; 35-mer, 5′-TCAGACAGTTGATCGGCTGACCAGCGCATCGTGCC-3′; 40-mer, 5′-TTTAGCACGTGCGGACAGGTTCGAGAGGGCACGTGCTAAA-3′; and 50-mer, 5′-CAGTTCGTGCTCGGGGCATATGACGGCATTCGTTCACGGGCTACTGTACA-3′. Oligonucleotides with sequences complementary to those of the 34-mer and 35-mer were also synthesized. After deprotection, oligonucleotides were purified by electrophoresis on 12% polyacrylamide gels containing 7 M urea and then recovered by electroelution using a Biotrap kit (Schleicher and Schuell, Dassel, Germany). Standard protocols (32) were employed to end-label oligonucleotides either at the 5′-terminus with [γ-32P]ATP and T4 polynucleotide kinase or at the 3′terminus by treatment with terminal deoxynucleotidyl transferase and [R-32P]ddATP. Unincorporated radioactivity was removed (33) by applying the reaction mixture to a 1 mL spin column of DNA grade Sephadex G-25 (Pharmacia Biotech) and eluting with 50 mM ammonium formate (pH 7.0). The collected

Chem. Res. Toxicol., Vol. 12, No. 1, 1999 39 samples were lyophilized, redissolved in 0.3 M sodium acetate (pH 5.2), and then precipitated at -20 °C after addition of 3 volumes of ethanol. The oligonucleotide pellets were freed from salt by washing them twice with ice-cold 90% ethanol and then dried in vacuo. To prepare double-stranded DNA molecules containing the 34-mer or 35-mer, they were annealed with a 1.5-fold molar excess of the unlabeled complementary strand by heating to 90 °C and cooling slowly to 4 °C. For this purpose, the 34-mer was dissolved in 25 mM sodium phosphate (pH 7.4), whereas the 35-mer was dissolved in 50 mM Tris-HCl (pH 7.5) containing 50 mM NaCl. The latter buffer was also used for annealing the 40-mer to produce a hairpin secondary structure. General Procedures. Isocratic HPLC was carried out with a solvent mix of acetonitrile and 25 mM ammonium formate (3:1, v/v) on a Waters µBondapak NH2 radial compression cartridge (8 mm × 100 mm) at a flow rate of 3 mL/min; the detection wavelength was 214 nm. Equilibrium dialysis was conducted at room temperature (23 ( 2 °C) by placing a dialysis cassette (Pierce), filled with 4 mL of a solution of DNA in 50 mM sodium phosphate buffer (pH 7.4), in a beaker containing 500 mL of a solution of PBSA in the same buffer. After the contents of the beaker were stirred for 24 h, the final concentrations of PBSA inside and outside the cassette were measured spectrophotometrically. Circular dichroism spectra were obtained with a Jasco model J720 spectropolarimeter and electrospray mass spectra with a Fisons VG Quattro instrument. Fluorometric measurements were made with a Perkin-Elmer MPF-44B spectrofluorimeter. UV Irradiation. Samples with a final volume of 200 µL, containing 20-40 pmol of end-labeled oligonucleotide in 5 mM sodium phosphate buffer (pH 7.4), were prepared by diluting small aliquots (typically 5 µL) of stock solutions with appropriate quantities of concentrated phosphate buffer and water; photosensitizers and quenchers were also added as required. The samples were placed in the inverted caps of 1.5 mL Eppendorf vials and positioned approximately 10 cm below a UV-B source and irradiated through a 295 nm cutoff glass filter (Schott, Mainz, Germany) at either 4 °C or room temperature. For experiments carried out at 4 °C, the UV-B source used was a model TM-15 transilluminator (UltraViolet Products, Upland, CA) with peak irradiance at 302 nm, while at room temperature, it was a VL-15M lamp (Vilber Lourmat, Torcy, France) with peak irradiance at 312 nm. In both cases, the incident fluence rate on the samples, measured with a UVX radiometer and UVX-31 sensor (UltraViolet Products), was in the range of 1012 J m-2 s-1. Samples irradiated with sunlight (midday, summer, latitude of 54° N) were contained in sealed 200 µL thinwalled clear plastic PCR tubes (Applied Biotechnologies). To recover the irradiated DNA, each 200 µL sample was mixed with 133 µL of a solution containing deproteinized calf thymus DNA (7.5 µg/mL) in 1.1 M sodium acetate (pH 5.2). Ethanol (1 mL) was then added, and the DNA that precipitated at -20 °C was pelleted, washed with 90% ethanol, and dried. Cleavage and Sequencing of DNA. Base-specific chemical modification reactions for DNA cleavage at G and C (18), A+G (33), and T.G (34) were carried out by standard methods. To effect strand scission, vacuum-dried samples of UV-irradiated or chemically modified DNA were heated with 1 M piperidine (100 µL) at 90 °C for 30 min. After extensive lyophilization to remove piperidine, samples were dissolved in loading solution and electrophoresed on sequencing gels containing 16% polyacrylamide and 7 M urea as described elsewhere (35). Gels were autoradiographed at -20 °C with medical X-ray film (Fuji), and profiles of individual lanes were recorded with a Bio-Rad model 620 video densitometer. Photooxidation of 2′-Deoxyguanosine (dGuo). In accordance with the procedure of Ravanat and Cadet (36), aliquots of a neutral aqueous solution containing dGuo (1 mM) and PBSA (0.5 mM) were irradiated (for 3 h at 10 °C) with UV-B, as described above, in a quartz cuvette (5 cm × 4 cm × 0.4 cm) through which air was constantly bubbled to maintain the concentration of dissolved oxygen. The residue obtained by

40 Chem. Res. Toxicol., Vol. 12, No. 1, 1999

Stevenson and Davies

Figure 1. Chemical structures of PBZ and PBSA and their UV absorption spectra at pH 7.4. evaporating 40 mL of the irradiated solution to dryness was extracted thoroughly with water (2 mL) and then centrifuged to remove undissolved starting material. The supernatant was passed through a micropore filter (0.45 µm) prior to fractionation by HPLC. Photooxidation with methylene blue (0.5 mM) was effected by irradiation with a tungsten lamp, and the products were isolated in a similar fashion. Single irradiations (at 312 nm, with bubbled oxygen and at room temperature) were employed to compare 1O2 generation by PBSA and perinaphthenone (PN). The sample solutions comprised dGuo (1 mM) and either PBSA (1 mM) or PN (1.5 mM) dissolved in 5 mM sodium phosphate (pH 7.4) containing 20% ethanol. After exposure to an incident UV-B fluence of 70 kJ m-2, the contents of the cuvette were lyophilized and the material extracted into 300 µL of water, by warming to 50 °C and vortexing, was analyzed by HPLC.

Results The photosensitizing properties of PBZ and PBSA are derived from their strong absorption in the UV-B region which is shown in Figure 1. In neutral aqueous solution PBZ, whose pKa equals 4.51 (37), is uncharged, but PBSA (whose basic pKa was determined by spectrophotometry to be 4.0) exists as the sulfonate monoanion. This accounts for the much greater solubility of PBSA (10 mM) as compared to PBZ (0.3 mM) in 5 mM phosphate buffer (pH 7.4) at 20 °C. Unless otherwise specified, the photosensitization experiments with DNA were carried out in the presence of 0.2 mM PBZ or PBSA. As judged by the constancy of their UV spectra, both compounds were stable toward photodecomposition under the irradiation conditions used. Neither PBZ nor PBSA showed measurable binding affinity for duplex DNA at 23 °C under the conditions

Figure 2. Gel autoradiogram for 5′-end-labeled 34-mer irradiated with UV-B in the presence of 0.2 mM PBSA. The incident fluence was 4 kJ/m2 for lanes 1-4 and 16 kJ/m2 for lanes 5-8: lanes 1, 2, 5, and 6, single-stranded oligomer before (2 and 6) and after (1 and 5) piperidine treatment; and lanes 3, 4, 7, and 8, duplex oligomer before (4 and 8) and after (3 and 7) piperidine treatment. The remaining lanes refer to the base-specific chemical sequencing reactions indicated.

employed for photosensitization. In equilibrium dialysis experiments [conducted in 50 mM phosphate buffer (pH 7.4)], no binding of PBSA by native calf thymus DNA could be detected at free PBSA concentrations of up to 6 mM. This finding was reinforced by the absence of an induced circular dichroism signal in the CD spectrum of a 2.5 mM solution of DNA containing 2.5 mM PBSA, in the same buffer. There was also no evidence for complex formation between 0.08 mM PBZ and DNA [in 5 mM phosphate buffer (pH 7.4)] since its UV absorption spectrum beyond 300 nm was unaltered in the presence of a 30-fold molar excess of native calf thymus DNA (expressed as moles of nucleotide phosphate per liter). Supporting this, the fluorescence emission of a 3 µM solution of PBZ (measured at 345 nm with excitation at 305 nm) was quenched by only 10% in the presence of a >100-fold excess of DNA. The investigations described below focus mainly on PBSA owing to its importance as a sunscreen agent. Figure 2 illustrates key features of the photodamage detected by gel sequencing in 5′-end-labeled single- and double-stranded DNA molecules exposed to UV-B in the presence of PBSA. Samples irradiated at 4 °C or at room


Chem. Res. Toxicol., Vol. 12, No. 1, 1999 41

Figure 3. Densitometric profiles illustrating guanine-specific cleavage within 5′-end-labeled oligonucleotides irradiated with 16 kJ/m2 UV-B in the presence of 0.2 mM PBSA and then heated with piperidine: (a) double-stranded 35-mer and (b) singlestranded 50-mer.

temperature gave indistinguishable results. No direct cleavage of the polynucleotide backbone occurs (see lanes 2, 4, 6, and 8), but subsequent treatment with hot piperidine causes selective chain scission at sites which map to guanine residues in the chemical sequencing ladder (see lanes 1, 3, 5, and 7). The intensity of cleavage increases with UV-B fluence (compare lane 1 with 5, and lane 3 with 7) being negligible in the absence of UV irradiation (not shown). Most notable is the striking difference in behavior exhibited by single-stranded and duplex DNA molecules. In the former, cleavage occurs fairly uniformly at all G residues in the sequence (lanes 1 and 5), but in the latter case, it is very largely confined to the 5′-G residue of GG doublets (lanes 3 and 7). The same pattern of photoreactivity was reproduced in several other 5′-end-labeled target oligonucleotides. When the duplex molecules contained GA doublets, they were subject to additional (though weaker) cleavage at the relevant 5′-G residues (Figure 3a). With single-stranded oligonucleotides, the cleavage intensity within runs of G bases tended to increase steadily at each successive G toward the 5′-end (Figure 3b). The influence of secondary structure on photoreactivity was examined with a 40-mer hairpin molecule comprising a stem of 12 base pairs and a G-rich loop of 16 bases. As shown in Figure 4, the PBSA-photosensitized production of piperidine-labile cleavage sites mapping to guanine was restricted to the loop region. All the (noncontiguous) G sites in the stem were unaffected. Cleavage was most pronounced at the two G residues situated at the junction of the stem and loop; unusually, one G residue in the loop was practically inert, and significant cleavage was evident at one of the A residues. When tested at the same concentration, PBZ gave results essentially identical to those with PBSA. This is illustrated in Figure 5 where samples containing either PBZ or PBSA were exposed for 40 min to the midday sun as a source of UV-B radiation. Both compounds act as highly effective photosensitizers when they are excited by sunlight. In this study, the target oligonucleotides were routinely radiolabeled by phosphorylating their 5′-termini. When cleaved by piperidine, they yielded discrete fragments

Figure 4. Gel autoradiogram showing piperidine-labile cleavage sites generated in a 40-mer hairpin DNA molecule by UV-B irradiation (30 kJ/m2) in the presence of increasing concentrations of PBSA. All samples except for that in lane 1 were heated with piperidine: lanes 1 and 2, unirradiated 40-mer; lane 3, 40-mer irradiated in absence of PBSA; lane 4, 40-mer and 100 µM PBSA, unirradiated; and lanes 5-7, 40-mer co-irradiated with 1, 10, and 100 µM PBSA, respectively. The remaining lanes refer to the indicated chemical sequencing reactions (note that lane C is overloaded).

whose electrophoretic mobility corresponded exactly to that of the fragments produced by chain scission at abasic sites arising from the removal of guanine bases. Experiments with a single-stranded 35-mer, photosensitized by PBSA, proved that 3′-end labeling of the oligonucleotide led to the same pattern of behavior on sequencing gel analysis (not shown). The effect of several commonly used quenchers and scavengers of reactive oxygen species on the photosensitization of DNA by PBSA was also examined in sequencing gel assays. With the single-stranded 34-mer (results not shown), the sites and intensity of cleavage were essentially unaltered following irradiation in the presence of mannitol or Tris (both 10 mM) or the bovine enzymes catalase (100 units/mL) and superoxide dismutase (200 units/mL). However, 10 mM concentrations of sodium azide, DABCO, or cysteine strongly suppressed the reaction. As shown in Figure 6, the same pattern of inhibition was obtained when the experiments were repeated with the double-stranded 34-mer. The ability of photoexcited PBSA to generate 1O2 in aerated aqueous solution was demonstrated by co-irradiating it with dGuo in the manner described by Ravanat and Cadet (36) for methylene blue. This led to the production of small (but equal) quantities of the 4R* and 4S* diastereomers of 4,8-dihydro-4-hydroxy-8-oxo2′-deoxyguanosine (4-OH-8-oxodGuo) which were isolated from the UV-B-irradiated reaction mixture using the recommended isocratic HPLC system (36). The individual diastereomers eluted as two well-resolved peaks having retention times (11.6 and 12.2 min) identical to those of


Figure 5. Gel autoradiogram showing induction of piperidinelabile cleavage sites in the 34-mer by exposure to sunlight for 40 min in the presence of PBZ or PBSA. All samples except for that in lane 1 were heated with piperidine; the estimated incident fluence of the UV-B radiation from sunlight for lanes 3-8 was ∼10 kJ/m2: lanes 1 and 2, unirradiated 34-mer; and lanes 3 and 4, 5 and 6, and 7 and 8, single-stranded and duplex 34-mer irradiated in absence of photosensitizer, in the presence of 0.2 mM PBSA, and in the presence of 0.2 mM PBZ, respectively. The remaining lanes refer to the indicated chemical sequencing reactions.

the same material synthesized independently from dGuo by photosensitization with methylene blue. The UV and circular dichroism spectra of the diastereomers were in full agreement with those reported by Ravanat and Cadet (36), and the expected molecular ions for 4-OH-8-oxodGuo were observed by positive and negative ion electrospray mass spectrometry: [M + H]+, m/z 300 (56% relative intensity); [M + Na]+, m/z 322 (46% relative intensity); and [M - H]-, m/z 298 (27% relative intensity). No formation of the diastereomers could be detected when dGuo was irradiated in the absence of PBSA, thus excluding a significant level of self-sensitized oxidation (38) under these reaction conditions. To assess the actual efficiency of PBSA as a photosensitizer of 1O2 production, its ability to oxidize dGuo to 4-OH-8-oxodGuo was compared with that of PN under the same irradiation conditions. In both cases, solutions absorbing >97% of the incident radiation at 312 nm were


Figure 6. Gel autoradiogram illustrating the effect of scavengers and quenchers of reactive oxygen species on the production of piperidine-labile cleavage sites in 5′-end-labeled doublestranded 34-mer irradiated with UV-B (25 kJ/m2), in the presence of 0.2 mM PBSA: lane 1, no PBSA; lane 2, singlestranded 34-mer and PBSA for comparison; and lanes 3-10, double-stranded 34-mer and no quencher (3), 10 mM sodium azide (4), 10 mM DABCO (5), 10 mM mannitol (6), superoxide dismutase (200 units/mL) (7), catalase (100 units/mL) (8), 10 mM L-cysteine (9), and 10 mM Tris (10). The remaining lane shows the G-specific chemical sequencing reaction.

exposed to a UV-B fluence of 70 kJ m-2. The 4-OH-8oxodGuo produced was then isolated by HPLC and its yield determined by spectrophotometry. There was a 46% conversion of dGuo to 4-OH-8-oxodGuo with PN as a sensitizer compared to 8% with PBSA, giving an efficiency ratio of approximately 6:1. Gel sequencing experiments (Figure 7) revealed that PN photosensitized the formation of alkali-labile lesions at all guanine residues in both the single- and doublestranded forms of the 34-mer oligonucleotide.

Discussion The foregoing results establish that PBZ and PBSA behave as efficient photosensitizers of DNA damage when they are exposed to narrow-band UV-B radiation or natural sunlight. This intrinsic photoreactivity, which may well extend to other 2-phenylbenzimidazole derivatives, does not appear to have been recognized hitherto, though photoaddition of PBZ to methyl acrylate has recently been reported (39). It is evident from the


Figure 7. Densitometric profiles of piperidine-induced cleavage patterns for 5′-end-labeled 34-mer irradiated, in 5 mM sodium phosphate buffer (pH 7.4) containing 20% ethanol, with UV-B (25 kJ/m2) in the presence of 0.2 mM PN: (a) single-stranded oligomer and (b) double-stranded oligomer.

sequencing gel assays that photosensitization of DNA does not cause direct chain cleavage (frank breaks) but engenders the formation of alkali-labile photolesions, predominantly at the sites of guanine nucleobases. However, it should be noted that covalent modifications to the structure of DNA that are stable toward hot piperidine would not have been detected. Comparison of the cleavage patterns observed for photosensitized single- and double-stranded DNA molecules (Figures 2, 3, and 5) reveals a remarkable dichotomy in the distribution of reactive guanines associated with alkali-labile sites. In single-stranded DNA, all the guanines are modified to an approximately equal extent. In duplex DNA, however, very distinct “hot spots” for modification occur which coincide with guanines situated 5′ to an adjacent guanine or (less prominently) an adenine residue. Similar conformation-dependent reactivity has been reported for a small number of other DNA photosensitizers, including riboflavin (20), a lysinenaphthalimide conjugate (40), and certain pterin derivatives (23). As now being argued, this contrasting behavior may be rationalized if there is a shift in the predominant pathway of photosensitization from a type II mechanism in single-stranded DNA to a type I mechanism, involving electron transfer, in double-stranded DNA. A role for singlet oxygen in the photodynamic action of PBSA is strongly supported by the finding that, under UV irradiation in aerated solution, PBSA is capable of sensitizing the formation of 4-OH-8-oxodGuo from dGuo. This reaction, which is regarded as being diagnostic of 1O production (36), did not occur in the absence of PBSA. 2 To gauge the relative efficiency of PBSA in generating 1O , its capacity to sensitize the formation of 4-OH-82 oxodGuo was compared with that of PN which is known (41) to produce 1O2 with a (solvent insensitive) quantum yield of ∼0.9. The same dose of UV-B radiation gave a 6-fold greater yield of 4-OH-8-oxodGuo when PN was employed as a photosensitizer, implying that (to a first approximation) the quantum efficiency for 1O2 production by PBSA was ∼0.15 under the experimental conditions. Because PBSA is negatively charged at neutral pH, and therefore has little or no binding affinity for DNA,

Chem. Res. Toxicol., Vol. 12, No. 1, 1999 43

photodamage mediated by 1O2 should be far more pronounced in single-stranded DNA (where the nucleobases are readily accessible for reaction) than in duplex DNA. Such a dependence of reactivity on DNA secondary structure has been convincingly demonstrated when 1O2 is generated by irradiating (anionic) hematoporphyrin (28) or an eosin-Tris complex (29), neither of which binds tightly to DNA. With hematoporphyrin, double-stranded DNA is effectively inert under conditions where all the guanines in single-stranded DNA are modified. With the eosin-Tris complex, denatured DNA is attacked some 20 times faster than native DNA. In the case of PBSA, this differential reactivity is clearly illustrated in Figure 4 by the 40-mer hairpin which incorporates single- and double-stranded regions in the same molecule. The piperidine-labile cleavage sites induced by photosensitization are confined to guanines (and one adenine) in the loop region; the guanines in the duplex stem (none of which is 5′ to G or A) are almost unaffected. Similar enhanced reactivity toward 1O2 by guanines in the loop region has been reported (42) for a hairpin target molecule complexed with a photosensitizing chlorinoligonucleotide conjugate. It is also noteworthy (see Figure 7a) that the 1O2 generator PN resembled PBSA (Figure 2) in uniformly attacking every guanine in the single-stranded 34-mer sequence. Unlike PBSA, however, PN also modified all guanines in the duplex 34-mer (see Figure 7b), though less evenly and with reduced intensity. Additional (but not definitive) support for 1O2 being the main source of base damage in single-stranded DNA was provided by the action of quenchers, though using D2O as solvent did not lead to significantly enhanced cleavage. As would be expected (43), the photosensitization by PBSA is strongly suppressed by sodium azide, DABCO, and cysteine. In contrast, the presence of scavengers for hydroxyl radicals (mannitol and Tris), hydrogen peroxide (catalase), and the superoxide anion (superoxide dismutase) had no significant effect. It is evident from inspection of Figures 2 and 5 that the intensity of the major cleavage bands associated with photosensitization by PBSA or PBZ is broadly comparable for single- and double-stranded DNA molecules exposed to the same fluence of UV-B radiation. On this basis, it may be inferred that photomodification leading to alkalilabile sites is similarly efficient in both conformations of DNA. In duplex DNA, however, the cleavage sites are essentially restricted to guanines that are situated 5′ to another guanine or, less noticeably, to adenine. This distinctive pattern of reactivity, which contrasts sharply with that of single-stranded DNA, is now considered (19, 22, 25) a hallmark of oxidative DNA damage initiated by one-electron transfer to a photosensitizer. The latter process is predicted, on energetic grounds, to favor the formation and localization of nucleobase radical cations at the 5′-guanines in GG doublets; these can act as precursors (44) to piperidine-sensitive alkali-labile sites. Several systems have been characterized (20-25) which exemplify this behavior, and although most of the relevant photosensitizers bind tightly to DNA, this is not a universal requirement. Thus, it has been shown that riboflavin (20) and certain pterins (23) photosensitize GGspecific lesions in a manner consistent with electron transfer but, like PBZ and PBSA, they bind only weakly, if at all, to duplex DNA. Consequently, these compounds provide a direct parallel and precedent for the observed


photosensitizing properties of the phenylbenzimidazoles [which, moreover, extends to their action on singlestranded DNA (20, 23)]. By analogy, therefore, it is probable that PBSA and PBZ also induce photodamage in duplex DNA primarily by a type I mechanism involving electron transfer. As postulated with respect to pterin photosensitization by Ito and Kawanishi (23), the abstraction of an electron from DNA by a photoexcited PBZ or PBSA molecule diffusing within close proximity of the double helix should occur most readily at the 5′-guanines of GG doublets because they have the lowest ionization potential of all its constituent nucleobases. The lack of reaction by guanines in other sequence contexts may be attributed to a rapid rate of back electron transfer within radical ion pairs formed from a sensitizer molecule and a single guanine residue. Routine studies with quenchers and scavengers of reactive oxygen species (Figure 6) showed that the amount of damage sensitized by PBSA in doublestranded DNA was reduced by 10 mM Tris but unaffected by 10 mM mannitol, or the enzymes catalase and superoxide dismutase. Curiously, the singlet oxygen quenchers sodium azide, DABCO, and cysteine all afforded complete protection; this could, however, stem from efficient quenching of the triplet excited state of PBSA. Confirmation that electron transfer can occur between photoexcited phenylbenzimidazoles and DNA nucleobases will require more definitive information that should be accessible through time-resolved optical spectroscopy (22) and ESR spin destruction experiments (23). Nonetheless, the marked dependence of the photocleavage pattern on the conformation of DNA is consistent with competition between the type I and type II processes described here. Singlet oxygen generated from unbound photosensitizers is known to modify denatured DNA far more rapidly than native DNA (28, 29), while electron transfer and hole migration are greatly enhanced by the regular base stacking of the double helix (25). The strong absorption of PBSA across the UV-B region has led to its use as a filter in a number of commercial sunscreen formulations. In this context, the photosensitizing properties of PBSA elicited by sunlight are of considerable interest and a potential source of concern. Its proven capacity to generate 1O2 upon UV irradiation could, in principle, pose a threat of oxidative damage to adjacent skin tissue and to cell membranes in particular (45). Nuclear DNA molecules would be at risk only if PBSA were able to enter the cells. While these possible outcomes deserve investigation, it must be stressed that extensive animal and human studies have demonstrated the efficacy and safety of PBSA and other sunscreens in protecting skin from the injurious effects of solar UV radiation (46). Nonetheless, there is scope for further in vitro evaluation of the photoreactivity of sunscreen ingredients toward cellular components. Although phototoxicity associated with individual agents may arise from damage caused to cellular constituents other than DNA, gel sequencing assays with end-labeled synthetic oligonucleotides provide a convenient and adaptable approach for assessing their potency as photosensitizers and probing their mechanism of action. As illustrated here for PBZ and PBSA, the system is generally applicable to investigating the photodynamic characteristics of water soluble compounds. It may be employed to determine whether the photosensitizing properties of PBZ and PBSA extend to structurally related compounds,


including some widely used benzimidazole fungicides and anthelmintics.

Acknowledgment. We gratefully acknowledge the skilful assistance of Damien McAleer during the preliminary stages of this work and financial support from Queen’s University.

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Photosensitization of Guanine-Specific DNA Damage by 2

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