Phototoxicity of Naphazoline. Evidence That Hydrated Electrons

Evidence That Hydrated Electrons, Nitrogen-Centered Radicals, and OH Radicals Trigger DNA Damage: A Combined Photocleavage and Laser Flash ...
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Chem. Res. Toxicol. 1999, 12, 971-978

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Phototoxicity of Naphazoline. Evidence That Hydrated Electrons, Nitrogen-Centered Radicals, and OH Radicals Trigger DNA Damage: A Combined Photocleavage and Laser Flash Photolysis Study Salvatore Sortino,*,†,‡ Salvatore Giuffrida,‡ and J. C. Scaiano† Department of Chemistry, University of Ottawa, 10 Marie Curie, Ottawa, Ontario K1N 6N5, Canada, and Dipartimento di Scienze Chimiche, Universita’ di Catania, Viale Andrea Doria 8, 95125 Catania, Italy Received March 26, 1999

The potential phototoxic activity of naphazoline (NP), 2-(1-naphthylmethyl)imidazoline, was investigated by studying its photoreactivity toward DNA. Photocleavage studies combined with laser flash photolysis experiments provide clear evidence that the transient species produced under NP photolysis react with DNA, thereby promoting its breakage under both aerobic and anaerobic conditions. Hydrated electrons and nitrogen-centered radicals are involved in the photodamage under anaerobic conditions. Hydroxyl radicals generated by Haber-Weiss reaction seem to initiate the photocleavage observed under aerobic conditions. A photodynamic mechanism involving the participation of singlet oxygen does not seem to play a crucial role in the photoinduced DNA breakage. The interaction between the NP and the biopolymer is also investigated by using both steady state and time-resolved spectroscopy.

Introduction The topic of drug phototoxicity has always received a considerable amount of attention, but recently, the level of interest has markedly increased due to the awareness among the scientific community of the increase in the UV portion of the sun spectrum reaching the earth. The majority of the work done in the past few years concerning the mechanisms of drug photosensitization is cited in some recent reviews (1-3). Reactions with DNA have been reported to be responsible for the phototoxicity of some drugs, including chloropromazine (4, 5), nonsteroidal anti-inflammatory drugs (6-8), and the recently reported fluoroquinolone antibacterials (9-11). Studies of photosensitized DNA damage are needed to gain an understanding of both phototoxicity and phototherapy. Such studies contribute to the knowledge of the mechanisms of degenerative skin diseases and of the cell toxicity of potential anticancer drugs. Naphazoline (NP),1 2-(1-naphthylmethyl)imidazoline, belongs to the vasoregulator class of drugs, present in the market as eye drops. It is widely used to relieve redness due to minor eye irritation caused by cold, dust, wind, smog, pollen, swimming, or wearing contact lenses. There are some reports in the literature concerning the in vivo toxicity of naphazoline (12-14), but they do not * To whom correspondence should be adressed: Dipartimento di Scienze Chimiche, Universita’ di Catania, Viale Andrea Doria 8, 95125 Catania, Italy. E-mail: [email protected]. † University of Ottawa. ‡ Universita’ di Catania. 1 Abbreviations: NP, naphazoline; GSH, reduced glutathione; SOD, bovine superoxide dismutase; sc-DNA, supercoiled DNA; ct-DNA, sonicated calf thymus DNA.

seem to be related to light exposure. Despite this, a warning not to expose to light is given when NP is purchased as part of an eye drop medication. This is not surprising given the presence of the naphthalene chromophore. Due to the protonation of the imidazoline ring, NP is present in its cationic form at physiological pH.

A recent study concerning the photochemistry of NP (15) pointed out that the photoreactivity of the drug is characterized by a photoionization process occurring through a mixture of mono- and biphotonic pathways. An intramolecular electron transfer involving both the imidazoline ring and the naphthalene radical cation followed by two steps of deprotonation led to the formation of nitrogen-centered radicals. The generation of singlet oxygen from the lowest excited triplet state of NP was also observed. These results demonstrated the potential of NP to act as both a type I and type II photosensitizer. In light of this and taking into account the fact that other drugs bearing the naphthalene moiety have been extensively described as phototoxic both in vivo and in vitro (8, 16, 17), we became interested in testing the phototoxicity of NP by analyzing its ability to induce DNA cleavage under UV-A irradiation. Further, to obtain more direct evidence about the role of the transient

10.1021/tx9900526 CCC: $18.00 © 1999 American Chemical Society Published on Web 09/11/1999

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species involved in the reaction with DNA, we decided to couple the steady state measurements with nanosecond laser flash photolysis experiments. We believe that the ideal approach to correlating the phototoxicity to the photochemical properties of a drug consists of studying the photochemistry of the photosensitizer in the presence of the biological target. Our results provide clear evidence of trapping of the hydrated electrons, produced during the NP photolysis, by DNA as well as the direct or indirect involvement of both nitrogen-centered radicals and OH radicals in the mechanism of DNA photodamage.

Experimental Procedures Chemicals. Naphazoline hydrochloride (molecular mass of 246.7 Da), sodium azide, mannitol, reduced glutathione (GSH), deferoxamine mesylate, catalase, bovine superoxide dismutase (SOD), thymine, and cytosine were obtained from Sigma Chemical Co. (St. Louis, MO). Water was purified with a Millipore Milli-Q system. All the experiments were performed in a phosphate-buffered saline solution (pH 7.4) consisting of a 1 × 10-2 M phosphate buffer, supplemented with 5 × 10-2 M NaCl. The pH of the solution was measured with a glass electrode. Supercoiled pBR322 DNA [sc-DNA, form I, molecular mass of 2.9 × 106 Da, 4365 base pairs (bp)] and sonicated calf thymus DNA (ct-DNA) (phenol extracted, lyophilized, average size of 2000 bases, range of 200-6000 bases) were obtained from Pharmacia (Milan, Italy) and Sigma Chemical Co. The percentage of relaxed form II of pBR322 was less than 12% in the starting material, and no linear form III was detected (the data were obtained from densitometric analysis of electrophoretic agarose gels). Irradiation Conditions. In all the photosensitization experiments, the irradiation was performed using a Rayonet photochemical reactor equipped with 16 “black light” phosphor lamps with emission in the 310-390 nm range with a maximum at 350 nm. The fluence at the irradiation position was about 800 µW/cm2. The incident photon flux on a 18 µL solution in the Eppendorf tubes (0.2 cm optical path) was 5 × 1015 quanta/ s. A “merry-go-round” irradiation apparatus was used to ensure that all the samples received equal radiation. The experimental procedures of irradiation and the light intensity measurements have been described previously (7). DNA Photocleavage Experiments. The samples containing pBR322 DNA and sensitizer with or without additives were prepared in a final volume of 18 µL, placed in Eppendorf tubes, and irradiated in a Rayonet photochemical reactor (see Irradiation Conditions). For experiments in a modified atmosphere, the merry-go-round apparatus was placed in a Lucite cylinder equipped with quartz walls. The chamber was then purged with humidified nitrogen, which was flushed continuously for the duration of the experiment. Agarose Gel Electrophoresis. Following irradiation, 4 µL of a mixture composed of 0.22% (w/v) bromophenol blue, 40% (w/v) sucrose, 0.1 mM EDTA (pH 8), and sodium lauryl sulfate (0.5% w/v) was added to the samples. Each sample (18 µL, 0.4 µg of DNA) was loaded onto a 5 mm thick 1% agarose gel (up to 22 wells). The electrophoretic analysis was performed in trisborate-EDTA buffer with a Pharmacia horizontal apparatus (model GNA-200). The power supply was set at 40 V for 15 h at 25 °C. Following electrophoresis, the gel was stained with ethidium bromide (1 µg/mL) for 30 min and rinsed with a MgCl2 solution (10 mM) for 20 min. DNA forms were detected by excitation of ethidium bromide fluorescence on a 300 nm UV transilluminator (Pharmacia). Photographs were taken with a Nikon F50 camera equipped with a 60 mm AF Micro lens (Nikkor) and a red filter. Quantitation of bands was achieved by microdensitometry of the negative produced from the gel photograph using a Beckman DU 650 spectrophotometer equipped with gel scan accessory.

Sortino et al. The fraction of sc-DNA after electrophoresis was calculated using eq 1:

sc-DNA )

areasc areasc +

∑

(1)

areacl/1.66

where areasc and Σareacl are the percentages of sc-DNA and cleaved DNA, respectively (forms II and III), which are both detected through densitometric analysis of fluorescent gels. The correct proportions of forms I-III in each sample were calculated by using the coefficient 1.66 for the lower efficiency of ethidium bromide in binding to the form I DNA with respect to forms II and III (18). In our photocleavage experiments, e10% form III DNA was produced. Nanosecond Laser Flash Photolysis. All the transient spectra and kinetics were recorded by employing a flow system with a 7 mm × 7 mm Suprasil quartz cell with a 2 mL capacity, and were purged in a storage tank with N2 for 30 min before as well as during the acquisition. The samples were excited with a Lumonics EX-530 laser with a Xe/HCl/Ne mixture generating pulses at 308 nm of ∼6 ns and e60 mJ/pulse. Care was taken to renew the solution at each laser shot. The signals from the monochromator/photomultiplier system were initially captured by a Tektronix 2440 digitizer and transferred to a PowerMacintosh computer that controlled the experiment with software developed in the LabView 3.1.1 environment from National Instruments. Further details have been described previously (19, 20). Luminescence spectra were recorded using a Perkin-Elmer LS 50 instrument. Fluorescence lifetime measurements were performed in the single-photon counting mode by exciting with the fourth harmonic (i.e., 266 nm) of a pulsed picosecond Nd: YAG laser and detecting the emission with a Hamamatsu C-4334 streak camera.

Results and Discussion Binding NP-DNA. We began our study with an investigation of the possible interaction between NP and DNA. Noncovalent interaction of small molecules with DNA often plays a dominant role in determining the efficiency of the DNA photocleavage as well as in the nature of the mechanisms involved in the DNA damage. Thus, it is very important to consider the biodistribution of the photosensitizing agent and its mode of binding to DNA. These kinds of interactions can be electrostatic binding, surface binding due to either hydrogen bonding or van der Waals forces along the grooves of the helix, and intercalation via π-stacking of aromatic heterocyclic groups between base pairs (21). Parameters such as hydrophobicity, charge, geometry, size, and shape permit the assessment of the affinity of the photosensitizer for DNA. The NP-DNA interaction was examined using fluorescence measurements. In Figure 1, we report the fluorescence spectra of NP recorded in the presence of various amounts of ct-DNA. The fluorescence maxima were not affected by the addition of the polynucleotide, but the emission intensity was efficiently quenched. Nevertheless, the NP fluorescence lifetime was influenced very little by the presence of DNA. This finding suggests that the contribution of a dynamic mechanism for the quenching process is negligible and that a static quenching occurring upon excitation is almost exclusively responsible for the behavior that is observed (22). The formation of ground state complexes between NP and DNA is most likely responsible for the static quenching process. Taking into account the fact that the imidazoline

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The quantum yields, Φ, for the photoinduced singlestrand breaks (ssb) related to the process hν

NP + sc-DNA 98 sc-DNA cleavage

(4)

were calculated from the initial rate of disappearance of starting sc-DNA, which equals

-

Figure 1. Influence of ct-DNA concentration on the fluorescence emission of NP in 10-2 M phosphate buffer at pH 7.4 (λexc ) 315 nm). The inset shows the Stern-Volmer plot related to the observed fluorescence quenching.

Figure 2. NP photoinduced sc-DNA cleavage under (9) anaerobic and (0) aerobic conditions. [NP] ) 1 × 10-3 M. [pBR322]bp ) 7 × 10-4 M. T ) 20 °C. The error estimate is (15%.

ring of NP is positively charged under our experimental conditions, we suggest that a strong electrostatic interaction between the NP and the negative charged backbone of DNA can account well for the static quenching that is observed. In light of this, when the existence of different types of fluorophore-DNA complexes is considered, with association constants Ki and relative fluorescence quantum yield Φi, the ratio of fluorescence intensities in the absence (I0) and presence (I) of DNA is given by the following equation (23):

I0/I ) (1 +

∑Ki[DNA])/(1 + ∑ΦiKi[DNA])

(2)

Since our results show that the fluorescence of the DNA-bound molecules is much lower than that of the free molecules, we can assume that the fluorophore-DNA complexes are basically nonfluorescent. As a consequence, eq 2 reduces to the familiar Stern-Volmer equation:

I0/I ) 1 + K[DNA]

(3)

where K (ΣKi) is related to the different types of complexes that cannot be distinguished by fluorescence. By a plot of I0/I versus DNA concentration (Inset Figure 1), we obtained a value for K of 1000 ( 100 M-1. Photocleavage Experiments. When samples that contain DNA were irradiated in the presence of NP, photocleavage was observed. Figure 2 shows the percentage of photoinduced sc-DNA cleavage at various irradiation times under both aerobic and anaerobic conditions.

d[sc-DNA] d[ssb] ΦFI ) ) dt dt v

(5)

where F ) 1 - 10-A, the fraction of photons absorbed by NP, I is the measured light intensity, and v is the volume of the irradiated sample. Values for Φ of 1.3 × 10-4 and 4.5 × 10-5 were obtained in nitrogen- and air-saturated solutions, respectively. These results suggest that a photodynamic mechanism involving the presence of activated species of oxygen plays a role in the photosensitizing process, albeit a minor one. Recent work has indicated that hydrated electrons, nitrogen-centered radicals, and singlet oxygen are the main transient species involved in the NP photolysis (15). All these species have the potential to damage DNA. Processes involving reduction of the most easily reduced DNA nucleobases such as cytosine and thymine by solvated electrons, oxidation of the most easily oxidized nucleobases such as adenine and guanine by radicals with oxidizing properties, and an oxidation process involving singlet oxygen and guanine are very wellknown in the literature (24, 25). On the basis of these considerations, we performed some photocleavage experiments in the presence of suitable additives to determine if some of these species are involved in the photocleavage process. The results are summarized in Table 1. The data obtained under anaerobic conditions show a strong inhibition of the photocleavage by cytosine and thymine. Since these additives are expected to react much more efficiently with the solvated electrons than when they are assembled in the DNA structure (26), it is reasonable to suggest that solvated electrons are involved in DNA photocleavage. Further, the high photocleavage quantum yield observed under anaerobic conditions and the considerable lowering of the extent of photoinduced cleavage in the presence of these two scavengers suggest that the hydrated electrons play an important role in the whole photosensitization process. The formation of radical anions of the pyrimidine bases is in fact a key step leading to DNA single-strand breakage by elimination of the C3′ ester phosphate of deoxyribose (27-29). The inhibition of the DNA damage effect exhibited by radical scavengers such as mannitol and GSH further confirmed the involvement of a type I mechanism in the DNA photocleavage. Under aerobic conditions, solvated electrons are very efficiently scavenged by oxygen, leading to the formation of superoxide anion (O2•-). Despite its name, this species is not a powerful oxidant (30), but the H2O2 which originates from its spontaneous disproportionation is able to promote DNA single-strand breaks via formation of hydroxyl radicals (31). Such transient species can be formed via the Haber-Weiss reaction involving hydrogen peroxide and traces of iron ions. The involvement of OH radicals, generated by the Haber-Weiss reaction, as triggers for DNA photocleavage under aerobic conditions, was demonstrated by using catalase and deferoxamine as an H2O2 quencher and iron sequesterer, respectively.

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Table 1. Effect of Additives on NP-Photoinduced sc-DNA Cleavage under Aerobic and Anaerobic Conditionsa aerobic additive none catalase inactivated catalase deferoxamine mannitol sodium azide SOD inactivated SOD

anaerobic

[additive] (M)

% cleavage

1 × 10-8 5 × 10-8 5 × 10-8 5 × 10-5 1 × 10-4 8 × 10-4 2 × 10-3 1 × 10-4 5 × 10-4 1 × 10-8 5 × 10-8 5 × 10-8

17.5 7.0 5.2 16.2 5.0 2.9 11.3 6.0 11.5 5.3 12.3 8.5 18.3

additive none cytosine thymine deferoxamine mannitol GSH

[additive] (M)

% cleavage

5 × 10-5 1 × 10-4 5 × 10-5 1 × 10-4 5 × 10-5 1 × 10-4 8 × 10-4 2 × 10-3 1 × 10-4 5 × 10-4

49.5 12.3 10.2 21.4 15.5 45.1 46.1 35.6 31.6 37.4 33.6

Irradiation time of 45 min. [NP] ) 1 × 10-3 M. [pBR322]bp ) 7 × 10-4 M. T ) 20 °C. Each point is the average of triplicate experiments. Error estimate of (15%. a

The data reported in Table 1 show clearly that a strong inhibition of the photoinduced cleavage is observed in the presence of these two additives. Furthemore, the lack of protection against the photoinduced cleavage exhibited by inactivated catalase under aerobic conditions and by deferoxamine under anaerobic conditions ruled out the possibility of nonspecific reactions, confirming the involvement of the Haber-Weiss reaction. The photoinduced cleavage was also inhibited by SOD because the enzyme removes the source of the reducing power, the O2•-, thereby removing the source of the reduced metal ion needed for formation of OH radicals. Also in this case, addition of inactivated enzyme did not provoke any protection effect on the amount of photoinduced cleavage, thus eliminating the involvement of nonspecific reactions. Finally, the considerable effect on the amount of damage observed by using mannitol as a hydrogen donor and, as a consequence, scavenger of OH radicals was in agreement with the proposed hypothesis. Usually, photocleavage processes triggered by OH radicals are expected to be more efficient than those involving direct reaction of hydrated electrons with DNA (32, 33). As a consequence, the higher photocleavage quantum yield under aerobic conditions is expected. We believe that since OH radicals are not produced directly by drug photolysis, the overall efficiency of the processes leading to their generation, such as quenching of the hydrated electrons by oxygen and the reactions involved in the Heber-Weiss cycle, may be responsible for the lower level of photoinduced damage observed under aerobic conditions. Moreover, we must consider the possibility that if OH radicals are generated in the bulk solution, other decay pathways may compete with the reactions with DNA. The reduction of the percentage of photocleavage observed in the presence of sodium azide (Table 1) suggests that a mechanism involving the participation of singlet oxygen (1O2) may also contribute to DNA damage. This result is expected on the basis of the fact that naphazoline was found to sensitize singlet oxygen formation with a quantum yield of 0.2 (15). Nevertheless, we think that 1O2 does not play an important role in the photosensitization process. We believe that the strong inhibition of the photoinduced cleavage displayed by sodium azide can be more reasonably attributed to a quenching of either OH radicals or OH-derived radical species rather than singlet oxygen. It is known that sodium azide is also able to efficiently quench radical

Figure 3. Transient absorption spectra observed in a 4 × 10-4 M NP nitrogen-saturated solution in 10-2 M phosphate buffer at pH 7.4 in the presence of 8 × 10-3 M ct-DNA upon 308 nm laser excitation: (b) 0.1 and (O) 2 µs after the laser pulse. Each point is the average of 10 laser shots. The signal-to-noise ratio is ca. 5 in the 700-800 nm region and ca. 20 in the 290-400 nm region.

species (34). Moreover, our hypothesis is in agreement with the fact that even though 1O2 is known to be responsible for guanine oxidation, DNA breakage induced by this transient species is not a very efficient process (35, 36). Time-resolved measurements (see next section) confirm our hypothesis. Laser Flash Photolysis Experiments. Figure 3 shows the transient spectra recorded in a nitrogensaturated solution containing NP in the presence of ctDNA upon 308 nm laser excitation at two delay times with respect to the laser pulse. A remarkable difference is observed when compared to the spectra recorded in the absence of DNA under the same experimental conditions (15): (i) the intensity of the entire spectrum is much lower; (ii) a maximum centered at 290 nm in place of the maximum at 330 nm, due to the nitrogen-centered radical produced by NP photolysis, was observed; and (iii) a growth around 310-320 nm was noticed. Experiments carried out at different DNA concentrations showed that the intensities of both the NP triplet, monitored at 410 nm, and the hydrated electrons, monitored at 720 nm, were strongly dependent on the amount of the polynucleotide until a limiting value was reached at a DNA/NP ratio of ca. 20 (Figure 4). This result is not surprising and is in agreement with the static quenching by DNA observed with the fluorescence measurements and discussed earlier. The results of the quenching occurring upon excitation show in fact a reduction of the efficiency of all the photochemical pathways (37).

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Figure 4. Dependence of the absorbances of (b) the triplettriplet, monitored at 410 nm, and (O) the solvated electrons, monitored at 720 nm, on the [ct-DNA]/[NP] ratio in a nitrogensaturated solution. [NP] ) 4 × 10-4 M. The phosphate buffer concentration was 10-2 M at pH 7.4. λexc ) 308 nm. ∆A taken 0.1 µs after the laser pulse. Each point is the average of 50 laser shots. The signal-to-noise ratio is ca. 5 at 720 nm and ca. 20 at 410 nm.

Figure 5. Time profile and biexponential fitting of the absorbance changes observed at 410 nm after 308 nm laser excitation of a 4 × 10-4 M NP nitrogen-saturated solution in the presence of of 8 × 10-3 M ct-DNA in 10-2 M phosphate buffer at pH 7.4.

Figure 5 shows the kinetic trace of the NP triplet at 410 nm observed with a DNA/NP ratio of 20. The decay is described well by a double-exponential function:

∆A(t) ) a1 exp(-k1t) + a2 exp(-k2t) + a3

(6)

with a rate constant k1 of ∼9.0 × 105 s-1 and a k2 of ∼2.5 × 104 s-1. These results reflect the fact that two different populations of triplet are monitored under these experimental conditions. Given the fact that these two rate constants were different compared to that of the decay of the NP triplet observed in the absence of ct-DNA (k0 ∼ 7 × 104 s-1), we believe that the oberved decays are due to two different NP-DNA complexes. Measurements of the triplet lifetime at different DNA/NP ratios confirmed this hypothesis. We observed a decrease in the lifetime of the slow component and an increase in the lifetime of the fast component as both lifetimes approached that observed for free NP (Figure 6). This result reflects the situation shown in Scheme 1. In the absence of ct-DNA, the observed kinetics are monoexponential and correspond to the triplet state of the unbound NP. In the presence of ct-DNA, the kinetics are always biexponential and the three NP species contribute to the observed kinetics: the two bound species and the free NP.2 For DNA/NP ratios of >20, the contribution of the unbound species is negligible and the biexponential decay

Figure 6. Dependence of the (9) slow and (b) fast components of the triplet of NP, monitored at 410 nm, on the [ct-DNA]/[NP] ratio in a nitrogen-saturated solution. [NP] ) 4 × 10-4 M. The phosphate buffer concentration was 10-2 M at pH 7.4. λexc ) 308 nm.

Scheme 1

mainly reflects the kinetics of the two NP-DNA complexes. Additionally, the observed changes can only be ascribed to the relationships in Scheme 1 as opposed to an equilibrium between the three species in their triplet states because in this case a monoexpontial decay would be observed at all DNA concentrations. Quenching experiments with oxygen performed with a DNA/NP ratio of ca. 20 showed that the two triplet components are quenched by oxygen with the following bimolecular rate constants: k1ox ) 1.6 × 109 M-1 s-1 and k2ox ) 8.7 × 107 M-1 s-1. Given the fact that the first quenching constant was exactly the same as that observed for the quenching of the NP triplet in the absence of DNA (15), we think that in the two different complexes with DNA, the naphthalene moiety is localized in an aqueous environment in the first case whereas it is incorporated in a deeper region of the DNA in the second case. Even though this latter complex could have the potential to sensitize singlet oxygen close to the nucleobases, the efficiency of the energy transfer process leading to 1O2 is low. Indeed, on the basis of the first-order time constant of this bound triplet (k2 ∼ 2.5 × 104 s-1) and the quenching constant for quenching by oxygen (k2ox ) 8.7 × 107 M-1 s-1), the fraction of triplet quenched by oxygen is ca. 50% lower than that observed in the absence of DNA. Likewise, on the basis of the values of k1 and k1ox, the fraction of the bound triplet localized in an aqueous environment quenched by oxygen is ca. 65% 2 Unfortunately, the small number of points of the kinetic traces obtained with the laser flash photolysis technique did not allow an accurate determination of the preexponential factors of the three different populations of triplets via a three-exponential analysis.

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photon process (39, 40). The non-zero intercept shows clearly that a mixture of mono- and biphotonic pathways are involved in the formation of both the 310-320 nm bands and the hydrated electrons, suggesting that these two transient species may be related. Under steady state irradiation conditions, the biphotonic pathway is not significant and the main component is due to the monophotonic process.

Figure 7. Time profiles and monoexponential fittings of the absorbance changes observed at (b) 720 and (O) 320 nm after 308 nm laser excitation of a 4 × 10-4 M NP nitrogen-saturated solution in the presence of 8 × 10-3 M ct-DNA. The phosphate buffer concentration was 10-2 M at pH 7.4.

Figure 8. Laser power dependence of the ratio between the absorbance changes and the laser intensity observed in a 4 × 10-4 M NP nitrogen-saturated solution in the presence of 8 × 10-3 M ct-DNA upon 308 nm laser excitation in 10-2 M phosphate buffer at pH 7.4 at (9) 720 nm 0.1 µs after the pulse, (b) 320 nm 2 µs after the pulse, and (O) 290 nm 0.1 µs after the pulse. Each point is the average of 50 laser shots. The signalto-noise ratio is ca. 5 at 720 nm and ca. 20 at 290-320 nm.

lower than that observed in the absence of DNA. These results are in good agreement with the previous assertion that a photodynamic process involving the participation of singlet oxygen plays only a minor role in DNA damage. As shown in Figure 3, a growth at 310-320 nm was observed, suggesting that some new species is formed. In Figure 7 is reported the decay of the solvated electrons, monitored at 720 nm, and the growth at 320 nm. The kinetic analysis shows that the first-order decay at 720 nm matches the growth observed at 320 nm with a rate constant k of ∼3.6 × 106 s-1. On the basis of these results, we believe that the absorption around 310-320 nm could be due to the product of a reaction between the solvated electrons and the pyrimidine bases of DNA. Reduction products of pyrimidine bases that originate after reaction with solvated electrons are characterized by absorptions in the observed range of wavelengths (38). To further confirm that the 310-320 nm band originated from the decay of the solvated electrons, we introduced a laser power effect. As shown in Figure 8, the data at 720 and 320 nm fit the equation

∆A/E ) a + bE

(7)

where E is the laser power, a is a coefficient depending on the quantum yield of the one-photon process, and b is a factor depending on the extinction coefficients and yields of the intermediate steps of the consecutive two-

A key experiment which poined out that the solvated electrons are quenched by DNA leading to the formation of reduction products of the bases was performed by analyzing the decay of the hydrated electrons in the presence of different concentrations of ct-DNA. A quenching constant for quenching by DNA of 1.2 × 108 M-1 s-1 per nucleotide unity was obtained. This value is very close to that of 1.4 × 108 M-1 s-1 reported in the literature (26), and accounts well for a direct reaction involving the solvated electrons and DNA. This direct evidence of a reaction involving this transient species and the polynucleotide is in agreement with what is observed in the photocleavage experiments, thereby confirming the proposed hypothesis of a dominant role played by the hydrated electrons in the photodamage observed under anaerobic conditions. As mentioned before, the transient spectrum recorded 0.1 µs after the laser pulse (Figure 3) exhibited a welldefined maximum at 290 nm instead of the band at 330 nm observed in the absence of DNA (15). This band was attributed to nitrogen-centered radicals located on the imidazoline ring, originating after electron transfer from the imidazoline ring to the naphthalene radical cation, formed after photoionization, followed by two steps of deprotonation (15). These radicals were characterized by a very strong oxidizing nature. We believe that a fast reaction involving this radical with the more easily oxidized nucleobases, either adenine or guanine, could be responsible for the formation of the maximum at 290 nm. The nitrogen-centered radicals were found to be reactive toward both adenine and guanine with a rate constant of ca. 1 × 107 M-1 s-1. The strong interaction between the NP and DNA would account for the higher reactivity of these two species, leading to the formation of the transient absorbing at 290 nm in less than 0.1 µs. Further, by following the transient absorbance change at 290 nm as a function of the laser intensity (Figure 8), we also noticed that in this case a mixture of mono- and biphotonic mechanisms is responsible for the formation of this band. This result is in accordance with the fact that this species is formed by quenching of the nitrogen-centered radicals which are generated through the same mechanistic pathway (15). Finally, oxidation products of purine bases are characterized by considerable absorption around 290 nm (41). We think that, even if not very efficiently, the reaction involving this nitrogen-centered radical with DNA bases could lead to DNA cleavage. Given that the highest concentration of the electron scavengers used in the photocleavage experiments was high enough to trap all the solvated electrons (26), the incomplete quenching of the photoinduced cleavage observed in the presence of these additives (Table 1) suggests that a minor contribution to the DNA cleavage due to oxidation processes involving the nitrogen-centered radicals cannot be excluded.

Naphazoline-Photosensitized DNA Cleavage

Chem. Res. Toxicol., Vol. 12, No. 10, 1999 977 Scheme 2

Conclusions We have shown the ability of transient species generated by naphazoline photolysis to react with DNA, promoting the cleavage of the polynucleotide. The higher photocleavage quantum yield found under anaerobic conditions was attributed mainly to the direct involvement of the hydrated electrons and, partially, to the nitrogen-centered radicals produced after NP photoionization in the reaction with DNA. Redox reactions involving these transient species and the DNA nucleobases are believed to be responsible for the DNA breakage. Nanosecond laser flash photolysis measurements provided clear evidence of reactions between these NP intermediates and the DNA. Hydroxyl radicals formed by the Haber-Weiss reaction were shown to initiate DNA photocleavage under aerobic conditions. The interaction between the photosensitizer and DNA played a key role in the photosensitization mechanism. The reaction involving the nitrogen-centered radicals and the DNA was believed to be very efficient due to this interaction. In contrast, the formation of two NP-DNA complexes characterized by triplet states that are scarcely quenchable by oxygen makes negligible the contribution of DNA oxidation processes mediated by singlet oxygen. A general mechanism for NP photocleavage according to the overall results is reported in Scheme 2.

Acknowledgment. Financial support from MURST (cofinanziamento di programmi di ricerca di rilevante interesse nazionale) and the Natural Sciences and Engineering Research Council of Canada is acknowledged.

We thank Dr. Daniel Yarosh for a kind gift of naphazoline-containing eye drops to J.C.S. in a time of need; this gift motivated the current study.

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