Photosensitization of DNA Strand Breaks by Three Phenothiazine

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Chem. Res. Toxicol. 2003, 16, 644-651

Photosensitization of DNA Strand Breaks by Three Phenothiazine Derivatives Giampietro Viola,*,† Loredana Latterini,‡ Daniela Vedaldi,† Gian Gaetano Aloisi,‡ Francesco Dall’Acqua,† Nadia Gabellini,§ Fausto Elisei,‡ and Arianna Barbafina‡ Department of Pharmaceutical Sciences, University of Padova, via Marzolo 5 Padova, Italy, Department of Chemistry, University of Perugia, 06123 Perugia, Italy, and Department of Biological Chemistry, University of Padova, via G. Colombo 3 Padova, Italy Received December 19, 2002

The interaction and the photosensitizing activity of three phenothiazine derivatives, fluphenazine hydrochloride (FP), thioridazine hydrochloride (TR), and perphenazine (PP), toward DNA were studied. Evidences obtained from various spectroscopic studies such as fluorimetric and linear dichroism measurements indicate that these derivatives bind to the DNA at least in two ways: intercalation and external stacking on the DNA helix, depending on their relative concentrations. Irradiation of supercoiled plasmid DNA in the presence of these phenothiazines leads to single strand breaks. The DNA photocleavage appears to be due to externally bound molecules rather than to those intercalated. The highest photocleavage activity was observed with PP and TR whereas FP was less efficient. The efficiency of the photocleavage in aerated and deaerated solutions does not change thus indicating that an involvement of singlet oxygen can be excluded. Primer extension analysis of plasmid DNA irradiated in the presence of phenothiazines indicates that photocleavage of DNA occurs predominantly at Gua and Cyt residues. Laser flash experiments carried out in the presence of 2′-deoxyguanosine 5′-monophosphate reveal an efficient electron transfer between the nucleotide and the radical cations produced by photoionization of the phenothiazines. In the presence of DNA, an electron transfer process takes place within the laser pulse from the lowest singlet state of phenothiazines to the DNA bases; the time-resolved measurements showed that the back-electron transfer is a negligible decay pathway for the charged species.

Introduction It is well-known that many drugs act as photosensitizers toward cells by interacting with various cellular components such as lipids, proteins, and nucleic acids (1, 2). The structural modifications of the cellular components may occur by direct interactions of the excited states (singlets or triplets) of the drugs with the biological substrate or indirectly, through reactive species of oxygen sensitized by the drug themselves. In particular, the phototoxic activity of various drugs is related with their potential mutagenic and carcinogenic effects, taking place through DNA modification. In some cases, the photoreaction of the drug with DNA may be used as a therapeutical tool as in the case of photoaddition of psoralens to DNA pyrimidine bases, which is used to treat psoriasis and other skin disorders (3). However, in most cases, the drug-DNA photoreaction causes undesired and dangerous effects. For various classes of drugs, such as antibacterial fluoroquinolones (4), nonsteroidal antiinflammatory drugs (5, 6), and phenotiazines (7), induced DNA photosensitization processes were observed. Phenotiazines represent a wide class of drugs used as antipsychotic in the therapy of many mental disorders * To whom correspondence should be addressed. Tel: +39 49 8275705. Fax: +39 49 8275366. E-mail: [email protected]. † Department of Pharmaceutical Sciences, University of Padova. ‡ Department of Chemistry, University of Perugia. § Department of Biological Chemistry, University of Padova.

such as the maniac depressive syndrome and schizophrenia. Chlorpromazine, a well-known phototoxic compound, is able to induce cell damages and photomutagenesis in both bacteria and mammalian cells and is the prototype of this class of drugs (8, 9). The phototoxic action of chlorpromazine toward DNA has been widely investigated. It is due to the formation of photoadducts with single- and double-stranded DNA, as well as the photosensitization of DNA strand breaks (7, 10, 11). We have reported previously the photochemical and photophysical properties of PP,1 FP, and TR together with a detailed investigation of phototoxic properties at the cellular level (12). In particular, we have clearly demonstrated that monophotonic ionization of phenothiazines takes place upon direct irradiation in aqueous solution, leading to the formation of radical cations and the solvated electrons. The results of phototoxicity tests excellently correlated with the involvement of radical cations in the photoreactivity of the phenothiazines. The aim of this work is to extend the knowledge of the phototoxicity mechanism responsible for the damage produced in the nucleic acids by three phenothiazine derivatives (Figure 1). In particular, the binding of these 1 Abbreviations: CAT, catalase; DMTU, N,N′-dimethyl thiourea; , absoption coefficient; FP, fluphenazine; GMP, 2′-deoxiguanosine 5′monophosphate; LD, linear dichroism; LDr, reduced linear dichroism; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide; PBS, phosphate-buffered saline; PP, perphenazine; SOD, superoxide dismutase; TR, thioridazine; τF, fluorescence lifetime; τT, triplet lifetime.

10.1021/tx025680t CCC: $25.00 © 2003 American Chemical Society Published on Web 04/09/2003

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Figure 1. Chemical structures of the examined compounds.

molecules to DNA was investigated by use of absorption and emission spectroscopy and flow LD. Moreover, the mechanism of the photosensitizing action of these drugs in the DNA strand breaks of double-stranded supercoiled pBR322 plasmid DNA was investigated. For this purpose, experiments of primer extension analysis combined with sequencing techniques were carried out to identify the preferential location of the photocleavage sites. Finally, to gain a deeper insight into the mechanism of DNA photocleavage, laser flash photolysis experiments of phenotiazines were performed in the presence of DNA and nucleotides.

Experimental Section Chemicals. FP, PP, and TR were kind gifts of Bristol Myers Squibb (Anagni, Italy), Schering Plough S.p.A. (Milano, Italy), and Novartis Pharma (Basel, Switzerland), respectively. DMTU, SOD, CAT, salmon testes DNA (sodium salt), GMP sodium salt, and agarose were Sigma products. pBR322 plasmid DNA and ethidium bromide solution were from Amersham Pharmacia Biotech AB (Uppsala, Sweden). Nucleic acid concentrations were determined spectrophotometrically at 260 nm using the value 6600 M-1 cm-1 for the molar absorption coefficient at this wavelength. DNA Binding Studies. Light absorption spectra were recorded with a Perkin-Elmer Lambda 15 spectrophotometer. Fluorescence spectra were recorded with a Perkin-Elmer LS50B spectrofluorimeter. The measurements were carried out in ETN buffer (TRIS, 10 mM; EDTA, 1 mM; and NaCl, 10 mM) at pH 7.0 and 25 °C. To obtain the intrinsic binding constant and the binding site size as well, absorption and emission spectra of solutions with different [DNA]/[drug] ratios and constant drug concentrations were recorded. The binding isotherms obtained were represented as Scatchard plots (13) and evaluated according to the McGhee and von Hippel model (14). The fluorescence lifetimes, τF (mean deviation of three independent experiments, ∼5%), were measured by a Spex Fluorolog-τ2 system, which uses the phase modulation technique (excitation wavelength modulated in the 1-300 MHz range; time resolution, ∼10 ps). The frequency domain intensity decays (phase angle and modulation vs frequency) were analyzed with the Globals Unlimited (rev. 3) global analysis software (15). LD. LD measurements were performed with a Jasco J500A spectropolarimeter equipped with an IBM PC and a Jasco J interface. The sample orientation was produced by a device designed by Wada and Kozawa (16) at a shear gradient of 700 rpm in ETN buffer. Steady State Irradiation Procedure. An HPW 125 Philips lamp with the emission profile centered at 365 nm was used for steady state irradiation experiments. The energy of the lamp light was measured by a radiometer from the Cole-Parmer Instrument Company (Niles, IL), equipped with a 365 CX sensor.

DNA Strand Breaks. Irradiation was performed on samples containing 20 ng/µL of pBR322 DNA dissolved in phosphate buffer (10 mM) at pH 7.2, and the examined compounds were in a quartz cuvette (0.1 cm path length) fitted with a plastic stopper through which a venting needle was inserted for nitrogen purging. After different irradiation times, 5 µL of the solution was removed and diluted with the loading buffer, and then, the samples were loaded on 1% agarose gel. The electrophoretic run was carried out in TAE buffer (0.04 M Tris-acetate, 1 mM EDTA) at 50 V for 4 h. Analogous experiments were performed in the same conditions by adding various scavengers to the solution. After staining in ethidium bromide solution, the gel was washed with water and the DNA bands were detected under UV radiation with a UV transilluminator. Photographs were taken with a digital photocamera Kodak DC256, and the quantitation of the bands was achieved by the image analyzer software Quantity One (BIO RAD). The fraction of supercoiled DNA was calculated as described by Ciulla et al. (17). Primer Extension Analysis. pUC18 plasmid DNA was purified using the QIAprep exctraction kit (QIAGEN, Hilden, Germany) and stored at 4 °C in elution buffer. Irradiation was performed as described before using 1.1 µg of plasmid DNA in a 50 µL reaction volume. The phenothiazine derivatives (PP and TR) were applied at a molar [drug]/[DNA] ratio of 0.4. Aliquots (10 µL) were taken after various irradiation intervals and kept on ice. Primer extension was performed with [γ-33P]-labeled M13 reverse primer and the pUC18 as the template, and the DNA synthesis was carried out by the Taq polymerase as described previously (18, 19). Amplified DNA fragments were separated electrophoretically on a 6% acrylamide, 50% urea gel. Laser Flash Photolysis. Triplet formation quantum yields and lifetimes were measured with a flash photolysis setup previously described (Nd:YAG Continuum, Surelite II, third harmonics, λexc ) 355 nm, pulse width ca. 7 ns, and energy e1 mJ pulse-1) (20, 21). First-order kinetics were observed for the decay of the lowest triplet state (T-T annihilation was prevented by the low excitation energy). The triplet lifetimes were measured at an absorbance of ca. 0.2; the concentration effect on τT was not investigated. The transient spectra were obtained by monitoring the optical density change at intervals of 5-10 nm over the 300-800 nm range and averaging at least 10 decays at each wavelength. All measurements were carried out at 22 ( 2 °C; the solutions were saturated by bubbling with argon. The experimental errors on τT were estimated to be about (10%.

Results DNA Binding Properties. The noncovalent binding of the phenotiazine derivatives to DNA in buffered aqueous solution was monitored by absorption and fluorescence measurements. The absorption spectra of the compounds were recorded in the absence and in the presence of DNA by adding the same amount of DNA in

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Figure 2. Spectrophotometric titration of DNA to PP (A), TR (B), and FP (C) in ETN buffer (0.01 M, pH 7.0, T ) 25 °C).

the reference cuvette. The spectra of the three derivatives, in the presence of increasing concentrations of DNA, presented in Figure 2, show pronounced changes, which indicate interactions between the drug and the DNA. In fact, absorption maxima of the three compounds alone are centered at 312 nm while at high DNA concentrations new maxima appeared at about 330 nm. The lack of a well-defined isosbestic point can be due to the formation of drug/DNA complexes with more than one structure. The interaction of the phenothiazine derivatives with DNA was also investigated by use of emission spectroscopy. Fluorescence emission of the drugs was significantly quenched upon increasing the concentration of DNA (Figure 3); the maximum quenching and the achievement of constant signals were observed for [drug]/[DNA] ratios of about 0.04-0.05 for the three compounds. The fluorescence titration data for the phenothiazine derivatives bound to DNA, binding isotherms plotted as Scatchard plot (13), gave the binding constant affinity (Ki) and the binding site size (n) values according to the McGhee and Von Hippel model (14). The binding constants obtained, whose values (2-5 × 104 M-1) are consistent with a significant affinity of these phenothiazines for the biological macromolecule, are shown in Table 1. Moreover, the high values of the binding site size (exclusion parameter, n ≈ 3) are not consistent with the nearest neighbor exclusion model for classical intercalation (14) but with a mixed binding mechanism, likely corresponding to intercalation plus external binding. Additional titrations were performed increasing the ionic strength of the buffer (i.e., ETN 100 mM). These experiments (data not shown) showed that the fluores-

Figure 3. Fluorimetric titrations of salmon testes DNA to PP (A), TR (B), and FP (C) in ETN buffer (0.01 M, pH 7.0, T ) 25 °C). The compounds were excited at the wavelength corresponding to the isosbestic points (325 nm) found in the spectrophotometric titrations. Table 1. Thermodynamic Parameters Obtained by Fluorimetric Titration of Salmon Testes DNA in ETN Buffer at pH 7.0

a

compds

Ki (× 104 M-1)

n(bp)a

PP TR FP

4.4 ( 1.2 2.1 ( 0.2 3.4 ( 0.2

3.0 ( 0.3 3.2 ( 0.2 3.2 ( 0.1

Exclusion parameter, i.e., binding site size in base pairs.

cence emission of the drugs are not efficaciously quenched as in low ionic strength buffer, indicating that at the higher ionic strength only a small fraction of the molecules binds to sites where the fluorescence is strongly quenched as in intercalatives sites. The interactions of drugs with DNA cause marked changes in the τF of phenothiazines, as shown in Table 2. In fact, in the absence of DNA, the fluorescence decay was well-fitted by a monoexponential function, while upon addition of DNA a second decay component was observed whose abundance increased significantly with the DNA concentration. A lifetime of 20-30 ps was measured for the latter component, while the lifetime value for the longer-lived fluorescence component was practically the same as measured for the drug alone. The preexponential factors shown in Table 2 are indicative of the different binding efficiency, which takes place between DNA and the three phenothiazines. In particular, DNA concentrations of 1.2 × 10-5 and 2.3 × 10-5 M

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Table 2. Fluorescence Lifetimes of Phenothiazines (ca. 1 × 10-4 M) in PBS compds PP

FP

TR

a

λexc (nm)

λem (nm)

[DNA] (× 10-5 M)

τF (ns)

A (%)

330 330

>370 >370

0 0.12

330

>370

1.2

330 330

>370 >370

0 1.2

330

>370

2.5

330 330

>370 >370

0 0.2

330

>370

2.3

0.43a 0.02 0.65 0.02 0.63 3.3a 0.02 3.2 0.03 3.3 1.7 0.02 1.4 0.02 1.1

100 63 37 98 2 100 57 43 65 35 100 79 21 98 2

Decay time values measured in water.

are sufficiently high to bind almost all PP and TR, respectively (98% of the fluorescence was due to the short component); instead, FP at a similar DNA concentration (2.5 × 10-5 M) is much less bound (only 65% of the fluorescence is due to the short-lived component). Even if the bound phenothiazines show a marked shortening of the τF with respect to the free molecules, only small changes were recorded in the emission spectra. This behavior suggests a small effect of the interactions with DNA on the energy of the lowest singlet state but a significant change of the decay rates, which become about 100 times faster in the complex systems. LD. To understand the binding nature between the drugs and the biological substrate, LD measurements (22, 23) were performed on solutions with salmon testes DNA and phenotiazines at various molar ratios. In a typical LD experiment, the long axis of the DNA double helix is aligned along the flow lines; thus, the DNA bases, which are perpendicularly oriented to the helix axis, give a negative LD signal. A negative contribution to the LD signal is also due to drug molecules intercalated in the DNA structure because of the aromatic plane almost perpendicular to the helix axis. Instead, drug molecules that are bound in the minor groove of DNA, generally exhibiting angles of 40-50° between the transition moment of the chromophore and the helix axis, give a positive contribution to the LD signal. The absorption, LD, and LDr spectra of PP, recorded at different [PP]/[DNA] ratios, are shown in Figure 4. All phenothiazine-DNA complexes give negative LD bands in the region of DNA absorption (230-300 nm). Here, a significant enhancement of the LD bands was observed for the three phenothiazines at [drug]/[DNA] ratios smaller than 0.08, thus indicating that the alignment of DNA in the hydrodynamic field becomes more distinct because of a stiffening of the helix upon binding of the ligand (22). For PP and TR at higher ratios (e.g., 0.2 in Figure 4 and in the Supporting Information), a decrease of the signal, probably due to the external binding, which increases the DNA flexibility, was observed. It can be noted that in the region of the drug absorption (300-400 nm), the LD signal is negative at each concentration ratio, as expected for a complex where the phenothiazine is intercalated in the DNA (22). Furthermore, the absorption and LD signals do not show the same maximum but there is a shift of about 15 nm (λmax ∼ 330 and ∼345 nm for absorption and LD, respectively).

Figure 4. Absorption (A), LD, and LDr spectra of PP in ETN buffer (0.01 M, pH 7.0, T ) 25 °C), recorded at [drug]/[DNA] ratios: 0.00 (a), 0.04 (b), 0.08 (c), and 0.2 (d).

This spectral behavior suggests that the drug molecules contributing to the absorption spectrum are located in different environments from those contributing to the LD signal; likely, the drug molecules externally bound to the DNA are mainly responsible for the 330 nm absorption while those intercalated in the DNA mainly contribute to the LD signal centered at 345 nm. Moreover, the LDr signal of the phenothiazine-DNA complexes recorded in the 300-400 nm range, which provide information on the relative orientation of the drug transition moments with respect to those of the DNA bases, is significantly wavelength-dependent, thus confirming the occurrence of an heterogeneous binding mode. Photoinduced DNA Cleavage. To investigate the DNA photocleavage activity of phenothiazines, buffered aqueous solutions of drugs were irradiated in the presence of supercoiled pBR322 plasmid DNA and analyzed by agarose gel electrophoresis. When kept in the dark, the phenothiazines do not promote any DNA damages, but under UVA irradiation, an increase of the nicked form of DNA with a parallel decrease of the supercoiled form was observed. The results obtained under our experimental conditions are consistent with single strand breaks of DNA. The correlation of the DNA strand breaks with the UVA dose in the presence of the three drugs at different concentrations and UVA dose is shown in Figure 5. For the three phenothiazines at low [drug]/[DNA] ratios, where an intercalation complex is mainly formed (see above), no significant DNA photocleavage was

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Figure 5. Effect of UVA doses on pBR322 plasmid DNA sensitized by PP (A), TR (B), and FP (C) at different [drug]/ [DNA] ratios: 0.02 (9), 0.04 (2), and 0.4 (b).

observed; instead, at larger [drug]/[DNA] ratios, where the drugs are also externally bound to DNA, a remarkable decrease of the supercoiled form took place. The results summarized in Figure 5 suggest that under our experimental conditions the photoreactivity of the three phenothiazines follows the order PP > TR > FP at any UVA dose. The percentage of supercoiled plasmid DNA detected after UVA irradiation (12 J cm-2) of PP-DNA and TRDNA systems in different experimental conditions (including air-equilibrated solutions and the presence of additives) is shown in Figure 6. All of the experiments suggest that reactive species of oxygen are not involved in photocleavage reaction of DNA. In fact, removal of oxygen did not reduce significantly the photocleavage efficiency; moreover, the experiments performed in the presence of SOD, CAT, and DMTU, scavengers of O2-, H2O2, and OH•, respectively (24), did not show significant effects on the DNA photocleavage. Determination of DNA Photocleavage Sites. To evaluate the sites of photocleavage in the DNA sensitized by the three phenothiazines, primer extension analysis was performed as described in the Experimental Section. The method is based on the ability of the DNA polymerase to build up a complementary copy of DNA, starting from a primer oligonucleotide, and was used to detect breakage and chemical modification sites at which the DNA polymerase stops (25). The pUC18 plasmid, used as a template, was irradiated in the presence of phenotiazines at a [drug]/[DNA] ratio of 0.4 in phosphate buffer. The sites of termination of DNA synthesis on the photoreacted template were precisely determined by running side by side the products of DNA sequencing reaction of the same DNA segment determined by the dideoxy nucleotide chain termination procedure (26). The synthesized DNA fragments that were separated, accord-

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Figure 6. Percentage of supercoiled pBR322 plasmid DNA upon irradiation in the presence of PP and TR for 60 min (12 J cm-2) in different experimental conditions (the data derived from densitometric analysis of agarose gels are the average of two different runs).

ing to their length, on a denaturing polyacrylamide gel are shown in Figure 7A. The autoradiogram shows that the photoreaction in the presence of PP and TR produced various bands whose intensity increases with the irradiation time. Only minor spots were detected in the control reactions carried out in the dark (data not shown). Moreover, the pattern of photocleavage exhibited by the two drugs was similar. Evaluation of the DNA cleavage sites in the examined sequence of 172 nucleotides (Figure 7B) indicated 33 break points, corresponding to the most frequent termination sites. Termination of DNA synthesis occurred at Gua (36%) and Cyt (36%), whereas strand break at the Ade (18%) and Thy residues (9%) occurred with a low frequency. No particular sequence selectivity was observed in the DNA photocleavage sensitized by the drugs. Laser Flash Photolysis. To reach a deeper insight on the reaction mechanism, laser flash photolysis experiments were carried out on phenothiazines in PBS in the presence of GMP and DNA. As already reported (12), direct excitation of the drugs in the absence of additives produced three transients assigned to the lower triplet state of phenothiazines (λmax ) 480 nm), to their radical cation (λmax ) 520, 530, and 630 nm for PP, FP, and TR, respectively), and to the solvated electron (λmax ) 710 nm) formed within the laser pulse. Figure 8 shows the timeresolved absorption spectra of the drugs in the presence of GMP. In all cases, addition of GMP quenched the radical cation with a rate constant in the range of 105-107 M-1 s-1 (see Table 3) while the lifetime of the lowest triplet state of phenothiazines was not affected. In the case of PP with GMP, the radical cation PP•+ (λmax ) 530 nm, τ ) 22 µs) was a precursor of a further transient, which appears at 360 and 620 nm (Figure 8A). In agreement with literature data (27, 28), this longerlived transient is assigned to the GMP radical cation formed by electron transfer to PP•+. Further decay reactions of the GMP radical cation led to stable photo-

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Figure 8. Time-resolved absorption spectra of the phenothiazines derivatives in PBS solution in the presence of 1 × 10-3 M of GMP. (A) PP spectra recorded at 0.08 (O), 0.2 (0), 1.0 (4), 5.0 (3), and 30 (]) µs after the laser pulse (λexc ) 355 nm). (B) TR spectra recorded at 0.8 (O), 3.2 (0), and 100 (4) µs after the laser pulse (λexc ) 355 nm). (C) FP spectra recorded at 0.8 (O), 2.0 (0), and 160 (]) after the laser pulse (λexc ) 355 nm). Table 3. Properties of the Transients of Phenothiazines in PBS

Figure 7. Identification of the sites of photocleavage by primer extension analysis. (A) The pUC18 template was irradiated in the presence of the phenothiazines derivatives PP and TR at a molar ratio of 0.4 for 0, 15, 30, and 60 min. The DNA fragments synthesized from the M13 primer by the Taq DNA polymerase were separated on a sequencing gel. (B) The sequence of the breakage sites can be directly identified by comparison with the sequence of the same DNA segment, determined as a reference (GATC). pUC18 plasmid that was analyzed from position 269439 is shown. The photodamaged nucleotides at which the DNA synthesis stops more frequently are shaded.

products with an absorption centered at ca. 520 nm, as also indicated by the spectrophotometric analysis of the PP-GMP solution irradiated in steady state conditions. The assignment of these photoproducts is still in progress. Instead, the quenching of the radical cation of FP and TR was not followed by the detection of further transients in the observed wavelength range (300-850 nm). The laser flash photolysis measurements were extended to the phenothiazine-DNA systems in airequilibrated Tris buffer at [drug]/[DNA] ≈ 0.13. The timeresolved absorption spectra obtained by direct irradiation of PP in the presence of DNA are shown in Figure 9 as an example. For this system, the absorption spectra recorded at short delay times (ca. 40 ns) show the presence of absorption bands centered at ca. 500 and 600 nm, which were formed within the laser pulse and decayed by first-order kinetics (τ ) 1 and 0.3 µs at ca.

λmax compds (nm) PP FP TR

490 530 480 520 480 630

τ (µs) 2.9 150 3.3 4 × 106 3.3 190

 (M-1 cm-1) 12 000 17 000 19 000

transient

kq (GMP) (M-1 s-1)

triplet cation radical 3.0 × 107 triplet cation radical 1.3 × 105 triplet cation radical 1.7 × 107

500 and 600 nm, respectively; see Table 4). At longer delay times, these absorptions were replaced by further bands with λmax at ca. 390 and 540 nm, which decayed by mixed-order kinetics (t1/2 ) 15 ms). The absorption band at ca. 500 nm is assigned to the lowest triplet state of PP by comparison with previously reported data (12); instead, the longer-lived transient, which absorbs at ca. 390 and 540 nm, is assigned to the cation radicals PP•+ (12). The short-lived absorption at 600 nm was then attributed to the anion radical of DNA basis formed by electron transfer from the singlet excited state of PP (in fact, its absorption was produced within the laser pulse). This is in agreement with the disappearance of the absorption of the hydrated electron detected in the absence of DNA and with the absorption spectra of the anion radicals of the DNA basis, which show absorption bands above 500 nm (29); under our experimental conditions, the spectral shape below 400 nm is strongly affected by the ground state absorption and then it is not

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Figure 9. Time-resolved absorption spectra of PP in PBS solution in the presence of DNA recorded at 0.04 (O), 0.11 (0), 1.5 (4), and 3.0 (3) µs after the laser pulse (λexc ) 355 nm). Table 4. Properties of the Transients of Phenothiazines in Air-Equilibrated PBS in the Presence of DNA compds PP FP TR

λmax (nm)

τ (µs)

transient

500 540 600 480 520 600 490 600 600

1.0 1.5 × 104 0.3 0.9 63 0.2 0.6 0.4 6.6

triplet cation radical anion radical triplet cation radical anion radical triplet anion radical cation radical

informative. For FP and TR, the results of the timeresolved measurements summarized in Table 4 are in substantial agreement with those obtained for PP, even if the spectral overlap of the ion radicals produced in the TR-DNA system makes it more difficult to analyze the decay kinetics in the 600 nm region.

Discussion The significant changes recorded by both the absorption (spectra) and the emission (fluorescence lifetimes and spectra) measurements upon addition of DNA clearly indicate associative interactions of phenothiazines with nucleic acids. The binding affinity shown by these phenothiazines with DNA is comparable to that already found in the case of the parent chlorpromazine (30). Noteworthy, the values of the exclusion parameter obtained by absorption and emission titrations (n ∼ 3) are higher than those expected from the neighbor exclusion model (n ) 2), thus suggesting that the binding is not consistent with classical intercalation. In fact, the flow LD spectroscopic investigations on the association of PP, TR, and FP with DNA are also consistent with the presence of at least two different binding modes: the intercalation that occurs predominantly at low [drug]/[DNA] ratios and at high [drug]/[DNA] ratios. The results, however, do not prove that the phenothiazine-DNA complexes are fully inserted between adjacent base pairs as in the classical intercalation mode. The presence of two binding modes suggested by the spectroscopic measurements is also in agreement with

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the photochemical behavior of these systems. In fact, phenothiazines lead to photoinduced DNA damage, which depends strongly on the drug concentration. In particular, at low [drug]/[DNA] ratios (