Photosensitizing Effect of Some Nonsteroidal Antiinflammatory Drugs

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Photosensitizing Effect of Some Nonsteroidal Antiinflammatory Drugs on Natural and Artificial Membranes: Dependence on Phospholipid Composition Guido De Guidi,*,† Santa Ragusa,‡ Maria T. Cambria,‡ Alessandra Belvedere,† Alfio Catalfo,† and Antonio Cambria‡ Department of Chemical Sciences and Section of Biochemistry, University of Catania, 95125 Catania, Italy Received July 1, 2004

Previous studies have clarified the molecular mechanism of photosensitization on red blood cell membranes induced by some drugs belonging to the class of nonsteroidal antiinflammatory drugs: ketoprofen, naproxen, and diflunisal. This process involves the participation of photodegradation products, free radicals, and reactive oxygen species. The aim of the present paper is to investigate the photohemolytic process using red blood cells of mammalian species, with different membrane phospholipid compositions. Human and bovine red blood cell membranes were selectively enriched with phosphatidylcholine and sphingomyelin. For this purpose, a new approach for phospholipid investigation was undertaken. Moreover, the phototoxic effect was tested with liposomes at different phospholipid compositions. A structurefunction relationship between the erythrocyte membrane phospholipid composition and the photohemolytic process induced by the sensitizers can be proposed. Indeed, the different contents of the photoperoxidable double bond and the variable architecture of the membrane bilayer, due to the different phosphatidylcholine and sphingomyelin contents, strongly influence the resistance of the cell to an osmotic shock induced by photogenerated transient species or by the lytic activity of drug photoproducts. The higher content of sphingomyelin, its asymmetric disposition at the outer surface of membrane bilayers, the high level of saturated acyl fatty chains, and the presence of photoperoxidable trans double bonds in the hydrophilic region greatly decrease the fluidity of bilayers and enhance the resistance of the membrane to phototoxic damage. On the other hand, an increase in the content of phosphatidylcholine, which is rich in species with unsaturated acyl fatty chains, decreases the membrane resistance, because these latter can be easily oxidized by drug-photogenerated reactive oxygen species.

Introduction

Chart 1

Nonsteroidal antiinflammatory drugs (NSAIDs)1 are widely used in the treatment of pain and inflammation of several diseases (1). Nevertheless, several of them were reported to cause adverse cutaneous reactions that can be associated with photosensitized reactions (2-7). Some of these compounds have been extensively studied in vitro as regards their photosensitizing properties in the presence of biological substrates. Several studies have been carried out with DNA (3, 5, 7, 8 and literature cited herein, 9, 10), proteins (7, 8, 11, 12), erythrocytes (4, 8, 13), and membrane components (7, 8, 13). Among them, ketoprofen (KPF), naproxen (NAP), and diflunisal (DFN) (see Chart 1) can be compared with regard to their photosensitizing properties on the basis of a structural criterion. They are all carboxylic acids; the latter is a * To whom correspondence should be addressed. Tel: +39-0957385061. Fax: +39-095-580138. E-mail: [email protected]. † Department of Chemical Sciences. ‡ Department of Chemical Sciences, Section of Biochemistry. 1 Abbreviations: t , delayed semihemolysis time; DFN, diflunisal; 50 G6P, glucose-6-phosphate; KPF, ketoprofen; NAP, naproxen; NSAIDs, nonsteroidal antiinflammatory drugs; 1O2, singlet oxygen; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PhP, DFN main photoproduct; PL, phospholipid; RBCs, red blood cells; ROS, reactive oxygen species; SIM, single ion monitoring; SM, sphingomyelin; SUV, small unilamellar liposomes; TIC, total ion chromatogram.

salicilyc acid derivative, whereas the others present a propionic unit. Moreover, the aromatic moiety consists of classic chromophores such as diaryl ketone, naphthalene, and diphenyl (14), with a well-known photochemistry and photophysics. KPF, the 2-(3-benzoylphenyl) propionic acid, shows a light-induced lytic activity greater than other propionic acid derivatives and is considered phototoxic in vitro (3-

10.1021/tx049824a CCC: $30.25 © 2005 American Chemical Society Published on Web 01/15/2005

Nonsteroidal Antiinflammatory Drug Effects on Membranes

6, 8). KPF photodegradation involves a triplet state; the photogenerated excited drug species undergo a photodecarboxylation reaction through a type I mechanism, with high quantum yield, via the key intermediacy of benzylic radical, a short-lived species (8, 15). No singlet oxygen (1O2) has been detected, whereas superoxide anion is produced in large amounts (8). In neutral aqueous medium, in addition to the known KPF photodecarboxylation product, dimers and several photoproducts are formed (16). This can be explained by the hydrogenabstracting ability of the excited carbonyl moiety, typical of benzophenone photochemistry. Free radicals, superoxide anion, and sensitizer photodegradation products with lytic activity are the species responsible for photohemolysis (4, 8, 16-20). In particular, the key role played by an oxygen-mediated type I mechanism in KPF photosensitization in the cell membrane was evident (4, 8, 16, 21). NAP, 6-methoxy-R-methyl-2-naphthalene acetic acid, is well-known to cause skin photosensitivity (8). Laser flash photolysis studies showed both photoionization (with production of solvated electrons) and triplet state production, which were generated approximately with the same quantum yield. 1O2 is produced by NAP irradiation, and its presence was confirmed by oxygen uptake measurements (8). NAP photolysis involves decarboxylation with production of free radicals; a photodegradation pathway involving a carbenium ion, whose formation can be explained by homolysis of a radical cation after photoionization, was also proposed (8). The molecular mechanism of photosensitization by NAP was studied through red blood cell (RBC) lysis, lipid peroxidation, and DNA photocleavage. The obtained data were consistent with a mechanism involving free radicals and, mostly, 1 O2, which is produced by 3NAP energy transfer with high quantum yield (8). DFN, 2′,4′-difluoro-4-hydroxy[1,1′-byphenyl]-3-carboxylic acid, was found to be quite phototoxic (8). Defluorination is the primary photochemical act, leading to the photosensitization processes; the noxious species involved were the consequently formed free radicals, superoxide anion, 1O2, and, mostly, the main photoproduct (PhP), a dimer of carboxylic acid formed through SRN1, which is a typical reaction for arylhalides. This PhP, quickly formed during DFN irradiation, was considered the main factor in the photosensitizing process (8). In light of these results, we have focused the study on the photosensitized membrane damage induced by these three drugs because it could be useful for a better comprehension in the phototoxicity and phototherapy fields, and it could shed more light on the mechanisms of both degenerative skin diseases and cell toxicity. In particular, we have compared their different abilities in inducing cell hemolysis and lipid peroxidation in natural and modified membranes. Indeed, some of them provided positive reactions in the photopatch tests (8, 22-24) and were involved in photocontact dermatitis (22, 25-27). This literature thus suggests a probable correlation between photosensitization and photoallergic response. Taking into account the involvement of intermediate radicals and reactive oxygen species (ROS) in the photoinduced membrane damage, we have carried out some experiments to assess the photosensitizing activity of these drugs in modified cells to clarify the mechanism of this process. Change of the membrane structure was focused on phospholipid (PL) composition (28), and the

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link up of RBC membrane PL modification with hemolysis was outlined. In particular, the roles played by phosphatidylcholine (PC) and sphingomyelin (SM), because of their quite different physical and chemical structures, were investigated. For this purpose, two mammalian RBC membranes of human and cow that differ strongly in natural composition with regard to PC and SM distribution (28) were studied, and the effect of selective enrichment was examined. In parallel, the effect of membrane PL composition was investigated by monitoring the photoinduced release of entrapped marker in a simpler model: the small unilamellar liposomes (SUV). This study was carried out on NAP and DFN, because KPF gave swelling but not marker release (8). A comparison between the trend of damage photoinduced by these drugs on cell membranes of natural and modified cells, as well as on SUV, clarified the effect of the membrane PL composition on photosensitization.

Experimental Procedures Materials. KPF, NAP, DFN, egg yolk PC, SM and other PLs, glucose-6-phosphate (G6P), G6P dehydrogenase, and nicotine amide adenine dinucleotide phosphate were obtained from the Sigma Chemical Company (St. Louis, MO). Methanol, acetonitrile, and H2O were of HPLC grade. All other chemicals were reagent grade. Krebs ringer phosphate buffer (KRP): 120 mmol/L NaCl, 4.8 mmol/L KCl, 1 mmol/L CaCl2, 1.2 mmol/L MgSO4, and 16.5 mmol/L NaH2PO4/Na2HPO4, pH 7.4. Phosphatebuffered saline (PBS): 138 mmol/l NaCl, 2.7 mmol/L KCl, and 10 mmol/L NaH2PO4/Na2HPO4, pH 7.4. Analytical Instrumentations. Ultraviolet-visible absorption spectra were recorded on a HP 8452A diode array spectrophotometer. The HPLC MS analyses of membrane extracts were performed on a Hewlett-Packard 1100 chromatograph equipped with an on-line diode array detector and a MSD supplied with an atmospheric pressure ionization electrospray interface. Irradiation Conditions. Irradiation of samples was performed using a Rayonet photochemical reactor equipped with four “black light” phosphor lamps (RPR-3500, Southern N. E. Ultraviolet Co.) with an emission in the 310-390 nm range with a maximum at 350 nm. The incident photon flux on 3 mL of investigated sample in quartz cuvettes (optical length, 1 cm) was 6 × 1015 quanta/s, which is of the same order as the solar fluence incident on skin. A “merry-go-round” irradiation apparatus was used to ensure that all parallel samples received equal radiation. The fluence rate at the irradiation position was about 800 µW/cm2. Further experimental procedures of irradiation and light intensity measurements have been described previously (29). RBC Preparation. Heparinized mammalian blood was kindly supplied by the local abattoir and by a veterinary structure. Blood (20 mL) was transferred to a 50 mL test tube and centrifuged at 1000g for 10 min. The plasma, the buffy coat, and the upper 30% of the cell pellet were removed, and the remaining cells were washed (three times) with 35 mL of KRP. RBCs from samples of out-of-date (not more than 15 days from the date stated) packed human erythrocytes were gently supplied by the local blood bank. However, all mammalian erythrocytes from out-of-date cells gave reproducible results over many days (29). RBCs were prepared by washing 20 mL of them three times with 35 mL of KRP, each time centrifuging the cells at 1000g for 10 min and carefully removing the supernatant. The cell pellets from all species were stored at 4 °C. Liposome Preparation and RBC Enrichment. The procedure of liposome preparation was performed as described by Bigelow et al. (30). The PLs used in the preparation were PC and SM from egg yolk. These PL classes were preferred due to their known and relative pure contents in unoxidized chains as certified by the manufacturer. SM contains mainly palmitic acid

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(16:0), giving amide species such as m/z ) 703. PC has about 50% of unsaturated fatty acids: (18:1)∼30%, (18:2)∼14%, (20: 4)∼4%. The species with sn-1 ) (16:0) and sn-2 ) (18:1) should give a m/z ) 760 [sn, lipid side chain, where, for example, (18: 1) means total number of carbons ) 18 and total number of double bonds ) 1]. As emphasized below, the SM ion species (m/z ) 703) and the PC ion species (m/z ) 760) were utilized for PL class quantification. The liposome suspensions were stored at 4 °C until RBCs enrichment. Thus, the RBC pellet (2 mL) was incubated with liposome suspension for 30 min at 30 °C (1:10 v/v packed RBCs to liposome suspension), centrifuged at 1000g for 10 min, and washed (three times) with 35 mL of KRP buffer. PC-enriched, SM-enriched and untreated cell pellets were stored at 4 °C until drug treatment. For details, see Bigelow et al. (30). Photohemolysis Experiments in Natural and PC or SM Enriched RBC. For all mammalian species, RBCs suspensions of 1.9 × 106 cells/mL (A650 ) 0.5) were prepared in PBS solution containing NSAIDs. In all experiments, the concentrations of NSAIDs were 32 µM KPF, 0.2 mM NAP, or 1 mM DFN, very close to blood serum levels after oral administration of these drugs (31). Each series of test was performed with aliquots from the same sample of blood. After addition of RBCs in PBS solution containing NSAIDs, the cell suspensions were irradiated within 2 h. Nonirradiated controls were placed in the dark. After irradiation, hemolysis was followed by measuring the decrease in the absorbance at 650 nm as a function of the time, measured from the beginning of the irradiation (delayed hemolysis time), since the optical density due to the scattering of cells is linearly proportional to the number of intact RBCs (32). All of the photohemolysis values were obtained by analysis of a sigmoid Boltzman fit. Results were expressed as the time needed to reach 50% delayed hemolysis after irradiation (t50), by comparison with a sample in which the cells had been completely hemolyzed by brief sonication. Further details can be found in a previous paper (29). The selective enrichment experiments were carried out on human and bovine RBCs incubated with SUV containing PC or SM. After natural RBC enrichment, the t50 of the samples (as well as that of their controls) was monitored as a function of the irradiation time (5, 10, and 15 min) and the experiments were carried out in the same conditions used for unmodified cells. RBC Membrane Lipid Extraction. For a thorough description of the procedure, see Rose and Oklander (33). Briefly, an equal volume of distilled water was added to a 1 mL cell pellet of both PC- and SM-enriched RBCs. The lysis proceeded for 15 min, and then, 11 mL of 2-propanol was added by mixing and the extraction continued for 60 min with occasional mixing. Then, 7 mL of chloroform was added and the mixture was incubated for an additional time of 60 min. The extract was centrifuged for 30 min at 500g, and the supernatant was filtered through a cotton-plugged Pasteur pipet. The lipid extract was dried on a rotavapor at 50 °C, washed down with 2 mL of chloroform/methanol 2:1 (v/v) to concentrate the sample, and dried under a stream of nitrogen. The membrane lipids were then dissolved in 0.5 mL of chloroform and stored in a freezer at -25 °C (for no more than 24 h) until analysis by TLC or HPLC/MS. Separation, Identification, and Quantification of PL by HPLC/MS. The membrane extracts were injected (20 µL loop) into an analytical Lichrospher Si 60 column protected by a precolumn (5 µm, 250 mm × 4.6 mm, Merck) and column prefilter (0.5 µm, Supelco). The experimental conditions were as follows: flow rate of 1 mL/min and linear gradient through eluent A, chloroform/methanol/ammonium hydroxide (800:195: 5, v/v), and eluent B, chloroform/methanol/water/ammonium hydroxide (600:340:55:5, v/v) (34): 0-14 min, 100% A to 100% B; 14-25 min, hold 100% B; 25-30 min, 100% B to 100% A; 30-45 min, hold 100% A (column regeneration). For qualitative measurement, the MSD with an ESI source was set as follows: positive ion mode, scan range 600-1000 amu; nebulizer and

De Guidi et al. curtain nitrogen gas, 60 psig and 13 L/min, respectively; drying gas at 350 °C; capillary voltage, 3.5 kV; and fragmentor, 100 V. For quantitative measurement, the detector was set as follows: positive ion mode, time programmed single ion monitoring (SIM) 0-12.5 for a species of PC ) 760 amu and 12.5-30 min for one of SM ) 703 amu. Results are reported as mg lipid species/mL solution after the correlation of the integrated peaks, of the opportune MSD trace, with the respective standard curve of genuine lipid class. Cell lipids were separated by means of TLC and quantified by the inorganic phosphorus assay as described by Bigelow et al (30). Aliquots were employed to build SIM calibration curves. For a quantitative measurement of PL with the ESI technique, the use of the identical PL class for calibration curve generation was necessary, because of the dissimilar amount of PL species present in the egg yolk standard with respect to that of the relevant class in RBCs. Peaks identification was assessed as described below. Detection in the negative ion mode resulted in low ion intensities (sensitivity) and no pseudomolecular signal. G6P Assay (Liposomes Preparation and Marker Efflux Assays). SUVs with entrapped marker G6P were prepared with the solvent injection method as described by Kremer and van der Esker (35). Details appear in Costanzo et al. (36). SUVs were irradiated in the presence of photosensitizers, and membrane damage was measured by efflux of the entrapped marker, which was determined spectrophotometrically at 340 nm, after addition of G6P dehydrogenase and nicotine amide adenine dinucleotide phosphate, according to the method of Girotti and Thomas (37). Other Assays. Samples of membrane preparation were assayed for cholesterol according to Allain et al. (38) and for protein as described by Lowry et al (39). Hemoglobin determination was performed to estimate the cell number, in parallel and during extraction of membrane lipids. Briefly, a 1 mL cell pellet was diluted with 9 mL of water. The suspension was incubated at room temperature for 15 min. Finally, 20 µL of solution was taken and diluted with 980 µL of PBS. Therefore, the human RBCs samples had an absorbance (at 540 nm) around 0.47 that corresponds to 0.008 µmol of hemoglobin (540 ) 53000).

Results and Discussion Membrane Resistance of RBCs to NSAIDs Induced Photosensitization in Various Mammalian Species: Effect of PL Composition. An accurate investigation about the membrane nature with particular regard to its PL composition allows us to focus the attention on the diversity of various PL membrane components and the characteristic response of these latter to the interaction with physical agents such as UVA light in the presence of the NSAID drugs chosen for this study: KPF, NAP, and DFN. The data of photosensitized hemolysis induced by 32 µM KPF in RBC suspensions of different mammalian species are reported in Figure 1a,b. After 10 min of irradiation, the t50 values were in the range of 80-275 min. Considering the RBC membrane composition in PL of several mammalian species, as reported by Nelson et al. (28), some of these were chosen. The RBCs of cow, sheep, and goat, which show no detectable PC content but have a very high content of SM (about 50% of all PL), present a lower level of photohemolysis, whereas the RBCs of horse and dog, which contain a low amount of SM (13.5 and 10.8%, respectively) and nearly 50% of PC (28), present a high level of photohemolysis. The RBCs of human, cat, and pig, which contain a nearly equal percent amount of PC and SM, present an intemediate level of photohemolysis. Similar results are obtained for irradiation of NAP and DFN solution in the presence of RBC suspensions of


Figure 1. Photosensitized hemolysis induced by KPF (32 µM) in 1.9 × 106 cells/mL RBCs suspension in PBS solution; UVA irradiation time, 10 min. Horse (0), cat (2), pig (4), goat ()), human ((), sheep (O), cow (b), and dog (9). (a) Dependence on natural SM composition; (b) dependence on natural PC composition. At least three independent sets of measurements, in duplicate, were carried out for each test. Mean values ( standard errors are reported. Table 1. t50 (min) for Irradiation of RBC Suspension of NSAID Solution for Various Mammalian Speciesa dog horse cat pig

KPF

NAP

DFN

90 80 125 110

90 70 125 120

75 40 130 90

man goat cow sheep

KPF

NAP

DFN

110 200 275 170

190 160 195 140

250 230 220 190

Irradiation time, 10 min; 1.9 × 106 cells/mL RBC. [KPF], 32 µM; [NAP], 0.2 mM NAP; [DFN], 0.1 mM. At least three independent sets of measurements, in duplicate, were carried out for each test. Significances of difference from control values were determined with the Student’s t-test, and the level of significance was set as p < 0.01. Mean values are reported. a

different mammalian species (Table 1). Considering the relative amount of SM and PC, respectively, a particular resistance against the damage in mammalian species with abundant membrane SM percentage can be observed. On the other hand, a lower resistance against damage in those species with abundant membrane PC percentage occurs. Thus, a dependence of natural PC and SM concentration to photoinduced membrane damage could be rationalized. Photohemolysis Experiments with PC and SM Enriched RBCs. To further demonstrate the effect of PL composition on NSAID-induced photohemolysis, assays were carried out on bovine and human RBCs where membranes were selectively enriched with SUV containing PC or SM. Bovine and human cells appear particularly suitable for this procedure. In fact, bovine cells lack of PC in their membranes vs a percentage of SM of about 46. Human cells have an intermediate content, with regard to the two investigated PLs, with respect to the other mam-


Figure 2. Photohemolysis of 1.9 × 106 cells/mL RBCs suspension of each mammalian species in a PBS solution of 32 µM KPF, as a function of the irradiation time. (a) Hemolysis assay of human cells of SM-enriched (2), natural (9), and PC-enriched (1) cells. (b) Hemolysis assay of bovine cells of SM-enriched (2), natural (9), and PC-enriched (1) cells. At least three independent sets of measurements, in duplicate, were carried out for each test. The significance of differences from control values was determined with the Student’s t-test, and the level of significance was set as p < 0.01. Mean values ( standard errors are reported.

malian species. Indeed, they show a percentage of PC and SM of 29 and 27, respectively (28). All of the membrane samples were assayed for protein and cholesterol contents: After selective PL enrichment, the contents of membrane protein and cholesterol did not substantially change (data not shown). For example, the human RBC cholesterol/PL ratio was assessed to be about 0.8 in all samples. In Figure 2a,b, the results concerning the KPF photoinduced osmotic shock in human- and bovine-enriched cells (as well as their controls), as a function of the irradiation time, are reported. The t50 values of the samples were in the range of 50-330 min. It is interesting to note that PC-enriched cells always show a decrease in the t50 value, i.e., an increase of photoinduced membrane damage with respect to the control. In human cells, a decrease of a maximum of 15% in t50 is obtained in PCenriched RBCs, whereas an increase of this value up to 17% is observed in SM-enriched samples indicating that SM always protects against photodamage. In bovine cells, the influence of PL enrichment on the photohemolysis rate, even if characterized by higher t50 values, shows a behavior comparable with that of human cells. The observed trend is similar for all irradiation times. The same behavior was observed in the case of NAP and DFN photoinduced osmotic shock in human and bovine cells (see Table 2), and it occurs at all irradiation times used.

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Table 2. t50 (min) for Irradiation of Human and Bovine RBC Suspension (1.9 × 106 Cells/mL) in the Absence and in the Presence of PC and SM Enrichmenta + SM

no enrichment

+ PC

human bovine

NAP (0.2 mM) 205 190 210 195

180 190

human bovine

125 290

KPF (32 µM) 110 275

105 270

human bovine

265 325

DFN (0.1 mM) 250 310

240 305

a Irradiation time, 10 min. At least three independent sets of measurements, in duplicate, were carried out for each test. Significances of difference from control values were determined with the Student’s t-test, and the level of significance was set as p < 0.01. Mean values are reported.

These results suggest that a higher content of a peroxidable PL such PC should greatly influence the photohemolysis course. As a consequence, it can be moreover deduced that cells with a selective increased PC content are more sensitive to injury, while those with a selective increased SM content show an enhanced resistance to membrane damage, with respect to the controls. This behavior is valid in the case of both investigated bovine and human RBCs. Indeed, the ROS formed during drug irradiation are believed to be involved in photohemolysis through modification of membrane components (37). The membrane is a cell constituent, which is mostly susceptible to oxidative damage. In particular, mono- and, most preferably, polyunsaturated acyl chains of lipids are involved. Oxidized PLs are largely a result of the attack of ROS at polyunsaturated fatty acyl substituents, which remove bisallylic hydrogen atoms forming a lipid carbon-centered radical that reacts with molecular oxygen (40). Lipid peroxidation in the membrane usually occurs as a chain reaction that can only be terminated by a chain breaking antioxidant. This final reaction results in the formation of lipid hydroperoxides. These species are relatively unstable and tend to break down to give a variety of products including hydroxy lipids, peroxydized lipids, alkenes, and aldehydes (41). The literature reports, as a consequence of the addition of one, two, and three molecules of dioxygen on lipids, the formation of oxidized chain shortened products (42, 43), epoxide products (44), and chlorohydrin products (45). Thus, the higher content of SM, its asymmetric disposition at the outer surface of membrane bilayers, the high level of saturated acyl fatty chains, and the presence of trans double bonds in the hydrophilic region greatly decrease the fluidity of bilayers and could enhance the resistance of the membrane to phototoxic damage. On the other hand, the higher content of PC, with a large amount of polynsaturated aliphatic groups, at the sn-1 and sn-2 positions, is a key factor in determining lower transition phase temperatures and increasing lateral diffusion of membranes (46). These conditions make the cells more vulnerable to lipid photoinduced peroxidation. Quantification of PL Enrichment in RBCs. To evaluate the extent of selective PL enrichment of the two mammalian species, the coupled use of the HPLC-ESIMS analysis was applied. This technique allows a quantitative and extremely sensitive and accurate separation

and characterization of both saturated and unsaturated species, such as intact PLs. The method has the ability to resolve the major amine containing PL classes or detect any of their species in a single run. Mass detection is relatively independent of the degree of unsaturation (see SM species) and has a sufficient sensitivity to allow fg quantification of PL species. Moreover, it is possible to correlate the amount of each PL species to the whole class. Indeed, in each native PL class, the species are present in a constant proportion (47). They were identified by comparison with previously published spectra (48, 49), and further information about molecular structure was obtained adopting collision-induced dissociation (in source CID) by increasing the ESI orifice potential, which gives high yields in fragments corresponding to the fatty acyl substituents esterified at sn-1 and sn-2 positions of the PL species (data not shown) (47). In any case, the external calibration was performed with genuine standards obtained from RBCs membranes (see Experimental Procedures). The use of other standards such as egg yolk gives false results because of the noncomparable quantity and distribution of species from these standards with respect to natural RBCs membrane species. Figure 3a shows the chromatogram of a qualitative analysis of human lipid extract. Ion peak spectra of SM and PC are reported in insets 1 and 2, respectively. Given that between the various ion species, the 760+ for PC and the 703+ for SM were some of the most abundant ones, these were chosen for the quantitative evaluation in the SIM analysis (Figure 3b). The calibration curves were obtained using genuine standards to correlate their concentration with the integrated area of MS response. Table 3 shows the quantitative ESI-MS results for membrane extracts specific enrichment, values in brackets. PC species with sn-1 ) (16:0) and sn-2 ) (18:1) should give a m/z ) 760. On the other hand, a large number of species with a lack of polyunsaturated centers in the acyl groups are present. Indeed, the large difference in the chain lengths of the fatty acid in the amide linkage, present in SM, could explain the separation into two fractions for both the human and the bovine species in the qualitative analysis (Figures 3a and 4a). Thus, in bovine RBCs, the first peak (at about 13 min) contains SM with long chain behenic (22:0) and lignoceric (24:0; 24:1) acid with relative amounts of 16.3 and 63%, respectively (50). The other peak (at about 14 min) consists of palmitic (16:0) acid with a relative amount of 85.8%, giving amide species such as m/z ) 703. Only (18: 2) and (22:2) chains in the 5% level are present as polyunsaturated acylic groups. Similar results are reported for human RBCs (51). Photosensitized G6P Release from SUV. Data regarding the photosensitized entrapped G6P release from SUV induced by NSAIDs as a function of PL membrane contents are reported in Figure 5. Because of the fact that KPF liposome photosensitization leads to a swelling without any release of marker (8), only NAP and DFN were used. The artificial membranes (about 1 mM PL) in 10 mM PBS solutions containing 0.2 mM NAP were subjected to UVA irradiation up to 20 min, whereas longer irradiation times (up to 40 min) were used for 1 mM DFN. This is justified by the different photodegradation quantum yields of the two drugs measured in homogeneous solution (8). The quantum yield of NAP photodegradation and DFN photoproduct formation (which is almost the only



Figure 3. (a) Representative total ion chromatogram (TIC) (scanning mode) of human RBCs membrane lipid extract. Inset 1: Ion peak spectrum of SM. Identification of the main ions of SM peak, i.e., lipid species of SM. Inset 2: Ion peak spectrum of PC. Identification of the main ions of PC peak, i.e., lipid species of PC. (b) TIC of time programmed SIM: 0-12 min, monitoring of PC 760+ species; 12-30 min, monitoring of SM 703+ ion species. Table 3. ESI-MS Results of Membrane Extractsa specific PL enrichment PL class % PC SM others PS PE

human

cow

control with PC with SM control with PC with SM 29 24 1.1 13 2

37 (+7) 20 0.9 11 2

27 32 (+28) 0.7 1 27

ND 46 3.3 20 29

6.3 (+6.3) 42 2.3 21 25

ND 53 (+7) 2.6 19 26

a The PL specific enrichment in modified cells is about 6% (values in brackets). Abbreviations: PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; LPC, lysophosphatidylcholine; X, unknown; ND, not detected. At least three independent sets of measurements, in duplicate, were carried out for each test. Significances of difference from control values were determined with the Student’s t-test, and the level of significance was set as p < 0.01. Mean values are reported.

responsible for photodamage) are 1 × 10-2 and about 3 × 10-3, respectively. Generally, the higher the irradiation time, the higher is the marker release. It must be noted that in each case an increase of SM artificial membrane content results in a decrease in marker release. On the contrary, it follows that a higher PC content in liposomes is needed

to increase the marker release. When SUVs with a different percent content (0-100%) of two PLs are irradiated in the presence of NAP, G6P release decreases with increases of SM content to reach no release at 100%. This behavior is independent of the irradiation time. In the case of DFN, when 100% PC liposomes are photosensitized, a maximum marker release of 40% decreases to 15% when the liposome is totally built of SM. Therefore, the light-induced G6P release from liposomes by NAP and DFN is dependent on the PC and SM concentration and confirms the relevance of SM content in the resistance to phototoxic effects observed in RBCs. Comparing Figures 2 and 5 as well as Table 2, it is important to note that the specific PL enrichment in cells (about 5%), when compared with a 5% variation of PL liposomes, results in damage or protection of the same order. Unfortunately, the tentative of obtaining enrichment higher then 8% in cells gave unreproducible results probably due to elevated perturbation in the cell bilayer. Some observations can be made on the basis of RBCs and SUV photosensitization experiments: (i) The trend of membrane resistance with PL composition is independent of the photosensitizer used. (ii) The higher observed damage for DFN photosensitization, as compared to NAP

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Figure 4. (a) Representative TIC (scanning mode) of bovine RBCs membrane lipid extract. Inset 1: Ion peak spectrum of SM. Identification of the main ions of SM peak, i.e., lipid species of SM. (b) TIC of time programmed SIM: 0-12 min, PC 760+ ion species, whereas 12-30 min, SM 703+ ion species.

Figure 5. Photosensitized G6P release from SUV suspension after UVA irradiation (about 1 mM PL, 0.1 M G6P entrapped) in 10 mM PBS solutions containing 0.2 mM NAP or 1 mM DFN. Irradiation times: 0 (/), 5 ((), 10 (2), 15 (1), and 20 ()) min for NAP and 30 (O) and 40 (0) min for DFN. At least three independent sets of measurements, in duplicate, were carried out for each test. Mean values ( standard errors are reported.

and KPF experiments, can be attributed to the presence of a photoproduct that was demonstrated to be a highly damaging species toward membranes also in the absence of UVA irradiation (8). (iii) On comparing the relative rates of photohemolysis in the two model species, bovine and human (Table 2), the inversion in the relative efficiency of RBC photosensitization induced by NAP and KPF can be ascribed to the fact that NAP-induced

De Guidi et al.

photosensitization is mainly attributed to 1O2 (8) whereas KPF photosensitizing activity can be ascribed mainly to oxygen radicals, in particular to superoxide anion and its decay product hydroxyl radical (4, 8, 16, 21). Indeed, the drug intermediate radicals provoke lipid peroxidation on the outer leaflet of the RBC bilayer and increase its permeability. This produces a favorable situation for the toxic activity of superoxide anion because it can diffuse into erythrocytes, where suitable conditions for a localized generation of OH• radicals via Haber-Weiss reactions, with a consequent destructive attack on membrane, are possible (41). On the contrary, this diffusion process is not favored in the case of 1O2 due to its shorter lifetime. On the basis of these remarks, a comparison among the photosensitizing activity of different drugs toward both RBCs and liposomes provides useful information about the correlation among the photochemistry of the drugs and their photosensitizing action. In addition, the study of photosensitization in artificial membranes suggests that possible influences of other cell components different from PL are negligible, at least for these models. The results of both cells and liposomes are in agreement and strongly support the fact that SM strengthens the membranes against photodamage, contrary to what was observed for PC. This can be explained also with the highly asymmetric distribution of PLs in biomembranes. In particular, the SM is localized completely on the outer surface of the bilayer in erythrocyte membranes and its compact structure protects the membranes against the photoinduced effect. Indeed, the “interface region” between the hydrophobic acyl chains and the zwitterion headgroup is more complex than the glycerol diester group in the PC. The presence of NH and OH groups could lead to hydrogen bond formation between the SM molecules or to intramolecular hydrogen bond formation between these groups and the phosphate of the headgroup. Thus, a structure-function relationship of erythrocyte membranes PL composition and the photohemolytic process induced by the sensitizers can be suggested. Consequently, the RBCs and SUV enrichment in PC, with a great number photooxidable species, can constitute a specific target involved in photohemolysis and marker release, respectively. Indeed, resultant oxidized PL can physically alter the cellular membrane due to the reorientation of the oxidized fatty acyl substituent toward the aqueous interface causing as much as a 50% increase in the membrane surface area (52) as well as inducing membrane shape change and alterations in membrane permeability (53), i.e., initiation of hemolysis process. The results obtained by photosensitized entrapped G6P release from liposomes with different PL composition confirmed on the other hand the relevance of SM content on the resistance to phototoxic effect. The mechanism of phototoxic damage can be explained by the formation, during drug irradiation, of photoproducts or ROS, which are believed to be involved in photohemolysis through peroxidation of membranes PLs. In conclusion, these observations can alert one about the choice of testing material (for both in vivo and in vitro experiments) in general screening or target analysis in photosensitization studies.

Acknowledgment. Financial support from the following institutions is gratefully acknowledged, the Italian MURST, in the framework of the “Programmi di ricerca


di rilevante interesse nazionale” (Project: Fotoprocessi di interesse applicativo), the Universita` degli studi di Catania “Progetto giovani ricercatori Anno 1999”, and the Consorzio Interuniversitario Nazionale “La Chimica per l’Ambiente Cluster 11, legge 488”.

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TX049824A

Photosensitizing Effect of Some Nonsteroidal Antiinflammatory Drugs

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