Photodynamic DNA damage mediated by {delta}-aminolevulinic acid-induced porphyrins

P. Duez1,, M. Hanocq and J. Dubois

Institut de Pharmacie, Service de Chimie Bioanalytique, de Toxicologie et de Chimie Physique Appliquée, Université Libre de Bruxelles, CP 205/1, Bd du Triomphe, 1050 Bruxelles, Belgium


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mutagenic properties of UVA are thought to be predominantly radical-mediated, which supposes endogenous sensitizers. In order to investigate a possible role of porphyrins, their synthesis was induced in a murine leukemia P388D1 cell model by treatment with {delta}-aminolevulinic acid ({delta}-ala). Intra-cellular protoporphyrin IX reached a plateau after about 2 h, whereas soluble porphyrins, probably the photostable uro- and/or coproporphyrins, were excreted. Irradiation of treated cells by UVA (tanning lamp) but also by visible light was found to generate in DNA a significant increase of 8-oxo-7,8-dihydro-2'-deoxyguanosine, a mutagenic marker of oxidative damage. The different parameters involved in this photodynamic effect are reported, namely {delta}-ala concentration and loading time, light dosage and the influence of intracellular and medium-excreted porphyrins. These results point to an implication of porphyrins in solar-induced carcinogenicity but also in possible adverse effects of the medical applications of photodynamic therapy and diagnosis.

Abbreviations: {delta}-ala, {delta}-aminolevulinic acid; dG, 2'-deoxyguanosine; G, guanosine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate buffered saline; PDD, photodynamic diagnosis; PDT, photodynamic therapy; PP IX, protoporphyrin IX; ROS, reactive oxygen species.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The current craze for tanning observed in Caucasian populations involves many hours of sun exposure and massive recourse to sun beds. The use of protective creams, the vast majority of which primarily filter UVB, markedly reduces the erythema warning signals, allowing mass exposure to unprecedented levels of UVA and visible light (1). Solar exposure is, however, a recognized carcinogenic factor and all UV portions of the light spectrum have been implicated (2,3). Whereas the energy of UVC (220–290 nm; practically absent in ground-level solar light) and UVB (290–315 nm) is directly absorbed by the nucleobase chromophores to produce pyrimidine dimers and 6–4 photoproducts, the UVA absorbance of DNA is considered negligible. The mutagenic properties of this spectral band would therefore be predominantly radical-mediated, through the production of reactive oxygen species (ROS) by photoactivation of cellular sensitizers.

Nucleic acids are indeed a major target for oxidative modifications (2,4). Previous work (Table IGo) on DNA in solution, cell culture, animal models and man has demonstrated that the entire UV spectrum can induce DNA oxidation, and a series of exogenous photosensitizers have been determined. For those exposure conditions in which no exogenous sensitizers are added, a radical-mediated mechanism of carcinogenicity, however, supposes endogenous sensitizers, which are still unidentified; likely candidates include NADP, NADPH, bilirubin (5), porphyrins (2,6) and riboflavin (7).


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Table I. Published data on 8-oxodG photo-induction
 
Among the numerous DNA base modifications and adducts (8) described, the highly mutagenic lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-hydroxy-2'-deoxyguanosine or 8-oxodG) (911) is increasingly considered as a marker of oxidative damage both in carcinogenesis (1214) and aging (15) studies. Although the exact steady-state levels of 8-oxodG as evaluated by different methods are still controversial (16), significant increases have been observed upon treatment with a large number of mutagens in a series of models (14).

To determine if endogenous porphyrins could be photosensitizers relevant for carcinogenesis, we have incubated in vitro a continuous cell line with {delta}-aminolevulinic acid ({delta}-ala) to induce porphyrin synthesis and, after exposure of treated cells to light, we assessed the DNA damage by measuring its 8-oxodG level. {delta}-Ala, a key intermediate in the porphyrin biosynthetic pathway, has been widely investigated as a topical or systemic drug for cancer detection [photodynamic diagnosis (PDD)] or treatment [photodynamic therapy (PDT)]. Upon administration, the major regulatory step of porphyrin synthesis, a feedback inhibition of {delta}-ala synthase by haem, is bypassed and porphyrins, mostly protoporphyrin IX, can be produced. For still obscure reasons, this synthesis is more active in tumour cells than normal cells, providing the means to selectively localize or destroy cancer tissues through a subsequent photoactivation.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Solutions of proteinase K (Tritirachium album; EC 3.4.21.14) and alkaline phosphatase (calf intestine; EC 3.1.3.1) were obtained from Böhringer Mannheim (Germany). Nuclease P1 (Penicillium citrinum; EC 3.1.30.1), 2'-deoxyguanosine (dG), guanosine (G), 8-oxodG, {delta}-aminolevulinic acid hydrochloride ({delta}-ala) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma (St Louis, USA). 8-OxoG was from Calbiochem (La Jolla, USA). Cell culture medium, phosphate buffered saline (PBS) and reagents were from Gibco Life Technologies (Paisley, UK).

Cell culture and irradiation
All the work was performed in darkened rooms. Murine P388D1 leukaemia cells (ATCC CCL-46) were maintained as a cell suspension in logarithmic growth at 37°C in a humidified atmosphere of 5% CO2 in air, in RPMI 1640 medium supplemented with 10 mM HEPES, 100 U/ml penicillin, 100 U/ml streptomycin and 9% fetal bovine serum (South American origin). One day before the experiment, cells were harvested by centrifugation (900 g, 2 min) and suspended at a density of 5x106 cells in 6 ml fresh medium in 25 cm2 aerated culture flasks. After a 22–26 h stay in the CO2 incubator, 100 µl of a fresh solution of {delta}-ala were added and the flask was maintained in the incubator for the indicated loading time. The stopper was hermetically sealed and the flask exposed for 5–60 min to light from either a typical UVA tanning lamp (total output of an array of four tanning tubes, Philips Cleo 15 W; continuous spectrum from 300 to 425 nm, with a maximum at about 354 nm and presenting the spikes of mercury lines at about 365, 406, 436, 546, 580 and 630 nm; UVB/UVA ratio, 0.012; fluence rate averaged 0.31 mW/cm2) or visible light (total output of three spots, Philips Spotone R95, 75 W; continuous spectrum starting at 370 nm and extending above 850 nm; very low emission below 400 nm and maximum at about 725 nm; fluence rate averaged 11.9 mW/cm2). The polyethylene of the flask presented a 50% UV cut-off at 308 nm, 70% transmittance at 355 nm and 85% transmittance above 380 nm. Power densities were measured at cell level, through a culture flask containing 6 ml culture medium, with a Nova Display equipped with a calibrated 2A-SH thermopile head (Ophir Optronics, Peabody, USA). During irradiation, the flask temperature was regulated at 4°C with the help of a circulating water bath. Sham controls were prepared in exactly the same way, but without light exposure.

Analysis of 8-oxodG
Immediately after treatment, the cells were isolated (900 g, 2 min), washed two times with cold PBS, transferred to a 1.5 ml Eppendorf tube, centrifuged (900 g, 2 min), gently overlaid with 250 µl HM buffer (300 mM sucrose, 25 mM Tris, 2 mM EDTA-Na2H2, 6 mM reduced glutathione, 2 mM desferoxamine mesylate, 8 mM DL-histidine, pH 7.3) and stored at –80°C for a maximum of 3 weeks. As described previously (8,17), the cells were digested by proteinase K, urea and SDS, and extracted with 24:1 (v/v) chloroform:isoamyl alcohol; after precipitation from the aqueous phase, washing, drying and redissolution, the nucleic acids were digested to nucleosides by successive treatments with nuclease P1 and alkaline phosphatase. The resulting mixture was analysed by HPLC (17) on a reverse phase 5 µm octadecyl column with serial UV (245 nm) and amperometric (glassy carbon, +700 mV vs Ag/AgCl) detection. The analytical method was fully validated (17), according to recognized quality criteria (18,19); the applied time schedule and procedure minimize the artifactual production of the analyte and result in a total (between-assays) relative standard deviation of 3%: (i) all work is under reduced light; (ii) ice-cold organic solvents are used; (iii) enzymatic digestion and chromatography are performed within the same day.

Fluorescence spectroscopy of porphyrins
The cells were loaded with {delta}-ala as described for the irradiation experiments and immediately centrifuged (900 g, 2 min); the supernatant and the pellet (washed two times with 3 ml PBS) were frozen at –20°C until spectroscopic measurements. For quantitative evaluation, the porphyrins were extracted with 1 M HClO4:CH3OH (1:1, v:v) as described (20); this procedure monomerizes porphyrins so that their concentration can be reliably determined by fluorescence measurements (21). Upon thawing, some samples were sonicated (2000 W, 30 s) in 1 ml PBS with a Soniprep 150 (MSE, Sanyo Gallenkamp PLC, UK) to record fluorescence spectra at physiological pH. The fluorescence spectra and measurements were recorded with a luminescence spectrometer Aminco-Bowman Series 2 (Sim Aminco, Urbana, IL, USA). The absolute fluorescence signals were standardized to the protein content of the sample, as determined by the method of Lowry (22).

MTT cytotoxicity testing
Cells (2x104) were seeded in 96-well plates; after a 22–26 h stay in the CO2 incubator, base 2 logarithmic dilutions of a fresh solution of {delta}-ala in culture medium were added. After a 3 h stay in the incubator, the plate was sealed with a teflon rubber and exposed to light as described (30 min, 4°C); the teflon was removed and the plate returned to the incubator for 48 h. The supernatant was then replaced by a MTT solution in PBS (5 mg/ml) and the plates left for a further 2 h in the incubator. The supernatant was replaced by dimethyl sulfoxide to dissolve the crystals of reduced formazan (15 min agitation, 700 r.p.m.) and the absorbances were measured with a Labsystems iEMS reader/dispenser MF (Labsystems, Finland) at 540 nm versus a 620 nm reference (23).


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Porphyrin synthesis by P388D1 cells
The expected porphyrins produced upon incubation with {delta}-ala are the water-insoluble protoporphyrin IX (PP IX) and the soluble uro- and/or coproporphyrins. The fluorescence properties of the intra-cellular porphyrin species (maxima, {lambda}exc., 413 nm, {lambda}em., 636 nm, switching to respectively 408 and 604 nm, with a second maximum at 664 nm, in HClO4: methanol) are in agreement with those published for protoporphyrin IX (PP IX) (20,21,24). The kinetics of the increase in fluorescence (Figure 1Go) over 8 h demonstrate that a plateau in PP IX production is reached after ~2 h. In the medium, there is continuous excretion of other porphyrin species (maxima, {lambda}exc., 399 nm; {lambda}em., 620 and 680 nm, switching to respectively 404 nm, 603 and 660 nm in HClO4:methanol); these fluorescence properties are consistent with the water soluble uro- and/or coproporphyrins (25).



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Fig. 1. Induction of porphyrins in P388D1 cells by {delta}-aminolevulinic acid (1 mM)—fluorescence kinetics (n = 3; mean ± SD). {circ}, Intracellular porphyrins ({lambda}exc, 408 nm; {lambda}em, 605 nm); •, porphyrins excreted in the medium ({lambda}exc, 404 nm; {lambda}em, 603 nm).

 
Light-induced nucleic acid damage
8-OxodG and 8-oxoG are produced in nucleic acids upon photodynamic activation of treated cells with either UVA or visible light (Figure 2Go; Table IIGo); this oxidation depends on {delta}-ala loading time (Figure 3Go) and is consistent with the time course of PP IX induction.



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Fig. 2. 8-OxodG production through light-irradiation—HPLC chromatograms of enzymatically digested nucleic acids. The cells were loaded with {delta}-ala (1 mM) for 3 h and irradiated for 30 min (4°C; 560 mJ/cm2 for the UVA tanning lamp; 21.4 J/cm2 for visible light). The separation was performed on an octadecyl column (Ultrasphere C18, 5 µm; 250x4.6 mm i.d. with guard cartridge, Beckman, Fullerton, USA) maintained at 30°C. The mobile phase was a mixture of an aqueous buffer (0.05 M NaH2PO4, 1 mM EDTA-Na2H2, pH `as is', 5.45) and methanol (92.5:7.5) at 1 ml/min; detection was amperometric on a glassy carbon electrode set at 700 mV vs Ag/AgCl.

 

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Table II. Comparison of DNA (8-oxodG) and RNA (8-oxoG) photo-oxidation after porphyrin induction in P388D1 cells
 


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Fig. 3. Effect of {delta}-ala loading time. {delta}-Ala concentration was 1 mM and irradiation conditions were as in Figure 2Go (n = 3; mean ± SD). (Student t-test versus the respective control; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

 
The 8-oxodG production increases with light exposure (Figure 4AGo). This may be due to an increase in the odds that individual cells in suspension absorb light as time increases; this may also suggest either a low bleaching rate of PP IX, a continuous resynthesis of fresh porphyrin from {delta}-ala in the medium or progressive cell death from the photodynamic treatment. There was no effect of the {delta}-ala concentration parameter in the investigated range (0.3–1.6 mM) (Figure 4BGo).



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Fig. 4. Influence of light dosage and {delta}-ala concentration in loading medium. {delta}-Ala loading time was 3 h and irradiation was performed at 4°C (n = 3; mean ± RANGE). (A) {delta}-Ala concentration was 1 mM. The fluence rates were 0.31 mW/cm2 for the UVA tanning lamp (•) and 11.9 mW/cm2 for visible light ({circ}). (B) Irradiation time was 30 min, corresponding to 560 mJ/cm2 for the UVA tanning lamp (•) and to 21.4 J/cm2 for visible light ({circ}).

 
Cell survival curves 48 h after UV exposure are shown in Figure 5Go; a comparison with Figures 3 and 4GoGo shows that at {delta}-ala and light dosages inducing a low cell death a significant DNA damaging effect is still observed.



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Fig. 5. MTT cytotoxicity testing of {delta}-ala doses combined with UVA (A) and visible light (B) irradiations. {delta}-Ala loading time was 3 h, irradiation was performed at 4°C for 30 min, corresponding to 560 mJ/cm2 for the UVA tanning lamp and to 21.4 J/cm2 for visible light. MTT test was performed 48 h after irradiation. All the experimental results obtained from two separate experiments in quadruplicate (• and {circ} for experiments 1 and 2, respectively) were fitted to a parametric function by means of an original simplex algorithm (63,64): N = N°. exp (–k.C), where C = concentration, N = percent of living cells at concentration C, = percentage of living cells at concentration 0 and k = parameter.

 
The influence of intracellular and medium-excreted porphyrins was also studied by exchanging, before UVA irradiation, the media of control cells and {delta}-ala loaded cells (Figure 5Go). When treated cells are irradiated in the presence of control medium, similar effects are observed as for treated cells irradiated in treating medium, until 30 min of irradiation. After that time, a reduced effect is observed for the control medium, which probably corresponds to cellular depletion of the porphyrin precursor, {delta}-ala. The supernatant of {delta}-ala treated cells had no photodynamic activity on control cells, indicating that only the intracellular porphyrins participate in the light-induced oxidation.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Potential consequences for solar carcinogenicity
The carcinogenic potential of natural sunlight is mostly a consequence of direct excitation of DNA chromophores by UVB, generating mutagenic dimeric photoproducts at bipyrimidine sites (26). Whereas the UVA contribution is estimated at around 10–20%, adding up arithmetically with UVB in the induction of skin carcinoma (27), no data are available yet on the possible contribution of additional massive doses of visible light, such as undergone under sunlight or in tanning salons.

UVA is furthermore a complete carcinogen by itself, acting as both initiator and promoter. This range of the light spectrum generates oxidative modifications in DNA such as 8-oxodG (Table IGo) and various lesions including strand breaks, abasic sites and DNA–protein cross-links (26). These mutagenic damages are radical-mediated and the major involvement of singlet oxygen (1O2), most probably originating through a type II photosensitizing mechanism, has been established (3,28); a possible contribution of hydroxyl radical (29) could not be excluded (28) or confirmed (3). In addition to these tumor initiating effects, UVA is known to induce intracellular signalling pathways and a number of transcription factors implied in cell proliferation and functions (30).

The light spectrum for the yield of 8-oxodG was determined in confluent primary skin fibroblasts, confirming that UVA (above 334 nm) and near visible radiations (405 nm) cause almost all of the guanine oxidation by natural sunlight (3). An alkaline elution assay with specific repair endonucleases established a wavelength spectrum for oxidative damage to DNA with two maxima ranges, 290–315 and 400–450 nm, and a minimum at about 350 nm (31). The growth status of skin cells was found to variously influence the 8-oxodG induction by UVA (3) (Table IGo), probably indicating additional unidentified factors.

From studies on point mutations, the role of 8-oxodG and other oxidized bases in solar mutagenesis, however, remains uncertain (3,32); reported mutation spectra as well indicate a very minor contribution of oxidative lesions in the overall solar UV mutagenic process (32). Even in the UVA region, bipyrimidic photolesions seem to play the major role, leading to GC->AT transitions, whereas the main landmark mutations expected for the 8-oxodG and other oxidative lesions, GC->TA transversions, represent only a minor fraction of induced mutations (3). Some data point out that, although pyrimidine dimers and 8-oxodG are produced at similar rates by the overall solar spectrum, the latter lesions would be more efficiently repaired (32).

Porphyrins have long been suspected of being endogenous photosensitizers. From studies on the general metabolic behaviour of cancer cells and descriptions of malignant cellular phenotypes, the deficiencies and abnormalities in haem synthesis have even been proposed (33) as a general initiating lesion for carcinogenesis. The present work demonstrates that light activation of porphyrins derived from exogenous {delta}-ala produces DNA oxidative damage, mostly depending on light dosage. Both UVA and visible light are found photoactive; as the two sources investigated in this work partly emit in the porphyrin Soret band around 410 nm, this major chromophore may be involved (6), which would be consistent with published action spectra for 8-oxodG induction (3,31).

DNA damage induced by porphyrins and light treatment have been mostly investigated for exogenous porphyrins on cell-free DNA (34,35). Haematoporphyrin derivative (HpD) was found to provoke single strand breaks and alkali-labile sites (34), whereas both DNA-binding and non-binding porphyrins induce guanine photodegradation to form different photoproducts, including 8-oxodG (35). HpD and light were found to induce guanine damage only in single-stranded DNA, which was attributed to the fact that this porphyrin does not bind to DNA (36). As DNA is protected from oxidative damage by its compaction in chromatin (37), RNA and single-stranded DNA, formed by double-stranded DNA unfolding during replication or transcription, may then be an important phototarget. From our data (Figure 2Go; Table IIGo), RNA indeed appears much more sensitive to photodynamic degradation than DNA. Since most RNA is cytoplasmic rather than nuclear, this may also indicate that the oxidizing radicals are generated primarily in the cytoplasm. Although this higher sensitivity of RNA to oxidative damage has been previously reported after UVA irradiation (38), no biological consequences have yet been described. In our opinion, it is most likely that strand breakage concomitant to oxidative damage may simply lead to oxidized RNA inactivation and further splicing.

Only cell-bound porphyrins are able to sensitize cells; the water-soluble uroporphyrin, which is able to efficiently generate 1O2 during light exposure, is completely ineffective as a cell photosensitizer (34). This is in agreement with our demonstration that medium-excreted porphyrins induce no DNA photodynamic damage (Figure 6Go).



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Fig. 6. Photodynamic effect of intra- and extra-cellular porphyrins. Cells from {delta}-ala treated (1 mM; 3 h loading) and control groups were centrifuged (900 g, 2 min) and supernatants exchanged before irradiation. Irradiation was performed at 4°C; the fluence rates were 0.31 mW/cm2 for the UVA tanning lamp and 11.9 mW/cm2 for visible light (n = 3; mean ± RANGE).

 
After synthesis in mitochondria, PP IX relocates in the cytoplasm, binds to membranes but does not accumulate in the nucleus (39,40). For HpD (34), the porphyrin fluorescence is seen at the nuclear membrane, while the nucleus itself does not exhibit any significant fluorescence; however, during the initial phase of light exposure, HpD seems to be relocalized in cells (41). Such a relocalization, which has also been proposed for phthalocyanine photosensitizers (42), has not been investigated for PP IX. It is then not yet known whether the photodynamic effect we demonstrate is direct (generation of 1O2 in the proximity of DNA) or indirect (amplification of generated radicals through lipid peroxidation of nuclear membrane). Purely cytoplasmic events are indeed able to induce mutations, a process mediated by ROS (43). As PPIX is synthesized in mitochondria, it is possible that some, perhaps most, of the 8-oxodG damage is in fact located in mitochondrial DNA (mtDNA); this DNA comprises about 1% of total cellular DNA and its recovery by nuclear DNA (nDNA) extraction methods is estimated at about 70%, implying a maximal possible contamination of 0.3% mtDNA in nDNA (44). Extraction of mtDNA for oxidative damage studies is, however, still a considerable challenge, many artefacts being encountered during organelles isolation; published values of background oxidative DNA damage in mtDNA represent a span of >60 000-fold (45) and biological consequences of mtDNA oxidation are still largely ignored.

Previous studies have shown, both in vitro and in vivo, that {delta}-ala by itself is a pro-oxidant (4650) able to yield an alkylating degradation product (51). {delta}-Ala is moreover able to mobilize iron from ferritin and so is assumed to promote its auto-oxidation (52,53). No dark toxicity was, however, found in our experimental model, either in terms of cell cytotoxicity (up to 6 mM, 48 h) or 8-oxodG production. It is nevertheless possible that {delta}-ala loading taxes the anti-oxidant defences of the cell so that a further pro-oxidant treatment, light irradiation on induced PP IX, overcomes these defences. This might lead to DNA damage, not from a direct porphyrin photodynamic action on DNA but through a general ROS overloading.

The fine regulation of porphyrin synthesis is still largely unknown, apart from the well-known feedback inhibition by haem. For example, the observed differences in {delta}-ala induced fluorescence according to the tissue or to the proliferative status of cells are still unexplained; similarly, factors triggering porphyrin synthesis are unidentified. Complex regulatory patterns, in the dark or under light, are only just being elucidated (54) but may have consequences for solar exposure carcinogenicity. We hypothesize that a skin basal layer cell that would be, for any reason, in an active porphyrin synthesis state is susceptible, through exposure to solar or sun bed light, to undergo PP IX bleaching with ROS production and oxidative DNA damage. In the absence of haem production, the endogenous synthesis of {delta}-ala is not expected to be a limiting factor; this is also the case in our experimental model for which {delta}-ala is continuously available during irradiation. This may then imply a continuous synthesis and bleaching of PP IX, DNA damage increasing with light dosage and possibly exceeding repair capacities. Previous studies (6,55) with alkaline elution coupled to specific repair endonucleases have shown that light-induced DNA oxidative damage depends on the cell type and may correlate with the basal concentration of porphyrins in the cells (55); visible light genotoxicity (as measured by micronuclei induction) and cytotoxicity were significant only after induction of porphyrin synthesis.

Possible implications for photodynamic therapy and photodynamic diagnostics
Photodynamic therapy
The clinical uses of photosensitizing therapy have led to quite a number of genotoxicity studies. From investigations on PDT with HpD and phthalocyanines, the mutagenicity was found to vary according to the target gene locus (56), the PDT dose, the identity of the photosensitizer, the time-course between photosensitizer addition and light application (42), but also to the cell's repair capacities, p53 status (57) and sensitivity to the lethal effects of treatment (58).

A recent extensive review (59) of all the data gathered on porphyrins leads to the conclusion that the overall risk for secondary skin carcinoma after topical PDT seems indeed low. The authors recall the clear pro-oxidant and genotoxic potential of this therapy, the known antioxidant and antimutagenic properties of porphyrins and the lack of evidence indicating higher rates of skin cancer in patients with photosensitivity diseases due to the presence of high PP IX levels in skin. All these data indicate that, when the combined dose of PP IX and light is sufficient to effectively kill cells, the probability of genetic damage being sustained is extremely low.

This may, however, not be the case for lower photodynamic dosages that could be encountered in cells normal or predisposed to cancer that synthesize physiological levels of porphyrins under normal or excessive solar exposure.

Although a possibly carcinogenic treatment is acceptable for cancer, the proposed applications of PDT to other pathologies, such as bacterial infections (60) or acne (61), certainly warrants further risk–benefit studies.

Photodynamic diagnosis
Upon {delta}-ala induction of porphyrins, a variety of protocols has been tested for in situ tumour detection through irradiation, most often by UVA. There is, however, a large variation in porphyrin synthesis in tissues, which may be due to various capacities in haem production, {delta}-ala uptake or different feedback control mechanisms (62). Depending then on organ, wavelengths, fluence and illumination schedule, there might be some risk of DNA oxidative damage in normal or pre-cancerous cells, and this should be further evaluated.

Conclusion
After a thorough review of solar mutagenesis, Kvam and Tyrrell (3) and Douki et al. (32) have concluded that the problem is extremely complex but that, although their involvement seems minor, monomeric oxidative lesions should not be excluded. Indeed some mechanisms of mutations, induced more efficiently by UVA and visible light than UVB and UVC, are still partly unexplained, for example at AT base pairs (3). Even if the role of 8-oxodG itself in solar mutagenesis were minor, this base is foremost considered a sensitive marker indicating that the defence systems of the cells have been so overwhelmed that oxidative damage of DNA could take place; other damaged bases, possibly occurring at greater magnitude than 8-oxodG, are then a likely possibility.

The demonstrated involvement of PP IX photobleaching in DNA damage certainly calls for further studies on the fine regulation of haem synthesis in order to ascertain the schedule of porphyrin synthesis during the cell cycle but also the risk of light exposure at presumably crucial moments.


    Notes
 
1 To whom correspondence should be addressed Email: pduez{at}ulb.ac.be Back


    Acknowledgments
 
We thank Prof. J.P.Dehaye and Dr N.Chaïb for help in fluorescence spectroscopy measurements and Prof. G.Atassi and Dr L.Badolo for a critical review of our manuscript.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 15, 2000; revised May 15, 2000; accepted January 29, 2001.





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