MUTAGENIC EFFECTS PHOTOINDUCED IN MAMMALIAN CELLS IN VITRO BY TWO MONOFUNCTIONAL PYRIDOPSORALENS

Abstract— The photobiological activity of the two monofunctional pyridopsoralens pyrido (3,4-c) psoralen (PyPs) and 7-methyl pyrido (3,4-c) psoralen (MePyPs) was studied in mammalian cells in vitro taking 8-methoxypsoralen (8-MOP) as a reference compound.
In the presence of 365-nm irradiation (UVA) MePyPs was found to be more effective than 8-MOP in terms of DNA photobinding capacity and inhibition of cell cloning ability in Chinese hamsterV–79 cells. As a function of UVA dose and of the number of total photoadducts induced MePyPs produced a higher frequency of 6-thioguanine resistant mutants than 8-MOP. PyPs showed an intermediate response for cell killing and mutation induction. At equal cytotoxic levels both monofunctional pyridopsoralens exhibited the same mutagenic activity as the Afunctional furocoumarin 8-MOP.
The antiproliferative effect being taken as indicative for an efficient photochemotherapeutic activity against psoriasis, the inhibition of cloning capacity induced by MePyPs plus UVA was studied in parallel on human skin fibroblasts. Such cells were more sensitive to 8-MOP photoadditions thanV–79 cells and even more so to MePyPs photoadditions. Data obtained on the rate of DNA semi conservative synthesis on both cell lines following treatments with the two compounds are in line with these observations.     

Tracing the Photoaddition of Pharmaceutical Psoralens to DNA

The psoralens 8-methoxypsoralen (8-MOP), 4,5′,8-trimethylpsoralen (TMP) and 5-methoxypsoralen (5-MOP) find clinical application in PUVA (psoralen + UVA) therapy. PUVA treats skin diseases like psoriasis and atopic eczema. Psoralens target the DNA of cells. Upon photo-excitation psoralens bind to the DNA base thymine. This photo-binding was studied using steady-state UV/Vis and IR spectroscopy as well as nanosecond transient UV/Vis absorption. The experiments show that the photo-addition of 8-MOP and TMP involve the psoralen triplet state and a biradical intermediate. 5-MOP forms a structurally different photo-product. Its formation could not be traced by the present spectroscopic technique.     

Furocoumarins photolysis products induce differentiation of human erythroid cells

Psoralens, also known as furocoumarins, are a well-known class of photosensitizers largely used in the therapy of various skin disease. In this study we have evaluated the effects of crude pre-irradiated solutions of furocoumarins derivatives on (a) erythroid differentiation and apoptosis of human leukemic K562 cells and (b) hemoglobin synthesis in cultures of human erythroid progenitors derived from the peripheral blood. To prove the activity of a mixture of photoproducts generated by UVA irradiation of the three psoralen derivatives 5-methoxypsoralen (5-MOP) 8-methoxypsoralen (8-MOP), and angelicin (ANG), we employed the human leukemic K562 cell line and the two-phase liquid culture procedure for growing erythroid progenitors. The results obtained demonstrate that pre-irradiated solutions of psoralen derivatives significantly induce erythroid differentiation of K562 cells irrespective of the type of derivative used, suggesting that the active photoproduct(s) share a common structure. Interestingly, solutions of psoralens irradiated in anaerobic conditions do not exhibits erythroid inducing ability, indicating that the effect is mostly due to photooxidized psoralen products. In erythroid precursor cells, psoralens photolysis products stimulates at low concentrations an increase of hemoglobin A and hemoglobin F. Altogether, these data suggest that photoproducts of psoralen warrant further evaluation as potential therapeutic drugs in beta-thalassaemia and sickle cell anaemia.     

8-Methoxypsoralen and long-wave ultraviolet A inhibit the release of proinflammatory mediators and cytokines from human Fc epsilon RI+ cells: an in vitro study

The in vitro effects of 8-MOP (concentrations of 20, 100 and 500 ng/ml) alone or in combination with UVA on mediator release from human basophils and skin mast cells (HSMC), activated with immunological and non-immunological stimuli, were investigated. With respect to basophils activated with anti-IgE serum, the results of this study show that: (i) 8-MOP alone inhibits histamine, LTC(4), IL-4 and IL-13 release concentration dependently with a maximal effect at 500 ng/ml (a concentration not reached in vivo); and (ii) UVA irradiation (5 J/cm(2)), after 8-MOP incubation, enhances this inhibitory effect on all released mediators, but for IL-4 and IL-13 the percentage inhibition is also significant for the 8-MOP concentrations (20-100 ng/ml) employed in vivo during PUVA treatment. Moreover, histamine release from basophils activated with non-immunological stimuli (FMLP and A23187) is inhibited by 8-MOP, alone or in combination with UVA. With respect to the HSMC activated with anti-IgE serum, the results show that: (i) 8-MOP alone reduces histamine release concentration dependently; and (ii) this inhibitory effect is enhanced by UVA irradiation (5 J/cm(2)). Histamine release from HSMC activated with A23187 is not modified either by 8-MOP alone or by 8-MOP plus UVA.     

SINGLET OXYGEN FORMATION BY SENSITIZATION OF FUROCOUMARINS COMPLEXED WITH, OR BOUND COVALENTLY TO DNA

Abstract— The formation of singlet molecular oxygen (¹O₂) by sensitization of the furocoumarins 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) and psoralen complexed with DNA was investigated. From the results it is concluded that 5-MOP complexed with native DNA is able to generate ¹O₂, even in a larger extent than 5-MOP free in solution. Also, with 8-MOP and especially with psoralen, ¹O₂ formation by the complexed compound could be observed. The ¹O₂ formation sensitized by covalently bound furocoumarin was demonstrated with psoralen as a model compound. 4',5'-Dihydropsoralen, a model compound for the UVA light absorbing 4',5'monoadducts of furocoumarins to DNA, is also able to generate ¹O₂.     

Turnover of phospholipids in HUT 102 lymphoblasts and chromatographic characterization of purified lecithins after their exposure to long-wave UV light, psoralen, and UV light and psoralen.

The turnover of 32P-labeled phospholipids in HUT 102 lymphoblasts was determined after a 2 h interaction of lymphoblasts with 8-methoxypsoralen (8-MOP) (15 micrograms ml-1), longwave UV light (UVA) irradiation and PUVA (8-MOP and UVA). In parallel experiments, micellar suspensions of lyso-phosphatidylcholine (PtdC), dipalmitoyl-PtdC and dilinoleoyl-PtdC, treated in a similar manner, served for the correlative assessments of cellular lipid changes. The dark reaction, UVA irradiation and PUVA all depressed total phospholipid levels in HUT 102 cells, although only PUVA induced a statistically significant decline. Thin layer chromatography (TLC) analysis revealed that neither UVA nor 8-MOP alone triggered any significant changes in the cellular content of phosphatidylinositol (PtdI), phosphatidylinositol 4-monophosphate (PtdIP) and phosphatidylinositol 4,5-bisphosphate (PtdIP2), whereas the lyso-PtdC and PtdI content of lymphoblasts showed a two-fold increase after PUVA. The TLC analysis of lyso-PtdC and micelles of dipalmitoyl-PtdC did not reveal any detectable changes after the dark reaction with 8-MOP, UVA irradiation and PUVA. In contrast, the derivatives of dark and UVA mediated reactions of 8-MOP with dilinoleoyl-PtdC were detected by TLC. These results suggest that the formation of 8-MOP derivatives of cellular phospholipids effected by PUVA, modulates the turnover of phosphoinositides and the rate of cellular proliferation.     

EFFECTS OF 8-METHOXYPSORALEN AND NEAR ULTRAVIOLET RADIATION ON THE SURVIVAL OF THE LOWER EUKARYOTE D. Discoideum

Abstract— Seven axenic wild-type and repair-deficient mutant strains of the cellular slime mold Dictyostelium discoideum have been treated with the furocoumarin 8-methoxypsoralen (8-MOP) up to 50 μg/mζ and then exposed to near ultraviolet light (UVA 320-400 nm) up to 21 kJ/m². Fluence-response survival curves exhibit shoulders at lower fluences and an exponential lethal response at higher fluences. Neither the psoralen alone nor the irradiation alone produced any measurable lethal effect. Wild-type strains, which show resistance to 254 nm UV and gamma radiation, also show resistance to psoralen plus UVA. The moderate sensitivity of a rad D repair-deficient mutant strain and the extreme sensitivity of a rad B mutant strain to 8-MOP plus UVA parallel their responses to UV and gamma radiation. However a rad C mutant which is sensitive to UV, exhibits wild-type response to photoactivated psoralen.     

Simple equilibrium dialysis-high-performance liquid chromatographic method for the in vitro assessment of 5-methoxypsoralen bound to human albumin

Increasingly used in therapeutics, 5-methoxypsoralen (5-MOP), a linear furocoumarin, associated with UVA irradiation (PUVA), is now an established treatment for skin diseases such as vitiligo, mycosis funcoides and particularly psoriasis. Successful PUVA therapy depends on a sufficiently high peak 5-MOP plasma concentration coinciding with the UVA irradiation. However, as with most drugs, only the free plasma fraction is able to enter the target cells and has a pharmacological effect. In this work, the binding of 5-MOP to human albumin was studied in vitro, using a dialysis chamber. Bound and free 5-MOP fractions were quantified by a modification of Stolk's high-performance liquid chromatographic method. Dialysis was performed at 37 degrees C and pH 7.4 for 2 h, against a 4% albumin solution in phosphate buffer. The 5-MOP concentrations used were from 5 x 10(-5) to 5 x 10(-2) g/l in 1 x 10(-1) g/l steps. The 5-MOP bound strongly to human albumin in an unsaturable way. The mean 5-MOP binding to albumin was 95.3%. These results are in accordance with those published by Artuc et al. and not with those of Veronese et al., who found a lower saturable fixation (91%). These two research groups used tritiated 5-MOP. The technique used in this work is simple and inexpensive. It can be employed easily in vivo, e.g., for the assessment of 5-MOP free fractions in different therapeutic conditions.     

Kinetic Analysis of Apoptosis Induction in Human Cell Lines by UVA and 8-MOP

Whereas previous studies have indicated that DNA damage as a result of 8-methoxypsoralen (8-MOP) and UVA treatment leads to cell death, this study establishes the minimum concentrations of 8-MOP and UVA necessary to induce apoptosis in human T-lymphocytic and mono-cytic cell lines. In order to assess apoptosis, we used fluorescent microscopy to examine changes in light scattering as well as internucleosomal DNA fragmentation. Generation of a dose response curve showed that the minimum combination of UVA and 8-MOP that was necessary to induce greater than background levels of apoptosis within 24 h of treatment was 0.5 J/cm² UVA and 12.5 ng/mL of 8-MOP. A striking observation was that UVA alone at doses 1.0 J/cm², but not 8-MOP alone (6300 ng/mL), induced significant apoptosis in the Sup-T1 cell line within 24 h. Although the percentage of apoptotic Sup-T1 cells induced by UVA alone was not as great as that of 8-MOP and UVA in combination, a highly significant correlation between the product of the concentration of 8-MOP (ng/mL) times the dose of UVA (J/ cm²) and the percentage of apoptotic cells was observed. This correlation provides an important tool for studying the relationship of UVA-induced DNA damage to apoptosis induction. Moreover, it will provide a means by which early events in the apoptotic pathway can be dissected.     

Effect on topical 5-methoxypsoralen on tumorigenesis induced in albino and pigmented hairless mouse skin by UV irradiation

A new line of the Skh:HRII hairless pigmented mouse (black juvenile coat) is described which has been selectively bred for the capacity to respond consistently to simulated solar UV radiation with a continuous and strong tan. This mouse demonstrates a degree of protection from chronic UV-induced tumorigenesis when compared with the Skh:HRI hairless albino mouse, and has been used here to study the effect of induced melanogenesis on phototumorigenesis. Mice were irradiated for 10 weeks with incremental doses of simulated solar UV radiation (UVA + B) from a fluorescent tube source which induced tumours in 100% of albino mice and 93% of black mice by 200 days (minimally oedemal), or with 60% of this dose (sub-oedemal) which induced tumours in 85% of albino mice and 65% of black mice. Mice were also exposed to the UVA component of these radiation sources, obtained by window glass filtration. The effect of topical 5-methoxypsoralen (5-MOP) was examined, at either 0.003% with minimally oedemal UVA + B or its UVA component alone, or at 0.01% with sub-oedemal UVA + B or its UVA component alone, in both albino and black mice. The 5-MOP concentrations were selected as the maximum concentration which did not increase the erythema and oedema responses after a single exposure to minimally oedemal or sub-oedemal UVA + B. At 200 days, the tumorigenic response to sub-oedemal UVA + B was significantly increased by topical 5-MOP, to 100% in albinos and 93% in black mice. In contrast, tumorigenesis in response to minimally oedemal UVA + B was unaffected by topical 5-MOP. The UVA component alone of either irradiation regime was not tumorigenic under these conditions. When combined with topical 5-MOP, the UVA of minimally oedemal UVA + B became moderately tumorigenic, and resulted in a tumour incidence of 23% in albinos and 14.5% in black mice. However, the UVA component of sub-oedemal UVA + B, when combined with topical 5-MOP, was highly tumorigenic specifically in albino mice, inducing tumours in 93% of albino mice but in only 27% of black mice. Tan intensity resulting from minimally oedemal UVA + B was not enhanced by topical 5-MOP, and its UVA component combined with 5-MOP resulted in only a minimal tan. However, the tan intensity resulting from sub-oedemal UVA + B with topical 5-MOP was strongly increased, although its UVA component combined with 5-MOP did not produce a perceptible tan.(ABSTRACT TRUNCATED AT 400 WORDS)     

4, 4'', 5''-TRIMETHYL-8-AZAPSORALEN, A NEW-PHOTOREACTIVE AND NON-SKIN-PHOTOTOXIC BIFUNCTIONAL BIOISOSTER OF PSORALEN

Photochemical and photobiological properties of a new isoster of psoralen, 4,4',5'-trimethyl-8-azapsoralen (4,4',5'-TMAP), have been studied. This compound shows a high DNA-photobinding rate, higher than that of 8-methoxypsoralen (8-MOP), forming both monoadducts and inter-strand cross-links. The yield of cross-links, however, is markedly lower than that of 8-MOP. Antiproliferative activity of 4,4',5'-TMAP, in terms of DNA synthesis inhibition in Ehrlich ascites tumor cells, is higher than that of 8-MOP. Mutagenic activity on E. coli WP2 R46+ cells appeared similar to or even lower than that of 8-MOP. This new compound applied on depilated guinea pig skin and irradiated with UVA did not show any skin-phototoxicity. On the basis of these properties 4,4',5'-TMAP appears to be a potential photochemotherapeutic agent.     

The Immunological Effect of 8-Methoxypsoralen and UVA Treatment on Murine T-Cell Leukemia

Abstract— 8-Methoxypsoralen (8-MOP) plus long-wavelength UV radiation (UVA, 320–400 nm) have been used to treat various diseases such as cutaneous T-cell lymphoma, systemic scleroderma, rheumatoid arthritis and rejection of heart transplants. However, the immunological mechanism of this treatment remains unknown. In this report, we investigated the effect of 8-MOP/UVA on the modulation of the immunogenicity of a T-cell leukemia cell line (RL ♂l cells). The results demonstrated that the stimulator function of the in vitro 8-MOP/UVA-treated RL♂ cells was enhanced in both RL ♂1-specific allogeneic and syngeneic immune responses. Furthermore, the enhancement of the immunogenicity of the 8-MOP/UVA-treated RL♂ cells was found to be strongly associated with the increase of intercellular adhesion molecule-1 expression on these 8-MOP/UVA-treated tumor cells. Therefore, our findings suggested that the alteration of the expression of the immune-related cell surface molecules might be an important effect of 8-MOP/UVA treatment on the elevation of the immunogenicity of the 8-MOP/UVA-treated tumor cells.     

Psoralen derivatives and longwave ultraviolet irradiation are active in vitro against human melanoma cell line

Cutaneous malignant melanoma is a very serious form of skin cancer that arises from melanocytes. Currently there is no effective treatment for metastatic melanoma so intense clinical trials are evaluating new drugs for this human malignancy. Psoralens are a group of compounds that bind to DNA in rapidly dividing cells and with ultraviolet light in the A band (UVA) cause DNA crosslinking, thereby preventing cellular division. They are used in the treatment of psoriasis and cutaneous T-cell lymphoma among other skin and blood diseases. We have investigated the cytotoxic potential of three psoralen derivatives plus UVA exposure (PUVA) on a established cell line of human melanoma. Cells were treated with different concentrations of 8-methoxypsoralen (8-MOP), 4,5',8-trimethylpsoralen (TMP) and 7-methylpyridopsoralen (MPP), for 1 h and after exposure to UVA light (0.3 J/cm(2)) were allowed to recover over a 24-72 h period. Viability was assessed by the microculture 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Cisplatin, one of the most important drugs in the chemotherapy of melanoma, was included for comparative studies. All the psoralen derivatives tested were markedly cytotoxic in a dose and post-exposure-time dependent manner. The IC(50) values for 72 h of post-exposure time were as follows: MPP=0.05+/-0.01, TMP=0.13+/-0.003 and 8-MOP=10.79+/-1.85 micromol/L. Regardless of the limitations of the in vitro model, our results suggested that the lower IC(50) values of TMP and MPP might be of clinical importance.     

Duck Hepatitis B Virus Inactivation and 8-Methoxypsoralen Photoadduct Formation in Human Platelet Concentrates*

Photochemical inactivation (PCI) of virus and bacteria in platelet concentrates (PC) has been demonstrated using 8-methoxypsoralen (8-MOP) and long-wavelength UV light (UVA). To study inactivation of blood-borne virus, we have employed duck hepatitis B virus (DHBV), a model for human hepatitis B virus. A specific hepatocyte culture infectivity assay, with PCR detection, could measure 5–6 log₁₀ virus kill. The DHBV inactivation in PC was dependent on UVA dose, was enhanced when plasma was reduced from 100% to 20% and was limited by 8-MOP solubility in the reduced-plasma medium. Optimum conditions for PCI were 100 μg/mL 8-MOP in 20% plasma and 80% synthetic platelet storage medium. A radiolabeling assay for 8-MOP photoadducts in hepatocytes seeded into PC confirmed that DHBV inactivation reflected DNA modification and indicated that adduct formation was insensitive to minor variations in conditions. Kinetic modeling indicated that optimum adduct formation was a compromise between 8-MOP dark binding and optical transmittance and that plasma proteins competed for 8-MOP binding. The PCI results in various media correlated with corresponding DNA modification densities and were compared to statistical models incorporating DHBV characteristics and predictions of 8-MOP crosslink formation between DNA strands. Behavior was consistent with one or a small number of lethal modifications per DNA strand, including monoadducts, but probably not crosslinks alone. A minor subpopulation of DHBV was found to be, somewhat more difficult to inactivate, consistent with three-fold lower modification, due possibly to single-stranded DNA character or host repair of photoadducts.     

Relative affinity of 5-methoxypsoralen and 8-methoxypsoralen towards beta-cyclodextrin: a fluorescence, circular dichroism and chromatographic study

The relative affinity of 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP) towards beta-cyclodextrin, a good model for the study of lipophilic interactions in biological systems and a potential drug carrier, has been investigated using spectroscopic and chromatographic methods. The fluorescence emission of 5-MOP in aqueous solution containing beta-cyclodextrin (10(-2) M) is found to be markedly blue shifted and enhanced by a factor of 6 whereas no significant changes are observed for 8-MOP. The existence of an induced circular dichroism is evidence for the formation of a 1:1 inclusion complex (association constant K = 400 +/- 50 M-1). Moreover, chromatographic results obtained with a beta-cyclodextrin linked stationary phase are consistent with our spectroscopic results and might have interesting analytical implications. These results clearly demonstrate that, in contrast to 8-MOP, 5-MOP exhibits a strong affinity for hydrophobic medium. Interesting pharmacological and analytical applications may result from the possible inclusion of psoralen derivatives into beta-cyclodextrin.     

Generation and Characterization of Psoralen Radical Cations

Abstract— Radical cations of psoralen, 8-methoxypsoralen(8-MOP) and 5-methoxypsoralen have been generated by photosensitized electron transfer in acetonitrile and aqueous buffer/acetonitrile (1:1) and have absorption maxima at 600, 650 and 550 nm, respectively. The radical cations have lifetimes of 5 p.s under these conditions, are unreactive toward oxygen and show behavior typical of ar-ylalkene radical cations in their reactivity toward nucle-ophiles and the precursor psoralens. Direct 355 nm excitation of 8-MOP in aqueous buffer at physiological pH results in monophotonic photoionization to give 8-MOP_*+ with a quantum yield of 0.015.The 8-MOP_*+ reacts with both guanosine and adenosine mononucleotides ( k = 2.5 times 10₉ and 3.4 times 10₇ M_-1 s₁, respectively) via electron transfer to give the purine radical cations, but does not react with pyrimidine mononucleotides. These results suggest that reactions of psoralen radical cations generated by electron transfer or photoionization may be involved in psoralen/UVA therapy.     

Effects of butylated hydroxytoluene upon PUVA-tumorigenesis and induction of ornithine decarboxylase activity in the mouse

Psoralen photochemotherapy (PUVA) is widely used in the treatment of psoriasis. Some therapy regimen have been associated with increased risk of skin cancer. Free radical species are thought to play a role in psoralen phototoxicity and photocarcinogenesis. It has been reported that the antioxidant butylated hydroxytoluene (BHT) inhibits acute phototoxicity by PUVA but does not reduce therapeutic efficacy. It has also been shown that BHT inhibits UVB-induced erythema, tumorigenesis and induction of ornithine decarboxylase (ODC) activity--ODC activity is thought by some to be associated with tumor promotion. Therefore, we have investigated the effect of BHT on psoralen tumorigenesis and PUVA-induced epidermal ODC activity. SKH-Hr-1 hairless albino mice were treated with topically applied 8-MOP and exposed to UVA (3X weekly) for 31 weeks with and without BHT administered either in the diet or topically. Induction of ODC activity was determined in similar experimental groups 24 h after a single exposure to UVA. Neither route of BHT administration had any effect on 8-MOP phototumorigenesis. However, BHT when administered in the diet reduced induction of ODC activity by 40% (p less than 0.05). These data indicate different mechanisms for UVB- and PUVA-induced carcinogenesis and again bring into question the relationship between induction of ODC activity and photocarcinogenesis.     

PHOTOADDITION OF 8-METHOXYPSORALEN TO E. coli DNA POLYMERASE I. ROLE OF PSORALEN PHOTO-ADDUCTS IN THE PHOTOSENSITIZED ALTERATIONS OF POL I ENZYMATIC ACTIVITIES

Abstract— We have previously demonstrated that 8-methoxypsoralen (8-MOP)‡ plus UVA is able to inactivate the three enzymatic activities of E. coli DNA polymerase I and that oxygen is required for these reactions (M. Granger et al. , (1982) Photochem. Photobiol. , 36 , 175–180). We now show that UV-A irradiation produces a covalent incorporation of the psoralen derivative into the enzyme either in the presence or in the absence of oxygen. The excited psoralen binds directly to the protein in an oxygen-independent reaction; no complex was detected in the absence of irradiation. Fluorescence measurements reveal that at least two photoadducts are formed.
The 8-MOP-photomodified enzyme is still fully active but further irradiation leads to an inhibition of the 5'→ 3' polymerase activity whereas the 5'→ 3' exonuclease activity is not affected. A major part of the inhibition reaction is shown to be oxygen-dependent but singlet oxygen quenchers have no effect on the kinetics. This oxygen-dependent reaction is attributed to a photosensitization, due to covalently bound 8-MOP, of neighbouring amino acids through an intermediate reactive oxygen species which is not singlet oxygen. The oxygen-independent reaction is attributed to a direct photosensitization through, for example, a radical mechanism.     

Dark interaction of 5-methoxypsoralen and 8-methoxypsoralen with erythrocyte ghosts: a fluorescence and circular dichroism study

The dark interaction of 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP) with plasma membranes was studied using human erythrocyte ghosts as a model. In the presence of ghosts, modifications of the fluorescence characteristics of 5-MOP were observed, together with a quenching of the fluorescence of the tryptophan (Trp) residues of membrane proteins (up to 25%). Moreover, the appearance of an induced circular dichroism indicates that 5-MOP is located in a chiral environment. In contrast, only slight effects were observed in the case of 8-MOP. It is concluded that 5-MOP molecules are located partly within chiral protein sites of the membrane in such a way that a F?rster energy transfer can occur from the Trp residues to the psoralen molecules.     

Evaluation of UVA-Mediated Oxidative Damage to Proteins and Lipids in Extracorporeal Photoimmunotherapy

The combination of UVA and 8-methoxypsoralen (8-MOP) is known for the ability to produce reactive oxygen species (ROS) that react subsequently with DNA, lipids and proteins. In most studies concerned with UVA effects mediated by free radicals, UVA doses higher than those exhibiting beneficial clinical results in extracorporeal photoimmunotherapy (ECPI) were used. The present study was undertaken to determine markers of oxidative stress in plasma and cells from the buffy coat using conditions relevant for ECPI (cumulative UVA dose at the sample level < or = 2 J/cm2). Plasma exposed to UVA of 20 J/cm2 resulted in protein oxidation as well in crosslinking and fragmentation revealed by electrophoresis. Exposure of the buffy coat and plasma to considerably lower doses of UVA (up to 2 J/cm2) combined with various 8-MOP concentrations resulted neither in an increase of malondialdehyde as a marker of lipid peroxidation nor in a changed electrophoretic protein pattern. In these same experiments the total antioxidative capacity decreased to 65% of the initial value, suggesting that the antioxidative defense of plasma is able to cope with oxidative stress under ECPI conditions. These results were confirmed by data from 10 patients with scleroderma or cutaneous T-cell lymphoma during ECPI treatment. The present results suggest that, although ROS are formed during ECPI, gross oxidative damage does not occur. It is, however, possible, that specific effects mediated by oxygen radicals may co-trigger the photoimmunomodulatory effects of ECPI.