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.     

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.     

ACTION SPECTRA AND CHROMOPHORES FOR LETHAL PHOTOSENSITIZATION OF Candida albicans BY DNA MONOADDUCTS FORMED BY 8-METHOXYPSORALEN AND MONOFUNCTIONAL FUROCOUMARINS

The red-shift of furocoumarin action spectra, compared with their absorption spectra, has been investigated. An action spectrum for 8-methoxypsoralen (8-MOP) monoadduct formation in the yeast Candida albicans has been determined. The yeast cells were initially exposed to sublethal doses of monochromatic UVA at different wavelengths. Monoadduct formation was monitored by growth inhibition induced, after washing out any unbound 8-MOP, by re-irradiation with a constant second (non-lethal) dose of 330 nm radiation. A comparison between this action spectrum and the absorption spectrum of the dark complex of 8-MOP and DNA was made. In addition, the action spectra of monoadduct formation of five monofunctional compounds including a coumarin derivative have been determined. These action spectra were compared with their respective DNA dark complex absorption spectra. In general, the peaks of the furocoumarin DNA dark complexes show a red-shift when compared with the free furocoumarin molecule and the action spectra show peaks which correspond with the peaks of the dark complexes. Such data indicate that the DNA dark complex is the chromophore for growth inhibition in yeast rather than the free furocoumarin. The similarity of the 8-MOP monoadduct formation action spectrum and 8-MOP action spectra suggests that spectral dependence for the photobiological effects (including the red-shift) is dependent on monoadduct formation rather than, as previously suggested by several authors, crosslink formation. The action spectrum for the coumarin derivative 4-methyl N-ethylpyrrolo (3,2-g) coumarin (PCNEt) correlated well with the free molecule absorption spectrum rather than DNA dark complex indicating that the free molecule is the chromophore.(ABSTRACT TRUNCATED AT 250 WORDS)     

HERPES VIRUS PRODUCTION IN MONKEY KIDNEY AND HUMAN SKIN CELLS TREATED WITH ANGELICIN OR 8-METHOXYPSORALEN PLUS 365 nm LIGHT

Abstract—At an cquimolar concentration of 50 μM the bifunctional furocoumarin, 8-methoxypsoralen (8-MOP), is about 36 times more efficient in inhibiting the colony forming ability of CV-I monkey kidney cells than the monofunctional furocoumarin angelicin. In contrast 8-MOP is only 7.5 times more efficient than angelicin for the inhibition of herpes simplex virus (HSV) production in CV-1 cells. This latter factor seems to reflect differences in photoreactivity of the two compounds with host cell DNA.
A substantial recovery of HSV production was seen when cells were infected at different time intervals after treatment with angelicin-plus-light, whereas recovery was very limited after 8-MOP plus light treatment. The recovery process was slow as compared to that observed after UV (254 nm)-irradiation.
The repair capacities of treated normal and xeroderma pigmentosum (group A) skin fibroblasts were estimated by measuring HSV production and unscheduled DNA synthesis. XP-A cells repaired angelicin induced damage less efficiently than did normal cells. Neither cell type showed any repair activity after 8-MOP plus light treatment.     

Lysosomes: a possible target for psoralen photodamage

Treatment in vitro of Ehrlich ascites tumor cells or human fibroblasts with 8-methoxypsoralen (8-MOP, 2.4 microM) and UVA irradiation results in a 30% and 60% respectively reduction in lysosomal beta-galactosidase activity in situ. Under identical conditions one 8-MOP adduct was formed per 2 X 10(4) bases of DNA, one 8-MOP adduct was formed per approximately 10(4) tRNA molecules and one per approximately 100 ribosomes. It is suggested that the decrease in lysosomal beta-galactosidase activity is a result of leakage through the lysosomal membrane caused by psoralen-UVA damage of the lipids in the membrane, since no effect was found on beta-galactosidase in vitro. These results indicate that the lysosomes may also be a target for cellular photodamage by 8-methoxy-psoralen.     

DNA damage induced by 4,6,8,9-tetramethyl-2H-furo[2,3-h]quinolin-2-one, a new furocoumarin analog: biological consequences

4,6,8,9-Tetramethyl-2H-furo[2,3-h]quinolin-2-one (HFQ) and its isomer FQ (1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2-one) showed very strong antiproliferative activity in mammalian cells, about two times greater than 8-methoxypsoralen (8-MOP). Both compounds induced DNA-protein cross-links (DPC) but not interstrand cross-links. The FQ generated DPC in a biphotonic process, yielding a new kind of diadduct, whereas HFQ induced DPC by a monophotonic one, probably without its physical participation in the covalent bridge. These lesions gave different toxic responses. Sensitization of FQ led to extensive DNA fragmentation and to a number of chromosomal aberrations. Conversely, HFQ seemed to be completely inactive and 8-MOP gave intermediate results. A strict relationship between DPC formation and induction of chromosomal aberrations was observed. The HFQ did not induce light skin erythemas, whereas FQ was more phototoxic than 8-MOP, thus suggesting that FQ lesions, DPC in particular, may be implicated in skin phototoxicity. Ehrlich ascites cells, a transplantable mouse tumor, inactivated by furoquinolinone sensitization and injected into healthy mice, protected them from a successive challenge by viable tumor cells. This response appeared to be based on an immune mechanism. Comparable amounts of base substitution revertants were scored when testing furoquinolinones and 8-MOP in bacteria but no DPC were detected. This suggests that classic mutagenesis tests on bacteria are insufficient to give adequate information on furocoumarin genotoxicity. Given its features, HFQ can be regarded as an interesting new agent for psoralen plus UVA photochemotherapy and photopheresis.     

Cell surface acid-base properties of Escherichia coli and Bacillus brevis and variation as a function of growth phase, nitrogen source and C:N ratio

Potentiometric titration has been conducted to systematically examine the acid–base properties of the cell surfaces of Escherichia coli K-12 and Bacillus brevis as a function of growth phase, nitrogen source (ammonium or nitrate), and carbon to nitrogen (C:N) ratio of the growth substrate. The two bacterial species revealed four distinct proton binding sites, with pK_a values in the range of 3.08–4.05 (pK₁), 4.62–5.57 (pK₂), 6.47–7.30 (pK₃), and 9.68–10.89 (pK₄) corresponding to phosphoric/carboxylic, carboxylic, phosphoric, and hydroxyl/amine groups, respectively. Two general observations in the data are that for B. brevis the first site concentration (N₁), corresponding to phosphoric/carboxylic groups (pK₁), varied as a function of nitrogen source, while for E. coli the fourth site concentration (N₄), corresponding to hydroxyl/amine groups (pK₄), varied as a function of C:N ratio. Correspondingly, it was found that N₁ was the highest of the four site concentrations for B. brevis and N₄ was the highest for E. coli. The concentrations of the remaining sites showed little variation. Finally, comparison between the titration data and a number of cell surface compositional studies in the literature indicates one distinct difference between the two bacteria is that pK₄ of the Gram-negative E. coli can be attributed to hydroxyl groups while that of the Gram-positive B. brevis can be attributed to amine groups.     

UVA-induced binding of 8-methoxypsoralen to cells of Saccharomyces cerevisiae: separation and characterization of DNA photoadducts

We present methods for the determination of UVA-induced binding of 8-methoxypsoralen (8-MOP) to nucleic acids and protein and for a quantitative assay of radioactively labelled 8-MOP plus UVA induced DNA photoproducts in the yeast Saccharomyces cerevisiae. For the dose range up to 60 kJ m-2, with a wild-type survival of 1% or higher, binding to DNA is 100-fold and to RNA 10- to 20-fold more efficient than that to protein. Between 20% and 65% of the 8-MOP binds to macromolecules that are neither nucleic acids nor protein. The number of DNA-bound 8-MOP molecules for the haploid genome rises from 14 (unirradiated control) to 349 at the highest UVA exposure dose (60 kJ m-2). Gel chromatography reveals three types of DNA thymine photoproduct, the pyrone-side monoadducts, the furan-side monoadducts and the diadducts. Among these, pyrone-side monoadducts always constitute the smallest fraction, regardless of whether the treatment is with in vitro or in vivo 8-MOP plus UVA.     

Reduction of Escherichia coli 0157:H7 in shredded iceberg lettuce by chlorination and gamma irradiation

Lettuce was inoculated with a six-strain cocktail of acid-adapted Escherichia coli 0157:H7 at a level of 1×10⁷ CFU/g. Following chlorination at 200 μg/ml, the lettuce was irradiated at 0.15, 0.38, or 0.55 kGy using a ⁶⁰Co source. Survival of E. coli 0157:H7, aerobic mesophiles and yeast and molds were measured over a period of 10 days. For quality analysis, chlorinated lettuce was subjected to irradiation at 0.33 and 0.53 kGy and stored at 1.0°C, 4.0°C or 7.0°C. Changes in texture and color were determined by instrumental means and changes in flavor, odor, and visual quality were determined by sensory testing.

Chlorination plus irradiation at 0.55 kGy produced a 5.4−log reduction in E. coli 0157:H7 levels. Chlorination alone reduced the E. coli 0157:H7 counts by 1–2 logs. Irradiation at 0.55 kGy was also effective in reducing standard plate counts and yeast and mold counts. Irradiation at this level did not cause softening of lettuce and sensory attributes were not adversely affected. In general, appearance and flavor were affected more by the length of storage than by temperature conditions. The 5+log reduction in E. coli counts and lack of adverse effects on sensory attributes indicate that low-dose irradiation can improve the safety and shelf-life of fresh-cut iceberg lettuce for retail sale or food service.     

Phototoxicity, distribution and kinetics of association of UVA-activated chlorpromazine, 8-methoxypsoralen, and 4,6,4'-trimethylangelicin in Jurkat cells

Extracorporeal phototherapy (ECP) is a therapeutic approach based on photobiological effects of 8-methoxypsoralen (8-MOP) on white blood cells isolated from the blood, exposed to UVA and then reinfused into the patient. 8-MOP is presently the only drug approved for clinical application of ECP; therefore, identification of other photosensitizers with better photochemical and pharmacokinetic properties might enhance the efficacy of this treatment modality. Among such alternative drugs are 4,6,4'-trimethylangelicin (TMA) and chlorpromazine (CPZ), which have previously been studied in an animal model for ECP. In this current study, cellular bioavailability of 8-MOP, TMA and CPZ was investigated in vitro, using low doses of UVA relevant for the clinical setting of ECP. Our fluorescence microscopy study revealed that 8-MOP and CPZ penetrated readily into the cells, where they accumulated with similar kinetics. No distinct fluorescence was observed in cells incubated with TMA. We found that the phototoxic efficiency of 8-MOP was an order of magnitude greater than that of CPZ, i.e., to obtain a similar reduction in survival of cells subjected to photosensitization by the drugs, the concentration of CPZ needed to be 10 times higher than that of 8-MOP. The photoactivated TMA exhibited the highest pro-apoptotic efficiency. A clear indication of photoinduced formation of reactive oxygen species and peroxidation of lipids was observed only in CPZ-sensitized cells, suggesting different mechanisms for phototoxicity mediated by CPZ and by the two furocoumarins.     

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.     

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.     

Investigations of coronatine biosynthesis. Overexpression and assay of CmaT, a thioesterase involved in coronamic acid biosynthesis

The protein (CmaT) encoded by the cmaT gene of the coronamic acid biosynthetic gene cluster has been overexpressed in Escherichia coli in soluble and active form fused to the carboxyl terminus of MalE, the maltose-binding protein. CmaT was also overexpressed in E. coli as an N-terminal His-tagged protein. The N-terminal His-tagged form of CmaT was produced in insoluble form, but it could be refolded to obtain CmaT in soluble and highly active form. Both the MalE-CmaT fusion protein and the refolded His-tagged CmaT protein exhibited esterase activity.     

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.     

PHOTOBIOLOGICAL ACTIVITY OF 3,4''-DIMETHYL-8-METHOXYPSORALEN, A LINEAR FUROCOUMARIN WITH UNUSUAL DNA-BINDING PROPERTIES

The furocoumarin derivative 3,4'-dimethyl-8-methoxypsoralen (DMe-8-MOP) exhibits remarkable antiproliferative activity, but is devoid of skin phototoxicity. To gain insight into this peculiar behaviour we investigated non-covalent and covalent binding of DMe-8-MOP to calf thymus DNA, along with DNA-synthesis inhibition and mutagenic activity. The non-covalent interaction of DMe-8-MOP with the nucleic acid is quite poor as shown by equilibrium dialysis, spectroscopic, chiroptical and hydrodynamic techniques. However, it exhibits relevant photobinding ability to DNA using both isolated nucleic acid samples and cellular systems. Unlike the large majority of congeners, DMe-8-MOP undergoes predominantly photochemical monoaddition to the double helical polynucleotide. Upon examination of the products obtained by enzymatic hydrolysis of DMe-8-MOP photomodified DNA, the formation of an unusual furan side adduct is proposed, which could account for the peculiar photochemical and photobiological properties of the 3,4'-dimethyl furocoumarin derivative.     

Mitochondrial alterations in fanconi anemia fibroblasts following ultraviolet A or psoralen photoactivation

The genetic disease Fanconi anemia (FA), generally considered to be a DNA repair defect, has also been related to a deficiency in cellular defense against reactive oxygen species (ROS). Results show that mitochondrial matrix densification occurs rapidly and transiently in FA fibroblasts following 8-methoxypsoralen (8-MOP) photoreaction or ultraviolet A (320 to 380 nm) (UVA) irradiation. This effect is oxygen dependent because it is more important under 20 than under 5% oxygen tension. In contrast, in normal fibroblasts very little, if any, densification of mitochondrial matrix is induced by treatments even at the highest oxygen tension. The changes in matrix density in FA cells are accompanied by some modifications in transmembrane potential, linked to a Fenton-like reaction, and in mitochondrial cardiolipin content, differing from the responses of normal cells. These data are indicative of some sort of membrane damage induced by 8-MOP photoreaction and UVA irradiation, to which FA cells appear to be particularly sensitive.     

4,4'',6-TRIMETHYLANGELICIN, A NEW VERY PHOTOREACTIVE AND NON SKIN-PHOTOTOXIC MONOFUNCTIONAL FUROCOUMARIN

Abstract— 4,4',6-Trimethylangelicin is a new monofunctional furocoumarin which appears to be a very promising potential agent for the photochemotherapy of psoriasis. Actually, it is capable of photoreacting with DNA to a large extent (four times more than 8-MOP), forming only monoadducts; it produces singlet oxygen to an insignificant extent. Its antiproliferative effect (tested in Ehrlich ascites tunior cells) appears to be several times higher than that induced by the most active angelicins now known and by 8-MOP itself. In spite of this high photosensitized effect, 4,4',6-trimethylangelicin seems to be non-phototoxic on guinea-pig skin and much less mutagenic than 8-MOP in E. coli WP2 uvr -A, a strain in which cross-links show lethality rather than mutations.     

Novel indirect enzyme-linked immunosorbent assay (ELISA) method to detect Total E. coli in water environment

In order to establish ELISA (enzyme-linked immunosorbent assay) method to detect Total E. coli in water environment, E. coli multi-characters antigens in water environment were prepared according to the characters of kinds of E. coli serotypes, including antigen of whole cell, antigen of disrupted whole cell, somatic antigen, flagellar antigen and fimbrial antigen. Total E. coli polyclonal antibodies were obtained from the New Zealand rabbits immunized with these five antigens, respectively. Antibodies generated in this research are with high titers and good purity, can conjugate with antigens, specifically, stably and strongly. Indirect ELISA shows the titers of antibody of whole cell and antibody of disrupted whole cell are both over 1 × 10⁵. The cross-reactivity of the antibody is from 12 to 30% which indicate the specificity of the antibody against Total E. coli. Based on these antibodies, we established indirect ELISA method to detect Total E. coli in water environment. The matrix effects were studied and the results show that there is no significant influence by all the factors. The ELISA result shows that the detection limitation could be 10⁴ CFU (colony forming units) L⁻¹. The indirect ELISA method developed in this study is well suited for Total E. coli analysis in real water samples as a rapid screen method.     

3-CARBETHOXYPYRANOCOUMARIN, A PHOTOREACTIVE DERIVATIVE OF XANTHYLETIN WITH INTERESTING PHOTOBIOLOGICAL PROPERTIES

Abstract— The photobiological activity of the newly synthesized pyranocoumarin derivative 3-carbethoxypyranocoumarin, so-called 3-carbethoxyhomopsoralen (3-CHPs) was studied in comparison to the known bifunctional furocoumarin 8-methoxypsoralen {8-MOP) and to the monofunctional furocoumarin 3-carbethoxypsoralen (3-CPs) in the presence of 365 nm irradiation using two eukaryotic cell systems, the yeast Saccharomyces cerevisiae and cultured normal human skin fibroblasts. 3-Carbethoxyhomopsoralen is shown to be a photobiologically active compound capable of effectively photoinducing cytoplasmic "petite" mutants (mitochondrial damage), nuclear reversions and mitotic gene conversion in the diploid yeast strain D₇. Per unit dose it is more effective than 8-MOP and 3-CPs for the induction of cytoplasmic "petite" mutants but less effective than 8-MOP for the induction of nuclear reversions and mitotic gene conversion. A very moderate effect on cell survival is accompanied by a relatively strong genetic activity per viable cell. In human fibroblasts 3-CHPs produces a stronger inhibition of DNA synthesis than 8-MOP and 3-CPs at low doses of 365 nm radiation. During post-treatment incubation human fibroblasts recovered more easily from DNA synthesis and growth inhibitions photoinduced by 3-CHPs than from those photoinduced by 8-MOP. The results are in accord with the notion that 3-CHPs is a highly photoreactive monofunctional compound inducing easily repairable lesions with a low lethal but significant mutagenic potential.     

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.