Photoinduced cutaneous inflammatory response by psoralens.

Our studies describe the inflammatory response in rabbit skin induced by topical application of 8-methoxypsoralen (8-MOP) and UVA-visible irradiation (320-700 nm). Increase in vascular permeability (iVP) and accumulation of polymorphonuclear leucocytes (aPMN) at the test sites were quantitated using 125I-albumin and 51Cr-labelled PMNs respectively. Erythema was graded visually. 8-MOP cream was applied topically and irradiated. The erythemal response, aPMN and iVP at the test sites were quantitated at 6, 24, 48 and 72 h post-irradiation. The iVP and aPMN were maximal at 24 h; the erythemal response was the same at 24-48 h. The responses were dependent on 8-MOP concentration and irradiation dose. Topical application of 200 micrograms 8-MOP cream followed by irradiation for 2 h (9.4 J cm-2) produced 3-7 times iVP, 2-4 times aPMN and intense erythema at the test sites after 24 h. Neither aPMN nor iVP was detected before 6 h and erythemal response was not observable up to 16 h after irradiation. The aPMN and iVP gradually subsided in 72 h, although the erythemal response was still present. The repeated exposure of 8-MOP-treated sites for three consecutive days 24 h apart did not produce appreciable iVP or aPMN at 72 h or 24 h after the last exposure; however, erythema persisted. The 8-MOP-treated sites previously exposed for three consecutive days on reapplication of 8-MOP cream plus irradiation showed significantly less response compared with non-pretreated sites. Our results suggest that the erythemal response is not directly related to either iVP or aPMN.     

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.     

SYNTHESIS AND PHOTOBIOLOGICAL PROPERTIES OF 4-HYDROXYMETHYL-4'-METHYLPSORALEN DERIVATIVES

The synthesis and the photobiological activity of two new hydroxymethyl derivatives of psoralen namely 4-hydroxymethyl-4'-methyl- and 4-hydroxymethyl-4'-methyl-8-rnethoxypsoralen are described. Both compounds exhibited efficient photobinding to DNA and RNA. The DNA-photobinding process was investigated using different nucleic acid structures such as double-helical DNA, ribosomal RNA, bacterial DNA and DNA organized in the nucleosomal arrangement. The test derivatives were able to induce cross-links to a similar extent as 8-methoxypsoralen (8-MOP), used as a reference photochemotherapeutic drug. In contrast to 8-MOP, they produced relatively high levels of ^lO₂. Most photobiological effects (DNA synthesis inhibition, T₂ phage sensitization, inhibition of tumor transmitting capacity) showed a good correlation with the extent of covalent photoaddition. On the other hand, the new 4-hydroxymethylpsoralens were unable to induce skin erythema, in striking contrast with 8-MOP. Thus, neither cross-linking of the nucleic acid nor ¹O₂ production were coupled with skin phototoxicity in this class of compounds. The new derivatives appear to represent an important beginning to development of new active photochemotherapeutic agents devoid of undesired phototoxic side effects.     

RELATION BETWEEN SOME PHOTOBIOLOGICAL PROPERTIES OF FUROCOUMARINS AND THEIR EXTENT OF SINGLET OXYGEN PRODUCTION

Abstract— The production of singlet oxygen (¹O₂) by a series of furocoumarins with different skin sensitizing abilities has been investigated with methods already proven to be suitable to establish the ability of 8-methoxypsoralen (8-MOP) to generate ¹O₂.
The following compounds: 5-methoxypsoralen (5-MOP), psoralen, 4,5',8-trimethylpsoralen (TMP) and 5,8–dimethoxypsoralen (5,8–DMOP), are able to generate ¹O₂ when irradiated with long–wave ultraviolet light. With the photobiologically inactive angelicin no ¹O₂ production has been found. The relative extent of ¹O₂ formation has been determined for the various furocoumarins and has been compared with literature data for the skin photosensitizing effect. The observed relation between experimental data on the one side and the literature data on the other side is discussed.     

THE TRIPLET STATE OF 8-METHOXYPSORALEN

Abstract— Transient absorption spectra produced by laser flash photolysis of an aqueous solution of 8-methoxypsoralen (8-MOP) have been studied. The biphotonic production of hydrated electrons and of the radical ions, 8-MOP ⁺ and 8-MOP^- is reported. The hydrated electron was found to react with ground state 8-MOP with k ˜ 3 × 10¹⁰ M ^-1 s^-1. In order to obtain a true triplet-triplet absorption spectrum. contributions from the radical ions were subtracted from the overall transient absorption. In addition, contributions from e^-_aq to the transient spectrum were removed by using N₂O, low laser intensity to minimize photoionization or by measuring the transient O.D. after the electron has decayed. These three methods each produced the same triplet-triplet spectrum which differs in the red region from previously reported spectra.     

PHOTOBINDING OF PSORALENS TO BACTERIAL MACROMOLECULES IN SITU AND INDUCTION OF GENETIC EFFECTS IN A BACTERIAL TEST SYSTEM. EFFECTS OF SINGLET OXYGEN DIAGNOSTIC AIDS D2O AND DABCO

The photobinding of radiolabeled psoralen and 8-methoxypsoralen (8-MOP) to biological macromolecules under conditions that affect the lifetime of singlet oxygen (¹O₂) is reported. These conditions are: increase of ¹O₂ lifetime in D₂O and ¹O₂ quenching with DABCO. The photobinding to calf thymus DNA was studied in vitro and the covalent photobinding to DNA and other biological macromolecules (RNA, proteins) was also studied in intact bacteria. The results of the DNA photobinding experiments have been related to the induction of genetic damage in a bacterial test system. In addition, laser flash photolysis has been used to measure the effect of D₂O and DABCO on the psoralen and 8-MOP triplet lifetimes. In general D₂O increases the triplet lifetimes and DABCO quenches the triplet states with the probable formation of radicals. The results suggest that the covalent photobinding of 8-MOP to various biological macromolecules in situ is a basis for cell damage occurring at various cellular targets. Analysis of the results of the mutagenicity test suggests that in the presence of D₂O the mechanism of induction of genetic lesions is not changed and therefore largely seems to be independent of singlet oxygen.     

THE PHOTOCHEMICAL INTERACTION BETWEEN THE TRIPLET STATE OF 8-METHOXYPSORALEN AND THE MELANIN PRECURSORL–3, 4 DIHYDROXYPHENYLALANINE

Abstract— The photochemical interaction between 8-methoxypsoralen (8-MOP) and the melanin precursorL–3,4-dihydroxyphenylalanine(dopaH₂) has been studied using laser flash photolysis. Triplet excited 8-MOP was thus found to abstract electrons from dopaH₂ ( k ∼ 2 × 10⁹ dm³ mol^-1 s^-1) to form semireduced 8-MOP and semioxidised dopaH₂.The technique of pulse radiolysis was used to establish separately the spectra of (a) the semi-reduced form of 8-MOP at pH 6.5 and (b) the semioxidised forms of dopaH₂ at pH 6.5, 5.8, 4.6 and 3.3. The corresponding λ_max and extinction coefficients found were: for 8-MOP at pH 6.5, λ_max= 350 nm (= 9050 dm³ mol^-1 cm^-1); for dopa at pH 6.5, λ_max= 305 nm (ε= 12000 dm³ mol^-1 cm^-1) and for dopaH at pH 3.3, λ= 305 nm (ε= 5900 dm³ mol^-1 cm^-1).     

PHOTOADDUCTS OF 8-METHOXYPSORALEN TO CYTOSINE IN DNA

Abstract— The isolation and partial characterization of several photoadducts formed between 8-methoxypsoralen (8-MOP) and cytosine is described. The formation of these adducts was analysed in E. coli DNA containing ³H-labeled cytosine and/or ¹⁴C-labeled thymine, and in oligonucleotides of defined sequence. The major initial adduct has been identified as an 8-MOP cytosine monoadduct, most likely forming at the pyrone end of the 8-MOP molecule. Further irradiation converts this adduct to several other species, including both cytosine:cytosine and cytosine:thymine diadducts, as well as a number of derivative monoadducts. One isomer of the C:T diadduct appears to undergo a reversible isomerization under the conditions normally used to analyse adduct mixtures by HPLC. The isomerization can cause this adduct to exhibit a retention time on reversed-phase HPLC closely resembling either that of a thymine-thymine crosslink or a thymine monoadduct.     

AN INSTRUMENT FOR ACTION SPECTRUM STUDIES IN DERMATOLOGY

Abstract— An instrument designed for convenient determination of action spectra for cutaneous photo-responses in man and experimental animals is described. Light from 450 W Xe lamp is dispersed by a concave holographic grating. The spectrum from 244 to 616 nm is projected as a planar strip (2 times 17 cm) intercepted by a grid with 31 ports. The bandwidth at each port is 12 nm and the size of the port increases from about 4 × 4 mm to 6 × 8 mm from the low to high wavelength limits, respectively. Typical fluence rates in quanta m^-2 s^-1 are 4.0 times 10¹⁹ at 298 nm, 16 times 10¹⁹ at 394nm and 22 times 10¹⁹ at 538 nm. Responses due to delayed erythema in normal skin and to musk ambrette photoallergy and solar urticaria in patients skin have been elicited with this instrument.     

THE TREATMENT OF MASTOCYTOMA CELLS WITH 8-METHOXYPSORALEN AND LONG-WAVELENGTH ULTRAVIOLET RADIATION ENHANCES CELLULAR IMMUNOGENICITY: PRELIMINARY RESULTS

Evidence for the increased immunogenicity of mastocytoma cells (P815) treated with 8-methoxypsoralen (8-MOP) and long-wavelength ultraviolet radiation (UVA) is presented. A highly tumorigenic clone (P1) became much less tumorigenic (tum^-) after repetitive phototreatments with 8-MOP (16 ng/mL) and UVA (1 J/cm²). The yield of tum^- clones was proportional to the number of phototreatments. In a pilot study in which P1 cells were treated with three successive rounds of 8-MOP/UVA, one clone out of 73 was tum^-. In a second series of experiments, the P1 cells were treated 10 times and 4 out of 100 clones were much less tumorigenic. When some of the tum^-clones were administered intraperitoneally to DBA/2 mice, significant protection against challenge with the original P1 clone was observed. In addition, the transfer of immune cells from tum^--treated mice allowed the transfer of resistance to other tum^- clones to immunosuppressed mice (650 rad). These results are consistent with earlier literature showing the potent mutagen, N -methyl- N' -nitrosoguanidine, led to mutations in P1 that altered the expression of new surface antigens, which stimulated the murine immune system such that there was also cross recognition of shared antigens on untreated P1 cells used to challenge the immunized mice. The increased immunogenicity that resulted from the less mutagenic 8-MOP/UVA treatment may arise by a similar mechanism and may be responsible in part for the efficacy of 8-MOP/UVA photochemotherapy for the treatment of cutaneous T cell lymphoma.     

MECHANISMS FOR DYE-MEDIATED PHOTODYNAMIC ACTION: SINGLET OXYGEN PRODUCTION, DEOXYGUANOSINE OXIDATION AND PHAGE INACTIVATING EFFICIENCIES

Abstract The production of singlet oxygen (¹O₂) upon irradiation of several dyes in aqueous solution at pH 9.0, was quantitatively analyzed on the basis of the appearance of stable nitroxide radicals using the amine 2,2,6,6-tetramethyl-4-piperidone as ¹O₂ acceptor. The dyes were checked for purity, their concentrations uniformized in terms of absorbance values and a correction factor was introduced which took into account the amount of photons absorbed. The rates of ¹O₂ production (in arbitrary units per min) were: 71 with rose Bengal, 70 with methylene blue, 61 with eosin Y, 18 with thiopyronine, 10 with proflavine and 9 with acridine yellow. Production of ¹O₂ was not observed with 9-aminoacridine, acridine orange, quinacrine and ethidium bromide. Irradiated lumichrome initiated, with the same amine, another type of reaction. The rates of two other photoreactions were also determined under similar experimental conditions by following (i) the deoxyguanosine decomposition in which case the reaction was found to be less sensitive but largely parallel to the ¹O₂ production and (ii) the bacteriophage ØX174 inactivation in which case the dyes showed differences in their relative efficiencies. The proteinic capsid of the phage appeared as an effective impermeability barrier towards externally generated ¹O₂. Moreover, some of the dyes studied intercalated into the phage DNA, a process known to favor radicalar reactions.     

Induction of tumor necrosis factor-alpha by UVB: a role for reactive oxygen intermediates and eicosanoids

UVB irradiation induces nuclear factor-kappaB (NF-kappaB) activation, tumor necrosis factor-alpha (TNF-alpha) expression and reactive oxygen intermediates (ROI) in keratinocytes. We investigated whether ROI play a role in UVB-induced TNF-alpha mRNA expression. The antioxidants N-acetyl cysteine, NAC, epigallocathin gallate, EGCG, butylated hydroxyanisole (BHA) and vitamin C could reduce UVB-induced TNF-alpha mRNA levels to various degrees; vitamin E (alpha-tocopherol) had no effect. BHA was the most potent inhibitor. The oxidant tertiary butylated hydroperoxide could effectively induce TNF-alpha mRNA expression. Nordihydroguaiaretic acid (NDGA) and MK-886, inhibitors of lipoxygenase (LOX), and indometacin and quinacrine, inhibitors of cyclooxygenase (COX) and phospholipase A2, respectively, could also reduce UVB-induced TNF-alpha mRNA expression. Inhibition by NDGA was in concordance with the results for BHA. NDGA, indometacin, quinacrine and BHA could also effectively inhibit the inhibitor of NF-kappaB degradation, thereby maintaining NF-kappaB inactivity. In conclusion, we show that ROI are implicated in the induction of TNF-alpha mRNA by UVB and that not all antioxidants are equally effective inhibitors. COX products and more importantly LOX products, which themselves are products of an oxidative metabolism, are the main ROI implicated in this induction of TNF-alpha expression by UVB probably via activation of NF-kappaB.     

MODULATION OF 8-METHOXYPSORALEN-DNA PHOTOADDUCT FORMATION BY CELL DIFFERENTIATION, MITOGENIC STIMULATION AND PHORBOL ESTER EXPOSURE IN MURINE T LYMPHOCYTES

Abstract The effects of cell differentiation and mitogen and phorbol ester stimulation on the formation of 8-methoxypsoralen (8-MOP)-DNA photoadducts in murine T lymphocytes were examined using ³H-8-MOP. While there were no significant differences in 8-MOP photoadduct formation among BALB/c thymocytes, splenocytes, splenic T cells and MRL/1pr lymph node cells, BALB/c bone marrow cells showed fewer photoadducts than did the lymphocytes. This suggested that proliferating progenitor cells may be resistant to 8-MOP photoadduct formation. Incubation of purified splenic T cells with lectin mitogens for 2 h or with phorbol 12-myristate 13-acetate (PMA) for 2–43 h resulted in reduction of 8-MOP photoadduct formation in the DNA, whereas 64 h cultivation with these agents augmented the photoadduct formation. The reduction of photoadduct formation induced by phytohemagglutinin was restored by the further addition of a protein kinase C (PKC) inhibitor, H-7, to the culture. Thus, it is assumed that the reduction of adduct formation evoked by mitogens and PMA is mediated in part by the activation of PKC in the cells. On the other hand, the augmentation of the adduct formation induced by the longer-period cultures with mitogens and PMA appeared to be caused by down-regulation of PKC. The present study showed that the stimulatory signals in which PKC is presumably involved affect the ability of cells to form 8-MOP-DNA photoadducts.     

SOME PHOTOPHYSICAL PROPERTIES OF 3-CARBETHOXYPSORALEN, 8-METHOXYPSORALEN and 5-METHOXYPSORALEN TRIPLET STATES

Abstract Triplet absorption spectra, extinction coefficients (ɛ_T), decay rates ( K ₁), oxygen quenching rates (k_q) and intersystem crossing yields (φ_T) for 3-carbethoxypsoralen (3-CPs). 8-methoxypsoralcn (8-MOP) and 5-methoxypsoralen (5-MOP) in methanol are reported. For 8-MOP and 3-CPs corresponding values are also reported with water as the solvent. Some photophysical data are also reported for 5-MOP in water, but ɛ_T and φ_T were not obtained.
The phosphorescence spectra for these furocoumarin derivatives in ethanol at 77 K are reported together with the corresponding lowest triplet energy and lifetime. The values of the various photophysical properties obtained are compared with values reported by previous workers.     

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.     

4'-AMINOMETHYL-4,5',8-TRIMETHYLPSORALEN PHOTOCHEMISTRY THE EFFECT OF CONCENTRATION AND UVA FLUENCE ON PHOTOADDUCT FORMATION IN POLY(dAdT) AND CALF THYMUS DNA

Abstract-The photochemistry of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with poly(dA-dT) and calf thymus DNA was studied. The extent of photoadduct formation and the distribution of photoadducts (3,4– and 4',5'-monoadducts and crosslinks) were determined by liquid scintillation analysis and HPLC, respectively. The adducts were characterized on the basis of their UV absorption spectra and mass spectral analysis. The high DNA binding constant for AMT (1.5 x 10⁵ M⁻¹ ) led to a high fraction of intercalated molecules, which contributed to the high level of AMT photoadduct formation, as many as 102 adducts per kilobase pair. In addition, there is a distinct difference in the adduct distribution compared to the previously studied 8-methoxypsoralen (8-MOP). Under the conditions employed for the photochemical studies, virtually all of the AMT molecules in solution are intercalated, occupying 25% of the base pair sites. Under similar conditions, 8-MOP molecules occupied 10 times fewer sites. Thus, for AMT, DNA base pair sites other than 5'TA, the well-characterized strong binding for psoralens in general, are an additional target for photomodification, which results in the formation of a higher percentage of monoadducts. The proportion of photoadducts formed was virtually independent of AMT concentration and UVA (320–400 nm radiation) fluence.     

FLUORESCENCE DETECTION OF AN 8-METHOXYPSORALEN METABOLITE IN HUMAN INTERSTITIAL FLUID

Abstract— A fluorescent method has been used to study the suction blister fluid of human volunteers collected after 8-methoxypsoralen (8-MOP) oral intake. A fluorescent chromophore with spectral characteristics (Λ_max= 390 nm, Λ_max=470nm) distinct from 8-MOP has been detected. Our results suggest the existence of a metabolite form of 8-MOP within the patients's skin prior to any UV irradiation. This form might result in the opening of the4–5' double bond of the 8-MOP molecule.     

NMR ANALYSES OF TRYPTOPHAN-8-METHOXYPSORALEN PHOTOREACTION PRODUCTS FORMED IN THE PRESENCE OF OXYGEN

Proton-made spectroscopy was performed on solutions of l -tryptophan and 8-mcthoxypsoralen (8-MOP) in either D₂O or DMSO-d₆ in the presence of oxygen before and after irradiation with 360 nm monochromatic light. Irradiation results in the loss of hydrogen atoms at the 3. 4 and 4'. 5' positions of 8-MOP and at the indole C₂ position of tryptophan. Changes in the aliphatic regions of the spectra also occur with irradiation. It is postulated that generation of photoreaction products between 8-MOP and tryptophan involves the 3.4 and 4'.5' positions of 8-MOP and the imidazole moiety of tryptophan.
Reprint requests to: Dr J. Megaw, Laboratory for Ophthalmic Research, Emory University School of Medicine, Atlanta. Georgia 30322. USA.     

INHIBITION OF DNA SYNTHESIS BY PSORALEN-INDUCED LESIONS IN XERODERMA PIGMENTOSUM AND FANCONI''S ANEMIA FIBROBLASTS

Abstract— Exposure of human cells to psoralens and near-UV light produces a mixture of monoadducts and crosslinks in DNA, which inhibit DNA synthesis by blocking replicon initiation and chain elongation. 8-Methoxypsoralen (8-MOP) has a greater effect than angelicin in normal, xeroderma pigmentosum, and Fanconi's anemia cells. Recovery of DNA synthesis is not detectable up to 8 h after exposure. The average distance between lesions that block replication in individual replicons was measured by means of bromodeoxyuridine photolysis. After exposure to 10 μg/mℓ of 8-MOP and 7500 J/m² of near-UV light, blocks were formed every 20 μm. Replicon initiation was inhibited by exposure to near-UV light alone in normal and xeroderma pigmentosum. Exposure to low concentrations of angelicin or 8-MOP plus near-UV light inhibited replicon initiation in normal and Fanconi's anemia cells, but not in xeroderma pigmentosum cells. Inhibition of initiation was not obvious after treatment with high concentrations of 8-MOP or angelicin because of the dominant effect of crosslinks in blocking chain elongation.     

QUENCHING OF THE FLUORESCENCE OF 8-METHOXYPSORALEN BY PROTONS

Abstract— The quenching of 8-methoxypsoralen (8-MOP) fluorescence by protons was observed to occur at the diffusion controlled rates in aqueous solutions at room temperature. Enhanced basicity of 8-MOP in the excited state compared to the ground state is expected on theoretical grounds. The fluorescence yield. which we determined as 6.3 × 10^--⁴ at pH 1 is surprisingly low and indicative of extremely fast radiationless decay pathways. The fluorescence lifetime of 8-MOP in neutral aqueous solution is on the order of 1–2 ns.