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.     

4',5'-SUBSTITUTED METHYLANGELICINS: PHOTOCYCLOADDUCTS WITH PYRIMIDINE BASES OF DNA

The isolation and characterization of photocycloadducts with pyrimidine bases from DNA samples irradiated (365 nm) in the presence of four 4',5'-substituted methylangelicins was performed. All these furocoumarins yielded mainly the cis-syn furan-side cycloadduct with thymine. For 4',5'-dimethyl-, 5,4',5'-trimethyl- and 6,4',5'-trimethylangelicin this adduct was accompanied by two pyrone-side adducts ( cis-syn and cis-anti ), whereas the 4,4',5'-trimethyl derivative gave the furan-side adduct with cytosine.
The characterization of the regio- and stereochemistry of the adducts was accomplished by ¹H NOE (nuclear Overhauser effect) and ¹H-¹³C HMBC (heteronuclear multiple-bond connectivity) spectroscopies.
The formation of different cycloadducts in DNA by the various derivatives highlights the role of the methyl groups in determining the regio- and stereochemistry of the cycloaddition.     

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.     

ALTERATIONS IN DNA IRRADIATED WITH ULTRAVIOLET RADIATION—I. THE FORMATION PROCESS OF CYCLOBUTYLPYRIMIDINE DIMERS: CROSS SECTIONS, ACTION SPECTRA and QUANTUM YIELDS

Abstract— We have determined the dimerization and monomerization cross sections of the Thy < > Thy (cyclobutyl dimer of thymine and thymine) and the Cyt < > Thy (cyclobutyl dimer of cytosine and thymine) dimers in Escherichia coti [³H]-DNA ([³H]-thymine labeled DNA) at five wavelengths in the range 240–300 nm. It may be concluded from the dimerization action spectra for the two dimers that the excitation of Thy (thymine) is mainly responsible for the photochemical dimerization reaction in both cases. The calculated quantum yields of dimerization and monomerization are also presented in this paper and several questions, raised by the results obtained at 300 nm, are 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).     

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.     

THE EFFECTS OF PHOTOOXIDATION BY PROFLAVINE ON HeLa CELLS—II. DAMAGE TO DNA

Abstract— HeLa cell suspensions, prelabeled with specific [¹⁴C]-nucleosides, were treated with proflavine and irradiated with visible light (400–500 nm). The DNA was isolated from the cells (as well as from the appropriate control cells) and examined for macromolecular and molecular changes. Although the UV absorbance spectrum of DNA from irradiated HeLa cells showed no discernible change, a fluorescence spectrum (excitation/emission: 305/405) indicated a molecular change in the DNA. Isolated DNA samples were hydrolyzed with 90% formic acid and chromatographed. There were no detectable differences between the irradiated and non-irradiated profile (R _f and radioactivity) for both guanine and adenine. However, the chromatograms of thymine and cytosine showed distinct changes. There was a loss of radioactivity in the [¹⁴C]-thymidine labeled samples, while the [¹⁴C]-cytidine labeled samples indicated the formation of a new compound, containing 10% of the radioactivity, running just ahead of cytosine. These data strongly suggest the formation of a new compound resulting from the photooxidation of cytosine when nuclear DNA was sensitized by proflavine.     

INTERACTIONS AND PHOTOREACTIVITY OF 8-ALKOXYPSORALENS WITH THYMINE. A MODEL APPROACH

Abstract— To investigate the interactions and the photoreactions in solution between thymine and psoralen, model compounds have been synthesized in which a thymine molecule is linked to a psoralen ring by a polymethylene bridge. Two series of compounds have been studied and compared as models for the two major drugs 5-MOP and 8-MOP. We report here the results obtained in the 8-alkoxypso-ralen series. In water, intramolecular ring-ring stacking interactions were evidenced using UV and ¹H NMR spectroscopies. In organic solvents such as ethanol, these interactions disappear completely. The photoreactivity of the models was examined in relation to their ground state interaction properties. in water and in ethanol. Slow photolysis of the pyrone ring of psoralen occurs whatever the length of the linking chain (3 to 12 methylenes). No intramolecular thymine-psoralen photoaddition nor psoralen photodimerization were observed. These results are discussed with regards to the behavior of the 5-alkoxy models which lead selectively to the 3,4 psoralen-thymine photoadduct.     

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.     

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.     

THE EFFECTS OF HIGH-INTENSITY UV-RADIATION ON NUCLEIC ACIDS AND THEIR COMPONENTS—I. THYMINE

Abstract— The set of final products of thymine conversion induced by high-intensity UV irradiation (λ= 266nm, intensity 10²⁴-5 × 10²⁹ photons·s⁻¹·m⁻², pulse duration 10ns) of the dilute aqueous solution to the first approximation is similar to that formed with ionizing irradiation (γ-irradiation of aqueous solution or autoradiolysis of a solid 2-[¹⁴C]-thymine). The data obtained suggest that high-intensity UV-induced photochemical conversion of thymine involves photoionization and/or photodissociation. These processes pass through the higher excited state(s) populated as a result of the second photon absorption by excited (most probably in the T₁ triplet state) thymine molecules.     

PHOTOADDITION OF CHLORPROMAZINE TO GUANOSINE-5'-MONOPHOSPHATE

Abstrart—The photochemistry of chlorpromazine (CPZ) with guanosine-5'-monophosphate (GMP) was studied as a model for the photoaddition of CPZ to DNA. Irradiation of CPZ with calf thymus DNA produced a product emitting at 520 nm, whereas with GMP emission was at 495 nm. HPLC separation of photolysis mixtures of [³H]CPZ with GMP and [¹⁴C]GMP with CPZ indicated that three photoadducts were formed. One of the adducts fluoresced at 500 nm and appeared to be the product detected but not separated by Fujita et al. (Photochem. Photobiol . 1981, 34 , 101–105). A second adduct emitted at 460 nm, and the third was nonfluorescent. The photoadduct emitting at 500 nm was characterized by UV, fluorescence, and NMR to be an adduct from coupling of the C-8 position of guanine to the C-2 position of the phenothiazine ring of CPZ. The cation radical of CPZ (CPZ ⁺) does not appear to be an intermediate since enzymatically generated CPZ ⁺ formed a product that eluted with a retention time close to that of the photoadducts, but did not emit at 520 nm.     

SOME PROPERTIES OF THE TRIPLET EXCITED STATE OF THE PHOTOSENSITIZING FUROCOUMARIN: 3-CARBETHOXYPSORALEN

Abstract— 3-Carbethoxypsoralen (3-CPs) has been tested in the photochemotherapy of psoriasis. It only forms monoadducts with DNA and is being considered as a non-carcinogenic alternative to 8-MOP which itself forms DNA crosslinks that arc difficult to repair. Using laser flash photolysis or pulse radiolysis, the triplet state of 3-CPs, a possible intermediate in photosensitization, has been generated in several solvents: ethanol, water and benzene. The triplet lifetime, extinction coefficient and quantum efficiency of formation have been measured. Triplet reactivities towards (i) the solvents used, (ii) 3-CPs, (iii) oxygen, (iv) tryptophan and (v) tyrosine, leading, respectively, to photoadditions with water, ethanol and 3-CPs, to ¹O₂, semioxidized tryptophan and semioxidized tyrosine, (vi) thymine and (vii) uracil have been investigated. The dark binding of 3-CPs to DNA has been studied by comparing the reactivity of e_aq^- with free 3-CPs, free DNA and the 3-CPs DNA complex. Some photophysical and photochemical properties of 4',5'di-hydro-3-carbethoxypsoralen(DH–3-CPs), model of the main fluorescent photo-product of 3-CPs, have also been investigated. Biological consequences of the photochemical properties of 3-CPs andDH–3-CPs have been studied in a cellular system (haploid yeast).     

ULTRAVIOLET-INDUCED REACTIONS OF THYMINE AND URACIL IN THE PRESENCE OF CYSTEINE

Abstract —Ultraviolet-radiation photolysis of thymine in the presence of cysteine gives rise to four isomeric dimers, dihydrothymine, and at least five cysteine addition products. Similar reactions occur for uracil but the products have not all been characterized in detail. The addition reactions arise from the triplet state of the pyrimidine. The initial step is production of a hydropyrimidine radical, which then reacts with cysteine to give the addition products. The triplet is quenched by cysteine with a rate constant of about 2 times 10⁸ M^-1 s^-1 for thymine and 2–9 times 10⁸ for uracil. The total yield of products gives a lower-limit estimate of the triplet yield and hence of the intersystem-crossing efficiency. These studies, combined with earlier determinations of dimer yields, show that 93% of the thymine triplets which interact with another thymine molecule are quenched without forming stable dimers. For uracil, the corresponding figure is 75%.     

Modulation of 8-Methoxypsoralen-Photoinduced Cutaneous Inflammatory Reactions by Various Chemotherapeutic Agents in vivo

Abstract— Exposure of albino rabbits to UVA-VIS (320-700 nm) radiation after the topical application of 8-methoxypsor-alen (8-MOP) cream is associated with acute cutaneous inflammatory reactions in situ. In the present studies the effects of various agents on 8-MOP plus light induced cutaneous inflammatory response viz. increase in vascular permeability (iVP), accumulation of polymorphonuclear leukocytes (aPMN) and erythema formation were investigated. The inflammatory reactions were induced by a single exposure of 8-MOP-sensitized sites to UVA-VIS (9.4J/cm²) light. Indomethacin, p-bromophenacyl bromide (BPAB), MK886 (trade name of Merck Sharpe & Dome), ibuprofen (IB), nordihydroguaiaretic acid (NDGA) or quinacrine were applied topically in cream base at various times prior to 8-MOP application. The iVP and aPMN were quantitated 24 h postirradiation using ^12SI-HSA and ⁵¹Cr-labeled PMN respectively, while erythema was graded visually. The rate of iVP, aPMN and erythema was inhibited almost completely by indomethacin (7.5-10%) when applied twice, 18 h and 3 h prior to 8-MOP. At lower concentrations of indomethacin (5%) iVP was inhibited whereas aPMN was augmented. The BPAB (0.25%) inhibited more than 90% of 8-MOP-photoinduced iVP and aPMN while there was partial reduction in erythema. The MK886 (0.1%) cream inhibited about 50% of iVP and aPMN but erythema persisted. The agents that are somewhat nonspecific such as IB, quinacrine and NDGA inhibited 8-MOP-photoinduced inflammation only marginally at the concentrations tested. The fact that iVP, aPMN and erythema can be dissociated suggests that there are independent variables in 8-MOP-photoinduced reactions, which involve multifactorial mechanisms probably controlled by different cell-signalling pathways and mediators.     

INTERACTIONS AND PHOTOREACTIVITY OF 5-ALKOXYPSORALENS WITH THYMINE. A MODEL APPROACH

Abstract— In order to investigate the interactions and the photoreactions in solution between the thymine (thy) and the psoralen (Pso) rings, we have prepared model compounds Thy-(CH₂)_n-Pso in which two aromatic chromophores Thy and Pso are linked by flexible polymethylene chains of varying length (CH₂)_n. Two series of compounds were examined and compared as models for the two important drugs 5-methoxypsoralen and 8-methoxypsoralen. Results concerning the 5-alkoxypsoralen series are reported here. In water, these model molecules exhibit intramolecular ring-ring stacking interactions as indicated by hypochromism in the UV and by shielding of the protons in ¹H NMR spectroscopy. These interactions disappear in organic solvents. The photochemical properties of the models were examined in relation with their ground state interaction properties. Irradiation at 365 nm carried out at the usual concentrations (10^-2-10^-3 M) leads exclusively to a stereoselective dimerization involving the psoralen moieties of the models at the 3,4 double bonds. However, when operating at exceedingly low concentrations (2 × 10^-5 M ), the psoralen photodimerization is avoided and a highly regio and stereo-selective psoralen thymine photoaddition is observed involving the 3,4 double bond of psoralen leading to the cis adduct. The same reaction occurs for all models under study being independent of the length of the (CH₂)_n polymethylene linking chain, n = 2 to 6, 12 and of the solvent used. This is unambiguous proof for the highest intrinsic photoreactivity of the 3,4 vs the 4',5' double bond in 5-alkoxy psoralen.     

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)     

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.