PHOTOPRODUCTS OF CHLORPROMAZINE WHICH CAUSE RED BLOOD CELL LYSIS

A mechanism for chlorpromazine (CPZ) phototoxicity has been proposed that attributes the response to formation of stable, toxic photoproducts which cause cell membrane disruption. We have characterized these toxic photoproducts as dimers and higher multimers of CPZ. Chlorpromazine solutions (3 or 10 mA/) were irradiated with a medium pressure Hg lamp filtered to exclude λ < 280 nm. Five low mol wt photoproducts were separated by high performance liquid chromatography. Two were identified as CPZ-sulfoxide and promazine. Higher mol wt photoproducts were separated by Sephadex G-50 chromatography into 3 broad bands which were characterized by their absorption and fluorescence spectra. Band A (mol wt > 800) had λ_max^abs= 263 nm, λ_max^fl= 490 nm and Band B (mol wt = 350-800) had λ_max^abs= 255 nm, λ_max^fl= 450 nm. Based on the mol wt of CPZ, Band A contained trimers and higher mol wt compounds and Band B was composed of dimeric structures. BandC(λ_max^abs= 255,310 nm; λ_max^fl= 445 nm) was composed of CPZ (mol wt = 315) and the low mol wt photoproducts. Red blood cell lysis was used as an assay for the ability of photoproducts to cause membrane disruption. Bands A and B, but not Band C, caused cell lysis. These data indicate that the CPZ photoproducts which cause cell membrane disruption are dimers (Band B) and higher multimers (Band A).     

Characterization of Photodegradation of Meta-tetra (Hydroxyphenyl)chlorin (mTHPC) in Solution: Biological Consequences in Human Tumor Cells

The photobleaching of meta -tetra(hydroxyphenyl)chlorin m THPC) (irradiation wavelength 413 nm) in protein-containing solution was evaluated by decay in absorbance in Soret band and in fluorescence (λ_exc= 423 nm, λ_em= 655 nm). Light exposure resulted in a decrease in absorption throughout the spectrum and simultaneous appearance of new absorption bands in the spectral region 325–450 nm. The rate of m THPC photodegradation, followed by decay in absorbance, was 15-fold lower than that observed in fluorescence. This fact reflects the photobleaching of presumably monomeric, fluorescing species of m THPC. In order to determine the consequences of photobleaching of fluorescing m THPC material on cellular uptake and photocytotoxicity, human HT29 colon adenocarcinoma cells were incubated with photobleached m THPC during 5 h with or without following irradiation with the fixed fluence. Surprisingly, but up to the time when the fluorescence decreased by 50%, only a slight decrease in photocytotoxicity was detected. Either aggregated forms that have been taken up undergo intracellular monomerization (but we did not observe increase in fluorescence in living cells) or the photodynamic activity is mostly due to aggregates. The discrepancy of m -THPC-photodynamic therapy (PDT) effect and fluorescence measurements may suggest that aggregated m -THPC plays an important role in m THPC-PDT.     

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).     

PHOTOACTIVABLE FLUOROPHORES FOR THE MEASUREMENT OF FLUENCE IN TURBID MEDIA

Knowledge of the fluence distribution in biological tissue is essential for applications of lasers and light in medicine. A method using a photoactivable fluorophore as a chemical actinometer is presented to investigate the fluence (J/cm²) distribution in tissue-simulating phantoms. Such a chemical actinometer provides high spatial resolution (≤20 μm) while minimizing the disturbance of the fluence distribution. The actinometer substance, nonfluorescent in its native state, is incorporated into an acrylamide gel. Upon absorption of 351 nm radiation (λ_act), the actinometer substance becomes a Ruorophorc, which is excited at λ_ex≤ 485 nm. Thus the spatial distribution of the emitted fluorescence (λ_em≤ 515 nm) in the actinometer represents the fluence distribution of the activating radiation. Using histological techniques, 20 μm sections are cut from gel-like optical phantoms containing the actinometric substance. The fluorescence intensity in the section is recorded under a standard fluorescence microscope equipped with a sensitive video camera. To simulate different biological tissues, the scattering and absorption properties of the gel phantoms arc varicd over a wide range. The experimentally obtained fluence distributions are compared with theoretical models of light distribution in turbid media.     

PHOTOBIOLOGICAL ACTIVITY IN YEAST OF DERIVATIVES OF PSORALEN SUBSTITUTED AT THE 3,4 AND/OR THE 4'',5'' REACTION SITE

Abstract— Derivatives of psoralen substituted at the position 3 and/or 5' with various substituents (CN, CO-R or COO-R) have been newly synthesized. Their photobiological reactivity was determined by measuring the effects on survival and the induction of cytoplasmic 'petite' mutations (rho-) in the haploid yeast Saccharomyces cereuisiae after treatment with 365-nm irradiation in the presence of the different compounds. The effects were compared to those of known mono-functional compounds such as angelicin, 3-carbethoxypsoralen and 5,7-dimethoxycoumarin and bi-functional compounds such as psoralen and 8-methoxypsoralen for which the photoaffinity to DNA is well known. The derivative carrying a CN group in position 3 of the psoralen molecule exhibited effects comparable to those of the bi-functional agent 8-methoxypsoralen. Three derivatives substituted at position 3 showed a biological activity comparable to those of the mono-functional agent 3-carbethoxypsoralen. Two compounds substituted at position 5' by COOH or COOCH₃-groups were much less photobiologically active than the corresponding derivatives substituted in position 3. This suggested that the furanic site of the psoralen molecule may be photobiologically more active than the 3,4 site. Four newly synthesized angular derivatives, i.e. furo (2,3 f, g) coumarins, were found to be two to three times less active than angelicin.
At the dose rate of 365-nm irradiation used the measure of survival and rho^- induction per viable cell allowed the classification of some of the compounds as mono- and some as bi-functional agents in analogy to known mono- and bi-functional compounds.     

FLUORESCENCE AND PHOTOCHEMISTRY OF OLIGOCYTIDYLIC AND POLYCYTIDYLIC ACIDS IN AQUEOUS SOLUTION

Abstract —In addition to the monomer-like fluorescence, a long-wavelength emission (Λ^max_em= 410 nm) has been detected in the dinucleoside 5'-5' pyrophosphate (CppC) at room temperature. This emission looks very similar to that previously reported for the acidic forms of Poly C (Poly C. Poly C⁺ and Poly I. Poly C. Poly C⁺). Only the monomer-like emission (Λ^max_em= 330 nm) can be detected in neutral Poly C, acidic CppC, and the neutral or protonated forms of the dinucleoside phosphate CpC.A correlation between the room temperature fluorescence of oligo and polycytidylic acids and their photochemical behaviour is found. Irradiation of all the polymeric samples at both neutral and acid pH results in the formation of minor photoproducts. They have been characterized by their absorbance (in the range 300–400 nm) and their fluorescence spectra. The same product is obtained in all cases where the monomer-like fluorescence only is detected. Distinct products are formed in neutral CppC and in the acidic Poly C forms.
The results are discussed with respect to the conformation of the oligo and polycytidylic acids and possible relationships between the 410–420 nm emission and adduct formation. An excimer is proposed as a common, intermediate excited state in both radiative deactivation and adduct formation in neutral CppC and the acidic Poly C forms.     

Depopulation of Highly Excited Singlet States of DNA Model Compounds: Quantum Yields of 193 and 245 nm Photoproducts of Pyrimidine Monomers and Dinucleoside Monophosphates

Abstract— Formation of uracil and orotic acid photodimers, uridine and 5'-UMP photohydrates, TpT photodimers and (6-4)photoproducts, dCpT photohydrates and (6-4)photo-products and UpU, CpC and CpU photohydrates were studied in neutral deoxygenated aqueous solution at room temperature upon irradiation at either 193 or 254 nm. The photoproducts were identified and quantified and the contribution from photoionization to substrate decomposition, using λ_irr= 193 nm, was separated. The ratio of the quantum yields of respective stable products,η=φ¹⁹³/φ²⁵⁴ is indicative of the yield of internal conversion from the second to the first excited singlet state, S₂→ S₁. For the observed photodimers η decreases from 0.94 for uracil to 0.7 for TpT and further to 0.55 for orotic acid. For the (6-4)photoproducts of TpT and dCpT T| = 0.5-0.8 and for the photohydrates in the cases of UpU, CpC, CpU and dCpT TJ ranges from 0.55 to 1.     

THE PHOTOPHYSICAL PROPERTIES OF SOME C15 LUPINE ALKALOIDS:

Abstract Sparteine, the tetracyclic saturated amine alkaloid, is highly fluorescent in n-hexane solution (Φ_f= 0.64; ζ= 63 ns) and has a large Stokes shift [λ_max (abs) = 203 nm; λ_max (fluor) = 325 nm]. Its isomer, α-isosparteine, has similar properties: Φ_f= 0.55; ζ_f= 50 ns; λ_max (fluor) = 338 nm. Oxidized derivatives, such as lupanine, thermopsine, and α-diplospartyrine, are weakly fluorescent. Based on a comparison with spectroscopic and photophysical properties of the monoamine, quinolizidine, it is concluded that the excitation energy is delocalized over the two N-atoms in starteine and a-isosparteine. The self quenching rate constant for sparteine, ca. 1 times 10⁷M^-1 s^-1, is about 100 times smaller than that for quinolizidine, consistent with excited state derealization. The significant fluorescence quenching in lupanine is rationalized by the fact that N-methyl-2-piperidone mfe/molecularly quenches the fluorescence of quinolizidine at nearly the diffusion controlled rate in -hexane. Comparisons are made with the fluorescence properties of other diamines such as N, N'-dimethylbispidine and N, N'-disubstituted piperazines.     

CHARACTERIZATION OF PHOTOCYCLOADDITION PRODUCTS FROM REACTION BETWEEN THYMIDINE and THE MONOFUNCTIONAL 3-CARBETHOXYPSORALEN

The photochemical reaction of 3-carbethoxypsoralen. a monofunctional furocoumarin. with thymidine was investigated as a model system for its photoaddition to DNA. Near UV irradiation (320 nm > λ > 400 nm) of a mixture of thymidine and 3-carbethoxypsoralen as a dry film gave rise to two main nucleoside diastereoisomers which were isolated by reverse phase high performance liquid chromatography. The structure of these products was assigned on the basis of UV absorption, fluorescence.'H-NMR and plasma desorption mass spectra analysis. The results are consistent with 1:1 C, cycloadducts involving the 5,6 double bond of thymine and the 4', 5'double bond of 3-carbethoxypsoralen. These two cycloadducts of cis-syn stereoconfiguration show opposite circular dichroism suggesting a diastereoisomeric relationship.     

A FLASH PHOTOLYSIS STUDY OF 5-METHOXYINDOLE

Abstract— The microsecond flash photolysis of 5-methoxyindole in aqueous solutions has been studied at γ_exc≥ 290 nm. Transients identified in this time realm in neutral solutions are: e_aq^-, the 5-methoxyindole radical cation (γ_max≅ 440 nm), the neutral transient with γ_max≅ 530 nm) and an unidentified oxygen sensitive transient with γ_max≅ 435 nm. Radical cations and e^-_aq are shown to be produced in equal amounts consistent with a photoionization process as the only source of both transients. H⁺ quenching of fluorescence and radical cation production gives equivalent Stern-Volmer constants indicating that photoionization occurs from the fluorescent state. The unidentified oxygen sensitive transient exhibits a pK _a of2–2.5 and is quenched at lower pH values indicating that it also has a fluorescent state precursor.     

The influence of water on the photophysical and photochemical properties of Piroxicam in AOT/iso-octane/water reversed micelles

The photophysical properties of Piroxicam , a nonsteroidal anti-inflammatory drug (NSAID), were investigated at different pH_ext values in reversed micelles of Aerosol-OT (AOT) in iso -octane, using both steady-state and picosecond time-resolved fluorescence spectroscopy. In contrast with the very complex data obtained in aqueous media, where several prototropic species are in equilibrium, the reversed micellar system essentially favors two species. The absorption spectra shows only one isosbestic point at λ= 348 nm. Excited-state intramolecular proton transfer (ESIPT), also detected in water, is promoted at low water pool contents measured by ω₀= [H₂O]/[AOT]. A strongly shifted (λ_em= 470 nm) tautomeric emission is found. Upon the gradual increase of ω₀, striking differences with pH_ext are found. At pH_ext= 4, the drug preferentially locates itself in the interfacial region partitioning between a hydrophobic and a hydrophilic domain. Global analysis was applied to the decay data and the results were interpreted by the "two-state excited-state" formalism. At pH_ext= 7, the anionic species is prevalent and the probe locates itself deeper inside the water core of the reversed micelles. Thus, a strong dependence on water content is detected, approaching a behavior similar to that observed in free aqueous solutions.     

TRIPLET, RADICAL ANION AND RADICAL CATION SPECTRA OF FUROCOUMARINS

Abstract— Absorption spectra of triplets, radical anions and radical cations of four furocoumarins, psoralen (Ps), 8-methoxypsoralen (8MOP), 5-methoxypsoralen (5MOP) and 3-carbethoxypsoralen (3CPs), have been determined by laser flash photolysis and pulse radiolysis. The triplet spectra of 8MOP, 5MOP and 3CPs are strongly modified when going from an H-bonding solvent such as water to a non H-bonding solvent as benzene or acetonitrile while the triplet spectrum of Ps is solvent independent. Theoretical considerations using a CNDO/S method are able to explain the existence of these two different triplets. For 8MOP, 5MOP and 3CPs in water the triplets might be considered as triplet exciplexes ³(FC^δ-. H₂O^δ+) consistent with these triplet spectra being similar to the spectra of the corresponding radical anions.     

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.     

FLUORESCENCE QUANTUM YIELDS OF 124-kDa PHYTOCHROME FROM OAT UPON EXCITATION WITHIN DIFFERENT ABSORPTION BANDS

Abstract— A fluorescence quantum yield (emission at650–850 nm) of π= (2.3 ± 0.3)10⁻³ was measured for the red-absorbing form (Pr) of 124-kDa phytochrome from etiolated oat seedlings ( Avena sativa ) upon excitation in the Soret band at Λ^exc= 380 nm. The small difference between this value and the previously determined quantum yield with Λ^exc= 640 nm, π= (3.5 ± 0.4)10⁻³is attributed to a blue-absorbing emitter responsible for the "anomalous" or "blue" emission of the chromoprotein in the region from ca. 400 to 550 nm. The absorption of Pr at 380 nm is consequently somewhat lower than that measured directly from the spectrum. Processes from upper excited states of the Pr phytochromobilin-derived chromophore other than rapid relaxation to the emitting state are not important. A quantum yield of Φ ' 1.2 times 10⁻³ is estimated for the blue fluorescence. The proportion of the blue emitters relative to Pr appears to be relatively high.     

PHOTOLESIONS IN DNA UPON 193 nm EXCITATION

Aqueous solutions of plasmid (pBR322 and pTZ18R) and calf thymus DNA were excited by 20 ns laser pulses at 193 nm. The quantum yields of single- and double-strand break formation, interstrand cross-links, locally denatured sites, (6–4)photoproducts and biological inactivation (Φ_ssb, Φ_dsb, Φ_icl, Φ_ids, Φ_6–4 and Φ_ina, respectively) were measured. The quantum yields are virtually independent of intensity, demonstrating a one-quantum process. The obtained values in aerated neutral solution in the absence of additives are Φ_ssb= 1.5 × 10^--³, Φ_dsb, = 0.06 × 10^--³ (dose: 10–200 J m^-2), Φ_icl=Φ_Ids= 0.1 × 10³ and Φ_6–4= 0.5 × 10^--³ Both Φ_ssb and Φ_dsb decrease strongly with increasing concentrations of TE buffer (0.01–10 m M ). Biological inactivation of the pTZ18R plasmid was determined from the transformation efficiency of Escherichia coli bacteria strains AB1157, AB1886 uvr and A82480 uvr rec; the Φ_ina values are 1.4 × 10³, 2.1 × 10³ and 3 × 10^--³, respectively. The monoexponential survival curves in all cases show that a single damage site leads to inactivation (one single hit). The biological consequences of different photoproducts are discussed.     

QUANTUM EFFICIENCY AND PHOTOSENSITIVITY OF THE RHODOPSIN ⇌ METARHODOPSIN CONVERSION IN CRAYFISH PHOTORECEPTORS

Photosensitivity (K_λ) of a visual pigment is the product of the molecular absorption coefficient (α_λ) and the quantum efficiency for photoconversion (γ). Among the invertebrates, many visual pigments are stable not only in the rhodopsin (R) conformation but also as the photoproduct, metarhodopsin (M), We here employ a method for determining the photosensitivities of the two stable pigments of a rhodopsin-metarhodopsin pair, using kinetic analysis of fluorescence from metarhodopsin combined with measurements of spectral absorption made before and after saturation at the isosbestic wavelength of the pigment pair. A curve fitting technique, in which a theoretical function is scaled for best fit to the measured absorption spectrum of the photosteady-state mixture, yields values for the photosensitivity of rhodopsin at λ._max, the ratio of quantum efficiencies for rhodopsin—metarhodopsin interconversion, and the fractional composition of the steady-state mixture. With knowledge of the molecular extinction coefficient, the absolute values of quantum efficiency can be calculated. For crayfish ( Orconectes, Procambarus ) rhodopsin, measured in isolated rhabdoms, K_max= 1.05 x 10^-16 cm² at 535 nm with >7λR→M0.69. These values are similar to the photosensitivity and quantum efficiency of bleaching of vertebrate rhodopsins in digitonin solution (Dartnall, 1972). For the metarhodopsin, K_max= 1.02 x 10^-16 cm² at 510 nm, and λ_M-R= 0.49.     

STRUCTURE OF 3-CARBETHOXYPSORALEN PHOTOLYSIS PRODUCTS

Abstract— Upon UV-A irradiation(320–400 nm), the photorcaction of psoralens either with a solvent molecule or with a second psoralen molecule competes with the well-known photoaddition of psoralens to DNA. In the present study, a structural assignment of the 3-carbethoxypsoralen (3-CPs) photolysis products is proposed on the basis of their chromatographic (high performance liquid chromatography) and of their spectroscopic (absorption, fluorescence, mass spectrometry and ¹H Nuclear Magnetic Resonance) properties. Four photolysis products have been isolated and identified. The first one results from a water molecule addition on the 4',5' double bond of 3-CPs, the second from an ethanol molecule addition on the 4',5' double bond. Two cyclobutane type dimers of 3-CPs have also been characterized. These results confirm that, in protic solvents, the furan(4',5') double bond of 3-CPs is more photoreactive than the pyrone(3,4) double bond.     

TRANSIENT PHENOMENA IN THE PULSE RADIOLYSIS OF RETINYL POLYENES—7. RADICAL ANIONS OF VITAMIN A AND ITS DERIVATIVES

Abstract— Upon e^--pulse irradiation in nonprotic solvents, all- trans retinol (ROH) and retinylmethyl ether (ROMe) form transient species (τ= 0.5–7μs, λ_max=575–590 nm) identifiable as radical anions. Similar species are also formed upon laser pulse photoexcitation of these retinyl derivatives in the presence of N,N-dimethylaniline in acetonitrile. In contrast, electron transfer or attachment to all- trans retinyl acetate (ROAc) and palmitate (ROPa) results in 'instantaneous' loss of carboxylate anions from electron adducts giving the retinylmethyl radical (R-, λ_max= 395 nm, τ_k > 100 μ,s); the radical anions in these cases are too short-lived to be detected by nanosecond pulse radiolysis. The lifetimes of radical anions of ROH and ROMe are very sensitive to water and alcohols (e.g. k_q = 10⁷ M ^-1 s^-1 with methanol as quencher for ROH^- in tetrahydrofuran). Based on these findings, the spectral dissimilarity of the one-electron reduction products from ROH and ROAc in alcohols and aqueous micelles becomes explainable in terms of fast formation of protonated radical anions (RH(OH), τ_1/2, > 100 μs, λ_max=370–375 nm) in the case of ROH and of retinylmethyl radical via loss of AcO^- from radical anion in the case of ROAc. In tetrahydrofuran, the complexation of ROH^- with cations such as Na⁺ and Bu₄N⁺ affects the relative importance of its major decay modes, namely, protonation and dehydroxylation, the latter process being significantly enhanced by the presence of Na⁺.     

3-CARBETHOXYPSORALEN-DNA PHOTOLESIONS: IDENTIFICATION AND QUANTITATIVE DETECTION IN YEAST AND MAMMALIAN CELLS OF THE TWO cis-syn DIASTEREOISOMERS FORMED WITH THYMIDINE

Abstract A chemical method for the identification and the quantitative detection of psoralen DNA furan-side photoadducts formed in cells is presented. It is based on an enzymatic digestion of the purified DNA extracted from the treated cells and a further separation by high performance liquid chromatography of the modified nucleosides coupled to a highly sensitive fluorescence analysis and detection. Using this method, 3-carbethoxypsoralen- DNA photoadducts formed in yeast and mammalian cells have been identified and quantified. The predominant photoadducts induced have been identified as two cis-syn dThd(⁵₆^4'_5')3-CPs diastereoisomers. In Chinese Hamster V79 cells treated with 3-carbethoxypsoralen at 50 μM and irradiated at 365 nm with an incident dose of 24 kJ/m², the two monoadducts could be quantitatively assessed at levels as low as 1.3 and 0.7 per 10 000 base pairs.     

REPAIR OF NEAR (365 nm)- AND FAR (254 nm)-UV DAMAGE TO BACTERIOPHAGE OF ESCHERICHIA COLI

Abstract: Intact bacteriophage have been irradiated at 365 nm or at 254 nm and then analysed for DNA photoproducts or injected into their bacterial host to test susceptibility of the damage to both phage and host-cell mediated repair systems. Both thymine dimers and single-strand breaks are induced in the phage DNA by 365 nm radiation. The dimers appear to be the major lethal lesion (approximately 2 dimers per lethal event) in both repair deficient bacteriophage T4 and bacteriophage λ. after irradiation with either 254 nm or 365 nm radiation. Damage induced in T4 by either wavelength is equally susceptible to x -gene reactivation (repair sector approximately 0.5). v -gene reactivation acts on a larger fraction of the near-UV damage (repair sector of 0.82 at 365 nm as against 0.66 at 254 nm). The host-cell mediated photoreactivation system is only slightly less effective for near-UV damage but host-cell reactivation (as measured by comparing survival of phage λ. on a uvr⁺ and a uvr^- host) is effective against a far smaller sector of near-UV damage (0.35) than far-UV damage (0.85). Weigle-reactivation (far-UV induced) of near-UV damage to phage λ is not observed. The results suggest that unless the near-UV damaged phage DNA is repaired immediately after injection. the lesions rapidly lose their susceptibility to repair with a consequent loss of activity of the phage particles.