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.     

A NEW REUSABLE CHEMICAL ACTINOMETER FOR UV IRRADIATION IN THE 248 - 334 nm RANGE

The highly reversible thermally stable photochromic system consisting of heterocoerdianthrone-endoperoxide (HCDPO) and its parent compounds HCD and 0₂ is proposed as a new reusable liquid chemical actinometer in the UV region 248 ≤λ≤ 334 nm. The foremost advantageous features of this new system are: (1) high reproducibility and accuracy, (2) high sensitivity, (3) almost wavelength independent quantum yields, (4) no loss in accuracy even after 100 repeated actinometric cycles and (5) very easy handling and straightforward calculability of the radiation quantum flux.     

SPECTROSCOPIC PROPERTIES OF PSORALEN DERIVATIVES SUBSTITUTED BY CARBETHOXY GROUPS AT THE 3,4 AND/OR 4,5' REACTION SITE

Abstract— The newly synthesized linear psoralen derivative 3-carbethoxypsoralen has been shown recently to behave as a monofunctional derivative and has attracted some interest in the psoriasis treatment. In a first attempt to understand, by the fluorescence technique, the molecular mechanism by which it interacts with DNA, a spectroscopic study of the molecule was undertaken. The fluorescence of 3-carbethoxypsoralen at room temperature resembles that of 8-methoxypsoralen with a ten times higher quantum yield. 365 nm irradiation of dilute solutions of 3-carbethoxypsoralen rapidly leads to the formation of two types of highly fluorescent photoproducts. Type 1 photoproducts (λ_em^max= 425 nm, λ_exc^max= 360 nm) have been identified as the result of the addition of a solvent molecule to the 4,5' reaction site of the molecule. Their fluorescence intensity is a hundred times higher for 3-carbethoxypsoralen than for 8-methoxypsoralen. They become negligible when the 4',5' reaction site carries also a carbethoxy group. Type 2 photoproducts exhibit a somewhat different emission (λ_em^{max =} 443 nm, λ_exc^max= 413 nm). They are probably the result of an opening of the furocoumarin molecule. The implications of the peculiar photochemical properties of 3-carbethoxypsoralen are discussed in view of its biological activity. In addition, the use of fluorescence in monitoring the photobinding of psoralens to DNA is also discussed     

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).     

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.     

BIOPHYSICAL AND BIOCHEMICAL ASPECTS OF PHENGODID (RAILROAD-WORM) BIOLUMINESCENCE

In studies of the bioluminescence of 11 species of phengodid collected in central and southeast Brazil, we have found that: (1) their lateral lanterns emit light in the yellow-green region (λ_max= 540–580 nm) and the head lantern color is shifted to the red region ( λ_max= 565–620 nm), (2) the luciferins of both types of lanterns are identical to that of lampyrids and elaterids and (3) the luciferase physicochemical properties are also similar to those of lampyrids and elaterids (optimum pH ca 8.1; K_m(ATP) = 260–370 μM , Kμ(luciferin) = 170–400 μM; molecular weight ca 60 kDa; apparent activation energy of in vitro bioluminescence ca 58 kJ/mol). Thus the bioluminescence system of phengodids appears to be essentially the same as that of lampyrids and elaterids. The different bioluminescence colors of the lanterns of Phrixothrix species (λ_head= 600–620 nm; λ_lateral= 535–565 nm) and other phengodid species are probably elicited by the presence of luciferase isoenzymes, as occurs in the case of elaterid prothoracic and abdominal lanterns.     

In situ COMPARISON OF 665 nm and 633 nm WAVELENGTH LIGHT PENETRATION IN THE HUMAN PROSTATE GLAND

Abstract— The depth of treatment in photodynamic therapy (PDT) of tumors varies with the wavelength of light activating the photosensitizer. New generation photosensitizers that are excited at longer wavelengths have the potential for increasing treatment depths. Tin ethyl etiopurpurin (SnET2), a promising second-generation photosensitizer is maximally activated at 665 nm, which may be significantly more penetrating than 633 nm light currently used with porphyrins in PDT. The penetration of 665 nm and 633 nm wavelength red light in the prostate gland was compared in 11 patients undergoing prostatic biopsies for suspected prostatic cancer. Interstitial optical fibers determined the light attenuation within the prostate gland. Of the 11 patients, 7 had dual wavelength and 4 had single wavelength studies. The mean attenuation coefficients, μ_eff, for 665 nm and 633 nm wavelength light were 0.32 ± 0.05 mm^-1 and 0.39 ± 0.05 mm^-1, respectively, showing a statistically significant difference (P = 0.0003). This represented a 22% increase in the mean penetration depth and at 10 mm from the delivery fiber there was 1.8 times as much 665 nm light fluence than 633 nm. The mean μ_eff at 665 nm for benign and malignant prostate tissue were similar ( P = 0.42), however, there was significant interpatient variation (μ_eff ranging from 0.24 to 0.42 mm^-1) reflecting biological differences of therapeutic importance. The enhanced light fluence and penetration depth with 665 nm light should allow significantly larger volumes of prostatic tissue to be treated with SnET2-mediated PDT.     

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.     

ANALYSIS OF MULTIPLE PHOTORECEPTOR PIGMENTS FOR PHOTOTROPISM IN A MUTANT OF Arabidopsis thaliana

Abstract
The shape of the fluence-response relationship for the phototropic response of the JK224 strain of Arabidopsis thaliana depends on the fluence rate and wavelength of the actinic light. At low fluence rate (0.1 μmol m^-2s^-1), the response to 450-nm light is characterized by a single maximum at about 9 μmol m^-2. At higher fluence rate (0.4 μmol m^-2s^-1), the response shows two maxima, at 4.5 and 9 μmol m^-2. The response to 510-nm light shows a single maximum at 4.5 μmol m^-2. Unilateral preirradiation with high fluence rate (25 μmol m^-2s^-1) 510-nm light eliminates the maximum at 4.5 μmol m^-2 in the fluence response curve to a subsequent unilateral 450-nm irradiation, while the second maximum at 9 μmol m^-2 is unaffected. Based on these results, it is concluded that a single photoreceptor pigment has been altered in the JK224 strain of Arabidopsis thaliana.     

QUANTUM YIELDS OF TRIPLET FORMATION OF SOME DERIVATIVES OF ANTHRAQUINONE

Abstract— Quantum yields of triplet formation for five amino substituted anthraquinones have been determined by the comparative actinometer method using laser flash photolysis. Substitution reduces the yields to values of ˜10^--². and the requirement of low laser intensities required high sensitivity of detection of triplet absorption. The ø_T values are compared with the quantum efficiencies of fluorescence and decomposition for the compounds, and the criteria for light stability discussed.     

THE ''Q-BAND'' ABSORPTION spECTRA OF HEMATOPORPHYRIN MONOMER AND AGGREGATE IN AQUEOUS SOLUTION

Abstract The resolution of the absorption spectra in the Q band (480 nm-620 nm) spectral region of monomeric and dimeric hematoporphyrin species present in aqueous solutions has been achieved using absorption, fluorescence and computer analysis methods. The absorption maxima of the dimer in this spectral region are red shifted about 12 nm with respect to those of the monomer. The significance of this finding in relationship to the well documented blue shift of hematoporphyrin aggregate observed in the Soret band region (λ_malx∼400 nm) of the absorption spectrum is discussed.     

USE OF POTASSIUM IODIDE AS A CHEMICAL ACTINOMETER

Abstract Aqueous solutions of KI were examined for use as chemical actinometers to measure 254 nm (germicidal) radiation. Irradiation results in electron ejection from iodide such that aqueous electrons and iodine atoms are formed. In the presence of N₂0, an electron scavenger, recombination of these two reactive species is eliminated and stoi-chiometric formation of triiodide occurs. The absorbance increase due to triiodide was followed and the quantum yield determined using either a radiometer or ferrioxalate actinometry to estimate the amount of energy absorbed by the KI solution. The quantum yield ( ø ) at 25°C was determined to be 0.26 using radiometry, and 0.224–0.233 using actinometry, depending on the radiation conditions. The following expression was used to measure the incident Huence rate for 254 radiation at a given temperature T for irradiation over a time interval Δt (s) fluence rate (W/m²) = 4. 96 × 10⁶ΔOD(λ)/ e(λ) [0.23+0.004(T-25)] δ t cm⁻¹
At temperatures other than 25°C, the temperature dependence of the quantum yield (0.004/°C) is taken into account by the term shown in the denominator. Because KI remains relatively blind to longer wavelengths such as those found in normal room light, measurements can be made in the presence of room light without having to work in a darkened room.     

SPECTROPHOTOMETRIC IDENTITY OF THE ENERGY TRANSFER CHROMOPHORES IN RENILLA AND AEQUOREA GREEN-FLUORESCENT PROTEINS

Abstract— Spectral properties of guanidine-denaturated and pronase-digested green-fluorescent proteins (GFP) from two species of bioluminescent coelenterates have been investigated. Spectrophotometric titrations of Renilla and Aequorea GFP, following denaturation in 6 M guanidine HCl at elevated temperature, revealed identical absorption peaks in acid (383–384 nm) and in alkali (447–448 nm) and a single isosbestic point in the visible region at 405 nm. Both proteins exhibited a spectrophotometric pK. of 8.1 in guanidine -HCl. Pronase digestion of the heat-denaturated GFP's generated a methanol-soluble blue-fluorescent peptide with identical fluorescence emission spectra (λ_max= 430 nm, uncorrected; φ_f1= 0.003) for both coelenterate species. These data suggest that the large absorption differences between native Renilla and Aequorea GFP molecules result from unique protein environments imported to a common chromophore.     

HALORHODOPSIN and PHOTOSENSORY BEHAVIOR IN Halobacterium halobium MUTANT STRAIN L-33

Halobacterium halobium , strain L-33, which is deficient in bacteriorhodopsin (BR) but synthesizes increased amounts of halorhodopsin (HR), shows behavioral responses upon changes in fluence rate with visible light or with UV light. The observations support the earlier report (Schimz et al. , 1982). that BR is not essential for photosensing in H. halobium. In the UV-range, changes in light intensity elicit the maximal response at λ= 370 nm. In the visible range, changes in light intensity show the maximal response at Δ= 565 nm and a secondary peak at Δ= 590 nm. The latter corresponds to the absorption maximum of HR (Δ_max= 588 nm). This light-energy converting retinal pigment of H. halobium thus appears to contribute to photosensory behavior.     

MECHANISM OF HIGH POWER PICOSECOND LASER UV INACTIVATION OF VIRUSES AND BACTERIAL PLASMIDS

Abstract— The mechanism of inactivating action of high-power picosecond laser UV radiation (λ= 266 nm) on the λ and φX174 bacteriophages and the pBR 322 plasmid has been studied. It has been shown that at UV radiation intensities from 10¹¹ to 10¹³ W/m², inactivation of viruses and bacterial plasmids occurs mainly on account of single-strand breaks in the DNA chain unlike the case of less powerful UV radiation where the inactivation is associated with the formation of pyrimidine dimers.     

Action spectrum for the suppression of arylalkylamine N-acetyltransferase activity in the two-spotted spider mite Tetranychus urticae

An action spectrum was obtained for the suppression of arylalkylamine N -acetyltransferase (NAT) activity in the two-spotted spider mite Tetranychus urticae by irradiating the mite with monochromatic lights of various wavelengths using the Okazaki Large Spectrograph at the National Institute for Basic Biology, Okazaki, Japan. Fluence–response curves were obtained for wavelengths between 300 and 650 nm by irradiating the mite for 4 h day⁻¹. The samples were frozen after the third exposure. A negative correlation between the logarithmic fluence rate and NAT activity was detected in the range of 0.01–1 μmol m⁻² s⁻¹ for wavelengths between 300 and 500 nm and in the range of 0.1–10 μmol m⁻² s⁻¹ for wavelengths between 550 and 650 nm. The constructed action spectrum indicated that the photoreceptors mediating the circadian and/or photoperiodic systems might be UV-A- and blue-type photoreceptors with absorption peaks at 350 and 450 nm.     

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⁺.     

RADICAL CATION PRODUCTION IN THE PHOTOLYSIS OF 5-M-ETHOXY-1-M-ETHYLINDOLE

Abstract— The flash photolysis of 5-m-ethoxy-1-m-ethylindole in aqueous media was studied for the purpose of assigning the absorption spectrum of the radical cation. Transients produced in this study were analogous to those formed in the photolysis of 5-m-ethoxyindole. The major transient observed with an absorption maximum of 460 nm was O₂-s_-ensitive and had a lifetime of 20 μs in nitrogen saturated solutions. One radical species is produced with absorption maxima at 445 and 530 nm. Ionic strength effects on the reaction of this species with I⁻ confirms that it is the radical cation of 5-m-ethoxy-l-methylindole. The effect of H⁺ and Br⁻ on the fluorescence, radical cation and triplet yields is discussed in relation to the mechanism of transient formation.     

IN VIVO FLUORESCENCE SPECTROSCOPY OF CHLOROPHYLL IN VARIOUS UNRIPE AND RIPE FRUIT

—Low temperature (77 K) fluorescence emission spectra of slices obtained from the peel and various layers of the pericarp were recorded for fruits which remain green or undergo color break during ripening.
Fluorescence emission peaks characteristic of the photosystem II antennae (λ_F 686 nm) and reaction center (λ_F 696 nm), as well as of the photosystem I antenna (λ_F 730-740 nm), were present in the peel and all parts of the green pericarp of ripe kiwi, avocado and cantaloupe, as well as in ripe tomato and tangerine after color break. The pattern of the fluorescence emission spectra of all samples except that of the kiwi fruit was similar to that obtained from green photosynthetic tissue of leaves, indicating a normal organization of the chlorophyll-containing complexes of thylakoidal membranes. This pattern is characterized by a significantly higher emission at 730-740 nm relative to that of the 696 and 686 nm peaks. In contradistinction, the fluorescence emission at 686 and 696 nm was higher than that at 730 nm in the kiwi fruit, indicating a reduction in the size of the photosystem I antenna chlorophyll. In the innermost yellowish layers of the kiwi pericarp, a further loss of this antenna occurred, as well as disorganization of the photosystem II complex. The above conclusions are suggested also by measurements of variable fluorescence kinetics.
The results presented here indicate that fluorescence spectroscopy might be used as a tool for the study of chlorophyll organization during the growth and ripening periods of fruit.     

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.