DETERMINANTS OF PHOTOSENSITIZATION BY PURPURINS

The human colon adenocarcinoma cell line, WiDr, was exposed to Photofrin II, hematoporphyrin derivative (HPD), hematoporphyrin (HP) or tetrasodium-meso-tetra(4-sulfonatophyenyl)porphine (TPPS4) followed by irradiation with light. Clonogenicity was determined and the resultant survival curves compared and shown to be qualitatively similar in shape. However, for equal amounts of drug in the medium, there were large differences in photosensitizing efficiency with Photofrin II approximately 5, 25 and 50 fold more effective than HPD, HP and TTPS4, respectively. For the same power used, all drugs were less efficient photosensitizers under red light (600-1100 nm) than under white light (300-110 nm). For all drugs this could be explained in terms of changes in light absorption over the two wavelength ranges. Differences in clonogenic cell survival could not be explained in terms of differences in singlet oxygen production (from published values). A reduction in drug uptake into the cells was sufficient to explain the differences between Photofrin II, HPD and HP, while TPPS4 was 5-fold less effective compared to other drugs than would be expected from drug uptake measurements. Two methods for measuring drug uptake were compared and shown to give different results for Photofrin II. Measurements of drug fluorescence in 0.1 N NaOH yielded 5-fold lower values than when measurements were in 1 N HCl following heat treatment to monomerise aggregated drug. Clearly the reliability of the method used in determining drug uptake must be carefully ascertained.     

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)     

PHOTOSENSITIZATION OF MITOCHONDRIA BY LIPOSOME-BOUND PORPHYRINS

We have compared the photodynamic activities of hematoporphyrin (HP) and protoporphyrin (PP) on isolated rat liver mitochondria by measuring the decline of the respiratory control ratio (RCR) after irradiation at 365 nm. Before addition to the respiratory mcdium, the dyes were dissolved in phosphate-buffered saline (PBS) or incorporated into unilamellar liposomes of dipalmitoyl-phosphatidylcholine (DPPC), sometimes enriched with cholesterol (Chol) or cardiolipin (Card), which are naturally present in mitochondrial membranes. Chol and especially Card strongly increase the porphyrin uptake by mitochondria. In all experimental conditions, PP is taken up by mitochondria to a higher extent than HP. Nevertheless, under conditions giving the same amount of mitochondriabound dye, HP is a morc efficient photosensitizer than PP. As the efficiency of singlet oxygen production has been shown to be equivalent for the two porphyrins in monomeric state, the resulting photobiological effects are explained in terms of different localization of HP and PP in the mitochondrial membrancs. In particular, HP preferentially localizes in the protein-rich polar domains of the inner mitochondrial membrane, whereas PP dissolvcs in the lipid regions of the mcmbrancs.     

SITES OF PHOTOSENSITIZATION BY DERIVATIVES OF HEMATOPORPHYRIN

Leukemia L1210 cells were incubated in vitro with the tumor-localizing product HPD (hem-atoporphyrin derivative) for 0.5. 4 and 18 h. Effects of subsequent irradiation on viability, membrane transport and integrity, DNA synthesis and intracellular ATP concentration were assessed. Intracellular porphyrin pools were analyzed by HPLC. A 30 min incubation led to concentration of a readily-exchangeable pool of monomeric HPD components at plasma membrane loci; irradiation resulted in photodamage to membrane transport and a loss in capacity for dye exclusion. In contrast, increasing the incubation time led to a corresponding increase in the size of a non-exchangeable intracellular pool of other HPD components. Subsequent irradiation led to depletion of intracellular ATP and loss of capacity for biosynthesis of DNA, but little plasma membrane damage.     

LASER PHOTOSENSITIZATION OF CELLS BY HYPERICIN

Abstract— Administering a light dose of 90 J/cm² at 599 nm during incubation with hypericin to a highly differentiated normal epithelial cell line(FRTL–5), derived from Fisher rat thyroid, and to a neoplastic cell line(MPTK–6), derived from the lung metastases of a thyroid carcinoma induced in Fisher rats, produces cell kill at drug doses 1000 times lower than those necessary to cause the same mortality in the dark. The photocytocidal activity of this polycyclic quinone drug on neoplastic cells is superior to that of antitumor anthraquinone drugs, such as daunomycin and mitoxanthrone, and to the photosensitized antiviral activity previously reported for hypericin.     

SYNTHESIS OF BENZOINS BY DYE PHOTOSENSITIZATION

Using heavy-atom-containing xanthene dyes, benzoins can be quanti-tatively prepared by photosensitized reduction from benzils with triethyla-mine.It is an important supplement to"benzoin condensation", esp .for thosebenzoins with electron-donating substituents.     

PHOTOSENSITIZATION OF PYRIMIDINE DIMER SPLITTING BY A COVALENTLY BOUND INDOLE

Abstract— Photosensitized pyrimidine dimer splitting characterizes the enzymatic process of DNA repair by the DNA photolyases. Possible pathways for the enzymatic reaction include photoinduced electron transfer to or from the dimer. To study the mechanistic photochemistry of splitting by a sensitizer representative of excited state electron donors, a compound in which an indole is covalently linked to a pyrimidine dimer has been synthesized. This compound allowed the quantitative measurement of the quantum efficiency of dimer splitting to be made without uncertainties resulting from lack of extensive preassociation of the unlinked dimer and sensitizer free in solution. Irradiation of the compound with light at wavelengths absorbed only by the indolyl group (approximately 280 nm) resulted in splitting of the attached dimer. The quantum yield of splitting of the linked system dissolved in N₂0-saturated aqueous solution was found to be 0.04 ± 0.01. The fluorescence typical of indoles was almost totally quenched by the attached dimer. A splitting mechanism in which an electron is efficiently transferred intramolecularly from photoexcited indole to ground state dimer has been formulated. The surprisingly low quantum yield of splitting has been attributed to inefficient splitting of the resulting dimer radical anion. Insights gained from this study have important mechanistic implications for the analogous reaction effected by the DNA photolyases.     

PHOTOSENSITIZATION BY METHOTREXATE PHOTOPRODUCTS

Abstract— Photoproducts induced upon excitation of methotrexate by UV light have been separated by ion exchange chromatography. They include 2,4-diamino-6-pteridinecarboxylic acid, 2,4-diamino-6-pteridine-carboxaldehyde and other unidentified pteridine derivatives. The same photoproducts can be also formed upon photodynamic reaction using hematoporphyrin as photosensitizer. In oxygen saturated aqueous solutions (pH∼7), methotrexate photoproducts sensitize the oxidation of histidine and tryptophan by UV light by a process involving singlet oxygen. In aqueous solutions containing albumin or in human serum, the same photoproducts are formed from free methotrexate but not from albumin-bound methotrexate. In the latter case the results may suggest that methotrexate covalently binds to albumin upon excitation with UV light either in absence or in presence of oxygen. These results could explain the photosensitization accompanying cancer chemotherapy with high dose methotrexate and also the synergistic effects of PUVA + low dose methotrexate in psoriasis therapy.     

DETERMINANTS OF PHOTOSENSITIZATION BY MONO-L-ASPARTYL CHLORIN e6

The mono-N-aspartyl derivative of chlorin e6 (MACE) is a new photosensitizer being examined for use in anti-neoplastic photodynamic therapy. Studies were carried out to identify unique aspects of MACE localization by murine leukemia L1210 cells in vitro. Octanol/water partitioning studies were used to quantitate the hydrophobicity of MACE and two analogs, chlorin e6 and mesochlorin. Sites of cellular localization of these dyes were probed by fluorescence studies, and by examining loci of photodamage. These studies indicate that MACE, a hydrophilic dye, partitions to cytoplasmic loci. Data obtained with chlorin e6, a more hydrophobic dye, are consistent with binding at both membrane and cytoplasmic sites. A substantially more hydrophobic product, meso-chlorin, binds primarily to the cell membrane. While the tumor-localizing porphyrin product HPD binds to plasma LDL less than HDL, MACE and CE are predominantly bound to plasma protein and HDL. Patterns of distribution and localization of MACE differ substantially from those observed with HPD and other hydrophobic sensitizers. Phototoxic effects of MACE could not be specifically attributed to membrane or mitochondrial damage.     

PHOTOSENSITIZATION BY ANTIMALARIAL DRUGS

Abstract The antimalarial drugs, chloroquine, hydroxychloroquine, quinine, quinacrine, amodiaquine and primaquine and the local anaesthetic, dibucaine, were tested for in vitro photosensitizing capability by irradiation with 365 nm UV light in aqueous solutions. The ability of these compounds to photosensitize the oxidation of 2,5-dimethylfuran, histidine, tryptophan or xanthine, and to initiate the free radical polymerization of acrylamide was examined in the pH range 2-12. Chloroquine and hydroxychloroquine show maximal photooxidative behaviour when in the monocation form at pH 9, in contrast to quinine which is extremely efficient as the dication below pH 4. This pattern appears to relate to the fluorescence yield as a function of pH. Chloroquine in the monocation or neutral form was found to undergo dechlorination upon irradiation, and this correlates directly with its ability to initiate photo-polymerization of acrylamide. Quinine also gives rise to small polymerization rates, attributed to photo-ionization in the quinoline ring, yielding a cation radical. Amodiaquine, primaquine and quinacrine do not have significant photochemical activity in aqueous solution. Dibucaine exhibits a strong photosensitizing capability at low pH, similar to quinine.     

PHOTOSENSITIZATION OF METHYL LINOLEATE OXIDATION BY TRYPTOPHAN IN PEPTIDES

The ability of tryptophan in peptides to photosensitize the oxidation of methyl linoleate (ML) was evaluated. Purified ML was irradiated (λ > 270 nm) alone or in the presence of a tryptophan-containing peptide in ethanol solution. Oxidation was monitored by measuring the dienc hydroperoxides formed from ML by high performance liquid chromatography. N-acetylphenylalanyltryptophan (NAPT) and N-acetyltryptophan were about 2-fold more effective as photosensitizers than leucyltryptophan and tryptophylleucine. N-acetylphenylalanyltryptophan photolyzed in ethanol to form multiple photoproducts including N-formylkynurenine-type compounds. However, the NAPT photoproducts did not photosensitize the oxidation of ML and N-formylkynurenine was about 8% as effective as NAPT. Sodium azide partially quenched the photooxidation sensitized by NAPT.     

ELECTRON TRANSFER IN PHOTOSENSITIZATION OF BIOMOLECULES BY ACRIDINE DYES

Abstract— The photosensitization of some biomolecules by proflavine was studied using the technique of electron paramagnetic resonance. It was shown that this process is directly influenced by the relative molar concentration of dye and substrate. For some values of these concentrations, a singlet associated with a radical form of proflavine is observed. The appearance of this signal is discussed in terms of electron transfer from an ionized proflavine molecule directly to another neutral dye molecule or via a molecule of substrate as an intermediate. It is shown that this transfer is facilitated by stacking. These results were compared with radiolysis of the same kind of complexes in frozen aqueous solution.     

GENETIC TOXICOLOGY OF THE PHOTOSENSITIZATION OF CHINESE HAMSTER CELLS BY PHTHALOCYANINES

Chloroaluminum phthalocyanine (CAPC) was recently shown to photosensitize cell killing in culture and tumor destruction in vivo. Because this compound is potentially useful in the photodynamic therapy of cancer, its properties as a genotoxic agent were evaluated. Applying the technique of alkaline elution to study DNA integrity, it was found that CAPC could produce single-strand breaks in the DNA of Chinese hamster cells after exposure to white fluorescent light. At equicytotoxic doses, the number of DNA strand breaks produced by CAPC photosensitization was about three times lower than that induced by X-irradiation. During incubation in growth medium after exposure to CAPC-plus-fluorescent light, cells rejoined DNA strand breaks at a rate similar to that observed after X-irradiation. Resistance to 6-thioguanine (6-TG') or to ouabain (OUA') were used as end points of mutagenic potential. Following a treatment that caused -90% cell killing, there was a slight mutagenic effect, i.e. the frequencies were increased by -40% above the background or spontaneous mutations. However, this enhancement was not statistically significant. Taken together, the foregoing, plus an earlier observation that there is no variation in the sensitivity of cells to CAPC + light through the cell cycle, lead to the inferences that DNA damage does not play a major role in cell killing and that the mutagenic potential of this treatment is small.     

MITOCHONDRIAL PHOTOSENSITIZATION BY PHOTOFRIN II

Abstract V-79 Chinese hamster cells grown as monolayers or as multicell spheroids were treated with Photofrin II (10 μ.g m⁻¹ for 16 h) and various doses of red light irradiation. The resulting biochemical and functional damage to cell mitochondria was studied. The activities of both succinic dehydrogenese and cytochrome c oxidase were found to decrease in a light dose-dependent manner. The respiratory control quotient (RC) decreased in parallel with a decrease in the activities of the respiratory chain proteins. Our data also showed a distinct temporal difference in the relative progression of mitochondrial damage and cell death as assessed by loss of discrete Rhodamine-123 (Rh-123) localization and trypan blue infiltration, respectively. Mitochondrial damage was detected immediately, as seen by derealization of Rh-123 resulting from dissipation of the electrochemical gradient in damaged mitochondria. Trypan blue infiltration occurs with a distinct time lag. These findings are consistent with the hypothesis that, at least for long Photofrin II incubation times, the mitochondrion is a primary target of photosensitization. The subsequent changes in cell membrane permeability may be a delayed result of decreased bioenergetics of the Photofrin II photosensitized cell.     

PHOTOSENSITIZATION OF HUMAN DIPLOID CELL CULTURES BY INTRACELLULAR FLAVINS AND PROTECTION BY ANTIOXIDANTS

Abstract— The damaging effects of near ultraviolet and visible light on WI-38 human diploid lung fibroblasts were investigated. WI-38 cells in culture were killed by light doses ranging from 2 to 10 × 10³ W/m² h. There was an inverse correlation between culture age, i.e. population doubling level and photosensitivity. However, this effect could not be related to capacity for DNA synthesis and cell division.
Flavins were clearly implicated as endogenous photosensitizers, and antioxidants such as d, l -α-tocopherol (vitamin E), BHT and ascorbic acid were found to afford the cells protection from light damage. Furthermore, products of lipid peroxidation could be detected in cell homogenates irradiated in the presence of ribofiavin.     

OLIGONUCLEOTIDE PHOTOPRODUCTS FORMED BY PHOTOLYSIS OF POLYRIBOBROMOURIDYLIC ACID

Abstract— Polyribobromouridylic acid was irradiated with 313 nm light at an exposure of ˜ 190 pE/cm*. Oligonucleotides found after RNase hydrolysis of the photolysed poly-rBrU were isolated by DEAE-cellulose chromatography and partially characterized. The dmucleo-tide fraction, found in highest amounts, was not susceptible to hydrolysis by KOH or snake venom phosphodiesterase and may contain a coupled photoproduct. The properties of the dinucleotide were not those of a molecule containing a cyclobutane-type dimer, but were compatible with the properties of a coupled product similar to 5–5'-diuracil. The trinucleotide fraction probably consisted of more than one component. One component may contain a dimeric photoproduct. The tetranucleotide material was sensitive to cleavage into fragments by KOH, and could consist of adjacent photoproducts of the types found in the di- and trinucleotide fractions. The photoproducts formed over a range of lower doses of light were found to have properties similar to those found at high doses.     

THE MECHANISM OF PHOTOSENSITIZATION IN PHOTODYNAMIC THERAPY: CHEMILUMINESCENCE CAUSED BY PHOTOSENSITIZATION OF PORPHYRINS IN SALINE CONTAINING HUMAN SERUM ALBUMIN

Chemiluminescence (CL) caused by photosensitization of porphyrins in phosphate buffered saline (PBS) solution containing 3% human serum albumin (HSA) was observed for the first time. Irrespective of porphyrins concerned, CL shows a spectrum ranging from 380 to 520 nm with a peak near 450 nm and decays almost single-exponentially with a lifetime of about 15 s. The intensity of CL depends on concentrations of porphyrins and HSA in PBS solution. We have examined a number of porphyrins and observed CL for the compounds with triplet lifetimes longer than 0.1 ms. The appearance and quenching of CL by photosensitization of porphyrin-HSA systems indicate that type II reaction by singlet oxygen occurs significantly in photodynamic therapy resulting in hypoxic regions in environments surrounding the sensitizer.     

MEMBRANE IONIC CURRENT PHOTOMODIFICATION BY ROSE BENGAL AND MENADIONE: ROLE OF SINGLET OXYGEN

Abstract: Photosensitized modification of ionic leak current and potassium current was studied in frog cardiac atrial cells using whole cell patch clamp techniques. Rose bengal (RB) and menadione (MQ) were used as photosensitizers. Separate photophysical studies of the photosensitizers in deuterium oxide solution demonstrated that MQ did not produce singlet oxygen as evidenced by the lack of luminescence at 1270 nm, whereas RB was an efficient singlet oxygen generator. Both photosensitizers sensitized block of potassium current in atrial cells, and both sensitized an increase of ionic leak current. However, when photosensitizer concentrations and illumination intensities were adjusted to match the rate of block of potassium current by the two photosensitizers, there were dramatic differences in leak current increase, both quantitatively and qualitatively. Menadione sensitized a much slower increase in leak current than did RB. Further, the leak current sensitized by MQ had a more positive reversal potential than that sensitized by RB, suggesting a less potassium-selective leak current pathway. The results suggest that, while the effects of singlet oxygen and non-singlet oxygen modification of cell membranes may be similar, there may also be significant differences in the resulting membrane permeabilities. The results also demonstrate that MQ and RB may be useful agents to study the role of singlet oxygen versus non-singlet oxygen modification of biological systems.     

PHOTOSENSITIZATION OF SUPERCOILED DNA DAMAGE BY 5,6-DIHYDROXYINDOLE-2-CARBOXYLIC ACID, A PRECURSOR OF EUMELANIN

The photosensitizing or photoprotecting action of 5,6-dihydroxyindole-2-carboxylic acid (DICA), an intermediate in the biosynthesis of eumelanins, was investigated. Under irradiation at 313 nm, aqueous buffered solutions of DICA (22.5 μW) photosensitized the cleavage of phage φX174 DNA. The number of single strand breaks (SSB) depended on the dose of irradiation and was more important in the absence than in the presence of oxygen. In the presence of oxygen, the quantum yield of SSB was around 6′10 ⁷ (Φ_SSB) The influence of specific scavengers, such as mannitol, sodium azide or superoxide dismutase, indicated that hydroxyl radicals, superoxide anions and perhaps singlet oxygen were involved in these processes. The increase in SSB in D₂O was also indicative of the participation of singlet oxygen. Comparative experiments performed with indole-2-carboxylic acid (IC), a dehydrox-ylated analog of DICA, showed that this compound, although lacking a phenol group, also photosensitized DNA cleavage via a mechanism involving hydroxyl radicals. Various sources of these radicals were envisioned. Furthermore, under our conditions, DICA was not found to photoinduce the formation of DNA dimers: No increase in SSB was observed in DNA irradiated in the presence of DICA, after treatment by phage T4 endonuclease V (an enzyme that selectively cuts DNA at dimer sites), whereas, in contrast, a significant increase in SSB was detected after treatment of DNA irradiated alone. So it appears that DICA may both photosensitize DNA cleavage and reduce UV-induced DNA dimer formation.     

MECHANISM OF PHOTOSENSITIZATION BY PHEOPHORBIDE a STUDIED BY PHOTOHEMOLYSIS OF ERYTHROCYTES AND ELECTRON spIN RESONANCE SPECTROSCOPY

Abstract Phcophorbide a (PPa), a causal substance of food intoxication, when excited by exposure to light wavelengths of over 600 nm, caused the photohemolysis of goat erythrocytes in proportion to the incubation time of the cells. The addition of N-₃, an effective scavenger of ¹O₂, to the medium markedly inhibited the hemolysis of erythrocytes in a concentration-dependent manner, whereas the addition of superoxide dismutase (SOD) and catalase, inhibitors of O^-₂ and H₂O₂ generation, respectively, to the medium had little effect on it.
Methods for converting ¹O₂ to a nitroxide radical by 2,2,6,6-tetramethyl-4-piperidone (TMPD) and for trapping O^-₂ and OH by 5,5-dimethyl-l-pyrroline-A'-oxide (DMPO) were employed to observe directly these activated oxygens by electron spin resonance (ESR). The methods provided evidence that only ¹O₂, was produced by PPa, which was excited by light wavelengths of over 600 nm. Both the addition of N₃ to the solution and the removal of oxygen from the solution inhibited the generation of ¹O₂.
These results led us to conclude that ¹O₂ was mainly responsible for the hemolysis of erythrocytes by photoexcited PPa.