MOLECULAR MECHANISM OF DRUG PHOTOSENSITIZATION–II. PHOTOHEMOLYSIS SENSITIZED BY KETOPROFEN

Red blood cell lysis photosensitized by ketoprofen (KPF) was investigated. The photohemolysis was inhibited by butylated hydroxyanisole, reduced glutathione, superoxide dismutase and mannitol, and was unaffected by sodium azide; the presence of oxygen markedly enhanced the lysis. Photohemolysis was also observed under anaerobic conditions. Ketoprofen, irradiated in aqueous buffer solution at pH 7.4, underwent a decarboxylation process via intermediate radicals, leading to the compounds (3-benzoylphenyl)ethane, (3-benzoylphenyl)ethyl hydroperoxide, (3-benzoylphenyl)ethanol and (3-benzoylphenyl)ethanone under aerobic conditions and only to the compound (3-benzoylphenyl)ethane under anaerobic conditions. The four photoproducts showed lytic activity, particularly high for the alcohol and hydroperoxide. The overall results suggest for KPF-photosensitized hemolysis a molecular mechanism involving free radicals, superoxide anion and sensitizer photodegradation products.     

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.     

NEAR-INFRARED DETECTION OF SINGLET MOLECULAR OXYGEN PRODUCED BY PHOTOSENSITIZATION WITH PROMAZINE and CHLORPROMAZINE

A sensitive near-infrared detection system has been used to study the steady-state emission of ¹O₂ at 1268 nra produced by promazine (PZ) and chlorpromazine (CPZ) during photo-illumination. Singlet molecular oxygen could be detected in a variety of ordinary and perdeuterated organic solvents, but was not detectable in water or deuterium oxide. The emission was enhanced in the perdeuterated organic solvents and could be eliminated by rigorous degassing or by addition of the singlet oxygen scavenger 2,3-dimethylfuran. Singlet oxygen could not be detected in any of the solvents during irradiation of the sulfoxides of PZ and CPZ. We conclude that in biological systems ¹O₂ production is not a major pathway to phototoxicity for the sulfoxides, while for the parent phenothiazines the formation of ¹O₂ is much more likely to be important in nonpolar environments such as cell membranes than in the aqueous parts of the cell.     

PHOTOSENSITIZATION OF CONDUCTION IN MOLECULAR SOLIDS*

Abstract— The mechanism of spectral sensitization of conduction in molecular photoconductors has been investigated in systems in which sensitizer and photoconductor form separate layers. The efficiency of sensitization is strongly influenced by external fields, approaching 100 per cent at 10⁶ V/cm in certain cases. The spectral response, efficiency, and field dependence are characteristic primarily of the sensitizer, while the transport characteristics are largely determined by the photoconductor. These results are considered in the light of available information concerning the molecular energy levels of the materials studied. They support the view that light absorption produces mobile electronic charge carriers in the sensitizer, which may be separated, injected, and transported across the photoconductor under an applied electric field. This mechanism is consistent with the interpretation of work on electrolyte-photoconductor contacts, and appears to account for all that is presently known about optical sensitization of molecularly bonded photoconductors.     

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.     

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.     

PHOTOCHEMISTRY OF QUINOLYLMETHYLISOTHIORONIUM SALTS. GUANINE SELECTIVE DNA PHOTOCLEAVAGE REAGENTS

Quinolylmethylisothioronium salts (1a and 4a) cleave DNA upon irradiation. The cleavage is more than 10-fold enhanced by piperidine treatment and subsequently shows a high preference for guanines. Photolysis of 1a, 2a and 4a in water at lambda greater than 300 nm resulted in photoheterolysis. Irradiation of 1a in 2-propanol gave only products from photohomolysis, irradiation of 1a in methanol and 2a and 4a in 2-propanol resulted in products from both photoheterolysis and photohomolysis. Quantum yields for the disappearance of 1a in water and 2-propanol were determined. The presence or absence of oxygen had no effect in water, whereas oxidation products were observed upon irradiation in methanol and 2-propanol in the presence of oxygen. The guanine specific DNA photoreaction is proposed to take place by alkylation at N7 via the quinolylmethyl carbocation and thus to represent a photoalkylation.     

PHOTOSENSITIZATION OF SINGLE-STRAND BREAKS IN pBR322 DNA BY ROSE BENGAL

Rose bengal photosensitized the formation of frank single-strand breaks (SSBs) in double-stranded, supercoiled pBR322 DNA as measured by neutral agarose electrophoresis. The yield of SSBs followed first order kinetics with respect to light fluence and dye concentration. The efficiency of cleavage was more than 20 times greater in an argon atmosphere than in an oxygen atmosphere. The quantum yield in an air atmosphere was 1.7 (+/- 0.3) X 10(-8). Sodium azide quenched the cleavage more efficiently in an oxygen atmosphere than when the oxygen concentration was reduced. Isopropanol and mannitol were poor quenchers; ribose-5-phosphate and guanosine-5'-monophosphate did not quench the cleavage. Substituting D2O for H2O increased the yield of SSBs in both oxygen and oxygen-depleted atmospheres. The results are consistent with initiation of cleavage by reaction of the triplet state of rose bengal (or a radical derived from it) with DNA. In the presence of oxygen, an additional mechanism is introduced.     

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.     

PHOTOCLEAVAGE OF DNA IN THE PRESENCE OF SYNTHETIC WATER-SOLUBLE PORPHYRINS

In the presence of oxygen and visible light, various synthetic water-soluble porphyrins cleave pBR 322 plasmid supercoiled DNA (form I) producing relaxed (form II) and linear (form III) DNA corresponding to single-strand and double-strand breaks respectively. Large variations are observed in the efficiency of the porphyrins containing a diamagnetic metal or no metal at all. Singlet oxygen (¹O₂) seems to be involved in the mechanism of cleavage consistent with the inhibitory effect of the azide anion, N^–₃. The higher efficiency of cationic porphyrins (as compared to anionic ones) is due to their greater affinity for DNA as shown by experiments carried out at either high ionic strength or in the presence of the surfactant, sodium dodecyl sulfate.     

PHOTOCLEAVAGE OF DNA: IRRADIATION OF QUINONE-CONTAINING REAGENTS CONVERTS SUPERCOILED TO LINEAR DNA

Irradiation (350 nm) of air-saturated solutions of reagents containing an anthraquinone group linked to quaternary alkyl ammonium groups converts supercoiled DNA to circular and to linear DNA. Generation of linear DNA does not occur by accumulation of numerous single-strand cuts but by coincident-site double-strand cleavage of DNA. Irradiation forms the triplet state of the anthraquinone, which reacts either by hydrogen atom abstraction from a sugar of DNA or by electron transfer from a base of the DNA. Subsequent reactions result in chain scission. The quinone is apparently reformed after this sequence and reirradiation leads to double-strand cleavage.     

2-甲基-1,4-萘醌对DNA光敏损伤的诱导作用

选择波长337 nm的激光作为激励光源,借助凝胶电泳研究了2-甲基-1,4-萘醌诱导的DNA光敏损伤.结果表明:在氧气饱和、脱氧条件下光敏损伤显著,DNA损伤主要与光子剂量、核酸与萘醌浓度比及DNA存在形式有关.     

INQUIRY OF PHOTOSENSITIZATION MECHANISM OF YANGZHOU HEMATOPORPHYRIN DERIVATIVE(YHPD)

By the use of the advanced ESR technique and through comparing with BHPD, the characteristic of YHPD photosensitization is discussed in this paper in the respect of the primary process of photosensitization. The experiment results showed: (ⅰ) not only ~1O_2, but also free radicals(O_2· OH and YHPD)can be formed by the aid of YHPD; and (ⅱ) as to the ability of producing ~1O_2, YHPDBHPD. Two points are indicated: first, the photosensitized damage of YHPD is interrelated to not only ~1O_2, but also free radicals (O_2. OH and YHPD); second, although the photosensitized damage of YHPD is stronger than that of BHPD, yet the photosensitized damage is negatively correlated to the yield of ~1O_2 but positively correlated to those of O_2 and OH. Based on these two points, it is suggested that activated oxygen free radicals(O_2 and. OH) instead of ~1O_2 play the main role as instantaneously activated material in the photosensitized damage of YHPD.     

SENSITIZED PHOTOOXIDATION OF PINENES BY 9,10-DICYANOANTHRACENE

Abstract —The products of 9,10-dicyanoanthracene sensitized photooxidation of α- and β-pinene were separated and identified. The mechanism of this reaction was investigated by fluorescence spectroscopy, solvent dependence and determination of oxidation potentials of olefins. The formation of an exciplex between excited dicyanoanthracene and pinenes was observed in non-polar solvents. On the bases of these observations, an electron transfer mechanism is proposed for the initial step of this reaction.     

PHOTOOXYGENATION OF CAFFEINE SENSITIZED BY HYPOCRELLIN B

The photooxygenation of caffeine sensitized by Hypocrellin B in alcohol solvents hasbeen investigated in this paper. The products have been isolated and identified. The mainproduct is 8--alkoxyl substituted caffeine derivatives. Deuterium. oxygen partial pressure andNaN_3-quenching experiments indicate that the reaction proceeds on singlet oxygen mechanism.     

PHOTOSENSITIZATION OF MELANIN: AN ELECTRON SPIN RESONANCE STUDY OF SENSITIZED RADICAL PRODUCTION AND OXYGEN CONSUMPTION

Abstract Rose Bengal is shown to photosensitize free-radical production and oxygen consumption in solutions of melanin from autooxidation of 3,4-dihydroxyphenylalanine (DOPA). In anaerobic solutions the sensitizer enhances rates of free-radical production by up to a factor of 20. In aerobic solutions, rates of oxygen consumption can be increased by a factor of several hundred. The reactions appear to involve the triplet state of the sensitizer. The effect of the sensitizer in increasing oxygen consumption is quenched by low concentrations of azide and enhanced by D₂O, suggesting that a singlet oxygen mechanism is involved.     

MOLECULAR BASIS OF DRUG PHOTOTOXICITY: PHOTOSENSITIZED CELL DAMAGE BY THE MAJOR PHOTOPRODUCT OF TIAPROFENIC ACID

Abstract Tiaprofenic acid is a photosensitizing nonsteroidal anti-inflammatory drug, whose major photoproduct (decarboxytiaprofenic acid) is also a potent photosensitizer. Because of the lack of the carboxylate moiety, this photoproduct is more lipophilic and might bind more efficiently to cell membranes, thereby causing phototoxic damage. To verify the feasibility of this hypothesis, we have prepared the ³H-labeled analogs of tiaprofenic acid and its photoproduct and examined the binding, persistence and phototoxicity of the photoproduct using poorly metabolizing (fibroblasts) and actively metabolizing cells (hepatocytes). The photoproduct of tiaprofenic acid accumulates in both cell types as it is formed. Upon removal of the photoproduct from the culture medium, it rapidly disappears from hepatocytes but not from fibroblasts. Consequently, irradiation of fibroblasts previously incubated with the photoproduct and kept in culture in the dark for 20 h results in generalized cell damage while this effect is not observed in hepatocytes. Because of its long persistence in poorly metabolizing skin cells and its reluctance to photobleaching, the formation of this photoproduct in skin may be of relevance to explain the in vivo phototoxicity of tiaprofenic acid.