A MICROSPECTROFLUOROMETRIC STUDY OF PORPHYRIN-PHOTOSENSITIZED SINGLE LIVING CELLS— I. MEMBRANE ALTERATIONS

Microspectrofluorometry on single living cells reveals that the primary effects of porphyrin-photosensitization on membranes results in the production of fluorescent lipofuscin-like pigments implying important lipid photoperoxidation. These fluorescent products (_max 450 nm) can also be formed in the dark after the irradiation of the cell. Their formation is abolished not only by crocetin and Tigason, two long-chain polyenes, but also by chloroquine. The latter, although a lysosomotropic drug, cannot inhibit the photosensitized permeation of lysosomal membranes which occurs at the beginning of the irradiation as shown by the splitting of fluorogenic substrates by lysosomal proteinases or by β-galactosidase when added before or after irradiation.     

PHOTOREACTIVATION ASSOCIATED WITH CHROMOSOMAL ABNORMALITY IN SEA URCHIN EGGS FERTILIZED WITH ULTRAVIOLET-IRRADIATED SPERM

Abstract— As a model for studies involving biologic systems, rates of hematoporphyrin and protopor-phyrin-sensitized photooxidation of tryptophan were measured in water-methanol mixtures. Absorption and fluorescence-emission spectra associated with solutions of porphyrins in different water-methano! mixtures were also examined. The optimal rate of photooxidation by hematoporphyrin occurred in 30% methanol; by protoporphyrin, in 60% methanol. Absorption spectra were examined to characterize dispersion of hematoporphyrin into monomeric form in 90% methanol, and the dimer formation which predominates in 30% methanol. With the more hydrophobic protoporphyrin, an absorption spectrum characteristic of drug monomer was seen in 100% methanol, a dimer spectrum was observed in 60% methanol. At methanol concentrations < 60%, we observed impaired photooxidation and another alteration in the absorption pattern apparently reflecting drug aggregation beyond the dimer stage. Fluorescence emission maxima of both porphyrins were blue-shifted with increasing solvent dielectric constant, but an additional blue shift was detected in an aqueous environment which was apparently related to porphyrin aggregation. Absorption and fluorescence-emission spectra appear helpful for characterizing environmental determinants of porphyrin-sensitized photooxidation.     

INTERACTION OF HUMAN SERUM LOW DENSITY LIPOPROTEINS WITH PORPHYRINS: A SPECTROSCOPIC AND PHOTOCHEMICAL STUDY

The incorporation of proto-, uro- and hematoporphyrin in low density lipoproteins (LDL) of human blood has been studied by equilibrium dialysis, fluorescence and absorption spectroscopy. The lipoproteins may efficiently compete with albumin in the binding of protoporphyrin to human blood proteins in patients suffering from protoporphyria. It can be concluded that hydrophobic porphyrins bind to blood proteins.
The complexation of hydrophobic porphyrins in LDL is responsible not only for efficient photodynamic effect at the lipoprotein level, but also for photoinduced lipid peroxidation and for consumption of β-carotene incorporated into LDL which are one of their natural carriers. The water-soluble uroporphyrin, although an efficient photosensitizer for the LDL apoprotein photoinactivation, is much less efficient for lipid peroxidation and β-carotene bleaching. The 353 nm laser flash photolysis shows that porphyrin triplet states are not affected by the physiological β-carotene content of LDL but are fully accessible to oxygen.     

MODIFICATION OF ε-AMINO GROUP OF LYSINES, CHOLESTEROL OXIDATION AND OXIDIZED LIPID-APOPROTEIN CROSS-LINK FORMATION BY PORPHYRIN-PHOTOSENSITIZED OXIDATION OF HUMAN LOW DENSITY LIPOPROTEINS

Abstract— When incorporated into LDL, protoporphyrin and the porphyrin mixture constituting the trade mark Photofrin II photosensitize peroxidations of cholesterol and lipids. Not only 5a-hydroperoxicholesterol, the specific product of cholesterol oxidation by ¹O₂ but also the epimeric 6-hydroperoxicholesterols are produced as shown by HPLC. In addition to malonaldehyde-like substances, the formation of 4-hydroxynonenal, a highly reactive and toxic aldehyde resulting from lipid peroxide breakdown is detected. These products are also formed by dark radical chain reactions of lipid photoperoxides induced by photosensitization. The involvement of lipid photoperoxidation in LDL apoprotein modification is demonstrated by following the derivatization of e-NH₂ groups of Lys residues which are necessary to binding to the LDL receptor. As a result, photosensitized LDL cannot bind to their receptors on fibroblasts. Lys residues are not sensitive to direct photodynamic reaction as confirmed by delipidation of LDL and solubilization of apoLDL in 1% SDS which totally inhibit Lys derivatization without affecting the degradation of Trp residues susceptible to the photodynamic attack. Another consequence of lipid peroxidation at the protein level is the formation of cross-links between apoLDL and photooxidized lipids as shown using LDL loaded with radioactive arachidonic acid. On the other hand, cholesterol photoperoxidation does not lead to protein-oxidized cholesterol cross-links. These reactions between peroxidized lipids and LDL proteins are also responsible for the formation of lipofuscin-like fluorescent pigments encountered in all aging processes. The biological and biomedical consequences of these results are discussed.     

ENERGY TRANSFER AND PHOTOOXIDATION KINETICS IN REACTION CENTERS ON THE PICOSECOND TIME SCALE

Abstract— Picosecond 530 nm actinic and 1242 nm probe light pulses have been used to measure the kinetics of energy transfer and photooxidation in Rhodopseudomonas sphaeroides R-26 reaction centers. The energy transfer rate between bacteriopheophytin and the bacteriochlorophyll dimer is 1.0 ± 0.3 ± 10¹¹s^-land photooxidation of the dimer occurs within 5 ps after the dimer reaches the first excited singlet state. Using these parameters in a simple model we are able to explain the odd result that the number of reaction centers oxidized by a saturating 530 nm actinic picopulse is only 60% of the number oxidized by a saturating CW light source.     

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.     

DESTRUCTION OF MICROSOMAL CYTOCHROME P-450 BY REACTIVE OXYGEN SPECIES GENERATED DURING PHOTOSENSITIZATION OF HEMATOPORPHYRIN DERIVATIVE

Rat liver microsomal cytochrome P-450 undergoes rapid destruction in the presence of hematoporphyrin derivative (HpD) and solar radiation (∼ 400 nm). Destruction of cytochrome P-450 is associated with the formation of cytochrome P-420 and significant loss of microsomal haem. Quenchers of singlet oxygen including 2,5-dimethylfuran, histidine, ß-carotene, and ascorbic acid and inhibitors of the hydroxyl radical such as benzoate, mannitol, and ethanol prevent deterioration of the microsomal haem-protein, whereas superoxide dismutase and catalase are ineffective in this regard. These results indicate that generation of singlet oxygen during hematoporphyrin photosensitization is associated with destruction of microsomal cytochrome P-450 and haem.     

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.     

PERMEATION OF LYSOSOMAL MEMBRANES IN THE COURSE OF PHOTOSENSITIZATION WITH METHYLENE BLUE AND HEMATOPORPHYRIN: STUDY BY CELLULAR MICROSPECTROFLUOROMETRY

Abstract— The photodynamically-induced liberation of lysosomal enzymes using ß-galactosidase as marker for the lysosomal enzymes has been studied by microspectrofluorometry on mouse L cells. Similar studies have been carried out using N-acetyl-ß-D-glucosaminidase as marker for the lysosomal enzymes of human fibroblasts. The high sensitivity of the fluorescence detection makes it possible to use 4-methylumbelliferyl substrates for the enzymes contained in a single cell. Methylene blue and hematoporphyrin readily incorporate into both cells and upon excitation, sensitize lysosomal membrane damages, leading to enzyme release accompanying strong morphological changes.     

PHOTOFRIN II UPTAKE BY ATHEROMA IN ATHEROSCLEROTIC RABBITS. FLUORESCENCE AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS ON POST-MORTEM AORTA

Atherosclerotic lesions were induced in normal and Watanabe rabbits by atherogenic diet and stripping of aorta endothelium. The rabbits were injected with Photofrin II and sacrificed two days later. Atheromatous aorta as well as normal aorta from control animals were characterized by their fluorescence spectra using front face excitation. Characteristic emission peaks at 631 and 694 nm were displayed at atheromatous plaques. The excitation spectrum shows a strong band at 394 nm and weaker bands at 446, 504, 536 and 574 nm. Although no fluorescence of normal aorta can be seen by visual inspection, emission with a maximum at 626 nm was detected by spectrofluorimetry. Normal phase high performance liquid chromatography analysis of extracts from atheroma and control aorta were also carried out. The specific labelling of atheroma involves mainly protoporphyrin, hematoporphyrin and also minor components of Photofrin II which are accumulated. Some other components are accumulated but do not appear to be specifically retained by atheroma.     

SINGLET OXYGEN GENERATION BY HEMATOPORPHYRIN IX, UROPORPHYRIN I and HEMATOPORPHYRIN DERIVATIVE AT 546 nm IN PHOSPHATE BUFFER and IN THE PRESENCE OF EGG PHOSPHATIDYLCHOLINE LIPOSOMES

Abstract— The quantum yield of singlet oxygen generation (φ) was measured at 546 nm with the p -nitrosodimethylaniline (RNO) method. The results obtained in pH 7.4 phosphate buffer (PB) were: φ(HP) = 0.44 ± 0.05: φ= 0.71 ± 0.07: φ(HpD-A) = 0.06 ± 0.02. The value of φ was constant from 3 to 67 μM , attributed to the dominance of HP dimers; φ (HP) increased to 0.77 ± 0.13 in the presence of small egg phosphatidylcholine liposomes (SUV), attributed to solubilization and monomerization: φ (HpD-A) increased to 0.87 ± 0.17 in the presence of SUV. attributed to monomerization of the impurity porphyrins and unfolding of the covalent dimer constituents. The quantum efficiency of the RNO system (100 μ M RNO plus 10 mM histidine) was approximately 0.015 at pH 7.4 and increased significantly at lower pH.     

INTRACELLULAR TARGET FOR α-TERTHIENYL PHOTOSENSITIZATION: INVOLVEMENT OF LYSOSOMAL MEMBRANE DAMAGE

Abstract— Intracellular targets for the photosensitizer α-terthienyl (αT) were examined by fluorescence microscopy and microfluorospectrometry using human nonkeratinized buccal cells. Intracellular distribution of αT was observed as fluorescent patches widely dispersed in the cytoplasm. The distribution of the fluorescent patches was compared with that of acid phosphatase activity visualized as an azo dye produced by the fast garnet 2-methyl-4-[(2-methyl-phenyl)azo]benzenediasonium sulfate reaction. Because both the distribution sites coincided, lysosomes were the likely sites of intracellular affinity of αT. However, because acid phosphatase is not a specific lysosomal marker, we tried to detect another lysosomal enzyme, β-galactosidase, to confirm if the fluorescent patches were lysosomes, using fluorescein-di-(β-D-galactopyranoside) (FDG) as a fluorogenic substrate. Without UV-A (320–400 nm) irradiation of the cells after uptake of αT and FDG, no significant fluorescence was observed. In contrast, with prior UV-A irradiation in the presence of αT and FDG, the bright yellow fluorescence of fluorescein, which is the digested product of FDG, was clearly detected in the cells by fluorescence microscopy. This observation implied that inflow of external FDG into the lysosomes is caused by lysosomal membrane damage on αT photosensitization. The present results indicated that lysosomes are the primary photosensitization site of αT.     

PRIMARY PHOTOCHEMICAL PROCESSES OF CATIONIC ACRIDINE ORANGE IN AQUEOUS SOLUTION STUDIED BY FLASH PHOTOLYSIS

Abstract— A transformation system in Escherichia coli was employed to verify the extent of the lesions caused by ultraviolet (UV) and ionizing radiations. DNA inactivated at 280 nm could be reactivated to some extent by exposing the transforming DNA solutions at 240 nm. This reactivation has been tested using more than one strain of E. coli as recipient. Transforming DNA inactivated by ionizing radiations (⁹⁰Sr beta rays and ⁶⁰Co gamma rays) was not reactivable. Low doses of beta rays, however, reactivated the DNA inactivated by 280 nm UV to a slight but significant extent.     

THE EFFECT OF PROTOPORPHYRIN ON HISTAMINE SECRETION BY RAT PERITONEAL MAST CELLS: A DUAL PHOTOTOXIC REACTION

Abstract— Protoporphyrin-induced phototoxicity in rat peritoneal mast cells was manifested either by inhibition of 48/80-stimulated histamine secretion or by cell lysis. At a protoporphyrin concentration of 100ng/m/ (0.17 μM), histamine secretion was completely inhibited after 30min illumination. After initiation, the inhibited state progressed in the dark, and was irreversible, however, it did not develop into cell lysis. More severe phototoxic reactions in mast cells could not be produced by increasing the PP concentration or the incubation time; however, cell lysis was evoked by increasing the light intensity between 180–950W/m², using a light source with emission maxima in the 350–470nm region. Dual phototoxic effects could also be demonstrated in erythrocytes by manipulating the illumination conditions. Increased resistance to osmotic lysis was seen under moderate conditions, and decreased resistance and cell lysis were seen under severe conditions. In the absence of protoporphyrin, the effect of light alone on mast cells was similar to protoporphyrin-phototoxicity, although the light intensities required were higher both for inhibition (60–130W/m²) and lysis (280–950W/m²). The data therefore indicate that certain cell functions can be specifically disrupted by phototoxic reactions that are not cytotoxic; however, phototoxic reactions that lead to severe membrane protein denaturation and cell lysis also occur. The manifestation of these dual effects depends on the intensity of illumination in the 350–470nm region.     

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.     

INACTIVATION BY MONOCHROMATIC NEAR-UV RADIATION OF AN Escherichia coli hemA8 MUTANT GROWN WITH AND WITHOUT δ-AMINOLEVULINIC ACID: THE ROLE OF DNA vs MEMBRANE DAMAGE

Abstract— Escherichia coli strain RT8 hemA8 [blocked in biosynthesis of δ-aminolevulinic acid (δ-ALA), and unable to manufacture porphyrins unless exogenously supplied with δ-ALA] is inactivated more efficiently by monochromatic 334- and 405-nm radiations if the cells are grown with δ-ALA supplementation. The fiuence enhancement factor for δ-ALA sensitization is larger for light at 405 nm than at 334 nm. Both irradiation conditions (plus or minus δ-ALA) showed prominent oxygen enhancement ratios, which were also larger at 405 nm than at 334 nm. At 334 nm, δ-ALA supplementation did not affect the accumulation of DNA breaks, while at 405 nm, the induction of DNA breaks doubled for cells supplemented with δ-ALA. Rubidium leakage caused by 405-nm radiation occurred at a smaller fiuence in cells supplemented with higher concentrations of δ-ALA than in cells supplemented with a lower concentration. The results suggest that (1) porphyrin derivatives may have a role in cell killing by near-UV radiations, and (2) damage to cytomembranes may be a critical lesion produced by these events, whereas DNA breakage may not.     

NEW PHOTO-CONVERTIBLE REACTIONS OF BLUE-FLUORESCENT CALF α-CRYSTALLIN

Abstract— Both native blue fluorescent α-crystallin from calf lenses and UV (300 nm)-irradiated blue-fluorescent α-crystallin, when further irradiated with 365 nm-UV light, produce photo-products capable of emitting a new fluorescence at 455 nm. Illumination of the photo-products with 420 nm visible light regenerates the original fluorescence at 420–425 nm. In addition, another fluorescence at 400 nm has also been found in UV (300 nm)-irradiated blue-fluorescent α-crystallin, when exposed to 365 nm-UV light.     

SUNLIGHT-INDUCED PYRIMIDINE DIMERS IN HUMAN SKIN FIBROBLASTS IN COMPARISON WITH DIMERIZATION AFTER ARTIFICIAL UV-IRRADIATION

Abstract— We compared artificial UV-sources such as germicidal- or sun-lamps with summer noon sunlight in Switzerland for selective efficiency in the induction of pyrimidine dimers in the DNA of human cells. In our studies we determined cytosine-thymine (C-T) as well as thymine-thymine dimer densities (T-T) by high pressure liquid chromatography in cultures of xeroderma pigmentosum cells of group A. Using far-UV light from a germicidal lamp, we found a rate of formation per Jirr² for C-T and T-T of 0.0019% and 0.0024%, respectively, of the total thymine radioactivity in hydrolysates of [³H]thymidine labeled cells. After irradiation with an unfiltered sunlamp we measured a rate of formation of 0.0005% per Jm-² both for C-T and T-T, based on the sunlamp emission of 297 ±4 nm wavelength. Utilization of Kodacel- or Mylar-filters lowered the rate of dimerization by a factor of 2 and 60, respectively. One hour of irradiation with noon summer sunlight induced 0.038 ±0.012% C-T and 0.036 ±0.011% T-T. This extent of dimer production is equivalent to 15 Jm^-2 of far-UV exposure at 254 nm.     

AN ULTRASTRUCTURAL COMPARATIVE EVALUATION OF TUMORS PHOTOSENSITIZED BY PORPHYRINS ADMINISTERED IN AQUEOUS SOLUTION, BOUND TO LIPOSOMES OR TO LIPOPROTEINS

Abstract— Balb/c mice bearing a transplantedMS–2 fibrosarcoma have been injected with 2 mg kg^-1 hematoporphyrin either dissolved in phosphate-buffered saline (Hp-aq), or incorporated into unilamellar liposomes of dipalmitoyl-phosphatidylcholine (Hp-lip), or precomplexed with low density lipoproteins (Hp-LDL). At 24 h after injection, the mice received 150 J cm^-2 of600–680 nm light irradiation. Electron microscopic studies performed on tumor tissues taken from mice sacrificed at different times after the phototreatment showed that, in the presence of Hp-aq. tumor necrosis is largely the consequence of vascular damage. On the other hand, in the presence of Hp-lip and Hp-LDL, the response of the tumor to the phototreatment occurs at a faster rate and is mainly determined by direct damage of neoplastic cells. These findings are correlated with the different distribution of the various hematoporphyrin forms (Hp-aq, Hp-lip, Hp-LDL) among the serum proteins and the modalities of hematoporphyrin delivery to tissues by the possible carriers.     

The photosensitizing potential of compact fluorescent vs incandescent light bulbs

Recently an article about the new energy‐saving compact fluorescent light (CFL) bulbs appeared in Parade magazine [Rosenfeld, I. (2008) Parade Feb 3 , 22]. Under the heading “Bright Lights, Bad Headache?” the writer states that “new research suggests some dangers” involving these lights because they are fluorescent and “can aggravate skin rashes in people with lups, eczema, dermatitis or porphyria.” We measured the emission spectrum of a 14 W compact fluorescent bulb (with the same luminous flux as a 60 W incandescent bulb) and compared it to 60 W soft white incandescent and cool white fluorescent (CWF) bulbs. Our results clearly show that the spectral irradiance of the compact fluorescent bulb is similar to that of the CWF bulb; both exhibit sharp Hg emission lines at 365 nm (very weak), 404 nm (weak), 435 nm (moderate) and 543 nm (strong). In contrast, the emission of the incandescent bulb begins at 375 nm and then increases monotonically to above 750 nm. From their respective absorption spectra we calculated the potential photosensitization indices of protoporphyrin IX (PPIX; a prototypic porphyria skin photosensitizer) and riboflavin (a putative lens photosensitizer) vs 14 W compact fluorescent, CWF and 60 W incandescent bulbs. A higher photosensitization index would indicate a greater chance that the light/photosensitizer combination would cause photosensitization of the skin or eyes. We found that for PPIX and riboflavin the photosensitization index of the compact fluorescent bulb is less than half that of the 60 W incandescent bulb. These results suggest that substitution of a compact fluorescent bulb for an incandescent bulb of the same luminous flux should not increase the phototoxicity of skin porphyrins or lens riboflavin.