CYTOTOXIC AND MUTAGENIC EFFECTS OF THE PHOTODYNAMIC ACTION OF CHLOROALUMINUM PHTHALOCYANINE AND VISIBLE LIGHT IN L5178Y CELLS

Abstract The cytotoxic and mutagenic effects of chloroaluminum phthalocyanine (CAPC) plus red light have been measured in strains of L5178Y mouse lymphoma cells which differ in their DNA repair capacities. Strain LY-R, deficient in the excision repair of UV-induced dimers, was found to be relatively more sensitive to the cytotoxic effects of CAPC plus light, whereas strain LY-S, deficienl in the repair of DNA double-strand breaks, was more sensitive than strain LY-R to the mutagenic effects of the treatment. Mutation frequencies were measured in LY-S and LY-R sub-strains which were heterozygous or hemizygous at the thymidine kinase (tk) locus. The mutation frequency at the tk locus induced in the heterozygous strain LY-SI by CAPC plus light was lower than that induced by an equitoxic dose of ionizing radiation but similar to that induced by an equitoxic dose of UVC radiation: The mutation frequency at the F., dose of CAPC plus light was approximately 1100 per 10⁶ surviving cells. The induced frequency in strain LY-S1 was much higher than in either tk+l-heterozygous or ik+10 hemizygous strains of LY-R. The rate and extent of incorporation of CAPC by the LY-R strains was somewhat greater than for strain LY-S1 at early times after CAPC addition, but by the time the cells were irradiated (18 h after CAPC addition) the difference was not great enough to account for the difference in cytotoxicity. It is possible that the cytotoxic and mutagenic lesions differ and that either the quantities of the respective lesions induced or the efficiencies of repair of the respective lesions differ inversely in the two strains. light have been measured in strains of L5178Y mouse lymphoma cells which differ in their DNA repair capacities. Strain LY-R, deficient in the excision repair of UV-induced dimers, was found to be relatively more sensitive to the cytotoxic effects of CAPC plus light, whereas strain LY-S, deficienl in the repair of DNA double-strand breaks, was more sensitive than strain LY-R to the mutagenic effects of the treatment. Mutation frequencies were measured in LY-S and LY-R sub-strains which were heterozygous or hemizygous at the thymidine kinase (tk) locus. The mutation frequency at the tk locus induced in the heterozygous strain LY-SI by CAPC plus light was lower than that induced by an equitoxic dose of ionizing radiation but similar to that induced by an equitoxic dose of UVC radiation: The mutation frequency at the F., dose of CAPC plus light was approximately 1100 per 10⁶ surviving cells. The induced frequency in strain LY-S1 was much higher than in either tk+l-heterozygous or ik+10 hemizygous strains of LY-R. The rate and extent of incorporation of CAPC by the LY-R strains was somewhat greater than for strain LY-S1 at early times after CAPC addition, but by the time the cells were irradiated (18 h after CAPC addition) the difference was not great enough to account for the difference in cytotoxicity. It is possible that the cytotoxic and mutagenic lesions differ and that either the quantities of the respective lesions induced or the efficiencies of repair of the respective lesions differ inversely in the two strains.     

THE CYTOTOXIC AND MUTAGENIC EFFECTS OF UVA RADIATION ON L5178Y MOUSE LYMPHOMA CELLS

Abstract— A broad-band UVA source that emits primarily350–400 nm radiation and no measurable radiation below 340 nm was used to test toxicity and mutagenicity at the thymidine kinase locus in L5178Y, subclone 3.7.2C (TK⁺/^-) mouse lymphoma cells. Cells were exposed to a fluence of 0 to 80 × 10⁴ J/m². The relationship between UVA fluence and survival was found to have a shoulder region followed by an exponential decrease in survival at higher fluence levels. An exposure-dependent increase in mutation was observed with increasing fluences from 0 to about 60 × 10⁴ J/m². An approximately 3- to 4-fold increase in mutations (trifluorothymidine resistance) over unexposed, control cells was seen at a fluence that resulted in 90% cell killing. We conclude that UVA radiation is a mutagen in the L5178Y mouse lymphoma cells used in this study.     

INDUCTION OF DNA-PROTEIN CROSS-LINKS IN CHINESE HAMSTER CELLS BY THE PHOTODYNAMIC ACTION OF CHLOROALUMINUM PHTHALOCYANINE AND VISIBLE LIGHT

Abstract— Chloroaluminum phthalocyanine (CAPC) is an efficient photosensitizer for the inactivation of Chinese hamster V79 cells. In order to investigate possible molecular mechanisms in the photo-dynamic action of CAPC and visible light, the induction and repair rate of two classes of DNA lesions have been determined, i.e. DNA single-strand breaks and DNA-protein cross-links. In cells pretreated with 1 μ.M CAPC, a fluence of 12 kJ/m² of red light (>600 nm) kills approximately 50% of the cells and induces 3 to 3.5 Gy-equivalents of single-strand breaks. The repair of these breaks was slower than the repair of single-strand breaks induced by -irradiation. The photodynamic action of CAPC also induces a large number of DNA-protein cross-links which, in contrast to -radiation-induced DNA-protein cross-links, do not appear to be repaired during 4 h of post-treatment incubation in fresh medium. These studies suggest that DNA may be an important target for the cytotoxicity of CAPC + red light.     

LARGE MUTAGENIC LESIONS ARE INDUCED BY PHOTODYNAMIC THERAPY IN MURINE L5178Y LYMPHOBLASTS*

Abstract— Mutagenic lesions at the thymidine kinase locus (tk) in mouse lymphoma L5178Y (LY) cells treated with red light and either Photofrin (PF) or chloroaluminurn phthalocyanine (AIPc) as the photosensitizer were compared in the relatively photodynamic therapy (PDT)-sensitive strain LY-R16 and the relatively resistant strains LY-S1 and LY-SR1. Southern blot analysis revealed that 92% (36/39) of the PDT-induced thymidine kinase (TK ^?/-) mutants of strains LY-R16 and LY-SR1 lost the entire active tk allele. (Strain LY-S1 lacks a known tk polymorphism and has not been analyzed for loss of the active tk allele.) A decrease in galactokinase (GK) activity in the TK^?/- mutants has been taken as an indication that the mutagenic lesion extends from the tk gene to the closely linked galactokinase gene (gk). Using PF as the photosensitizer, GK activity was decreased in 45% of the LY-R16 mutants and in 22% of the LY-S1 and LY-SR1 mutants. With photoactivated AIPc, 59% of the TK ^?/- mutants of strains LY-S1 and LY-SR1 showed GK inactivation. (LY-R16 mutants were not analyzed because of the low LY-R16 mutant frequency induced by PDT with AlPc.) Thus, many of the TK^?/- mutants of LY cells induced by PDT with either PF or AlPc harbor multilocus lesions.     

THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET LIGHT—INDUCED CELL KILLING AND MUTAGENESIS IN L5178Y MOUSE LYMPHOMA CELLS

Abstract The wavelength dependence of ultraviolet radiation-induced cell killing and mutagenicity in L5178Y mouse lymphoma cells has been determined from 235 nm to 313 nm. Cells were irradiated in phosphate buffered saline at 20°C. The amount of cell killing was determined by cloning in soft agar medium immediately after irradiation. Mutation frequency was determined, after a 3-day expression time, by cloning in soft agar medium in the presence and the absence of 5-bromo-2'-deoxyuridine (BrdUrd). The endpoint used to quantitate lethal effects was the exposure necessary to reduce the surviving fraction to 10%, while the endpoint for mutagenesis was the exposure necessary to increase the frequency of BrdUrd-resistant colonies ten-fold over the background level. Data were corrected for quantum energy and the action spectra for cell killing and mutagenesis were plotted as relative biological effectiveness per quantum vs wavelength, relative to the effect at 265.2 nm. Both action spectra show broad maxima at 270 nm, and are very similar to the action spectra determined by Rothman and Setlow (1979) for pyrimidine dimer formation and cell killing in V-79 cells.     

PHOTODYNAMIC THERAPY OF CHEMICALLY- AND ULTRAVIOLET B RADIATION-INDUCED MURINE SKIN PAPILLOMAS BY CHLOROALUMINUM PHTHALOCYANINE TETRASULFONATE

Photodynamic therapy (PDT) of cancer combines irradiation of tumors with visible light following selective uptake of the photosensitizer by the tumor cells. PhotofrinR-II (Pf-II) is the only photosensitizer which is in clinical use in PDT, whereas chloroaluminum phthalocyanine tetrasulfonate (AlPcTS) has also shown promise in preclinical studies. In most such studies, the effectiveness of the photosensitizers has been assessed in implanted tumor model systems rather than in model systems where tumors are allowed to grow in their own connective tissue matrix. In this study the pharmacokinetics, tumor ablation capability and cutaneous photosensitization response of AlPcTS have been assessed in mice bearing chemically- and ultraviolet B radiation (UVB)-induced benign skin papillomas. When tumor-bearing animals were injected intraperitoneally with AlPcTS (5 mg/kg body wt), maximum tumor:normal skin ratio of 2.4 was observed at 48 h, at which time the mice were irradiated within the absorption spectrum of the photosensitizer. In tumor ablation studies with SENCAR mice bearing chemically-induced skin tumors, AlPcTS resulted in greater than 80% ablation in tumor volume at 20 days post-irradiation. In cutaneous photosensitization response, AlPcTS produced only transient effects (no effect after 24 h) in SENCAR mice. Pharmacokinetics data, tumor ablation effects and cutaneous photosensitization response of AlPcTS were comparable in SKH-1 hairless mice bearing UVB-induced skin tumors. Our data indicate that AlPcTS produces significant photodynamic effects towards the ablation of murine skin tumors, and that it does not produce prolonged cutaneous photosensitivity.     

PHOTOFRIN II PHOTOSENSITIZATION IS MUTAGENIC AT THE tk LOCUS IN MOUSE L5178Y CELLS

Photosensitization mediated by Photofrin II (PFII) was found to be mutagenic at the heterozygous thymidine kinase (tk) locus in mouse L5178Y lymphoma strains LY-S1 and LY-R16 but not in strain LY-R83 which is hemizygous at the tk locus. After treatments yielding 37% survival, the mutagenicity of photosensitization with PFII in strain LY-S1 was similar to that of other mutagenic agents including x-radiation, ethyl methanesulfonate, and photosensitization with chloroaluminum phthalocyanine (AlPcCl). Although both strain LY-S1 and strain LY-R16 were mutagenized by photosensitization with PFII, only strain LY-S1 was mutagenized by photosensitization with AlPcCl. The non-mutability of strain LY-R83 following photodynamic treatment with either sensitizer may be because of the poor recovery of mutants with intergenic mutations in this TK+/0 hemizygous strain, whereas the non-mutability of strain LY-R16 subjected to photodynamic treatment with AlPcCl may be because LY-R16 cells sustaining mutagenic damage do not survive for reasons other than the loss of an essential gene.     

THE EFFECT OF IRRADIANCE ON VIRUS STERILIZATION AND PHOTODYNAMIC DAMAGE IN RED BLOOD CELLS SENSITIZED BY PHTHALOCYANINES

Abstract— Phthalocyanines are being studied as photosensitizers for virus sterilization of red blood cells (RBC). During optimization of the reaction conditions, we observed a marked effect of the irradiance on production of RBC damage. Using a broad-band light source (600–700 nm) between 5 and 80 mW/ cm², there was an inverse relationship between irradiance and rate of photohemolysis. This effect was observed with aluminum sulfonated phthalocyanine (AlPcS_n) and cationic silicon (HOSiPc-OSi[CH₃]₂ [CH₂]₃N⁺[CH₃]₃I^- phthalocyanine (Pc5) photosensitizers. The same effect occurred when the reduction of RBC negative surface charges was used as an endpoint. Under the same treatment conditions, vesicular stomatitis virus inactivation rate was unaffected by changes in the irradiance. Reduction in oxygen availability for the photochemical reaction at high irradiance could explain the effect. However, theoretical estimates suggest that oxygen depletion is minimal under our conditions. In addition, because the rate of photohemolysis at 80 mW/cm² was not increased when irradiations were carried out under an oxygen atmosphere this seems unlikely. Likewise, formation of singlet oxygen dimoles at high irradiances does not appear to be involved because the effect was unchanged when light exposure was in D₂O. While there is no ready explanation for this irradiance effect, it could be used to increase the safety margin of RBC virucidal treatment by employing exposure at high irradiance, thus minimizing the damage to RBC.     

CYTOTOXICITY AND MUTAGENICITY OF OPHTHALMIC SOLUTION PRESERVATIVES AND UVA RADIATION IN L5178Y CELLS

Four chemical preservatives commonly used in ophthalmic solutions were tested for their toxic and mutagenic potential in mouse lymphoma cells with and without exposure of the cells to ultraviolet A (UVA) radiation. The preservatives tested were benzalkonium chloride (BAK), chlorhexidine, thimerosal and ethylenediaminetetraacetic acid (EDTA). Cell survival and mutagenesis were measured using the L5178Y mouse lymphoma (TK +/-) system. Cells were exposed to varying amounts of preservatives for 1 h at 37 degrees C, and then aliquots were irradiated with UVA radiation (during the exposure to preservative). Cells were then assayed for survival, and for mutagenesis at the thymidine kinase (TK) locus. In concentrations commonly found in ophthalmic solutions, BAK, chlorhexidine, and thimerosal were toxic to cells, and thimerosal was slightly mutagenic. When cells were exposed to preservative and UVA radiation, chlorhexidine was mutagenic and the mutagenic activity of thimerosal was enhanced.     

THE ROLE OF SINGLET OXYGEN IN THE PHOTOHEMOLYSIS OF RED BLOOD CELLS SENSITIZED BY PHTHALOCYANINE SULFONATES

Abstract The post-illumination photohemolysis of human red blood cells was used as a model system for studying membrane damage sensitized by various phthalocyanine sulfonates. With a constant fluence and photolysis time at 670 ± 10 nm and with a fixed optical density of the sensitizer, the percentage of hemolysis was measured as a function of time after illumination; and from the usual sigmoidal shaped curves, the times for 50% hemolysis were determined. The most effective central cation was Al, followed by Zn; the metal-free compound and those containing the paramagnetic ions Cu, Fe and Co were inactive. The sensitizer bound to the red blood cells was found to be effective for photohemolysis. Studies of the Al compound in 98.0% deuterated media (as measured by NMR) showed a definite deuterium isotope effect, suggesting the participation of singlet oxygen. This result was supported by the large protective effects of 3.3 raM and 13.3 mM tryptophan present during photolysis. Tryptophan reacts rapidly with singlet oxygen and with OH radicals. The much smaller effects observed with the same concentrations of mannitol and glycerol, OH radical scavengers which do not react with singlet oxygen, are consistent with the predominant role of singlet oxygen in photohemolysis sensitized by Al phthalocyanine sulfonate.     

INTRACELLULAR DAMAGE IN YEAST CELLS CAUSED BY PHOTODYNAMIC TREATMENT WITH TOLUIDINE BLUE

Abstract— The positively charged photosensitizer toluidine blue (TB) can induce loss of clonogenicity in Kluyveromyces marxianus. Previous studies have revealed that, as a consequence of the localization of this dye at the cell surface, photodynamic action results in extensive damage at the level of the plasma membrane. In this paper, a study is reported on the effect of photodynamic treatment with TB on intracellular enzymes. It is shown that treatment with TB and light resulted in the inhibition of alcohol dehydrogenase, cytochrome c oxidase, glyceraldehyde-3-phosphate dehydrogenase and hexokinase. Photodynamic treatment also lowered the ATP levels. The ATP levels could be partially restored in the presence of glucose but not with ethanol. Toluidine blue binding experiments revealed that photodynamic treatment caused a rapid increase in the amount of cell-associated dye. Moreover, it also appeared that this treatment decreased the binding of TB to the cell surface. It is concluded that TB enters the cell during the first minutes of illumination, whereafter intracellular enzymes are inactivated. The data indicate that photodynamic damage of intracellular sites contributes to the loss of viability.     

NON-NUCLEAR DAMAGE AND CELL LYSIS ARE INDUCED BY UVA, BUT NOT UVB OR UVC, RADIATION IN THREE STRAINS OF L5178Y CELLS

The potential to induce non-nuclear changes in mammalian cells has been examined for (1) UVA1 radiation (340–400 nm, UVASUN 2000 lamp), (2) UVA + UVB (peak at 313 nm) radiation (FS20 lamp), and (3) UVC (254 nm) radiation (GI5T8 lamp). The effects of irradiation were monitored in vitro using three strains of L5178Y (LY) mouse lymphoma cells that markedly differ in sensitivity to UV radiation. Comparisons were made for the effects of approximately equitoxic fluences that reduced cell survival to 1–15%. Depending on the cell strain, the fluences ranged from 830 to 1600 kJ/m² for the UVASUN lamp, 75 to 390 J/m² for the FS20 lamp and 3.8 to 17.2 J/m² for the G15T8 lamp. At the exposure level used in this study, irradiation with the UVASUN, but not the FS20 or G15T8, lamp induced a variety of non-nuclear changes including damage to cytoplasmic organelles and increased plasma membrane permeability and cell lysis. Cell lysis and membrane permeabilization were induced by the UVA1 emission of the UVASUN lamp, but not by its visible + IR components (>400 nm). The results show that the plasma membrane and other organelles of LY cells are highly sensitive to UVA1 but not to UVB or UVC radiation. Also UVA1, but not UVB or UVC radiation, causes rapid and extensive lysis of LY cells. In conclusion, non-nuclear damage contributes substantially to UVA cytotoxicity in all three strains of LY cells.     

INHIBITION OF DNA SYNTHESIS BY PSORALEN-INDUCED LESIONS IN XERODERMA PIGMENTOSUM AND FANCONI''S ANEMIA FIBROBLASTS

Abstract— Exposure of human cells to psoralens and near-UV light produces a mixture of monoadducts and crosslinks in DNA, which inhibit DNA synthesis by blocking replicon initiation and chain elongation. 8-Methoxypsoralen (8-MOP) has a greater effect than angelicin in normal, xeroderma pigmentosum, and Fanconi's anemia cells. Recovery of DNA synthesis is not detectable up to 8 h after exposure. The average distance between lesions that block replication in individual replicons was measured by means of bromodeoxyuridine photolysis. After exposure to 10 μg/mℓ of 8-MOP and 7500 J/m² of near-UV light, blocks were formed every 20 μm. Replicon initiation was inhibited by exposure to near-UV light alone in normal and xeroderma pigmentosum. Exposure to low concentrations of angelicin or 8-MOP plus near-UV light inhibited replicon initiation in normal and Fanconi's anemia cells, but not in xeroderma pigmentosum cells. Inhibition of initiation was not obvious after treatment with high concentrations of 8-MOP or angelicin because of the dominant effect of crosslinks in blocking chain elongation.     

DNA SINGLE-STRAND LESIONS DUE TO ''SUNLIGHT'' AND UV LIGHT: A COMPARISON OF THEIR INDUCTION IN CHINESE HAMSTER AND HUMAN CELLS, AND THEIR FATE IN CHINESE HAMSTER CELLS

Abstract— It has been shown that the lethal properties of germicidal UV light (254 nm) and sunlight-simulating near UV light are qualitatively different (Elkind et al ., 1978). Further to compare these two radiations, the induction of single-strand DNA breaks (i.e. frank breaks plus alkali-labile lesions) was measured in two cell lines. Equal numbers of breaks in Chinese hamster cells require a dose of UV 5.5% of a near UV dose but in HeLa cells a UV dose of 7.6% of a near-UV dose is required. The rate of break production by these radiations is about 1/10-th of that due to X-rays when a comparison is made on an equal killing dose basis. The inventory of breaks in Chinese hamster cells was also followed and was found to be characteristically different for UV compared to near UV light. These data indicate that significant differences exist, at a molecular level, in the effects produced by ultraviolet and sunlight-simulating light, and further emphasize the need for caution in attempting to extrapolate from observed molecular or biological effects due to the former to those to be expected from the latter.     

DNA DAMAGE IN RAT 9L CELLS TREATED WITH 8-METHOXYPSORALEN AND NEAR-UV LIGHT ASSAYED BY VISCOELASTOMETRY AND S1 NUCLEASE

–The techniques of viscoelastometry and S1 nuclease digestion were applied to the analysis of DNA damage in rat 9L cells treated with the combination of 8-MOP (8-methoxysporalen) and near-UV light. Treatment of cells with near-UV light alone resulted in a decrease in the viscoelastic retardation time under both denaturing and nondenaturing conditons. Exposure of cells to 8-MOP alone yielded a maximum in the plot of retardation time vs dose under nondenaturing conditions, similar to that found with ionizing radition. This observation suggests that treatment with 8-MOP alone leads to DNA strand breaks. Viscoelastic analysis of cell lysates under denaturing conditions demonstrated that treatment of cells with 8-MOP and UV radiation led to substantial increases in both the viscoelastic retardation time and recoil, consistent with formation of DNA interstrand cross-links. Viscoelastic analysis of cell lysates under nondenaturing conditions showed that exposure to long wavelength UV light in the presence of 8-MOP produced a decrease in retardation time. This decrease reflects the combined effect of strand breaks and interstrand cross-links. Results from the S1 nuclease assay confirmed these observations and permitted quantitation of DNA damage arising from single-strand breaks and DNA interstrand crosslinks. The importance of including the effect of strand breaks in the quantitation of cross-link formation is discussed.     

EVALUATION OF DNA CROSSLINKS AND MONOADDUCTS IN MOUSE EMBRYO FIBROBLASTS AFTER TREATMENT WITH MONO-AND BIFUNCTIONAL FUROCOUMARINS AND 365 nm (UVA) IRRADIATION. POSSIBLE RELATIONSHIP TO CARCINOGENICITY

Abstract— Formation of crosslinks in DNA by various mono- and bifunctional furocoumarins plus UVA light in mouse embryo fibroblasts was evaluated by a NaI density gradient centrifugation method. Angelicin and 3-carbethoxypsoralen did not form any crosslinks: however, angelicin was slightly carcinogenic for mouse skin, whereas 3-carbethoxypsoralen was not carcinogenic. Psoralen induced DNA crosslinking, in a dose-dependent fashion and was highly carcinogenic for mouse skin. In contrast to the psoralen-induced photoadducts (120 per 10⁶ nucleotides) which were left unrepaired, 41% of the 3-carbethoxypsoralen adducts (30 per 10⁶ nucleotides) were removed during 1 h of dark post-incubation.     

UV-INDUCED ALKALINE LABILE DNA DAMAGE IN HUMAN ADENOVIRUS AND ITS REPAIR AFTER INFECTION OF HUMAN CELLS

Abstract— UV-induced alkaline labile viral DNA damage was detected following irradiation of adenovirus type 2 and found to be repaired following the infection of human KB cells. Human adenovirus type 2 was irradiated with various doses of UV and subsequently used to infect human KB cells in tissue culture at approximately 2 × 10³ particles per cell. Before, and at various times after infection, the viral DNA was examined on alkaline sucrose gradients. Irradiated free virus DNA showed a dose dependent decrease in molecular weight compared to unirradiated virus DNA, indicating the presence of UV-induced alkaline labile lesions. Furthermore, an increase in the molecular weight of the irradiated virus DNA was found after infection indicating that alkaline labile lesions were removed from the viral DNA by a host mediated repair mechanism. After infection, the molecular weight of the irradiated virus DNA reached a value similar to that of unirradiated virus DNA for all the UV doses studied.     

IMPAIRED REPAIR OF UVC-INDUCED DNA DAMAGE IN L5178Y-R CELLS: SEDIMENTATION STUDIES WITH THE USE OF 5''-BROMODEOXYURIDINE PHOTOLYSIS

Abstract Relative to their L5178Y-S counterparts, L5178Y-R cells have an impaired capacity to form patches in DNA after exposure to UVC radiation. The photolysis of 5'-bromodeoxyuridine (BrdUrd) incorporated into DNA was used to estimate the number of 'repair patches'formed in response to a 254 nm UV (UVC) exposure. L5178Y-S cells, typical of rodent cell lines, formed a small number of patches in exposed DNA (1-2 patches per 1 times 10⁸ dalton during a 6 h recovery after an exposure of 20 J/m²). In contrast, DNA extracted from L5178Y-R cells exposed to UVC and subsequently incubated with BrdUrd for 6 h showed no evidence of BrdUrd incorporation indicating no capacity to form sites of repair (fewer than 0.5 sites of BrdUrd incorporation per 1 times 10⁸ dalton). Moreover, in L5178Y-R cells high fluences of UVC caused an extensive DNA degradation. Such degradation was not observed in L5178Y-S cells during the 24-h post-exposure period. These results are consistent with the notion that L5178Y-R cells have a reduced capacity to repair DNA damage induced by UVC radiation.