EFFECTS OF 8-METHOXYPSORALEN AND NEAR ULTRAVIOLET RADIATION ON THE SURVIVAL OF THE LOWER EUKARYOTE D. Discoideum

Abstract— Seven axenic wild-type and repair-deficient mutant strains of the cellular slime mold Dictyostelium discoideum have been treated with the furocoumarin 8-methoxypsoralen (8-MOP) up to 50 μg/mζ and then exposed to near ultraviolet light (UVA 320-400 nm) up to 21 kJ/m². Fluence-response survival curves exhibit shoulders at lower fluences and an exponential lethal response at higher fluences. Neither the psoralen alone nor the irradiation alone produced any measurable lethal effect. Wild-type strains, which show resistance to 254 nm UV and gamma radiation, also show resistance to psoralen plus UVA. The moderate sensitivity of a rad D repair-deficient mutant strain and the extreme sensitivity of a rad B mutant strain to 8-MOP plus UVA parallel their responses to UV and gamma radiation. However a rad C mutant which is sensitive to UV, exhibits wild-type response to photoactivated psoralen.     

INACTIVATION OF WILD-TYPE AND rad MUTANT Caenorhabditis elegans BY 8-METHOXYPSORALEN AND NEAR ULTRAVIOLET RADIATION

Survival of wild-type and four radiation-sensitive (rad) mutants of the nematode Caenorhabditis elegans was determined after near-UV irradiation in the presence of 8-methoxypsoralen (8-MOP). Three sets of inactivation profiles were generated for each strain by irradiating synchronous populations of either early embryos, late embryos or first-stage larvae (L1s). Late embryos were consistently the most sensitive. Curiously, none of the four rad mutants were even moderately hypersensitive. Split-dose experiments indicated that DNA-DNA crosslinks were primarily responsible for lethality. Crosslink induction and repair were determined using two different assays. In both cases, little if any repair was observed in wild-type. This lack of repair thus explains why the rad mutants were not hypersensitive to 8-MOP photoinactivation. Since early embryos undergo extensive cell cycling, their resistance to 8-MOP photoinactivation suggests that replication is highly refractory to both monoadducts and crosslinks, as has been demonstrated previously for UV radiation-induced photoproducts (Hartman et al., 1991, Mutat. Res., 255, pp. 163-173).     

THE EFFECTS OF ULTRAVIOLET AND VISIBLE RADIATION ON MOUSE ASCITES TUMOUR CELLS

Abstract— Effects of ultraviolet and visible radiation on the viability of Landschutz ascites tumour cells have been tested by growing control and treated tumour samples in adult mice. The tumour cells were irradiated as a dilute suspension in isotonic buffered salt solution, and were equilibrated at 0°C with oxygen or with nitrogen before irradiation.
Tumour cell proliferation was measured by a variety of techniques. The preferred assay-method was the growth of solid tumours in the axillae and groins of mice after sub-cutaneous inoculation of varying dilutions of treated or control ascites tumour cells. The immune response of the mice to the injected cells was reduced by whole body irradiation with a 300r dose of x-rays two days before inoculation. Results were calculated from parallel line assays using the reciprocal of the delay in appearance of the solid tumours up to 30 days post-innoculation. This reciprocal (1/T) was linearly related to the logarithm of the number of cells inoculated.
Photoreactivation has been demonstrated for this system, in which both U.V. and visible radiations were absorbed by the same cells. Light delivered alone in oxygen or in nitrogen was without effect on cell-viability, but it increased cell-survival after u.v.-irradiation in nitrogen and decreased survival after u.v.-irradiation in oxygen. Ultraviolet radiation alone was not significantly more lethal in oxygen than in nitrogen. A further observation in this work was an interaction between irradiated and control tumour cells injected into the same animal.
It is suggested that the radiation used may affect the antigenic character of the tumour cells as well as their reproductive capadity.     

THE PHOTOREACTIONS OF 8-METHOXYPSORALEN WITH TRYPTOPHAN AND LENS PROTEINS*

Abstract— We have previously demonstrated that 8-methoxypsoralen (8-MOP) can be found in the lenses of rats injected (i.p.) with this drug, and that its presence can lead to a photosensitized enhancement of lenticular fluorescence. The cutaneous photosensitizing properties of psoralens are thought to be mediated via their excited triplet states, resulting in photoaddition cyclobutane products between pyri-midine bases and 8-MOP. We have now investigated the possibility that similar types of photoadducts could be generated between 8-MOP and the aromatic amino acid residues in lens proteins. Our experiments involved in vitro irradiation (at 360 nm) of aqueous solutions of 0.1 mM 8-MOP plus purified alpha, beta, or gamma crystallins from calf or normal human (under 20 years of age) lenses. UV absorption and fluorescence emission spectra were measured before and after radiation, and aliquots from all experiments were frozen and kept in the dark for subsequent phosphorescence and EPR spectroscopy. Similar experiments were performed with irradiated aqueous solutions of tryptophan or thymine plus 8-MOP. All controls consisted of solutions kept in the dark. NMR spectra demonstrated that the hydrogen atoms at the 3,4 and 4',5' positions of the 8-MOP molecule were lost following irradiation, suggesting that these two sites were involved in the photoproduct formed between tryptophan and 8-MOP. These studies strongly suggest that 8-MOP is capable of forming photoaddition products with tryptophan and with lens proteins as well as DNA in vivo, resulting in its permanent retention within the ocular lens.     

THE WAVELENGTH DEPENDENCE OF THE EFFECT OF 8-METHOXYPSORALEN PLUS ULTRAVIOLET RADIATION ON THE INDUCTION OF LATENT SIMIAN VIRUS 40 FROM A MAMMALIAN CELL

Abstract— The effect of 8-methoxypsoralen (8-MOP) plus ultraviolet radiation (UV) of different wavelengths in the region 238–365 nm on the induction of SV40 from SV40-transformed Syrian hamster kidney cells was investigated. Results indicate that 8-MOP + UV treatment activates as much as 1000-fold more virus than UV alone at wavelengths in the region 302–365 nm. At wavelengths below 302 nm, 8-MOP addition to cells prior to irradiation shows little, if any, effect. A wavelength dependence for this viral induction is presented.     

THE EFFECTS OF ULTRAVIOLET RADIATION EXPOSURE OF ADJACENT CELLS ON PLAQUE FORMATION WITH Herpes simplex VIRUS TYPE I

African green monkey kidney cells (CV-1P) were exposed to low fluences of 254 nm germicidal radiation and then infected with Herpes simplex virus, type I. The result of this treatment was an increase in viral plaque development rate, the large plaque effect (LPE). A measurement of the kinetics of plaque development suggested that a large portion of the effect could be due to events occurring in those cells that are adjacent to the initially infected cell. An infectious center assay was employed in order to isolate the effects of ultraviolet radiation (UV) on the initially infected cells from those effects on the adjacent cells that became infected as the plaque spread radially outward. Plaque development began earlier in UV irradiated cells and progressed at a uniformly accelerated rate compared to untreated cells. Results indicate that although the initially infected cell contributes to the LPE, the major effect is due to events that occur in the adjacent cells. Each round of viral replication appears to contribute equally to the LPE. The virally induced rate of fusion of the initially infected cell with its immediate neighbors is not affected by UV.     

SV40 INDUCTION FROM A MAMMALIAN CELL LINE BY ULTRAVIOLET RADIATION and THE PHOTOSENSITIZERS 8-METHOXYPSORALEN and ANGELICIN

Abstract SV40-transformed weanling Syrian hamster kidney cells were exposed to long wavelength ultraviolet radiation in the presence of 8-methoxypsoralen or angelicin. The number of viruses induced as a result of this treatment was quantitated on CV-1P cells. The results indicate that angelicin, which is generally believed to form only monoadducts, is a potent viral inducing agent even when compared with 8-methoxypsoralen (8-MOP), which forms both monoadducts and crosslinks. At low radiation exposures 8-MOP is more effective than angelicin; as the radiation exposure is increased angelicin is capable of inducing the same peak level of virus expression as was 8-MOP. It appears that the induction of SV40 is a phenomenon possibly related to the repair of monoadducts.     

THE EFFECT OF CHANGES IN CELL GEOMETRY ON THE SENSITIVITY TO ULTRAVIOLET RADIATION OF MAMMALIAN CELLULAR CAPACITY

Abstract— We have measured a calcium and magnesium dependent change in cell shape when mammalian cell monolayers are being prepared for irradiation by replacing their growth medium with certain buffers. In some cases, flattened cells (umbonate) assumed a spherical configuration. In order to assume a centrally located target molecule, we used a DNA-dependent cellular function–pacity for herpes viral growth–as the parameter to measure ultraviolet (UV) sensitivity of cells irradiated while in either of the two shapes. Umbonate cells were more sensitive to UV than were spherical cells. Exposures to the cell that lowered the cellular capacity of umbonate cells to the 10% survival level only lowered spherical cells to the 50% level. Twenty-seven per cent additional UV exposure to spherical cells was required to get the same effect as with umbonate cells. Included in the text are photographs of both cell types, survival curves for cellular capacity, a measure of the absorbance of cell homogenates, and a calculation of the relative number of photons absorbed by each cell nucleus.     

INTERACTION OF 8-METHOXYPSORALEN AND NEAR-UV LIGHT CAUSES MUTATION AND CYTOTOXICITY IN MAMMALIAN CELLS

Abstract The interaction of near-UV light and a photosensitizer, 8-methoxypsoralen (8-MOP), was studied in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase system; cell survival (cloning efficiency) and mutation induction (resistance to 6-thioguanine) were quantified. Exposure of cells to either 8-MOP up to 20 μg/m l (93 μ M ) or near–UV light up to 40000 J/m² had no effect on either survival or mutation frequency. Preincubation of cells with 8–MOP from 5 to 120 min prior to irradiation with various fluences did not affect cell survival or mutation frequency. Survival decreased and mutation frequency increased linearly when either the 8-MOP concentration or fluence was increased while the other factor was held as a constant. Mutation frequency appears to show reciprocity relative to the product of 8-MOP concentration times fluence of near–UV light [(μg/m l )·(J/m²)] throughout a range apparently limited by high cell lethality. The observed pooled data on mutation, f (x), as a function of (μg/m l )·(J/m²), x , fit a linear dose–response line, f (x) = (34.2 + 0.05 x ) × 10^-6. Cell survival, however, does not appear to exhibit such reciprocity.     

THE EFFECT OF DARK COMPLEXING ON THE PHOTOSENSITIZED FORMATION OF 8-METHOXYPSORALEN CROSS-LINKS WITH DNA*

The near-UV dose at 365 nm required for the formation of 8-methoxypsoralen (8-MOP) cross-links with calf thymus DNA was found to be insensitive to the concentration ratio of nucleotides to 8-MOP over the range 100:1 to 5:1. The proposed explanation is that only the dark-complexed 8-MOP contributes to the photosensitized formation of monoadducts and cross-links. A kinetics analysis indicates that the fluence required for cross-linking should be inversely proportional to the square root of the fraction of dark-complexed 8-MOP per nucleotide (the binding ratio), a quantity that is insensitive to the ratio of nucleotides to 8-MOP. These findings predict a small dependence of photosensitivity on the total 8-MOP concentration in cellular systems in which cross-links are the major lethal lesion.     

MUTAGENICITY OF MONOADDUCTS AND CROSS-LINKS INDUCED IN ASPERGILLUS NIDULANS BY 8-METHOXYPSORALEN PLUS 365 NM RADIATION

Abstract— 8-Methoxypsoralen plus 365 nm radiation induces mutation at the methionine supressor loci of Aspergillus inhibitor-deficient conidia at low doses of near-UV radiation with one-hit kinetics and at higher near-UV radiation doses with two-hit kinetics. These results and others suggest that both monoadducts and cross-links, formed by 8-methoxypsoralen and DNA upon exposure to UV radiation, are capable of inducing mutation. Evidence is also presented that induced furocoumarin cross-links are responsible for the inactivation of the Aspergillus conidium.     

COMPARISON OF THE EFFECTS OF ULTRAVIOLET RADIATION ON THE INTRACELLULAR CATALASE OF AEROBIC AND ANAEROBIC YEAST

Abstract— Suspensioris of aerobic and anaerobic yeast were subjected to ultraviolet radiation (principally 254 mµ ) under closely comparable experimental conditions, and changes in the level and in the temperature dependence of their catalase activity were determined. Qualita tively, the effects of U.V. on the enzyme of the anaerobic cells were similar to those on that of the aerobic cells. The effect of U.V. on the anaerobic catalase differed from that on the aerobic enzyme in the following respects: I, a considerably greater dose of U.V. was necessary in order to attain the maximum activity and the minimum activation energy of the enzyme-substrate system; 2, a far greater dose was required before appreciable photoinactivation of the maxi mally active enzyme occurred; 3, photoinactivation proceeded at less than one-half the rate; 4, the u.v.-induced increase in the catalase activity of the suspension was virtually complete before appreciable reduction in activation energy occured. The first three of these differences were interpreted in terms of a model, which pictures the anaerohic catalase as being tightly bound to an intracellular chromophore group.     

THE INTERACTION OF MELANIN WITH 8-METHOXYPSORALEN: EFFECT ON RADIATIVE AND NONRADIATTVE TRANSITIONS. A FLUORESCENCE AND PULSED PHOTOACOUSTIC STUDY

Abstract: The interaction of 8-methoxypsoralen (8-MOP) with synthetic eumelanin was investigated using static and time-resolved fluorescence and pulsed photoacoustic calorimetry. Due to the strong overlap of the absorption bands of melanin and 8-MOP, a method is presented to account for the systematic errors introduced by the optical filter effect exerted by each absorbing species in the fluorescence and the photoacoustic measurements. As a preliminary step to the understanding of the nonradiative behavior of the psoralen-melanin complexes, the photoacoustic parameters of 8-MOP in various solvents were determined. Spectroscopic data indicate the absence of interaction at the ground-state level, whereas the singlet excited state of 8-MOP is quenched by the pigment; the average fluorescence lifetimes are independent of the melanin concentration, thus indicating a static quenching mechanism. The photoacoustic data show that the quenching process involves an increased intersystem crossing probability, which is almost unaffected by the presence of oxygen, as expected for a molecule essentially acting as a type I photosensitizing agent.     

THE EXCITATION OF 8-METHOXYPSORALEN WITH VISIBLE LIGHT: REVERSED PHASE HPLC QUANTITATION OF MONOADDUCTS and CROSS-LINKS

Abstract— The formation of 8-methoxypsoralen-DNA monoadducts and cross-links is presumed to be responsible for the efficacy of photochemotherapies that employ 8-methoxypsoralen activated with long-wavelength ultraviolet radiation (UVA,320–400 nm). In this report it is shown that 8-methoxypsoralen can also be activated with visible light (419 nm). Bovine aorta smooth muscle cells were treated with 8-methoxypsoralen (1000 ng/mL) and 419 nm light (up to 12 J/cm²). Cellular DNA was isolated, hydrolyzed using nucleolytic enzymes and then analyzed by reversed-phase high-performance liquid chromatography. The primary effect of using visible light instead of long-wavelength ultraviolet radiation is a more than 10-fold reduction in the extent of cross-link formation. Because the extent of monoadduct and cross-link formation has not been routinely measured in experiments in which cellular assays have been performed, it is difficult to correlate cell response to the presence of a particular type of 8-methoxypsoralen photoadduct (monoadduct or cross-link). Thus, the use of visible light allows the study of cells containing nearly 100% monoadducts. In addition, the reduction in cross-link formation when visible light is used to activate the compound may also reduce the mutagenicity of 8-methoxypsoralen and hence enhance its therapeutic efficacy.