Effects of glutathione peroxidase and catalase on hemolysis and methemoglobin modifications induced by photooxidized psoralen

Psoralen photooxidation products (POP products) were obtained by UVA irradiation (365 nm, 180-640 W/m2) of an aqueous psoralen solution with fluences of 0-800 kJ/m2. Preincubation of POP products with glutathione peroxidase (GSHPer) or catalase, as well as presence of catalase during UVA irradiation of the aqueous psoralen solution did not influence their hemolytic activity. However, both GSHPer and catalase inhibited POP-induced conversion of methemoglobin. This indicates that hydrogen peroxide and psoralen peroxides destructible by GSHPer, which are being produced during psoralen photooxidation, do not possess hemolytic activity. Furthermore, hydrogen peroxide does not appear to serve as an intermediate in the process of hemolysin formation. Hydrogen peroxide generated during psoralen photooxidation is apparently the main POP product responsible for MetHb conversion.     

In vitro Fluence Rate Effects in Photodynamic Reactions with AIPcS4 as Sensitizer

Abstract— It has been shown previously that the efficiency of photodynamic therapy (PDT) both in vivo and in vitro is dependent on fluence rate. In this study, different in vitro experiments showed that tetrasulfonated aluminum phthalocyanine (AlPcS₄) is more efficient in photosensi-tization if the light is delivered at low fluence rate. Erythrocyte damage, virus inactivation and photooxidation of reduced glutathione (GSH) and histidine were all enhanced if light was delivered at 100 W/m² as compared to 500 W/m². Bleaching did not occur under these conditions. Oxygen depletion, shown to be important in fluence rate effects observed in vivo , does not seem to be involved. On theoretical grounds saturation of the triplet state is not likely under these conditions. A possible explanation for the observed fluence rate effects might be found in different reaction pathways, that are favored under high or low fluence rate illuminations. These reactions might involve uni- or bimolecular reactions of intermediate products, resulting in less efficiency at higher fluence rate. It proves to be important, under all circumstances, to monitor fluence rate, because a change in fluence rate, even with similar total fluences, might influence photobiological results in an unexpected way.     

PHOTOINDUCTION OF PROTOPERITHECIA IN NEUROSPORA CRASSA BY BLUE LIGHT

Blue light induces the formation of Neurospora crassa protoperithecia.This photoinduction is completed in less than 24 h. Its threshold is about 4.2 J/m². Red light is ineffective. The Bunsen-Roscoe law is obeyed at the fluence of 12.6 J/m² for fluence rates from 5.25 × 10 ² to 1.05 W/m².     

SKIN DAMAGE IN ADULT AMPHIBIANS AFTER CHRONIC EXPOSURE TO ULTRAVIOLET RADIATION

Abstract— The effects of repeated UV exposure on the skin of the European crested newt, Triturus cristatus carnifex , have been investigated. The animals were irradiated 3 times per week with a Westing-house FS40T12 fluorescent sun lamp (wavelength spectrum 275–350 nm). Two groups of animals received the same total fluence of 1.3 × 10⁵ J/m² in single fluences of either 1570 J/m² (group A) or 9430 J/m² (group C), and one group received a total fluence of 2.6 × 10⁵ J/m² in single fluences of 4710 J/m² (group B). All the animals were killed 7 months after the first UV exposure, but at different intervals after the last exposure. Striking epidermal hyperplasia was found in the newts irradiated at the lower fluence rate (group A). In the animals given the higher total fluence (group B), the most prominent skin changes were dermal fibrosis and irregular thinning and thickening of the epidermis. No significant skin changes were found in group C., in which if there had been UV lesions, they had been repaired during the 5 month interval between the last irradiation and the killing of the animals. No skin tumors developed in any experimental group.     

EFFECTS OF INTENSITY AND FLUENCE UPON DNA SINGLE-STRAND BREAKS INDUCED BY EXCIMER LASER RADIATION

Abstract— A Xenon-chloride excimer laser emitting energy at 308 nm was used to induce single-strand breaks (SSBs, frank breaks plus alkali-labile lesions as assayed by alkaline sucrose sedimentation techniques) in purified DNA from Bacillus subtilis . A fluence response study and a peak pulse intensity study were performed. At a pulse energy of 0.1 mJ/pulse, the radiation induced SSBs in a linear fashion (91 SSB/10⁸ Da per MJ/m²) to a maximum exprimental fluence of 1.28 MJ/m². The pulse intensity study showed that there were no significant changes in DNA breakage (105 SSB/10⁸ Da) between 2.93 times 10⁹ and 5.86 times 10¹¹ W/m² (0.11 and 22.0 mJ/pulse) at a constant total fluence of 1.1 MJ/m² (27000 mJ dose). This study has verified and extended previous work by quantifying the yield of SSBs induced in DNA by this laser radiation.     

Photochemical and Photobiological Studies of a Furonaphthopyranone as a Benzo-spaced Psoralen Analog in Cell-free and Cellular DNA

Abstract— Photobiological activities of the benzo-spaced psoralen analog furonaphthopyranone 3 have been investigated in cell-free and cellular DNA. The molecular geometry parameters of 3 suggest that it should not form interstrand crosslinks with DNA. With cell-free DNA no evidence for crosslinking but also not for monoadduct formation was obtained; rather, the unnatural furocoumarin 3 induces oxidative DNA modifications under near-UVA irradiation. The enzymatic assay of the photosensitized damage in cell-free PM2 DNA revealed the significant formation of lesions sensitive to formamidopyrimidine DNA glyco-sylase (Fpg protein). In the photooxidation of calf thymus DNA by the furonaphthopyranone 3, 0.29±0.02% 8-oxo-7,8-dihydroguanine (8-oxoGua) was observed. With 2'-deoxyguanosine (dGuo), the guanidine-releasing photooxidation products oxazolone and oxoimidazolidine were formed predominately, while 8-oxodGuo and 4-HO-8-oxodGuo were obtained in minor amounts. The lack of a significant D₂O effect in the photooxidation of DNA and dGuo reveals that singlet oxygen (type II process) plays a minor role; control experiments with tert -butanol and mannitol confirm the absence of hydroxyl radicals as oxidizing species. The furonaphthopyranone 3 (E_red= -1.93±0.03V) should act in its singlet-excited state as electron acceptor for the photooxidation of dGuo (δG_ET ca – kcal/mol), which corroborates photoinduced electron transfer (type I) as a major DNA-oxidizing mechanism. A comet assay in Chinese hamster ovary (CHO) AS52 cells demonstrated that the psoralen analog 3 damages cellular DNA upon near-UVA irradiation; however, no photosensitized mutagenicity was observed in CHO AS52 cell cultures     

IMPACT OF ENHANCED SIMULATED SOLAR ULTRAVIOLET RADIATION UPON A MARINE COMMUNITY

Abstract—There is evidence to indicate that an increased exposure to solar radiation in the UV-B region (specifically, 290–320 nm) may occur as a result of anthropogenic degradation of stratospheric ozone. The fact that present levels of solar UV radiation can detrimentally affect marine organisms led to experiments to quantify the impact of increased UV radiation upon a marine community. Two 720–l seawater chambers (continuous flow-through design) were exposed to simulated solar UV radiation. Fluorescent sunlamps filtered by a 290 nm cutoff filter (a 0.13 mm thickness of cellulose triacetate film) were used as the radiation source. Utilization of three different weighting factors for the spectral irradiances at the surface of the chambers yielded differences of 18%, 35% and 40% in biologically effective fluence rate between the two chambers. Analysis of attached forms of algae at various depths demonstrated that a surface exposure of 1.4W/m² in the 290–315nm waveband as contrasted with the chamber receiving a surface exposure of 1.0W/m² resulted in depressed Chl a concentrations, reduced biomass, increased autotrophic indices, and decreased community diversity. These results indicate a potential for adverse effects of increased solar UV-8 radiation: decreased community diversity, community structure shifts, and decreased productivity.     

MECHANISM OF HIGH POWER PICOSECOND LASER UV INACTIVATION OF VIRUSES AND BACTERIAL PLASMIDS

Abstract— The mechanism of inactivating action of high-power picosecond laser UV radiation (λ= 266 nm) on the λ and φX174 bacteriophages and the pBR 322 plasmid has been studied. It has been shown that at UV radiation intensities from 10¹¹ to 10¹³ W/m², inactivation of viruses and bacterial plasmids occurs mainly on account of single-strand breaks in the DNA chain unlike the case of less powerful UV radiation where the inactivation is associated with the formation of pyrimidine dimers.     

PHOTOCHEMICAL ALTERATIONS IN DNA REVEALED BY DNA-BASED LIQUID CRYSTALS

Abstract The preparations of chicken erythrocyte linear double-stranded DNA and superhelical plasmid pBR322 DNA were irradiated by continuous low-intensity UV radiation (I = 25-50 W/m², λ= 254 nm) as well as by highintensity picosecond laser UV radiation (I = 10¹¹-10¹³ W/m², λ= 266 nm). The effect of DNA secondary structure alterations on the formation of liquid-crystalline dispersions from UV-irradiated DNA preparations was studied. It was shown that in the case of linear DNA, watching the disappearance of abnormal optical activity characteristic for cholesteric liquid crystal we managed to detect the presence of photochemical alterations in DNA irradiated by low-intensity UV radiation at an absorbed energy of more than 20 quanta per nucleotide. In the case of superhelical DNA using enzyme treatment of liquid-crystalline dispersions and monitoring the appearance of abnormal optical activity, we detected the presence of photochemical alterations in DNA molecules after low-intensity UV irradiation at an absorbed energy of less than 4 quanta per nucleotide. Under the latter approach using picosecond UV laser irradiation at three different light intensities we were able to distinguish the different mechanisms of fine alterations in DNA secondary structure at an absorbed energy value of about 3 quanta per nucleotide.     

THE EFFECT OF ULTRAVIOLET RADIATION ON WHEAT ROOT VESICLES ENRICHED IN PLASMA MEMBRANE

Abstract— The irradiation of plant cells with UV radiation (254nm) causes various solutes to leak from the cells. Vesicles enriched in plasma membranes were prepared from wheat roots. These were used to determine whether UV radiation alters membrane function by direct action on the membranes and to distinguish between the chemical effects produced by high and low fluences of UV. The plasma membrane-associated K⁺-stimulated ATPase was very sensitive to UV radiation (100% inhibition with 1.35kJ/m²). ATPase activity measured in the absence of K⁺ and K⁺-stimulated ATPase activity measured in the presence of diethylstilbestrol were much less sensitive. Lipid breakdown, as measured by malondialdehyde production, occurred only at UV fluences greater than 1.8 kJ/m².     

POSTREPLICATION REPAIR IN uvrA AND uvrB STRAINS OF ESCHERICHIA COLI K-12 IS INHIBITED BY RICH GROWTH MEDIUM

Abstract Escherichia coli K-12 uvrA or uvrB strains grown to logarithmic phase in minimal medium showed higher survival after ultraviolet (UV) irradiation (254 nm) if plated on minimal medium (MM) instead of rich medium. This'minimal medium recovery'(MMR) was largely blocked by additional recA56 (92% inhibition) or lexA101 (77%) mutations, was partially blocked by additional recB21 (54%), uvrD3 (31%) or recF143 (22%) mutations, but additional polA1 or polA5 mutations had no effect on MMR. When incubated in MM after UV irradiation, the uvrB5 and uvrB5 uvrD3 strains showed essentially complete repair of DNA daughter-strand gaps (DSG) produced after UV radiation fluences up to ∼ 6 J/m² and ∼1 J/m², respectively, and then they accumulated unrepaired DSG as a linear function of UV radiation fluence. However, when they were incubated in rich growth medium after UV irradiation, they did not show the complete repair of DSG and unrepaired DSG accumulated as a linear function of UV radiation fluence. The fluence-dependent correlation observed for the uvrB and uvrB uvrD cells between UV radiation-induced killing and the accumulation of unrepaired DSG, indicates that the molecular basis of MMR is the partial inhibition of postreplication repair by rich growth medium. Rich growth medium can be just MM plus Casamino Acids or the 13 pure amino acids therein in order to have an adverse effect on survival, regardless of whether the cells were grown in rich medium or not before UV irradiation.     

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.     

PHYSIOLOGICAL EFFECTS OF ULTRAVIOLET LIGHTON DICTYOSTELIUM DISCOIDEUM SPORE GERMINATION

Abstract— The spore germination in Dictyostelium discoideum consists of four stages: activation, postactivation lag, swelling and emergence. Ultraviolet irradiation (total fluence of 250 J/m²) of spores at any time prior to late spore swelling allows full swelling, but inhibits the emergence of myxamoebae. In the case of freshly activated spores, a UV exposure time of 30 s (total fluence of 50 J/m²) is sufficient to reduce emergence to about 6% when measured after 24 h of incubation. This same fluence results in about 10% viability as measured by plaque forming ability. Experiments utilizing 'fractionated exposures' result in the same percentage inhibition of emergence as that found for 'single exposures' provided the total fluence is equivalent. The higher fluences (250 J/m²) which completely prevent emergence, do not affect the endogenous oxygen uptake of spores during swelling. Ultraviolet light irradiated spores respond to the same activation and deactivation treatments as control unirradiated spores. Ultraviolet irradiation after late spore swelling allows emergence to occur in only a small fraction of the population. This fraction of cells which can emerge after UV treatment is said to have passed a 'competence point', which is believed to be the time when all the events necessary for emergence have been completed. Though the sites of UV inactivation in spores can only be postulated at present, it is apparent that the initial stages of germination (activation, postactivation lag and spore swelling) occur independently of the UV sensitive sites. The final stage of germination (emergence), however, is dependent on UV sensitive functions.     

USE OF METABOLIC INHIBITORS TO INVESTIGATE THE EXCISION REPAIR OF PYRIMIDINE DIMERS AND NON-DIMER DNA DAMAGES INDUCED IN HUMAN AND ICR 2A FROG CELLS BY SOLAR ULTRAVIOLET RADIATION

Abstract— ICR 2A frog and normal human skin fibroblasts were exposed to either 5 J/m² of 254 nm UV or 50 kJ/m² of the Mylar-filtered solar UV wavelengths produced by a fluorescent sunlamp. Following these approximately equitoxic treatments, cells were incubated in medium containing the DNA synthesis inhibitors hydroxyurea (HU) and 1–β-D-arabinofuranosyl cytosine (ara C) for 0–20 min (human fibroblasts) or 0–4 h (frog cells) to accumulate DNA breaks resulting from enzymatic incision during excision repair. It was found that breaks were formed in human cells at about a 200-f-old higher rate compared with the ICR 2A cells indicating a relatively low capacity for excision repair in the frog cells. In addition, the rate of DNA break formation in solar UV-irradiated cells was only one-third of the level detected in 254 nm-irradiated cells. This result is consistent with the conclusion that the pathway(s) involved in the repair of solar UV-induced DNA damages differs from the repair of lesions produced in cells exposed to 254 nm UV.     

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.     

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 SKIN TUMORS IN THE RAT BY SINGLE EXPOSURE TO ULTRAVIOLET RADIATION

Abstract— The dose response for tumor induction in albino rat skin by single exposures of UV radiation has been characterized. The shaved dorsal skin of 202 animals was exposed to either of two sources: one emitting a broad spectrum of wavelengths from 275 to 375 nm, and the other emitting at 254 nm. Skin tumors began to appear within 10 weeks of exposure and continued to appear for 70 weeks. The highest tumor yield was 5.5 tumors per rat and occurred when the rats were exposed to 13.0 times 10⁴ J/m² of the 275–375 nm UV. The 275–375 nm UV was about eight times as effective as the 254 nm UV for the induction of tumors throughout the exposure range from 0.8 times 10⁴ to 26.0 times 10⁴J/m². Tissue destruction and hair follicle damage was found at the highest exposure to 275–375 nm UV but at none of the exposures to 254 nm UV. Repeated weekly exposures to 275–375 nm UV proved less effective than an equivalent single exposure for inducing tumors, even though the multiple exposures caused more severe skin damage. The transmission of the UV through excised samples of rat epidermis indicated that the exposure to the basal cell layer was about 3% of the surface exposure at 254 nm and about 15% of the surface exposure between 275 and 320 nm. The dependence of tumor yield on UV exposure was linear for 254 nm UV but was more complex for the 275–375 nm UV. For the latter more tumors were produced per unit exposure at lower exposures than at higher exposures.     

PHOTOBIOLOGICAL ACTIVITY OF MARMESIN (5-B-HYDROXYISOPROPYL-4–5 DIHYDROFUROCOUMARIN) IN CHINESE HAMSTER V79 CELLS

Abstract— Marmesin was isolated from the medicinal plant, Afraegle paniculata. Its cytotoxicity and mutagenicity in Chinese hamster V79 cells when sensitized to near ultraviolet (NUV) and long wavelength ultraviolet light or black light (BL) were assayed.
Marmesin was extremely cytotoxic in the dark. This cytotoxicity was photoenhanced in NUV and BL; the photoenhanced lethality being higher in NUV than in BL. The LD₅₀ of marmesin under NUV and BL photosensitization were 0.002 μ M and (0.012 μ M ), respectively. In the absence of NUV and BL, marmesin's LD₅₀ was 0.013 μ M . NUV and BL without marmesin were not significantly cytotoxic at the fluence rates of 0.29 W/m² and 4.2 W/m², respectively, for up to 20 min. In contrast to the observed high cytotoxicity of marmesin, its mutagenicity at the HGPRT locus (A_zG^r) was weak. The implication of this result in the high incidence of skin cancer in Nigeria in which A. paniculata is used as a medicinal plant is discussed.     

UV-INDUCED REVERSION OF ADENOVIRUS 5ts2 INFECTING HUMAN CELLS

Abstract— Among the progeny produced by UV-irradiated adenovirus 5ts2 when infecting either A498 (human kidney tumor) or CRL1187 (normal human fibroblast) strains at 32° C were a certain number (UV–fluence dependent) of revertants able to grow at 39° C. Within experimental error, the induced reversion frequency was independent of whether or not the cells received UV (5 J/m²) 20 h prior to infection, indicating that induced error-prone repair was not observed in these experiments.     

CHLOROPHYLL METABOLISM IN ETIOLATED GHERKIN SEEDLINGS I. PHOTOINHIBITION OF CHLOROPHYLL ACCUMULATION

Abstract. Cotyledons of etiolated gherkin seedlings do not turn green upon transfer to high intensity red light (about 25 W/m²). A pre-irradiation with high intensity red light has an after-effect as chlorophyll accumulation during a subsequent exposure to white light (20 W/m²) is inhibited.
The capacity of protochlorophyll regeneration during a dark period depends on the length of a previous light period but is hardly affected by the light intensity. At high intensity light the rate of protochlorophyll regeneration, which also depends on the length of the foregoing irradiation, is lower than that at low intensity light only during the first 1.5h of the light period. It is concluded that high intensity red light inhibits chlorophyll accumulation mainly by photo-bleaching of chlorophyll. The after-effect is the result of a photooxidation which may lead to photo-bleaching of newly formed chlorophyll in relatively low intensity light.
Photoinhibition of chlorophyll accumulation is accompanied by a disturbed development of etioplasts into chloroplasts.