INDUCTION OF DIRECT AND INDIRECT SINGLE-STRAND BREAKS IN HUMAN CELL DNA BY FAR- AND NEAR-ULTRAVIOLET RADIATIONS: ACTION SPECTRUM AND MECHANISMS

Abstract— An action spectrum for the immediate induction in DNA of single-strand breaks (SSBs, frank breaks plus alkali-labile sites) in human P3 teratoma cells in culture by monochromatic 254-, 270-, 290-, 313-, 334-, 365-, and 405-nm radiation is described. The cells were held at +0.5C during irradiation and were Iysed immediately for alkaline sedimentation analysis following the irradiation treatments. Linear fluence responses were observed over the fluence ranges studied for all energies. Irradiation of the cells in a D₂O environment (compared with the normal H₂O environment) did not alter the rate of induction of SSBs by 290-nm radiation, whereas the D₂O environment enhanced the induction of SSBs by 365- and 405-nm irradiation. Analysis of the relative efficiencies for the induction of SSBs, corrected for quantum efficiency and cellular shielding, revealed a spectrum that coincided closely with nucleic acid absorption below 313 nm. At longer wavelengths, the plot of relative efficiency vs . wavelength contained a minor shoulder in the same wavelength region as that observed in a previously obtained action spectrum for stationary phase Bacillus subtilis cells. Far-UV radiation induced few breaks relative to pyrimidine dimers, whereas in the near-UV region of radiation, SSBs account for a significant proportion of the lesions relative to dimers, with a maximum number of SSBs per lethal event occurring at 365-nm radiation.     

INDUCTION OF DNA-PROTEIN CROSSLINKS IN HUMAN CELLS BY ULTRAVIOLET and VISIBLE RADIATIONS: ACTION SPECTRUM

Abstract— DNA-protein crosslinking was induced in cultured human P3 teratocarcinoma cells by irradiation with monochromatic radiation with wavelengths in the range254–434 nm (far-UV, near-UV, and blue light). Wavelength 545 nm green light did not induce these crosslinks, using the method of alkaline elution of the DNA from membrane filters. The action spectrum for the formation of DNA-protein crosslinks revealed two maxima, one in the far-UV spectrum that closely coincided with the relative spectrum of DNA at 254 and 290 nm, and one in the visible light spectrum at 405 nm, which has no counterpart in the DNA spectrum. The primary events for the formation of DNA-protein crosslinks by such long-wavelength radiation probably involve photosensitizers. This dual mechanism for DNA-protein crosslink formation is in strong contrast to the single mechanism for pyrimidine dimer formation in DNA, which apparently has no component in the visible light spectrum.     

IMPROVED QUANTITATION OF DNA-PROTEIN CROSSLINKING CAUSED BY 405-nm MONOCHROMATIC NEAR-UV RADIATION IN HUMAN CELLS

An improved alkaline elution analysis has been used to estimate the yield of DNA-protein crosslinks induced in human P3 cells by monochromatic 405-nm near-ultraviolet radiation. Crosslinks are induced linearly over the fluence range studied, at a rate of 1.22 crosslinks per 10¹⁰ daltons per MJ m^-2± 0.066 (one standard error of the slope, N = 37). This rate is equivalent to 1360 crosslinks per cell genome per lethal event.     

SINGLET OXYGEN INVOLVEMENT IN THE INACTIVATION OF CULTURED HUMAN FIBROBLASTS BY UVA (334 nm, 365 nm) AND NEAR-VISIBLE (405 nm) RADIATIONS

The UVA (320-380 nm) radiation inactivation of mammalian cells is dependent upon the presence of oxygen. In order to examine the intermediates involved, we have irradiated cells in the presence of chemical probes which are able to modify the activity of various oxygen species. We have also examined the possibility that UVA inactivates cultured human fibroblasts via generation of intracellular hydrogen peroxide. An iron scavenger (desferrioxamine) and a hydroxyl radical scavenger (dimethylsulfoxide) protect the cells against hydrogen peroxide. Diethyldithiocarbamate (a superoxide dismutase inhibitor) and aminotriazole (a catalase inhibitor) sensitize the cells to this oxidizing agent. These data support previous reports that hydrogen peroxide inactivates as a result of the iron-catalyzed generation of hydroxyl radical. None of these agents significantly alter the fluence-dependent inactivation of cell populations by radiation at 365 nm. In contrast, the cells are sensitized to radiation at 334, 365 and 405 nm in the presence of deuterium (an enhancer of singlet oxygen lifetime) and are protected against radiation at 365 nm by sodium azide (a quencher of singlet oxygen). These results are consistent with the conclusion that the generation of singlet oxygen, but not hydrogen peroxide or hydroxyl radical, plays an important role in the inactivation of cultured human cells by UVA and near-visible radiations.     

MUTATION INDUCTION BY AND MUTATIONAL INTERACTION BETWEEN MONOCHROMATIC WAVELENGTH RADIATIONS IN THE NEAR-ULTRAVIOLET AND VISIBLE RANGES

Abstract— The induction of mutations (reversion to tryptophan independence) by various UV (254, 313, 334 and 365 nm) and visible (405 and 434 nm) wavelengths was measured in exponential phase populations of Escherichia coli B/r thy trp and B/r thy trp uvrA by assay of irradiated populations on semi-enriched media. No mutations were induced in the repair proficient strain at wavelengths longer than 313 nm. Mutations were induced in the excisionless strain at wavelengths as long as 405 nm but less than expected from the known amount of DNA damage induced. Irradiation at the longer wavelengths (434, 405, 365 and 334 nm) suppressed the appearance of 254- or 313-nm-induced mutations in the repair competent strain but not in the excision deficient strain. The relative dose-requirement for mutation suppression was related to the relative efficiency of these wavelengths in inducing growth delay. These results suggest that the growth delay induced by near-UV and visible wavelengths allows more time for the 'error-free" excision repair process to act on the potentially mutagenic lesions induced by 254- and 313-nm radiations, thereby reducing the mutation frequency observed in the repair-proficient strain. The level of near-UV mutation induced in the excision deficient strain is lower than expected from the DNA damage known to be induced. It is possible that near-UV radiation induces a class of lethal lesions that are not susceptible to error-prone repair.     

DIFFERENT PHOTOINACTIVATION MECHANISMS IN Propionibacterium acnes FOR NEAR-ULTRAVIOLET and VISIBLE LIGHT

The light sensitivity of Propionibacterium acnes was investigated when the cells were exposed to anoxia, sodium azide, D₂O or superoxide dismutase in combination with visible light (broad band red light and 415 nm) and near-ultraviolet irradiation (360 and 320 nm). During anoxia the cells became less sensitive when the irradiation wavelength increased. Oxygen increased the photodamage to a greater extent in the case of visible light than of near-UV light. The photosensitization effects were, however, more or less oxygen dependent at all wavelengths used. An effect of azide and D₂O on the light sensitivity was observed for visible light, while superoxide dismutase was effective only at 320 nm.
The results support the hypothesis that inactivation of P. acnes with near-UV and visible light is based on different mechanisms. Porphyrin photosensitization, accomplished by singlet oxygen, is the most important mechanism when visible light is used. At shorter wavelengths (320 and 360 nm) singlet oxygen is not involved and for 320 nm the destruction might occur via superoxide anion formation.     

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.     

CONVEX CURVATURES OF ALKALINE ELUTION PROFILES OF DNA FROM HUMAN CELLS IRRADIATED WITH 405 nm UVA: EVIDENCE FOR INDUCTION OF SLOWLY DEVELOPING ALKALI-LABILE SITES

The alkaline (pH 12.1) elution profiles of DNA from human P3 cells exposed to monochromatic 405 nm UVA radiation deviate from exponential: on a logarithmic plot of eluted fraction of DNA vs time of elution, the rate of elution accelerates for the first 6 h. Following this period, the profiles become exponential. In contrast, the elution profiles of DNA after 520 nm green light or ionizing radiation exposures (x- and gamma rays, and fission spectrum neutrons) are always strictly exponential, evidence that the convex profiles were not due to an artifact caused by elution technique. Holding the DNA at pH 12.1 for 6 h after 405-nm exposures before initiating elution resulted in profiles that were close to exponential, with slopes similar to the final slopes observed following the 6-h elution period in the original experiments. This is evidence that some DNA breaks develop slowly during the first 6 h of elution, as a result of exposure to alkali. Therefore, the DNA lesions induced by 405-nm light as measured by the alkaline elution technique are apparently heterogeneous and include a major class of alkali-labile sites that develop slowly during incubation at pH 12.1. Convex profiles also occur following exposure of the cells to visible light at 434 and 512 nm.     

TEMPERATURE DEPENDENCE OF INDUCTION OF CYCLOBUTANE-TYPE PYRIMIDINE PHOTODIMERS IN HUMAN FIBROBLASTS BY 313 nm LIGHT

The rate of cyclobutane-type pyrimidine photodimerization with 313 nm light was determined in confluent cultures of xeroderma pigmentosum cells of group A. A new method was developed for the determination of sodium borohydride reduced thymine-thymine (TT) and cytosine-thymine (CT) dirners by high pressure liquid chromatography. It was found that the yield of dimerization andtheratioof CT/TT depended on the irradiation temperature in the physiological fluence range of 2.25 to 15 kJ m⁻². Both were significantly higher at 37 than at 0°C.     

PHOTOSENSITIZED PRODUCTION OF SUPEROXIDE ANION BY MONOCHROMATIC (290–405 nm) ULTRAVIOLET IRRADIATION OF NADH and NADPH COENZYMES

Abstract— The reduced pyridine coenzymes NADPH and NADH produced superoxide anion("CK") from ground state molecular oxygen when irradiated by ultraviolet (UV) radiation extending from 290 to 405 nm as detected by cytochrome c reduction. Superoxide dismutase (SOD), but not catalase or heat-inactivated SOD, decreased the amount of cytochrome c reduced, indicating that O₂⁻ was responsible for the reduction of cytochrome c. Decreased oxygen tension during irradiation also inhibited production of O₂⁻. Quantum yields for the production of the anion were in the region of 10⁻⁷ to 10⁻⁹ mol per photon. These data indicate that NADH and NADPH can act as type II photosensitizers of both far-and near-UV radiation, and that the deleterious biological effects of exposure to these radiations such as erythema and dermal carcinogenesis may be mediated at least in part through the generation of O₂⁻.     

INDUCTION OF DNA–PROTEIN CROSS-LINKING IN CHINESE HAMSTER CELLS BY MONOCHROMATIC 365 AND 405 NM ULTRAVIOLET LIGHT

Abstract— The survival, the induction of DNA-protein cross-linking, and the number of T4-endonuclease sensitive sites were measured in Chinese hamster cells that had been irradiated with 365 and 405 nm monochromatic light. The survival measurements show that cells are somewhat less sensitive to 405 nm light than to 365 nm light. The difference is expressed predominantly in the shoulder widths of the survival curves, whereas the slopes of the two curves are about the same. Induction of pyrimidine dimers, as indicated by the number of endonuclease-sensitive sites, after exposures that produce about 10% survival is very low at 365 nm (˜ 4 endonuclease sites per 2 × 10⁸ daltons), while no dimers are detected at 405 nm. In contrast, DNA-protein cross-links are induced rather effectively at either wavelength even after exposures that result in a relatively high survival (60-20%). Our measurements support the conclusion that lethality in mammalian cells after irradiations with 365 or 405 nm light is caused by a nondimer damage, possibly DNA-protein cross-links.     

RECOVERY FROM INHIBITION BY UV-IRRADIATION OF ORNITHINE DECARBOXYLASE INDUCTION IN HUMAN CELLS: IMPLICATION OF EXCISION REPAIR

Abstract— Exposure of stationary-phase human breast carcinoma(T–47D) cells to far-UV light (254 nm) inhibited the appearance of induced ornithine decarboxylase (ODC) activity. The fluence response curve had a shoulder (D₄= 2 J m_-2) followed by an exponential decline (D₀= 4.2 Jm_-2). The cells could recover from this inhibition when the stimulus of induction of ODC was delayed for20–24 h after irradiation. Hydroxyurea (HU) when present at 3 mM during the recovery period eliminated completely the ability of the cells to recover. This effect of HU on ODC induction was partially reversed by 50 nM of the four deoxyribonucleosides required for DNA synthesis. Neither HU nor the deoxyribonucleosides by themselves affected ODC induction in unirradiated cells. Since HU inhibited the recovery from potentially lethal UV damage and is a known inhibitor of excision repair, we interpret the above results to mean that recovery from UV-induced inhibition of ODC induction depends on excision-repair of DNA damage. This interpretation is strongly supported by the finding that specific photolysis of 5-bromodeoxyuridine, incorporated into DNA during the recovery period, inhibited recovery of ODC induction from inhibition by UV light.     

REDUCTION BY NEAR-ULTRAVIOLET (334 nm) LIGHT OF E. COLI CAPACITY FOR PHAGE GROWTH DEPENDS UPON THE rel GENE AND 4-THIOURIDINE

Abstract— Near-ultraviolet radiation (near UV; 300–380 nm) has long been known to produce a transient reduction of the capacity of bacteria to support phage growth. The present work shows that, at high fluenœs (40–100 kJ/m²), 85% of 334-nm-induced reduction of capacity in Escherichia coli B/r requires the rel gene; that is, it results from rel -gene activity caused by the near-UV treatment. This rel -gene activity leads to (1) a bacterial growth delay and concomitantly lowered bacterial metabolism, and (2) a parallel delay in phage development, with a considerable depression of burst size. We propose that the observed effects on phage development are a consequence primarily of the lowered bacterial metabolism, but they may also result partly from a direct inhibition of phage DNA synthesis by the rel gene product, these effects together leading to the observed reduction of capacity in a rel ⁺ strain. The remaining 15% of capacity reduction, observed in a rel strain, has an unknown mechanism, but does appear to involve a delay in phage development.
At least 95% of the total capacity reduction observed in the rel ⁺ strain in the range 40–100 kJ/m² requires the presence of 4-thiouridine, an unusual base in E. coli transfer RNA, which is presumably both the chromophore and the target for near-UV-induced capacity reduction.     

MEASUREMENT OF DNA CROSSLINKS BY S1 NUCLEASE: INDUCTION AND REPAIR IN PSORALEN-PLUS-360 nm LIGHT TREATED ESCHERICHIA COLI

Abstract—DNA crosslinks in Escherichia coli cells. exposed to 4.5',8-trimethylpsoralen plus 360 nm light, were measured using a rapid and sensitive new approach. The assay is based on the specificity of S₁ nuclease from Aspergillus oryzae to single-stranded DNA. Bacterial cells were lysed and the DNA denatured by alkali. Following acid neutralization. crosslinked DNA undergoes spontaneous renaturation and is rendered S₁-nuclease resistant and therefore acid-precipitable. The single-stranded fraction after denaturation by alkali decreases with increasing near UV light exposure in the presence of TMP following first order kinetics. The kinetics were faster when exposure was at 4°C rather than at 20°C. This suggests that excision of crosslinks occurs during exposure at the higher temperature. Indeed. since the rate of DNA crosslinking in a uvr B mutant which is excision-deficient was higher than in wild type bacteria at 4°C, some excision must have occurred even in the cold. DNA from excision-proficient cells incubated at 37°C following exposure to TMP-plus-near UV at 4° showed a greater single stranded fraction than that from non-incubated cells. This indicates repair of DNA crosslinks. which proceeded with a half-time of 8 min at 37°C and was unaffected by substitution of thymine in DNA by 5-bromouracil.     

ON THE INDUCTION OF PROTECTIVE RESPONSES IN Salmonella typhimurium STRAIN TA1535/pSK1002 BY UVA (365 nm)

Exposure to UVA (365 nm) led to growth delay, loss of viability and inhibition of ³H-thymidine incorporation into the cells of Salmonella typhimurium strain TA1535 containing multiple copies of a plasmid pSK1002 carrying a umuC-'lacZ fusion gene. Ultraviolet-A induced umu gene expression, as monitored by the estimation of β-galactosidase, in a linear fluence-dependent manner. The induction of umu gene expression increased with the increase of postirradiation incubation period of the cells in the LB-ampicillin (LB A) medium at 37° C and leveled off from 2 h onward. The induction of gene expression depended on concomitant protein synthesis and represented the induction of the SOS response in the particular S. typhimurium cells used. The exposure to low fluences (sublethal) of UVA also led to the induction of an adaptive response in the same bacterial cells, which made them resistant to subsequent challenge by a much higher fluence of the same radiation. The adaptive response, as monitored by the assays of viability and β-galactosidase units, increased with the period of exposure to sublethal fluences of UVA, attained a maximum at the UVA exposure of 4.5 kj/m² (15 min) and thereafter gradually decreased with further increase of UVA exposure period. Modulation studies involving D₂O, LBA growth medium, different scavengers of free radicals and quenchers of activated oxygen species indicated the involvement of both hydroxyl free radicals and singlet oxygen in the UVA-induced umu gene expression.     

直接滴定法测定葡萄酒中糖含量操作条件的探讨

通过分析滴定温度、滴定碱度、热源温度、沸腾时间和滴定速度等操作条件,探讨了操作条件对直接滴定法测定葡萄酒中糖含量的影响,确定了最佳操作条件,提高了测定结果的准确度。     

UPTAKE AND RETENTION OF PHOTOFRIN® BY CULTIVATED HUMAN LYMPHOBLASTIC CELLS (Reh6): PREFERENTIAL AFFINITY OF THE CELLS FOR A MINOR COMPONENT DEMONSTRATED BY NORMAL PHASE CHROMATOGRAPHY

The uptake of Photofrin by the human cultivated lymphoblastic cell line Reh6 was studied using normal phase high performance liquid chromatography (HPLC) techniques. Relative cellular uptake of eight fractions (uptake/amount of component initially present in the incubation solution) was determined. After 4 h of incubation, protoporphyrin and a small fraction (denoted 4) were incorporated to a greater relative extent than the other fractions. Weakly incorporated components (hematoporphyrin and aggregate-like components) were better retained by cells than the hydrophobic monomeric porphyrins (protoporphyrin and hydroxyethylvinyldeuteroporphyrin). Thus, any benefit gained from a higher uptake was mostly cancelled by a fast release--a situation observed for all fractions except for fraction 4, which displayed both high uptake and good cellular retention. This pattern was not modified when Photofrin concentration or serum percentage was changed. Fraction 4 was further resolved using a gradient system on normal silica. A single component appeared to be mostly responsible for the favorable properties presented by fraction 4, i.e. high uptake and retention within cells. This component was found to correspond to a late eluted peak in the typical reverse-phase HPLC profile of Photofrin. These results emphasize the possible role of minor Photofrin components.     

FORMATION OF PHOTOPRODUCTS LETHAL FOR HUMAN CELLS IN CULTURE BY DAYLIGHT FLUORESCENT LIGHT AND BILIRUBIN LIGHT

Abstract. Irradiation of Dulbecco's modified Eagle's tissue culture medium with "Daylight,""Special Blue," or "Bilirubin" fluorescent light produces photoproducts lethal to human cells. Killing is abolished when (1) riboflavin, (2) tryptophan and tyrosine, or (3) riboflavin, tryptophan and tyrosine are deleted from medium prior to irradiation with any of the above fluorescent lamps. Toxic photoproducts are also formed when buffered salt solutions containing (a) riboflavin and tryptophan, (b) riboflavin and tyrosine, or (c) riboflavin, tryptophan and tyrosine are exposed to any of these light sources.