Effects of photo-activated bleomycin on deoxyribonuclease I, exonuclease III and deoxyribonucleic acid polymerase I reactions

The activity of bleomycin to break the strand of deoxyribonucleic acid (DNA) in the presence of 2-hydroxy-1-ethanethiol (2-mercaptoethanol) was enhanced by ultraviolet (UV) irradiation. Photo-activated bleomycin stimulated the action of deoxyribonuclease I (DNase I) to degrade DNA and the DNA synthesis by DNA polymerase I with DNase I. On the other hand, although UV-irradiated bleomycin scarcely broke the DNA strand in the presence of 1,2-benzenediol (catechol), it stimulated the action of DNase I to degrade DNA in the presence of catechol. In accordance with the inhibition by catechol, when DNA treated with UV-irradiated bleomycin in the presence of catechol was employed as a primer for the DNA synthesis, the incorporation of precursor into the acid-insoluble fraction by DNA polymerase I with exonuclease III was reduced to about one-half of the incorporation into DNA treated with unirradiated bleomycin. These findings suggest that the ability of bleomycin to bind to double-helical DNA forming regions sensitive to DNase I was increased by an appropriate dose of UV irradiation and that catechol inhibited the activity of the UV-irradiated bleomycin to break the DNA strand rather than to bind to DNA.     

INHIBITION OF 12-O-TETRADECANOYLPHORBOL-13-ACETATE-INDUCED TUMOR PROMOTION IN MURINE SKIN BY SYSTEMIC EFFECTS OF ULTRAVIOLET IRRADIATION

Systemic effects of UVB irradiation (280-320 nm) have been shown to prevent subsequent chemical tumorigenesis induced by an initiation-promotion protocol. The present investigation was designed to determine whether initiation or promotion is prevented by UV irradiation. Groups of 25 B6D2F1/J mice received 12 weeks of intermittent dorsal UVB radiation treatments administered before, or 3 weeks after, initiation with a single application of 7,12-dimethylbenz[a]anthracene on the ventral skin. All mice were promoted ventrally with 5 micrograms 12-O-tetradecanoylphorbol-13-acetate (TPA) applied three times weekly throughout the experiment. UV irradiation consisted of five 30-min exposures per week to a bank of 6 Westinghouse FS40 sunlamps. UV irradiation applied before or after initiation resulted in a decrease of 18-16 tumors per group of 25 mice, for a reduction of 61 and 50%, respectively, at 24 weeks after the first TPA treatment. Thus, prevention of tumor development was similar whether the UV influence was present or not during initiation. This finding suggests that the UV prevention of promotion could account for UV inhibition of skin tumors induced by an initiation-promotion regimen. Consistent with this concept, pretreatment of mice with dorsal UVB radiation was found to reduce DNA synthesis after exposure to TPA by 46%, although it did not decrease tritiated benzo[a]pyrene binding to DNA, in ventral epidermis. Thus, UVB irradiation systemically reduced TPA-induced tumor promotion in murine skin.     

Inhibition of angiogenesis by bleomycin and its copper complex.

The effects of bleomycin (BLM) and its copper complex on embryonic angiogenesis were studied in the chorioallantoic membranes of 4.5-day-old chick embryos. Copper-free BLM inhibited embryonic angiogenesis in a dose-dependent manner, with activity detectable at 1 ng/egg and maximal at 1000 ng/egg. Also, treatment with copper-BLM complex dose-dependently caused inhibition of embryonic angiogenesis in a lower dose range. Since tumor growth is believed to depend on angiogenesis, the present results may indicate that the antiangiogenic activity of BLM is, at least in part, implicated in the antitumor activity of the drug.     

Synthesis and Evaluation of Nicotinoylamino Acid Compounds as Radiosensitizer

Nicotinoylamino acid compounds 4, 5, 9a, 9b, 9c, 10a and 10b were synthesized with nicotinoyl chloride or nicotinoyl azide as acetylating agents of amino acid esters or amino acids. The compounds were tested for their radiosensitizing activity in Leukemia cell line（L1210） and compared with nicotinamide; among them, compounds 9a and 9c showed significant radiosensitizing effects, the sensitizer enhancement ratio（SER） was 1.64 and 1.58, respectively, while nicotinamide did not show good radiosensitizing effect under the same conditions. Compound 9c was alone tested for radiosensitization in LA 795 cell-bearing T-739 mice, or hyperthermia and breathing carbogen（5%CO2＋95%O2） were together tested for radiosensitization. The results showed that radiation-induced growth delay was enhanced by 9c alone or by the combination of hypertheimia and carbogen. The tumor-bearing mice were irradiated locally by total 10 Gy, and the tumors grew to three times that of the original volume in an average of 5.8 d. The mice were given i.p. compound 9c at 1000 mg/kg 60 min before irradiation and treated at 43 ℃ for 30 min after irradiation or treated with breathing carbogen for 5 min before radiation or with hyperthmia（43 ℃） for 30 min after irradiation; the time required for the tumor to grow to three times the orginal volume was in an average of 12.9 and 13 d, respectively.     

THYMIDINE UPTAKE, INCORPORATION AND DNA POLYMERASE ACTIVITY IN MURINE BLADDER TUMOR CELL,MBT–2, EXPOSED TO UV ACTIVATED DIHEMATOPORPHYRIN ETHER

Abstract— Photodynamic therapy (PDT) is a new modality for treatment of malignancy. In this paper, we reported the effect of UV activated dihematoporphyrin ether (DHE) on [³H] thymidine uptake and DNA synthesis in murine bladder tumor cells,MBT–2. Exponentially growing cells were pretreated with 0.05–5 μg/ml of DHE for 30 min in complete darkness prior to irradiation with 0.15-0.90 J/cm² of UV light (265 nm). The rates of thymidine uptake and DNA synthesis were suppressed in a DHE concentration and photic energy dependent manner. Double reciprocal analysis on the kinetics of the thymidine uptake and DNA synthesis indicated that the inhibition was non-competitive, i.e. decrease in both the apparent K^m value and maximum velocity in DHE plus UV light treated cells. The activities of DNA polymerase a and (3 were determined by [*H] dATP incorporation into DNA of permeabilizedMBT–2 cells. DNA polymerase a activity was approximately 60% of the control after 0.45 J/cm² of UV light exposure; a further inhibition of DNA polymerase a was observed when 0.5–5ng/W of DHE and UV photoradiation were combined. In contrast, a slight stimulation of DNA polymerase fJ was noted after a similar treatment. This study demonstrates that photodynamic therapy-induced suppression of DNA synthesis inMBT–2 cells is a complex process involving in reduction of thymidine transport as well as the perturbation of the enzymes involved in DNA synthesis.     

UVB-INDUCED IMMUNE SUPPRESSION AND INFECTION WITH SCHISTOSOMA MANSONI

Abstract— Irradiation with ultraviolet B (UVB, 290–320 nm) causes a systemic immunosuppression of cell-mediated immunity. The question of whether UV immunosuppression modulates the course of infectious diseases is important becauseUVB levels in sunlight are sufficient to predict significant UV-induced immunosuppression at most latitudes. We have investigated the effect of immunosuppressive doses of UVB on the disease caused by the helminth parasite Schistosoma mansoni. C57BL/6 mice were irradiated once or three times weekly over 60–80 days with UV from a bank of FS40 sunlamps. Each UV treatment consisted of an immunosuppressive UV dose, as determined by suppression of contact hypersensitivity to trinitrochlorobenzene, corresponding to about 15–30 min of noonday tropical sunlight exposure under ideal clear sky conditions. Cumulative UV doses were between 80 and 170 kJ/m2. Worm and egg burdens, liver granuloma diameters and liver fibrosis showed minimal changes(> 20%) compared with parameters in unirradiated animals. Ultraviolet irradiation (a total of 55 kJ/m2 administered in six treatments) did not impair the resistance to rechallenge conferred by vaccination with ⁶⁰Co-irradiated cercariae. We have thus observed a dichotomy between UV immunosuppression and both disease and vaccination in this helminth infection, in contrast to the effects of UVB shown in other infectious diseases.     

ENHANCED GROWTH AND EXPERIMENTAL METASTASIS OF CHEMICALLY INDUCED TUMOR IN ULTRAVIOLET IRRADIATED SYNGENEIC MICE

Abstract— Recent studies have shown that ultraviolet (UV) irradiation induces a systemic effect which enhances subsequent tumor induction by benzo[a]pyrene in a manner which is dependent on the dose of benzo[a]pyrene. The present study was designed to test whether UV-B irradiation renders mice susceptible to subcutaneous or intravenous injection of a regressor tumor induced by benzo[a]pyrene. The sources of UV-B irradiation were banks of 6 Westinghouse FS-40 sunlamps, situated 20 cm above the mouse cages. Female BALB/cAnNHsd received five 30-min dorsal UV-B radiation treatments per week for 12 weeks, resulting in a total dose of approx. 6.4 × 10⁵ J m^-2. Two to seven days after termination of UV treatments, syngeneic regressor tumor cells (BP2) induced by benzo[a]pyrene were injected subcutaneously or intravenously into irradiated mice and unirradiated controls. By 38 days post subcutaneous implantation, 24/30 and 3/30 BP2 implants were detectable in the irradiated and unirradiated mice, respectively. Ultraviolet irradiated mice were also unable to reject lung colonies resulting from intravenous administration of BP2 cells, although they were rejected by unirradiated mice. The mean number of lung colonies per mouse was 16- to 35-fold greater in UV irradiated mice than in unirradiated controls, at 14 to 17 days post injection. Thus, UV irradiation rendered mice, with no known exposure to benzo[a]pyrene, susceptible to a subcutaneous or intravenous injection of a regressor tumor induced by benzo[a]pyrene.     

Isolation of lipid and 2-alkylcyclobutanones from irradiated foods by supercritical fluid extraction.

The 2-alkylcyclobutanone method was adopted as a European Standard (EN1785) and MAFF Validated Method (MAFF V37) in 1996 for the detection of irradiated food containing fat. As the method requires a relatively long period (ca 2 days) of time for extraction of the 2-alkylcyclobutanones from a foodstuff, a means was sought to increase the speed at which these irradiation markers could be isolated while at the same time decreasing the amount of organic solvents required. Thus, the technique of supercritical fluid extraction (SFE) was investigated. Results showed that SFE can be used for the rapid extraction (60 min) of lipid from irradiated foods such as chicken, pork, liquid whole egg, ground beef, and from the seeds of irradiated mango and papaya with only 10 mL n-hexane being necessary for collection of the extracted sample. A method was also developed whereby the 2-alkylcyclobutanones can be selectively extracted from irradiated foods without prior extraction of the lipid. The sample extract, in 10 mL n-hexane, is purified through a Florisil SPE cartridge which is washed with 10 mL n-hexane and the 2-alkylcyclobutanones eluted with 10 mL 2% diethyl ether in n-hexane before analysis by gas chromatography/mass spectrometry. 2-Dodecylcyclobutanone and 2-tetradecylcyclobutanone were selectively extracted from irradiated chicken meat, liquid whole egg, ground beef, and mango as well as from beef burgers and baked products containing irradiated ground beef and liquid whole egg, respectively. Using this method, samples can be analyzed for irradiation treatment within 6 h as opposed to the 2-day period required for the EN1785/MAFF V37 validated method.     

RESISTANCE OF PIGMENTED HUMAN CELLS TO KILLING BY SUNLIGHT AND OXYGEN RADICALS

Solar irradiation of a panel of human cell lines revealed three phenomena relevant to understanding the biological role of melanin; a heavily melanised melanoma line (MM418) was considerably more resistant to solar killing compared with HeLa and amelanotic melanoma cells of similar size and DNA content; MM418 cells were also resistant to killing by artificial UVB and by hydrogen peroxide generated in situ with extracellular glucose oxidase; and no difference in survival between the cell lines was found using 254 nm UV or gamma radiation. MM418 cells were resistant to sunlight when irradiated as attached monolayers but not when irradiated in suspension. Further studies showed that resistance to solar radiation in MM418 cells was not due to less DNA damage, as judged by inhibition of semiconservative DNA synthesis, or to enhanced constitutive or induced repair determined by reactivation of irradiated adenovirus. These results indicate that melanisation protects human cells from solar UVB in vitro and that the mechanism is associated with protection from hydrogen peroxide-type damage rather than direct shielding of DNA.     

Capillary electrophoretic study of the synergistic biological effects of alkaloids from Chelidonium majus L. in normal and cancer cells

In this study, the synergistic biological action of five celandine alkaloids in normal and cancer cells was investigated by capillary electrophoresis with light-emitting diode-induced native fluorescence detection. The specific capacity of each alkaloid to penetrate into the cells was estimated by monitoring alkaloid concentration decreases in the cell medium during incubation with murine fibroblast NIH/3T3, mouse melanoma B16F10, and human breast cancer MCF7 cell lines. Mixtures of isoquinoline alkaloids containing protopine, chelidonine, sanguinarine, allocryptopine, and stylopine were applied to cell cultures for 20 and 40 min, and the content of alkaloids in the cell media was measured by capillary electrophoresis (CE). CE separation of isoquinoline alkaloids was performed in 30 mM phosphate buffer (pH 2.5). As these alkaloids have native fluorescence, they were directly detected using the commercially available UV light-emitting diode without troublesome fluorescent derivatization. The results showed a differential ability of celandine alkaloids to penetrate into the normal and cancer cell interior, which was inversely proportional to their cytotoxic activity. While the most effective transport of celandine alkaloids from the cell medium to the cell interior was observed for normal murine fibroblast NIH/3T3 cells (about 55% of total content), cytotoxicity tests demonstrated selective and profound apoptotic effects of a five-alkaloid combination in the mouse melanoma B16F10 cell line.     

Effects of ultraviolet irradiation on melanogenesis from tyrosine, Dopa and dopamine: a matrix-assisted laser desorption/ionization mass spectrometric study

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry experiments were applied to study the influence of ultraviolet (UV) irradiation in melanogenesis. Samples were prepared starting from three different precursors, tyrosine, Dopa and dopamine, in the presence or absence of tyrosinase, the enzyme responsible for the synthesis of melanin. Enzymatic reactions were carried out for 10, 30, 60 and 120 min under UV irradiation at 365 nm, and aliquots were then immediately ultrafiltered and lyophilized. Samples obtained by irradiation of tyrosine solution revealed the formation of 5,6-dihydroxyindole (DHI) oligomers up to pentamers at 120 min; the reaction kinetics were markedly enhanced in the presence of tyrosinase. In the case of Dopa, UV irradiation favored melanogenesis only in the presence of the enzyme; in this case, many reaction pathways were activated, originating various oligomeric species of Dopa, DHI and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Conversely, when dopamine was used as tyrosinase substrate under UV light, mechanisms of melanogenesis different from those generated by simple enzymatic reaction without irradiation were not activated, as the same oligomeric species were present.     

Wavelength-specific activation of MAP kinase family proteins by monochromatic UV irradiation

The depletion of stratospheric ozone causes related increase in UV light below about 310 nm, which significantly affects biological and ecological systems. To understand the wavelength-specific effects of UV light, Molt4 cells (human T lymphoma cells) were irradiated with a series of monochromatic UV lights and the activities of three members of the mitogen-activated protein (MAP) kinase group were examined. Extracellular signal-regulated kinase was specifically activated within 1 min after UV irradiation in the range 320-360 nm. In contrast, P38 kinase was activated by 270-280 nm light with a peak at 1 min after irradiation. c-Jun N-terminal kinase activation was observed in a narrow range of UV light with a sharp peak at 280 nm occurring in 10 min. JNK translocated from the cytosol to the nucleus upon irradiation, while P38 remained in the cytosol even after UV irradiation. The activation of three MAP kinases was prevented by antioxidant reagents, suggesting that an oxidative signal initiates these responses. These results confirm that UV light affects various cellular functions through the activation of intracellular signaling systems including MAP kinase family proteins. However, the UV-induced activities of the separate MAP kinases show distinctly different dose, time and wavelength dependencies.     

Simultaneous Determination of Cadmium,Copper, Lead and Zinc in Amino Acid Parenteral Nutrition Solutions by Anodic Stripping Voltammetry and Sample Digestion by UV Irradiation

Abstract

Irradiation with ultraviolet (UV) energy was investigated to assay cadmium, copper, lead, and zinc by anodic stripping voltammetry (ASV) in amino acid parenteral nutrition (PN) solutions. Sample digestion by UV irradiation showed the best performances to liberate the metals from the samples (metal recoveries between 90% and 102%) in comparison with classical oxidative wet digestion methods. The best UV digestion condition was obtained with 1:10 diluted PN samples irradiated during 10 h at 90±3°C with the addition of one aliquot of 50 µL concentrated H₂SO₄ and repeated additions of 50 µL 30% (v/v) H₂O₂ at each 60 min irradiation interval. By using the UV digestion procedure cadmium, copper, lead, and zinc were simultaneously assayed in commercial amino acid PN solutions by ASV. The metal concentrations ranged between 1.3 to 4.4 for cadmium, 2.9 to 40.8 for copper, 4.4 to 16.8 for lead, and 1.4 to 208.5 for zinc. The ASV method correlated well with atomic absorption spectrometry measurements to assay the investigated analytes in amino acid PN samples after the UV digestion.     

Effects of gamma radiation on codling moth, Cydia pomonella (L.), eggs

The radiosensitivity of codling moth, Cydia pomonella (L.), eggs in different stages of development was studied. Eggs ranging in age from 1–24 to 97–120 h were exposed, at 24 h intervals, to gamma radiation doses ranging from 10 to 350 Gy. The effects of gamma radiation on egg hatch, pupation and adult emergence was examined. Results showed that the radiosensitivity of codling moth eggs decreased with increasing age. Egg hatch in 1–24 h old eggs was significantly affected at 20 Gy dose and at 60 Gy dose, egg hatch decreased to about 1%. At the age of 25–48 h, however, egg hatch at 60 Gy dose was about 10%, and egg sensitivity to gamma irradiation decreased significantly in the 49–72 h age group; 60 Gy dose had no significant effect on egg hatch. Eggs irradiated few hours before hatch (at the blackhead stage), were the most resistant ones; 100 Gy had no significant effect on egg hatch and at 350 Gy dose over 56% of the eggs hatched. When adult emergence was used as a criterion for measuring effectiveness, however, the effect of gamma radiation was very sever. A dose of 60 Gy completely prevented adult emergence and at 100 Gy dose all resulted larvae died before pupation.     

THE EFFECT OF ULTRAVIOLET LIGHT ON THE CYCLIC NUCLEOTIDE SYSTEM OF HUMAN FIBROBLASTS

Abstract— The concentrations of cyclic AMP and cyclic GMP in human skin fibroblasts in culture were determined after exposing the cells to varying fluences of UV (254 nm) light. The cyclic nucleotide concentrations of cells irradiated in the log phase of growth were unchanged relative to controls. In contrast, there was a rise in the concentration of cyclic AMP in cells irradiated after they reached confluency. The increase in concentration was observed as early as 30 min after irradiation, reached a maximum of about 200% of control at 4 to 6 h after exposure, and returned to control values by 24 h after irradiation. The effect was proportional to a UV fluence from 5 to 20 J/m², and was blocked by the addition of the UV absorbing agent para-aminobenzoic acid. In contrast, our results indicated that UV light had no effect on the concentration of cyclic GMP in human fibroblast cell cultures. Because of the importance of cyclic nucleotides in the regulation of cellular function, it is reasonable to hypothesize that changes in cyclic AMP induced by UV light may affect the extranuclear functions of irradiated cells.     

The Relative UV Sensitizer Activity of Purified Advanced Glycation Endproducts

Abstract— The oxidation products of ascorbic acid react with lens proteins to form advanced glycation endproducts (AGE) that are capable of generating reactive oxygen species when irradiated with UVA light. L-Threose, the most active of these oxidation products, was reacted with N -acetyl lysine and six AGE peaks were isolated by RP-HPLC. Each peak exhibited fluorescence and generated superoxide anion and singlet oxygen in response to UV light. Solutions of these AGE peaks (50 μg/mL) generated5–10 nmol/mL of superoxide anion during a 30 min irradiation. This activity was 100-fold less than the superoxide anion generated by kynurenic acid and 400-fold less than riboflavin.
Ultraviolet irradiation generated from 1.2 to 2.7 μmol/mL of singlet oxygen with the purified threose AGE compounds. This activity was similar to that seen with other purified AGE compounds (pentosidine, LM-1 and Ac-FTP) and with kynurenine and 3-OH kynurenine. This considerable singlet oxygen formation, however, was still 40-fold less than that obtained with kynurenic acid and 100-fold less than riboflavin under the same irradiation conditions. In spite of this lower sensitizer efficiency, the purified AGE generated20–60-fold more singlet oxygen on a weight basis than either crude ascorbic acid glycated proteins or a preparation of water-insoluble proteins from aged normal human lenses. On a molar basis, therefore, AGE could account for the sensitizer activity in these protein preparations if they represented less than 1% of the total amino acids.     

Structural changes in self-assembled monolayers initiated by ultraviolet light

Self-assembled monolayers of 2-anthracenethiol and 2-naphthalenethiol on gold (111) were irradiated with low-power UV light. Scanning tunneling microscope images recorded in situ show unusual structural changes. In the case of 2-anthracenethiol, structures measuring 4-7 nm wide and 30-40 nm in length are formed. Images taken 10 min after irradiation ceased to show further surface reorganization. With 2-naphthalenethiol SAMs, smaller structures form upon irradiation, which subsequently revert to resemble the original structure after time.     

Oxygen evolution loss and structural transitions in photosystem II induced by low intensity UV-B radiation of 280 nm wavelength

UV-B radiation of 280 nm wavelength (UV280) and low intensity (2.0 W/m2) gives rise to an important oxygen evolution (OE) loss in photosystem II (PSII) particles isolated from the thylakoid membrane of plant chloroplasts on the one hand, and to structural changes, or transitions, in the proteins of the PSII complex on the other hand. The latter UV280 effect was studied in this work by Fourier transform infrared (FT-IR) spectroscopy. First, irradiation of the PSII particles with UV280 for about 40 min causes an almost complete loss of OE activity. The remaining OE after 15, 20, 30 and 40 min is respectively 52, 44, 27 and 12% of the OE activity in control PSH particles kept in darkness. Secondly, difference FT-IR spectra of PSII particles irradiated for 30 min, i.e., [PSII irradiated with UV280]-minus-[PSII non-irradiated], show that the UV280 light is at the origin of significant IR absorbance changes in several spectral regions: (i) amide I (1696-1620 cm(-1)) and amide II (1580-1520 cm(-1)), (ii) tyrosine side chain (1620-1580 cm(-1) and 1520-1500 cm(-1), i.e., the v8a, v8b and v19a vibrational modes, respectively), and (iii) chlorophylls (1750-1696 cm(-1)). Thirdly, comparison of the UV-B effect reported here with structural changes induced by heat-stress in PSII proteins [M. Joshi, M. Fragata, Z. Naturforsch. 54c (1999) 35-43] clearly indicates that the stability of the functional centers in the PSII complex is dependent on a dynamic equilibrium between a-helix conformers and extended chain (beta-strand) structures. In this framework, transient 'alpha-helix-to-beta-strand transitions' are susceptible of giving rise in vivo to recurrent changes in the activity of the PSII complex, and as such act as a control mechanism of the photosynthetic function in the thylakoid membrane.     

多钼酸盐–柠檬酸复合膜的多色光致变色

制备了多钼酸盐–柠檬酸光致变色复合膜，紫外光照后发现不同摩尔比的复合膜呈现不同的颜色。当摩尔比为1.0，0.3和0.2时，变色后的薄膜分别显深蓝色，深黄褐色和淡海绿色。通过对薄膜的拉曼光谱分析证实呈现不同的颜色是由于在变色后的膜中生成了不同的物种。柠檬酸在光致变色过程中起着重要的作用, 在紫外光的照射下它作为空穴的捕获剂, 抑制了光生电子和空穴的复合, 使多钼酸盐呈现紫外光致变色现象。