Oxidative DNA damage in cucumber cotyledons irradiated with ultraviolet light

DNA was isolated from the cotyledons of cucumber seedlings irradiated with ultraviolet (UV)-C (254 nm) or UV-B+UV-A (280–360 nm; maximum energy at 312 nm) at various fluence rates and durations. Following enzymatic hydrolysis of DNA, the content of 8-hydroxy-2-deoxyguanosine [(8-OHdG), 8-oxo-7,8-dihydro-2-deoxyguanosine], a well-established biomarker closely identified with carcinogenesis and aging in animal cells, was determined using a high-performance liquid chromatograph equipped with an electrochemical detector. The levels of 8-OHdG increased with UV-C and UV-B irradiation in a fluence-dependent manner. This increase was also observed in etiolated cotyledons that had been excised from dark-grown cucumber seedlings and then cultured in vitro under UV light: monochromatic UV light at 270 nm or 290 nm increased the 8-OHdG level considerably, while UV at wavelengths above 310 nm had only small effects. In situ detection of H₂O₂ and quantification of H₂O₂ in plant extracts revealed that H₂O₂ accumulated in cotyledons irradiated with UV light. These results suggest that UV irradiation induces oxidative DNA damage in plant cells.     

A molecular hybrid producing simultaneously singlet oxygen and nitric oxide by single photon excitation with green light

Combination of photosensitizers (PS) for photodynamic therapy with NO photodonors (NOPD) is opening intriguing horizons towards new and still underexplored multimodal anticancer and antibacterial treatments not based on “conventional” drugs and entirely controlled by light stimuli. In this contribution, we report an intriguing molecular hybrid based on a BODIPY light-harvesting antenna that acts simultaneously as PS and NOPD upon single photon excitation with the highly biocompatible green light. The presented hybrid offers a combination of superior advantages with respect to the other rare cases reported to date, meeting most of the key criteria for both PSs and NOPDs in the same molecular entity such as: (i) capability to generate ¹O₂ and NO with single photon excitation of biocompatible visible light, (ii) excellent ¹O₂ quantum yield and NO quantum efficiency, (iii) photogeneration of NO independent from the presence of oxygen, (iv) large light harvesting properties in the green region. Furthermore, this compound together with its stable photoproduct, is well tolerated by both normal and cancer cells in the dark and exhibits bimodal photomortality of cancer cells under green light excitation due to the combined action of the cytotoxic ¹O₂ and NO.     

Photocytotoxicity and near-IR light DNA cleavage activity of oxovanadium(IV) Schiff base complexes having phenanthroline bases

Oxovanadium(IV) complexes [VO(L)(B)] (1-3), where H₂L is a Schiff base ligand 2-(2-hydroxybenzylideneamino)phenol and B is 1,10-phenanthroline (phen for 1), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq for 2) or dipyrido[3,2-a:2′,3′-c]phenazine (dppz for 3), have been prepared, characterized and their DNA binding property and photo-induced DNA cleavage activity studied. Complex 3 which is structurally characterized by X-ray crystallography shows the presence of an oxovanadium(IV) moiety in a six coordinate VO₃N₃ coordination geometry. The complexes show a d-d band within 800-850 nm in DMF. The complexes display an oxidative response near 0.7 V versus SCE for V(V)-V(IV) and a reductive response within −1.1 to −1.3 V due to V(IV)-V(III) couple in DMF-0.1 M TBAP. The complexes are avid binders to calf thymus DNA giving binding constant values of 4.2 × 10⁴ to 1.2 × 10⁵ M⁻¹. The complexes do not show any “chemical nuclease” activity in dark. The dpq and dppz complexes are photocleavers of plasmid DNA in UV-A light of 365 nm via ¹O₂ pathway and in near-IR light (752.5 to 799.3 nm IR optics) by HO^• pathway. Complex 3 exhibits significant photocytotoxicity in visible light in HeLa cells giving IC₅₀ value of 13 μM, while it is less toxic in dark (IC₅₀ = 97 μM).     

Fluorescent somatostatin receptor probes for the intraoperative detection of tumor tissue with long-wavelength visible light

Targeted fluorescent dyes are of substantial value for the intraoperative delineation of primary tumors and metastatic lesions. For this purpose long-wavelength red light (lambda=550-650 nm) offers advantages because of good tissue penetration and direct visibility. Since somatostatin receptors (SSTR) are overexpressed in a number of tumors, a series of potentially tumor-selective peptide-dye conjugates were synthesized by solid-phase peptide synthesis (SPPS). The octapeptides octreotate, Tyr(3)-octreotate and Tyr(3)-octreotide were employed and exhibited high affinity for somatostatin receptors (SSTR). The fluorescent dyes rhodamine 101, sulforhodamine B acid chloride, sulforhodamine 101 or rhodamine B isothiocyanate were conjugated either directly or via spacers, for example the peptidase-labile pentapeptide sequence Ala-Leu-Ala-Leu-Ala. The conjugates were completely assembled on the solid support: Fmoc-SPPS, cyclization via a disulfide linkage, N-terminal attachment of a spacer, and linkage to the fluorescent dye. An in vitro competition assay revealed that the conjugates bind to SSTRs with IC(50) values between 0.7 and 89 nM. The conjugates were generally stable to hydrolysis at pH 7-8 in buffer or serum. However, the rhodamine 101 conjugates revealed a loss of absorption at alkaline pH due to conversion to a neutral spirolactam form, as characterized by NMR.     

Iron release analyses from ferritin by visible light irradiation

We investigated the iron release from ferritin by irradiation from a white fluorescent light in the absence or presence of ADP. Irradiation of a ferritin solution at 17,000 lx in the absence of ADP slightly induces iron release from ferritin but only at acidic pH conditions (pH 5.0 or pH 6.0). Irradiation in the presence of ADP markedly enhances iron release from ferritin under the same conditions. In the absence of irradiation, the iron release from ferritin was low even in the presence of ADP. The induction of the iron release by irradiation in the presence of ADP was also affected by various factors such as irradiation dose and acidity, but not temperature (4-47°C), oxygen concentration, or free radical generations during the irradiation. The iron release during the irradiation ceased to increase by turning off the light and was found to increase again after additional irradiation. These results suggest that visible light directly induces iron release from ferritin via the photoreduction of iron stored inside ferritin.     

Oxidative cleavage of DNA by tridentate copper (II) complex

Copper (II) complex 1 having planar tridentate ligand, bzimpy, where bzimpy is 2,6-bis(benzimidazo-2-yl) pyridine was synthesized and characterized by UV-visible, FAB (fast atom bombardment) mass and infrared spectroscopy. From absorption titration data, the binding constant of Cu(II) with DNA was calculated to be (1.8+/-0.02)x10(4) M(-1). Thermal denaturation study of DNA with 1 revealed deltaT(m) of 5+/-0.5 degrees C. Viscosity measurement showed that complex binds with DNA through intercalative mode. Copper (II) complex induces cleavage in plasmid DNA in the presence of coreductants such as ascorbic acid or glutathione.     

Proliferation and tumorigenity of murine hepatoma cells irradiated with polychromatic visible and infrared light

In experiments in vitro, the effects of polychromatic visible (VIS) light combined with polychromatic infrared light (VIS-IR, 480–3400 nm) and the effects of the entire spectrum of VIS radiation (385–750 nm) on viability and proliferative activity of the murine hepatoma cells MH22a were studied. In experiments in vivo, changes in the tumorigenic properties of cells MH22a were studied after the same kinds of light exposure. It was shown that irradiation of hepatoma cells with two kinds of polychromatic light at a wide range of doses (4.8–38.4 J/cm²) did not lead to an increase in the number of dead cells for 24–72 h of cultivation and did not cause deceleration of the hepatoma cell proliferation; moreover, the VIS-IR light at a dose of 4.8 J/cm² and the VIS light at a dose 38.4 J/cm² even promoted more intense cell proliferation after 24 h. In cells irradiated with VIS-IR and VIS light, the proliferation index rose by 1.6 and 1.4 times, respectively, and the time of the cells’ number doubling decreased as compared with control. Studying the tumorigenic properties of irradiated tumor cells has shown that, for 30 days after transplantation to syngenic mice C3HA of hepatoma cells 24 h after their irradiation with VIS-IR light at a dose of 4.8 J/cm², the tumor volume decreased significantly (2.6–4.1 times) at all periods of observation, while the incidence of tumor formation decreased, whereas the survival of the tumor-bearing mice did not change. Transplantation of cells irradiated with the same light at a dose of 9.6 J/cm² did not lead to significant changes in the tumor volume, the tumor formation incidence, and animal survival. The main contribution to the antitumor effect of VIS-IR light seems to be made by the VIS component, as transplantation into mice of cells irradiated with VIS light alone at a dose of 38.4 J/cm² also stimulating proliferation of hepatoma cells in vitro resulted in a decrease of their tumorigenic properties. However, the IR component in the combined VIS-IR radiation enhanced the antitumor effect of the VIS light; as a result, it was manifested after use of doses eight times lower (4.8 J/cm²) than in the case of VIS light alone (38.4 J/cm²). Mechanisms of the decrease of tumorigenic properties of hepatoma cells after irradiation with polychromatic light at doses stimulating their proliferation in vitro are studied.     

Oxidative cleavage of DNA mediated by hybrid metalloporphyrin-ellipticine molecules and functionalized metalloporphyrin precursors

The nuclease activity of functionalized metalloporphyrins 1-8 and hybrid metalloporphyrin-ellipticine molecules 10-16 in the presence of potassium monopersulfate (KHSO5) or magnesium monoperoxyphthalate (MMPP), water-soluble oxygen atom donors at physiological pH, toward double-stranded phi X174 DNA is reported. The DNA cleavage efficiency as a function of the nature of functionalized metalloporphyrins, the length of the linkage between the two parts of the hybrid molecule, viz., metalloporphyrin and 9-methoxyellipticine, the nature of the central metal atom (Mn, Fe, or Zn) the ionic strength, and the nature of the oxygen donor has been studied. Single-strand breaks (SSBs) are observed on double-stranded DNA with a short incubation time of 2 min in the presence of manganese derivatives of both metalloporphyrins and hybrid molecules. Owing to their cytotoxic and nuclease activity, these new water-soluble hybrid molecules may be considered as efficient bleomycin models based on cationic metalloporphyrins.     

Mediation of 8-hydroxy-guanine formation in DNA by thiazin dyes plus light

We have discovered that methylene blue plus light mediates the formation of 8-OHdG in DNA. Methylene blue is one of several thiazin dyes and we report here that the other thiazin dyes tested, in combination with white light, are effective in mediating 8-OHdG formation in DNA. The effectiveness of light plus the thiazin dyes in forming 8-OHdG in DNA were as follows: methylene blue greater than azure B greater than azure A greater than toluidine blue greater than thionin. Two other compounds tested; riboflavin and fuschin acid, in combination with light, caused formation of very little, if any, 8-OHdG in DNA. Thiazin dye mediated formation of 8-OHdG in DNA was not inhibited by the spin trap alpha-phenyl-t-butyl nitrone, which supports our previous observations that oxygen free radical scavengers did not inhibit methylene blue plus light mediated 8-OHdG formation in DNA. Ascorbate addition to methylene blue plus DNA, in the absence of light, was ineffective in mediating 8-OHdG formation in DNA.     

Oxidative DNA cleavage by Schiff base tetraazamacrocyclic oxamido nickel(II) complexes

Nickel is considered a weak carcinogen. Some researches have shown that bound proteins or synthetic ligands may increase the toxic effect of nickel ions. A systematic study of ligand effects on the interaction between nickel complexes and DNA is necessary. Here, we compared the interactions between DNA and six closely related Schiff base tetraazamacrocyclic oxamido nickel(II) complexes NiL(1-3a,1-3b). The structure of one of the six complexes, NiL(3b) has been characterized by single crystal X-ray analysis. All of the complexes can cleave plasmid DNA under physiological conditions in the presence of H(2)O(2). NiL(3b) shows the highest DNA cleavage activity. It can convert supercoiled DNA to nicked DNA then linear DNA in a sequential manner as the complex concentration or reaction time is increased. The cleavage reaction is a typical pseudo-first-order consecutive reaction with the rate constants of 3.27+/-0.14h(-1) (k(1)) and 0.0966+/-0.0042h(-1) (k(2)), respectively, when a complex concentration of 0.6mM is used. The cleavage mechanism between the complex and plasmid DNA is likely to involve hydroxyl radicals as reactive oxygen species. Circular dichronism (CD), fluorescence spectroscopy and gel electrophoresis indicate that the complexes bind to DNA by partial intercalative and groove binding modes, but these binding interactions are not the dominant factor in determining the DNA cleavage abilities of the complexes.     

Shining light on nanotechnology to help repair and regeneration

Phototherapy can be used in two completely different but complementary therapeutic applications. While low level laser (or light) therapy (LLLT) uses red or near-infrared light alone to reduce inflammation, pain and stimulate tissue repair and regeneration, photodynamic therapy (PDT) uses the combination of light plus non-toxic dyes (called photosensitizers) to produce reactive oxygen species that can kill infectious microorganisms and cancer cells or destroy unwanted tissue (neo-vascularization in the choroid, atherosclerotic plaques in the arteries). The recent development of nanotechnology applied to medicine (nanomedicine) has opened a new front of advancement in the field of phototherapy and has provided hope for the development of nanoscale drug delivery platforms for effective killing of pathological cells and to promote repair and regeneration. Despite the well-known beneficial effects of phototherapy and nanomaterials in producing the killing of unwanted cells and promoting repair and regeneration, there are few reports that combine all three elements i.e. phototherapy, nanotechnology and, tissue repair and regeneration. However, these areas in all possible binary combinations have been addressed by many workers. The present review aims at highlighting the combined multi-model applications of phototherapy, nanotechnology and, reparative and regeneration medicine and outlines current strategies, future applications and limitations of nanoscale-assisted phototherapy for the management of cancers, microbial infections and other diseases, and to promote tissue repair and regeneration.     

Tissue oximetry by diffusive reflective visible light spectroscopy: Comparison of algorithms and their robustness

It is essential to measure tissue oxygen saturation (StO₂) locally and in thin layers of tissue, for example, the bronchial mucosa, skin flaps and small bones. Visible light spectroscopy (VLS) with a shallow penetration depth is suitable method. Although several VLS algorithms have been developed and described, they have not yet been compared to each other. This hinders attempts to compare the clinical results obtained by different algorithms. To address this issue, we compared the algorithms of Harrison, Knoefel, Pittman‐Duling, Sato and our OxyVLS oximeter, which applies the algorithm from Wodick and Lübbers, in a liquid phantom with optical properties of human tissue. We generally observed considerable differences between the algorithms, which were StO₂ dependent. Exceptions were OxyVLS and Sato, showing a high level of agreement with negligible StO₂ dependency. In spite of the considerable deviation between the other algorithms, the difference of StO₂ between them in clinically normal StO₂ was <10%. We did not observe any dependency of the algorithms on hemoglobin content of the phantom or temperature.      

Labeling with fluorescent carbocyanine dyes of cultured endothelial and smooth muscle cells by growth in dye-containing medium

Summary We describe a method for labeling cultured endothelial cells (ECs) and smooth muscle cells (SMCs) by letting the cells grow for three days in culture medium containing a low concentration of the fluorescent carbocyanine dyes DiI and DiO. We show that good labeling can be obtained with considerably lower concentrations (2.5 g/ml) than has previously been described. With optimal concentration the labeling is very strong and seems to label all membranous structures in the cells. It was possible to clearly distinguish differentially pre-labeled cells both in coculture and seeded on denaturated vascular grafts. The cells remain fluorescent for more than seven days and may be passaged with retained proliferative capability. We suggest that DiI/DiO-labeling using dye-containing medium may be used for several cell types and is applicable in tissue culture and in the detection of implanted cells in vivo.     

Bio-CO2 fixation with formate dehydrogenase from Saccharomyces cerevisiae and water-soluble zinc porphyrin by visible light

The CO₂ fixation system, that is formic acid production from CO₂ with FDH from Saccharomyces cerevisiae and reduced Methyl viologen produced by the visible light photosensitization of zinc tetrakis(4-methylpyridyl) porphyrin, was investigated. When the sample solution containing triethanolamine, zinc tetrakis(4-methylpyridyl) porphyrin, Methyl viologen, NaHCO₃ and FDH in phosphate buffer was irradiated, formic acid was produced. The amount of formic acid production and the yield of CO₂ to formic acid were estimated to be 0.104 ± 0.008 mM and 10.4%, respectively.     

Photoinduced H2 production with Mg chlorophyll-a from Spirulina and colloidal platinum by visible light

Photoinduced H₂ production with Mg chlorophyll-a from Spirulina as a visible light photosensitizer by use of a three component system consisting of NADPH as an electron donor, Methyl Viologen as electron relay and colloidal platinum as catalyst was investigated. By using this system, the H₂ production rate was estimated to be 0.70 ± 0.03 × 10^–6 mol h^–1.     

Reduction in accumulation of [3H]triphenylmethylphosphonium cation in neuroblastoma cells caused by optical probes of membrane potential: Evidence for interactions between carbocyanine dyes and lipophilic anions

The accumulation of [³H]triphenylmethylphosphonium cation in neuroblastoma N1E 115 cells in the presence of tetraphenylboron is reduced by 3,3′-diethylthiadicarbocyanine iodide and by 3,3′-dipropylthiadicarbocyanine iodide. This reduction in uptake of the lipophilic cation is not due to the carbocyanine dyes depolarizing the plasma membrane of these cells but due to an interaction between the carbocyanine dyes and tetraphenylboron leaving less of the lipophilic anion free in solution to assist uptake of the lipophilic cation. This interaction is shown to have a 1:1 stoicheiometry.     

The bactericidal effect of ultraviolet and visible light on Escherichia coli

The bactericidal radiation dosages at specific wavelengths in the ultraviolet (UV)-visible spectrum are not well documented. Such information is important for the development of new monochromatic bactericidal devices to be operated at different wavelengths. In this study, radiation dosages required to cause mortality of an Escherichia coli strain, ATCC 25922, at various wavelengths between 250 and 532 nm in the UV and visible spectrum were determined. Radiation at 265 nm in the UV region was most efficient in killing the E. coli cells and 100% mortality was achieved at a dose of 1.17 log mJ/cm(2). In the visible spectrum, the radiation dosages required for a one-log reduction of the E. coli cell density at 458 and 488 nm were 5.5 and 6.9 log mJ/cm(2), respectively. However, at 515 and 532 nm, significant killing was not observed at radiation dosage up to 7 log mJ/cm(2). Based on the cell survival data at various radiation dosages between 250 and 488 nm, a predictive equation for the survival of E. coli cells is derived, namely log(S/S(0)) = -(1.089 x 10(7) e(-0.0633lambda))D. The symbols, S(0), S, lambda, and D, represent initial cell density, cell density after irradiation, wavelength of the radiation and radiation dosage, respectively. The proportion of the surviving E. coli cells decreases exponentially with the increase in radiation dosage at a given wavelength. In addition, the radiation dose required for killing a certain fraction of the E. coli cells increases exponentially as the wavelength of radiation increases.