Biomodulative effects induced by 805 nm laser light irradiation of normal and tumor cells

The influence of light emitted from a diode laser centred at λ = 805 nm was investigated on murine skeletal myotubes (C2), normal urothelial cells (HCV29), human squamous carcinoma cells of the gingival muscosa (ZMK) and urothelial carcinoma cells (J82) in a computer-controlled irradiation chamber. Cells were treated with varying fluences between 0 and 20 J cm⁻². The response was tested by analysis of the mitotic index using single cell counting after Orcein staining and proliferation index based on BrdU incorporation during DNA synthesis. While the mitotic index of C2, HCV29 and J82 cells increased at a fluence of 4 J cm⁻², irradiation with fluences of 20 J cm⁻² resulted in a slight decrease. ZMK tumor cells showed a decrease of the mitotic index with both fluences. No significant differences could be determined when using irradiances between 10 mW cm⁻² and 150 mW cm⁻². The BrdU test after irradiation showed no significant effects compared to the controls in each cell line.     

Different cell death mechanisms are induced by a hydrophobic flavin in human tumor cells after visible light irradiation

Riboflavin (RF) is an endogenous cell component and an efficient photosensitizer that can act by both types I and II photochemical mechanisms. Human tumor cells lines cultured in vitro, were used as model to study the effect of a photosensitizer synthesized from riboflavin, the 2',3',4',5'-riboflavin-tetrabutyrate (RTB), to increase the flavin concentration in the human promyelocytic leukemia cell line HL-60 and the human epithelial cervical cancer cell line HeLa. We demonstrate that this compound, alone or with Trp, has a toxic dose-response effect evidenced by abnormal cell morphology and a decrease in the cell proliferation rate. The mechanism of cell death was investigated and the experimental evidence indicates that it proceeds primarily via apoptosis; however, autophagy cannot be discarded. Nuclear fluorescent staining with Hoechst 33258 and transmission electron microscopy of the cells showed condensed chromatin margination at the nuclear periphery and the formation of apoptotic bodies. Furthermore, Caspase-3 activity was demonstrated in both cell lines. In addition, the characteristic apoptotic DNA ladder was observed in HL-60 cells. On the other hand, a high cytoplasmic vacuolization was observed by electron transmission and confocal microscopy. LysoTraker-red localization in the vacuoles was observed by fluorescence microscopy, and a significant decrease in the number of vacuoles and in the cell proliferation rate diminution was observed when irradiation was performed in the presence of the autophagy inhibitor 3-methyladenine. Considering that both cell death mechanisms have a dual role in the killing of tumor cells in vivo, a harmful effect that does not cause inflammation leading to tumor prophylaxis, we conclude that RTB could have potential clinical applications.     

Bilirubin- and light induced cell death in a murine lymphoma cell line

Cells from the mouse lymphoma cell line L5178Y-R were exposed to blue light from phototherapy lamps in the presence of solutions of 160 microM bilirubin supplemented with serum albumin. HPLC analysis showed that the bilirubin solution was photooxidised as a function of increasing light dose. The cells were stained with trypan blue to score necrosis, and apoptosis was assayed by the terminal deoxynucleotide transferase assay (TdT) or by studying the nuclear structure in cells stained with propidium iodide. A rapidly developing apoptosis was observed after light doses killing 60-80% of the cells as judged from the trypan blue exclusion test. The fraction of apoptotic cells was smaller than the fraction of necrotic cells. Exposure of the cells to fractions of light at a high dose rate was compared to the effect of the same total dose at a lower dose rate given as a single fraction. No large differences were found, however, there was a tendency of a higher degree of necrosis as well as apoptosis in the cells receiving the light in fractions at a high dose rate.     

Calcium oscillations in plant cell cytoplasm induced by red and far-red light irradiation

Light-mediated oscillations of cytoplasmic Ca²⁺ concentration in oat protoplasts were demonstrated using the fluorescence probe quin2 loaded into the cells. The oscillations are not due to an increase in cytoplasmic Ca²⁺ concentration and have no connection with the mitochondrial Ca⁺ pool. A possible association between the Ca⁺ oscillations and phosphoinositide metabolism in plant cell membranes is analysed.     

Proteomic analysis of the cellular proteins induced by adaptive concentrations of hydrogen peroxide in human U937 cells

When cells are first exposed to low levels of oxidative stress, they develop a resistance to a subsequent challenge of the same stress, even at higher levels. Although some protein(s) induced by oxidative stress likely mediated this adaptive response, the nature of these proteins is unknown. In this study, the total proteins extracted from human U937 leukemia cells exposed to 50 micromM H(2)O(2) for 24 h to induce an optimal protective response were analyzed by two-dimensional polyacrylamide gel electrophoresis. H(2)O(2) treatment induced elevation of level of 34 protein spots. An analysis of these spots by a matrix associated laser desorption/ionization time-of-flight mass spectrometry identified 28 of the H(2)O(2)-induced proteins. These include proteins involved in energy metabolism, translation and RNA processing, chaperoning or mediating protein folding, cellular signaling, and redox regulation, as well as a mitochondrial channel component, and an actin-bundling protein. Therefore, it appears that the cellular adaptation to oxidative stress is a complex process, and is accompanied by a modulation of diverse cellular functions.     

Photo-activation of hypericin with low doses of light promotes apparent photo-resistance in human histiocytic lymphoma U937 cells

We have observed that exposure of U937 cells, pre-incubated for 18 h with 0.2 μM hypericin, to 599 nm laser radiation with a fluence of 2.5 J/cm² renders them insensitive to higher light doses. In fact, pre-sensitized cells appear to be fully resistant to light doses that normally determine massive cellular apoptosis in experimental photo-dynamic therapy. The appearance of the photo-resistance, as measured by evaluating the changes in levels of expression of pro and anti apoptotic proteins, PARP fragmentation and cell viability is exclusively observed with exposure to light doses not exceeding 5–6 J/cm². Above this energy, necrosis replaces apoptosis upon photo-stimulation of U937 cells. Here, we describe the appearance of photo-resistance in hypericin-loaded U937 cells, but could not fully unravel the molecular mechanism underlying this process. However, the observed stimulation of the expression of the HSP-70 protein upon photo-induced stress may suggest its involvement in this process.     

Formation of a novel porphyrin-gold nanoparticle network film induced by IR light irradiation

IR light irradiation of a mixed toluene solution of ammonium salt-stabilized gold nanoparticles with 3.8 +/- 0.8 nm core diameter and a porphyrin thioacetate derivative affords a thin photoactive film of the cluster-porphyrin network.     

Asymmetric Stark broadening of the Fe I 538.34 nm emission line in a laser induced plasma

In this work asymmetries along with shifts in the line profiles of neutral iron emission lines coming from a laser induced plasma have been detected. The plasma was produced in air at atmospheric pressure on a 50% Fe–Ni alloy and the emission was collected at a temporal window of (2.5, 3) μs. To avoid the effect of spatial inhomogeneity on the profiles, a deconvolution procedure was applied to obtain the spatially resolved emissivity. Asymmetric theoretical Stark profiles, which take into account the effect of static ions, were used to be fitted to the experimental data of the emission profile of the line Fe I 538.34 nm. The fitting of the theoretical profile to the experimental data was carried out by means of the least squares method using genetic algorithms to automatically solve the optimization problem. The correlation coefficient was higher for the asymmetric fits than for the symmetric ones. From the fit, the quasistatic ion broadening parameter α, the electron broadening parameter w_e, and the total shift of the maximum of the line d_t, were obtained. The ion parameter α varied in a range (0.2–0.3) for an electron density between (4–15) × 10¹⁶ cm^− 3. The ion influence on the total broadening was of 15–20%. The total shift varied in the range (0.01–0.06) nm and it was mainly given by the ion shift, the electron shift being negligible. For the electron density range in this work, approximated linear behaviors of the total width and shift with electron density have been obtained.     

Combined inhibition of HDAC and DNMT1 induces p85α/MEKmediated cell cycle arrest by dual target inhibitor 208 in U937 cells

Herein we reported an efficient dual DNMT and HDAC inhibitor 208 with great antiproliferative activity against U937 cells. Further studies revealed 208 affected the whole proteome profile and could induce G1 cell cycle arrest and apoptosis in U937 cells through upregulating CDK inhibitor p16 and downregulating cyclin-dependent kinases and their activators.     

Blue light irradiation inhibits the production of HGF by human retinal pigment epithelium cells in vitro

Blue visible light damage to retinal pigment epithelial cells occurs through a photooxidative mechanism and the resultant damage is hypothesized to induce or exacerbate age-related macular degeneration. The purpose of the present study was to identify changes in the cell growth and the expression of hepatocyte growth factor (HGF) in cultured human retinal pigment epithelium (RPE) cells as a result of both blue and red light irradiation. HGF is a growth factor and neurotrophic factor that stimulates growth of various ocular cells and promotes the survival of RPE and retinal neurons. Early passages of human RPE cells were exposed to blue light (460 nm) and red light (640 nm). Nonirradiated cells were used as controls. After 24 and 48 h, conditioned medium was collected and the amount of HGF was measured by ELISA. Cells were detached from the well and counted. Cell viability was evaluated by trypan-blue exclusion study. Blue light at dosage of 63 J/cm(2) significantly inhibited the growth of RPE cells without affecting of cell viability. Amounts of HGF in the culture medium were significantly inhibited by blue-light irradiation at the dosage from 32 to 63 J/cm(2). Red light at a dose of 174 J/cm(2) causes a nonsignificant inhibition of growth of RPE cells and a slight decrease of secretion of HGF. As HGF promotes survival of RPE cells and retinal neurons, the inhibition of production of HGF by visible light, especially by blue light, may enhance the phototoxic effects of visible light on the RPE and retinal neurons.     

Analysis of a photosensitive lesion induced by sunlamp UV greater than 315 nm exposure of 254-nm-irradiated human cells

Normal human skin fibroblasts were exposed to 0-10 J m-2 of 254 nm UV, incubated 0-16 h and then treated with 0-150 kJ m-2 of sunlamp UV greater than 315 nm. For each treatment, the cells were subjected to alkaline elution in order to measure the yield of single strand breaks (ssb) produced. It was found that treatment of 254-nm-irradiated cells with sunlamp UV greater than 315 nm resulted in the production of a higher level of ssb than that produced by separate exposures. Hence, lesions are produced by the 254 nm irradiation that are photolyzed through exposure to sunlamp UV greater than 315 nm. Approximately 50% of these lesions are removed following a 2-4 h incubation of the 254-nm-irradiated cells and nearly complete removal is achieved by 16 h. In addition, the profiles for elutions performed at pH 12.8 with cells exposed to the combined treatment were indicative of the presence of alkali labile sites. The repair kinetics of this lesion and alkaline lability of the photolysis product suggest that this photosensitive lesion may represent pyrimidine(6-4)pyrimidone photoproducts. Hence, this approach may represent a relatively simple and sensitive assay for the measurement of this DNA damage.     

Humidity-sensitive electrical conductivity of a Langmuir-Blodgett film of titania nanosheets: surface modification as induced by light irradiation under humid conditions

Electrical conductivity of titania nanosheets was investigated for a single-layered Langmuir-Blodgett (LB) film deposited onto a comb-type electrode (5 or 10 microm (electrode spacing) x 8 mm (electrode width)). The photoresponsive electrical properties of the film were investigated by irradiating with a Xe lamp under various atmospheric conditions. The atmosphere was controlled by introducing either oxygen or nitrogen gases containing different amounts of water vapor. As a result, the LB film behaved as an insulator with little photoresponse under dry atmospheric conditions. It became conductive on illuminating with a Xe lamp under a wet oxygen atmosphere. Conductivity increased with the increase of irradiation time (0-30 min) to attain a stationary value in 1 h. The highest conductive state thus attained lasted for several hours in the dark. The impedance of the film was measured over the frequency range of 1 MHz to 50 Hz by varying the relative humidity of an atmosphere from 0 to 100%. The results were analyzed by assuming an equivalent circuit consisting of one resistance (R) with constant Warburg component (W) and one capacitance (C) in parallel. The R component depended remarkably on the relative humidity, while the C component stayed nearly at the constant value. The dependence of R on water vapor (PH2O) was expressed by R = A[PH2O]n with A = constant and n = -2.9. The results were rationalized in terms of the surface modification of titania nanosheets to hydrophilic nature under the illumination of UV light.     

Cytosolic accumulation of gammaH2AX is associated with tropomyosin-related kinase A-induced cell death in U2OS cells

Tropomyosin-related kinase A (TrkA) plays an important role in cell survival, differentiation, and apoptosis in various neuronal and nonneuronal cell types. Here we show that TrkA overexpression by the Tet-On system mimics NGF-mediated activation pathways in the absence of nerve growth factor (NGF) stimulation in U2OS cells. In addition, p53 upregulation upon DNA damage was inhibited by TrkA, and p21 was upregulated by TrkA in a p53-independent manner. TrkA overexpression caused cell death by interrupting cell cycle progression, and TrkA-induced cell death was diminished in the presence of its specific inhibitor GW441756. Interestingly, TrkA-mediated cell death was strongly related to gammaH2AX production and poly (ADP-ribose) polymerase cleavage in the absence of DNA damage inducer. In this study, we also reveal that gammaH2AX production by TrkA is blocked by TrkA kinase inhibitors K-252a and GW441756, and it is also significantly inhibited by JNK inhibitor SP600125. Moreover, reduction of cell viability by TrkA was strongly suppressed by SP600125 treatment, suggesting a critical role of JNK in TrkA-induced cell death. We also found that gammaH2AX and TrkA were colocalized in cytosol in the absence of DNA damage, and the nuclear localization of gammaH2AX induced by DNA damage was partly altered to cytosol by TrkA overexpression. Our results suggest that the abnormal cytosolic accumulation of gammaH2AX is implicated in TrkA-induced cell death in the absence of DNA damage.     

Polymerization induced by electron-beam irradiation of octadecyl methacrylate in the form of a multilayer or monolayer

In continuation of the previous series of studies, polymerization of octadecyl methacrylate (ODMA) induced by electron beams has been investigated in a form of its multilayer or monolayer in an attempt to prepare a stable thin polymer film having a regular layer structure. ODMA multilayers were prepared by the Langmuir-Blodgett technique and irradiated with electron beams from a Van de Graaff accelerator. Multiple reflection infrared spectroscopy and x-ray diffractometry revealed that the ODMA multilayer was polymerized to give a thin polymer film having a regular layered structure when irradiated in nitrogen atmosphere, but no indication of polymerization was observed when irradiated in air. A preliminary study on the ODMA monolayer at a nitrogen–water interface indicated that the monolayer was polymerized.     

Determination of mercury in fish tissue using a minianalyzer based on cold vapor atomic absorption spectrometry at the 184.9 nm line.

A sensitive method was proposed and optimized for the determination of total mercury in fish tissue by using wet digestion, followed by cold vapor atomic absorption spectrometry (CVAAS) at the main resonance line of mercury (184.9 nm). The measurements were made using a new type of a non-dispersive mercury minianalyzer. This instrument was initially designed and built for atmospheric mercury-vapor detection. For determining mercury in aqueous samples, the minianalyzer was linked with a mercury/hydride system, Perkin Elmer Model MHS-10. To check the method, the analyzed samples were spiked with a standard solution of mercury. The recoveries of mercury spiked to wet fish tissue were >90% for 0.5 - 0.8 g samples. The results showed a better sensitivity (about 2.5 times higher) when using the mercury absorption line at 184.9 nm compared with the sensitivity obtained by conventional CVAAS at 253.7 nm.     

Measurements of light scattering changes induced by a strong optical field in solutions of tRNA

Changes in light scattering induced by a strong laser beam, as predicted theoretically by Kielich, were measured for unfractionated yeast transfer ribonucleic acid (tRNA) solutions. The vertically polarized electric field of a strong laser pulse (λ = 1060nm) amounted to 4.5 × 10³ esu cgs; its duration was 10 nsec. A weak incident laser beam (λ = 630nm) was also polarized vertically and the vertical and horizontal intensity components of the light scattered through 90° at the latter wavelength were measured. These measurements together with previous results from measurements of Rayleigh light scattering and light scattering in a magnetic field permitted evaluation of the tensor of third-order polarizability (c = 3 × 10^?30 esu cgs, c = ?373 × 10^?30 esu cgs) and the anisotropy of the third-order polarizability components with its sign (δ_c = +56 × 10^?2, δ_c = +0.25 × 10^?2 for tRNA monomer and aggregate, respectively). The new method described may be useful for studies of macromolecules and macromolecular complexes of biological importance.     

Inter-pulse delay optimization in dual-pulse laser induced breakdown vacuum ultraviolet spectroscopy of a steel sample in ambient gases at low pressure

Time-integrated spatially-resolved Laser Induced Breakdown Spectroscopy (LIBS) has been used to investigate spectral emissions from laser-induced plasmas generated on steel targets. Instead of detecting spectral lines in the visible/near ultraviolet (UV), as investigated in conventional LIBS, this work explored the use of spectral lines emitted by ions in the shorter wavelength vacuum ultraviolet (VUV) spectral region. Single-pulse (SP) and dual-pulse LIBS (DP-LIBS) experiments were performed on standardized steel samples. In the case of the double-pulse scheme, two synchronized lasers were used, an ablation laser (200 mJ/15 ns), and a reheating laser (665 mJ/6 ns) in a collinear beam geometry. Spatially resolved and temporally integrated laser induced plasma VUV emission in the DP scheme and its dependence on inter-pulse delay time were studied. The VUV spectral line intensities were found to be enhanced in the DP mode and were significantly affected by the inter-pulse delay time. Additionally, the influence of ambient conditions was investigated by employing low pressure nitrogen, argon or helium as buffer gases in the ablation chamber. The results clearly demonstrate the existence of a sharp ubiquitous emission intensity peak at 100 ns and a wider peak, in the multi-microsecond range of inter-pulse time delay, dependent on the ambient gas conditions.     

A distinct intermediate of RNase A is induced by sodium dodecyl sulfate at its pK(a)

The chemical denaturation of RNase A was found to be mediated by sodium dodecyl sulfate (SDS) at various pH. The characterization of the unfolding pathway was investigated by spectrophotometry and differential scanning calorimetry (DSC), and was analyzed by multivariate curve resolution (MCR) as a chemometric method. The spectrophotometric titration curve of RNase A upon interaction with SDS indicated a distinct complex intermediate in glycine buffer at pH 3.3. This was accompanied with the catalytic activation of the enzyme and was concurrent with maximum population of the intermediate, determined by MCR. This was confirmed by the DSC profile of RNase A in the presence of SDS, indicated by two transitions in thermal unfolding. The kinetic data on the unfolding process of RNase A upon addition of SDS showed a two-phase pathway under the same conditions. The intermediate appeared at low pH especially at the pK_a of SDS (pH 3.3). These results provide strong evidence of the influence of low pH (around the pK_a of SDS) on the existence of an intermediate upon interaction of RNase A with SDS.     

Chronology of the apoptotic events induced in the K562 cell line by photodynamic treatment with hematoporphyrin and monoglucosylporphyrin

Photodynamic treatment of promyelocytic K562 cells in the presence of a monoglucosylporphyrin or hematoporphyrin leads to a sequence of events recognized as hallmarks of apoptosis: a drop in mitochondrial potential, concurrent with a drop in ATP level and a decrease in cell respiration, translocation of phosphatidylserine of the plasma membrane, DNA fragmentation, appearance of apoptotic bodies and eventually loss of plasma membrane integrity. The chronology of these events is in accordance with sequential events induced by other known proapoptotic agents; in contrast to these agents that induce apoptosis in a restricted part of the cell population, we observed that the entire cell population (apart from a small percentage of cells that endured rapid necrosis during phototreatment) underwent apoptosis after irradiation in the presence of porphyrins. It appears that photodynamic treatment allows the bypass of early apoptotic signals in K562 cells that are otherwise renowned for their resistance to drug-induced apoptosis (A. McGahon, R. Bissonnette, M. Schmitt, K. M. Cotter, D. R. Green and T. G. Cotter, Blood 83, 1179-1187, 1994). Singlet oxygen is believed to be the proximate reactive species generated by porphyrin illumination. Because this molecule reacts with almost every cellular constituent, the way that singlet oxygen or its reactive oxygen species byproducts trigger apoptosis remains to be elucidated.