Role of nitric oxide in the response to photooxidative stress in prostate cancer cells.

A continuous state of oxidative stress during inflammation contributes to the development of 25% of human cancers. Epithelial and inflammatory cells release reactive oxygen species (ROS) and reactive nitrogen species (RNS) that can damage DNA. ROS/RNS have biological implications in both chemoresistance and tumor recurrence. As several clinically employed anticancer drugs can generate ROS/RNS, we have addressed herein how inducible nitric oxide synthase and nitric oxide (iNOS/•NO) affect the molecular pathways implicated in the tumor response to oxidative stress. To mimic the oxidative stress associated with chemotherapy, we used a photosensitizer (pheophorbide a) that can generate ROS/RNS in a controlled manner. We investigated how iNOS/•NO modulates the tumor response to oxidative stress by involving the NF-κB and Nrf2 molecular pathways. We found that low levels of iNOS induce the development of a more aggressive tumor population, leading to survival, recurrence and resistance. By contrast, high levels of iNOS/•NO sensitize tumor cells to oxidative treatment, causing cell growth arrest. Our analysis showed that NF-κB and Nrf2, which are activated in response to oxidative stress, communicate with each other through RKIP. For this critical role, RKIP could be an interesting target for anticancer drugs. Our study provides insight into the complex signaling response of cancer cells to oxidative treatments as well as new possibilities for the rational design of new therapeutic strategies.

Fluorimetric-Based Method to Detect and Quantify Total S-Nitrosothiols (SNOs) in Plant Samples.

Accumulating experimental evidence indicates that S-nitrosylation (technically S-nitrosation) events have a central role in plant biology, presumably accounting for much of the widespread influence of nitric oxide (NO) on developmental, metabolic, and stress-related plant responses. Therefore, the accurate detection and quantification of S-nitrosylated proteins and peptides can be particularly useful to determine the relevance of this class of compounds in the ever-increasing number of NO-dependent signaling events described in plant systems. Up to now, the quantification of S-nitrosothiols (SNOs) in plant samples has mostly relied on the Saville reaction and the ozone-based chemiluminescence method, which lacks sensitivity and are very time-consuming, respectively. Taking advantage of the photolytic properties of S-nitrosylated proteins and peptides, the method described in this chapter allows simple, fast, and high-throughput detection of SNOs in plant samples.

Blue light (λ=453 nm) nitric oxide dependently induces ß-endorphin production of human skin keratinocytes in-vitro and increases systemic ß-endorphin levels in humans in-vivo.

ß-Endorphin exerts a broad spectrum of physiological activity on mood, immune functions, pain management, reward effects, and behavioral stability. ß-Endorphin is produced in certain neurons within the central and peripheral nervous system but also in the skin, especially in response to ultraviolet radiation. In the present study we have investigated the impact of visible blue light at λ = 453 nm (BL) on ß-endorphin production of primary human skin keratinocytes (hKC) in-vitro as well as on systemic ß-endorphin formation of whole-body exposed subjects in-vivo. We found that BL irradiation significantly enhanced both keratinocytic ß-endorphin production of hKC cultures as well as systemic ß-endorphin concentrations in light exposed healthy subjects. Interestingly, in hKC cultures elevated ß-endorphin formation was paralleled by significantly increased levels of non-enzymatically generated nitric oxide (NO), whereas elevated systemic ß-endorphin values of BL-exposed subjects were accompanied by enhanced systemic concentration of bioactive NO-derivates. These findings point to a pivotal role of NO in the molecular mechanism of the observed BL-induced effects, and indeed, exogenously applied NO was able to significantly enhance ß-endorphin production in hKC cultures. Thus, our finding of BL-induced increases in systemic ß-endorphin concentration in-vivo can be plausibly explained by an event sequence comprising 1.) BL-driven non-enzymatic formation of NO in the exposed skin tissue, 2.) systemic distribution of cutaneously produced NO in the form of bioactive nitroso compounds, 3.) a subsequent NO-dependent induction of ß-endorphin synthesis in epidermal keratinocytes, and 4.) probably also a NO-dependent modulation of ß-endorphin synthesis in specialized neurons within the central and peripheral nervous system.

Contact Lenses Delivering Nitric Oxide under Daylight for Reduction of Bacterial Contamination.

Ocular infection due to microbial contamination is one of the main risks associated with the wearing of contact lens, which demands novel straightforward strategies to find reliable solutions. This contribution reports the preparation, characterization and biological evaluation of soft contact lenses (CL) releasing nitric oxide (NO), as an unconventional antibacterial agent, under daylight exposure. A tailored NO photodonor (NOPD) was embedded into commercial CL leading to doped CL with an excellent optical transparency (transmittance = 100%) at λ ≥ 450 nm. The NOPD results homogeneously distributed in the CL matrix where it fully preserves the photobehavior exhibited in solution. In particular, NO release from the CL and its diffusion in the supernatant physiological solution is observed upon visible light illumination. The presence of a blue fluorescent reporting functionality into the molecular skeleton of the NOPD, which activates concomitantly to the NO photorelease, allows the easy monitoring of the NO delivery in real-time and confirms that the doped CL work under daylight exposure. The NO photoreleasing CL are well-tolerated in both dark and light conditions by corneal cells while being able to induce good growth inhibition of Staphylococcus aureus under visible light irradiation. These results may pave the way to further engineering of the CL with NOPD as innovative ocular devices activatable by sunlight.

Protective Effects and Mechanisms of N-Phenethyl Caffeamide from UVA-Induced Skin Damage in Human Epidermal Keratinocytes through Nrf2/HO-1 Regulation.

The skin provides an effective barrier against physical, chemical, and microbial invasion; however, overexposure to ultraviolet (UV) radiation causes excessive cellular oxidative stress, which leads to skin damage, DNA damage, mutations, and skin cancer. This study investigated the protective effects of N-phenethyl caffeamide (K36) from UVA damage on human epidermal keratinocytes. We found that K36 reduced UVA-induced intracellular reactive oxygen species (ROS) production and induced the expression of the intrinsic antioxidant enzyme heme oxygenase-1 (HO-1) by increasing the translocation of nuclear factor erythroid 2⁻related factor 2 (Nrf2). K36 could inhibit the phosphorylation of extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinases (JNK) and reduce UVA-induced matrix metalloproteinase (MMP)-1 and MMP-2 overexpression; it could also elevate the expression of tissue inhibitors of metalloproteinases (TIMP). In addition, K36 ameliorated 8-hydroxy-2'-deoxyguanosine (8-OHdG) induced by UVA irradiation. Furthermore, K36 could downregulate the expression of inducible nitric oxide synthase (iNOS) and interleukin-6 (IL-6) and the subsequent production of nitric oxide (NO) and prostaglandin E2 (PGE2). Based on our findings, K36 possessed potent antioxidant, anti-inflammatory, antiphotodamage, and even antiphotocarcinogenesis activities. Thus, K36 has the potential to be used to multifunctional skin care products and drugs.

Peculiar Effects of Electromagnetic Millimeter Waves on Tumor Development in BALB/c Mice.

The study examined the effects of millimeter electromagnetic waves at a frequency of 130 GHz corresponding to the molecular absorption and radiation spectra of NO and O2 with the total exposition time of 6 h on tumor morphogenesis in 3- and 6-month-old tumor-prone BALB/c mice of both sexes. In experimental mice exposed to electromagnetic radiation, the development of cancer process was slowed down throughout the observation period; moreover, no macroscopic signs of the tumors were revealed. However, in contrast to control mice, experimental animals demonstrated the formation of pathological reactions reflected by hepatic biochemical indices accompanied by the development of dystrophic and microcirculatory alterations in the liver tissue.

Mesoporous nanoplate multi-directional assembled Bi2WO6 for high efficient photocatalytic oxidation of NO.

Herein, a mesoporous nanoplate multi-directional assembled Bi2WO6 architecture was successfully prepared and applied for the photocatalytic removal of NOx pollutants at low concentrations under visible light and simulated solar light irradiation. Bi2WO6-180-C synthesized at a hydrothermal temperature of 180 °C with calcination exhibited an excellent conversion efficiency in the photocatalytic oxidation of gaseous NO. The crystallinity, morphology, specific surface area, pore environment, light absorption, and separation of photogenerated electrons and holes were investigated by various techniques; the excellent photocatalytic performance of Bi2WO6-180-C was attributed to its special hierarchical mesoporous structure with an appropriate pore size and interconnected porous network, which imparted good gas permeability and fast mass transfer of reaction intermediates and final products of NO oxidation. Furthermore, hierarchical mesoporous Bi2WO6 showed excellent photocatalytic durability and reusability.

Effects of different-intensity laser acupuncture at two adjacent same-meridian acupoints on nitric oxide and soluble guanylate cyclase releases in human.

OBJECTIVES: The aim of this study was to detect the influences of LA at nonacupoint and two adjacent acupoints of pericardium meridian on the releases of NO and sGC in 20 healthy subjects. METHODS: Different intensities (12, 24, 48 mW) of infrared laser were used for irradiating Jianshi (PC5), Ximen (PC4) acupoints and nonacupoint for 20, 40 minutes, respectively. Semi-circular tubes were taped to the skin surface and filled with NO-scavenging compound for 20 minutes to capture NO and sGC, which were measured using spectrophotometry in a blinded fashion. RESULTS: As the increase in the intensity of LA stimulation, the levels of NO releases over acupoints all were significantly increased, NO releases in nonacupoints following the same treatment only changed slightly, sGC amounts were observably enhanced over acupoints, but did not any change in nonacupoint area. Different intensities of LA treatments can sensitively affect the NO and sGC releases over acupoints. This indicated that LA-induced releases of the NO and sGC were specific to acupoints. CONCLUSIONS: This is the first evidence reporting that LA induced significant elevations of NO-sGC releases over acupoints, and the enhanced signal molecules contribute to local circulation, which improves the beneficial effects of the therapy.

Effect of the transdermal low-level laser therapy on endothelial function.

The effect of low-level laser therapy (LLLT) on the cardiovascular system is not fully established. Since the endothelium is an important endocrine element, establishing the mechanisms of LLLT action is an important issue.The aim of the study was to evaluate the effect of transdermal LLLT on endothelial function.In this study, healthy volunteers (n = 40, age = 20-40 years) were enrolled. N = 30 (14 female, 16 male, mean age 30 ± 5 years) constituted the laser-irradiated group (LG). The remaining 10 subjects (6 women, 4 men, mean age 28 ± 5 years) constituted the control group (CG). Participants were subjected to LLLT once a day for three consecutive days. Blood for biochemical assessments was drawn before the first irradiation and 24 h after the last session. In the LG, transdermal illumination of radial artery was conducted (a semiconductor laser λ = 808 nm, irradiation 50 mW, energy density 1.6 W/cm(2) and a dose 20 J/day, a total dose of 60 J). Biochemical parameters (reflecting angiogenesis: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), angiostatin; antioxidative status: glutathione (GSH) and the nitric oxide metabolic pathway: symmetric dimethylarginine (SDMA), asymmetric dimethylarginine (ADMA) and L-arginine) were assessed. In the LG, a significant increase in GSH levels and considerable decrease in angiostatin concentration following the LLLT were observed. No significant differences in levels of the VEGF, FGF, SDMA, ADMA were observed.LLLT modifies vascular endothelial function by increasing its antioxidant and angiogenic potential. We found no significant differences in levels of the nitric oxide pathway metabolites within 24 h following the LLLT irradiation.

Effects of cell phone radiation on lipid peroxidation, glutathione and nitric oxide levels in mouse brain during epileptic seizure.

The objective of the this study was to evaluate the effects of cellular phone radiation on oxidative stress parameters and oxide levels in mouse brain during pentylenetetrazole (PTZ) induced epileptic seizure. Eight weeks old mice were used in the study. Animals were distributed in the following groups: Group I: Control group treated with PTZ, Group II: 15min cellular phone radiation+PTZ treatment+30min cellular phone radiation, Group III: 30min cellular phone radiation+PTZ treatment+30min cellular phone radiation. The RF radiation was produced by a 900MHz cellular phone. Lipid peroxidation, which is the indicator of oxidative stress was quantified by measuring the formation of thiobarbituric acid reactive substances (TBARS). The glutathione (GSH) levels were determined by the Ellman method. Tissue total nitric oxide (NOx) levels were obtained using the Griess assay. Lipid peroxidation and NOx levels of brain tissue increased significantly in group II and III compared to group I. On the contrary, GSH levels were significantly lower in group II and III than group I. However, no statistically significant alterations in any of the endpoints were noted between group II and Group III. Overall, the experimental findings demonstrated that cellular phone radiation may increase the oxidative damage and NOx level during epileptic activity in mouse brain.

Effects of short term and long term Extremely Low Frequency Magnetic Field on depressive disorder in mice: Involvement of nitric oxide pathway.

AIMS: Previous reports on the possible effects of Extremely Low Frequency Magnetic Fields (ELF MF) on mood have been paradoxical in different settings while no study has yet been conducted on animal behavior. In addition, it was shown that ELF MF exposure makes an increase in brain nitric oxide level. Therefore, in the current study, we aimed to assess the possible effect(s) of ELF MF exposure on mice Forced Swimming Test (FST) and evaluate the probable role of the increased level of nitric oxide in the observed behavior. MAIN METHODS: Male adult mice NMRI were recruited to investigate the short term and long term ELF MF exposure (0.5 mT and 50 Hz, single 2h and 2 weeks 2h a day). Locomotor behavior was assessed by using open-field test (OFT) followed by FST to evaluate the immobility time. Accordingly, NΩ-nitro-l-arginine methyl ester 30 mg/kg was used to exert anti-depressant like effect. KEY FINDINGS: According to the results, short term exposure did not alter the immobility time, whereas long term exposure significantly reduces immobility time (p<0.01). However, it was revealed that the locomotion did not differ among all experimental groups. Short term exposure reversed the anti-depressant like effect resulting from 30 mg/kg of NΩ-nitro-l-arginine methyl ester (p<0.01). SIGNIFICANCE: It has been concluded that long term exposure could alter the depressive disorder in mice, whereas short term exposure has no significant effect. Also, reversing the anti-depressant activity of L-NAME indicates a probable increase in the brain nitric oxide.

[INFLUENCE OF MILLIMETER-WAVE ELECTROMAGNETIC EMISSION ON NITRIC OXIDE SYNTHESIS DURING VESSEL ENDOTHELIUM AGING IN VITRO].

The applying of millimeter-wave electromagnetic emission (EHF-therapy) is an effective method for various age-related pathologies treatment, among other cardio-vascular diseases. During the EHF-emission of aging human endothelial cell cultures it was obtained changing of NO-synthase (eNOS), endothelin-1, angiotensin-2 and vasopressin expression dependence of irradiation exposition. These data have shown that EHF-emission has activated endothelium functional activity, which can play the important role to search for approaches to treatment of arterial hypertension and atherosclerosis.

Visible light-induced nitric oxide release from a novel nitrobenzene derivative cross-conjugated with a coumarin fluorophore.

Nitric oxide (NO) is a well-known free-radical molecule which is endogenously biosynthesised and shows various functions in mammals. To investigate NO functions, photocontrollable NO donors, compounds which release NO in response to light, are expected to be potentially useful. However, most of the conventional NO donors require harmful ultra-violet light for NO release. In this study, two dimethylnitrobenzene derivatives conjugated with coumarins were designed, synthesized and evaluated as photocontrollable NO donors. The optical properties and efficiency of photo-induced NO release were dependent upon the nature of the conjugation system. One of these compounds, Bhc-DNB (1), showed spatiotemporally well-controlled NO release in cultured cells upon exposure to light in the less-cytotoxic visible wavelength range (400-430 nm).

Single-field slice-imaging with a movable repeller: photodissociation of N2O from a hot nozzle.

We present a new photo-fragment imaging spectrometer, which employs a movable repeller in a single field imaging geometry. This innovation offers two principal advantages. First, the optimal fields for velocity mapping can easily be achieved even using a large molecular beam diameter (5 mm); the velocity resolution (better than 1%) is sufficient to easily resolve photo-electron recoil in (2 + 1) resonant enhanced multiphoton ionization of N2 photoproducts from N2O or from molecular beam cooled N2. Second, rapid changes between spatial imaging, velocity mapping, and slice imaging are straightforward. We demonstrate this technique's utility in a re-investigation of the photodissociation of N2O. Using a hot nozzle, we observe slice images that strongly depend on nozzle temperature. Our data indicate that in our hot nozzle expansion, only pure bending vibrations--(0, v2, 0)--are populated, as vibrational excitation in pure stretching or bend-stretch combination modes are quenched via collisional near-resonant V-V energy transfer to the nearly degenerate bending states. We derive vibrationally state resolved absolute absorption cross-sections for (0, v2 ≤ 7, 0). These results agree well with previous work at lower values of v2, both experimental and theoretical. The dissociation energy of N2O with respect to the O((1)D) + N2¹Σ(g)⁺ asymptote was determined to be 3.65 ± 0.02 eV.

Irradiation by gallium-aluminum-arsenate diode laser enhances the induction of nitric oxide by Porphyromonas gingivalis in RAW 264.7 cells.

BACKGROUND: Low-level laser irradiation promotes cell viability and wound healing in periodontal tissue. However, its effect on periodontal pathogenic bacteria is unknown. The purpose of this study is to investigate the biologic effect of low-level laser irradiation on Porphyromonas gingivalis. METHODS: A murine macrophage cell line (RAW 264.7) was cultured and treated with gallium-aluminum-arsenate (GaAlAs) laser-irradiated P. gingivalis with varying levels of energy fluency. Gene expression of monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), interferon-ß (IFN-ß), and inducible nitric oxide synthase (iNOS) was examined by reverse transcription-polymerase chain reaction. Production of iNOS was determined by Western blot analysis, and nitric oxide (NO) release was assessed using Griess reagent. Flow cytometric analysis was performed to determine the activation of Toll-like receptors (TLRs) in response to P. gingivalis. RESULTS: The laser-irradiated P. gingivalis significantly enhanced messenger RNA and protein levels of iNOS in RAW 264.7. Although the laser irradiation on P. gingivalis did not alter the expression level of MCP-1, IL-6, and IFN-ß, it showed a noticeable effect on NO production in RAW 264.7. Furthermore, the laser-irradiated P. gingivalis accelerated TLR2 activation, but not TLR4 activation. CONCLUSIONS: This study reveals that GaAlAs laser irradiation on P. gingivalis induced iNOS expression at the transcriptional and translation levels and increased NO release in macrophages. Moreover, it is confirmed that this process was mediated specifically by TLR2 activation. These findings suggest that low-level laser irradiation to periodontal pathogenic bacteria could be detrimental to periodontal treatments.

Radiation-induced nitric oxide mitigates tumor hypoxia and radioresistance in a murine SCCVII tumor model.

Tumor hypoxia, which occurs mainly as a result of inadequate tissue perfusion in solid tumors, is a well-known challenge for successful radiotherapy. Recent evidence suggests that ionizing radiation (IR) upregulates nitric oxide (NO) production and that IR-induced NO has the potential to increase intratumoral circulation. However, the kinetics of NO production and the responsible isoforms for NO synthase in tumors exposed to IR remain unclear. In this study, we aimed to elucidate the mechanism by which IR stimulates NO production in tumors and the effect of IR-induced NO on tumor radiosensitivity. Hoechst33342 perfusion assay and electron spin resonance oxymetry showed that IR increased tissue perfusion and pO2 in tumor tissue. Immunohistochemical analysis using two different hypoxic probes showed that IR decreased hypoxic regions in tumors; treatment with a nitric oxide synthase (NOS) inhibitor, L-NAME, abrogated the effects of IR. Moreover, IR increased endothelial NOS (eNOS) activity without affecting its mRNA or protein expression levels in SCCVII-transplanted tumors. Tumor growth delay assay showed that L-NAME decreased the anti-tumor effect of fractionated radiation (10Gy×2). These results suggested that IR increased eNOS activity and subsequent tissue perfusion in tumors. Increases in intratumoral circulation simultaneously decreased tumor hypoxia. As a result, IR-induced NO increased tumor radiosensitivity. Our study provides a new insight into the NO-dependent mechanism for efficient fractionated radiotherapy.

Multi-photon excitation in uncaging the small molecule bioregulator nitric oxide.

Multi-photon excitation allows one to use tissue transmitting near-infrared (NIR) light to access excited states with energies corresponding to single-photon excitation in the visible or ultraviolet wavelength ranges. Here, we present an overview of the application of both simultaneous and sequential multi-photon excitation in studies directed towards the photochemical delivery ('uncaging') of bioactive small molecules such as nitric oxide (NO) to physiological targets. Particular focus will be directed towards the use of dyes with high two-photon absorption cross sections and lanthanide ion-doped upconverting nanoparticles as sensitizers to facilitate the uncaging of NO using NIR excitation.

Selective X-ray-induced NO photodissociation in haemoglobin crystals: evidence from a Raman-assisted crystallographic study.

Despite their high physiological relevance, haemoglobin crystal structures with NO bound to haem constitute less than 1% of the total ligated haemoglobins (Hbs) deposited in the Protein Data Bank. The major difficulty in obtaining NO-ligated Hbs is most likely to be related to the oxidative denitrosylation caused by the high reactivity of the nitrosylated species with O(2). Here, using Raman-assisted X-ray crystallography, it is shown that under X-ray exposure (at four different radiation doses) crystals of nitrosylated haemoglobin from Trematomus bernacchii undergo a transition, mainly in the ß chains, that generates a pentacoordinate species owing to photodissociation of the Fe-NO bond. These data provide a physical explanation for the low number of nitrosylated Hb structures available in the literature.

Modulation of cytokine and nitric oxide production by keratinocytes, epithelial cells, and mononuclear phagocytes in a co-culture model of inflammatory acne.

INTRODUCTION: Ultraviolet B (UVB, 290 nm to 320 nm) has been reported to modulate the cytokine-mediated inflammatory process in various inflammatory skin conditions, including production of TNF-α, IL-1α, IL-6, IL-8, and IL-10. We constructed an in vitro model system involving co-culture of different cell types to study the effect of UVB on the inflammatory process using nitric oxide (NO) and tumor necrosis factor (TNF)-α as markers of inflammation. OBJECTIVE: This study was conducted to quantitatively assess the products secreted by human epithelial keratinocytes in the presence and absence of macrophages/monocytes. METHODS: Cells were exposed to UVB radiation (50 mJ to 200 mJ per cm2) or treated with bacterial lipopolysaccharide (LPS) as stimulator of inflammatory response. Nitric oxide (NO) was measured by modified Griess assay and TNF-α was measured by quantitative ELISA. For the co-culture system, SC monocytes were seeded in a 24-well Transwell tissue culture plate whereas irradiated keratinocytes were seeded in the individual baskets subsequently placed on top of the monocyte cultures, and samples of culture supernatants were collected at 1 to 6 days. RESULTS: When primary human epidermal keratinocytes (NHEK) were irradiated with UVB, a dose-dependent stimulation of TNF-α production was observed (33% to 200% increase). TNF-α production was not changed significantly in SC monocytes/NHEK co-culture. In contrast, when macrophages were irradiated with UVB, significant inhibition of NO production (40% suppression, P<0.001) was seen. CONCLUSION: This improved model of cutaneous inflammation could use multiple cells to study their interactions and to offer convenience, reproducibility, and a closer approximation of in vivo conditions.