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.

Differential modulation of inflammatory markers in plasma and skin after single exposures to UVA or UVB radiation in vivo.

BACKGROUND: Solar light generates inflammatory responses in exposed skin. These effects are generally attributed to UVB light. However, skin is expose d to a huge quantum of UVA photons as UVA is a predominant part of sunlight and the radiation used in tanning beds. We examined the effects of a single exposure to UVA and UVB wavebands on cytokine levels in skin and plasma, myeloperoxidase (MPO) activity, expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in skin. METHODS: Hairless mice were irradiated with either UVA (10 or 20 J/cm²) or UVB (200 or 800 mJ/cm²). The effects were assessed after 4/24 h. Plasma cytokine levels were evaluated using a Bio-Plex cytokine assay. Cytokine, iNOS and COX-2 levels in skin were determined by Western blot. Skin MPO activity was monitored spectrophotometrically. RESULTS: UVB induced up-regulation of interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) and decrease in interleukin-10 (IL-10) mainly after 4 h. In contrast, UVA caused increase in levels of tumor necrosis factor-alpha (TNF-α) and IL-6 after 4 h and up-regulated IL-10 and interleukin-12 (IL-12) after 24 h. The increase in MPO activity from infiltrated leucocytes was observed only in UVB irradiated animals. iNOS was up-regulated 4 h after UVA and UVB treatment. No significant effect on COX-2 expression was detected. CONCLUSIONS: UVA and UVB light affected several inflammatory markers. For individual waveband, changes in plasma parameters did not correlate with those in skin. Thus evaluation of plasma samples cannot simply be replaced by determination in skin specimens and vice versa.

Early changes in L-arginine-nitric oxide metabolic pathways in response to the whole-body gamma irradiation of rats.

PURPOSE: Nitric oxide (NO), a reactive radical, is formed in higher amounts from L-arginine by inducible NO synthase (iNOS) during early response to ionizing radiation presumably as a part of signal transduction pathways. This study investigated the changes in L-arginine-NO metabolic pathways within a 24-hour period after whole-body gamma irradiation of rats with the range of low to supra-lethal doses. MATERIALS AND METHODS: Young adult female Wistar rats received either 0-50 Gy whole-body irradiation or an intraperitoneal injection of bacterial lipopolysaccharide (LPS, 10 mg/kg). Exhaled NO was monitored using chemiluminiscence, nitrite + nitrate (NO(x)) and L-arginine were assayed by high-performance liquid chromatography, and expression of iNOS was determined using Western blot. RESULTS: Irradiation resulted in a dose-dependent increase of plasma NO(x) to maximum levels which were 4-fold higher compared to controls (p < 0.001). The NO(x) levels increased less in the bronchoalveolar lavage fluid (BAL) (1.7-fold, p < 0.001) and liver homogenate (2.5-fold, p < 0.05), respectively, and were dose-independent. Exhaled NO, lung NO(x), plasma and BAL L-arginine, and the expression of iNOS in lung and liver tissues of irradiated rats and controls were similar. LPS caused a considerable increase (p < 0.001) in exhaled NO (61-fold), NO(x) levels (plasma 34-fold, BAL 6-fold, lung 5-fold, liver 4-fold), and in iNOS expression, respectively. CONCLUSION: In contrast to the LPS treatment of rats, the radiation-induced changes in L-arginine-NO metabolic pathways are modest, particularly in the airways and lungs. Noninvasive measurement of exhaled NO within a 24-h period following the exposure of rats to ionizing radiation has no value for biodosimetry.

Nanosecond photoreduction of inducible nitric oxide synthase by a Ru-diimine electron tunneling wire bound distant from the active site.

A Ru-diimine wire, [(4,4',5,5'-tetramethylbipyridine)2Ru(F9bp)]2+ (tmRu-F9bp, where F9bp is 4-methyl-4'-methylperfluorobiphenylbipyridine), binds tightly to the oxidase domain of inducible nitric oxide synthase (iNOSoxy). The binding of tmRu-F9bp is independent of tetrahydrobiopterin, arginine, and imidazole, indicating that the wire resides on the surface of the enzyme, distant from the active-site heme. Photoreduction of an imidazole-bound active-site heme iron in the enzyme-wire conjugate (k(ET) = 2(1) x 10(7) s(-1)) is fully seven orders of magnitude faster than the in vivo process.

In vivo effects of low level laser therapy on inducible nitric oxide synthase.

BACKGROUND AND OBJECTIVE: Low level laser therapy (LLLT) has been demonstrated to modulate inflammatory processes with evidence suggesting that treatment protocol, such as wavelength, total energy, and number of treatments determine the clinical efficacy. In this study, the effects of LLLT mediated by different wavelengths and continuous versus pulsed delivery mode were quantified in a transgenic murine model with the luciferase gene under control of the inducible nitric oxide synthase (iNOS) expression. STUDY DESIGN/MATERIALS AND METHODS: LLLT modulated iNOS gene expressed in the acute Zymosan-induced inflammation model is quantified using transgenic mice (FVB/N-Tg(iNOS-luc)). Here an energy density of 5 J cm(-2) at either 635, 660, 690, and 905 nm in continuous wave mode and at 905 nm for short pulse delivery were evaluated. Age of the animals was determined as additional modulating the inflammatory response and the LLLT efficacy for some treatment protocols. RESULTS: Animals younger than 15 weeks showed mostly reduction of iNOS expression, while older animals showed increased iNOS expression for some LLLT protocols. Intensity and time course of inducible nitric oxide expression was found to not only depend on wavelength, but also on the mode of delivery, continuous, or pulsed irradiation. CONCLUSION: LLLT exhibit different effects in induced inflammatory process according to different wavelengths and wave mode. Upregulation of iNOS gene following 905 nm pulsed wave suggests a different mechanism in activating the inflammatory pathway response when compared to the continuous wave.

The role of nitric oxide in radiation damage.

This study investigated the role of nitric oxide in radiation-induced damage by examining changes in mouse serum nitrate concentrations after irradiation. In addition, the contribution of S-2-aminoethylisothiourea 2HBr (AET) to the mechanisms of radiation damage protection was also clarified. The serum nitrate concentration increased as soon as 1.5 h after irradiation, and after 2.5 to 3.0 h the concentrations were significantly higher compared with normal levels. Normal levels were re-established after 12 h. Post-irradiation serum nitrate concentrations increased dose-dependently with irradiation dose (19.6-31.5 Gy). AET suppressed increases in the serum nitrate concentration following irradiation while 2-mercaptoethylamine HCl (MEA) did not. AET has an inhibitory effect on inducible nitric oxide synthase (iNOS); therefore, the increase in nitric oxide after irradiation may be produced by iNOS. Combined administration of irradiation and lipopolysaccharide (LPS) induced a significant increase in serum nitrate concentration, and a significant decrease in survival rate, compared with irradiation alone. The administration of AET or aminoguanidine increased survival rate following irradiation. In contrast to findings after LPS administration, IL-1beta and IFN-gamma were not determined in serum following irradiation. Existing iNOS is activated by irradiation, and nitric oxide production appears to increase without iNOS induction. Thus, the irradiation-induced increase in nitric oxide may be related to lethal injury.

Nitric oxide increases in the rat retina after continuous illumination.

Continuous illumination (CI) of the retina induces an oxidative stress followed by the degeneration of photoreceptors. This phenomenon may be partially related to the excessive production of nitric oxide (NO). In order to confirm this hypothesis, the aims of this work are to determine NO levels during the illumination of the retina by electron paramagnetic resonance (EPR), and if an increase of NO is found, to characterize the NOS isoform responsible of the increment by using Western blot. Sprague-Dawley rats were continuously illuminated with white light (12,000 lux) for 2, 24, 48 h, 5 and 7 days while control rats were maintained at light/dark cycles of 12/12 h. Using EPR, an increase of NO signal was observed in the light exposed retinas peaking at 24 h of CI. Western blot analysis showed the expression of iNOS in the illuminated retinas with a peak after 24 h of CI, but did not show significant differences of nNOS among illuminated and control retinas. In summary, there is an increase of NO during CI. Further studies will reveal whether this mechanism is responsible for light induced photoreceptor degeneration.

Effects of low-level laser therapy (LLLT) on the nuclear factor (NF)-kappaB signaling pathway in traumatized muscle.

BACKGROUND AND OBJECTIVE: To investigate the effects of low-level laser therapy (LLLT) on nuclear factor kappa B (NF-kappaB) activation and inducible nitric oxide synthase (iNOS) expression in an experimental model of muscle trauma. STUDY DESIGN/MATERIALS AND METHODS: Injury to the gastrocnemius muscle in the rat was produced by a single impact blunt trauma. A low-level galium arsenide (Ga-As) laser (904 nm, 45 mW, and 5 J/cm2) was applied for 35 seconds duration, continuously. RESULTS: Histological abnormalities with increase in collagen concentration, and oxidative stress were observed after trauma. This was accompanied by activation of NF-kappaB and upregulation of iNOS expression, whereas protein concentration of I kappa B alpha decreased. These effects were blocked by LLLT. CONCLUSION: LLLT reduced the inflammatory response induced by trauma and was able to block the effects of reactive oxygen species (ROS) release and the activation of NF-kappaB. The associated reduction of iNOS overexpression and collagen production suggest that the NF-kappaB pathway may be a signaling route involved in the pathogenesis of muscle trauma.

Modulations of VEGF and iNOS in the rat heart by low level laser therapy are associated with cardioprotection and enhanced angiogenesis.

BACKGROUND AND OBJECTIVES: It has been shown previously that low-level laser therapy (LLLT) significantly reduces infarct size following induction of myocardial infarction in rats and dogs. The aim of the present study was to investigate the effect of LLLT on the expression of vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). STUDY DESIGN AND MATERIAL AND METHODS: Myocardial infarction was induced by occlusion of the left descending artery in 87 rats. LLLT was applied to intact and post-infarction. VEGF, iNOS, and angiogenesis were determined. RESULTS: Both the laser-irradiated rat hearts post-infarction and intact hearts demonstrated a significant increase in VEGF and iNOS expression compared to non-laser-irradiated hearts. LLLT also caused a significant elevation in angiogenesis. CONCLUSIONS: It is concluded that VEGF and iNOS expression in the infarcted rat heart is markedly upregulated by LLLT and is associated with enhanced angiogenesis and cardioprotection.

Decrease in salivary secretion by radiation mediated by nitric oxide and prostaglandins.

OBJECTIVE: In the present work, we evaluated the effect of exposing the submandibular glands (SMG) to radiation, studying different functional parameters such as salivary secretion, nitric oxide (NO) production, reactive oxygen species formation, prostaglandin (PGE) content and apoptosis. METHODS: We irradiated rats in the head and neck region with a single dose of gamma-ray radiation of 15 Gy. Two hours after radiation, we measured norepinephrine-induced salivary secretion. After that, the SMG were dissected, and in this tissue, we measured the activity of NO synthase (NOS), the PGE content, the amount of reactive oxygen species, apoptotic cells and mitochondrial inducible NOS (iNOS) expression. RESULTS: We found that radiation decreased salivary secretion when 10 and 30 microg/kg of norepinephrine was administered via the right femoral vein. We observed that iNOS activity was reduced and PGE content increased after radiation in SMG, indicating that NO and PGEs may participate in salivary secretion. The expression of mitochondrial NOS was increased after radiation leading to the formation of large amounts of NO that acts as a proapoptotic signal. In fact, we observed an augmentation in apoptotic cells. In this study, we also observed an increase in lipid peroxidation induced by radiation that may contribute to tissue damage. CONCLUSIONS: Our results indicate that radiation induced a decrease in salivary secretion and SMG iNOS activity, meanwhile the PGE content, the lipid peroxidation and apoptosis increased in the tissue. These modifications decrease salivary secretion.

Modulation of MCP-1 and iNOS by 50-Hz sinusoidal electromagnetic field.

The purpose of this study was to investigate whether overnight exposure to 1 mT-50 Hz extremely low-frequency sinusoidal electromagnetic field (EMF) affects the expression and production of inducible nitric oxide synthase (iNOS) and monocyte chemotactic protein-1 (MCP-1) in human monocytes. RT-PCR and Western blot analysis demonstrate that EMF exposure affects the expression of iNOS and MCP-1 in cultured human mononuclear cells at the mRNA level and protein synthesis. Interestingly, the effects of EMF exposure clearly differed with respect to the potentiation and inhibition of iNOS and MCP-1 expression. Whereas iNOS was down-regulated both at the mRNA level and at the protein level, MCP-1 was up-regulated. These results provide helpful information regarding the EMF-mediated modulation of the inflammatory response in vivo. However, additional studies are necessary to demonstrate that EMF acts as a nonpharmacological inhibitor of NO and inducer of MCP-1 in some diseases where the balance of MCP-1 and NO may be important.

Picosecond photoreduction of inducible nitric oxide synthase by rhenium(I)-diimine wires.

In a continuing effort to unravel mechanistic questions associated with metalloenzymes, we are developing methods for rapid delivery of electrons to deeply buried active sites. Herein, we report picosecond reduction of the heme active site of inducible nitric oxide synthase bound to a series of rhenium-diimine electron-tunneling wires, [Re(CO)3LL']+, where L is 4,7-dimethylphenanthroline and L' is a perfluorinated biphenyl bridge connecting a rhenium-ligated imidazole or aminopropylimidazole to a distal imidazole (F8bp-im (1) and C3-F8bp-im (2)) or F (F9bp (3) and C3-F9bp (4)). All four wires bind tightly (Kd in the micromolar to nanomolar range) to the tetrahydrobiopterin-free oxidase domain of inducible nitric oxide synthase (iNOSoxy). The two fluorine-terminated wires displace water from the active site, and the two imidazole-terminated wires ligate the heme iron. Upon 355-nm excitation of iNOSoxy conjugates with 1 and 2, the active site Fe(III) is reduced to Fe(II) within 300 ps, almost 10 orders of magnitude faster than the naturally occurring reduction.