Effects of extremely low frequency electromagnetic field (ELF-EMF) on catalase, cytochrome P450 and nitric oxide synthase in erythro-leukemic cells.

AIMS: Extremely low frequency electromagnetic fields (ELF-EMFs) are widely employed in electrical appliances and different equipment such as television sets, mobile phones, computers and microwaves. The molecular mechanism through which ELF-EMFs can influence cellular behavior is still unclear. A hypothesis is that ELF-EMFs could interfere with chemical reactions involving free radical production. Under physiologic conditions, cells maintain redox balance through production of ROS/RNS and antioxidant molecules. The altered balance between ROS generation and elimination plays a critical role in a variety of pathologic conditions including neurodegenerative diseases, aging and cancer. Actually, there is a disagreement as to whether there is a causal or coincidental relationship between ELF-EMF exposure and leukemia development. Increased ROS levels have been observed in several hematopoietic malignancies including acute and chronic myeloid leukemias. MAIN METHODS: In our study, the effect of ELF-EMF exposure on catalase, cytochrome P450 and inducible nitric oxide synthase activity and their expression by Western blot analysis in myelogenous leukemia cell line K562 was evaluated. KEY FINDINGS: A significant modulation of iNOS, CAT and Cyt P450 protein expression was recorded as a result of ELF-EMF exposure in both phorbol 12-myristate 13-acetate (PMA)-stimulated and non-stimulated cell lines. Modulation in kinetic parameters of CAT, CYP-450 and iNOS enzymes in response to ELF-EMF indicates an interaction between the ELF-EMF and the enzymological system. SIGNIFICANCE: These new insights might be important in establishing a mechanistic framework at the molecular level within which the possible effects of ELF-EMF on health can be understood.

Nitric oxide synthase-like dependent NO production enhances heme oxygenase up-regulation in ultraviolet-B-irradiated soybean plants.

Heme oxygenase (HO) has antioxidant properties and is up-regulated by reactive oxygen species (ROS) in ultraviolet-B-irradiated soybean plants. This study shows that nitric oxide (NO) protects against oxidative damage and that nitric oxide synthase (NOS)-like activity is also required for HO-1 induction under UV-B radiation. Pre-treatments with sodium nitroprussiate (SNP), a NO-donor, prevented chlorophyll loss, H(2)O(2) and O(2)(*-) accumulation, and ion leakage in UV-B-treated plants. HO activity was significantly enhanced by NO and showed a positive correlation with HO-1 transcript levels. In fact, HO-1 mRNA levels were increased 2.1-fold in 0.8 mM SNP-treated plants, whereas subsequent UV-B irradiation augmented this expression up to 3.5-fold with respect to controls. This response was not observed using ferrocyanide, a SNP inactive analog, and was effectively blocked by 2-(4-carboxyphenil)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a specific NO-scavenger. In addition, experiments carried out in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME) or tungsten, well-known inhibitors of NOS and nitrate reductase, showed that NOS is the endogenous source of NO that mediates HO-1 expression. In summary, we found that NO is involved in the signaling pathway leading to HO-1 up-regulation under UV-B, and that a balance between NO and ROS is important to trigger the antioxidant response against oxidative stress.

Change of nitric oxide concentration in men exposed to a 1.5 T constant magnetic field.

This study was carried out in order to determine nitric oxide (NO) production immediately after a 1.5 T magnetic field 30 min exposure to an experimental group, comprising 33 healthy young male volunteers aged 18-26 years old. In addition, a control group, comprising 30 healthy male volunteers aged 19-26 years old, was not exposed to the magnetic field and their NO levels were also measured. The experimental group was exposed using a magnetic resonance imaging (MRI) apparatus. Nitrite and nitrate concentrations were determined by UV-VIS spectrophotometer. The results, related to the parameters measured in this study, were analyzed by one-way ANOVA. Total nitrite concentration in post-magnetic field samples was found to be higher than in pre-magnetic field samples (P < .05).

Altered nitric oxide synthase and PKC activities in cerebellum of gamma-irradiated neonatal rats.

In this study, we show that one single dose of gamma-irradiation at birth induces an inhibition of the cerebellar calcium dependent nitric oxide synthase (NOS) activity, probably correlated to the motor abnormalities and the disarrangement in the cerebellar cytoarchitecture observed in adult rats. This decrease in calcium dependent NOS activity could be associated with an increased protein kinase C (PKC) activity. PKC inhibition partially restores calcium dependent NOS activity, indicating that PKC activity could be negatively modulating the catalytic activity of calcium dependent NOS. These findings suggest that a decrease in nitric oxide (NO) production and the related increase in PKC activity could be intracellular events that participate in the onset of motor and cerebellar abnormalities induced by postnatal gamma-irradiation at early stages of life.

Photoperiod affects neuronal nitric oxide synthase and aggressive behaviour in male Siberian hamsters (Phodopus sungorus).

Many nontropical animals display physiological and behavioural changes in response to seasonal environmental cues including photoperiod (day length). Male Siberian hamsters (Phodopus sungorus) housed in short photoperiod undergo testicular regression accompanied by reduced circulating testosterone and decreased reproductive behaviour. By contrast to the majority of small mammals studied, aggressive behaviour is elevated in short-day Siberian hamsters when blood testosterone concentrations are not detectable. Because gonadal steroid hormones influence neuronal nitric oxide synthase (nNOS), and this enzyme has been implicated in aggressive behaviour, we hypothesized that nNOS expression would be decreased in short-day male Siberian hamsters and negatively correlated with the display of territorial aggression. Adult male Siberian hamsters were individually housed in either long (LD 16:8 h) or short (LD 8:16 h) photoperiods for 10 weeks. Hamsters were assigned to one of two categories by assessing testicular volume and plasma testosterone values: (i) photoperiodic responsive (i.e. regressed testes and low testosterone concentrations) or (ii) photoperiodic nonresponsive (i.e. testes size and circulating testosterone concentrations equivalent to hamsters maintained in long days). At week 10, aggression was assessed using a resident-intruder test. Latency to initial attack, frequency of attacks and duration of total attacks were recorded during a 10-min aggression trial. Brains were collected immediately after behavioural testing and stained for nNOS expression using immunohistochemistry. All short day-housed hamsters were significantly more aggressive than long-day animals, regardless of gonadal size or testosterone concentrations. Short-day animals, both reproductively responsive and nonresponsive morphs, also had significantly less nNOS-immunoreactive cells in the anterior and basolateral amygdaloid areas and paraventricular nuclei compared to long-day hamsters. Together, these results suggest that seasonal aggression in male Siberian hamsters is regulated by photoperiod, through mechanisms that are likely independent from gonadal steroid hormones.

Use of the radiation-inducible WAF1 promoter to drive iNOS gene therapy as a novel anti-cancer treatment.

BACKGROUND: Inducible nitric oxide synthase (iNOS) gene therapy has been identified as a potential anti-tumour strategy. A major problem common to most gene therapy strategies is targeting of treatment to the tumour volume. In this study we report on the use of the X-ray-inducible WAF1 promoter to achieve targeting of iNOS expression to the tumour volume. METHODS: A WAF1/iNOS/liposome complex was injected directly into RIF-1 and HT29 tumours in mice. A 4 Gy dose of X-rays was applied to induce the WAF1 promoter followed, 8 h later, by treatment doses of 10 or 20 Gy. Tumour volume was measured, and growth curves plotted. RESULTS: Intra-tumoural injection of WAF1/iNOS combined with a priming dose of X-rays to induce the WAF1 promoter, followed by a treatment dose, resulted in sensitiser enhancement ratios of 2.0 and 1.3 in RIF-1 and HT29 tumours, respectively, compared with radiation treatment alone. PCR analysis of organ tissue after intra-tumoural injection of WAF1/iNOS showed that vector sequences were detected in all tissue tested; however, Western blot analysis revealed that iNOS protein levels were significantly increased only in tumour and the surrounding dermal tissue that had been exposed to the 4 Gy inducing dose. CONCLUSIONS: iNOS gene therapy in combination with an inducible promoter results in significant tumour cell radiosensitisation. The WAF1 promoter may be a good candidate for a gene therapy as it is silent in normal tissue yet can be induced by the tumour environment.

Low-intensity ultrasound increases endothelial cell nitric oxide synthase activity and nitric oxide synthesis.

Low-intensity ultrasound (US) increases tissue perfusion in ischemic muscle through a nitric oxide (NO)-dependent mechanism. We have developed a model to expose endothelial cells to well-characterized acoustic fields in vitro and investigate the physical and biological mechanisms involved. Human umbilical vein endothelial cells (HUVEC) or bovine aortic endothelial cells (BAEC) were grown in tissue culture plates suspended in a temperature-controlled water bath and exposed to US. Exposure to 27 kHz continuous wave US at 0.25 W cm(-2) for 10 min increased HUVEC media NO by 102 +/- 19% (P < 0.05) and BAEC by 117 +/- 23% (P < 0.01). Endothelial cell NO synthase activity increased by 27 +/- 24% in HUVEC and by 32 +/- 16% in BAEC (P < 0.05 for each). The cell response was rapid with a significant increase in NO synthesis by 10 s and a maximum increase after exposure for 1 min. By 30 min post-exposure NO synthesis declined to baseline, indicating that the response was transient. Unexpectedly, pulsing at a 10% duty cycle resulted in a 46% increase in NO synthesis over the response seen with continuous wave US, resulting in an increase of 147 +/- 18%. Cells responded to very low intensity US, with a significant increase at 0.075 W cm(-2) (P < 0.01) and a maximum response at 0.125 W cm(-2). US caused minor reversible changes in cell morphology but did not alter proliferative capacity, indicating absence of injury. We conclude that exposure of endothelial cells to low-intensity, low-frequency US increases NO synthase activity and NO production, which could be used to induce vasodilatation experimentally or therapeutically.

[Biological oxidative stress induced by electromagnetic irradiation].

Biological effects of electromagnetic irradiation is a hot topic at present. From the point of oxidative stress in biology, this article reviewed the progress of researches of the effects of electromagnetic irradiation on reactive oxygen species (ROS), antioxidase and antioxidant, nitric oxide synthase (NOS), as well as oxidative stress related cell signal transduction in biology.

Inhibition of the iNOS pathway in inflammatory macrophages by low-dose X-irradiation in vitro. Is there a time dependence?

BACKGROUND: Low radiation doses (< or = 1.25 Gy), if applied 6 h before or after stimulation, are known to inhibit the inducible nitric oxide synthase (iNOS) pathway in inflammatory macrophages in vitro. We therefore investigated the time dependence and the underlying molecular mechanism of this effect, since it may be involved in the clinically observed anti-inflammatory and analgesic efficacy of low-dose radiotherapy. MATERIAL AND METHODS: Metabolic activity, nitric oxide (NO) production, iNOS- and hemoxygenase 1-(HO-1-)protein and -mRNA expression by macrophages in vitro after stimulation with LPS/IFN-gamma (0.1 microg ml(-1)/100 U ml(-1)) were investigated. Irradiation was performed at 6, 4, 2 h before and 0, 2, 4, 6 h after stimulation with doses ranging from 0.3 to 10 Gy. For each group, three independent experiments were performed over a period of 30 h with sampling intervals of 3 h. RESULTS: In stimulated macrophages, metabolic activity was not affected by radiation doses up to 10 Gy. A dose-dependent modulation of the cumulative NO production was observed with significant inhibition by low radiation doses < or = 1.25 Gy) and return to control level and even higher concentrations by higher doses (< or = 5 Gy). The degree of inhibition did not show any significant time dependence within the experimental time window used. The iNOS-mRNA expression 3-18 h following stimulation and subsequent irradiation was not affected by doses < or = 1.25 Gy. The iNOS-protein expression 6-24 h following stimulation and subsequent irradiation was reduced by doses < or = 1.25 Gy. By contrast, neither HO-1-protein nor HO-1-mRNA expression at the same time points was influenced by these low doses. CONCLUSION: The inhibitory interference of low radiation doses with the iNOS pathway in inflammatory macrophages appears to be based on radiation effects on the translational and posttranslational control mechanisms of iNOS activity. However, contrary to our working hypothesis this is not related to radiation-induced induction of HO-1 expression and thereby increased degradation of heme which is essential for iNOS activity. Thus, other posttranslational modifications such as the proteasome degradation pathway might be involved.

A photosensitive vascular smooth muscle store of nitric oxide in mouse aorta: no dependence on expression of endothelial nitric oxide synthase.

(1) Photorelaxation is the reversible relaxation of vascular smooth muscle (VSM) when irradiated with ultraviolet (UV) light resulting from the release of nitric oxide (NO). In this study we characterize the involvement of endothelial nitric oxide synthase (eNOS) in the photorelaxation response of thoracic aorta from endothelial NOS deficient (-/-) and control (C57BL/6j) mice. (2) Cirazoline contracted aortae were repeatedly exposed to 30 s of UV light every 3-4 min. Equal levels of photorelaxation (45+/-2%; n=34) was observed in both strains. (3) 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), K(+), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), 4-aminopyridine (4-AP) and ethacrynic acid significantly reduced the photorelaxation response. In C57BL/6j mice diethyldithiocarbamate (DETCA) also reduced photorelaxation. (4) Control endothelium-intact and -denuded aorta and L-NAME (100 micro M) treated and untreated eNOS (-/-) aortae were repeatedly exposed to UV light for 5 min every 10 min until no photorelaxation response was observed. After 1 h of rest in the dark the vessels showed between 30-70% recovery of the photorelaxation response indicating regeneration of the store in the absence of the endothelium and eNOS. (5) The results of this study suggest that photorelaxation in mouse aorta VSM results from the release of NO from a stable store of RSNOs, which activates soluble guanylate cyclase (sGC), leading to cGMP-dependent relaxation that is partially mediated by an increase in K(V) channel activation and hyperpolarization. In addition, the eNOS isoform is not essential for the formation of the photorelaxation store and a non-NOS source of NO may be involved in the maintenance of this store.

Treatment of experimentally induced transient cerebral ischemia with low energy laser inhibits nitric oxide synthase activity and up-regulates the expression of transforming growth factor-beta 1.

BACKGROUND AND OBJECTIVES: Nitric oxide (NO) has been shown to be neurotoxic while transforming growth factor-beta 1 (TGF-beta1) is neuroprotective in the stroke model. The present study investigates the effects of low energy laser on nitric oxide synthase (NOS) and TGF-beta1 activities after cerebral ischemia and reperfusion injury. STUDY DESIGN/MATERIALS AND METHODS: Cerebral ischemia was induced for 1 hour in male adult Sprague-Dawley (S.D.) rats with unilateral occlusion of middle cerebral artery (MCAO). Low energy laser irradiation was then applied to the cerebrum at different durations (1, 5, or 10 minutes). The activity of NOS and the expression of TGF-beta1 were evaluated in groups with different durations of laser irradiation. RESULTS: After ischemia, the activity of NOS was gradually increased from day 3, became significantly higher from day 4 to 6 (P < 0.001), but returned to the normal level after day 7. The activity and expression of the three isoforms of NOS were significantly suppressed (P < 0.001) to different extents after laser irradiation. In addition, laser irradiation was shown to trigger the expression of TGF-beta1 (P < 0.001). CONCLUSIONS: Low energy laser could suppress the activity of NOS and up-regulate the expression of TGF-beta1 after stroke in rats.

Role of inducible nitric oxide synthase in pulmonary microvascular protein leak in murine sepsis.

The effects of nitric oxide (NO) from calcium-independent NO synthase (iNOS) on microvascular protein leak in acute lung injury (ALI) are uncertain, possibly because of disparate effects of iNOS-derived NO from different cells. We assessed the contribution of iNOS from inflammatory versus parenchymal cells to pulmonary protein leak in murine cecal ligation and perforation-induced ALI. We studied iNOS+/+, iNOS-/-, and two reciprocally bone marrow-transplanted iNOS chimeric mice groups: + to - (iNOS+/+ donor bone marrow-transplanted into iNOS-/- recipient mice) and - to +. Sepsis-induced ALI was characterized by pulmonary leukocyte infiltration, increased pulmonary iNOS activity, and increased pulmonary microvascular protein leak, as assessed by Evans blue (EB) dye. Despite equal neutrophil infiltration, sepsis-induced EB-protein leak was eliminated in iNOS-/- mice and in - to + iNOS chimeras (parenchymal cell-localized iNOS) but was preserved in + to - chimeric mice (inflammatory cell-localized iNOS). EB-protein leak was also prevented by pretreatment with allopurinol and superoxide dismutase. Microvascular protein leak in sepsis-induced ALI is uniquely dependent on iNOS in inflammatory cells with no obvious contribution of iNOS in pulmonary parenchymal cells. Pulmonary protein leak is also dependent on superoxide, suggesting an effect of peroxynitrite rather than NO itself.

Local vasodilator response to mobile phones.

OBJECTIVES: The use of mobile phones with the resulting generation of potentially harmful electromagnetic fields (EMF) is the focus of public interest. Heat generation and the activation of the inducible form of nitric oxide (NO) synthase may be possible causes of the biological effects of EMF exposure. We investigated if a mobile telephone conversation can modify skin temperature, NO, and nasal resistance. METHODS: We studied the effect of an EMF (900 MHz) generated by a commercially available cellular phone during a 30-minute telephone conversation on skin temperature, nasal NO measured by chemiluminescence, and nasal minimal cross-sectional area (MCA) measured by rhinometry. Eleven normal subjects (mean age +/- standard error of mean [SEM], 32 +/- 5 y; 10 male) were studied. RESULTS: There was a similar and significant increase in skin temperature of the nostril and occipital area on the same side as the telephone (maximal increase 2.3 +/- 0.2 degrees C at 6 min) as well as a tendency for higher nasal NO levels (maximal increase 12.9 +/- 4.9% at 10 min), whereas the MCA was significantly reduced (maximal decrease -27 +/- 6% at 15 min). Such changes were not recorded when an earpiece was used to avoid the direct exposure to the electromagnetic field. There were no changes in the skin temperature and nasal NO measured on the opposite side to the mobile phone, whereas the MCA was significantly increased (38 +/- 10%). CONCLUSIONS: Exposure to EMF produced by a mobile phone produces biological effects that can be easily measured. Microwaves may increase skin temperature and therefore cause vasodilation and reduce MCA. Further studies are needed to study the long-term effects of mobile phone use and the relation among NO production, vasodilation, and temperature.

Hyperactivity caused by a nitric oxide synthase inhibitor is countered by ultra-wideband pulses.

Potential action of ultra-wideband (UWB) electromagnetic field pulses on effects of N(G)-nitro- L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), on nociception and locomotor activity was investigated in CF-1 mice. Animals were injected IP with saline or 50 mg/kg L-NAME and exposed for 30 min to no pulses (sham exposure) or UWB pulses with electric field parameters of 102+/-1 kV/m peak amplitude, 0.90+/-0.05 ns duration, and 160+/-5 ps rise time (mean+/-S.D.) at 600/s. Animals were tested for thermal nociceptive responses on a 50 degrees C surface and for spontaneous locomotor activity for 5 min. L-NAME by itself increased mean first-response (paw lift, shake, or lick; jump) and back-paw-lick response latencies and mean locomotor activity. Exposure to UWB pulses reduced the L-NAME-induced increase in back-paw-lick latency by 22%, but this change was not statistically significant. The L-NAME-induced hyperactivity was not present after UWB exposure. Reduction and cancellation of effects of L-NAME suggest activation of opposing mechanism(s) by the UWB pulses, possibly including increase of nitric oxide production by NOS. The action, or actions, of UWB pulses appears to be more effective on locomotor activity than on thermal nociception in CF-1 mice.

Activation of inducible nitric oxide synthase results in nitric oxide-mediated radiosensitization of hypoxic EMT-6 tumor cells.

EMT-6 cells treated for 16 h with 1-10 units/ml IFN-gamma showed a gradual activation of inducible nitric oxide synthase (iNOS) in Western and Northern blots, a simultaneous raise in NO output, and an increase in hypoxic cell radiosensitivity almost to the level of aerobic cells. Both the NO signal and radiosensitization were counteracted by the NO scavenger oxyhemoglobin, by the specific iNOS inhibitor aminoguanidine, and by the L-arginine analogue N(G)-monomethyl-L-arginine. Collectively, these data demonstrate that IFN-gamma can radiosensitize EMT-6 cells through iNOS induction and that NO is the effector molecule responsible for radiosensitization. Compared with the spontaneous NO releaser (2)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino)diazen-1-ium -1,2-diolate], the iNOS-generated NO signal appeared to be 10 times lower yet resulting in the same enhancement ratio of 2.4. Direct stimulation of NO synthesis in tumor cells through the L-arginine/iNOS pathway represents a novel approach to exploit the radiosensitizing properties of NO.

Photic control of nitric oxide synthase activity in golden hamster retina.

A characterization of nitric oxide synthase (NOS) in the golden hamster retina was performed. Enzymatic activity was partially Ca2+ and calmodulin-dependent, required NADPH, and was inhibited by L-arginine analogs. Retinal NOS activity was higher at midnight than at midday. When the hamster were placed under constant darkness for 24 or 48 h, and killed at equivalent time points, representing subjective day and subjective night respectively, the differences in NOS activity disappeared. One hour of darkness during the day increased, while the same period of light during the night decreased, retinal NOS activity. From these results, it might be presumed that hamster retinal NOS activity is regulated by the photic stimulus.

Alterations of nitric oxide synthase and xanthine oxidase activities of human keratinocytes by ultraviolet B radiation. Potential role for peroxynitrite in skin inflammation.

In the present study, we demonstrated that NO synthase (cNOS) and xanthine oxidase (XO) of human keratinocytes can be activated to release NO, superoxide (O2-) and peroxynitrite (ONOO-) following exposure to ultraviolet B (UVB) radiation. We defined that this photo induced response may be involved in the pathogenesis of sunburn erythema and inflammation. Treatment of human keratinocytes with UVB (290-320 nm) radiation (up to 200 mJ/cm2) resulted in a dose-dependent increase in NO and ONOO- release that was inhibited by N-monomethyl-L-arginine (L-NMMA). NO and ONOO- release from keratinocytes was accompanied by an increase in intracellular cGMP levels. Treatment of human keratinocyte cytosol with various doses of UVB (up to 100 mJ/cm2) resulted in an increase in XO activity that was inhibited by oxypurinol. UVB radiation (up to 100 mJ/cm2) of keratinocytes resulted in a 15-fold increase in S-nitrosothiol formation, which directly increased purified soluble guanylate cyclase (sGC) activity by a mechanism characteristic of release of NO from a carrier molecule. In reconstitution experiments, when UVB-irradiated (20 mJ/cm2) purified cNOS isolated from keratinocyte cytosol was combined with UVB-irradiated (20 mJ/cm2) purified XO, a 4-fold increase in ONOO- production, as compared to nonirradiated enzymes, was observed. ONOO- synthesized by NO and O2- following UVB radiation of cNOS and XO was inhibited by oxypurinol (100 microM). UVB radiation of keratinocyte cytosol resulted in an increase in oxygen free radical production, consistent with the increased production of ONOO- by UVB-irradiated keratinocyte cytosol. In in vivo experiments, when experimental animals were subjected to UVB radiation, a protection factor (PF) of 6.5 +/- 1.8 was calculated when an emulsified cream formulation containing nitro-L-arginine (L-NA) (2%) and L-NMMA (2%) was applied to their skin. The present study indicates that UVB radiation acts as a potent stimulator of cNOS and XO activities in human keratinocytes. NO and ONOO- may exert cytotoxic effects in keratinocytes themselves, as well as in their neighboring endothelial and smooth muscle cells. This may be a major part of the integrated response leading to erythema production and the inflammation process.