Effects of 50 Hz electromagnetic fields on rat cortical synaptosomes.

Nerve cells are very responsive to weak pulsed electromagnetic fields (EMFs). Such non-ionizing radiation, with frequencies of 0-300 Hz and 0.1-100 mT, can affect several cellular activities, with unusual dose-response characteristics. The present study examined the effect of a 2-h exposure of synaptosomes on a system generating a peak magnetic field of 2 mT. We evaluated the changes of the synaptosomal mitochondrial respiration rate and ATP production, membrane potential, intrasynaptosomal Ca2+ concentration, and the release of free iron and F2-isoprostanes. O2 consumption and ATP production remained unchanged in exposed synaptosomes. The intrasynaptosomal Ca2+ concentration decreased slowly and no depolarization of the synaptosomal membrane was detected. Finally, the release of free iron and F2-isoprostanes by synaptosomal suspensions also remained unchanged after EMF exposure. These results indicate that the physiological behavior of cortical synaptosomes was unaffected by weak pulsed EMFs.

Physiopathological effects of the NO donor 3-morpholinosydnonimine on rat cortical synaptosomes.

The NO donor 3-Morpholinosydnonimine (SIN-1) releases NO in the presence of molecular oxygen. In this study, we evaluated the effect of SIN-1 on mitochondria of rat cortical synaptosomes. We demonstrated in vitro that the amount of ONOO(-) generated and H(2)O(2) formation directly correlated with SIN-1 concentration. The mean oxygen consumption by synaptosomal mitochondria was approximately 3.8 nmol of O(2) min(-1) mg(-1) protein, which decreased significantly in the presence of SIN-1 1 mM to 2.5 nmol O(2) min(-1) mg(-1). This decrease was not modified by catalase or Trolox, demonstrating that ONOO(-) was responsible for the effect. The same concentration of SIN-1 caused a significant decrease of ATP production by synaptosomal mitochondria and depolarized the mitochondrial membrane. Moreover, ROS production increased progressively and was completely inhibited by pre-incubation of synaptosomes with Trolox. Finally, phosphatidylserine was externalized and, at the same time, intrasynaptosomal lactate dehydrogenase decreased confirming both, the external membrane breakdown after the addition of SIN-1 and the damage to the synaptosomes.

Synaptosome behaviour is unaffected by weak pulsed electromagnetic fields.

The present study examined the effect on rat cortical synaptosomes of a 2 h exposure to 50-Hz electromagnetic fields (EMFs) with a peak magnetic field of 2 mT. We measured modifications of synaptosomal mitochondrial respiration rate, ATP production, membrane potential, intrasynaptosomal Ca(2+) concentration and free iron release. The O(2) consumption remained unvaried in exposed synaptosomes at about 2 nM O(2)/min/mg proteins; ATP production was also unchanged. The intrasynaptosomal Ca(2+) concentration decreased slowly and there was a slight, but non-significant, depolarisation of the synaptosomal membrane. Finally, the free iron release by synaptosomal suspensions, a useful predictor of neuro-developmental outcome, remained unchanged after EMF exposure. On the whole, our results indicate that the physiological behaviour of cortical synaptosomes is not affected by weak pulsed EMFs.

The effect of mild and severe hypoxia on rat cortical synaptosomes.

Brain ischemia results in neuronal injury and neurological disability. The present study examined the effect of mild (6% O2) and severe (2% O2) hypoxia on mitochondria of rat cortical synaptosomes. During mild and severe hypoxia, JO2 and ATP production significantly decreased and mitochondrial membranes depolarized. Synaptosomal calcium concentration increased slightly, albeit not significantly. After a 1 h re-oxygenation period, JO2, ATP production and mitochondrial membrane potential returned to control levels in synaptosomes incubated in 6% O2. In synaptosomes incubated in 2% O2, however, the ATP production was not restored after re-oxygenation and intrasynaptosomal Ca2+ significantly increased. The results indicate that both mild and severe hypoxia influence the physiology of synaptosomal mitochondria; the modifications are reversible after mild hypoxia and but partly irreversible after severe hypoxia.

LDL and HDL affect nitric oxide metabolism in human astrocytoma cells.

Astrocytes provide structural, trophic and metabolic support to neurons and modulate synaptic activity. Under physiological conditions, neuronal-derived nitric oxide (NO) plays an important role in the modulation of a variety of central nervous system (CNS) functions. NO, although short lived, can travel sufficient distances to be able to act as an intercellular messenger in the brain. Its targets include adjacent neurons and astrocytes. The aim of the present study was performed in order to investigate the effects produced by incubation of lipoproteins, at different times, with human astrocytoma cells and thus measuring NO and its metabolite production. NO and peroxynitrite production, iNOS and nNOS expression by Western immunoblot were evaluated. The LDL and HDL-treated cells showed an increased production of NO, more evident after 12 h, compared to basal levels; concerning peroxynitrite production, LDL and HDL-treated cells showed a higher fluorescence, more evident at 3 h. nNOS and iNOS protein levels were significantly higher in the cells incubated with control LDL and HDL. The present work supports the hypothesis that lipoproteins can induce the formation of reactive astrocytes, inducing iNOS as reported by other authors, giving experimental support to a role played by LDL and HDL inducing a reactive response.

Pulsed electromagnetic fields affect the intracellular calcium concentrations in human astrocytoma cells.

Experiments assessed whether long term exposure to 50 Hz pulsed electromagnetic fields with a peak magnetic field of 3 mT can alter the dynamics of intracellular calcium in human astrocytoma U-373 MG cells. Pretreatment of cells with 1.2 microM substance P significantly increased the [Ca(2+)](i). The same effect was also observed when [Ca(2+)](i) was evaluated in the presence of 20 mM caffeine. After exposure to electromagnetic fields the basal [Ca(2+)](i) levels increased significantly from 143 +/- 46 nM to 278 +/- 125 nM. The increase was also evident after caffeine addition, but in cells treated with substance P and substance P + caffeine we observed a [Ca(2+)](i) decrease after exposure. When we substituted calcium-free medium for normal medium immediately before the [Ca(2+)](i) measurements, the [Ca(2+)](i) was similar to that measured in the presence of Ca(2+). In this case, after EMFs exposure of cells treated with substance P, the [Ca(2+)](i), measured without and with addition of caffeine, declined from 824 +/- 425 to 38 +/- 13 nM and from 1369 +/- 700 to 11 +/- 4 nM, respectively, indicating that electromagnetic fields act either on intracellular Ca(2+) stores or on the plasma membrane. Moreover the electromagnetic fields that affected [Ca(2+)](i) did not cause cell proliferation or cell death and the proliferation indexes remained unchanged after exposure.

The effect of pulsed electromagnetic fields on the physiologic behaviour of a human astrocytoma cell line.

We evaluated the effects of 50 Hz pulsed electromagnetic fields (EMFs) with a peak magnetic field of 3 mT on human astrocytoma cells. Our results clearly demonstrate that, after the cells were exposed to EMFs for 24 h, the basal [Ca(2+)](i) levels increased significantly from 124+/-51 nM to 200+/-79 nM. Pretreatment of the cells with 1.2 microM substance P increased the [Ca(2+)](i) to 555+/-278 nM, while EMF exposure caused a significant drop in [Ca(2+)](i) to 327+/-146 nM. The overall effect of EMFs probably depends on the prevailing Ca(2+) conditions of the cells. After exposure, the proliferative responses of both normal and substance P-pretreated cells increased slightly from 1.03 to 1.07 and 1.04 to 1.06, respectively. U-373 MG cells spontaneously released about 10 pg/ml of interleukin-6 which was significantly increased after the addition of substance P. Moreover, immediately after EMF exposure and 24 h thereafter, the interleukin-6 levels were more elevated (about 40%) than in controls. On the whole, our data suggest that, by changing the properties of cell membranes, EMFs can influence Ca(2+) transport processes and hence Ca(2+) homeostasis. The increased levels of interleukin-6 after 24 h of EMF exposure may confirm the complex connection between Ca(2+) levels, substance P and the cytokine network.

Pulsed electromagnetic fields enhance the induction of cytokines by peripheral blood mononuclear cells challenged with phytohemagglutinin.

We evaluated the effects of a 50-Hz pulsed electromagnetic field on the production of cytokines by both resting and mitogen-treated peripheral blood mononuclear cells. Our results demonstrate that after exposure of normal cells to EMFs for 12 h, the levels of neither interleukin-1beta, nor interleukin-2 were increased. Indeed, the concentration of tumor necrosis factor alpha decreased significantly immediately after the exposure period. The results were, however, markedly different when cells were stimulated with phytohemagglutinin immediately before the exposure to EMFs. In this case the levels of cytokines, measured 24 and 48 h after the treatment, were 630 +/- 440 pg/ml and 910 +/- 530 pg/ml for interleukin-1beta, 530 +/- 330 pg/ml, and 860 +/- 560 pg/ml for tumor necrosis factor alpha, respectively. These values were significantly higher (P < 0.05) when compared with the controls. Interleukin-2 levels were significantly higher at the end of the EMF exposure only in supernatants of phytohemagglutinin-stimulated cells and, as a consequence of this increase, the proliferation indexes also were significantly increased 48 h after the EMFs' treatment. The comparison between biological activity and the cytokine antigen present in our samples indicated that the amount of antigen was paralleled by an equal recovery of biological activity. This suggests either the absence of qualitative differences in these proteins or the impairment of both the transcriptional and translational processes.

Short cycles of both static and pulsed electromagnetic fields have no effect on the induction of cytokines by peripheral blood mononuclear cells.

We evaluated the effect of short cycles of static and pulsed electromagnetic field exposure on the eventual activation of peripheral blood mononuclear cells. The cells were subjected to three 15-min cycles of EMF, each exposure being followed by 105 min without a field, for a total of 6 hr. The results clearly demonstrate that the proliferative responses of both normal cells and cells stimulated with 1 microg/ml phytohemagglutinin were not distinguishable from control cells not exposed to EMF. Moreover, although the production of interleukin-2, interferon gamma and tumor necrosis factor alpha increased during the first 48 hr of incubation, the values remained unchanged with respect to controls. This indicates that brief exposure to an electromagnetic field has no significant effect on peripheral blood mononuclear cells. The comparison between biological activity and the cytokine antigen present in our samples indicated that the recovery of antigen corresponded to an equal recovery of biological activity, suggesting the absence of either qualitative differences in these proteins or the impairment of transcriptional and translational processes.

Pulmonary catabolism of interleukin 6 evaluated by lung perfusion of normal and smoker rats.

Cytokines such as interleukin 6 are involved in the pulmonary inflammation arising as a result of smoking. By use of isolated and perfused lung preparations we have evaluated the role of the lungs in the catabolism of human recombinant interleukin 6 both in normal rats and in rats subjected to an acute cigarette smoking episode. When interleukin 6 was incorporated into the lung perfusion medium, neither control nor smoke-exposed rat lungs cleared the cytokine and only 0.1 +/- 0.2% of the total dose was recovered in the bronchoalveolar lavage fluid. When, on the other hand, the same amount of interleukin 6 was instilled into the bronchoalveolar tree, concentrations of the cytokine in the perfusate increased progressively so that after 3 h up to 70.1 +/- 9.8% and 40.9 +/- 22.5% of the administered dose, as measured by immunoenzymatic test, had been transferred from the bronchial lumen to the perfusion medium of either control or smoker rat lungs, respectively, indicating significantly (P < or = 0.05) different behaviour of the cytokine in the two experimental groups. Total recoveries of the administered interleukin 6 evaluated in smoke-exposed rat lungs were 55.3 +/- 23.2%, significantly lower than those for control rat lungs (83.9 +/- 11%). Determination of biological activity gave values always lower than those measured by immunoenzymatic test, indicating loss of biological activity during the transalveolar transit. It appears that the transfer of interleukin 6, especially in smokers, is almost exclusively unidirectional, from the alveolar space to the plasmatic pool with degradation during the transalveolar passage.

Pharmacokinetics and catabolism of tumor necrosis factor-alpha in rat lungs.

The role of the lungs in the catabolism of tumor necrosis factor alpha (TNF-alpha) either in normal rats, or in rats subjected to an acute cigarette smoking episode has been evaluated by using isolated and perfused lung preparations. After administration of TNF-alpha into the lung perfusion medium, there was no clearance of the cytokine in both control and smoker rat lungs and only 0.2 +/- 0.1% of the administered dose was recovered in the bronchoalveolar lavage fluid. When TNF-alpha was instilled into the bronchoalveolar tree, concentrations of the cytokine in the perfusate increased progressively so that after 3 h up to 68.8 +/- 8% and 52.7 +/- 11.4% of the administered dose had been transferred from the bronchial lumen to the perfusion medium of either control or smoker rat lungs, respectively, the latter values being significantly lower (p < or = 0.05) than those obtained in control lungs. Moreover, total recoveries of TNF-alpha evaluated in smoker rat lungs (65.5 +/- 10.2%) were also significantly lower than those observed in control rat lungs (82.8 +/- 7.1%). In conclusion, it appears that transfer of TNF-alpha is almost exclusively unidirectional, from the alveolar space to the plasma pool with partial degradation during the transalveolar passage. These results may be useful when attempting to deliver TNF-alpha by aerosol.

Pulmonary catabolism of interferon-gamma evaluated by lung perfusion of both normal and smoke-exposed rats.

The role of the lungs in the catabolism of rat recombinant interferon-gamma, either in normal rats or in rats subjected to an acute cigarette smoking episode, was evaluated using an isolated and perfused lung preparation. After administration of interferon-gamma into the lung perfusion medium, there was no clearance of the cytokine in either control or smoke-exposed rat lungs, and only 0.1 +/- 0.2% of the total dose was recovered in the bronchoalveolar lavage fluid. When the same amount of interferon-gamma was instilled into the bronchial alveolar tree, concentrations of the cytokine in the perfusate increased progressively so that after 3 h up to 71.2 +/- 4.3 and 62 +/- 5.7% of the administered dose, as measured by ELISA test, had been transferred from the bronchial lumen to the perfusion medium of either control or smoke-exposed rat lungs, respectively, the latter values being significantly lower (p < or = 0.05) than those obtained in control lungs. Moreover, total recoveries of interferon-gamma evaluated in smoke-exposed rat lungs (78.4 +/- 8.6%) were also significantly lower than those observed in control rat lungs (91.4 +/- 11.8%). Biologic activity evaluations on the same samples gave values significantly lower than those obtained using ELISA, indicating a partial loss of biologic activity during transalveolar transit. In conclusion, it appears that the transfer of interferon-gamma is almost exclusively unidirectional from the alveolar space to the plasmatic pool, with partial degradation during transalveolar passage.

The lymphatic route. IX. Distribution of recombinant interferon-alpha 2 administered subcutaneously with oedematogenic drugs.

We have evaluated whether the addition of either bradykinin or histamine favours the lymphatic absorption of human recombinant interferon-alpha 2 (IFN-alpha 2) administered by the subcutaneous route. Subcutaneous administration of IFN-alpha 2 with bradykinin enhances IFN absorption via both capillaries and lymphatics, so that either the plasma or lymph areas under the concentration curves (AUC) increase significantly up to 1751 +/- 483 and 1319 +/- 608 IU/ml/min respectively as compared to the respective AUC values (613 +/- 208 and 483 +/- 213 IU/ml/min) obtained after IFN injection in normal saline. Since the lymph AUC/plasma AUC ratios remain unaltered, there is no preferential lymphatic absorption of IFN-alpha 2 after bradykinin administration. Dual-label experiments, 125I-IFN-alpha 2 in saline and 131I-IFN-alpha in saline containing 200 micrograms histamine were injected subcutaneously into the left and into the right shank of the same animal, gave similar results. The kinetics of 125I and 131I acid-soluble radioactivity confirm that histamine favours both plasmatic and lymphatic absorption.

Effects of acute cigarette smoke exposure on macrophage kinetics and release of tumour necrosis factor alpha in rats.

Some biological parameters before and after an acute episode of cigarette smoking in rats have been evaluated. The carboxyhaemoglobin levels depended either on the number of cigarettes, or on the time of exposure to cigarette smoke and returned to pre-smoking values in about 2 h. The evaluation of the kinetics of alveolar and peritoneal macrophages in rats after a smoking session of three cigarettes within an hour, indicated that alveolar macrophages in the bronchoalveolar lavage fluid significantly increased 8 h after the smoking, whereas the number of peritoneal macrophages remained practically constant. The incubation of these cells for various times at 37( degrees )C in a humidified atmosphere, resulted in a spontaneous release, 24 h thereafter, of variable amounts of tumour necrosis factor alpha (TNFalpha), which remained practically constant during the following days. Neither alveolar macrophages of control rats, nor peritoneal macrophages of both control and smoking rats were able to release TNFalpha. Moreover, after lipopolysaccharide induction of alveolar macrophages of both control and smoking rats, an increased release of TNFalpha was observed, indicating that these cells were in an active state.

Chronic cigarette smoking enhances spontaneous release of tumour necrosis factor-alpha from alveolar macrophages of rats.

Some biological effects of chronic cigarette smoking (two cigarettes for 2 h, daily for 4 months) in rats were evaluated. During the smoking period, body weight of smoker rats was always significantly lower than that of control rats. Immediately after the last smoking session the carboxyhaemoglobin concentration in the blood was about 8.5% and the polymorphonuclear cells in the bronchoalveolar fluid increased significantly. At the same time, enzymatic analyses on the supernatants of bronchoalveolar fluid revealed a significant increase of beta-glucuronidase in the smoker group. Alveolar macrophages, collected 0, 8 and 24 h after the last smoking session, significantly increased the generation of superoxide anion and, after incubation for 24 h at 37( degrees ) C in a humidified atmosphere, released significantly high amounts of TNF-alpha. When challenged with lipopolysaccharide, alveolar macrophages of smoker rats released much more TNF-alpha but, in such a case, TNF-alpha release was about one half of that observed in the control group. Peritoneal macrophages of both control and smoker rats were unable either to generate high levels of superoxide anion or to release significant amounts of TNF-alpha. The results clearly demonstrated the activated state of alveolar macrophages and the resting state of peritoneal macrophages.

Production of tumor necrosis factor alpha by rat alveolar macrophages collected after acute cigarette smoking.

In this study we evaluated the effect of cigarette smoke on the activation of alveolar macrophages of the rat lungs exposed to an episode of acute passive cigarette smoking. Our experiments were carried out in rats that, after undergoing smoking (3 cigarettes within 1 h) showed a COHb increase of about 16%. The evaluation of the kinetics of alveolar and peritoneal macrophages, indicated that the number of alveolar macrophages in the bronchoalveolar lavage fluids significantly increased 8 h after the smoking session, whereas the number of peritoneal macrophages remained practically constant. Alveolar macrophages collected 0.8 and 24 h after smoking and incubated for 24 h at 37 degrees C in an atmosphere of 5% CO2 in air spontaneously released 5 +/- 1, 48 +/- 14 and 15 +/- 9 units of TNF-alpha per 10(6) cells, respectively. Moreover, neither alveolar macrophages collected from smokers, nor those collected from controls, released IFN, and both cytokines were also absent either in bronchoalveolar lavage and peritoneal lavage fluids or in plasma. Alveolar macrophages collected from controls rats, when challenged with lipopolysaccharide (LPS), released more TNF than those collected from smoke exposed rats. Thus, it seemed that macrophages of experimental animals were activated but at the same time were somewhat depressed and responded less well to LPS.

Human beta-interferon incubated with muscle homogenate is protected by albumin but not by proteinase inhibitors.

The scarce bioavailability of beta-interferon (IFN-beta) after intramuscular administration is probably due either to the binding of IFN-beta to interstitial matrix, or to lymphatic absorption and/or to local breakdown by lysosomal proteinases from muscle. In this work, we first showed that after intramuscular injection, the apparent bioavailability of natural human IFN-beta is about 10% of that of recombinant IFN-alpha 2 and then we evaluated the effects of proteinase inhibitors and albumin on IFN-beta incubated at 37 degrees C with muscle homogenate. IFN biological activity decreased spontaneously by about 20% after incubation for 6 hr at 37 degrees C in Hanks' solution, but it was almost completely lost after incubation with muscle homogenate. Proteinase inhibitors (alpha 1-antitrypsin, alpha 2-macroglobulin, aprotinin, soybean trypsin inhibitor, leupeptin, EP-459, and EP-475) failed to block the inactivation of IFN-beta by muscle proteinases, whereas albumin exerted a partial but consistent protection.

Interferon induction in rabbits after intraduodenal administration of a phosphorylated glucomannan-protein fraction of the cell wall of Candida albicans.

The aim of this work was to demonstrate whether a glucomannan protein fraction (GMP) of Candida albicans cell wall could induce interferon after intraduodenal administration in normal rabbits and rabbits immunized against C. albicans. For this purpose we collected simultaneously plasma and abdominal lymph for 10 h after the administration of the inducer. We observed a peak of antiviral activity in the lymph 4 h after intraduodenal administration of 20 mg GMP dissolved in saline to 6 normal rabbits. Immunized rabbits (anti-GMP titres greater than 1024) responded earlier (peak after 2 h) and more intensely; analysis of the values of the areas under the curve indicated that the IFN response in the lymph of immunized rabbits was significantly higher (P less than 0.0025) than in normal rabbits. Antiviral activity was absent in plasma in all cases. Preliminary characterization of the IFN activity has shown it to be trypsin-sensitive, acid and heat stable, and species-specific.

The lymphatic route. VII. Distribution of recombinant human interleukin-2 in rabbit plasma and lymph.

Human recombinant (R) interleukin-2 (IL-2) has been administered through intravenous (i.v.), intramuscular (i.m.) and subcutaneous (s.c.) routes and its distribution in lymph and plasma has been evaluated in rabbits. It has been shown that after i.m. administration of RIL-2 in saline, the lymphokine is preferentially absorbed via lymphatics. A similar result has been obtained after s.c. administration when RIL-2 was injected with a high concentration (12.5%) of human albumin, which acts as a retarder and a promoter of lymphatic absorption. These routes may be valid alternatives to i.v. administration.