Microwave bioeffects in the erythrocyte are temperature and pO2 dependent: Cation permeability and protein shedding occur at the membrane phase transition

Microwave exposure (2450 MHz, 60 mW/g, CW) of rabbit erythrocytes increases Na passive transport only at membrane phase transition temperatures (T_c) of 17–19°C. This permeability effect is enhanced for relative hypoxia which is characteristic of intracellular oxygen tension (pO₂ ? 5 mm Hg). Neither the permeability nor the pO₂ effects are observed in temperature-matched (± 0.05°C), sham-exposed controls. In addition, at T_c, microwave exposure is observed to induce the shedding or release of two erythrocyte proteins not seen in sham-exposed controls. Moreover, the enhanced shedding of at least seven other proteins all of molecular weight ? 28,000 D was detected in the microwave-treated samples. Using sensitive silver staining we estimate that approximately 450 fg of protein were shed per erythrocyte. These results demonstrate that temperature and pO₂ are important influences on both functional and structural responses of cell membranes to microwave radiation.     

Fe and Fe initiated peroxidation of sonicated and non-sonicated liposomes made of retinal lipids in different aqueous media

Retina is highly susceptible to oxidative damage due to its high content of polyunsaturated fatty acids (PUFAs), mainly docosahexaenoic acid (22:6 n3). Lipid peroxidation process is thought to be involved in many physiological and pathological events. Many model membranes can be used to learn more about issues that cannot be studied in biological membranes. Sonicated liposomes (SL) and non-sonicated liposomes (NSL) prepared with lipids isolated from bovine retina and characterized by dynamic light-scattering, were submitted to lipid peroxidation, under air atmosphere at 22 °C, with Fe²⁺ or Fe³⁺ as initiator, in different aqueous media. Conjugated dienes and trienes, determined by absorption at 234 and 270 nm respectively, and thiobarbituric acid-reactive substances were measured as a function of time. Peroxidation of SL or NSL initiated with 25 μM FeSO₄ in 20 mM Tris-HCl pH 7.4 resulted in an increase in TBARS production after a lag phase of 60 min. Incubation of both types of liposomes in water resulted in shortening of the lag phase at 30 min. When lipid peroxidation was performed in 0.15 M NaCl, lag phase completely disappeared. On the other hand, FeCl₃ (25 μM) induced a limited production of TBARS only just after 30 min of incubation. When Fe²⁺- or Fe³⁺-lipid peroxidation of both types of liposomes was carried out in water or 0.15 M NaCl, formation of conjugated dienes and conjugated trienes were higher than in reactions carried out in 20 mM Tris-HCl pH 7.4.Our results established that both liposome types were susceptible to Fe²⁺- and Fe³⁺-initiated lipid peroxidation. However, Fe²⁺ showed a clearly enhanced effect on peroxidation rate and steady state concentration of oxidation products.We verified that peroxidation of liposomes made of retinal lipids is affected not only by type of initiator but also by aqueous media. This model constitutes a useful system to study formation of lipid peroxidation intermediaries and products in an aqueous environment.     

Direct linkage of thrombin to its cell surface receptors in different cell types

When ¹²⁵I-thrombin was incubated with foreskin fibroblasts, cervical carcinoma cells or fibrosarcoma cells of human origin, or with secondary chick embryo cells or Chinese hamster lung cells, it became directly linked to its cell surface receptors. The thrombin-receptor complex (TH-R) was derived exclusively from a pool of ¹²⁵I-thrombin that had become specifically bound to the cell surface. The linkage was probably covalent, since the complex was resistant to boiling in sodium dodecyl sulfate and 2-mercaptoethanol. Raising the pH to 12 disrupted TH-R, but did not affect a similar complex between epidermal growth factor and its receptor, suggesting that the linkage of these mitogens to their receptors was different. Mild trypsin treatment removed the ability of cells to form TH-R; however, after a 24-h incubation in serum-free medium, trypsin-treated cells recovered the capacity to form TH-R, suggesting that TH-R resulted from interaction of ¹²⁵I-thrombin with a cellular rather than a serum component. The mitogenic response of cells to thrombin was inversely related to the fraction of specifically bound ¹²⁵I-thrombin represented by TH-R. The role of TH-R in mitogenesis may be clarified in future studies by obtaining clones of Chinese hamster lung cells that vary in their capacities to form TH-R and to respond to the mitogenic action of thrombin.     

Influence of lipids on ice formation in cryoprotective media

Cryomicroscopic analysis demonstrated that two lipid preparations from marine vertebrates (<0.1%) and liposomes prepared from rainbow trout sperm lipids (<0.5%) efficiently hindered the growth of ice crystals during freezing of multicomponent cryoprotective media used for trout sperm cryopreservation. At higher lipid concentrations, crystals either did not form at all or had altered shape and blurred boundaries. Addition of egg yolk (10%) together with these lipids increased the size of crystal structures and markedly changed their shape.     

Effects of phytic acid on the myoglobin-t-butylhydroperoxide-catalysed oxidation of uric acid and peroxidation of erythrocyte membrane lipids

Phytic acid stimulated the myoglobin-t-butylhydroperoxide (TBHP)-catalysed oxidation of uric acid, but inhibited the peroxidation of erythrocyte membrane lipids induced by the same system. Butylated hydroxy-toluene, a free radical chain reaction-terminating antioxidant, also suppressed the myoglobin-TBHP-induced lipid peroxidation. Moreover, phytic acid inhibited the hydroxyl radical-induced degradation of deoxyribose, but the extent of inhibition in this system was reduced by increasing the ferric ion concentration, suggesting that these effects of phytic acid on the myoglobin-TBHP-mediated oxidation are more likely attributable to its metal chelating properties rather than to a free radical scavenging action. The effectiveness of phytic acid, a naturally occurring antioxidant, in the inhibition of both iron- (as previously shown) and myoglobin-dependent lipid peroxidation suggests its possible therapeutic application as a non-toxic antioxidant for ameliorating the extent of oxy-radical-mediated myocardial ischemia/reperfusion damage.Abbreviations ASC Ascorbic acid - BHT Butylated Hydroxytoluene - DMSO Dimethyl Sulfoxide - TBHP t-Butylhydroperoxide - TBA Thiobarbituric Acid - TBARS Thiobarbituric Acid-reactive Substances     

Influence of salt concentration on membrane lipids of halophilic bacteria

Abstract A review of salt-dependent changes in membrane lipid composition of halotolerant, moderately halophilic, and extremely halophilic bacteria is presented. The biosynthetic and regulatory mechanisms underlying the observed changes are discussed. Possible implications for the evolution of extreme halophiles and other Archaebacteria are also discussed.     

Influence of N-phthaloyl gamma-aminobutyric acid on circadian rhythms of lipid peroxidation products and antioxidants

N-Phthaloyl GABA was administrated daily (50 mg/kg body weight-i.p) to Wistar rats for 21 days and circadian rhythms of thiobarbituric acid reactive substances (TBARS) and antioxidants such as reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD) were studied. N-Phthaloyl GABA was found to delay TBARS and to advance GSH, CAT and SOD acrophases. Amplitude and mesor values of these rhythms were found to be altered during N-Phthaloyl GABA treatment. Since GABA is hypothesized to be involved in conveying dark information to clock, exogenous administration of P-GABA might alter the photic information received by the clock. Our study shows that P-GABA administration alters the temporal patterns of lipid peroxidation and antioxidants in Wistar rats. But the exact mechanism remains to be explored and further research is needed.     

The effects of hyperthermia and hyperthermia plus microwaves on rat brain energy metabolism

The effects of hyperthermia, alone and in conjunction with microwave exposure, on brain energetics were studied in anesthetized male Sprague-Dawley rats. The effect of temperature on adenosine triphosphate concentration [ATP] and creatine phosphate concentration [CP] was determined in the brains of rats that were maintained at 35.6, 37.0, 39.0, and 41.0 degrees C. At 37, 39, and 41 degrees C brain [ATP] and [CP] were down 6.0, 10.8, and 29.2%, and 19.6, 28.7, and 44%, respectively, from the 35.6 degrees C control concentrations. Exposure of the brain to 591-MHz radiation at 13.8 mW/cm2 for 0.5, 1.0, 3.0, and 5.0 min caused further decreases (below those observed for 30 degrees C hyperthermia only) of 16.0, 29.8, 22.5, and 12.3% in brain [ATP], and of 15.6, 25.1, 21.4, and 25.9% in brain [CP] after 0.5, 1.0, 3.0, and 5.0 min, respectively. Recording of brain reduced nicotinamide adenine dinucleotide (NADH) fluorescence before, during, and after microwave exposure showed an increase in NADH fluorescence during microwave exposure that returned to preexposure levels within 1 min postexposure. Continuous recording of brain temperatures during microwave exposures showed that brain temperature varied between -0.1 and +0.05 degrees C. Since the microwave exposures did not induce tissue hyperthermia, it is concluded that direct microwave interaction at the subcellular level is responsible for the observed decrease in [ATP] and [CP].     

Effects of continuous-wave, pulsed, and sinusoidal-amplitude-modulated microwaves on brain energy metabolism

A comparison of the effects of continuous-wave, sinusoidal-amplitude-modulated, and pulsed square-wave-modulated 591-MHz microwave exposures on brain energy metabolism was made in male Sprague-Dawley rats (175-225 g). Brain NADH fluorescence, adenosine triphosphate (ATP) concentration, and creatine phosphate (CP) concentration were determined as a function of modulation frequency. Brain temperatures of animals were maintained between -0.1 and -0.4 degrees C from the preexposure temperature when subjected to as much as 20 mW/cm2 (average power) CW, pulsed, or sinusoidal-amplitude modulated 591-MHz radiation for 5 min. Sinusoidal-amplitude-modulated exposures at 16-24 Hz showed a trend toward preferential modulation frequency response in inducing an increase in brain NADH fluorescence. The pulse-modulated and sinusoidal-amplitude-modulated (16 Hz) microwaves were not significantly different from CW exposures in inducing increased brain NADH fluorescence and decreased ATP and CP concentrations. When the pulse-modulation frequency was decreased from 500 to 250 pulses per second the average incident power density threshold for inducing an increase in brain NADH fluorescence increased by a factor of 4--ie, from about 0.45 to about 1.85 mW/cm2. Since brain temperature did not increase, the microwave-induced increase in brain NADH and decrease in ATP and CP concentrations was not due to hyperthermia. This suggests a direct interaction mechanism and is consistent with the hypothesis of microwave inhibition of mitochondrial electron transport chain function of ATP production.     

Effect of selenium on lipids, lipid peroxidation, and sulfhydryl group in neuroendocrine centers of rats

The effects of various doses of sodium selenite (0.05, 0.1, and 0.2 mg/kg body weight, ip) were studied on the content of phospholipids, cholesterol, esterified fatty acids (EFA), gangliosides, thiobarbituric acid reactive substance (TBARS), and sulfhydryl group in neuroendocrine centers of male Wistar rats for 7 d. The lowest dose of Se (0.05 mg/kg) did not alter the above parameters significantly in neuroendocrine centers. The content of phospholipids was depleted significantly in the pituitary and depletion in the pineal was 80.22% with a 0.1-mg/kg dose of Se, but this dose elevated its level significantly in the hypothalamus. Conversely, a 0.2-mg/kg dose of selenium elevated the level of phospholipids significantly in the pituitary and hypothalamus, the elevation in the pineal was 70%. Selenium, 0.1 mg/kg, elevated the level of cholesterol in the pituitary but depleted its level in the pineal (56.8%) and hypothalamus (13.60%). Selenium, 0.2 mg/kg, elevated the level of cholesterol significantly in the hypothalamus but its level was not significant in the pituitary and pineal. The depletion of esterified fatty acid in the pituitary and pineal with doses of 0.1 and 0.2 mg/kg was significant in the pituitary, whereas its depletion in the pineal was 85.4% and 69.26%, respectively. Selenium, 0.1 and 0.2 mg/kg, depleted the level of gangliosides significantly and dose dependently in the pituitary but has elevated its level significantly and dose dependently in the hypothalamus. Its depletion in the pineal was 87.1% and 67.8% with the 0.1- and 0.2-mg/kg dose of selenium, respectively. Selenium, 0.1 mg/kg, increased the content of TBARS significantly in neuroendocrine centers and its elevation in the pineal was 703.8%. Selenium, 0.2 mg/kg, elevated its level in the pituitary and it was 126.9% in the pineal, but this dose depleted its level significantly in the hypothalamus. The content of the sulfhydryl group with a 0.1-mg/kg dose of selenite was depleted significantly in neuroendocrine centers and it was 55.9% in the pineal. Selenium, 0.2 mg/kg, depleted the level of the sulfhydryl group more significantly in the pituitary and pineal, but its elevation in hypothalamus was significant.     

Comparative effects of pulsed and continuous-wave 2.8-GHz microwaves on temporally defined behavior

The effects of pulsed-(PW) and continuous-wave (CW) 2.8-GHz microwaves were compared on the performance of rodents maintained by a temporally defined schedule of positive reinforcement. The schedule involved food-pellet reinforcement of behavior according to a differential-reinforcement-of-low-rate (DRL) contingency. The rats were independently exposed to PW and to CW fields at power densities ranging from 1 to 15 mW/cm². Alterations of normal performance were more pronounced after a 30-minute exposure to the PW field than to the CW field. The rate of emission of appropriately timed responses declined after exposure to PW at 10 and 15 mW/cm², whereas exposure at the same power levels to the CW field did not consistently affect the rate of responding. Change in performance associated with microwave exposure was not necessarily related to a general decline in responding: in some instances, increases in overall rates of responding were observed.     

Protective role of ascorbic acid against lipid peroxidation and myocardial injury

Ascorbic acid (AH₂) is a potential scavenger of superoxide radical and singlet oxygen. In the guinea pig, marginal AH₂ deficiency results in intracellular oxidative damage in the cardiac tissue as evidenced by lipid peroxidation, formation of fluorescent pigment and loss of structural integrity of the microsomal membranes. The oxidative damage does not occur due to lack of enzymatic scavengers of reactive oxygen species such as superoxide dismutase, catalase and glutathione peroxidase. Also, glutathione transferase activity is not decreased in AH₂ deficiency. Lipid peroxidation, fluorescent pigment formation and protein modification disappear after AH₂ therapy. These results, if extra-polated to human beings, would indicate that chronic subclinical AH₂ deficiency may result in progressive oxidative damage which in the long run may lead to permanent degenerative diseases in the heart.     

The role of membrane lipids in the induction of macrophage apoptosis by microparticles

Microparticles are membrane-derived vesicles that are released from cells during activation or cell death. These particles can serve as mediators of intercellular cross-talk and induce a variety of cellular responses. Previous studies have shown that macrophages undergo apoptosis after phagocytosing microparticles. Here, we have addressed the hypothesis that microparticles trigger this process via lipid pathways. In these experiments, microparticles induced apoptosis in primary macrophage cells or cell lines (RAW 264.7 or U937) with up to a 5-fold increase. Preincubation of macrophages with phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)BP) reduced the microparticle-induced apoptosis in a dose-dependent manner. PtdIns(3,5)BP is a specific inhibitor of the acid sphingomyelinase and thus can block the generation of pro-apoptotic ceramides. Similarly, the pre-incubation of macrophages with PtdIns(3,5)BP prevented microparticle-induced upregulation of caspase 8, which is a major target molecule of ceramide action in the apoptosis pathway. PtdIns(3,5)BP, however, had no effect on the spontaneous rate of apoptosis. To evaluate further signaling pathways induced by microparticles, the extracellular signal regulated kinase (ERK-) 1 was investigated. This kinase plays a role in activating phospholipases A2 which cleaves membrane phospholipids into arachidonic acid; microparticles have been suggested to be a preferred substrate for phospholipases A2. As shown in our experiments, microparticles strongly increased the amount of phosphorylated ERK1/2 in RAW 264.7 macrophages in a time-dependent manner, peaking 15 min after co-incubation. Addition of PD98059, a specific inhibitor of ERK1, prevented the increase in apoptosis of RAW 264.7 macrophages. Together, these data suggest that microparticles perturb lipid homeostasis of macrophages and thereby induce apoptosis. These results emphasize the importance of biolipids in the cellular cross-talk of immune cells. Based on the fact that in clinical situations with excessive cell death such as malignancies, autoimmune diseases and following chemotherapies high levels of circulating microparticles might modulate phagocytosing cells, a suppression of the immune response might occur due to loss of macrophages.     

Single vs. repeated microwave exposure: effects on benzodiazepine receptors in the brain of the rat.

We studied the effects of single (45 min) and repeated (ten daily 45-min sessions) microwave exposures (2450-MHz, 1 mW/cm2, average whole-body SAR of 0.6 W/kg, pulsed at 500 pps with pulse width of 2 microseconds) on the concentration and affinity of benzodiazepine receptors in the cerebral cortex, hippocampus, and cerebellum of the rat. We used a receptor-binding assay with 3H-flunitrazepam as ligand. Immediately after a single exposure, an increase in the concentration of receptor was observed in the cerebral cortex, but no significant effect was observed in the hippocampus or cerebellum. No significant change in binding affinity of the receptors was observed in any of the brain-regions studied. In rats subjected to repeated exposures, no significant change in receptor concentration was found in the cerebral cortex immediately after the last exposure, which may indicate an adaptation to repeated exposures. Our data also show that handling and exposure procedures in our experiments did not significantly affect benzodiazepine receptors in the brain. Because benzodiazepine receptors in the brain are responsive to anxiety and stress, our data support the hypothesis that low-intensity microwave irradiation can be a source of stress.     

Investigation of the effects of continuous-wave,pulse- and amplitude-modulated microwaves on single excitable cells of chara corallina

Single internodal excitable cells of Chara corallina were exposed to CW, pulse-modulated and sinusoidally modulated S-band microwave fields in a temperature-controled waveguide exposure chamber. All electrical measurements were made external to the waveguide (ie, under no impressed microwave field). The dependent variables measured before, during, and after exposure to the S-band microwave fields included: resting potential, amplitude of the action potential, rise and decay time of the action potential, conduction velocity, and excitability. Cells maintained at 22 ± 0.1 °C during exposure showed no consistent or statistically significant microwave-dependent alterations in any of the dependent variables.     

Selenium altered the levels of lipids, lipid peroxidation, and sulfhydryl groups in straitum and thalamus of rat

The effect of sodium selenite (0.05, 0.1, and 0.2 mg/kg body weight, ip) on the lipid levels (total lipids, phospholipids, cholesterol, gangliosides), thiobarbituric acid reactive substance (TBARS), and sulfhydryl group (-SH) in the straitum and thalamus of a male Wistar rat was studied after 7 d of treatment. The level of total lipids and cholesterol was significantly and dose-dependently elevated in the straitum and thalamus with 0.1 and 0.2 mg/kg of sodium selenite. However, the cholesterol level was significantly increased only with 0.2 mg/kg of sodium selenite in the thalamus. The level of phospholipids and gangliosides was more significant with 0.1 mg/kg of sodium selenite as compared to 0.2 mg. No significant alteration on the gangliosides level was observed in the thalamus with various doses of sodium selenite although the elevation with 0.2 mg dose was 25.9%. The content of TBARS was elevated dose dependently in straitum, but its level was depleted significantly with 0.1-mg/kg dose of sodium selenite in the thalamus. The level of the -SH group was significantly depleted in the straitum with 0.1-mg/kg dose of sodium selenite; conversely, this dose has significantly elevated the levels of-SH group in the thalamus.     

Oxidative stress induced by beta-amyloid peptide(1-42) is involved in the altered composition of cellular membrane lipids and the decreased expression of nicotinic receptors in human SH-SY5Y neuroblastoma cells

The neurotoxic effects and influence of beta-amyloid peptide (Aβ)_1–42 on membrane lipids and nicotinic acetylcholine receptors (nAChRs) in human SH-SY5Y neuroblastoma cells were investigated in parallel. Exposure of the cultured cells to varying concentrations of Aβ_1–42 evoked a significantly decrease in cellular reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyl tetrazolium bromide), together with enhanced lipid peroxidation and protein oxidation. Significant reductions in the total contents of phospholipid and unbiquinone-10, as well as in the levels of the 3 and 7 subunit proteins of nAChRs were detected in cells exposed to Aβ_1–42. In contrast, such treatment had no effect on the total cellular content of cholesterol. Among these alterations, increased lipid peroxidation and decreased levels of cellular phospholipids were most sensitive to Aβ_1–42, occurring at lower concentrations. In addition, when SH-SY5Y cells were pretreated with the antioxidant Vitamin E, prior to the addition of Aβ_1–42, these alterations in neurotoxicity, oxidative stress, composition of membrane lipids and expression of nAChRs were partially prevented. These findings suggest that stimulation of lipid peroxidation by Aβ may be involved in eliciting the alterations in membrane lipid composition and the reduced expression of nAChRs associated with the pathogenesis of AD.     

Effect of 9.6-GHz pulsed microwaves on the orb web spinning ability of the cross spider (Araneus diadematus)

Eight cross spiders (Araneus diadematus) were exposed overnight (16 h) during web-building activity to pulsed 9.6-GHz microwaves at average power densities of 10, 1, and 0.1 mW/cm2 (estimated SARs 40, 4, and 0.4 mW/g). Under these conditions, 9.6-GHz pulsed microwaves did not affect the web-spinning ability of the cross spider.     

Cell density-dependent regulation of cell surface expression of two types of human tumor necrosis factor receptors and its effect on cellular response

Tumor necrosis factor (TNF) is a multipotential cytokine known to regulate the growth of a wide variety of normal and tumor cells. It has been shown that the density of cells in culture can modulate the growth regulatory activities of TNF, the mechanism of which, however, is not understood. In this report, we investigated the effect of cell density on the expression of TNF receptors. The receptors were examined on epithelial cells (e.g., HeLa), which primarily express the p60 form, and on myeloid cells (e.g., HL-60) known to express mainly the p80 form. We observed that binding of TNF to both cell lines decreased with increase in cell density. Scatchard analysis of binding on HeLa and HL-60 cells revealed a 4- to 5-fold reduction in the number of TNF receptors without any significant change in receptor affinity in both cell types at high density. The decrease in TNF receptor numbers at high cell density was also observed in several other epithelial and myeloid cell lines. The downmodulation at high cell density was unique to TNF receptors, since minimum change in other cell surface proteins was observed as revealed by fluorescent activated cell sorter analysis. Neutralization of binding with antibodies specific to each type of the receptors revealed that both the p60 and p80 forms of the TNF receptor were equally downmodulated. A decrease in leucine incorporation into proteins was observed with increase in cell density, suggesting a reduction in protein synthesis. Since inhibition of protein synthesis by cycloheximide also leads to a decrease in TNF receptors, it is possible that the density-dependent reduction in TNF receptor number is due to an overall decrease in protein synthesis. The density-dependent decrease in TNF receptors was accompanied by a decrease in intracellular reduced glutathione levels. A reduction in the number of receptors on TNF sensitive tumor cells induced by cell-density correlated with increase in resistance to the cytokine.