Electric field-induced changes in agonist-stimulated calcium fluxes of human HL-60 leukemia cells.

The mechanism of biological effects of extremely-low-frequency electric and magnetic fields may involve induced changes of Ca2+ transport through plasma membrane ion channels. In this study we investigated the effects of externally applied, low-intensity 60 Hz electric (E) fields (0.5 V/m, current density 0.8 A/m2) on the agonist-induced Ca2+ fluxes of HL-60 leukemia cells. The suspensions of HL-60 cells received E-field or sham exposure for 60 min and were simultaneously stimulated either by 1 microM ATP or by 100 microM histamine or were not stimulated at all. After E-field or sham exposure, the responses of the intracellular calcium levels of the cells to different concentrations of ATP (0.2-100 microM) were assessed. Compared with control cells, exposure of ATP-activated cells to an E-field resulted in a 20-30% decrease in the magnitude of [Ca2+]i elevation induced by a low concentration of ATP (<1 microM). In contrast, exposure of histamine-activated HL-60 cells resulted in a 20-40% increase of ATP-induced elevation of [Ca2+]i. E-field exposure had no effect on non-activated cells. Kinetic analysis of concentration-response plots also showed that compared with control cells, exposure to the E-field resulted in increases of the Michaelis constant, Km, value in ATP-treated cells and of the maximal [Ca2+]i peak rise in histamine-treated HL-60 cells. The observed effects were reversible, indicating the absence of permanent structural damages induced by acute 60 min exposure to electric fields. These results demonstrate that low-intensity electric fields can alter calcium distribution in cells, most probably due to the effect on receptor-operated Ca2+ and/or ion channels.

Second cancers following pediatric Hodgkin's disease.

PURPOSE: To define the magnitude of second cancer risk among pediatric Hodgkin's disease survivors and to determine which factors influence this risk. PATIENTS AND METHODS: At Stanford,694 children and teenagers were monitored for 1 to 31.6 years (mean, 13.1) after treatment for Hodgkin's disease. Relative risks (RRs), actuarial risks, and absolute excess risks for second malignancies were calculated. The influences of sex, age, stage, splenectomy, treatment and relapse were assessed by multivariate analysis. RESULTS: Fifty-six patients developed 59 secondary malignancies: 48 solid tumors, eight leukemias, and three non-Hodgkin's lymphomas. The RR of developing a second cancer was 15.4 (95% confidence interval [CI], 10.6 to 21.5) for females and 10.6 (95% CI, 6.6 to 16.0) for males. Breast cancer (n = 16) and sarcoma (n = 13) were the most common solid tumors. The actuarial risk at 20 years follow-up evaluation was 9.7% for males, 16.8% for females, and 9.2% for breast cancer. The median interval to diagnosis of a second malignancy was shortest for leukemia, 4.3 years, and longest for lung cancer, 18.4 years. Relapse of Hodgkin's disease increased the risk of second malignancy (hazards ratio [HR] = 2.6, P < .001). Hodgkin's disease stage, patient age, splenectomy, and treatment modality did not appear to alter overall risk, although chemotherapy was associated with subsequent leukemia. CONCLUSION: Aggressive Hodgkin's disease therapy is successful, but patients have a significant risk of second malignancy. Newer treatment programs focus on obtaining a relapse-free cure of Hodgkin's disease with judicious use of radiation and alkylating agent chemotherapy. Survivors of pediatric Hodgkin's disease require lifelong evaluation and cancer screening.

Stress proteins are not induced in mammalian cells exposed to radiofrequency or microwave radiation.

The induction of stress proteins in HeLa and CHO cells was investigated following a 2 h exposure to radiofrequency (RF) or microwave radiation. Cells were exposed or sham exposed in vitro under isothermal (37 +/- 0.2 degrees C) conditions. HeLa cells were exposed to 27- or 2450 MHz continuous wave (CW) radiation at a specific absorption rate (SAR) of 25 W/kg. CHO cells were exposed to CW 27 MHz radiation at a SAR of 100 W/kg. Parallel positive control studies included 2 h exposure of HeLa or CHO cells to 40 degrees C or to 45 microM cadmium sulfate. Stress protein induction was assayed 24 h after treatment by electrophoresis of whole-cell extracted protein labeled with [35S]-methionine. Both cell types exhibited well-characterized responses to the positive control stresses. Under these exposure conditions, neither microwave nor RF radiation had a detectable effect on stress protein induction as determined by either comparison of RF-exposed cells with sham-exposed cells or comparison with heat-stressed or Cd++ positive control cells.

Effect of isothermal radiofrequency radiation on cytolytic T lymphocytes.

Previous in vitro studies provide evidence that RF electromagnetic radiation modulates proliferation of human glioma, lymphocytes, and other cell types. The mechanism of RF radiation cell proliferation modulation, as well as mechanisms for effects on other cell physiologic endpoints, are not well understood. To obtain insight regarding interaction mechanisms, we investigated effects of RF radiation exposure on interleukin 2 (IL-2) -dependent proliferation of cytolytic T lymphocytes (CTLL-2). After exposure to RF radiation in the presence or absence of IL-2 cells were cultured at various physiological concentrations of IL-2. Treatment effects on CTLL-2 proliferation were determined by tritiated thymidine incorporation immediately or 24 h after exposure. Exposure to 2450 MHz RIF radiation at specific absorption rates (SARs) of greater than 25 W/kg (induced E-field strength 98.4 V/m) induced a consistent, statistically significant reduction in CTLL-2 proliferation, especially at low IL-2 concentrations. At lower SARs, 2450 MHz exposure increased CTLL-2 proliferation immediately after exposure but reduced 24 h postexposure proliferation. RF radiation effects depended on the mitotic state of the cells at the time of exposure. Comparison of the effects of temperature elevation and RF radiation indicated significant qualitative and quantitative differences.

Absorbed energy distribution from radiofrequency electromagnetic radiation in a mammalian cell model: effect of membrane-bound water.

The spatial distributions of induced 27 or 2450 MHz radiofrequency (RF) electric fields (E-fields) and specific absorption rates (SARs) in a three-component spherical cell model (cytoplasm, membrane, extracellular space) were determined by Mie scattering theory. The results were compared to results for the same cell model but with 0.5 nm thick of bound water on the inner (cytoplasmic) and outer (extracellular) membrane surfaces (i.e., five-component cell model). The results provide insight regarding direct frequency-dependent RF radiation effects at the cellular level. Induced E-fields and SARs were calculated for two bound-water characteristic frequencies (400 or 1000 MHz) and ionic conductivities (1-1000 mS/m). In order to estimate the dependence of the results on bound water within the membrane per se, the model was revised to include bound water within the inner and outer membrane surfaces. The results were as follows: 1) on the x-axis, the y- and z-components of the induced E-field were of insignificant magnitude compared to the x-component for an incident E-field parallel to the x-axis; 2) the ratio of transmembrane E-fields induced by 2450 MHz vs. 27 MHz RF [i.e., Ex (2450 MHz)/Ex (27 MHz)] was 0.1; 3) for the three-component cell model, the corresponding SAR ratios [SAR (2450 MHz)/SAR (27MHz)] in the cytoplasm and extracellular space were 1.66 and 5.0, respectively; 4) the SAR rations [SAR (2450 MHz)/SAR (27 MHz)] for the cytoplasm and extracellular space for the five-component cell model were 1.66 and 5.0, respectively; 5) the ratio of the E-fields induced in the cytoplasmic and extracellular layers of bound water in the five-component cell model [E (2450 MHz)/ E (27Mhz)] were 0.62 and 0.63, respectively; 6) the SAR ratios [SAR (2450 MHz)/SAR (27 MHz)] for the cytoplasmic and extracellular bound-water layers were 66 and 65.3, respectively; and 7) variation of bound-water characteristic frequency, ionic conductivity, or bound-water incorporation inside the membrane surfaces, per se, did not significantly affect the E-field or SAR ratios. These results indicate that frequency-dependent nonuniformities may occur in the distribution of induced RF E-fields and SARs at the cellular level.

Hodgkin's disease in the very young.

PURPOSE: A retrospective review of patients with Hodgkin's disease treated at Stanford University Medical Center was undertaken to determine if, within the pediatric population, children < or = 10 years of age have a unique prognosis and response to treatment. METHODS AND MATERIALS: Records of all patients treated for Hodgkin's disease at SUMC between 1961 and 1991 were reviewed. RESULTS: Of 2238 patients with Hodgkin's disease, 91 (4%) were < or = 10 years of age. There is a predominance of male patients (80%) and a higher percentage of mixed cellularity (33%) and lymphocyte predominance (13%) histologies among the very young patients compared to adolescents and adults. The 5 and 10-year survival is 94 +/- 3% and 92 +/- 3%, respectively, for children < or = 10 vs. 93 +/- 2% and 86 +/- 3% for adolescents and 84 +/- 1% and 73 +/- 1% for adults. Five and 10-year freedom from relapse is also higher in the youngest children (88 +/- 4% and 85 +/- 4%, respectively) compared to adolescents (78 +/- 3% and 74 +/- 3%, respectively) and adults (70 +/- 1% and 67 +/- 1%, respectively). Actuarial survival at 25 years for children < or = 10 years is 78%, which is slightly better than for adolescents (67%) and significantly better than for adults (41%) (p = 0.001). Actuarial 25-year freedom from relapse is also significantly better for children < or = 10 (78%) compared to adolescents (74% [p = 0.05]) and adults (65% [p = 0.001]). For all stages of disease, children < or = 10 fare similarly to or slightly better than adolescents and substantially better than adults. For those with Stage I or II disease, survival at 5, 10, and 25 years is 98 +/- 2%, 93 +/- 4% and 73%, respectively, for children aged < or = 10; 98 +/- 1%, 91 +/- 3%, and 79%, respectively, for adolescents and 89 +/- 1%, 80 +/- 1%, and 45%, respectively, for adults. The greatest difference between age groups is seen for Stage III and IV patients. Those aged < or = 10 have an 89 +/- 5% 5 and 10-year survival, and 89% actuarial 25-year survival compared to 87 +/- 4%, 80 +/- 5%, and 28%, respectively, for adolescents and 77 +/- 2%, 64 +/- 2%, and 41%, respectively, for adults. Of patients < or = 10 years of age, 28 (31%) were treated with primary external beam radiotherapy, and 59 (65%) received combined modality therapy consisting of low-dose radiation and chemotherapy. With a median follow-up of 11 years, freedom from relapse is 64% and survival 75% for the radiotherapy group, compared to 97% (p = 0.000) and 93% (p = 0.21) for those treated with combined modality therapy. CONCLUSION: Results indicate that young age is a favorable prognostic factor in Hodgkin's disease. Combined modality therapy has led to improved freedom from relapse and survival rates for all stages of disease and is currently the treatment of choice for the majority of very young children.

A review of in vitro studies: low-frequency electromagnetic fields.

In vitro studies of effects of low-frequency (LF) electromagnetic (EM) fields have revealed a variety of sensitive cell-physiologic end-points. Effects have been reported on (1) DNA, RNA, and protein synthesis; (2) cell proliferation; (3) cation fluxes and binding; (4) immune responses; and (5) membrane signal transduction (i.e., hormones, enzymes, and neuro-transmitters). Typically such effects occurred as a result of short-term exposure of cells to EM at frequencies of 100 Hz or less and at low field intensities. The dependency on frequency or modulation, as well as the apparent weak cellular interaction of these LF EM fields, lacks theoretic explanation. It has not been determined whether effects are induced by electric or magnetic fields. Confounding interpretation of the results are phenomena such as (1) transient or time-delayed responses; (2) modulation- and intensity-specific effects, referred to as modulation or intensity "windows;" and (3) general lack of dose- (or dose-rate) response data or EM field thresholds. Consequently, although it is well-established that LF EM fields affect biological systems in vitro, use of these data to assess human health effects is limited. This paper reviews selected published reports of LF EM fields on in vitro systems. Where possible, relevance of the findings to occupational exposures will be assessed, principally by considering the consistency of in vitro and in vivo EM exposure effects and comparison of EM field intensities that affect in vitro systems with occupational EM exposure intensities. Finally, suggestions will be made for the direction of future in vitro research of direct pertinence to potential occupational exposure problems.

Glioma proliferation modulated in vitro by isothermal radiofrequency radiation exposure.

Isothermal (37 +/- 0.2 degrees C) exposure of glioma cells (LN71) for 2 h to 27 or 2450 MHz continuous-wave radiofrequency (RF) radiation in vitro modulated the rates of DNA and RNA synthesis 1, 3, and 5 days after exposure. The alterations indicate effects on cell proliferation and were not caused by RF-induced cell heating. The dose response for either frequency of the radiation was biphasic. Exposure to specific absorption rates (SARs) of 50 W/kg or less stimulated incorporation rates of tritiated thymidine (3H-TdR) and tritiated uridine (3H-UdR), whereas higher SARs suppressed DNA and RNA synthesis. Statistically significant time-dependent alterations were detected for up to 5 days postexposure, suggesting a kinetic cellular response to RF radiation and the possibility of cumulative effects on cell proliferation. General mechanisms of effects are discussed.

In vitro lymphocyte proliferation induced by radio-frequency electromagnetic radiation under isothermal conditions.

Whole human blood was exposed or sham-exposed in vitro for 2 h to 27 or 2,450 MHz radio-frequency electromagnetic (RF) radiation under isothermal conditions (i.e., 37 +/- 0.2 degrees C). Immediately after exposure, mononuclear cells were separated from blood by Ficoll density-gradient centrifugation and cultured for 3 days at 37 degrees C with or without mitogenic stimulation by phytohemagglutinin (PHA). Lymphocyte proliferation was assayed at the end of the culture period by 6 h of pulse labeling with 3H-thymidine (3H-TdR). Exposure to radiation at either frequency at specific absorption rates (SARs) below 50 W/kg resulted in a dose-dependent, statistically significant increase of 3H-TdR uptake in PHA-activated or unstimulated lymphocytes. Exposure at 50 W/kg or higher suppressed 3H-TdR uptake relative to that of sham-exposed cells. There were no detectable effects of RF radiation on lymphocyte morphology or viability. Notwithstanding the characteristic temperature dependence of lymphocyte activation in vitro, the isothermal exposure conditions of this study warrant the conclusion that the biphasic, dose-dependent effects of the radiation on lymphocyte proliferation were not dependent on heating.

In vitro fertilization of mouse ova by spermatozoa exposed isothermally to radio-frequency radiation.

Mouse spermatozoa were exposed in vitro for 1 h to 27- or 2,450-MHz CW RF radiation at SARs of 0 to 90 W/kg under isothermal (37 +/- 0.2 degrees C) conditions. Exposure at either frequency to RF radiation at SARs of 50 W/kg or greater resulted in a statistically significant reduction in the ability of irradiated sperm to fertilize mouse ova in vitro (P less than .05). Over the range of SARs there was no apparent difference in the effects of 27- vs. 2,450-MHz RF radiation. There were no readily detectable exposure effects on spermatozoan morphology, ultrastructure, or capacitation. The reduction of in vitro fertilization is attributed to a direct effect of RF radiation on spermatozoa rather than to heating.

Effects of 2.45-GHz microwave and 100-MHz radiofrequency radiation on liposome permeability at the phase transition temperature.

Large unilamellar dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) liposomes loaded with an aqueous chemotherapeutic drug, cytosine arabinofuranoside (ARA-C), were exposed for 30 min to 60 W/kg continuous-wave (CW) 100-MHz or 2.45-GHz radiation in vitro at temperatures between 37 degrees C and 43 degrees C. Liposomes were exposed in HEPES buffer or in HEPES buffer supplemented with 44% by volume fetal calf serum (FCS). Characteristic phase transition responses were detected in the range of 39 degrees C to 40 degrees C with the presence of FCS, increasing maximum % release of 3H-ARA-C by 20% relative to HEPES suspension. Neither frequency of electromagnetic radiation had any detectable effect on liposome permeability or the location of the phase transition in the presence or absence of FCS.

Soluble amoebicidal factors mediate cytolysis of Naegleria fowleri by activated macrophages.

Murine peritoneal macrophages activated in vivo with Corynebacterium parvum or bacille Calmette-Guérin, in contrast to resident macrophages, demonstrated significant cytolysis of the amoeba, Naegleria fowleri. Catalase and superoxide dismutase, both alone and in combination, failed to inhibit cytolysis of amoebae. N. fowleri amoebae demonstrated significant resistance to exogenously added hydrogen peroxide. The hydroxyl radical scavengers mannitol, thiourea, and dimethyl sulfoxide, as well as anaerobic conditions, failed to inhibit the amoebicidal activity of activated macrophages. Actinomycin D, cycloheximide, and puromycin blocked macrophage amoebicidal activity. Conditioned medium (CM) from lipopolysaccharide-stimulated, but not unstimulated, cultures of activated macrophages was capable of mediating cytolysis of N. fowleri amoebae. Cytolytic activity was recovered by ammonium sulfate precipitation of CM. Heat treatment of the CM inactivated cytolytic activity. Results indicate soluble proteins of activated macrophage origin to be responsible for the amoebicidal activity.

Activated macrophages demonstrate direct cytotoxicity, antibody-dependent cellular cytotoxicity, and enhanced binding of Naegleria fowleri amoebae.

Macrophages activated in vivo by injection of Corynebacterium parvum or bacillus Calmette-Guérin caused direct cytolysis of the pathogenic free-living amoeba, Naegleria fowleri, in vitro. Amoebicidal activity was time and cell density-dependent but was not dependent on the presence of specific antibody. Antibody-dependent cellular cytotoxicity for amoebae was also expressed by activated macrophages. Resident and thioglycolate-elicited macrophages demonstrated low cytolytic activity under all conditions tested. From scanning electron microscopy it appears that the degree of target cell binding is directly related to the degree of cytolysis expressed by the macrophage populations. Cell-cell contact was required for cytolysis of amoebae by activated macrophages since cytolysis did not occur when contact was blocked by a porous filter. For each macrophage population, the levels of amoebicidal activity and tumoricidal activity were comparable.

Viability and phagocytosis of neutrophils exposed in vitro to 100-MHz radiofrequency radiation.

Rabbit polymorphonuclear leucocytes (PMN, neutrophils) obtained from peritoneal exudate were exposed in vitro for one-half or one hour to continuous wave or amplitude-modulated (20-Hz) 100-MHz RF radiation in a temperature-controlled coaxial exposure chamber at field strengths from 2.5 to 4.1 V/cm (SARs of 120 to 341 W/kg). RF exposure at 37 +/- 0.2 degrees C had no detectable effect on PMN viability or phagocytosis compared to sham-exposed cells simultaneously subjected to the same time-temperature regime. Temperature control studies indicated that at 37 degrees C no effect on PMN viability would be expected but phagocytosis would be reduced by approximately 6%/degrees C temperature increase. The absence of an effect of RF exposure suggests that there was minimal undetected intrasample heating and that phagocytosis was not affected by 100-MHz RF radiation under the conditions of this study.

Studies of exposure of rabbits to electromagnetic pulsed fields.

Dutch rabbits were acutely exposed to electromagnetic pulsed (EMP) fields (pulse duration 0.4 mus, field strengths of 1--2 kV/cm and pulse repetition rates in the range of 10 to 38 Hz) for periods of up to two hours. The dependent variables investigated were pentobarbital-induced sleeping time and serum chemistry (including serum triglycerides, creatine phosphokinase (CPK) isoenzymes, and sodium and potassium). Core temperature measured immediately pre-exposure and postexposure revealed no exposure-related alterations. Over the range of field strengths and pulse durations investigated no consistent, statistically significant alterations were found in the end-points investigated.

The in vivo effects of 2.45 GHz microwave radiation of rabbit serum components and sleeping times.

An investigation was conducted to determine the effects of relatively low power density microwave exposures on various serum components of the Dutch rabbit. Both continous wave and pulsed mode exposures at 2.45 GHzwere used at power densities of 25,10 and 5 mW/cm2. Studies of 10 serum components were performed. Additional studies were conducted on changes in sleeping times of pentobarbital-sedated rabbits at various power densities. Gross and histopathological examinations were performed on representative samples of animals. Changes in the blood chemistry of irradiated animals were consistent with a dose-dependent response to a non-specific thermal stress at all power densities used. Observed physiological response, as well as rectal temperature measurements, indicated that the thermoregulatory capability of the rabbits was sufficient to compensate for the thermal burden at 5 and 10 mW/cm2, but could be overridden by a 2 h exposure at 25 mW/cm2. Pathology findings included a mild, repairable nephrosis in animals exposed at a power density of 25 mW/cm2. A further investigation of analeptic effects at power densities varying from 5 mW/cm2 to 50 mW/cm2 resulted in a statistically significant decrease in sleeping times, apparently proportional to power density below 15 mW/cm2.