In vitro and in vivo genotoxicity of radiofrequency fields.

There has been growing concern about the possibility of adverse health effects resulting from exposure to radiofrequency radiations (RFR), such as those emitted by wireless communication devices. Since the introduction of mobile phones many studies have been conducted regarding alleged health effects but there is still some uncertainty and no definitive conclusions have been reached so far. Although thermal effects are well understood they are not of great concern as they are unlikely to result from the typical low-level RFR exposures. Concern rests essentially with the possibility that RFR-exposure may induce non-thermal and/or long-term health effects such as an increased cancer risk. Consequently, possible genetic effects have often been studied but with mixed results. In this paper we review the data on alleged RFR-induced genetic effects from in vitro and in vivo investigations as well as from human cytogenetic biomonitoring surveys. Attention is also paid to combined exposures of RFR with chemical or physical agents. Again, however, no entirely consistent picture emerges. Many of the positive studies may well be due to thermal exposures, but a few studies suggest that biological effects can be seen at low levels of exposure. Overall, however, the evidence for low-level genotoxic effects is very weak.

Phosphomannosyl receptors may participate in the adhesive interaction between lymphocytes and high endothelial venules.

Normal and malignant lymphocytes can migrate from the bloodstream into lymph nodes and Peyer's patches. This process helps distribute normal lymphocytes throughout the lymphoid system and may provide a portal of entry for circulating malignant cells. An adhesive interaction between lymphocytes and the endothelium of postcapillary venules is the first step in the migratory process. We have recently shown that the simple sugars L-fucose and D-mannose, and an L-fucose-rich polysaccharide (fucoidin), can inhibit this adhesive interaction in vitro. We now report that mannose-6-phosphate, the structurally related sugar fructose-1-phosphate, and a phosphomannan, core polysaccharide from the yeast Hansenula holstii (PPME) are also potent inhibitors. Inhibitory activity was assessed by incubating freshly prepared suspensions of lymphocytes, containing the various additives, over air-dried, frozen sections of syngeneic lymph nodes at 7-10 degrees C. Sections were then evaluated in the light microscope for the binding of lymphocytes to postcapillary venules. Mannose-6-phosphate and fructose-1-phosphate were potent inhibitors of lymphocyte attachment (one-half maximal inhibition at 2-3 mM). Mannose-1-phosphate and fructose-6-phosphate had slight inhibitory activity, while glucose-1-phosphate, glucose-6-phosphate, galactose-1-phosphate, and galactose-6-phosphate had no significant activity (at 10 mM). In addition, the phosphomannan core polysaccharide was a potent inhibitor (one-half maximal inhibition at 10-20 micrograms/ml); dephosphorylation with alkaline phosphatase resulted in loss of its inhibitory activity. Preincubation of the lymphocytes, but not the lymph node frozen sections, with PPME resulted in persistent inhibition of binding. Neither the monosaccharides nor the polysaccharide suppressed protein synthesis nor decreased the viability of the lymphocytes. Furthermore, inhibitory activity did not correlate with an increase in negative charge on the lymphocyte surface (as measured by cellular electrophoresis). These data suggest that a carbohydrate-binding molecule on the lymphocyte surface, with specificity for mannose-phosphates and structurally related carbohydrates, may be involved in the adhesive interaction mediating lymphocyte recirculation.

Recognition and invasion of human erythrocytes by malarial parasites: contribution of sialoglycoproteins to attachment and host specificity.

The receptivity of human erythrocytes to invasion by Plasmodium falciparum merozoites can be decreased by neuraminidase or trypsin treatment, an observation that supports a role for the erythrocyte sialoglycoproteins (glycophorins) in invasion. We have found that alpha 1-acid glycoprotein (AGP), added to in vitro cultures, can restore invasion of enzyme-treated human erythrocytes. AGP is structurally different from the glycophorins although it does carry 12% sialic acid. Its ability to restore receptivity to desialylated cells is dependent on its sialic acid complement, its concentration, and its binding to the erythrocyte surface. We present evidence that AGP forms a bridge between the merozoite and the enzyme-treated erythrocyte that allows the stronger and more complex interactions of invasion to proceed. We suggest that the glycophorins play the same role on the surface of the intact erythrocyte.

Carbodiimide enhancement of complement-dependent antibody-mediated tumor cell lysis in vitro and antitumor activity in vivo.

The water-soluble carbodiimide salt 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide. HCl (EDCI-HCl) has been shown to increase the complement-dependent lysis of cultured mouse neuroblastoma C1300 cells by two types of antibody: (1) natural antibodies in the sera of normal (nonimmunized) rabbits, and (b) serum antibodies from snygeneic tumor-bearing A/HeJ mice. In the latter case, both the level of serum antibodies and the extent of carbodiimide enhancement of immune lysis were demonstrated in vitro to be substantially greater with sera from mice bearing 21-day-old tumors relative to 4-day-old tumors. The carbodiimide EDCI-HCl has also been found to increase the complement-dependent lysis of cultured TA3 carcinoma cells by serum antibodies from isogeneic LAF1/J mice bearing ascites tumors in advanced stages of growth. Finally, it has been shown that EDCI-HCl exerts an antitumor activity in vivo that is significantly greater against 21-day-old than against 4-day-old neuroblastoma c1300 tumors. The increase in EDCI-HCl activity with tumor age is contrary to the response that would be expected if this drug were serving as an antimetabolite. This is evidenced by data showing that the antimetabolite 6-thioguanine is most effective against young rapidly growing neuroblastoma C1300 tumors. The correlation between carbodiimide antitumor activity and host production of cytotoxic antibodies suggests that EDCI-HCl may operate in vivo by an immunological mechanism comparalbe to that demonstrated in vitro.

Hypoxic cell sensitizers and heavy charged particle beams may play complementary roles in killing hypoxic tumor cells.

The analysis of growth delay data of a rat rhabdomyosarcoma tumor system with and without misonidazole and irradiated with spread-peak heavy-ion radiation yields two conclusions that bear on the relative efficacy of the two modes of treatment and imply a complementary role of the two modes which enhances the effects of either given separately. 1. For both carbon and neon ion peak radiation given in four fractions, RBE values for tumor growth delay are significantly greater than the enhancement ratio for an X ray plus misonidazole fractionation scheme [2.0-2.3 (carbon) and 2.6-2.8 (neon) vs. 1.2-1.5 (X rays plus misonidazole)]. This implies that high LET killing is considerably more effective in this tumor system (hypoxic fraction of about 35%) than the hypoxic cell sensitization caused by misonidazole. 2. When misonidazole is given in conjunction with the heavy ion beam irradiations, an increased growth delay is seen, greater than when either heavy ions or misonidazole plus X rays are given separately. The product of the sensitizer enhancement ratio for heavy ions and the RBE for no sensitizer yields a measure of the overall enhancement of effect relative to an X ray treatment. The values of this product for the carbon beam (2.4-2.5) and neon beam (3.4) show high effectiveness for either beam plus misonidazole. The interpretation is that heavy ion beams reach and kill hypoxic cells not penetrated by the misonidazole, and some hypoxic cells not killed by the high LET component receive low LET damage which is made lethal by the drug. Thus, the net hypoxic cell killing is enhanced by the high LET beams and in a complementary way by the combination of the drug and the low LET portion of the radiation.

Reoxygenation in a rat rhabdomyosarcoma tumor following X-irradiation.

The paired survival curve technique was used to characterize the rate at which the fraction of hypoxic cells in rat rhabdomyosarcoma R-1 tumors returns to the preirradiation value of 37% following a single dose of 225-kVp X rays. Tumors were administered a conditioning x-ray dose of 15-Gy, followed at 0, 3, 6, 12, 24, or 48 hr by a 5-Gy, 10-Gy, or 15-Gy dose of X rays under air-breathing conditions or under hypoxic conditions produced by nitrogen-gas asphyxiation 5 min prior to irradiation. Cellular surviving fractions were determined by the tumor excision assay following in vivo irradiation. From the ratio of the survival fractions measured for tumor cells from air-breathing and hypoxic animals, the fraction of hypoxic cells was determined as a function of time postirradiation. These results indicated that immediately following a 15-Gy dose of X rays, essentially 100% of the viable cells remaining were hypoxic. The tumors reoxygenated rapidly, returning to the preirradiation level of 37% during the first 6 hr postirradiation.

Response of rat rhabdomyosarcoma tumors to split doses of mixed high- and low-let radiation.

Radiation-induced growth delay was measured in rat rhabdomyosarcoma tumors exposed to split doses of high-LET (linear energy transfer) neon ions in the extended-peak ionization region and low-LET X rays. Top-off doses of 7.5, 15, and 25 Gy of 225-kVp X rays were administered to the tumors at 0.5, 4.0, and 24.0 hr following priming doses of either peak neon ions or X rays. The priming doses used were 7 Gy of peak neon ions and 20 Gy of X rays, both of which produced a 10 day delay in tumor regrowth to a volume twice that measured on the day of irradiation. The tumor response to split doses of X rays indicated rapid repair of sublethal damage, with significant recovery occurring at 0.5 hr and complete recovery by 4 hr after the initial 20-Gy X ray dose. The top-off doses of X rays required to produce an additional 10 or 20 days of tumor growth delay were 18 and 7% larger, respectively, when the priming dose was 20 Gy of X rays as compared to 7 Gy of peak neon ions. This result indicates that relatively little interaction of the neon-ion and X ray radiations occurred, even when the time interval between split-dose irradiations was as short as 0.5 hr. Our data indicate that the interaction of high- and low-LET radiation modalities is small, and approaches a simple additivity of effects when the tumors repair a major portion of the sublethal radiation injury imparted by a priming dose before the second dose is administered.

Molecular and cellular radiobiology of heavy ions.

Quantitative studies at the BEVALAC have demonstrated some of the physical and radiobiological factors that promise to make accelerated heavy ions important for the therapy of cancer. The measured physical dose-biological effect relationships allow the safe and effective delivery of therapeutic schedules of heavy ions. Among the charged particle beams available, carbon, neon and helium ions in the "extended Bragg peak mode" have optimal physical and biological effectiveness for delivery of therapy to deep seated tumors. The depth-dose profiles of these beams protect intervening and adjacent tissues as well as tissues beyond the range of the particles. For the treatment of hypoxic tumors, silicon and argon beams are being considered because they significantly depress the radiobiological oxygen effect in the region of the extended Bragg ionization peak. The depth-effectiveness of the argon beam is somewhat limited, however, because of primary particle fragmentation. Silicon beams have a depth-dose profile which is intermediate between that of neon and argon, and are candidates to become the particle of choice for maximizing high LET particle effects. Heavy accelerated ions depress enzymatic repair mechanisms, decrease variations of radiosensitivity during the cell division cycle, cause greater than expected delays in cell division, and decrease the protective effects of neighboring cells in organized systems. Near the Bragg peak, enhancement of heavy particle effects are observed in split dose schedules. Late and carcinogenic effects are being studied. With the newly developed Repair-Misrepair theory we can quantitatively model most observations.

Treatment of cancer with heavy charged particles.

A clinical radiotherapeutic trial using heavy charged particles in the treatment of human cancers has accrued over 400 patients since 1975, 378 of whom were treated with particles and 28 with low LET photons as control patients. Heavy charged particle radiotherapy offers the potential advantages of improved dose localization and/or enhanced biologic effect, depending on particle selected for treatment. Target sites have included selected head and neck tumors, ocular melanomata, malignant gliomata of the brain, carcinoma of the esophagus, carcinoma of the stomach, carcinoma of the pancreas, selected juxtaspinal tumors and other locally advanced, unresectable tumors. A Phase III prospective clinical trial has been started in carcinoma of the pancreas using helium ions. Phase I-II studies are underway with heavier particles such as carbon, neon and argon ions in order to prepare for prospective Phase III trials. Silicon ions are also under consideration for clinical trial. These studies are supported by the United States Department of Energy and National Institutes of Health.

Magnetic field-induced drug permeability in liposome vesicles.

Liposome vesicles maintained in a uniform static magnetic field release a chemotherapeutic drug (ARA-C, MW = 243) at temperatures approaching the phase-transition region where these liposomes are not normally leaky. Drug release is rapid, and a maximum difference between treated and unexposed liposomes of 30% of the total maximal release of ARA-C was observed within 1 min in a magnetic field. Dose-effect studies conducted between 0.01 and 7.5 T (1 T = 10(4) G) reveal that this permeability effect has a sigmoidal dependence on magnetic flux density. The ED50 is 15 mT, with a 95% confidence interval of 6.50-34.9 mT. Magnetic field exposures were conducted using a superconducting magnet with the liposomes maintained at +/- 0.08 degrees C. For comparison, samarium-cobalt permanent magnets induced a comparable drug release at 0.4 T. These results indicate that a static magnetic field of 10 mT or greater can increase passive transport in phospholipid membrane bilayers maintained at or near their membrane phase-transition temperature. Lipid clustering which occurs at prephase-transition temperatures may predispose phospholipid domains to diamagnetic orientation in a magnetic field and thereby facilitate drug release.

Repopulation kinetics of rat rhabdomyosarcoma tumors following single and fractionated doses of low-LET and high-LET radiation.

Measurements were made of clonogenic cell survival in rat rhabdomyosarcoma tumors as a function of time following in situ irradiation with single or fractionated doses of 225-kVp X rays or with 557-MeV/u neon ions in the distal position of a 4-cm extended-peak ionization region. Single doses of 20 Gy of X rays or 7 Gy of peak neon ions reduced the initial surviving fraction to approximately 0.025 for each modality. Daily fractionated doses (four fractions in 3 days) of either peak neon ions (1.75 Gy per fraction) or X rays (6 Gy per fraction) achieved a cell survival of approximately 0.02-0.03 after the fourth dose of radiation. In the single-dose experiments, significant 5- and 10-fold decreases in the fraction of clonogenic cells were observed between the third and fourth days after irradiation with peak neon ions and X rays, respectively. After the sixth day postirradiation, the residual clonogenic cells exhibited a rapid burst of proliferation leading to doubling times for the surviving cell fractions of approximately 1.5 days. Radiation-induced growth delay was consistent with the cellular repopulation dynamics. In the fractionated-dose experiments with both radiation modalities, a large delayed decrease in cell survival was observed at 1-3 days after completion of the fractionated-dose schedule. Cellular repopulation was consistent with postirradiation tumor volume regression and regrowth for both radiation modalities. The extent of decrease in survival following the four-fraction radiation schedule was approximately two times greater in X-irradiated than in neon-ion-irradiated tumors that produced the same survival level immediately after the fourth dose. Mechanisms underlying the marked reduction in cell survival 3-4 days postirradiation are discussed, including the possible role of a toxic host cell response against the irradiated tumor cells.

PLD repair in rat rhabdomyosarcoma tumor cells irradiated in vivo and in vitro with high-LET and low-LET radiation.

Results are reported of studies to measure the extent of recovery of potentially lethal damage (PLD) in rat rhabdomyosarcoma tumor cells after irradiation both in vivo and in vitro with either high-LET or low-LET radiation. Stationary-phase cultures were found to exhibit repair of PLD following irradiation in vitro either with low-LET X rays or with high-LET neon ions in the extended-peak ionization region. Following a 9-Gy dose of 225-kVp X rays or a 3.5-Gy dose of peak neon ions, both of which reduced the initial cell survival to 6-8%, the maximum PLD recovery factors were 3.4 and 1.6, respectively. In contrast, the standard tumor excision assay procedure failed to reveal any recovery from PLD in tumors irradiated in situ with either X rays or peak neon ions. PLD repair by the in vivo tumor cells could be observed, however, when the excision assay procedure was altered by the addition of a known PLD repair inhibitor beta-arabinofuranosyladenine (beta-ara-A). When a noncytotoxic 50 microM concentration of beta-ara-A was added to the excised tumor cells immediately following a 14.5-Gy in situ dose of X rays, cell survival in the inhibitor-treated cells was lower than in the untreated cells (0.018 compared to 0.056), resulting in a PLD repair inhibition factor of 3.1. Delaying the addition of beta-ara-A for 1, 2, or 3 h following tumor excision reduced the PLD repair inhibition factor to 1.6, 1.5, and 0.9, respectively. Following tumor irradiation in situ with neon ions in the extended-peak ionization region (median LET = 145 keV/micron), less PLD repair was observed than after X irradiation. For 5.8 Gy of peak neon ions, the PLD repair inhibition factors were 2.1, 1.5, 1.3, and 1.1 at 0, 1, 2, and 3 h, respectively. We interpret the absence of measurable PLD repair using the standard tumor excision assay procedure as resulting from undetectable repair occurring during the long interval (about 2 h) required for the cell dissociation and plating procedures. We conclude that at least for our tumor system, PLD repair does occur after irradiation of tumors in situ, even though it is not detectable using the standard tumor excision assay procedure. Thus a failure to measure such repair by this assay in a given tumor system does not necessarily mean the cells are incapable of PLD repair.

Host cell cytotoxicity, cellular repopulation dynamics, and phase-specific cell survival in X-irradiated rat rhabdomyosarcoma tumors.

Postirradiation tumor volume response, cellular repopulation dynamics, cell-cycle perturbations, and phase-specific cell survival were characterized in rat rhabdomyosarcoma R-1 tumors (the R2C5 subline) following an in situ 10-Gy dose of 225-kVp X rays. This X-ray dose produced a 7.5-day delay in tumor growth to twice the volume measured at the time of irradiation, and reduced the initial surviving fraction of R2C5 cells to 0.17 as measured by the excision assay procedure. The surviving fraction of R2C5 cells returned to unity by the 16th day after tumor irradiation. On the basis of flow cytometry measurements of DNA content in tumor cells stained with a noncytotoxic concentration of Hoechst 33342 (5 microM, 2 h, 37 degrees C), a transient G2 block was observed 1 day after irradiation. Flow cytometry measurements also demonstrated that the tetraploid R2C5 cells constituted only 30% of the total tumor cell population, with the remainder being diploid host cells comprised of macrophages, monocytes, lymphocytes, and granulocytes. Large numbers of host cells infiltrated the irradiated tumors, leading to an increase in the percentage of diploid cells by Day 2 and reaching a level of more than 80% of the total tumor cell population by 4 to 8 days after irradiation. The influx of host cells into irradiated tumors was correlated temporally with a significant 12-fold decrease in the surviving fraction of R2C5 cells that occurred between Days 2 and 4 postirradiation. When the diploid host cell population was removed by cell sorting procedures, the surviving fraction of R2C5 cells at Day 4 was substantially greater than that in the presence of the host cells. Experiments involving the mixing of 4/1 and 12/1 ratios of diploid host cells and tetraploid tumor cells isolated from irradiated and unirradiated tumors demonstrated that the cytotoxic effect of the host cells was specific for the irradiated tumor cells. The significant toxic effect of host cells on irradiated tumor cells was observed only at 2 to 4 days after irradiation, and not at earlier or later times. The data obtained in these experiments indicate that the immunogenicity of R2C5 cells is increased significantly by irradiation, and a resultant cell-mediated host immune response produced a delayed decrease in tumor cell survival that is most pronounced 4 days after irradiation. The cell survival characteristics of R2C5 cells in different cell-cycle phases were found to be similar through the 16-day postirradiation interval that was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of extremely low frequency electric and magnetic fields with humans.

The dosimetry and physical interaction mechanisms of electromagnetic fields with frequencies in the extremely low frequency (ELF) range (below 300 Hz) are described. The mechanisms through which ELF electric and magnetic fields induce electric currents in living organisms are summarized, with particular emphasis on humans. Topics that are discussed include: (1) sources and measurements of ELF electric and magnetic fields; (2) direct and indirect coupling of these fields to humans; (3) transient discharges and contact currents, and the thresholds for human response to these phenomena; (4) protective measures for the mitigation of potential ELF field effects on humans; and (5) mechanisms of interaction of ELF fields with cellular and tissue systems, with emphasis on field transduction mechanisms involving the cell membrane.