LaVerne, J. A. Track Effects of Heavy Ions in Liquid Water.

The various types of ionizing radiation can have widely differing radiation effects due to their variation in track structure. The effects of track structure on the radiation chemistry of water are particularly important because of the fundamental aspects and the wide range of practical applications. This review gives an overview of how the physics of energy-loss processes are responsible for the geometry of the particle track and thereby the final product yields. The radiation chemical effects are discussed in qualitative terms to show how the fundamental relationship between the physics and chemistry of particle tracks leads to the observed products. Special phenomena at very high rates of energy deposition are also covered. Finally, a discussion on the future research trends is given.

Lisfi, D., Bonnefont-Rousselot, D., Fernet, M., Jore, D., Delattre, J. and Gardès-Albert, M. Protection of Endogenous Vitamin E and Beta-Carotene by Aminoguanidine upon Oxidation of Human Low-Density Lipoproteins by ^·OH/O₂^·−.

This study was designed to evaluate the antioxidant effect of aminoguanidine toward human low-density lipoproteins (LDLs) initiated by oxygenated free radicals (^·OH/O₂^·−) generated by gamma radiolysis. Initial radiolytic yields related to the markers of lipid peroxidation [i.e. decrease in endogenous α-tocopherol and β-carotene, formation of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes] were determined in 3 g liter⁻¹ LDLs (expressed as total LDL concentration) in the absence and presence of 10 different concentrations of aminoguanidine (from 0.04 to 5 mmol liter⁻¹). Fluorescence and relative electrophoretic mobility of oxidized LDLs were also studied as markers that indirectly reflect the attack of the protein moiety of LDLs (namely apolipoprotein B). Our data clearly showed the inhibitory effect of aminoguanidine on lipid peroxidation induced in LDLs by ^·OH/O₂^·− in a concentration-dependent manner. This effect probably resulted from a scavenging activity of aminoguanidine toward ^·OH. In contrast, aminoguanidine did not appear to react significantly with O₂^·−, which resulted in a poor residual lipid peroxidation. Our data led us to determine an optimum [aminoguanidine]/[LDL] ratio ranging from 250 to 500 to obtain the best in vitro protection of LDLs under our experimental conditions. It is also of great interest that aminoguanidine was able to protect endogenous α-tocopherol and β-carotene of LDLs upon ^·OH/O₂^·−-induced oxidation.

Seymour, C. B. and Mothersill, C. Relative Contribution of Bystander and Targeted Cell Killing to the Low-Dose Region of the Radiation Dose–Response Curve.

Human keratinocytes show a bystander effect when exposed to low doses of low-LET radiation. In this paper, data are presented showing a method of correcting the overall survival curve to enable analysis of the relative contributions of the bystander effect and the effect attributable to direct interaction of the radiation with the target cell. The technique used is to obtain a standard clonogenic survival curve using the assay of Puck and Marcus and, with a different set of flasks containing cloning densities of unirradiated cells, to assay the cell killing caused by medium harvested from 2 × 10⁵ cells irradiated with the same doses. The data show that for this human epithelial cell line, doses of 0.01–0.5 Gy show clonogenic death by the bystander effect only, if maximum potential bystander killing is assumed. The magnitude of the effect is relatively constant, and it appears to saturate at doses in the range of 0.03–0.05 Gy. After doses greater than 0.5 Gy, the curves for clonogenic death are the result of a dose-dependent non-bystander effect and a dose-independent bystander effect. If these particular dose–response effects occur in epithelial cells in vivo, they may have important consequences for therapy and studies of low-dose risk.

Hengstler, J. G., Bockisch, A., Fuchs, J., Grimm, W., Görges, R., Oesch-Bartlomowicz, B., Zapf, A-O., Lade, K., Tanner, B., Teichmann, E., Thelen, M., Gebhard, S. and Oesch, F. Induction of DNA Single-Strand Breaks by ¹³¹I and ^99mTc in Human Mononuclear Blood Cells In Vitro and Extrapolation to the In Vivo Situation.

The radionuclides ¹³¹I and ^99mTc are frequently used for therapy of benign and malignant thyroid disease (¹³¹I) and for diagnosis of thyroid and other diseases (^99mTc). However, the levels of DNA single-strand breaks (SSBs) induced in cells of patients after administration of ¹³¹I and ^99mTc are not known. In this study, we measured the number of SSBs per cell induced by ¹³¹I and ^99mTc in vitro, extrapolated the results to the clinical situation, and assessed their biological relevance by comparing levels of SSBs induced after therapeutic administration of ¹³¹I and ^99mTc to those induced by endogenous processes or by occupational exposure to genotoxic substances. A linear dose–response relationship between the radioactivity concentrations of ¹³¹I and ^99mTc and SSBs in human mononuclear blood cells (determined by alkaline elution) was obtained after incubation at 4 and 37°C. At 4°C, where almost no repair of SSBs takes place, ¹³¹I and ^99mTc induced 81 and 7 SSBs per cell per hour/(MBq/ml), respectively. At 37°C, only 20 and 1.6 SSBs per cell per hour/(MBq/ml) were observed after incubation with ¹³¹I and ^99mTc. To estimate the induction of SSBs in vivo in cells of patients after administration of 3700 MBq ¹³¹I (oral) or 60 MBq ^99mTc (i.v.), the rates of induction of SSBs obtained in vitro were extrapolated to the concentrations of ¹³¹I and ^99mTc measured in blood of patients. The total number of SSBs (mean ± standard deviation) accumulated after oral administration of 3700 MBq ¹³¹I up to 70 h after administration was calculated as 200 ± 59 SSBs/cell. After administration of 60 MBq ^99mTc (i.v.), 0.032 ± 0.009 SSBs per cell (total SSBs up to 2 h after administration) were cumulated. The induction of SSBs by endogenous processes (estimated 2,000 SSBs per cell per hour) and by occupational exposure to genotoxic substances (125–430 SSBs per cell) has been estimated in earlier studies. In conclusion, the frequency of SSBs induced by thyroid diagnosis with 60 MBq ^99mTc is approximately 5 orders of magnitude smaller than the frequency of spontaneous SSBs and thus is most probably without biological relevance. Since the frequency of induction of SSBs by therapy with ¹³¹I (3700 MBq) is about 6000-fold higher compared to thyroid diagnosis by ^99mTc, its biological relevance is more difficult to assess. Nevertheless, the number of SSBs induced by therapy with ¹³¹I is substantially lower than that induced by endogenous processes.

Pross, H. D., Casares, A. and Kiefer, J. Induction and Repair of DNA Double-Strand Breaks under Irradiation and Microgravity.

The influence of microgravity on induction and repair of double-strand breaks was studied in the yeast mutant rad54-3, which is temperature-conditional for the repair of DNA double-strand breaks. The experiment was performed on the shuttle Atlantis flight STS-84. Cell samples were kept at 0–4°C until they reached orbit, where they were transferred to 22 (permissive temperature for repair) and 37°C (restrictive temperature). They were exposed to graded doses of β particles from an in-built ⁶³Ni source during the repair period. After 152 h in microgravity, the radiation exposure was stopped, and the samples were returned to low-temperature conditions, where they remained until final evaluation in the home laboratory. The amount of double-strand breaks remaining was estimated from the differences in survival after plating and incubation at the restrictive temperature. The results show that there is no significant difference for both the induction and the repair of double-strand breaks between microgravity and terrestrial conditions.

Bottollier-Depois, J. F., Chau, Q., Bouisset, P., Kerlau, G., Plawinski, L. and Lebaron-Jacobs, L. Assessing Exposure to Cosmic Radiation during Long-haul Flights.

The assessment of exposure to cosmic radiation on board aircraft is one of the concerns of organizations responsible for radiation protection. Cosmic-particle flux increases with altitude and latitude and depends on solar activity. To illustrate the effect of these parameters, exposure has been estimated on several airlines operating subsonic and supersonic aircraft on transatlantic, Siberian and transequatorial routes. Measurements have been made with a tissue-equivalent proportional counter using the microdosimetric technique. This type of system provides the absorbed dose, the ambient dose equivalent, the mean quality factor, and the dose distribution as a function of lineal energy. Data were collected at maximum solar activity in 1991–1992 and at minimum activity in 1996–1998. The lowest mean dose rate measured was 3 μSv h^–1 during a Paris–Buenos Aires flight in 1991. The highest rates were 6.6 μSv h^–1 during a Paris–Tokyo flight on a Siberian route and 9.7 μSv h^–1 on Concorde in 1996–1997. The mean quality factor is around 1.8. The corresponding annual effective dose, based on 700 h of flight for subsonic aircraft and 300 h for Concorde, can be estimated at between 2 mSv for the least-exposed routes and 5 mSv for the more-exposed routes.

Zheng, H., Wang, J. and Hauer-Jensen, M. Role of Mast Cells in Early and Delayed Radiation Injury in Rat Intestine.

Mast cell hyperplasia is a characteristic feature of many inflammatory and fibrotic conditions, including intestinal radiation injury (radiation enteropathy). This study used mast cell-deficient rats to define the role of mast cells in the mechanisms underlying early radiation-induced mucosal injury and delayed intestinal wall fibrosis. Mast cell-deficient (Ws/Ws) mutant rats and mast cell-competent ( / ) littermates were used. A 4-cm loop of ileum was exposed to 21 Gy single-dose radiation. Irradiated and unirradiated intestine were examined at 2 or 26 weeks using quantitative histology and morphometry. Quantitative immunohistochemistry was used to assess transforming growth factor β (Tgfb), myeloperoxidase, and epithelial and smooth muscle cell proliferation. Collagen content was measured colorimetrically, and steady-state Tgfb1 mRNA was determined with fluorogenic probe RT-PCR. Compared to / rats, Ws/Ws animals exhibited strikingly exacerbated mucosal injury but minimal reactive intestinal wall fibrosis. Ws/Ws rats exhibited less radiation-induced intestinal smooth muscle cell proliferation and collagen accumulation than / littermates. Tgfb expression increased to a similar extent in Ws/Ws and / rats. Unirradiated intestine from Ws/Ws and / rats did not differ significantly. Mast cells protected the intestinal mucosa during the early phase of radiation enteropathy and promoted intestinal fibrosis after the breakdown of the mucosal barrier. Mast cells may be required for Tgfb to exert its full fibrogenic effect in radiation enteropathy.

Gao, M., Shirato, H., Miyasaka, K., Kuwabara, M. and Koyama, T. Induction of Growth Factors in Rat Cardiac Tissue by X Irradiation.

To investigate the relationship between angiogenic growth factors and endothelial enzyme activity in capillaries after injury of rat cardiomyocytes caused by X irradiation, 7-week-old male Wistar rats were anesthetized with pentobarbitone and their hearts irradiated (X rays, 20 Gy) through a hole in the lead casing in which they were enclosed. The hearts were excised at 1 h, 1 week and 3 weeks after irradiation. Left ventricular cross sections were stained for capillary enzymes by double staining for two endothelial enzymes, alkaline phosphatase (AP) and dipeptidylpeptidase IV (DPP), immunohistochemically stained for basic fibroblast growth factor (Fgf, also known as bFgf) and vascular endothelial growth factor (Vegf), and stained for nick end-labeling of DNA by the TUNEL method. Staining for distribution of AP in the arteriolar portion was reduced at both 1 and 3 weeks after irradiation with 20 Gy, but staining for DPP in the venular portion was unchanged, suggesting a close relationship between growth factors and injury of the arteriolar capillary portion. Fgf and Vegf proteins were present within the cytoplasm of the cardiomyocytes, or around capillaries, 1 h, 1 week and 3 weeks after irradiation. Many TUNEL-stained cardiomyocyte nuclei were observed at 1 h, but they had decreased markedly at 1 week and had almost disappeared by 3 weeks after irradiation. Thus Fgf and Vegf were induced concomitantly with the decrease in the staining for endothelial AP by 20 Gy X irradiation, which also caused microeffects as indicated by TUNEL staining of many nuclei at 1 h postirradiation.

Kalia, V. K., Al-Nabulsi, I., Wallen, C. A., Zhang, H. and Wheeler, K. T. Radiation-Induced DNA Damage in Tumors and Normal Tissues. VI. Estimation of the Hypoxic Fraction of Experimental Tumors.

For several years, we have concentrated our efforts on validating the use of radiation-induced DNA strand breaks and DNA–protein crosslinks to assess the oxygenation status of tumors and normal tissues. We have demonstrated that (1) the oxygen dependence of strand break formation is identical to that of radiation-induced cell killing; (2) the oxygen dependence of DNA–protein crosslink formation is the mirror image of that of radiation-induced cell killing; and (3) the formation of these radiation-induced DNA lesions is predominantly dependent on the oxygen concentration near the DNA and is independent of the cell type, metabolic status, proliferative status, pH of the surrounding environment, and composition or properties of the proteins tightly associated with the DNA. In the present study, the hypoxic fraction of three experimental tumors was estimated using our assay of radiation-induced DNA damage. The average hypoxic fraction of a large number of tumors estimated with this assay of radiation-induced DNA damage for (1) WiDR human colorectal carcinoma xenografts (40.8 ± 4.2%), (2) 66 mouse mammary adenocarcinoma tumors (41.8 ± 3.1%), and (3) subcutaneous tumors grown from 9L rat brain tumor cells (95% CI =−8.2–4.2%) was not statistically different from that of a large number of tumors measured for each of these tumor models by the paired survival curve method (38.3 ± 6.3%, 28.9 ± 5.5%, 95% CI = 2.2–4.4%, respectively). When the hypoxic fraction measured by the alkaline elution method on one half of an individual tumor was compared to that measured by the paired survival curve method on the other half of the same tumor, no statistical correlation was found for either 66 or WiDR tumors. Although this assay of radiation-induced DNA damage can be used effectively in the laboratory to answer a number of important questions about the oxygenation status of animal tumors and normal tissues, failure to reliably estimate the hypoxic fraction of individual tumors and technical considerations make it unlikely that the assay can be used in the clinic to estimate the hypoxic fraction of human tumors.

Bartstra, R. W., Bentvelzen, P. A. J., Zoetelief, J., Mulder, A. H., Broerse, J. J. and van Bekkum, D. W. The Effects of Fractionated Gamma Irradiation on Induction of Mammary Carcinoma in Normal and Estrogen-Treated Rats.

The effects of dose fractionation on induction of mammary carcinoma were studied in normal and estrogen-treated female rats of the inbred WAG/Rij strain. Groups of 40 animals received total-body doses of 1 or 2 Gy of ¹³⁷Cs γ radiation, administered in fractions of 2.5, 10 or 40 mGy with intervals of 12 h, or in fractions of 10 mGy with intervals of 2, 5 or 24 h. The irradiations were started at the age of 8 weeks. Estrogen treatment was accomplished by implantation of a pellet containing estrogen at the age of 6 weeks. All mammary tumors were resected and classified histologically as carcinoma or fibroadenoma. The age-specific incidence of mammary carcinoma was compared with that in control groups of unirradiated normal or estrogen-treated rats and was expressed as excess normalized risk, using lifetime statistical analysis with both parametric and nonparametric methods. The data were also compared to the results of single-dose experiments reported in previous papers. Fractionated irradiation increased the risk of mammary cancer in both normal and estrogen-treated rats compared to the corresponding unirradiated control group. The excess normalized risk per unit of total dose was approximately equal with or without estrogen treatment. Without estrogen treatment, the effects of the single-dose and fractionated irradiations were approximately equal. In estrogen-treated animals, however, single-dose irradiation was up to 15 times more carcinogenic than the fractionated exposures. This fractionation effect appeared to vanish for total doses below approximately 0.3 Gy. With estrogen treatment, the excess normalized risk was significantly higher for dose fractions of 40 mGy than for fractions of 10 mGy. The risk was also markedly higher for fractionation intervals of 2 or 5 h than for intervals of 12 or 24 h. The results of these experiments show that the effects of dose fractionation on the induction of mammary carcinoma may depend on hormonal status, the total dose delivered, the dose per fraction, and the fractionation interval.

Barber, J. B. P., West, C. M. L., Kiltie, A. E., Roberts, S. A. and Scott, D. Detection of Individual Differences in Radiation-Induced Apoptosis of Peripheral Blood Lymphocytes in Normal Individuals, Ataxia Telangiectasia Homozygotes and Heterozygotes, and Breast Cancer Patients after Radiotherapy.

Quantification of radiation-induced apoptosis in peripheral blood lymphocytes (PBLs) has been proposed as a possible screening test for cancer-prone individuals and also for the prediction of normal tissue responses after radiotherapy. We have used the TUNEL assay (terminal transferase nick-end labeling) 24 h after irradiation with 4 Gy at high dose rate to assess interindividual differences in radiation-induced apoptosis between (1) a panel of normal individuals, (2) ataxia telangiectasia (AT) homozygotes and heterozygotes, and (3) breast cancer patients who had received radiotherapy 8–13 years ago, including a number of patients who had suffered adverse responses to radiation. With this protocol, we show clear differences in radiation-induced apoptosis between individuals, and good reproducibility in the assay. In agreement with previous reports using EBV-transformed lymphoblasts, we show a very poor induction of apoptosis in AT homozygotes and a reduced level in AT heterozygotes compared to normal individuals. A similar reduced level compared to normal individuals was seen in the breast cancer patients. Despite a wide range of values in the breast cancer patients and good reproducibility on repeat samples, there was no correlation of rates of apoptosis with the severity of breast fibrosis, retraction or telangiectasia. The reduced rate of apoptosis observed in the breast cancer cases may be associated with genetic predisposition to breast cancer; however, we conclude that assays of lymphocyte apoptosis are unlikely to be of use in predicting normal tissue tolerance to radiotherapy.

Boreham, D. R., Dolling, J-A., Maves, S. R., Siwarungsun, N. and Mitchel, R. E. J. Dose-Rate Effects for Apoptosis and Micronucleus Formation in Gamma-Irradiated Human Lymphocytes.

We have compared dose-rate effects for γ-radiation-induced apoptosis and micronucleus formation in human lymphocytes. Long-term assessment of individual radiation-induced apoptosis showed little intraindividual variation but significant interindividual variation. The effectiveness of radiation exposure to cause apoptosis or micronucleus formation was reduced by low-dose-rate exposures, but the reduction was apparent at different dose rates for these two end points. Micronucleus formation showed a dose-rate effect when the dose rate was lowered to 0.29 cGy/min, but there was no accompanying cell cycle delay. A further increase in the dose-rate effect was seen at 0.15 cGy/min, but was now accompanied by cell cycle delay. There was no dose-rate effect for the induction of apoptosis until the dose rate was reduced to 0.15 cGy/min, indicating that the mechanisms or signals for processing radiation-induced lesions for these two end points must be different at least in part. There appear to be two mechanisms that contribute to the dose-rate effect for micronucleus formation. One of these does not affect binucleate cell frequency and occurs at dose rates higher than that required to produce a dose-rate effect for apoptosis, and one affects binucleate cell frequency, induced only at the very low dose rate which coincidentally produces a dose-rate effect for apoptosis. Since the dose rate at which cells showed reduced apoptosis as well as a further reduction in micronucleus formation was very low, we conclude that the processing of the radiation-induced lesions that induce apoptosis, and some micronuclei, is very slow in quiescent and PHA-stimulated lymphocytes, respectively.

Kajioka, E. H., Andres, M. L., Li, J., Mao, X. W., Moyers, M. F., Nelson, G. A., Slater, J. M. and Gridley, D. S. Acute Effects of Whole-Body Proton Irradiation on the Immune System of the Mouse.

The acute effects of proton whole-body irradiation on the distribution and function of leukocyte populations in the spleen and blood were examined and compared to the effects of photons derived from a ⁶⁰Co γ-ray source. Adult female C57BL/6 mice were exposed to a single dose (3 Gy at 0.4 Gy/min) of protons at spread-out Bragg peak (SOBP), protons at the distal entry (E) region, or γ rays and killed humanely at six different times thereafter. Specific differences were noted in the results, thereby suggesting that the kinetics of the response may be variable. However, the lack of significant differences in most assays at most times suggests that the RBE for both entry and peak regions of the Bragg curve was essentially 1.0 under the conditions of this study. The greatest immunodepression was observed at 4 days postexposure. Flow cytometry and mitogenic stimulation analyses of the spleen and peripheral blood demonstrated that lymphocyte populations differ in radiosensitivity, with B (CD19 ) cells being most sensitive, T (CD3 ) cells being moderately sensitive, and natural killer (NK1.1 ) cells being most resistant. B lymphocytes showed the most rapid recovery. Comparison of the T-lymphocyte subsets showed that CD4 T helper/inducer cells were more radiosensitive than the CD8 T cytotoxic/suppressor cells. These findings should have an impact on future studies designed to maximize protection of normal tissue during and after proton-radiation exposure.

Hillebrandt, S. and Streffer, C. Relative Biological Effectiveness of Neutrons: Induction of Malformation in a Mouse Strain with Genetic Predisposition.

Female hybrid mice (C57BL/6J × HLG) were exposed to 0.5 Gy of cyclotron neutrons (mean energy 5.8 MeV) after mating with HLG males when embryos were at the early zygote stage. Uterine contents were examined on gestation day 19 for prenatal mortality and malformed development. The results were compared to those obtained after irradiation of zygotes with 1.0 and 1.7 Gy X rays. The number of malformed fetuses was greater than for the unirradiated controls. Gastroschisis was the main observation, but exencephaly was also observed. The neutron relative biological effectiveness (RBE) for the induction of lethal events and malformations in the [(C57BL/6J × HLG)F₁× HLG] mice was estimated. This value is higher than that for the RBE calculated for the HLG strain, which is known to be more sensitive to radiation than the hybrids.

Safwat, A. The Immunobiology of Low-Dose Total-Body Irradiation: More Questions than Answers.

Low-dose total-body irradiation (TBI) is used in the treatment of chronic lymphocytic leukemia and low-grade non-Hodgkin's lymphoma. The usual practice is to give very low individual fractions (0.1 to 0.25 Gy) several times a week, to a total dose of 1.5 to 2 Gy. Despite this low dose, low-dose TBI can induce long-term remissions in the majority of patients. Immune enhancement, rather than direct radiation cell killing, is one of the suggested mechanisms by which low-dose TBI can exert its effect. Data from animal experiments have shown that low-dose TBI could enhance the immune response through (1) augmenting the proliferative reactive response of the T cells to mitogenic stimulation; (2) altering cytokine release, particularly the activation of interferon γ and Il2 production; (3) increasing the expression of Il2 receptors on the T-cell surface; (4) facilitating signal transduction in T lymphocytes; (5) increasing splenic catecholamine content and lowering the serum corticosterone level; and (6) eliminating a particularly radiosensitive subset of the suppressor T cells. Data for humans, though scarce, suggest that at least some of these mechanisms occur in patients treated with low-dose TBI. Whether these immunomodulatory effects are responsible for the clinical outcome is not yet clear. Much is still unknown about the immunobiology of low-dose TBI, its clinical potential, and the possible synergism with chemotherapy, biological response modifiers, or immunotherapy. This lack of comprehensive knowledge hampers the optimal and widespread use of this intriguing and potentially useful treatment modality in clinical practice.

Moulder, J. E. The Electric and Magnetic Fields Research and Public Information Dissemination (EMF-RAPID) Program.

In the United States, public concern that exposure to power-line fields was linked to cancer led to the establishment of a Congressionally mandated program, the Electric and Magnetic Fields Research and Public Information Dissemination (EMF-RAPID) Program. A major goal of the program was to “determine whether or not exposures to electric and magnetic fields produced by the generation, transmission, and use of electrical energy affect human health”. Between 1994 and 1998, the EMF-RAPID program spent approximately $41 million on biological research. Much of the work funded by the EMF-RAPID program has not yet been published in the peer-reviewed literature. The U.S. National Institute of Environmental Health Sciences (NIEHS) asked that Radiation Research publish this special issue in an attempt to remedy this publication gap. The issue includes reviews of studies that were done to assess the biological plausibility of claims that power-frequency fields caused leukemia and breast cancer. The issue continues with two teratology studies and one immunology study. The section of the issue covering in vitro studies begins with an overview of the efforts NIEHS made to replicate a wide range of reported effects of power-frequency fields and continues with four papers reporting the absence of effects of power-frequency fields on the expression of stress-response genes and oncogenes. Other reports of in vitro studies and studies of mechanisms cover cytotoxicity, gap junction intracellular communication, calcium ion transport across the plasma membrane, and intracellular electric fields.

Boorman, G.A., McCormick, D.L., Ward, J.M., Haseman, J.K. and Sills, R.C. Magnetic Fields and Mammary Cancer in Rodents: A Critical Review and Evaluation of Published Literature.

Epidemiological data suggesting a possible increase in breast cancer risk in male electricians have raised concerns about the relationship between exposure to power-frequency magnetic fields and breast cancer. In this paper, we review the results of animal studies that are relevant to identifying possible increases in breast cancer risk resulting from exposure to 50 or 60 Hz magnetic fields. Three large-scale chronic bioassays of carcinogenesis in rats or mice exposed to magnetic fields for 2 years demonstrated no increases in the incidence of mammary cancer; it is generally accepted that power-frequency magnetic fields have little or no activity as a complete carcinogen in the rodent mammary gland. Findings from one laboratory, though inconsistent, suggest that magnetic fields may stimulate mammary neoplasia in rats treated with a chemical carcinogen. However, studies conducted in two other laboratories failed to confirm these findings; rats exposed to magnetic fields demonstrated patterns of tumor incidence, multiplicity, size and latency that were generally similar to those in sham-exposed controls. Where differences were seen, the groups exposed to magnetic fields generally had fewer mammary tumors than did sham-exposed controls. On this basis, evaluations of the activity of 50 or 60 Hz magnetic fields in models of multistage mammary cancer in rodents have generally been negative; positive findings have been reported from only one laboratory. The totality of rodent data does not support the hypothesis that power-frequency magnetic-field exposure enhances mammary cancer in rodents, nor does it provide experimental support for possible epidemiological associations between magnetic-field exposure and increased breast cancer risk.

Boorman, G.A., Rafferty, C.N., Ward, J.M. and Sills, R.C. Leukemia and Lymphoma Incidence in Rodents Exposed to Low-Frequency Magnetic Fields.

A weak association between residential or occupational exposure to electric and magnetic fields (50/60 Hz fields) and an increased incidence of leukemia has been reported. Numerous animal studies have evaluated the potential association between magnetic-field exposure and leukemia. These include long-term (up to 2½ years) bioassays, initiation/promotion studies, investigations in transgenic models, and tumor growth studies. Exposure to 60 Hz circularly polarized magnetic fields at 1,400 μT for 28 months did not affect lymphoma incidence in mice. The study included over 2000 C57BL/6J mice. In another study, 1000 B6C3F₁ mice exposed to 60 Hz magnetic fields up to 1000 μT for 2 years showed no increase in lymphomas. Approximately 400 transgenic Eμ-Pim1 mice exposed to 50 Hz fields up to 1000 μT for up to 18 months had no increased incidence of leukemia. Similarly, Trp53 ^/− mice and Pim1transgenic mice exposed to 60 Hz magnetic fields for 23 weeks showed no increased incidence of lymphoma. Three studies in F344 rats exposed to 50 or 60 Hz magnetic fields up to 5 mT showed no increased incidence of leukemia. The combined animal bioassay results are nearly uniformly negative for magnetic-field exposures enhancing leukemia and weaken the possible epidemiological association between magnetic-field exposures and leukemia in humans as suggested by epidemiological data.

Ryan, B. M., Polen, M., Gauger, J. R., Mallett, B., Jr., Kearns, M. E., Bryan, T. E. and McCormick, D. L. Evaluation of the Developmental Toxicity of 60 Hz Magnetic Fields and Harmonic Frequencies in Sprague-Dawley Rats.

Experimental data suggest that exposure to the 50 and 60 Hz sinusoidal components of power-frequency magnetic fields (MFs) does not have an adverse impact on fetal development. However, the possible developmental toxicity of MF harmonics has not been investigated. This study was designed to determine whether exposure to 180 Hz MFs (third harmonic), alone or in combination with 60 Hz MFs, induces birth defects in Sprague-Dawley rats. Groups of sperm-positive dams (≥20/group) were exposed for 18.5 h per day from gestation days 6 through 19 to (1) ambient MFs only (<0.0001 mT; sham controls); (2) 60 Hz MFs at 0.2 mT; (3) 180 Hz MFs at 0.2 mT; or (4) 60 Hz 180 Hz MFs (10% third harmonic; total field strength = 0.2 mT). Litter size, litter weight, percentage live births, sex ratio, and number of resorption sites were determined for each dam, and gross external, visceral, cephalic and skeletal examinations were performed on all fetuses. MF exposure had no significant effects on litter size, litter weight, or fetal development. With the exception of common rib variants, the incidence of fetal anomalies was comparable in all groups. A small increase in the incidence of rib variants was seen in the group exposed to 60 Hz 180 Hz MFs; however, the incidence of rib variants in this group was similar to that in historical controls from our laboratory. These data extend the existing database on developmental toxicity of MFs by demonstrating that exposure to 180 Hz MFs, either alone or superimposed on an underlying 60 Hz signal, does not induce biologically significant developmental toxicity. These data do not support the hypothesis that exposure to power-frequency MFs is an important risk factor for fetal development.

Harry, G. J., Bartenbach, M., Haines, W. and Bruccoleri, A. Developmental Profiles of Growth-Associated Protein (Gap43), Ngfb, Bndf and Ntf4 mRNA Levels in the Rat Forebrain after Exposure to 60 Hz Magnetic Fields.

Fischer 344 rats were exposed to 60 Hz magnetic fields (EMFs) during gestation and lactation. Rats received continuous exposure to 2-, 200- or 1000-μT magnetic fields for 18.5 h per day, 7 days a week, or sham exposure (sham controls). During postnatal development, on postnatal days 1, 3, 6, 9, 15 and 20, forebrain tissue from male pups was examined for alterations in mRNA level for developmentally regulated central nervous system-specific proteins. Alterations in these factors during critical periods of development could result in alterations in the final neural network. Gap43 (growth-associated protein 43) mRNA was measured by Northern hybridization as a developmental indicator of axonal growth during the development of the neuron. Between postnatal days 1 and 9, detectable levels of Gap43 mRNA displayed a similar pattern across all sham control and exposure groups. In addition to Gap43, mRNA levels for the nervous system-specific growth factors ciliary neurotrophic factor (Cntf), brain-derived neurotrophic factor (Bdnf), β nerve growth factor (Ngfb), neurotrophin-3 (Ntf3), and neurotrophin-4 (Ntf4) were examined by RNase protection assay. While there is public concern for developmental neurotoxicity associated with exposure to EMFs, these data, generated from animals exposed to 2-, 200- or 1000-μT magnetic fields during both gestational and lactational periods of development, suggest that under these conditions no significant alterations in these critical factors for brain development occur.

Boorman, G. A., Owen, R. D., Lotz, W. G. and Galvin, M. J., Jr. Evaluation of In Vitro Effects of 50 and 60 Hz Magnetic Fields in Regional EMF Exposure Facilities.

A weak association between magnetic-field exposure and increased incidences of cancer has been reported. While alterations in cellular processes after in vitro magnetic-field exposures have also been reported to provide plausibility for this association, other laboratories have been unable to repeat the findings. As part of an accelerated electric- and magnetic-field (EMF) research program, the National Institute of Environmental Health Sciences with the Department of Energy identified the replication of the published positive effects as a priority. Regional EMF exposure facilities were established to investigate major in vitro effects from the literature. These included effects on gene expression, intracellular calcium, colony growth in soft agar, and ornithine decarboxylase activity. The laboratories that first reported these effects provided experimental protocols, cell lines, and other relevant experiment details. Regional facility studies included sham/sham exposures (no applied field in either chamber) and were done in a blinded fashion to minimize investigator bias. In nearly all experiments, no effects of magnetic-field exposure were found. The effort provided insight into dealing with the difficulty of replication of subtle effects in complex biological systems. Experimental techniques provided some clues for the differences in experimental results between the regional facility and the original investigator. Studies of subtle effects require extraordinary efforts to confirm that the effect can be attributed to the applied exposure.

Morehouse, C. A. and Owen, R. D. Exposure to Low-Frequency Electromagnetic Fields Does Not Alter HSP70 Expression or HSF-HSE Binding in HL60 Cells.

Environmental exposure to extremely low-frequency electromagnetic fields (ELF EMFs) has been identified as a possible contributor to increased cancer incidence and other diseases. In vitro studies designed to probe for biological mechanisms that might explain such relationships have included several studies of gene expression. While gene expression studies have focused on MYC, effects of ELF EMFs on the expression of β-actin, histone H2B, β-tubulin, SRC, FOS and JUN have also been reported. In addition, some investigators have reported both an induction of HSP70 expression and an increase in HSF-HSE binding in HL60 (human promyelocytic leukemia) cells after exposure to a 60 Hz magnetic field. In this study, HL60 cells were exposed to a weak 60 Hz magnetic field (6.3 or 8.0 μT) or to a positive control heat shock (42 or 44°C). While heat shock led to reproducible induction of HSP70 expression and HSF-HSE binding, no significant effect of exposure to ELF EMFs on either of these end points was observed.

Morehouse, C. A. and Owen, R. D. Exposure of Daudi Cells to Low-Frequency Magnetic Fields Does Not Elevate MYC Steady-State mRNA Levels.

The effect of extremely low-frequency electromagnetic field (ELF EMF) exposures to human health has been widely debated. Epidemiological studies have found a possible correlation between increased cancer incidence and environmental ELF EMF exposures. Results from in vitro studies performed to examine the possible underlying bioeffects of ELF EMFs have varied greatly. Reported effects range from robust and reproducible effects to undetectable. In this study, Daudi cells were exposed to 60 Hz magnetic fields for 20, 40 or 60 min at flux densities of 12.5, 50, 100 or 500 μT. Exposures were performed in the Regional ELF-EMF Exposure Facility (Rockville, MD) to minimize variables that might contribute to a false positive effect. Exposures included sham/sham, exposed/sham or sham/exposed, and were performed with blinding with respect to type of exposure. 12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment was used as a positive control. Total cellular RNA was isolated using a single-step technique. Human MYC expression was measured by northern blot hybridization as an indicator of the responsiveness of Daudi cells to experimental conditions. Beta-2-microglobulin (B2M) expression was measured simultaneously as an internal control. Exposure to a 60 Hz magnetic field did not significantly alter MYC expression in Daudi cells under any of the exposure conditions.

Balcer-Kubiczek, E. K., Harrison, G. H., Davis, C. C., Haas, M. L. and Koffman, B. H. Expression Analysis of Human HL60 Cells Exposed to 60 Hz Square- or Sine-Wave Magnetic Fields.

A total of 960 complementary DNA (cDNA) clones from an HL60 cell cDNA library were screened to discover genes that were differentially expressed in HL60 cells exposed to 60 Hz square-wave magnetic fields (MFs) compared to sham-exposed cells. Square-wave fields are rich in odd harmonic frequency content. We used a two-gel cDNA library screening method (BIGEL) to identify treatment-induced alterations in gene expression. Four cDNA clones were tentatively identified as differentially expressed after exposure to square-wave MFs at 2 mT for 24 h. BIGEL-identified genes (GenBank accession number) corresponding to these clones were: TI227H (D50525), EST Homo sapiens partial cDNA (Z17814), human ribosomal protein S13 (L01124), and AICAR transformylase mRNAs (D82348). The differences in mRNA levels were not confirmed in test compared to experimental cells by Northern analysis. In other experiments, we used concurrent exposure to 60 Hz sine- or square-wave MFs (0 or 2 mT, duration of 3 or 24 h, no postexposure delay). In addition to the four BIGEL genes, we also investigated MYC, HSP70, RAN and SOD1. In the case of MYC and HSP70, square-wave MFs appeared to exhibit more marked alterations when compared to sinusoidal waveforms, but the overall results indicated no effect of possible differential magnetic-field-induced expression of all eight genes. In contrast, alterations of mRNA levels were observed for seven genes after exposure to X irradiation, hyperthermia and TPA. These results are contrary to previously proposed similarities between the action of these agents and MF effects on gene transcription.

Loberg, L. I., Engdahl, W. R., Gauger, J. R. and McCormick, D. L. Expression of Cancer-Related Genes in Human Cells Exposed to 60 Hz Magnetic Fields.

Exposure to 60 Hz magnetic fields (MFs) may be a risk factor for human cancer. One mechanism through which MFs could influence neoplastic development is through alterations in the expression of cancer-related genes. Previous molecular studies of the action of MFs have measured effects on a limited number of genes. In the present studies, arrays containing cDNAs for 588 cancer-related genes were used to approach the hypothesis that the biological activity of MFs is mediated by alterations in gene expression. Cultures of normal (HME) and transformed (HBL-100) human mammary epithelial cells and human promyelocytic leukemia (HL60) cells were exposed to MFs at field strengths of 0, 0.01 or 1.0 mT for 24 h. Several genes were identified in MF-exposed cells whose expression was increased by at least twofold or decreased by 50% or more. However, no gene was found to be differentially expressed in each of three independent exposures for any cell type, and no relationship between exposure intensity and differential gene expression was found. These studies failed to identify a plausible genetic target for the action of MFs in human cells, and they provide no support for the hypothesis that MF exposure alters the expression of genes that are involved in cancer development.

Loberg, L. I., Luther, M. J., Gauger, J. R. and McCormick, D. L. 60 Hz Magnetic Fields Do Not Enhance Cell Killing by Genotoxic Chemicals in Ataxia Telangiectasia and Normal Lymphoblastoid Cells.

Ataxia telangiectasia (AT) is an inherited autosomal recessive disease characterized by increased risk of cancer, immune deficiency, and neurodegeneration. Cells cultured from AT patients are highly sensitive to genotoxic agents and are deficient in cell cycle arrest after exposure to ionizing radiation. In consideration of their sensitivity to both ionizing and nonionizing radiation, AT cells may provide a sensitive model system to study the biological activity of other components of the electromagnetic spectrum. To characterize the effects of power-frequency (60 Hz) magnetic fields (MFs) in AT cells, we compared responses of AT and normal lymphoblast cells to sinusoidal MFs at 1.0 mT, either alone or in combination with the genotoxic agents mitomycin C or streptonigrin. The MF alone had no effect on cell growth or survival in a clonogenic assay in either AT or normal cells. The MF also had no effect on induction of cell death by mitomycin C or streptonigrin in either cell type. AT cells do not demonstrate differential sensitivity to MF exposure. These results do not support the hypothesis that MFs interact with genotoxic agents to induce adverse biological effects in either normal or genetically susceptible human cells.

Griffin, G. D., Williams, M. W. and Gailey, P. C. Cellular Communication in Clone 9 Cells Exposed to Magnetic Fields.

Magnetic-field exposure (45 Hz B_a.c. over a flux density range of 7.7 to 49.9 μT r.m.s. with parallel B_d.c. of 36.6 μT) has been reported by Blackman and coworkers to inhibit gap junction intercellular communication in Clone 9 cells treated with chloral hydrate for 24 h prior to field exposure in accord with predictions of the ion parametric resonance model. The study reported here is an attempt to reproduce this effect. Baseline experiments showed that growth in culture and state of confluence at time of addition of chloral hydrate were comparable in both laboratories. PMA inhibited cell–cell communication in a dose-dependent manner, similar to the results of Blackman and coworkers, whereas cells in the present study were somewhat more sensitive to chloral hydrate than reported by Blackman and coworkers. A total of 38 exposure experiments were undertaken using a 45 Hz magnetic field with a flux density of 23.8 μT r.m.s., in parallel with a 36.6-μT static magnetic field for 40 to 45 min, after pretreatment with 2.5 mM chloral hydrate for 24 h. In 14 unblinded experiments, a small but statistically significant effect of magnetic-field exposure was observed, but due to the subjective nature of the assay, it was deemed essential to carry out blinded experiments. The remaining 24 experiments were blinded. In 15 blinded experiments, cells purchased from the American Type Culture Collection and grown only in this laboratory were used, while in 9 experiments, the cells had originally been grown in Blackman's laboratory and were subsequently sent to this laboratory. There was no statistically significant effect of magnetic-field exposure on gap junction intercellular communication in these blinded experiments using either cell line.

Sisken, J. E. and DeRemer, D. Power-Frequency Electromagnetic Fields and the Capacitative Calcium Entry System in SV40-Transformed Swiss 3T3 Cells.

The present study was designed to test the hypothesis that a 60 Hz electromagnetic field could affect the influx of calcium ions across the plasma membrane through the so-called capacitative calcium entry system. Recordings of cytosolic calcium-ion concentrations in SV40-transformed Swiss 3T3 cells were obtained in real time during exposure to magnetic fields ranging from 0.3–50 mT or to sham conditions using the calcium-sensitive photoprotein aequorin. This was done for cell populations whose capacitative entry system was activated by either bradykinin or thapsigargin under a variety of experimental conditions. No effects of the magnetic field were observed on bradykinin-induced calcium transients and, with the exception of a small but statistically significant increase observed in experiments performed at 50 mT, no effects of the fields were observed on baseline calcium levels prior to or after such transients. The magnetic fields also had no effects on the size or kinetics of any of the thapsigargin-induced calcium transients. Overall, the data fail to support the hypothesis tested in this work.

McLeod, K. J. and Collazo, L. Suppression of a Differentiation Response in MC-3T3-E1 Osteoblast-like Cells by Sustained, Low-Level, 30 Hz Magnetic-Field Exposure.

Extremely low-frequency (ELF) magnetic fields have been reported to be capable of influencing both tissue remodeling and cell phenotypic expression in culture. However, whether the cells or tissues respond directly to the magnetic flux or to the electric field induced by the time-changing magnetic flux remains a controversial topic. To address this question, we developed an osteoblast cell assay based on the activity of alkaline phosphatase, an enzyme whose activity is up-regulated during the differentiation of bone cells. MC-3T3-E1 cells plated at a confluent density were allowed to proceed through the differentiation process for 3 days, after which they were exposed to a 30 Hz, 1.8-mT r.m.s. magnetic field inducing a spatially varying electric field with a maximum intensity of 0.9 mV/m r.m.s. In situ assays of alkaline phosphatase activity at 4, 8, 16 and 64 h demonstrated a progressive inhibition of enzyme activity, the pattern of which maps to the intensity of the induced electric field (R² = 0.5, P < 0.001). We interpret these results to indicate that cells are capable of responding to ELF induced electric fields at intensities below 1 mV/m, and that the principal effect on cells is an inhibition of differentiation.

King, R. W. P. and Wu, T. T. Calculated Electric Field Induced in Bundles of Long Cells in the Human Body When It Is Exposed to Low-Frequency Electric Fields.

A theoretical analysis is made of the electric field induced in the interior of long cylindrical cells arranged in bundles of parallel cells embedded in the saline fluid in the human body exposed to extremely low-frequency (ELF) and very low-frequency (VLF) electric fields parallel to the length of the cells. Such bundles characterize muscle. The analysis parallels in many respects the earlier study of isolated long cells (King and Wu, Phys. Rev. E 58, 2363–2369, 1998) but has important added complications. It leads to a similar result: The field inside the membrane of each long cell is the same as the field just outside the membrane in the saline fluid; i.e., the membrane provides no shielding. While one might expect the presence of the neighboring cells to provide a shielding effect for each cell, the results of this study show quantitatively that this is not the case. In fact, the electric field in the interior of each of a bundle of long cells is seen to increase very slightly (of the order of 0.4%) when the cells in the bundle are moved close enough to be practically in contact.

House, R. V. and McCormick, D. L. Modulation of Natural Killer Cell Function after Exposure to 60 Hz Magnetic Fields: Confirmation of the Effect in Mature B6C3F₁ Mice.

In a previous study, we demonstrated that subchronic exposure to pure, linearly polarized 60 Hz magnetic fields (MFs) at flux densities ranging from 0.002 to 1.0 mT induced a modest but statistically significant and reproducible suppression of NK cell activity in young adult B6C3F₁ mice. NK cell activity in mice declines with age and is known to be suboptimal in older animals. The present study was designed to determine if the same MF exposure regimens will suppress NK cell activity in mature (i.e. more than 1 year old) animals. Extending our previous findings, a modest suppressive effect of MFs on NK cell activity in B6C3F₁ mice was found when subchronic exposure was initiated in animals held in quarantine for 1 year prior to exposure. These data demonstrate that MF exposure suppresses NK cell activity in both young and mature adult B6C3F₁ mice. However, because chronic exposure to the same MF parameters used in the NK function studies does not increase the incidence of neoplasia in B6C3F₁ mice, this statistically significant inhibition of NK cell function appears to be of limited biological significance.

Loberg, L. I., Engdahl, W. R., Gauger, J. R. and McCormick, D. L. Cell Viability and Growth in a Battery of Human Breast Cancer Cell Lines Exposed to 60 Hz Magnetic Fields.

Epidemiological data suggest that exposure to power-frequency (50/60 Hz) magnetic fields (MFs) may be a risk factor for breast cancer in humans. To determine whether MFs affect human breast cancer cells, we measured viability, growth and cytotoxicity in a battery of breast cancer cell lines after in vitro MF and sham exposure. Cells of three estrogen receptor-positive human breast cancer cell lines (MCF-7, ZR-75-1 and T-47D) and one estrogen receptor-negative human breast cancer cell line (MDA-MB-231) and normal (nontransformed) human breast epithelial cells were exposed to MFs (1 mT) or sham fields (<0.0001 mT) for 72 h. Cell viability was determined using the sulforhodamine B (SRB) assay at 0 and 72 h after the MF exposure period. Cell growth was measured as the change in SRB dye uptake over 72 h after MF exposure. MF exposure had no effect on cell viability or growth in any cell type examined. Similarly, MF exposure had no effect on cytotoxicity induced by exposure to the retinoid N-(4-hydroxyphenyl)retinamide. These data do not support the hypothesis that MF exposure stimulates growth of breast cancer cells.