Song, C. W., Park, H. and Griffin, R. J. Improvement of Tumor Oxygenation by Mild Hyperthermia.

There is now abundant evidence that oxygenation in rodent, canine and human tumors is improved during and for up to 1–2 days after heating at mild temperatures. An increase in tumor blood perfusion along with a decline in the oxygen consumption rate appears to account for the improvement of tumor oxygenation by mild hyperthermia. The magnitude of the increase in tumor pO₂, determined with oxygen-sensitive microelectrodes, caused by mild hyperthermia is less than that caused by carbogen breathing. However, mild hyperthermia is far more effective than carbogen breathing in increasing the radiation response of experimental tumors, probably because mild hyperthermia oxygenates both (diffusion-limited) chronically hypoxic and (perfusion-limited) acutely hypoxic cells, whereas carbogen breathing oxygenates only the chronically hypoxic cells. Mild hyperthermia is also more effective than nicotinamide, which is known to oxygenate acutely hypoxic cells, in enhancing the radiation response of experimental tumors. The combination of mild hyperthermia with carbogen or nicotinamide is highly effective in reducing the hypoxic cell fraction in tumors and increasing the radiation response of experimental tumors. A primary rationale for the use of hyperthermia in combination with radiotherapy has been that hyperthermia is equally cytotoxic toward fully oxygenated and hypoxic cells and that it directly sensitizes both fully oxygenated and hypoxic cells to radiation. Such cytotoxicity and such a radiosensitizing effect may be expected to be significant when the tumor temperature is elevated to at least 42–43°C. Unfortunately, it is often impossible to uniformly raise the temperature of human tumors to this level using the hyperthermia devices currently available. However, it is relatively easy to raise the temperature of human tumors into the range of 39–42°C, which is a temperature that can improve tumor oxygenation for up to 1–2 days. The potential usefulness of mild hyperthermia to enhance the response of human tumors to radiotherapy by improving tumor oxygenation merits continued investigation.

Schneiderman, M. H., Hofer, K. G. and Schneiderman, G. S. Targets for Radiation-Induced Cell Death: When DNA Damage Doesn't Kill.

Chinese hamster ovary (CHO) K1 and radiosensitive CHO irs-20 cells were synchronized in S phase and labeled for 10 min with 5-[¹²⁵I]-iodo-2′-deoxyuridine (¹²⁵IdU). The cells were washed, incubated in fresh medium for 1 h for incorporation of the intracellular radionucleotides into DNA, and then frozen (–80°C) for accumulation of ¹²⁵I decays. At intervals after freezing, when the cells had accumulated the desired number of decays, aliquots of the frozen cells were thawed and plated to determine survival. The survival curves for K1 and irs-20 cells were similar from 100% to 30% survival. At higher ¹²⁵I doses (more decays/cell), the survival of K1 cells continued to decline exponentially, but the survival of X-ray-sensitive irs- 20 cells remained at approximately 30% even after the cells had accumulated 1265 decays/cell. The results contradict the notion that increased DNA damage inevitably causes increased cell death. To account for these findings, we propose a model that postulates the existence of a second radiation target. According to this model, radiation damage to DNA may be necessary to induce cell death, but DNA damage alone is not sufficient to kill cells. We infer from the survival response of irs-20 cells that damage to a second (non-DNA) structure is involved in cell death, and that this structure directly affects the repair of DNA and cell survival.

Mansur, D. B., Kataoka, Y., Grdina, D. J. and Diamond, A. M. Radiosensitivity of Mammalian Cell Lines Engineered to Overexpress Cytosolic Glutathione Peroxidase.

Reactive oxygen species are believed to be involved in radiation lethality. Glutathione peroxidase is an intracellular enzyme with antioxidant functions. To determine whether increasing the cellular antioxidant capacity can confer radiation resistance, the effect of overexpression of glutathione peroxidase on radiosensitivity was determined in two different cell types. An expression construct including the bovine cytosolic glutathione peroxidase cDNA was used to overexpress this enzyme in cells of the human lymphoblast cell line Sup-T1 as well as the Chinese hamster ovary cell line AA8. Supplementation of the culture media with 30 nM sodium selenite was included to obtain optimal glutathione peroxidase activity. Northern blot analysis confirmed the presence of the construct mRNA, and a standard coupled spectrophotometric assay demonstrated significantly increased glutathione peroxidase activity in the transfected cell lines. An approximately 8-fold increase was found in the Sup-T1 cells, and an approximately 30-fold increase was obtained in the Chinese hamster ovary AA8 cells. Clonogenic survival was assayed in the overexpressing cells and compared to that in control cells transfected with vector alone. Despite significantly increased glutathione peroxidase activity, no observable radioprotection was conferred in either of the two cell lines studied, indicating that increased glutathione peroxidase activity is insufficient to confer radioresistance in the two cell types examined. These data are discussed in the context of using antioxidants as adjuncts to clinical radiotherapy.

Li, Z., Xia, L., Lee, L. M., Khaletskiy, A., Wang, J., Wong, J. Y. C. and Li, J-J. Effector Genes Altered in MCF-7 Human Breast Cancer Cells after Exposure to Fractionated Ionizing Radiation.

Understanding the molecular mechanisms involved in the response of tumors to fractionated exposures to ionizing radiation is important for improving radiotherapy and/or radiochemotherapy. In the present study, we examined the expression of stress-related genes in an MCF-7 cell population (MCF-IR20) that has been derived through treatment with fractionated irradiation (2 Gy per fraction with a total dose of 40 Gy). MCF-IR20 cells showed a 1.6-fold increase in sensitization with dose at 10% isosurvival in a clonogenic assay, and a reduced growth delay (∼15 h compared to ∼27 h), compared to the parental MCF-7 cells treated with a single dose of 5 Gy. To determine which effector genes were altered in the MCF-IR20 cells, the expression of stress-related effector genes was measured using a filter with 588 genes (Clontech) that included major elements involved in cell cycle control, DNA repair, and apoptosis. Compared to MCF-7 cells that were not exposed to fractionated radiation, 19 genes were up- regulated (2.2–5.1-fold) and 4 were down-regulated (2.7–3.4- fold) in the MCF-IR20 cells. In agreement with the array results, 6 up-regulated genes tested by RT-PCR showed elevated expression. Also, activities of the stress-related transcription factors NFKB, TP53 and AP1 showed a 1.2–4.5-fold increase after a single dose of 5 Gy in MCF-IR20 cells compared with parental MCF-7 cells. However, when the radioresistant MCF-IR20 cell were cultured for more than 12 passages after fractionated irradiation (MCF-RV), radioresistance was lost, with the radiosensitivity being the same as the parental MCF- 7 cells. Interestingly, expression levels of CCNB1, CD9 and CDKN1A in MCF-RV cells returned to levels expressed by the parental cells, whereas the expression levels of three other genes, MSH2, MSH6 and RPA remained elevated. To determine if any of the changes in gene expression could be responsible for the induced radioresistance, CCNB1 and CDKN1A, both of which were up-regulated in MCF-IR20 cells and down-regulated in MCF-RV cells, were studied further by transfection with antisense oligonucleotides. Antisense of CCNB1 significantly reduced the clonogenic survival of MCF- IR20 cells at doses of 5 and 10 Gy, from 42% to 26% and from 5.7% to 1.0%, respectively. Antisense of CDKN1A, however, had no effect on radiation survival of MCF-IR20 cells. In summary, these results suggest that stress-related effector genes are altered in cells after treatment with fractionated irradiation, and that up-regulation of CCNB1 is responsible, at least in part, for radioresistance after fractionated irradiation.

Savell, J., Rao, S., Pledger, W. J. and Wharton, W. Permanent Growth Arrest in Irradiated Human Fibroblasts.

Exposure of human fibroblasts to doses of ionizing radiation sufficient to cause a permanent growth arrest repressed the expression of genes induced late during G₀/G₁-phase traverse, including both cyclin A and cyclin E. In addition, radiation prevented the cell cycle-dependent activation of cyclin D1-associated kinase activity and the subsequent phosphorylation of the RB tumor suppressor protein. Exposure to radiation did not alter the cellular levels of cyclin D1 protein, nor did it alter the formation of cyclin D1-CDK4 complexes. Surprisingly, the repression of cyclin D1-associated kinase activity in damaged mitogen-stimulated quiescent cells could not be accounted for by a relative increase in the association of CDKN1A (also known as p21^Cip1) with cyclin D1 complexes, nor was cyclin D1 activity targeted by increased levels of CDKN1A in irradiated, logarithmically growing cultures under conditions where cyclin A activity was acutely repressed. Therefore, a radiation-induced permanent growth arrest is mediated by pathways that are distinct from those that cause cell cycle delay in damaged cells involving repression of cyclin-dependent kinase activity by CDKN1A.

Banáth, J. P., Kim, A. and Olive, P. L. Overnight Lysis Improves the Efficiency of Detection of DNA Damage in the Alkaline Comet Assay.

The ability to detect DNA damage using the alkaline comet assay depends on pH, lysis time and temperature during lysis. However, it is not known whether different lysis conditions identify different types of DNA damage or simply measure the same damage with different efficiencies. Results support the latter interpretation for radiation, but not for the alkylating agent MNNG. For X-ray-induced damage, cells showed the same amount of damage, regardless of lysis pH (12.3 compared to >13). However, increasing the duration of lysis at 5°C from 1 h to more than 6 h increased the amount of DNA damage detected by almost twofold. Another twofold increase in apparent damage was observed by conducting lysis at room temperature (22°C) for 6 h, but at the expense of a higher background level of DNA damage. The oxygen enhancement ratio and the rate of rejoining of single-strand breaks after irradiation were similar regardless of pH and lysis time, consistent with more efficient detection of strand breaks rather than detection of damage to the DNA bases. Conversely, after MNNG treatment, DNA damage was dependent on both lysis time and pH. With the higher-pH lysis, there was a reduction in the ratio of oxidative base damage to strand breaks as revealed using treatment with endonuclease III and formamidopyrimidine glycosylase. Therefore, our current results support the hypothesis that the increased sensitivity of longer lysis at higher pH for detecting radiation-induced DNA damage is due primarily to an increase in efficiency for detecting strand breaks, probably by allowing more time for DNA unwinding and diffusion before electrophoresis.

Zook, B. C. and Simmens, S. J. The Effects of 860 MHz Radiofrequency Radiation on the Induction or Promotion of Brain Tumors and Other Neoplasms in Rats.

Sprague-Dawley rats were irradiated with a continuous- wave (CW) or a pulsed-wave (P) radiofrequency (RF) for 6 h/day, 5 days/week from 2 up to 24 months of age. The RFs emanated from dipole antennas (1 W average output) 2.0 ± 0.5 cm from the tip of each rat's nose. The RFs had an 860 MHz frequency, and the specific absorption rate was 1.0 W/ kg averaged over the brain. Fifteen groups of 60 rats (900 total) were formed from offspring of females injected i.v. with 0 (groups 1, 2, 9, 10, 13), 2.5 (groups 5, 6, 7, 8, 11, 12, 14) or 10 mg/kg (groups 3, 4, 15) ethylnitrosourea (ENU) to induce brain tumors. Groups 1, 3, 5 and 7 received the PRF, and groups 9 and 11 the CWRF; groups 2, 4, 6, 8, 10 and 12 were sham-irradiated, and groups 13–15 were cage controls. All rats but 2, totaling 898, were necropsied, and major tissues were studied histopathologically. There was no statistically significant evidence that the PRF or CWRF induced neoplasia in any tissues. Additionally, there was no significant evidence of promotion of cranial or spinal nerve or spinal cord tumors. The PRF or CWRF had no statistically significant effect on the number, volume, location, multiplicity, histological type, malignancy or fatality of brain tumors. There was a trend for the group that received a high dose of ENU and was exposed to the PRF to develop fatal brain tumors at a higher rate than its sham group; however, the result was not significant using the log-rank test (P = 0.14, 2-tailed). No statistically significant differences were related to the PRF or CWRF compared to controls in the low- or zero-dose groups regarding tumors of any kind.

Stagg, R. B., Hawel, L. H., III, Pastorian, K., Cain, C., Adey, W. R. and Byus, C. V. Effect of Immobilization and Concurrent Exposure to a Pulse-Modulated Microwave Field on Core Body Temperature, Plasma ACTH and Corticosteroid, and Brain Ornithine Decarboxylase, Fos and Jun mRNA.

Exposure of humans and rodents to radiofrequency (RF) cell phone fields has been reported to alter a number of stress- related parameters. To study this potential relationship in more detail, tube-restrained immobilized Fischer 344 rats were exposed in the near field in a dose-dependent manner to pulse-modulated (11 packets/s) digital cell phone microwave fields at 1.6 GHz in accordance with the Iridium protocol. Core body temperatures, plasma levels of the stress-induced hormones adrenocorticotrophic hormone (ACTH) and corticosterone, and brain levels of ornithine decarboxylase (Odc), Fos and Jun mRNAs were measured as potential markers of stress responses mediated by RF radiation. We tested the effects of the loose-tube immobilization with and without prior conditioning throughout a 2-h period (required for near-field head exposure to RF fields), on core body temperature, plasma ACTH and corticosteroids. Core body temperature increased transiently (±0.3°C) during the initial 30 min of loose- tube restraint in conditioned animals. When conditioned/tube- trained animals were followed as a function of time after immobilization, both the ACTH and corticosterone levels were increased by nearly 10-fold. For example, within 2–3 min, ACTH increased to 83.2 ± 31.0 pg/dl, compared to 28.1 ± 7.7 pg/dl for cage controls, reaching a maximum at 15–30 min (254.6 ± 46.8 pg/dl) before returning to near resting levels by 120 min (31.2 ± 10.2 pg/dl). However, when non-tube-trained animals were submitted to loose-tube immobilization, these animals demonstrated significantly higher (3–10-fold greater) hormone levels at 120 min than their tube-trained counterparts (313.5 ± 54.8 compared to 31.2 ± 10.2 pg/dl; corticosterone, 12.2 ± 6.2 μg/dl compared to 37.1 ± 6.4 μg/dl). Hormone levels in exposed animals were also compared to those in swim-stressed animals. Swimming stress also resulted in marked elevation in both ACTH and corticosterone levels, which were 10–20 fold higher (541.8 compared to 27.2–59.1 pg/dl for ACTH) and 2–5 fold higher (45.7 compared to 8.4– 20.0 μg/dl for corticosteroids) than the cage control animals. Three time-averaged brain SAR levels of 0.16, 1.6 and 5 W/ kg were tested in a single 2-h RF-field exposure to the Iridium cell phone field. When RF-exposed and sham-exposed (immobilized) animals were compared, no differences were seen in core body temperature, corticosterone or ACTH that could be attributed to near-field RF radiation. Levels of Odc, Fos and Jun mRNA were also monitored in brains of animals exposed to the RF field for 2 h, and they showed no differences from sham-exposed (loose-tube immobilized) animals that were due to RF-field exposure. These data suggest that a significant stress response, indicated by a transient increase in core body temperature, ACTH and corticosterone, occurred in animals placed in even the mild loose-tube immobilization required for near-field RF exposure employed here and in our other studies. Failure to adequately characterize and control this immobilization response with appropriate cage control animals, as described previously, could significantly mask any potential effects mediated by the RF field on these and other stress-related parameters. We conclude that the pulse-modulated digital Iridium RF field at SARs up to 5 W/kg is incapable of altering these stress-related responses. This conclusion is further supported by our use of an RF-field exposure apparatus that minimized immobilization stress; the use of conditioned/tube-trained animals and the measurement of hormonal and molecular markers after 2 h RF

Levin-Plotnik, D., Hamilton, R. J., Niemierko, A. and Akselrod, S. A Model for Optimizing Normal Tissue Complication Probability in the Spinal Cord Using a Generalized Incomplete Repair Scheme.

The purpose of this study was to determine the treatment protocol, in terms of dose fractions and interfraction intervals, which minimizes normal tissue complication probability in the spinal cord for a given total treatment dose and treatment time. We generalize the concept of incomplete repair in the linear-quadratic model, allowing for arbitrary dose fractions and interfraction intervals. This is incorporated into a previously presented model of normal tissue complication probability for the spinal cord. Equations are derived for both mono-exponential and bi-exponential repair schemes, regarding each dose fraction and interfraction interval as an independent parameter, subject to the constraints of fixed total treatment dose and treatment time. When the interfraction intervals are fixed and equal, an exact analytical solution is found. The general problem is nonlinear and is solved numerically using simulated annealing. For constant interfraction intervals and varying dose fractions, we find that optimal normal tissue complication probability is obtained by two large and equal doses at the start and conclusion of the treatment, with the rest of the doses equal to one another and smaller than the two dose spikes. A similar result is obtained for bi-exponential repair. For the general case where the interfraction intervals are discrete and also vary, the pattern of two large dose spikes is maintained, while the interfraction intervals oscillate between the smallest two values. As the minimum interfraction interval is reduced, the normal tissue complication probability decreases, indicating that the global minimum is achieved in the continuum limit, where the dose delivered by the “middle” fractions is given continuously at a low dose rate. Furthermore, for bi-exponential repair, it is seen that as the slow component of repair becomes increasingly dominant as the magnitude of the dose spikes decreases. Continuous low-dose-rate irradiation with dose spikes at the start and end of treatment yields the lowest normal tissue complication probability in the spinal cord, given a fixed total dose and total treatment time, for both mono-exponential and bi-exponential repair. The magnitudes of the dose spikes can be calculated analytically, and are in close agreement with the numerical results.

Miura, M., Morris, G. M., Micca, P. L., Lombardo, D. T., Youngs, K. M., Kalef-Ezra, J. A., Hoch, D. A., Slatkin, D. N., Ma, R. and Coderre, J. A. Boron Neutron Capture Therapy of a Murine Tumor using a Lipophilic Carboranyltetraphenylporphyrin.

The first control of a malignant tumor in vivo by porphyrin- mediated boron neutron capture therapy (BNCT) is described. In mice bearing implanted EMT-6 mammary carcinomas, boron uptake using a single injection of either p-boronophenylalanine (BPA) or mercaptoundecahydrododecaborane (BSH) was compared with either a single injection or multiple injections of the carboranylporphyrin CuTCPH. The BSH and BPA doses used were comparable to the highest doses of these compounds previously administered in a single injection to rodents. For BNCT, boron concentrations averaged 85 μg ¹⁰B/g in the tumor and 4 μg ¹⁰B/g in blood 2 days after the last of six injections (over 32 h) that delivered a total of 190 μg CuTCPH/g body weight. During a single 15, 20, 25 or 30 MW-min exposure to the thermalized neutron beam of the Brookhaven Medical Research Reactor, a tumor received average absorbed doses of approximately 39, 52, 66 or 79 Gy, respectively. A long-term (>200 days) tumor control rate of 71% was achieved at a dose of 66 Gy with minimal damage to the leg. Equivalent long-term tumor control by a single exposure to 42 Gy X rays was achieved, but with greater damage to the irradiated leg.

Kiger, W. S., III, Palmer, M. R., Riley, K. J., Zamenhof, R. G. and Busse, P. M. A Pharmacokinetic Model for the Concentration of ¹⁰B in Blood after Boronophenylalanine- Fructose Administration in Humans.

An open two-compartment model has been developed for predicting ¹⁰B concentrations in blood after intravenous infusion of the l-p-boronophenylalanine-fructose complex (BPA-F) in humans and derived from studies of pharmacokinetics in 24 patients in the Harvard-MIT Phase I clinical trials of BNCT. The ¹⁰B concentration profile in blood exhibits a characteristic rise during the infusion to a peak of ∼32 μg/g (for infusion of 350 mg/kg over 90 min) followed by a biphasic exponential clearance profile with half-lives of 0.34 ± 0.12 and 9.0 ± 2.7 h, due to redistribution and primarily renal elimination, respectively. The model rate constants k₁, k₂ and k₃ are 0.0227 ± 0.0064, 0.0099 ± 0.0027 and 0.0052 ± 0.0016 min^–1, respectively, and the central compartment volume of distribution, V₁, is 0.235 ± 0.042 kg/kg. The validity of this model was demonstrated by successfully predicting the average pharmacokinetic response for a cohort of patients who were administered BPA-F using an infusion schedule different from those used to derive the parameters of the model. Furthermore, the mean parameters of the model do not differ for cohorts of patients infused using different schedules.

Strzelczak, G., Vanhaelewyn, G., Stachowicz, W., Goovaerts, E., Callens, F. and Michalik, J. Multifrequency EPR Study of Carbonate- and Sulfate-Derived Radicals Produced by Radiation in Shells and Corallite.

Shells of two sea mollusks (Venus sp.), pearl oyster (Meleagrina vulgaris) and corallite (white coral) were exposed to ionizing radiation (γ and X rays) and then examined by EPR spectroscopy in X, Q and W band. The resulting spectra were analyzed and the g values of the EPR lines in the multicomponent spectra were determined. The increased resolution in Q- and W-band spectra allowed us to assign the observed lines to CO₂^– ion radicals (isotropic and orthorhombic), SO₂^– isotropic, SO₃^– (isotropic and axial), and Mn² species. The assignments were confirmed by simulations of the spectra. Practical implications for the use of Q and/or W band in low-dose quantitative EPR measurements for dating and for accidental dose estimation are discussed.

Abdoul-Carime, H., Cloutier P. and Sanche, L. Low-Energy (5–40 eV) Electron-Stimulated Desorption of Anions from Physisorbed DNA Bases.

We present the results of experiments on anion desorption from the physisorbed DNA bases adenine, thymine, guanine and cytosine induced by the impact of low-energy (5–40 eV) electrons. Electron bombardment of DNA base films induces ring fragmentation and desorption of H^–, O^–, OH^–, CN^–, OCN^– and CH₂^– anions through either single or complex multibond dissociation. We designate the variation of the yield of an anion with electron energy as the yield function. Below 15 eV incident electron energy, bond cleavage is controlled mainly by dissociative electron attachment. Above 15 eV, the portion of a yield function that increases linearly is attributed to nonresonant processes, such as dipolar dissociation. A resonant structure is superimposed on this signal around 20 eV in the anion yield functions. This structure implicates dissociative electron attachment and/or resonant decay of the transient anion into the dipolar dissociation channel, with a minimal contribution from multiple inelastic electron scattering. The yields of all desorbing anions clearly show that electron resonances contribute to the damage of all DNA bases bombarded with 5–40 eV electrons. Comparison of the ion yields indicates that adenine is the least sensitive base to slow electron attack. Electron-irradiated guanine films exhibit the largest yields of desorbed anions.

Patrzyc, H. B., Dawidzik, J. B., Budzinski, E. E., Iijima, H. and Box, H. C. Double Lesions are Produced in DNA Oligomer by Ionizing Radiation and by Metal-Catalyzed H₂O₂ Reactions.

It was demonstrated previously that double lesions are produced in DNA by ionizing radiation. These double lesions consist of adjacent nucleotides each bearing a modified base. The goal of the present investigation was to determine whether Fenton chemistry can generate the same kind of lesions. DNA oligomers were exposed to metal-catalyzed H₂O₂ reactions, and the products were characterized by chromatography and by mass spectrometry. Double lesions are produced by this treatment in which deoxyguanosine is oxidized to 8-oxo-7,8- dihydrodeoxyguanosine and an adjacent pyrimidine nucleoside is degraded to a formamido remnant.

Brocklehurst, B. Radiation Damage in DNA: Possible Role of Higher Triplet States.

Triplet states of deoxyribose are expected to dissociate efficiently into radicals, leading to strand breaks. Such states could be excited by slow secondary electrons (A) or result from ion recombination in spurs containing two or more ion pairs (B). Estimates of the efficiencies of these processes are presented and the mechanisms discussed in the light of recent work with electrons, vacuum ultraviolet (VUV) photons, and X rays. Route B could play a significant role in producing double-strand breaks, while route A may be a better approach to characterizing the process experimentally.