Eifel, P. Chemoradiation for Carcinoma of the Cervix: Advances and Opportunities.

Although it is possible to cure many patients with locally advanced cervical cancer using radiation therapy alone, loco-regional relapse continues to be a component of most recurrences. To improve control rates, clinicians have investigated ways of combining chemotherapy and radiation for more than 30 years. Despite encouraging results from phase II trials of neoadjuvant chemotherapy, randomized trials failed to improve on the results with radiation therapy alone. For a number of reasons, early trials of concurrent chemoradiation were inconclusive. However, recent reports of five large prospective randomized trials demonstrated dramatic improvements in survival and local control rates when cisplatin-containing chemotherapy was given during radiation therapy. These results also suggest a number of avenues for future research.

Koshurnikova, N. A., Gilbert, E. S., Sokolnikov, M., Khokhryakov, V. F., Miller, S., Preston, D. L., Romanov, S. A., Shilnikova, N. S., Suslova, K. G. and Vostrotin, V. V. Bone Cancers in Mayak Workers.

Bone cancer mortality risks were evaluated in 11,000 workers who started working at the “Mayak” Production Association in 1948–1958 and who were exposed to both internally deposited plutonium and external γ radiation. Comparisons with Russian and U.S. general population rates indicate excess mortality, especially among females, plutonium plant workers, and workers with external doses exceeding 1 Sv. Comparisons within the Mayak worker cohort, which evaluate the role of plutonium body burden with adjustment for cumulative external dose, indicate excess mortality among workers with burdens estimated to exceed 7.4 kBq (relative risk = 7.9; 95% CI = 1.6–32) and among workers in the plutonium plant who did not have routine plutonium monitoring data based on urine measurements (relative risk = 4.1; 95% CI = 1.2–14). In addition, analyses treating the estimated plutonium body burden as a continuous variable indicate increasing risk with increasing burden (P < 0.001). Because of limitations in current plutonium dosimetry, no attempt was made to quantify bone cancer risks from plutonium in terms of organ dose, and risk from external dose could not be reliably evaluated.

Gilbert, E. S., Koshurnikova, N. A., Sokolnikov, M., Khokhryakov, V. F., Miller, S., Preston, D. L., Romanov, S. A., Shilnikova, N. S., Suslova, K. G. and Vostrotin, V. V. Liver Cancers in Mayak Workers.

Liver cancer mortality risks were evaluated in 11,000 workers who started working at the “Mayak” Production Association in 1948–1958 and who were exposed to both internally deposited plutonium and external γ radiation. Comparisons with Russian liver cancer incidence rates indicate excess risk, especially among those with detectable plutonium body burdens and among female workers in the plutonium plant. Comparisons within the Mayak worker cohort which evaluate the role of plutonium body burden with adjustment for cumulative external dose indicate excess risk among workers with burdens estimated to exceed 7.4 kBq (relative risk = 17; 95% CI = 8.0–36) and among workers in the plutonium plant who did not have routine plutonium monitoring data based on urine measurements (relative risk = 2.8; 95% CI = 1.3–6.2). In addition, analyses treating the estimated plutonium body burden as a continuous variable indicate increasing risk with increasing burden (P < 0.001). Relative risks tended to be higher for females than for males, probably because of the lower baseline risk and the higher levels of plutonium measured in females. Because of limitations in current plutonium dosimetry, no attempt was made to quantify liver cancer risks from plutonium in terms of organ dose, and risk from external dose could not be reliably evaluated.

Oghiso, Y. and Yamada, Y. Pathogenetic Process of Lung Tumors Induced by Inhalation Exposures of Rats to Plutonium Dioxide Aerosols.

Sequential examinations were done on the pulmonary cytokinetics and pulmonary lesions in rats after inhalation exposure to ²³⁹PuO₂ aerosols to investigate the pathogenesis of lung tumors. Total cell yields of lavaged bronchoalveolar cells as well as the estimated numbers of pulmonary alveolar macrophages were significantly reduced from 1 to 3 months after exposure but recovered thereafter to the control levels. The proportions of multinucleated or micronucleated pulmonary alveolar macrophages increased significantly in lavaged cells from 1 month, and the increase was sustained up to 18 months after exposure. Both tumor necrosis factor and nitric oxide were shown to be differentially released from stimulated cultures of pulmonary alveolar macrophages during the period from 6 to 18 months after exposure. The labeling indices of alveolar and bronchiolar epithelial cells treated with 5-bromo-2′-deoxyuridine increased significantly in lungs from 3 months and were sustained up to 18 months after exposure. Histopathological examinations revealed that after the early inflammation, hyperplasia and metaplasia of the lining of the bronchioloalveolar epithelium were predominant from 3 to 6 months, while adenomatous or adenocarcinomatous lesions appeared and developed from 12 months after exposure. The appearance of primary lung tumors, almost all of which were adenomas and adenocarcinomas, was found in the dose range of 1 to 2 Gy from 12 months after exposures. These results indicate that the pathogenetic process initiated by early cellular damage and alterations associated with inflammation is followed by the proliferative and metaplastic lesions of pulmonary epithelium, leading to the appearance and development of pulmonary neoplasms from 1 year after the inhalation exposures in rats that received a minimum lung dose of more than 1 Gy.

Ishizuka, S., Ito, S., Kasai, T. and Hara, H. Dietary Sugar Beet Fiber Ameliorates Diarrhea as an Acute Gamma-Radiation Injury in Rats.

Gamma radiation induces diarrhea as an acute injury. We have studied whether ingestion of sugar beet fiber influences radiation-induced diarrhea. Abdominal irradiation with γ rays induced diarrhea in male Wistar/ST rats from 2 to 7 days after a single sublethal dose. The body weight of the irradiated rats was decreased temporarily at 4 days after irradiation regardless of the ingestion of sugar beet fiber. At day 8, it returned to almost the same level as that of unirradiated rats. A change in daily food intake resulted in a pattern similar to that for body weight. Dietary sugar beet fiber had little significant effect on the changes in body weight and daily food intake, and its ingestion significantly decreased γ-ray-induced diarrhea. Changes in biochemical and histological parameters in intestinal mucosa (small intestine, cecum and colon) were not greatly influenced by the ingestion of sugar beet fiber through the periods of diarrhea. It was concluded that dietary sugar beet fiber ameliorated the diarrhea induced by abdominal irradiation. We suggest that the inhibitory effect of the ingestion of sugar beet fiber is due to its effects on the luminal environment, such as support for bacterial function in the luminal contents in the colon of animals that ingest sugar beet fiber.

Li, Y. Q. and Wong, C. S. Radiation-Induced Apoptosis in the Neonatal and Adult Rat Spinal Cord.

This study was designed to characterize radiation-induced apoptosis in the spinal cord of the neonatal and young adult rat. Spinal cords (C2–T2) of 1-, 2- and 10-week-old rats were irradiated with a single dose of 8, 18 or 22 Gy. Apoptosis was assessed histologically according to its specific morphological features or by using the TUNEL assay. Cell proliferation was assessed immunohistochemically using BrdU. Identities of cell types undergoing apoptosis were assessed using immunohistochemistry or in situ hybridization using markers for neurons, glial progenitor cells, microglia, oligodendrocytes and astrocytes. The time course of radiation-induced apoptosis in 1- or 2-week-old rat spinal cord was similar to that in the young adult rat spinal cord. A peak response was observed at about 8 h after irradiation, and the apoptosis index returned to the levels in nonirradiated spinal cords at 24 h. The neonatal rat spinal cord demonstrated increased apoptosis compared to the adult. Values for total yield of apoptosis over 24 h induced by 8 Gy in the neonatal rat spinal cord were significantly greater than that in the adult. Immunohistochemistry studies using Leu7, galactocerebroside, Rip and adenomatous polyposis coli tumor suppressor protein indicated that most apoptotic cells were cells of the oligodendroglial lineage regardless of the age of the animal. No evidence of Gfap or factor VIII-related antigen-positive apoptotic cells was observed, and there was a small number of apoptotic microglial cells (lectin-Rca1 positive) in the neonatal and adult rat spinal cord. In the neonatal but not adult rat spinal cord, about 10% of the apoptotic cells appeared to be neurons and were immunoreactive for synaptophysin. Labeling indices (LI) for BrdU in nonirradiated 1- and 2-week-old rat spinal cord were 20.0 and 16.3%, respectively, significantly greater than the LI of 1.0% in the 10-week-old rat spinal cord. At 8 h after a single dose of 8 Gy, 13.4% of the apoptotic cells were BrdU-positive in 10-week-old rat spinal cord, whereas 62.4 and 44.1% of the apoptotic cells showed BrdU incorporation in 1- and 2-week-old rat spinal cord, respectively. Regardless of the age of the animal, the apoptosis indices in BrdU-positive cells were greater than those in BrdU-negative cells. We conclude that the neonatal spinal cord demonstrates a greater level of apoptosis after exposure to ionizing radiation than the young adult spinal cord. This increase in apoptosis may be associated in part with the greater percentage of proliferating cells in the neonatal spinal cord, which demonstrate a greater level of radiation-induced apoptosis than nonproliferating cells.

Wang, B., Ohyama, H., Haginoya, K., Odaka, T., Itsukaichi, H., Yukawa, O., Yamada, T. and Hayata, I. Adaptive Response in Embryogenesis. III. Relationship to Radiation-Induced Apoptosis and Trp53 Gene Status.

We reported previously that a radiation-induced adaptive response existed in the late period of embryogenesis, and that radiation-induced apoptosis in the predigital regions was responsible for digital defects in embryonic ICR mice. To investigate the possible involvement of the Trp53 gene and radiation-induced apoptosis in radiation-induced adaptive responses in embryogenesis, the present study was conducted using Trp53 wild-type (Trp53 ^/ ) and Trp53 heterozygous (Trp53 ^/–) embryonic mice of the C57BL/6 strain. The existence of a radioadaptive response in the Trp53 ^/ embryonic mice was demonstrated by irradiating the embryos with 5 or 30 cGy on embryonic day 11 prior to a challenging irradiation at 3 Gy on embryonic day 12. The two conditioning doses at 5 and 30 cGy significantly suppressed the induction of apoptosis by the challenging dose in the predigital regions of limb buds in the Trp53 ^/ embryonic mice, while no such effect was found in the Trp53 ^/– embryonic mice. These findings indicate that induction of a radioadaptive response in embryogenesis is related to Trp53 gene status and the occurrence of radiation-induced apoptosis.

Giver, C. R., Moore, D. H. II and Pallavicini, M. G. Radiation-Induced Translocations in Mice: Persistence, Chromosome Specificity, and Influence of Genetic Background.

The translocation frequency response in the chromosomes of peripheral blood lymphocytes is widely used for radiation biomonitoring and dose estimation. However, this assay is based upon several assumptions that have not been rigorously tested. It is typically assumed that the translocation frequency in blood lymphocytes reflects the level of genomic damage in other hemopoietic tissues and is independent of the chromosome probe and genetic background. We conducted studies to evaluate these assumptions using mice with different genetic backgrounds. Six different whole-chromosome fluorescence in situ hybridization (FISH) probes were used to detect translocations in peripheral blood lymphocytes at multiple times after whole-body irradiation. Translocation frequencies were chromosome-independent at 6 and 16 weeks after exposure but were chromosome-dependent at 1.5 years after exposure. Similar translocation frequencies were observed in blood, bone marrow and spleen at 1.5 years, supporting previous suggestions that genetically aberrant peripheral blood lymphocytes may derive from precursor populations in hemopoietic tissues. Translocations measured 66 h after irradiation differed among some strains. We conclude that the translocation frequency response is a complex phenotype that is influenced not only by exposure dose but also by genetic background, the choice of chromosome analyzed, and time after exposure. These results raise important considerations for the use of the FISH-based translocation frequency response for radiation dosimetry and biomonitoring.

Shimada, Y., Nishimura, M., Kakinuma, S., Okumoto, M., Shiroishi, T., Clifton, K. H. and Wakana, S. Radiation-Associated Loss of Heterozygosity at the Znfn1a1 (Ikaros) Locus on Chromosome 11 in Murine Thymic Lymphomas.

Although information on the molecular pathways in radiation carcinogenesis is accumulating, the data are still relatively scanty. To find the tumor suppressor locus associated with radiation carcinogenesis, we determined the frequency and distribution of loss of heterozygosity (LOH) of X-ray-induced thymic lymphomas of B6C3F₁ mice using 58 microsatellite markers and compared the results with those for spontaneous lymphomas and N-ethylnitrosourea (ENU)-induced lymphomas. Based on the results, we describe a unique locus with frequent LOH in the centromeric region of chromosome 11 of X-ray-induced lymphomas. This locus has never been observed to be altered similarly in either ENU-induced or spontaneous lymphomas, suggesting radiation-specific molecular alteration. The LOH patterns of individual thymic lymphomas indicated that the common region of LOH was located within 1.6 cM between D11Mit62 and D11Mit204, a region syntenic to human chromosome 7p13. Linkage analysis revealed that the markers of the common LOH region were genetically linked to Ikaros (now known as Znfn1a1), a master gene of lymphopoiesis. Although the presence of radiation-associated LOH in other loci cannot be ruled out, these results suggest a novel molecular pathway in induction of thymic lymphomas by ionizing radiation.

Holl, V., Coelho, D., Weltin, D., Denis, J. M., Dufour, P., Gueulette, J. and Bischoff, P. Ex Vivo Determination of the Effect of Whole-Body Exposure to Fast Neutrons on Murine Spleen Cell Viability and Apoptosis.

The effects of high-linear energy transfer (LET) radiations on lymphoid tissues and lymphocytes are not well understood. As a first approach to delineate these effects, the present work was conducted to assess the effects of high-LET radiations on murine spleen cells ex vivo and in vitro. BALB/c mice were irradiated whole-body with 65 MeV neutrons or 15 MV X rays at doses ranging from 0.2 to 3 Gy. Spleens were removed 1 day postirradiation and weighed, and single cell suspensions were prepared and cultured for several days. Apoptosis occurring in vitro was determined at different times by flow cytometry analysis of cells labeled with propidium iodide. It was found that irradiation with fast neutrons reduced spleen weight and cellularity to a greater extent than photons. Considering the spleen cellularity as end point, the relative biological effectiveness (RBE) of fast neutrons was 2. However, for both modes of irradiation, apoptosis of recovered spleen cells in vitro increased as a function of dose and the duration of culture. The level of apoptosis occurring at various times postirradiation was found to be identical for high- and low-LET radiations. Taken together, these results suggest that external as well as cellular factors might differentially modulate the sensitivity of lymphocytes to fast neutrons and photons.

Schmid, E., Regulla, D., Guldbakke, S., Schlegel, D. and Bauchinger, M. The Effectiveness of Monoenergetic Neutrons at 565 keV in Producing Dicentric Chromosomes in Human Lymphocytes at Low Doses.

The induction of dicentric chromosomes in human lymphocytes from one individual irradiated in vitro with monoenergetic neutrons at 565 keV was examined to provide additional data for an improved evaluation of neutrons with respect to radiation risk in radioprotection. The resulting linear dose–response relationship obtained (0.813 ± 0.052 dicentrics per cell per gray) over the dose range of 0.0213–0.167 Gy is consistent with published results obtained for irradiation with neutrons from different sources and with different spectra at energies lower than 1000 keV. Comparing this value to previously published “average” dose–response curves obtained by different laboratories for ⁶⁰Co γ rays and orthovoltage X rays resulted in maximum RBEs (RBE_m) of about 37 ± 8 and 16 ± 4, respectively. However, when our neutron data were matched to low-LET dose responses that were constructed several years earlier for lymphocytes from the same individual, higher values of RBE_m resulted: 76.0 ± 29.5 for ⁶⁰Co γ rays and 54.2 ± 18.4 for ¹³⁷Cs γ rays; differentially filtered 220 kV X rays produced values of RBE_m between 20.3 ± 2.0 or 37.0 ± 7.1. The results highlight the dependence of RBE_m on the choice of low-LET reference radiation and raise the possibility that differential individual response to low-LET radiations may need to be examined more fully in this context.

Kinashi, Y., Sakurai, Y., Masunaga, S., Suzuki, M., Takagaki, M., Akaboshi, M. and Ono, K. Molecular Structural Analysis of HPRT Mutations Induced by Thermal and Epithermal Neutrons in Chinese Hamster Ovary Cells.

Chinese hamster ovary (CHO) cells were exposed to thermal and epithermal neutrons, and the occurrence of mutations at the HPRT locus was investigated. The Kyoto University Research Reactor (KUR), which has been improved for use in neutron capture therapy, was the neutron source. Neutron energy spectra ranging from nearly pure thermal to epithermal can be chosen using the spectrum shifters and thermal neutron filters. To determine mutant frequency and cell survival, cells were irradiated with thermal and epithermal neutrons under three conditions: thermal neutron mode, mixed mode with thermal and epithermal neutrons, and epithermal neutron mode. The mutagenicity was different among the three irradiation modes, with the epithermal neutrons showing a mutation frequency about 5-fold that of the thermal neutrons and about 1.5-fold that of the mixed mode. In the thermal neutron and mixed mode, boron did not significantly increase the frequency of the mutants at the same dose. Therefore, the effect of boron as used in boron neutron capture therapy (BNCT) is quantitatively minimal in terms of mutation induction. Over 300 independent neutron-induced mutant clones were isolated from 12 experiments. The molecular structure of HPRT mutations was determined by analysis of all nine exons by multiplex polymerase chain reaction. In the thermal neutron and mixed modes, total and partial deletions were dominant and the fraction of total deletions was increased in the presence of boron. In the epithermal neutron mode, more than half of the mutations observed were total deletions. Our results suggest that there are clear differences between thermal and epithermal neutron beams in their mutagenicity and in the structural pattern of the mutants that they induce. Mapping of deletion breakpoints of 173 partial-deletion mutants showed that regions of introns 3–4, 7/8–9 and 9–0 are sensitive to the induction of mutants by neutron irradiation.

Razskazovskiy, Y., Roginskaya, M., Jacobs, A. and Sevilla, M. D. Reductively Activated Cleavage of DNA Mediated by o,o′-Diphenylenehalonium Compounds.

o,o′-Diphenylenehalonium (DPH) cations represent a novel class of DNA-affinic compounds characterized by binding constants within the range of 10⁵–10⁶ M^–1. The maximum binding capacity of 2–2.5 base pairs per DPH cation and about 30% hypochromic reduction in the optical absorption of DPH cations upon binding to DNA suggest intercalation as a likely binding mode. In a DNA-bound form, DPH cations induce strand breaks upon reduction by radiation-produced electrons in aqueous solutions. In keeping with this mechanism, the cleavage is strongly inhibited by oxygen and is not affected by OH radical scavengers in the bulk. The yields of DPH-mediated base release significantly exceed the yield of base release caused by hydroxyl radical (in the absence of scavenger) in anoxic solutions. The yields are weakly dependent on DNA loading within the range from 5 to 50 base pairs per intercalator, which indicates the ability of excess electrons in DNA to react with a scavenger separated by tens of base pairs from the electron attachment site. The question regarding the mechanism by which the distant reactants reach each other in DNA remains unanswered, although it most likely involves electron hopping rather than a single-step long-distance tunneling. The latter conclusion is based on our finding that the electron affinity of DPH cations does not affect their properties as electron scavengers in DNA as would be expected if the direct long-distance tunneling is involved.

Walicka, M. A., Ding, Y., Adelstein, S. J. and Kassis, A. I. Toxicity of DNA-Incorporated Iodine-125: Quantifying the Direct and Indirect Effects.

To understand the biophysical mechanism(s) underlying the induction of cell death by the decay of the Auger electron emitter iodine-125 in DNA, Chinese hamster V79 lung fibroblasts were labeled with 5-[¹²⁵I]iodo-2′-deoxyuridine (¹²⁵IdU) for two doubling times and frozen and stored at −135°C in the presence of 0.26–3.0 M dimethyl sulfoxide (DMSO), which acts simultaneously as a cryoprotector and a hydroxyl radical scavenger. After the accumulation of ¹²⁵I decays, the cells were defrosted and their survival was determined. Within the range of the number of decays examined (up to 470 disintegrations per cell), the survival curves are exponential. The dependence of the D₃₇ on DMSO concentration is triphasic and seems to reach a plateau at ∼1.3 M. By extrapolating to infinite DMSO concentration, we estimate the D₃₇ for maximal hydroxyl radical scavenging to be 411 ± 36 disintegrations per cell. To determine the D₃₇ in the absence of DMSO, we extrapolate the D₃₇ curve to zero concentration, and a D₃₇ of 54 ± 5 disintegrations per cell is obtained. The maximal dose modification factor, calculated as the ratio of the D₃₇ at infinite DMSO concentration (i.e. direct effects only) to the D₃₇ at zero DMSO concentration (i.e. direct and indirect effects), is 7.6 ± 1.0. By inference, ∼90% of the radiotoxic effects of DNA-incorporated ¹²⁵I are due to indirect mechanisms.

Dahle, J., Mikalsen, S-O., Rivedal, E. and Steen, H. B. Gap Junctional Intercellular Communication is not a Major Mediator in the Bystander Effect in Photodynamic Treatment of MDCK II Cells.

Photodynamic treatment (PDT) of confluent MDCK II cells resulted in a noticeable clustering of dead cells, consistent with a significant bystander effect. Likewise, PDT of cells in microcolonies resulted in an overabundance of microcolonies that had responded to the treatment as a single unit, that is, in which either all or no cells were dead. Confluent MDCK II cells appeared to communicate via gap junction channels, while cells in microcolonies did not. Monte Carlo simulation models were fitted to the distributions of dead cells in confluent monolayers and in microcolonies. The simulations showed that the degree of the bystander effect was higher in microcolonies than in confluent cells, suggesting that gap junction communication may be involved in the bystander effect. However, when the gap junction hypothesis was tested by treatment of microcolonies with 30 μM dieldrin, an inhibitor of gap junctional intercellular communication, there was no reduction of the bystander effect, indicating that this effect was not mediated by gap junctional intercellular communication. PDT influenced phosphorylation of tyrosine residues in several proteins in the cells. Protein phosphorylation is important in cellular signaling pathways and may be involved in the bystander effect, for example by influencing the mode of cell death.

Amundson, S. A., Do, K. T., Shahab, S., Bittner, M., Meltzer, P., Trent, J. and Fornace, A. J., Jr. Identification of Potential mRNA Biomarkers in Peripheral Blood Lymphocytes for Human Exposure to Ionizing Radiation.

Since early in the Atomic Age, biological indicators of radiation exposure have been sought, but currently available methods are not entirely satisfactory. Using cDNA microarray hybridization to discover new potential biomarkers, we have identified genes expressed at increased levels in human peripheral blood lymphocytes after ex vivo irradiation. We recently used this technique to identify a large set of ionizing radiation-responsive genes in a human cell line (Oncogene 18, 3666–3672, 1999). The present set of radiation markers in peripheral blood lymphocytes was identified 24 h after treatment, and while the magnitude of mRNA induction generally decreased over time, many markers were still significantly elevated up to 72 h after irradiation. In all donors, the most highly responsive gene identified was DDB2, which codes for the p48 subunit of XPE, a protein known to play a crucial role in repair of ultraviolet (UV) radiation damage in DNA. Induction of DDB2, CDKN1A (also known as CIP1/WAF1) and XPC showed a linear dose–response relationship between 0.2 and 2 Gy at 24 and 48 h after irradiation, with less linearity at earlier or later times. These results suggest that relative levels of gene expression in peripheral blood cells may provide estimates of environmental radiation exposures.

Sugimoto, M. and Utsumi, H. Proficient Repair of Potentially Lethal Damage Sensitive to Hypertonic Treatment in Osteosarcoma Cells.

Fast-repairing potentially lethal damage (PLD) in seven osteosarcoma cell lines was analyzed after treatment with a hypertonic 0.5 M NaCl solution for 20 min and compared to that in seven human fibroblast strains. Fixation of PLD after exposure to ionizing radiation was observed without exception in both the osteosarcoma cells and the fibroblast strains. The percentages by which the D₀'s of the osteosarcoma cells decreased were significantly higher than the percentage decreases in the D₀'s of the fibroblast strains (P < 0.01). Hypertonic treatment resulted in radiosensitization due to fixation of PLD in all of the osteosarcoma cell lines, demonstrating that osteosarcoma cells can repair PLD better than normal fibroblast cells. The radiobiological response of the osteosarcoma cells, with enhanced killing after hypertonic treatment, was similar to that of normal untreated fibroblast cells.