Potten, C. S. Radiation, the Ideal Cytotoxic Agent for Studying the Cell Biology of Tissues such as the Small Intestine. Radiat. Res. 161, 123–136 (2004).

Epithelial tissues are highly polarized, with the proliferative compartment subdivided into units of proliferation in many instances. My interests have been in trying to understand how many cellular constituents exist, what their function is, and what the intercommunicants are that ensure appropriate steady-state cell replacement rates. Radiation has proven to be a valuable tool to induce cell death, reproductive sterilization, and regenerative proliferation in these systems, the responses to which can provide information on the number of regenerative cells (a function associated with stem cells). Such studies have helped define the epidermal proliferative units and the structurally similar units on the dorsal surface of the tongue. The radiation responses considered in conjunction with a wide range of cell kinetic, lineage tracking and somatic mutation studies together with complex mathematical modeling provide insights into the functioning of the proliferative units (crypts) of the small intestine. Comparative studies have then been undertaken with the crypts in the large bowel. In the small intestine, in which cancer rarely develops, various protective mechanisms have evolved to ensure the genetic integrity of the stem cell compartment. Stem cells in the small intestinal crypts are intolerant of genotoxic damage (including that induced by very low doses of radiation); they do not undergo cell cycle arrest and repair but commit an altruistic TP53-dependent cell suicide (apoptosis). This process is compromised in the large bowel by BCL2 expression. Recent studies have suggested a second genome protection mechanism operating in the stem cells of the small intestinal crypts that may also have a TP53 dependence. Such studies have allowed the cell lineages and genome protection mechanisms operating the small intestinal crypts to be defined.

Kim, J. H., Brown, S. L., Kolozsvary, A., Jenrow, K. A., Ryu, S., Rosenblum, M. L. and Carretero, O. A. Modification of Radiation Injury by Ramipril, Inhibitor of Angiotensin-Converting Enzyme, on Optic Neuropathy in the Rat. Radiat. Res. 161, 137–142 (2004).

Inhibitors of angiotensin-converting enzyme (ACE) have been used to reduce radiation-induced normal tissue injury. The present study was carried out to determine whether ramipril, one of the inhibitors of ACE, would ameliorate radiation-induced brain damage, using a well-characterized optic neuropathy model in the rat, one of the most critical and radiosensitive structures in the brain. The brains of adult Fischer rats were irradiated stereotactically with 30 Gy using a single collimated beam. Six months after irradiation and 1.5 mg/kg day⁻¹ ramipril (started 2 weeks after irradiation), rats were assessed for optic nerve damage functionally, using visual evoked potential, and histologically. Results show that ramipril conferred significant modification of radiation injury, since rats receiving radiation alone showed a threefold lengthening in the mean peak latency in the visual evoked potential, whereas 75% of rats receiving radiation followed by ramipril had evoked potentials that resembled those of normal untreated control rats. The histology of irradiated and ramipril-treated optic nerves appeared nearly normal, while there was significant demyelination in both optic nerves of irradiated rats. The study represents the first demonstration of prophylaxis of radiation injury by a carboxyl-containing ACE inhibitor, providing a pharmacological strategy designed to reduce radiation-induced normal tissue damage.

Li, Y. Q., Chen, P., Jain, V., Reilly, R. M. and Wong, C. S. Early Radiation-Induced Endothelial Cell Loss and Blood–Spinal Cord Barrier Breakdown in the Rat Spinal Cord. Radiat. Res. 161, 143–152 (2004).

Using a rat spinal cord model, this study was designed to characterize radiation-induced vascular endothelial cell loss and its relationship to early blood–brain barrier disruption in the central nervous system. Adult rats were given a single dose of 0, 2, 8, 19.5, 22, 30 or 50 Gy to the cervical spinal cord. At various times up to 2 weeks after irradiation, the spinal cord was processed for histological and immunohistochemical analysis. Radiation-induced apoptosis was assessed by morphology and TdT-mediated dUTP nick end labeling combined with immunohistochemical markers for endothelial and glial cells. Image analysis was performed to determine endothelial cell and microvessel density using immunohistochemistry with endothelial markers, namely endothelial barrier antigen, glucose transporter isoform 1, laminin and zonula occludens 1. Blood–spinal cord barrier permeability was assessed using immunohistochemistry for albumin and ^99mTc-diethylenetriamine pentaacetic acid as a vascular tracer. Endothelial cell proliferation was assessed using in vivo BrdU labeling. During the first 24 h after irradiation, apoptotic endothelial cells were observed in the rat spinal cord. The decrease in endothelial cell density at 24 h after irradiation was associated with an increase in albumin immunostaining around microvessels. The decrease in the number of endothelial cells persisted for 7 days and recovery of endothelial density was apparent by day 14. A similar pattern of blood–spinal cord barrier disruption and recovery of permeability was observed over the 2 weeks, and an increase in BrdU-labeled endothelial cells was seen at day 3. These results are consistent with an association between endothelial cell death and acute blood–spinal cord barrier disruption in the rat spinal cord after irradiation.

Moore, A. H., Olschowka, J. A., Williams, J. P., Paige, S. L. and O'Banion, M. K. Radiation-Induced Edema is Dependent on Cyclooxygenase 2 Activity in Mouse Brain. Radiat. Res. 161, 153–160 (2004).

Cerebrovascular dysfunction, characterized by compromise of the blood–brain barrier and formation of cerebral edema, is common during the acute period after brain irradiation and may contribute to delayed pathology (e.g. vascular collapse, white matter necrosis) that leads to functional deficits. Another response of normal brain tissue to radiation is the induction of inflammatory markers, such as cytokine expression and glial activation. In particular, radiation-induced neuroinflammation is associated with an elevation in cyclooxygenase 2 (COX2), one of two isoforms of the obligate enzyme in prostanoid synthesis and the principal target of non-steroid anti-inflammatory drugs. Since prostanoids serve as autocrine and paracrine mediators in numerous physiological and pathological processes, including vasoregulation, we investigated COX2 protein expression and COX2-mediated prostanoid production in radiation-induced cerebral edema in male C57/BL6 mice. We found that radiation induces COX2 protein that is accompanied by specific increases in prostaglandin E₂ and thromboxane A₂ within 4 and 24 h after brain irradiation. Furthermore, we showed that treatment with NS-398, a selective COX2 inhibitor, attenuated prostanoid induction and edema formation. These results suggest that radiation-induced changes in vascular permeability are dependent on COX2 activity, implicating this enzyme and its products as targets for potential therapeutic treatment/protection from the effects of radiation on normal brain tissue.

Yonezawa, M., Horie, K., Kondo, H. and Kubo, K. Increase in Endogenous Spleen Colonies without Recovery of Blood Cell Counts in Radioadaptive Survival Response in C57BL/6 Mice. Radiat. Res. 161, 161–167 (2004).

The radioadaptive survival response induced by a conditioning exposure to 0.45 Gy and measured as an increase in 30-day survival after mid-lethal X irradiation was studied in C57BL/6N mice. The acquired radioresistance appeared on day 9 after the conditioning exposure, reached a maximum on days 12–14, and disappeared on day 21. The conditioning exposure 14 days prior to the challenge exposure increased the number of endogenous spleen colonies (CFU-S) on days 12–13 after the exposure to 5 Gy. On day 12 after irradiation, the conditioning exposure also increased the number of endogenous CFU-S to about five times that seen in animals exposed to 4.25–6.75 Gy without preirradiation. The effect of the interval between the preirradiation and the challenge irradiation on the increase in endogenous CFU-S was also examined. A significant increase in endogenous CFU-S was observed when the interval was 14 days, but not 9 days. This result corresponded to the increase in survival observed on day 14 after the challenge irradiation. Radiation-inducted resistance to radiation-induced lethality in mice appears to be closely related to the marked recovery of endogenous CFU-S in the surviving hematopoietic stem cells that acquired radioresistance by preirradiation. Preirradiation enhanced the recovery of the numbers of erythrocytes, leukocytes and thrombocytes very slightly in mice exposed to a sublethal dose of 5 Gy, a dose that does not cause bone marrow death. There appears to be no correlation between the marked increase in endogenous CFU-S and the slight increase or no increase in peripheral blood cells induced by the radioadaptive response. The possible contribution by some factor, such as Il4 or Il11, that has been reported to protect irradiated animals without stimulating hematopoiesis is discussed.

Ina, Y. and Sakai, K. Prolongation of Life Span Associated with Immunological Modification by Chronic Low-Dose-Rate Irradiation in MRL-lpr/lpr Mice. Radiat. Res. 161, 168–173 (2004).

Chronic low-dose-rate γ irradiation at 0.35 or 1.2 mGy/h prolonged the life span of MRL-lpr/lpr mice carrying a deletion in the apoptosis-regulating Fas gene that markedly shortens life due to severe autoimmune disease. Immunological modifications as indicated by a significant increase of CD8 T cells and a significant decrease of CD3 CD45R/B220 as well as CD45R/B220 CD40 cells were found in parallel with amelioration of total-body lymphadenopathy, splenomegaly, proteinuria, and kidney and brain syndromes.

Vorotnikova, E., Tries, M. and Braunhut, S. J. Retinoids and TIMP1 Prevent Radiation-Induced Apoptosis of Capillary Endothelial Cells. Radiat. Res. 161, 174–184 (2004).

Radiation-induced changes in capillaries constitute a basic injury in the pathogenesis of chronic radiation damage to the heart, lung, liver, kidney and brain. It is important to identify new radioprotectors for capillary endothelial cells for use during radiotherapy to minimize normal tissue damage and possibly to increase the deliverable dose. Previously we demonstrated that exposure to ionizing radiation (10 Gy) results in death of bovine adrenal capillary endothelial cells in confluent monolayers by apoptosis. We also showed that retinoids inhibit the growth of endothelial cells, induce their differentiation, down-regulate matrix metalloproteinase (MMP) production, and up-regulate tissue inhibitors of matrix metalloproteinases (TIMPs). In the present studies, we demonstrated that radiation (10 Gy) induced an immediate increase in the amounts and activation of MMP1 and MMP2 in the cell fraction and medium of bovine capillary endothelial cells followed by an incidence of apoptosis. We also obtained data indicating that radiation-induced apoptosis can be inhibited by exposing bovine capillary endothelial cells to all-trans-retinol or all-trans-retinoic acid for 6 days before irradiation, even when the vitamins were removed 24 h before irradiation. Finally, we determined that inhibition of MMPs by TIMP was sufficient to block radiation-induced apoptosis, suggesting that the mechanism of protection by retinoids is through the alteration of levels of MMPs and TIMPs produced by the cells.

Alsbeih, G., Torres, M., Al-Harbi, N. and Alsubael, M. Loss of Wild-Type Trp53 Protein in Mouse Fibroblasts Leads to Increased Radioresistance with Consequent Decrease in Repair of Potentially Lethal Damage. Radiat. Res. 161, 185–192 (2004).

It has been reported that the loss of function of Trp53 protein is associated with a reduction in the expression of radiation-induced potentially lethal damage (PLD). These studies, however, were carried out using either transformed or transfected cell lines, and other factors may have existed that could interfere with PLD repair. In this study, we used isogenic fibroblasts derived from Trp53 knockout mice to study radiation sensitivity, PLD repair, and repair of DNA double-strand breaks (DSBs). Experiments were carried out using wild-type (Trp53 ^/ ), heterozygous (Trp53 ^/−) and homozygous mutant (Trp53^−/−) cells. This is an ideal system because the only difference in the three cell strains is the status of the Trp53 protein. DSB repair was measured by pulsed-field-gel electrophoresis (PFGE), while radiosensitivity and PLD repair were studied using the clonogenic survival assay. Cells were irradiated in plateau phase and then trypsinized and plated either immediately or 24 h later to allow for PLD repair. The results of Western blot analyses showed that Trp53^−/− cells expressed a putative mutant form of Trp53 that was unable to transcriptionally activate Cdkn1a (p21) protein in response to irradiation. The Trp53^−/− cells were significantly more radioresistant than the Trp53 ^/ cells, and this was associated with a moderate reduction in PLD repair. DNA repair experiments showed no difference in DSB rejoining capability between the two cell lines. In conclusion, our results show that loss of wild-type Trp53 leads to increased radioresistance with consequent reduction in PLD repair but with no effect on DNA DSB repair.

Hook, G. J., Zhang, P., Lagroye, I., Li, L., Higashikubo, R., Moros, E. G., Straube, W. L., Pickard, W. F., Baty, J. D. and Roti Roti, J. L. Measurement of DNA Damage and Apoptosis in Molt-4 Cells after In Vitro Exposure to Radiofrequency Radiation. Radiat. Res. 161, 193–200 (2004).

To determine whether exposure to radiofrequency (RF) radiation can induce DNA damage or apoptosis, Molt-4 T lymphoblastoid cells were exposed with RF fields at frequencies and modulations of the type used by wireless communication devices. Four types of frequency/modulation forms were studied: 847.74 MHz code-division multiple-access (CDMA), 835.62 MHz frequency-division multiple-access (FDMA), 813.56 MHz iDEN® (iDEN), and 836.55 MHz time-division multiple-access (TDMA). Exponentially growing cells were exposed to RF radiation for periods up to 24 h using a radial transmission line (RTL) exposure system. The specific absorption rates used were 3.2 W/kg for CDMA and FDMA, 2.4 or 24 mW/kg for iDEN, and 2.6 or 26 mW/kg for TDMA. The temperature in the RTLs was maintained at 37°C ± 0.3°C. DNA damage was measured using the single-cell gel electrophoresis assay. The annexin V affinity assay was used to detect apoptosis. No statistically significant difference in the level of DNA damage or apoptosis was observed between sham-treated cells and cells exposed to RF radiation for any frequency, modulation or exposure time. Our results show that exposure of Molt-4 cells to CDMA, FDMA, iDEN or TDMA modulated RF radiation does not induce alterations in level of DNA damage or induce apoptosis.

Lagroye, I., Hook, G. J., Wettring, B. A., Baty, J. D., Moros, E. G., Straube, W. L. and Roti Roti, J. L. Measurements of Alkali-Labile DNA Damage and Protein–DNA Crosslinks after 2450 MHz Microwave and Low-Dose Gamma Irradiation In Vitro. Radiat. Res. 161, 201–214 (2004).

In vitro experiments were performed to determine whether 2450 MHz microwave radiation induces alkali-labile DNA damage and/or DNA–protein or DNA–DNA crosslinks in C3H 10T½ cells. After a 2-h exposure to either 2450 MHz continuous-wave (CW) microwaves at an SAR of 1.9 W/kg or 1 mM cisplatinum (CDDP, a positive control for DNA crosslinks), C3H 10T½ cells were irradiated with 4 Gy of γ rays (¹³⁷Cs). Immediately after γ irradiation, the single-cell gel electrophoresis assay was performed to detect DNA damage. For each exposure condition, one set of samples was treated with proteinase K (1 mg/ml) to remove any possible DNA–protein crosslinks. To measure DNA–protein crosslinks independent of DNA–DNA crosslinks, we quantified the proteins that were recovered with DNA after microwave exposure, using CDDP and γ irradiation, positive controls for DNA–protein crosslinks. Ionizing radiation (4 Gy) induced significant DNA damage. However, no DNA damage could be detected after exposure to 2450 MHz CW microwaves alone. The crosslinking agent CDDP significantly reduced both the comet length and the normalized comet moment in C3H 10T½ cells irradiated with 4 Gy γ rays. In contrast, 2450 MHz microwaves did not impede the DNA migration induced by γ rays. When control cells were treated with proteinase K, both parameters increased in the absence of any DNA damage. However, no additional effect of proteinase K was seen in samples exposed to 2450 MHz microwaves or in samples treated with the combination of microwaves and radiation. On the other hand, proteinase K treatment was ineffective in restoring any migration of the DNA in cells pretreated with CDDP and irradiated with γ rays. When DNA–protein crosslinks were specifically measured, we found no evidence for the induction of DNA–protein crosslinks or changes in amount of the protein associated with DNA by 2450 MHz CW microwave exposure. Thus 2-h exposures to 1.9 W/ kg of 2450 MHz CW microwaves did not induce measurable alkali-labile DNA damage or DNA–DNA or DNA–protein crosslinks.

Raaphorst, G. P., Maude-Leblanc, J. and Li, L. Evaluation of Recombination Repair Pathways in Thermal Radiosensitization. Radiat. Res. 161, 215–218 (2004).

Thermal radiosensitization has been shown to cause inhibition of repair of sublethal and potentially lethal damage and DNA DSBs. In this study we assessed thermal radiosensitization in mutants deficient in homologous recombinational (HR) repair and nonhomologous end joining repair (NHEJ). Using cells of the mouse wild-type embryo fibroblast cell line MEF and its Ku80^–/– derivative that is deficient in NHEJ, we showed that thermal radiosensitization is the same in both cell lines. Further studies with cells of the wild-type CHO-AA8 cell line and its derivative IRS_ISF, which is deficient in HR, also showed comparable thermal radiosensitization in both cell lines. Further experiments using cells of chicken DT40 cell lines also showed comparable thermal radiosensitization between the wild-type HR mutant Rad54, the NHEJ mutant Ku70, and the double mutant Rad 54-Ku70. These results indicate that the HR and NHEJ pathways may not be targets for thermal radiosensitization.

Staab, A., Zukowski, D., Walenta, S., Scholz, M. and Mueller-Klieser, W. Response of Chinese Hamster V79 Multicellular Spheroids Exposed to High-Energy Carbon Ions. Radiat. Res. 161, 219–227 (2004).

Chinese hamster V79-379A spheroids 200 ± 30 μm (± SD) in diameter were irradiated in agitated medium in different oxygen atmospheres with (1) 227 MeV/nucleon ¹²C ⁶ ions (plateau region) to model tissue in the entrance channel during therapy, (2) carbon ions in the extended Bragg peak modeling tissue in the target volume, or (3) X rays as a reference modality. Cell survival curves were similar for modes (1) and (3), indicating the absence of a contact effect and the presence of a pronounced oxygen effect with oxygen enhancement ratios (OERs) of 2.8 and 2.9, respectively. In contrast, the oxygen effect was substantially smaller in mode (2) with an OER of 1.4. Under normal or restricted oxygen supply conditions (external pO₂ = 145 or 0 mmHg), the relative biological effectiveness (RBE) was 2.1 or 4.3, respectively, for Bragg-peak irradiation. This modality induced apoptosis and a dose-dependent accumulation of cells in G₂/M phase even at pO₂ = 0 mmHg. The volume ratios of treated to untreated spheroids exhibited cyclic variations after heavy-particle treatment that were not directly attributable to cell cycle synchronization. In summary, the results suggest that carbon ions in the extended Bragg peak are more effective than conventional X rays, particularly under hypoxic conditions.

Goblirsch, M., Mathews, W., Lynch, C., Alaei, P., Gerbi, B. J., Mantyh, P. W. and Clohisy, D. R. Radiation Treatment Decreases Bone Cancer Pain, Osteolysis and Tumor Size. Radiat. Res. 161, 228–234 (2004).

Radiotherapy is the cornerstone of palliative treatment for primary bone cancer in animals and metastatic bone cancer in humans. However, the mechanism(s) responsible for pain relief after irradiation is unknown. To identify the mechanism through which radiation treatment decreases bone cancer pain, the effect of radiation on mice with painful bone cancer was studied. Analysis of the effects of a 20-Gy treatment on localized sites of painful bone cancers was performed through assessments of animal behavior, radiographs and histological analysis. The findings indicated that radiation treatment reduced bone pain and supported reduced cancer burden and reduced osteolysis as mechanisms through which radiation reduces bone cancer pain.

Liu, D., Wada, I., Tateno, H., Ogino, D., Suzuki, M., Li, L., Lu, W., Kojiro, M., Fukayama, M., Okabe, H. and Fukumoto, M. Allelotypic Characteristics of Thorotrast-Induced Intrahepatic Cholangiocarcinoma: Comparison to Liver Cancers not Associated with Thorotrast. Radiat. Res. 161, 235–243 (2004).

To elucidate the genetic alterations that are specific to Thorotrast-induced liver cancers and their possible roles in tumorigenesis, we analyzed loss of heterozygosity (LOH) at 37 loci. Our previous study of liver cancers that were not associated with Thorotrast found LOH at 9 of these loci to be characteristic of intrahepatic cholangiocarcinoma (ICC), at 19 to be characteristic of hepatocellular carcinoma (HCC), and at 9 to be common to both ICC and HCC. LOH analysis was also performed in tissues of cholangiolocellular carcinoma, which is thought to originate from a common stem cell progenitor of hepatocytes and bile duct epithelial cells. We found frequent LOH at D4S1538, D16S2624 and D17S1303 to be common to all the subtypes of liver cancers, independent of the specific carcinogenic agent. In contrast, LOH at D4S1652 generally was not observed in Thorotrast-induced ICC. LOH analysis revealed that Thorotrast-induced ICC shares some LOH features with both ICC and HCC that were not induced by Thorotrast; however, it is more similar to ICC than to HCC in terms of genetic changes. This study could narrow down the crucial chromosomal loci whose deletions are relevant to hepatobiliary carcinogenesis irrespective of the carcinogenic agent. The study of LOH at loci other the those crucial ones may help us understand how the phenotype of liver cancers is determined.