Gaillard, S., Pusset, D., de Toledo, S. M., Fromm, M. and Azzam, E. I. Propagation Distance of the α-Particle-Induced Bystander Effect: The Role of Nuclear Traversal and Gap Junction Communication. Radiat. Res. 171, 513–520 (2009).

When cell populations are exposed to low-dose α-particle radiation, a significant fraction of the cells will not be traversed by a radiation track. However, stressful effects occur in both irradiated and bystander cells in the population. Characterizing these effects, and investigating their underlying mechanism(s), is critical to understanding human health risks associated with exposure to α particles. To this end, confluent normal human fibroblast cultures were grown on polyethylene terephthalate foil grafted to an ultrathin solid-state nuclear track detector and exposed under non-perturbing conditions to low-fluence α particles from a broadbeam irradiator. Irradiated and affected bystander cells were localized with micrometer precision. The stress-responsive protein p21^Waf1 (also known as CDKN1A) was induced in bystander cells within a 100-μm radius from an irradiated cell. The mean propagation distance ranged from 20 to 40 μm around the intranuclear α-particle impact point, which corresponds to a set of ∼30 cells. Nuclear traversal, induced DNA damage, and gap junction communication were critical contributors to propagation of this stressful effect. The strategy described here may be ideal to investigate the size of radiation-affected target and the relative contribution of different cellular organelles to bystander effects induced by energetic particles, which is relevant to radioprotection and cancer radiotherapy.

Howe, O., O'Sullivan, J., Nolan, B., Vaughan, J., Gorman,S., Clarke, C., McClean, B., and Lyng, F. M. Do Radiation-Induced Bystander Effects Correlate to the Intrinsic Radiosensitivity of Individuals and Have Clinical Significance? Radiat. Res. 171, 521-529 (2009).

It is well known that patients can vary in their normal tissue response to radiotherapy, and this can be problematic. As a result, radiobiologists have been using in vitro models to assess variation in response and elucidate the genetic determinants of this variation. However, the clinical relevance of these models is currently unknown. In this study, blood samples from healthy controls (n  =  20) and colorectal carcinoma patients (n  =  60) were cultured in vitro to assess two radiobiological end points in parallel: intrinsic radiosensitivity assayed by chromosomal aberrations (G₂ scores) and radiation-induced bystander effects assayed by viability testing. Increased intrinsic radiosensitivity was observed in colorectal carcinoma donors (55%) compared to the healthy donors (5%) (P < 0.005). Similarly, more pronounced radiation-induced bystander effects were observed in the colorectal carcinoma donors compared to the healthy donors after 24 h exposure but not after 96 h exposure to donor irradiated cell conditioned medium (ICCM) (P < 0.05). All scores were tested for correlation with the age, sex and clinical stage of the colorectal carcinoma patients. The only statistically significant correlation was found in samples from severe Dukes D patients (P < 0.005), which had low/radioresistant G₂ scores. No correlation was found between radiation-induced intrinsic sensitivity and bystander effects, which suggests that they may have separate underlying molecular mechanisms, but they both show clinical relevance in individual patient samples.

Fournier, C., Barberet, P., Pouthier, T., Ritter, S., Fischer, B., Voss, K. O., Funayama, T., Hamada, N., Kobayashi, Y. and Taucher-Scholz, G. No Evidence for DNA and Early Cytogenetic Damage in Bystander Cells after Heavy-Ion Microirradiation at Two Facilities. Radiat. Res. 171, 530–540 (2009).

The occurrence of bystander effects has challenged the evaluation of risk for heavy ions, mainly in the context of space exploration and the increasing application of carbon ions in radiotherapy. In the present study, we addressed whether heavy-ion-induced DNA and cytogenetic damage is detectable in bystander cells. The formation of γ-H2AX foci, sister chromatid exchanges and micronuclei were used as markers of damage to DNA. Normal human fibroblasts were exposed to low fluences of carbon and uranium ions, and alternatively single cells were targeted with heavy ions using the GSI microbeam. We did not observe a significant increase in the bystander formation of γ-H2AX foci, sister chromatid exchanges or micronuclei. In addition, we performed for the first time parallel experiments at two microbeam facilities (GSI, JAEA) using the same cell line, culture conditions and irradiation protocols. No significant enhancement of the micronucleus frequencies in bystander cells was detected after targeted carbon-ion irradiation, confirming the results. Details regarding the history, culture conditions or support of the cells might be affecting the detection of bystander effects. On the other hand, the potential X-ray- and heavy-ion-induced bystander effects investigated herein clearly do not exceed the experimental error and thus are either lacking or are less pronounced than the effects reported in the literature for similar end points after α-particle and X-ray exposure.

Vaurijoux, A., Gruel, G., Pouzoulet, F., Grégoire, E., Martin, C., Roch-Lefèvre, S., Voisin, P., Voisin, P. and Roy, L. Strategy for Population Triage Based on Dicentric Analysis. Radiat. Res. 171, 541–548 (2009).

After large-scale accidental overexposure to ionizing radiation, a rapid triage of the exposed population can be performed by scoring dicentrics and ring chromosomes among 50 metaphases. This is rapid but is not accurate because the sensitivity is around 0.5 Gy. After the triage step, dose can be estimated by scoring 500 metaphases. This is lengthy but very accurate because the sensitivity is between 0.1 and 0.2 Gy. To improve the methodology, we propose the use of software for automatic dicentric scoring that was tested on victims of an accident in Dakar. Manual scoring of 50 metaphases was carried out, then manual scoring of 500 metaphases, and automatic scoring. Comparison between the dose classifications obtained with manual scoring on 50 metaphases and 500 metaphases showed 50% misclassification with the manual scoring on 50 metaphases. Comparison between the dose classifications obtained with the automatic scoring and manual scoring on 500 metaphases showed only 4.35% misclassification with the automatic scoring. The automatic scoring method is more accurate than the manual scoring on 50 metaphases and can therefore be used for triage, and in place of the manual scoring on 500 metaphases method for individual dose estimation, because it is as accurate and much faster.

Ivey, R. G., Subramanian, O., Lorentzen, T. D., and Paulovich, A. G. Antibody-Based Screen for Ionizing Radiation-Dependent Changes in the Mammalian Proteome for Use in Biodosimetry. Radiat. Res. 171, 549-561 (2009).

In an effort to identify proteomic changes that may be useful for radiation biodosimetry, human cells of hematological origin were treated with ionizing radiation or mock-irradiated and then harvested at different times after treatment. Protein lysates were generated from these cells and evaluated by Western blotting using a panel of 301 commercially available antibodies targeting 161 unique proteins. From this screen, we identified 55 ionizing radiation-responsive proteins, including 14 proteins not previously reported to be radiation-responsive at the protein level. The data from this large-scale screen have been assembled into a public website ( http://labs.fhcrc.org/paulovich/biodose_index.html) that may be of value to the radiation community both as a source of putative biomarkers for biodosimetry and also as a source of validation data on commercially available antibodies that detect radiation-responsive proteins. Using a panel of candidate radiation biomarkers in human cell lines, we demonstrate the feasibility of assembling a complementary panel of radiation-responsive proteins. Furthermore, we demonstrate the feasibility of using blood cell-based proteomic changes for biodosimetry by demonstrating detection of protein changes in circulating cells after total-body irradiation in a canine model.

Adamsen, B. L., Kravik, L. and De Angelis, P. M. Cellular Response to Chemoradiotherapy, Radiotherapy and Chemotherapy in Two Colorectal Cancer Cell Lines. Radiat. Res. 171, 562–571 (2009).

The cellular response to chemoradiotherapy was investigated in cells of the HCT116 (wild-type TP53) and HT29 (mutated TP53) human colorectal cancer cell lines to better understand how the chemotherapeutic agent 5-fluorouracil (5-FU) acts as a radiosensitizer in vitro and how it contributes to the well-documented greater efficacy of chemoradiotherapy compared to radiotherapy (or chemotherapy) alone. A bolus 5-FU treatment protocol that simulated actual clinical clearance kinetics was used with a radiation dose given within 90 min after drug addition. The involvements of key signaling pathways (DNA damage response, cell cycle progression, cell proliferation, cell death) in cell responses were investigated concurrently, allowing for direct correlations of numerous treatment response phenotypes. Early DNA damage response, substantial cell death, loss of clonogenicity, and senescence characterized both radiotherapy- and chemoradiotherapy-treated cultures but not chemotherapy-treated cultures. The largest G₂/M arrests and strongest correlation of senescence with non-clonogenicity were seen in radiotherapy- and chemoradiotherapy-treated HCT116 cell cultures, suggesting that functional TP53 could play a role in maintaining/inducing these cellular phenotypes. Overall, chemoradiotherapy proved to be the most effective treatment modality since it resulted in the strongest growth inhibitions, largest G₂/M arrests, largest fractions of senescent cells, and complete loss of clonogenicity in both cell lines.

Singh, S., Cowen, R. L., Chinje, E. C. and Stratford, I. J. The Impact of Intracellular Generation of Nitric Oxide on the Radiation Response of Human Tumor Cells. Radiat. Res. 171, 572–580 (2009).

Nitric oxide (NO) is a potent radiosensitizer of hypoxic mammalian cells. There have been many reports demonstrating radiosensitization in vitro and in vivo by the use of NO donors to generate NO by chemical means or by producing agents that mimic the free radical mechanism(s) of NO for potentiating radiosensitivity. However, much of this work has been done without taking account of the endogenous NO that is generated in tumor cells by NO synthase (NOS) in vitro or in tumor cells and host cells in solid tumors in vivo. To evaluate the contribution of intracellular generated NO to cellular radiosensitivity, we exposed human HT1080 and MDA231 tumor cells to a cytokine cocktail that results in an increase in cellular NOS expression to a level that is seen in many human solid tumors. We also carried out parallel studies to determine the radiosensitivity of HT1080 and MDA231 cells engineered to constitutively overexpress the iNOS gene. When cells are treated with cytokines under anoxic conditions (<0.01% O₂), there is up to a 9–15-fold increase in NOS expression, but no detectable NO is generated (since O₂ is required for the generation of NO via the NOS-mediated conversion of arginine to citrulline). As a consequence, when these cells are irradiated under hypoxic conditions, no radiosensitization is observed. However, as the oxygen tension was increased, the amount of NO generated also increased, and we show that this NO then contributes to an overall increase in the radiosensitivity of cells. For example, at 1% O₂ in control HT1080 cells, with little measurable NOS activity, the dose of radiation required to reduce survival by 90% was 6 Gy compared to 10 Gy in anoxic conditions. After cytokine treatment, the level of NO generated at 1% O₂ was significantly increased and the dose of radiation needed for 90% cell killing was reduced further to 4 Gy. The presence of the NOS inhibitor N^G-methyl-l-arginine (NMLA) shortly before and during irradiation ablated this increase in radiosensitivity, confirming that the effect was due to the generation of NO. We conclude that cytokine-mediated up-regulation of the NOS expression in tumor cells can produce sufficient NO to significantly increase the cytotoxic effect of radiation and that this is particularly apparent at intermediate oxygen concentrations.

Cloos, C. R., Daniels, D. H., Kalen, A., Matthews, K., Du, J., Goswami, P. C. and Cullen, J. J. Mitochondrial DNA Depletion Induces Radioresistance by Suppressing G₂ Checkpoint Activation in Human Pancreatic Cancer Cells. Radiat. Res. 171, 581–587 (2009).

We hypothesized that mitochondrial function regulates cell cycle checkpoint activation and radiosensitivity. Human pancreatic tumor cells (MiaPaCa-2, rho ) were depleted of mitochondrial DNA (rho°) by culturing cells in the presence of ethidium bromide. Depletion of mitochondrial DNA was verified by PCR amplification of total DNA using primer pairs specific for mitochondrial DNA. Loss of mitochondrial DNA decreased plating efficiency and the percentage of cells in S phase. Exponential cultures were irradiated with 2, 4 and 6 Gy (dose rate: 0.83 Gy/min) of ionizing radiation and harvested for determination of cell viability, growth and cell cycle phase distributions. Rho° cells were radioresistant compared to rho cells, with a dose-modifying factor (DMF) of 1.6. Although cell growth was significantly inhibited in irradiated rho cells compared to unirradiated control cells, the inhibition in Rho° cells was minimal. In addition, mitochondrial DNA depletion suppressed radiation-induced G₂ checkpoint activation, which was accompanied by increases in both cyclin B1 and CDK1. These results suggest that mitochondrial function may regulate cell cycle checkpoint activation and radiosensitivity in pancreatic cancer cells.

Epperly, M. W., Melendez, J. A., Zhang, X., Nie, S, Pearce, L., Peterson, J., Franicola, D., Dixon, T., Greenberger, B. A., Komanduri, P., Wang, H. and Greenberger, J. S. Mitochondrial Targeting of a Catalase Transgene Product by Plasmid Liposomes Increases Radioresistance In Vitro and In Vivo. Radiat. Res. 171, 588-595 (2009).

To determine whether increased mitochondrially localized catalase was radioprotective, a human catalase transgene was cloned into a small pSVZeo plasmid and localized to the mitochondria of 32D cl 3 cells by adding the mitochondrial localization sequence of MnSOD (mt-catalase). The cell lines 32D-Cat and 32D-mt-Cat had increased catalase biochemical activity as confirmed by Western blot analysis compared to the 32D cl 3 parent cells. The MnSOD-overexpressing 32D cl 3 cell line, 2C6, had decreased baseline catalase activity that was increased in 2C6-Cat and 2C6-mt-Cat subclonal cell lines. 32D-mt-Cat cells were more radioresistant than 32D-Cat cells, but both were radioresistant relative to 32D cl 3 cells. 2C6-mt-Cat cells but not 2C6-Cat cells were radioresistant compared to 2C6 cells. Intratracheal injection of the mt-catalase-plasmid liposome complex (mt-Cat-PL) but not the catalase-plasmid liposome complex (Cat-PL) increased the resistance of C57BL/6NHsd female mice to 20 Gy thoracic irradiation compared to MnSOD-plasmid liposomes. Thus mitochondrially targeted overexpression of the catalase transgene is radioprotective in vitro and in vivo.

Berbée, M., Fu, Q., Boerma, M., Wang, J., Kumar, K. S. and Hauer-Jensen, M. γ-Tocotrienol Ameliorates Intestinal Radiation Injury and Reduces Vascular Oxidative Stress after Total-Body Irradiation by an HMG-CoA Reductase-Dependent Mechanism. Radiat. Res. 171, 596–605 (2009).

Analogs of vitamin E (tocols) are under development as radioprophylactic agents because of their high efficacy and lack of toxicity. Gamma-tocotrienol (GT3) is of particular interest because, in addition to being an antioxidant, it also inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and accumulates to greater extent in endothelial cells than other tocols. We addressed in vivo whether HMG-CoA reductase inhibition contributes to the radioprotection conferred by GT3. Groups of mice were treated with vehicle, mevalonate (the product of the reaction catalyzed by HMG-CoA reductase), GT3 alone or GT3 in combination with mevalonate. Lethality and standard parameters of injury to the hematopoietic, intestinal and vascular/endothelial systems were assessed after exposure to total-body irradiation. GT3 improved postirradiation survival and decreased radiation-induced vascular oxidative stress, an effect that was reversible by mevalonate. GT3 also enhanced hematopoietic recovery, reduced intestinal radiation injury, and accelerated the recovery of soluble markers of endothelial function. These parameters were not reversed by mevalonate co-administration. Our data confirm GT3's radioprophylactic properties against hematopoietic injury and, for the first time, demonstrate benefits in terms of protection against gastrointestinal and vascular injury. The radioprotective efficacy of GT3 against vascular injury is related to its properties as an HMG-CoA reductase inhibitor.

Fenton, B. M. and Paoni, S. F. Alterations in Daily Sequencing of Axitinib and Fractionated Radiotherapy do not Affect Tumor Growth Inhibition or Pathophysiological Response. Radiat. Res. 171, 606–614 (2009).

A variety of antiangiogenic strategies have proven effective in preclinical tumor models, either as single agents or in combination with radiation. Clinical gains have been relatively modest, however, and questions remain regarding optimal scheduling. The objectives of the current work were to evaluate whether the sequencing of acute treatment critically affects tumor pathophysiological and therapeutic response. Axitinib (Pfizer Global Research & Development), an inhibitor that predominantly targets vascular endothelial growth factor receptors, was administered either before or after each daily radiation fraction in two human prostate xenograft tumor models. Tumors were frozen at sequential times to monitor changes in (1) vascular spacing, (2) pericyte and basement membrane coverage, and (3) hypoxia. Although similar reductions in blood vessel counts were observed with each tumor model, tumor vasculature was not functionally normalized. Instead, tumor hypoxia increased, accompanied by a progressive dissociation of pericytes and basement membranes. Ultimately, tumor growth inhibition was found to be equivalent for each of the combination schedules. These studies illustrate a clear advantage to combining axitinib with fractionated therapy but argue against an acute radiosensitization or radioprotection of either the tumor cells or tumor vasculature. Instead, post- and preirradiation daily drug administration serve equally well in supplementing the response to radiotherapy.

McQuade, J. M., Merritt, J. H., Miller, S. A., Scholin, T., Cook, M. C., Salazar, A., Rahimi, O. B., Murphy, M. R. and Mason, P. A. Radiofrequency-Radiation Exposure Does Not Induce Detectable Leakage of Albumin Across the Blood-Brain Barrier. Radiat. Res. 171, 615–621 (2009).

The blood-brain barrier (BBB) consists of tight junctions between the endothelial cells that line the capillaries in the central nervous system. This structure protects the brain, and neurological damage could occur if it is compromised. Several publications by researchers at Lund University have reported alterations in the BBB after exposure to low-power 915 MHz energy. These publications increased the level of concern regarding the safety of wireless communication devices such as mobile phones. We performed a confirmation study designed to determine whether the BBB is altered in rats exposed in a transverse electromagnetic (TEM) transmission line cell to 915 MHz energy at parameters similar to those in the Lund University studies. Unanesthetized rats were exposed for 30 min to either continuous-wave or modulated (16 or 217 Hz) 915 MHz energy at power levels resulting in whole-body specific absorption rates (SARs) of 0.0018–20 W/kg. Albumin immunohistochemistry was performed on perfused brain tissue sections to determine the integrity of the BBB. Chi-square analysis revealed no significant increase in albumin extravasation in any of the exposed animals compared to the sham-exposed or home cage control animals.

Sid Ahmed-Adrar, N., Collin, F., Couturier, M., Vitrac, H., Bonnefont-Rousselot, D., Jore, D. and Gardès-Albert, M. Radiolytic Yield of Cardiolipin Peroxidation by Gamma Rays in Large Unilamellar Vesicles of Phosphatidylcholine. Radiat. Res. 171, 622–630 (2009).

Large unilamellar vesicles of 1-hexanoyl-2-(9Z-12Z-octadecadienoyl)-sn-glycero-3-phosphocholine (PLPC) have been used as model membrane to investigate the effect of increasing amount of cardiolipin (1′,3′-bis-[1,2-Di-(9Z-12Z-octadecadienoyl)-sn-glycero-3-phospho]-sn-glycerol, CL) on the peroxidizability of the lipid phase. Hydroxyl radicals generated by gamma radiolysis of water initiated the lipid peroxidation. Both peroxidation products (conjugated dienes and hydroperoxides of PLPC, mono- and dihydroperoxides of CL) and disappearance of CL and PLPC were assessed as a function of the radiation dose (25 to 400 Gy, I = 10 Gy min⁻¹). Our results show that the addition of 5% to 15% CL to large unilamellar vesicles (concentration ratio) produces almost complete inhibition of PLPC peroxidation. Thus, for 15% CL (known to be the proportion of CL in the inner mitochondrial membrane), the radiolytic yield of formation of PLPC hydroperoxides is reduced to zero, whereas it is equal to (3.1 ± 0.2) × 10⁻⁷ mol J⁻¹ for CL hydroperoxides, showing the importance of the targeted CL. For this concentration ratio (CL/ PLPC 15%), we have established the balance equation between the consumption of CL [G(–CL) = (2.8 ± 0.1) × 10⁻⁷ mol J⁻¹] and the formation of CL hydroperoxides [G(CLOOH_T) = (3.1 ± 0.2) × 10⁻⁷ mol J⁻¹]. In addition, the radiolytic yields of disappearance of PLPC and CL have been determined [(1.5 ± 0.1) × 10⁻⁷ mol J⁻¹ and (2.8 ± 0.1) × 10⁻⁷ mol J⁻¹, respectively], their sum [(4.3 ± 0.2) × 10⁻⁷ mol J⁻¹] being higher than G_HO· (2.8 × 10⁻⁷ mol J⁻¹). However, there is no balance between the radiolytic yield of formation of PLPC hydroperoxides [G (PCOOH_T) ∼ 0] and the yield of disappearance of PLPC [(1.5 ± 0.1) × 10⁻⁷ mol J⁻¹], likely because lipid fragments (not measured in this work) could be generated from HO^· reaction on the polar head of PLPC. These results have been interpreted by assuming that the hydroxyl radicals attack in competition both lipid targets, i.e. PLPC and CL, with a higher sensitivity to CL oxidation. It can be concluded that a little amount of CL (10–15% CL/ PLPC concentration ratio) may exert a strong protective effect against the HO^·-induced peroxidation of PLPC.