An understanding of proton radiobiology is critical for optimization of both proton radiotherapy and assessment of carcinogenesis risk from space radiation. Although the physical aspects of proton beam radiobiology is well understood, the biological aspects, particularly the complex biological end points, have been underexplored and underexploited. This review focuses on the biological responses observed to date, across various scales, molecular, cellular and especially tissue levels. Proton-induced perturbations of gene expression, along with signaling and functional alterations in cell cycle, invasion, angiogenesis and metastasis are included. Particular emphasis is placed on differences noted in the literature between biological effects induced by protons and those induced by high-energy photons. An appreciation of the unique physical and biological characteristics of proton radiobiology should augment current strategies both to enhance therapeutic effectiveness and to quantify risk related to proton irradiation.

Since 1957, broad proton beam radiotherapy with a spread out Bragg peak has been used for cancer treatment. More recently, studies on the use of proton therapy in the treatment of non-small cell lung cancer (NSCLC) were performed and although the benefit of using protons for the treatment of NSCLC is recognized, more work is needed to gather additional data for the understanding of cell response. Human A549 cell survival was evaluated by colony forming assay 11 days after 10 keV/μm proton beam irradiation at 0.1 and 1 Gy/min. The residual energy of the proton beam at the location of the irradiated cells was 3.9 MeV. In parallel, early effects on the cell viability and DNA damage were assessed and DNA synthesis was measured. The survival curve obtained was fitted with both the linear and the induced-repair models, as a hyper-radiosensitivity was evidenced at very low doses. Above 0.5 Gy, a linear shape was observed with the α parameter equal to 0.824 ± 0.029 Gy⁻¹. In addition, early cell death and cell proliferation arrest were enhanced. Moreover, a clear correlation between DNA damage and surviving fraction was observed. Finally, comparisons with X ray results indicate that proton irradiation at 10 keV/μm enhanced the tumor radiosensitivity with a significant dose-dependent decrease in the survival fraction. The RBE value of 1.9 ± 0.4 obtained for a 10% survival support this observation.

The use of radiation-inducible promoters to drive transgene expression offers the possibility of temporal and spatial regulation of gene activation. This study assessed the potential of one such promoter element, p21^WAF1/CIP1 (WAF1), to drive expression of the noradrenaline transporter (NAT) gene, which conveys sensitivity to radioiodinated meta-iodobenzylguanidine (MIBG). An expression vector containing NAT under the control of the radiation-inducible WAF1 promoter (pWAF/NAT) was produced. The non-NAT expressing cell lines UVW (glioma) and HCT116 (colorectal cancer) were transfected with this construct to assess radiation-controlled WAF1 activation of the NAT gene. Transfection of UVW and HCT cells with pWAF/NAT conferred upon them the ability to accumulate [¹³¹I]MIBG, which led to increased sensitivity to the radiopharmaceutical. Pretreatment of transfected cells with γ radiation or the radiopharmaceuticals [¹²³I]MIBG or [¹³¹I]MIBG induced dose- and time-dependent increases in subsequent [¹³¹I]MIBG uptake and led to enhanced efficacy of [¹³¹I]MIBG-mediated cell kill. Gene therapy using WAF1-driven expression of NAT has the potential to expand the use of this therapeutic modality to tumors that lack a radio-targetable feature.

Restriction Landmark Genome Scanning (RLGS) is a method that uses end-labeled ³²P NotI sites that are mostly associated with coding genes to visualizes thousands of DNA fragments as spots in two-dimensional autoradiograms. This approach allows direct detection of autosomal deletions as spots with half normal intensity. The method was applied to mouse offspring derived from spermatogonia exposed to 4 Gy of X rays. A genome-wide assessment of the mutation induction rate was estimated from the detected deletions. Examinations were made of 1,007 progeny (502 derived from control males and 505 from irradiated males) and 1,190 paternal and 1,240 maternal spots for each mouse. The results showed one deletion mutation in the unirradiated paternal genomes of 502 offspring (0.2%) and 5 deletions in the irradiated paternal genomes of 505 offspring (1%). The difference was marginally significant, with the deletion sizes ranged from 2–13 Mb. If the frequencies are taken at face value, the net increase was 0.8% after an exposure of 4 Gy, or 0.2% per Gy per individual if a linear dose response is assumed. Since the present RLGS analysis examined 1,190 NotI sites, while the mouse genome contains ∼25,000 genes, the genomic probability of any gene undergoing a deletion mutation would be 25× 0.2%, or 5% per Gy. Furthermore, since the present RLGS screened about 0.2% of the total genome, the probability of detecting a deletion anywhere in the total genome would be estimated to be 500 times 0.2% or 100% (i.e., 1 deletion per Gy). These results are discussed with reference to copy number variation in the human genome.

Our earlier studies indicated that ionizing radiation (IR) induces NF-κB-dependent clonal expansion of therapy resistant tumor cells. Herein, we investigated whether mitigation of NF-κB-dependent telomerase activation by EGFR tyrosine kinase inhibitor can enhance IR-induced celling killing. SCC-4 and SCC-9 cells exposed to IR with or without Pelitinib were examined for NF-κB and hTERT transcription using luciferase reporter assays. NF-κB-dependent hTERT transcription was confirmed by either muting NF-κB or by using hTERT constructs lacking NF-κB binding sites. hTERT, mRNA, telomerase activity and cell survival of tumor cells were analyzed using QPCR, TRAP and clonogenic assay, respectively. Pelitinib inhibited IR-induced NF-κB, telomerase activity and hTERT transactivation. Ionizing radiation-induced telomerase activity is regulated at the transcriptional level by triggering TERT promoter activation. Functional NF-κB mediates telomerase activity by binding to the κB binding region in the promoter region of TERT. Elimination of the NF-κB recognition site on telomerase or muting NF-κB compromises IR-induced telomerase promoter activation. We found that Pelitinib inhibited IR-induced TERT transcription, transactivation and telomerase activation in IR-exposed and NF-κB-overexpressed cells. Furthermore, Pelitinib potentiates IR-induced cell killing. Our results strongly suggest that IR-induced NF-κB-mediated cell survival is supported by telomerase activation. We propose that if this pathway can be inhibited with Pelitinib treatment, one could further enhance therapeutic outcome in squamous cell carcinoma.

Radicals generated in trehalose single crystals by X radiation at room temperature were investigated by electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR) and ENDOR-induced EPR measurements, together with periodic density functional theory calculations. In the first days after irradiation, three radical species (I1, I2 and I3) were detected, two of which (I1 and I2) dominate the EPR spectrum and could be identified as H-abstracted species centered at C3′ (I1) and C2 (I2), the latter with additional formation of a carbonyl group at C3. Annealing the sample at 40°C for 3 days or storing it in ambient conditions for three months resulted in another, more stable EPR spectrum. Two major species could be characterized in this stage (S1 and S2), only one of which was tentatively identified as an H-abstracted, C2-centered species (S1). Our findings disagree with a previous EPR study [Gräslund and Löfroth (23)] on several accounts. This work stresses the need for caution when interpreting composite EPR spectra and thermally induced spectral changes of radiation-induced species, even in these relatively simple carbohydrates. It also provides further evidence that the pathways for radiation damage critically depend on the specific conformation of a molecule and its environment, but also that carbonyl group formation is a common process in the radiation chemistry of sugars and related compounds.

The synergistic interaction of cisplatin with ionizing radiation is the clinical rationale for the treatment of several cancers including head and neck, cervical and lung cancer. The underlying molecular mechanism of the synergy has not yet been identified, although both DNA damage and repair processes are likely involved. Here, we investigate the indirect effect of γ rays on strand break formation in a supercoiled plasmid DNA (pGEM–3Zf-) covalently modified by cisplatin. The yields of single- and double-strand breaks were determined by irradiation of DNA and cisplatin/DNA samples with ⁶⁰Co γ rays under four different scavenging conditions to examine the involvement of hydrated electrons and hydroxyl radicals in inducing the DNA damage. At 5 mM tris in an N₂ atmosphere, the presence of an average of two cisplatins per plasmid increased the yields of single- and double-strand breaks by factors of 1.9 and 2.2, respectively, relative to the irradiated unmodified DNA samples. Given that each plasmid of 3,200 base pairs contained an average of two cisplatins, this represents an increase in radiosensitivity of 3,200-fold on a per base pair basis. When hydrated electrons were scavenged by saturating the samples with N₂O, these enhancement factors decreased to 1.5 and 1.2, respectively, for single- and double-strand breaks. When hydroxyl radicals were scavenged using 200 mM tris, the respective enhancement factors were 1.2 and 1.6 for single- and double-strand breaks, respectively. Furthermore, no enhancement in DNA damage by cisplatin was observed after scavenging both hydroxyl radicals and hydrated electrons. These findings show that hydrated electrons can induce both single- and double-strand breaks in the platinated DNA, but not in unmodified DNA. In addition, cisplatin modification is clearly an extremely efficient means of increasing the formation of both single- and double-strand breaks by the hydrated electrons and hydroxyl radicals created by ionizing radiation.

Workers at the Mayak nuclear facility in the Russian Federation offer a unique opportunity to evaluate health risks from exposure to inhaled plutonium. Risks of mortality from lung cancer, the most serious carcinogenic effect of plutonium, were evaluated in 14,621 Mayak workers who were hired in the period from 1948–1982, followed for at least 5 years, and either monitored for plutonium or never worked with plutonium. Over the follow-up period from 1953–2008, there were 486 deaths from lung cancer, 446 of them in men. In analyses that were adjusted for external radiation dose and smoking, the plutonium excess relative risk (ERR) per Gy declined with attained age and was higher for females than for males. The ERR per Gy for males at age 60 was 7.4 (95% CI: 5.0–11) while that for females was 24 (95% CI: 11–56). When analyses were restricted to plutonium doses <0.2 Gy, the ERR per Gy for males at age 60 was similar: 7.0 (95% CI: 2.5–13). Of the 486 lung cancer deaths, 105 (22%) were attributed to plutonium exposure and 29 (6%) to external exposure. Analyses of the 12,708 workers with information on smoking indicated that the relationship of plutonium exposure and smoking was likely sub-multiplicative (P = 0.011) and strongly indicated that it was super-additive (P < 0.001). Although extensive efforts have been made to improve plutonium dose estimates in this cohort, they are nevertheless subject to large uncertainties. Large bioassay measurement errors alone are likely to have resulted in serious underestimation of risks, whereas other sources of uncertainty may have biased results in ways that are difficult to predict.

Tumor hypoxia impedes the outcome of radiotherapy. As the extent of hypoxia in solid tumors varies during the course of radiotherapy, methods that can provide repeated assessment of tumor pO₂ such as EPR oximetry may enhance the efficacy of radiotherapy by scheduling irradiations when the tumors are oxygenated. The repeated measurements of tumor pO₂ may also identify responders, and thereby facilitate the design of better treatment plans for nonresponding tumors. We have investigated the temporal changes in the ectopic 9L and C6 glioma pO₂ irradiated with single radiation doses less than 10 Gy by EPR oximetry. The 9L and C6 tumors were hypoxic with pO₂ of approximately 5–9 mmHg. The pO₂ of C6 tumors increased significantly with irradiation of 4.8–9.3 Gy. However, no change in the 9L tumor pO₂ was observed. The irradiation of the oxygenated C6 tumors with a second dose of 4.8 Gy resulted in a significant delay in growth compared to hypoxic and 2 Gy × 5 treatment groups. The C6 tumors with an increase in pO₂ of greater than 50% from the baseline of irradiation with 4.8 Gy (responders) had a significant tumor growth delay compared to nonresponders. These results indicate that the ectopic 9L and C6 tumors responded differently to radiotherapy. We propose that the repeated measurement of the oxygen levels in the tumors during radiotherapy can be used to identify responders and to design tumor oxygen guided treatment plans to improve the outcome.

Many epidemiologic studies have shown that the exposure to high external radiation doses increases thyroid neoplastic frequency, especially when given during childhood or adolescence. The use of radioprotective drugs may decrease the damage caused by radiation therapy and therefore could be useful to prevent the development of thyroid tumors. The aim of this study was to investigate the possible application of 6-propyl-2-thiouracil (PTU) as a radioprotector in the thyroid gland. Rat thyroid epithelial cells (FRTL-5) were exposed to different doses of γ irradiation with or without the addition of PTU, methimazole (MMI), reduced glutathione (GSH) and perchlorate (KClO₄). Radiation response was analyzed by clonogenic survival assay. Cyclic AMP (cAMP) levels were measured by radioimmunoassay (RIA). Apoptosis was quantified by nuclear cell morphology and caspase 3 activity assays. Intracellular reactive oxygen species (ROS) levels were measured using the fluorescent dye 2′,7′-dichlorofluorescein-diacetate. Catalase, superoxide dismutase and glutathione peroxidase activities were also determined. Pretreatment with PTU, MMI and GSH prior to irradiation significantly increased the surviving cell fraction (SF) at 2 Gy (P < 0.05), while no effect was observed with KClO₄. An increase in extracellular levels of cAMP was found only in PTU treated cells in a dose and time-dependent manner. Cells incubated with agents that stimulate cAMP (forskolin and dibutyril cAMP) mimicked the effect of PTU on SF. Moreover, pretreatment with the inhibitor of protein kinase A, H-89, abolished the radioprotective effect of PTU. PTU treatment diminished radiation-induced apoptosis and protected cells against radiation-induced ROS elevation and suppression of the antioxidant enzyme's activity. PTU was found to radioprotect normal thyroid cells through cAMP elevation and reduction in both apoptosis and radiation-induced oxidative stress damage.

A marked increase in leukemia risks was the first and most striking late effect of radiation exposure seen among the Hiroshima and Nagasaki atomic bomb survivors. This article presents analyses of radiation effects on leukemia, lymphoma and multiple myeloma incidence in the Life Span Study cohort of atomic bomb survivors updated 14 years since the last comprehensive report on these malignancies. These analyses make use of tumor- and leukemia-registry based incidence data on 113,011 cohort members with 3.6 million person-years of follow-up from late 1950 through the end of 2001. In addition to a detailed analysis of the excess risk for all leukemias other than chronic lymphocytic leukemia or adult T-cell leukemia (neither of which appear to be radiation-related), we present results for the major hematopoietic malignancy types: acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, adult T-cell leukemia, Hodgkin and non-Hodgkin lymphoma and multiple myeloma. Poisson regression methods were used to characterize the shape of the radiation dose-response relationship and, to the extent the data allowed, to investigate variation in the excess risks with gender, attained age, exposure age and time since exposure. In contrast to the previous report that focused on describing excess absolute rates, we considered both excess absolute rate (EAR) and excess relative risk (ERR) models and found that ERR models can often provide equivalent and sometimes more parsimonious descriptions of the excess risk than EAR models. The leukemia results indicated that there was a nonlinear dose response for leukemias other than chronic lymphocytic leukemia or adult T-cell leukemia, which varied markedly with time and age at exposure, with much of the evidence for this nonlinearity arising from the acute myeloid leukemia risks. Although the leukemia excess risks generally declined with attained age or time since exposure, there was evidence that the radiation-associated excess leukemia risks, especially for acute myeloid leukemia, had persisted throughout the follow-up period out to 55 years after the bombings. As in earlier analyses, there was a weak suggestion of a radiation dose response for non-Hodgkin lymphoma among men, with no indication of such an effect among women. There was no evidence of radiation-associated excess risks for either Hodgkin lymphoma or multiple myeloma.