In the event of an improvised nuclear device or “dirty bomb” in a highly populated area, potentially hundreds of thousands of people will require screening to ensure that exposed individuals receive appropriate treatment. For this reason, there is a need to develop tools for high-throughput radiation biodosimetry. Gene expression represents an emerging approach to biodosimetry and could potentially provide an estimate of both absorbed dose and individual radiation-induced injury. Since approximately 2–4% of humans are thought to be radiosensitive, and would suffer greater radiological injury at a given dose than members of the general population, it is of interest to explore the potential impact of such sensitivity on the biodosimetric gene expression signatures being developed. In this study, we used wild-type mice and genetically engineered mouse models deficient in two DNA repair pathways that can contribute to radiation sensitivity to estimate the maximum effect of differences in radiosensitivity. We compared gene expression in response to a roughly equitoxic (LD_50/30) dose of gamma rays in wild-type C57BL/6 (8 Gy) and DNA double-strand break repair-deficient Atm^–/– (4 Gy) and Prkdc^scid (3 Gy) mutants of C57BL/6. Overall, 780 genes were significantly differentially expressed in wild-type mice one day postirradiation, 232 in Atm^–/– and 269 in Prkdc^scid. Upstream regulators including TP53 and NFκB were predicted to be activated by radiation exposure in the wild-type mice, but not in either of the DNA repair-deficient mutant strains. There was also a significant muting of the apparent inflammatory response triggered by radiation in both mutant strains. These differences impacted the ability of gene expression signatures developed in wild-type mice to detect potentially fatal radiation exposure in the DNA repair-deficient mice, with the greatest impact on Atm^–/– mice. However, the inclusion of mutant mice in gene selection vastly improved performance of the classifiers.

Clinical management of primary and secondary central nervous system (CNS) malignancies frequently includes radiotherapy to forestall tumor growth and recurrence after surgical resection. While cranial radiotherapy remains beneficial, adult and pediatric brain tumor survivors suffer from a wide range of debilitating and progressive cognitive deficits. Although this has been recognized as a significant problem for decades, there remains no clinical recourse for the unintended neurocognitive sequelae associated with these types of cancer treatments. In previous work, multiple mechanisms have been identified that contribute to radiation-induced cognitive dysfunction, including the inhibition of neurogenesis caused by the depletion of radiosensitive populations of stem and progenitor cells in the hippocampus. To explore the potential neuroprotective properties of a pro-neurogenic compound NSI-189, Long-Evans rats were subjected to a clinically relevant fractionated irradiation protocol followed by four weeks of NSI-189 administered daily by oral gavage. Animals were then subjected to five different behavioral tasks followed by an analysis of neurogenesis, hippocampal volume and neuroinflammation. Irradiated cohorts manifested significant behavioral decrements on all four spontaneous exploration tasks. Importantly, NSI-189 treatment resulted in significantly improved performance in four of these tasks: novel place recognition, novel object recognition, object in place and temporal order. In addition, there was a trend of improved performance in the contextual phase of the fear conditioning task. Importantly, enhanced cognition in the NSI-189-treated cohort was found to persist one month after the cessation of drug treatment. These neurocognitive benefits of NSI-189 coincided with a significant increase in neurogenesis and a significant decrease in the numbers of activated microglia compared to the irradiated cohort that was given vehicle alone. The foregoing changes were not accompanied by major changes in hippocampal volume. These data demonstrate that oral administration of a pro-neurogenic compound exhibiting anti-inflammatory indications could impart long-term neurocognitive benefits in the irradiated brain.

Nuclear factor kappaB (NF-κB) is a central transcription factor in the immune system and modulates cell survival in response to radiotherapy. Activation of NF-κB was shown to be an early step in the cellular response to ultraviolet A (UVA) and ionizing radiation exposure in human cells. NF-κB activation by the genotoxic stress-dependent sub-pathway after exposure to different radiation qualities had been evaluated to a very limited extent. In addition, the resulting gene expression profile, which shapes the cellular and tissue response, is unknown. Therefore, in this study the activation of NF-κB after exposure to low- and high-linear energy transfer (LET) radiation and the expression of its target genes were analyzed in human embryonic kidney (HEK) cells. The activation of NF-κB via canonical and genotoxic stress-induced pathways was visualized by the cell line HEK-pNF-κB-d2EGFP/Neo L2 carrying the destabilized enhanced green fluorescent protein (d2EGFP) as reporter. The NF-κB-dependent d2EGFP expression after irradiation with X rays and heavy ions was evaluated by flow cytometry. Because of differences in the extent of NF-κB activation after irradiation with X rays (significant NF-κB activation for doses >4 Gy) and heavy ions (significant NF-κB activation at doses as low as 1 Gy), it was expected that radiation quality (LET) played an important role in the cellular radiation response. In addition, the relative biological effectiveness (RBE) of NF-κB activation and reduction of cellular survival were compared for heavy ions having a broad LET range (∼0.3–9,674 keV/μm). Furthermore, the effect of LET on NF-κB target gene expression was analyzed by real-time reverse transcriptase quantitative PCR (RT-qPCR). The maximal RBE for NF-κB activation and cell killing occurred at an LET value of 80 and 175 keV/μm, respectively. There was a dose-dependent increase in expression of NF-κB target genes NF-κB1A and CXCL8. A qPCR array of 84 NF-κB target genes revealed that TNF and a set of CXCL genes (CXCL1, CXCL2, CXCL8, CXCL10), CCL2, VCAM1, CD83, NF-κB1, NF-κB2 and NF-κBIA were strongly upregulated after exposure to X rays and neon ions (LET 92 keV/μm). After heavy-ion irradiations, it was noted that the expression of NF-κB target genes such as chemokines and CD83 was highest at an LET value that coincided with the LET resulting in maximal NF-κB activation, whereas expression of the NF-κB inhibitory gene NFKBIA was induced transiently by all radiation qualities investigated. Taken together, these findings clearly demonstrate that NF-κB activation and NF-κB-dependent gene expression by heavy ions are highest in the LET range of ∼50–200 keV/μm. The upregulated chemokines and cytokines (CXCL1, CXCL2, CXCL10, CXCL8/IL-8 and TNF) could be important for cell–cell communication among hit as well as nonhit cells (bystander effect).

Mortality from circulatory disease (CD), ischemic heart disease (IHD) and cerebrovascular disease (CeVD) was investigated in relationship to cumulative doses of external gamma radiation and internal alpha radiation to the liver from deposited plutonium over long follow-up periods in two large cohorts of nuclear workers: the Russian Mayak Worker Cohort (MWC) and the UK Sellafield Worker Cohort (SWC). The MWC comprised 22,374 workers (74.6% males) with 5,123 CD deaths registered during 842,538 person-years of follow-up, while the SWC comprised 23,443 workers (87.8% males) with 2,322 CD deaths registered during 602,311 person-years of follow-up. Dose estimates for external gamma radiation and internal alpha radiation to the liver were calculated via a common methodology, in accordance with an agreed protocol. The mean cumulative external H_p(10) dose was 0.52 Sv for the MWC and 0.07 Sv for the SWC, while the mean cumulative internal dose was 0.19 Gy for the MWC and 0.01 Gy for the SWC. Categorical relative risks (RR) and excess relative risks (ERR) per unit dose were estimated for each cohort and for the pooled cohort when appropriate. The dose responses for CD, IHD and CeVD in relationship to internal alpha-particle dose did not differ significantly from the null for either the MWC, the SWC or the pooled plutonium worker cohort. The ERR/Sv estimates in relationship to external exposure were significantly raised for both cohorts (marginally so for the MWC) for CD and IHD (but not for CeVD), but differed significantly between the two cohorts, the estimate for the SWC being approximately ten times greater than that for the MWC. Examination of the ERR/Sv estimates for two periods of first employment at the two facilities revealed that the significant heterogeneity was confined to the earlier sub-cohorts, and that the estimates for the later sub-cohorts were compatible. The two sub-cohorts for the later first-employment periods were pooled, producing risk estimates that were raised, but not significantly so: ERR/Sv for CD, IHD and CeVD of 0.22 (95% CI: –0.01, 0.49), 0.22 (95% CI: –0.06, 0.57) and 0.24 (95% CI: –0.17, 0.80), respectively. The reasons for the complex pattern of results found in this study are unclear. Among potential explanations are the influence of differences in background CD mortality rates, an effect of other occupational factors, substantial uncertainties in doses, particularly during earlier periods of operations, as well as confounding and/or modifying factors that were not taken into account in the current analysis.

The research for high-throughput diagnostic tests for victims of radio/nuclear incidents remains ongoing. In this context, we have previously identified candidate genes that predict risk of late-occurring hematologic acute radiation syndrome (HARS) in a baboon model. The goal of the current study was to validate these genes after radiation exposure in humans. We also examined ex vivo relative to in vivo measurements in both species and describe dose-response relationships. Eighteen baboons were irradiated in vivo to simulate different patterns of partial- or total-body irradiation (TBI), corresponding to an equivalent dose of 2.5 or 5 Sv. Human in vivo blood samples were obtained from patients exposed to different dose ranges: diagnostic computerized tomography (CT; 0.004–0.018 Sv); radiotherapy for prostate cancer (0.25–0.3 Sv); and TBI of leukemia patients (2 × 1.5 or 2 × 2 Sv, five patients each). Peripheral whole blood of another five baboons and human samples from five healthy donors were cultivated ex vivo and irradiated with 0–4 Sv. RNA was isolated pairwise before and 24 h after irradiation and converted into cDNA. Gene expression of six promising candidate genes found previously by us in a baboon model (WNT3, POU2AF1, CCR7, ARG2, CD177, WLS), as well as three genes commonly used in ex vivo whole blood experiments (FDXR, PCNA, DDB2) was measured using qRT-PCR. We confirmed the six baboon candidate genes in leukemia patients. However, expression for the candidate gene FDXR showed an inverse relationship, as it was downregulated in baboons and upregulated in human samples. Comparisons among the in vivo and ex vivo experiments revealed the same pattern in both species and indicated peripheral blood cells to represent the radiation-responsive targets causing WNT3 and POU2AF1 gene expression changes. CCR7, ARG2, CD177 and WLS appeared to be altered due to radiation-responsive targets other than the whole blood cells. Linear dose-response relationships of FDXR, WNT3 and POU2AF1 using human ex vivo samples corresponded with human in vivo samples, suggesting that ex vivo models for in vivo dose estimates can be used over a wide dose range (0.001–5 Sv for POU2AF1). In summary, we validated six baboon candidate genes in humans, but the FDXR measurements underscored the importance of independent assessments even when candidates from animal models have striking gene sequence homology to humans. Since whole blood cells represented the same radiation-responsive targets for FDXR, WNT3 and POU2AF1 gene expression changes, ex vivo cell culture models can be utilized for in vivo dose estimates over a dose range covering up to 3.5 log scales. These findings might be a step forward in the development of a gene expression-based high-throughput diagnostic test for populations involved in large-scale radio/nuclear incidents.

This study describes the retrospective lens dose calculation methods developed and applied within the European epidemiological study on radiation-induced lens opacities among interventional cardiologists. While one approach focuses on self-reported data regarding working practice in combination with available procedure-specific eye lens dose values, the second approach focuses on the conversion of the individual whole-body dose to eye lens dose. In contrast with usual dose reconstruction methods within an epidemiological study, a protocol is applied resulting in an individual distribution of possible cumulative lens doses for each recruited cardiologist, rather than a single dose estimate. In this way, the uncertainty in the dose estimate (from measurement uncertainty and variability among cardiologists) is represented for each individual. Eye lens dose and whole-body dose measurements have been performed in clinical practice to validate both methods, and it was concluded that both produce acceptable results in the framework of a dose-risk evaluation study. Optimal results were obtained for the dose to the left eye using procedure-specific lens dose data in combination with information collected on working practice. This method has been applied to 421 interventional cardiologists resulting in a median cumulative eye lens dose of 15.1 cSv for the left eye and 11.4 cSv for the right eye. From the individual cumulative eye lens dose distributions obtained for each cardiologist, maxima up to 9–10 Sv were observed, although with low probability. Since whole-body dose values above the lead apron are available for only a small fraction of the cohort and in many cases not for the entire working career, the second method has only been used to benchmark the results from the first approach. This study succeeded in improving the retrospective calculation of cumulative eye lens doses in the framework of radiation-induced risk assessment of lens opacities, but it remains dependent on self-reported information, which is not always reliable for early years. However, the calculation tools developed can also be used to make an assessment of the eye lens dose in current practice.

For dendritic cells (DCs) to initiate an immune response, their ability to migrate and to produce interleukin-12 (IL-12) is crucial. It has been previously shown that low-dose radiation (LDR) promoted IL-12 production by DCs, resulting in increased DC activity that contributed to LDR hormesis in the immune system. However, the molecular mechanism of LDR-induced IL-12 production, as well as the effect of LDR on DC migration capacity require further elucidation. Using the JAWSII immortalized mouse dendritic cell line, we showed that in vitro X-ray irradiation (0.2 Gy) of DCs significantly increased DC migration and IL-12 production, and upregulated CCR7. The neutralizing antibody against CCR7 has been shown to abolish LDR-enhanced DC migration, demonstrating that CCR7 mediates LDR-promoting DC migration. We identified nuclear factor kappaB (NF-κB) as the central signaling pathway that mediated LDR-enhanced expression of IL-12 and CCR7 based on findings that 0.2 Gy X-ray irradiation activated NF-κB, showing increased nuclear p65 translocation and NF-κB DNA-binding activity, while an NF-κB inhibitor blocked LDR-enhanced expression of IL-12 and CCR7, as well as DC migration. Finally, we demonstrated that 0.2 Gy X-ray irradiation promoted ATM phosphorylation and reactive oxygen species generation; however, only the ATM inhibitor abolished the LDR-induced NF-κB-mediated expression of IL-12 and CCR7. Altogether, our data show that exposure to LDR resulted in a hormetic effect on DCs regarding CCR7-mediated migration and IL-12 production by activating the ATM/NF-κB pathway.

Nontargeted late effects of radiation include an increased risk of cardiovascular disease, although this is still debatable in the context of low-dose radiation. Tinea capitis patients treated in childhood with X rays to induce scalp epilation received a low dose of radiation to their carotids. To better clarify this issue, we evaluated carotid atherosclerosis in a cohort of such patients treated in 1950–1963 in Portugal. A group of 454 individuals randomly chosen from previously observed Portuguese tinea capitis patients and a control group mainly composed of their spouses (n = 280) were enrolled. Cardiovascular risk factors such as waist circumference, body mass index, blood pressure and tobacco consumption, as well as biochemical measurements were obtained. Ultrasound imaging of carotid arteries for intima media thickness and stenosis evaluation were performed according to a standardized protocol. In comparison to the control group, the irradiated cohort members were significantly older, more frequently never smokers, hypertensive, and presented higher glycated hemoglobin and alkaline phosphatase levels. In addition, the irradiated cohort showed a higher frequency of carotid stenosis ≥30% than the nonirradiated group (13.9% vs. 10.7%), although this was not significant (P = 0.20). Stenosis was ≥50% in 2.9% of the irradiated group and 0.4% of the nonirradiated group (P = 0.02). Likewise, the frequency of intima media thickness ≥1 mm was significantly higher in the irradiated group (16.8% vs. 10.7%; P = 0.02). Multivariate analysis, including other cardiovascular risk factors, showed that exposure to low-dose radiation increased the risk of carotid stenosis by ≥50% [odds ratio (OR) = 8.85; P = 0.04] and intima media thickness by ≥1 mm (OR = 1.82; P = 0.02). These findings confirm that low-dose exposure is a risk factor of carotid atherosclerotic disease.

Pregnant C57BL/6JJcl mice were exposed to γ rays at low dose rate (20 mGy/day, LDR) or medium dose rate (200 and 400 mGy/day, MDR) from gestation day (GD) 0–18 to total accumulated doses of 360, 3,600 and 7,200 mGy, respectively. An additional group of pregnant mice were acutely exposed to 2 Gy at high dose rate (HDR) of 0.77 Gy/min on GD 11. In experiment 1, fetuses collected via cesarean section on GD 18 were examined for external and skeletal abnormalities. While the results of LDR exposure (20 mGy/day) did not significantly differ from the nonirradiated controls in all parameters examined, MDR (200 and 400 mGy/day) and acute HDR (2 Gy) exposure caused growth retardation and significantly increased incidence of unossified bones. Increased incidence of external abnormalities was observed only in the acute HDR group. In experiment 2, the dams were allowed to give birth and the pups were clinically monitored and weighed periodically until 10 weeks of age when they were sacrificed and subjected to pathological examination. Pups exposed at MDRs of 200 and 400 mGy/dayand at acute HDR of 0.77 Gy/min had lower bodyweights from weaning (3 weeks) to 10 weeks of age except for females exposed to 400 mGy/day MDR. None of the pups exposed to an acute 2 Gy dose on GD 11 survived to 10 weeks of age. Histopathological changes were not significantly different between the nonirradiated control and the 20 mGy/day LDR groups. However, at both MDR exposures of 200 and 400 mGy/day. gonadal (testes and ovary) hypoplasia/atrophy was observed in all the 10-week-old pups. Our results show that in utero LDR exposure to 20 mGy/day for the entire gestation period did not cause any significant effect in pups when compared to the nonirradiated controls up to 10 weeks of age. However, pups exposed in utero to MDRs showed dose-related growth retardation with delayed ossifications (400 mGy/day) and gonadal hypoplasia/atrophy. These findings suggest that increased post-implantation loss in dams exposed at MDR is due to early embryonic deaths resulting in early resorption.

In a scenario involving a nuclear detonation during war or a terrorist attack, acute radiation exposure combined with thermal and blast effects results in severe skin injury. Although the cutaneous injury in such a scenario may not be lethal, it may lead to inflammation, delayed wound healing and loss of the skin barrier, resulting in an increased risk of infection. In this study, we tested the potential use of timolol, a beta-adrenergic receptor antagonist, to improve epidermal wound closure after combined burn and radiation injury using an ex vivo human skin culture model. Daily application of 10 μM timolol after combined injury (burn and 10 Gy ex vivo irradiation) increased wound epithelialization by 5–20%. In addition, exposure to 10 Gy significantly suppressed epidermal keratinocyte proliferation by 46% at 48 h postirradiation. Similar to what has been observed in a thermal burn injury, the enzyme phenylethanolamine N-methyltransferase (PNMT), which generates epinephrine, was elevated in the combined thermal burn and radiation wounds. This likely resulted in elevated tissue levels of this catecholamine, which has been shown to delay healing. Thus, with the addition of timolol to the wound to block the binding of locally generated epinephrine to the beta-adrenergic receptor, healing is improved. This work suggests that by antagonizing local epinephrine action within the wound, a beta-adrenergic receptor antagonist such as timolol may be a useful adjunctive treatment to improve healing in the combined burn and radiation injury.