Broustas et al. BMC Genomics (2018) 19:504 https://doi.org/10.1186/s12864-018-4884-6 RESEARCHARTICLE Open Access Identification of differentially expressed genes and pathways in mice exposed to mixed field neutron/photon radiation Constantinos G. Broustas1* , Andrew D. Harken2, Guy Garty2 and Sally A. Amundson1 Abstract Background: Radiation exposure due to the detonation of an improvised nuclear device remains a major security concern. Radiation from such a device involves a combination of photons and neutrons. Although photons will make the greater contribution to the total dose, neutrons will certainly have an impact on the severity of the exposure as they have high relative biological effectiveness. Results: We investigated the gene expression signatures in the blood of mice exposed to 3 Gy x-rays, 0.75 Gy of neutrons, or to mixed field photon/neutron with the neutron fraction contributing 5, 15%, or 25% of a total 3 Gy radiation dose. Gene ontology and pathway analysis revealed that genes involved in protein ubiquitination pathways were significantly overrepresented in all radiation doses and qualities. On the other hand, eukaryotic initiation factor 2 (EIF2) signaling pathway was identified as one of the top 10 ranked canonical pathways in neutron, but not pure x-ray, exposures. In addition, the related mTOR and regulation of EIF4/p70S6K pathways were also significantly underrepresented in the exposures with a neutron component, but not in x-ray radiation. The majority of the changed genes in these pathways belonged to the ribosome biogenesis and translation machinery and included several translation initiation factors (e.g. Eif2ak4, Eif3f), as well as 40S and 60S ribosomal subunits (e.g. Rsp19, Rpl19, Rpl27). Many of the differentially downregulated ribosomal genes (e.g. RPS19, RPS28) have been causally associated with human bone marrow failure syndromes and hematologic malignancies. We also observed downregulation of transfer RNA processes, in the neutron-only exposure (p < 0.005). Ingenuity Pathway Analysis (p < 0.05) of differentially expressed genes predicted significantly suppressed activity of the upstream regulators c-Myc and Mycn, transcription factors known to control ribosome biogenesis. Conclusions: We describe the gene expression profile of mouse blood following exposure to mixed field neutron/ photon irradiation. We have discovered that pathways related to protein translation are significantly underrepresented in the exposures containing a neutron component. Our results highlight the significance of neutron exposures that even the smallest percentage can have profound biological effects that will affect medical management and treatment decisions in case of a radiological emergency. Keywords: Mixed field neutron/photon, Gene expression, Radiation biodosimetry, Mouse blood * Correspondence: [email protected] 1Center for Radiological Research, Columbia University Medical Center, 630 West 168th Street, New York, NY 10032, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Broustas et al. BMC Genomics (2018) 19:504 Page 2 of 14 Background There have been a limited number of publications de- Radiation exposure due to the detonation of an impro- scribing the effect of mixed neutron/photon field irradi- vised nuclear device poses a growing threat to human ation on the survival of various strains of mice and health. In such a scenario, large populations will be ex- non-human primates [12–17]. In these studies the neu- posed to various doses of radiation. A number of fatal- tron component ranges from 50 to 70% of the total radi- ities will occur immediately after the incident, whereas ation dose, somewhat higher than the proportion of an even larger number of individuals will experience neutron dose expected in an IND. These studies have long-term effects such as higher incidence of cancer, in- shown that exposure to neutron/photon irradiation results fections and other diseases. However, many people at a in increased defects in T cell populations, greater suscepti- distance from the directly affected area will also receive bility to bacterial infections, and reduced overall survival radiation that poses no immediate health risk. The goal compared with animals irradiated with pure photons. of radiation biodosimetry is to quickly and accurately In the present study, we analyzed transcriptomic evaluate radiation dose as a means to assess radiological changes over a range of mixed neutron/photon radiation injury. A wide variety of radiation biodosimetry methods doses with the neutron component contributing 5, 15, has been developed, ranging from cytogenetic assays [1] 25%, of the total dose of 3 Gy. For comparison, we also to the more recently proposed miRNA [2], metabolo- included mice exposed to 3 Gy of pure x-rays or 0.75 Gy mics [3], and gene expression analyses. Unbiased tran- neutron radiations (equivalent to the neutron compo- scriptomic profiling has been used to assess individual nent in the 83% neutron exposure). To mimic the radi- exposure to radiation. We and others have focused on ation conditions of an IND detonation, we used the peripheral blood cells for the development of transcrip- broad-spectrum neutron irradiator at the Columbia Uni- tomic approaches. Global gene expression in blood from versity Radiological Research Accelerator Facility mice exposed to photons (x-rays, γ-rays) has shown that (RARAF) that is capable of generating neutrons with an gene expression changes with radiation dose, time, and energy spectrum mirroring that produced at 1–1.5 km dose rate [4–6]. Although radiation quality is expected from the epicenter of the Hiroshima blast [18, 19]. A to play an important role in driving gene expression, and small percentage of photons (~ 17%) contributes to total different signaling pathways may be triggered in re- radiation dose generated by the neutron source [19]. sponse to the different types of irradiation, a limited To our knowledge this is the first description of the number of such studies have been performed. For ex- gene expression patterns in mouse peripheral blood after ample, a small number of studies have focused on com- a mixed-field radiation exposure. Our ultimate goal is to paring gene expression profiles in various tissues investigate whether gene expression signatures can ac- between photon and heavy ions or α-particles, which curately identify the neutron component in a mixed field could be relevant to a radiological dispersal device. De- exposure. pending on the cell type, similar or distinct gene expres- sion profiles have been described [7–9]. Methods The detonation of an improvised nuclear device could Animals and irradiation expose individuals to a mixture of photon and neutron ir- All animal experiments were conducted in accordance radiation. Although most of the radiation dose will come with applicable federal and state guidelines and were ap- from photons, a small part of it will be due to neutrons; proved by the Columbia University Institutional Animal however, neutron’s potential of radiological injury will be Care and Use Committee (IACUC) (approval number disproportionally large, since neutrons have high relative AC-AAAG4356). Male C57BL/6 mice were received biological effectiveness (RBE), as they are able to generate from Charles River Laboratories (Frederick, MD) and DNA damage that is irreparable or difficult to repair [10]. quarantined for 2 weeks before irradiation at 8–10 weeks We previously compared the gene expression signa- of age. Six mice were used for each treatment and sacri- tures in blood of mice exposed separately to either ficed 7 days after treatment. x-rays or neutrons at 1 and 7 days post-irradiation [11]. Mice were either sham-irradiated or exposed to x-ray, Our analysis showed differential gene expression effects neutron or mixed field neutron/x-ray radiation as de- of neutrons versus photons. Gene ontology analysis re- tailed in Table 1. The neutron source delivers a small vealed that genes involved in nucleic acid metabolism percentage of photons, as well. Therefore the maximum processes were downregulated in neutron-irradiated achievable dose of neutrons is approximately 83%. Neu- mice, whereas genes involved in lipid metabolism were tron irradiations were performed using the upregulated in x-ray irradiated animals. Cell cycle pro- accelerator-based IND-spectrum neutron source [19]at cesses were significant among down-regulated genes at the Radiological Research Accelerator Facility (RARAF). day 1, but among up-regulated genes at day 7 after ex- The characteristics of the neutron source and the mouse posure to either neutron or x-rays. exposure protocol have been detailed previously [11]. Broustas et al. BMC Genomics (2018) 19:504 Page 3 of 14 Table 1 Protocol of mixed field neutron/x-ray exposure using default parameters for background correction and Neutron Photon x-ray Total Neutron flagging non-uniform features. Dose, Gy dose dose dose Fraction Background-corrected hybridization intensities were 0 0 3.00 3.00 0% imported into BRB-ArrayTools, version 4.5.0, 0.15 0.03 2.82 3.00 5% log2-transformed and median normalized. Non-uniform 0.45 0.08 2.47 3.00 15% outliers or features not significantly above background intensity in 25% or more of the hybridizations were fil- 0.75 0.13 2.12 3.00 25% tered out. In addition, a minimum 1.5-fold change in at 0.75 0.13 0 0.75 83% least 20% of the hybridizations was set as a requirement.