bioRxiv preprint doi: https://doi.org/10.1101/207951; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 2 3 4 Elucidation of dose-dependent transcriptional events immediately following 5 ionizing radiation exposure 6 7 Eric C. Rouchka1,2,*, Robert M. Flight3, Brigitte H. Fasciotto4, Rosendo Estrada5, John W. 8 Eaton6,7,8, Phani K. Patibandla5, Sabine J. Waigel8, Dazhuo Li1, John K. Kirtley1, Palaniappan 9 Sethu9,10, and Robert S. Keynton5 10 11 1Department of Computer Engineering and Computer Science, University of Louisville, 12 Louisville, Kentucky, United States of America 13 14 2Kentucky Biomedical Research Infrastructure Network Bioinformatics Core, University of 15 Louisville, Louisville, Kentucky, United States of America 16 17 3Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, 18 Kentucky, United States of America 19 20 4The ElectroOptics Research Institute and Nanotechnology Center, University of Louisville, 21 Louisville, Kentucky, United States of America 22 23 5Department of Bioengineering, University of Louisville, Louisville, Kentucky, United States of 24 America 25 26 6Department of Medicine, University of Louisville, Louisville, Kentucky, United States of 27 America 28 29 7Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky, 30 United States of America 31 32 8James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, United 33 States of America 34 35 9Division of Cardiovascular Disease, Department of Medicine, University of Alabama at 36 Birmingham, Birmingham, Alabama, United States of America 37 38 10Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, 39 Alabama, United States of America 1 bioRxiv preprint doi: https://doi.org/10.1101/207951; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 40 41 *Corresponding author 42 E-mail: [email protected] (ECR) 43 44 Author contributions 45 ECR, RMF, DL, and JKK designed and performed all computational analyses. BHF, RE, JWE, 46 PKP and PS designed the wet lab experiments and oversaw blood sample collection. SJW 47 prepared and ran the microarray experiments. ECR drafted manuscript. RSK provided oversight 48 and input into the overall experimental design. 49 50 Short Title: Transcriptional events following early radiation exposure 2 bioRxiv preprint doi: https://doi.org/10.1101/207951; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 51 Abstract 52 53 Long duration space missions expose astronauts to ionizing radiation events associated with 54 highly energetic and charged heavy particles. Such exposure can result in chromosomal 55 aberrations increasing the likelihood of the development of cancer. Early detection and 56 mitigation of these events is critical in providing positive outcomes. In order to aid in the 57 development of portable devices used to measure radiation exposure, we constructed a genome- 58 wide screen to detect transcriptional changes in peripheral blood lymphocytes shortly after 59 (approximately 1 hour) radiation exposure at low (0.3 Gy), medium (1.5 Gy) and high (3.0 Gy) 60 doses compared to control (0.0 Gy) using Affymetrix® Human Gene 1.0 ST v1 microarrays. 61 Our results indicate a number of sensitive and specific transcriptional profiles induced by 62 radiation exposure that can potentially be implemented as biomarkers for radiation exposure as 63 well as dose effect. For overall immediate radiation exposure, KDELC1, MRPS30, RARS, and 64 HEXIM1 were determined to be effective biomarkers while PRDM9, CHST4, and SLC26A10 65 were determined to be biomarkers specific to 0.3 Gy exposure; RPH, CCDC96, WDYHV1, and 66 IFNA16 were identified for 1.5 Gy exposure; and CWC15, CHCHD7, and DNAAF2 were 67 determined to be sensitive and specific to 3.0 Gy exposure. The resulting raw and analyzed data 68 are publicly available through NCBI’s Gene Expression Ominibus via accession GSE64375. 69 70 Introduction 71 The National Aeronautics and Space Administration Authorization Act of 2010 (1) and the 72 National Space Policy of the United States of America (2010) (2) set in motion the goals for 73 cislunar and deep space exploration. Among these goals are manned missions for the Asteroid 74 Redirect Mission (3) and Journey to Mars (4). The long duration of these missions (up to 1,100 3 bioRxiv preprint doi: https://doi.org/10.1101/207951; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 75 days), necessitates the development of lightweight and portable devices for monitoring health. 76 The top health risk for astronauts on such missions beyond low earth orbit (LEO) is exposure to 77 ionizing radiation associated with highly energetic and charged heavy particles originating from 78 constant galactic cosmic rays and sporadic solar particle events (5-7). This exposure can lead to 79 increased risk of cancer (8-14); deficits in the central nervous system (15-23); degenerative 80 tissue effects (24, 25) including changes attributed to the increase in oxidative stress that are 81 normally associated with aging, such as cataract formation (26-28) and vascular degeneration 82 (29, 30); and acute radiation syndrome (31-34) marked by decreased circulating blood cells (35, 83 36), lung damage (37), decreased cardiac function (38, 39), and immune system suppression (5). 84 Current methodologies for measuring radiation exposure still have a high degree of 85 uncertainty when it comes to determining the level of radiation to which an astronaut crew 86 member has been exposed (5, 40-42). Most of the methods to date have focused on detecting 87 chromosomal aberrations, including single- (SSB) and double-stranded (DSB) breaks, 88 translocations, and exchanges. DSBs have been used extensively, due to their direct relation 89 with radio-induced biological effects, including unequal crossover, chromosomal rings (43), 90 inversions, and dicentric chromosomes (44-46). Detection of these aberrations using either 91 Giemsa staining techniques or fluorescent in-situ hybridization (FISH) (7, 47) have become the 92 de facto gold standard for biological dosimetry, showing a dosage exposure accuracy rate of ± 93 10% (48). While these are currently the most reliable methods in detecting chromosomal 94 aberrations, it is likely the development of high-throughput genome-wide screens of radiation 95 exposure will allow for the detection of sensitive and specific transcriptional biomarkers which 96 can be used in biodosimeters for detecting changes in response to radiation (47). 4 bioRxiv preprint doi: https://doi.org/10.1101/207951; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 97 On an individual gene level, a number of genes have been used as biomarkers for 98 radiation exposure at either the transcript, protein, or modified protein level, including 99 phosphorylated H2A Histone Family, Member X (γH2AFX), Tumor Protein 53 (TP53), and 100 Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A). Detection of the phosphorylation of H2AFX 101 has been used in assays to determine radiation exposure due to its role in DNA double-stranded 102 break repair (49-53) and is perhaps the best example of a single protein modification used for 103 radiation detection. TP53 is known to function as a transcription factor, which is radiation- 104 modulated (54-59), and CDKN1A is a downstream target of TP53, which regulates progression 105 through the cell cycle (60-62). 106 While each of these biomarkers have been shown to be sensitive to radiation exposure, 107 they fall short in their specificity at a transcriptional level. Therefore, the purpose of this work is 108 to identify additional transcriptional biomarkers which are both sensitive and specific to low, 109 medium, and high levels of radiation at 1 hr post-exposure. The focus of our study is specifically 110 on ionizing radiation from γ-rays and does not consider the specific effects of other sources of 111 ionizing radiation (such as α-particles, β-particles, and positrons) which may also have an effect 112 on transcriptional activity. 113 114 Materials and methods 115 116 Experimental design 117 Details of the study design have been previous described in Rouchka et al. (63). In summary, the 118 design was constructed to determine transcriptional changes within white blood cells (WBC) at 119 an early time point (1 hr) following ionizing radiation exposure. Whole blood was drawn from 120 four volunteers prior to irradiation. Samples were maintained at room temperature throughout 121 the radiation and WBC isolation process. The blood samples were then irradiated with γ- 5 bioRxiv preprint doi: https://doi.org/10.1101/207951; this version posted October 23, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.